PKC/PKA Regulation in Prostate Cancer by SSeCKS/Gravin/AKAP12

SSeCKS/Gravin/AKAP12 对前列腺癌的 PKC/PKA 调节

• 批准号: 7883003
• 负责人: IRWIN H. GELMAN
• 金额: $ 28.98万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7883003
• 关键词: Actomyosin; Apoptosis; Attenuated; Binding; Biochemical; CDK4 gene; Cell Aging; Cell Line; Cells; Complex; Conflict (Psychology); Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Cytokinesis; DNA; Data; Defect; Disease Progression; Epithelial Cells; Equilibrium; Excision; Exhibits; F-Actin; Family; Fibroblasts; Figs - dietary; Fluorescence; Focal Dysplasia; Genetic; Gleason Grade for Prostate Cancer; Gravin; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Human papillomavirus 16; Hyperplasia; In Vitro; Isoenzymes; Knock-out; Knockout Mice; LIMK1 gene; Lead; MEKs; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Mediator of activation protein; Microscopy; Mitotic; Modeling; Mus; Neoplasm Metastasis; Oncogenes; Oncogenic; Pathway interactions; Phorbol Esters; Phosphotransferases; Polyploidy; Prostate; Prostatic Intraepithelial Neoplasias; Prostatic hypertrophy; Protein Kinase; Protein Kinase C; Proteins; Reactive Oxygen Species; Regulation; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; TP53 gene; Techniques; Time; Tissues; Transgenic Mice; Tumor Suppressor Proteins; Vascular Endothelial Growth Factors; base; cofilin; in vivo Model; mouse model; neovascularization; novel; overexpression; premature; promoter; public health relevance; research study; rho; scaffold; senescence; telophase; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): Activation of protein kinase (PK) C has been considered cancer-promoting based on early data showing that phorbol esters could induce oncogenes is through the activation of so called classic and novel PKC isozymes. Although many human cancers exhibit increased levels of specific PKC isozymes there are conflicting data which show that specific isozymes can act as either promoters or suppressors of oncogenesis depending on tissue context. In human prostate cancer (CaP), PKC, levels increase with malignancy whereas PKC* levels are constant, yet only the overexpression of PKC, is sufficient to induce prostatic intraepithelial neoplasia (PIN). Therefore, characterization of the pro-oncogenic PKC-induced signaling pathways and mediators is paramount to our overall understanding of CaP disease progression. Our lab identified a novel PKC substrate, SSeCKS/Gravin/AKAP12 ("SSeCKS") that is also a scaffold for both PKC and PKA, capable of attenuating PKC kinase activity and altering its cellular compartmentalization, while regulating PKA through compartmentalization only. SSeCKS displays many of the hallmarks of a tumor suppressor in prostate cancer: it is severely downregulated in human CaP cell lines and tissues with Gleason sums =6, SSeCKS reexpression suppresses macroscopic CaP metastasis growth by inhibiting VEGF-induced neovascularization, genetic knockout (KO) in mice induces prostatic hyperplasia and focal dysplasia with evidence of epithelial cell senescence, and KO mouse fibroblasts (MEF) display premature senescence correlating with polyploidy and binucleation. Premature senescence seems Rb-dependent because it can be suppressed by HPV-16E7orpRb-siRNA but not byHPV-16 E6 or p53-siRNA. In keeping with SSeCKS' scaffolding function for PKC, KO-MEF have >2-fold higher total PKC activity than WT-MEF, with a >3-fold increase in PKC* activity. Based on preliminary data from expression microarray and biochemical experiments, we hypothesize the loss of SSeCKS induces hyperactive PKC*/,,which in turn induce i) cytokinesis defects through the activation of RhoA and LIMK, and through the suppression of the mitotic exit network kinase, WARTS, and ii) Rb-dependent senescence by increasing reactive oxygen species (ROS) mediators such as p47phox. We believe SSeCKS controls cytokinesis directly because a pool of SSeCKS enriches in the cleavage furrow during telophase where PKC* and , are known to normally regulate abscission by coordinating the timing of RhoA and LIMK activation, and thus, formation and contraction of the actomyosin abscission ring. Our overall aim is to use genetic, biochemical, and fluorescence and time-lapse microscopy techniques to dissect this novel SSeCKS-PKC pathway in WT vs. KO MEF and murine prostate epithelial cells (PrEC), and in human PrEC vs. CaP cell lines, using readouts of senescence/ polyploidy/ binucleation and cytokinesis completion (Aim 1), podosome formation and tumor invasiveness (Aim 2), and CaP formation, invasiveness and metastatic potential in transgenic mouse models (Aim 3). The experiments proposed are envisioned to elucidate the mechanism and pathways controlled by the SSeCKS/PKC scaffold complex and how dysregulation by the loss of SSeCKS contributes to CaP progression. PUBLIC HEALTH RELEVANCE: Our project focuses on SSeCKS, a protein that seems to suppress prostate cancer progression especially metastasis, by binding two opposing signaling proteins, PKA and PKC, and controlling their function by regulating when and where in the cell they are activated. We now propose to elaborate on pro-cancer pathways that PKC induces, yet which are normally regulated by SSeCKS. These studies will elucidate how the loss of SSeCKS expression in prostate cancer increases disease progression by allowing PKC to be hyperactivated and dysregulated.

描述（由申请人提供）：根据早期数据显示，磷酸酯可以通过激活所谓的经典和新型PKC同工酶诱导致癌基因，活化蛋白激酶（PK） C被认为是促进癌症的。尽管许多人类癌症表现出特异性PKC同工酶水平的增加，但有相互矛盾的数据表明，特异性同工酶可以根据组织环境作为肿瘤发生的促进因子或抑制因子。在人类前列腺癌（CaP）中，PKC水平随着恶性肿瘤而升高，而PKC*水平不变，但只有PKC的过表达才足以诱导前列腺上皮内瘤变（PIN）。因此，表征pkc诱导的促癌信号通路和介质对我们全面了解CaP疾病进展至关重要。我们的实验室发现了一种新的PKC底物SSeCKS/Gravin/AKAP12（“SSeCKS”），它也是PKC和PKA的支架，能够减弱PKC激酶活性并改变其细胞区隔化，同时仅通过区隔化调节PKA。SSeCKS在前列腺癌中表现出许多肿瘤抑制因子的特征：SSeCKS在人CaP细胞系和组织中严重下调，Gleason sum =6； SSeCKS重表达通过抑制vegf诱导的新生血管形成抑制宏观CaP转移生长；小鼠基因敲除（KO）诱导前列腺增生和局灶性不典型增生，并伴有上皮细胞衰老；KO小鼠成纤维细胞（MEF）表现出与多倍体和双核相关的过早衰老。过早衰老似乎依赖于rb，因为它可以被HPV-16E7orpRb-siRNA抑制，但不能被hpv - 16e6或p53-siRNA抑制。根据SSeCKS对PKC的脚手架功能，KO-MEF的PKC总活性比WT-MEF高100倍，PKC*活性比WT-MEF高100倍。基于表达芯片和生化实验的初步数据，我们假设SSeCKS的缺失导致PKC*/过度活跃，进而通过RhoA和LIMK的激活，以及通过抑制有丝分裂退出网络激酶、疣，诱导i)细胞分裂缺陷，以及ii)通过增加活性氧（ROS）介质（如p47phox）诱导rb依赖性衰老。我们认为SSeCKS直接控制细胞分裂，因为在分裂末期，PKC*和PKC通常通过协调RhoA和LIMK激活的时间来调节分离，从而调节肌动球蛋白脱落环的形成和收缩。我们的总体目标是使用遗传，生化，荧光和延时显微镜技术来解剖WT与KO MEF和小鼠前列腺上皮细胞（PrEC）以及人类PrEC与CaP细胞系中的这种新型SSeCKS-PKC途径，使用衰老/多倍体/双核和细胞分裂完成（aim 1）， podosome形成和肿瘤侵袭性（aim 2），以及转基因小鼠模型中的CaP形成，侵袭性和转移潜力（aim 3）。所提出的实验旨在阐明SSeCKS/PKC支架复合物控制的机制和途径，以及SSeCKS缺失导致的失调如何促进CaP进展。