ETS1 and ETS2 role in Ras oncogenic transformation in mouse embryonic fibroblasts

Human soft-tissue sarcoma is a malignant tumor that is usually found in children and young adults. In the United States more than 12000 new cases are diagnosed each year with soft tissue sarcomas. Oncogenic Ras has been shown to be involved in soft-tissue sarcoma development and K-Ras, H-Ras and N-Ras missense mutations at codons 12, 13 and 61 have been reported in 23 to 30% of soft tissue sarcoma tumors.;The MAPK pathway is one of the main mediators of the Ras response, and it has been shown to be deregulated in cancer in general and soft tissue sarcoma in particular. Ets1 and Ets2 are the downstream effectors of the Ras/ERK pathway and have been shown to be over expressed in cancer and activated by Ras/ERK through the phosphorylation of their conserved MAPK phosphorylation domain next to the PNT domain. This phosphorylation is essential for Ets1 and Ets2 to regulate many genes that are important for diverse biological processes.;Over expression of constitutively active Ras in mouse embryonic fibroblasts leads to cellular transformation, and injection of Ras transformed fibroblasts into nude mice leads to formation of tumors similar to fibroblastic soft tissue tumor, which makes the Ras transformation in vitro model a suitable system for studying soft-tissue sarcoma. The Ets families of transcription factors have been shown to play an essential role in Ras transformation, but the identity of the Ets family members that are essential for Ras transformation is still unkown.;Using the Cre-lox system to delete Ets2 from Mouse Embryonic Fibroblasts (MEFs) that are Ets1-/- and have the Ets1 null allele, we showed that the deletion of both Ets1 and Ets2 is necessary to inhibit Ras oncogenic transformation and tumorigenesis in MEFs.;Here we show that Ets1 and Ets2 play a post transcriptional repression role in Ras transformation through a C-myc dependent up regulation of miR17-92 microRNA. Ets1 and Ets2 bind to the C-myc promoter and regulate C-myc RNA and protein expression. We also show that Ets2 similar to C-myc binds to the promoter of miR17-92, and Ets1 and Ets2 through transient transfection are able to upregulate the miR17-92 cluster expression in C-myc -/- MEFs. C-myc and miR17-92 Cluster over expression in Ets1/Ets2 double knockout MEFs rescued Ras tumorigenesis in vivo upon injection in nude mice.;Also we show that Ets1 and Ets2 play a transcriptional repression role through a c-myc dependent repression of Sfrp1, Fas and Lox tumor suppressor genes during Ras transformation. Fas, Lox and Sfrp1 are not repressed in Ets1/Ets2 double knockout MEFs even after Ras retroviral infection, but over expression of C-myc repressed Fas, Lox and Sfrp1 again in the Ets1/Ets2 double knockout MEFs. We found that Ets1, Ets2 and C-myc bind to the promoter of Fas, Sfrp1 and Lox genes during Ras transformation, and we show an enrichment of these promoters with H3K9 and H3K27 trymethylation markers which are usually present on silent promoters.