Novel Cell Cycle Therapeutic Targets in Pancreatic Cancer

胰腺癌的新细胞周期治疗靶点

基本信息

批准号：
8511187
负责人：
STEVEN F DOWDY
金额：
$ 20.23万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-03-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8511187
关键词：
Acute Affect Biochemical Biological Assay C-terminal Cancer Etiology Cell Cycle Cell Cycle Checkpoint Cell Cycle Progression Cell Extracts Cells Complex Cyclin A Cyclin B Cyclin D1 Cyclin E Cyclins DNA Polymerase II Diagnosis Disease Genes Genetic Goals Grant Human Human Genome In Vitro Libraries MADH4 gene Maintenance Malignant neoplasm of pancreas Mass Spectrum Analysis Mitosis Molecular Weight Mutation Oncogenes Pancreas Phosphorylation Phosphotransferases Positioning Attribute RNA RNA Interference RNA Polymerase II Regimen Regulator Genes Role S Phase Survival Rate Therapeutic Therapeutic Intervention Threonine Transcription Initiation Work cellular targeting cyclin H cyclin-dependent kinase-activating kinase genetic analysis in vivo mortality neoplastic cell new therapeutic target novel overexpression pancreatic cancer cells public health relevance research study small hairpin RNA therapeutic target transcription factor TFIIH tumor

项目摘要

DESCRIPTION (provided by applicant): With a 4% survival rate at 5 years after diagnosis, pancreatic cancer is a devastating disease that remains essentially incurable with current therapeutic regimens and is the fourth leading cause of cancer mortality. Thus, there is a great need to identify novel cellular targets in pancreatic cancer cells that can be exploited for therapeutic benefit. In all tumor cells, including pancreatic cancer cells, transition across cell cycle checkpoints is dependent on activation of cyclin:Cdk complexes by Cdk Activating Kinase (CAK). CAK represents a potential novel Achilles' heel type of target for therapeutic intervention treatment in pancreatic cancer that would impact many positions in the cell cycle. However, the identity of CAK remains unclear. A high molecular weight CAK complex was previously identified as cyclin H:Cdk7; however, cyclin H:cdk7 is also the bona fide kinase in TFIIH, a kinase that activates RNA polymerase II for initiation of transcription. Consequently, all experiments that disrupted the putative CAK activity of Cdk7 also resulted in global disruption of transcriptional elongation by Cdk7 TFIIH, thereby making interpretation problematic. Moreover, we find that selective inactivation of Cdk7 in cells results in the maintenance of in vivo CAK activity and thereby questions Cdk7's role as a CAK. Previously, we have detected a second Low Molecular Weight CAK activity that is entirely independent of Cdk7. Our central hypothesis is that the low molecular weight complex contains the relevant Cdk Activating Kinase (CAK) that phosphorylates and activates cyclin:Cdk complexes required to drive pancreatic cancer cells across cell cycle checkpoints. If correct, this CAK is a potential novel therapeutic target for the treatment of pancreatic cancer whose inactivation would simultaneously affect multiple cell cycle checkpoints. Our goal for this R21 exploratory application is to isolate the Low Mw CAK gene(s) and determine if CAK is rate-limiting for cyclin:Cdk activation. Aim 1: Biochemically Purify and Identify the Low Mw CAK Gene(s) We will isolate the Low Mw CAK complex from Panc1 pancreatic cancer cells that contain constitutively active Cdk2, and hence, by definition, also contain constitutively express CAK, and identify it's sequence by mass spectrometry and confirm by RNAi. Aim 2. Unbiased RNAi Screen to Identify the CAK Gene(s) from Pancreatic Cells. We will perform an unbiased kinome RNAi screen in Panc1 pancreatic cancer cells. There are ~448 proven and putative Ser/Thr kinases in the human genome and therefore, this is manageable as a low throughput, but high information generating RNAi screen using a high validated, high titer lentiviral shRNA library.

描述（由申请人提供）：胰腺癌诊断后 5 年生存率为 4%，它是一种毁灭性的疾病，目前的治疗方案基本上无法治愈，并且是癌症死亡的第四大原因。因此，非常需要鉴定胰腺癌细胞中可用于治疗益处的新细胞靶点。在包括胰腺癌细胞在内的所有肿瘤细胞中，跨细胞周期检查点的转变取决于 Cdk 激活激酶 (CAK) 对细胞周期蛋白：Cdk 复合物的激活。 CAK 代表了胰腺癌治疗干预治疗的潜在新型致命弱点类型，它将影响细胞周期中的许多位置。然而，CAK 的身份仍不清楚。高分子量 CAK 复合物先前被鉴定为细胞周期蛋白 H:Cdk7；然而，cyclin H:cdk7 也是 TFIIH 中真正的激酶，TFIIH 是一种激活 RNA 聚合酶 II 以启动转录的激酶。因此，所有破坏 Cdk7 推定 CAK 活性的实验也会导致 Cdk7 TFIIH 转录延伸的整体破坏，从而导致解释出现问题。此外，我们发现细胞中 Cdk7 的选择性失活导致体内 CAK 活性的维持，从而质疑 Cdk7 作为 CAK 的作用。此前，我们检测到了第二种完全独立于 Cdk7 的低分子量 CAK 活性。我们的中心假设是，低分子量复合物含有相关的 Cdk 激活激酶 (CAK)，它可以磷酸化并激活细胞周期蛋白：驱动胰腺癌细胞跨过细胞周期检查点所需的 Cdk 复合物。如果正确的话，该 CAK 是一个潜在的新型治疗靶点胰腺癌的治疗，其失活会同时影响多个细胞周期检查点。我们此 R21 探索性应用的目标是分离低分子量 CAK 基因并确定 CAK 是否是细胞周期蛋白：Cdk 激活的限速剂。目标 1：生化纯化和鉴定低分子量 CAK 基因我们将从含有组成型活性 Cdk2 的 Panc1 胰腺癌细胞中分离出低分子量 CAK 复合物，因此，根据定义，也含有组成型表达 CAK，并鉴定其序列通过质谱分析并通过 RNAi 进行确认。目标 2. 通过无偏 RNAi 筛选来识别胰腺细胞中的 CAK 基因。我们将在 Panc1 胰腺癌细胞中进行公正的激酶组 RNAi 筛选。人类基因组中有约 448 个经过验证和推定的 Ser/Thr 激酶，因此，可以使用高验证、高滴度慢病毒 shRNA 文库作为低通量但高信息生成的 RNAi 筛选进行管理。