Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women

Tissue Specific DNA Methylation in Normal Human Breast Epithelium and in Breast Cancer

<div><p>Cancer is a heterogeneous and tissue-specific disease. Thus, the tissue of origin reflects on the natural history of the disease and dictates the therapeutic approach. It is suggested that tissue differentiation, mediated mostly by epigenetic modifications, could guide tissue-specific susceptibility and protective mechanisms against cancer. Here we studied breast specific methylation in purified normal epithelium and its reflection in breast cancers. We established genome wide methylation profiles of various normal epithelial tissues and identified 110 genes that were differentially methylated in normal breast epithelium. A number of these genes also showed methylation alterations in breast cancers. We elaborated on one of them, TRIM29 (ATDC), and showed that its promoter was hypo-methylated in normal breast epithelium and heavily methylated in other normal epithelial tissues. Moreover, in breast carcinomas methylation increased and expression decreased whereas the reverse was noted for multiple other carcinomas. Interestingly, TRIM29 regulation in breast tumors clustered according to the PAM50 classification. Thus, it was repressed in the estrogen receptor positive tumors, particularly in the more proliferative luminal B subtype. This goes in line with previous reports indicating tumor suppressive activity of TRIM29 in estrogen receptor positive luminal breast cells in contrast to oncogenic function in pancreatic and lung cancers. Overall, these findings emphasize the linkage between breast specific epigenetic regulation and tissue specificity of cancer.</p></div

TRIM29 gene expression and promoter methylation varies in breast cancer subtypes.

<p>Analysis of TCGA data base shows (<b>A</b>) variable gene expression of TRIM29 in normal breast tissues (n = 97) and in breast tumor subtypes defined by the PAM50 classification: basal (n = 75), luminal A (n = 187), luminal B (n = 104), HER2 (n = 46), normal-like (n = 6). Heat map scale ranges from red (high) to green (low). (<b>B</b>) Variable methylation at the promoter of TRIM29 (Illumina probe #cg13625403) in breast tumor subtypes. Expression inversely correlates with promoter methylation. Data was selected from TCGA data base as described in methods. Number of samples indicated at the bottom, <b>***</b>p<0.001, <b>*</b>p<0.01, P-values between groups were calculated using Welch's corrected t-test.</p

HOXB7 is an ERα cofactor in the activation of HER2 and multiple ER target genes leading to endocrine resistance

Why breast cancers become resistant to tamoxifen despite continued expression of the estrogen receptor alpha (ERα) and what factors are responsible for high HER2 expression in these tumors remains an enigma. HOXB7 ChIP analysis followed by validation showed that HOXB7 physically interacts with ERα, and that the HOXB7-ERα complex enhances transcription of many ERα target genes including HER2. Investigating strategies for controlling HOXB7, our studies revealed that MYC, stabilized via phosphorylation mediated by EGFR-HER2 signaling, inhibits transcription of miRNA-196a, a HOXB7 repressor. This leads to increased expression of HOXB7, ER-target genes and HER2. Repressing MYC using small molecule inhibitors reverses these events, and causes regression of breast cancer xenografts. The MYC-HOXB7-HER2 signaling pathway is eminently targetable in endocrine-resistant breast cancer

TRIM29 promoter methylation and gene expression varies in normal epithelial tissues.

<p>(<b>A&C</b>) Q-MSP at TRIM29 promoter in (<b>A</b>) various human normal tissues enriched for epithelium and (<b>C</b>) purified human normal epithelial cells. The same tissue samples studied by the Illumina methylation array were included in (A). Q-MSP amplicon overlapped with the sequence of Illumina probe #cg13625403 used for array analysis (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091805#pone.0091805.s005" target="_blank">Table S4</a></b>). (<b>B&D</b>) The same samples were used for gene expression analysis by qRT-PCR that showed inverse correlation with promoter methylation (RNA was not available for fallopian tube and urinary bladder). HMEC- human mammary epithelial cells, HPEpiC-prostate, OvEC-ovarian, HCEpiC- colon, HUtEpiC-uterine, HREC-renal. (<b>E</b>) Data from Illumina Infinium methylation450K array obtained from ENCODE showed similar pattern of TRIM29 promoter methylation in additional purified human normal epithelial cells. Methylation at 5 Illumina probes is shown: 1.cg20655548 2.cg12201660 3.cg17971587 4.cg13285004 5.cg13625403 (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091805#pone.0091805.s005" target="_blank">Table S4B</a></b>). Each row refers to one individual.</p

Bidirectional “switch” in TRIM29 regulation from normal epithelium to the respective carcinomas.

<p>TCGA data analysis: (<b>A</b>) TRIM29 promoter methylation (average of 5 Illumina probes, <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091805#pone.0091805.s005" target="_blank">Table S4B</a></b>) in normal and in cancer tissues. Partial methylation was noted in breast and prostate normal tissues that increased in the respective cancers. In contrast, for the other tissue-types, methylation was high in the normal tissues and decreased in the respective carcinomas. (<b>B</b>) For breast and prostate cancers, increase in promoter methylation was associated with decrease in gene expression whereas the opposite was noted for the other tissues. BRCA = breast carcinoma, PRAD = prostate adenocarcinoma, LUSC = lung squamous cell carcinoma, BLCA = bladder carcinoma, PAAD = pancreatic adenocarcinoma, COAD = colon adenocarcinoma, READ = rectal adenocarcinoma UCEC = uterine-cervix carcinoma. Number of samples indicated at the bottom, <b>***</b>p<0.0001, NS – not significant, P-values between groups were calculated using Welch's corrected t-test.</p

Genome-wide methylation analysis of various normal human epithelial tissues.

<p>Pearson correlation coefficient was calculated (<b>A</b>) between the right and the left breast and (<b>B</b>) between breast epithelium and WBC of the same woman. (<b>C</b>) Principal Component Analysis (PCA) grouped 15 tissue samples by their global methylation resemblance into 5 clusters that corresponded with the origin of the tissues.</p