Role of Stroma in Mammary Gland Cell Proliferation

描述（由申请人提供）：孕激素(P)是成人乳房中的主要有丝分裂原，对乳腺癌风险有重要影响。由于P广泛用于口服避孕药和绝经后激素替代治疗，因此了解其作用机制至关重要。本研究的目的是阐明正常乳腺中p依赖性增殖的机制。在目前的资助期内，我们利用小鼠乳腺模型开发了一种微量补充、无血清胶原凝胶原代培养系统，在该系统中，由腔上皮细胞和肌上皮细胞组成的乳腺上皮类器官在P的作用下增殖并经历肺泡形态形成。除了P，这些反应还需要乳腺基质源性肝细胞生长因子（HGF）。单独使用HGF可诱导细胞增殖和小管导管形态发生，而单独使用P不能诱导细胞增殖，但可诱导与细胞凋亡增加相关的管腔形成。HGF+P比单独HGF增加增殖，并引起从导管到肺泡形态的变化。这是一项新的体外实验，证明了有丝分裂和形态学对P的反应，再现了体内对P的增殖和肺泡形成反应。此外，正常乳腺细胞对P有促凋亡反应。在本研究中，我们将确定P是否以及如何在细胞质中起作用，可能通过孕激素受体（PR）上的SH3结合域，影响与乳腺特异性管生成（FAK, SHIP-1, PI3-K）和增殖（ERK, Src/Myc）相关的HGF/ met激活通路，从而抑制管生成，促进增殖并引起肺泡生成。我们还将确定PR在细胞核中的作用，通过其转录激活功能，对与肺泡形成（Wnt-4）和增殖（Met, cyclin D1, Myc）相关的p应答靶基因表达的影响。此外，还将确定特定的细胞类型（管腔上皮细胞，肌上皮细胞），其中P的这些作用发生。使用来自PRA缺失或PRB缺失小鼠的培养物，我们还将确定两种PR亚型（A和B）在增殖和肺泡形成中的作用。这项提议的长期目标是描述成人正常乳腺中p依赖性生长调节的机制，这可能导致预防和治疗乳腺癌的新策略。

DESCRIPTION (provided by applicant): Progestins (P) are major mitogens in the adult human breast and contribute significantly to breast cancer risk. Since P are widely used in oral contraceptives and in postmenopausal hormone replacement therapy it is critically important that their mechanims of action be understood. It is the goal of the proposed research to delineate the mechanisms of P-dependent proliferation in the normal mammary gland. In the current grant period, using the mouse mammary gland model, we have developed a minimally-supplemented, serum-free collagen gel primary culture system in which mammary epithelial organoids, made up from both luminal epithelial and myoepithelial cells, proliferate and undergo alveolar morphogenesis in response to P. In addition to P, these responses require mammary stroma derived-hepatocyte growth factor (HGF). Treatment with HGF alone induces proliferation and tubulo-ductal morphogenesis, whereas P alone does not induce proliferation but induces lumen formation that is associated with increased apoptosis. HGF+P increase proliferation above HGF alone and cause a change from ductal to alveolar morphology. This is a novel in vitro demonstration of a mitogenic and morphological response to P that recapitulates the proliferative and alveologenesis response to P in vivo. In addition a pro-apoptotic response to P in normal mammary cells has been identified. In this proposal we will determine whether and how P acts in the cytoplasm, possibly through an SH3 binding domain on the progesterone receptor (PR), to affect HGF/Met-activated pathways associated with mammary-specific tublogenesis (FAK, SHIP-1, PI3-K) and proliferation (ERK, Src/Myc) to inhibit tubulogenesis, promote proliferation and cause alveologenesis. We will also determine the effect of PR action in the nucleus, via its transcriptional activation function, on the expression of P-responsive target genes relevant to alveologenesis (Wnt-4) and proliferation (Met, cyclin D1, Myc). In addition will also identify the specific cells types (luminal epithelial, myoepithelial cells) in which these effects of P occur. Using cultures derived from PRA null or PRB null mice we will also determine the role of the two PR isoforms (A and B) in proliferation and alveologenesis. The long-term goal of this proposal is the delineation of the mechanisms of P-dependent growth regulation in the adult normal mammary gland that may lead to novel strategies for the prevention and treatment of breast cancer.