Mammary Gland Morphogenesis and Breast Cancer

乳腺形态发生与乳腺癌

• 批准号: 8446157
• 负责人: IAN G MACARA
• 金额: $ 33.87万
• 依托单位: VANDERBILT UNIVERSITY
• 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/8446157
• 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; 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; screening; 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.

描述（由申请人提供）：超过90%的乳腺癌起源于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的不对称分裂是必不可少的，并且极化失败可导致肿瘤发生。Par极性蛋白是细胞不对称分裂的关键调节因子。Par蛋白还控制分化的上皮细胞的极化。然而，乳腺形态发生中极性的分子机制和Par蛋白在乳腺癌中的作用仍然是未知的。我们已经开发出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，并在宿主小鼠中从这些细胞再生乳腺。慢病毒转导能够通过敲低/敲入方法快速分析分子机制。使用这些方法，发现了Par 3极性蛋白在乳腺形态发生中的重要作用。此外，Par 3的沉默增强肿瘤生长和转移。具体目标如下：1. Par 3如何调节乳腺形态发生过程中干/祖细胞的自我更新和分化？在不存在Par 3的情况下，非典型蛋白激酶C（aPKC）从管腔顶端表面错误定位。敲低/敲入策略将用于询问命运决定是否可以通过顶端系留的aPKC和Par 3突变体来挽救。一种新的miRNA传感器将用于识别干细胞，并询问Par 3是否是不对称分裂所必需的。Par 3在管腔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. Par 3是如何抑制肿瘤生长和转移的？初步数据表明，Par 3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单独控制细胞谱系确定和转移。慢病毒将用于操纵原代细胞中的基因表达，使用体内移植模型、尾静脉注射和体外乳腺球培养物对原代细胞进行检测。3.我们能否通过体内筛选鉴定多种额外的转移抑制剂/诱导剂？来自ErbB 2小鼠的乳腺干细胞将用表达cDNA文库的混合慢病毒感染，然后植入野生型宿主小鼠的清洁脂肪垫中。这些病毒还表达红色荧光蛋白。将分离RFP阳性肺转移瘤，并通过PCR进行筛选，以鉴定与每个转移集落相关的cDNA。将根据人肿瘤中的表达模式对阳性进行优先排序，以进行机制研究。总之，这些研究将为干/祖细胞自我更新和谱系确定过程中的极性蛋白信号传导提供见解，并确定乳腺肿瘤转移的新参与者。