Study of the role of transcription factors ETV4 and ETV1 of the ETS family in the tumor process of hormone-dependent cancers : breast cancer, metastatic progression of prostate cancer

Les facteurs ETV1, 4, 5 sont souvent associée au développement de cancers. Le 1èr projet porte sur ETV4 et MMP13 en tant que gène cible et relais potentiel de l’effet pro-tumorigène d’ETV4 dans la tumorigenèse mammaire. ETV4 est surexprimé dans le cancer du sein et est associé à un mauvais pronostic, mais les événements impliqués sont encore peu connus. ETV4 contrôle l’expression de nombreux gènes comme MMP13. Cette étude a permis de montrer que MMP13 est un gène cible d’ETV4. En effet, la surexpression de MMP13 contribue aux effets pro-tumorigène et l’inhibition de MMP13 dans un contexte de surexpression de ETV4 diminue son impact. Enfin, l’étude effectuée dans la cohorte de patiente associe la surexpression d’ETV4 et de MMP13 à un cancer de mauvais pronostic. Le 2ème projet porte sur l’implication d’ETV1 dans la progression métastatique du cancer de la prostate (CaP). Les fusions de gènes impliquant les facteurs ERG et ETV1 sont présentes dans ≈50% et 10% des cas respectivement, ETV1 est présent à 50% sous sa forme pleine longueur et à 50% sous une forme tronquée. Les études ne présageant pas de différences entre les fusions ERG et ETV1. Dans ce contexte, mon 2ème sujet d’étude porte sur la recherche de l’implication du facteur ETV1 (pleine longueur ou tronquée) dans la formation des métastases du CaP et la recherche de gènes cibles impliqués, le tout en comparaison avec les données sur ERG. Cette étude nous a permis de mettre en évidence une différence en terme d’agressivité entre les 2 formes d’ETV1 in vitro et in vivo. Cependant la fusion étant peut représentée, les résultats récolté dans la cohorte ne peuvent permettre de confirmer cette différence d’agressivité.ETV1, 4, 5 transcription factor are overexpress in various cancer. The first part is focus in MMP13 like target gene, potentially implicated in the mammary tumorigenesis induced by ETV4. Thus we show, the regulation of MMP13 expression by ETV4. Next we show that ETV4 promotes the mammary tumorigenesis and that MMP13 is a relay. In fact, the overexpression of MMP13 is implicate in pro-tumorigenesis effect and the repression of MMP13 in context of ETV4 overexpression decreases this effect. This approach was completed by the injection of cells in mice and show MMP13 mediate the pro-tomorigene effect of ETV4. We analysed expression of MMP13 and ETV4 in primary breast tumors and show the overexpression concomintant of ETV4 and MMP13 are associated with a poor prognosis. The second part of our study is about the ETV1 factor, in the progression of metastasis of prostate cancer (PCa) and the research of involved target genes. Gene fusion involving ERG and ETV1 and their overexpression are frequent in PCa and occur in 50% and 10% of cases respectively. To understand the role of ETV1, in comparison with available data on ERG, differents PCa cells overexpress or repress ETV1 were used. We have shown that the ETV1 factor enhance tumorigenesis capacities PCa cell lines and her truncated form has opposite effects. ETV1 full-lenght (FL) induces more bones metastasis formation than her truncated form. The expression of ETV1, and his truncated form was study in PCa samples. We confirmed the pro-tumorigenic statute of ETV1 factor in PCa and we defined differences beetwen FL and truncated length and complet the comprehension of the ETV1 function in the formation of bones metastasis of PCa

ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Abstract Background The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis. Methods Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples. Results Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer. Conclusion MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression

Additional file 7: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S5. Expression of ETV4 in MMT-shMMP13-repressing cells. a Relative ETV4 mRNA expression in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl + shCtrl cells was arbitrarily = 1. Error bars indicate SD. The results were not statistically significant. b Western blot analysis of ETV4 protein expression (61 kDa) in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells. GAPDH expression served as the loading control. (PDF 71 kb

Additional file 8: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S6. The repression of MMP13 reduces the anchorage-independent growth capacity of MMT-ETV4-overexpressing cells. a Relative MMP13 mRNA expression in the transiently transfected MMT-siCtrl and MMT-siMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-siCtrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. b Anchorage-independent growth. MMT-ETV4-siCtrl and MMT-ETV4-siMMP13 cells were cultured for 10 days in soft agar. This histogram represents the number of clones counted for experimental time points. Soft agar assays were conducted three times in triplicate. Magnification × 5. Error bars indicate SD. ****P ≤ 0.0001. (PDF 45 kb

Additional file 6: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S4. Expression of MMP13 in MMT cells overexpressing or repressing MMP13. a and b Relative MMP13 mRNA expression in the MMT-Ctrl and MMT-MMP13 (a) or MMT-shCtrl and MMT-shMMP13 cells (b) determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. c Western blot analysis of MMP13 protein expression (60 kDa) in the MMT-Ctrl and MMT-MMP13 cells. GAPDH expression served as the loading control. d Zymographic analysis of MMP13 protein activity (55 kDa) from the supernatant of MMT-Ctrl and MMT-MMP13 cells. (PDF 72 kb

Additional file 10: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S8. Metastasis-free survival analysis from the publicly available NKI datasets of breast tumors. a Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 12), High-ETV4 and Low-MMP13 (n = 243), or High-ETV4 and High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. b Metastasis-free survival (MFS) curves for breast tumor patients according to Low-MMP13 (n = 255) or High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. c Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 13) and High-ETV4 (n = 251) mRNA levels. ****P ≤ 0.0001. (PDF 19 kb

Additional file 2: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Table S1. Pathological and clinical characteristics of patients in relation to metastasis-free survival (MFS). (PDF 33 kb

Additional file 11: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Table S2. Multivariate Cox proportional hazards analysis of MFS for MMP13 and ETV4 expression levels in the series of 456 breast tumors. (PDF 42 kb