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，一个是适度生长的初始阶段（胚胎期至青春期），另一个是稳健扩张的后期阶段。毫不奇怪，对早期相对“静止”阶段的研究被后者大大掩盖了。然而，在早期阶段不断变化的条件有重要的后果，后来的乳腺发育和致癌潜力。当发现5天雌激素处理的新生小鼠暴露于雌激素导致增殖反应时，这一点得到了明确的确认，但这一反应发生在青春期后期。旁分泌雌激素受体（ER 1）刺激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，从而诱导上皮增殖反应，但延迟的原因尚不清楚。我们发表的初步结果和其他人的结果提出了同源框转录因子HoxC 6可能是这种延迟反应的基础的可能性：1）在我们的HoxC 6缺陷动物中，乳腺导管树显示出经历出生后扩张的严重失败; 2）在正常动物中，HoxC 6在上皮和间质中表达，而间质表达被雌激素抑制; 3）HoxC 6是IGF结合蛋白3（IGFBP 3）的直接转录抑制因子，IGFBP 3在HoxC 6下调的乳腺上皮细胞系和HoxC 6缺失的乳腺中上调。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IGFBP 3稳定。这表明新生儿暴露于雌激素确实会刺激IGF-I的产生（通过ER 1），但也会抑制HoxC 6，导致IGFBP 3合成和稳定性增加。IGFBP 3的存在将隔离并稳定基质中的IGF-I，并钝化立即的上皮反应。在青春期，ER 1过度刺激会使IGFBP 3不稳定，从而释放IGF-I的储存用于过度生长/存活和导管扩张。未分化的乳腺上皮干细胞过度扩增与风险有关。我们的总体假设是，HoxC 6介导的雌激素诱导的乳腺上皮细胞增殖的延迟，通过去抑制IGFBP 3的表达，钝化IGF-I基质上皮信号在早期阶段。这将在一个特定目标中进行实验测试。AIM 1的目标是确定HoxC 6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。这将使用以下方法完成：1）细胞类型特异性培养物; 2）全器官培养物;和3）体内模型。在所有研究中，将测量乳腺基质和上皮中雌激素暴露和HoxC 6表达状态对IGF-I信号传导、基因表达（IGF-I、HoxC 6、IGFBP 3、FGFR 2）和生长/存活、迁移、分化和分支的细胞特征的影响。本研究的一个实际目标是利用体外HoxC 6基因表达水平作为评估影响出生后乳腺上皮致癌潜力的人为和天然化合物的替代物。 公共卫生相关性：初潮和乳房发育早是乳腺癌的危险因素，乳腺癌是妇女癌症相关死亡的第二大原因，早期接触天然和人为雌激素可使乳腺对早期发育和癌症高度敏感。在这项研究中，我们将测试我们的模型，即早期暴露于雌激素刺激产生过量的生长因子，这些生长因子被隔离和稳定，从而消除早期月经初潮和过度刺激乳房生长/存活和扩张。这项工作的实际应用是对促进或预防乳腺癌发生的生物活性化合物的高通量鉴定的发展。

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.