Mammary Gland Morphogenesis and Breast Cancer

乳腺形态发生与乳腺癌

• 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
• 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

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% 的乳腺癌源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。 Par 极性蛋白是不对称细胞分裂的关键调节因子。 Par 蛋白还控制分化上皮细胞的极化。然而，乳腺形态发生中极性的分子机制以及 Par 蛋白在乳腺癌中的作用仍然很大程度上未知。我们开发了促进乳腺形态发生和肿瘤发生研究的方法。为了操纵乳腺中的基因表达，用慢病毒转导干细胞，并利用这些细胞在宿主小鼠中再生乳腺。慢病毒转导可以通过敲低/敲入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 的沉默会增强肿瘤的生长和转移。目标如下： 1. Par3如何在乳腺形态发生过程中调节干细胞/祖细胞的自我更新和分化？在缺少 Par3 的情况下，非典型蛋白激酶 C (aPKC) 从管腔顶端表面错误定位。将使用击倒/敲入策略来询问命运决定是否可以通过顶端束缚的 aPKC 和 Par3 突变体来挽救。一种新型 miRNA 传感器将用于识别干细胞并询问 Par3 是否是不对称分裂所必需的。 Par3 在管腔细胞和肌上皮细胞中的差异功能将使用细胞类型特异性 RNAi 沉默的新方法进行测试。 2. Par3如何发挥抑制肿瘤生长和转移的作用？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增殖，同时通过 aPKC 单独控制细胞谱系决定和转移。慢病毒将用于操纵原代细胞中的基因表达，并使用体内移植模型、尾静脉注射和体外乳腺球培养物进行测试。 3. 我们能否通过体内筛选来鉴定多种额外的转移抑制因子/诱导因子？来自 ErbB2 小鼠的乳腺干细胞将被表达 cDNA 文库的慢病毒混合感染，然后植入野生型宿主小鼠的透明脂肪垫中。这些病毒还表达红色荧光蛋白。 RFP 阳性肺转移瘤将被分离并通过 PCR 进行筛选，以鉴定与每个转移集落相关的 cDNA。将根据人类肿瘤的表达模式对阳性进行优先排序，以进行机制研究。总之，这些研究将深入了解干/祖细胞自我更新和谱系决定过程中的极性蛋白信号传导，并确定乳腺肿瘤转移中的新参与者。 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既昂贵又耗时。细胞培养的研究并没有概括构建器官或肿瘤进展所涉及的复杂过程。需要新的方法来解决这些问题。我们开发了有助于快速分析乳腺发育和癌症中基因功能的方法。我们将使用这些方法来识别新的癌症基因，并了解乳腺癌如何发展和转移。