IGF-1 Receptor Signaling in Mammary Gland Development

描述(申请人提供)：末端芽细胞(TEB)是哺乳动物出生后发育过程中发现的最具增殖和侵袭性的细胞。这些细胞通过其巨大的增殖能力，推动整个乳腺导管系统的发育，作为乳腺前体细胞的来源，是乳腺内致癌物质的最重要靶点。内分泌、旁分泌和细胞外基质来源的信号利用这种巨大的再生潜力并将其引导到高度有序的发育模式的能力，使TEB成为研究细胞内信号的内分泌调节的重要和非常有趣的系统。我们对来自基因敲除小鼠模型的移植乳腺组织的分析表明，IGF-I受体(Lgf1r)基因的定向失活突变极大地减少了乳腺导管的发育，并降低了TEB细胞的增殖[1]。然而，这种增殖性缺陷可在早期妊娠时部分修复。在乳腺癌细胞模型中，IGF-I受体以及少数其他激素受体通过胰岛素受体底物(IRS)蛋白和两个丝氨酸苏氨酸激酶ERK和Akt传递信号。本研究提出的总体假设是IGF-I受体通过胰岛素反应底物(IRS)依赖的ERK和Akt在处女期导管发育过程中激活ERK和Akt来刺激TEB细胞的增殖，妊娠或卵巢类固醇激素雌二醇(E2)和孕酮(P)增加了IRS依赖的信号通路对IGF1r-/-TEB中胰岛素受体依赖的激活的敏感性。通过使用共聚焦显微镜结合新近发展的抗体原位检测细胞信号，我们将了解信号通路在TEB中是如何调节的，以及这些通路对正常乳腺功能和乳腺癌的重要过程有何意义。其具体目的是：1)确定动情周期、卵巢切除、早孕或外源性E2+P是否改变正常小鼠乳腺IGF-I-，或胰岛素诱导的IRS-1、-2、ERK和Akt的磷酸化；2)确定IGF-IR或IR依赖的ERK激活，Akt在lgf1r/-移植物的TEB中减弱，以及早期怀孕或外源性E2+P能否恢复这种激活；3)确定IRS-1或IRS-2在乳腺上皮细胞中的过表达是否恢复Igf1r/乳腺上皮的发育，并增强ERK和Akt的胰岛素依赖的后磷酸化。

DESCRIPTION (provided by applicant): The cells of the terminal end bud (TEB) are the most proliferative and invasive cells found during postnatal development in mammals. These cells, through their enormous proliferative capacity, drive the development of the entire mammary ductal system, serve as a source of mammary progenitor cells, and are the single most important target for carcinogens within the mammary gland. The ability of endocrine-, paracrine- and extracellular matrix-derived signals to harness this enormous regenerative potential and direct it into a highly ordered developmental pattern makes the TEB both an important and very fascinating system with which to study the endocrine regulation of intracellular signaling. Our analysis of grafted mammary tissue from a knockout mouse model has demonstrated that a targeted inactivating mutation of the gene for the IGF-I receptor (lgf1r) dramatically reduces mammary ductal development and decreases TEB cell proliferation(1). This proliferative defect, however, is partial restored by early pregnancy. In breast cancer cell models the IGF-I receptor as well as a handful of other hormone receptors signal through insulin receptor substrate (IRS) proteins, and two serine threonine kinases, ERK and Akt. The overall hypothesis addressed in this proposal is that IGF-I receptor stimulates TEB cell proliferation through insulin responsive substrate (IRS)-dependent activation of ERK and Akt during virgin ductal development and that pregnancy or the ovarian steroid hormones estradiol (E2) and progesterone (P) increase the sensitivity of IRS-dependent signaling pathways to insulin receptor-dependent activation in Igf1r-/- TEB. Through the use confocal microscopy with recently developed antibodies to detect cell signaling in-situ, we will learn how signaling pathways are regulated in TEBs and what the significance of these pathways is to processes important to both normal mammary gland function and breast cancer. The specific aims are; 1) determine if estrus cycle, ovarectomy, early pregnancy, or exogenous E2+P alters IGF-I-, or insulin-induced phosphorylation of IRS-1, -2, ERK and Akt in mammary glands of normal mice, 2) determine if IGF-IR or IR dependent activation of ERK, and Akt is attenuated in the TEB of lgf1r -/- grafts and if early pregnancy or exogenous E2+P can restore this activation, and 3) determine if overexpression of IRS-1 or IRS-2 within the mammary epithelium restores development of Igf1r-/- mammary epithelium and enhances insulin-dependent posphorylation of ERK and Akt.