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Title: A genome-wide Drosophila epithelial tumorigenesis screen identifies Tetraspanin 29Fb as an evolutionarily conserved suppressor of Ras-driven cancer}
Article:
Abstract:
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.
Introduction:
Mutations in Ha-RAS, N-RAS, and K-RAS genes are frequent in human tumors and represent oncogenic drivers of cell proliferation and survival. However, tissue growth induced by oncogenic Ras is restrained by the induction of cellular senescence[1, 2] and additional mutations are needed to promote tumorigenesis and metastasis[3]. Metastatic dissemination represents a complex process[4, 5] that appears to be regulated via multiple biological pathways[6, 7]. Considering millions of cancer patients, in particular in an aging population, it is, therefore, paramount to identify novel pathways and genes involved in cancer growth and metastasis. Many developmental pathways and signaling cascades were originally identified in Drosophila melanogaster[8, 9]. Among the most studied conserved signal transduction pathways involved in tumorigenesis are Ras and Notch[8, 10–12]. In landmark studies, invasion-metastasis has also been successfully modeled in fly larvae[13]. Using the Drosophila system, cell polarity genes were identified as suppressors of Ras-driven tumor growth and invasion[14, 15] implicating high inter-species conservation of fundamental genetic networks controlling transformation and metastasis[16]. Given the accessibility of Drosophila to in vivo RNAi screening approaches and conservation of major developmental pathways including Ras, the fly offers a valuable opportunity to genetically dissect cancer-relevant signaling networks in the context of a whole animal, which could lead to the identification of novel biomarkers and new drug targets for a diverse range of epithelial tumors. Moreover, whereas mammals have three RAS genes, K-, N- and Ha-Ras, the Drosophila has only two: D-Ras1 or Ras85D and D-Ras2 or Ras64B[17]. Ras85D has been shown to be the authentic human ortholog[18, 19], making the fly a suitable model system to genetically dissect Ras-regulated networks. One of the most frequent Ras mutations is RasV12, an activating point mutation in codon 12[20, 21]. To identify novel conserved genes involved in RasV12-driven tumor progression and metastases, we performed a dRas( Ras85DV12)-driven tumorigenesis screen using Drosophila genetics, stratified the mammalian orthologs of the fly hits in thousands of human cancers, and further validated the top 100 candidate tumor suppressor genes in secondary Drosophila assays. Our screen identified many known cancer genes and multiple novel, evolutionary conserved candidate genes that cooperate with Ras in tumorigenesis. We also validated and analyzed the mechanism of action of one of these novel tumor suppressors, the Tetraspanin Tsp29Fb, showing that it inhibits Ras signaling and acts to maintain epithelial tissue architecture.
Discussion:
We have performed a large-scale screen in Drosophila melanogaster to identify genes involved in suppression of RasV12-induced epithelial tumors and tested 10, 777 UAS-RNAi lines corresponding to evolutionarily conserved 6675 genes. For this screen, we expressed dRasV12 and UAS-RNAi constructs in the eye imaginal disc of fly larvae, which allowed us to monitor tumor formation, invasion, and formation of distant site “metastases”. Moreover, RNAi-mediated knockdown allowed us to assay a large number of previously unexplored genes in epithelial tumorigenesis in vivo. Using this experimental set-up, we identified multiple known tumor suppressor pathways as well as many novel genes and pathways potentially involved in the suppression of RasV12-driven epithelial tumorigenesis. Importantly, human genes orthologous to the primary fly hits that, as an entire data set, were massively downregulated when we assayed gene expression in thousands of human cancers, supporting the concept that genes controlling epithelial growth, fate and integrity are highly conserved across phyla. The top 100 candidate genes stratified in human cancers were then further validated in secondary Drosophila Ras-driven eye hyperplasia assays, confirming that 80 of these genes were cooperating tumor suppressors with dRasV12 in epithelial tissues. This screen allowed us to construct an evolutionarily conserved network of genes and pathways predicted to cooperate with Ras in tumor growth and invasion-metastasis. This data set should provide a starting point for the identification of novel genes and pathways involved in tumor suppression and metastasis of Ras-driven epithelial cancers. Loss of function screens have previously been performed in Drosophila, identifying genes that modify Ras function[59, 60]. However, only 20–30% of genes in Drosophila genome can be mutated to give a detectable phenotype[61], leaving the function of a majority of expressed gene unexplored. In addition, these screens have been largely conducted in differentiating eye cells of the Drosophila larvae/pupae that display limited or no mitotic potential and undergo final steps of differentiation[60]. Moreover, in previous studies, Drosophila has been established as a tool to screen for invasive tumor growth[14, 15]; but these screens relied on genetic mutants and required 3 crosses to analyze the effects due to homozygous lethality of the mutants and pupal stage lethality of dRasV12 expression alone. We therefore established a model that circumvents dRasV12-induced lethality by using a CyO, tub-GAL80 balancer[62], offering a system that can be used for rapid screening and to model hyperplastic tumor growth, and formation of distant site “metastases” in a pool of epithelial progenitor cells. Using this experimental system, we tested genes that are conserved between Drosophila and human using RNAi-mediated knockdown, which allowed us to assay the role of a large number of previously unexplored genes in Ras-driven epithelial tumorigenesis in vivo. Since hallmarks of cancer such as migration and proliferation are regulated by evolutionarily conserved pathways, e. g. Ras/MAPK, Wnt or Hedgehog signaling, our high primary hit rate can be explained by the selected set of conserved genes[63]. C2 and KEGG analysis of our hit list revealed that our screen had markedly enriched for known genes and pathways implicated in mammalian tumorigenesis including cell adhesion, cell polarity, phosphoinositide signaling, metabolism, or Wnt, Hedgehog, and, as one must expect, Ras signaling. Interestingly, several genes and pathways identified in our primary screen have an effect on oxidative stress levels or proper mitochondrial function: detoxifying enzymes such as Gutathione S Transferases or Wwox[64], and many mitochondrial genes, the loss of which can potentially impede proper mitochondrial function and generate oxidative stress. Interestingly, we have recently shown that increased oxidative stress levels induced by autophagy impairment can indeed cooperate with oncogenic Ras by triggering the JNK stress response pathway[28]. Perhaps not surprisingly, giving the central role of Ras in conveying intracellular signals, we find that oncogenic Ras cooperates with a wide range of cellular processes scattered across all the different cellular compartments. However, we can attempt to group the Ras-cooperating tumor suppressors identified and validated in our screen into a few pivotal mechanisms that influence Ras tumorigenic outcome: i) modification of the DNA structure, replication or regulation( eg chromatin remodelling, cell cycle regulators, DNA repair enzymes); ii) regulation of Ras and other signalling pathways( eg Wnt, Notch, Hippo, Jak/Stat, TGFβ, PI3K); iii) regulation of apoptosis; iv) disruption of metabolic homeostasis and increase of oxidative stress( eg glycogen breakdown, glucose transport, TCA cycle, autophagy); iv) disruption of plasma membrane organisation or homeostasis( eg cell junction organization, endocytosis). Indeed in this study, in line with recent data suggesting that Tetraspanins are enriched in membrane micro-domains and regulate receptor signaling[65–67], we found that Tsp29Fb genetically interacts with EGFR and dRas and regulates EGFR-Ras signaling. Whilst we have concentrated here on the validated top 80 genes, future analysis of the remaining hits from our screen, may reveal new signaling pathways, or reveal further novel tumor suppressors, involved in Ras-driven tumorigenesis. Of the 80 confirmed genes, 55 belonged to known signaling networks, whereas 25 could not be assigned to any known functional module. Expression as well as methylation analysis of these genes accurately stratified high and low risk survival groups of patients diagnosed with pancreatic cancer, and also correlated with the mutational status for RASG12 in the pancreas tumors of these patients. Given our findings, it will be important to assess whether low expression of the 80 confirmed genes is correlated with poor patient survival in other cancers where RAS signaling is elevated, such as EGFR or RAS-GAP( NF1) tumors. The top hit among the novel 25 tumor suppressor genes was the Tetraspanin family member, Tsp29Fb. Mechanistically, our genetic and biochemical experiments show that Drosophila Tsp29Fb modulates Ras activation and in genetic epistasis experiments affects EGFR-Ras signaling. Whether Tsp29Fb regulates other signaling pathways, alone or together with dRasV12, needs to be further explored. Interestingly, depletion of Tsp29Fb resulted in mislocalized Dlg at cell junctions, and ommatidial patterning defects in terminally-differentiating photoreceptor cells, implicating this protein in epithelial apico-basal polarity regulation and the control of epithelial architecture. It is possible that these effects of Tsp29Fb depletion on epithelial architecture directly affect EGFR/Ras activation, as Scrib depletion does in mammalian cells[68, 69] and in Drosophila epithelial tissue[70]. Indeed, our results showing that Tsp29Fb genetically interacts with scrib support that notion that Tsp29Fb regulates, or functions with, Scrib/Dlg in cell polarity regulation in epithelial development and in the control of signaling pathways. Further analysis of how Tsp29Fb, as well as the other novel tumor suppressor genes identified in our screen, cooperate with dRasV12 in tumorigenesis, will potentially reveal the importance of cell polarity regulation, as well as other novel pathways, in Ras-driven cancer.
Cancer involves the cooperative interaction of many gene mutations . The Ras signaling pathway is upregulated in many human cancers , but upregulated Ras signaling alone is not sufficient to induce malignant tumors . We have undertaken a genome-wide genetic screen using a transgenic RNAi library in the vinegar fly , Drosophila melanogaster , to identify tumor suppressor genes that cooperate with the Ras oncogene ( RasV12 ) in conferring overgrown invasive tumors . We stratified the hits by analyzing the expression of human orthologs of these genes in human epithelial cancers , revealing genes that were strongly downregulated in human cancer . By conducting secondary genetic interaction tests , we validated 80 of the top 100 genes . Pathway analysis of these genes revealed that 55 fell into known pathways involved in human cancer , whereas 25 were unique genes . We then confirmed the tumor suppressor properties of one of these genes , Tsp29Fb , encoding a Tetraspanin membrane protein , and showed that Tsp29Fb functions as a tumor suppressor by inhibiting Ras signaling and by maintaining epithelial cell polarity . Altogether , our study has revealed novel Ras-cooperating tumor suppressors in Drosophila and suggests that these genes may also be involved in human cancer .