REGULATION OF PAR 4 BY ONCOGENIC RAS

前列腺凋亡反应-4 （Par-4）是一种含亮氨酸拉链结构域的蛋白，在前列腺和非前列腺细胞的刺激依赖性凋亡中都是必需的。这种广泛表达的蛋白本身不足以诱导细胞凋亡，但可以使细胞对凋亡刺激的作用敏感。我们检测了各种肿瘤和相应的正常组织中Par-4蛋白的表达，发现相对于相应的正常细胞，Par-4水平在肾细胞癌（RCCs）和胰腺癌中定量降低，而在前列腺癌中则没有。这提示par4下调可能在某些癌症的恶性转化过程中发挥作用。表达ras癌基因的永生化成纤维细胞是研究转化和肿瘤发生所必需的信号通路的良好模型。我们对永生化成纤维细胞的初步研究表明，内源性的Par-4蛋白表达被致癌的Ras降低，并且Par-4的恢复抑制了致癌的Ras诱导的转化，而不改变细胞的活力。这些发现导致了一种假设，即致癌Ras下调Par-4对转化是必要的，因为Par-4可能抑制转化所需的关键致癌Ras诱导途径。为了验证这一假设，提出了三个具体目标：(1)确定致癌ras下调Par-4的分子机制；(2)确定补充Par- 4阻止转化过程的分子机制；(3)确定调控转化抑制的Par-4结构域。在体外实验中，研究人员将在成纤维细胞中阐明致癌Ras和Par-4激活的信号机制，并进一步研究在体内成纤维细胞和上皮细胞背景下，抑制转化的Par-4结构域是否也能阻止致癌Ras诱导的肿瘤生长。提出的研究将有助于剖析par4的功能域，这些功能域调节蛋白质的抗转化和凋亡致敏特性。因此，这些研究有望揭示致癌Ras用来克服干扰转化的机制的信号通路，并确定Par-4用来阻断细胞转化或肿瘤生长的新机制。这一发现可能有助于未来的研究探索利用par4过表达来控制肿瘤生长的可行性。

Prostate apoptosis response-4 (Par-4) is a leucine zipper domain- containing protein that is required for stimulus-dependent apoptosis in both prostatic and non-prostatic cells. This widely expressed protein is not sufficient on its own to induce apoptosis, but can sensitize the cells to the action of apoptotic stimuli. We examined various tumors and corresponding normal tissues for Par-4 protein expression, and found that Par-4 levels are quantitatively decreased in renal cell carcinomas (RCCs) and pancreatic cancer but not in prostate cancer relative to the corresponding normal cells. This suggested that Par-4 down-regulation may play a role in the process of malignant transformation in certain cancers. Immortalized fibroblasts expressing the ras oncogene serve as an excellent model to study signaling pathways essential for transformation and tumorigenesis. Our preliminary studies in immortalized fibroblasts suggested that endogenous Par-4 protein expression is diminished by oncogenic Ras, and that restoration of Par-4 inhibits oncogenic Ras- inducible transformation without altering cell viability. These findings led to the hypothesis that down-regulation of Par-4 by oncogenic Ras is necessary for transformation because Par-4 may inhibit key oncogenic Ras-inducible pathway(s) that are required for transformation. Three specific aims proposed to test this hypothesis are: (1) To determine the molecular mechanism(s) by which oncogenic ras down-regulates Par-4; (2) To determine the molecular mechanism(s) by which replenishment of Par- 4 prevents the process of transformation; and (3) To identify the structural domains of Par-4 that regulate suppression of transformation. The signaling mechanisms invoked by oncogenic Ras and Par-4 will be elucidated in fibroblasts in vitro, and the findings will be extended to examine whether the structural domains of Par-4 that inhibit transformation, also prevent oncogenic ras-inducible tumor growth in fibroblast and epithelial cell backgrounds in vivo. The proposed studies will help dissect out the functional domains of Par-4 that regulate the anti- transformation and apoptosis-sensitization properties of the protein. Thus, these studies are expected to uncover the signaling pathways used by oncogenic Ras to overcome the mechanisms that interfere with transformation and identify novel mechanisms that are utilized by Par-4 to block cellular transformation or tumor growth. The findings may help future studies to explore the feasibility of using Par-4 over-expression for tumor growth control.