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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）：胰腺癌是一种破坏性疾病，诊断后5年的生存率为4%，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，非常需要在胰腺癌细胞中鉴定可以用于治疗益处的新的细胞靶点。在包括胰腺癌细胞在内的所有肿瘤细胞中，跨越细胞周期检查点的转变依赖于Cdk活化激酶（CAK）对细胞周期蛋白：Cdk复合物的活化。CAK代表了胰腺癌治疗性干预治疗的潜在新型阿基里斯之踵型靶点，其将影响细胞周期中的许多位置。然而，CAK的身份仍然不清楚。高分子量CAK复合物之前被鉴定为细胞周期蛋白H：cdk 7;然而，细胞周期蛋白H：cdk 7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Cdk 7的假定CAK活性的实验也导致Cdk 7 TFIIH对转录延伸的全局破坏，从而使解释成为问题。此外，我们发现，Cdk 7在细胞中的选择性失活的结果在体内CAK活性的维护，从而质疑Cdk 7的作用作为CAK。以前，我们已经检测到第二个低分子量CAK活性，这是完全独立的Cdk 7。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），磷酸化和激活细胞周期蛋白：Cdk复合物需要驱动胰腺癌细胞通过细胞周期检查点。如果正确的话，这种CAK是一种潜在的新的治疗靶点，治疗胰腺癌，其失活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激活的限速因子。目标1：生物化学纯化和鉴定低分子量CAK基因我们将从含有组成型活性Cdk 2的Panc 1胰腺癌细胞中分离低分子量CAK复合物，因此，根据定义，也含有组成型表达的CAK，并通过质谱鉴定其序列并通过RNAi证实。目标二。无偏RNAi筛选以鉴定来自胰腺细胞的CAK基因。我们将在Panc 1胰腺癌细胞中进行无偏倚的激酶组RNAi筛选。在人类基因组中有约448种已证实和推定的Ser/Thr激酶，因此，这是可管理的，作为低通量，但使用高验证的高滴度慢病毒shRNA文库产生高信息的RNAi筛选。