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 data 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, to facilitate selection and monitoring of women for breast cancer prevention, and to eliminate disparities in breast cancer outcomes for women in different ethnic groups. 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 breast tissue at normal risk and at high risk for breast cancer, in hyperplasia, and in atypical hyperplasia to define the accumulation of molecular changes in early breast carcinogenesis, and to guide molecular studies which will define the breast carcinogenic pathway. d.) Regulation of proliferation and DNA damage repair in breast epithelium by the endogenous risk factors estradiol and IGF-1. 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. Ninety-one women have been studied, 52 high risk subjects and 39 subjects at normal risk. Cytologic atypia of ductal epithelial cells was identified and correlated with ductal endoscopic findings of architectural changes. A new method for acquiring ductal epithelial cellular samples for cytologic and molecular analysis was developed which provides multiple aliquots of significantly increased cellular yields ( 90% pure ductal epithelial cells. Quantitative and qualitative assessment is available for each aliquot. Individual cell pellets and ductal fluid for each aliquot are frozen for future studies. A novel method of whole genome amplification of DNA from cytopathologic slides was developed. Molecular studies to define the gene expression profile, and numerical and structural chromosome abnormalities of at-risk breast epithelial cells are in progress. Gene expression profiling is performed on total RNA extracted from epithelial cells with the PicoPure RNA extraction kit (Invitrogen). Total RNA (10 25 ng) undergoes one round of amplification [Low Input Quick Amp Kit, Agilent) (45-47)], and amplified RNA hybridized to Agilent Human Gene Expression 4 x 44 microarray (G4845A), using a one color labeling technique. Universal Reference total RNA from normal tissues (Clontech) will be treated similarly and serve as an additional control. Gene expression will be analyzed first by unsupervised hierarchical clustering to identify innate expression differences between the two ethnic groups, followed by supervised analysis to identify specific gene expression patterns and signaling pathways to delineate differences between risk and ethnic groups, and changes consistent with breast carcinogenesis within and between these groups. Gene expression at specific loci will be confirmed by qRT-PCR. Chromosomal numerical and structural changes will be studied on Genomic DNA (10 ng) which will be whole-genome amplified with Relpli-g (Qiagen) and studied on Human Omni-Quad 1 M HD SNP array (probe spacing 2.4 kb;Illumina) using a paired sample mode of analysis with matched peripheral blood lymphocyte DNA (similarly amplified) from each subject. The log2R ratio and B allele frequency will be calculated for each array, and ploidy, LOH, and copy number aberrations determined. DNA copy numbers for each allelic loss or gain will be validated with gene-specific TaqMan Copy Number Assays (Applied Biosystems), utilizing a TaqMan Copy Number Reference Assay RNase P. Quantitative assessment of telomere length will be determined by qRT-PCR using telomere specific primers according to the methods of Cawthon. Centrosome duplication: quantitative cellular centrosome determinations will be made by indirect immunofluorescence staining on ThinPrep cytopath slides using a monoclonal antibody against gamma-tubulin, a protein localized to centrosomes. An increase in the number or size of centrosomes is considered to represent centrosome hyperamplification. The identification of telomeric shortening may indicate cells at significant risk for chromosomal instability and progression in the carcinogenic pathway. Quantitative assessment of telomere length will be determined by QPCR using telomere specific primers according to the methods of Cawthon. MicroRNA. Breast ductal fluid and ductal epithelial cells are 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. Protocol, 02-C-0144 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 Gail model risk estimate of breast cancer of >1.67%, or BRCA1 and BRCA2 mutation carriers. Mortal epithelial, fibroblast, and adipose cell lines are developed from normal breast tissue to allow for a wide range of phenotypic, metabolic, and molecular studies. Demographic data is collected for each subject, and all specimens are stored in an NIH repository. 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 were 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 acted synergistically with estradiol 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. The role of IGF-1 in enhancing chromosomal instability in early carcinogenesis will be defined by studying its modulation of DNA double-strand break induction and repair and signaling in normal and increased risk breast epithelium.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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