PLEKHA7 and beta-catenin interact to regulate mutant KRas

 描述(由申请人提供)：KRAS癌基因在许多癌症类型的25%的患者肿瘤中被发现。尽管自40多年前发现KRAS以来，人们做出了广泛的努力，但目前还没有有效的治疗方法来治疗突变的KRAS，据估计，2014年美国有35万名肿瘤中KRAS突变的患者将死于这种疾病。突变的KRAS在推动肿瘤生长和抵抗治疗方面起着关键作用。它的作用是如此强大，以至于我超过了许多今天正在为癌症开发的新的分子靶向信号药物的活性。因此，寻找新的药物来抑制突变的KRAS的影响是当今癌症的一个关键的未得到满足的需求。然而，野生型KRAS蛋白的胞内信号控制着正常细胞功能的许多方面，因此针对抑制突变KRAS的治疗应该理想地使野生型KRAS功能不受影响。利用全局siRNA功能筛选和野生型或突变型KRAS的等基因细胞系对，我们鉴定了PLEKHA7(Pleckstrin Homology DOMAIN，家族A7)是一种蛋白质，当被击倒时，它可以抑制突变KRAS的结肠癌细胞的增殖，但不能显著地抑制野生型KRAS细胞的增殖。突变的KRAS表型的其他方面也受到抑制，包括锚定非依赖性(3D)生长、细胞侵袭和体内肿瘤生长。我们还表明，PLEKHA7基因敲除降低了突变型KRAS的活性(GTP结合)，但不减少野生型KRAS的活性，并抑制了下游突变型KRAS信号。PLEKHA7通常存在于正常上皮细胞的粘连连接处。在癌细胞中，它存在于质膜紧密连接中，但其功能尚不清楚。PLEKHA7是一组信号蛋白中的一员，它包含一个独特的3D蛋白质折叠，即Pleckstrin Homology(PH)结构域，它与膜上的磷脂酰肌醇结合，将亲本蛋白定位在膜上对其功能至关重要的特定位置。我们以前的研究表明，PH结构域可以被小分子选择性地下药，从而抑制蛋白质的信号功能。我们有证据表明，在癌细胞中，PLEKHA7与质膜结合的KRAS信号纳米簇的蛋白有关。因此，我们假设PLEKHA7选择性地调节信号纳米簇中突变的KRAS活性，因此PLEKHA7的PH域是小分子抑制剂的靶点，用于PLEKHA7功能的机制研究，以及选择性地阻断突变的KRAS癌细胞的生长作为潜在的治疗方法。这代表了一种通过抑制相关的调控信号纳米簇蛋白来攻击RAS的新范式。我们的目标是：1)探讨PLEKHA7的S选择性抑制突变的KRAS的能力的机制；2)对PLEKHA7的PH结构域和两个潜在的“铰链”区域进行结构研究，以确定PLEKHA7的S与KRAS信号纳米簇的质膜和其他蛋白质相关的功能；以及3)寻找PLEKHA7 PH结构域的小分子抑制剂作为药理探针，研究PLEKHA7在KRAS调控中的作用，并为潜在的治疗突变的KRAS肿瘤的药物提供线索。

 DESCRIPTION (provided by applicant): The KRAS oncogene is found in 25% of patient tumors across many cancer types. Despite extensive effort since its discovery over 40 years ago there is no effective treatment for mutated KRAS, and an estimated 350,000 patients in the US in 2014 with mutated KRas in their tumors will die of their disease. Mutated KRAS plays a critical role in driving tumor growth and resistance to therapy. Its effects are so powerful that i overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today. Thus, finding new agents that inhibit the effects of mutated KRas is a critical unmet need in cancer today. However, intracellular signaling by wild type KRas protein controls many aspects of normal cell function, so that a therapy directed at inhibiting mutant KRas should ideally leave wild type KRas function unaffected. Using a global siRNA functional screen and isogenic cell line pairs of mutant or wild type KRas we identified PLEKHA7 (pleckstrin homology domain containing, family A7) as a protein that when knocked down inhibits the proliferation of colon cancer cells with mutant KRas, but remarkably not wild type KRas cells. Other aspects of the mutant KRas phenotype were also inhibited including anchorage independent (3D) growth, cell invasion, and in vivo tumor growth. We have also shown that PLEKHA7 knockdown decreases the active (GTP bound) form of mutant but not of wild type KRas, with inhibition of downstream mutant KRas signaling. PLEKHA7 is normally found in the adherens junction of normal epithelial cells. In cancer cells it is found in plasma membrane tight junctions but its function is not known. PLEKHA7 is a member of a group of signaling proteins containing a distinctive 3D protein fold, the pleckstrin homology (PH)-domain, that binds to membrane phosphoinositides to position the parent proteins at specific sites on the membrane important for their function. Our previous studies have shown that PH domains can be selectively drugged by small molecules, thus inhibiting the signaling function of the proteins. We have evidence that in cancer cells PLEKHA7 is associated with proteins of the plasma membrane-bound KRas signaling nanocluster. We thus hypothesize that PLEKHA7 selectively regulates mutated KRas activity in the signaling nanocluster, and therefore that the PH- domain of PLEKHA7 is a target for small molecules inhibitors as pharmacological probes for mechanistic studies of PLEKHA7 function, as well as selectively blocking the growth of mutated KRas cancer cells as potential therapy. This represents a new paradigm for attacking Ras via inhibition of associated regulatory signaling nanocluster proteins. The objectives of our study are: 1) to investigate the mechanism for PLEKHA7's ability to selectively inhibit mutated KRas; 2) to conduct structural studies of the PLEKHA7 PH domain and two potential "hinge" regions, to delineate PLEKHA7's function in associating with the plasma membrane and other proteins of the KRas signaling nanocluster; and 3) to identify small molecule inhibitors of the PLEKHA7 PH- domain as pharmacological probes to study the role of PLEKHA7 in KRas regulation, and as leads for potential agents to treat mutated KRas tumors.