Abstract
Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells in vitro and in vivo However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27. To explore additional players that are regulated downstream of E6AP, we combined a transcriptomic and proteomic approach. We identified and quantified 16,130 transcripts and 7,209 proteins in castration resistant prostate cancer cell line, DU145. A total of 2,763 transcripts and 308 proteins were significantly altered on knockdown of E6AP. Pathway analyses supported the known phenotypic effects of E6AP knockdown in prostate cancer cells and in parallel exposed novel potential links of E6AP with cancer metabolism, DNA damage repair and immune response. Changes in expression of the top candidates were confirmed using real-time polymerase chain reaction. Of these, clusterin, a stress-induced chaperone protein, commonly deregulated in prostate cancer, was pursued further. Knockdown of E6AP resulted in increased clusterin transcript and protein levels in vitro and in vivo Concomitant knockdown of E6AP and clusterin supported the contribution of clusterin to the phenotype induced by E6AP. Overall, results from this study provide insight into the potential biological pathways controlled by E6AP in prostate cancer cells and identifies clusterin as a novel target of E6AP.
Highlights
Prostate cancer (PC)1 is one of the leading causes of cancer-related mortality in men, with one in five men newly diagnosed with prostate cancer present locally advanced or metastatic form of the disease [1]
Experimental Design and Statistical Rationale—PC cell line DU145 was chosen for the discovery-based approach as the phenotypic effects of E6-associated protein (E6AP) knockdown both in vitro and in vivo have been explored in DU145 cells in our laboratory [17, 20]
PC cell line DU145 was chosen for this discovery-based approach as the phenotypic effects of E6AP knockdown both in vitro and in vivo have been explored in DU145 cells in our laboratory [17, 20]
Summary
Prostate cancer (PC) is one of the leading causes of cancer-related mortality in men, with one in five men newly diagnosed with prostate cancer present locally advanced or metastatic form of the disease [1]. The functions of E6AP, mostly attributed to its ligase activity include cellular growth, cell cycle, apoptosis, cellular senescence, cellular stress response and proteasomal regulation (10 – 16). We have recently demonstrated that this is partially explained by the effect of E6AP on the key tumor suppressors, promyelocytic leukemia protein (PML; [20]) and p27 [17]. We identify clusterin, a stress-induced chaperon protein, as a novel tumor suppressor whose expression is regulated by E6AP
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