前列腺癌SPOP基因高频突变导致肿瘤的表观遗传学机制研究

Characterization of the exome and genome of prostate cancers by next-generation sequencing has identified numerous genetic alternations. SPOP was identified as one of the most frequently affected genes by somatic point mutations in prostate cancer(6-15%), suggesting SPOP is potentially a key driver for prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer remains to be elucidated. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex, selectively recruits substrates for their ubiquitination and subsequent degradation. The bromodomain and extra-terminal (BET) protein family members, including BRD2,3,4 and BRDT, bind to acetylated lysines on histones and regulate mitosis and cell proliferation. In this study, we identified BRD2/3/4 as a bona fide substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes a Ser/Thr (S/T)-rich degron in the transactivation domain of BRD2/3/4 and triggers BRD2/3/4 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting BRD2/3/4 degradation. This study revealed novel molecular events underlying the regulation of BRD2/3/4 protein homeostasis, and provided insights in understanding the relationship between SPOP mutations and epigenetics dysregulation in prostate cancer.

最近的几项高通量测序研究发现SPOP基因在前列腺癌中发生高频突变（6-15%）。但SPOP突变对于前列腺癌发生、发展的生物学意义还不清楚。SPOP是Cullin3家族泛素E3连接酶复合体的一个底物识别亚基，介导底物的泛素化降解。BET蛋白（BRD2、3、4和BRDT）是一类可以结合乙酰化组蛋白的表观遗传调控因子，在肿瘤细胞的恶性增殖和侵袭过程中发挥了重要的促进作用，是抗肿瘤药物设计的“明星”靶点。我们通过酵母双杂交方法发现BRD2/3/4是SPOP的互作蛋白。SPOP可促进BRD2/3/4的泛素化降解，但前列腺癌来源的SPOP突变体丧失了对BRD2/3/4的泛素化降解作用。本项研究将在分子、细胞、模式动物和肿瘤样本等多个层次，阐明前列腺癌中SPOP突变导致的BRD2/3/4蛋白异常积累所引起的表观遗传学效应和生物学表型。并为BET蛋白抑制剂应用于前列腺癌的个性化治疗提供理论依据。

前列腺癌是目前全世界男性最常见的癌症之一。近些年来，前列腺癌的高通量测序研究发现SPOP基因在前列腺癌中发生高频率的点突变。野生型SPOP能抑制前列腺癌细胞的增殖和侵袭，而突变型SPOP如SPOP-F102C、F133V则能显著促进细胞的增殖和侵袭。SPOP是Cul3泛素E3连接酶复合体的底物识别亚基。BET蛋白（BRD2、3和4）是一类可以和乙酰化组蛋白结合的表观遗传学调控蛋白，它控制了C-Myc、PIM1和BCL2等促生长、抗凋亡靶基因的转录。BET蛋白是目前以表观遗传蛋白为靶点的抗肿瘤药物设计的“明星分子”。BET小分子抑制剂如JQ1、iBET对多种肿瘤细胞表现出极佳的杀伤效果，已在前列腺癌中开展一期临床试验。我们发现野生型SPOP能通过识别它们共同的降解决定子序列与BET蛋白（BRD2，BRD3和BRD4）结合，泛素化并通过蛋白酶体降解BET蛋白。相反，前列腺癌来源的SPOP突变大大减弱与BET蛋白的结合，导致这些蛋白在在前列腺癌细胞和病人标本中积累，引起细胞对BET抑制剂的原发耐药。 转录组和BRD4顺反子分析显示由于BRD4蛋白稳定而导致GTPase结合蛋白 RAC1和胆固醇合成相关基因的表达增强和AKT-mTORC1信号的激活。我们的数据表明，与AKT抑制剂联用可以克服SPOP突变的前列腺癌对BET抑制剂的耐药性，可被联合使用AKT抑制剂而逆转。这些结论进一步支持SPOP突变可作为生物标记物指导BET抑制剂为导向的前列腺癌病人的治疗。