RNA-Seq Reveals Deregulation of G2/M and p53-p21-DREAM-CDE/CHR Pathways in HMEC^BRCA1+/- Upon DNA Damage

Introduction

Women who carry a heterozygous mutation in BRCA1/2 face an extremely high risk of breast and ovarian cancer but rarely of other cancers. The BRCA1/2 proteins play a key role in the maintenance of genomic integrity and DNA repair. To further elucidate the molecular mechanisms that stand behind the marked susceptibility of the mammary epithelium to cancer in BRCA mutation carriers, we compared the transcriptional profiles of primary human mammary epithelial cells from BRCA1 mutation carriers (HMEC^BRCA1+/-) and non-carriers (HMEC^BRCA1+/+) at baseline and following DNA damage.

Methods

Primary normal human mammary epithelial cells were isolated from surgical specimens of BRCA1 mutation carriers and non-carriers (n=3 per group). The cells were cultured for 1-3 passages and exposed to a single dose of doxorubicin for 24 hrs to induce DNA damage. Total RNA was extracted and subjected to RNA-sequencing. Differentially expressed genes were calculated using DESeq2, and were tested for enrichment of pathways and functional gene groups.

Results

RNA-Seq analysis identified 2823 genes in HMEC^BRCA1+/+ and 2508 in HMEC^BRCA1+/- that were either up or down regulated following DNA damage. Pathway analysis of these gene sets revealed enrichment of DNA damage response pathways including homologous recombination, DNA repair and chromosomal maintenance in both HMEC groups. Interestingly, we found enrichment of the G2/M checkpoint cell cycle control pathway for HMEC^BRCA1+/-. We further noted enrichment of the ATM/ATR- p53-p21-DREAM-CDE/CHR pathway, known to be inhibited upon DNA damage. This pathway includes key G2/M regulatory genes such as CDK1, cyclins A2/B1, PLK1, CDC25B, WEE1 and Aurora B, which were over-repressed by DNA damage particularly in HMEC^BRCA1+/-.

Discussion and Conclusion

It was previously shown in various cellular models that BRCA1 deficiency may result in compromised G2/M checkpoint, disrupted G2 arrest and increased entry to mitosis upon DNA damage. Here we point to key regulatory genes involved in the G2/M checkpoint that are transcriptionally deregulated in HMEC^BRCA1+/- upon DNA damage. Given the defective DNA repair in these cells, this could result in accumulation of genetic aberrations and evasion of apoptosis during mitosis, and thus contribute to the marked susceptibility of these cells to cancer. Targeting specific genes in this pathway is therefore a potential innovative approach for prevention of breast cancer in high risk BRCA mutation carriers.