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，最初阶段为适度发育(胚胎至青春期)，后期为强劲扩张阶段。不足为奇的是，对早期相对“静止”阶段的研究被后者大大盖过。然而，早期条件的变化对后来的乳房发育和致癌潜力有重要的影响。当发现雌激素治疗5天的新生小鼠雌激素暴露导致增殖反应时，这一点被清楚地识别出来，但这种反应发生在青春期较晚的时候。旁分泌雌激素受体(ER1)刺激增加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。我们已发表的和初步的结果以及其他人的结果提出了这样一种可能性，即同源盒转录因子HoxC6可能是这种延迟反应的基础：1)在HoxC6缺乏的动物中，乳腺导管树显示出严重的出生后扩张；2)在正常动物中，HoxC6在上皮和间质中表达，间质表达被雌激素抑制；3)HoxC6是IGF结合蛋白3(IGFBP3)的直接转录抑制因子，在HoxC6下调表达的乳腺上皮细胞系和HoxC6缺失的乳腺中发生IGFBP3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抑制物的上调，表明IGFBP3稳定。这表明，新生儿暴露于雌激素确实会刺激IGF-I的产生(通过ER1)，但也会抑制HoxC6，导致IGFBP3合成和稳定性的增加。IGFBP3的存在将隔离和稳定间质中的IGF-I，并钝化即刻的上皮反应。在青春期，ER1的过度刺激会破坏IGFBP3的稳定，从而释放储存的IGF-I，以促进过度生长/存活和导管扩张。未分化的乳腺上皮干细胞过度扩张是风险的一个环节。我们的总体假设是，HoxC6通过抑制IGFBP3的表达，在早期阶段钝化IGF-I基质到上皮的信号转导，从而介导雌激素诱导的乳腺上皮细胞增殖延迟。这将在一个特定的目标上进行实验测试。AIM 1的目标是明确HoxC6在调控乳腺上皮生长/存活和分支中调节IGF-I基质-上皮信号转导的机制。这将使用：1)特定细胞类型的培养；2)整个器官培养；以及3)活体模型。在所有研究中，将从IGF-I信号、基因表达(IGF-I、HoxC6、IGFBP3、FGFR2)以及生长/存活、迁移、分化和分支的细胞特征来衡量雌激素暴露和HoxC6在乳腺基质和上皮中的表达状态的影响。这项研究的一个实际目标是利用体外HoxC6基因表达水平作为替代，评估人为和天然化合物对出生后乳腺上皮细胞致癌潜力的影响。 公共卫生相关性：月经初潮早期和乳房发育是乳腺癌的风险因素，乳腺癌是女性癌症相关死亡的第二大原因，早期暴露于自然和人为雌激素可使乳腺对早期发育和癌症非常敏感。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会刺激过量生长因子的产生，这些生长因子被隔离和稳定，从而导致月经初潮早期和对乳房生长/存活和扩张的过度刺激。这项工作的实际应用是开发高通量鉴定促进或预防乳腺癌发生的生物活性化合物。

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.