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Posts Tagged 'tumorigenesis'
Curbing Cancer Cell Growth & Metastasis-on-a-Chip' Models Cancer's Spread
Posted in 3D Plotting Scaffolds, Anaerobic Glycolysis, BioIT: BioInformatics, NGS, Clinical & Translational, Pharmaceutical R&D Informatics, Clinical Genomics, Cancer Informatics, Biological Networks, BioPrinting in Regenerative Medicine, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Clinical Genomics, Drug Development using MultiOrgan Chip, MicroEngineering Cell-Tissue & Systems, Oxidative phosphorylation, Warburg effect, tagged 3D printing, Acute Leukemia, anti-tumor pharma, antibody-drug conjugates, ASP2215, Cancer - General, Chemotherapy, Clonal evolution, Drug discovery, FMS-like tyrosine kinase-3 (FLT3) inhibitors, Hypoxia-inducible factors, Immunotherapy, metastasis, monitoring, nanoparticle, organ on a chip, Philadelphia chromosome (BCR-ABL), pluripotency factors, Ponatinib, reporter, self-renewal, serine metabolism, tumorigenesis, Wolf Prize in Chemistry on March 19, 2016| Leave a Comment »
Curator: Larry H. Bernstein, MD, FCAP
New Approach to Curbing Cancer Cell Growth
http://www.technologynetworks.com/Metabolomics/news.aspx?ID=189342
Using a new approach, scientists at The Scripps Research Institute (TSRI) and collaborating institutions have discovered a novel drug candidate that could be used to treat certain types of breast cancer, lung cancer and melanoma.
The new study focused on serine, one of the 20 amino acids (protein building blocks) found in nature. Many types of cancer require synthesis of serine to sustain rapid, constant and unregulated growth.
To find a drug candidate that interfered with this pathway, the team screened a large library of compounds from a variety of sources, searching for molecules that inhibited a specific enzyme known as 3-phosphoglycerate dehydrogenase (PHGDH), which is responsible for the first committed step in serine biosynthesis.
"In addition to discovering an inhibitor that targets cancer metabolism, we also now have a tool to help answer interesting questions about serine metabolism," said Luke L. Lairson, assistant professor of chemistry at TSRI and principal investigator of cell biology at the California Institute for Biomedical Research (CALIBR).
Lairson was senior author of the study, published recently in the Proceedings of the National Academy of Sciences (PNAS), with Lewis Cantley of Weill Cornell Medical College and Costas Lyssiotis of the University of Michigan.
Addicted to Serine
Serine is necessary for nucleotide, protein and lipid biosynthesis in all cells. Cells use two main routes for acquiring serine: through import from the extracellular environment or through conversion of 3-phosphoglycerate (a glycolytic intermediate) by PHGDH.
"Since the late 1950s, it has been known that cancer cells use the process of aerobic glycolysis to generate metabolites needed for proliferative growth," said Lairson.
This process can lead to an overproduction of serine. The genetic basis for this abundance had remained mysterious until recently, when it was demonstrated that some cancers acquire mutations that increased the expression of PHGDH; reducing PHGDH in these "serine-addicted" cancer cells also inhibited their growth.
The labs of Lewis C. Cantley at Weill Cornell Medical College (in work published in Nature Genetics) and David Sabatini at the Whitehead Institute (in work published in Nature) suggested PHGDH as a potential drug target for cancer types that overexpress the enzyme.
Lairson and colleagues hypothesized that a small molecule drug candidate that inhibited PHGDH could interfere with cancer metabolism and point the way to the development of an effective cancer therapeutic. Importantly, this drug candidate would be inactive against normal cells because they would be able to import enough serine to support ordinary growth.
As Easy as 1-2-800,000
Lairson, in collaboration with colleagues including Cantley, Lyssiotis, Edouard Mullarky of Weill Cornell and Harvard Medical School and Natasha Lucki of CALIBR, screened through a library of 800,000 small molecules using a high-throughput in vitro enzyme assay to detect inhibition of PHGDH. The group identified 408 candidates and further narrowed this list down based on cell-type specific anti-proliferative activity and by eliminating those inhibitors that broadly targeted other dehydrogenases.
With the successful identification of seven candidate inhibitors, the team sought to determine if these molecules could inhibit PHGDH in the complex cellular environment. To do so, the team used a mass spectrometry-based assay (test) to measure newly synthesized serine in a cell in the presence of the drug candidates.
One of the seven small molecules tested, named CBR-5884, was able to specifically inhibit serine synthesis by 30 percent, suggesting that the molecule specifically targeted PHGDH. The group went on to show that CBR-5884 was able to inhibit cell proliferation of breast cancer and melanoma cells lines that overexpress PHGDH.
As expected, CBR-5884 did not inhibit cancer cells that did not overexpress PHGDH, as they can import serine; however, when incubated in media lacking serine, the presence of CBR-5884 decreased growth in these cells.
The group anticipates much optimization work before this drug candidate can become an effective therapeutic. In pursuit of this goal, the researchers plan to take a medicinal chemistry approach to improve potency and metabolic stability.
How Cancer Stem Cells Thrive When Oxygen Is Scarce
3/29/2016 Johns Hopkins University http://www.biosciencetechnology.com/news/2016/03/how-cancer-stem-cells-thrive-when-oxygen-scarce
Working with human breast cancer cells and mice, scientists at The Johns Hopkins University say new experiments explain how certain cancer stem cells thrive in low oxygen conditions. Proliferation of such cells, which tend to resist chemotherapy and help tumors spread, are considered a major roadblock to successful cancer treatment.
The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.
"There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells," said Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center. "That gives us a few more possible targets for drugs that diminish their threat in human cancer."
A summary of the findings was published online March 21 in the Proceedings of the National Academy of Sciences.
"Aggressive cancers contain regions where the cancer cells are starved for oxygen and die off, yet patients with these tumors generally have the worst outcome. Our new findings tell us that low oxygen conditions actually encourage certain cancer stem cells to multiply through the same mechanism used by embryonic stem cells."
All stem cells are immature cells known for their ability to multiply indefinitely and give rise to progenitor cells that mature into specific cell types that populate the body's tissues during embryonic development. They also replenish tissues throughout the life of an organism. But stem cells found in tumors use those same attributes and twist them to maintain and enhance the survival of cancers.
Recent studies showed that low oxygen conditions increase levels of a family of proteins known as HIFs, or hypoxia-inducible factors, that turn on hundreds of genes, including one called NANOG that instructs cells to become stem cells.
Studies of embryonic stem cells revealed that NANOG protein levels can be lowered by a chemical process known as methylation, which involves putting a methyl group chemical tag on a protein's messenger RNA (mRNA) precursor. Semenza said methylation leads to the destruction of NANOG's mRNA so that no protein is made, which in turn causes the embryonic stem cells to abandon their stem cell state and mature into different cell types.
Zeroing in on NANOG, the scientists found that low oxygen conditions increased NANOG's mRNA levels through the action of HIF proteins, which turned on the gene for ALKBH5, which decreased the methylation and subsequent destruction of NANOG's mRNA. When they prevented the cells from making ALKBH5, NANOG levels and the number of cancer stem cells decreased. When the researchers manipulated the cell's genetics to increase levels of ALKBH5 without exposing them to low oxygen, they found this also decreased methylation of NANOG mRNA and increased the numbers of breast cancer stem cells.
Finally, using live mice, the scientists injected 1,000 triple-negative breast cancer cells into their mammary fat pads, where the mouse version of breast cancer forms. Unaltered cells created tumors in all seven mice injected with such cells, but when cells missing ALKBH5 were used, they caused tumors in only 43 percent (six out of 14) of mice. "That confirmed for us that ALKBH5 helps preserve cancer stem cells and their tumor-forming abilities," Semenza said.
How cancer stem cells thrive when oxygen is scarce https://www.sciencedaily.com/releases/2016/03/160328100159.htm
"There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells," says Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center.
Chuanzhao Zhang, Debangshu Samanta, Haiquan Lu, John W. Bullen, Huimin Zhang, Ivan Chen, Xiaoshun He, Gregg L. Semenza.
Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.
Proceedings of the National Academy of Sciences, 2016; 201602883 DOI: 10.1073/pnas.1602883113
Pluripotency factors, such as NANOG, play a critical role in the maintenance and specification of cancer stem cells, which are required for primary tumor formation and metastasis. In this study, we report that exposure of breast cancer cells to hypoxia (i.e., reduced O2 availability), which is a critical feature of the tumor microenvironment, induces N6-methyladenosine (m6A) demethylation and stabilization of NANOG mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype. We show that inhibiting the expression of AlkB homolog 5 (ALKBH5), which demethylates m6A, or the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α, which activate ALKBH5 gene transcription in hypoxic breast cancer cells, is an effective strategy to decrease NANOG expression and target BCSCs in vivo.
N6-methyladenosine (m6A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α–dependent expression of AlkB homolog 5 (ALKBH5), an m6A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m6A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.
Specific Proteins Found to Jump Start Spread of Cancer Cells
http://www.genengnews.com/gen-news-highlights/specific-proteins-found-to-jump-start-spread-of-cancer-cells/81252417/
Metastatic breast cancer cells. [National Cancer Institute]
http://www.genengnews.com/Media/images/GENHighlight/thumb_Feb29_2016_NCI_MetastaticBreastCancerCells1797514764.jpg
Scientists at the University of California, San Diego School of Medicine and Moores Cancer Center, with colleagues in Spain and Germany, have discovered how elevated levels of particular proteins in cancer cells trigger hyperactivity in other proteins, fueling the growth and spread of a variety of cancers. Their study ("Prognostic Impact of Modulators of G Proteins in Circulating Tumor Cells from Patients with Metastatic Colorectal Cancer") is published in Scientific Reports.
Specifically, the international team, led by senior author Pradipta Ghosh, M.D., associate professor at the University of California San Diego School of Medicine, found that increased levels of expression of some members of a protein family called guanine nucleotide exchange factors (GEFs) triggered unsuspected hyperactivation of G proteins and subsequent progression or metastasis of cancer.
The discovery suggests GEFs offer a new and more precise indicator of disease state and prognosis. "We found that elevated expression of each GEF is associated with a shorter, progression-free survival in patients with metastatic colorectal cancer," said Dr. Ghosh. "The GEFs fared better as prognostic markers than two well-known markers of cancer progression, and the clustering of all GEFs together improved the predictive accuracy of each individual family member."
In recent years, circulating tumor cells (CTCs), which are shed from primary tumors into the bloodstream and act as seeds for new tumors taking root in other parts of the body, have become a prognostic and predictive biomarker. The presence of CTCs is used to monitor the efficacy of therapies and detect early signs of metastasis.
But counting CTCs in the bloodstream has limited utility, said Dr. Ghosh. "Enumeration alone does not capture the particular characteristics of CTCs that are actually tumorigenic and most likely to cause additional malignancies."
Numerous efforts are underway to improve the value and precision of CTC analysis. According to Dr. Ghosh the new findings are a step in that direction. First, GEFs activate trimeric G proteins, and second, G protein signaling is involved in CTCs. G proteins are ubiquitous and essential molecular switches involved in transmitting external signals from stimuli into cells' interiors. They have been a subject of heightened scientific interest for many years.
Dr. Ghosh and colleagues found that elevated expression of nonreceptor GEFs activates Gαi proteins, fueling CTCs and ultimately impacting the disease course and survival of cancer patients.
"Our work shows the prognostic impact of elevated expression of individual and clustered GEFs on survival and the benefit of transcriptome analysis of G protein regulatory proteins in cancer biology," said Dr. Ghosh. "The next step will be to carry this technology into the clinic where it can be applied directly to deciphering a patient's state of cancer and how best to treat."
Metastasis-on-a-Chip' Models Cancer's Spread
http://www.mdtmag.com/news/2016/03/metastasis-chip-models-cancers-spread?et_cid=5200644&et_rid=461755519
In the journal Biotechnology Bioengineering, the team reports on its "metastasis-on-a-chip" system believed to be one of the first laboratory models of cancer spreading from one 3D tissue to another.
The current version of the system models a colorectal tumor spreading from the colon to the liver, the most common site of metastasis. Skardal said future versions could include additional organs, such as the lung and bone marrow, which are also potential sites of metastasis. The team also plans to model other types of cancer, such as the deadly brain tumor glioblastoma
To create the system, researchers encapsulated human intestine and colorectal cancer cells inside a biocompatible gel-like material to make a mini-organ. A mini-liver composed of human liver cells was made in the same way. These organoids were placed in a "chip" system made up of a set of micro-channels and chambers etched into the chip's surface to mimic a simplified version of the body's circulatory system. The tumor cells were tagged with fluorescent molecules so their activity could be viewed under a microscope.
To test whether the system could model metastasis, the researchers first used highly aggressive cancer cells in the colon organoid. Under the microscope, they saw the tumor grow in the colon organoid until the cells broke free, entered the circulatory system and then invaded the liver tissue, where another tumor formed and grew. When a less aggressive form of colon cancer was used in the system, the tumor did not metastasize, but continued to grow in the colon.
To test the system's potential for screening drugs, the team introduced Marimastat, a drug used to inhibit metastasis in human patients, into the system and found that it significantly prevented the migration of metastatic cells over a 10-day period. Likewise, the team also tested 5-fluorouracil, a common colorectal cancer drug, which reduced the metabolic activity of the tumor cells.
"We are currently exploring whether other established anti-cancer drugs have the same effects in the system as they do in patients," said Skardal. "If this link can be validated and expanded, we believe the system can be used to screen drug candidates for patients as a tool in personalized medicine. If we can create the same model systems, only with tumor cells from an actual patient, then we believe we can use this platform to determine the best therapy for any individual patient."
The scientists are currently working to refine their system. They plan to use 3D printing to create organoids more similar in function to natural organs. And they aim to make the process of metastasis more realistic. When cancer spreads in the human body, the tumor cells must break through blood vessels to enter the blood steam and reach other organs. The scientists plan to add a barrier of endothelial cells, the cells that line blood vessels, to the model.
This concept of modeling the body's processes on a miniature level is made possible because of advances in micro-tissue engineering and micro-fluidics technologies. It is similar to advances in the electronics industry made possible by miniaturizing electronics on a chip.
Scientists Synthesize Anti-Cancer Agent
Fri, 03/11/2016 Rice University
http://www.dddmag.com/news/2016/03/scientists-synthesize-anti-cancer-agent
A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University http://www.dddmag.com/sites/dddmag.com/files/ddd1603_rice-anticancer.jpg
A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.
The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.
Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.
"Not only does this synthesis render these valuable molecules readily available for biological investigation, but it also allows the previously unknown full structural elucidation of one of them," Nicolaou said. "The newly developed synthetic technologies will allow us to construct variations for biological evaluation as part of a program to optimize their pharmacological profiles."
At present, there are no drugs based on trioxacarcins, which damage DNA through a novel mechanism, Nicolaou said.
Trioxacarcins were discovered in the fermentation broth of the bacterial strain Streptomyces bottropensis. They disrupt the replication of cancer cells by binding and chemically modifying their genetic material.
"These molecules are endowed with powerful anti-tumor properties," Nicolaou said. "They are not as potent as shishijimicin, which we also synthesized recently, but they are more powerful than taxol, the widely used anti-cancer drug. Our objective is to make it more powerful through fine-tuning its structure."
He said his lab is working with a biotechnology partner to pair these cytotoxic compounds (called payloads) to cancer cell-targeting antibodies through chemical linkers. The process produces so-called antibody-drug conjugates as drugs to treat cancer patients. "It's one of the latest frontiers in personalized targeting chemotherapies," said Nicolaou, who earlier this year won the prestigious Wolf Prize in Chemistry.
Fluorescent Nanoparticle Tracks Cancer Treatment's Effectiveness in Hours
Bevin Fletcher, Associate Editor http://www.biosciencetechnology.com/news/2016/03/fluorescent-nanoparticle-tracks-cancer-treatments-effectiveness-hours
Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women's Hospital)
Bioengineers at Brigham and Women's Hospital have developed a new technique to help determine if chemotherapy is working in as few as eight hours after treatment. The new approach, which can also be used for monitoring the effectiveness of immunotherapy, has shown success in pre-clinical models.
The technology utilizes a nanoparticle, carrying anti-cancer drugs, that glows green when cancer cells begin dying. Researchers, using the "reporter nanoparticles" that responds to a particular enzyme known as caspase, which is activated when cells die, were able to distinguish between a tumor that is drug-sensitive or drug-resistant much faster than conventional detection methods such as PET scans, CT and MRI. The findings were published online March 28 in the Proceedings of the National Academy of Sciences.
"Using this approach, the cells light up the moment a cancer drug starts working," co-corresponding author Shiladitya Sengupta, Ph.D., principal investigator in BWH's Division of Bioengineering, said in a prepared statement. "We can determine if a cancer therapy is effective within hours of treatment. Our long-term goal is to find a way to monitor outcomes very early so that we don't give a chemotherapy drug to patients who are not responding to it."
Cancer killers send signal of success
Nanoparticles deliver drug, then give real-time feedback when tumor cells die BY SARAH SCHWARTZ
New lab-made nanoparticles deliver cancer drugs into tumors, then report their effects in real time by lighting up in response to proteins produced by dying cells. More light (right, green) indicates a tumor is responding to chemotherapy.
Tiny biochemical bundles carry chemotherapy drugs into tumors and light up when surrounding cancer cells start dying. Future iterations of these lab-made particles could allow doctors to monitor the effects of cancer treatment in real time, researchers report the week of March 28 in theProceedings of the National Academy of Sciences.
"This is the first system that allows you to read out whether your drug is working or not," says study coauthor Shiladitya Sengupta, a bioengineer at Brigham and Women's Hospital in Boston.
Each roughly 100-nanometer-wide particle consists of a drug and a fluorescent dye linked to a coiled molecular chain. Before the particles enter cells, the dye is tethered to a "quencher" molecule that prevents it from lighting up. When injected into the bloodstream of a mouse with cancer, the nanoparticles accumulate in tumor cells and release the drug, which activates a protein that tears a cancer cell apart. This cell-splitting protein not only kills the tumor cell, but also severs the link between the dye and the quencher, allowing the nanoparticles to glow under infrared light.
Reporter nanoparticle that monitors its anticancer efficacy in real time
Ashish Kulkarnia,b,1,Poornima Raoa,b,Siva Natarajana,b,Aaron Goldman, et al.
The ability to identify responders and nonresponders very early during chemotherapy by direct visualization of the activity of the anticancer treatment and to switch, if necessary, to a regimen that is effective can have a significant effect on the outcome as well as quality of life. Current approaches to quantify response rely on imaging techniques that fail to detect very early responses. In the case of immunotherapy, the early anatomical readout is often discordant with the biological response. This study describes a self-reporting nanomedicine that not only delivers chemotherapy or immunotherapy to the tumor but also reports back on its efficacy in real time, thereby identifying responders and nonresponders early on
The ability to monitor the efficacy of an anticancer treatment in real time can have a critical effect on the outcome. Currently, clinical readouts of efficacy rely on indirect or anatomic measurements, which occur over prolonged time scales postchemotherapy or postimmunotherapy and may not be concordant with the actual effect. Here we describe the biology-inspired engineering of a simple 2-in-1 reporter nanoparticle that not only delivers a cytotoxic or an immunotherapy payload to the tumor but also reports back on the efficacy in real time. The reporter nanoparticles are engineered from a novel two-staged stimuli-responsive polymeric material with an optimal ratio of an enzyme-cleavable drug or immunotherapy (effector elements) and a drug function-activatable reporter element. The spatiotemporally constrained delivery of the effector and the reporter elements in a single nanoparticle produces maximum signal enhancement due to the availability of the reporter element in the same cell as the drug, thereby effectively capturing the temporal apoptosis process. Using chemotherapy-sensitive and chemotherapy-resistant tumors in vivo, we show that the reporter nanoparticles can provide a real-time noninvasive readout of tumor response to chemotherapy. The reporter nanoparticle can also monitor the efficacy of immune checkpoint inhibition in melanoma. The self-reporting capability, for the first time to our knowledge, captures an anticancer nanoparticle in action in vivo.
Cancer Treatment's New Direction
Genetic testing helps oncologists target tumors and tailor treatments
http://www.wsj.com/articles/cancer-treatments-new-direction-1459193085
Evan Johnson had battled a cold for weeks, endured occasional nosebleeds and felt so fatigued he struggled to finish his workouts at the gym. But it was the unexplained bruises and chest pain that ultimately sent the then 23-year-old senior at the University of North Dakota to the Mayo Clinic. There a genetic test revealed a particularly aggressive form of acute myeloid leukemia. That was two years ago.
The harrowing roller-coaster that followed for Mr. Johnson and his family highlights new directions oncologists are taking with genetic testing to find and attack cancer. Tumors can evolve to resist treatments, and doctors are beginning to turn such setbacks into possible advantages by identifying new targets to attack as the tumors change.
His course involved a failed stem cell transplant, a half-dozen different drug regimens, four relapses and life-threatening side effects related to his treatment.
Nine months in, his leukemia had evolved to develop a surprising new mutation. The change meant the cancer escaped one treatment, but the new anomaly provided doctors with a fresh target, one susceptible to drugs approved for other cancers. Doctors adjusted Mr. Johnson's treatment accordingly, knocked out the disease and paved the way for a second, more successful stem cell transplant. He has now been free of leukemia for a year.
Now patients with advanced cancer who are treated at major centers can expect to have their tumors sequenced, in hopes of finding a match in a growing medicine chest of drugs that precisely target mutations that drive cancer's growth. When they work, such matches can have a dramatic effect on tumors. But these "precision medicines" aren't cures. They are often foiled when tumors evolve, pushing doctors to take the next step to identify new mutations in hopes of attacking them with an effective treatment.
Dr. Kasi and his Mayo colleagues—Naseema Gangat, a hematologist, and Shahrukh Hashmi, a transplant specialist—are among the authors of an account of Mr. Johnson's case published in January in the journal Leukemia Research Reports.
Before qualifying for a transplant, a patient's blasts need to be under 5%.
To get under 5%, he started on a standard chemotherapy regimen and almost immediately, things went south. His blast cells plummeted, but "the chemo just wiped out my immune system,"
Then as mysteriously as it began, a serious mycotic throat infection stopped. But Mr. Johnson couldn't tolerate the chemo, and his blast cells were on the rise. A two-drug combination that included the liver cancer drug Nexavar, which targets the FLT3 mutation, knocked back the blast cells. But the stem cell transplant in May, which came from one of his brothers, failed to take, and he relapsed after 67 days, around late July.
He was put into a clinical trial of an experimental AML drug being developed by Astellas Pharma of Japan. He started to regain weight. In November 2014, doctors spotted the initial signs in blood tests that Mr. Johnson's cancer was evolving to acquire a new mutation. By late January, he relapsed again , but there was a Philadelphia chromosome mutation, a well-known genetic alteration associated with chronic myeloid leukemia. It also is a target of the blockbuster cancer drug Gleevec and several other medicines.
Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3) inhibitor therapy with evolving actionable targets
Naseema Gangat, Mark R. Litzow, Mrinal M. Patnaik, Shahrukh K. Hashmi, Naseema Gangat
Leukemia Research Reports Volume 5, 2016, Pages 7–10 doi:10.1016/j.lrr.2016.01.002
• The article reports on a case of AML that underwent clonal evolution.
• We report on novel acquisition of the Philadelphia t(9;22) translocation in AML.
• Next generation sequencing maybe helpful in these refractory/relapse cases.
• Novel FLT3-inhibitor targeted therapies are another option in patients with AML.
• Personalizing cancer treatment based on evolving targets is a viable option.
For acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215) to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉)
The development of kinase inhibitors for the treatment of leukemia has revolutionized the care of these patients. Since the introduction of imatinib for the treatment of chronic myeloid leukemia, multiple other tyrosine kinase inhibitors (TKIs) have become available[1]. Additionally, for acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors [2], [3], [4] and [5]. The article herein reports a unique case of AML that underwent clonal evolution while on a novel FLT3-inhibitor clinical trial.
Our work herein presents clinical and next generation sequencing data at the time of progression to illustrate these important concepts stemming from Darwinian evolution [6]. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process.
Our work focuses on a 23-year-old male who presented with 3 months history of fatigue and easy bruising, a white blood count of 22.0×109/L with 51% circulating blasts, hemoglobin 7.6 g/dL, and a platelet count of 43×109/L. A bone marrow biopsy confirmed a diagnosis of AML. Initial cytogenetic studies identified trisomy 8 in all the twenty metaphases examined. Mutational analysis revealed an internal tandem duplication of the FLT3 gene (FLT3-ITD).
He received standard induction chemotherapy (7+3) with cytarabine (ARA-C; 100 mg/m2for 7 days) and daunorubicin (DNM; 60 mg/m2 for 3 days). His induction chemotherapy was complicated by severe palatine and uvular necrosis of indeterminate etiology (possible mucormycosis).
Bone marrow biopsy at day 28 demonstrated persistent disease with 10% bone marrow blasts (Fig. 1). Due to his complicated clinical course and the presence of a FLT3-ITD, salvage therapy with 5-azacitidine (5-AZA) and sorafenib (SFN) was instituted. Table 1.
The highlighted therapies were employed in this particular case at various time points as shown in Fig. 1.
http://ars.els-cdn.com/content/image/1-s2.0-S221304891530025X-gr1.jpg
J.E. Cortes, D.W. Kim, J. Pinilla-Ibarz, et al.
A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias
New Engl. J. Med., 369 (19) (2013), pp. 1783–1796
F. Ravandi, M.L. Alattar, M.R. Grunwald, et al.
Phase 2 study of azacytidine plus sorafenib in patients with acute myeloid leukemia and FLT-3 internal tandem duplication mutation
Blood, 121 (23) (2013), pp. 4655–4662
N.P. Shah, M. Talpaz, M.W. Deininger, et al.
Ponatinib in patients with refractory acute myeloid leukaemia: findings from a phase 1 study
Br. J. Haematol., 162 (4) (2013), pp. 548–552
Y. Alvarado, H.M. Kantarjian, R. Luthra, et al.
Treatment with FLT3 inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated with development of secondary FLT3-tyrosine kinase domain mutations
Cancer, 120 (14) (2014), pp. 2142–2149
C.C. Smith, C. Zhang, K.C. Lin, et al.
Characterizing and overriding the structural mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L mutation with PLX3397
Cancer Discov. (2015)
M. Greaves, C.C. Maley
Clonal evolution in cancer
Nature, 481 (7381) (2012), pp. 306–313
Pathway Specific Targeting in Anticancer Therapies [7.7]
Posted in Academic Publishing, Amino acids, Biochemical pathways, Biological Networks, Biomarkers & Medical Diagnostics, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Clinical Diagnostics, Curation, Cytoskeleton, Disease Biology, Fatty acids, Gene Regulation, Genome Biology, Innovations, Lipid metabolism, Lipids, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Oxidative phosphorylation, Pharmaceutical Discovery, Proteins, Proteomics, RNA Biology, Warburg effect, tagged acylation, Cancer - General, cancer stem cell (CSC), cell adhesion, cell membrane, cell mobility, dysmetabolism, fatty acid acyl Coa Synthase, fatty acid synthesis, Glycolysis, Hepatocellular carcinoma, intercellular bridging, mass spectrometry, Metabolomics, metastasis, novel therapeutic targets, ovarian cancer, Progenitor cell, protein-protein interaction networks, tumorigenesis on April 9, 2015| 2 Comments »
Pathway Specific Targeting in Anticancer Therapies
Writer and Curator: Larry H. Bernstein, MD, FCAP
7.7 Pathway specific targeting in anticancer therapies
7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism
7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.
7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells
7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells
7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling
7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth
7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis
7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells
7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation
Thangavelua, CQ Pana, …, BC Lowa, and J. Sivaramana
Proc Nat Acad Sci 2012; 109(20):7705–7710
http://dx.doi.org:/10.1073/pnas.1116573109
Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.
The Warburg effect in cancer biology describes the tendency of cancer cells to take up more glucose than most normal cells, despite the availability of oxygen (1, 2). In addition to altered glucose metabolism, glutaminolysis (catabolism of glutamine to ATP and lactate) is another hallmark of cancer cells (2, 3). In glutaminolysis, mitochondrial glutaminase catalyzes the conversion of glutamine to glutamate (4), which is further catabolized in the Krebs cycle for the production of ATP, nucleotides, certain amino acids, lipids, and glutathione (2, 5).
Humans express two glutaminase isoforms: KGA (kidney-type) and LGA (liver-type) from two closely related genes (6). Although KGA is important for promoting growth, nothing is known about the precise mechanism of its activation or inhibition and how its functions are regulated under physiological or pathophysiological conditions. Inhibition of rat KGA activity by antisense mRNA results in decreased growth and tumorigenicity of Ehrlich ascites tumor cells (7), reduced level of glutathione, and induced apoptosis (8), whereas Myc, an oncogenic transcription factor, stimulates KGA expression and glutamine metabolism (5). Interestingly, direct suppression of miR23a and miR23b (9) or activation of TGF-β (10) enhances KGA expression. Similarly, Rho GTPase that controls cytoskeleton and cell division also up-regulates KGA expression in an NF-κB–dependent manner (11). In addition, KGA is a substrate for the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1, linking glutaminolysis to cell cycle progression (12). In comparison, function and regulation of LGA is not well studied, although it was recently shown to be linked to p53 pathway (13, 14). Although intense efforts are being made to develop a specific KGA inhibitor such as BPTES [bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] (15), its mechanism of inhibition and selectivity is not yet understood. Equally important is to understand how KGA function is regulated in normal and cancer cells so that a better treatment strategy can be considered.
The previous crystal structures of microbial (Mglu) and Escherichia coli glutaminases show a conserved catalytic domain of KGA (16, 17). However, detailed structural information and regulation are not available for human glutaminases especially the KGA, and this has hindered our strategies to develop inhibitors. Here we report the crystal structure of the catalytic domain of human apo KGA and its complexes with substrate (L-glutamine), product (L-glutamate), BPTES, and its derived inhibitors. Further, Raf-Mek-Erk module is identified as the regulator of KGA activity. Although BPTES is not recognized in the active site, its binding confers a drastic conformational change of a key loop (Glu312-Pro329), which is essential in stabilizing the catalytic pocket. Significantly, EGF activates KGA activity, which can be abolished by the kinase-dead, dominant negative mutants of Mek2 (Mek2-K101A) or its upstream activator Raf-1 (Raf-1-K375M), which are the kinase components of the growth-promoting Raf-Mek2-Erk signaling node. Furthermore, coexpression of phosphatase PP2A and treatment with Mek-specific inhibitor or alkaline phosphatase all abolished enhanced KGA activity inside the cells and in vitro, indicating that stimulation of KGA is phosphorylation dependent. Our results therefore provide mechanistic insights into KGA inhibition by BPTES and its regulation by EGF-mediated Raf-Mek-Erk module in cell growth and possibly cancer manifestation.
Structures of cKGA and Its Complexes with L-Glutamine and L-Glutamate.
The human KGA consists of 669 amino acids. We refer to Ile221-Leu533 as the catalytic domain of KGA (cKGA) (Fig. 1A). The crystal structures of the apo cKGA and in complex with L-glutamine or L-glutamate were determined (Table S1). The structure of cKGA has two domains with the active site located at the interface. Domain I comprises (Ile221-Pro281 and Cys424 -Leu533) of a five-stranded anti-parallel β-sheet (β2↓β1↑β5↓β4↑β3↓) surrounded by six α-helices and several loops. The domain II (Phe282-Thr423) mainly consists of seven α-helices. L-Glutamine/L-glutamate is bound in the active site cleft (Fig. 1B and Fig. S1B). Overall the active site is highly basic, and the bound ligand makes several hydrogen-bonding contacts to Gln285, Ser286, Asn335, Glu381, Asn388, Tyr414, Tyr466, and Val484 (Fig. 1C and Fig. S1C), and these residues are highly conserved among KGA homologs (Fig. S1D). Notably, the putative serine-lysine catalytic dyad (286-SCVK-289), corresponding to the SXXK motif of class D β-lactamase (17), is located in close proximity to the bound ligand. In the apo structure, two water molecules were located in the active site, one of them being displaced by glutamine in the substrate complex. The substrate side chain is within hydrogen-bonding distance (2.9 Å) to the active site Ser286. Other key residues involved in catalysis, such as Lys289, Tyr414, and Tyr466, are in the vicinity of the active site. Lys289 is within hydrogen-bonding distance to Ser286 (3.1 Å) and acts as a general base for the nucleophilic attack by accepting the proton from Ser286. Tyr466, which is close to Ser286 and in hydrogen-bonding contact (3.2 Å) with glutamine, is involved in proton transfer during catalysis. Moreover, the carbonyl oxygen of the glutamine is hydrogen-bonded with the main chain amino groups of Ser286 and Val484, forming the oxyanion hole. Thus, we propose that in addition to the putative catalytic dyad (Ser286 XX Lys289), Tyr466 could play an important role in the catalysis (Fig. 1Cand Fig. S2).
structure of the cKGA-L-glutamine complex
http://www.pnas.org/content/109/20/7705/F1.medium.gif
Fig. 1. Schematic view and structure of the cKGA-L-glutamine complex. (A) Human KGA domains and signature motifs (refer to Fig. S1A for details). (B) Structure of the of cKGA and bound substrate (L-glutamine) is shown as a cyan stick. (C) Fourier 2Fo-Fc electron density map (contoured at 1 σ) for L-glutamine, that makes hydrogen bonds with active site residues are shown.
Allosteric Binding Pocket for BPTES. The chemical structure of BPTES has an internal symmetry, with two exactly equivalent parts including a thiadiazole, amide, and a phenyl group (Fig. S3A), and it equally interacts with each monomer. The thiadiazole group and the aliphatic linker are well buried in a hydrophobic cluster that consists of Leu321, Phe322, Leu323, and Tyr394 from both monomers, which forms the allosteric pocket (Fig. 2 B–E). The side chain of Phe322 is found at the bottom of the allosteric pocket. The phenyl-acetamido moiety of BPTES is partially exposed on the loop (Asn324-Glu325), where it interacts with Phe318, Asn324, and the aliphatic part of the Glu325 side chain. On the basis of our observations we synthesized a series of BPTES-derived inhibitors (compounds2–5) (Fig. S3 A–F and SI Results) and solved their cocrystal structure of compounds 2–4. Similar to BPTES, compounds 2–4 all resides within the hydrophobic cluster of the allosteric pocket (Fig. S3 C–F).
Fig. 2. Structure of cKGA: BPTES complex and the allosteric binding mode of BPTES.
Allosteric Binding of BPTES Triggers Major Conformational Change in the Key Loop Near the Active Site. The overall structure of these inhibitor complexes superimposes well with apo cKGA. However, a major conformational change at the Glu312 to Pro329 loop was observed in the BPTES complex (Fig. 2F). The most conformational changes of the backbone atoms that moved away from the active site region are found at the center of the loop (Leu316-Lys320). The backbone of the residues Phe318 and Asn319 is moved ≈9 Å and ≈7 Å, respectively, compared with the apo structure, whereas the side chain of these residues moved ≈14 Å and ≈12 Å, respectively. This loop rearrangement in turn brings Phe318 closer to the phenyl group of the inhibitor and forms the inhibitor binding pocket, whereas in the apo structure the same loop region (Leu316-Lys320) was found to be adjacent to the active site and forms a closed conformation of the active site.
Binding of BPTES Stabilizes the Inactive Tetramers of cKGA. To understand the role of oligomerization in KGA function, dimers and tetramers of cKGA were generated using the symmetry-related monomers (Fig. 2 A–E and Fig. S4 D and E). The dimer interface in the cKGA: BPTES complex is formed by residues from the helix Asp386-Lys398 of both monomers and involves hydrogen bonding, salt bridges, and hydrophobic interactions (Phe389, Ala390, Tyr393, and Tyr394), besides two sulfate ions located in the interface (Fig. 2E). The dimers are further stabilized by binding of BPTES, where it binds to loop residues (Glu312-Pro329) and Tyr394 from both monomers (Fig. 2 D and E). Similarly, residues from Lys311-Asn319 loop and Arg454, His461, Gln471, and Asn529-Leu533 are involved in the interface with neighboring monomers to form the tetramer in the BPTES complex.
BPTES Induces Allosteric Conformational Changes That Destabilize Catalytic Function of KGA
Fig. 3A shows that 293T cells overexpressing KGA produced higher level of glutamate compared with the vector control cells. Most significantly, all of these mutants, except Phe322Ala, greatly diminished the KGA activity.
Fig. 3. Mutations at allosteric loop and BPTES binding pocket abrogate KGA activity and BPTES sensitivity.
Raf-Mek-Erk Signaling Module Regulates KGA Activity. Because KGA supports cell growth and proliferation, we first validated that treatment of cells with BPTES indeed inhibits KGA activity and cell proliferation (Fig. S5 A–D and SI Results). Next, as cells respond to various physiological stimuli to regulate their metabolism, with many of the metabolic enzymes being the primary targets of modulation (18), we examined whether KGA activity can be regulated by physiological stimuli, in particular EGF, which is important for cell growth and proliferation. Cells overexpressing KGA were made quiescent and then stimulated with EGF for various time points. Fig. 4A shows that the basal KGA activity remained unchanged 30 min after EGF stimulation, but the activity was substantially enhanced after 1 h and then gradually returned to the basal level after 4 h. Because EGF activates the Raf-Mek-Erk signaling module (19), treatment of cells with Mek-specific inhibitor U0126 could block the enhanced KGA activity with parallel inhibition of Erk phosphorylation (Fig. 4A). Interestingly, such Mek-induced KGA activity is specific to EGF and lysophosphatidic acid (LPA) but not with other growth factors, such as PDGF, TGF-β, and basic FGF (bFGF), despite activation of Mek-Erk by bFGF (Fig. S6A).
The results show that KGA could interact equally well with the wild-type or mutant forms of Raf-1 and Mek2 (Fig. 4C). Importantly, endogenous Raf-1 or Erk1/2, including the phosphorylated Erk1/2 (Fig. 4 C and D), could be detected in the KGA complex. Taken together, these results indicate that the activity of KGA is directly regulated by Raf-Mek-Erk downstream of EGF receptor. To further show that Mek2-enhanced KGA activity requires both the kinase activity of Mek2 and the core residues for KGA catalysis, wild-type or triple mutant (Leu321Ala/Phe322Ala/Leu323Ala) of KGA was coexpressed with dominant negative Mek2-KA or the constitutive active Mek2-SD and their KGA activities measured. The result shows that the presence of Mek2-KA blocks KGA activity, whereas the triple mutant still remains inert even in the presence of the constitutively active Mek2 (Fig. 4E), and despite Mek2 binding to the KGA triple mutant (Fig. S7B). Consequently, expressing triple mutant did not support cell proliferation as well as the wild-type control (Fig. S7C).
Fig. 4. EGFR-Raf-Mek-Erk signaling stimulates KGA activity.
When cells expressing both KGA and Mek2-K101A were treated with subthreshold levels of BPTES, there was a synergistic reduction in cell proliferation (Fig. S6C and SI Results). Lastly, to determine whether regulation of KGA by Raf-Mek-Erk depends on its phosphorylation status, cells were transfected with KGA with or without the protein phosphatase PP2A and assayed for the KGA activity. PP2A is a ubiquitous and conserved serine/threonine phosphatase with broad substrate specificity. The results indicate that KGA activity was reduced down to the basal level in the presence of PP2A (Fig. 5A). Coimmunoprecipitation study also revealed that KGA interacts with PP2A (Fig. 5B), suggesting a negative feedback regulation by this protein phosphatase. Furthermore, treatment of immunoprecipitated and purified KGA with calf-intestine alkaline phosphatase (CIAP) almost completely abolished the KGA activity in vitro (Fig. S6D). Taken together, these results indicate that KGA activity is regulated by Raf-Mek2, and KGA activation by EGF could be part of the EGF-stimulated Raf-Mek-Erk signaling program in controlling cell growth and proliferation (Fig. 5C).
KGA activity is regulated by phosphorylation
Fig. 5. KGA activity is regulated by phosphorylation. (C) Schematic model depicting the synergistic cross-talk between KGA-mediated glutaminolysis and EGF-activated Raf-Mek-Erk signaling. Exogenous glutamine can be transported across the membrane and converted to glutamate by glutaminase (KGA), thus feeding the metabolite to the ATP-producing tricarboxylic acid (TCA) cycle. This process can be stimulated by EGF receptor-mediated Raf-Mek-Erk signaling via their phosphorylation-dependent pathway, as evidenced by the inhibition of KGA activity by the kinase-dead and dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-specific inhibitor U0126. Consequently, inhibiting KGA with BPTES and blocking Raf-Mek pathway with Mek2-K101A provide a synergistic inhibition on cell proliferation.
Small-molecule inhibitors that target glutaminase activity in cancer cells are under development. Earlier efforts targeting glutaminase using glutamine analogs have been unsuccessful owing to their toxicities (2). BPTES has attracted much attention as a selective, nontoxic inhibitor of KGA (15), and preclinical testing of BPTES toward human cancers has just begun (20). BPTES selectively suppresses the growth of glioma cells (21) and inhibits the growth of lymphoma tumor growth in animal model studies (22). Wang et al. (11) reported a small molecule that targets glutaminase activity and oncogenic transformation. Despite extensive studies, nothing is known about the structural and molecular basis for KGA inhibitory mechanisms and how their function is regulated during normal and cancer cell metabolism. Such limited information impedes our effort in producing better generations of inhibitors for better treatment regimens.
Comparison of the complex structures with apo cKGA structure, which has well-defined electron density for the key loop, we provide the atomic view of an allosteric binding pocket for BPTES and elucidate the inhibitory mechanism of KGA by BPTES. The key residues of the loop (Glu312-Pro329) undergo major conformational changes upon binding of BPTES. In addition, structure-based mutagenesis studies suggest that this loop is essential for stabilizing the active site. Therefore, by binding in an allosteric pocket, BPTES inhibits the enzymatic activity of KGA through (i) triggering a major conformational change on the key residues that would normally be involved in stabilizing the active sites and regulating its enzymatic activity; and (ii) forming a stable inactive tetrameric KGA form. Our findings are further supported by two very recent reports on KGA isoform (GAC) (23, 24), although these studies lack full details owing to limitation of their electron density maps. BPTES is specific to KGA but not to LGA (15). Sequence comparison of KGA with LGA (Fig. S8A) reveals two unique residues on KGA, Phe318 and Phe322, which upon mutation to LGA counterparts, become resistant to BPTES. Thus, our study provides the molecular basis of BPTES specificity.
Islam SS, Mokhtari RB, Noman AS, …, van der Kwast T, Yeger H, Farhat WA.
Molec Carcinogenesis mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22300
shh sonic hedgehog signaling pathway nri2151-f1
Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis
Sonic_hedgehog_pathway
Gaikwad SM, Thakur B, Sakpal A, Singh RK, Ray P.
Int J Biochem Cell Biol. 2015 Apr; 61:90-102
http://dx.doi.org:/10.1016/j.biocel.2015.02.001
Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.
Kuboki M, Ito A, Simizu S, Umezawa K.
Biochem Biophys Res Commun. 2015 Jan 16; 456(3):810-4
http://dx.doi.org:/10.1016/j.bbrc.2014.12.024
Activation of caspase 3 and caspase-dependent apoptosis nrmicro2071-f1
We have prepared RelB mutants that are resistant to caspase 3-induced scission.
Vinblastine induced caspase 3-dependent site-specific RelB cleavage in cancer cells.
Cancer cells expressing cleavage-resistant RelB showed less sensitivity to vinblastine.
Caspase 3-induced RelB cleavage may provide positive feedback mechanism in apoptosis.
DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.
apoptotic-caspases-control-microglia-activation-cdd2011107f3
He G, Dhar D, Nakagawa H, Font-Burgada J, Ogata H, Jiang Y, et al.
Cell. 2013 Oct 10; 155(2):384-96
http://dx.doi.org/10.1016%2Fj.cell.2013.09.031
Il-6 signaling in cancer cells
Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from pre-malignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.
Clonal evolution and selective pressure may cause some descendants of the initial progenitor to cross the bridge of no return and form a premalignant lesion. Cancer genome sequencing indicates that most cancers require at least five genetic changes to evolve (Wood et al., 2007). It has been difficult to isolate and propagate cancer progenitors prior to detection of tumor masses. Further, it is not clear whether cancer progenitors are the precursors for the cancer stem cells (CSCs)isolated from cancers. An answer to these critical questions depends on identification and isolation of cancer progenitors, which may also enable definition of molecular markers and signaling pathways suitable for early detection and treatment.
Hepatocellular carcinoma (HCC), the end product of chronic liver diseases, requires several decades to evolve (El-Serag, 2011). It is the third most deadly and fifth most common cancer worldwide, and in the United States its incidence has doubled in the past two decades. Furthermore, 8% of the world's population are chronically infected with hepatitis B or C viruses (HBV and HCV) and are at a high risk of new HCC development (El-Serag, 2011). Up to 5% of HCV patients will develop HCC in their lifetime, and the yearly HCC incidence in patients with cirrhosis is 3%–5%. These tumors may arise from premalignant lesions, ranging from dysplastic foci to dysplastic hepatocyte nodules that are often seen in damaged and cirrhotic livers and are more proliferative than the surrounding parenchyma (Hytiroglou et al., 2007). There is no effective treatment for HCC and, upon diagnosis, most patients with advanced disease have a remaining lifespan of 4–6 months. Premalignant lesions, called foci of altered hepatocytes (FAH), were described in chemically induced HCC models (Pitot, 1990), but it was questioned whether these lesions harbor tumor progenitors or result from compensatory proliferation (Sell and Leffert, 2008). The aim of this study was to determine whether HCC progenitor cells (HcPCs) exist and if so, to isolate these cells and identify some of the signaling networks that are involved in their maintenance and progression.
We now describe HcPC isolation from mice treated with the procarcinogen diethyl nitrosamine (DEN), which induces poorly differentiated HCC nodules within 8 to 9 months (Verna et al., 1996). The use of a chemical carcinogen is justified because the finding of up to 121 mutations per HCC genome suggests that carcinogens may be responsible for human HCC induction (Guichard et al., 2012). Furthermore, 20%–30% of HCC, especially in HBV-infected individuals, evolve in noncirrhotic livers (El-Serag, 2011). Nonetheless, we also isolated HcPCs fromTak1Δhep mice, which develop spontaneous HCC as a result of progressive liver damage, inflammation, and fibrosis caused by ablation of TAK1 (Inokuchi et al., 2010). Although the etiology of each model is distinct, both contain HcPCs that express marker genes and signaling pathways previously identified in human HCC stem cells (Marquardt and Thorgeirsson, 2010) long before visible tumors are detected. Furthermore, DEN-induced premalignant lesions and HcPCs exhibit autocrine IL-6 production that is critical for tumorigenic progression. Circulating IL-6 is a risk indicator in several human pathologies and is strongly correlated with adverse prognosis in HCC and cholangiocarcinoma (Porta et al., 2008; Soresi et al., 2006). IL-6 produced by in-vitro-induced CSCs was suggested to be important for their maintenance (Iliopoulos et al., 2009). Little is known about the source of IL-6 in HCC.
DEN-Induced Collagenase-Resistant Aggregates of HCC Progenitors
A single intraperitoneal (i.p.) injection of DEN into 15-day-old BL/6 mice induces HCC nodules first detected 8 to 9 months later. However, hepatocytes prepared from macroscopically normal livers 3 months after DEN administration already contain cells that progress to HCC when transplanted into the permissive liver environment of MUP-uPA mice (He et al., 2010), which express urokinase plasminogen activator (uPA) from a mouse liver-specific major urinary protein (MUP) promoter and undergo chronic liver damage and compensatory proliferation (Rhim et al., 1994). HCC markers such as α fetoprotein (AFP), glypican 3 (Gpc3), and Ly6D, whose expression in mouse liver cancer was reported (Meyer et al., 2003), were upregulated in aggregates from DEN-treated livers, but not in nonaggregated hepatocytes or aggregates from control livers (Figure S1A). Using 70 μm and 40 μm sieves, we separated aggregated from nonaggregated hepatocytes (Figure 1A) and tested their tumorigenic potential by transplantation into MUP-uPA mice (Figure 1B). To facilitate transplantation, the aggregates were mechanically dispersed and suspended in Dulbecco's modified Eagle's medium (DMEM). Five months after intrasplenic (i.s.) injection of 104 viable cells, mice receiving cells from aggregates developed about 18 liver tumors per mouse, whereas mice receiving nonaggregated hepatocytes developed less than 1 tumor each (Figure 1B). The tumors exhibited typical trabecular HCC morphology and contained cells that abundantly express AFP (Figure S1B).
Only liver tumors were formed by the transplanted cells. Other organs, including the spleen into which the cells were injected, remained tumor free (Figure 1B), suggesting that HcPCs progress to cancer only in the proper microenvironment. Indeed, no tumors appeared after HcPC transplantation into normal BL/6 mice. But, if BL/6 mice were first treated with retrorsine (a chemical that permanently inhibits hepatocyte proliferation [Laconi et al., 1998]), intrasplenically transplanted with HcPC-containing aggregates, and challenged with CCl4 to induce liver injury and compensatory proliferation (Guo et al., 2002), HCCs readily appeared (Figure 1C). CCl4 omission prevented tumor development. Notably, MUP-uPA or CCl4-treated livers are fragile, rendering direct intrahepatic transplantation difficult. CCl4-induced liver damage, especially within a male liver, generates a microenvironment that drives HcPC proliferation and malignant progression. To examine this point, we transplanted GFP-labeled HcPC-containing aggregates into retrorsine-treated BL/6 mice and examined their ability to proliferate with or without subsequent CCl4 treatment. Indeed, the GFP+ cells formed clusters that grew in size only in CCl4-treated host livers (Figure S1E). Omission of CC14 prevented their expansion.
Because CD44 is expressed by HCC stem cells (Yang et al., 2008; Zhu et al., 2010), we dispersed the aggregates and separated CD44+ from CD44− cells and transplanted both into MUP-uPA mice. Whereas as few as 103 CD44+ cells gave rise to HCCs in 100% of recipients, no tumors were detected after transplantation of CD44− cells (Figure 1E). Remarkably, 50% of recipients developed at least one HCC after receiving as few as 102 CD44+ cells.
HcPC-Containing Aggregates in Tak1Δhep Mice
We applied the same HcPC isolation protocol to Tak1Δhep mice, which develop HCC of different etiology from DEN-induced HCC. Importantly, Tak1Δhep mice develop HCC as a consequence of chronic liver injury and fibrosis without carcinogen or toxicant exposure (Inokuchi et al., 2010). Indeed, whole-tumor exome sequencing revealed that DEN-induced HCC contained about 24 mutations per 106 bases (Mb) sequenced, with B-RafV637E being the most recurrent, whereas 1.4 mutations per Mb were detected inTak1Δhep HCC's exome (Table S1). By contrast, Tak1Δhep HCC exhibited gene copy number changes. HCC developed in 75% of MUP-uPA mice that received dispersed Tak1Δhep aggregates, but no tumors appeared in mice receiving nonaggregated Tak1Δhep or totalTak1f/f hepatocytes (Figure 2B). bile duct ligation (BDL) or feeding with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), treatments that cause cholestatic liver injuries and oval cell expansion (Dorrell et al., 2011), did increase the number of small hepatocytic cell aggregates (Figure S2A). Nonetheless, no tumors were observed 5 months after injection of such aggregates into MUP-uPA mice (Figure S2B). Thus, not all hepatocytic aggregates contain HcPCs, and HcPCs only appear under tumorigenic conditions.
The HcPC Transcriptome Is Similar to that of HCC and Oval Cells
To determine the relationship between DEN-induced HcPCs, normal hepatocytes, and fully transformed HCC cells, we analyzed the transcriptomes of aggregated and nonaggregated hepatocytes from male littermates 5 months after DEN administration, HCC epithelial cells from DEN-induced tumors, and normal hepatocytes from age- and gender-matched littermate controls. Clustering analysis distinguished the HCC samples from other samples and revealed that the aggregated hepatocyte samples did not cluster with each other but rather with nonaggregated hepatocytes derived from the same mouse (Figure S3A). 57% (583/1,020) of genes differentially expressed in aggregated relative to nonaggregated hepatocytes are also differentially expressed in HCC relative to normal hepatocytes (Figure 3B, top), a value that is highly significant (p < 7.13 × 10−243). More specifically, 85% (494/583) of these genes are overexpressed in both HCC and HcPC-containing aggregates (Figure 3B, bottom table). Thus, hepatocyte aggregates isolated 5 months after DEN injection contain cells that are related in their gene expression profile to HCC cells isolated from fully developed tumor nodules.
Figure 3 Aggregated Hepatocytes Exhibit an Altered Transcriptome Similar to that of HCC Cells
We examined which biological processes or cellular compartments were significantly overrepresented in the induced or repressed genes in both pairwise comparisons (Gene Ontology Analysis). As expected, processes and compartments that were enriched in aggregated hepatocytes relative to nonaggregated hepatocytes were almost identical to those that were enriched in HCC relative to normal hepatocytes (Figure 3C). Several human HCC markers, including AFP, Gpc3 and H19, were upregulated in aggregated hepatocytes (Figures 3D and 3E). Aggregated hepatocytes also expressed more Tetraspanin 8 (Tspan8), a cell-surface glycoprotein that complexes with integrins and is overexpressed in human carcinomas (Zöller, 2009). Another cell-surface molecule highly expressed in aggregated cells is Ly6D (Figures 3D and 3E). Immunofluorescence (IF) analysis revealed that Ly6D was undetectable in normal liver but was elevated in FAH and ubiquitously expressed in most HCC cells (Figure S3C). A fluorescent-labeled Ly6D antibody injected into HCC-bearing mice specifically stained tumor nodules (Figure S3D). Other cell-surface molecules that were upregulated in aggregated cells included syndecan 3 (Sdc3), integrin α 9 (Itga9), claudin 5 (Cldn5), and cadherin 5 (Cdh5) (Figure 3D). Aggregated hepatocytes also exhibited elevated expression of extracellular matrix proteins (TIF3 and Reln1) and a serine protease inhibitor (Spink3). Elevated expression of such proteins may explain aggregate formation. Aggregated hepatocytes also expressed progenitor cell markers, including the epithelial cell adhesion molecule (EpCAM) (Figure 3E) and Dlk1 (Figure 3D). We compared the HcPC and HCC (Figure 3A) to the transcriptome of DDC-induced oval cells (Shin et al., 2011). This analysis revealed a striking similarity between the HCC, HcPC, and the oval cell transcriptomes (Figure S3B). Despite these similarities, some genes that were upregulated in HcPC-containing aggregates and HCC were not upregulated in oval cells. Such genes may account for the tumorigenic properties of HcPC and HCC.
Figure 4 DEN-Induced HcPC Aggregates Express Pathways and Markers Characteristic of HCC and Hepatobiliary Stem Cells
We examined the aggregates for signaling pathways and transcription factors involved in hepatocarcinogenesis. Many aggregated cells were positive for phosphorylated c-Jun and STAT3 (Figure 4A), transcription factors involved in DEN-induced hepatocarcinogenesis (Eferl et al., 2003; He et al., 2010). Sox9, a transcription factor that marks hepatobiliary progenitors (Dorrell et al., 2011), was also expressed by many of the aggregated cells, which were also positive for phosphorylated c-Met (Figure 4A), a receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF) and is essential for liver development (Bladt et al., 1995) and hepatocarcinogenesis (Wang et al., 2001). Few of the nonaggregated hepatocytes exhibited activation of these signaling pathways. Despite different etiology, HcPC-containing aggregates from Tak1Δhep mice exhibit upregulation of many of the same markers and pathways that are upregulated in DEN-induced HcPC-containing aggregates. Flow cytometry confirmed enrichment of CD44+ cells as well as CD44+/CD90+ and CD44+/EpCAM+ double-positive cells in the HcPC-containing aggregates from either DEN-treated or Tak1Δhep livers (Figure S4B).
HcPC-Containing Aggregates Originate from Premalignant Dysplastic Lesions
FAH are dysplastic lesions occurring in rodent livers exposed to hepatic carcinogens (Su et al., 1990). Similar lesions are present in premalignant human livers (Su et al., 1997). Yet, it is still debated whether FAH correspond to premalignant lesions or are a reaction to liver injury that does not lead to cancer (Sell and Leffert, 2008). In DEN-treated males, FAH were detected as early as 3 months after DEN administration (Figure 5A), concomitant with the time at which HcPC-containing aggregates were detected. In females, FAH development was delayed. FAH contained cells positive for the same progenitor cell markers and activated signaling pathways present in HcPC-containing aggregates, including AFP, CD44, and EpCAM (Figure 5C). FAH also contained cells positive for activated STAT3, c-Jun, and PCNA (Figure 5C).
HcPCs Exhibit Autocrine IL-6 Expression Necessary for HCC Progression
In situ hybridization (ISH) and immunohistochemistry (IHC) revealed that DEN-induced FAH contained IL-6-expressing cells (Figures 6A, 6B, and S5), and freshly isolated DEN-induced aggregates contained more IL-6 messenger RNA (mRNA) than nonaggregated hepatocytes (Figure 6C). We examined several factors that control IL-6 expression and found that LIN28A and B were significantly upregulated in HcPCs and HCC (Figures 6D and 6E). LIN28-expressing cells were also detected within FAH (Figure 6F). As reported (Iliopoulos et al., 2009), knockdown of LIN28B in cultured HcPC or HCC cell lines decreased IL-6 expression (Figure 6G). LIN28 exerts its effects through downregulation of the microRNA (miRNA) Let-7 (Iliopoulos et al., 2009).
Figure 6 Liver Premalignant Lesions and HcPCs Exhibit Elevated IL-6 and LIN28 Expression
Figure 7 HCC Growth Depends on Autocrine IL-6 Production
The isolation and characterization of cells that can give rise to HCC only after transplantation into an appropriate host liver undergoing chronic injury demonstrates that cancer arises from progenitor cells that are yet to become fully malignant. Importantly, unlike fully malignant HCC cells, the HcPCs we isolated cannot form s.c. tumors or even liver tumors when introduced into a nondamaged liver. Liver damage induced by uPA expression or CCl4 treatment provides HcPCs with the proper cytokine and growth factor milieu needed for their proliferation. Although HcPCs produce IL-6, they may also depend on other cytokines such as TNF, which is produced by macrophages that are recruited to the damaged liver. In addition, uPA expression and CCl4 treatment may enhance HcPC growth and progression through their fibrogenic effect on hepatic stellate cells. Although HCC and other cancers have been suspected to arise from premalignant/dysplastic lesions (Hruban et al., 2007; Hytiroglou et al., 2007), a direct demonstration that such lesions progress into malignant tumors has been lacking. Based on expression of common markers—EpCAM, CD44, AFP, activated STAT3, and IL-6—that are not expressed in normal hepatocytes, we postulate that HcPCs originate from FAH or dysplastic foci, which are first observed in male mice within 3 months of DEN exposure.
Lin R1, Tao R, Gao X, Li T, Zhou X, Guan KL, Xiong Y, Lei QY.
Mol Cell. 2013 Aug 22; 51(4):506-18
http://dx.doi.org:/10.1016/j.molcel.2013.07.002
Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.
Fatty acid synthesis occurs at low rates in most nondividing cells of normal tissues that primarily uptake lipids from circulation. In contrast, increased lipogenesis, especially de novo lipid synthesis, is a key characteristic of cancer cells. Many studies have demonstrated that in cancer cells, fatty acids are preferred to be derived from de novo synthesis instead of extracellular lipid supply (Medes et al., 1953; Menendez and Lupu, 2007;Ookhtens et al., 1984; Sabine et al., 1967). Fatty acids are key building blocks for membrane biogenesis, and glucose serves as a major carbon source for de novo fatty acid synthesis (Kuhajda, 2000; McAndrew, 1986;Swinnen et al., 2006). In rapidly proliferating cells, citrate generated by the tricarboxylic acid (TCA) cycle, either from glucose by glycolysis or glutamine by anaplerosis, is preferentially exported from mitochondria to cytosol and then cleaved by ATP citrate lyase (ACLY) (Icard et al., 2012) to produce cytosolic acetyl coenzyme A (acetyl-CoA), which is the building block for de novo lipid synthesis. As such, ACLY couples energy metabolism with fatty acids synthesis and plays a critical role in supporting cell growth. The function of ACLY in cell growth is supported by the observation that inhibition of ACLY by chemical inhibitors or RNAi dramatically suppresses tumor cell proliferation and induces differentiation in vitro and in vivo (Bauer et al., 2005; Hatzivassiliou et al., 2005). In addition, ACLY activity may link metabolic status to histone acetylation by providing acetyl-CoA and, therefore, gene expression (Wellen et al., 2009).
While ACLY is transcriptionally regulated by sterol regulatory element-binding protein 1 (SREBP-1) (Kim et al., 2010), ACLY activity is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Berwick et al., 2002; Migita et al., 2008; Pierce et al., 1982). Akt can directly phosphorylate and activate ACLY (Bauer et al., 2005; Berwick et al., 2002; Migita et al., 2008; Potapova et al., 2000). Covalent lysine acetylation has recently been found to play a broad and critical role in the regulation of multiple metabolic enzymes (Choudhary et al., 2009; Zhao et al., 2010). In this study, we demonstrate that ACLY protein is acetylated on multiple lysine residues in response to high glucose. Acetylation of ACLY blocks its ubiquitinylation and degradation, thus leading to ACLY accumulation and increased fatty acid synthesis. Our observations reveal a crosstalk between protein acetylation and ubiquitylation in the regulation of fatty acid synthesis and cell growth.
Acetylation of ACLY at Lysines 540, 546, and 554
Recent mass spectrometry-based proteomic analyses have potentially identified a large number of acetylated proteins, including ACLY (Figure S1A available online; Choudhary et al., 2009, Zhao et al., 2010). We detected the acetylation level of ectopically expressed ACLY followed by western blot using pan-specific anti-acetylated lysine antibody. ACLY was indeed acetylated, and its acetylation was increased by nearly 3-fold after treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases, and trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) class I and class II (Figure 1A). Experiments with endogenous ACLY also showed that TSA and NAM treatment enhanced ACLY acetylation (Figure 1B).
Figure 1 ACLY Is Acetylated at Lysines 540, 546, and 554
Ten putative acetylation sites were identified by mass spectrometry analyses (Table S1). We singly mutated each lysine to either a glutamine (Q) or an arginine (R) and found that no single mutation resulted in a significant reduction of ACLY acetylation (data not shown), indicating that ACLY may be acetylated at multiple lysine residues. Three lysine residues, K540, K546, and K554, received high scores in the acetylation proteomic screen and are evolutionarily conserved from C. elegans to mammals (Figure S1A). We generated triple Q and R mutants of K540, K546, and K554 (3KQ and 3KR) and found that both 3KQ and 3KR mutations resulted in a significant (~60%) decrease in ACLY acetylation (Figure 1C), indicating that 3K are the major acetylation sites of ACLY. Further, we found that the acetylation of endogenous ACLY is clearly increased after treatment of cells with NAM and TSA (Figure 1D). These results demonstrate that ACLY is acetylated at K540, K546, and K554.
Glucose Promotes ACLY Acetylation to Stabilize ACLY
In mammalian cells, glucose is the main carbon source for de novo lipid synthesis. We found that ACLY levels increased with increasing glucose concentration, which also correlated with increased ACLY 3K acetylation (Figure 1E). Furthermore, to confirm whether the glucose level affects ACLY protein stability in vivo, we intraperitoneally injected glucose in BALB/c mice and found that high glucose resulted in a significant increase of ACLY protein levels (Figure 1F).
To determine whether ACLY acetylation affects its protein levels, we treated HeLa and Chang liver cells with NAM and TSA and found an increase in ACLY protein levels (Figure S1G, upper panel). ACLY mRNA levels were not significantly changed by the treatment of NAM and TSA (Figure S1G, lower panel), indicating that this upregulation of ACLY is mostly achieved at the posttranscriptional level. Indeed, ACLY protein was also accumulated in cells treated with the proteasome inhibitor MG132, indicating that ACLY stability could be regulated by the ubiquitin-proteasome pathway (Figure 1G). Blocking deacetylase activity stabilized ACLY (Figure S1H). The stabilization of ACLY induced by high glucose was associated with an increase of ACLY acetylation at K540, K546, and K554. Together, these data support a notion that high glucose induces both ACLY acetylation and protein stabilization and prompted us to ask whether acetylation directly regulates ACLY stability. We then generated ACLYWT, ACLY3KQ, and ACLY3KRstable cells after knocking down the endogenous ACLY. We found that the ACLY3KR or ACLY3KQmutant was more stable than the ACLYWT (Figures 1I and S1I). Collectively, our results suggest that glucose induces acetylation at K540, 546, and 554 to stabilize ACLY.
Acetylation Stabilizes ACLY by Inhibiting Ubiquitylation
To determine the mechanism underlying the acetylation and ACLY protein stability, we first examined ACLY ubiquitylation and found that it was actively ubiquitylated (Figure 2A). Previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). In order to identify the ubiquitylation sites, we tested the ubiquitylation levels of double mutants 540R–546R and 546–554R (Figure S2A). We found that the ubiquitylation of the 540R-546R and 546R-554R mutants is partially decreased, while mutation of K540, K546, and K554 (3KR), which changes all three putative acetylation lysine residues of ACLY to arginine residues, dramatically reduced the ACLY ubiquitylation level (Figures 2B and S2A), indicating that 3K lysines might also be the ubiquitylation target residues. Moreover, inhibition of deacetylases by NAM and TSA decreased ubiquitylation of WT but not 3KQ or 3KR mutant ACLY (Figure 2C). These results implicate an antagonizing role of the acetylation towards the ubiquitylation of ACLY at these three lysine residues.
Figure 2 Acetylation Protects ACLY from Proteasome Degradation by Inhibiting Ubiquitylation
We found that ACLY acetylation was only detected in the nonubiquitylated, but not the ubiquitylated (high-molecular-weight), ACLY species. This result indicates that ACLY acetylation and ubiquitylation are mutually exclusive and is consistent with the model that K540, K546, and K554 are the sites of both ubiquitylation and acetylation. Therefore, acetylation of these lysines would block ubiquitylation.
We also found that glucose upregulates ACLY acetylation at 3K and decreases its ubiquitylation (Figure S2B). High glucose (25 mM) effectively decreased ACLY ubiquitylation, while inhibition of deacetylases clearly diminished its ubiquitylation (Figure 2E). We conclude that acetylation and ubiquitylation occur mutually exclusively at K540, K546, and K554 and that high-glucose-induced acetylation at these three sites blocks ACLY ubiquitylation and degradation.
UBR4 Targets ACLY for Degradation
UBR4 was identified as a putative ACLY-interacting protein by affinity purification coupled with mass spectrometry analysis (data not shown). To address if UBR4 is a potential ACLY E3 ligase, we determined the interaction between ACLY and UBR4 and found that ACLY interacted with the E3 ligase domain of UBR4; this interaction was enhanced by MG132 treatment (Figure 3A). UBR4 knockdown in A549 cells resulted in an increase of endogenous ACLY protein level (Figure 3C). Moreover, UBR4 knockdown significantly stabilized ACLY (Figure 3D) and decreased ACLY ubiquitylation (Figure 3E). Taken together, these results indicate that UBR4 is an ACLY E3 ligase that responds to glucose regulation.
Figure 3 UBR4 Is the E3 Ligase of ACLY
PCAF Acetylates ACLY
PCAF knockdown significantly reduced acetylation of 3K, indicating that PCAF is a potential 3K acetyltransferase in vivo (Figure 4C, upper panel). Furthermore, PCAF knockdown decreased the steady-state level of endogenous ACLY, but not ACLY mRNA (Figure 4C, middle and lower panels). Moreover, we found that PCAF knockdown destabilized ACLY (Figure 4D). In addition, overexpression of PCAF decreases ACLY ubiquitylation (Figure 4E), while PCAF inhibition increases the interaction between UBR4 E3 ligase domain and wild-type ACLY, but not 3KR (Figure 4F). Together, our results indicate that PCAF increases ACLY protein level, possibly via acetylating ACLY at 3K.
Figure 4 PCAF Is the Acetylase of ACLY
SIRT2 Deacetylates ACLY
Figure 5 SIRT2 Decreases ACLY Acetylation and Increases Its Protein Levels In Vivo
Acetylation of ACLY Promotes Cell Proliferation and De Novo Lipid Synthesis
The protein levels of ACLY 3KQ and 3KR were accumulated to a level higher than the wild-type cells upon extended culture in low-glucose medium (Figure S6A, right panel), indicating a growth advantage conferred by ACLY stabilization resulting from the disruption of both acetylation and ubiquitylation at K540, K546, and K554. Cellular acetyl-CoA assay showed that cells expressing 3KQ or 3KR mutant ACLY produce more acetyl-CoA than cells expressing the wild-type ACLY under low glucose (Figures 6B and S6B), further supporting the conclusion that 3KQ or 3KR mutation stabilizes ACLY.
Figure 6 Acetylation of ACLY at 3K Promotes Lipogenesis and Tumor Cell Proliferation
ACLY is a key enzyme in de novo lipid synthesis. Silencing ACLY inhibited the proliferation of multiple cancer cell lines, and this inhibition can be partially rescued by adding extra fatty acids or cholesterol into the culture media (Zaidi et al., 2012). This prompted us to measure extracellular lipid incorporation in A549 cells after knockdown and ectopic expression of ACLY. We found that when cultured in low glucose (2.5 mM), cells expressing wild-type ACLY uptake significantly more phospholipids compared to cells expressing 3KQ or 3KR mutant ACLY (Figures 6C, 6D, and S6D). When cultured in the presence of high glucose (25 mM), however, cells expressing either the wild-type, 3KQ, or 3KR mutant ACLY all have reduced, but similar, uptake of extracellular phospholipids (Figures 6C, 6D, and S6D). The above results are consistent with a model that acetylation of ACLY induced by high glucose increases its stability and stimulates de novo lipid synthesis.
3K Acetylation of ACLY Is Increased in Lung Cancer
ACLY is reported to be upregulated in human lung cancer (Migita et al., 2008). Many small chemicals targeting ACLY have been designed for cancer treatment (Zu et al., 2012). The finding that 3KQ or 3KR mutant increased the ability of ACLY to support A549 lung cancer cell proliferation prompted us to examine 3K acetylation in human lung cancers. We collected a total of 54 pairs of primary human lung cancer samples with adjacent normal lung tissues and performed immunoblotting for ACLY protein levels. This analysis revealed that, when compared to the matched normal lung tissues, 29 pairs showed a significant increase of total ACLY protein using b-actin as a loading control (Figures 7A and S7A). The tumor sample analyses demonstrate that ACLY protein levels are elevated in lung cancers, and 3K acetylation positively correlates with the elevated ACLY protein. These data also indicate that ACLY with 3K acetylation may be potential biomarker for lung cancer diagnosis.
Figure 7 Acetylation of ACLY at 3K Is Upregulated in Human Lung Carcinoma
Dysregulation of cellular metabolism is a hallmark of cancer (Hanahan and Weinberg, 2011; Vander Heiden et al., 2009). Besides elevated glycolysis, increased lipogenesis, especially de novo lipid synthesis, also plays an important role in tumor growth. Because most carbon sources for fatty acid synthesis are from glucose in mammalian cells (Wellen et al., 2009), the channeling of carbon into de novo lipid synthesis as building blocks for tumor cell growth is primarily linked to acetyl-CoA production by ACLY. Moreover, the ACLY-catalyzed reaction consumes ATP. Therefore, as the key cellular energy and carbon source, one may expect a role for glucose in ACLY regulation. In the present study, we have uncovered a mechanism of ACLY regulation by glucose that increases ACLY protein level to meet the enhanced demand of lipogenesis in growing cells, such as tumor cells (Figure 7C). Glucose increases ACLY protein levels by stimulating its acetylation.
Upregulation of ACLY is common in many cancers (Kuhajda, 2000; Milgraum et al., 1997; Swinnen et al., 2004; Yahagi et al., 2005). This is in part due to the transcriptional activation by SREBP-1 resulting from the activation of the PI3K/AKT pathway in cancers (Kim et al., 2010; Nadler et al., 2001; Wang and Dey, 2006). In this study, we report a mechanism of ACLY regulation at the posttranscriptional level. We propose that acetylation modulated by glucose status plays a crucial role in coordinating the intracellular level of ACLY, hence fatty acid synthesis, and glucose availability. When glucose is sufficient, lipogenesis is enhanced. This can be achieved, at least in part, by the glucose-induced stabilization of ACLY. High glucose increases ACLY acetylation, which inhibits its ubiquitylation and degradation, leading to the accumulation of ACLY and enhanced lipogenesis. In contrast, when glucose is limited, ACLY is not acetylated and thus can be ubiquitylated, leading to ACLY degradation and reduced lipogenesis. Moreover, our data indicate that acetylation and ubiquitylation in ACLY may compete with each other by targeting the same lysine residues at K540, K546, and K554. Consistently, previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). Similar models of different modifications on the same lysine residues have been reported in the regulation of other proteins (Grönroos et al., 2002; Li et al., 2002, 2012). We propose that acetylation and ubiquitylation have opposing effects in the regulation of ACLY by competitively modifying the same lysine residues. The acetylation-mimetic 3KQ and the acetylation-deficient 3KR mutants behaved indistinguishably in most biochemical and functional assays, mainly due to the fact that these mutations disrupt lysine ubiquitylation that primarily occurs on these three residues.
ACLY is increased in lung cancer tissues compared to adjacent tissues. Consistently, ACLY acetylation at 3K is also significantly increased in lung cancer tissues. These observations not only confirm ACLY acetylation in vivo, but also suggest that ACLY 3K acetylation may play a role in lung cancer development. Our study reveals a mechanism of ACLY regulation in response to glucose signals.
Nomura DK1, Long JZ, Niessen S, Hoover HS, Ng SW, Cravatt BF.
Cell. 2010 Jan 8; 140(1):49-61
http://dx.doi.org/10.1016.2Fj.cell.2009.11.027
Monoacylglycerol lipase (MAGL) is elevated in aggressive human cancer cells
Loss of MAGL lowers fatty acid levels in cancer cells and impairs pathogenicity
MAGL controls a signaling network enriched in protumorigenic lipids
A high-fat diet can restore the growth of tumors lacking MAGL in vivo
monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors
http://www.cell.com/cms/attachment/1082768/7977146/fx1.jpg
Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity—phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.
We show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in non-aggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity — phenotypes that are reversed by an MAGL inhibitor. Interestingly, impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of pro-tumorigenic signals.
The conversion of cells from a normal to cancerous state is accompanied by reprogramming of metabolic pathways (Deberardinis et al., 2008; Jones and Thompson, 2009; Kroemer and Pouyssegur, 2008), including those that regulate glycolysis (Christofk et al., 2008; Gatenby and Gillies, 2004), glutamine-dependent anaplerosis (DeBerardinis et al., 2008; DeBerardinis et al., 2007; Wise et al., 2008), and the production of lipids (DeBerardinis et al., 2008; Menendez and Lupu, 2007). Despite a growing appreciation that dysregulated metabolism is a defining feature of cancer, it remains unclear, in many instances, how such biochemical changes occur and whether they play crucial roles in disease progression and malignancy.
Among dysregulated metabolic pathways, heightened de novo lipid biosynthesis, or the development a "lipogenic" phenotype (Menendez and Lupu, 2007), has been posited to play a major role in cancer. For instance, elevated levels of fatty acid synthase (FAS), the enzyme responsible for fatty acid biosynthesis from acetate and malonyl CoA, are correlated with poor prognosis in breast cancer patients, and inhibition of FAS results in decreased cell proliferation, loss of cell viability, and decreased tumor growth in vivo (Kuhajda et al., 2000; Menendez and Lupu, 2007; Zhou et al., 2007). FAS may support cancer growth, at least in part, by providing metabolic substrates for energy production (via fatty acid oxidation) (Buzzai et al., 2005; Buzzai et al., 2007; Liu, 2006). Many other features of lipid biochemistry, however, are also critical for supporting the malignancy of cancer cells, including:
the generation of building blocks for newly synthesized membranes to accommodate high rates of proliferation (DeBerardinis et al., 2008; Deberardinis et al., 2008),
the composition and regulation of membrane structures that coordinate signal transduction and motility [e.g., lipid rafts (Gao and Zhang, 2008), invadopodia (Stylli et al., 2008), blebs (Fackler and Grosse, 2008)] and
the biosynthesis of an array of pro-tumorigenic lipid signaling molecules.
Prominent examples of lipid messengers that contribute to cancer include:
phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3], which is formed by the action of phosphatidylinositol-3-kinase and activates protein kinase B/Akt to promote cell proliferation and survival (Yuan and Cantley, 2008; Zunder et al., 2008);
lysophosphatidic acid (LPA), which signals through a family of G-protein coupled receptors to stimulate cancer aggressiveness (Mills and Moolenaar, 2003; Ren et al., 2006); and
prostaglandins formed by cyclooxygenases, which support migration and tumor-host interactions (Gupta et al., 2007; Marnett, 1992).
Here, we use functional proteomic methods to discover a lipolytic enzyme, monoacylglycerol lipase (MAGL), that is highly elevated in aggressive cancer cells from multiple tissues of origin. We show that MAGL, through hydrolysis of monoacylglycerols (MAGs), controls free fatty acid (FFA) levels in cancer cells. The resulting MAGL-FFA pathway feeds into a diverse lipid network enriched in pro-tumorigenic signaling molecules and promotes migration, survival, and in vivo tumor growth. Aggressive cancer cells thus pair lipogenesis with high lipolytic activity to generate an array of pro-tumorigenic signals that support their malignant behavior.
Activity-Based Proteomic Analysis of Hydrolytic Enzymes in Human Cancer Cells
To identify enzyme activities that contribute to cancer pathogenesis, we conducted a functional proteomic analysis of a panel of aggressive and non-aggressive human cancer cell lines from multiple tumors of origin, including melanoma [aggressive (C8161, MUM2B), non-aggressive (MUM2C)], ovarian [aggressive (SKOV3), non-aggressive (OVCAR3)], and breast [aggressive (231MFP), non-aggressive (MCF7)] cancer. Aggressive cancer lines were confirmed to display much greater in vitro migration and in vivo tumor-growth rates compared to their non-aggressive counterparts (Figure S1), as previously shown (Jessani et al., 2004;Jessani et al., 2002; Seftor et al., 2002; Welch et al., 1991). Proteomes from these cancer lines were screened by activity-based protein profiling (ABPP) using serine hydrolase-directed fluorophosphonate (FP) activity-based probes (Jessani et al., 2002; Patricelli et al., 2001). Serine hydrolases are one of the largest and most diverse enzyme classes in the human proteome (representing ~ 1–1.5% of all human proteins) and play important roles in many biochemical processes of potential relevance to cancer, such as proteolysis (McMahon and Kwaan, 2008; Puustinen et al., 2009), signal transduction (Puustinen et al., 2009), and lipid metabolism (Menendez and Lupu, 2007; Zechner et al., 2005). The goal of this study was to identify hydrolytic enzyme activities that were consistently altered in aggressive versus non-aggressive cancer lines, working under the hypothesis that these conserved enzymatic changes would have a high probability of contributing to the pathogenic state of cancer cells.
Among the more than 50 serine hydrolases detected in this analysis (Tables S1–3), two enzymes, KIAA1363 and MAGL, were found to be consistently elevated in aggressive cancer cells relative to their non-aggressive counterparts, as judged by spectral counting (Jessani et al., 2005; Liu et al., 2004). We confirmed elevations in KIAA1363 and MAGL in aggressive cancer cells by gel-based ABPP, where proteomes are treated with a rhodamine-tagged FP probe and resolved by 1D-SDS-PAGE and in-gel fluorescence scanning (Figure 1A). In both cases, two forms of each enzyme were detected (Figure 1A), due to differential glycoslyation for KIAA1363 (Jessani et al., 2002), and possibly alternative splicing for MAGL (Karlsson et al., 2001). We have previously shown that KIAA1363 plays a role in regulating ether lipid signaling pathways in aggressive cancer cells (Chiang et al., 2006). On the other hand, very little was known about the function of MAGL in cancer.
Figure 1 MAGL is elevated in aggressive cancer cells, where the enzyme regulates monoacylgycerol (MAG) and free fatty acid (FFA) levels
The heightened activity of MAGL in aggressive cancer cells was confirmed using the substrate C20:4 MAG (Figure 1B). Since several enzymes have been shown to display MAG hydrolytic activity (Blankman et al., 2007), we confirmed the contribution that MAGL makes to this process in cancer cells using the potent and selective MAGL inhibitor JZL184 (Long et al., 2009a).
MAGL Regulates Free Fatty Acid Levels in Aggressive Cancer Cells
MAGL is perhaps best recognized for its role in degrading the endogenous cannabinoid 2-arachidonoylglycerol (2-AG, C20:4 MAG), as well as other MAGs, in brain and peripheral tissues (Dinh et al., 2002; Long et al., 2009a; Long et al., 2009b; Nomura et al., 2008). Consistent with this established function, blockade of MAGL by JZL184 (1 μM, 4 hr) produced significant elevations in the levels of several MAGs, including 2-AG, in each of the aggressive cancer cell lines (Figure 1C and Figure S2). Interestingly, however, MAGL inhibition also caused significant reductions in the levels of FFAs in aggressive cancer cells (Figure 1D and Figure S2). This surprising finding contrasts with the function of MAGL in normal tissues, where the enzyme does not, in general, control the levels of FFAs (Long et al., 2009a; Long et al., 2009b;Nomura et al., 2008).
Metabolic labeling studies using the non-natural C17:0-MAG confirmed that MAGs are converted to LPC and LPE by aggressive cancer cells, and that this metabolic transformation is significantly enhanced by treatment with JZL184 (Figure S1). Finally, JZL184 treatment did not affect the levels of MAGs and FFAs in non-aggressive cancer lines (Figure 1C, D), consistent with the negligible expression of MAGL in these cells (Figure 1A, B).
We next stably knocked down MAGL expression by RNA interference technology using two independent shRNA probes (shMAGL1, shMAGL2), both of which reduced MAGL activity by 70–80% in aggressive cancer lines (Figure 2A, D and Figure S2). Other serine hydrolase activities were unaffected by shMAGL probes (Figure 2A, D and Figures S2), confirming the specificity of these reagents. Both shMAGL probes caused significant elevations in MAGs and corresponding reductions in FFAs in aggressive melanoma (Figure 2B, C), ovarian (Figure 2E, F), and breast cancer cells (Figure S2).
Figure 2 Stable shRNA-mediated knockdown of MAGL lowers FFA levels in aggressive cancer cells.
Together, these data demonstrate that both acute (pharmacological) and stable (shRNA) blockade of MAGL cause elevations in MAGs and reductions in FFAs in aggressive cancer cells. These intriguing findings indicate that MAGL is the principal regulator of FFA levels in aggressive cancer cells. Finally, we confirmed that MAGL activity (Figure 3A, B) and FFA levels (Figure 3C) are also elevated in high-grade primary human ovarian tumors compared to benign or low-grade tumors. Thus, heightened expression of the MAGL-FFA pathway is a prominent feature of both aggressive human cancer cell lines and primary tumors.
Figure 3 High-grade primary human ovarian tumors possess elevated MAGL activity and FFAs compared to benign tumors.
Disruption of MAGL Expression and Activity Impairs Cancer Pathogenicity
shMAGL cancer lines were next examined for alterations in pathogenicity using a set of in vitro and in vivo assays. shMAGL-melanoma (C8161), ovarian (SKOV3), and breast (231MFP) cancer cells exhibited significantly reduced in vitro migration (Figure 4A, F and Figure S2), invasion (Figure 4B, G and Figure S2), and cell survival under serum-starvation conditions (Figure 4C, H and Figure S2). Acute pharmacological blockade of MAGL by JZL184 also decreased cancer cell migration (Figure S2), but not survival, possibly indicating that maximal impairments in cancer aggressiveness require sustained inhibition of MAGL.
Figure 4 shRNA-mediated knockdown and pharmacological inhibition of MAGL impair cancer aggressiveness.
MAGL Overexpression Increases FFAs and the Aggressiveness of Cancer Cells
Stable MAGL-overexpressing (MAGL-OE) and control [expressing an empty vector or a catalytically inactive version of MAGL, where the serine nucleophile was mutated to alanine (S122A)] variants of MUM2C and OVCAR3 cells were generated by retroviral infection and evaluated for their respective MAGL activities by ABPP and C20:4 MAG substrate assays. Both assays confirmed that MAGL-OE cells possess greater than 10-fold elevations in MAGL activity compared to control cells (Figure 5A and Figure S4). MAGL-OE cells also showed significant reductions in MAGs (Figure 5B andFigure S4) and elevated FFAs (Figure 5C and Figure S4). This altered metabolic profile was accompanied by increased migration (Figure 5D and Figure S4), invasion (Figure 5E and Figure S4), and survival (Figure S4) in MAGL-OE cells. None of these effects were observed in cancer cells expressing the S122A MAGL mutant, indicating that they require MAGL activity. MAGL-OE MUM2C cells also showed enhanced tumor growth in vivo compared to control cells (Figure 5F). Notably, the increased tumor growth rate of MAGL-OE MUM2C cells nearly matched that of aggressive C8161 cells (Figure S4). These data indicate that the ectopic expression of MAGL in non-aggressive cancer cells is sufficient to elevate their FFA levels and promote pathogenicity both in vitro and in vivo.
Figure 5 Ectopic expression of MAGL elevates FFA levels and enhances the in vitro and in vivo pathogenicity of MUM2C melanoma cells.
Metabolic Rescue of Impaired Pathogenicity in MAGL-Disrupted Cancer Cells
MAGL could support the aggressiveness of cancer cells by either reducing the levels of its MAG substrates, elevating the levels of its FFA products, or both. Among MAGs, the principal signaling molecule is the endocannabinoid 2-AG, which activates the CB1 and CB2 receptors (Ahn et al., 2008; Mackie and Stella, 2006). The endocannabinoid system has been implicated previously in cancer progression and, depending on the specific study, shown to promote (Sarnataro et al., 2006; Zhao et al., 2005) or suppress (Endsley et al., 2007; Wang et al., 2008) cancer pathogenesis. Neither a CB1 or CB2 antagonist rescued the migratory defects of shMAGL cancer cells (Figure S5). CB1 and CB2 antagonists also did not affect the levels of MAGs or FFAs in cancer cells (Figure S5).
We then determined whether increased FFA delivery could rectify the tumor growth defect observed for shMAGL cells in vivo. Immune-deficient mice were fed either a normal chow or high-fat diet throughout the duration of a xenograft tumor growth experiment. Notably, the impaired tumor growth rate of shMAGL-C8161 cells was completely rescued in mice fed a high-fat diet. In contrast, shControl-C8161 cells showed equivalent tumor growth rates on a normal versus high-fat diet. The recovery in tumor growth for shMAGL-C8161 cells in the high-fat diet group correlated with significantly increases levels of FFAs in excised tumors (Figure 6D). Collectively, these results indicate that MAGL supports the pathogenic properties of cancer cells by maintaining tonically elevated levels of FFAs.
Figure 6 Recovery of the pathogenic properties of shMAGL cancer cells by treatment with exogenous fatty acids.
MAGL Regulates a Fatty Acid Network Enriched in Pro-Tumorigenic Signals
Studies revealed that neither
the MAGL-FFA pathway might serve as a means to regenerate NAD+ (via continual fatty acyl glyceride/FFA recycling) to fuel glycolysis, or
increased lipolysis could be to generate FFA substrates for β-oxidation, which may serve as an important energy source for cancer cells (Buzzai et al., 2005), or
CPT1 blockade (reduced expression of CPT1 in aggressive cancer cells (data not shown) has been reported previously (Deberardinis et al., 2006))
providing evidence against a role for β-oxidation as a downstream mediator of the pathogenic effects of the MAGL-fatty acid pathway.
Considering that FFAs are fundamental building blocks for the production and remodeling of membrane structures and signaling molecules, perturbations in MAGL might be expected to affect several lipid-dependent biochemical networks important for malignancy. To test this hypothesis, we performed lipidomic analyses of cancer cell models with altered MAGL activity, including comparisons of:
MAGL-OE versus control cancer cells (OVCAR3, MUM2C), and
shMAGL versus shControl cancer cells (SKOV3, C8161).
Complementing these global profiles, we also conducted targeted measurements of specific bioactive lipids (e.g., prostaglandins) that are too low in abundance for detection by standard lipidomic methods. The resulting data sets were then mined to identify a common signature of lipid metabolites regulated by MAGL, which we defined as metabolites that were significantly increased or reduced in MAGL–OE cells and showed the opposite change in shMAGL cells relative to their respective control groups (Figure 7A, B and Table S4).
Figure 7 MAGL regulates a lipid network enriched in pro-tumorigenic signaling molecules.
Most of the lipids in the MAGL-fatty acid network, including several lysophospholipids (LPC, LPA, LPE), ether lipids (MAGE, alkyl LPE), phosphatidic acid (PA), and prostaglandin E2 (PGE2), displayed similar profiles to FFAs, being consistently elevated and reduced in MAGL-OE and shMAGL cells, respectively. Only MAGs were found to show the opposite profile (elevated and reduced in shMAGL and MAGL-OE cells, respectively). Interestingly, virtually this entire lipidomic signature was also observed in aggressive cancer cells when compared to their non-aggressive counterparts (e.g., C8161 versus MUM2C and SKOV3 versus OVCAR3, respectively; Table S4). These findings demonstrate that MAGL regulates a lipid network in aggressive cancer cells that consists of not only FFAs and MAGs, but also a host of secondary lipid metabolites. Increases (rather than decreases) in LPCs and LPEs were observed in JZL184-treated cells (Figure S1 and Table S4). These data indicate that acute and chronic blockade of MAGL generate distinct metabolomic effects in cancer cells, likely reflecting the differential outcomes of short- versus long-term depletion of FFAs.
Within the MAGL-fatty acid network are several pro-tumorigenic lipid messengers, including LPA and PGE2, that have been reported to promote the aggressiveness of cancer cells (Gupta et al., 2007; Mills and Moolenaar, 2003). Metabolic labeling studies confirmed that aggressive cancer cells can convert both MAGs and FFAs (Figure S1) to LPA and PGE2 and, for MAGs, this conversion was blocked by JZL184 (Figure S1). Interestingly, treatment with either LPA or PGE2 (100 nM, 4 hr) rescued the impaired migration of shMAGL cancer cells at concentrations that did not affect the migration of shControl cells (Figure 7E).
Heightened lipogenesis is an established early hallmark of dysregulated metabolism and pathogenicity in cancer (Menendez and Lupu, 2007). Cancer lipogenesis appears to be driven principally by FAS, which is elevated in most transformed cells and important for survival and proliferation (De Schrijver et al., 2003;Kuhajda et al., 2000; Vazquez-Martin et al., 2008). It is not yet clear how FAS supports cancer growth, but most of the proposed mechanisms invoke pro-tumorigenic functions for the enzyme s fatty acid products and their lipid derivatives (Menendez and Lupu, 2007). This creates a conundrum, since the fatty acid molecules produced by FAS are thought to be rapidly incorporated into neutral- and phospho-lipids, pointing to the need for complementary lipolytic pathways in cancer cells to release stored fatty acids for metabolic and signaling purposes (Prentki and Madiraju, 2008; Przybytkowski et al., 2007). Consistent with this hypothesis, we found that acute treatment with the FAS inhibitor C75 (40 μM, 4 h) did not reduce FFA levels in cancer cells (data not shown). Furthermore, aggressive and non-aggressive cancer cells exhibited similar levels of FAS (data not shown), indicating that lipogenesis in the absence of paired lipolysis may be insufficient to confer high levels of malignancy.
Here we show that aggressive cancer cells do indeed acquire the ability to liberate FFAs from neutral lipid stores as a consequence of heightened expression of MAGL. MAGL and its FFA products were found to be elevated in aggressive human cancer cell lines from multiple tissues of origin, as well as in high-grade primary human ovarian tumors. These data suggest that the MAGL-FFA pathway may be a conserved feature of advanced forms of many types of cancer. Further evidence in support of this premise originates from gene expression profiling studies, which have identified increased levels of MAGL in primary human ductal breast tumors compared to less malignant medullary breast tumors (Gjerstorff et al., 2006). The key role that MAGL plays in regulating FFA levels in aggressive cancer cells contrasts with the function of this enzyme in normal tissues, where it mainly controls the levels of MAGs, but not FFAs (Long et al., 2009b). These data thus provide a striking example of the co-opting of an enzyme by cancer cells to serve a distinct metabolic purpose that supports their pathogenic behavior.
Taken together, our results indicate that MAGL serves as key metabolic hub in aggressive cancer cells, where the enzyme regulates a fatty acid network that feeds into a number of pro-tumorigenic signaling pathways.
7.7.8.1 Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling
Komai K1, Niwa Y1, Sasazawa Y1, Simizu S2.
FEBS Lett. 2015 Mar 12; 589(6):738-43
http://dx.doi.org:/10.1016/j.febslet.2015.01.040
Pirin decreases E-cadherin expression and induces EMT.
The induction of EMT by Pirin is achieved through a Bcl3 independent pathway.
Pirin may be a novel target for cancer therapy.
Epithelial to mesenchymal transition (EMT) is an important mechanism for the initial step of metastasis. Proteomic analysis indicates that Pirin is involved in metastasis. However, there are no reports demonstrating its direct contribution. Here we investigated the involvement of Pirin in EMT. In HeLa cells, Pirin suppressed E-cadherin expression and regulated the expression of other EMT markers. Furthermore, cells expressing Pirin exhibited a spindle-like morphology, which is reminiscent of EMT. A Pirin mutant defective for Bcl3 binding decreased E-cadherin expression similar to wild-type, suggesting that Pirin regulates E-cadherin independently of Bcl3-Slug signaling. These data provide direct evidence that Pirin contributes to cancer metastasis.
Pirin regulates the expression of E-cadherin and EMT markers
In melanoma, Pirin enhances NF-jB activity and increases Slug expression by binding Bcl3 [31], and it may also be involved in adenoid cystic tumor metastasis [23]. Since Slug suppresses E-cadherin transcription and is recognized as a major EMT inducer, we hypothesized that Pirin may regulate EMT through inducing Slug expression. To investigate whether Pirin regulates EMT, we measured E-cadherin expression following Pirin knockdown. As shown in Fig. 1A and B, E-cadherin expression was significantly increased following Pirin knockdown indicating that it may promote EMT. To confirm this, we established Pirin-expressing HeLa cells (Fig. 1C), which inhibited the expression of E-cadherin (Fig. 1D). Additionally, the expression of Occludin, an epithelial marker, was decreased, and several mesenchymal markers, including Fibronectin, N-cadherin, and Vimentin, were increased by Pirin expression (Fig. 1D). These data suggest that Pirin promotes EMT.
Pirin induces EMT-associated cell morphological changes
As mentioned above, cells undergo morphological changes during EMT. Therefore, we next analyzed whether Pirin expression affects cell morphology. Quantitative analysis of morphological changes was based on cell circularity, {4p(area)/(perimeter)2}100, which decreases during EMT-associated morphological changes [34–36]. Indeed, TGF-b or TNF-a exposure induced EMTassociated cell morphological changes in HeLa cells (data not shown). Employing this parameter of circularity, we compared the morphology of our established HeLa/Pirin-GFP cells with control HeLa/GFP cells. Although the control HeLa/GFP cells displayed a cobblestone-like morphology, HeLa/Pirin-GFP cells were elongated in shape (Fig. 2A). Indeed, compared with control cells, the circularity of HeLa/Pirin-GFP cells was significantly decreased (Fig. 2B). To confirm that these observations were dependent on Pirin expression, HeLa/Pirin-GFP cells were treated with an siRNA targeting Pirin. HeLa/Pirin-GFP cells recovered a cobblestone-like morphology (Fig. 2C) and circularity (Fig. 2D) when treated with Pirin siRNA indicating that Pirin expression induces EMT.
Pirin induces cell migration
During EMT cells acquire migratory capabilities. Therefore, we analyzed whether Pirin affects cell migration. HeLa cells were treated with an siRNA targeting Pirin and migration was assessed using a wound healing assay. Although Pirin knockdown had no effect on cell proliferation (data not shown), wound repair was inhibited in Pirin-depleted HeLa cells (Fig. 3A and B) suggesting that Pirin promoted cell migration. Furthermore, camptothecin treatment of HeLa/GFP cells caused decreased cell viability in a dose-dependent manner, whereas HeLa/Pirin-GFP cells were more resistantto drugtreatment (datanot shown).These results suggest that Pirin induces EMT-like phenotypes, such as cell migration and anticancer drug resistance.
Pirin regulates EMT independently of Bcl3-Slug signaling
To investigate whether Pirin controls E-cadherin expression at the transcriptional level, we measured E-cadherin promoter activity with a reporter assay. Indeed, the luciferase reporter analysis indicated that Pirin inhibited E-cadherin promoter activity (Fig. 4A and B). To determine if Bcl3 is involved in Pirin-induced EMT, we tested whether a Pirin mutant defective in Bcl3 binding could inhibit E-cadherin expression. We generated a mutation in the metal-binding cavity of Pirin(E103A) and confirmed that it disrupted Bcl3 binding. In vitro GST pull-down analysis using recombinant Pirin and Bcl3/ARD demonstrated that the Pirin mutant was defective for Bcl3 binding compared to wild-type (Fig. 5A). Interestingly, expression of both wild-type Pirin and the mutant defective in Bcl3 binding reduced E-cadherin gene and protein expression (Fig. 5B and C). Taken together these results indicate that Pirin decreases E-cadherin expression without binding Bcl3, and suggest that Pirin regulates EMT independently of Bcl3-Slug signaling.
A characteristic feature of EMT is the disruption of epithelial cell–cell contact, which is achieved by reduced E-cadherin expression. Therefore, revealing the regulatory pathways controlling E-cadherin expression may elucidate the mechanisms of EMT. Several transcription factors regulate E-cadherin transcription. For instance,Snail,Slug,Twist,and Zebact as mastertranscriptional regulators that bind the consensus E-box sequence in the E-cadherin gene promoter and decrease the transcriptional activity [38]. Since Pirin regulates the transcription of Slug [31], we hypothesized that Pirin may also regulate EMT. In this study we demonstrated that Pirin decreases E-cadherin expression, and induces EMT and cancer malignant phenotypes. Since EMT is an initial step of metastasis, Pirin may contribute to cancer progression. We next examined whether the regulation of EMT by Pirin is attributed to Bcl3 binding and the induction of Slug. To this end, we generated a Pirin mutant (E103A) defective for Bcl3 binding (Fig. 5A). Single Fe2+ ion chelating is coordinated by His56, His58, His101, and Glu103 of Pirin, and the N-terminal domain containing these residues is highly conserved between mammals, plants, fungi, and prokaryotic organisms [15,27]. Therefore, it has been predicted that this N-terminal domain containing the metal-binding cavity is important for Pirin function [20,26,31]. Indeed, TPh A inserts into the metal-binding cavity and inhibits binding to Bcl3 suggesting that the interaction occurs with the metal-binding cavity of Pirin [31]. In contrast, Hai Pang suggests that a Pirin–Bcl3– (p50)2 complex forms between acidic regions of the N-terminal Pirin domain at residues 77–82, 97–103 and 124–128 with a basic patch of Bcl3 [27]. In this study, we mutated Glutamic acid 103, a residue common between Hai Pang's model and Pirin's metalbinding cavity. Pull-down analysis indicated that an E103A mutant is defectiveinfor Bcl3binding(Fig.5A). Thisis the firstexperimental demonstration showing that Glu103 of Pirin is important Bcl3 binding. However, expression of the E103A mutant suppressed Ecadherin gene expression similarly to wild-type Pirin (Fig. 5B and C). Although the Bcl3–(p50)2 complex participates in oncogene addiction in cervical cells [39,40], expression of Pirin in HeLa cells did not increase Slug expression (data not shown). Therefore, we concludethatPirindecreasesE-cadherinexpressionindependently of Bcl3-Slug signaling. To understand how Pirin suppresses E-cadherin gene expression, we analyzed E-cadherin promoter activity (Fig. 4). Since Pirin decreased the activity of the E-cadherin promoter (995+1), we constructed a series of promoter deletion mutants (795+1, 565+1, 365+1, 175+1) to identify a region important for Pirin-mediated regulation. Expression of Pirin decreased the transcriptional activity of all constructs (Supplementary Fig. S1A), suggesting that Pirin may suppress E-cadherin expression through element(s) in region 175+1. Yan-Nan Liu and colleagues proposed that this region contains four Sp1-binding sites and two E-boxes that regulate E-cadherin expression.
Fig. 1. Pirin regulates E-cadherin gene expression. (A, B) HeLa cells were transfected with siRNA targeting Pirin (siPirin#1 or #2) or control siRNA (siCTRL). Forty-eight hours after transfection, cDNA was used for PCR using primer sets specific against Pirin, E-cadherin and GAPDH (A). Forty-eight hours after transfection, HeLa cells were lysed and the lysates were analyzed by Western blot with the indicated antibodies (B). (C) Lysates from HeLa/Pirin-GFP and HeLa/GFP cells were analyzed by Western blot with the indicated antibodies. (D) cDNA from HeLa/GFP or HeLa/Pirin-GFP cells was used for PCR to determine the effect of Pirin on the expression of EMT marker genes.
Fig. 2. Pirin induces cell morphological changes associated with EMT. (A) Phase contrast and fluorescence microscopic images were taken of HeLa/GFP and HeLa/Pirin-GFP cells. (B) Cell circularity was defined as form factor, {4p(area)/(perimeter)2}100 [%], and calculated using Image J software. A random selection of 100 cells from each condition was measured. (C, D) Phase contrast and fluorescence microscopic images were taken of siRNA-treated HeLa/GFP and HeLa/Pirin-GFP cells. Each cell line was transfected with siPirin#2 or siCTRL. Cells were observed by microscopy 48 h after transfection (C) and circularity was measured (D). Data shown are means ± s.d. ⁄P <0.05, bars 100lm.
Fig. 3. Pirin knockdown suppresses cell migration. (A, B) HeLa cells were transfected with siPirin#2 or siCTRL. An artificial wound was created with a tip 24h after transfection and cells were cultured for an additional 12 h. For quantification, the cells were photographed after 12h of incubation (A) and the area covered by cells was measured using Image J and normalized to control cells (B).
Fig. 4. Pirin regulates E-cadherin promoter activity.(A). HeLacells were transfected with siPirin#2 or siGFP (control) and cultured for 24 h. The E-cadherin promoter construct (995+1) and phRL-TK vectorwere transfected and cellswere cultured for an additional 24 h. Luciferase activities were measured and normalized to Renilla luciferase activity. (B) HeLa cells were transfected with the promoter construct (995+1), phRL-TK vector, and a Pirin expression vector. After 24 h, luciferase activities were measured and normalized to Renilla luciferase activity. Data are the mean ± s.d. ⁄P < 0.05.
Fig. 5. Pirin decreases E-cadherin expression in a Bcl3-independent manner. (A) Purified His6-Pirin and His6-Pirin(E103A) were incubated with Glutathione-Sepharose beads conjugated to GST or GST-Bcl3/ARD. The samples were analyzed by Western blot. (B, C) HeLa cells were transfected with vectors encoding GFP, Pirin-GFP, or Pirin(E103A)GFP. Cells were lysed 48 h after transfection and lysates were analyzed by Western blot (B). RNA collected at 48h was used for RT-PCR with the specified primer sets for each gene (C).
7.7.8.2 1324 PIRIN DOWN-REGULATES THE EAF2/U19 SIGNALING AND RETARDS THE GROWTH INHIBITION INDUCED BY EAF2/U19 IN PROSTATE CANCER CELLS
Zhongjie Qiao, Dan Wang, Zhou Wang
The Journal of Urology Apr 2013; 189(4), Supplement: e541
http://dx.doi.org/10.1016/j.juro.2013.02.2678
EAF2/U19, as the tumor suppressor, has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth in vivo, and its expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification and characterization of the binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells.
7.7.8.3 Pirin Inhibits Cellular Senescence in Melanocytic Cells
Silvia Licciulli, Chiara Luise, Gaia Scafetta, Maria Capra
Am J Pathol. 2011 May; 178(5): 2397–2406.
http://dx.doi.org/10.1016%2Fj.ajpath.2011.01.019
Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression
Cellular senescence is a physiological process through which normal somatic cells lose their ability to divide and enter an irreversible state of cell cycle arrest, although they remain viable and metabolically active.1,2The specific molecular circuitry underlying the onset of cellular senescence is dependent on the type of stimulus and on the cellular context. A central role is held by the activation of the tumor suppressor proteins p53 and retinoblastoma susceptibility protein (pRB),3–5 which act by interfering with the transcriptional program of the cell and ultimately arresting cell cycle progression.
In the last decade, senescence has been recognized as a major barrier against the development of tumors in mammals.6–8 One of the most prominent in vivo examples is represented by nevi, in which cells proliferate after oncogene activation and then become senescent. Melanoma is a highly aggressive form of neoplasm often observed to derive from nevi, and the transition implies suppression of the mechanisms that sustain the onset and maintenance of senescence.9 In fact, many of the melanoma-associated tumor suppressor genes identified to date are themselves involved in control of senescence, including BRAF (encoding serine/threonine-protein kinase B-raf), CKD4 (cyclin-dependent kinase 4), and CDKN2A (encoding cyclin-dependent kinase inhibitor 2A isoforms p16INK4a and p19ARF).3,10
Nevi frequently harbor oncogenic mutations of the tyrosine kinase BRAF gene, particularly V600E,11 andBRAFV600E is also found in approximately 70% of cutaneous melanomas.12 Expression of BRAFV600E in human melanocytes leads to oncogene-induced senescence,8 which can be considered as a mechanism that protects from malignant progression. In time, some cells may eventually escape senescence, probably through the acquisition of additional genetic abnormalities, thus favoring transformation to melanoma.13
Pirin (PIR) is a highly conserved nuclear protein belonging to the Cupin superfamily14 whose function is, to date, poorly characterized. It has been described as a putative transcriptional regulator on the basis of its physical association with the nuclear I/CCAAT box transcription factor NFI/CTF115 and with the B-cell lymphoma protein, BCL-3, a regulator of NF-κB/Rel activity. A recent report shows that PIR controls melanoma cell migration through the transcriptional regulation of snail homolog 2, SNAI2 (previously SLUG).16 Other reports described quercetinase enzymatic activity,17 and regulation of apoptosis18,19 and stress response, unveiling a high degree of cell-type and species specificity in PIR function.
There is evidence of variations in PIR expression levels in different types of malignancies, but a systematic analysis of PIR expression in human tumors has been lacking. We analyzed PIR expression pattern in a collection of normal and neoplastic human tissues and found that it is expressed in scattered melanocytes, virtually absent in more mature regions of nevi, and present at high levels in a subset of melanomas. Functional studies performed in normal and transformed melanocytic cells revealed that PIR ablation results in cellular senescence, and that PIR levels decrease in response to senescence stimuli. Our results suggest that PIR may be a relevant player in the negative control of cellular senescence in PIR-expressing melanomas.
Figure 3 PIR overexpression in PIR− melanoma cells has no effect on proliferation.
PIR Expression Is Down-Regulated by BRAF Activation and Camptothecin Treatment
BRAF mutations are frequent in nevi, and are directly linked to the induction of oncogene-induced senescence. Variations in PIR expression levels were therefore investigated in an experimental model of senescence induced by oncogenic BRAF. Human diploid fibroblasts (TIG3–hTERT) expressing a conditional form of constitutively activated BRAF fused to the ligand-binding domain of the estrogen receptor (ER) rapidly undergo oncogene-induced senescence on treatment with 4-hydroxytamoxifen (OHT).28,29 PIR protein and mRNA levels were measured in TIG3-BRAF-ER cells at different time points of treatment with 800 nmol/L OHT. PIR expression was significantly repressed both at the mRNA and at the protein level after BRAF activation (Figure 6A), and remained at low levels after 120 hours, suggesting that a significant reduction of PIR expression is associated with the establishment of oncogene-induced senescence in different cell types.
Brian A. Lewis
Biochim et Biophys Acta (BBA) – Gene Regulatory Mechanisms Nov 2013; 1829(11): 1202–1206
http://dx.doi.org/10.1016/j.bbagrm.2013.09.003
This review article discusses recent advances in the links between O-GlcNAc and transcriptional regulation.
Discusses several systems to illustrate O-GlcNAc dynamics: Tet proteins, MLL complexes, circadian clock proteins and RNA pol II.
Suggests that promoters are nutrient sensors.
Post-translational modifications play important roles in transcriptional regulation. Among the less understood PTMs is O-GlcNAcylation. Nevertheless, O-GlcNAcylation in the nucleus is found on hundreds of transcription factors and coactivators and is often found in a mutually exclusive ying–yang relationship with phosphorylation. O-GlcNAcylation also links cellular metabolism directly to the proteome, serving as a conduit of metabolic information to the nucleus. This review serves as a brief introduction to O-GlcNAcylation, emphasizing its important thematic roles in transcriptional regulation, and highlights several recent and important additions to the literature that illustrate the connections between O-GlcNAc and transcription.
links between O-GlcNAc and transcriptional regulation.
http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr1.sml
systems to illustrate O-GlcNAc dynamics
7.7.10 O-GlcNAcylation in cellular functions and human diseases
Yang YR1, Suh PG2.
Adv Biol Regul. 2014 Jan; 54:68-73
http://dx.doi.org:/10.1016/j.jbior.2013.09.007
O-GlcNAcylation is dynamic and a ubiquitous post-translational modification. O-GlcNAcylated proteins influence fundamental functions of proteins such as protein-protein interactions, altering protein stability, and changing protein activity. Thus, aberrant regulation of O-GlcNAcylation contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, metabolic disorders, and Alzheimer's disease. Diverse cellular signaling systems are involved in pathogenesis of these diseases. O-GlcNAcylated proteins occur in many different tissues and cellular compartments and affect specific cell signaling. This review focuses on the O-GlcNAcylation in basic cellular functions and human diseases.
O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions
aberrant regulation of O-GlcNAcylation in disease
Body of review in energetic metabolic pathways in malignant T cells
Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-B is required for T cell proliferation and differentiation of effector cells.
The TCR-to-NF-B pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-B.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation.
Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.
Dr. Aurel,
Manipulate Signaling Pathways [7.6]
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Manipulate Signaling Pathways
Writer and Curator: Larry H Bernstein, MD, FCAP
7.6 Manipulate Signaling Pathways
7.6.1 The Dynamics of Signaling as a Pharmacological Target
7.6.2 A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging
7.6.3 IQGAPs choreograph cellular signaling from the membrane to the nucleus
7.6.4 Signaling cell death from the endoplasmic reticulum stress response
7.6.5 An Enzyme that Regulates Ether Lipid Signaling Pathways in Cancer Annotated by Multidimensional Profiling
7.6.6 Peroxisomes – A Nexus for Lipid Metabolism and Cellular Signaling
7.6.7 A nexus for cellular homeostasis- the interplay between metabolic and signal transduction pathways
7.6.8 Mechanisms-of-intercellular-signaling
7.6.9 Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis
Marcelo Behar, Derren Barken, Shannon L. Werner, Alexander Hoffmann
Cell 10 Oct 2013; 155(2):448–461
Drugs targeting signaling hubs may block specific dynamic features of the signal
Specific inhibition of dynamic features may introduce pathway selectivity
Phase space analysis reveals principles for drug targeting signaling dynamics
Based on these principles, NFκB dynamics can be manipulated with specificity
Highly networked signaling hubs are often associated with disease, but targeting them pharmacologically has largely been unsuccessful in the clinic because of their functional pleiotropy. Motivated by the hypothesis that a dynamic signaling code confers functional specificity, we investigated whether dynamic features may be targeted pharmacologically to achieve therapeutic specificity. With a virtual screen, we identified combinations of signaling hub topologies and dynamic signal profiles that are amenable to selective inhibition. Mathematical analysis revealed principles that may guide stimulus-specific inhibition of signaling hubs, even in the absence of detailed mathematical models. Using the NFκB signaling module as a test bed, we identified perturbations that selectively affect the response to cytokines or pathogen components. Together, our results demonstrate that the dynamics of signaling may serve as a pharmacological target, and we reveal principles that delineate the opportunities and constraints of developing stimulus-specific therapeutic agents aimed at pleiotropic signaling hubs.
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Intracellular signals link the cell's genome to the environment. Misregulation of such signals often cause or exacerbate disease (Lin and Karin, 2007 and Weinberg, 2007) (so-called "signaling diseases"), and their rectification has been a major focus of biomedical and pharmaceutical research (Cohen, 2002, Frelin et al., 2005 and Ghoreschi et al., 2009). For the identification of therapeutic targets, the concept of discrete signaling pathways that transmit intracellular signals to connect cellular sensor/receptors with cellular core machineries has been influential. In this framework, molecular specificity of therapeutic agents correlates well with their functional or phenotypic specificity. However, in practice, clinical outcomes for many drugs with high molecular specificity has been disappointing (e.g., inhibitors of IKK, MAPK, and JNK; Berger and Iyengar, 2011, DiDonato et al., 2012, Röring and Brummer, 2012 and Seki et al., 2012).
Many prominent signaling mediators are functionally pleiotropic, playing roles in multiple physiological functions (Chavali et al., 2010 and Gandhi et al., 2006). Indeed, signals triggered by different stimuli often travel through shared network segments that operate as hubs before reaching the effectors of the cellular response (Bitterman and Polunovsky, 2012 and Gao and Chen, 2010). Hubs' inherent pleiotropy means that their inhibition may have broad and likely undesired effects (Karin, 2008, Berger and Iyengar, 2011,Force et al., 2007, Oda and Kitano, 2006 and Zhang et al., 2008); this is a major obstacle for the efficacy of drugs targeting prominent signaling hubs such as p53, MAPK, or IKK.
Recent studies have begun to address how signaling networks generate stimulus-specific responses (Bardwell, 2006, Haney et al., 2010, Hao et al., 2008 and Zalatan et al., 2012). For example, the activity of some pleiotropic kinases may be steered to particular targets by scaffold proteins (Park et al., 2003,Schröfelbauer et al., 2012 and Zalatan et al., 2012). Alternatively, or in addition, some signaling hubs may rely on stimulus-specific signal dynamics to activate selective downstream branches in a stimulus-specific manner in a process known as temporal or dynamic coding or multiplexing (Behar and Hoffmann, 2010,Chalmers et al., 2007, Hoffmann et al., 2002, Kubota et al., 2012, Marshall, 1995 and Purvis et al., 2012;Purvis and Lahav, 2013, Schneider et al., 2012 and Werner et al., 2005).
Although the importance of signaling scaffolds and their pharmacological promise is widely appreciated (Klussmann et al., 2008 and Zalatan et al., 2012) and isolated studies have altered the stimulus-responsive signal dynamics (Purvis et al., 2012, Park et al., 2003, Sung et al., 2008 and Sung and Simon, 2004), the capacity for modulating signal dynamics for pharmacological gain has not been addressed in a systematic manner. In this work, we demonstrate by theoretical means that, when signal dynamics are targeted, pharmacological perturbations can produce stimulus-selective results. Specifically, we identify combinations of signaling hub topology and input-signal dynamics that allow for pharmacological perturbations with dynamic feature-specific or input-specific effects. Then, we investigate stimulus-specific drug targeting in the IKK-NFκB signaling hub both in silico and in vivo. Together, our work begins to define the opportunities for pharmacological targeting of signaling dynamics to achieve therapeutic specificity.
Dynamic Signaling Hubs May Be Manipulated to Mute Specific Signals
Previous work has shown how stimulus-specific signal dynamics may allow a signaling hub to selectively route effector functions to different downstream branches (Behar et al., 2007). Here, we investigated the capacity of simple perturbations to kinetic parameters (caused for example by drug treatments) to produce stimulus-specific effects. For this, we examined a simple model of an idealized signaling hub (Figure 1A), reminiscent of the NFκB p53 or of MAPK signaling modules. The hub X∗ reacts with strong but transient activity to stimulus S1 and sustained, slowly rising activity to stimulus S2. These stimulus-specific signaling dynamics are decoded by two effector modules, regulating transcription factors TF1 and TF2. TF1, regulated by a strongly adaptive negative feedback, is sensitive only to fast-changing signals, whereas TF2, regulated by a slowly activating two-state switch, requires sustained signals for activation (Figure 1B). We found it useful to characterize the X∗, TF1, and TF2 responses in terms of two dynamic features, namely the maximum early amplitude ("E," time < 15′) and the average late amplitude ("L," 15′ < t < 6 hr). These features, calculated using a mathematical model of the network (see Experimental Procedures) show good fidelity and specificity (Komarova et al., 2005) (Figure 1C), as S1 causes strong activation of TF1 with minimal crosstalk to TF2, and vice versa for S2.
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Figure 1. Pharmacologic Perturbations with Stimulus-Specific Effects
(A) A negative-feedback module transduces input signals S1 and S2, producing outputs that are decoded by downstream effectors circuits that may distinguish between different dynamics.
(B) Unperturbed dynamics of X∗, TF1∗, and TF2∗ in response to S1 (red) and S2 (blue). Definition of early (E) and late (L) parts of the signal is indicated.
(C) Specificity and fidelity of E and L for TF1∗ and TF2∗, as defined in Komarova et al., 2005).
(D) Partial inhibition of X∗ activation (A) abolishes the response to S1, but not S2, whereas a perturbation targeting the feedback regulator (FBR) suppresses the response to S2, but not S1.
(E) Perturbation phenotypes defined as difference between unperturbed and perturbed values of the indicated quantities (arbitrary scales for X∗, TF1∗, and TF2∗). Perturbation A inhibits E and TF1∗, but not TF2∗; perturbation FBR inhibits L and TF2∗, but not TF1∗.
(F) Virtual screening pipeline showing the experimental design and the two analysis branches for characterizing feature- and input-specific effects.
See also in Experimental Procedures and Table S1.
Seeking simple (affecting a single reaction) perturbations that selectively inhibit signaling by S1 or S2, we found that perturbation A, partially inhibiting the activation of X, was capable of suppressing hub activity in response to a range of S1 amplitudes while still allowing for activity in response to S2 (Figure 1D). Consequently, this perturbation significantly reduced TF1 activity in response to S1 but had little effect on TF2 activity elicited by S2. We also found that the most effective way to inhibit S2 signaling was by targeting the deactivation of negative feedback regulator Y (FBR). This perturbation caused almost complete abrogation of late X activity yet allows for significant levels of early activity. As a result, TF2 was nearly completely abrogated in response to S2, but stimulus S1 still produced a solid TF1 response. The early (E) and late (L) amplitudes could be used to quantify the input-signal-specific effects of these perturbations (Figure 1E).
This numerical experiment showed that it is possible to selectively suppress transient or sustained dynamic signals transduced through a common negative-feedback-containing signaling hub. Moreover, the dynamic features E and L could be independently inhibited. To study how prevalent such opportunities for selective inhibition are, we established a computational pipeline for screening reaction perturbations within multiple network topologies and in response to multiple dynamic input signals; the simulation results were analyzed to identify cases of either "input-signal-specific" inhibition or "dynamic feature-specific" inhibition (Figure 1F).
A Computational Screen to Identify Opportunities for Input-Signal-Specific Inhibition
The computational screen involved small libraries of one- and two-component regulatory modules and temporal profiles of input signals (Figure 2A), both commonly found in intracellular signaling networks. All modules (M1–M7, column on left) contained a species X that, upon stimulation by an input signal, is converted into an active form X∗ (the output) that propagates the signal to downstream effectors. One-component modules included a reversible two-state switch (M1) and a three-state cycle with a refractory state (M2). Two-component modules contained a species Y that, upon activation via a feedback (M3 and M5) or feedforward (M4 and M6) loop, either deactivates X (M3 and M4) or inhibits (M5 and M6) its activation. We also included the afore-described topology that mimics the IκB-NFκB or the Mdm2-p53 modules (M7). Mathematical descriptions may be found in the Experimental Procedures. Although many biological signaling networks may conform to one of these simple topologies, others may be abstracted to one that recapitulates the physiologically relevant emergent properties
Figure 2. A Virtual Screen for Stimulus Specificity in Pharmacologic Perturbations
(A) Signaling modules (left) and input library (top) used in the screen. Dotted lines indicate enzymatic reactions (perturbation names indicated in letter code). Time courses of hub activity for each module/input combination for the unperturbed (black) and perturbed cases (blue indicates a decrease, red an increase in parameter value).
(B) Relative sensitivity of the stimulus response to the indicated perturbation (defined as the perturbation's effect on the area under the curve), normalized per row.
See also Experimental Procedures, Figure S1, and Tables S2 and S3.
The library of stimuli (S1–S10; Figure 2A, top row) comprises ten input functions with different combinations of "fast" and "slow" initiation and decay phases (see Experimental Procedures). The virtual screen was performed by varying the kinetic parameter for each reaction over a range of values, thereby modeling simple perturbations of different strengths and recording the temporal profile of X∗ abundance. To quantify stimulus-specific inhibition, we measured the area under the normalized dose-response curves (time average of X∗ versus perturbation dose) for each module-input combination (Experimental Procedures, Figure 2B, and Figure S1 available online).
Phase Space Analysis Reveals Underlying Regulatory Principles
To understand the origin of dynamic feature-specific inhibition, we investigated the perturbation effects analytically on each module's phase space, i.e., the space defined by X∗ and Y∗ quasi-equilibrium surfaces (Figures 4 and S4). These surfaces ("q.e. surfaces") represent the dose response of X∗ as a function of Y∗ and a stationary input signal S ("X surface") and the dose response of Y∗ as a function of X∗ and S ("Y surface") (Figure 4A). The points at which the surfaces intersect correspond to the concentrations of X∗ and Y∗ in equilibrium for a given value of S. In the basal state, when S is low, the system is resting at an equilibrium point close to the origin of coordinates. When S increases, the concentrations of X∗ and Y∗ adjust until the signal settles at some stationary value (Figure 4A). Gradually, changing input signals cause the concentrations to follow trajectories close to the q.e. surfaces (quasi-equilibrium dynamics), following the line defined by the intersection of the surfaces ("q.e. line") in the extreme of infinitely slow inputs. Fast-changing stimuli drive the system out of equilibrium, causing the trajectories to deviate markedly from the q.e. surfaces.
Two main principles emerged: (1) perturbations that primarily affect the shape of a q.e. surface tend to affect steady-state levels or responses that evolve close to quasi-equilibrium, and (2) perturbations that primarily affect the balance of timescales (X∗, Y∗ activation, and S) tend to affect transient out-of-equilibrium parts of the response. These principles reflect the fact that out-of-equilibrium parts of a signal are largely insensitive to the precise shape of the underlying dose-response surfaces (they may still be bounded by them) but depend on the balance between the timescales of the biochemical processes involved. Perturbation of these balances affects how a system approaches steady state (thus affecting out-of-equilibrium and quasi-equilibrium dynamics), but not steady-state levels. To illustrate these principles, we present selected results for modules M3 and M4 and discuss additional cases in the supplement (Figure S3).
Detailed Analysis of Modules M3 and M4, Related to Figure 4
Time courses and projections of the phase space for modules M3 and M4. Color coding similar to Figure 4.
In the feedback-based modules (M3 and M5), the early peak of activity in response to rapidly changing signals is an out-of-equilibrium feature that occurs when the timescale of Y activation is significantly slower than that of X. Under these conditions, the concentration of X∗ increases rapidly (out of equilibrium) before decaying along the X∗ surface (in quasi-equilibrium) as more Y gets activated (Figure 4A, parameters modified to better illustrate the effects being discussed; see Table S2). For input signals that settle at some stationary level of S, Y activation eventually catches up and the concentration of X∗ settles at the equilibrium point where the X∗ and Y∗ curves intersect. Gradually changing signals allow X∗ and Y∗activation to continuously adapt, and the system evolves closer to the q.e. line.
In such modules, perturbation A (X∗ activation) changes both the shape of the q.e. surface for X∗ and the kinetics of activation. When in the unperturbed system Y∗ saturates, perturbation A primarily reduces X∗steady-state level (Figures 4B and 4C, left and center). When Y∗ does not saturate in the unperturbed system, the primary effect is the reduced activation kinetics. Thus the perturbation affects the out-of-equilibrium peak (Figures 4B and 4C, center and right), with only minor reduction of steady-state levels (especially when Y∗'s dose response respect to X∗ is steep). The transition from saturated to not-saturated feedback (as well as the perturbation strength) underlies the dose-dependent switch from L to E observed in the screen. In both saturated and unsaturated regimes, the shift in the shape of the surfaces does change the q.e. line and thus affects responses occurring in quasi-equilibrium. In contrast, perturbation of the feedback recovery (FBR) shifts the Y∗ surface vertically (Figure 4D), specifically affecting the steady-state levels and late signaling; the effect on Y∗ kinetics is limited because the reaction is relatively slow. Perturbation FBA also shifts the Y∗ surface, but the net effect is less specific because the associated increase in the rate of Y∗ activation tends to equalize X∗ and Y∗ kinetics affecting also the out-of-equilibrium peak.
In resting cells, NFκB is held inactive through its association with inhibitors IκBα, β, and ε. Upon stimulation, these proteins are phosphorylated by the kinase IKK triggering their degradation. Free nuclear NFκB activates the expression of target genes, including IκB-encoding genes, which thereby provide negative feedback (Figure 5A). The IκB-NFκB-signaling module is a complex dynamic system; however, by abstracting the control mechanism to its essentials, we show below that the above-described principles can be applied profitably.
IκB-NFκB signaling module
Figure 5. Modulating NFκB Signaling Dynamics
(A) The IκB-NFκB signaling module.
(B) Equilibrium dose-response relationship for NFκB versus IKK.
(C) Three IKK curves representative of three stimulation regimes; TNFc (red), TNFp (green), and LPS (blue) function as inputs into the model, which computes the corresponding NFκB activity dynamics (bottom). The quasi-equilibrium line (black) was obtained by transforming the IKK temporal profiles by the dose response in (B). Deviation from the quasi-equilibrium line for the TNF response indicates out-of-equilibrium dynamics.
(D) Coarse-grained model of the IκB-NFκB module and predicted effects of perturbations.
(E) Selected perturbations with specific effects on out-of-equilibrium (top three) or steady state (bottom two). (Left to right) Feature maps in the E-L space (E: t < 60 ′, L: 120′ < t < 300′), tangent angle at the unperturbed point (θ > 0 indicates L is more suppressed than E and vice versa), and time courses (green, TNF chronic; red, TNF pulse; blue, LPS). Only inhibitory perturbations are shown. Additional perturbations are shown in Figure S4.
See also Experimental Procedures and Table S7.
Here, we delineate the potential of achieving stimulus-specific inhibition when targeting molecular reactions within pleiotropic signaling hubs. We found that it is theoretically possible to design perturbations that (1) selectively attenuate signaling in response to one stimulus but not another, (2) selectively attenuate undesirable features of dynamic signals or enhance desirable ones, or (3) remodulate output signals to fit a dynamic profile normally associated with a different stimulus.
These opportunities—not all of them possible for every signaling module topology or biological scenario—are governed by two general principles based on timescale and dose-response relationships between upstream signal dynamics and intramodule reaction kinetics (Figure 4 and Table S4). In short, a steady-state or quasi-equilibrium part of a response may be selectively affected by perturbations that introduce changes in the relevant dose-response surfaces. Out-of-equilibrium responses that are not sensitive to the precise shape of a dose-response curve may be selectively attenuated by perturbations that modify the relative timescales. Dose responses and timescales cannot, in general, be modified independently by simple perturbations (combination treatments are required), but as we show, in some cases, one effect dominates resulting in feature or stimulus specificity.
The degree to which specific dynamic features of a signaling profile or the dynamic responses to specific stimuli can be selectively inhibited depends on how distinctly they rely on quasi-equilibrium and out-of-equilibrium control. Signals that contain both features may be partially inhibited by both types of perturbation, limiting the specific inhibition achievable by simple perturbations. In practice, this limited the degree to which NFκB signaling could be inhibited in a stimulus-specific manner (Figure 5) and the associated therapeutic dose window (Figure 6). The most selective stimulus-specific effects can be introduced when a signal is heavily dependent on a particular dynamic feature; for example, suppression of out-of-equilibrium transients will abrogate the response to stimuli that produce such transients. For a selected group of target genes, this specificity at the signal level translated directly to expression patterns (Figure 6B, middle). More generally, selective inhibition of early or late phases of a signal may allow for specific control of early and late response genes (Figure 6C), a concept that remains to be studied at genomic scales. Though the principles are general, how they apply to specific signaling pathways depends not only on the regulatory topology, but also on the dynamic regime determined by the parameters. As demonstrated with the IκB-NFκB module, analysis of a coarse-grained topology in terms of the principles may allow the prediction of perturbations with a desired specificity.
Marvin E. Tanenbaum, Luke A. Gilbert, Lei S. Qi, Jonathan S. Weissman, Ronald D. Vale
Cell 23 Oct 2014; 159(3): 635–646
SunTag allows controlled protein multimerization on a protein scaffold
SunTag enables long-term single-molecule imaging in living cells
SunTag greatly improves CRISPR-based activation of gene expression
Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.
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SunTag, which can recruit multiple copies of an antibody-fusion protein
Development of the SunTag, a System for Recruiting Multiple Protein Copies to a Polypeptide Scaffold Protein multimerization on a single RNA or DNA template is made possible by identifying protein domains that bind with high affinity to a relatively short nucleic acid motif. We therefore sought a protein-based system with similar properties, specifically a protein that can bind tightly to a short peptide sequence (Figures 1A and1B).Antibodies arecapable ofbindingto short,unstructured peptide sequences with high affinity and specificity, and, importantly, peptide epitopes can be designed that differ from naturally occurring sequences in the genome. Furthermore, whereas antibodies generally do not fold properly in the cytoplasm, single-chain variable fragment (scFv) antibodies, in which the epitope-binding regions of the light and heavy chains of the antibody are fused to forma single polypeptide, have been successfully expressed in soluble form in cells (Colby et al., 2004; Lecerf et al., 2001; Wo ¨rn et al., 2000).
We expressed three previously developed single-chain antibodies (Colby et al., 2004; Lecerf et al., 2001; Wo ¨rn et al., 2000) fused to EGFP in U2OS cells and coexpressed their cognate peptides (multimerized in four tandem copies) fused to the cytoplasmic side of the mitochondrial protein mitoNEET (Colca et al., 2004) (referred to here as Mito, Figure S1A). We then assayed whether the antibody-GFP fusion proteins would be recruited to the mitochondria by fluorescence microscopy, which would indicate binding between antibody and peptide (Figure 1B). Of the three antibody-peptide pairs tested, only the GCN4 antibody-peptide pair showed robust and specific binding while not disrupting normal mitochondrial morphology (Figures 1C and S1B). Thus, we focused our further efforts on the GCN4 antibody-peptide pair. The GCN4 antibody was optimized to allow intracellular expression in yeast (Wo ¨rn et al., 2000). In human cells, however, we still observed some protein aggregates of scFv-GCN4-GFP at high expression levels (Figure S2A). To improve scFv-GCN4 stability, we added a variety of N- and C-terminal fusion proteins known to enhance protein solubility and found that fusion of superfolder-GFP (sfGFP) alone
(Pe'delacq et al., 2006) or along with the small solubility tag GB1 (Gronenborn et al., 1991) to the C terminus of the GCN4 antibody almost completely eliminated protein aggregation, even at high expression levels (Figure S2A). Thus, we performed all further experiments with scFv-GCN4-sfGFP-GB1 (hereafter referred to as scFvGCN4-GFP). Very tight binding of the antibody-peptide pair in vivo is critical fortheformation ofmultimersonaproteinscaffoldbackbone.To determine the dissociation rate of the GCN4 antibody-peptide interaction, we performed fluorescence recovery after photobleaching (FRAP) experiments on scFv-GCN4-GFP bound to the mitochondrial-localized mito-mCherry-4xGCN4pep. After photobleaching, very slow GFP recovery was observed (halflife of 5–10 min [Figures 2A and 2B]), indicating that the antibody bound very tightly to the peptide. It is also important to optimize the spacing of the scFv-GCN4 binding sites within the protein scaffold so that they could be saturated by scFvGCN4 because steric hindrance of neighboring peptide binding sites is a concern. We varied the spacing between neighboring GCN4 peptides and quantified the antibody occupancy on the peptide array.
Figure 1. Identification of an Antibody-Peptide Pair that Binds Tightly In Vivo (A) Schematic of the antibody-peptide labeling strategy. (B) Schematic of the experiment described in (C) in which the mitochondrial targeting domain of mitoNEET (yellow box, mito) fused to mCherry and four tandem copies of a peptide recruits a GFP-tagged intracellular antibody to mitochondria. (C) ScFv-GCN4-GFP was coexpressed with either mito-mCherry-4xGCN4peptide (bottom) or mito-mCherry-FKBP as a control (top) in U2OS cells, and cells were imaged using spinning-disk confocal microscopy. Scale bars, 10 mm. See also Figure S1.
Figure 2. Characterization of the Off Rate and Stoichiometry of the Binding Interaction between the scFv-GCN4 Antibody and the GCN4 Peptide Array In Vivo (A) Mito-mCherry-24xGCN4pep was cotransfected with scFv-GCN4-GFP in HEK293 cells, and their colocalization on mitochondria in a single cell is shown (10 s). At 0 s, the mitochondria-localized GFP signal was photobleached in a single z plane using a 472 nm laser, and fluorescence recovery was followed by time-lapse microscopy. Scale bar, 5 mm. (B) The FRAP was quantified for 20 cells. (C–E) Indicated constructs were transfected in HEK293 cells, and images were acquired 24 hr after transfection with identical image acquisition settings. Representative images are shown in (C). Note that the GFP signal intensity in the mito-mCherry-24xGCN4pep + scFv-GCN4-GFP is highly saturated when the same scaling is used as in the other panels. Bottom row shows a zoom of a region of interest: dynamic scaling was different for the GFP and mCherry signals, so that both could be observed. Scale bars, 10 mm. (D and E) Quantifications of the GFP:mCherry fluorescence intensity ratio on mitochondria after normalization. Eachdot represents a single cell, and dashed lines indicates the average value. See also Figure S2.
Figure 3. The SunTag Allows Long-Term Single-Molecule Fluorescence Imaging in the Cytoplasm (A–H) U2OS cells were transfected with indicated SunTag24x constructs together with the scFv-GCN4-GFP-NLS and were imaged by spinning-disk confocal microscopy 24 hr after transfection. (A) A representative image of SunTag24x-CAAX-GFP is shown (left), as well as the fluorescence intensities quantification of the foci (right, blue bars). As a control, U2OS were transfected with sfGFP-CAAX and fluorescence intensities of single sfGFP-CAAX molecules were also quantified (red bars). The average fluorescence intensity of the single sfGFP-CAAX was set to 1. Dotted line marks the outline of the cell (left). Scale bar, 10 mm. (B) Cells expressing K560-SunTag24x-GFP were imaged by spinning disk confocal microscopy (image acquisition every 200 ms). Movement is revealed by a maximum intensity projection of 50 time points (left) and a kymograph (right). Scale bar, 10 mm. (C and D) Cells expressing both EB3-tdTomato and K560-SunTag24x-GFP were imaged, and moving particles were tracked manually. Red and blue tracks (bottom) indicate movement toward the cell interior and periphery, respectively (C). The duration of the movie was 20 s. Scale bar, 5 mm. Dots in (D) represent individual cells with between 5 and 20 moving particles scored per cell. The mean and SD are indicated. (E and F) Cells expressing Kif18b-SunTag24x-GFP were imaged with a 250 ms time interval. Images in (E) show a maximum intensity projection (50 time- points, left) and a kymograph (right). Speeds of moving molecules were quantified from ten different cells (F). Scale bar, 10 mm. (G and H) Cells expressing both mCherry-a-tubulin and K560rig-SunTag24x-GFP were imaged with a 600 ms time interval.The entire cell is shown in (G), whereas H shows zoomed-instills of atime series from the same cell. Open circlestrack two foci on the same microtubule,which is indicated bythe dashed line. Asterisks indicate stationary foci. Scale bars, 10 and 2 mm (G and H), respectively. See also Figure S3 and Movies S1, S2, S3, S4, S5, and S6.
The GCN4 peptide contains many hydrophobic residues (Figure 4B) and is largely unstructured in solution (Berger et al., 1999); thus, the poor expression of the peptide array could be due to its unstructured and hydrophobic nature. To test this idea, we designed several modified peptide sequence that were predicted to increase a-helical propensity and reduce hydrophobicity. One of these optimized peptides (v4, Figure 4B) was expressed moderately well as a 243 peptide array, and even higher expression was achieved with a 103 peptide array (Figure 4C). Importantly, fluorescence imaging revealed that thescFv-GCN4antibody robustlyboundto theGCN4v4peptide array in vivo and FRAP analysis suggests that the scFv-GCN4 antibody dissociates with a similar slow off rate from the GCN4
v4 peptide array as the original peptide (Figures 4D and 4E). Furthermore, K560 motility could be observed when it was tagged with the optimized v4 243 peptide array, indicating that the optimized v4 peptide array did not interfere with protein function (Movie S7). Together, these results identify a second version of the peptide array that can be used for applications requiring higher expression.
Activation of Gene Transcription Using Cas9-SunTag Because the SunTag system could be used for amplification of a fluorescence signal, we tested whether it also could be used to amplify regulatory signals involved in gene expression. Transcription of a gene is strongly enhanced by recruiting multiple copies of transcriptional activators to endogenous or artificial gene promoters (Anderson and Freytag, 1991; Chen et al., 1992; Pettersson and Schaffner, 1990). Thus, we thought that robust, artificial activation of gene transcription might also be achieved by recruiting multiple copies of a synthetic transcriptional activator to a gene using the SunTag.
Figure 4. An Optimized Peptide Array for High Expression (A) Indicated constructs were transfected in HEK293 cells and imaged 24 hr after transfection using wide-field microscopy. All images were acquired using identical acquisition parameters. Representative images are shown (left), and fluorescence intensities were quantified (n = 3) (right). (B) Sequence of the first and second generation GCN4 peptide (modified or added residues are colored blue, hydrophobic residues are red, and linker residues are yellow). (C–E) Indicated constructs were transfected in HEK293 cells and imaged 24 hr after transfection using wide-field (C) or spinning-disk confocal (D and E) microscopy. (C) Representative images are shown (left), and fluorescence intensities were quantified (n = 3) (right). (D and E) GFP signal on mitochondria was photobleached, and fluorescence recovery was determined over time. The graph (E) represents an average of six cells per condition. (E) shows an image of a representative cell before photobleaching. Scale bars in (A) and (C), 50 mm; scale bars in (D) and (E), 10 mm. Error bars in (A) and (C) represent SDs. See also Movie S7.
Figure 5. dCas9-SunTag Allows Genetic Rewiring of Cells through Activation of Endogenous Genes (A) Schematic of gene activation by dCas9-VP64 and dCas9-SunTag-VP64. dCas9 binds to a gene promoter through its sequence-specific sgRNA (red line). Direct fusion of VP64 to dCas9 (top) results in a single VP64 domain at the promoter, which poorly activates transcription of the downstream gene. In contrast, recruitment of many VP64 domains using the SunTag potently activates transcription of the gene (bottom). (B–D) K562 cells stably expressing dCas9-VP64 or dCas9-SunTag-VP64 were infected with lentiviral particles encoding indicated sgRNAs, as well as BFP and a puromycin resistance gene and selected with 0.7 mg/ml puromycin for 3 days to kill uninfected cells. (B and C) Cells were stained for CXCR4 using adirectlylabeleda-CXCR4 antibody, and fluorescence was analyzed by FACS. (D) Trans-well migration assays (see Experimental Procedures) were performed with indicated sgRNAs. Results are displayed as the fold change in directional migrating cells over control cell migration. (E) dCas9-VP64 or dCas9-SunTag-VP64 induced transcription of CDKN1B with several sgRNAs. mRNA levels were quantified by qPCR. (F) Doubling timeofcontrolcells orcells expressing indicated sgRNAs was determined (see Experimental Procedures section). Graphs in (C), (D), and (F) are averages of three independent experiments. Graph in (E) is average of two biological replicates, each with two or three technical replicates. All error bars indicate SEM. See also Figure S4
Jessica M. Smith, Andrew C. Hedman, David B. Sacks
Trends Cell Biol Mar 2015; 25(3): 171–184
http://dx.doi.org/10.1016/j.tcb.2014.12.005
IQGAP proteins scaffold diverse signaling molecules.
IQGAPs mediate crosstalk between signaling pathways.
IQGAP1 regulates nuclear processes, including transcription.
Since its discovery in 1994, recognized cellular functions for the scaffold protein IQGAP1 have expanded immensely. Over 100 unique IQGAP1-interacting proteins have been identified, implicating IQGAP1 as a critical integrator of cellular signaling pathways. Initial research established functions for IQGAP1 in cell–cell adhesion, cell migration, and cell signaling. Recent studies have revealed additional IQGAP1 binding partners, expanding the biological roles of IQGAP1. These include crosstalk between signaling cascades, regulation of nuclear function, and Wnt pathway potentiation. Investigation of the IQGAP2 and IQGAP3 homologs demonstrates unique functions, some of which differ from those of IQGAP1. Summarized here are recent observations that enhance our understanding of IQGAP proteins in the integration of diverse signaling pathways.
Shore GC1, Papa FR, Oakes SA
Curr Opin Cell Biol. 2011 Apr; 23(2):143-9
http://dx.doi.org/10.1016%2Fj.ceb.2010.11.003
Inability to meet protein folding demands within the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), a signaling pathway with both adaptive and apoptotic outputs. While some secretory cell types have a remarkable ability to increase protein folding capacity, their upper limits can be reached when pathological conditions overwhelm the fidelity and/or output of the secretory pathway.
The lumen of the ER is a unique cellular environment optimized to carry out the three primary tasks of this organelle:
calcium storage and release,
protein folding and secretion, and
lipid biogenesis [1].
A range of cellular disturbances lead to accumulation of misfolded proteins in the ER, including
point mutations in secreted proteins that disrupt their proper folding,
sustained secretory demands on endocrine cells,
viral infection with ER overload of virus-encoding protein, and
loss of calcium homeostasis with detrimental effects on ER-resident calcium-dependent chaperones [2–4].
The tripartite UPR consists of three ER transmembrane proteins (IRE1α, PERK, ATF6) that
alert the cell to the presence of misfolded proteins in the ER and
attempt to restore homeostasis in this organelle through increasing ER biogenesis,
decreasing the influx of new proteins into the ER,
promoting the transport of damaged proteins from the ER to the cytosol for degradation, and
upregulating protein folding chaperones [5].
The adaptive responses of the UPR can markedly expand the protein folding capacity of the cell and restore ER homeostasis [6]. However, if these adaptive outputs fail to compensate because ER stress is excessive or prolonged, the UPR induces cell death.
The cell death pathways collectively triggered by the UPR include both caspase-dependent apoptosis and caspase-independent necrosis. While many details remain unknown, we are beginning to understand how cells determine when ER stress is beyond repair and communicate this information to the cell death machinery. For the purposes of this review, we focus on the apoptotic outputs triggered by the UPR under irremediable ER stress.
Connections from the UPR to the Mitochondrial Apoptotic Pathway
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078187/bin/nihms256554f1.jpg
Figure 1 Connections from the UPR to the Mitochondrial Apoptotic Pathway
Under excessive ER stress, the ER transmembrane sensors IRE1α and PERK send signals through the BCL-2 family of proteins to activate the mitochondrial apoptotic pathway. In response to unfolded proteins, IRE1α oligomerizes and induces endonucleolytic decay of hundreds of ER-localized mRNAs, depleting ER protein folding components and leading to worsening ER stress. Phosphorylated IRE1α also recruits TNF receptor-associated factor 2 (TRAF2) and activates apoptosis signaling kinase 1 (ASK1) and its downstream target c-Jun NH2-terminal kinase (JNK). JNK then activates pro-apoptotic BIM and inhibits anti-apoptotic BCL-2. These conditions result in dimerization of PERK and activation of its kinase domain to phosphorylate eukaryotic translation initiation factor 2α (eIF2α), which causes selective translation of activating transcription factor-4 (ATF4). ATF4 upregulates expression of the CHOP/GADD153 transcription factor, which inhibits the gene encoding anti-apoptotic BCL-2 while inducing expression of pro-apoptotic BIM. ER stress also promotes p53-dependent transcriptional upregulation of Noxa and Puma, two additional pro-apoptotic BH3-only proteins. Furthermore, high levels of UPR signaling induce initiator caspase-2 to proteolytically cleave and activate pro-apoptotic BID upstream of the mitochondrion. In addition to antagonizing pro-survival BCL-2 members, cleaved BID, BIM and PUMA activate Bax and/or Bak. Hence, in response to excessive UPR signaling, the balance of BCL-2 family proteins shifts in the direction of apoptosis and leads to the oligomerization of BAX and BAK, two multi-domain pro-apoptotic BCL-2 family proteins that then drive the permeabilization of the outer mitochondrial membrane, apoptosome formation and activation of executioner caspases such as Caspase-3. Figure adapted with permission from the Journal of Cell Science [58].
The proximal unfolded protein response sensors
UPR signaling is initiated by three ER transmembrane proteins:
IRE1α,
PERK, and
The most ancient ER stress sensor, IRE1α, contains
an ER lumenal domain,
a cytosolic kinase domain and
a cytosolic RNase domain [9,10].
In the presence of unfolded proteins, IRE1α's ER lumenal domains homo-oligomerize, leading
first to kinase trans-autophosphorylation and
subsequent RNase activation.
Dissociation of the ER chaperone BiP from IRE1α's lumenal domain in order to engage unfolded proteins may facilitate IRE1α oligomerization [11]; alternatively, the lumenal domain may bind unfolded proteins directly [12]. PERK's ER lumenal domain is thought to be activated similarly [13,14]. The ATF6 activation mechanism is less clear. Under ER stress, ATF6 translocates to the Golgi and is cleaved by Site-1 and Site-2 proteases to generate the ATF6(N) transcription factor [15].
All three UPR sensors have outputs that attempt to tilt protein folding demand and capacity back into homeostasis. PERK contains a cytosolic kinase that phosphorylates eukaryotic translation initiation factor 2α (eIF2α), which impedes translation initiation to reduce the protein load on the ER [16]. IRE1α splices XBP1mRNA, to produce the homeostatic transcription factor XBP1s [17,18]. Together with ATF6(N), XBP1s increases transcription of genes that augment ER size and function[19]. When eIF2α is phosphorylated, the translation of the activating transcription factor-4 (ATF4) is actively promoted and leads to the transcription of many pro-survival genes [20]. Together, these transcriptional events act as homeostatic feedback loops to reduce ER stress. If successful in reducing the amount of unfolded proteins, the UPR attenuates.
However, when these adaptive responses prove insufficient, the UPR switches into an alternate mode that promotes apoptosis. Under irremediable ER stress, PERK signaling can induce ATF-4-dependent upregulation of the CHOP/GADD153 transcription factor, which inhibits expression of the gene encoding anti-apoptotic BCL-2 while upregulating the expression of oxidase ERO1α to induce damaging ER oxidation [21,22]. Sustained IRE1α oligomerization leads to activation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream target c-Jun NH2-terminal kinase (JNK) [23,24]. Phosphorylation by JNK has been reported to both activate pro-apoptotic BIM and inhibit anti-apoptotic BCL-2 (see below). Small molecule modulators of ASK1 have been shown to protect cultured cells against ER stress-induced apoptosis, emphasizing the importance of the IRE1α-ASK1-JNK output as a death signal in this pathway [25]. In response to sustained oligomerization, the IRE1α RNase also causes endonucleolytic decay of hundreds of ER-localized mRNAs [26]. By depleting ER cargo and protein folding components, IRE1α-mediated mRNA decay may worsen ER stress, and could be a key aspect of IRE1α's pro-apoptotic program [27]. Recently, inhibitors of IRE1α's kinase pocket have been shown to conformationally activate its adjacent RNase domain in a manner that enforces homeostatic XBP1s without causing destructive mRNA decay [27], a potentially exciting strategy for preventing ER stress-induced cell loss.
The BCL-2 family and the Mitochondrial Apoptotic Pathway
A wealth of genetic and biochemical data argues that the intrinsic (mitochondrial) apoptotic pathway is the major cell death pathway induced by the UPR, at least in most cell types. This apoptotic pathway is set in motion when several toxic proteins (e.g., cytochrome c, Smac/Diablo) are released from mitochondria into the cytosol where they lead to activation of downstream effector caspases (e.g., Caspase-3) [30]. The BCL-2 family, a large class of both pro- and anti- survival proteins, tightly regulates the intrinsic apoptotic pathway by controlling the integrity of the outer mitochondrial membrane [31]. This pathway is set in motion when cell injury leads to the transcriptional and/or post-translational activation of one or more BH3-only proteins that share sequence similarity in a short alpha helix (~9–12 a.a.) known as the Bcl-2 homology 3 (BH3) domain [32]. Once activated, BH3-only proteins lead to loss of mitochondrial integrity by disabling mitochondrial protecting proteins that drive the permeabilization of the outer mitochondrial membrane.
ER stress has been reported to activate at least four distinct BH3-only proteins (BID, BIM, NOXA, PUMA) that then signal the mitochondrial apoptotic machinery (i.e., BAX/BAK) [33–35]. Each of these BH3-only proteins is activated by ER stress in a unique way. Cells individually deficient in any of these BH3-only proteins are modestly protected against ER stress-inducing agents, but not nearly as resistant as cells null for their common downstream targets BAX and BAK [36]—the essential gatekeepers to the mitochondrial apoptotic pathway. Moreover, cells genetically deficient in both Bim andPuma are more protected against ER stress-induced apoptosis than Bim or Puma single knockout cells [37].
The ER stress sensor that signals these BH3-only proteins is known in a few cases (i.e., BIM is downstream of PERK); however, we do not yet understand how the UPR communicates with most of the BH3-only proteins. Moreover, it is not known if all of the above BH3-only proteins are simultaneously set in motion by all forms of ER stress or if a subset is activated under specific pathological stimuli that injure this organelle. Understanding the molecular details of how ER damage is communicated to the mitochondrial apoptotic machinery is critical if we want to target disease specific apoptotic signals sent from the ER.
Initiator and Executor Caspases
Caspases, or cysteine-dependent aspartate-directed proteases, play essential roles in both initiating apoptotic signaling (initiator caspases- 2, 4, 8, 12) and executing the final stages of cell demise (executioner caspases- 3, 7, 9) [38]. It is not surprising that the executioner caspases (casp-3,7,9) are critical for cell death resulting from damage to this organelle. Caspase 12 was the first caspase reported to localize to the ER downstream of BAX/BAK-dependent mitochondrial permeabilization becomes activated by UPR signaling in murine cells [39],but humans fail to express a functional Caspase 12 [41. Genetic knockdown or pharmacological inhibition of caspase-2 confers resistance to ER stress-induced apoptosis [42]. How the UPR activates caspase-2 and whether other initiator caspasesare also involved remains to be determined.
Calcium and Cell Death
Although an extreme depletion of ER luminal Ca2+ concentrations is a well-documented initiator of the UPR and ER stress-induced apoptosis or necrosis, it represents a relatively non-physiological stimulus. Ca2+ signaling from the ER is likely coupled to most pathways leading to apoptosis. UPR-induced activation of ERO1-α via CHOP in macrophages results in stimulation of inositol 1,4,5-triphosphate receptor (IP3R) [43]. All three sub-groups of the Bcl-2 family at the ER regulate IP3R activity. A significant fraction of IP3R is a constituent of highly specialized tethers that physically attach ER cisternae to mitochondria (mitochondrial-associated membrane) and regulate local Ca2+ dynamics at the ER-mitochondrion interface [45–46]. This results in propagation of privileged IP3R-mediated Ca2+ oscillations into mitochondria. In an extreme scenario, massive transmission of Ca2+ into mitochondria results in Ca2+ overload and cell death by caspase-dependent and –independent means [46,47]. More refined transmission regulated by the Bcl-2 axis at the ER can influence cristae junctions and the availability of cytochrome c for its release across the outer mitochondrial membrane [48]. Finally, such regulated Ca2+transmission to mitochondria is a key determinant of mitochondrial bioenergetics [49].
ER Stress-Induced Cell Loss and Disease
Mounting evidence suggests that ER stress-induced apoptosis contributes to a range of human diseases of cell loss, including diabetes, neurodegeneration, stroke, and heart disease, to name a few (reviewed in REF [50]). The cause of ER stress in these distinct diseases varies depending on the cell type affected and the intracellular and/or extracellular conditions that disrupt proteostasis. Both mutant SOD1 and mutant huntingtin proteins aggregate, exhaust proteasome activity, and result in secondary accumulations of misfolded proteins in the ER [51–52].
In the case of IRE1α, it may be possible to use kinase inhibitors to activate its cytoprotective signaling and shut down its apoptotic outputs [27]. Whether similar strategies will work for PERK and/or ATF6 remains to be seen. Alternatively, blocking the specific apoptotic signals that emerge from the UPR is perhaps a more straightforward strategy to prevent ER stress-induced cell loss. To this end, small molecular inhibitors of ASK and JNK are currently being tested in a variety preclinical models of ER stress [52–53,56–57]. This is just the beginning, and much work needs to be done to validate the best drugs targets in the ER stress pathway.
The UPR is a highly complex signaling pathway activated by ER stress that sends out both adaptive and apoptotic signals. All three transmembrane ER stress sensors (IRE1α, PERK, AFT6) have outputs that initially decrease the load and increase capacity of the ER secretory pathway in an effort to restore ER homeostasis. However, under extreme ER stress, continuous engagement of IRE1α and PERK results in events that simultaneously exacerbate protein misfolding and signal death, the latter involving caspase-dependent apoptosis and caspase-independent necrosis. Advances in our molecular understanding of how these stress sensors switch from life to death signaling will hopefully lead to new strategies to prevent diseases caused by ER stress-induced cell loss.
Chiang KP, Niessen S, Saghatelian A, Cravatt BF.
Chem Biol. 2006 Oct; 13(10):1041-50.
http://dx.doi.org/10.1016/j.chembiol.2006.08.008
Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.
Elucidation of the metabolic and signaling networks that regulate health and disease stands as a principal goal of postgenomic research. The remarkable complexity of these molecular pathways has inspired the advancement of "systems biology" methods for their characterization [1]. Toward this end, global profiling technologies, such as DNA microarrays 2 and 3 and mass spectrometry (MS)-based proteomics 4 and 5, have succeeded in generating gene and protein signatures that depict key features of many human diseases. However, extricating from these associative relationships the roles that specific biomolecules play in cell physiology and pathology remains problematic, especially for proteins of unknown biochemical or cellular function.
The functions of certain proteins, such as adaptor or scaffolding proteins, can be gleaned from large-scale protein-interaction maps generated by technologies like yeast two-hybrid 6 and 7, protein microarrays [8], and MS analysis of immunoprecipitated protein complexes 9 and 10. In contrast, enzymes contribute to biological processes principally through catalysis. Thus, elucidation of the activities of the many thousands of enzymes encoded by eukaryotic and prokaryotic genomes requires knowledge of their endogenous substrates and products. The functional annotation of enzymes in prokaryotic systems has been facilitated by the clever analysis of gene clusters or operons 11 and 12, which correspond to sets of genes adjacently located in the genome that encode for enzymes participating in the same metabolic cascade. The assembly of eukaryotic enzymes into metabolic pathways is more problematic, however, as their corresponding genes are not, in general, physically organized into operons, but rather are scattered randomly throughout the genome.
We hypothesized that the determination of endogenous catalytic activities for uncharacterized enzymes could be accomplished directly in living systems by the integrated application of global profiling technologies that survey both the enzymatic proteome and its primary biochemical output (i.e., the metabolome). Here, we have tested this premise by utilizing multidimensional profiling to characterize an integral membrane enzyme of unknown function that is highly elevated in human cancer.
Development of a Selective Inhibitor for the Uncharacterized Enzyme KIAA1363
Previous studies using the chemical proteomic technology activity-based protein profiling (ABPP) 15, 16 and 17 have identified enzyme activity signatures that distinguish human cancer cells based on their biological properties, including tumor of origin and state of invasiveness [18]. A primary component of these signatures was the protein KIAA1363, an uncharacterized integral membrane hydrolase found to be upregulated in aggressive cancer cells from multiple tissues of origin. To investigate the role that KIAA1363 plays in cancer cell metabolism and signaling, a selective inhibitor of this enzyme was generated by competitive ABPP 20 and 21.
Previous competitive ABPP screens that target the serine hydrolase superfamily identified a set of trifluoromethyl ketone (TFMK) inhibitors that showed activity in mouse brain extracts [20]. These TFMK inhibitors showed only limited activity in living human cells (data not shown). We postulated that the activity of KIAA1363 inhibitors could be enhanced by replacing the TFMK group with a carbamate, which inactivates serine hydrolases via a covalent mechanism (Figure S1; see the Supplemental Data available with this article online). Carbamate AS115 (Figure 1A) was synthesized and tested for its effects on the invasive ovarian cancer cell line SKOV-3 by competitive ABPP (Figure 1B). AS115 was found to potently and selectively inactivate KIAA1363, displaying an IC50 value of 150 nM, while other serine hydrolase activities were not affected by this agent (IC50 values > 10 μM) (Figures 1B and 1C). AS115 also selectively inhibited KIAA1363 in other aggressive cancer cell lines that possess high levels of this enzyme, including the melanoma lines C8161 and MUM-2B (Figure S2B).
Figure 1. Characterization of AS115, a Selective Inhibitor of the Cancer-Related Enzyme KIAA1363
Profiling the Metabolic Effects of KIAA1363 Inactivation in Cancer Cells
We next compared the global metabolite profiles of SKOV-3 cells treated with AS115 to identify endogenous small molecules regulated by KIAA1363, using a recently described, untargeted liquid chromatography-mass spectrometry (LC-MS) platform for comparative metabolomics [22]. AS115 (10 μM, 4 hr) was found to cause a dramatic reduction in the levels of a specific set of lipophilic metabolites (m/z 317, 343, and 345) in SKOV-3 cells ( Figure 2A). These metabolites did not correspond to any of the typical lipid species found in cells, none of which were significantly altered by AS115 treatment ( Table S1). High-resolution MS of the m/z 317 metabolite provided a molecular formula of C19H40O3 ( Figure 2B), which suggests that this compound might represent a monoalkylglycerol ether bearing a C16:0 alkyl chain (C16:0 MAGE). This structure assignment was corroborated by tandem MS and LC analysis, in which the endogenous m/z 317 product and synthetic C16:0 MAGE displayed equivalent fragmentation and migration patterns, respectively ( Figure S3). By extension, the m/z 343 and 345 metabolites were interpreted to represent the C18:1 and C18:0 MAGEs, respectively. A control carbamate inhibitor, URB597, which targets other hydrolytic enzymes [23], but not KIAA1363, did not affect MAGE levels in cancer cells ( Figure S4).
Pharmacological Inhibition of KIAA1363 Reduces Monoalkylglycerol Ether, MAGE, Levels in Human Cancer Cells
Figure 2. Pharmacological Inhibition of KIAA1363 Reduces Monoalkylglycerol Ether, MAGE, Levels in Human Cancer Cells
(A) Global metabolite profiling of AS115-treated SKOV-3 cells (10 μM AS115, 4 hr) with untargeted LC-MS methods [22]revealed a specific reduction in a set of structurally related metabolites with m/z values of 317, 343, and 345 (p < 0.001 for AS115- versus DMSO-treated SKOV-3 cells). Results represent the average fold change for three independent experiments. See Table S1for a more complete list of metabolite levels.
(B) High-resolution MS analysis of the sodium adduct of the purified m/z 317 metabolite provided a molecular formula of C19H40O3, which, in combination with tandem MS and LC analysis ( Figure S3), led to the determination of the structure of this small molecule as C16:0 monoalkylglycerol ether (C16:0 MAGE).
Biochemical Characterization of KIAA1363 as a 2-Acetyl MAGE Hydrolase
The correlation between KIAA1363 inactivation and reduced MAGE levels suggests that these lipids are products of a KIAA1363-catalyzed reaction. A primary route for the biosynthesis of MAGEs has been proposed to occur via the enzymatic hydrolysis of their 2-acetyl precursors 24 and 25. This 2-acetyl MAGE hydrolysis activity was first detected in cancer cell extracts over a decade ago [25], but, to date, it has eluded molecular characterization. To test whether KIAA1363 functions as a 2-acetyl MAGE hydrolase, this enzyme was transiently transfected into COS7 cells. KIAA1363-transfected cells possessed significantly higher 2-acetyl MAGE hydrolase activity compared to mock-transfected cells, and this elevated activity was blocked by treatment with AS115 (Figure 3A). In contrast, KIAA1363- and mock-transfected cells showed no differences in their respective hydrolytic activity for 2-oleoyl MAGE, monoacylglycerols, or phospholipids (e.g., platelet-activating factor [PAF], phosphatidylcholine) (Figure S5A). These data indicate that KIAA1363 selectively catalyzes the hydrolysis of 2-acetyl MAGEs to MAGEs.
KIAA1363 Regulates an Ether Lipid Signaling Network that Bridges Platelet-Activating Factor and the Lysophospholipids
Examination of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [26] suggests that the KIAA1363-MAGE pathway might serve as a unique metabolic node linking the PAF [27] and lysophospholipid [28] signaling systems in cancer cells (Figure 4A). Consistent with a direct pathway leading from MAGEs to these lysophospholipids, addition of 13C-MAGE to SKOV-3 cells resulted in the formation of 13C-labeled alkyl-LPC and alkyl-LPA (Figure 4C).
Conversely, the levels of 2-acetyl MAGE in SKOV-3 cells, as judged by metabolic labeling experiments, were significantly stabilized by treatment with AS115, which, in turn, led to an accumulation of PAF (Figure 4D). A comparison of the metabolite profiles of SKOV-3 and OVCAR-3 cells revealed significantly higher levels of MAGE, alkyl-LPC, and alkyl-LPA in the former line (Figure 4E). These data indicate that the lysophospholipid branch of the MAGE network is elevated in aggressive cancer cells, and that this metabolic shift is regulated by KIAA1363.
Figure 4. KIAA1363 Serves as a Key Enzymatic Node in a Metabolic Network that Connects the PAF and Lysophospholipid Families of Signaling Lipids
Stable Knockdown of KIAA1363 Impairs Tumor Growth In Vivo
Figure 6. KIAA1363 Contributes to Ovarian Tumor Growth and Cancer Cell Migration
The decrease in tumorigenic potential of shKIAA1363 cells was not associated with a change in proliferation potential in vitro (Figure S8). shKIAA1363 cells were, however, impaired in their in vitro migration capacity compared to control cells (Figure 6B). Neither MAGE nor alkyl-LPC impacted cancer cell migration at concentrations up to 1 μM (Figure 6B). In contrast, alkyl-LPA (10 nM) completely rescued the reduced migratory activity of shKIAA1363 cells. Collectively, these results indicate that KIAA1363 contributes to the pathogenic properties of cancer cells in vitro and in vivo, possibly through regulating the levels of the bioactive lipid LPA.
We have determined by integrated enzyme and small-molecule profiling that KIAA1363, a protein of previously unknown function, is a 2-acetyl MAGE hydrolase that serves as a key regulator of a lipid signaling network that contributes to cancer pathogenesis. Although we cannot yet conclude which of the specific metabolites regulated by KIAA1363 supports tumor growth in vivo, the rescue of the reduced migratory phenotype of shKIAA1363 cancer cells by LPA is consistent with previous reports showing that this lipid signals through a family of G protein-coupled receptors to promote cancer cell migration and invasion 28, 29 and 30. LPA is also an established biomarker in ovarian cancer, and the levels of this metabolite are elevated nearly 10-fold in ascites fluid and plasma of patients with ovarian cancer [31]. Our results suggest that additional components in the KIAA1363-ether lipid network, including MAGE, alkyl LPC, and KIAA1363 itself, might also merit consideration as potential diagnostic markers for ovarian cancer. Consistent with this premise, our preliminary analyses have revealed highly elevated levels of KIAA1363 in primary human ovarian tumors compared to normal ovarian tissues (data not shown). The heightened expression of KIAA1363 in several other cancers, including breast 18 and 32, melanoma [18], and pancreatic cancer [33], indicates that alterations in the KIAA1363-ether lipid network may be a conserved feature of tumorigenesis. Considering further that reductions in KIAA1363 activity were found to impair tumor growth of both ovarian and breast cancer cells, it is possible that inhibitors of this enzyme may prove to be of value for the treatment of multiple types of cancer.
Lodhi IJ, Semenkovich CF
Cell Metab. 2014 Mar 4; 19(3):380-92
http://dx.doi.org/10.1016%2Fj.cmet.2014.01.002
Peroxisomes are often dismissed as the cellular hoi polloi, relegated to cleaning up reactive oxygen chemical debris discarded by other organelles. However, their functions extend far beyond hydrogen peroxide metabolism. Peroxisomes are intimately associated with lipid droplets and mitochondria, and their ability to carry out fatty acid oxidation and lipid synthesis, especially the production of ether lipids, may be critical for generating cellular signals required for normal physiology. Here we review the biology of peroxisomes and their potential relevance to human disorders including cancer, obesity-related diabetes, and degenerative neurologic disease.
Peroxisomes are multifunctional organelles present in virtually all eukaryotic cells. In addition to being ubiquitous, they are also highly plastic, responding rapidly to cellular or environmental cues by modifying their size, number, morphology, and function (Schrader et al., 2013). Early ultrastructural studies of kidney and liver cells revealed cytoplasmic particles enclosed by a single membrane containing granular matrix and a crystalline core (Rhodin, 1958). These particles were linked with the term "peroxisome" by Christian de Duve, who first identified the organelle in mammalian cells when enzymes such as oxidases and catalases involved in hydrogen peroxide metabolism co-sedimented in equilibrium density gradients (De Duve and Baudhuin, 1966). Based on these studies, it was originally thought that the primary function of these organelles was the metabolism of hydrogen peroxide. Novikoff and colleagues observed a large number of peroxisomes in tissues active in lipid metabolism such as liver, brain, intestinal mucosa, and adipose tissue (Novikoff and Novikoff, 1982;Novikoff et al., 1980). Peroxisomes in different tissues vary greatly in shape and size, ranging from 0.1-0.5 μM in diameter. In adipocytes, peroxisomes tend to be small in size and localized in the vicinity of lipid droplets. Notably, a striking increase in the number of peroxisomes was observed during differentiation of adipogenic cells in culture (Novikoff and Novikoff, 1982). These findings suggest that peroxisomes may be involved in lipid metabolism.
Lazarow and de Duve hypothesized that peroxisomes in animal cells were capable of carrying out fatty acid oxidation. This was confirmed when they showed that purified rat liver peroxisomes contained fatty acid oxidation activity that was robustly increased by treatment of animals with clofibrate (Lazarow and De Duve, 1976). In a series of experiments, Hajra and colleagues discovered that peroxisomes were also capable of lipid synthesis (Hajra and Das, 1996). Over the past three decades, multiple lines of evidence have solidified the concept that peroxisomes play fundamentally important roles in lipid metabolism. In addition to removal of reactive oxygen species, metabolic functions of peroxisomes in mammalian cells include β-oxidation of very long chain fatty acids, α-oxidation of branched chain fatty acids, and synthesis of ether-linked phospholipids as well as bile acids (Figure 1). β-oxidation also occurs in mitochondria, but peroxisomal β-oxidation involves distinctive substrates and complements mitochondrial function; the processes of α-oxidation and ether lipid synthesis are unique to peroxisomes and important for metabolic homeostasis.
Structure and functions of peroxisomes
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951609/bin/nihms-555068-f0001.jpg
Figure 1 Structure and functions of peroxisomes
The peroxisome is a single membrane-enclosed organelle that plays an important role in metabolism. The main metabolic functions of peroxisomes in mammalian cells include β-oxidation of very long chain fatty acids, α-oxidation of branched chain fatty acids, synthesis of bile acids and ether-linked phospholipids and removal of reactive oxygen species. Peroxisomes in many, but not all, cell types contain a dense crystalline core of oxidative enzymes.
Here we highlight the established role of peroxisomes in lipid metabolism and their emerging role in cellular signaling relevant to metabolism. We describe the origin of peroxisomes and factors involved in their assembly, division, and function. We address the interaction of peroxisomes with lipid droplets and implications of this interaction for lipid metabolism. We consider fatty acid oxidation and lipid synthesis in peroxisomes and their importance in brown and white adipose tissue (sites relevant to lipid oxidation and synthesis) and disease pathogenesis.
peroxisomal biogenesis and protein import
Potential pathways to peroxisomal biogenesis. Peroxisomes are generated autonomously through division of pre-existing organelles (top) or through a de novo process involving budding from the ER followed by import of matrix proteins (bottom). B. Peroxisomal membrane protein import. Peroxisomal membrane proteins (PMPs) are imported post-translationally to the peroxisomal membrane. Pex19 is a soluble chaperone that binds to PMPs and transports them to the peroxisomal membrane, where it docks with a complex containing Pex16 and Pex3. Following insertion of the PMP, Pex19 is recycled back to the cytosol.
Regardless of their origin, peroxisomes require a group of proteins called peroxins for their assembly, division, and inheritance. Over 30 peroxins, encoded by Pex genes, have been identified in yeast (Dimitrov et al., 2013). At least a dozen of these proteins are conserved in mammals, where they regulate various aspects of peroxisomal biogenesis, including factors that control assembly of the peroxisomal membrane, factors that interact with peroxisomal targeting sequences allowing proteins to be shuttled to peroxisomes, and factors that act as docking receptors for peroxisomal proteins.
At least three peroxins (Pex3, Pex16 and Pex19) appear to be critical for assembly of the peroxisomal membrane and import of peroxisomal membrane proteins (PMPs) (Figure 2B). Pex19 is a soluble chaperone and import receptor for newly synthesized PMPs (Jones et al., 2004). Pex3 buds from the ER in a pre-peroxisomal vesicle and functions as a docking receptor for Pex19 (Fang et al., 2004). Pex16 acts as a docking site on the peroxisomal membrane for recruitment of Pex3 (Matsuzaki and Fujiki, 2008). Peroxisomal matrix proteins are translated on free ribosomes in the cytoplasm prior to their import. These proteins have specific peroxisomal targeting sequences (PTS) located either at the carboxyl (PTS1) or amino (PTS2) terminus (Gould et al., 1987; Swinkels et al., 1991).
Ana P Gomes, John Blenis
Current Opinion in Biotechnology Aug 2015; 34:110–117
http://dx.doi.org/10.1016/j.copbio.2014.12.007
Signaling networks sense intracellular and extracellular cues to maintain homeostasis.
PI3K/AKT and Ras/ERK signaling induces anabolic reprogramming.
mTORC1 is a master node of signaling integration that promotes anabolism.
AMPK and SIRT1 fine tune signaling networks in response to energetic status.
In multicellular organisms, individual cells have evolved to sense external and internal cues in order to maintain cellular homeostasis and survive under different environmental conditions. Cells efficiently adjust their metabolism to reflect the abundance of nutrients, energy and growth factors. The ability to rewire cellular metabolism between anabolic and catabolic processes is crucial for cells to thrive. Thus, cells have developed, through evolution, metabolic networks that are highly plastic and tightly regulated to meet the requirements necessary to maintain cellular homeostasis. The plasticity of these cellular systems is tightly regulated by complex signaling networks that integrate the intracellular and extracellular information. The coordination of signal transduction and metabolic pathways is essential in maintaining a healthy and rapidly responsive cellular state.
AMPK and SIRT1 fine tune signaling networks in response to energetic status
Fine-tuning signaling networks
PI3K/Akt signaling-induced anabolic reprogramming
Growth factors and other ligands activate PI3K signaling upon binding and consequent activation of their cell surface receptors, such as receptor tyrosine kinases (RTKs) and G protein-coupled
receptors (GPCRs). This leads to the phosphorylation of membrane phosphatidylinositiol lipids and the recruitment and activation of several protein kinases, which perpetuate the extracellular
signals to modulate intracellular processes [3,4]. One of the most crucial signal propagators regulated by PI3K signaling is protein kinase B/Akt [3,4]. Indeed, Akt rewires metabolism in response
to environmental cues by three distinct means;
(i) by the direct phosphorylation and regulation of metabolic enzymes,
(ii) by activating/inactivating metabolism altering transcriptional factors, and
(iii) by modulating other kinases that themselves regulate metabolism [5].
Akt regulates glucose metabolism, inducing both glucose uptake and glycolytic flux by increasing the expression of the glucose transporter genes and regulating the activity of glycolytic enzymes,
respectively [6–8]. Moreover, the ability of Akt to induce glycolysis is also mediated by the regulation of Hexokinase (HK). HK performs the first step of glycolysis.
Figure 1 Anabolic rewiring induced by PI3K/Akt, Ras/ERK and mTORC1 signaling.
Extracellular signals activate two major signaling cascades controlled by the activation of PI3K and Ras. PI3K and Ras regulate Akt and ERK, which in turn induce changes in intermediate metabolism
to promote anabolic processes. In addition, they also induce the activation of mTORC1, thus further supporting the rewiring of cellular metabolism towards anabolic processes. Through various mechanisms
Akt, ERK and mTORC1 stimulate mRNA translation, aerobic glycolysis, glutamine anaplerosis, lipid synthesis, the pentose phosphate and pyrimidine synthesis, thus producing the major components
necessary for cell growth and proliferation.
Figure 2. Regulation of intermediate metabolism by nutrient and energy sensors.
Nutrient and energy-responsive pathways fine-tune the output of signaling cascades, allowing for the correct balance between the availability of nutrients and the cellular capacity to use them effectively.
AMPK and SIRT1 respond to the energy status of the cells through sensing of AMP and NAD+ levels respectively. When energy is scarce, these sensors are activated inducing a rewiring of intermediate
metabolism to catabolic processes in order to produce energy and restore homeostasis. When nutrients (such as glucose and amino acids) and energy are available, AMPK, SIRT1, SIRT3 and SIRT6 are
repressed and mTORC1 is active, thus promoting a shift towards anabolic processes and energy production. These networks of signaling cascades, their interconnection and regulation allow the cells
to maintain energetic balance and allow for the physiological adaptation to the ever-changing environment.
7.6.8.1 Activation and signaling of the p38 MAP kinase pathway
Tyler Zarubin1 and Jiahuai Han
Cell Research (2005) 15, 11–18
http://dx.doi.org:/10.1038/sj.cr.7290257
The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.
Cellular behavior in response to extracellular stimuli is mediated through intracellular signaling pathways such as the mitogen-activated protein (MAP) kinase pathways 1. MAP kinases are members of discrete signaling cascades and serve as focal points in response to a variety of extracellular stimuli. Four distinct subgroups within the MAP kinase family have been described:
extracellular signal-regulated kinases (ERKs),
c-jun N-terminal or stress-activated protein kinases (JNK/SAPK),
ERK/big MAP kinase 1 (BMK1), and
the p38 group of protein kinases.
The focus of this review will be to highlight the characteristics of
the p38 kinases,
components of this kinase cascade,
activation of this pathway, and
the biological consequences of its activation.
p38 (p38) was first isolated as a 38-kDa protein rapidly tyrosine phosphorylated in response to LPS stimulation 2, 3. p38 cDNA was also cloned as a molecule that binds puridinyl imidazole derivatives which are known to inhibit biosynthesis of inflammatory cytokines such as interleukin-1 (IL-1) and tumor-necrosis factor (TNF) in LPS stimulated monocytes 4. To date, four splice variants of the p38 family have been identified: p38, p38 5, p38 (ERK6, SAPK3) 6, 7, and p38(SAPK4) 8, 9. Of these, p38 and p38 are ubiquitously expressed while p38 and p38 are differentially expressed depending on tissue type. All p38 kinases can be categorized by a Thr-Gly-Tyr (TGY) dual phosphorylation motif 10. Sequence comparisons have revealed that each p38 isoform shares 60% identity within the p38 group but only 40–45% to the other three MAP kinase family members.
Mammalian p38s activation has been shown to occur in response to extracellular stimuli such as UV light, heat, osmotic shock, inflammatory cytokines (TNF- & IL-1), and growth factors (CSF-1) 1, 3, 15, 16, 17,18, 19, 20, 21. This plethora of activators conveys the complexity of the p38 pathway and this matter is further complicated by the observation that activation of p38 is not only dependent on stimulus, but on cell type as well. For example, insulin can stimulate p38 in 3T3-L1 adipocytes 22, but downregulates p38 activity in chick forebrain neuron cells 23. The activation of p38 isoforms can be specifically controlled through different regulators and coactivated by various combinations of upstream regulators 24, 26.
Like all MAP kinases, p38 kinases are activated by dual kinases termed the MAP kinase kinases (MKKs). However, despite conserved dual phosphorylation sites among p38 isoforms, selective activation by distinct MKKs has been observed. There are two main MAPKKs that are known to activate p38, MKK3 and MKK6. It is proposed that upstream kinases can differentially regulate p38 isoforms as evidenced by the inability of MKK3 to effectively activate p38 while MKK6 is a potent activator despite 80% homology between these two MKKs 27. Also, it has been shown that MKK4, an upstream kinase of JNK, can aid in the activation of p38 and p38 in specific cell types 8. This data suggests then, that activation of p38 isoforms can be specifically controlled through different regulators and coactivated by various combinations of upstream regulators. Furthermore, substrate selectivity may be a reason why each MKK has a distinct function. In addition to the activation by upstream kinases, there is a MAPKK-independent mechanism of p38 MAPK activation involving TAB1 (transforming growth factor–activated protein kinase 1 (TAK1)-binding protein) 28. The activation of p38 in this pathway is achieved by the autophosphorylation of p38 after interaction with TAB1.
The activation of p38 in response to the wide range of extracellular stimuli can be seen in part by the diverse range of MKK kinases (MAP3K) that participate in p38 activation. These include TAK1 33, ASK1/MAPKKK5 34, DLK/MUK/ZPK 35, 36, and MEKK4 35, 37, 38. Overexpression of these MAP3Ks leads to activation of both p38 and JNK pathways which is possibly one reason why these two pathways are often co-activated. Also contributing to p38 activation upstream of MAPK kinases are low molecular weight GTP-binding proteins in the Rho family such as Rac1 and Cdc42 40, 41. Rac1 can bind to MEKK1 or MLK1 while Cdc42 can only bind to MLK1 and both result in activation of p38 via MAP3Ks 35, 42.
Dephosphorylation, would seem to play a major role in the downregulation of MAP kinase activity. Many dual-specificity phosphatases have been identified that act upon various members of the MAP kinase pathway and are grouped as the MAP kinase phosphatase (MKP) family 45. Several members can efficiently dephosphorylate p38 and p38 46, 47; however, p38 and p38 are resistant to all known MKP family members.
The first p38 substrate identified was the MAP kinase-activated protein kinase 2 (MAPKAPK2 or MK2) 1, 15, 52. This substrate, along with its closely related family member MK3 (3pk), were both shown to activate various substrates including small heat shock protein 27 (HSP27) 53, lymphocyte-specific protein 1 (LSP1) 54, cAMP response element-binding protein (CREB) 55, transcription factor ATF1 55, SRF 56, and tyrosine hydroxylase 57. p38 regulated/activated kinase (PRAK) is a p38 and/or p38activated kinase that shares 20-30% sequence identity to MK2 and is thought to regulate heat shock protein 27 (HSP27) 61. Mitogen- and stress-activated protein kinase-1 (MSK1) can be directly activated by p38 and ERK, and may mediate activation of CREB 62, 63, 64.
Another group of substrates that are activated by p38 comprise transcription factors. Many transcription factors encompassing a broad range of action have been shown to be phosphorylated and subsequently activated by p38. Examples include activating transcription factor 1, 2 & 6 (ATF-1/2/6), SRF accessory protein (Sap1), CHOP (growth arrest and DNA damage inducible gene 153, or GADD153), p53, C/EBP, myocyte enhance factor 2C (MEF2C), MEF2A, MITF1, DDIT3, ELK1, NFAT, and high mobility group-box protein 1 (HBP1) 17, 55, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76. An important cis-element, AP-1 appears to be influenced by p38 through several different mechanisms. Taken together, all the data suggest that the p38 pathway has a wide variety of functions.
Abundant evidence for p38 involvement in apoptosis exists to date and is based on concomitant activation of p38 and apoptosis induced by a variety of agents such as NGF withdrawal and Fas ligation 95, 96, 97. Cysteine proteases (caspases) are central to the apoptotic pathway and are expressed as inactive zymogens 98,99. Caspase inhibitors then can block p38 activation through Fas cross-linking, suggesting p38 functions downstream of caspase activation 97, 100. However, overexpression of dominant active MKK6b can also induce caspase activity and cell death thus implying that p38 may function both upstream and downstream of caspases in apoptosis 101, 102. It must be mentioned that the role of p38 in apoptosis is cell type and stimulus dependent. While p38 signaling has been shown to promote cell death in some cell lines, in different cell lines p38 has been shown to enhance survival, cell growth, and differentiation.
p38 now seems to have a role in tumorigenesis and sensescence. There have been reports that activation of MKK6 and MKK3 led to a senescent phenotype dependent upon p38 MAPK activity. Also, p38 MAPK activity was shown responsible for senescence in response to telomere shortening, H2O2 exposure, and chronic RAS oncogene signaling 117, 118, 119. A common feature of tumor cells is a loss of senescence and p38 may be linked to tumorigenesis in certain cells. It has been reported that p38 activation may be reduced in tumors and that loss of components of the p38 pathway such as MKK3 and MKK6 resulted in increased proliferation and likelihood of tumorigenic conversion regardless of the cell line or the tumor induction agent used in these studies 29.
Although all research done on the p38 pathway cannot be reviewed here, certain conclusions can still be made regarding the operation of p38 as a signal transduction mediator. The p38 family (,,,) is activated by both stress and mitogenic stimuli in a cell dependent manner and certain isoforms can either directly or indirectly target proteins to control pre/post transcription. p38 MAPKs also have the ability to activate other kinases and consequently regulate numerous cellular responses. Because p38 signaling has been implicated in cellular responses including inflammation, cell cycle, cell death, development, cell differentiation, senescence, and tumorigenesis, emphasis must be placed on p38 function with respect to specific cell types.
Regulation of the p38 pathway is not an isolated cascade and many different upstream signals can lead to p38 activation. These signals may be p38 specific (MKK3/6), general MAPKKs (MKK4), or MAPKK independent signals (TAB1). Downstream signaling pathways of p38 are quite divergent and each component may interact with other cellular components, both upstream and downstream, to coordinate cellular processes such as feedback mechanisms. Furthermore, in vivo p38 is not an isolated event and exists in the presence of other MAP kinases and a plethora of other signaling pathways. The subcellular location of p38 activation may also play a critical role determining the resulting effect and may add yet another order of complexity to the investigation of p38 function.
7.6.8.2 Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases
Gary L. Johnson and Razvan Lapadat
Science 6 Dec 2002; 298: 1911-1912.
Multicellular organisms have three well-characterized subfamilies of mitogen activated protein kinases (MAPKs) that control a vast array of physiological processes. These enzymes are regulated by a characteristic phosphorelay system in which a series of three protein kinases phosphorylate and activate one another. The extracellular signal–regulated kinases (ERKs) function in the control of cell division, and inhibitors of these enzymes are being explored as anticancer agents. The c-Jun amino-terminal kinases ( JNKs) are critical regulators of transcription, and JNK inhibitors may be effective in control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses.
Protein kinases are enzymes that covalently attach phosphate to the side chain of either serine, threonine, or tyrosine of specific proteins inside cells. Such phosphorylation of proteins can control their enzymatic activity, their interaction with other proteins and molecules, their location in the cell, and their propensity for degradation by proteases. Mitogen-activated protein kinases (MAPKs) compose a family of protein kinases whose function and regulation have been conserved during evolution from unicellular organisms such as brewers' yeast to complex organisms including humans (1). MAPKs phosphorylate specific serines and threonines of target protein substrates and regulate cellular activities ranging from gene expression, mitosis, movement, metabolism, and programmed death. Because of the many important cellular functions controlled by MAPKs, they have been studied extensively to define their roles in physiology and human disease. MAPK-catalyzed phosphorylation of substrate proteins functions as a switch to turn on or off the activity of the substrate protein.
MAPKs are part of a phosphorelay system composed of three sequentially activated kinases, and, like their substrates, MAPKs are regulated by phosphorylation (Fig. 1) (2). MKK-catalyzed phosphorylation activates the MAPK and increases its activity in catalyzing the phosphorylation of its own substrates. MAPK phosphatases reverse the phosphorylation and return the MAPK to an inactive state. MKKs are highly selective in phosphorylating specific MAPKs. MAPK kinase kinases (MKKKs) are the third component of the phosphorelay system. MKKKs phosphorylate and activate specific MKKs. MKKKs have distinct motifs in their sequences that selectively confer their activation in response to different stimuli.
Fig. 1. MAPK phosphorelay systems.
The modules shown are representative of pathway connections for the respective MAPK phosphorelay systems.There are multiple component MKKKs, MKKs, and MAPKs for each system.For example, there are three Raf proteins (c-Raf1, B-Raf, A-Raf), two MKKs (MKK1 and MKK2), and two ERKs (ERK1 and ERK2) that can compose MAPK phosphorelay systems responsive to growth factors.The ERK, JNK, and p39 pathways in the STKE Connections Map demonstrate the potential complexity of these systems.
ERKs 1 and 2 are both components of a three-kinase phosphorelay module that includes the MKKK c-Raf1, B-Raf, or A-Raf, which can be activated by the proto-oncogene Ras. Mutations that convert Ras to an activated oncogene are common oncogenic mutations in many human tumors. Oncogenic Ras persistently activates the ERK1 and ERK2 pathways, which contributes to the increased proliferative rate of tumor cells. For this reason, inhibitors of the ERK pathways are entering clinical trials as potential anticancer agents.
Regulation of the JNK pathway is extremely complex and is influenced by many MKKKs. As depicted in the STKE JNK Pathway Connections Map, there are 13 MKKKs that regulate the JNKs. This diversity of MKKKs allows a wide range of stimuli to activate this MAPK pathway. JNKs are important in controlling programmed cell death or apoptosis (9). The inhibition of JNKs enhances chemotherapy-induced inhibition of tumor cell growth, suggesting that JNKs may provide a molecular target for the treatment of cancer. The pharmaceutical industry is bringing JNK inhibitors into clinical trials.
Recently, a major paradigm shift for MAPK regulation was developed for p38. The p38 enzyme is activated by the protein TAB1 (12), but TAB1 is not a MKK. Rather, TAB1 appears to be an adaptor or scaffolding protein and has no known catalytic activity. This is the first demonstration that another mechanism exists for the regulation of MAPKs in addition to the MKKK-MKKMAPK regulatory module.
The importance of MAPKs in controlling cellular responses to the environment and in regulating gene expression, cell growth, and apoptosis has made them a priority for research related to many human diseases. The ERK, JNK, and p38 pathways are all molecular targets for drug development, and inhibitors of MAPKs will undoubtedly be one of the next group of drugs developed for the treatment of human disease (13).
B Bian, S Mongrain, S Cagnol, Marie-Josée Langlois, J Boulanger, et al.
Molec Carcinogen 25 Mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22312
Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non-permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27Kip1 were observed in cathepsin B-deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy
Colorectal cancer (CRC),a major malignancy worldwide and the second leading cause of cancer death in North America, develops through multiple steps. The ability of cancers to invade and metastasize depends on the action of proteases actively taking center stage in extracellular proteolysis [2]. Of all the proteases, the cysteine protease cathepsin B is of significant importance [3]. Cathepsin B primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during malignant transformation cathepsin B can be upregulated [3, 4]. Cathepsin B in tumors can either be secreted, bound to the cell membrane or released by shedding vesicles [4]. Expression and redistribution of active cathepsin B to the basal plasma membrane occurs in late colon adenomas [5, 6] coincident with the activation of KRAS [1]. In line with these results, Cavallo-Medved et al. [7] have demonstrated that trafficking of cathepsin B to caveolae and its secretion are regulated by active KRAS in CRC cells in culture. Accordingly, secretion of cathepsin B, increased in the extracellular environment of CRC [8, 9], is suspected to play an essential role in disrupting extracellular matrix barriers, facilitating invasion and metastasis [10-12]. These data are consistent with the link between cathepsin B protein expression in colorectal carcinomas and shortened patient survival [6].
In a recent prospective cohort study of 558 men and women with colonic tumors [13] 82% of patients had tumors that expressed cathepsin B, irrespective of stage, while the remaining 18% had tumors that did not express cathepsin B. Other studies have suggested that cathepsin B expression or activity may actually peak during early stage cancer and subsequently decline with advanced disease [14, 15]. This points to a possible role of cathepsin B in both early and late alterations leading to colonic cancer.
This study used two strategies to specifically counteract the action of cathepsin B. The first involved the use of RNA interference (RNAi) to inhibit the expression of cathepsin B protein into CRC cells while the second approach employed the highly selective cathepsin inhibitor Ca074 to block extracellular cathepsin B activity. Results suggest that extracellular cathepsin B is involved in cell invasion whereas intracellular cathepsin B controls malignant properties of CRC cells. Further, biochemical analysis suggests that intracellular cathepsin B regulates tumorigenesis by degrading the p27Kip1 cell cycle inhibitor.
mRNA and Activated Levels of Cathepsin B Are Increased in Adenomas and in Colorectal Tumors of All Stages
Cathepsin B expression was analyzed at both the mRNA and protein levels in a series of human paired specimens at various tumor stages. As shown in Figure 1A, increased transcript levels of cathepsin B were observed in colorectal tumors, regardless of tumor stage, including in adenomas. Of note, increased cathepsin B expression was more prominent in tumors exhibiting APC mutations. By contrast, there did not appear to be a significant difference relative to KRAS mutations (Figure 1B). To establish whether these increased mRNA levels could be correlated with increased cathepsin B protein levels and more importantly with increased activity, expression of the active processed forms of the protease (25 and 30 kDa) was analyzed by Western blot. Both pro-cathepsin B and active cathepsin B were also increased in colorectal tumors compared to normal tissues (Figure 1C and D). These data hence suggest that increased transcription contributes to a greater expression of active cathepsin B in CRC.
Extracellular Cathepsin B Contributes to Invasiveness of Human CRC Cells but is Dispensable for Their Growth in Soft Agar
Cathepsin B protein levels were next examined in lysates obtained from various human CRC cell lines. As shown in Figure 2A, the proactive and catalytically active processed forms of cathepsin B were detected at various levels in CRC cell lines. Selected cathepsin B presence was also confirmed in conditioned culture medium of CRC cells, again at various levels (Figure 2A, lower panel). However, while the pro-form of cathepsin B was readily observed in conditioned culture medium of all CRC cells, the catalytically-active processed forms of cathepsin B were not detected in Western blot analyses. Additionally, using a fluorescence-based enzymatic assay, no cathepsin B enzyme activity was detected in conditioned medium. Since the pro-protease form might be activated under acidic pH conditions (peri- or extracellular) and by extracellular components of the extracellular matrix, the impact of extracellular inhibition of cathepsin B activation on CRC cell invasion was verified using Biocoat Matrigel chambers. HT-29, DLD1, and SW480 CRC cell lines secreting different levels of pro-cathepsin B (Figure 2A) were tested. Experiments were performed using the highly selective and non-permeant inhibitor Ca074 to reduce extracellular cathepsin B activity. At 10 μM, Ca074 produced a >99% inhibition of recombinant cathepsin B levels while barely reducing intracellular cathepsin B, that is, 5–8%, even upon 12 h exposure to the inhibitor (data not shown). Of note, treatment with 10 μM Ca074 significantly inhibited Matrigel invasion by approximately 45–60% in HT29, DLD1, and SW480 CRC cell lines (Figure 2B). By contrast, treatment with Ca074 had no significant effect on their capacity to form colonies in soft agarose (Figure 2C).
Cathepsin B Silencing in Human CRC Cells Inhibits Tumorigenicity and Metastasis in Immunodeficient Mice
Suppression of cathepsin B expression was found to significantly attenuate the metastatic potential of CRC cells in vivo in experimental metastasis assays. Indeed, immunodeficient mice injected with control CRC cells into the tail vein showed extensive lung metastasis within 28 d, whereas cells expressing shRNA against cathepsin B exhibited reduced lung colonization (Figure 4A). Cathepsin B silencing also altered the capacity of CRC cells to form tumors in mice as assessed by subcutaneous xenograft assays. HT29 cells induced palpable tumors with a short latency period of 9 d after their injection while downregulation of cathepsin B expression in these cells severely impaired their capacity to grow as tumors (Figure 4B).
Cathepsin B Silencing in Human CRC Cells Inhibits Growth in Soft Agar and Invasion Capacity
Recombinant lentiviruses encoding anti-cathepsin B short hairpin RNA (shRNA) were developed in order to stably suppress cathepsin B expression in CRC cells. As shown in Figure 3A, intracellular cathepsin B mRNA and protein levels were decreased in HT29 and DLD1 cells in comparison to a control shRNA which had no effect. Reduction of cathepsin B expression modestly slowed the proliferation rate of HT29 and DLD1 populations in 2D cell culture (Figure 3B). Conversely, cathepsin B silencing significantly reduced the ability of HT29 and DLD1 cells to form colonies in soft agarose (Figure 3C). This indicates that intracellular cathepsin B controls anchorage-independent growth of CRC cells given the absence of Ca074 effect (Figure 2C). Moreover, cathepsin B silencing also reduced the number of invading HT29 and DLD1 cells to a similar extent as Ca074 treatment (Figure 3D vs. Figure 2B).
Cathepsin B Cleaves the Cell Cycle Inhibitor p27Kip1
In order to verify whether p27Kip1 is in fact a substrate for cathepsin B, both proteins were first overexpressed in 293 T cells and cells subsequently lysed 2 d later for Western blot analysis of their respective expression. As shown in Figure 5A, forced expression of cathepsin B in 293 T cells dose-dependently reduced p27Kip1 protein levels. Next, to determine whether p27Kip1 could be degraded by cathepsin B in vitro, lysates from 293 T cells overexpressing HA-tagged p27Kip1 were incubated with purified cathepsin B and analyzed by Western blot. Figure 5B and C shows that cathepsin B degraded p27Kip1 in a time-dependent manner as visualized by the accumulation of three lower molecular mass species (26, 20, and 12 kDa) in addition to the full-length p27Kip1 protein (see arrows versus arrowhead).
Cathepsin B is capable of endopeptidase, peptidyl-dipeptidase, and carboxydipeptidase activities [18-20]. Cathepsin B also possesses a basic amino acid in the catalytic subsite in position S2 enabling the protease to preferentially split its substrates after Arg–Arg or Lys–Arg or Arg–Lys sequences. At least five of these sequences can be found within the human p27Kip1 sequence (Figure 5D). Therefore, the first amino acid of these doublets was mutated into alanine to test whether it would affect the degradation by cathepsin B. Mutation of arginine 58 (Figure 5E) and lysine 189 (Figure 5F) did not alter the cleavage profile of p27Kip1 by cathepsin B. Mutation of lysine 165 and arginine 194 also had no altering effect (not shown). On the other hand, mutation of arginine 152 into alanine markedly reduced the detection of the 20-kDa fragment (Figure 5E).
The protein stability of wild-type p27Kip1 was then compared to that of the p27Kip1 R152A/Δ189–198 mutant, which is more resistant to cathepsin B cleavage. 293T cells were transiently transfected with either wild-type p27Kip1 or p27Kip1 mutant and subsequently treated with cycloheximide to inhibit protein neosynthesis. Thereafter, cells were lysed at different time intervals in order to analyze protein expression levels of p27Kip1 forms. As shown in Figure 6A, following cycloheximide treatment, protein levels of the p27Kip1 mutant decreased much more slowly than that of wild-type protein. Specifically, 10 h after cycloheximide addition, expression of p27Kip1 protein was clearly decreased while expression of the p27Kip1 mutant remained at control (time 0) levels. Of note, forced expression of cathepsin B in 293 T cells dose-dependently reduced the wild-type form of p27Kip1 protein levels while expression of p27Kip1 R152A/Δ189–198 mutant was only very slightly affected (Figure 6B).
Colocalization of Endogenous p27Kip1 With Cathepsin B Into Lysosomes
As shown in Figure 7A, the anti-cathepsin B antibody confirmed the colocalization of cathepsin B (in green) with the lysosomal acidotropic probe LysoTracker (in red). As expected, most of p27Kip1 staining (in green) was observed in the cell nucleus (Figure 7B). However, certain areas of colocalization were observed between endogenous p27Kip1 (in green) and cathepsin B (in red) (Figure 7B, asterisks). Moreover, Western blot analyses revealed the presence of p27Kip1 protein in lysosome-enriched fractions obtained from differential centrifugation of Caco-2/15 and SW480 cell lysates (Figure 7C and D). These lysosomal fractions were enriched in lysosome-associated membrane protein 1 (LAMP1) and exhibited very low or undetectable levels of the nuclear lamin B protein.
The most extensive literature to date regarding cathepsin B highlights a key role of this protease in the invasiveness and metastasis of various carcinoma cells [3, 8, 10-12]. The present findings demonstrate that cathepsin B has not only a role in facilitating CRC invasion and metastasis, but also in mediating early premalignant processes. Results herein show that cathepsin B promotes anchorage-independent CRC cell growth, which translates in vivo to enhanced tumor growth. In addition, cathepsin B was identified as a new protease capable of proteolytic cleavage of the cell cycle inhibitor p27Kip1. This is especially relevant since the loss of p27Kip1 expression has been strongly associated with aggressive tumor behavior and poor clinical outcome in CRC [22, 23].
These data are reminiscent of the immunohistochemistry data reported by Chan et al. [13] showing that cathepsin B protein was expressed in the vast majority of colon cancers analyzed (558 tumors), which was also independent of tumor stage. The present data also revealed that increased transcription of cathepsin B was associated with the presence of mutations in APC but not in KRAS, thus emphasizing the fact that cathepsin B gene expression is already deregulated in early stages of colorectal carcinoma. Indeed, most CRCs acquire loss-of-function mutations in both copies of the APC gene, resulting in inefficient breakdown of intracellular β-catenin and enhanced nuclear signaling [27]. Given the importance of the Wnt/APC/β-catenin pathway in human tumorigenesis initiation, the present data showing an association between cathepsin B expression and APC mutations are particularly noteworthy.
Cancer Mutations Across the Landscape
Posted in Biological Networks, Gene Regulation and Evolution, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, tagged Cancer - General, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Genome Atlas, cell signaling, clinical associations, clusters, Cox Proportional Hazards model, DNA, DNA repair, driver mutations, functional associations, gene mutations, mutations across cancer types, prognostic analysis, The Cancer Genome Atlas (TCGA), tumorigenesis, Washington University in St. Louis on November 1, 2013| 2 Comments »
This is an up-to-date article about the significance of mutations found in 12 major types of cancer.
Mutational landscape and significance across 12 major cancer types
Cyriac Kandoth1*, Michael D. McLellan1*, Fabio Vandin2, Kai Ye1,3, Beifang Niu1, Charles Lu1, et al.
1The Genome Institute, Washington University in St Louis, Missouri 63108, USA. 2Department of Computer Science, Brown University, Providence, Rhode Island 02912, USA. 3Department of Genetics, Washington University in St Louis, Missouri 63108, USA. 4Department of Medicine, Washington University in St Louis, Missouri 63108, USA. 5Siteman Cancer Center, Washington University in St Louis, Missouri 63108, USA. 6Department of Mathematics, Washington University in St Louis, Missouri 63108, USA.
NATURE 17 Oct 2013; 5 0 2 http://dx.doi.org/10.1038/nature12634
The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate
the distributions of mutation frequencies,
types and contexts across tumour types, and
establish their links to tissues of origin,
environmental/ carcinogen influences, and
DNA repair defects.
Using the integrated data sets, we identified 127 significantly mutated genes from well-knownand emerging cellular processes in cancer.
(for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase,Wnt/b-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control)
(for example, histone, histone modification, splicing, metabolism and proteolysis)
The average number of mutations in these significantly mutated genes varies across tumour types;
most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small.
Mutations in transcriptional factors/regulators show tissue specificity, whereas
histone modifiers are often mutated across several cancer types.
Clinical association analysis identifies genes having a significant effect on survival, and
investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis.
Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment
The advancement of DNA sequencing technologies now enables the processing of thousands of tumours of many types for systematic mutation discovery. This expansion of scope, coupled with appreciable progress in algorithms1–5, has led directly to characterization of significant functional mutations, genes and pathways6–18. Cancer encompasses more than 100 related diseases19, making it crucial to understand the commonalities and differences among various types and subtypes. TCGA was founded to address these needs, and its large data sets are providing unprecedented opportunities for systematic, integrated analysis.
We performed a systematic analysis of 3,281 tumours from 12 cancer types to investigate underlying mechanisms of cancer initiation and progression. We describe variable mutation frequencies and contexts and their associations with environmental factors and defects in DNA repair. We identify 127 significantlymutated genes (SMGs) from diverse signalling and enzymatic processes. The finding of a TP53-driven breast, head and neck, and ovarian cancer cluster with a dearth of other mutations in SMGs suggests common therapeutic strategies might be applied for these tumours. We determined interactions among mutations and correlated mutations in BAP1, FBXW7 and TP53 with detrimental phenotypes across several cancer types. The subclonal structure and transcription status of underlying somatic mutations reveal the trajectory of tumour progression in patients with cancer.
Standardization of mutation data
Stringent filters (Methods) were applied to ensure high quality mutation calls for 12 cancer types: breast adenocarcinoma (BRCA), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), uterine corpus endometrial carcinoma (UCEC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), colon and rectal carcinoma (COAD, READ),bladder urothelial carcinoma (BLCA), kidney renal clear cell carcinoma (KIRC), ovarian serous carcinoma (OV) and acute myeloid leukaemia (LAML; conventionally called AML) (Supplementary Table 1). A total of 617,354 somatic mutations, consisting of
398,750 missense,
145,488 silent,
36,443 nonsense,
9,778 splice site,
7,693 non-coding RNA,
523 non-stop/readthrough,
15,141 frameshift insertions/deletions (indels) and
3,538 inframe indels,
were included for downstream analyses (Supplementary Table 2).
Distinct mutation frequencies and sequence context
Figure 1a shows that AML has the lowest median mutation frequency and LUSC the highest (0.28 and 8.15 mutations per megabase (Mb), respectively). Besides AML, all types average over 1 mutation per Mb, substantially higher than in pediatric tumours20. Clustering21 illustrates that
mutation frequencies for KIRC, BRCA, OV and AML are normally distributed within a single cluster, whereas
other types have several clusters (for example, 5 and 6 clusters in UCEC and COAD/ READ, respectively) (Fig. 1a and Supplementary Table 3a, b).
In UCEC, the largest patient cluster has a frequency of approximately 1.5 mutations per Mb, and
the cluster with the highest frequency is more than 150 times greater.
Multiple clusters suggest that factors other than age contribute to development in these tumours14,16. Indeed,
there is a significant correlation between high mutation frequency and DNA repair pathway genes (for example, PRKDC, TP53 and MSH6) (Supplementary Table 3c). Notably,
PRKDC mutations are associated with high frequency in BLCA, COAD/READ, LUAD and UCEC, whereas
TP53 mutations are related with higher frequencies in AML, BLCA, BRCA, HNSC, LUAD, LUSC and UCEC (all P < 0.05).
Mutations in POLQ and POLE associate with high frequencies in multiple cancer types; POLE association in UCEC is consistent with previous observations14.
Comparison of spectra across the 12 types (Fig. 1b and Supplementary Table 3d) reveals that LUSC and LUAD contain increased C>A transversions, a signature of cigarette smoke exposure10. Sequence context analysis across 12 types revealed
the largest difference being in C>T transitions and C>G transversions (Fig. 1c).
The frequency of thymine 1-bp (base pair) upstream of C>G transversions is markedly higher in BLCA, BRCA and HNSC than in other cancer types (Extended Data Fig. 1). GBM, AML, COAD/READ and UCEC have similar contexts in that
the proportions of guanine 1 base downstream of C>T transitions are between
59% and 67%, substantially higher than the approximately 40% in other cancer types.
Higher frequencies of transition mutations at CpG in gastrointestinal tumours, including colorectal, were previously reported22. We found three additional cancer types (GBM, AML and UCEC) clustered in the C>T mutation at CpG, consistent with previous findings of
aberrant DNA methylation in endometrial cancer23 and glioblastoma24.
BLCA has a unique signature for C>T transitions compared to the other types (enriched for TC) (Extended Data Fig. 1).
Significantly mutated genes
Genes under positive selection, either in individual or multiple tumour types, tend to display higher mutation frequencies above background. Our statistical analysis3, guided by expression data and curation (Methods), identified 127 such genes (SMGs; Supplementary Table 4). These SMGs are involved in a wide range of cellular processes, broadly classified into 20 categories (Fig. 2), including
transcription factors/regulators, histone modifiers, genome integrity, receptor tyrosine kinase signalling, cell cycle, mitogen-activated protein kinases (MAPK) signalling, phosphatidylinositol-3-OH kinase (PI(3)K) signalling, Wnt/ -catenin signalling, histones, ubiquitin-mediatedproteolysis, and splicing (Fig. 2).
The identification of MAPK, PI(3)K and Wnt/ -catenin signaling pathways is consistent with classical cancer studies. Notably, newer categories (for example, splicing, transcription regulators, metabolism, proteolysis and histones) emerge as exciting guides for the development of new therapeutic targets. Genes categorized as histone modifiers (Z = 0.57), PI(3)K signalling (Z = 1.03), and genome integrity (Z = 0.66) all relate to more than one cancer type, whereas
transcription factor/regulator (Z = 0.40), TGF- signalling (Z = 0.66), and Wnt/ -catenin signalling (Z = 0.55) genes tend to associate with single types (Methods).
Notably, 3,053 out of 3,281 total samples (93%) across the Pan-Cancer collection had at least one non-synonymous mutation in at least one SMG. The average number of point mutations and small indels in these genes varies across tumour types, with the highest (,6 mutations per tumour) in UCEC, LUAD and LUSC, and the lowest (,2 mutations per tumour) in AML, BRCA, KIRC and OV. This suggests that the numbers of both cancer-related genes (only 127 identified in this study) and cooperating driver mutations required during oncogenesis are small (most cases only had 2–6) (Fig. 3), although large-scale structural rearrangements were not included in this analysis.
Common mutations
The most frequently mutated gene in the Pan-Cancer cohort is TP53 (42% of samples). Its mutations predominate in serous ovarian (95%) and serous endometrial carcinomas (89%) (Fig. 2). TP53 mutations are also associated with basal subtype breast tumours. PIK3CA is the second most commonly mutated gene, occurring frequently (>10%) in most cancer types except OV, KIRC, LUAD and AML. PIK3CA mutations frequented UCEC (52%) and BRCA (33.6%), being specifically enriched in luminal subtype tumours. Tumours lacking PIK3CA mutations often had mutations in PIK3R1, with the highest occurrences in UCEC (31%) and GBM (11%) (Fig. 2).
Many cancer types carried mutations in chromatin re-modelling genes. In particular, histone-lysine N-methyltransferase genes (MLL2 (also known as KMT2D), MLL3 (KMT2C) and MLL4 (KMT2B)) cluster in bladder, lung and endometrial cancers, whereas the lysine (K)-specific demethylase KDM5C is prevalently mutated in KIRC (7%). Mutations in ARID1A are frequent in BLCA, UCEC, LUAD and LUSC, whereas mutations in ARID5B predominate in UCEC (10%) (Fig. 2).
Fig. 1. | Distribution of mutation frequencies across 12 cancer types.
Dashed grey and solid white lines denote average across cancer types and median for each type, respectively. b, Mutation spectrum of six transition (Ti) and transversion (Tv) categories for each cancer type. c, Hierarchically clustered mutation context (defined by the proportion of A, T, C and G nucleotides within ±2bp of variant site) for six mutation categories. Cancer types correspond to colours in a. Colour denotes degree of correlation: yellow (r = 0.75) and red (r = 1).
Fig. 2. The 127 SMGs from 20 cellular processes in cancer identified in and Pan-Cancer are shown, with the highest percentage in each gene among 12 (not shown)
Fig. 3. | Distribution of mutations in 127 SMGs across Pan-Cancer cohort.
Box plot displays median numbers of non-synonymous mutations, with outliers shown as dots. In total, 3,210 tumours were used for this analysis (hypermutators excluded).
Figure 4 | Unsupervised clustering based on mutation status of SMGs. Tumours having no mutation or more than 500 mutations were excluded. A mutation status matrix was constructed for 2,611 tumours. Major clusters of mutations detected in UCEC, COAD, GBM, AML, KIRC, OV and BRCA were highlighted.
Complete gene list shown in Extended Data Fig. 3. (not shown)
Figure 5 | Driver initiation and progression mutations and tumour clonal mutation is in the subclone
We examined which genes correlate with survival using the Cox proportional hazards model, first analysing individual cancer types using age and gender as covariates; an average of 2 genes (range: 0–4) with mutation frequency 2% were significant (P<_0.05) in each type (Supplementary Table 10a and Extended Data Fig. 6). KDM6A and ARID1A mutations correlate with better survival in BLCA (P = 0.03, hazard ratio (HR) = 0.36, 95% confidence interval (CI): 0.14–0.92) and UCEC (P = 0.03, HR = 0.11, 95% CI: 0.01–0.84), respectively, but mutations in SETBP1, recently identified with worse prognosis in atypical chronic myeloid leukaemia (aCML)31, have a significant detrimental effect in HNSC (P = 0.006, HR = 3.21, 95% CI: 1.39–7.44). BAP1 strongly correlates with poor survival (P = 0.00079, HR = 2.17, 95% CI: 1.38–3.41) in KIRC. Conversely, BRCA2 mutations (P = 0.02, HR = 0.31, 95% CI: 0.12–0.85) associate with better survival in ovarian cancer, consistent with previous reports32,33; BRCA1 mutations showed positive correlation with better survival, but did not reach significance here.
We extended our survival analysis across cancer types, restricting our attention to the subset of 97 SMGs whose mutations appeared in 2% of patients having survival data in 2 tumour types. Taking type, age and gender as covariates, we found 7 significant genes: BAP1, DNMT3A, HGF, KDM5C, FBXW7, BRCA2 and TP53 (Extended Data Table 1). In particular, BAP1 was highly significant (P = 0.00013, HR = 2.20, 95% CI: 1.47–3.29, more than 53 mutated tumours out of 888 total), with mutations associating with detrimental outcome in four tumour types and notable associations in KIRC (P = 0.00079), consistent with a recent report28, and in UCEC(P = 0.066). Mutations in several other genes are detrimental, including DNMT3A (HR = 1.59), previously identified with poor prognosis in AML34, and KDM5C (HR = 1.63), FBXW7 (HR = 1.57) and TP53 (HR = 1.19). TP53 has significant associations with poor outcome in KIRC (P = 0.012), AML (P = 0.0007) and HNSC (P = 0.00007). Conversely, BRCA2 (P = 0.05, HR = 0.62, 95% CI: 0.38 to 0.99) correlates with survival benefit in six types, including OV and UCEC (Supplementary Table 10a, b). IDH1 mutations are associated with improved prognosis across the Pan-Cancer set (HR = 0.67, P = 0.16) and also in GBM (HR = 0.42, P = 0.09) (Supplementary Table 10a, b), consistent with previous work.35
Driver mutations and tumour clonal architecture
To understand the temporal order of somatic events, we analysed the variant allele fraction (VAF) distribution of mutations in SMGs across AML, BRCA and UCEC (Fig. 5a and Supplementary Table 11a) and other tumour types (Extended Data Fig. 7). To minimize the effect of copy number alterations, we focused on mutations in copy neutral segments. Mutations in TP53 have higher VAFs on average in all three cancer types, suggesting early appearance during tumorigenesis.
It is worth noting that copy neutral loss of heterozygosity is commonly found in classical tumour suppressors such as TP53, BRCA1, BRCA2 and PTEN, leading to increased VAFs in these genes. In AML, DNMT3A (permutation test P = 0), RUNX1 (P = 0.0003) and SMC3 (P = 0.05) have significantly higher VAFs than average among SMGs (Fig. 5a and Supplementary Table 11b). In breast cancer, AKT1, CBFB, MAP2K4, ARID1A, FOXA1 and PIK3CA have relatively high average VAFs. For endometrial cancer, multiple SMGs (for example, PIK3CA, PIK3R1, PTEN, FOXA2 and ARID1A) have similar median VAFs. Conversely, KRAS and/or NRAS mutations tend to have lower VAFs in all three tumour types (Fig. 5a), suggesting NRAS (for example, P = 0 in AML) and KRAS (for example, P = 0.02 in BRCA) have a progression role in a subset of AML, BRCA and UCEC tumours. For all three cancer types, we clearly observed a shift towards higher expression VAFs in SMGs versus non-SMGs, most apparent in BRCA and UCEC (Extended Data Fig. 8a and Methods).
Previous analysis using whole-genome sequencing (WGS) detected subclones in approximately 50% of AML cases15,36,37; however, analysis is difficult using AML exome owing to its relatively few coding mutations. Using 50 AML WGS cases, sciClone (http://github.com/ genome/sciclone) detected DNMT3A mutations in the founding clone for 100% (8 out of 8) of cases and NRAS mutations in the subclone for 75% (3 out of 4) of cases (Extended Data Fig. 8b). Among 304 and 160 of BRCA and UCEC tumours, respectively, with enough coding mutations for clustering, 35% BRCA and 44% UCEC tumours contained subclones. Our analysis provides the lower bound for tumour heterogeneity, because only coding mutations were used for clustering. In BRCA, 95% (62 out of 65) of cases contained PIK3CA mutations in the founding clone, whereas 33% (3 out of 9) of cases had MLL3 mutations in the subclone. Similar patterns were found in UCEC tumours, with 96% (65 out of 68) and 95% (62 out of 65) of tumours containing PIK3CA and PTEN mutations, respectively, in the founding clone, and 9% (2 out of22) ofKRAS and 14% (1 out of 7) ofNRAS mutations in the subclone (Extended Data Fig. 8b and Supplementary Table 12).
Mutation context (-2 to +2 bp) was calculated for each somatic variant in each mutation category, and hierarchical clustering was then performed using the pairwise mutation context correlation across all cancer types. The mutational significance in cancer (MuSiC)3 package was used to identify significant genes for both individual tumour types and the Pan-Cancer collective. An R function 'hclust' was used for complete-linkage hierarchical clustering across mutations and samples, and Dendrix30 was used to identify sets of approximately mutual exclusive mutations. Cross-cancer survival analysis was based on the Cox proportional hazards model, as implemented in the R package 'survival' (http://cran.r-project.org/web/ packages/survival/), and the sciClone algorithm (http://github.com/genome/sci-clone) generated mutation clusters using point mutations from copy number neutral segments. A complete description of the materials and methods used to generate this data set and its results is provided in the Methods.
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Crucial role of Nitric Oxide in Cancer
Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Metabolomics, Nitric Oxide in Health and Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged Akt, angiogenesis, anti-apoptotic, Apoptosis, Bcl-2, C-Src, Cancer - General, cancer risk, carcinogenesis, cell cycle, cell proliferation, Cisplatin, conformational, cytotoxicity, DNA damage, DNA repair, Endothelium, eNOS, genotoxicity, HIF-1, HIF-1a, inflammation, inflammatory, iNOS, intracellular localization, macrophages, MMP, nitric oxide, Nitric oxide synthase, nNOS, NO, NO donors, NOS, p53, PI3K, post translational modifications, pro-apoptotic, proteasomal, PTEN, RAW264.7, S-nitrosylation, transcription, tumorigenesis, Ubiquitin on October 16, 2012| 21 Comments »
Author and Curator: Ritu Saxena, Ph.D.
Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al
NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.
Regulation by Nitric oxide
NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.
The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.
Role of Nitric oxide in Cancer
One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.
NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.
S-nitrosylation and Cancer
The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.
Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.
NO mediated aberrant proteins in Cancer
Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.
p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.
HIF-1a
Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.
Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.
C-Src
C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.
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Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9. http://www.ncbi.nlm.nih.gov/pubmed/22425962
Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.http://www.ncbi.nlm.nih.gov/pubmed/20167555
Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34. http://www.ncbi.nlm.nih.gov/pubmed/11518674