Defining Mechanisms of Pathological Trans-Lesion Synthesis During Carcinogenesis

总结 我们对细胞如何耐受遗传毒性和内在DNA的理解存在根本性的差距 复制压力，同时积累突变，煽动和燃料致癌。不幸的是， 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损伤耐受性和诱变方面的知识差距限制了我们的研究。 对致癌作用的认识不足，阻碍了癌症的有效预防和治疗。我们的长期 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， 在致癌过程中基因组发生突变。这里的目标是实现这一目标的关键一步， 从机制上定义一种新的癌细胞特异性DNA损伤耐受性和易错DNA合成 途径，并确定其在致癌作用。值得注意的是，我们发现黑色素瘤抗原A4 （MAGE-A4，癌症/睾丸抗原或'CTA'）是DNA修复蛋白的活化结合配偶体 RAD18（一种E3泛素连接酶）在癌细胞中的作用。MAGE-A4在正常体细胞中缺失， 在癌细胞中以非常高的水平表达。MAGE-A4表达也与患者的不良反应有关。 预后，但其对肿瘤发生的潜在作用尚未得到证实。基于令人兴奋和引人注目的 在初步研究中，我们将检验MAGE-A4结合RAD18重编程泛素的中心假设 信号传导，导致病理性跨损伤合成（TLS），DNA损伤耐受性和突变性， 驱动致癌作用。基本原理是，确定MAGE-A4对肿瘤发生的贡献将允许 特别针对肿瘤细胞的脆弱性的治疗策略。具体目标是：（1） 定义MAGE-A4/RAD18介导的DNA损伤耐受性和诱变的分子机制。（二） 确定MAGE-A4/Rad18对体内DNA损伤敏感性、诱变和致癌作用的影响。 (3)确定MAGE-A4/Rad18对致癌应激耐受性的贡献。为了支持SA1，我们将 确定MAGE-A4影响无错和易错（致突变）复制的机制 受损和未受损的DNA模板。对于SA2，我们将使用新的小鼠模型（已经在手）， 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/RAD 18信号传导如何影响耐受性 癌基因诱导的复制应激（在培养细胞中）和癌基因诱导的肿瘤发生（在体内）。的 提出的想法和研究是创新的，因为没有人测试过CTA如何影响基因组 维持、致癌或癌症治疗。这项工作很重要，因为我们将提供新的 与环境暴露、诱变、肿瘤发生相关的基因组维持范例 和癌症治疗。这项工作可能会导致靶向DNA损伤耐受的新策略 和突变性，从而增强化疗的功效和选择性。

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.