Defining the genetic requirements for maintenance of colorectal cancer

简介(申请人提供)：结直肠癌(CRC)是一种极其常见且难以治疗的癌症。在疾病的晚期，几乎没有有效的治疗选择，使其成为发达国家与癌症相关的死亡的第二大原因。与受益于靶向药物开发的许多其他癌症治疗策略不同，结直肠癌的护理标准仍然是细胞毒性化疗的组合。迫切需要更有针对性、毒性更低的治疗方法。明确癌症生长和进展的肿瘤特异性要求为发现药物靶点提供了一条潜在的途径。这项建议整合了新的基因技术和体外培养系统，以表征CRC维持和进展的遗传要求，目的是确定可用于癌症治疗的肿瘤特异性弱点。腺瘤性结肠息肉病(APC)抑癌基因的突变失活被认为是大多数家族性和散发性结肠癌的始发事件。APC的破坏导致Wnt通路的失控激活，正因为如此，目前有大量的努力开发用于治疗CRC的选择性Wnt信号抑制剂。然而，从良性腺瘤到结直肠癌的进展涉及许多额外的遗传和表观遗传学改变，最常见的是P53肿瘤抑制基因缺失(60%)和/或Kras癌基因激活(45%)。鉴于疾病的复杂性质，目前尚不清楚癌细胞是否仍然依赖APC缺失和Wnt过度激活来生长和存活，因此Wnt靶向治疗在治疗晚期恶性肿瘤方面是否有效。在这里，我将使用我开发的一种独特的转基因小鼠模型来解决这个突出但重要的问题，该模型允许对APC的表达进行时间、空间和可逆控制。这种方法结合了现有的和新的小鼠品系，能够使多西环素诱导的shRNA介导的APC沉默，特别是在成年小鼠的肠道和结肠中。最重要的是，APC的丢失可以通过停用多西环素来逆转，恢复内源性肿瘤抑制网络。在目标1中，我将定义维持良性和发育不良病变的持续APC丢失的要求，这代表了结肠癌变的开始，并反映了家族性腺瘤性息肉病(FAP)患者发展的早期肿瘤。此外，我将使用相同的转基因小鼠模型来开发、表征和替代体外培养系统，以检测APC修复在早期疾病中的分子后果。目的2扩展在目标1中开发的系统，以研究在进展为侵袭性和转移性结直肠癌过程中获得的反复遗传改变(致癌Kras突变和P53丢失)如何影响对Wnt抑制的反应。由于在约80%的APC突变肿瘤中观察到Kras和/或p53突变，了解这些事件如何导致WNT依赖对于WNT靶向治疗的未来临床应用至关重要。最后，通过表征肿瘤对APC修复的反应，以及对耐药或复发的、Wnt非依赖性疾病的详细分析，我的目标是识别介导对Wnt抑制的抵抗的候选基因/途径，并展望它们将代表具有吸引力的联合治疗方法的靶向。这项提案中概述的工作将提供对常见突变基因在结直肠癌中的作用的关键见解，并确定对结直肠癌细胞的生长和生存至关重要的因素，代表Rational的潜在靶点 药物开发。通过这项工作开发的试剂、工具和知识将构成灵活和创新的研究计划的基础，该计划有可能为理解和治疗多种形式的癌症做出重大贡献。

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is an extremely prevalent and difficult to treat cancer. In the advanced stage of disease there are virtually no effective treatment options rendering it the second-leading cause of cancer- related deaths in the developed world. Unlike a number of other cancer treatment strategies, which have benefited from the development of targeted agents, the standard of care for CRC remains a combination of cytotoxic chemotherapies. There is an urgent need for more targeted, less toxic, treatments. Defining the tumor-specific requirements for cancer growth and progression offers one potential avenue for drug target discovery. This proposal integrates new genetic technologies and ex vivo cultures systems to characterize the genetic requirements for CRC maintenance and progression, with the goal of identifying tumor-specific weaknesses that could be exploited for cancer therapy. Mutational inactivation of the Adenomatous Polyposis Coli (APC) tumor suppressor is thought to be the initiating event in most familial and sporadic colon cancers. APC disruption results in uncontrolled activation of the Wnt pathway and because of this there is currently significant effort to develop selective Wnt signalling inhibitors for treatment of CRC. However, progression from benign adenoma to CRC involves many additional genetic and epigenetic alterations, most commonly, loss of the p53 tumor suppressor (60%) and/or oncogenic activation of Kras (45%). Given the complex nature of the disease it is unclear if CRCs remain dependent on APC loss and Wnt hyperactivation for growth and survival, and thus whether Wnt- targeted therapies will be effective in treating advanced malignancies. Here I will address this outstanding, but important question, using a unique transgenic mouse model I developed that allows temporal, spatial and reversible control of APC expression. The approach described combines existing and novel mouse strains to enable doxycycline-inducible shRNA-mediated APC silencing specifically in the intestine and colon of adult mice. Most importantly, APC loss can be reversed simply by withdrawal of doxycycline, restoring the endogenous tumor suppressor network. In Aim 1 I will define the requirement for sustained APC loss in the maintenance of benign and dysplastic lesions, which represent the initiation of carcinogenesis in the colon and mirror the early stage tumors that develop in patients with Familial Adenomatous Polyposis (FAP). In addition, I will use the same transgenic mouse model to develop and characterize and surrogate ex vivo culture system to examine the molecular consequences of APC restoration in early stage disease. Aim 2 expands on the systems developed in Aim 1 to investigate how recurrent genetic alterations acquired during progression to invasive and metastatic CRC (oncogenic Kras mutation and loss of p53) affect the response to Wnt inhibition. As Kras and/or p53 mutations are observed in ~80% of APC mutant tumors, understanding how these events contribute to Wnt- dependency is critical for future clinical applications of Wnt-targeted therapy. Finally, through characterisation of the tumor response to APC restoration and detailed analysis of resistant or relapsed, Wnt-independent disease, I aim to identify candidate genes/pathways that mediate resistance to Wnt inhibition with the vision that they will represent attractive targeted for combinatorial therapeutic approaches. The work outlined in this proposal will provide key insights into the role of commonly mutated genes in CRC and define factors that are essential for the growth and survival of CRC cells, representing potential targets for rational drug development. The reagents, tools and knowledge developed through this work will form the foundation of a flexible and innovative research program that has the potential to contribute significantly to the understanding and treatment of many forms of cancer.