p53-independent PUMA induction in anti-cancer therapies

描述（由申请方提供）：BH 3 A是一种仅BH 3的蛋白质，在遗传毒性和非遗传毒性应激反应中引发细胞凋亡。 BclA与抗凋亡Bcl-2蛋白家族的成员（如Bcl-2、Bcl-XL和Mcl-1）结合，以释放Bax并触发导致凋亡的大量下游事件。 由基因毒性应激引起的p53和凋亡诱导依赖于p53，p53是一种在约50%的癌症中失活的肿瘤抑制因子。 最近，我们的实验室发现，cDNAA是必要的非遗传毒性剂，如激酶抑制剂staurosporine及其类似物UCN-01，通过p53非依赖性机制诱导的细胞凋亡。 初步研究表明，转录因子叉头盒O3 A（FoxO 3a）负责激酶抑制剂激活的p53 A。 我建议测试的假设，FoxO 3a介导的cDNAA诱导可用于恢复p53缺陷的人类癌细胞的凋亡调控。 目的1探讨激酶抑制剂对大肠癌细胞中p53非依赖性p53蛋白表达的诱导机制。 我将使用siRNA，蛋白质印迹，荧光素酶测定和染色质免疫沉淀，以确定FoxO 3a是否在激酶抑制剂治疗后的BRA诱导中发挥直接作用。 我还将探索激酶抑制激活FoxO 3a和FoxA的上游途径。 目的2：探讨在激酶抑制剂诱导的细胞凋亡中，CD 45 A的功能作用及其在体内的治疗作用。 我将确定激酶抑制剂UCN-01诱导的细胞凋亡事件是否需要CD 4A，以及UCN- 01的化学增敏作用是否需要CD 4A。 我还将研究是否UCN-01在体内介导的抗肿瘤作用，通过促进细胞凋亡。 目的3旨在开发一种用于鉴定在p53缺陷的人癌细胞中激活p53 A的新试剂的筛选测定。 我将设计一个稳定的p53缺陷细胞系，表达由FoxO 3a反应元件驱动的荧光素酶报告基因。 与我的共同赞助者的实验室合作，我将使用该细胞系通过高通量测定筛选FDA批准的药物，以鉴定独立于p53诱导p53的药物。 将进一步测试阳性试剂在其他结直肠癌细胞系中诱导CD 45 A和凋亡的能力。 公共卫生相关性：选择性杀死癌细胞对于抗癌疗法至关重要。 通过激活p53-非依赖性的p53 A诱导途径恢复人癌细胞的凋亡可能提供改进的治疗策略和药物。

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.