Proteotranscriptomic profiling of potential E6AP targets in prostate cancer cells

Prostate cancer is a common cause of cancer-related death in men. E6AP, 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 tumour 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 approaches. 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 upon 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.

前列腺癌是男性癌症相关死亡的常见病因。E6AP作为一种E3泛素连接酶（E3 ubiquitin ligase）与转录辅因子（transcription cofactor），在部分前列腺癌患者体内表达上调。对前列腺癌细胞中的E6AP进行基因操作的实验显示，E6AP可在体外与体内环境中促进前列腺癌细胞的生长与存活。然而，E6AP对前列腺癌细胞的调控作用范围广泛，无法仅通过此前已确认的E6AP肿瘤抑制靶标——早幼粒细胞白血病蛋白（promyelocytic leukemia protein）与p27——得到完整解释。为探究E6AP下游调控的其他效应分子，本研究联合采用转录组学（transcriptomic）与蛋白质组学（proteomic）技术。我们在去势抵抗性前列腺癌细胞系DU145中鉴定并定量了16130条转录本与7209种蛋白质。敲低E6AP后，共有2763条转录本与308种蛋白质的表达发生显著改变。通路分析验证了E6AP敲低对前列腺癌细胞已知的表型效应，同时揭示了E6AP与癌症代谢、DNA损伤修复及免疫应答之间全新的潜在关联。通过实时聚合酶链反应（real-time polymerase chain reaction）验证了部分核心候选分子的表达变化。其中，在前列腺癌中常发生表达失调的应激诱导型分子伴侣蛋白簇蛋白（clusterin）被选为进一步研究对象。体外与体内实验均证实，敲低E6AP可导致簇蛋白的转录本与蛋白质水平升高。共敲低E6AP与簇蛋白的实验结果支持了簇蛋白参与E6AP诱导的表型调控这一结论。综上，本研究结果揭示了E6AP在前列腺癌细胞中调控的潜在生物学通路，并将簇蛋白鉴定为E6AP的新型靶标。