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: 8449707
• 负责人: IRWIN H. GELMAN
• 金额: $ 27.43万
• 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/8449707
• 关键词: AKAP12 gene; 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; Fluorescence; Focal Dysplasia; Genetic; Gleason Grade for Prostate Cancer; Gravin; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Human Papillomavirus; 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 C; Proteins; Reactive Oxygen Species; Regulation; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; 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.

描述（由申请人提供）：蛋白激酶（PK）C的激活被认为是促癌的，基于早期数据显示佛波醇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。尽管许多人类癌症表现出特定PKC同工酶水平的增加，但存在相互矛盾的数据，这些数据表明特定同工酶可以作为肿瘤发生的促进剂或抑制剂，这取决于组织环境。在人前列腺癌（CaP）中，PKC水平随着恶性程度增加而增加，而PKC* 水平是恒定的，然而仅PKC的过表达足以诱导前列腺上皮内瘤形成（PIN）。因此，表征的原癌PKC诱导的信号通路和介质是至关重要的，我们的整体理解CaP疾病的进展。我们的实验室鉴定了一种新的PKC底物SSeCKS/Gravin/AKAP 12（“SSeCKS”），其也是PKC和PKA的支架，能够减弱PKC激酶活性并改变其细胞区室化，同时仅通过区室化调节PKA。SSeCKS显示出前列腺癌中肿瘤抑制因子的许多特征：它在人CaP细胞系和组织中严重下调，Gleasonsum =6，SSeCKS再表达通过抑制VEGF诱导的新血管形成抑制宏观CaP转移生长，小鼠中的基因敲除（KO）诱导前列腺增生和局灶性发育异常，伴有上皮细胞衰老的证据，和KO小鼠成纤维细胞（MEF）显示与多倍性和双核相关的过早衰老。早衰似乎是Rb依赖性的，因为HPV-16 E7或pRb-siRNA可以抑制早衰，而HPV-16 E6或p53-siRNA不能抑制早衰。与SSeCKS对PKC的支架功能一致，KO-MEF具有比WT-MEF高>2倍的总PKC活性，其中PKC* 活性增加>3倍。基于来自表达微阵列和生物化学实验的初步数据，我们假设SSeCKS的损失诱导过度活跃的PKC* i，其进而诱导i）通过RhoA和LIMK的活化以及通过有丝分裂出口网络激酶WARTS的抑制的胞质分裂缺陷，和ii）通过增加活性氧类（ROS）介质如p47 phox的Rb依赖性衰老。我们相信SSeCKS直接控制胞质分裂，因为在分裂末期，SSeCKS池在分裂沟中富集，其中已知PKC* 和，通常通过协调RhoA和LIMK激活的时间来调节分裂，从而调节肌动球蛋白分裂环的形成和收缩。我们的总体目标是使用遗传、生物化学、荧光和延时显微镜技术来解剖WT与KO MEF和鼠前列腺上皮细胞（PrEC）以及人PrEC与CaP细胞系中的这种新型SSeCKS-PKC通路，使用衰老/多倍性/双核化和胞质分裂完成（Aim 1）、足体形成和肿瘤侵袭性（Aim 2）以及CaP形成的读数，在转基因小鼠模型中的侵袭性和转移潜力（目的3）。设想所提出的实验来阐明SSeCKS/PKC支架复合物控制的机制和途径，以及SSeCKS的丢失如何导致CaP进展的失调。