Establishing the molecular and cellular mechanisms of Lgr5 signaling for controlling cancer stem cell behavior

摘要 结肠癌是男性和女性癌症死亡的第二大原因。因此，我们认为， 替代疗法是迫切需要的。在正常组织中，肠干细胞的作用是使肠上皮细胞能够更新。 整个上皮细胞。目前已知干细胞转化为结肠癌干细胞 是致癌作用的主要诱因。癌症干细胞是肿瘤起始细胞， 难治性，并与晚期结肠癌相关的极差预后有关 诊断.如果能够消除结肠癌干细胞，可能会产生更好的治疗方法。然而，根除这种 由于结肠癌干细胞的内在能力， 是长寿的，自我更新的，高度增殖的。对这个问题的一个建议的解决方案是 使癌症干细胞命运偏向于终末分化细胞命运。换句话说，开车 癌症干细胞采用不增殖的细胞命运，具有短的预期寿命， 对化疗敏感这种分化治疗的实现需要癌症的身份 干细胞是已知且存在用于治疗进入点的候选分子靶。为此中央 已发现富含亮氨酸的G蛋白偶联受体-5（Lgr 5）可作为结肠癌干细胞的标记物 并且是诱人的药理学靶点。Lgr 5与其同系物Lgr 4一起作为调节剂， Wnt/β连环蛋白信号通路。Wnt/β catenin信号通路是调控干细胞的关键通路 肠道内的稳态。该通路中的突变导致过度信号传导并强烈地偏向干细胞 向长寿和高度增殖的癌症干细胞的行为。因此，LGR 5是一个有吸引力的 药理学靶点，以抵消这种不平衡和恢复正常的细胞命运动态。但 Lgr 5信号传导的机制及其在体内的作用仍然是模糊的，代表了一个主要的知识空白。 因此，本提案的目标将是确定Lgr 5信号传导如何控制干细胞和癌症 干细胞命运为了实现这一目标，我将测试中心假设，抑制Lgr 5 内化减弱Wnt/β连环蛋白信号传导并促进癌症干细胞分化。三个具体 已经提出了目的来验证这一假设，并将在（目的1）阐明Lgr 5介导的 Wnt/β catenin信号传导，（Aim 2）阐明Lgr 5依赖性信号传导机制协调茎 细胞行为，以及（目标3）确定驱动癌症干细胞的小分子干预措施 分化本研究计划中描述了关键的初步结果， 核心假设和具体目标。生物化学、细胞生物学、药理学和癌症的整合 生物学使这成为一个创新的研究方案。这项提议意义重大，因为它将改变LGR 5 从干细胞标记物转化为高价值的候选药物， 癌症干细胞的命运我的研究和职业发展计划展示了跨学科的培训 我收到的。这跨越了生物医学科学的范围，包括干细胞生物学、分子生物学和细胞生物学。 生物学、基因工程、癌症研究、生物化学、细胞命运分析和高通量筛选。 这个NCI K22提案将巩固这些技能的整合，并促进我自己的独立研究 这是我的短期职业目标。通过完成这份提案，我将能够获得 R 01资金和工作，以实现我的长期职业目标，使高影响力和基础 这些发现可以转化为治疗和治愈癌症的方法。这样做，我将成为一个 NCI的使命的仪器组成部分“，以加快科学发现的速度，减少 美国和全世界的癌症负担。”

ABSTRACT Colon cancer is the second leading cause of cancer deaths among men and women combined. Therefore, alternative therapies are of critical need. In normal tissue, intestinal stem cells act to enable renewal of the entire epithelium every week. It is now known that the transformation of stem cells into colon cancer stem cells is a major inciting event that underlies carcinogenesis. Cancer stem cells are tumor-initiating cells, are refractory to therapy, and are linked to the very poor prognosis associated with late-stage colon cancer diagnosis. Better therapies may result if colon cancer stem cells could be eliminated. However, eradicating this population of cells is an extremely challenging problem, due to the intrinsic capacity of colon cancer stem cells to be long-lived, self-renewing, and highly proliferative. One proposed solution to this problem is to pharmacologically bias cancer stem cell fate toward a terminally differentiated cell fate. In other words, drive cancer stem cells to adopt a cell fate that does not proliferate, that has a short life expectancy, and that is sensitive to chemotherapy. Realization of this differentiation therapy requires that the identity of the cancer stem cell be known and that a candidate molecular target exists for a therapeutic entry point. To this end, the leucine-rich G protein coupled receptor-5 (Lgr5) has been found to act as a marker for colon cancer stem cells and is a tantalizing pharmacological target. Lgr5, together with its homologue Lgr4, act as modulators for the Wnt/ßcatenin signaling pathway. Wnt/ßcatenin signaling is a critical pathway that regulates stem cell homeostasis in the intestine. Mutations in this pathway result in excessive signaling and strongly bias stem cell behavior toward that of a long-lived and highly proliferative cancer stem cell. Lgr5 is therefore an attractive pharmacological target for counteracting this imbalance and restoring normal cell fate dynamics. However, the mechanisms of Lgr5-signaling and its roles in vivo are still vague and represent a major knowledge gap. Therefore, the objective of this proposal will be to determine how Lgr5-signaling controls stem and cancer stem cell fate. To accomplish this objective, I will test the central hypothesis that inhibition of Lgr5 internalization attenuates Wnt/ßcatenin signaling and promotes cancer stem cell differentiation. Three specific aims have been proposed to test this hypothesis and will in (Aim 1) Clarify the mechanism of Lgr5-mediated Wnt/ßcatenin signaling, (Aim 2) Elucidate Lgr5-dependent signaling mechanisms coordinating stem cell behavior, and (Aim 3) Identify small molecule interventions for driving cancer stem cell differentiation. Key preliminary findings are described in this research plan that enable formulation of the central hypothesis and specific aims. The integration of biochemistry, cell biology, pharmacology, and cancer biology make this an innovative research proposal. This proposal is significant because it will transform Lgr5 from a stem cell marker into a high-value drug candidate that can be pharmacologically targeted to control cancer stem cell fate. My research and career development plan demonstrates the interdisciplinary training that I have received. This spans the biomedical science spectrum and includes stem cell biology, molecular biology, genetic engineering, cancer research, biochemistry, cell-fate analysis, and high-throughput screening. This NCI K22 proposal will solidify the integration of these skills and facilitate my own independent research program, which is my short-term career objective. By completing this proposal I will be in a position to obtain R01 funding and work towards my long-term career objective of making high-impact and foundational discoveries that can be translated into treatments and cures for cancer. In so doing, I will become an instrumental component of the NCI's mission “to accelerate the rate of scientific discovery and reduce the burden of cancer in the United States and around the world.”