Study of Ras-Mediated Apoptosis

描述(由申请人提供)：抗癌策略的一个有吸引力的范例是以癌基因为靶点，使肿瘤细胞对凋亡敏感。其基本原理是癌蛋白 依靠其他因素来维持动态平衡或促进癌细胞存活。其中一种协调蛋白的破坏会引发癌细胞的凋亡危机。在大约30-40%的人类癌症中发现了致癌的RAS。然而，针对突变的RAS的抑制剂已被证明在临床上并不有效。最近，人们的重点一直放在寻找RAS下游或与RAS平行的信号通路上，这些信号通路对癌细胞的生存至关重要。在努力确定突变的RAS的凋亡伙伴的过程中，研究表明，PKC的缺失与突变的RAS一起，在许多类型的肿瘤中是综合致命的。最近，我们做了一个新的观察，即不同的PKC亚型的缺失会使表达突变的ras的细胞对细胞凋亡产生不同的敏感性。特别是，我们确定了PKC？/？的共抑制。其中PI3K/Akt在RAS下游发挥作用，动员两条信号通路：一条是通过上调ROS和ER应激介导的UPR来启动凋亡机制；另一条是通过激活PKC？，导致p73的磷酸化和PUMA/NOXA的上调。在这一新的应用中，我们将研究我们的新观察的分子机制，即当PKC？/？丢失时，RAS如何不同地利用其下游效应途径选择性地使肿瘤细胞对凋亡敏感，而对正常细胞或周围组织的毒性较小或没有毒性。我们将确定ROS和PKC？/p73通路如何被异常的RAS/PI3K/Akt重定向，从而在共抑制PKC？？/？后激活细胞死亡程序。因此，假设PI3K/Akt在共敲除PKC？/？后，通过扰乱氧化还原动态平衡和激活PKC？/p73介导的细胞凋亡网络，在RAS介导的细胞凋亡的诱导过程中起关键作用。据此，形成了4个具体目标。目的1研究在表达异常ras或PI3K/Akt的细胞中，通过共同抑制PKC？/？上调ROS的机制。目的研究ROS通过共同抑制PKC？/？，在表达异常ras或PI3K/Akt的细胞中诱导ER应激/UPR活化的机制。在目标3中，我们将确定p73在表达异常ras或PI3K/Akt的细胞在共敲除PKC？？/？后对细胞凋亡的敏感性中的作用。在目标4中，我们将在体外和体内检测共抑制PKC？/？后对表达异常ras或PI3K/Akt的细胞的促凋亡作用。我们的研究结果将揭示RAS突变和PKC？？/？缺失之间的这种合成致死相互作用的分子，这将为设计更有效的药物来治疗携带RAS或PI3K/Akt异常的人类癌症提供潜在的信息。

DESCRIPTION (provided by applicant): One attractive paradigm of anticancer strategies is to target oncogenes for sensitization of tumor cells to apoptosis. The rationale is that onco-proteins rely on other factors to maintain homeostasis or promote cancer cell survival. Disruption of one of coordinating proteins would trigger an apoptotic crisis in cancer cells. Oncogenic Ras has been discovered in about 30-40% of human cancers. However, inhibitors targeting mutated Ras have proved not to be effective clinically. Recently, the focus has been on finding signaling pathways that are downstream of or parallel with Ras, and are essential for cancer cells to survive. In the effort to identify the apoptotic partners of mutated Ras, studies showed that loss of PKC, together with mutated Ras, are synthetically lethal in many types of tumors. Recently, we made the novel observation that loss of different PKC isoforms differentially sensitized cells expressing mutated ras to apoptosis. In particular, we identified that co-suppression of PKC?/? were critical for the induction of apoptosis in cancer cells harboring mutated ras, in which PI3K/Akt function downstream of Ras to mobilized two signaling pathways: one was via upregulating ROS and ER stress-mediated UPR for switching on the apoptotic machinery; and another one was through activating PKC?, resulting in p73 phosphorylation and PUMA/NOXA upregulation. In this renewal application, we will investigate the molecular mechanisms of our novel observation of how Ras, upon loss of PKC??/?, differentially utilizes its downstream effector pathways to selectively sensitize tumor cells to apoptosis, with less or no toxicity to normal cells or surrounding tissues. We will determine how ROS and PKC??/p73 pathways are being redirected by aberrant Ras/PI3K/Akt to activate cell death program after co-suppression of PKC??/?. Thus, the hypothesis to be tested is that upon co-knockdown of PKC??/?, PI3K/Akt are key players in the induction of Ras-mediated apoptosis, which is via perturbing redox homeostasis as well as through activating PKC??/ p73-mediated apoptotic network. Accordingly, 4 Specific Aims are formed. Aim 1 will study the mechanisms by which ROS is upregulated in cells expressing aberrant ras or PI3K/Akt upon co- suppression of PKC??/?. Aim 2 will determine the mechanisms by which ROS induces ER-stress/UPR activation in cells expressing aberrant ras or PI3K/Akt upon co-suppression of PKC??/?. In Aim 3, we will determine the role of p73 in the sensitization of cells expressing aberrant ras or PI3K/Akt to apoptosis after co- knockdown of PKC??/?. In Aim 4, we will determine the pro-apoptotic effect in cells expressing aberrant ras or PI3K/Akt in vitro and in vivo after co-suppression of PKC??/?. The outcomes of our research will reveal molecules involved in this synthetic lethal interaction between Ras mutations and loss of PKC??/?, which will provide the potential information for designing more effective drugs to treat human cancers harboring aberrant ras or PI3K/Akt.