Role of Autophagy in Bortezomib-induced HNSCC Apoptosis

 描述（由申请人提供）：头颈部鳞状细胞癌（HNSCC）占头颈部癌症的90%，通常预后不良且对化疗耐药。因此，开发更有效的治疗策略至关重要。以前，硼替佐米已被提议作为HNSCC中常规化疗的替代化疗剂，但通过一个很大程度上未知的途径推断化疗耐药性。本申请的主要目的是研究曲古抑菌素A（TSA）和硼替佐米（又名PS-341）联合方案治疗HNSCC的潜在用途，并了解TSA增强硼替佐米诱导的细胞凋亡以有效克服化疗耐药性的分子机制。我们的初步研究表明，组蛋白去乙酰化酶6（HDAC 6）促进自噬，以减少硼替佐米的细胞毒性。当我们在HNSCC细胞中消除HDAC 6的表达时，自噬诱导被抑制，硼替佐米的细胞毒性显著增强。基于这些新的发现，我们假设TSA可能通过抑制HDAC 6活性增强硼替佐米的抗肿瘤作用，HDAC 6活性可能是HNSCC中自噬、凋亡和细胞存活反应之间的关键因果关系。我们的假设将在以下具体目标中得到解决：（1）确定TSA是否增强硼替佐米诱导的细胞凋亡，同时抑制HDAC 6介导的自噬;和（2）研究HDAC 6在HNSCC中启动硼替佐米诱导的自噬的分子机制。该计划将利用基础科学和转化医学方面的几个特征：绿色荧光蛋白（GFP）-LC 3斑点形成试验、流式细胞术、蛋白质印迹、实时qRT-PCR、染色质免疫沉淀和免疫荧光染色将用于检查与HDAC 6诱导的自噬和细胞毒性蛋白聚集体积累相关的分子表达变化。此外，我们计划利用HDAC 6缺失的人HNSCC细胞在裸鼠中的异种移植来研究与自噬、内质网应激和凋亡相关的分子和细胞变化。该研究策略是研究培训计划的一个组成部分，旨在培养受训者在头颈部致癌、耐药性和细胞凋亡的分子/细胞生物学和遗传学方面的专业知识，并为受训者作为独立研究人员的学术生涯做好准备。

 DESCRIPTION (provided by applicant): Head and Neck Squamous Cell Carcinoma (HNSCC) comprise 90% of head and neck cancer and typically presents poor prognosis and resistant to chemotherapy. Hence, the development of more effective therapeutic strategy is critical. Previously, Bortezomib has been proposed as an alternate chemotherapeutic agent to conventional chemotherapy in HNSCC, but infers chemoresistance through a largely unknown pathway. The main objectives of this application are to investigate the potential utilization of Trichostatin A (TSA) and Bortezomib (aka PS-341) combinatory regimen to treat HNSCC and to understand the molecular mechanisms by which TSA enhances Bortezomib-induced apoptosis to effectively overcome chemoresistance. Our preliminary studies suggest that histone deacetylase 6 (HDAC6) promotes autophagy to reduce the Bortezomib cytotoxicity. When we abolished expression of HDAC6 in HNSCC cells, autophagy induction was inhibited and the Bortezomib cytotoxicity was significantly enhanced. Based on these novel findings, we hypothesize that TSA may enhance antitumor effects of Bortezomib through inhibition of HDAC6 activity, which may serve as a critical causal link between autophagy, apoptosis, and cellular survival response in HNSCC. Our hypotheses will be addressed in the following specific aims: (1) Determine if TSA enhances Bortezomib- induced apoptosis while inhibiting HDAC6-mediated autophagy; and (2) Investigate the molecular mechanisms by which HDAC6 initiates autophagy induced by Bortezomib in HNSCC. This plan will utilize several features from both basic science and translational medicine aspects: Green fluorescent protein (GFP) - LC3 puncta formation assay, flow cytometry, western blotting, real-time qRT-PCR, chromatin immunoprecipitation, and immunofluorescence staining will be used to examine changes in expression of molecules associated with HDAC6-induced autophagy and accumulation of cytotoxic protein aggregates. Further, we plan to utilize xenotransplantation of HDAC6-depleted human HNSCC cells in nude mice to investigate molecular and cellular changes associated with autophagy, endoplasmic reticulum stress, and apoptosis. This research strategy is an integral component of a research training plan designed to develop the trainee's expertise in molecular/cellular biology and genetics of head and neck carcinogenesis, chemoresistance, and apoptosis and will prepare the trainee for an academic career as an independent researcher.