Characterizing the relationship between the DNA repair proteins in the Classical NHEJ and the ALT NHEJ pathways and investigating the role of homeobox genes in abnormal DNA repair in breast cancer

Abstract

Cancer cells, including the sporadic breast cancers, often show defects in DNA double strand break (DSB) repair that can lead to accumulation of genomic aberrations. However, mechanisms leading to these defects are not well understood. Recent studies in Rassool lab have showed that relative to non-tumorigenic cells, estrogen receptor- and progesterone receptor-negative (ER/PR-) breast cancer cells have reduced state levels of Ku70, a component of the classical non-homologous end-joining (C-NHEJ) pathway. However, components of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, PARP1 and DNA Ligase III, are increased. The study also found that downregulation of Ku70 in ER/PR+ MCF7 cells leads to an upregulation of ALT NHEJ proteins. Herein, we have conducted preliminary studies to determine the mechanisms that regulate the switch between C-NHEJ and ALT NHEJ. To determine whether the expression of ALT NHEJ proteins is inversely correlated to more than one component of C-NHEJ, we generated individual transient knockdown (KD) of Ku70 and XRCC4 in MCF7 cells. Whereas Ku70 KD resulted in an increase in steady state levels of ALT NHEJ components, no significant changes were observed following XRCC4 KD, suggesting that this is a Ku-specific effect. We also investigated the role of homeobox family of transcription factors, particularly HOXB7, in enhancing C-NHEJ, or ALT-NHEJ, or both pathways. A recent study has indicated an important role of HOXB7 in regulating C-NHEJ activity by functional interaction with Ku70. Our in vivo plasmid-based NHEJ repair assay in ER/PR- SKBR3, generated for overexpression of HOXB7 (SKBR3-B7), revealed that these cells have a 2-fold increase in NHEJ repair efficiency as compared to vector-transfected cells (SKBR3-Vec). However, sequencing of repair junctions revealed no significant changes in size of deletions and occurrence of microhomologies at the break sites of repaired plasmids between SKBR3-B7 and SKBR3-Vec cells. These results suggest that although HOXB7 increases the repair capacity, it is not a critical regulator in dictating fate between C-NHEJ and ALT-NHEJ. In order to provide insights into pathways that lead to genomic instability, further studies are required to establish whether HOXB7 is indeed a master regulator of activity of both C-NHEJ and ALT NHEJ pathways.