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）：诊断后5年的存活率为4％，胰腺癌是一种毁灭性疾病，与当前的治疗方案基本无法治愈，并且是癌症死亡率的第四个主要原因。因此，非常需要鉴定胰腺癌细胞中的新细胞靶标，这些细胞可以被用来用于治疗益处。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过渡取决于细胞周期蛋白的激活：通过CDK激活激酶（CAK）的CDK复合物的激活。 CAK代表了一种潜在的新型阿喀琉斯的脚跟类型，用于胰腺癌治疗治疗的靶标，这将影响细胞周期中的许多位置。但是，CAK的身份尚不清楚。以前，高分子量CAK复合物已被鉴定为Cyclin H：CDK7；但是，Cyclin H：CDK7也是TFIIH中的真正的FIDE激酶，这是一种激活RNA聚合酶II的激酶，用于启动转录。因此，所有破坏CDK7推定CAK活性的实验也导致CDK7 TFIIH对转录伸长的全球破坏，从而使解释问题有问题。此外，我们发现CDK7在细胞中的选择性失活导致维持体内CAK活性，从而质疑CDK7作为CAK的作用。以前，我们已经检测到第二个低分子量CAK活性，该活性完全独立于CDK7。我们的中心假设是，低分子量复合物包含相关的CDK激活激酶（CAK），该激活激酶（CAK）磷酸化和激活细胞周期蛋白：跨细胞周期检查点驱动胰腺癌细胞所需的CDK复合物。如果正确，此CAK是潜在的新型治疗靶标胰腺癌的治疗将同时影响多个细胞周期检查点。我们的R21探索性应用的目标是隔离低MW CAK基因，并确定CAK是否是细胞周期蛋白的速率限制：CDK激活。 AIM 1：在生化上纯化并鉴定低MW CAK基因（S）我们将从PANC1胰腺癌细胞中分离出低MW CAK复合物，这些胰腺癌细胞包含组成型活性CDK2，因此，根据定义，还包含组成型表达CAK，并通过质谱和RNAi确认其序列。 AIM 2。无偏的RNAi屏幕，以鉴定胰腺细胞中的CAK基因。我们将在PANC1胰腺癌细胞中执行无偏的Kinome RNAi筛选。在人类基因组中有〜448个经过证明的和推定的Ser/Thr激酶，因此，使用高验证的高滴度慢病毒ShRNA库可以作为低通量，但高信息生成RNAi屏幕。