COBRE PROJ 7: CONTROL OF TUMOR GROWTH BY RAS-RELATED PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 具体目的1：研究Ras效应子的RASSF家族在转化中的作用 Ras癌基因在超过三分之一的人类癌症的发展中起着关键作用。 Ras似乎通过激活多种异源效应蛋白来发挥作用，这些效应蛋白调节控制生长和发育的协同信号传导途径。 在实验上，过量的Ras激活导致剧烈的转化，并且最佳表征的Ras效应蛋白本身是癌蛋白。 然而，Ras的过度活化也可导致细胞经历生长停滞和凋亡。 这表明Ras蛋白可能激活效应子的子集，而不是促进转化，介导生长抑制。 这似乎是合理的假设，这样的效应系统将不得不在转化过程中被破坏，以允许进展到致瘤性。 具体目标2：开发Ras作用的新型小分子抑制剂 二十多年来，Ras已被确定为靶向抗癌治疗的主要候选者，但迄今为止，开发Ras特异性抑制剂的尝试被证明是无效的。 最著名的尝试涉及一系列法尼基转移酶抑制剂，这些抑制剂实际上对H-Ras有效，但对家族中最重要的成员K-Ras无效。 最近的工作表明，在人类系统中最重要的Ras效应器的转化是RalGDS组的效应器。 这与鼠系统中的结果形成对比，鼠系统中涉及Rafs作为关键效应子。 尚未描述RalGDS功能的抑制剂。 研究和结果 1. 艾伦等人，Oncogene，2007年出版（epub 2007年4月）。 在本文中，我们已经确定和特点的RASSF家族的新成员，指定RASSF 6。 我们发现RASSF 6具有Ras效应子/肿瘤抑制子的特征，但与其他家族成员相比，它具有几个独特的功能。 这些包括负调节NFkB途径的能力。 这可能为最近证实的RASSF 6与对严重呼吸道合胞病毒感染的敏感性之间的遗传联系提供了一种机制解释。 2. 我们已经证明RASSF 2家族成员在前列腺癌中经常下调，并且RASSF 2的敲低增强前列腺肿瘤细胞系的致瘤表型。 此外，我们已经表明，RASSF 2与肿瘤抑制因子PAR-4（前列腺凋亡反应基因4）形成内源性复合物。 因此，我们似乎已经确定了RASSF 2参与前列腺癌的作用和新机制。 3. 我们已经确定了新的蛋白质萨尔瓦多作为RASSF 1A的结合伴侣，并表明萨尔瓦多表现出肿瘤抑制特性。 我们已经有证据表明RASSF 1A使用萨尔瓦多来促进LATs肿瘤抑制因子的激活。 4. 我们已经使用微阵列分析来证明RASSF 5（Nore 1a）激活p21 cdk抑制剂。 我们已经通过Western分析证实了这一点，并表明p21 -/- MEFs对Nore 1a介导的生长抑制具有抗性。 5. 我们已经启动了一个虚拟屏幕的Ras/RalGDS相互作用与分子建模核心的抑制剂。 意义：我们是第一个鉴定和表征RASSF 6作为一种新型肿瘤抑制因子的人。 此外，我们已经证明了一个潜在的机制，它调节肿瘤抑制和呼吸道合胞病毒感染的致病性。 我们已经确定了三个不同的新的信号通路利用不同的RASSF家族成员。 它们是RASSF 1A的萨尔瓦多肿瘤抑制剂、RASSF 2的PAR-4肿瘤抑制剂和Nore 1a的p21 WAF/CIP cdk抑制剂。 在每种情况下，该途径都与转化表型的发展密切相关。 这些发现开始解释了RASSF家族成员用来阻碍转化的机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aim 1: Investigation of the RASSF family of Ras effectors in transformation The Ras oncogene has been implicated as a key player in the development of more than a third of human cancers. Ras appears to function by activating multiple, heterologous effector proteins that regulate synergistic signaling pathways controlling growth and development. Experimentally, excess Ras activation leads to vigorous transformation and the best characterized Ras effector proteins are themselves oncoproteins. However, excessive activation of Ras can also cause cells to undergo growth arrest and apoptosis. This suggests that Ras proteins may activate a sub-set of effectors that, rather than promoting transformation, mediate growth inhibition. It would seem reasonable to suppose that such effector systems would have to be subverted during the transformation process to allow progression to tumorigenicity. Specific Aim 2: Development of novel small molecule inhibitors of Ras action Ras has been identified as a prime candidate for targeted anti-cancer therapy for more than two decades, but attempts to develop specific inhibitors of Ras have so far proved ineffective. The best known attempt involved a series of Farnesyl transferase inhibitors that actually do work well on H-Ras but are ineffective against the most important member of the family, K-Ras. Recent work has shown that the most important Ras effector for transformation in human systems is the RalGDS group of effectors. This contrasts with results in murine systems which have implicated Rafs as the key effectors. No inhibitors of RalGDS function have been described. Studies and Results 1. Allen et al, Oncogene, 2007 in press (epub April 2007). In this paper we have identified and characterized a novel member of the RASSF family, designated RASSF6. We show that RASSF6 exhibits the characteristics of a Ras effector/tumor suppressor but that it has several unique features compared to other family members. These include the ability to negatively modulate the NFkB pathway. This may provide a mechanistic explanation for the genetic link that has recently been demonstrated between RASSF6 and sensitivity to severe Respiratory Syncytial Virus infection. 2. We have shown that the RASSF2 family member is frequently down-regulated in prostate cancer and that knockdown of RASSF2 enhances the tumorigenic phenotype of prostate tumor cell lines. Moreover, we have shown that RASSF2 forms an endogenous complex with the tumor suppressor PAR-4 (Prostate apoptosis response gene 4). Thus, we appear to have identified a role and a novel mechanism for the involvement of RASSF2 in prostate cancer. 3. We have identified the novel protein Salvador as a binding partner for RASSF1A and have shown that Salvador exhibits tumor suppressor properties. We have developed evidence that RASSF1A uses Salvador to promote the activation of the LATs tumor suppressors. 4. We have used micro array analysis to demonstrate that RASSF5 (Nore1a) activates the p21 cdk inhibitor. We have confirmed this by Western analysis and shown that p21 -/- MEFs are resistant to Nore1a mediated growth inhibition. 5. We have initiated a virtual screen for inhibitors of the Ras/RalGDS interaction with the Molecular Modeling Core. Significance: We were the first to identify and characterize RASSF6 as a novel tumor suppressor. Moreover, we have demonstrated a potential mechanism by which it modulates both tumor suppression and contributes to the pathogenicity of Respiratory Syncytial Virus infection. We have identified three distinct novel signaling pathways utilized by different RASSF family members. These are the Salvador tumor suppressor for RASSF1A, the PAR-4 tumor suppressor for RASSF2 and the p21 WAF/CIP cdk inhibitor for Nore1a. In each case, the pathway is deeply implicated in the development of the transformed phenotype. These findings begin to explain the mechanisms that RASSF family members use to impede transformation.