Endocrine Status of MG Stroma Alters Breast CA Invasion

描述(申请人提供)：生殖与生殖之间的关系 病史和乳腺癌是复杂的。年轻女性的足月怀孕是 在生命后期预防乳腺癌，而第一个完整的足月 40岁以后怀孕的人患癌症的几率更高。在……里面 此外，在前两年内，乳腺癌的发病率有所增加。 怀孕，不分年龄。与妊娠相关的乳腺癌也表现为 在较晚的临床阶段，比非妊娠相关的更具侵袭性 乳腺癌。我们假设怀孕对癌症的促进作用 是由于乳腺间质生理性重塑所致 怀孕了。在分娩或断奶引起的乳腺退化期间， 乳腺间质降解与基质中基质金属蛋白酶水平的关系 促炎症介质的含量也在上升。我们建议这些 乳腺间质退行性变促进上皮细胞向 原有肿瘤细胞的间质转化(EMT)。由此产生的 激活的肿瘤细胞，加上减弱的间质屏障，增加了 肿瘤细胞渗出的可能性。因为有可能存在超自然现象 肿瘤细胞随年龄增长而增多，妊娠相关腺体退化较多 可能会影响老年女性的EMT和转移。《目标1》中的假设 肿瘤细胞的转移潜能受生殖状态的调节 在体内使用异种移植模型进行测试。人乳腺肿瘤细胞将被 直接注射到免疫缺陷小鼠的乳头或乳房脂肪垫中 不同的生殖阶段(即未分娩、“妊娠相关”和 帕鲁斯)。肿瘤细胞离开腺体的能力被预测为 取决于注射时组织重塑的程度：低 未经产和经产，腺体退化程度高。原发肿瘤将是 组织学评价与肺、肝、脑、长骨转移 这条腿将用一种独特的人类基因进行半定量聚合酶链式反应 序列，CCR5基因。在目标2中，内分泌诱导的假设 间质成分的变化可以促进或抑制EMT 使用重建模型进行体外评估。人乳腺上皮细胞， 从非致瘤性到转移性的转移潜能不同，将 与不同生殖类型大鼠分离的乳腺基质结合 各州。预计静止的乳腺间质(未产妇和 PARUS)会抑制EMT，而内化基质会高度促进EMT 转移细胞。EMT的功能证据将通过测量获得 基质依赖的细胞黏附、运动和侵袭力的变化 改良的Boyden小室分析。子宫内膜异位症的形态和生化证据 将在肿瘤形成的三维模型中进行评估。的影响 乳腺基质中EMT、肌动蛋白纤维组织和表达的关键标志物 E-钙粘蛋白、波形蛋白、明胶蛋白和Fspl蛋白的含量将被测定 生化方面的。在目标3中，乳房基质退化引起的假说， 而未产妇和临产乳房基质则抑制基质金属蛋白酶的表达 肿瘤细胞将通过报告基因分析进行评估。基于荧光素酶的 含有基质分解酶(MMP3)、明胶酶A的报告构建体 明胶酶B启动子(MMP2)或明胶酶B(MMP9)启动子在肿瘤中的活性将被测试 细胞在不同的乳房基质上电镀。这些研究的结果将 有助于我们理解与怀孕相关的变化对 乳腺间质对乳腺癌进展的影响，并应提供见解 进入描述怀孕对乳房双重影响的流行病学数据 癌症。

DESCRIPTION (provided by applicant): The relationship between reproductive history and breast cancer is complex. A full term pregnancy in younger women is protective against breast cancer later in life, while a first full term pregnancy after 40 years of age is associated with higher cancer occurrence. In addition, there is an increase in breast cancer within the first two years of pregnancy, regardless of age. Pregnancy associated breast cancer also presents at a later clinical stage and is more invasive than non-pregnancy associated breast cancers. We hypothesize that the cancer promotional effect of pregnancy is due to physiologic remodeling of the mammary stroma that occurs after pregnancy. During parturition or weaning-induced mammary gland involution, mammary stroma is degraded and stroma levels of matrix metalloproteinases (MMP) and pro-inflammatory mediators are elevated. We propose that these involution-associated changes in mammary stroma promote an epithelial to mesenchymal transition (EMT) in pre-existing neoplastic cells. The resulting activated tumor cells, coupled with the weakened stromal barrier, increases the likelihood of tumor cell extravasation. Since the likelihood of having occult tumor cells increases with age, pregnancy associated gland involution is more likely to effect EMT and metastases in older women. In Aim 1. the hypothesis that tumor cell metastatic potential is modulated by reproductive status will be tested in vivo using xenograft models. Human mammary tumor cells will be injected directly into teats or mammary fat pads of immunodeficient mice at different reproductive stages (i.e. nulliparous, 'pregnancy-associated', and parous). The ability of tumor cells to exit the gland is predicted to be dependent on the degree of tissue remodeling at the time of injection: low in nulliparous and parous, and high in regressing glands. Primary tumors will be evaluated histologically and metastases to lung, liver, brain and long bone of the leg will be measured by semi-quantitative PCR using a unique human sequence, the CCR5 gene. In Aim 2, the hypothesis that endocrine-induced changes in stroma composition can either promote or suppress EMT will be evaluated in vitro using reconstitution models. Human mammary epithelial cells, which differ in metastatic potential from non-tumorigenic to metastatic, will be combined with mammary stroma isolated from rats with different reproductive states. It is anticipated that quiescent mammary stroma (nulliparous and parous) will inhibit, while involuting matrix will promote EMT in highly metastatic cells. Functional evidence for EMT will be obtained by measuring stroma-dependent changes in cell adhesion, motility and invasiveness using modified Boyden chambers assays. Morphological and biochemical evidence for EMT will be evaluated in a 3-dimensional model of tumor formation. The effects of mammary matrix on key markers of EMT, actin fiber organization, and expression of E-cadherin, vimentin, gelsolin and Fspl proteins, will be determined biochemically. In Aim 3, the hypothesis that involuting mammary matrix induces, while nulliparous and parous mammary matrices suppress expression of MMP in tumor cells will be evaluated by reporter gene assays. Luciferase-based reporter constructs containing either the stromelysin (MMP-3), gelatinase A (MMP-2), or gelatinase B (MMP-9) promoters will be tested for activity in tumor cells plated on different mammary matrices. The results of these studies will contribute to our understanding of the effects pregnancy-associated changes in mammary stroma have on breast cancer progression, and should provide insights into epidemiological data describing the dual effect of pregnancy on breast cancer.