Cell death regulation in proteotoxic therapy

描述（由申请人提供）：抑制蛋白质降解是一种新兴的抗癌策略。蛋白酶体抑制剂硼替佐米已被FDA批准用于治疗多发性骨髓瘤，并正在许多癌症中进行试验。然而，潜在的分子机制仍然难以捉摸。此外，目前还不清楚某些分子特征是否可用于预测基于蛋白酶体转运蛋白的治疗的结果。我们最近报道，蛋白酶体抑制剂可以诱导细胞内聚集和激活caspase-8和随后的凋亡。这种半胱天冬酶-8的激活是通过其与泛素结合蛋白SQSTM 1/p62和自噬相关蛋白微管相关蛋白轻链3（LC 3）的结合来介导的。这些发现促使我们假设存在一种以前未表征的细胞死亡机制，该机制涉及蛋白质聚集体形成和细胞内caspase-8的活化。沿着这个方向，我们的额外的初步结果表明，p62本身进行泛素化。p62的这种新修饰可能在介导聚集体形成和凋亡中发挥关键作用。 我们还报道了内源性丝氨酸/半胱氨酸蛋白酶抑制剂SerpinB 3（也称为鳞状细胞癌抗原1，SCCA 1）可能作为预测基于蛋白毒性的抗癌治疗疗效的分子特征。SCCA 1是一种细胞内丝氨酸蛋白酶抑制剂，可抑制溶酶体蛋白酶。SCCA 1被认为限制了由溶酶体蛋白酶的非计划性激活引起的细胞损伤，这对细胞是有害的，因此可能有助于肿瘤发生和化学抗性。包括我实验室最近的研究在内的研究表明，SCCA 1表达升高与许多晚期人类癌症（如肺、头颈部和食管鳞状细胞癌以及肝细胞癌和乳腺癌）的预后较差相关。事实上，在分子水平上，我们发现SCCA 1保护细胞免受DNA烷基化损伤和氧化应激诱导的溶酶体损伤。另一方面，我们还发现SCCA 1在蛋白毒性应激中促进细胞凋亡。因此，SCCA 1一方面可以通过保护细胞免受溶酶体损伤而赋予对化疗的抗性，另一方面，它可以使癌细胞对蛋白毒性敏感。 该提案旨在了解蛋白酶体抑制剂抗肿瘤作用的分子机制，并确定癌细胞中的某些分子变化（如LC 3或SCCA 1表达升高）是否会使肿瘤细胞对体内蛋白毒性药物敏感。我们提出了三个分子和临床相关的具体目标：1）表征caspase-8在抑制蛋白酶体降解时的活化。2)研究p62调节聚集体形成和caspase-8激活的机制。3)检查某些分子如LC 3和SCCA 1可以使肿瘤对体内蛋白毒性剂敏感的假设。完成这一项目将对理解基于蛋白毒性的抗癌治疗的分子基础以及蛋白质聚集体在许多病理条件下的生理相关性产生普遍影响。在临床水平上，它可以建立LC 3或SCCA 1作为建议用蛋白毒性剂治疗的分子标记。

DESCRIPTION (provided by applicant): Inhibition of protein degradation is an emerging anti-cancer strategy. The proteasome inhibitor Bortezomib has been approved by FDA for treatment of multiple myeloma, and is being trialed in numerous cancers. However, the underlying molecular mechanisms remain elusive. Moreover, it has been unclear whether certain molecular signatures can be used to predict the outcome of proteasome inhibitor-based therapy. We recently reported that proteasome inhibitors can induce an intracellular aggregation and activation of caspase-8 and subsequent apoptosis. This caspase-8 activation is mediated by its association with a ubiquitin-binding protein SQSTM1/p62 and an autophagy-related protein microtubule-associated protein light chain 3 (LC3). These findings prompt us to hypothesize that there exists a previously uncharacterized cell death mechanism that involves protein aggregate formation and intracellular activation of caspase-8. Along this direction, our additional preliminary results show that p62 itself undergoes ubiquitination. This novel modification of p62 may play a critical function in mediating aggregate formation and apoptosis. We also reported that an endogenous serine/cysteine protease inhibitor SerpinB3 (also termed squamous cell carcinoma antigen 1, SCCA1) may function as a molecular signature for predicting efficacy with proteotoxicity-based anti-cancer therapy. SCCA1 is an intracellular serpin that inhibits lysosomal proteases. SCCA1 is believed to limit cellular damage resulting from unscheduled activation of lysosomal protease that is detrimental to the cell, hence may contribute to tumorigenesis and chemo-resistance. Studies including those recently from my laboratory have demonstrated that elevated SCCA1 expression is associated with poorer prognosis in numerous advanced human cancers such as squamous cell carcinomas of lung, head and neck, and esophagus, as well as hepatocellular carcinoma and breast carcinoma. Indeed, at the molecular level, we found that SCCA1 protects cells from lysosomal injury induced by DNA alkylating damage and oxidative stress. On the other hand, we also found that SCCA1 promotes apoptosis in response to proteotoxic stress. Therefore, SCCA1 on one hand may confer resistance to chemotherapy by protecting cells against lysosomal injury, on the other hand, it may sensitize cancer cells to proteotoxicity. This proposal is designed to understand the molecular mechanisms underlying the anti-tumor effect of proteasome inhibitors, and to determine whether certain molecular changes in cancer cells such as elevated expression of LC3 or SCCA1 can confer tumor cell sensitive to proteotoxic agents in vivo. We propose three molecularly and clinically related Specific Aims: 1) Characterize the activation of caspase-8 upon the inhibition of proteasome degradation. 2) Study the mechanisms through which p62 regulates aggregate formation and caspase-8 activation. 3) Examine the hypothesis that certain molecules such as LC3 and SCCA1 can sensitize tumors to proteotoxic agents in vivo. Accomplishing this project will have a general impact on the understanding of the molecular basis for proteotoxicity-based anti-cancer therapy, as well as on the physiological relevance of protein aggregates in many pathological conditions. At the clinical level, it may establish LC3 or SCCA1 as a molecular signature for suggesting treatment with proteotoxic agents.