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: 7735368
• 负责人: DAVID DANFORTH
• 金额: $ 43.23万
• 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/7735368
• 关键词: 17p13.1; Adipocytes; Allelic Imbalance; Apoptosis; Atypia; Atypical hyperplasia; BRCA1 gene; BRCA2 Mutation; Base Excision Repairs; Benign; Biological Assay; Breast; Breast Cancer Prevention; Breast Cancer Risk Assessment Tool; Breast Cancer Risk Factor; Cell Cycle Progression; Cell Line; Cell Proliferation; Cells; Characteristics; Chromosomal Instability; Chromosomal Loss; Chromosomes; Chromosomes, Human, Pair 17; Classification; Clinical; Clonal Expansion; Contralateral; CpG Islands; Cytologic Atypia; Cytology; DNA Methylation; DNA Repair; DNA Sequence; DNA amplification; Data; Development; Devices; Dose; Double Strand Break Repair; Down-Regulation; Duct (organ) structure; Ductal; Ductal Epithelial Cell; Ductal Epithelium; Endoscopy; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Evaluation; Event; Family history of; Fibroblasts; Gene Amplification; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Proteins; Genes; Genetic; Genome; Genotype; Growth; High Risk Woman; Histologic; Human; Hybridization Array; Hyperplasia; In Vitro; Individual; Insulin-Like Growth Factor I; Irrigation; Laboratory Study; Loss of Heterozygosity; Lymphoma; Mammary Gland Parenchyma; Mediastinal; Metabolic; Metabolism; Methods; Mismatch Repair; Mitogens; Modeling; Molecular; Molecular Abnormality; Molecular Analysis; Molecular Profiling; Monitor; Mutation; Nucleotide Excision Repair; Numbers; Oligonucleotides; Pathway interactions; Pattern; Phase; Phosphorylation; Progesterone; Progesterone Receptors; Protocols documentation; RNA amplification; Radiation induced double strand break; Range; Regulation; Review Literature; Risk; Risk Assessment; Risk Estimate; Risk Factors; Role; SNP genotyping; Sampling; Signal Transduction; Site; Slide; Specimen; Staging; Standards of Weights and Measures; TP53 gene; Time; Tissues; Tumor Suppressor Genes; Tumor Suppressor Proteins; United States National Institutes of Health; Woman; carcinogenesis; comparative genomic hybridization; design; improved; irradiation; malignant breast neoplasm; mutation carrier; novel; protein expression; repository; synergism; tissue processing; tumorigenesis

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. This information is needed to define early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, and to identify new targets, and facilitate selection and monitoring of women, for breast cancer prevention. This project is developed by 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 normal risk or at high risk for breast cancer, in hyperplasia, and in atypical hyperplasia. d.) Regulation of proliferation and DNA damage repair in breast epithelium by endogenous risk factors. Protocol 02-C-0077 examines and characterizes by ductal lavage and ductal endoscopy the ducts and ductal epithelium of women at normal risk and increased risk for breast cancer. Fifty-four women have been studied, 39 high risk subjects and 15 at subjects at normal risk. Cytologic atypia of ductal epithelial cells was identified and correlated with ductal endoscopic findings of architectural changes. Repeat ductal lavage and endoscopy was performed on all subjects with atypia to define the persistence of cellular and ductal changes. A new method for acquisition of ductal epithelial cellular samples for cytologic and molecular analysis was developed: ductal endoscopic sampling with brush sampling devices of normal epithelium. This provided significantly increased cellular yields and samples of more than 90% pure ductal epithelial cells. Molecular characteristics of high risk breast epithelium is in progress, and a novel method of whole genome amplification of DNA from cytopathologic slides has been developed. Initial studies will define abnormalities of the p53 gene, a major factor in breast carcinogenesis. P53 mutations will be defined by whole gene sequencing, segmental chromosomal loss in the 17p13.1 region by fine tiling CGH of chromosome 17, and allelic imbalance and intragenic loss of heterozygosity by SNP Taqman genotyping assay. Molecular analysis will then be expanded to include DNA methylation array to define tumor suppressor gene loss, an early change in breast tumorigenesis. (Danforth DN et al. Jour Surg Oncol, 94:555-564, 2006). Protocol, 02-C-0144 (DN Danforth, PI) establishes a tissue and cell line repository from all major sites of normal breast tissue at increased risk for breast cancer, including the contralateral normal breast, tissue adjacent to a breast cancer, women with a strong family history of breast cancer (including BRCA1 and BRCA2 mutation carriers), a Gail model risk estimate of breast cancer of more than 1.67%, or women with prior mediastinal irradiation for lymphoma. Tissues are processed to allow for a wide spectrum of molecular studies. Mortal epithelial, fibroblast and adipose cell lines are developed to allow for a wide range of phenotypic, metabolic, and molecular studies. Demographic data is collected for each subject, and all specimens stored in an NIH repository. A comprehensive literature review of the major molecular changes in preneoplastic breast tissue to define the carcinogenic pathway and aid in the molecular analysis of normal risk and high risk breast tissue has been conducted. Four types of preneoplastic breast tissue - normal/benign breast tissue at low risk for breast cancer, normal/benign tissue at high risk, epithelial hyperplasia, and epithelial hyperplasia with atypia were reviewed to define numerical chromosomal changes, structural chromosome changes, and changes in expression of individual genes. Patterns of molecular changes have been correlated with risk and with progression between histologic/morphologic subtypes. This review indicated that the earliest molecular changes in breast carcinogenesis are loss of heterozygosity and DNA methylation of tumor suppressor loci, present in morphologically normal low risk, and more frequently high risk breast tissue, while aneusomy is a later event and is first identified in high risk normal breast tissue. Gene amplification is an uncommon early event in preneoplastic tissues. This characterization provides an important standard for whole genome analysis of human breast tissue at risk for breast cancer. Proliferation of normal breast epithelial cells at increased risk for breast cancer is a critical determinant for clonal expansion and for the accumulation of genetic abnormalities in breast carcinogenesis. To understand regulation of this proliferation, the effects of two prominent stimulatory and risk factors for breast cancer, estradiol (E2) and insulin-like growth factor-1 (IGF-1) on normal and high risk breast epithelial cells was studied. IGF-1 stimulated growth of all breast epithelial cells in a time-dependent and dose-dependent manner without modulation of apoptosis. This proliferative action by IGF-1 was accompanied by the rapid stimulation of phosphorylation of IGF-1R and IRS-1, and by downregulation of IRS-1 at the posttranscriptional level (protein expression) and of IRS-2 at the transcriptional level (gene and protein expression). These cells express estrogen receptor alpha (ERalpha) and beta (ERbeta) and progesterone (PR) receptors, however estradiol did not stimulate proliferation or cell cycle progression and did not modulate ER or PR in any of these cells. Importantly, IGF-1 did not act synergistically with estradiol to stimulate any of these growth or metabolic processes, in contrast to the synergism observed in breast cancer. These findings indicate that IGF-1 is the dominant mitogen in early breast carcinogenesis. Estrogen responsiveness of normal breast epithelial cells and synergism with IGF-1 occur later in the carcinogenic pathway. The role of IGF-1 in enhancing chromosomal instability in early carcinogenesis will next be defined by studying its modulation of DNA damage repair and signaling in normal and increased risk breast epithelium.

该项目旨在定义正常风险和乳腺癌风险增加的乳腺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，开发改进的癌前乳腺组织分类和分子特征以进行风险评估，并确定新的目标，并促进对女性的选择和监测，以预防乳腺癌。该项目是通过以下临床和实验室研究开发的：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.) 对乳腺癌正常风险或高风险、增生和非典型增生的正常乳腺组织的分子变化进行全面的文献综述。 d.) 内源性危险因素对乳腺上皮增殖和 DNA 损伤修复的调节。方案 02-C-0077 通过导管灌洗和导管内窥镜检查和表征处于正常乳腺癌风险和增加乳腺癌风险的女性的导管和导管上皮。对 54 名女性进行了研究，其中 39 名是高风险受试者，15 名是正常风险受试者。确定了导管上皮细胞的细胞学异型性，并将其与导管内窥镜结构变化的发现相关联。 对所有异型性受试者进行重复导管灌洗和内窥镜检查，以确定细胞和导管变化的持续性。开发了一种获取导管上皮细胞样本进行细胞学和分子分析的新方法：使用正常上皮刷采样装置进行导管内窥镜采样。这显着增加了细胞产量和超过 90% 纯导管上皮细胞的样本。高风险乳腺上皮的分子特征正在进行中，并且已经开发出一种从细胞病理切片中扩增 DNA 全基因组的新方法。初步研究将确定 p53 基因的异常，这是乳腺癌发生的主要因素。 P53 突变将通过全基因测序、通过 17 号染色体的细平铺 CGH 确定 17p13.1 区域的节段染色体丢失以及通过 SNP Taqman 基因分型测定确定等位基因不平衡和杂合性基因内丢失来确定。随后，分子分析将扩展到包括 DNA 甲基化阵列，以定义肿瘤抑制基因的丢失，这是乳腺肿瘤发生的早期变化。 (Danforth DN 等人，Jour Surg Oncol，94：555-564，2006)。方案，02-C-0144（DN Danforth，PI）建立了一个组织和细胞系存储库，其中包括乳腺癌风险增加的正常乳腺组织的所有主要部位，包括对侧正常乳房、邻近乳腺癌的组织、患有乳腺癌的女性。有明显乳腺癌家族史（包括 BRCA1 和 BRCA2 突变携带者）、盖尔模型乳腺癌风险估计超过 1.67%，或既往接受过淋巴瘤纵隔放射治疗的女性。对组织进行处理以进行广泛的分子研究。开发人类上皮细胞、成纤维细胞和脂肪细胞系以进行广泛的表型、代谢和分子研究。收集每个受试者的人口统计数据，并将所有样本存储在 NIH 存储库中。对癌前乳腺组织的主要分子变化进行了全面的文献综述，以确定致癌途径并帮助对正常风险和高风险乳腺组织进行分子分析。回顾了四种类型的癌前乳腺组织——乳腺癌低风险的正常/良性乳腺组织、高风险的正常/良性组织、上皮增生和异型性上皮增生，以定义染色体数量变化、染色体结构变化和染色体结构变化。个体基因的表达。分子变化模式与组织学/形态学亚型之间的风险和进展相关。该综述表明，乳腺癌发生中最早的分子变化是肿瘤抑制基因座的杂合性丧失和 DNA 甲基化，存在于形态正常的低风险乳腺组织中，更常见的是高风险乳腺组织，而异型是较晚发生的事件，首先在高风险乳腺组织中发现。危害正常乳腺组织。基因扩增是癌前组织中罕见的早期事件。这一特征为有乳腺癌风险的人类乳腺组织的全基因组分析提供了重要标准。正常乳腺上皮细胞在乳腺癌风险增加时的增殖是克隆扩张和乳腺癌发生过程中遗传异常积累的关键决定因素。 为了了解这种增殖的调节，研究了乳腺癌的两种重要刺激和危险因素——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。 IGF-1 以时间依赖性和剂量依赖性方式刺激所有乳腺上皮细胞的生长，而不调节细胞凋亡。 IGF-1 的这种增殖作用伴随着 IGF-1R 和 IRS-1 磷酸化的快速刺激，以及 IRS-1 在转录后水平（蛋白质表达）和 IRS-2 在转录水平（基因）的下调。和蛋白质表达）。这些细胞表达雌激素受体 α (ERα) 和 β (ERβ) 以及孕激素 (PR) 受体，但雌二醇不会刺激增殖或细胞周期进展，也不会调节任何这些细胞中的 ER 或 PR。 重要的是，IGF-1 不会与雌二醇协同作用来刺激任何这些生长或代谢过程，这与在乳腺癌中观察到的协同作用相反。这些发现表明 IGF-1 是早期乳腺癌发生中的主要有丝分裂原。正常乳腺上皮细胞的雌激素反应以及与 IGF-1 的协同作用发生在致癌途径的后期。接下来将通过研究 IGF-1 在正常和高风险乳腺上皮中对 DNA 损伤修复和信号传导的调节来确定 IGF-1 在增强早期癌发生过程中染色体不稳定性中的作用。