Mechanism of Apoptosis and Inhibition of Tumor Progression and Metastasis by Par-

描述（由申请人提供）：前列腺凋亡反应-4蛋白（Par-4;也称为PAWR）是一种在脊椎动物中保守的亮氨酸拉链结构域蛋白。在之前的资助期间，我们发现Par-4的核心效应结构域（氨基酸137-195，命名为SAC结构域）是诱导细胞凋亡所必需和足够的。有趣的是，Par-4及其SAC结构域都选择性地在癌细胞中诱导凋亡，但在正常细胞中不诱导凋亡。尽管异位Par-4和SAC结构域长期以来被认为通过在细胞核内作用以抑制NF-κ B来影响凋亡，但我们最近的瞬时转染研究意外地显示了不表达Par-4或SAC质粒的邻近细胞中的凋亡。此外，来自Par-4转染细胞的条件培养基（CM）在应用于癌细胞而非正常细胞时诱导凋亡;这意味着Par-4的过表达导致促凋亡活性的分泌。通过Western印迹分析在CM中检测到Par-4和SAC蛋白，并且该CM中的凋亡活性被针对Par-4的抗体中和。来自用布雷菲德菌素A处理的Par-4转染子的CM中的凋亡活性降低，表明Par-4和SAC分泌（或分别为sPar-4和sSAC）通过经典途径（即，从内质网到高尔基体，然后到细胞膜）。重要的是，来自正常细胞和癌细胞的CM含有内源性Par-4，其水平在用TRAIL处理（不足以产生细胞凋亡的短期暴露）后增加，从而表明分泌是生理学相关的，而不是与Par-4和SAC构建体的过表达相关的伪影。基于这些数据，我们假设内源性和外源性sPar-4和sSAC通过细胞内和细胞外机制发挥癌症特异性凋亡作用。我们将从机制的角度解决这一假设，表征前列腺癌细胞中分泌的Par-4和SAC，并阐明它们选择性启动癌细胞凋亡的方法。具体而言，我们将1）描述前列腺癌细胞中sPar-4/sSac分泌Par-4/SAC和凋亡的机制; 2）测试前列腺癌模型中sPar-4和sSAC结构域的生长抑制作用;以及3）研究细胞外和细胞内Par-4在诱导癌细胞凋亡中的相互依赖性。由于我们最近的研究表明Par- 4及其SAC结构域是分泌的，并且重组（r）Par-4或rSAC在外源性应用于细胞时可以诱导细胞凋亡，因此这些数据可能具有显著的临床相关性。我们的研究结果介绍了新的前景，使用sPar-4或sSAC诱导凋亡，不仅在局部或表面局限的肿瘤，其中Par-4或SAC可以很容易地交付到肿瘤内，而且通过静脉注射或骨髓移植，我们将探讨在这个建议的远处转移。公共卫生相关性：我们的研究集中在Par-4的分泌形式及其核心效应结构域SAC，这两者都选择性地诱导癌细胞凋亡。我们将从体外和体内机制的角度研究这两种蛋白质，表征前列腺癌细胞中分泌的Par-4和SAC，并阐明它们选择性启动癌细胞凋亡的方法。这项研究将探索使用sPar-4或sSAC诱导细胞凋亡的新前景，不仅在局部或表面局限的肿瘤中，Par-4或SAC可以很容易地在肿瘤内递送，而且还可以通过静脉注射或骨髓移植诱导远处转移。

DESCRIPTION (provided by applicant): Prostate apoptosis response-4 protein (Par-4; also known as PAWR) is a leucine zipper domain protein that is conserved in vertebrates. During the previous funding period, we showed the core effector domain of Par-4 (amino acids 137-195, designated the SAC domain) is necessary and sufficient to induce apoptosis. Interestingly, both Par-4 and its SAC domain induce apoptosis selectively in cancer cells, but not in normal cells. Although ectopic Par-4 and the SAC domain have long been thought to effect apoptosis by acting within the cell nucleus to inhibit NF-kappaB, our recent transient transfection studies unexpectedly displayed apoptosis in neighboring cells that did not express the Par-4 or SAC plasmid. Moreover, conditioned medium (CM) from Par-4 transfected cells induced apoptosis when applied to cancer cells, but not to normal cells; this implies over-expression of Par-4 leads to the secretion of a pro-apoptotic activity. Both Par-4 and the SAC protein were detected in the CM by Western blot analysis, and the apoptotic activity in this CM was neutralized by antibodies against Par-4. The apoptotic activity was diminished in the CM from Par-4 transfectants treated with brefeldin A, indicating Par-4 and SAC secretion (or sPar-4 and sSAC, respectively) occurs by the classical pathway (i.e., from the endoplasmic reticulum to the Golgi, and then to the cell membrane). Importantly, CM from normal and cancer cells contains endogenous Par-4, of which the levels increase upon treatment with TRAIL (a short term exposure that is insufficient to produce apoptosis), thereby indicating secretion is physiologically relevant, and not an artifact associated with over-expression of the Par-4 and SAC constructs. Based on these data, we hypothesize endogenous and exogenous sPar-4 and sSAC exert cancer-specific apoptotic effects via both intracellular and extracellular mechanisms. We will address this hypothesis from a mechanistic perspective, characterizing secreted Par-4 and SAC in prostate cancer cells, and clarifying the means by which they selectively initiate apoptosis in cancer cells. Specifically, we will 1) Delineate the mechanism of Par-4/SAC secretion and apoptosis by sPar-4/sSac in prostate cancer cells; 2) Test the growth suppressive effects of sPar-4 and sSAC domain in prostate cancer models; and 3) Study the inter-dependence of extracellular and intracellular Par-4 in inducing apoptosis in cancer cells. As our recent studies indicate Par- 4 and its SAC domain are secreted, and that recombinant (r) Par-4 or rSAC can induce apoptosis when applied exogenously to cells, these data may hold significant clinical relevance. Our results introduce the novel prospect of using sPar-4 or sSAC to induce apoptosis, not only in local or superficially confined tumors into which Par-4 or SAC can be readily delivered intratumorally, but also to distant metastases by intravenous injection or bone-marrow transplantation, which we will explore in this proposal. PUBLIC HEALTH RELEVANCE: Our research focuses on the secreted forms of Par-4 and it core effector domain SAC, both of which selectively induce apoptosis in cancer cells. We will study these two proteins from a mechanistic perspective both in vitro and in vivo, characterizing secreted Par-4 and SAC in prostate cancer cells, and clarifying the means by which they selectively initiate apoptosis in cancer cells. The proposed science will explore the novel prospect of using sPar-4 or sSAC to induce apoptosis, not only in local or superficially confined tumors into which Par-4 or SAC can be readily delivered intratumorally, but also to distant metastases by intravenous injection or bone-marrow transplantation.