Dawei Bu

DNA Methylation in Benign Breast Epithelium in Relation to Age and Breast Cancer Risk

Background: Many established breast cancer risk factors are related to the timing and duration of exposure to reproductive hormones, which are known to drive breast epithelial cell proliferation. The epigenetic molecular clock hypothesis suggests that CpG island methylation records the cell division history of benign epithelium. In proliferative epithelium, such as breast, this may provide an individualized cell-based measure of cancer risk. Methods: Methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-β2 was measured by quantitative multiplex methylation-specific PCR in 290 benign and malignant breast epithelial cell samples obtained by palpation-directed fine-needle aspiration biopsy from 164 women. Univariate, multivariate, and unsupervised cluster analysis was used to establish the relationship between TSG methylation and a personal history of breast cancer, predicted breast cancer risk, and specific breast cancer risk factors. Results: RASSF1A methylation was highly correlated with breast cancer risk [odds ratio (OR), 5.28; 95% confidence interval (95% CI), 1.95-14.32; P = 0.001], atypical cytology (OR, 4.11; 95% CI, 1.30-12.98; P = 0.016), and benign breast disease requiring biopsy (OR, 6.12; 95% CI, 1.41-26.51; P = 0.016). RASSF1A methylation increased linearly between ages 32 and 55. Increasing parity was associated with decreased APC methylation. Conclusions: TSG methylation increases in benign breast epithelium with increasing age. Because it is independently related to a personal history of benign or malignant breast disease and to predicted breast cancer risk, it may have value for breast cancer risk stratification and as a surrogate endpoint marker in prevention trials. (Cancer Epidemiol Biomarkers Prev 2008;17(5):1051–9)

Role of Physical Activity in Modulating Breast Cancer Risk as Defined by APC and RASSF1A Promoter Hypermethylation in Nonmalignant Breast Tissue

Physical activity reduces breast cancer risk. Promoter hypermethylation of the tumor suppressor genes APC and RASSF1A, which is potentially reversible, is associated with breast cancer risk. We conducted a cross-sectional study in 45 women without breast cancer to determine the association of physical activity with promoter hypermethylation of APC and RASSF1A in breast tissue. We used quantitative methylation-specific PCR to test the methylation status of APC and RASSF1A, and questionnaires to assess study covariates and physical activity (measured in metabolic equivalent hours per week). In univariate analyses, the study covariate, benign breast biopsy number, was positively associated with promoter hypermethylation of APC (P = 0.01) but not RASSF1A. Mulitvariate logistic regression indicated that, although not significant, physical activities for a lifetime [odds ratio (OR), 0.57; 95% confidence interval (95% CI), 0.22-1.45; P = 0.24], previous 5 years (OR, 0.62; 95% CI, 0.34-1.12; P = 0.11), and previous year (OR, 0.72; 95% CI, 0.43-1.22; P = 0.22) were inversely related to promoter hypermethylation of APC but not RASSF1A for all physical activity measures. Univariate logistic regression indicated that physical activities for a lifetime, previous 5 years, and previous year were inversely associated with benign breast biopsy number, and these results were approaching significance for lifetime physical activity (OR, 0.41; 95% CI, 0.16-1.01; P = 0.05) and significant for physical activity in the previous 5 years (OR, 0.57; 95% CI, 0.34-0.94; P = 0.03). The study provides indirect evidence supporting the hypothesis that physical activity is inversely associated with promoter hypermethylation of tumor suppressor genes, such as APC, in nonmalignant breast tissue. (Cancer Epidemiol Biomarkers Prev 2007;16(2):192–6)

Atypia and DNA Methylation in Nipple Duct Lavage in Relation to Predicted Breast Cancer Risk

Background: Tumor suppressor gene (TSG) methylation is identified more frequently in random periareolar fine needle aspiration samples from women at high risk for breast cancer than women at lower risk. It is not known whether TSG methylation or atypia in nipple duct lavage (NDL) samples is related to predicted breast cancer risk. Methods: 514 NDL samples obtained from 150 women selected to represent a wide range of breast cancer risk were evaluated cytologically and by quantitative multiplex methylation-specific PCR for methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-β2. Results: Based on methylation patterns and cytology, NDL retrieved cancer cells from only 9% of breasts ipsilateral to a breast cancer. Methylation of ≥2 genes correlated with marked atypia by univariate analysis, but not multivariate analysis, that adjusted for sample cellularity and risk group classification. Both marked atypia and TSG methylation independently predicted abundant cellularity in multivariate analyses. Discrimination between Gail lower-risk ducts and Gail high-risk ducts was similar for marked atypia [odds ratio (OR), 3.48; P = 0.06] and measures of TSG methylation (OR, 3.51; P = 0.03). However, marked atypia provided better discrimination between Gail lower-risk ducts and ducts contralateral to a breast cancer (OR, 6.91; P = 0.003, compared with methylation OR, 4.21; P = 0.02). Conclusions: TSG methylation in NDL samples does not predict marked atypia after correcting for sample cellularity and risk group classification. Rather, both methylation and marked atypia are independently associated with highly cellular samples, Gail model risk classifications, and a personal history of breast cancer. This suggests the existence of related, but independent, pathogenic pathways in breast epithelium. (Cancer Epidemiol Biomarkers Prev 2007;16(9):1812–21)

Telomerase immortalization of human mammary epithelial cells derived from a BRCA2 mutation carrier

SummaryA novel human mammary epithelial cell line, HME348, was established from benign breast tissue from a 44-year-old germ-line BRCA2 mutation carrier with a history of stage 1 breast cancer. Mutation analysis showed that the patient had a known 6872del4 BRCA2 heterozygous mutation. The human mammary epithelial cells passaged in culture exhibited cellular replicative aging as evidenced by telomere shortening, lack of telomerase activity, and senescence. Ectopic expression of telomerase (hTERT) reconstituted telomerase activity in these cells and led to the immortalization of the cells. When grown on glass, the majority of immortalized HME348 cells expressed ESA and p63 with a small population also expressing EMA. In three-dimensional Matrigel® culture, HME348 cells formed complex branching acini structures that expressed luminal (EMA, CK18) and myoepithelial (p63, CALLA, CK14) markers. Three clones derived from this culture were also p63+/ESA+/EMA+/− on glass but formed similar acinar structures with both luminal and myoepithelial cell differentiation in Matrigel® confirming the mammary progenitor nature of these cells. Additionally, the experimentally immortalized HME348 cells formed acini in cleared mammary fat pads in vivo. As this is the first report establishing and characterizing a benign human mammary epithelial cell line derived from a BRCA2 patient without the use of viral oncogenes, these cells may be useful for the study of BRCA2 function in breast morphogenesis and carcinogenesis.

Tamoxifen Downregulates Ets Oncogene Family Members ETV4 and ETV5 in Benign Breast Tissue: Implications for Durable Risk Reduction

Five years of tamoxifen reduces breast cancer risk by nearly 50% but is associated with significant side effects and toxicities. A better understanding of the direct and indirect effects of tamoxifen in benign breast tissue could elucidate new mechanisms of breast carcinogenesis, suggest novel chemoprevention targets, and provide relevant early response biomarkers for phase II prevention trials. Seventy-three women at increased risk for breast cancer were randomized to tamoxifen (20 mg daily) or placebo for 3 months. Blood and breast tissue samples were collected at baseline and posttreatment. Sixty-nine women completed all study activities (37 tamoxifen and 32 placebo). The selected biomarkers focused on estradiol and IGFs in the blood; DNA methylation and cytology in random periareolar fine-needle aspirates; and tissue morphometry, proliferation, apoptosis, and gene expression (microarray and reverse transcriptase PCR) in the tissue core samples. Tamoxifen downregulated Ets oncogene transcription factor family members ETV4 and ETV5 and reduced breast epithelial cell proliferation independent of CYP2D6 genotypes or effects on estradiol, ESR1, or IGFs. Reduction in proliferation was correlated with downregulation of ETV4 and DNAJC12. Tamoxifen reduced the expression of ETV4- and ETV5-regulated genes implicated in epithelial-stromal interaction and tissue remodeling. Three months of tamoxifen did not affect breast tissue composition, cytologic atypia, preneoplasia, or apoptosis. A plausible mechanism for the chemopreventive effects of tamoxifen is restriction of lobular expansion into stroma through downregulation of ETV4 and ETV5. The human equivalent of murine multipotential progenitor cap cells of terminal end buds may be the primary target. Cancer Prev Res; 4(11); 1852–62. ©2011 AACR.

Identification of Breast Cancer DNA Methylation Markers Optimized for Fine-Needle Aspiration Samples

Background: Random periareolar fine-needle aspiration (RP-FNA) is increasingly used in trials of breast cancer prevention for biomarker assessments. DNA methylation markers may have value as surrogate endpoint biomarkers, but this requires identification of biologically relevant markers suitable for paucicellular, lymphocyte-contaminated clinical samples. Methods: Unbiased whole-genome 5-aza-2′-deoxycytidine (5AZA)–induced gene expression assays, followed by several phases of qualitative and quantitative methylation-specific PCR (MSP) testing, were used to identify novel breast cancer DNA methylation markers optimized for clinical FNA samples. Results: The initial 5AZA experiment identified 453 genes whose expression was potentially regulated by promoter region methylation. Informatics filters excluded 273 genes unlikely to yield useful DNA methylation markers. MSP assays were designed for 271 of the remaining genes and, ultimately, 33 genes were identified that were differentially methylated in clinical breast cancer samples, as compared with benign RP-FNA samples, and never methylated in lymphocytes. A subset of these markers was validated by quantitative multiplex MSP in extended clinical sample sets. Using a novel permutation method for analysis of quantitative methylation data, PSAT1, GNE, CPNE8, and CXCL14 were found to correlate strongly with specific clinical and pathologic features of breast cancer. In general, our approach identified markers methylated in a smaller subpopulation of tumor cells than those identified in published methylation array studies. Conclusions: Clinically relevant DNA methylation markers were identified using a 5AZA-induced gene expression approach. Impact: These breast cancer-relevant, FNA-optimized DNA methylation markers may have value as surrogate endpoint biomarkers in RP-FNA studies. Cancer Epidemiol Biomarkers Prev; 22(12); 2212–21. ©2013 AACR.

A Variant of DNA Polymerase β Is Not Cancer Specific

DNA polymerase β (pol β) carries out base-excision repair (BER) required for DNA maintenance, replication, and recombination in eukaryotic cells. A variant characterized by a deletion of exon 11, an 87-bp region in the catalytic domain (pol β Δ 208–236), was previously described as a possible cause of genomic instability in cancer. The variant form was hypothesized to act in a dominant negative fashion, due to the fact that the variant inhibits the gap filling and DNA binding activities of the wild-type pol β protein. DNA polymerase β transcripts were analyzed in 8 breast cancer cell lines, snap-frozen benign breast tissues from 10 women, and lymphocytes from 10 normal controls, using reverse-transcription polymerase chain reaction (RT-PCR) and three separate primer pairs. The exon 10–12 splice site (variant) was identified using a primer designed to span the spliced exons and by sequencing RT-PCR products that included exon 10, exon 11 (if present), and exon 12. In all of the samples tested, we found both the wild-type and exon 11 87-bp deleted variant mRNAs expressed. We conclude that expression of the DNA polymerase β variant (pol β Δ 208–236) is ubiquitous and not breast cancer specific.

Abstract 843: Spontaneous immortalization of human mammary epithelial cells from a woman with a germline STK11 mutation

Spontaneous immortalization of benign mammary epithelial cells (MEC) in culture is very rare. Models of MEC immortalization suggest that cells escape M0 growth arrest (selection) by silencing p16 expression after which they encounter a second growth arrest, M1 (agonescence), characterized by telomere shortening and profound genomic instability. Pre-selection, post-selection, and spontaneously immortalized MEC from a woman with a germline STK11 mutation were subjected to a comprehensive “OMICS” evaluation including miRNA, transcriptome, methylome, exome, and comparative genomic hybridization (CGH) in order to understand the molecular events associated with MEC immortalization. Principle component analysis of gene expression identified 4 significant components, the first of which showed the greatest separation between the different time points. The most notable expression changes were upregulation of FOXQ1, IL1-related genes, Ras-related genes, and fibronectin and downregulation of COL1, COL6, CK19, and CDH1. MEC gradually acquired global hypomethylation of non-CpG DNA and regional hypermethylation of CpG islands (including p16). A classifier based on methylation pattern showed that immortalized cells moved away from identity with The Cancer Genome Atlas (TCGA) benign samples towards TCGA breast cancer samples. Immortalization was associated with a modest accumulation of mutations and a modestly greater number of DNA copy number alterations than was observed in earlier passage cells but no evidence of general genomic instability. Immortal cells showed a large deletion at 10q13-15 and deletions at 10p11-15 and 17p11-13 that were associated with reduced expression of genes in those regions. Post-selection bulk cell populations are quite distinct from early passage cells, but are not direct precursors of the immortal cells as some DNA copy number alterations acquired by post-selection cells were not observed in the immortalized cells. Rather, in this instance, it is likely that post-selection cells established conditions conducive to the immortalization of a rare cell in the mix. This has implications for the development of tissue-based approaches for breast cancer risk stratification. Citation Format: David Euhus, Dawei Bu, Michael Considine, Leslie Cope. Spontaneous immortalization of human mammary epithelial cells from a woman with a germline STK11 mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 843.

Abstract 4934: Promoter methylation biomarkers for breast cancer risk stratification using an unbiased whole genome approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Silencing of tumor suppressor gene expression by promoter methylation occurs frequently in breast cancer and can affect the expression of >100 genes per tumor. Since tumorigenesis is a multistep process and occurs over a large period of time, promoter methylation of some genes may occur very early in this process and can be used for risk stratification or occur very late and be used for early detection. The objective of our study is to identify genes that are frequently methylated in breast cancers and can be used for individualized breast cancer risk stratification. Methods and Results: We have used an unbiased whole genome approach to identify breast cancer methylation markers in addition to 18 candidate markers obtained from breast cancer literature. Six breast cancer cell lines and 6 short term benign breast primary cultures were exposed to 0.5 µM 5-aza-2′-deoxycytidine or DMSO for 5 days; total RNA was isolated and expression differences were determined by gene expression profiling using Illumina arrays. After normalization and bioinformatic analysis, 288 genes of interest were identified. The methylation status of each promoter was analyzed by methylation specific PCR (MSP) using an independent set of 10 breast cancer cell lines, 6 benign breast primary cultures, and 4 lymphocyte samples. Of the 288 genes tested, 204 genes were excluded because they were: not methylated in breast cancer cell lines (109), methylated in lymphocytes (91), or methylated in all benign breast cultures at the same intensity as the cancer samples (4). Of the remaining 84 genes, 58 genes with the highest methylation frequency in the breast cancer cell lines were analyzed by MSP with a panel of 15 primary breast cancers and 15 random periareolar fine needle aspirate (RP-FNA) samples from benign breast tissue (5 breast cancer patients, 5 unaffected Gail high-risk patients, and 5 unaffected Gail average-risk patients). Thirty seven genes were methylated in at least 4 breast cancer samples and had a cancer/RP-FNA ratio of >2. Unsupervised hierarchical clustering separated the genes into 3 groups based on the primary breast tumor data. One group containing 10 genes was highly methylated in all primary tumors with an average of 7.6/10 methylated genes per tumor (range 3-10). 21 genes representing all groups have been selected for quantitative MSP analysis. We are currently testing these genes in a panel of 200 RP-FNA samples including primary breast tumors and benign breast tissue from cancer and unaffected patients to identify biomarkers for breast cancer risk stratification. These data will be presented at the meeting. Conclusions: We have identified 37 candidate genes that are highly methylated in breast cancer and less frequently in benign breast tissue. Quantitative MSP analysis is underway using clinical samples to determine the best biomarkers for individualized breast cancer risk assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4934.

Abstract A107: Epigenetic markers for tissue-based breast cancer risk stratification

Effective breast cancer risk management requires accurate breast cancer risk stratification. The Gail model is the most widely used and best validated epidemiological risk assessment model. Though well calibrated, this model is not ideal for individualized risk counseling as it is only 8% better than chance for discriminating between women with breast cancer and unaffected women. Biologically based approaches, such as cytological assessment of nipple aspirate fluid or assessment of mammographic density, only marginally improve the discrimination of the Gail model. Promoter region methylation is one of the earliest identifiable molecular changes during breast carcinogenesis. We have previously shown that RASSF1A methylation, detected in benign breast epithelium obtained by random periareolar fine needle aspiration biopsy (RP-FNA), is associated with increased breast cancer risk, but this was based primarily on correlation with Gail risk and not cancer case prediction. A clinically useful epigenetic risk stratification panel would be ideally suited to benign RP-FNA samples and would provide a high degree of discrimination between benign samples from women with cancer and unaffected women. We performed a genome-wide 5-aza-2’-deoxycytidine (5-aza) induced gene re-expression screen in 6 breast cancer cell lines and 6 early passage primary benign breast epithelial cell cultures to identify potential epigenetic markers of breast cancer risk. MSP assays were designed for the 286 genes that were 1) expressed in benign breast epithelium, 2) not expressed in cancer, and 3) induced by 5-aza in cancer, or 4) had previously shown some potential for breast cancer risk stratification. 20 genes were identified that were frequently methylated in primary breast cancer, rarely methylated in benign RP-FNA samples and never methylated in lymphocytes. Quantitative multiplex methylation-specific PCR assays were designed and optimized for the 17 best genes and then assessed in an archival RP-FNA sample set that included 146 samples from unaffected women, 59 benign samples from breast cancer patients, and 52 primary breast cancers. A three marker panel including PSAT1, HS3ST2, and GNE provided the best discrimination between benign samples from cancer patients and unaffected women. One or more of these markers was scored as positive in 46% of benign samples from cancer patients, 8% of samples from unaffected patients, and 72% of primary cancers. The odds ratio for cancer case prediction based on assessment of benign RP-FNA samples was 9.42 (95% CI 4.31 — 20.59, p Citation Information: Cancer Prev Res 2010;3(12 Suppl):A107.