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.

Genome-wide analysis of androgen receptor binding and gene regulation in two CWR22-derived prostate cancer cell lines.

Prostate carcinoma (CaP) is a heterogeneous multifocal disease where gene expression and regulation are altered not only with disease progression but also between metastatic lesions. The androgen receptor (AR) regulates the growth of metastatic CaPs; however, sensitivity to androgen ablation is short lived, yielding to emergence of castrate-resistant CaP (CRCaP). CRCaP prostate cancers continue to express the AR, a pivotal prostate regulator, but it is not known whether the AR targets similar or different genes in different castrate-resistant cells. In this study, we investigated AR binding and AR-dependent transcription in two related castrate-resistant cell lines derived from androgen-dependent CWR22-relapsed tumors: CWR22Rv1 (Rv1) and CWR-R1 (R1). Expression microarray analysis revealed that R1 and Rv1 cells had significantly different gene expression profiles individually and in response to androgen. In contrast, AR chromatin immunoprecipitation (ChIP) combined with promoter DNA microarrays (ChIP-on-chip) studies showed that they have a similar AR-binding profile. Coupling of the microarray study with ChIP-on-chip analysis identified direct AR targets. The most prominent function of transcripts that were direct AR targets was transcriptional regulation, although only one transcriptional regulator, CCAAT/enhancer binding protein δ, was commonly regulated in both lines. Our results indicate that the AR regulates the expression of different transcripts in the two lines, and demonstrate the versatility of the AR-regulated gene expression program in prostate tumors.