In vitro model for DNA double‐strand break repair analysis in breast cancer reveals cell type–specific associations with age and prognosis

Abstract

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

“…Breast cancer cell lines were grown on cover slips, fixed with 3.7% formaldehyde in PBS, permeabilized with 0.5% TritonX-100, and incubated with antibodies and nuclear DAPI-stain as described in Deniz et al [49]. Primary antibodies used for immunostaining were polyclonal rabbit antibodies against 53BP1 (NB100-304, Novus Biologicals) and mouse mAb anti-phospho-Histone H2A.X (Ser139, clone JBW301, Invitrogen, Darmstadt, Germany).…”

Increased single-strand annealing rather than non-homologous end-joining predicts hereditary ovarian carcinoma

“…In our study presented here, we did not obtain evidence for altered PARP inhibitor responsiveness in PBLs from high-risk individuals or ovarian cancer patients as compared to healthy controls. Previously, we observed a highly significant response in homozygously BRCA1 - or BRCA2 -mutated cells, but either failed to detect a significant change or determined marginally significantly reduced IC50 values in heterozygously BRCA1 -, BRCA2 - and PALB2 - mutated cells [ 21 , 25 ]. In agreement with our findings, clinical PARP inhibitor use was originally based on the concept that loss of the wild-type BRCA allele represents an obligatory step for specific tumor cell killing [ 53 ].…”

“…Changes in various DNA repair genes in tumor biopsies were further demonstrated to correlate with clinical PARP inhibitor responses in metastatic prostate cancer patients [ 27 ]. In our previous studies, we demonstrated increased sensitivity to PARP inhibitor 1,5-isoquinolinediol (IQD) in immortalized lymphocytes (LCLs) with homozygously mutated BRCA2 and in mammary epithelial cells with homozygously mutated BRCA1 [ 21 , 25 ]. Therefore, we asked whether DSB repair defects in our case groups as indicated by SSA increases also translate into altered responses to the PARP inhibitor IQD in PBLs (Figure 5 ).…”

“…The combined deficiencies from hereditary predisposition and ageing may thus generate a DNA damage response reaching the sensitivity level of PARP activity measurements in human PBLs. Notably, we previously observed increasing PARP inhibitor sensitivities and a deregulation of DSB repair in epithelial cells from breast cancer specimens with age [ 21 ]. In our study presented here, we did not find differences in PARP inhibitor sensitivities or DSB repair activities in high-risk individuals or ovarian cancer patients with age.…”

Poly(ADP‐ribose) polymerase‐1 promotes recruitment of meiotic recombination‐11 to chromatin and DNA double‐strand break repair in Ku70‐deficient breast cancer cells

“…However, the potential effect of Alt-EJ and PARP-1 on breast cancer development and progression has been the subject of few studies, although it could be an important mechanism. Alt-EJ is an extremely error-prone DNA repair pathway that has elevated activity in high-risk breast cancer (27) and breast cancer is one of the most genomically unstable cancers. PARP-1 inhibition is effective in breast cancer therapy, especially in patients with BRCA1/2 mutations.…”