Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP

Abstract

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells

“…Consistently, mice over-expressing E6AP develop enlarged prostate glands and show prostatic intra-epithelial neoplasia (18,19). Moreover, knockdown of E6AP in PC cells attenuates cellular growth in vitro and in vivo (20), further supporting an oncogenic function of E6AP in PC. 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).…”

“…Moreover, knockdown of E6AP in PC cells attenuates cellular growth in vitro and in vivo (20), further supporting an oncogenic function of E6AP in PC. 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). This in vitro and in vivo work is further supported by tissue microarray data demonstrating that patients with localized PC expressing high levels of E6AP and low levels of PML have the shortest clinical relapse-free survival and this correlation is a significant predictor of prostate cancer-associated death (21).…”

“…Plasmid and Lentivirus Generation-The sequences for lentiviruses expressing shRNA against E6AP (shE6AP) and its control (shControl) and methodology for viral production and infection have previously been described (20). The sequence of lentivirus expressing shRNA against clusterin (shCLU) was F 5Ј-TCCCGCTCAGCAACCTAGAA-GAATTCAAGAGATTCTTCTAGG TTGCTGAGCTTTTTC-3Ј and R 5Ј-TCGAGAAAAAGCTCAGCAACCTAGAAGAATCTCTTGAATTCTTCT-AGGTTGCTGAGC-3Ј and a control against clusterin (shControl-CLU) was F 5Ј-TCCCGCTCAGCTGACTAGAAGAATTCAAGAGATTCTTC-TAGTCAGCTGAGCTTTTTC-3Ј and R 5Ј-TCGAGAAAAAGCTCAGCT-GACTAGAA GAATCTCTTGAATTCTTCTAGTCAGCTGAGC-3Ј.…”

“…Immunoblotting-Immunoblotting was performed as previously described (20). Primary antibodies against E6AP (E6AP-330, Sigma-Aldrich), clusterin (41D, Millipore, Melbourne, Victoria, Australia), CDK6 (C21, Santa Cruz, Dallas, TX), HSP60 (H-300, Santa Cruz), HA (3F10, Roche, Melbourne, Victoria, Australia) and GFP (B2, Santa Cruz) were used.…”

“…Xenografts Study-DU145 xenografts experiments were performed as explained in (20). The animal experiments were performed with the ethics approval of the Peter MacCallum Institute Animal Experimentation Committee.…”