The interleukin 6 receptor is a direct transcriptional target of E2F3 in prostate tumor derived cells

BACKGROUND The E2F/RB pathway is frequently disrupted in multiple human cancers. E2F3 levels are elevated in prostate tumors and E2F3 overexpression independently predicts clinical outcome. The goals of this study were to identify direct transcriptional targets of E2F3 in prostate tumor derived cells. METHODS. Expression array studies identified the interleukin 6 receptor (IL-6R) as an E2F3 target. E2F3-dependent expression of IL-6R was analyzed by real time PCR and Western immunoblot analysis in several cell lines. Chromatin immunoprecipitation (ChIP) and IL-6R-luciferase reporter plasmid studies were used to characterize the IL-6R promoter. RESULTS. Expression array studies identified genes that were regulated by E2F3 in prostate tumor derived cell lines. The network most significantly associated with E2F3-regulated transcripts was cytokine signaling and the IL-6R was a component of several of the most prominent E2F3-regulated pathways. The transcriptional regulation of IL-6R by E2F3 knockdown was validated in several prostate tumor-derived cell lines at the RNA level and protein level. The IL-6R regulatory region containing ChIP-identified E2F3 binding sites was cloned into a reporter and co-transfected with an E2F3a expression plasmid. The luciferase assay showed that E2F3a transactivated the IL-6R promoter in a dose dependent manner. The functional consequence of IL-6R decrease was a reduction in the levels of ERK1/2 phosphorylation, indicating that IL-6R initiated signaling was altered. CONCLUSION. These studies connect the E2F and IL-6 signaling cascade, thus providing the mechanistic link between two major regulatory networks that are perturbed during prostate tumorigenesis. Prostate 72:649-660, 2012. (C) 2011 Wiley Periodicals, Inc.