REGULATION OF PAR 4 BY ONCOGENIC RAS

前列腺凋亡反应-4（Par-4）是前列腺和非前列腺细胞中刺激依赖性凋亡所需的含亮氨酸拉链结构域的蛋白。这种广泛表达的蛋白质本身不足以诱导凋亡，但可以使细胞对凋亡刺激物的作用敏感。我们检测了各种肿瘤和相应的正常组织的Par-4蛋白表达，发现Par-4水平在肾细胞癌（RCC）和胰腺癌中定量降低，但相对于相应的正常细胞，在前列腺癌中不降低。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极好模型。我们在永生化成纤维细胞中的初步研究表明，内源性Par-4蛋白表达被致癌Ras减少，并且Par-4的恢复抑制致癌Ras诱导的转化而不改变细胞活力。这些发现导致了这样的假设，即致癌Ras下调Par-4是转化所必需的，因为Par-4可能抑制转化所需的关键致癌Ras诱导途径。为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Par- 4补充阻止转化过程的分子机制;（3）鉴定Par-4调节转化抑制的结构域。致癌Ras和Par-4引起的信号传导机制将在体外成纤维细胞中阐明，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。这些研究将有助于剖析Par-4的功能结构域，这些功能结构域调节蛋白质的抗转化和细胞凋亡致敏特性。 因此，这些研究有望揭示致癌Ras用于克服干扰转化的机制的信号通路，并确定Par-4用于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使用Par-4过表达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.