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.

该项目旨在定义乳腺导管和导管上皮细胞正常风险和乳腺癌风险增加的形态学，分子和代谢特征。需要此信息来定义乳腺癌致癌途径的早期变化，以开发出肿瘤性乳房组织的分类和分子特征，以评估风险评估，并确定新的靶标，并促进对妇女的选择和监测，以预防乳腺癌。该项目由以下临床和实验室研究开发：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（DN Danforth，pi）来表征高风险乳房导管上皮的表征。 b。）方案02-C-0144，建立正常的乳房上皮细胞培养物，以及来自乳腺癌高风险的乳房组织的高风险细胞系和组织仓库（DN Danforth，PI）。 c。）综合文献回顾正常乳腺组织的分子变化，正常风险或乳腺癌，增生和非典型增生的高风险。 d。）通过内源性危险因素调节乳房上皮的增殖和DNA损伤修复。协议02-C-0077检查和特征是导管灌洗和导管内窥镜检查，处于正常风险和乳腺癌风险增加的女性导管和导管上皮。对54名妇女进行了研究，有39名高风险受试者和15名受试者正常风险。鉴定了导管上皮细胞的细胞学异型，并与导管内镜的结构变化发现相关。 对具有异型的所有受试者进行重复的导管灌洗和内窥镜检查，以定义细胞和导管变化的持久性。开发了一种新方法，用于采集导管上皮细胞样品进行细胞学和分子分析：使用正常上皮的刷子采样装置进行导管内镜采样。这提供了超过90％纯导管上皮细胞的细胞产量和样品的显着增加。高风险乳房上皮的分子特征正在进行中，并且已经开发了一种新型的细胞病理载玻片DNA基因组扩增的方法。最初的研究将定义p53基因的异常，这是乳腺癌发生的主要因素。 p53突变将通过整个基因测序，通过17p13.1区域的分段染色体损失来定义，通过染色体17的细瓷砖CGH以及SNP TAQMAN Genotyping Assay通过SNP Taqman Genoteging Assay的等位基因失衡和杂合性丧失。然后，将扩展分子分析以包括DNA甲基化阵列以定义肿瘤抑制基因丧失，这是乳腺肿瘤发生的早期变化。 （Danforth DN等人，Jour Oncol，94：555-564，2006）。协议，02-C-0144（DN DANFORTH，PI）建立了来自正常乳腺组织的所有主要部位的组织和细胞系存储库，其乳腺癌风险增加，包括乳腺癌的对侧正常乳腺癌，与乳腺癌的妇女相邻的乳腺癌妇女，患有乳腺癌的家族史强大（包括BRCA1和BRCA2突变携带者），或者是乳腺癌的风险。淋巴瘤的辐照。处理组织以允许大量分子研究。开发了致命上皮，成纤维细胞和脂肪细胞系，以允许多种表型，代谢和分子研究。为每个受试者收集人口统计数据，并将所有标本存储在NIH存储库中。对癌性乳腺组织的主要分子变化进行了全面的文献综述，以定义致癌途径，并有助于对正常风险和高风险乳腺组织的分子分析。乳腺癌风险低的正常/良性乳腺组织的四种类型的乳腺癌，高风险的正常/良性组织，上皮增生和与阿比亚的上皮增生相关，以定义数值染色体变化，结构性染色体变化，以及单个基因表达的变化。分子变化的模式与风险和组织学/形态学亚型之间的进展相关。这篇综述表明，乳腺癌发生的最早分子变化是杂合性的丧失和肿瘤抑制基因座的DNA甲基化的丧失，存在于形态上正常的低风险中，而较高的风险乳房组织较高，而动脉症是后来的事件，首先在高风险正常的正常乳房组织中鉴定出来。基因扩增是核组织组织中不常见的早期事件。这种特征为对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。乳腺癌风险增加的正常乳腺上皮细胞的增殖对于克隆扩张和乳腺癌发生中遗传异常的积累是至关重要的决定因素。 为了了解这种增殖的调节，研究了两个突出的刺激性和危险因素对乳腺癌，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，而无需调节凋亡。 IGF-1的这种增​​殖作用伴随着IGF-1R和IRS-1的磷酸化的快速刺激，以及在转录后水平（蛋白质表达）和在转录水平（基因和蛋白质表达）下IRS-1的下调（蛋白质表达）和IRS-2。这些细胞表达雌激素受体α（Eralpha）和β（Erbeta）和孕激素（PR）受体，但是雌二醇并未刺激增殖或细胞周期进展，并且在任何这些细胞中都没有调节ER或PR。 重要的是，与在乳腺癌中观察到的协同作用相反，IGF-1与雌二醇无协同作用以刺激这些生长或代谢过程。这些发现表明，IGF-1是早期乳腺癌作用中的主要有丝分裂原。正常乳腺上皮细胞和与IGF-1协同作用的雌激素反应性发生在致癌途径中。接下来，IGF-1在早期癌变中增强染色体不稳定性中的作用将通过研究其对正常和风险增加的乳腺上皮细胞中DNA损伤修复和信号传导的调节来定义。