Smoothened function as a G-Protein Coupled Receptor in mammary epithelial cells

描述(申请人提供)：Hedgehog(HH)信号网络调节胚胎发育和成人干细胞维持。HH信号改变与大约25%的人类癌症有关，包括皮肤癌、前列腺癌、脑癌和乳腺癌。SMO是HH通路的重要组成部分，在约70%的导管原位癌(DCIS)和约30%的浸润性乳腺癌中过表达。在小鼠中，SMO的过度表达增加了细胞分裂并破坏了乳腺的形态。SMO还与ErbB2(Her2)癌基因合作，促进小鼠肿瘤的形成。总而言之，这些数据表明了HH信号在乳腺癌中的重要性。从机制上讲，SMO驱动的过度增殖似乎并不完全是由已知的GLI转录因子激活机制介导的。相反，这种快速的细胞分裂可以被百日咳毒素完全阻断，百日咳毒素是一种“异源三聚体G蛋白”的特异性抑制剂。这一发现表明，我们的假设是，SmoM2的激活通过一种独特的G蛋白介导的机制促进乳腺上皮细胞的增殖。为了检验这一假说并确定G蛋白下游的关键调节因子(S)，具体目的是：特定目的I：确定G1I亚基在我们的SMO突变小鼠的乳腺中表达，并使用转录检测和干扰方法结合移植来测试此类亚单位(S)的功能中断是否可以阻止SMO突变小鼠乳腺表型的改变。特定目的II：利用蛋白表达和检测方法，检测SMO介导的乳腺上皮细胞增生症是否是23个亚基通过PTX敏感机制传递信号的结果。具体目的III：使用药物干预方法，在我们的模型中，确定G蛋白激活下游驱动SMO介导的乳腺增生表型的分子机制。意义：由于目前正在开发的小分子抑制剂仅设计用于抑制SMO下游的GLI介导的信号转导，而不一定是G蛋白介导的信号转导，因此拟议中的HH信号转导抑制剂临床开发研究的意义可能是令人震惊的。培训计划：贝勒医学院的莱斯特和苏·史密斯乳房中心允许在合作和丰富的环境中进行培训，在这种环境中，基础科学、翻译和临床研究同等重要。这种形式的培训通过每两周一次的研究和开发研讨会、杂志俱乐部系列、每周一次的乳腺癌研讨会和一年一度的乳房中心务虚会来加强。除了乳房中心提供的培训外，分子和细胞生物学系还举办了一年一度的学生研究研讨会、每周一系列的研讨会和一年一度的研究生研讨会。所有这些活动都创造了一个完美的环境，在这个环境中，我不仅可以向我的同事和教职员工展示研究成果，还可以收到宝贵的反馈，这只会将我的研究推向更高的水平。 公共卫生相关性：这项拟议的研究旨在确定特定的G蛋白亚基参与平滑(SMO)驱动的增生性疾病，以及确定G蛋白下游发挥作用的其他关键介质(S)。这些新靶点在促进细胞增殖方面很重要，而细胞增殖在乳腺癌的早期阶段可能是重要的。由于目前正在开发的小分子抑制剂仅设计用于抑制SMO下游的GLI介导的信号转导，而不一定是G蛋白介导的信号转导，因此拟议的Hedgehog信号转导抑制剂临床开发研究的意义可能是令人惊讶的。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) signaling network regulates embryonic development and adult stem cell maintenance. Altered Hh signaling has been implicated in approximately 25% of all human cancers including those of skin, prostate brain and breast. Smoothened (SMO), a critical component of the Hh pathway, is overexpressed in about 70% of ductal carcinoma in situ (DCIS), and about 30% of invasive breast cancer. In mice, SMO overexpression increases cell division and disrupts mammary gland morphology. SMO also cooperates with the ErbB2 (Her2) oncogene to promote tumor formation in mice. Collectively, these data suggest the importance of Hh signaling in breast cancer. Mechanistically, SMO-driven hyperproliferation does not appear to be completely mediated by the known mechanism of GLI transcription factor activation. Rather, this rapid cell division can be completely blocked by pertussis toxin, a specific inhibitor of "heterotrimeric G- proteins." This finding suggests our hypothesis that SmoM2 activation increases cell proliferation through a unique G-protein mediated mechanism in mammary epithelial cells. To test this hypothesis and to identify critical mediators functioning downstream of the G protein(s), the specific aims are: Specific Aim I: To determine which G1i subunits are expressed in the mammary gland of our SMO mutant mice and to test whether disruption of such subunit(s) function can block the altered mammary gland phenotype in SMO mutant mice using transcript detection & disruption methods coupled with transplantation. Specific Aim II: To test whether SMO-mediated mammary epithelial hyperplasias are a result of 23 subunit signaling through a PTX-sensitive mechanism using protein expression & detection methods. Specific Aim III: To define the molecular mechanism downstream of G-protein activation that drives the SMO- - mediated mammary hyperplastic phenotype in our model using a pharmacological intervention approach. Significance: Since small molecule inhibitors currently under development were designed to inhibit only the GLI-mediated signaling, but not necessarily G protein-mediated signaling, downstream of SMO, the implications of the proposed research on clinical development of Hh signaling inhibitors may be astounding. Training Program: The Lester and Sue Smith Breast Center at Baylor College of Medicine allows training in a collaborative and enriching environment in which basic science, translational, and clinical research are valued equally. This form of training is reinforced by the bi-weekly Research and Development workshops, journal club series, weekly breast cancer seminars and the annual Breast Center retreat. In addition to the training provided by the Breast Center, the department of Molecular and Cellular Biology holds its annual student research symposium, a weekly seminar series and the annual graduate student symposium. All of these events create the perfect environment in which to not only present research to my peers and faculty, but to receive valuable feedback which will only advance my research to a higher level. PUBLIC HEALTH RELEVANCE: The proposed study aims to identify the specific G protein subunits involved in Smoothened (SMO) driven hyperplasias, as well as to identify other critical mediators functioning downstream of the G protein(s). These new targets are important in promoting increased cell proliferation that could be important in the initial stages of breast cancer. Since small molecule inhibitors currently under development were designed to inhibit only the GLI-mediated signaling, but not necessarily G protein-mediated signaling, downstream of SMO, the implications of the proposed research on clinical development of Hedgehog signaling inhibitors may be astonishing.