Growth and Carcinogenic Potential in the Postnatal Mammary Gland

描述（由申请人提供）：乳房发育分为两个不同的阶段，一个适度生长的初始阶段（胚胎到青春期）和一个强健扩张的后期阶段。毫不奇怪，对早期相对“静止”阶段的研究在很大程度上被后者所掩盖。然而，在早期阶段改变条件对后期乳房发育和致癌潜力有重要影响。当发现雌激素暴露于5天雌激素治疗的新生小鼠导致增殖反应时，这一点得到了明确的确认，但这种反应发生在青春期的后期。旁分泌雌激素受体（ER1）刺激可提高生长激素诱导的胰岛素样生长因子1 （IGF-I）的产生和释放，从而诱导上皮细胞增殖反应，但延迟的原因尚不清楚。我们发表的和初步的研究结果以及其他人的研究结果提出了同源盒转录因子HoxC6可能是这种延迟反应的基础：1)HoxC6缺陷动物的乳腺导管树显示出严重的产后扩张失败；2)正常动物中，HoxC6在上皮和间质中表达，间质表达受雌激素抑制；3) HoxC6是IGF结合蛋白3 （IGFBP3）的直接转录抑制因子，IGFBP3的上调发生在HoxC6下调的乳腺上皮细胞系和HoxC6缺失的乳腺中。在后一种情况下，我们还观察到基质金属蛋白酶（MMPs）的下调和MMPs抑制剂的上调，表明IGFBP3稳定。这表明，新生儿暴露于雌激素确实会刺激igf - 1的产生（通过ER1），但也会抑制HoxC6，导致IGFBP3合成和稳定性增加。IGFBP3的存在会隔离和稳定间质中的igf - 1，并减弱上皮细胞的即时反应。在青春期，ER1的过度刺激会破坏IGFBP3的稳定，从而释放igf - 1的储存，导致过度生长/存活和导管扩张。未分化乳腺上皮干细胞的过度扩张与风险有关。我们的总体假设是，HoxC6通过抑制IGFBP3表达，在早期阶段减弱IGF-I基质-上皮信号传导，介导雌激素诱导的乳腺上皮细胞增殖延迟。这将在一个特定目标中进行实验测试。AIM 1的目的是确定HoxC6在调节乳腺上皮生长/存活和分支中调节IGF-I基质-上皮信号传导的机制。这将通过以下方法实现：1)细胞类型特异性培养；2)全器官培养；3)体内模型。在所有的研究中，雌激素暴露和乳腺间质和上皮中HoxC6表达状态的影响将测量IGF-I信号、基因表达（IGF-I、HoxC6、IGFBP3、FGFR2）以及细胞生长/存活、迁移、分化和分支的特征。本研究的一个实际目标是利用HoxC6基因在体外的表达水平作为评估影响产后乳腺上皮致癌潜力的人为和天然化合物的替代物。

DESCRIPTION (provided by applicant): Mammary development occurs in two distinct phases, an initial phase of modest growth (embryonic to puberty) and a later phase of robust expansion. Not surprisingly, studies on the early, relatively "quiescent" phase are greatly overshadowed by the latter. Nevertheless, changing conditions during the early phase have important consequences on later mammary development and carcinogenic potential. This was clearly identified when it was found that estrogenic exposure in 5-day estrogen-treated newborn mice resulted in a proliferative response, but one that occurred later at puberty. Paracrine estrogen receptor (ER1) stimulation elevates growth hormone-induced production and release of insulin-like growth factor 1 (IGF-I) and thereby induces an epithelial proliferative response, but the reason for the delay is unclear. Our published and preliminary results and those of others raise the possibility that the homeobox transcription factor HoxC6 may underlie this delayed response: 1) Mammary ductal trees in our HoxC6 deficient animals display a profound failure to undergo postnatal expansion; 2) In normal animals, HoxC6 is expressed in epithelium and stroma and stromal expression is repressed by estrogen; 3) HoxC6 is a direct transcriptional repressor of IGF binding protein 3 (IGFBP3), and IGFBP3 upregulation occurs in HoxC6-downregulated mammary epithelial cell lines and HoxC6 null mammary glands. In the latter case, we also observed downregulation of matrix metalloproteinases (MMPs) and upregulation of inhibitors of MMPs, indicating IGFBP3 stabilization. This would suggest that neonatal exposure to estrogen would indeed stimulate production of IGF-I (via ER1), but also repress HoxC6, resulting in an increase in IGFBP3 synthesis and stability. The presence of IGFBP3 would sequester and stabilize IGF-I in the stroma and blunt an immediate epithelial response. At puberty, ER1 hyper-stimulation would destabilize IGFBP3, thereby releasing stores of IGF-I for excessive growth/survival and ductal expansion. Excessive expansion of undifferentiated mammary epithelial stem cells being a link to risk. Our overall hypothesis is that HoxC6 mediates the estrogen-induced delay in mammary epithelial cell proliferation by de-repressing IGFBP3 expression, blunting IGF-I stromal-to-epithelial signaling in the early phase. This will be tested experimentally in one Specific Aim. The goal of AIM 1 is to define the mechanism by which HoxC6 modulates IGF-I stromal-to-epithelial signaling in the regulation of mammary epithelial growth/survival & branching. This will be accomplished using: 1) cell type-specific cultures; 2) whole organ cultures; and 3) in vivo models. In all studies, the impacts of estrogen exposure and HoxC6 expression status in mammary stroma and epithelium will be measured regarding IGF-I signaling, gene expression (IGF-I, HoxC6, IGFBP3, FGFR2) and cellular characteristics of growth/survival, migration, differentiation and branching. A practical goal of this study is to utilize HoxC6 gene expression levels in vitro as a surrogate for the assessment of anthropogenic and natural compounds that impact on postnatal mammary epithelial carcinogenic potential. PUBLIC HEALTH RELEVANCE: Early menarche and breast development are risk factors for breast cancer, which is the second leading cause of cancer-related deaths in women, and early exposure to natural and anthropogenic estrogens can profoundly sensitize the mammary gland to early development and cancer. In this study, we will test our model that early exposure to estrogens stimulate production of excess growth factor that is sequestered and stabilized and thereby elicits early menarche and hyper-stimulation of mammary growth/survival and expansion. The practical application of this work is toward the development of high throughput identification of bioactive compounds that promote or prevent breast carcinogenesis.