SIAH2-Dependent Proteolysis in Cell Migration, Tumor Growth and Cancer Metastasis

描述（由申请人提供）：诊断为浸润性和转移性癌症的患者预后不良，表明我们有效的抗癌疗法有限。EGFR/HER 2/RAS通路激活在肿瘤转化和肿瘤发生中的核心重要性已经得到充分证实。癌症生物学的一个重要目标是确定抵消激活的EGFR/HER 2/RAS信号和逆转恶性转化的方法。果蝇SEVEN-IN-ABSENTIA（SINA）及其人类同源物SIAH属于进化上保守的RING结构域E3连接酶家族，其是RAS信号转导所需的必需下游信号传导组分。我们已经进行了三项观察，这些观察构成了本申请的基础：（1）我们表明SIAH是人类癌症中的新生物标志物;（2）我们证明了适当的SIAH功能对RAS信号通路至关重要。通过抑制SIAH 2功能，我们已经完全阻断了11种已知最具侵袭性的人类癌细胞系的肿瘤发生。(3)通过使用抗SIAH 2分子，我们还完全抑制了裸鼠中四种最具侵袭性和转移性的人类癌细胞系的转移（外渗）。当然，我们认识到，由于异种移植模型的不完善，结果仍然是初步的。为了解决这些缺陷并扩大这些观察结果，我们建议在此修订的拨款提案中实现以下两个目标：（I）为了描述SIAH 2在癌症中的功能，通过蛋白质组学方法，我们从所使用的三种最具侵袭性的人类癌细胞系中鉴定了三种LIM结构域蛋白TRIP 6/FHL 2/LPXN作为新型SIAH 2相互作用蛋白。我们提供了大量的生化证据表明TRIP 6/FHL 2/LPXN是真正的SIAH 2底物。这些新的SIAH 2底物可以成功地挽救在SIAH 2缺陷癌细胞中观察到的细胞运动性和活力缺陷，这表明这些粘着斑蛋白可能代表SIAH 2下游介导RAS依赖性信号传导方面的许多不同信号传导模块之一。这一发现可能为解释ERBB/RAS激活如何降低细胞粘附、增加细胞运动性以及促进肿瘤细胞的侵袭和转移提供了一种新的机制。(II)在目标2中，我们将在裸鼠中进行的观察扩展到更稳健的体内系统，以测试抗SIAH分子在转基因小鼠癌症模型中的抗肿瘤功效。这些临床前研究对于证明基于抗SIAH 2的抗癌策略在动物模型中的有效性是必要的。我们的初步数据表明，SIAH 2不足阻断K-RAS介导的小鼠肺肿瘤形成。我们将使用诱导型小鼠模型验证SIAH 2-TRIP 6/FHL 2/LPXN相互作用的分子调控，以了解SIAH 2在调节小鼠中响应RAS激活的细胞连接、粘附和迁移中的分子动力学。最终，我们希望从这些研究中获得的知识将有助于确定新的基于抗SIAH 2的分子机制，以抑制未来的肿瘤生长和转移。

DESCRIPTION (provided by applicant): The dismal prognosis of patients diagnosed with invasive and metastatic cancer points to our limited arsenal of effective anti-cancer therapies. The central importance of EGFR/HER2/RAS pathway activation has been well established in neoplastic transformation and tumorigenesis. An important goal in cancer biology is to identify means of countervailing activated EGFR/HER2/RAS signals and reverse malignant transformation. Drosophila SEVEN-IN-ABSENTIA (SINA) and its human homolog SIAHs belong to an evolutionarily conserved family of the RING domain E3 ligases that are an essential downstream signaling component required for RAS signal transduction. We have made three observations that form the basis for this application: (1) we show that SIAH is a novel biomarker in human cancer; (2) we demonstrate and that proper SIAH function is critical for RAS signaling pathway. By inhibiting SIAH2 function, we have completely blocked tumorigenesis in eleven of the most aggressive human cancer cell lines known. (3) By using anti-SIAH2 molecules, we have also completely inhibited metastasis (extravasation) in four of the most aggressive and metastatic human cancer cell lines in nude mice. Certainly, we are cognizant that the results are still preliminary due to the imperfections of the xenograft models. To address the imperfections and expand these observations, we propose to accomplish the following two aims in this revised grant proposal: (I) To delineate SIAH2 function in cancer, through a proteomic approach, we identified three LIM domain proteins, TRIP6/FHL2/LPXN, as novel SIAH2-interacting proteins from three of the most aggressive human cancer cell lines used. We provide extensive biochemical evidence to show that TRIP6/FHL2/LPXN are bona fide SIAH2 substrates. These new SIAH2 substrates can successfully rescue cell motility and viability defects observed in SIAH2-deficient cancer cells, suggesting that these focal adhesion proteins may represent one of many diverse signaling modules that function downstream of SIAH2 in mediating aspects of RAS-dependent signaling in normal development and cancer. This finding may provide a novel mechanism to explain how ERBB/RAS activation reduces cell adhesion, increases cell motility and promotes invasion and metastasis in tumor cells. (II) In Aim 2, we will extend the observations made in nude mice to a more robust in vivo system to test the anti-tumor efficacy of anti-SIAH molecules in transgenic mouse cancer models. These preclinical studies are necessary to demonstrate the effectiveness of anti-SIAH2-based anticancer strategy in animal models. Our preliminary data indicate that SIAH2-insufficiency blocks K-RAS-mediated lung tumor formation in mice. We will validate the molecular regulation of SIAH2- TRIP6/FHL2/LPXN interaction using the inducible mouse models to understand the molecular dynamics of SIAH2 action in modulating cell junction, adhesion and migration in response to RAS activation in mice. Ultimately, we hope that the knowledge gained from these studies will be useful to identify novel anti-SIAH2- based molecular mechanism(s) to inhibit tumor growth and metastasis in the future.