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Constitutively Active Type I Insulin-Like Growth Factor Receptor Causes Transformation and Xenograft Growth of Immortalized Mammary Epithelial Cells and Is Accompanied by an Epithelial-to-Mesenchymal Transition Mediated by NF-κB and Snail
Hyun-Jung Kim, Beate C. Litzenburger, Xiaojiang Cui, David A. Delgado, Brian C. Grabiner, Xin Lin, Michael T. Lewis, Marco M. Gottardis, Tai W. Wong, Ricardo M. Attar, Joan M. Carboni, Adrian V. Lee
Hyun-Jung Kim
Breast Center, Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine and the Methodist Hospital, Houston, Texas
Beate C. Litzenburger
Xiaojiang Cui
David A. Delgado
Brian C. Grabiner
Department of Molecular and Cellular Oncology, University of Texas, M. D. Anderson Cancer Center, Houston, Texas
Xin Lin
Michael T. Lewis
Marco M. Gottardis
Oncology Drug Discovery, Bristol-Myers Squibb Research Institute, Princeton, New Jersey
Tai W. Wong
Ricardo M. Attar
Joan M. Carboni
Adrian V. Lee
For correspondence: avlee@breastcenter.tmc.edu
DOI: 10.1128/MCB.01315-06
Type I insulin-like growth factor receptor (IGF-IR) can transform mouse fibroblasts; however, little is known about the transforming potential of IGF-IR in human fibroblasts or epithelial cells. We found that overexpression of a constitutively activated IGF-IR (CD8-IGF-IR) was sufficient to cause transformation of immortalized human mammary epithelial cells and growth in immunocompromised mice. Furthermore, CD8-IGF-IR caused cells to undergo an epithelial-to-mesenchymal transition (EMT) which was associated with dramatically increased migration and invasion. The EMT was mediated by the induction of the transcriptional repressor Snail and downregulation of E-cadherin. NF-κB was highly active in CD8-IGF-IR-MCF10A cells, and both increased levels of Snail and the EMT were partially reversed by blocking NF-κB or IGF-IR activity. This study places IGF-IR among a small group of oncogenes that, when overexpressed alone, can confer in vivo tumorigenic growth of MCF10A cells and indicates the hierarchy in the mechanism of IGF-IR-induced EMT.
Numerous studies have shown the importance of type I insulin-like growth factor receptor (IGF-IR) signaling in mouse mammary gland development and cancer. IGF-IR-null mice have markedly reduced mammary gland development (6), and overexpression of a constitutively active IGF-IR in the mammary gland caused rapid mammary tumorigenesis (8). Similar mammary tumorigenesis was recently shown using inducible overexpression of wild-type IGF-IR (25). Interestingly, transgenic overexpression of the IGF-IR downstream signaling intermediates insulin receptor substrate 1 (IRS1) and IRS2 also caused mammary tumorigenesis (10), and targeted deletion of IRS2 blocked metastasis (32).
IGF-IR is elevated and autophosphorylated in human breast cancer (41). Several strategies have been reported that block IGF-IR activity and inhibit breast cancer growth and metastasis (38), and numerous pharmaceutical companies have agents that target IGF-IR in clinical trials (15). Although IGF-IR signaling is implicated in breast cancer, the molecular mechanisms of IGF-IR-mediated tumorigenesis and metastasis remain unclear.
The role of IGF-IR in malignant transformation has been addressed mainly by studies of mouse fibroblasts. In this setting, IGF-IR acts as a classic oncogene, with overexpression causing transformation (26). Conversely, mouse embryo fibroblasts with a targeted disruption of the IGF-IR gene are resistant to transformation by a variety of viral and cellular oncogenes (35). Reintroduction of IGF-IR renders these cells susceptible to transformation. These results have led to the concept that IGF-IR is itself not only an oncogene, but also "quasi-necessary" for transformation (5). However, recent studies have highlighted important differences between transformation of mouse and human cells and, more importantly, between fibroblasts and epithelial cells (14). To this point, few data exist on IGF-IR-mediated transformation of human epithelial cells.
Two recent studies have used the human MCF10A immortalized mammary epithelial cell line to examine the effect of elevated IGF-IR levels on mammary acinus formation in three-dimensional (3D) culture (24, 47). Both groups found that overexpressed IGF-IR remained ligand dependent, but when stimulated by IGF-I, caused hyperproliferation, decreased apoptosis, and altered polarity, resulting in large, complex, disrupted acini. Blockade of phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2 (ERK1/2) blocked the formation of disrupted acini by MCF10A-IGF-IR cells (47).
Epithelial-to-mesenchymal transition (EMT) has been recognized as a cellular mechanism in normal development as well as, recently, in tumorigenesis (7, 21, 42), and reports strongly indicate that both invasion and metastasis may be dependent on the acquisition of EMT features by primary cancer cells (22, 46). Several transcription factors are central to EMT, including Snail, Slug, Twist, and Zeb1 (4, 22, 46).
IGF-I stimulation of breast cancer cells overexpressing IGF-IR has been shown to cause depolarization and a mesenchyme-like transition (20). Irie et al. noted that MCF10A cells overexpressing IGF-IR showed a subtle conversion, from a cuboidal epithelial morphology to a more spindle-shaped morphology (24), and Yanochko and Eckhart noted that MCF10A-IGF-IR cells showed altered E-cadherin localization (47).
The transcription factor nuclear factor κB (NF-κB) was recently shown to be essential for EMT and metastasis in a breast cancer cell model (22). NF-κB has recently been implicated in cell proliferation and various cancers, including breast cancer (9, 33). High levels of nuclear NF-κB were found in the majority of primary human and rodent breast tumor tissue samples, breast cancer cell lines, and carcinogen-transformed mammary epithelial cells (9, 27). IGF-IR can activate the NF-κB signaling pathway (29). IGF-IR can modulate glycogen synthase kinase 3β (GSK-3β) activity via Akt; GSK-3β regulates Snail via NF-κB (1); and NF-κB binds Snail promoter and increases its activity (3), suggesting a potential IGF-IR-GS3Kβ-NF-κB-Snail signaling pathway in EMT in MCF10A cells.
In this study, we established MCF10A human mammary epithelial cells that overexpress a constitutively active IGF-IR (CD8-IGF-IR-MCF10A). CD8-IGF-IR-MCF10A cells showed numerous features of transformation, including growth factor-independent proliferation, lack of contact inhibition, anchorage-independent growth, invasion, and tumorigenesis in vivo. This study is significant, as relatively few oncogenes, when expressed alone, have been shown to cause in vivo tumorigenic potential in MCF10A cells. In addition, CD8-IGF-IR-MCF10A cells showed an EMT which was associated with the downregulation of multiple epithelial cell markers and concomitant upregulation of many mesenchymal markers. Transcriptional repressor Snail mRNA levels were dramatically induced in CD8-IGF-IR-MCF10A cells, despite no change in Slug, Twist, or Zeb1. NF-κB was highly activated in CD8-IGF-IR-MCF10A cells, and the EMT and upregulation of Snail gene expression were reversed by the blockade of NF-κB or IGF-IR signaling. Therefore, constitutively activated IGF-IR activates NF-κB to induce Snail and cause EMT in MCF10A cells.
Plasmid cloning.Construction of the CD8-IGF-IR chimera has been described previously (8). For this study, we isolated CD8-IGF-IR from pcDNA3.1 using NheI and linearized pBabe-puro with BamHI. After the 5′ overhangs of each plasmid were filled in with Klenow enzyme (Promega, Madison, WI), they were ligated with T4 DNA ligase (Promega). Positive clones were identified by restriction digestion and confirmed by sequencing. For the cloning of the dominant negative human Snail (SnaDN), the human Snail gene including zinc finger domains and flanking regions was amplified with a forward primer (NM_005985.2; 5′-ccggatcctctgaggccaaggatctccag-3′) and reverse primer of the T7 promoter region of pCMV-Tag 2B. Flag-Snail-pCMV-Tag 2B was used as the template for PCR. A 459-bp PCR fragment was digested with BamHI and XhoI, resulting in a 380-bp fragment, and ligated with linearized pCMV-Tag 2B vector. Then, Flag-SnaDN was excised with BstXI and XhoI and blunted with Klenow enzyme. pLNCX2 vector was digested with HindIII and then blunted with Klenow enzyme. They were ligated with T4 DNA ligase and positive clones were confirmed by sequencing.
Cell culture, retrovirus production, and establishment of stable cell lines.The immortalized human breast epithelial cell line MCF10A was cultured in Dulbecco's modified Eagle medium (DMEM)-F-12 (Invitrogen) supplemented with 5% horse serum (Sigma, St. Louis, MO), 20 ng/ml epidermal growth factor (Sigma), 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 100 ng/ml cholera toxin (Sigma), and 100 units/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA). The retrovirus was generated by transfection of CD8-IGF-IR-pBabe-puro or pBabe-puro plasmid into PT67 packaging cells (BD Biosciences, San Jose, CA) using Lipofectamine reagent (Invitrogen). The supernatant was harvested 48 h after transfection and MCF10A cells were plated on 60-mm dishes the day before infection and were infected for 48 h with 1 ml of the supernatant containing retrovirus encoding CD8-IGF-IR or empty vector (pBabe-puro) as a control. Infected cells were selected with 5 μg/ml puromycin (Sigma) 48 h after infection, and individual clones were isolated 10 days later. The overexpression of CD8-IGF-IR was confirmed by immunoblot analysis with anti-IGF-IRβ antibody. Twelve individual positive clones were pooled together to rule out clonal artifacts and were named the CD8-IGF-IR-MCF10A pool. For the control clones, isolated individual clones (n = 5) were pooled together (vector-MCF10A). For CD8-IGF-IR-MCF10A cells overexpressing SnaDN, retrovirus was made by the same method as described above and used to infect CD8-IGF-IR-MCF10A cells. Infected cells were selected with 1 mg/ml G418 (Invitrogen), and positive clones were detected with anti-Flag antibody (Sigma).
Monolayer growth assay.For growth curve experiments, cells were plated in 12-well plates at a concentration of 25,000 cells/well and the next day after cells were plated was designated day 0. For measuring cell numbers in serum-free conditions, cells were placed in serum-free media at day 0. Cell numbers were counted every 2 days using a Beckman Coulter Z series counter (Beckman Coulter, Fullerton, CA).
Anchorage-independent growth assay.For anchorage-independent colony formation, 1 × 104 cells were suspended in their growth medium containing 0.35% SeaPlaque agarose (Cambrex Bio Science, Rockland, ME) and plated in 6-well plates over a base layer of complete medium containing 0.7% agarose. Colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/ml in phosphate-buffered saline [PBS]) by incubation for 4 h at 37°C. Cells were photographed using an Alpha Imager 7000 (Alpha Innotech, San Leandro, CA).
3D Matrigel culture.3D Matrigel culture was performed as described previously (12). Briefly, cells were treated with trypsin and first resuspended in DMEM-F-12 medium supplemented with 20% horse serum. The cells were centrifuged and resuspended in assay medium (DMEM-F-12 supplemented with 2% horse serum, 10 μg/ml insulin, 1 ng/ml cholera toxin, 100 μg/ml hydrocortisone, 50 U/ml penicillin, and 50 μg/ml streptomycin). The cells were counted and diluted in assay medium to achieve a final concentration of 25,000 cells/ml. Eight-chambered glass slides were coated with 60 μl Matrigel (BD Biosciences) per well and left to solidify for 30 min. The cells were mixed 1:1 with assay medium containing 4% Matrigel and 10 ng/ml epidermal growth factor (EGF), and 400 μl was added to each chamber of the Matrigel-coated slide. This corresponds to a final overlay solution of 5,000 cells/well in medium containing 2% Matrigel and 5 ng/ml EGF. Assay medium containing 5 ng/ml EGF and 2% Matrigel was replaced every 4 days. Immunostaining of acinar structures with antibodies to Ki-67 (Zymed) or laminin V (Chemicon) was performed as described previously (12). For nuclear staining, the acinar structures were fixed in 2% paraformaldehyde at room temperature for 20 min. Fixed structures were washed three times in PBS for 10 min each time. Nuclear staining was performed by incubation for 15 min with PBS containing 5 μM TOPRO-3 (Invitrogen) before cells were mounted with the antifade agent VECTASHIELD (Vector Laboratories, Burlingame, CA). Confocal analyses were performed with a Nikon Eclipse E1000 confocal microscopy system.
Migration and invasion assay.The migration of cells was analyzed both through an in vitro wound-healing assay and a transwell chamber assay. After overnight serum starvation, monolayers of confluent cultures were lightly scratched with a pipette tip, washed with serum-free medium to remove detached cells, and then incubated with serum-free or serum-containing medium. Cultures were observed regularly for up to 24 h. For the transwell chamber assay, the polycarbonate membranes (8-μm pore size) (Becton Dickinson Labware) of the upper compartments of transwell culture chambers were coated with 10 μg/μl fibronectin. For the invasion assay, a BD Matrigel invasion chamber (BD biosciences) was used. A total of 3 × 104 cells were resuspended in 500 μl serum-free medium and placed in the upper compartment, and the lower compartment was immediately filled with 500 μl growth medium. After 24 h, the cells on the upper surface of the transwell were carefully removed with a cotton swab, the membrane was stained using a Hema 3 Stat pack (Fisher), and cells that passed through the membrane to the lower surface were counted in four different fields at ×10 magnification.
Xenograft studies.All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of Baylor College of Medicine. Mice were maintained on a 12-h light, 12-h dark schedule with ad libitum access to laboratory chow (Picolab rodent diet 20 and lab diet 5053; PMI Nutrition International Inc., Brentwood, MO) and water. Vector-MCF10A and CD8-IGF-IR-MCF10A cells were harvested and 5 × 106 cells were injected orthotopically into the number 3 mammary fat pads of athymic nu/nu or scid/beige mice (Charles River, Wilmington, MA). The tumor sizes were measured with digital calipers twice a week, and tumor volumes were estimated according to the formula for an ellipse, (short dimension)2 × (long dimension)/2.
Immunoblot analysis.Forty micrograms of total protein was resuspended in denaturing sample loading buffer (3% dithiothreitol, 0.1 M Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol), separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrophoretically transferred to a nitrocellulose membrane overnight at 4°C. The remaining steps were all performed at room temperature. The membrane was blocked with PBS plus 0.05% Tween 20 (PBST) containing 5% nonfat milk (blocking solution) for 1 h followed by incubation with anti-pY1162/1163-IGF-IR (no. 44-804G; Biosource, Camarillo, CA), anti-IGF-IR beta (no. sc-713; Santa Cruz Biotechnology, Santa Cruz, CA), anti-N-cadherin (no. 610920; BD Transduction Laboratories), anti-vimentin (no. 550513; BD Transduction Laboratories), anti-beta-catenin (no. 610154; BD Transduction Laboratories), anti-alpha-catenin (no. sc-7894; Santa Cruz Biotechnology), anti-E-cadherin (no. sc-8426; Santa Cruz Biotechnology), anti-fibronectin (no. sc-9068; Santa Cruz Biotechnology), and anti-β-actin (no. A1978; Sigma) antibodies in blocking solution overnight at 4°C. Subsequently, the membrane was washed three times for 5 min each with PBST and then incubated with a horseradish peroxidase-linked secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) at a dilution of 1:4,000 in blocking solution. After the membrane was washed three times for 5 min each with PBST, bands were visualized by enhanced chemiluminescence according to the manufacturer's protocol (Pierce Biotechnology, Rockford, IL) and captured using an Alpha Innotech 7000 imaging system (Alpha Innotech, San Leandro, CA).
Immunofluoresence.1 × 105 cells were seeded onto two chamber slides (Falcon), and 2 days later, the medium was changed, with or without 1 μM BMS-536924 (45), and the incubation was continued for an additional 24 h. The cells were washed with PBS, fixed for 20 min in 2% paraformaldehyde, and then washed three times with PBS. The cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min, washed three times with PBS, and then blocked with 10% normal goat serum (Jackson Immunoresearch Laboratories Inc., West Grove, PA) in PBST. The cells were stained using monoclonal anti-E-cadherin antibody (BD Transduction Laboratories) and anti-NF-κB antibody (Santa Cruz Biotechnology) at 1:200 dilution in blocking solution overnight at 4°C, and then washed three times for 10 min in PBST buffer. Cells were then stained with goat anti-mouse Alexa Fluor 488 (Invitrogen) at 1:200 dilution in blocking solution, followed by three washes of 10 min in PBST. The slides were mounted using mounting medium (Vectorshield; Vector Laboratories, Burlingame, CA).
Immunohistochemistry.Vector-MCF10A and CD8-IGF-IR-MCF10A cells were cultured in maintenance media and then placed in serum-free medium (SFM) for 24 h. Cells were trypsinized, fixed briefly with 4% paraformaldehyde, and processed into paraffin. Slides were cut at 3 to 4 microns, baked overnight at 58°C, and deparaffinized using a Shandon-Lipshaw Varistain (program 2). Heat-induced antigen retrieval was performed in 1 mM EDTA (pH 8.0) (pY-IGF-IR) or in 0.1 M Tris-HCl (pH 9.0) (total IGF-IR) for 5 min. All further incubations were performed at room temperature, and all washing was performed with TBST (0.15 M NaCl, 0.01 M Tris-HCl [pH 7.4], 0.05% Tween 20) unless otherwise stated. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide solution for 5 min. The slides were then incubated with anti-pY1131-IGF-IR (catalogue no. 3021; Cell Signaling Technology) or anti-IGF-IR beta (catalogue no. sc-713; Santa Cruz) for 1 h, biotinylated secondary antibody (1:200) for 30 min, and then horseradish peroxidase-labeled avidin (1:200) for 30 min. As a negative control, the slides were incubated with purified rabbit immunoglobulin (Jackson Laboratories). Detection was achieved by incubation with diaminobenzidine (Dako) for 15 min, followed by enhancement with 0.2% osmium tetroxide for 30 s. The slides were counterstained with hematoxylin, dehydrated, and mounted using cytoseal.
RNA isolation and quantitative real-time PCR.Total RNA was prepared with an RNeasy mini kit (QIAGEN, CA) according to the instruction manual. Total RNA was reverse transcribed in a final volume of 100 μl using 0.5 μg of total RNA, 0.5 μl random primers, and 25 mM deoxynucleoside triphosphate. After the samples were heated for 5 min at 65°C, 20 μl of 5× first-strand buffer (Invitrogen), 5 μl 0.1 M dithiothreitol, and 0.5 μl of Superscript II RNase H reverse transcriptase (Invitrogen) were added. Reverse transcription was performed on a 9600 GeneAmp PCR system at 25 °C for 5 min, 48°C for 30 min, and 70°C for 10 min. The cDNA was stored at −20°C. Quantitative real-time PCR (Q-PCR) was performed using primers that were already published for human E-cadherin, Twist, and N-cadherin (37); fibronectin (36); Snail and ZEB1 (34); alpha-smooth muscle actin (α-SMA) (44); and Slug (40); and we designed primers for vimentin which consisted of forward 5′-CAACCTGGCCGAGGACAT-3′ and reverse 5′-ACGCATTGTCAACATCCTGTCT-3′. Q-PCR was performed in an ABI PRISM 7700 sequence detector (PE Biosystems) using the power SYBR green PCR master mix (Applied Biosystems). Each reaction mixture contained 5 μl of template cDNA, a final concentration of 0.15 μM of forward and reverse primers, 12.50 μl of 2× SYBR green buffer, and RNase-free H2O to make a final volume of 25 μl. The PCR for each gene consisted of the following: 50°C for 2 min, denaturing at 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 30 s. To determine the primer specificity, three stages (95°C for 15 s, 60°C for 20 s, and 95°C for 15 s, with a ramping time of 20 min) were added to the end of the PCR to obtain dissociation curves for each gene. Analysis of Q-PCR data was performed using Sequence Detector v. 1.7 software (ABI). Relative transcript levels were determined using the 2−ΔΔCT method and normalized to β-actin (28).
Preparation of nuclear extracts and electrophoretic mobility shift assays.Nuclear extracts and electrophoretic mobility shift assays were prepared as previously described (43). Nuclear extracts (10 μg) were incubated with 32P-labeled NF-κB or OCt-1 probe (Promega, Madison, WI) for 15 min at room temperature. The mixtures were then subjected to electrophoresis in 6% polyacrylamide gel and autoradiography.
Characterization of immortalized mammary epithelial MCF10A cells expressing a CD8-IGF-IR fusion gene.We recently reported the generation of a chimeric and constitutively activated IGF-IR that consists of a fusion of the human CD8α extracellular domain with the human IGF-IRβ subunit. CD8-IGF-IR was created to study the effect of IGF-IR in vivo without the complexity of IGF-I ligand and binding protein interactions. Expression of this chimeric receptor in the mammary glands of transgenic mice resulted in rapid mammary tumorigenesis (8) that was associated with an apparent EMT (data not shown). To further study the role of CD8-IGF-IR in transformation and EMT, we utilized MCF10A cells, an immortalized but nontransformed human mammary epithelial cell line (11). These cells exhibit numerous features of normal breast epithelium, including lack of tumorigenicity in nude mice, lack of anchorage-independent growth, and dependence on growth factors and hormones for proliferation and survival. MCF10A cells form polarized functional acini in 3D Matrigel culture and have proven to be a useful model for the study of oncogene disruption of polarity and acinus formation (11). To determine the effect of constitutive receptor activation on MCF10A immortalized human mammary epithelial cells, we generated stable cell lines by retroviral infection. Multiple clones expressing CD8-IGF-IR were identified, and these were combined together to generate a pool (CD8-IGF-IR-MCF10A) for further experiments. Clones expressing empty vector were combined to make a control pool clone (vector-MCF10A). Next we examined whether the overexpressed CD8-IGF-IR was constitutively active in the absence of ligand, IGF-I. Vector-MCF10A cells stimulated with IGF-I showed weak tyrosine phosphorylation of the β-subunit of IGF-IR (P∼IGF-IR) (Fig. 1A). CD8-IGF-IR has a molecular mass of approximately 75 kDa and exists as a doublet below the endogenous IGF-IRβ subunit (95 kDa), and it was constitutively phosphorylated without ligand. The activation was highly dominant because there was no additive or synergistic effect of IGF-I on these cells. Cells were then cultured in serum-free media, placed in paraffin, and immunohistochemistry (IHC) was performed to determine P∼IGF-IR and total IGF-IR levels. CD8-IGF-IR-MCF10A cells had readily detectable P∼IGF-IR staining that was mainly membranous and was completely absent in vector-MCF10A cells (Fig. 1B). IHC for total IGF-IR showed an approximately fivefold increase in their levels, which was consistent with the results of the immunoblotting (Fig. 1A).
Establishment of MCF10A stable cell lines overexpressing CD8-IGF-IR. (A) Vector-MCF10A and CD8-IGF-IR-MCF10A pools were incubated in SFM for 24 h and then stimulated with IGF-I (10 nM) for 10 min. Forty-microgram amounts of cell lysates were immunoblotted with anti-pY-IGF-IR (P∼IGF-IR) or total IGF-IRβ antibodies. The open and black arrows indicate endogenous IGF-IRβ and CD8-IGF-IR, respectively. β-Actin was used as a loading control. − and + indicate the absence or presence of IGF-I. (B) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were cultured in SFM, harvested, and processed in paraffin. Five-micrometer sections were then processed for IHC using anti-pY-IGF-IR (P∼IGF-IR) or total IGF-IRβ antibodies.
CD8-IGF-IR disrupts normal mammary acinus formation in 3D Matrigel culture.MCF10A cells grown in 3D Matrigel recapitulate numerous features of breast epithelium in vivo, including the formation of acinus-like spheroids with a hollow lumen, apicobasal polarization, and the basal deposition of basement membrane components (12). Therefore, we examined whether overexpressing CD8-IGF-IR would disrupt the normal mammary acinar morphogenesis of MCF10A cells in 3D Matrigel culture. As shown in Fig. 2A and B, vector-MCF10A cells formed small, well-formed acini, often with empty lumen. In contrast, CD8-IGF-IR-MCF10A acini appeared larger and disrupted, with cells filling the luminal space. The acini sometimes showed protrusions into the Matrigel (Fig. 2A; day 8) that are characteristic of invasive cells (Fig. 2A; arrows). Ki-67, a marker for proliferation, was highly expressed in the multiacinus structure of CD8-IGF-IR-MCF10A cells on day 12, while it was hard to detect Ki-67 staining in the growth-arrested acini of vector-MCF10A cells on the same day (Fig. 2B, upper panels), suggesting that constitutively active IGF-IR signaling induces hyperproliferation of the mammary epithelial cells. Vector-MCF10A acini often had cells in the center of the lumen that were undergoing apoptosis and stained positive for cleaved caspase-3 (Fig. 2B, left middle panel). CD8-IGF-IR cells showed no apoptosis as assessed by cleaved caspase-3 staining, consistent with the observed filling of the lumen (Fig. 2B, right middle panel). The staining of basement membranes of acinar structures with laminin V, a marker of epithelial cell polarity, showed that vector-MCF10A cells formed well-organized acinar structure with apical/basal polarity, while the multiacini of CD8-IGF-IR-MCF10A cells had disrupted polarity, with laminin V staining within the lumen (Fig. 2B, lower panels).
Constitutively active IGF-IR disrupted acinar morphogenesis of MCF10A cells on 3D Matrigel culture. (A) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were plated in Matrigel and cultured for 8 days or 15 days. Phase-contrast images of acini are shown in ×20 magnification. The arrow indicates invasive protrusions. (B) The cells were cultured on Matrigel for 8 to 12 days and then stained with antibodies to Ki-67 (green), cleaved caspase-3 (green) or laminin V (red). The nuclei (blue) were labeled with TOPRO-3 and visualized by confocal microscopy with ×40 magnification.
CD8-IGF-IR causes transformation of MCF10A cells.To evaluate whether the overexpression of CD8-IGF-IR caused transformation of MCF10A cells, we measured several hallmarks of the transformed cell (Fig. 3). Several experiments repeatedly indicated that the monolayer proliferation of CD8-IGF-IR-MCF10A cells was slightly slower than that of vector-MCF10A cells in normal growth media (Fig. 3A). In contrast, under SFM conditions, vector-MCF10A cells underwent growth arrest, whereas CD8-IGF-IR-MCF10A cells continued to proliferate. Furthermore, CD8-IGF-IR-MCF10A failed to exhibit contact inhibition and formed foci after long-term culture (Fig. 3B). Consistent with their nontransformed phenotype, vector-MCF10A cells failed to grow in soft agar, whereas CD8-IGF-IR-MCF10A cells repeatedly formed numerous colonies (Fig. 3B). Similarly, vector-MCF10A cells showed very poor ability to invade through Matrigel when a modified Boyden chamber was used, whereas CD8-IGF-IR-MCF10A cells were highly invasive (Fig. 3C).
Constitutive IGF-IR activation induced cellular transformation of nontransformed MCF10A cells. (A) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were plated on 12-well plates, and the next day, the medium was changed for serum-free medium to compare the growth rates of these cells in serum-free conditions. The next day after plating was designated day 0 (d0). Cell numbers were counted every 2 days. The error bars represent the standard errors of the means. (B) For monolayer culture, 2 × 105 cells were plated on 6-well plates and cultured for 10 days. Phase-contrast images of focus formation at day 10 are shown in the left panels. For the anchorage-independent growth assay, 1 × 104 cells were suspended in their growth medium containing 0.35% agarose and plated on 6-well plates over a basal layer of complete medium containing 0.7% agarose. Two weeks later, colonies were stained with MTT (right panels). (C) The Matrigel invasion assay showed that the number of CD8-IGF-IR-MCF10A cells that penetrated the membrane was significantly higher than that of vector-MCF10A cells. Cells were counted in four different microscopic fields (×10 magnification). The error bars represent the standard errors of the means. (D) Six-week-old mice were injected with CD8-IGF-IR-MCF10A cells in the number 3 mammary gland. The left panel is a representative photograph of a xenograft at 15 days after injection. The right panel is a representative section of tumor stained with hematoxylin and eosin.
Numerous oncogenes are able to disrupt MCF10A acinus formation and transform MCF10A cells in vitro. However, oncogenes such as ErbB2 disrupt acinus formation (11) but fail to convey in vivo xenograft growth (16). We therefore examined whether CD8-IGF-IR-MCF10A cells could grow in immunocompromised mice. In an initial pilot experiment, we injected vector-MCF10A and CD8-IGF-IR-MCF10A cells with or without Matrigel orthotopically into athymic nude mice. We found that vector-MCF10A cells injected into athymic nude mice were unable to form a palpable mass when injected with (0/3 mice) or without (0/3 mice) Matrigel. In contrast, CD8-IGF-IR-MCF10A cells formed a palpable mass within 7 to 10 days (3/3 mice with Matrigel and 1/3 mice without Matrigel) (Fig. 3D). Histologic analysis of the tumors by hematoxylin and eosin staining revealed them to be undifferentiated carcinomas. Next, we repeated the experiment, and in results similar to those of our pilot experiment, Matrigel caused an increase in tumor take and tumor size after 10 days, with palpable tumors appearing in 7/10 mice with Matrigel and 5/10 without Matrigel. After 3 weeks, xenografts injected with Matrigel had reached a volume of 120.3 ± 3.3 mm3, whereas the xenografts without Matrigel were only 56 ± 21.8 mm3. Taken together, these data indicate that overexpression of a single oncogene (CD8-IGF-IR) in MCF10A cells is sufficient to cause transformation.
CD8-IGF-IR causes EMT in MCF10A cells.The expression of CD8-IGF-IR in the mammary glands of transgenic mice resulted in rapid mammary tumorigenesis (8) that was associated with an apparent EMT (data not shown). Interestingly, when MCF10A cells were infected with CD8-IGF-IR and were screened to identify those with stable CD8-IGF-IR expression, it was noted that the majority of the puromycin-resistant clones exhibited a morphological change that resembled an EMT. As shown in Fig. 4A, vector-MCF10A cells showed highly organized cell-cell adhesion and cell contact, whereas CD8-IGF-IR-MCF10A cells had an elongated and refractive appearance, with cell scattering and loss of cell-cell contacts. The cobblestone-like morphology of MCF10A cells at confluence was replaced in CD8-IGF-IR-MCF10A cells by a spindle-like fibroblastic morphology. To determine the molecular alterations that occurred in these cells, we examined the expression of the epithelial adherence junction protein E-cadherin and the mesenchymal proteins vimentin and N-cadherin. In 3D culture, vector-MCF10A cells showed high levels of membrane-localized E-cadherin and little or no expression of vimentin or N-cadherin. In stark contrast, CD8-IGF-IR-MCF10A acini showed lower levels of E-cadherin, and the E-cadherin was mislocalized, showing punctate staining. In addition, the CD8-IGF-IR-MCF10A acini showed strong staining of both vimentin and N-cadherin. Immunoblot analysis showed that epithelial adherence junction proteins were dramatically downregulated in CD8-IGF-IR-MCF10A cells compared to their strong expression in vector-MCF10A cells (Fig. 4B). E-cadherin mRNA levels were reduced 10-fold in the CD8-IGF-IR-MCF10A cells compared to their levels in vector-MCF10A cells (Fig. 5A), and this correlated with a twofold reduction in E-cadherin promoter activity (Fig. 4C). The expression of mesenchymal markers, including vimentin, N-cadherin, fibronectin, and α-SMA, were all low or absent in vector-MCF10A cells but highly induced in CD8-IGF-IR-MCF10A cells (Fig. 4B).
EMT was induced in CD8-IGF-IR-MCF10A cells. (A) The morphologies of MCF10A cells expressing either the control vector or CD8-IGF-IR were revealed by phase-contrast microscopy (×20 magnification) at day 3. Vector-MCF10A and CD8-IGF-IR-MCF10A cells were cultured on Matrigel for 12 days and then stained with antibodies to E-cadherin (green), vimentin (red), or N-cadherin (red). The nuclei (blue) were labeled with TOPRO-3 and visualized by confocal microscopy with ×40 magnification. (B) Levels of epithelial proteins, including E-cadherin, β-catenin, and α-catenin, and mesenchymal proteins, including N-cadherin, vimentin, fibronectin, and α-SMA, in vector-MCF10A and CD8-IGF-IR-MCF10A cells were examined by immunoblotting. β-Actin was used as a loading control. (C) The activity of a 2-kb fragment of the E-cadherin promoter upstream of a luciferase reporter (E-cad-luc) was compared to that of the control plasmid (pGL2-basic) by transient transfection. The luciferase (luc) activity was normalized to the cotransfected β-galactosidase activity. The error bars represent the standard errors of the means. (D) Results of the transwell migration assay in which vector-MCF10A and CD8-IGF-IR-MCF10A cells were induced to migrate toward growth media. The migrated cells that passed through the membrane to the lower surface were counted in four different microscopic fields at ×10 magnification. (E) Confluent vector-MCF10A and CD8-IGF-IR-MCF10A cells were scratched and photographs were taken immediately (0 h) and 24 h postscratch at ×10 magnification.
Blocking of constitutive IGF-IR activation by a new small-molecule inhibitor of IGF-IR, BMS-536924, partially reversed the mesenchyme-like morphological changes and the downregulation of E-cadherin levels. (A) CD8-IGF-IR-MCF10A cells were incubated with the indicated concentration of BMS-536924 for 1 h after 16 h of serum starvation, and the phosphorylation of CD8-IGF-IR was analyzed by immunoblotting with anti-P∼IGF-IR antibody. β-Actin was used as a loading control. (B) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were incubated with or without 1 μM BMS-536924 for 24 h and were either visualized by phase-contrast microscopy at ×20 magnification (upper panels) or stained with E-cadherin (E-cad) (×40 magnification; green) (lower panels). The nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI). DMSO, dimethyl sulfoxide. (C) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were incubated with (+) or without (−) 1 μM BMS-536924 for 24 h in growth media. The levels of E-cadherin mRNA were examined by Q-PCR. The results are presented as relative transcript levels compared to vector-MCF10A by using the ΔΔCT method, with β-actin mRNA levels used as the normalization control. The error bars represent the standard errors of the means. (D) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were incubated with (+) or without (−) BMS-536924 for 24 h and lysed, and the levels of epithelial (E-cadherin) or mesenchymal markers (vimentin and α-SMA) were measured. β-Actin was used as a loading control.
Therefore, both the morphological and molecular changes in the CD8-IGF-IR-MCF10A cells demonstrated that these cells had undergone EMT. The functional consequences of EMT are enhanced cell scattering and migration. Indeed, both the fibronectin-coated transwell chamber assay and the wound-healing assay showed that CD8-IGF-IR-MCF10A cells migrated much faster than vector-MCF10A cells, which were generally unable to migrate (Fig. 4D and E).
EMT and E-cadherin downregulation are reversed by blockade of IGF-IR activity with BMS-536924.To investigate whether the EMT phenotype of CD8-IGF-IR-MCF10A cells is a specific event mediated by the constitutively active IGF-IR, cells were treated with a small-molecule inhibitor of IGF-IR (BMS-536924) (45). Immunoblot analysis showed that constitutive phosphorylation of CD8-IGF-IR was inhibited in a dose-dependent manner by BMS-536924, with an observed effect at 100 nM of the drug and maximal inhibition at 1 μM (Fig. 5A). CD8-IGF-IR-MCF10A cells treated with BMS-536924 for 24 h showed a partial reversion to a cobblestone-like epithelial morphology, and immunofluorescence results showed that E-cadherin levels were also partially reversed; however, the E-cadherin was not completely properly localized, having some cytoplasmic localization (Fig. 5B). Consistent with a decrease in E-cadherin protein and promoter activity in CD8-IGF-IR-MCF10A cells (Fig. 4C), E-cadherin mRNA levels were reduced 10-fold compared to its levels in vector-MCF10A cells (Fig. 5C). Importantly, this downregulation of E-cadherin mRNA was reversed (by up to 80%) with BMS-536924 (Fig. 5B). We also examined the ability of BMS-536924 to reverse changes in the protein levels of epithelial and mesenchymal markers in CD8-IGF-IR-MCF10A cells; we found that BMS-536924 reversed the reduction of E-cadherin protein levels and also reversed the induction of vimentin and α-SMA (Fig. 5D). It should be noted that, despite the ability of IGF-IR inhibition to reverse the levels of these proteins, the morphological reversion was incomplete, suggesting that there may be irreversible changes that have caused the EMT, or that other pathways, in addition to IGF-IR, need to be inhibited.
Snail transcription factor is an essential regulator of constitutively active IGF-IR-induced EMT.Since it is known that Snail, Slug, Twist, and Zeb1 are repressors of E-cadherin promoter activity and the subsequent reduction of E-cadherin mRNA and protein levels is a central component of EMT (23), we examined the mRNA expression levels of Snail, Slug, Twist, and Zeb1 by Q-PCR. Snail mRNA was dramatically induced (up to 80-fold) in CD8-IGF-IR-MCF10A cells, whereas no changes were observed for Slug, Twist, or Zeb1, even though mRNA was detected for all three genes (Fig. 6A). Next, we tested whether IGF-I can also induce Snail mRNA in vector-MCF10A cells. Figure 6B shows that IGF-I treatment induced Snail mRNA, even though the induction was not as strong as that in CD8-IGF-IR-MCF10A cells. The induction of Snail mRNA by IGF-I treatment, or in CD8-IGF-IR-MCF10A cells, was mostly reversed by treatment with BMS-536924. However, it should be noted that the inhibitor did not fully reverse the induction by IGF-I or CD8-IGF-IR, suggesting either incomplete blockade of IGF-IR, or that there are other pathways mediating the induction. To show whether Snail was necessary for the downregulation of E-cadherin and the EMT, we generated a SnaDN that consisted of only the zinc finger domains and small flanking regions of wild-type Snail and introduced this construct into CD8-IGF-IR-MCF10A cells by retroviral infection (CD8-IGF-IR/SnaDN-MCF10A cells). Monolayer growth of these cells showed that they had an epithelium-like morphology that was somewhat similar to that of vector-MCF10A cells (Fig. 6C, top panels). The expression of SnaDN in CD8-IGF-IR-MCF10A cells also reversed the abnormal 3D growth of CD8-IGF-IR-MCF10A cells, with acini now appearing smaller and similar to those of vector-MCF10A cells (Fig. 6C). Consistent with this partial morphological reversion, the CD8-IGF-IR/SnaDN-MCF10A cells showed elevated levels of E-cadherin (Fig. 6D).
Snail is the essential mediator of the EMT induced by the constitutively active IGF-IR signaling. (A) The expression of Snail, Slug, Twist, and Zeb1 mRNA in vector-MCF10A (open bar) and CD8-IGF-IR-MCF10A (shaded bar) cells was examined by Q-PCR. The results are presented as transcript levels relative to the level in vector-MCF10A cells by using the ΔΔCT method, with β-actin mRNA levels used as the normalization control. (B) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were incubated with (+) or without (−) 100 ng/ml IGF-I and 1 μM BMS-536924 for 24 h in growth media. The expression of Snail mRNA was examined by Q-PCR as described in Materials and Methods. The values shown are relative to those of vector-MCF10A cells without IGF-I stimulation. The error bars represent the standard errors of the means. (C) Vector-MCF10A, CD8-IGF-IR-MCF10A, and CD8-IGF-IR/SnaDN-MCF10A cells were grown in their growth media for 2 days. The cells were visualized by phase-contrast microscopy at ×20 magnification (upper panels). The cells were cultured on Matrigel for 8 days, and then the nuclei were labeled with TOPRO-3 and visualized by confocal microscopy with ×40 magnification (lower panels). (D) Vector-MCF10A, CD8-IGF-IR-MCF10A, and CD8-IGF-IR/SnaDN-MCF10A cells were immunoblotted for E-cadherin (E-cad) and Flag. β-Actin was used as a loading control.
NF-κB is the upstream regulator of Snail expression in the regulation of CD8-IGF-IR-induced EMT.We next examined pathways downstream of CD8-IGF-IR, which caused the dramatic elevation of Snail mRNA levels. The most-studied regulator of Snail promoter and mRNA levels is ERK1/2 (13). However, we found that incubation of CD8-IGF-IR-MCF10A cells with the ERK1/2 inhibitor U0126 (10 μM) had absolutely no effect on the 80-fold elevation of Snail mRNA levels in CD8-IGF-IR-MCF10A cells (Fig. 7A). U0126 also had no effect upon the morphology of CD8-IGF-IR-MCF10A cells (Fig. 7E). This is consistent with our observation that ERK1/2 is not active in these cells (data not shown). Recent reports indicated that NF-κB is an essential regulator of EMT and metastasis in a breast cancer cell model (23) and that NF-κB can bind the human Snail promoter and increase its activity (3). It was also reported that IGF-I stimulated sustained activation of NF-κB in human multiple myeloma cells (29). To determine whether the increased NF-κB was involved in the induction of Snail and EMT, we used inhibitors of the NF-κB signaling pathway. Treatment of CD8-IGF-IR-MCF10A cells for 24 h with two different inhibitors of the NF-κB pathway (IκB kinase [IKK] inhibitor II and Helenalin) partially reversed the induction of Snail mRNA in CD8-IGF-IR-MCF10A cells, and this reversion was similar to the results observed with BMS-536924 treatment (Fig. 7B).
NF-κB is the upstream regulator of Snail in CD8-IGF-IR-induced EMT. (A) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were incubated with dimethyl sulfoxide (DMSO) (open bars) or 10 μM U0126 (shaded bars) for 24 h, and then Snail mRNA levels were measured by Q-PCR as described in Materials and Methods. Representative levels are compared to those of vector-MCF10A cells incubated with DMSO. The bars represent the averages ± standard errors of the means of three measurements. (B) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were plated and the next day, cells were incubated with or without 1 μM BMS-536924 (BMS) and two NF-κB pathway inhibitors, including 20 μM IKK inhibitor II (IKK) and 20 μM Helenalin (Hele), for 24 h. Snail mRNA levels were measured by Q-PCR as described above for panel A. DMSO was used as the vehicle for the reagents. The transcript levels are relative to the level in vector-MCF10A cells incubated with DMSO. The bars represent the averages ± standard errors of the means of three measurements. (C) The cells were cultured for 2 days and then stained with anti-NF-κB p65 antibody (green). The nuclei (blue) were stained with TOPRO-3 and visualized by confocal microscopy (×60 magnification with oil). (D) The cells were harvested at day 1 or day 2, and nuclear extracts were prepared and incubated with oligonucleotides corresponding to NF-κB or OCt-1 consensus sequences. OCt-1 binding was used as a loading control. (E) Vector-MCF10A (a) and CD8-IGF-IR-MCF10A (b to e) cells were incubated with DMSO (a and b), BMS-536924 (c), 20 μM IKK inhibitor II (d), or 10 μM U0126 (e) for 24 h, and photographs were taken with phase-contrast microscopy at ×20 magnification. (F) Vector-MCF10A (a) and CD8-IGF-IR-MCF10A (b to d) cells were grown in 3D Matrigel and then incubated with DMSO (a and b), 1 μM BMS (c), or 20 μM IKK inhibitor II (d). They were harvested at day 12 and then stained with TOPRO-3 for nuclear staining.
We next examined NF-κB activity in CD8-IGF-IR-MCF10A cells. First, we compared the nuclear localization of the p65 subunit of NF-κB in both vector-MCF10A cells and CD8-IGF-IR-MCF10A cells, because activated NF-κB translocates from the cytoplasm to the nucleus. Immunofluorescence staining of p65 showed exclusive cytoplasmic staining in vector-MCF10A cells; however, p65 was detected in the nuclei of CD8-IGF-IR-MCF10A cells (Fig. 7C). To confirm whether nuclear NF-κB is active, gel mobility shift assays were performed. The results shown in Figure 7D show that NF-κB binding activity was highly induced in CD8-IGF-IR-MCF10A cells compared to the activity in vector-MCF10A cells.
Finally, we examined whether blockade of NF-κB activity could reverse the EMT phenotype of CD8-IGF-IR-MCF10A cells in a manner similar to that observed with blockade with the IGF-IR inhibitor BMS-536924. CD8-IGF-IR-MCF10A cells in monolayer culture were elongated and refractile compared to MCF10A cells, which showed a classic cobblestone epithelial appearance. Treatment of CD8-IGF-IR-MCF10A cells with IKK inhibitor II partially reversed the EMT phenotype, with cells now showing more cell attachment (Fig. 7E). This partial reversion was similar to that seen with BMS-536924 and was not seen with U0126 (Fig. 7E). We also tested the same inhibitors in 3D culture and found that both IKK and BMS-536924 caused marked alterations in the growth of CD8-IGF-IR-MCF10A cells, with smaller acini that resembled those of vector-MCF10A cells (Fig. 7F).
IGF-IR is a critical regulator of cell proliferation and survival. Consistent with this, this receptor also has a role in tumorigenesis and metastasis and is an attractive target for cancer therapy (38). We recently reported the generation and characterization of the first transgenic mouse overexpressing IGF-IR (8). In this model, we found that overexpression of a constitutively active IGF-IR chimera (CD8-IGF-IR) in the mouse mammary gland led to rapid mammary tumorigenesis (8), finally resulting in metastasis to the lungs (unpublished data). In this report, we show that overexpression of the same CD8-IGF-IR chimera in MCF10A immortalized mammary epithelial cells caused transformation when cells were grown in immunocompromised mice. Furthermore, the cells underwent EMT, which was associated with increased NF-κB activity, induction of Snail transcriptional repressor, and downregulation of E-cadherin. The EMT was correlated with dramatically enhanced migration and invasion, and the induction of the Snail and EMT phenotypes could be reversed by small-molecule inhibitors of IGF-IR and NF-κB.
MCF10A cells are a useful tool for examining the effects of oncogenes on cell polarity and transformation. Several "classic" oncogenes, such as HER-2, activated Ras, and cyclin D1, have been shown to disrupt cell polarity and cause transformation in vitro; however, all are unable to confer xenograft growth in vivo (11). In contrast to those results, other, less-studied oncogenes, including these for growth hormone (51), ephA2 (49), and αB-crystallin (31), have recently been shown to confer xenograft growth on MCF10A cells. Similarly, we have shown that CD8-IGF-IR alone can cause transformation of MCF10A cells in vitro and that it also confers growth in athymic nude mice when the cells are injected, with or without Matrigel.
During this study, two groups reported on the overexpression of wild-type IGF-IR in MCF10A cells (24, 47). Both groups found that overexpressed IGF-IR remained ligand dependent but, when hyperstimulated by IGF-I, caused increased proliferation, decreased apoptosis, and loss of polarity. This resulted in large, complex, disrupted acini; however, both studies found that cells were noninvasive. We found a similar induction of proliferation, absence of apoptosis, and loss of polarity that resulted in disrupted acinus formation in our 3D cultures of CD8-IGF-IR-MCF10A cells. However, CD8-IGF-IR-MCF10A cells also showed invasive properties in Matrigel culture and, after 15 days of culture, were found to grow together. This invasion in Matrigel was supported by the ability of the cells to invade when the modified Boyden chamber assay was used. This suggests that CD8-IGF-IR may be activating selected pathways to a greater and more sustained level than the hyperstimulated overexpressed wild-type receptor or that CD8-IGF-IR may be activating alternate pathways.
Irie et al. noted a subtle conversion from a cuboidal epithelial morphology to a more spindle-shaped morphology (24). Most intriguingly, downregulation of Akt1, but not Akt2, in IGF-IR-overexpressing clones caused cells to undergo a profound EMT, similar to the results with our CD8-IGF-IR clones. The EMT caused by Akt1 downregulation in the presence of overexpressed IGF-IR was attributed to a subsequent increase in ERK1/2 activity. In contrast to the results of the studies of Irie et al. (24), we found that CD8-IGF-IR caused EMT even though there was little or no activation of ERK1/2 (data not shown). Yanochko and Eckhart (47) did not comment on the morphology of their MCF10A-IGF-IR cells, but did indicate that in 3D culture, the cells showed areas of punctate E-cadherin staining, suggesting changes in cell adhesion. Interestingly, we noted that CD8-IGF-IR-MCF10A cells showed a dramatic change in E-cadherin staining, with a distinct punctate pattern perhaps somewhat similar, but exaggerated, compared to that of Yanochko and Eckhart (47). It is possible that the constitutive IGF-IR we have used is able to cause EMT due to higher and more-prolonged constitutive activation of downstream signaling pathways, or perhaps via the induction of novel signaling pathways not normally activated by the wild-type overexpressed receptor.
IGF-I stimulation of breast cancer cells overexpressing IGF-IR has been shown to cause depolarization and a mesenchyme-like transition (20). Other growth factor receptors, such as fibroblast growth factor receptor (39), epidermal growth factor receptor (17), and transforming growth factor β-receptor (48), are known to induce EMT. Furthermore, there is considerable cross-talk between these receptors and downstream signaling pathways and transcription factors that lead to EMT (19). Several transcription factors have been implicated in the repression of E-cadherin and EMT, including Snail/Slug, Twist, and Zeb1 (23). We found that CD8-IGF-IR-MCF10A cells showed a specific and dramatic elevation of Snail mRNA levels. Recent studies have implicated Snail as a critical regulator of both the EMT process in MCF10A cells (1) and breast cancer recurrence (30).
Snail expression has been shown previously to be regulated by numerous growth factors (13); however, ours is the first study to show that IGF-I and IGF-IR can induce Snail mRNA. The mechanism of Snail induction by receptor tyrosine kinases has generally focused on the role of Ras signaling (13). We found that CD8-IGF-IR caused an increase in Snail mRNA levels; however, this increase was not due to ERK1/2 activity, as levels of ERK1/2 were low in CD8-IGF-IR-MCF10A cells, and blockade of ERK1/2 with U0126 did not lower Snail levels. Phosphatidylinositol 3-kinase and Akt are required for transforming growth factor β-induced EMT (2), and constitutive Akt can induce EMT (18). GSK-3β has also been shown to repress EMT, via repression of Snail mRNA (1) and by phosphorylating Snail on two motifs that cause its proteasome-dependent degradation and also modulate its nuclear localization (50). We have found constitutive P∼Akt and loss of P∼GSK-3β in CD8-IGF-IR-MCF10A cells (data not shown), indicating that this may be a mechanism for the regulation of Snail. However, recent evidence has implicated the IGF-IR signaling cascade in the activation of the NF-κB signaling pathway (29), and inhibition of GSK-3β has been shown to activate NF-κB and increase Snail mRNA levels (1). Indeed, we found that NF-κB was highly active in CD8-IGF-IR-MCF10A cells and that blockade of NF-κB reduced Snail levels in these cells. This is consistent with previous data indicating that NF-κB binds Snail promoter and increases its activity (3) and strongly suggests a signaling scenario whereby IGF-IR leads to activation of Akt, loss of GSK-3β, increased NF-κB activity, increased levels of Snail, loss of E-cadherin, and subsequent EMT (Fig. 8). This model is supported by the numerous other studies described above.
Schematic indicating how CD8-IGF-IR leads to EMT. This model is based upon studies presented in this report indicating that CD8-IGF-IR induces NF-κB, Snail, repression of E-cadherin mRNA and protein levels, and subsequently, EMT. The hierarchy is shown by the reversion of Snail mRNA levels and EMT by IGF-IR or NF-κB inhibitors. The model is supported by studies showing that IGF-IR activates Akt to repress GSK-3β, GSK-3β is a repressor of NF-κB activity and Snail mRNA and protein levels, IGF-IR activates NF-κB, and NF-κB binds the Snail promoter and increases Snail mRNA levels. PI3K, phosphatidylinositol 3-kinase.
In summary, we show that the expression of a constitutively active IGF-IR in human mammary epithelial cells results in tumorigenic conversion that is accompanied by a transition to a mesenchyme-like phenotype. This study is novel in that relatively few single oncogenes have been shown to confer xenograft growth on MCF10A cells and thus provides a new model for examining MCF10A cell growth in vivo. This is also the first study to show that IGF-I and its receptor can modulate Snail levels and cause EMT in mammary epithelial cells.
We thank Ora Britton for help with the xenograft studies, Craig Allred and the Pathology core of the Breast Center at Baylor College of Medicine for IHC, and Steffi Oesterreich for critical reading of the manuscript. Flag-Snail-pCMV-Tag 2B was a gift from Mien-Chie Hung at the M. D. Anderson Cancer Center, Houston, TX.
This work was supported in part by Public Health Service grants R01CA94118 (A.V.L.) and P01CA30195 (C.K.O./A.V.L.). A.V.L. is the recipient of a T. T. Chao Scholar award (Department of Medicine, Baylor College of Medicine). X.C. is the recipient of a Career Development Award (P50CA58183).
Received 18 July 2006.
↵▿ Published ahead of print on 12 February 2007.
Bachelder, R. E., S. O. Yoon, C. Franci, A. G. de Herreros, and A. M. Mercurio. 2005. Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition. J. Cell Biol. 168:29-33.
Bakin, A. V., A. K. Tomlinson, N. A. Bhowmick, H. L. Moses, and C. L. Arteaga. 2000. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration. J. Biol. Chem. 275:36803-36810.
Barbera, M. J., I. Puig, D. Dominguez, S. Julien-Grille, S. Guaita-Esteruelas, S. Peiro, J. Baulida, C. Franci, S. Dedhar, L. Larue, and A. Garcia de Herreros. 2004. Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells. Oncogene 23:7345-7354.
Barrallo-Gimeno, A., and M. A. Nieto. 2005. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132:3151-3161.
Baserga, R. 1999. The IGF-I receptor in cancer research. Exp. Cell Res. 253:1-6.
Bonnette, S. G., and D. L. Hadsell. 2001. Targeted disruption of the IGF-I receptor gene decreases cellular proliferation in mammary terminal end buds. Endocrinology 142:4937-4945.
Boyer, B., A. M. Valles, and N. Edme. 2000. Induction and regulation of epithelial-mesenchymal transitions. Biochem. Pharmacol. 60:1091-1099.
Carboni, J. M., A. V. Lee, D. L. Hadsell, B. R. Rowley, F. Y. Lee, D. K. Bol, A. E. Camuso, M. Gottardis, A. F. Greer, C. P. Ho, W. Hurlburt, A. Li, M. Saulnier, U. Velaparthi, C. Wang, M. L. Wen, R. A. Westhouse, M. Wittman, K. Zimmermann, B. A. Rupnow, and T. W. Wong. 2005. Tumor development by transgenic expression of a constitutively active insulin-like growth factor I receptor. Cancer Res. 65:3781-3787.
Cogswell, P. C., D. C. Guttridge, W. K. Funkhouser, and A. S. Baldwin, Jr. 2000. Selective activation of NF-kappa B subunits in human breast cancer: potential roles for NF-kappa B2/p52 and for Bcl-3. Oncogene 19:1123-1131.
Dearth, R. K., X. Cui, H. J. Kim, I. Kuiatse, N. A. Lawrence, X. Zhang, J. Divisova, O. L. Britton, S. Mohsin, D. C. Allred, D. L. Hadsell, and A. V. Lee. 2006. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Mol. Cell. Biol. 26:9302-9314.
Debnath, J., and J. S. Brugge. 2005. Modelling glandular epithelial cancers in three-dimensional cultures. Nat. Rev. Cancer 5:675-688.
Debnath, J., S. K. Muthuswamy, and J. S. Brugge. 2003. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30:256-268.
De Craene, B., F. van Roy, and G. Berx. 2005. Unraveling signalling cascades for the Snail family of transcription factors. Cell Signal. 17:535-547.
Dimri, G., H. Band, and V. Band. 2005. Mammary epithelial cell transformation: insights from cell culture and mouse models. Breast Cancer Res. 7:171-179.
Garber, K. 2005. IGF-1: old growth factor shines as new drug target. J. Natl. Cancer Inst. 97:790-792.
Giunciuglio, D., M. Culty, G. Fassina, L. Masiello, A. Melchiori, G. Paglialunga, G. Arand, F. Ciardiello, F. Basolo, E. W. Thompson, et al. 1995. Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes. Int. J. Cancer 63:815-822.
Grande, M., A. Franzen, J. O. Karlsson, L. E. Ericson, N. E. Heldin, and M. Nilsson. 2002. Transforming growth factor-beta and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes. J. Cell Sci. 115:4227-4236.
Grille, S. J., A. Bellacosa, J. Upson, A. J. Klein-Szanto, F. van Roy, W. Lee-Kwon, M. Donowitz, P. N. Tsichlis, and L. Larue. 2003. The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res. 63:2172-2178.
Grunert, S., M. Jechlinger, and H. Beug. 2003. Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat. Rev. Mol. Cell. Biol. 4:657-665.
Guvakova, M. A., and E. Surmacz. 1999. The activated insulin-like growth factor I receptor induces depolarization in breast epithelial cells characterized by actin filament disassembly and tyrosine dephosphorylation of FAK, Cas, and paxillin. Exp. Cell Res. 251:244-255.
Hay, E. D. 1995. An overview of epithelio-mesenchymal transformation. Acta Anat. 154:8-20.
Huber, M. A., N. Azoitei, B. Baumann, S. Grunert, A. Sommer, H. Pehamberger, N. Kraut, H. Beug, and T. Wirth. 2004. NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J. Clin. Investig. 114:569-581.
Huber, M. A., N. Kraut, and H. Beug. 2005. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr. Opin. Cell Biol. 17:548-558.
Irie, H. Y., R. V. Pearline, D. Grueneberg, M. Hsia, P. Ravichandran, N. Kothari, S. Natesan, and J. S. Brugge. 2005. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J. Cell Biol. 171:1023-1034.
Jones, R. A., C. I. Campbell, E. J. Gunther, L. A. Chodosh, J. J. Petrik, R. Khokha, and R. A. Moorehead. 4 September 2006. Transgenic overexpression of IGF-IR disrupts mammary ductal morphogenesis and induces tumor formation. Oncogene doi:10.1038/sj.onc.1209955. [Epub ahead of print.]
Kaleko, M., W. Rutter, and A. Miller. 1990. Overexpression of the human insulin-like growth factor I receptor promotes ligand-dependent neoplastic transformation. Mol. Cell. Biol. 10:464-473.
Kim, D. W., M. A. Sovak, G. Zanieski, G. Nonet, R. Romieu-Mourez, A. W. Lau, L. J. Hafer, P. Yaswen, M. Stampfer, A. E. Rogers, J. Russo, and G. E. Sonenshein. 2000. Activation of NF-kappaB/Rel occurs early during neoplastic transformation of mammary cells. Carcinogenesis 21:871-879.
Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402-408.
Mitsiades, C. S., N. Mitsiades, V. Poulaki, R. Schlossman, M. Akiyama, D. Chauhan, T. Hideshima, S. P. Treon, N. C. Munshi, P. G. Richardson, and K. C. Anderson. 2002. Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications. Oncogene 21:5673-5683.
Moody, S. E., D. Perez, T. C. Pan, C. J. Sarkisian, C. P. Portocarrero, C. J. Sterner, K. L. Notorfrancesco, R. D. Cardiff, and L. A. Chodosh. 2005. The transcriptional repressor Snail promotes mammary tumor recurrence. Cancer Cell 8:197-209.
Moyano, J. V., J. R. Evans, F. Chen, M. Lu, M. E. Werner, F. Yehiely, L. K. Diaz, D. Turbin, G. Karaca, E. Wiley, T. O. Nielsen, C. M. Perou, and V. L. Cryns. 2006. αB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer. J. Clin. Investig. 116:261-270.
Nagle, J. A., Z. Ma, M. A. Byrne, M. F. White, and L. M. Shaw. 2004. Involvement of insulin receptor substrate 2 in mammary tumor metastasis. Mol. Cell. Biol. 24:9726-9735.
Orlowski, R. Z., and A. S. Baldwin, Jr. 2002. NF-kappaB as a therapeutic target in cancer. Trends Mol. Med. 8:385-389.
Pena, C., J. M. Garcia, V. Garcia, J. Silva, G. Dominguez, R. Rodriguez, C. Maximiano, A. Garcia de Herreros, A. Munoz, and F. Bonilla. 2006. The expression levels of the transcriptional regulators p300 and CtBP modulate the correlations between SNAIL, ZEB1, E-cadherin and vitamin D receptor in human colon carcinomas. Int. J. Cancer 119:2098-2104.
Resnicoff, M., and R. Baserga. 1998. The role of the insulin-like growth factor I receptor in transformation and apoptosis. Ann. N. Y. Acad. Sci. 842:76-81.
Rodgers, K., B. McMahon, D. Mitchell, D. Sadlier, and C. Godson. 2005. Lipoxin A4 modifies platelet-derived growth factor-induced pro-fibrotic gene expression in human renal mesangial cells. Am. J. Pathol. 167:683-694.
Rosivatz, E., I. Becker, K. Specht, E. Fricke, B. Luber, R. Busch, H. Hofler, and K. F. Becker. 2002. Differential expression of the epithelial-mesenchymal transition regulators Snail, SIP1, and twist in gastric cancer. Am. J. Pathol. 161:1881-1891.
Sachdev, D., and D. Yee. 2006. Inhibitors of insulin-like growth factor signaling: a therapeutic approach for breast cancer. J. Mammary Gland Biol. Neoplasia 11:27-39.
Strutz, F., M. Zeisberg, F. N. Ziyadeh, C. Q. Yang, R. Kalluri, G. A. Muller, and E. G. Neilson. 2002. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int. 61:1714-1728.
Sugimachi, K., S. Tanaka, T. Kameyama, K. Taguchi, S. Aishima, M. Shimada, K. Sugimachi, and M. Tsuneyoshi. 2003. Transcriptional repressor Snail and progression of human hepatocellular carcinoma. Clin. Cancer Res. 9:2657-2664.
Surmacz, E. 2000. Function of the IGF-I receptor in breast cancer. J. Mammary Gland Biol. Neoplasia 5:95-105.
Thiery, J. P. 2003. Epithelial-mesenchymal transitions in development and pathologies. Curr. Opin. Cell Biol. 15:740-746.
Wang, D., Y. You, S. M. Case, L. M. McAllister-Lucas, L. Wang, P. S. DiStefano, G. Nunez, J. Bertin, and X. Lin. 2002. A requirement for CARMA1 in TCR-induced NF-kappa B activation. Nat. Immunol. 3:830-835.
Wang, L., J. Wang, B. E. Wang, P. G. Xiao, Y. J. Qiao, and X. H. Tan. 2004. Effects of herbal compound 861 on human hepatic stellate cell proliferation and activation. World J. Gastroenterol. 10:2831-2835.
Wittman, M., J. Carboni, R. Attar, B. Balasubramanian, P. Balimane, P. Brassil, F. Beaulieu, C. Chang, W. Clarke, J. Dell, J. Eummer, D. Frennesson, M. Gottardis, A. Greer, S. Hansel, W. Hurlburt, B. Jacobson, S. Krishnananthan, F. Y. Lee, A. Li, T. A. Lin, P. Liu, C. Ouellet, X. Sang, M. G. Saulnier, K. Stoffan, Y. Sun, U. Velaparthi, H. Wong, Z. Yang, K. Zimmermann, M. Zoeckler, and D. Vyas. 2005. Discovery of a (1H-benzoimidazol-2-yl)-1H-pyridin-2-one (BMS-536924) inhibitor of insulin-like growth factor I receptor kinase with in vivo antitumor activity. J. Med. Chem. 48:5639-5643.
Yang, J., S. A. Mani, J. L. Donaher, S. Ramaswamy, R. A. Itzykson, C. Come, P. Savagner, I. Gitelman, A. Richardson, and R. A. Weinberg. 2004. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117:927-939.
Yanochko, G. M., and W. Eckhart. 2006. Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells. Breast Cancer Res. 8:R18.
Zavadil, J., and E. P. Bottinger. 2005. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24:5764-5774.
Zelinski, D. P., N. D. Zantek, J. C. Stewart, A. R. Irizarry, and M. S. Kinch. 2001. EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res. 61:2301-2306.
Zhou, B. P., J. Deng, W. Xia, J. Xu, Y. M. Li, M. Gunduz, and M. C. Hung. 2004. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat. Cell Biol. 6:931-940.
Zhu, T., B. Starling-Emerald, X. Zhang, K. O. Lee, P. D. Gluckman, H. C. Mertani, and P. E. Lobie. 2005. Oncogenic transformation of human mammary epithelial cells by autocrine human growth hormone. Cancer Res. 65:317-324.
Molecular and Cellular Biology Mar 2007, 27 (8) 3165-3175; DOI: 10.1128/MCB.01315-06
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Submission of BBL postponed again
The first schedule to submit the proposed Bangsamoro Basic Law (BBL) was on June 1, but during that time, the Bangsamoro Transition Commission (BTC) was still hotly debating the proposed Bangsamoro Basic Law (BBL). June 14 and 15 were proposed as alternative dates. They were suggested by BTC Chair Ghazali Jaafar during the meeting with President Rodrigo Roa Duterte on May 29. He estimated that by that time the final draft of the BBL would be finalized and ready for signatures by all the commissioners.
However, there was no concurrence on either of the two dates by the Office of the President (OP), which has the final say on the scheduling. Then later the dates June 16, 22 and 24 were also mentioned, and then finally 28. The last one officially came from the OP.
For this historic event, members of the BTC and its Secretariat made simple but sentimental preparation for the submission. Like the previous submission on September 14, 2014 by the first BTC chaired by Mohagher Iqbal, now a member of the current commission, a modest ceremony is expected in Malacañang Palace. Senate President Franklin Drilon and House Speaker Feliciano Belmonte, and other members of Congress, as well as members of the international community attended.
Again, the submission was postponed to July 10 – and this is still tentative. The reason given for this is the uncertainty of the presence of House Speaker Pantaleon Alvarez and Senate President Koko Pimentel, the two most important personalities as far as legislation, and for this matter, the passage of BBL in Congress, are concerned.
So be it! The government has the final say on this. The BTC has to wait; after all, it is mainly part of the official family.
Nevertheless, for ordinary Moro, such a litany of postponements do not bode well. The ghost of the previous BBL still lingers well in their minds. If there was Mamasapano then, now we have Marawi City. What an unhappy consequence!
Of course, we do not question intention; in fact, we say here that the postponement is for good, or for reasons only the inner circle of government knows. With our trust to the president, the MILF entertains no doubt about the pristine intention of the president to pass and solve the Bangsamoro Problem or Question during his time. We see no reason at all why he has other intention than to be truthful to our people. He is not only so consistent, but he is also very frank and forthright.
But we still believe things can still be improved, especially in setting up of schedules. The best system is when it can still fly under turbulent times. We hope July 10 will be the final rendezvous for submission of the BBL.

redpajama_set_name: RedPajamaCommonCrawl


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By Danial Martinus September 10, 2021
We all learn how to swim when we're little. Well, most of us at least.
And when we're first starting out, it can be more than little intimidating, especially when you don't have floaties to keep you above the surface of the water.
But eventually we'll find ourselves doing the breast stroke or butterfly, or simply staying submerged for the fun of it.
These Singapore otter pups can probably relate to all the nerves we feel when swimming is a completely alien task.
A group of otter pups were spotted at the Singapore Botanic Gardens being given a somewhat chaotic swimming lesson from their elder otter chaperones, much to the adoration of onlookers.
According to Marjorie Chong, the person who filmed this swimming lesson, the otter pups were visibly nervous, as 'kids' are in such a new situation. The video, shared on the Ottercity Facebook page on September 8, has already garnered over 41 million views and over 707,000 reactions:
Despite their aquatic nature, otters aren't born with the natural ability to swim. They have to be taught at a young age, usually at around two months old.
As seen in the video, the elder otters are egging the little pups into the water, constantly jumping in and climbing back out to show them it's safe. Some of the pups have no trouble following their lead.
IMAGE: Marjorie Chong / Ottercity
Others wind up having a little bit of a tough time, understandably. This little guy's face says it all:
However, it's important to note that adult otters aren't as patient with their young as humans are. They're not just going to sit there and wait all day for the pups to muster the courage to jump in.
Let's just say, some need to be literally dragged into the water. But it's okay, they can handle it:
"Being social animals, every otter in the family helps out in the chaotic swim lesson," Chong told Storyful. All in all, a very relatable family outing."
There are an estimated 90 otters in Singapore, made up of at least 19 different families scattered throughout the country.
Check out Ottercity on Facebook for more otter sightings in Singapore.
Read more wildlife stories:
Komodo dragons are now an endangered species thanks to useless humans
16 hilarious wildlife photos that show nature can be goofy too
Pink dolphin appears off Vietnam beach, locals call it a 'good omen'
Follow Mashable SEA on Facebook, Twitter, Instagram, YouTube, and Telegram.
Cover image sourced from Marjorie Chong / Ottercity.
Topics: Science, singapore, Otters, Wildlife, Nature, Pup
Animals are growing larger ears and mouths to adapt to global warming
But this isn't necessarily a good thing.
Sept. 8, 2021, 12:07 p.m.


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Miracle claims Chukchansi title
Tyler Takeda/The Madera Tribune
Madera South's Dylan Miracle works his finals opponent for more points Saturday to win the Chukchansi Invitational 170-pound championship.
After three years of wrestling at the Chukchansi Invitational wrestling tournament, Madera South wrestler Dylan Miracle wanted to capture a title at his home tournament.
Miracle defeated Bullard-Fresno's David Vardanyan 5-2 to help the Stallions place fifth overall at the tournament that drew more than 50 teams to Madera South.
"It went really well," Stallions wrestling coach Sal Gonzalez said. "We didn't get any negative feedback. We had a slight hiccup with starting the finals an hour late. From here on out, it will be a 5 p.m. start time."
Miracle was joined in the Chukchansi Invitational finals by Augustin Garcia, at 162 pounds, and Carlos Posas, at 106 pounds.
Also placing for the Stallions were Florencio Hernandez, sixth at 134 pounds and Nathan Chavez, fourth at 197 pounds.
"We did pretty good," Gonzalez said. "We started the day in 17th place and ended in fifth place. We've been faced with some adversity the last couple of weeks. Our 115 pound starter broke his finger. Without three that would have placed, we did pretty well to place fifth."
The Madera Coyotes were just 10.5 points behind Madera South in sixth place.
Abraham Del Toro led the Coyotes with a finals run at 140 pounds.
Also placing for the Coyotes were Mario Moreno, fifth at 122 pounds; Moses Contreras, third at 147 pounds; Jorge Gonzalez, sixth at 154 pounds and Nathan Alvarado, seventh at 154 pounds.
Meanwhile, the Liberty Hawks were led by heavyweight Javier Lopez, who was the only Hawks wrestler to place (eighth).
On the road to the championship rounds, Posas recorded a 9-8 victory over Manuel Bermudez of Mission Oak-Tulare. Posas led 8-5 after the second period. He allowed an early reversal, but got an escape to get a three-point lead and held on for the victory.
In the finals, Posas faced Pitman-Turlock's Isriel Tubera. In club season, Posas was pinned by Tubera and saught a measure of revenge. Posas took the lead with a takedown 38 seconds into the match. However, he fell for a reversal and a near fall at the end of the period and trailed 4-2.
After a scoreless second, Tubera recorded an escape and a takedown for a 7-2 victory.
"For Carlos, it was a matter of going to the finals," Gonzalez said. "He faced the No. 10 wrestler in the state in the finals. He was satisfied with his performance."
At 162 pounds, Garcia came as one of the favorites. He recorded two pins on his way to the semifinals. After taking a 2-0 lead in the first period against Mission Oak's Gabriel Salcedo, Garcia recorded an escape and a reversal in the second period. He capped his semifinal win with a reversal and a 3-point near fall for a 10-0 major victory.
Garcia faced top seed Jonathan Garcia from Frontier-Bakersfield in the championship match. He fell quickly to a takedown and a near fall and couldn't get out of the first period when Garcia pinned him with 37 seconds left in the period.
"Auggie ran into a horse," Gonzalez said. "His opponent defeated Dylan in the finals last year."
Miracle made the most of his last Chukchansi Invitational. He pinned his first two opponents to reach the semifinals. After a scoreless first period against Mariposa County's Antonio Hernandez, Miracle got an escape 15 seconds into the period and recorded a takedown. Hernandez got an escape early in the third period, but Miracle was able to stay away from a takedown for a 3-1 win.
Miracle faced County/Metro Athletic Conference rival Vardanyan and picked up an early takedown when he pulled Vardanyan back into the circle to complete a pin with 31 seconds left in the first period.
Miracle then got an escape early in the second period, but Vardanyan got an escape in the third period for a 3-1 Miracle lead. Miracle then recorded a takedown for a 5-1 lead before Vardanyan recorded an escape with 26 second left in the match for a 5-1 Miracle win.
"Dylan Miracle did phenomenal this year," Gonzalez said.
Del Toro was the top seed at 140 pounds and recorded three pins heading into his semifinal match against Buchanan-Clovis' Trenton Stuart. After the first period, the two wrestlers were tied at three.
After Stuart scored two points, Del Toro tied it again with a takedown with 25 seconds left in the period. Del Toro recorded a reversal 38 seconds into the third period, but Stuart got an escape with 34 seconds left. However, Del Toro clinched the match with a takedown with 12 seconds left in the match for a 9-6 win.
In the finals, Del Toro was hit with an early illegal hold penalty for a point and then got into a near fall for three more points and couldn't score any against Buchanan's Wyatt Peverill for a 4-0 loss.
Liberty's Lopez recorded two pins on the first day of the competition to head to the quarterfinals on Saturday.
He lost by pin to eventual champion, Armando Barcenos of Hanford.
In the seventh place match, Lopez lost in sudden death overtime, 3-2, to Pitman's Julian Barrera.
Before the finals, Gonzalez honored former Madera and Fresno City College wrestling coach Robert Arballo and named an award after him for the toughest wrestler in the finals. For the first award, Arballo gave it to 134-pound champion Isaiah Perez of Dinuba.


Bombay Grill Food Restaurant Menu Page.
To keep vegetarian guests safe, our kitchen is designed separately (for vegetarian cooking) to avoid contamination with meat products.
Garden fresh cauliflower tossed with fantastic and aromatic spices.
Jumbo shrimp cooked in clay oven.
Garden fresh peas and chunks of tofu cooked in aromatic spices.
Delicious corn and spinach or tofu cooked in mild spices.
Smoked baby eggplant cooked in aromatic spices.
Garden fresh mushroom and cottage cheese or tofu cooked in fresh spices.
Boneless lamb cooked in medium spicy mixture of blended aromatic gravy.


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Scottish Design Awards 2021 > ARCHITECTURE: Future Building or Project > John O'Groats Mill
John O'Groats Mill
Direct link: https://www.youtube.com/watch?v=FEa3z2q1hDk&t=650s
Direct link: https://www.youtube.com/watch?v=NlxOb23S0tY
ARCHITECTURE: Future Building or Project
McGregor Bowes
John O'Groats Mill Trust
John O'Groats Mill is the last of the great Caithness corn mills and was in operation until around 2001 when Magnus Houston last worked the mill. In 2016 a new group, the John O'Groats Mill Trust, was created with the vision of restoring the mill buildings, machinery and water systems to full working order, enabling visitors to see a working Caithness mill, and for the wider John O'Groats community to make use of the building for a range of community driven activities. The Trust raised the funds to purchase the mill buildings and adjacent lands and this was carried out with a formal handover in August 2020.
The proposal is to restore the mill building and add an extension on the rear side. The reception is at ground level in the middle section of the building and a lift is enclosed within the reception area. The west wing will contain the mill and interpretation. The new build extension is a single storey building located to the north of the existing mill, the extension comprises a mixed use/education room, service area and a viewing terrace. It has a "green roof", which merges with the adjacent landscape.
The design principle for the extension is that the building is tucked into the landscape and the greenery extends over the building like a rug of grass. The extension opens up on the west side with a wall of glazing. The roof of the extension overhangs this glazing to limit solar gain during the summer, but enable it in the winter and shoulder periods. The floor of the extension is made of Caithness slabs to match the floor of the original mill and in the extension it increases the opportunity for creating thermal mass.
On the mill first floor, mill interpretation and mixed use will continue in the west wing. An escape stair route to the north is shown from the main building exiting over the new extension.
The second floor continues the theme of mill use, interpretation and mixed uses. A new stainless steel mesh floor will be installed above the kiln area to replace the life-expired cast iron plates. The space can be used for temporary exhibitions when it is not in use for drying grain. There is also an escape route from this area that leads via a new bridge to a banked up stepped area of the adjacent field.
https://www.facebook.com/johnogroatsmill/


Mac Derscheid 1936 Corona Standard Serial # 1C70018 1936 Corona Standard typewriter, Serial # 1C70018 Mac Derscheid's 1936 Corona Standard typewriter. 2016-12-10 2018-02-13 From the Virtual Typewriter Collection of Mac Derscheid: 1936 Corona Standard Serial # 1C70018 Very nice machine. This is my favourite machine in my collection. It was not in wonderful cosmetic shape when I got it, however I purchased a second one that did not work and put the best shell pieces onto this machine. It now is in very nice condition, and types wonderfully. The only issue with the machine is that the K key appears to have almost melted, and is the only bad part of the machine.
Very nice machine. This is my favourite machine in my collection. It was not in wonderful cosmetic shape when I got it, however I purchased a second one that did not work and put the best shell pieces onto this machine. It now is in very nice condition, and types wonderfully. The only issue with the machine is that the K key appears to have almost melted, and is the only bad part of the machine.
A fun old time effect I like to apply to my machines. Works best on the oldest ones, but I think it looks great on most.


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Dr Jo Harrold
Dr Jo Harrold is a Senior Lecturer in Appetite and Obesity in the Department of Psychological Sciences, University of Liverpool.
She is a behavioural neuroendocrinologist with a...Read More
Professor Jason Halford
Chair in Biological Psychology and Health Behaviour, Head of Department Psychological Sciences
Prof Jason Halford is Head of the Department of Psychological Sciences at the University of Liverpool, President Elect of the European Association of Obesity (EASO), and former Cha...Read More
Professor Anne Raben
Department of Nutrition, Exercise and Sports University of Copenhagen
Science: More than 25 years of research in the prevention and treatment of obesity and the related diseases, type-2 diabetes, and cardiovascular diseases. Main focus has been on nu...Read More


Pictures stick with us, sometimes for decades. For me it was the first time I went to Ethiopia, I knew the famine was long over, but those images during the 80's were in my head.
In my head I knew that wouldnt be the reality anymore. But the imagery was very strong. It was the single picture I carried.
How many pictures of Africa do you think you have seen? How many pictures of aid work? We have all seen 10's of thousands of images of international aid workers. What story do they tell?
What is the story that is being told through this collection of pictures?
The first time I walked into an African village my perspective of the quaint village shifted.
When I think of living in the country, living rurally, the picture that comes to mind is of "Anne of Green Gables" and "Little House on the Prarie". Those were the shows I watched as a kid. Sod-busters. Barn-raising. Ice cream socials and square dancing. So when I think of village life I think rustic, hard-working strapping men and women who may be poor, but by using their few resources they pull themselves up by hard work and gumption. If there are any problems, they were solved in about 22 minutes – or 44 if it was a two part episode.
Villages are also filled with hard working men and women. They work together and share complex full lives. But the village there is very different than the village in my mind, the one I learned about from TV. In a developing country, living in a village usually means you are poor. Nothing wrong with being poor of course, but most people don't want to stay that way.
What picture comes to your mind when you think village?
6 responses on "89 reasons why you think about poverty the way you do"
Hey Jonathan, I think many, if not most, aid agencies actually do share positive pictures when they highlight a disaster. My question and the resulting video, was more about the total number of pictures we see.
Even if we are telling a positive story about people in a disaster, we still need to remember that there are 88 other pictures (or lifetime of images) that people think about when they think about our work!
1. There is an urgent need.
2. It is not hopeless and we are doing something about it.
3. We really did do something good with your money.


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Q: getUserMedia is not working in chrome version 48.0.2560.0 while working in 46.0 my confusion is that i had used the gerUSerMedia() in my page which is not working  from our our server while in some of the machine had chrome with version of 48.0.2560.0 in those chrome browser it will give following warning :
getUserMedia() no longer works on insecure origins. To use this feature, you should consider switching your application to a secure origin, such as HTTPS. See https://goo.gl/rStTGz for more details.
while in with chrome 46.0 its working fine with the same code and with same request which is server from our server.
how do we proceed ? so the same thing will working fine in all browser and in all versions.

A: The link provided to the Security Proposal suggests that you can still use this feature on localhost, or you can pass these flags to Chrome, replacing example.com with the site you need to access:
--unsafely-treat-insecure-origin-as-secure="http://example.com" --user-data-dir=/test/only/profile/dir
If you have some local filesystem content you are working with, then you'll still need to run a webserver on localhost rather than access directly on file://. A simple way to run a webserver with python is to run:
python -m SimpleHTTPServer 8080
inside the directory, then your content will be available on http://localhost:8080 and getUserMedia() will work.

redpajama_set_name: RedPajamaStackExchange


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a broad and her brood living the life abroad: Seriously? SERIOUSLY?
Seriously. I cannot believe a health professional just asked me if I had pee pee'd or poo poo'd yet today. Do I look like I am 3 years old?
Only in the Philippines folks, ONLY-IN-THE-PHILIPPINES.
I think if I hadn't been so desperate to be out of pain I would have walked out thinking there was no way he had a medical degree.
I have a head cold, a lovely little thing that won't go away nor can I drown it in nyquil. I thought I would play through the misery today but as I bent over to grab a tennis skirt off the shelf I had a shooting pain something fierce run up my back and down my legs. All I did was bend over, my heck, 30 is creeping up fast! Nate sat in shock on the bed as I hobbled in half bent over screaming in pain. I called the chiropractor who conveniently is in Beijing treating Olympic athletes (GIVE ME A BREAK, I definitely need him more than they do!) Long story short, I went to his colleague and THAT is what he SERIOUSLY asked me. I spent 2 hours 2800 pesos and I still can't move or breathe. I am a jolly camper right now. Jolly, jolly, jolly.
Pee Pee or Poo Poo? Wow. DOES he have a medical degree?
And you are the woman for playing so much tennis in your "state." How many weeks/months are you?
Just over 5 months, but it's looking as if the tennis days are dwindling. Sad.


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Marjorie Kinnan Rawlings Historic State Park
Pathway to the front door of the Marjorie Kinnan Rawlings home
Marjorie Kinnan Rawlings is most famous for winning the Pulitzer Prize after writing The Yearling in the 1930's. She lived and wrote at this Cracker-style home and farm for twenty-five years. The property has been restored to what it was like when she lived here.
In addition to the home and the restored outbuildings, the property offers ornamental gardens and two short nature trails. Adjacent to the state park is the county park where you'll find a playground and picnic facilities.
Marjorie Kinnan Rawlings Historic State Park is open daily from October through July, from 9 am to 5 pm, except on Thanksgiving Day and Christmas Day. Access inside the home is by guided tour only, Thursday through Sunday, at 10 am, 11 am, 1 pm, 2 pm, 3 pm and 4 pm. Marjorie Kinnan Rawlings Historic State Park is located off County Road 325 in Cross Creek (between Gainesville and Ocala on the eastern side of Orange Lake).
The old barn where Marjorie used to keep her livestock
Map of Marjorie Kinnan Rawlings Historic State Park
Marjorie Kinnan Rawlings Historic State Park area map
18700 S County Road 325, Cross Creek, FL 32640
Florida State Parks: Marjorie Kinnan Rawlings Historic State Park
Marjorie Kinnan Rawlings Historic State Park Related Pages
Florida State Parks - Paynes Prairie Preserve State Park - Old Florida Heritage Highway
Gainesville-Hawthorne State Trail
Dudley Farm Historic State Park - Devils Millhopper Geological State Park
San Felasco Hammock Preserve State Park - River Rise Preserve State Park
O'Leno State Park - Mike Roess Gold Head Branch State Park
Lochloosa Wildlife Management Area - Grove Park Wildlife Management Area
Photos and map courtesy of Florida State Parks. Area map courtesy of National Geographic Topo!


We all hear people telling us not to give up of our dream or whatever you're doing. But how many people would sit down next to a struggling person and tell him/her that they understand their urges to throw up the towels and call for the day!
– Do you know how many times when I walk to my work table, all I want is to go to the beach and join all the holiday makers around me to have fun? (Yes, here in Australia it is summer holiday!) Most of the time!