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批准用于多发性骨髓瘤的治疗，并正在进行多种癌症的试验。然而，潜在的分子机制仍然难以捉摸。此外，目前尚不清楚某些分子特征是否可用于预测基于蛋白酶体抑制剂的治疗的结果。我们最近报道蛋白酶体抑制剂可以诱导细胞内聚集和caspase-8的激活以及随后的细胞凋亡。caspase-8的激活是通过其与泛素结合蛋白SQSTM1/p62和自噬相关蛋白微管相关蛋白轻链3 （LC3）的关联介导的。这些发现促使我们假设存在一种以前未被表征的细胞死亡机制，该机制涉及蛋白质聚集形成和细胞内caspase-8的激活。沿着这个方向，我们额外的初步结果表明p62本身经历了泛素化。这种新的p62修饰可能在介导聚集体形成和细胞凋亡中起关键作用。我们还报道了内源性丝氨酸/半胱氨酸蛋白酶抑制剂SerpinB3（也称为鳞状细胞癌抗原1，SCCA1）可能作为预测基于蛋白质毒性的抗癌治疗疗效的分子标记。SCCA1是一种抑制溶酶体蛋白酶的细胞内丝氨酸蛋白酶。SCCA1被认为限制了由溶酶体蛋白酶的非预定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耐药。包括我的实验室最近的研究表明，SCCA1表达升高与许多晚期人类癌症（如肺癌、头颈癌、食道癌、肝细胞癌和乳腺癌）的预后较差有关。事实上，在分子水平上，我们发现SCCA1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SCCA1在响应蛋白毒性应激时促进细胞凋亡。因此，SCCA1一方面可能通过保护细胞免受溶酶体损伤而赋予化疗耐药性，另一方面，它可能使癌细胞对蛋白质毒性敏感。本研究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LC3或SCCA1的表达升高）是否会赋予肿瘤细胞对体内蛋白质毒性药物的敏感性。我们提出了三个分子和临床相关的特定目标：1)表征caspase-8在抑制蛋白酶体降解时的激活。2)研究p62调控聚集体形成和caspase-8活化的机制。3)验证LC3、SCCA1等分子在体内对蛋白毒性药物致敏的假设。完成这个项目将对理解基于蛋白质毒性的抗癌治疗的分子基础以及在许多病理条件下蛋白质聚集的生理相关性产生普遍影响。在临床水平上，它可能建立LC3或SCCA1作为建议使用蛋白毒性药物治疗的分子标志。

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.