Mechanisms of novel cancer targets in ARF-mediated tumor suppression

描述(申请人提供)：E3泛素连接酶MDM2是人类癌细胞中P53肿瘤抑制因子降解的关键因素，抑制MDM2活性是在癌症治疗中激活P53功能的有效方法。然而，MDM2的抑制剂如Nutlin-3有许多局限性，表明这一途径中的其他靶点需要进一步阐明。这些研究旨在剖析两个新发现的泛素连接酶在ARF/P53介导的肿瘤抑制中的关键作用机制，并为在癌症治疗中开发这两个潜在关键靶点的抑制剂提供明确的证据。ARF最初被鉴定为Ink4a/ARF肿瘤抑制基因的另一种转录本。目前公认，ARF在激活P53功能，特别是在应对致癌压力方面发挥着重要作用。令人惊讶的是，通过uSig小鼠模型，在体内P53状态可以在功能和非活动状态之间进行可逆切换，已经发现对肿瘤形成的保护完全依赖于体内ARF介导的P53激活。因此，ARF在抑制肿瘤发生方面的作用显然比最初预期的要重要得多。ARF-P53功能的恢复仍然是寻求更有效的癌症治疗方法的一个重要目标。事实上，像ARF一样，MDM2的小分子抑制剂Nutlin-3能够激活P53，并在表达野生型P53的癌细胞中显示出抗肿瘤效果。然而，虽然Nutlin-3可以有效地阻断MDM2和P53的相互作用，但Nutlin-3在促进MDM2 mRNA的合成和保护MDM2不被降解方面也非常有效。Nutlin-3对MDM2的这些作用导致癌细胞在暂时移除化合物后迅速恢复生长，破坏Nutlin-3在癌症治疗中的疗效。因此，为了达到更有效的治疗目的，显然需要针对这一途径的更多癌症靶点。我们的初步研究表明，靶向ARF-P53途径中的两个新的泛素连接酶(ARF-BP1和ULF)是一种在癌症治疗中激活ARF介导的肿瘤抑制的特别有吸引力的方法。这里要检验的中心假设是，ARF-BP1或ULF的失活可以有效地激活ARF介导的功能，并在体内抑制肿瘤的发生。它包括以下两个具体目标。在目标1中，我们将通过使用小鼠模型来研究ULF活性的失活是否足以抑制肿瘤的发生。在目标2中，为了验证ARF-BP1是否确实是一个潜在的癌症靶点，我们将使用ARF-BP1突变小鼠来检查抑制ARF-BP1是否以一种类似于ARF激活的方式抑制体内的肿瘤形成。

DESCRIPTION (provided by applicant): The E3 ubiquitin ligase Mdm2 acts as a key factor in the degradation of p53 tumor suppressor in human cancer cells and inhibition of Mdm2 activity is a validated approach to activate p53 function in cancer therapy. Nevertheless, inhibitors of Mdm2 such as Nutlin-3 has many limitations, suggesting that additional targets in this pathway need to be further elucidated. The proposed studies aim to dissect the mechanisms of two newly identified ubiquitin ligases critically involved in ARF/p53-mediated tumor suppression and provide the unequivocal evidence for "proof of concept" to develop the inhibitors of these two potential critical targets in cancer therapy. ARF was originally identified as an alternative transcript of the Ink4a/ARF tumor suppressor locus. It is well accepted that ARF plays a major role in activating p53 function, specifically, in respond to oncogenic stress. Surprisingly, by usig a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, it has been found that the protection from tumorigenesis is absolutely dependent on ARF-mediated p53 activation in vivo. Thus, ARF apparently plays a much more important role in suppressing tumorigenesis in general, than originally anticipated. Restoration of the ARF-p53 function remains an important goal in the quest for more effective cancer therapeutics. Indeed, like ARF, Nutlin-3, a small molecule inhibitor of Mdm2, is able to activate p53, and exhibits antitumor efficacy in cancer cells that express wild-type p53. Nevertheless, although Nutlin-3 can effectively block the interaction of Mdm2 and p53, Nutlin-3 is also very effective at increasing the synthesis of Mdm2 mRNA and protecting Mdm2 from degradation. These effects of Nutlin-3 on Mdm2 lead to a rapid restore of cancer cell growth upon temporary removal of the compound, sabotaging the efficacy of nutlin-3 in cancer therapy. Thus additional cancer targets aiming at this pathway are clearly needed for more effective therapeutic purpose. Our preliminary studies reveal that targeting two novel ubiquitin ligases (ARF-BP1 and ULF) in the ARF-p53 pathway represents a particularly attractive approach to activate ARF-mediated tumor suppression in cancer therapy. The central hypothesis to be tested here is that inactivation of ARF-BP1 or ULF can effectively activate ARF-mediated function and suppress tumorigenesis in vivo. It includes the following two specific aims. In Aim 1, we will investigate whether inactivatin of ULF activity is sufficient to suppress tumorigenesis by using mouse models. In Aim 2, to validate whether ARF-BP1 is indeed a potential cancer target, we will use the ARF-BP1 mutant mice to examine whether inhibition of ARF-BP1 suppresses tumorigenesis in vivo in a manner reminiscent of ARF activation.