Defining the genetic requirements for maintenance of colorectal cancer

描述（申请人提供）：结直肠癌（Colorectal cancer， CRC）是一种非常普遍和难以治疗的癌症。在疾病的晚期，几乎没有有效的治疗选择，使其成为发达国家癌症相关死亡的第二大原因。与许多其他受益于靶向药物发展的癌症治疗策略不同，CRC的标准治疗仍然是细胞毒性化疗的组合。迫切需要更有针对性、毒性更小的治疗方法。确定肿瘤生长和进展的肿瘤特异性需求为发现药物靶点提供了一条潜在途径。该建议整合了新的遗传技术和体外培养系统，以表征结直肠癌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。大肠腺瘤性息肉病（APC）肿瘤抑制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起始事件。APC的破坏导致Wnt通路的不受控制的激活，正因为如此，目前人们正在努力开发用于治疗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改变，最常见的是p53肿瘤抑制因子的缺失（60%）和/或Kras的致癌激活（45%）。鉴于该疾病的复杂性，目前尚不清楚crc是否仍然依赖APC丢失和Wnt过度激活来生长和存活，因此也不清楚Wnt靶向治疗是否能有效治疗晚期恶性肿瘤。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APC表达的时间，空间和可逆控制。所描述的方法结合了现有的和新的小鼠菌株，使多西环素诱导的shrna介导的APC在成年小鼠的肠道和结肠中特异性沉默。最重要的是，APC的丢失可以简单地通过停用强力霉素来逆转，恢复内源性肿瘤抑制网络。在目标1中，我将定义在维持良性和发育不良病变中持续APC损失的要求，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）患者中发展的早期肿瘤。此外，我将使用相同的转基因小鼠模型来开发和表征和替代离体培养系统，以检查APC在早期疾病中恢复的分子后果。Aim 2扩展了Aim 1中开发的系统，以研究在侵袭性和转移性CRC进展过程中获得的复发性遗传改变（致癌Kras突变和p53缺失）如何影响对Wnt抑制的反应。由于Kras和/或p53突变在约80%的APC突变肿瘤中观察到，了解这些事件如何导致Wnt依赖性对于未来Wnt靶向治疗的临床应用至关重要。最后，通过表征肿瘤对APC恢复的反应以及对耐药或复发的Wnt独立疾病的详细分析，我的目标是确定介导对Wnt抑制的抗性的候选基因/途径，以期它们将成为组合治疗方法的有吸引力的靶点。本提案中概述的工作将为CRC中常见突变基因的作用提供关键见解，并确定对CRC细胞生长和存活至关重要的因素，代表了合理治疗的潜在目标

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.