p53-independent PUMA induction in anti-cancer therapies

描述(由申请人提供)：PUMA是一种仅与BH3结合的蛋白质，可在遗传毒性和非遗传毒性压力下引发细胞凋亡。Puma与抗凋亡蛋白Bcl2家族的成员如Bcl2、Bclxl和Mcl-1结合，释放Bax并触发多种下游事件导致细胞凋亡。基因毒性应激诱导的PUMA和细胞凋亡依赖于P53，P53是一种肿瘤抑制因子，在约50%的癌症中处于失活状态。最近，我们的实验室发现PUMA在非遗传毒性药物诱导的细胞凋亡中是必需的，如激酶抑制剂星形孢子素及其类似物UCN-01，通过不依赖于P53的机制。初步研究表明，转录因子Forkhead box O3a(FOXO3a)负责PUMA被激酶抑制剂激活。我建议测试FOXO3a介导的PUMA诱导可以用来恢复p53缺失的人类癌细胞的凋亡调控的假设。目的1探讨蛋白激酶抑制剂诱导结直肠癌细胞PUMA非P53依赖性表达的机制。我将使用siRNA、Western blotting、荧光素酶分析和染色质免疫沉淀来确定FOXO3a是否在激酶抑制剂治疗后PUMA的诱导中起直接作用。我还将探索激酶抑制激活FOXO3a和PUMA的上游途径。目的2研究PUMA在蛋白激酶抑制剂诱导的细胞凋亡中的作用及其体内治疗作用。我将确定UCN-01诱导的细胞凋亡事件是否需要PUMA，以及UCN-01的化疗增敏作用是否需要PUMA。我还将研究PUMA是否通过促进细胞凋亡来介导UCN-01的体内抗肿瘤作用。AIM 3旨在开发一种筛选试验，以确定在P53缺失的人类癌细胞中激活PUMA的新药物。我将设计一个稳定的P53缺失细胞系，表达由PUMA启动子的FOXO3a反应元件驱动的荧光素酶报告基因。在与我的联合赞助商的实验室的合作下，我将使用这个细胞系通过高通量测试来筛选FDA批准的药物，以识别独立于p53的诱导PUMA的药物。这些阳性试剂将进一步测试它们诱导PUMA和其他结直肠癌细胞株凋亡的能力。与公共卫生相关：选择性杀灭癌细胞是抗癌治疗的关键。通过激活不依赖于p53的PUMA诱导通路恢复人类癌细胞的凋亡，可能会提供更好的治疗策略和药物。

DESCRIPTION (provided by applicant): PUMA is a BH3-only protein that elicits the initiation of apoptosis in response to genotoxic and non-genotoxic stresses. PUMA binds to members of the anti-apoptotic Bcl-2 family of proteins, such as Bcl-2, Bcl-XL, and Mcl-1, to release Bax and trigger a multitude of downstream events leading to apoptosis. PUMA and apoptosis induction by genotoxic stress is dependent on p53, a tumor suppressor inactivated in ~50% of cancers. Recently, our laboratory discovered that PUMA is necessary for apoptosis induced by non-genotoxic agents, such as the kinase inhibitor staurosporine and its analogue UCN-01, through p53-independent mechanisms. Preliminary studies suggest that the transcription factor forkhead box O3A (FoxO3a) is responsible for PUMA activation by kinase inhibitors. I propose to test the hypothesis that FoxO3a-mediated PUMA induction can be used to restore apoptosis regulation in p53-deficient human cancer cells. Aim 1 will investigate the mechanism of p53-independent induction of PUMA by kinase inhibitors in colorectal cancer cells. I will use siRNA, western blotting, luciferase assays, and chromatin immunoprecipitation to determine whether FoxO3a plays a direct role in PUMA induction following kinase inhibitor treatment. I will also explore the upstream pathways through which kinase inhibition activates FoxO3a and PUMA. Aim 2 will determine the functional role of PUMA in apoptosis induced by kinase inhibitors and its therapeutic effect in vivo. I will determine if PUMA is required for the apoptotic events induced by the kinase inhibitor UCN-01, and if PUMA is required for the chemosensitization effect of UCN- 01. I will also investigate if PUMA mediates the anti-tumor effect of UCN-01 in vivo by promoting apoptosis. Aim 3 is designed to develop a screening assay for identifying novel agents that activate PUMA in p53-deficient human cancer cells. I will engineer a stable p53-deficient cell line expressing a luciferase reporter driven by the FoxO3a response elements from the PUMA promoter. In collaboration with my Co-Sponsor's laboratory, I will use this cell line to screen FDA-approved drugs via a high-throughput assay to identify agents that induce PUMA independently of p53. The positive agents will be further tested for their ability to induce PUMA and apoptosis in other colorectal cancer cell lines. PUBLIC HEALTH RELEVANCE: Selective killing of cancer cells is essential for anti-cancer therapies. Restoring apoptosis in human cancer cells by activating p53-independent PUMA induction pathways may afford improved therapeutic strategies and agents.