Calcitonin in Prostate Growth and Neplasia

描述(申请人提供)：前列腺癌(PC)是美国最常见的癌症类型，也是导致男性癌症死亡的第二大原因。我们和其他人的研究表明，所有原代PC和PC细胞株都表达降钙素(CT)和/或其受体(CTR)，并且它们的共同表达与原代PC的肿瘤分级和PC细胞系的生长/侵袭呈正相关。此外，非侵袭性、非致瘤性LNCaP细胞中CT-CTR轴的激活可诱导侵袭性和致瘤性表型。相比之下，在高转移PC-3M细胞中沉默CT/CTR表达显著降低了它们的致瘤性，并取消了它们在裸鼠体内形成远处转移的能力。此外，我们还发现，CTR的胞质(C)尾部含有一个典型的I型PSD-95，Discs-Large，Zona Occludens-1(PDZ)配体基序，该基序的突变可以消除CT诱导的PC细胞生长和侵袭力的增加。在第二个重大发现中，我们发现CTR的PDZ配体与膜蛋白闭锁带(ZO-1)的PDZ结构域结合，形成一个“转移接受体”。我们的研究还表明，CTR激活了cAMP依赖的蛋白激酶(PKA)，而激活的PKA通过磷酸化关键的TJ蛋白来促进紧密连接(TJ)的分解。在第三个重要发现中，我们证明了A激酶锚定蛋白2(AKAP2)通过在TJ复合体的局部亚区内将PKA靶向CTR而在CTR介导的致癌作用中发挥关键作用。我们的中心假设是，CTR-ZO-1相互作用和PKA在TJ复合体中的局部作用是CT诱导的连接复合体解体所必需的，允许细胞-细胞接触的松动，促进PC细胞系侵袭性的增加。我们将在三个特定的目标上验证这一假说：1)利用定点突变，我们将确定与ZO-1结合所需的CTR-C-PDZ配体的关键氨基酸(S)，并研究PDZ突变(S)对CTR在TJ组装、侵袭和体内肿瘤生长/转移中的作用；2)利用ZO-1的PDZ缺失构建体，我们将鉴定ZO-1的三个PDZ结构域中的哪一个与CTR结合，并探讨ZO-1在介导CTR在TJ组装、侵袭和体内肿瘤生长/转移中的作用；3)我们将研究AKAP2在靶向TJ复合体的PKA及其对ZO-1和claudin 3的磷酸化中的作用，这可能在TJ的分解、侵袭和体内肿瘤的生长/转移中发挥关键作用。我们在PC细胞系中发现了CTR激活的致癌信号的一种新的机制，建立了各种研究工具、细胞系和实验模型，并组建了一支研究团队来成功地实现这些目标。我们相信，这项研究将揭示与PC进展相关的重要细胞内机制，并为开发治疗晚期PC的诊断工具和治疗剂提供新的靶点。公共卫生相关性：前列腺癌(PC)是美国最常见的癌症，也是导致男性癌症死亡的第二大原因。从可治疗的局限性PC发展为相对不可治疗的转移性PC的相关机制尚未阐明。我们的结果表明，降钙素(CT)-CT受体(CTR)轴的激活在前列腺癌的肿瘤生长和转移中起着重要作用。最近，我们发现CTR与PC细胞紧密连接之间的相互作用是CTR致癌作用的先决条件，并导致紧密连接的解体。这项应用的目的是阐明与CTR刺激的TJ分解相关的早期分子事件，这些事件导致肿瘤生长/转移增加。

DESCRIPTION (provided by applicant): Prostate cancer (PC) is the most commonly diagnosed cancer type and the second leading cause of male cancer deaths in the United States. We and others have shown that all primary PCs and PC cell lines express calcitonin (CT) and/or its receptor (CTR), and their co-expression positively correlates with the tumor grade of primary PCs and growth/invasiveness of PC cell lines. Moreover, activation of the CT-CTR axis in non-invasive, non-tumorigenic LNCaP cells induces an invasive and tumorigenic phenotype. In contrast, silencing of CT/CTR expression in highly metastatic PC-3M cells markedly reduces their tumorigenicity and abolishes their ability to form distant metastases in nude mice. Furthermore, we made the key discovery that the cytoplasmic (C) tail of CTR contains a canonical class I type PSD-95, Discs-large, Zona Occludens-1 (PDZ) ligand motif, mutation of which abrogates the CT-elicited increase in growth and invasiveness of PC cell lines. In a second major discovery, we showed that the PDZ ligand of the CTR binds to a PDZ domain of the membrane protein zonula occludens (ZO-1) to form a "metastasis receptosome". Our studies also showed that CTR activates cyclic AMP-dependent protein kinase (PKA), and that activated PKA facilitates disassembly of tight junctions (TJs) by phosphorylating key TJ proteins. In a third important discovery, we showed that A kinase anchoring protein 2 (AKAP2) plays a key role in CTR-mediated oncogenic actions by targeting PKA to CTR within a localized sub-region of the TJ complex. Our central hypothesis is that the CTR-ZO-1 interaction and localized action of PKA within the TJ complex is required for CT-induced disassembly of junctional complexes, permitting a loosening of cell-cell contacts and facilitating increased invasiveness of PC cell lines. We will test this hypothesis in three Specific Aims: 1) Using site-directed mutagenesis, we will identify the key amino acid(s) of the CTR-C-PDZ ligand required for ZO-1 binding, and investigate the effect of PDZ mutation(s) on the actions of CTR on TJ assembly, invasion, and in vivo tumor growth/metastasis; 2) Using ?PDZ deletion constructs of ZO-1, we will identify which of the three PDZ domains of ZO-1 binds to CTR and investigate the role of ZO-1 in mediating the actions of CTR on TJ assembly, invasion, and in vivo tumor growth/metastasis; 3) We will investigate the role of AKAP2 in both targeting PKA to the TJ complex and its phosphorylation of ZO-1 and claudin 3, which may play a key role in TJ disassembly, invasion, and in vivo tumor growth/metastasis. We have identified a novel mechanism for CTR-activated oncogenic signaling in PC cell lines, generated a variety of research tools, cell lines and experimental models, and assembled a team of investigators to successfully accomplish these aims. We believe this study will uncover important intracellular mechanisms associated with PC progression, and provide new targets for the development of diagnostic tools and therapeutic agent for the treatment of advanced PCs. PUBLIC HEALTH RELEVANCE: Prostate Cancer (PC) is the most commonly diagnosed cancer and the second leading cause of cancer deaths in men in America. The mechanisms associated with the progression from treatable, localized PC to relatively untreatable, metastatic form have not been elucidated. Our results suggest that the activation of the calcitonin (CT)-CT receptor (CTR) axis plays a major role in tumor growth and metastasis of prostate cancer. Recently, we have discovered that the interaction between CTR and tight junctions of PC cell lines is prerequisite for oncogenic actions of CTR, and it leads to the disassembly of tight junctions. The goal of this application is to elucidate early molecular events associated with CTR-stimulated TJ disassembly that lead to increased tumor growth/metastasis.