Mammary Gland Morphogenesis and Breast Cancer

描述(申请人提供)：超过90%的乳腺癌来源于上皮细胞或具有干细胞特性的祖细胞。细胞的极性对干细胞的不对称分裂是必不可少的，极化失败可能导致肿瘤的发生。PAR极性蛋白是细胞不对称分裂的关键调节因子。PAR蛋白也控制分化上皮细胞的极化。然而，极性在乳腺形态发生中的潜在分子机制以及PAR蛋白在乳腺癌中的作用在很大程度上仍不清楚。我们已经开发出有助于研究乳腺形态发生和肿瘤发生的方法。为了控制乳腺中的基因表达，用慢病毒转导干细胞，然后在宿主小鼠身上从这些细胞再生乳腺。慢病毒转导可以通过敲击/敲入的方法快速分析分子机制。利用这些方法，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。目的如下：1.在乳腺形态发生过程中，Par3如何调控干/祖细胞的自我更新和分化？在缺乏Par3的情况下，非典型蛋白激酶C(APKC)从管腔根尖表面错位。将使用敲击/敲入策略来询问是否可以通过顶部拴系的aPKC和Par3突变体来拯救命运决定。一种新型的miRNA传感器将被用来识别干细胞，并询问不对称分裂是否需要Par3。将使用新的细胞类型特异性RNAi沉默方法来测试Par3在腔上皮细胞和肌上皮细胞中的不同功能。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的增殖，同时通过aPKC单独控制细胞的谱系决定和转移。慢病毒将被用于操纵原代细胞中的基因表达，这些原代细胞通过体内移植模型、尾静脉注射和体外乳房培养进行测试。3.我们能否通过体内筛选确定多个额外的转移抑制/诱导因子？来自ErbB2小鼠的乳腺干细胞将被感染表达cDNA文库的慢病毒池，然后被植入野生型宿主小鼠的透明脂肪垫中。这些病毒还表达一种红色荧光蛋白。RFP阳性的肺转移瘤将被分离并通过聚合酶链式反应进行筛选，以确定与每个转移克隆相关的cDNAs。阳性将根据人类肿瘤的表达模式进行优先处理，以进行机制研究。总之，这些研究将为干细胞/祖细胞自我更新和谱系确定过程中的极性蛋白信号提供深入的见解，并确定在乳腺肿瘤转移中的新角色。

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.