Alterations to the expression and activity of androgen receptor (AR) co-regulators in prostate cancer is an important mechanism driving disease progression and therapy resistance. Using a novel proteomic technique, we identified a new AR co-regulator, the transcription factor Grainyhead-like 2 (GRHL2), and demonstrated its essential role in the oncogenic AR signaling axis. GRHL2 colocalized with AR in prostate tumors and was frequently amplified and upregulated in prostate cancer. Importantly, GRHL2 maintained AR expression in multiple prostate cancer model systems, was required for cell proliferation, enhanced AR's transcriptional activity, and co-located with AR at specific sites on chromatin to regulate genes relevant to disease progression. GRHL2 is itself an AR-regulated gene, creating a positive feedback loop between the two factors. The link between GRHL2 and AR also applied to constitutively active truncated AR variants (ARVs), as GRHL2 interacted with and regulated ARVs and vice versa. These oncogenic functions of GRHL2 were counterbalanced by its ability to suppress epithelial-mesenchymal transition and cell invasion. Mechanistic evidence suggested that AR assisted GRHL2 in maintaining the epithelial phenotype. In summary, this study has identified a new AR co-regulator with a multifaceted role in prostate cancer, functioning as an enhancer of the oncogenic AR signaling pathway but also a suppressor of metastasis-related phenotypes.

Hypoxia-inducible factor 1a (HIF-1a) degradation under normoxia is critical to modulating vascular growth. This degradation is mediated during normoxia by the von Hippel-Lindau tumour suppressor protein (VHL)-E3 ubiquitin ligase in partnership with the E2 enzyme UbcH5. In current models of the functionally similar Skp1, cullin, F-box (SCF)-E3 ligase, the E3 binds the target protein and the E2 catalyses ubiquitin transfer to lysines in an appropriately positioned domain. In the present study, we report that for efficient ubiquitination of HIF-1a to occur, three conserved lysines are required in both the HIF-1a and endothelial Per-ARNT-Sim domain protein (EPAS) sequences. The site of ubiquitin attachment via UbcH5 was mapped, and is shown to involve three HIF-1a lysines, K532, K538 and K547, and the same aligned lysines in EPAS. Only one of these lysines need to be intact for full ubiquitination to occur, analogous to the mechanism of Sic1 ubiquitination by the SCF/Cdc34 complex and further strengthening the functional link between the VHL and SCF-E3 ubiquitin ligases. We also report that lysines can be moved around the HIF-1a sequence with only minor losses in ubiquitination efficiency, thus suggesting HIF-1a and EPAS regulation by hypoxia depends primarily on an interaction with VHL per se, rather than the highly specific positioning of flanking lysine acceptors.

Hypoxia-inducible factor-1a (HIF-1a) induction and associated transcription were investigated during high cell density, focusing on the negative regulator of HIF-1a expression, the von Hippel-Lindau (VHL) protein. In 293T and HeLa cells, HIF-1a protein levels and associated transcription were induced as cells approached confluence. To determine whether these changes were due to a deficit in nuclear VHL-mediated ubiquitination of HIF-1a at confluence, cells were stably transfected with VHL. Overexpression of VHL in 293T cells had no demonstrable effect on the induction and nuclear accumulation of HIF-1a during high cell density or associated transcription. Moreover, RCC cells stably transfected with full-length VHL failed to exhibit the cell-densitydependent induction of HIF-1a noted in other cell lines. Investigation of both N-terminal and C-terminal (aa 727-826) oxygen-regulated proline and asparagine hydroxylation of HIF-1a revealed that both are inhibited during high cell density, as determined by impaired capture of HIF-1a by VHL and enhanced C-terminal transactivation. Finally, cell-density-mediated induction of HIF-1a and GLUT1 in RCC cells could be completely reconstituted by mutations in VHL binding affinity, suggesting that celldensity dependent induction of HIF-1a and transactivation may underpin some of the deregulated gene expression observed in VHL disease.