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

项目摘要 肠干细胞项目的失败是结直肠癌（CRC）发病机制的基础， 2015年，该疾病影响了超过13万人。因此，进一步定义规范 基本的干细胞过程可能导致新的治疗目标。因此，Wnt信号传导 在维持肠道内稳态中起着重要作用， 与结肠癌的发展有着千丝万缕的联系。β-连环蛋白是Wnt信号传导的关键效应子， 将信号传导至细胞核或作为细胞膜上钙粘蛋白复合物的组分。 CRC中Wnt突变的优势最终增加了细胞内β-连环蛋白的水平。然而，在这方面， 靶向该途径迄今尚未产生治疗效果。 血管心外膜物质（BVES）是一种紧密连接相关蛋白， 心脏发育的屏幕。BVES在结直肠癌中表达下调，BVES过度表达损害肿瘤 生长，确定BVES在致癌作用中的肿瘤抑制作用。BVES也被证明会影响 Wnt活性。BVES表达降低了Wnt报告基因活性，并在小鼠炎症模型中 在致癌作用中，Bves-/-肿瘤具有增加的β-连环蛋白水平。由于Wnt信号传导在维持细胞的增殖中至关重要， 肠干细胞龛，Bves-/-小鼠也有激活的干细胞程序。综合来看， 假设BVES主要通过对β-连环蛋白的影响来调节Wnt信号传导。这一假设 将通过两个集中的，机械的，假设驱动的具体目标，将扩大我们的测试 了解BVES如何调节Wnt信号传导。首先，BVES改变β-连环蛋白的机制 水平将被确定。BVES与蛋白磷酸酶2A调节亚基PR 61 α相互作用， 假设BVES通过这种相互作用调节β-连环蛋白。或者，由于BVES可以定位到 膜β-连环蛋白活性可以通过膜螯合来控制。第二，BVES 将采用条件性基因敲除小鼠模型来确定肠干细胞中BVES的丧失和 随后Wnt信号传导的改变影响肠隐窝动力学。新的3D肠样培养物将是 用于扩展这些研究，并进一步表征BVES的功能结构域， 调节Wnt依赖性表型。这些研究将转化为人源性类肿瘤 培养以确定恢复BVES功能是否可以调节肿瘤生长。我们提出的研究将确定 BVES在Wnt信号传导、肠干细胞生物学和结直肠癌中的作用。重要的是，澄清 BVES的作用可能阐明靶向这种促肿瘤发生途径的新机制。

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.