Mammary Gland Morphogenesis and Breast Cancer

乳腺形态发生与乳腺癌

基本信息

批准号：
8135217
负责人：
IAN G MACARA
金额：
$ 34.75万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8135217
关键词：
Address Animal Model Apical Cause of Death Cell Culture Techniques Cell Lineage Cell Polarity Cell Proliferation Cell division Cells Complementary DNA Complex Data Epithelial Cells Failure Fatty acid glycerol esters Gene Expression Genes Genetic Transcription Genetically Engineered Mouse Goals Human Implant In Vitro Injection of therapeutic agent Knock-in Mouse Knockout Mice Lung Malignant Neoplasms Mammary Neoplasms Mammary Tumorigenesis Mammary gland Methodology Methods MicroRNAs Modeling Molecular Molecular Analysis Mus Mutation Myoepithelial cell Natural regeneration Neoplasm Metastasis Oncogenes Organ Pattern Phosphorylation Population Primary Neoplasm Process Property Proteins RNA Interference Role Screening procedure Signaling Protein Stem cells Subfamily lentivirinae Surface Tail Testing Time Transplantation Veins Venus Virus Woman Work atypical protein kinase C base cDNA Library cancer stem cell cell type gene function in vivo insight knock-down malignant breast neoplasm mammary gland development mouse model mutant notch protein novel novel strategies progenitor programs public health relevance rapid growth red fluorescent protein self-renewal sensor stem stemness tumor tumor growth tumor progression tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): More than 90% of breast cancers arise from epithelial cells or from progenitors with stem cell properties. Cell polarity is essential for stem cell asymmetric divisions, and failures in polarization can result in tumorigenesis. The Par polarity proteins are key regulators of asymmetric cell division. Par proteins also control polarization of differentiated epithelial cells. Yet the molecular mechanisms underlying polarity in mammary morphogenesis and the role of Par proteins in breast cancer remain largely unknown. We have developed methods that facilitate the study of mammary morphogenesis and tumorigenesis. To manipulate gene expression in mammary glands, stem cells are transduced with lentivirus, and mammary glands are regenerated in host mice from these cells. Lentiviral transduction enables rapid analysis of molecular mechanism through a knock-down/knock-in approach. Using these methods, an essential role for the Par3 polarity protein in mammary morphogenesis was discovered. In addition, silencing of Par3 potentiated tumor growth and metastasis. The goals are as follows: 1. How does Par3 regulate stem/progenitor self-renewal and differentiation during mammary gland morphogenesis? In the absence of Par3, atypical protein kinase C (aPKC) is mislocalized from the luminal apical surface. A knock-down/knock-in strategy will be used to ask if fate determination can be rescued by apically-tethered aPKC and by Par3 mutants. A novel miRNA sensor will be used to identify stem cells and to ask if Par3 is required for asymmetric division. Differential functions of Par3 in luminal and myoepithelial cells will be tested using new methods for cell-type specific RNAi silencing. 2. How does Par3 function to suppress tumor growth and metastasis? Preliminary data suggest that Par3 might limit mammary cell proliferation by suppressing Rac activity in the NICD cells, while separately controlling cell lineage determination and metastasis through aPKC. Lentivirus will be used to manipulate gene expression in primary cells, which are tested using the in vivo transplant model, tail vein injections, and in vitro mammosphere cultures. 3. Can we identify multiple additional metastasis suppressors/inducers by in vivo screening?. Mammary stem cells from ErbB2 mice will be infected with pooled lentiviruses that express a library of cDNAs, then implanted into cleared fat pads of wild type host mice. The viruses also express a red fluorescent protein. RFP-positive lung metastases will be isolated and screened by PCR to identify the cDNA associated with each metastatic colony. Positives will be prioritized based on expression patterns in human tumors for mechanistic studies. Together, these studies will provide insights into polarity protein signaling during stem/progenitor cell self-renewal and lineage determination, and identify new players in mammary tumor metastasis. PUBLIC HEALTH RELEVANCE: Breast cancer is one of the leading causes of death among women. Traditional approaches to studying the molecular basis for breast cancer, using genetically-engineered mice, are expensive and time-consuming; and studies in cell culture do not recapitulate the complex processes involved in building an organ or in tumor progression. New approaches are needed to address these issues. We have developed methods that facilitate the rapid analysis of gene function in mammary gland development and cancer. We will use these methods to identify new cancer genes, and to understand how breast cancers develop and metastasize.

描述（由申请人提供）：超过90％的乳腺癌来自上皮细胞或具有干细胞特性的祖细胞。细胞极性对于干细胞不对称分裂至关重要，极化失败可能导致肿瘤发生。偏极蛋白是不对称细胞分裂的关键调节剂。 PAR蛋白还控制分化上皮细胞的极化。然而，乳腺形态发生的极性和PAR蛋白在乳腺癌中的作用的分子机制在很大程度上尚不清楚。我们开发了促进乳腺形态发生和肿瘤发生的方法。为了操纵乳腺中的基因表达，干细胞用慢病毒转导，并在这些细胞的宿主小鼠中再生乳腺。慢病毒转导可以通过敲门/敲门方法快速分析分子机制。使用这些方法，发现了PAR3极性蛋白在乳腺形态发生中的重要作用。此外，PAR3的沉默增强了肿瘤的生长和转移。目标如下：1。PAR3如何调节乳腺形态发生过程中的茎/祖细胞自我更新和分化？在没有PAR3的情况下，非典型蛋白激酶C（APKC）从腔顶端表面错误地定位。将使用击倒/敲门策略来询问命运的确定是否可以通过顶管的APKC和PAR3突变体挽救。新型的miRNA传感器将用于识别干细胞，并询问是否需要不对称分裂的PAR3。 PAR3在腔内和肌上皮细胞中的差异功能将使用用于细胞类型的特异性RNAI沉默的新方法进行测试。 2。PAR3如何抑制肿瘤生长和转移？初步数据表明，PAR3可能通过抑制NICD细胞中的RAC活性来限制乳腺细胞的增殖，同时通过APKC分别控制细胞谱系测定和转移。慢病毒将用于操纵原代细胞中的基因表达，这些基因表达使用体内移植模型，尾静脉注射和体外乳液培养物进行了测试。 3。我们可以通过体内筛选来识别多个额外的转移抑制剂/诱导剂吗？来自ERBB2小鼠的乳腺干细胞将感染表达CDNA库的合并慢病毒，然后植入野生型宿主小鼠的清除脂肪垫中。病毒还表达红色荧光蛋白。 RFP阳性的肺转移将被PCR分离和筛选，以鉴定与每个转移性菌落相关的cDNA。基于人类肿瘤中的表达模式，将优先考虑阳性。总之，这些研究将提供对茎/祖细胞自我更新和谱系测定期间极性蛋白信号传导的见解，并确定乳腺肿瘤转移的新参与者。公共卫生相关性：乳腺癌是女性死亡的主要原因之一。使用基因工程小鼠研究乳腺癌分子基础的传统方法是昂贵且耗时的。细胞培养的研究并未概括构建器官或肿瘤进展中涉及的复杂过程。需要新的方法来解决这些问题。我们开发了促进乳腺发育和癌症基因功能快速分析的方法。我们将使用这些方法来识别新的癌症基因，并了解乳腺癌的发展和转移。