Biophysical Regulation of Breast Differentiation

描述（由申请人提供）：乳腺组织x光片密度的增加与患乳腺癌的风险增加4至6倍相关，使其成为乳腺癌的单一最大危险因素。尽管如此，乳腺密度与癌形成之间的分子机制尚不清楚。我们最近的研究表明，在胶原沉积增加的小鼠模型中，乳腺肿瘤的发病率、侵袭和转移增加了三倍，这表明胶原蛋白本身的增加是乳腺密度增加与人类风险增加相关的机制的一部分。本提案的目的是了解外基质的物理性质如何调节乳腺细胞的行为。我们的假设是，局部致密的ECM增强了基质粘附的形成，从而激活与FAK相关的信号通路，并且FAK是基质密度调节基因表达以促进增殖和侵袭的中心介质。这一假设将在以下具体目标中得到验证：目标1：确定由胶原密度和排列调节的增殖和转移相关基因的表达变化。利用与患者预后相关的组织阵列，以及DCIS和微创DCIS，将在人类乳腺癌样本中确定增殖和转移基因特征的基因表达。在体外和体内，通过siRNA和shRNA敲除来筛选转移基因在介导三维基质入侵中的作用。目的2：验证FAK根据胶原密度和排列调节增殖和侵袭的假设，即使在FAK+/+基质的情况下，FAK-/-的肿瘤在体内也不会侵袭或转移。我们将使用体外3D侵袭实验和我们的体内FAK-/-小鼠来研究FAK在细胞增殖、基质排列和3D侵袭中的作用。条件FAK敲除小鼠将用于确定FAK在具有致密胶原基质的抗胶原酶转基因小鼠模型（Col1a1tm1Jae）中促进肿瘤进展中的作用。将在Col1a1小鼠、小鼠肿瘤和人类病理样本的乳腺组织中检测FAK pY397位点的磷酸化，以确定pY397FAK是否与体内致密乳腺组织相关。目的3：测试胶原密度，FAK和下游信号事件，ERK， Src和PI3K在调节细胞增殖和侵袭中的联系。FAK-/-细胞失去ERK和PI3K通路的激活。使用药物抑制剂和siRNA/shRNA方法，我们将抑制这些途径，并确定它们在体外介导人乳腺细胞增殖和侵入致密排列的胶原基质中的作用。我们将在体内用药理学抑制剂治疗人类肿瘤的异种移植物，以及wt和致密胶原基质（Col1a1小鼠模型）中携带肿瘤的小鼠，以测试这些分子在体内肿瘤进展中的作用。

DESCRIPTION (provided by applicant): An increase in the mammographic density of breast tissue is correlated to a four to six-fold increased risk of developing breast carcinoma, making it the single biggest risk factor for breast carcinoma. Despite this, the molecular mechanism by which breast density links to carcinoma formation is unknown. We have recently shown that in a mouse model of increased collagen deposition, mammary tumor incidence, invasion, and metastasis increase three-fold, suggesting that an increase in collagen per se is part of the mechanism by which increased breast density is correlated with increased risk in humans. The purpose of this proposal is to understand how physical properties of the ECM regulate breast cell behavior. Our hypothesis is that locally dense ECM enhances the formation of matrix adhesions that result in activation of signaling pathways linked to FAK, and that FAK is a central mediator by which matrix density regulates gene expression to promote proliferation and invasion. This hypothesis will be tested in the following Specific Aims: Aim 1: Define changes in the expression of proliferation and metastasis-associated genes regulated by collagen density and alignment. Gene expression of the proliferation and the metastasis gene signatures, will be determined in human breast carcinoma samples, using tissue arrays linked to patient outcome, and with DCIS and microinvasive DCIS. Metastasis genes will be screened by siRNA and shRNA knock-down for their role in mediating invasion into 3D matrices in vitro and in vivo. Aim 2: Test the hypothesis that FAK regulates proliferation and invasion in response to collagen density and alignment Tumors that are FAK-/- do not invade or metastasize in vivo, even in the context of a FAK+/+ stroma. We will use in vitro 3D invasion assays and our in vivo FAK-/- mice to investigate the role of FAK in cell proliferation, matrix alignment and 3D invasion. Conditional FAK knockout mice will be used to determine the role of FAK in enhanced tumor progression in the collagenase-resistant transgenic mouse model (Col1a1tm1Jae) having a dense collagen matrix. Phosphorylation of FAK at pY397 will be determined in mammary tissues from Col1a1 mice, mouse tumor, and human pathologic samples to determine if pY397FAK correlates to dense breast tissue in vivo. Aim 3: Test the link between collagen density, FAK and downstream signaling events, ERK, Src, and PI3K, in regulating cell proliferation and invasion. FAK-/- cells lose activation of ERK and PI3K pathways. Using pharmacologic inhibitors and siRNA/shRNA approaches, we will inhibit each of these pathways and determine their role in mediating the proliferation and invasion of human breast cells into dense and aligned collagen matrices in vitro. Xenografts of human carcinoma, and mice bearing tumors in wt and dense collagen stroma (Col1a1 mouse model) will be treated in vivo with pharmacologic inhibitors to test the role of these molecules in tumor progression in vivo. PUBLIC HEALTH RELEVANCE: Understanding the role the physical properties of the extracellular matrix plays in cancer progression is of great health relevance as breast density accounts for a 4-6 fold increase in carcinoma risk. Moreover, we have found that collagen alignment carries a 5 fold risk of disease relapse, suggesting that understanding how collagen alignment occurs, and how it contributes to progression will help us understand breast carcinoma. These experiments are designed to understand the underlying molecular mechanisms by which the dense extracellular matrix regulates breast cell behavior and invasion and progression, and could suggest future targets for therapy.