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.

. Schematic structure of the TP73 gene.
1.2 Figure S2 a, b. Cell cytotoxicity in the various human normal primary epithelial cells following treatment with cisplatin and doxorubicin.
1.3 Figure S2 c. Induction of PUMA, p21 and NOXA by DNA damage in HMECs and in other types of human epithelial cells.
1.4 Figure S3 a, b The deltaNp73 promoter is unmethylated in HMECs and fully methylated in white blood cells of BRCA1 mutation carriers and non-carriers.
1.5 Figure S3 c. Linear mixed model analysis compared the decrease in cell viability in HMEC BRCA1-wt and in HMECs BRCA1-mut.
1.6 Figure S4 a-c. DNA methylation and induction of the deltaNp73 and the TAp73 gene isoforms by DNA damage in MCF10A.
1.8 Figure S4 e. Linear mixed model analysis compared the decrease in cell viability in MCF10A and in MCF10A/BRCA-KD following deltaNp73 inhibition and treatment with cisplatin.
1.9 Figure S4 f, g. Inhibition of deltaNp73 by siRNA caused a decrease in cell viability (XTT) following exposure to doxorubicin in MCF10Aand this effect was augmented in MCF10A/BRCA1-KD.
1.10 Figure S5 a, b. Expression of BRCA1 in MCF10A clones stably transfected with shBRCA #1 and #2 and flag-i∆Np73. Comparison of cell viability curves in response to cisplatin between these clones.
1.11 Figure S6a, b. Expression of BRCA1 and deltaNp73 mRNA and protein in HMECs in response to cisplatin.
1.12 Figure S7. DNA methylation across the entire TP73 gene among breast cancer subtypes -TCGA analysis.
1.14 Full images of all western blots without processing.

Figure S1
Adapted from:   The cells were exposed to increasing doses of cisplatin or doxorubicin for 24 hours and cell viability was measured by XTT.
HMECsmammary, HRECrenal, SAECsmall airways, PRECprostate, OVECovary. The data represent average of 3 replicates in cells originated from 3 individuals (cisplatin) or 1 individual (doxorubicin). Error bars represent standard error of the mean (S.E.M). The tables on the right indicate that deltaNp73 induction by cisplatin or doxorubicin differed between cell types and these differences did not correlate with cell cytotoxicity.  mutation carriers (BRCA1-mut, n=4) and non-carriers (BRCA1-wt, n=5).
Methylation was measured by quantitative methylation specific PCR (qMSP). Ttest showed no significant difference between the groups.
Note: The Y-axis scale is different in each panel.    Corresponding author: Ayelet Avraham, email: AyeletA@shamir.gov.il

General remarks
1. All blots were cut to pieces horizontally or vertically before incubation with the specific antibodies according to the expected molecular weights of the corresponding proteins.
2. Most images contain more than one blot of more than one gel that were exposed together. These are annotated.       Marker Fluorescent 2.