BVES modulation of intestinal homeostasis and tumorigenesis via regulation of Wnt signaling

项目摘要 肠道干细胞计划的异常是结直肠癌(CRC)发病机制的基础， 这种疾病在2015年影响了超过13万人。因此，进一步定义规范 基本的干细胞过程可能会导致新的治疗靶点。因此，WNT信令 途径在维持肠道内环境稳定中起着重要作用，而这一途径的破坏 与结肠癌的发展有着千丝万缕的联系。β-连环蛋白是Wnt信号转导的关键效应器， 将信号传递到细胞核或作为细胞膜上钙粘附素复合体的一个组成部分。 Wnt突变在结直肠癌中的优势最终增加了细胞内β-连环蛋白的水平。然而， 到目前为止，针对这一途径还没有产生治疗效果。 血管心外膜物质(BVES)是在基因中发现的一种紧密连接相关蛋白 发展中的心灵的屏风。BVES在结直肠癌中下调，BVES过表达损害肿瘤 生长，确定BVES在致癌过程中的肿瘤抑制作用。BVES也被证明会影响 WNT活动。BVES的表达降低了Wnt报告蛋白的活性，并在炎性小鼠模型中 在癌变过程中，BVES-/-肿瘤中β-连环蛋白水平升高。由于WNT信令在维护 肠道干细胞利基BVES-/-小鼠也有激活的干细胞计划。综上所述，它是 假设BVES主要通过对β-连环蛋白的作用来调节Wnt信号。这一假设 将通过两个集中的、机械性的和假设驱动的特定目标进行测试，这两个目标将扩展我们的 了解BVES如何调节Wnt信号。首先，BVES改变β-连锁素的机制 级别将被确定。BVES与蛋白磷酸酶2A调节亚基Pr61α相互作用，它是 假设BVES通过这种相互作用调节β-连环蛋白。或者，由于BVES可以本地化到 膜的β-连环蛋白活性可通过膜隔离来调控。第二，BVES 将采用条件性基因敲除小鼠模型来确定肠道干细胞和 随后Wnt信号的改变会影响肠道隐窝的动力学。新的3D肠状培养将是 用来扩展这些研究并进一步描述BVES负责 调节Wnt依赖的表型。然后，这些研究将被转化为人类衍生的类癌 培养以确定恢复BVES功能是否可以调节肿瘤生长。我们提议的研究将界定 BVES在Wnt信号、肠道干细胞生物学和结直肠癌中的作用。重要的是，澄清 BVES的作用可能阐明针对这一促肿瘤途径的新机制。

Project Summary Abnormalities in intestinal stem cell programs underlie the pathogenesis of colorectal cancer (CRC), a disease that affected over 130,000 individuals in 2015. Therefore, further defining programs that regulate fundamental stem cell processes may lead to novel therapeutic targets. Accordingly, the Wnt signaling pathway plays a fundamental role in maintaining intestinal homeostasis and disruptions in this pathway are inextricably linked to the development of colon cancer. β-catenin is the key effector of Wnt signaling, transducing signals to the nucleus or acting as a component of the cadherin complex at the cell membrane. The preponderance of Wnt mutations in CRC ultimately increase levels of β-catenin within the cell. However, targeting this pathway has, as of yet, not yielded therapeutic efficacy. Blood vessel epicardial substance (BVES) is a tight junction-associated protein discovered in a cDNA screen of the developing heart. BVES is downregulated in CRC and overexpression of BVES impairs tumor growth, identifying a tumor suppressive role for BVES in carcinogenesis. BVES has also been shown to affect Wnt activity. BVES expression reduces Wnt reporter activity and in mouse models of inflammatory carcinogenesis, Bves-/- tumors have increased levels of β-catenin. As Wnt signaling is critical in maintenance of the intestinal stem cell niche, Bves-/- mice also have activated stem cell programs. Taken together, it is hypothesized that BVES modulates Wnt signaling, principally through an effect on β-catenin. This hypothesis will be tested via two focused, mechanistic, and hypothesis driven specific aims that will expand our understanding of how BVES regulates Wnt signaling. First, the mechanism by which BVES alters β-catenin levels will be determined. BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, and it is hypothesized that through this interaction BVES modulates β-catenin. Alternatively, as BVES can localize to the membrane, β-catenin activity may be controlled through membrane sequestration. Second, a BVES conditional knockout mouse model will be employed to determine if loss of BVES in intestinal stem cells and subsequent alterations in Wnt signaling affect intestinal crypt dynamics. Novel 3D enteroid cultures will be employed to expand these studies and further characterize the functional domain of BVES responsible for modulating Wnt dependent phenotypes. These studies will then be translated into human derived tumoroid cultures to determine if restoring BVES function can modulate tumor growth. Our proposed studies will define the role of BVES in Wnt signaling, intestinal stem cell biology, and colorectal cancer. Importantly, clarifying the role of BVES may elucidate novel mechanisms to target this pro-tumorigenic pathway.