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

描述(申请人提供)：根据早期数据显示佛波酯可以通过激活所谓的经典和新的PKC同工酶来诱导癌基因，因此蛋白激酶(PK)C的激活被认为是促癌作用。虽然许多人类癌症表现出特定的PKC同工酶水平升高，但有相互矛盾的数据表明，特定的同工酶可以作为肿瘤发生的促进者或抑制者，这取决于组织背景。在前列腺癌(CAP)中，PKC水平随恶性程度的增加而升高，而PKC*水平不变，但只有PKC的过度表达才足以诱发前列腺上皮内瘤变(PIN)。因此，表征致癌的PKC诱导的信号通路和介体对于我们全面了解CAP疾病的进展是至关重要的。我们的实验室发现了一种新的PKC底物SSeCKS/Gravin/AKAP12(SSeCKS)，它同时也是PKC和PKA的支架，能够减弱PKC激酶的活性并改变其细胞区划，同时仅通过区划来调节PKA。SSeCKS在前列腺癌中显示了许多肿瘤抑制因子的特征：它在Gleason sum=6的人CAP细胞系和组织中表达严重下调；SSeCKS重新表达通过抑制血管内皮生长因子诱导的新生血管抑制宏观CAP转移生长；小鼠的基因敲除(KO)诱导前列腺增生和局灶性异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早衰。早衰似乎依赖于Rb，因为它可以被HPV-16E7orpRb-siRNA抑制，但不能被HPV-16E6或P53-siRNA抑制。与SSeCKS对PKC的支架功能一致，KO-MEF的总PKC活性是WT-MEF的2倍，PKC*活性是WT-MEF的3倍。根据表达芯片和生化实验的初步数据，我们假设SSeCKS的缺失会诱导高活性的PKC*/，进而通过激活RhoA和LIMK，以及通过抑制有丝分裂退出网络激酶、疣，以及通过增加活性氧类(ROS)介质(如p47Phox)而导致依赖Rb的衰老。我们认为SSeCKS直接控制细胞质分裂，因为在末期分裂沟中SSeCKS池丰富，已知PKC*和，通常通过协调RhoA和LIMK激活的时间来调节脱落，从而调节肌动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在WT与KO MEF和小鼠前列腺上皮细胞(PREC)以及人PREC与CAP细胞系中剖析这一新的SSeCKS-PKC途径，使用衰老/多倍体/双核和胞质分裂完成(目标1)、足体形成与肿瘤侵袭性(目标2)以及转基因小鼠模型中的帽形成、侵袭和转移潜力(目标3)的读数。拟议的实验旨在阐明SSeCKS/PKC支架复合体控制的机制和途径，以及SSeCKS缺失如何导致CAP进展。 公共卫生相关性：我们的项目专注于SSeCKS，一种似乎可以抑制前列腺癌进展，特别是转移的蛋白质，它通过结合两个相反的信号蛋白PKA和PKC，并通过调节它们在细胞中何时何地被激活来控制它们的功能。我们现在建议详细阐述PKC诱导的致癌途径，但这些途径通常由SSeCKS调控。这些研究将阐明前列腺癌中SSeCKS表达的缺失如何通过允许PKC过度激活和失调来促进疾病进展。

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.