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

乳腺上皮细胞中 G 蛋白偶联受体的平滑功能

• 批准号: 8505416
• 负责人: Hugo Villanueva
• 金额: $ 3.94万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505416
• 关键词: 3-Dimensional; Adenylate Cyclase; Attenuated; Basic Science; Biological Assay; Brain; Breast; Cell Culture Techniques; Cell Maintenance; Cell Proliferation; Cell division; Cells; Clinical; Clinical Research; Coupled; Coupling; Data; Detection; Development; Educational workshop; Embryonic Development; Environment; Epithelial; Epithelial Cells; Erinaceidae; Event; Faculty; Family; Feedback; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Human; Hyperplasia; Immunofluorescence Microscopy; In Vitro; Intervention; Journals; Knockout Mice; Lead; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Measures; Mediating; Mediator of activation protein; Medicine; Methods; Modeling; Molecular; Molecular and Cellular Biology; Morphology; Mouse Mammary Tumor Virus; Mus; Muscle; Mutant Strains Mice; Noninfiltrating Intraductal Carcinoma; Oncogenes; Organ Culture Techniques; Outcome; Pathway interactions; Peptides; Pertussis Toxin; Phenotype; Prostate; Protein Subunits; Proteins; Research; Research Design; SCID Beige Mouse; Series; Signal Transduction; Skin; Staging; Students; Techniques; Testing; Training; Training Programs; Transcript; Transgenic Organisms; Transplantation; adult stem cell; base; cellular transduction; college; design; graduate student; human SMO protein; inhibitor/antagonist; malignant breast neoplasm; member; mouse model; mutant; overexpression; peer; protein activation; protein expression; receptor; receptor coupling; research and development; small hairpin RNA; small molecule; smoothened signaling pathway; src-Family Kinases; symposium; transcription factor; tumor

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.

描述（由申请人提供）：Hedgehog （Hh）信号网络调节胚胎发育和成体干细胞维持。Hh信号的改变与大约25%的人类癌症有关，包括皮肤癌、前列腺癌、脑癌和乳腺癌。Smoothened （SMO）是Hh通路的关键组成部分，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过表达增加细胞分裂并破坏乳腺形态。SMO还与ErbB2 （Her2）癌基因合作，促进小鼠肿瘤的形成。总的来说，这些数据表明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由已知的GLI转录因子激活机制介导。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。这一发现支持了我们的假设，即SmoM2激活通过一种独特的g蛋白介导的机制在乳腺上皮细胞中增加细胞增殖。为了验证这一假设并确定在G蛋白下游发挥作用的关键介质，具体目的如下：具体目的1：确定哪些G1i亚基在我们的SMO突变小鼠的乳腺中表达，并使用转录检测和破坏方法结合移植测试这些亚基功能的破坏是否可以阻断SMO突变小鼠改变的乳腺表型。特异性目的二：通过蛋白表达和检测方法检测smo介导的乳腺上皮增生是否为23个亚单位信号通过ptx敏感机制的结果。特定目的III：在我们的模型中，使用药物干预方法确定驱动SMO介导的乳腺增厚表型的g蛋白激活下游的分子机制。意义：由于目前正在开发的小分子抑制剂仅用于抑制SMO下游的glii介导的信号传导，而不一定是G蛋白介导的信号传导，因此拟议研究对Hh信号传导抑制剂临床开发的影响可能是惊人的。培训项目：贝勒医学院的莱斯特和苏·史密斯乳房中心提供协作和丰富的培训环境，在这里基础科学、转化和临床研究同等重要。这种形式的培训通过双周研究和发展讲习班、期刊俱乐部系列、每周乳腺癌研讨会和年度乳房中心静修活动得到加强。除了乳房中心提供的培训外，分子和细胞生物学系还举办年度学生研究研讨会、每周系列研讨会和年度研究生研讨会。所有这些活动都创造了一个完美的环境，不仅可以向我的同事和老师展示我的研究，还可以获得宝贵的反馈，这将使我的研究更上一层楼。