Role of Stroma in Mammary Gland Cell Proliferation

描述（由申请人提供）：孕激素（P）是成人乳腺中的主要有丝分裂原，对乳腺癌风险有显著影响。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，使用小鼠乳腺模型，我们已经开发了一种最低限度补充的无血清胶原凝胶原代培养系统，其中由管腔上皮细胞和肌上皮细胞组成的乳腺上皮类器官增殖并经历肺泡形态发生以响应P。除了P之外，这些响应还需要乳腺基质源性肝细胞生长因子（HGF）。单独用HGF处理诱导增殖和小管-导管形态发生，而单独用P不诱导增殖，但诱导管腔形成，其与增加的凋亡相关。HGF+P比单独HGF增加增殖，并引起从导管到肺泡形态的变化。这是一个新的体外证明的促有丝分裂和形态学反应P，概括的增殖和肺泡生成反应P在体内。此外，在正常乳腺细胞中，已经确定了对P的促凋亡反应。在这个提议中，我们将确定P是否以及如何在细胞质中起作用，可能通过孕酮受体（PR）上的SH 3结合结构域来影响与乳腺特异性小管形成（FAK，SHIP-1，PI 3-K）和增殖（ERK，Src/Myc）相关的HGF/Met激活的通路，以抑制小管形成，促进增殖并引起肺泡形成。我们还将确定PR作用在细胞核中的作用，通过其转录激活功能，对与肺泡形成（Wnt-4）和增殖（Met，细胞周期蛋白D1，Myc）相关的P-反应靶基因的表达。此外，还将确定P的这些作用发生的特定细胞类型（管腔上皮细胞、肌上皮细胞）。使用来自PRA无效或PR B无效小鼠的培养物，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。该提案的长期目标是描绘成年正常乳腺中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.