Defining Mechanisms of Pathological Trans-Lesion Synthesis During Carcinogenesis

摘要 在我们对细胞如何耐受遗传毒性和固有DNA的理解上存在着根本的差距 复制应激，同时积累刺激和助长癌症发生的突变。不幸的是，DNA 在癌变过程中获得的损伤耐受性和突变性也使癌细胞能够抵抗 化疗。因此，目前我们在DNA损伤耐受性和突变方面的知识空白限制了我们 对癌症发生的了解，排除了癌症的有效预防和治疗。我们的长期合作 目的是解决肿瘤细胞如何耐受DNA损伤和复制应激的相关问题 在致癌过程中突变他们的基因组。这里的目标，是追求这一目标的关键一步，是 从机制上确定一种新的癌细胞特异性DNA损伤耐受性和易出错的DNA合成 并确定其在癌症发生中的作用。值得注意的是，我们发现黑色素瘤抗原-A4 (MAGE-A4，一种癌症/睾丸抗原或CTA)是DNA修复蛋白的激活结合伙伴 RAD18(一种E3泛素连接酶)在癌细胞中的表达。MAGE-A4在正常体细胞中缺失，并发生异常 在癌细胞中高水平表达。MAGE-A4的表达也与贫困患者相关 然而，它对肿瘤发生的潜在贡献还没有得到检验。基于令人兴奋和令人信服的 初步研究，我们将检验MAGE-A4与RAD18结合以重新编程泛素的中心假设 信号，导致病理性跨损伤合成(TLS)，DNA损伤耐受性和突变性， 导致癌症的发生。其基本原理是，定义MAGE-A4在肿瘤发生中的作用将允许 针对肿瘤细胞脆弱性的治疗策略。具体目标是：(1) 明确MAGE-A4/RAD18介导的DNA损伤耐受和突变的分子机制。(2) 检测MAGE-A4/RAD18对DNA损伤敏感性、体内诱变和致癌的影响。 (3)确定MAGE-A4/RAD18在肿瘤应激耐受中的作用。为了支持SA1，我们将 确定MAGE-A4影响无错误和易出错(突变)复制的机制(S) 受损和未受损的DNA模板。对于SA2，我们将使用新的鼠标模型(已有)来 确定条件性MAGE-A4表达如何影响DNA损伤耐受性、基因组稳定性和 体内化学致癌作用。对于SA3，我们将确定MAGE-A4/RAD18信号如何影响耐受性 致癌基因诱导的复制应激(在培养细胞中)和致癌基因诱导的肿瘤形成(在体内)。这个 提出的想法和研究是创新的，因为还没有人测试过CTA如何影响基因组 维持、致癌或癌症治疗。拟议的工作意义重大，因为我们将提供新的 与环境暴露、突变、肿瘤发生有关的基因组维护范例 以及人类的癌症治疗。这项工作可能导致针对DNA损伤耐受性的新策略 和突变性，特别是在癌细胞中，从而提高化疗的有效性和选择性。

SUMMARY There are fundamental gaps in our understanding of how cells tolerate genotoxicity and intrinsic DNA replication stress while accumulating the mutations that incite and fuel carcinogenesis. Unfortunately, the DNA damage tolerance and mutability acquired during carcinogenesis also allow cancer cells to resist chemotherapy. Thus, current gaps in our knowledge of DNA damage tolerance and mutagenesis limit our understanding of carcinogenesis and preclude effective prevention and treatment of cancer. Our long-term goal is to solve the related problems of how neoplastic cells tolerate DNA damage and replication stress while mutating their genomes during carcinogenesis. The objective here, a critical step in the pursuit of that goal, is to mechanistically define a novel cancer cell-specific DNA damage-tolerance and error-prone DNA synthesis pathway and determine its role in carcinogenesis. Remarkably, we discovered that Melanoma Antigen-A4 (MAGE-A4, a Cancer/Testes Antigen or 'CTA') is an activating binding partner of the DNA repair protein RAD18 (an E3 ubiquitin ligase) in cancer cells. MAGE-A4 is absent from normal somatic cells and aberrantly expressed at very high levels in cancer cells. MAGE-A4 expression is also associated with poor patient prognosis, yet its potential contribution to tumorigenesis is untested. Based on exciting and compelling preliminary studies, we will test the central hypothesis that MAGE-A4 binds RAD18 to reprogram ubiquitin signaling, leading to pathological Trans-Lesion Synthesis (TLS), DNA damage-tolerance and mutability that drive carcinogenesis. The rationale is that defining the contribution of MAGE-A4 to tumorigenesis will allow therapeutic strategies that specifically target vulnerabilities of neoplastic cells. The Specific Aims are: (1) Define molecular mechanisms of MAGE-A4/RAD18-mediated DNA damage tolerance and mutagenesis. (2) Determine impact of MAGE-A4/Rad18 on DNA damage-sensitivity, mutagenesis and carcinogenesis in vivo. (3) Determine contribution of MAGE-A4/Rad18 to tolerance of oncogenic stress. In support of SA1 we will determine the mechanism(s) by which MAGE-A4 influences error-free and error-prone (mutagenic) replication of damaged and undamaged DNA templates. For SA2 we will use novel mouse models (already in hand) to determine how conditional MAGE-A4 expression impacts DNA damage tolerance, genome stability and chemical carcinogenesis in vivo. For SA3 we will determine how MAGE-A4/RAD18 signaling affects tolerance of oncogene-induced replication stress (in cultured cells) and oncogene-induced tumorigenesis (in vivo). The proposed ideas and research are innovative because nobody has ever tested how CTAs affect genome maintenance, carcinogenesis or cancer therapy. The proposed work is significant because we will provide new paradigms for genome maintenance that are relevant to environmental exposures, mutagenesis, tumorigenesis and cancer therapy in humans. This work may lead to novel strategies for targeting DNA damage tolerance and mutability specifically in cancer cells, thereby enhancing the efficacy and selectivity of chemotherapy.