Regulation of Apical Polarity in Breast Cancer

描述（由申请人提供）：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性因素。在乳腺中，基底-顶端组织极性通过与基底膜接触的细胞基底极和描绘上皮结构或腺泡内腔的细胞顶端极之间的膜蛋白的不对称分布来说明。基底极性的改变伴随着侵袭性表型。尽管在乳腺癌中经常报告顶端极性的改变，但关于这种改变的控制机制、后果以及诊断和预后价值知之甚少。紧密连接是粘附复合物，在顶端极性的建立和维持中起关键作用。我们的初步研究结果表明，紧密连接组织者ZO-1的分布在浸润性癌和某些类型的原位癌中发生了改变，证实了文献报道，即顶端极性的丧失伴随着肿瘤的进展。此外，我们有证据表明，DNA甲基化是顶端极性的表观遗传控制的主要机制。长期假设是，参与控制顶端极性的基因的鉴定将为乳腺肿瘤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。提出了两个目标：（1）使用非肿瘤性乳腺上皮SI细胞的三维（3D）培养物的Affytron微阵列分析来鉴定作为顶端极性的潜在调节剂的统计学显著基因，所述SI细胞在基质胶存在下形成表型正常的乳腺腺泡，并且在通过5-氮杂胞苷进行DNA低甲基化、在胶原I中培养或用间隙连接阻断剂处理后形成顶端极性受损的腺泡。将使用微阵列数据的3种不同的基于模型的统计分析来选择与对照相比在顶端极性受损腺泡的所有模型中显示改变的表达的基因。将通过改变所选基因在由不同乳腺细胞模型形成的极化和顶端极性受损结构中的表达来研究所选基因对顶端极性控制（CAP基因）的影响。(2)探索已知的顶端极性标记物的分布和CAP基因在正常和肿瘤性乳腺组织的存档活检中的表达谱，并将CAP基因的表达谱与顶端极性标记物分布和肿瘤进展的改变相关联。将对非肿瘤性、癌前病变和浸润前乳腺病变进行一系列紧密连接和心尖下CRB复合物蛋白的免疫染色，并评估这些心尖标志物的分布模式与组织学分级或心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组织学分类和顶端标志物的分布图进行比较。该项目应能更好地了解乳腺癌进展中顶端极性丧失的参与情况，并为乳腺癌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.