Regulation of Apical Polarity in Breast Cancer

描述(申请人提供)：癌症与组织结构和功能的改变有关。值得注意的是，组织极性的改变可能是上皮癌进展的决定因素。在乳腺，基尖组织的极性表现为膜蛋白在与基底膜接触的细胞的基极和描绘上皮结构或腺泡的管腔的细胞的顶极之间的不对称分布。基底极性的改变伴随着侵袭性表型。尽管根尖极改变在乳腺癌中经常被报道，但对这种改变的控制机制、后果以及诊断和预后价值知之甚少。紧密连接是一种黏附复合体，在根尖极性的建立和维持中起着关键作用。我们的初步结果表明，紧密连接组织者ZO-1在浸润性癌和某些类型的原位癌中的分布发生了变化，证实了文献报道的顶极丢失伴随着肿瘤的进展。此外，我们有证据表明，DNA甲基化是表观遗传控制顶端极性的主要机制。长期的假设是，识别参与控制心尖极性的基因将为乳腺癌的诊断和预后以及帮助预防乳腺癌进展的新策略的建立带来宝贵的信息。(1)利用Affymetrix微阵列分析非肿瘤性乳腺上皮SI细胞在Matrigel存在的情况下形成表型正常的乳腺腺泡细胞，以及经5-azacytidine低甲基化、I型胶原培养或缝隙连接阻滞剂处理后形成表型正常的乳腺腺泡细胞和顶极性受损的腺泡细胞，利用Affymetrix微阵列分析识别可能调节根尖极性的基因。与对照组相比，在所有根尖极性受损的腺泡模型中表现出表达变化的基因将通过对微阵列数据的3种不同的基于模型的统计分析来选择。将通过改变不同乳腺细胞模型形成的极化和顶极受损结构中CAP基因的表达来研究所选基因对顶极控制(CAP基因)的影响。(2)探讨已知的根尖极性标志物的分布和CAP基因在正常乳腺组织和肿瘤组织中的表达谱，并将CAP基因的表达谱与根尖极性标志物的分布变化和肿瘤进展相关联。对非肿瘤性、癌前和侵袭性乳腺病变进行一系列紧密连接和根尖下CRB复合体蛋白的免疫染色，并评估这些根尖标志物的分布模式与组织学分级或根尖极性丧失的组织标准之间的相关性。CAP基因在非侵袭性和侵袭性乳腺病变中的表达将与根尖标志物的组织学分类和分布图进行比较。这一项目将使人们更好地了解心尖部极性丢失在乳腺癌进展中的作用，并为预后相关研究和预防肿瘤进展的靶点确定新的标志物。

DESCRIPTION (provided by applicant): Cancer is associated with alterations in tissue organization and function. Notably, the alteration of tissue polarity may be determinant for the progression of epithelial cancers. In the mammary gland, baso-apical tissue polarity is illustrated by the asymmetrical distribution of membrane proteins between the basal pole of cells in contact with the basement membrane, and the apical pole of cells that delineates the lumen of the epithelial structures or acini. Alterations in basal polarity accompany the invasive phenotype. Although alterations in apical polarity have been frequently reported in breast cancer, little is known regarding the control mechanisms, and the consequences as well as diagnosis and prognosis values of such alterations. Tight junctions are adhesion complexes that play a critical role in the establishment and maintenance of apical polarity. Our preliminary results show that the distribution of tight junction organizer ZO-1 is altered in invasive carcinomas and in certain types of carcinoma in situ, confirming literature reports that suggest that loss of apical polarity accompany tumor progression. In addition, we have evidence to show that DNA methylation is a major mechanism of epigenetic control of apical polarity. The long-term hypothesis is that the identification of genes involved in the control of apical polarity will bring invaluable information for the diagnosis and prognosis of breast neoplasias, and the establishment of new strategies to help prevent breast cancer progression. 2 aims are proposed: (1) to identify statistically significant genes that are potential regulators of apical polarity using Affymetrix microarray analysis of three-dimension (3D) culture of non-neoplastic mammary epithelial SI cells that form phenotypically normal breast acini in the presence of matrigel, and apical polarity-impaired acini upon DNA-hypomethylation by 5-azacytidine, culture in collagen I, or treatment with a gap junction blocker. Genes showing altered expression in all models of apical polarity-impaired acini compared to controls will be selected using 3 different model-based statistical analyses of the microarray data. The influence of the selected genes on the control of apical polarity (CAP genes) will be investigated by altering their expression in polarized and apical polarity-impaired structures formed by different breast cell models. (2) To explore the distribution of known apical polarity markers and the expression profile of CAP genes on archival biopsies of normal and neoplastic breast tissues and relate the expression profile of CAP genes to alterations in the distribution of apical polarity markers and tumor progression. Non-neoplastic, pre-malignant and pre-invasive breast lesions will be immunostained for a series of tight junction and subapical CRB complex proteins and the correlation between the distribution patterns of these apical markers and histological grade or tissue criteria for apical polarity loss will be assessed. CAP genes expression in non-invasive and invasive breast lesions will be compared to histological classification and distribution maps of apical markers. This project should bring a better knowledge of the involvement of apical polarity loss in breast cancer progression and identify novel markers for prognosis-related studies and targets to prevent tumor progression.