The Role Of Brca1 In The Regulation Of Telomeres: Implications For Genomic Stability And Malignant Transformation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background The precise role of the BRCA1 gene product in breast carcinogenesis is not fully understood. BRCA1 protein is a part of the DNA damage repair machinery and may have a role in telomere biology. Telomere maintenance is crucial for malignant transformation. The connection between BRCA1 and telomeres was reported in very few studies so far, mainly in cancer cells. Here we study the crosstalk between telomeres and BRCA1 by knocking down its expression in breast epithelial cells. We also assessed telomere/telomerase parameters in BRCA1 mutation carriers compared to normal age-matched controls. Methods BRCA1 knockdown was performed in HB-2 cells and validated by Western blot. Telomere length was measured by Southern blot. Telomerase activity was detected by the Q-TRAP assay and its gene expression was followed by Q-PCR. The binding of the shelterin proteins to telomeres was evaluated by the ChIP assay; their cellular levels were assessed by Western blot. DNA damage was indicated by following the levels of γH2AX. Pyro-sequencing was employed to decipher the methylation status of the sub-telomeric regions. Telomeres G-strand overhang length was measured by the G-tail assay. Total gene expression was evaluated by Affymetrix whole exome microarrays. Similar methods were employed to study the telomere-telomerase parameters in the BRCA1/2 carriers. Results Telomeres became incrementally longer and dysfunction, as γH2AX protein's expression increased, after silencing of BRCA1. Telomerase activity or hTERT expression remained unchanged, implicating their involvement in our setting. The binding of 5 shelterin members to telomeres did not change; however TRF1 increased its binding to telomeres after BRCA1 knock-down, an increase which preceded telomeres elongation. While the methylation of chromosomes 5 was similar in the knock-downs and controls cells, it incrementally increased in the sub-telomere region of chromosome 10. The G overhang was shorter after BRCA1 knockdown. The knock-down of BRCA1 promoted the expression of a distinct set of genes, some of which may relate to the pre-disposition to malignancy after BRCA1 silencing. BRCA1/2 mutations carriers have different telomere dynamics compared with to that of the normal population. They possess a constant DNA damage, differences in methylation pattern of the sub-telomeric regions and a shorter telomeric G- overhang length. Conclusions BRCA1 has a role in telomere length homeostasis in breast epithelial cells. Its knockdown causes telomeres elongation accompanied by their loss of function which promoted DNA damage in the cells. TRF1 increased its binding to telomeres, contributing to their elongation by a yet unknown mechanism. BRCA1 mutation carriers are more prone to malignant transformation probably due to luck of telomere protection which together with BRCA1 loss of function may promote the accumulation of cancerous mutations. Citation Format: Orit Uziel, Lital Zuriano, Rinat Yerushalmi, Einat Beery, Yardena Nordenberg, Dan Frumkin, Meir Lahav. The role of BRCA1 in the regulation of telomeres: Implications for genomic stability and malignant transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3508. doi:10.1158/1538-7445.AM2014-3508