REGULATION OF PAR 4 BY ONCOGENIC RAS

前列腺细胞凋亡反应 4 (Par-4) 是一种含有亮氨酸拉链结构域的蛋白质，是前列腺和非前列腺细胞中刺激依赖性细胞凋亡所必需的。这种广泛表达的蛋白质本身不足以诱导细胞凋亡，但可以使细胞对细胞凋亡刺激的作用敏感。我们检查了各种肿瘤和相应正常组织的 Par-4 蛋白表达，发现相对于相应的正常细胞，Par-4 水平在肾细胞癌 (RCC) 和胰腺癌中定量降低，但在前列腺癌中则没有。这表明Par-4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绝佳模型。我们对永生化成纤维细胞的初步研究表明，致癌Ras会减少内源性Par-4蛋白表达，并且Par-4的恢复会抑制致癌Ras诱导的转化，而不改变细胞活力。这些发现得出这样的假设：致癌 Ras 下调 Par-4 对于转化是必要的，因为 Par-4 可能抑制转化所需的关键致癌 Ras 诱导途径。为检验这一假设而提出的三个具体目标是： (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Par-4调节转化抑制的结构域。致癌 Ras 和 Par-4 引发的信号传导机制将在体外成纤维细胞中得到阐明，并且研究结果将扩展到检查 Par-4 抑制转化的结构域是否也能阻止体内成纤维细胞和上皮细胞背景中致癌 ras 诱导的肿瘤生长。拟议的研究将有助于剖析 Par-4 调节该蛋白的抗转化和凋亡敏化特性的功能域。 因此，这些研究有望揭示致癌 Ras 所使用的信号通路，以克服干扰转化的机制，并确定 Par-4 用于阻止细胞转化或肿瘤生长的新机制。 这些发现可能有助于未来的研究探索利用 Par-4 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.