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Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

Supplementary Figure S7: Fibroblast transformation with each of the 8 somatic-mutation-harboring RTKs

PDF file - 42K, Relative luciferase activity measured for the human cyclin d2 promoter. Alignment of the 55 bp Mouse, Rat and Human proximal cyclin d2 promoter region.

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PDF file - 283K, Wound healing assay for TAC-V and TAC-Pea3-V cells transfected with ctrl or pea3 siRNAs and treated or not with TGF-β1. Western blot analysis of p27kip1 protein expression.

Supplementary Table S1: Detailed annotation of the somatic mutations in the 58 RTKs, KRAS, PI3KCA, and TP53

The Ets family transcription factor Pea3 (ETV4) is involved in tumorigenesis especially during the metastatic process. Pea3 is known to induce migration and invasion in mammary epithelial cell model systems. However, the molecular pathways regulated by Pea3 are still misunderstood. In the current study, using in vivo and in vitro assays, Pea3 increased the morphogenetic and tumorigenic capacity of mammary epithelial cells by modulating their cell morphology, proliferation, and migration potential. In addition, Pea3 overexpression favored an epithelial–mesenchymal transition (EMT) triggered by TGF-β1. During investigation for molecular events downstream of Pea3, Cyclin D2 (CCND2) was identified as a new Pea3 target gene involved in the control of cellular proliferation and migration, a finding that highlights a new negative regulatory loop between Pea3 and Cyclin D2. Furthermore, Cyclin D2 expression was lost during TGF-β1–induced EMT and Pea3-induced tumorigenesis. Finally, restored Cyclin D2 expression in Pea3-dependent mammary tumorigenic cells decreased cell migration in an opposite manner to Pea3. As such, these data demonstrate that loss of the negative feedback loop between Cyclin D2 and Pea3 contributes to Pea3-induced tumorigenesis.Implications: This study reveals molecular insight into how the Ets family transcription factor Pea3 favors EMT and contributes to tumorigenesis via a negative regulatory loop with Cyclin D2, a new Pea3 target gene. Mol Cancer Res; 11(11); 1412–24. ©2013 AACR.

PDF file - 22K, siRNA sequences used for the cyclin d2 AND pea3 knock-down experiments.

PDF file - 71K, Western blot analysis of Pea3 protein expression in the MMT and TAC cells. Effect of Pea3 and TGF-β1 on the expression of cell cycle related genes.

PDF file - 270K, Relative pea3 mRNA and protein expression in TAC-V vs TAC-Pea3-V. Wound healing assay for TAC-V and TAC-Pea3-V cells treated or not with TGF-β1 and with or without Mitomycin C. Apoptosis measurement.

PDF file - 69K, Primers sets used for Q-PCR and ChIP experiments.

Supplementary Table 1 from Autocrine Induction of Invasive and Metastatic Phenotypes by the MIF-CXCR4 Axis in Drug-Resistant Human Colon Cancer Cells

Supplementary Figure 1 from A Genetic Screen Identifies Topoisomerase 1 as a Regulator of Senescence

Supplementary Table 1 from Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age. [Cancer Res 2009;69(20):7917–24]

Supplementary Figure Legends 1-3 from A Genetic Screen Identifies Topoisomerase 1 as a Regulator of Senescence

Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil– and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2α and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells. Cancer Res; 70(11); 4644–54. ©2010 AACR.

Supplementary Figure 3 from A Genetic Screen Identifies Topoisomerase 1 as a Regulator of Senescence

Supplementary Table 2 from Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Supplementary Methods, Figure Legends 1 from Autocrine Induction of Invasive and Metastatic Phenotypes by the MIF-CXCR4 Axis in Drug-Resistant Human Colon Cancer Cells

Supplementary Figures 1-8 from Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Supplementary Methods from Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Supplementary Figure 2 from A Genetic Screen Identifies Topoisomerase 1 as a Regulator of Senescence

PDF file - 326K, Supplementary Figure 1: The knockdown of PLA2R1 favors OIS escape. Supplementary Figure 2: The knockdown of PLA2R1 favors transformation. Supplementary Figure 3: The knockdown of PLA2R1 favors OIS escape in human normal keratinocytes. Supplementary Figure 4: DMBA/TPA treatment induces senescence and its escape correlates with papillomas formation. Supplementary Figure 5: Validation of H-Ras mutation in papilloma. Supplementary Figure 6: JAK2 is involved in PLA2R1-mediated cell growth regulation. Supplementary Figure 7: PLA2R1 regulates JAK2 activity. -Supplementary Figure 8: JAK2 mediates PLA2R1 tumor suppressive effects.

Supplementary Figure 1 from Autocrine Induction of Invasive and Metastatic Phenotypes by the MIF-CXCR4 Axis in Drug-Resistant Human Colon Cancer Cells

International audience; Abstract : Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells.

International audience; Colorectal cancer (CRC) remains the most cancer type related to chronic inflammation; however, the mechanisms that link inflammation to CRC development and progression are still poorly understood. Our study aimed to investigate one of the prominent inflammatory response in cancers, iNOS/NO system. In this regard, we evaluated the link between the iNOS/NO system and CRC progression, its relation with the host immune responses and its response to cetuximab combined with chemotherapy. We found that the nitrite levels were nearly twice as high in metastatic CRC plasma and culture supernatants from PBMCs and tumor explants compared with those without metastases and healthy controls. Interestingly, we showed that the highest iNOS expression and NO levels are present in the damaged CRC tissues that have highest leukocyte infiltration. Our findings highlight the implication of iNOS/NO system in tissue alteration and leukocyte invasion. Thus, we observed imbalance between effector/memory T cell markers and Treg transcription factor (Foxp3). Accordingly, we detected higher IFNγ and T-bet expression levels in colorectal tumor tissues at early stage. In contrast, consistent with iNOS and Foxp3 expression, TGFβ, CTLA-4 and IL-10 were significantly related to the tumor stage progression. Furthermore, our study revealed that Cetuximab combined with chemotherapy treatment markedly down-regulates iNOS/NO system as well as IL-10 and TGFβ levels. Altogether, we conclude that cetuximab can potentiate the efficacy of chemotherapy, particularly by iNOS/NO system and immunosuppressive cytokines modulation. Thus, we suggest that iNOS/NO system may represent an attractive candidate biomarker for monitoring CRC progression, malignity and response to therapy.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

Colorectal cancer (CRC) remains the most cancer type related to chronic inflammation; however, the mechanisms that link inflammation to CRC development and progression are still poorly understood. Our study aimed to investigate one of the prominent inflammatory response in cancers, iNOS/NO system. In this regard, we evaluated the link between the iNOS/NO system and CRC progression, its relation with the host immune responses and its response to cetuximab combined with chemotherapy. We found that the nitrite levels were nearly twice as high in metastatic CRC plasma and culture supernatants from PBMCs and tumor explants compared with those without metastases and healthy controls. Interestingly, we showed that the highest iNOS expression and NO levels are present in the damaged CRC tissues that have highest leukocyte infiltration. Our findings highlight the implication of iNOS/NO system in tissue alteration and leukocyte invasion. Thus, we observed imbalance between effector/memory T cell markers and Treg transcription factor (Foxp3). Accordingly, we detected higher IFNγ and T-bet expression levels in colorectal tumor tissues at early stage. In contrast, consistent with iNOS and Foxp3 expression, TGFβ, CTLA-4 and IL-10 were significantly related to the tumor stage progression. Furthermore, our study revealed that Cetuximab combined with chemotherapy treatment markedly down-regulates iNOS/NO system as well as IL-10 and TGFβ levels. Altogether, we conclude that cetuximab can potentiate the efficacy of chemotherapy, particularly by iNOS/NO system and immunosuppressive cytokines modulation. Thus, we suggest that iNOS/NO system may represent an attractive candidate biomarker for monitoring CRC progression, malignity and response to therapy.

Bone metastasis is the major deleterious event in prostate cancer (PCa). TMPRSS2-ERG fusion is one of the most common chromosomic rearrangements in PCa. However, its implication in bone metastasis development is still unclear. Since bone metastasis starts with the tropism of cancer cells to bone through specific migratory and invasive processes involving osteomimetic capabilities, it is crucial to better our understanding of the influence of TMPRSS2-ERG expression in the mechanisms underlying the bone tropism properties of PCa cells. We developed bioluminescent cell lines expressing the TMPRSS2-ERG fusion in order to assess its role in tumor growth and bone metastasis appearance in a mouse model. First, we showed that the TMPRSS2-ERG fusion increases cell migration and subcutaneous tumor size. Second, using intracardiac injection experiments in mice, we showed that the expression of TMPRSS2-ERG fusion increases the number of metastases in bone. Moreover, TMPRSS2-ERG affects the pattern of metastatic spread by increasing the incidence of tumors in hind limbs and spine, which are two of the most frequent sites of human PCa metastases. Finally, transcriptome analysis highlighted a series of genes regulated by the fusion and involved in the metastatic process. Altogether, our work indicates that TMPRSS2-ERG increases bone tropism of PCa cells and metastasis development., info:eu-repo/semantics/published

Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF-κB signaling in development and progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-αmodulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-αinduction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-αexpression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-αexpression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.

Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF-κB signaling in development and progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-α induction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.

International audience; The transcription factor E-twenty-six version 5 (ETV5) has been linked with obesity in genome-wide association studies. Moreover, ETV5-deficient mice (knockout; KO) have reduced body weight, lower fat mass, and are resistant to diet-induced obesity, directly linking ETV5 to the regulation of energy balance and metabolism. ETV5 is expressed in hypothalamic brain regions that regulate both metabolism and HPA axis activity, suggesting that ETV5 may also modulate HPA axis function. In order to test this possibility, plasma corticosterone levels were measured in ETV5 KO and wildtype (WT) mice before (pre-stress) and after (post-stress) a mild stressor (intraperitoneal injection). ETV5 deficiency increased both pre- and post-stress plasma corticosterone, suggesting that loss of ETV5 elevated glucocorticoid tone. Consistent with this idea, ETV5 KO mice have reduced thymus weight, suggestive of increased glucocorticoid-induced thymic involution. ETV5 deficiency also decreased the mRNA expression of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and vasopressin receptor 1A in the hypothalamus, without altering vasopressin, corticotropin-releasing hormone, or oxytocin mRNA expression. In order to test whether reduced MR and GR expression affected glucocorticoid negative feedback, a dexamethasone suppression test was performed. Dexamethasone reduced plasma corticosterone in both ETV5 KO and WT mice, suggesting that glucocorticoid negative feedback was unaltered by ETV5 deficiency. In summary, these data suggest that the obesity-associated transcription factor ETV5 normally acts to diminish circulating glucocorticoids. This might occur directly via ETV5 actions on HPA-regulatory brain circuitry, and/or indirectly via ETV5-induced alterations in metabolic factors that then influence the HPA axis.

International audience; Various solid tumors including lung or gastric carcinomas display aberrant activation of the Met receptor which correlates with aggressive phenotypes and poor prognosis. Although downstream signaling of Met is well described, its integration at the transcriptional level is poorly understood. We demonstrate here that in cancer cells harboring met gene amplification, inhibition of Met activity with tyrosine kinase inhibitors or specific siRNA drastically decreased expression of ETV1, ETV4 and ETV5, three transcription factors constituting the PEA3 subgroup of the ETS family, while expression of the other members of the family were less or not affected. Similar link between Met activity and PEA3 factors expression was found in lung cancer cells displaying resistance to EGFR targeted therapy involving met gene amplification. Using silencing experiments, we demonstrate that the PEA3 factors are required for efficient migration and invasion mediated by Met, while other biological responses such as proliferation or unanchored growth remain unaffected. PEA3 overexpression or silencing revealed that they participated in the regulation of the MMP2 target gene involved in extracellular matrix remodeling. Our results demonstrated that PEA3-subgroup transcription factors are key players of the Met signaling integration involved in regulation of migration and invasiveness.

Comment in :- RE: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells. [J Natl Cancer Inst. 2015]- Response. [J Natl Cancer Inst. 2015]; International audience; BACKGROUND: Regulatory T cells (Treg) and tumor-exosomes are thought to play a role in preventing the rejection of malignant cells in patients bearing nasopharyngeal carcinoma (NPC).METHODS: Treg recruitment by exosomes derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients' plasma (Patient-Exo), and CCL20 were tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model (n = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA, and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). The Student's t test was used for group comparisons. All statistical tests were two-sided.RESULTS: CCL20 allowed the intratumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34 fold increase, P < .001), which was statistically significantly inhibited (P < .001) by an anti-CCL20 blocking mAb. NPC-Exo also recruited conventional CD4(+)CD25(-) T cells and mediated their conversion into inhibitory CD4(+)CD25(high) cells. Moreover, NPC-Exo enhanced (P = .0048) the expansion of human Treg, inducing the generation of Tim3(Low) Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75 (P < .001). These results were consistent with a stronger suppression of responder cells' proliferation and the secretion of immunosuppressive cytokines (IL10, TGFB1).CONCLUSION: Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.

Background: Bone metastases are frequent and severe complications of prostate cancer (PCa), the most common malignancy that affects men in Western countries. Recurrent gene fusions, involving the ERG gene and the androgen-regulated promoter of the TMPRSS2 gene, occur in over 50% of PCa. The abnormal over-expression of the ERG transcription factor disturbs prostate cell transcriptome. We previously identified a series of genes directly and positively regulated by the ERG-fusion in vitro and in vivo. Among them can be found Plexin A2 (PLXNA2), which is a semaphorin receptor family member involved in axon guidance and in many pathophysiological processes, including cancer and bone disorders. We showed that PLXNA2 is involved in TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. These results prompted us to look for genes functionally related to PLXNA2 and involved in ERG-associated PCa. Methods: Using a PCa cell line (PC3c) stably over-expressing the TMPRSS2:ERG fusion, we first used Ingenuity Pathway Analysis® in silico studies to analyze transcriptomic results and identify axon guidance genes associated with ERG-fusion expression in PCa. Their expression was validated in stable clones by RT-PCR and Immunofluorescence. Secondly, using RNAs of a human PCa sample cohort, we performed qRT-PCR to detect and correlate candidate genes to fusion expression. Finally, to validate the relevance of the identified genes, using immunohistochemistry experiments, we studied the expression of these genes in tissue samples, including primary tumors and lymph nodes or bone metastases. Results: Using transcriptomic analyzes and expression validation, we identified neuropilin (NRP1 and NRP2) genes, plexin and semaphorin genes deregulated by the ERG fusion expression in stable clones. In human samples, PLXNA2, NRP1 and NRP2 expression were strongly associated to PCa, and particularly to metastastic PCa. We established a significant correlation between both PLXNA2 and NRP1 gene expression and the presence of TMPRSS2-ERG fusion. At protein level, NRP1 and NRP2 were highly detected in human lymph nodes and bone metastasis samples. Since neuropilin proteins are known to interact with plexin to act as coreceptors for semaphorin ligands, protein colocalization in stable clones suggested a functional association of PLXNA2 and NRPs in tumor cells. Conclusion: Taken together, our results reveal that TMPRSS2-ERG fusion gene expression is able to deregulate axon guidance genes such as NRP1 and PLXNA2 in PCa. Therefore, since the interaction between plexins and their NRP1 and 2 coreceptors often determines the functional specificity of their semaphorin ligand effects, we hypothesize that the ERG fusion expression imbalances the plexin/neuropilin/semaphorin signaling to contribute to PCa progression. These results reinforce the recent interest in targeting neuropilins for cancer therapy. Citation Format: Anthony Turpin, Carine Delliaux, Tian Tian, Nathalie Vanpouille, Anne Flourens, Rachel Deplus, Xavier Leroy, Yvan de Launoit, Martine Duterque-Coquillaud. Axon guidance neuropilin and plexin A2 genes are involved in ERG-associated prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 694.

Background: Bone metastases are the major cause of morbidity and mortality in prostate cancer (PCa) patients. Recently, TMPRSS2:ERG gene fusions produced by rearrangements along chromosome 21 was found in more than 50% of PCa samples, resulting in androgen-dependent aberrant expression of the functional ERG transcription factor. Interestingly, ERG transcription factor has been previously shown to be involved in bone development. This study is therefore focused on investigating whether TMPRSS2:ERG gene fusions are involved in PCa bone metastasis development. Methods: We previously established cell clones overexpressing TMPRSS2:ERG from two PCa cell lines, PC3 and PC3c. We first studied induced bone lesion phenotype using in vivo intra-tibial injection models. Secondly, we analyzed transcriptional changes induced by ERG transcription factors in PC3c clones to identify potential target genes. Then, direct target genes were further validated and investigated in vitro using RT-qPCR, ELISA, siRNA and ChIP techniques. Importantly, using a cohort of prostate carcinoma samples, we validated the expression correlation between ERG and its target target genes expression in human pathology. Results: Bone lesions induced in vivo by intra-tibial injections of PC3 or PC3c cells are known to be osteolytic or mixed (osteoblastic/osteolytic) respectively. Interestingly, intra-tibial injections of PC3c clones expressing the fusion showed a statistically significant increase of osteoblastic phenotype compared to control cells. Furthermore, intra-tibial injections of PC3 clones expressing the fusion showed a strong decrease of osteolytic phenotype, reinforcing our previous result in PC3c clones. Among the genes identified by transcriptomic study and dysregulated in PC3c clones expressing the fusion, we have identified the ERG candidate target Endothelin-1 (ET-1), which is known to be involved in osteoblast proliferation and in osteoblastic metastasis formation in PCa. Indeed, we found that ET-1 expression was up-regulated in PC3c-TMPRSS2:ERG clones, and the up-regulation was dependent on ERG expression levels. Importantly, silencing of ERG resulted in decreased expression of ET-1. In silico analysis of the promoter of ET-1 revealed the presence of several potential binding sites of ERG. ChIP experiments followed by qPCR demonstrated a direct binding to one of them. Moreover, in human PCa samples, there was a significant expression correlation between ET-1 and fusion gene TMPRSS2:ERG, reinforcing the direct functional link between ET-1 and the fusion in PCa. Conclusion: Taken together, these results strongly suggest that the TMPRSS2:ERG gene fusion contributes to the osteoblastic phenotype of PCa bone metastases and ET-1 is one of the involved factors, directly regulated by the transcription factor ERG. Citation Format: Carine Delliaux, Tian V. Tian, Mathilde Bouchet, Anaïs Fradet, Nathalie Vanpouille, Anne Flourens, Rachel Deplus, Xavier Leroy, Yvan de Launoit, Edith Bonnelye, Martine Duterque-Coquillaud. TMPRSS2:ERG fusion enhances osteoblastic phenotype of prostate cancer bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1691.