Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer

乳腺癌风险中乳腺导管上皮的分子特征

• 批准号: 8938390
• 负责人: DAVID DANFORTH
• 金额: $ 46.41万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8938390
• 关键词: Address; African American; Age; Age of Onset; Aneuploidy; Atypia; Binding; Biological; Biological Assay; Breast; Breast Cancer Cell; Breast Cancer Prevention; Breast Cancer Risk Factor; Breast Carcinogenesis; Breast Epithelial Cells; Cancer cell line; Categories; Caucasians; Caucasoid Race; Cell Culture Techniques; Cell Cycle; Cell Cycle Progression; Cell Line; Cells; Cellularity; Characteristics; Chemoprevention; Childbirth; Chromosomal Instability; Chromosome abnormality; Classification; Clinical Research; Clinical Trials; Control Groups; Copy Number Polymorphism; Cytology; DNA; DNA Methylation; DNA amplification; Development; Doctor of Medicine; Duct (organ) structure; Ductal; Ductal Epithelial Cell; Ductal Epithelium; Early identification; Endoscopy; Epithelial; Epithelial Cells; Epithelium; Estradiol; Estrogens; Ethnic group; Exhibits; Freezing; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Silencing; Genes; Genome; Goals; Growth; Healthcare; High Risk Woman; Hispanic Americans; Hispanics; Histology; Hormonal; Hyperplasia; In Vitro; Insulin-Like Growth Factor I; Irrigation; Laboratory Study; Length; Liquid substance; Loss of Heterozygosity; MCF10A cells; Mammary Gland Parenchyma; Messenger RNA; Metabolic; Methods; MicroRNAs; Mitogens; Mitotic Checkpoint; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Monitor; Nulliparity; Obesity; Outcome; Pathway interactions; Ploidies; Protein Analysis; Proteins; Protocols documentation; Publishing; RNA; Regulation; Regulator Genes; Reproductive Health; Reverse Transcriptase Polymerase Chain Reaction; Review Literature; Risk; Risk Assessment; Risk Factors; S Phase; Sampling; Signal Pathway; Signal Transduction; Staging; Telomerase; Telomere Shortening; Time; Tissues; Transcript; Tumor Suppressor Genes; Woman; anticancer research; base; cancer risk; carcinogenesis; caucasian American; cell growth; design; high risk; improved; malignant breast neoplasm; repository; research and development; socioeconomics; synergism; telomere

This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and increased risk for breast cancer among Caucasian, Hispanic and African American women. This information is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets and to facilitate selection and monitoring of women for breast cancer prevention, and to define the molecular basis for disparities in the development and presentation of breast cancer. This project includes the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) Protocol 02-C-0144, Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer (DN Danforth, PI). c.) Comprehensive literature review of molecular changes in normal breast tissue at risk for breast cancer to define molecular changes in early breast carcinogenesis and guide the molecular characterization of breast epithelial cells collected from women at risk for breast cancer. d.) Comprehensive literature review of molecular changes contributing to disparities in development, presentation and outcomes of breast cancer between Caucasian, Hispanic, and African American women. e.) Regulation of breast epithelial proliferation by the endogenous risk factors estradiol and IGF-1. A comprehensive review has recently been published proposing for the first time a model describing the relationship of biological and nonbiological factors to the initiation and development of the major disparities in breast cancer between African American and Caucasian women (Danforth, DN Breast Cancer Research, 15:208, 2013). This model identifies multiple molecular differences in breast cancer between African American and Caucasian women, and these differences are the major drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African American vs. Caucasian women. Multiple socioeconomic, reproductive and health care factors influence the outcome of the disparities through their influence on the breast cancer molecular characteristics. This model also emphasizes the need for defining the molecular characteristics of early carcinogenesis in these ethnic groups. Protocol 02-C-0077 characterizes by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of Caucasian, African American, and Hispanic women at normal risk and at increased risk for breast cancer. One hundred twenty-four women have been studied, 54 high risk subjects and 70 subjects at normal risk. The ductal endoscopic architectural characteristics of breasts have been defined and correlated with the presence of epithelial cell atypia. A significantly improved method of ductal epithelial cell sampling has been developed which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. Extraction of a single intact ductal lavage sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA suitable for multiple downstream studies including RT-PCR of miRNA species, qPCR of the telomerase gene, whole genome DNA amplification and arrayCGH analysis. This method significantly expands the acquisition and molecular analysis of breast ductal epithelium in women at normal risk or at high risk for breast cancer. Molecular studies to define numerical and structural chromosome abnormalities and gene and microRNA expression of normal at-risk breast epithelial cells from Caucasian, Hispanic and African American women are in progress. Chromosomal numerical and structural changes willbe studied by CGH array and ploidy, loss of heterozygosity, hemizygous and homozygous deletions, and copy number aberrations determined. Allelic losses or gains will be validated with gene-specific TaqMan Copy Number Assays (Applied Biosystems). Telomere shortening is considered an early change in breast carcinogenesis, and its identification in normal epithelial cells may indicate cells at significant risk for chromosomal instability and progression in the carcinogenic pathway. Quantitative assessment of telomere length will be determined by qRT-PCR according to the methods of Das et al. Breast ductal fluid and ductal epithelial cells are also being studied for the presence of miRNA, noncoding transcripts which bind to mRNA and result in gene silencing. miRNA has been identified in exosomes of breast ductal fluid, suggesting an important mechanism for the expansion of the cancerized field within the breast and enhancement of progression through the carcinogenic pathway. To further define the molecular characteristics associated with increased risk for breast cancer we have collected under protocol 02-C-0077 breast epithelial cells from from women at increased risk from two additional important risk groups a.) women at increased risk because of hormonal anthropomorphic factors (nulliparity, late age of first childbirth, obesity [RR = 1.5 - 2.0), and b.) women at increased risk because of the presence of atypical ductal epithelial cells (RR = 2.0 - 4.0). Identification of the molecular characteristics associated with these two categories of risk factors may provide critical information for defining progression in the carcinogenic pathway and for development of a molecular signature for risk assessment. Importantly, this study has also allowed us to a comprise a control group of ductal epithelial cells from women who lack all of these risk factors, an important component for all of the above comparisons. The effects of two prominent endogenous mitogenic risk factors for breast cancer, estradiol (E2) and insulin-like growth factor-1 (IGF-1) on normal and high risk breast epithelial cells are being studied in vitro to further define regulation of proliferation in early carcinogenesis. IGF-1 acted synergistically with E2 to stimulate growth in a high risk breast epithelial cell line (MCF12A) but not in any normal risk cell lines (MCF10A, AG11132, AG11134), suggesting that the transition to estradiol responsiveness and synergism with IGF-1 may occur at or beyond the level of hyperplasia in the carcinogenic pathway. Further, these findings indicate that IGF-1 is the dominant mitogen in early breast carcinogenesis, and estrogen responsiveness of normal breast epithelial cells and modulation of IGF-1 signaling occur later in the carcinogenic pathway. Recent studies have identified important abnormalities in G1/S phase proteins and related tumor suppressor genes in African American breast cancer which may contribute to the earlier age of onset and more aggressive histology in these women, two major disparities in breast cancer. To further define these changes the structural chromosomal and gene expression changes of breast cancer cell lines developed from breast cancer in African Americans will be compared to those of Caucasian women. Analysis of proteins involved in cell cycle progression, mitotic checkpoint pathway and spindle assembly checkpoint pathways will be emphasized. The identification of specific abnormalities between Caucasian and African American breast cancer will also provide important correlative information for analysis of normal and high risk breast epithelium, and facilitate characterization of carcinogenic changes in at-risk breast epithelium.

该项目旨在确定白种人、西班牙裔和非裔美国女性乳腺癌正常风险和高风险乳腺癌导管和导管上皮细胞的形态、分子和代谢特征。需要这些信息来定义乳腺癌致癌途径的早期变化，开发用于风险评估的癌前乳腺组织的改进分类和分子特征，确定新目标并促进选择和监测女性预防乳腺癌，并确定乳腺癌发展和表现差异的分子基础。该项目包括以下临床和实验室研究：a.) 方案 02-C-0077，通过细胞学、乳腺导管内窥镜检查和基因表达谱表征高风险乳腺导管上皮（DN Danforth，PI）。 b.) 方案 02-C-0144，从乳腺癌高危女性的乳腺组织中建立正常乳腺上皮细胞培养物以及高危细胞系和组织库（DN Danforth，PI）。 c.) 对有乳腺癌风险的正常乳腺组织的分子变化进行全面的文献综述，以定义早期乳腺癌发生的分子变化，并指导从有乳腺癌风险的女性收集的乳腺上皮细胞的分子特征。 d.) 对导致白人、西班牙裔和非裔美国女性乳腺癌发展、表现和结果差异的分子变化进行全面文献综述。 e.) 内源性危险因素雌二醇和 IGF-1 对乳腺上皮增殖的调节。最近发表了一篇综合综述，首次提出了一个模型来描述生物和非生物因素与非裔美国人和白人妇女之间乳腺癌主要差异的发生和发展之间的关系（Danforth，DN Breast Cancer Research，15： 208，2013）。该模型确定了非裔美国人和白人女性乳腺癌的多种分子差异，这些差异是非裔美国人与白人女性发病年龄、晚期阶段、更具侵袭性的组织学和较差生存率差异的主要驱动因素。多种社会经济、生殖和医疗保健因素通过影响乳腺癌分子特征来影响差异的结果。该模型还强调需要定义这些种族群体早期致癌的分子特征。方案 02-C-0077 通过导管灌洗和导管内窥镜检查白种人、非裔美国人和西班牙裔女性的乳腺导管和导管上皮，这些妇女的乳腺癌风险正常，并且风险较高。对 124 名女性进行了研究，其中 54 名是高风险受试者，70 名是正常风险受试者。乳房的导管内窥镜结构特征已被定义并与上皮细胞异型性的存在相关。已经开发出一种显着改进的导管上皮细胞取样方法，该方法提供具有高细胞结构的纯（90％）导管上皮细胞的多个样品。提取单个完整的导管灌洗样本（液体和细胞）或单独的冷冻细胞成分可提供适合多种下游研究的 DNA 和 RNA，包括 miRNA 种类的 RT-PCR、端粒酶基因的 qPCR、全基因组 DNA 扩增和 arrayCGH 分析。该方法显着扩大了乳腺癌正常风险或高风险女性乳腺导管上皮的采集和分子分析。确定白种人、西班牙裔和非裔美国女性正常高危乳腺上皮细胞的数量和结构染色体异常以及基因和 microRNA 表达的分子研究正在进行中。将通过 CGH 阵列和倍性、杂合性丧失、半合子和纯合子缺失以及确定的拷贝数畸变来研究染色体数量和结构变化。等位基因的损失或增加将通过基因特异性 TaqMan 拷贝数测定（Applied Biosystems）进行验证。端粒缩短被认为是乳腺癌发生的早期变化，其在正常上皮细胞中的识别可能表明细胞处于染色体不稳定和致癌途径进展的显着风险中。端粒长度的定量评估将根据 Das 等人的方法通过 qRT-PCR 确定。乳腺导管液和导管上皮细胞也正在研究 miRNA 的存在，这种非编码转录物与 mRNA 结合并导致基因沉默。乳腺导管液的外泌体中已鉴定出 miRNA，这表明乳腺内癌变区域扩张和通过致癌途径增强进展的重要机制。为了进一步确定与乳腺癌风险增加相关的分子特征，我们根据方案 02-C-0077 收集了来自另外两个重要风险组的风险增加女性的乳腺上皮细胞：a.) 由于激素拟人化因素而风险增加的女性（未产、初产年龄晚、肥胖 [RR = 1.5 - 2.0），以及 b.) 由于非典型导管上皮细胞的存在而导致风险增加的女性 (RR = 2.0 - 4.0）。与这两类风险因素相关的分子特征的识别可以为定义致癌途径的进展和开发风险评估的分子特征提供关键信息。重要的是，这项研究还使我们能够包含来自缺乏所有这些危险因素的女性的导管上皮细胞的对照组，这是所有上述比较的重要组成部分。正在体外研究乳腺癌的两种重要的内源性有丝分裂危险因素——雌二醇（E2）和胰岛素样生长因子-1（IGF-1）对正常和高风险乳腺上皮细胞的影响，以进一步明确乳腺癌中增殖的调节。早期癌变。 IGF-1 与 E2 协同作用，刺激高风险乳腺上皮细胞系 (MCF12A) 的生长，但不刺激任何正常风险细胞系 (MCF10A、AG11132、AG11134)，这表明向雌二醇反应性的转变以及与 IGF-1 的协同作用可能发生在致癌途径中的增生水平或以上。此外，这些发现表明 IGF-1 是早期乳腺癌发生过程中的主要有丝分裂原，正常乳腺上皮细胞的雌激素反应和 IGF-1 信号传导的调节发生在致癌途径的后期。最近的研究发现，非裔美国人乳腺癌中 G1/S 期蛋白和相关肿瘤抑制基因存在重要异常，这可能导致这些女性的发病年龄较早，组织学更具侵袭性，这是乳腺癌的两个主要差异。为了进一步明确这些变化，将从非裔美国人乳腺癌中发展而来的乳腺癌细胞系的结构染色体和基因表达变化与白人女性的乳腺癌细胞系进行比较。将重点分析参与细胞周期进程、有丝分裂检查点途径和纺锤体组装检查点途径的蛋白质。白种人和非裔美国人乳腺癌之间特定异常的识别也将为分析正常和高风险乳腺上皮提供重要的相关信息，并有助于表征高危乳腺上皮的致癌变化。