Muc4 Involvement in Mammary Tumor Progression

描述(申请人提供)：建议研究的总体目的是阐明膜粘蛋白Muc4在促进乳腺肿瘤发生和发展中的作用。MUC4在培养的细胞中表达时具有强大的抗黏附和生长信号活性，原则上可以在健康上皮向早期增生表型的初始转变以及后来向高度侵袭和转移状态的转变中发挥作用。与此一致的是，有报道称，与患者匹配的正常组织样本相比，Muc4在多达20%的局部乳腺肿瘤中过度表达和定位错误，与匹配的原发肿瘤相比，多达40%的淋巴结转移病例中Muc4过度表达和定位错误。这个项目将通过分析Muc4在体外和体内逐渐更具攻击性状态的发展中的作用来整合这些观察结果。驱动假说是Muc4蛋白在乳腺肿瘤中的异常表达促进了肿瘤的恶变。目的1将分析从MCF10A细胞衍生的一系列等基因培养的乳腺上皮细胞系中诱导Muc4过表达的结果，该细胞系模拟正常、增生性和恶性乳腺组织，以及在模拟ErbB2阳性、ER阳性和三阴性疾病的乳腺肿瘤细胞系中。当细胞在2D和3D培养中生长时，将进行细胞生长特性和信号通路使用的分析。将全长MUC4与缺失其VNTR结构域的肿瘤相关剪接变异体进行比较，将揭示MUC4抗黏附活性在调节细胞生长特性中的作用。在Ai 2中，Muc4将在转基因小鼠的乳腺中诱导表达，并将这些表达交叉进入乳腺癌小鼠模型。在肿瘤发展的两个不同时间点诱导Muc4的表达将允许在体内评估Muc4在疾病不同阶段对肿瘤进展的贡献。还将分析Muc4基因敲除对小鼠乳腺肿瘤发生和进展的影响。AIMS 1和AIMS 2的研究将共同确定在患者肿瘤中观察到的Muc4过表达是否有助于乳腺癌的进展。目的3将研究患者样本中Muc4失调的机制，并将开始评估一种新的内质网定位的、由转化生长因子β调节的蛋白质降解途径在调控细胞Muc4水平中的作用。总体而言，这些研究将证实Muc4是乳腺癌进展的新贡献者，并将开始揭开最终可能被用于治疗干预的生化途径。

DESCRIPTION (provided by applicant): The overall purpose of the proposed studies is to elucidate the role of the membrane mucin Muc4 in promoting breast tumor initiation and progression. Muc4 harbors potent anti-adhesive and growth signaling activities when expressed in cultured cells, which in principle could play roles in both the initial transformation of health epithelia to early hyperplastic phenotypes and in later transitions to highly invasive and metastatic states. Consistent with this, it has been reported that Muc4 is overexpressed and mislocalized in up to 20% of local breast tumors compared to patient-matched normal tissue samples, and up to 40% of lymph node metastases relative to matched primary tumor. This project will integrate these observations by providing an analysis of Muc4 contribution to the development of progressively more aggressive states in vitro and in vivo. The driving hypothesis is that the aberrant expression of Muc4 protein in breast tumors facilitates their progression to malignancy. Aim 1 will analyze the outcome of inducible Muc4 overexpression in a series of isogenic cultured breast epithelial cell lines derived from MCF10A cells that model normal, hyperplastic, and malignant breast tissue, as well as in breast tumor cell lines that model ErbB2-positive, ER-positive and triple-negative disease. Analysis of cellular growth properties and signaling pathway usage will be carried out when cells are grown in 2D and 3D culture. Comparison of full-length Muc4 with a tumor-associated splice variant lacking its VNTR domain will reveal the role of Muc4 anti-adhesive activity in regulating cellular growth properties. In Ai 2, Muc4 will be inducibly expressed in the mammary glands of transgenic mice, and these will be crossed into mouse models of breast cancer. Induction of Muc4 expression at two different points in tumor development will permit the in vivo assessment of Muc4 contribution to tumor progression at different stages of disease. The impact of Muc4 knockout on mouse mammary tumor development and progression will also be analyzed. Together the studies of Aims 1 and 2 will determine whether the Muc4 overexpression observed in patient tumors contributes to breast cancer progression. Aim 3 will examine the mechanisms of Muc4 dysregulation in patient samples, and will begin to assess the role of a novel endoplasmic reticulum-localized, TGFß-regulated protein degradation pathway in governing cellular Muc4 levels. Overall, these studies will validate Muc4 as a novel contributor to breast cancer progression, and will begin to unravel biochemical pathways that might ultimately be exploited for therapeutic intervention.