RhoB in cancer pathogenesis and as a target in combinatorial therapy

描述（由申请人提供）：在检查作为候选癌症治疗剂的有效的新型高亲和力PPAR 3激动剂RS 5444时，我们发现RhoB mRNA和蛋白质被RS 5444快速上调，与观察到的抑制作用一致。这促使我们研究新的假设，即下游RhoB效应部分介导了PPAR 3对癌症发病机制的贡献。RhoB是一种调节肌动蛋白组织和囊泡转运的小GT3，在癌症中未发生突变。然而，RhoB抑制增殖。我们之前已经表明RS 5444和紫杉醇可以在体外和体内的间变性甲状腺癌（ATC）模型中协同组合，并且细胞周期蛋白激酶抑制剂p21 WAF 1/CIP 1（p21）对于PPAR 3介导的生长抑制和组合诱导的细胞凋亡协同作用是必需的。我们现在报道RhoB对RS 5444/PPAR 3介导的p21 mRNA和蛋白诱导至关重要。在我们的实验室中沉默RhoB导致RS 5444的生长抑制活性丧失，这支持了我们的假设，并使我们首次证明强制RhoB上调导致ATC中的癌细胞死亡。这些数据，加上我们档案ATC组织中RhoB下调的初步数据，使我们假设RhoB在ATC的化学敏感性和ATC发病机制中很重要。基于这些发现，ATC正在进行RS 5444和紫杉醇联合的1/2期临床试验，以改善紫杉醇3在这种几乎一致致命的癌症中的单药作用。与本试验结合获得的肿瘤活检将允许测量RhoB和p21诱导，因为早期反应与患者结局相关。RS 5444单独诱导ATC中G 0/G1细胞周期停滞而无凋亡，并且RhoB（泰特RhoB）的强制表达证明足以诱导p21、G2/M停滞和凋亡。这些观察结果与另一个发现相结合，即RhoB错误定位于细胞核，与p21共定位于包括蛋白激酶C相关激酶1（PRK 1）（一种已知的RhoB相互作用蛋白和丝氨酸/苏氨酸激酶）的核复合物中。该复合物的形成是RS 5444以及强制RhoB表达的结果，这使我们假设这种新型核复合物通过与细胞周期机制复合物的直接相互作用介导RhoB抗肿瘤活性，并且p21的翻译后修饰指导其与G 0/G1或G2/M细胞周期机制的物理关联，从而指定细胞周期停滞或凋亡。在目标1中，我们将详细阐述RhoB通过这种新型核复合物介导细胞周期阻滞和凋亡的机制。在目标2中，RhoB在细胞凋亡和抗肿瘤协同作用中的作用将在动物模型中确定，以及使用其他新发现的上调RhoB的药物，这些药物可能对PPAR 3阴性ATC患者有用。在目标3中，我们将开发治疗反应的预后指标。我们的数据表明RhoB是一种决定细胞命运的新型分子开关。因此，拟议的研究不仅有可能提高对RhoB信号传导的理解，而且最终导致ATC的治疗方法得到改善。

DESCRIPTION (Provided By Applicant): In examining the potent novel high affinity PPAR3 agonist, RS5444, as a candidate cancer therapeutic, we discovered that RhoB mRNA and protein were rapidly upregulated by RS5444 in concert with observed antineoplastic effects. This prompted us to examine the novel hypothesis that downstream RhoB effects in part mediate the contributions of PPAR3 to cancer pathogenesis. RhoB is a small GTPase regulating actin organization and vesicle transport not mutated in cancer. RhoB, however, inhibits proliferation. We had previously showed RS5444 and paclitaxel can be combined synergistically in anaplastic thyroid cancer (ATC) models in vitro and in vivo and that the cyclin kinase inhibitor, p21WAF1/CIP1 (p21), is necessary for PPAR3- mediated growth inhibition and for the apoptotic synergy induced by the combination. We now report that RhoB is critical for RS5444/PPAR3-mediated p21 mRNA and protein induction. Silencing RhoB in our laboratory resulted in loss of growth inhibitory activity by RS5444, bolstering our hypothesis, and leading us to demonstrate for the first time that forced RhoB upregulation leads to cancer cell death in ATC. These data, coupled with preliminary data of RhoB down-regulation in our archival ATC tissues, led us to hypothesize that RhoB is important in the chemosensitivity of ATC and in ATC pathogenesis. Based upon these findings, a Phase 1/2 clinical trial combining RS5444 and paclitaxel is under way in ATC in efforts to improve the single agent effects of paclitaxel 3 in this nearly uniformly fatal cancer. Tumor biopsies obtained in conjunction with this trial will allow measurement of RhoB and p21 induction as early response correlates to patient outcome. RS5444 alone induces G0/G1 cell cycle arrest without apoptosis in ATC and forced expression of RhoB (Tet RhoB) proves sufficient for induction of p21, G2/M arrest and apoptosis. These observations coupled with another discovery that RhoB mislocalizes to the nucleus, colocalizing with p21 in a nuclear complex that includes protein kinase C-related kinase one (PRK1), a known RhoB interacting protein and serine/threonine kinase. This complex forms as a result of RS5444 as well as forced RhoB expression leading us to hypothesize that this novel nuclear complex mediates RhoB antitumor activity via direct interaction with cell cycle machinery complexes and that post-translational modifications of p21 direct its physical association with either G0/G1 or G2/M cell cycle machinery thereby specifying cell cycle arrest or apoptosis. In Aim 1, we will elaborate the mechanism(s) by which RhoB mediates cell cycle arrest and apoptosis via this novel nuclear complex. In Aim 2, the role of RhoB in apoptotic and antitumor synergy will be defined in animal models as well as using other newly discovered drugs upregulating RhoB that may be useful for patients with PPAR3- negative ATC. In Aim 3, we will develop prognostic indicators for response to therapy. Our data indicate RhoB as a novel molecular switch dictating cell fate. Thusly, proposed studies have potential not only to improve understanding of RhoB signaling, but to ultimately lead to improved therapeutic approaches in ATC.