Hypermethylator phenotype in human breast cancer: therapeutic target and mechanism of DNMT3b regulation

A subset of primary breast cancers and breast cancer cell lines express a hypermethylation defect characterized by DNMT hyperactivity and DNMT3b overexpression. The objectives of this project were (i) to determine if targeting the methylome enhances the sensitivity of breast cancer cells to chemotherapy, and (ii) to elucidate the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer. To address the first objective, hypermethylator breast cancer cell lines were treated with demethylating agent (5-aza-2'-deoxycytidine) and/or were subjected to RNAi mediated DNMT3b knockdown (KD), and then tested for sensitivity to doxorubicin hydrochloride, paclitaxel, and 5-fluorouracil. The results show that pharmacologic demethylating pretreatment sensitizes hypermethylator breast cancer cells to cell killing by cytotoxic drugs, and provide proof of concept that direct targeting of DNMT3b also improves cell kill by these drugs. These findings suggest that targeting the methylome improves chemotherapeutic efficacy of cytotoxic drugs against hypermethylator breast cancer cells as a function of dose and duration of exposure to demethylating treatment. To address the second objective, the expression of microRNAs (miRs) that regulate or are predicted to regulate DNMT3b were examined in hypermethylator or non-hypermethylator breast cancer cell lines and in primary breast cancers. Hypermethylator cell lines express diminished levels of regulatory miRs compared to non-hypermethylator cell lines. Mechanistic studies were conducted to establish the role of miR expression in the hypermethylation defect. Antagomir-mediated knockdown of regulatory miRs in nonhypermethylator cell lines resulted in increased DNMT3b mRNA and forced reexpression of regulatory miRs reduced DNMT3b mRNA in hypermethylator cell lines. In primary breast cancers, miR expression patterns revealed two distinct subsets among the basal-like subtype. Most hypermethylator basal like cancers exhibit diminished expression of regulatory miRs. These findings strongly suggest that diminished expression of multiple regulatory miRs contributes to DNMT3b overexpression. Together, these results support the conclusion that the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer cells is the loss of post-transcriptional regulation of DNMT3b by regulatory miRs, and that combined epigenetic and cytotoxic treatment will improve the efficacy of breast cancer chemotherapy

Loss of post-transcriptional regulation of DNMT3b by microRNAs: A possible molecular mechanism for the hypermethylation defect observed in a subset of breast cancer cell lines

A hypermethylation defect associated with DNMT hyperactivity and DNMT3b overexpression characterizes a subset of breast cancers and breast cancer cell lines. We analyzed breast cancer cell lines for differential expression of regulatory miRs to determine if loss of miR-mediated post-transcriptional regulation of DNMT3b represents the molecular mechanism that governs the overexpression of DNMT3b that drives the hypermethylation defect in breast cancer. MicroRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a, miR-148b) or are predicted (miR-26a, miR-26b, miR-203, miR-222) to regulate DNMT3b were examined among 10 hypermethylator and 6 non-hypermethylator breast cancer cell lines. Hypermethylator cell lines express diminished levels of miR-29c, miR-148a, miR-148b, miR-26a, miR-26b, and miR-203 compared to non-hypermethylator cell lines. miR expression patterns correlate inversely with methylation-sensitive gene expression (r=−0.66, p=0.0056) and directly with the methylation status of these genes (r=0.72, p=0.002). To determine the mechanistic role of specific miRs in the dysregulation of DNMT3b among breast cancer cell lines, miR levels were modulated by transfection of pre-miR precursors for miR-148b, miR-26b, and miR-29c into hypermethylator cell lines (Hs578T, HCC1937, SUM185) and transfection of antagomirs directed against miR-148b, miR-26b, and miR-29c into non-hypermethylator cell lines (BT20, MDA-MB-415, MDA-MB-468). Antagomir-mediated knock-down of miR-148b, miR-29c, and miR-26b significantly increased DNMT3b mRNA in non-hypermethylator cell lines, and re-expression of miR-148b, miR-29c, and miR-26b following transfection of pre-miR precursors significantly reduced DNMT3b mRNA in hypermethylator cell lines. These findings strongly suggest that: i) post-transcriptional regulation of DNMT3b is combinatorial, ii) diminished expression of regulatory miRs contributes to DNMT3b overexpression, iii) re-expression of regulatory miRs reduces DNMT3b mRNA levels in hypermethylator breast cancer cell lines, and iv) down-regulation of regulatory miRs increases DNMT3b mRNA levels in non-hypermethylator breast cancer cell lines. In conlcusion, the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer cell lines involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs

Digital histologic analysis reveals morphometric patterns of age-related involution in breast epithelium and stroma

Complete age-related regression of mammary epithelium, often termed post-menopausal involution, is associated with decreased breast cancer risk. However, most studies have qualitatively assessed involution. We quantitatively analyzed epithelium, stroma, and adipose tissue from histologically normal breast tissue of 454 patients in the Normal Breast Study (NBS). High-resolution digital images of normal breast Hematoxylin & Eosin stained slides were partitioned into epithelium, adipose tissue, and non-fatty stroma. Percentage area and nuclei per unit area (nuclear density) were calculated for each component. Quantitative data were evaluated in association with age using linear regression and cubic spline models Stromal area decreased (p=0.0002) and adipose tissue area increased (p<0.0001), with an approximate 0.7% change in area for each component, until age 55 when these area measures reached a steady state. While epithelial area did not show linear changes with age, epithelial nuclear density decreased linearly beginning in the third decade of life. No significant age-related trends were observed for stromal or adipose nuclear density. Digital image analysis offers a high-throughput method for quantitatively measuring tissue morphometry and for objectively assessing age-related changes in adipose tissue, stroma, and epithelium. Epithelial nuclear density is a quantitative measure of age-related breast involution that begins to decline in the early premenopausal period

Imaging Extracellular Matrix Remodeling In Vitro by Diffusion-Sensitive Optical Coherence Tomography

The mammary gland extracellular matrix (ECM) is comprised of biopolymers, primarily collagen I, that are created and maintained by stromal fibroblasts. ECM remodeling by fibroblasts results in changes in ECM fiber spacing (pores) that have been shown to play a critical role in the aggressiveness of breast cancer. However, minimally invasive methods to measure the spatial distribution of ECM pore areas within tissues and in vitro 3D culture models are currently lacking. We introduce diffusion-sensitive optical coherence tomography (DS-OCT) to image the nanoscale porosity of ECM by sensing weakly constrained diffusion of gold nanorods (GNRs). DS-OCT combines the principles of low-coherence interferometry and heterodyne dynamic light scattering. By collecting co- and cross-polarized light backscattered from GNRs within tissue culture, the ensemble-averaged translational self-diffusion rate, DT, of GNRs is resolved within ∼3 coherence volumes (10 × 5 μm, x × z). As GNRs are slowed by intermittent collisions with ECM fibers, DT is sensitive to ECM porosity on the size scale of their hydrodynamic diameter (∼46 nm). Here, we validate the utility of DS-OCT using pure collagen I gels and 3D mammary fibroblast cultures seeded in collagen/Matrigel, and associate differences in artificial ECM pore areas with gel concentration and cell seed density. Across all samples, DT was highly correlated with pore area obtained by scanning electron microscopy (R2 = 0.968). We also demonstrate that DS-OCT can accurately map the spatial heterogeneity of layered samples. Importantly, DS-OCT of 3D mammary fibroblast cultures revealed the impact of fibroblast remodeling, where the spatial heterogeneity of matrix porosity was found to increase with cell density. This provides an unprecedented view into nanoscale changes in artificial ECM porosity over effective pore diameters ranging from ∼43 to 360 nm using a micron-scale optical imaging technique. In combination with the topical deposition of GNRs, the minimally invasive nature of DS-OCT makes this a promising technology for studying tissue remodeling processes

Dysregulation of microRNA expression drives aberrant DNA hypermethylation in basal-like breast cancer

Basal-like breast cancers frequently express aberrant DNA hypermethylation associated with concurrent silencing of specific genes secondary to DNMT3b overexpression and DNMT hyperactivity. DNMT3b is known to be post-transcriptionally regulated by microRNAs. The objective of the current study was to determine the role of microRNA dysregulation in the molecular mechanism governing DNMT3b overexpression in primary breast cancers that express aberrant DNA hypermethylation. The expression of microRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a and miR-148b) or are predicted to regulate DNMT3b (miR-26a, miR-26b, miR-203 and miR-222) were evaluated among 70 primary breast cancers (36 luminal A-like, 13 luminal B-like, 5 HER2-enriched, 16 basal-like) and 18 normal mammoplasty tissues. Significantly reduced expression of miR-29c distinguished basal-like breast cancers from other breast cancer molecular subtypes. The expression of aberrant DNA hypermethylation was determined in a subset of 33 breast cancers (6 luminal A-like, 6 luminal B-like, 5 HER2-enriched and 16 basal-like) through examination of methylation-sensitive biomarker gene expression (CEACAM6, CDH1, CST6, ESR1, GNA11, MUC1, MYB, TFF3 and SCNN1A), 11/33 (33%) cancers exhibited aberrant DNA hypermethylation including 9/16 (56%) basal-like cancers, but only 2/17 (12%) non-basal-like cancers (luminal A-like, n=1; HER2-enriched, n=1). Breast cancers with aberrant DNA hypermethylation express diminished levels of miR-29a, miR-29b, miR-26a, miR-26b, miR-148a and miR-148b compared to cancers lacking aberrant DNA hypermethylation. A total of 7/9 (78%) basal-like breast cancers with aberrant DNA hypermethylation exhibit diminished levels of ≥6 regulatory miRs. The results show that i) reduced expression of miR-29c is characteristic of basal-like breast cancers, ii) miR and methylation-sensitive gene expression patterns identify two subsets of basal-like breast cancers, and iii) the subset of basal-like breast cancers with reduced expression of multiple regulatory miRs express aberrant DNA hypermethylation. Together, these findings strongly suggest that the molecular mechanism governing the DNMT3b-mediated aberrant DNA hypermethylation in primary breast cancer involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs

Overexpression of miR-146a in basal-like breast cancer cells confers enhanced tumorigenic potential in association with altered p53 status

The tumor suppressor p53 is the most frequently mutated gene in human cancers, mutated in 25–30% of breast cancers. However, mutation rates differ according to breast cancer subtype, being more prevalent in aggressive estrogen receptor-negative tumors and basal-like and HER2-amplified subtypes. This heterogeneity suggests that p53 may function differently across breast cancer subtypes. We used RNAi-mediated p53 knockdown (KD) and antagomir-mediated KD of microRNAs to study how gene expression and cellular response to p53 loss differ in luminal versus basal-like breast cancer. As expected, p53 loss caused downregulation of established p53 targets (e.g. p21 and miR-34 family) and increased proliferation in both luminal and basal-like cell lines. However, some p53-dependent changes were subtype specific, including expression of miR-134, miR-146a and miR-181b. To study the cellular response to miR-146a upregulation in p53-impaired basal-like lines, antagomir KD of miR-146a was performed. KD of miR-146a caused decreased proliferation and increased apoptosis, effectively ablating the effects of p53 loss. Furthermore, we found that miR-146a upregulation decreased NF-κB expression and downregulated the NF-κB-dependent extrinsic apoptotic pathway (including tumor necrosis factor, FADD and TRADD) and antagomir-mediated miR-146a KD restored expression of these components, suggesting a plausible mechanism for miR-146a-dependent cellular responses. These findings are relevant to human basal-like tumor progression in vivo, since miR-146a is highly expressed in p53 mutant basal-like breast cancers. These findings suggest that targeting miR-146a expression may have value for altering the aggressiveness of p53 mutant basal-like tumors

Benign Breast Tissue Composition in Breast Cancer Patients: Association with Risk Factors, Clinical Variables, and Gene Expression

Breast tissue composition (epithelium, non-fatty stroma, and adipose) changes qualitatively and quantitatively throughout the lifespan, and may mediate relationships between risk factors and breast cancer initiation. We sought to identify relationships between tissue composition, risk factors, tumor characteristics, and gene expression

Body mass index associated with genome-wide methylation in breast tissue

Gene expression studies indicate that body mass index (BMI) is associated with molecular pathways involved in inflammation, insulin-like growth factor activation, and other carcinogenic processes in breast tissue. The goal of this study was to determine whether BMI is associated with gene methylation in breast tissue and to identify pathways that are commonly methylated in association with high BMI. Epigenome-wide methylation profiles were determined using the Illumina HumanMethylation450 BeadChip array in the non-diseased breast tissue of 81 women undergoing breast surgery between 2009 and 2013 at the University of North Carolina Hospitals. Multivariable, robust linear regression was performed to identify methylation sites associated with BMI at a false discovery rate q value <0.05. Gene expression microarray data was used to identify which of the BMI-associated methylation sites also showed correlation with gene expression. Gene set enrichment analysis was conducted to assess which pathways were enriched among the BMI-associated methylation sites. Of the 431,568 methylation sites analyzed, 2573 were associated with BMI (q value <0.05), 57 % of which showed an inverse correlation with BMI. Pathways enriched among the 2573 probe sites included those involved in inflammation, insulin receptor signaling, and leptin signaling. We were able to map 1251 of the BMI-associated methylation sites to gene expression data, and, of these, 226 (18 %) showed substantial correlations with gene expression. Our results suggest that BMI is associated with genome-wide methylation in non-diseased breast tissue and may influence epigenetic pathways involved in inflammatory and other carcinogenic processes

Tumor Intrinsic Subtype Is Reflected in Cancer-Adjacent Tissue

Overall survival of early-stage breast cancer (BC) patients is similar for those who undergo breast conserving therapy (BCT) and mastectomy, however, 10-15% of women undergoing BCT suffer ipsilateral breast tumor recurrence. The risk of recurrence may vary with BC subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk

Relationship of Mammographic Density and Gene Expression: Analysis of Normal Breast Tissue Surrounding Breast Cancer

Previous studies of breast tissue gene expression have demonstrated that the extratumoral microenvironment has substantial variability across individuals, some of which can be attributed to epidemiologic factors. To evaluate how mammographic density (MD) and breast tissue composition relate to extratumoral microenvironment gene expression, we used data on 121 breast cancer patients from the population-based Polish Women's Breast Cancer Study