ETV4 over-expression in prostate cells down-regulates p21 both in vitro and in vivo

Background The ETS transcription factors are deregulated in several tumors. In most prostate cancer ETS proteins (ERG, ETV1, ETV4, ETV5) are over-expressed because of recurrent chromosomal translocations that bring an ETS gene under the control of a promoter of a gene highly expressed in the prostate. We have previously found that, at variance with other ETS protein, ETV4 promotes cell proliferation in human prostate cell lines. Here we have investigated, in vitro and in vivo, how ETV4 expression drives an increased proliferation rate in prostate cells. Material and Methods We have assessed the proliferation rate of prostate cells in: (1) human cell lines by either over-expression (RWPE1, PNT1) or silencing (PC3) of ETV4; (2) mice with specific ETV4 prostate expression. The expression level of genes involved in cell cycle regulation has been measured by real time RT-PCR and Western blot. The regulation of the CDK1A gene (codifying for p21-WAF1/CIP1) by ETV4 has been investigated by ChIP and Dual-Luciferase assays. Result and discussion We confirmed that in human prostate cell lines ETV4 expression is associated with an increased proliferation rate and with variation in cell cycle. In addition, we found that the proportion of Ki67+ prostate cells was slightly but significantly increased also in 5 months-old ETV4-transgenic mice compared to wild type mice.We have quantified the expression of several cell-cycle genes; among them we found that the mRNA levels of CDK1A is reduced not only in prostate cell lines over-expressing ETV4 but also in vivo in the ETV4 mice. To determine how ETV4 regulates the expression of CDKN1A we performed ChIP and luciferase assays. In the 2 kb upstream the transcriptional start site there are 2 putative ETV4 binding sites (BS): distal (-1409/-1403) and proximal (-704/-696) BS. ChIP assay suggested that ETV4 binds the proximal but not the distal BS of CDKN1A promoter. The Luciferase assay performed in several cell lines using a vector, in which the firefly luciferase was expressed under the control of the WT proximal ETV4 BS, showed that ETV4 over-expression induce a reduction of the relative luciferase expression, while the normal level is restored when this site is mutated. Conclusion ETV4 over-expression increases the proliferation rate of prostate cell in vitro and also in vivo. This increased proliferation rate is associated with the down regulation of p21, that seems to result from the binding of ETV4 to the p21 promoter.