Detection of Impaired Homologous Recombination Repair in NSCLC Cells and Tissues

Abstract

Introduction Homologous recombination repair (HRR) is a critical pathway for the repair of DNA damage caused by cisplatin or PARP inhibitors. HRR may be impaired by multiple mechanisms in cancer, which complicates assessing the functional HRR status in cells. Here, we monitored the ability of non-small cell lung cancer (NSCLC) cells to form subnuclear foci of DNA repair proteins as a surrogate of HRR proficiency. Methods We assessed clonogenic survival of 16 NSCLC cell lines in response to cisplatin, mitomycin C (MMC), and the PARP inhibitor olaparib. Thirteen tumor explants from NSCLC patients were subjected to cisplatin ex-vivo. Cells were assayed for foci of repair-associated proteins such as BRCA1, FANCD2, RAD51, and γ-H2AX. Results Four cell lines (25%) showed an impaired RAD51 foci forming ability in response to cisplatin. Impaired foci formation correlated with cellular sensitivity to cisplatin and MMC as well as olaparib. Foci responses complemented or superseded genomic information suggesting alterations in the ATM/ATR and FA/BRCA pathways. Because baseline foci in untreated cells did not predict drug sensitivity we adapted an ex-vivo biomarker assay to monitor damage-induced RAD51 foci in NSCLC explants from patients. Ex-vivo cisplatin treatment of explants identified 2 tumors (15%) exhibiting compromised RAD51 foci induction. Conclusions A fraction of NSCLC harbors HRR defects that may sensitize the affected tumors to DNA damaging agents including PARP inhibitors. We propose that foci-based functional biomarker assays represent a powerful tool for prospective determination of treatment sensitivity, but will require ex-vivo techniques for induction of DNA damage to unmask the underlying HRR defect.

The role of DNA repair pathways in cisplatin resistant lung cancer

“…Over-expression of XRCC3 in MCF-7 cells resulted in a 2-fold increase in Rad51 foci formation in response to cisplatin (a marker of HR activity) with a concomitant 4-fold increase in cisplatin resistance [68]. A more recent analysis of 16 NSCLC cell lines revealed impaired RAD51 activity in 25% of cell lines examined, and correlated with increased sensitivity to cisplatin [69]. Rad51 expression may be a useful prognostic marker in NSCLC, with high expression levels indicative of significantly reduced survival.…”

DNA Damage Response Assessments in Human Tumor Samples Provide Functional Biomarkers of Radiosensitivity

“…Many investigators now believe that a functional assessment of the DDR in human tumor tissues can be used to predict treatment sensitivities 2,4,5,8 . Such functional assays most often rely on the detection of subnuclear foci of DDR proteins (reviewed in ref.…”

New therapeutic perspectives in CCDC6 deficient lung cancer cells

“…Nevertheless, treatment with inhibitors of the DNA repair enzymes, like the PARP inhibitors, restores the sensitivity to DNA damage inducers. 11,12,39,40 H460 cells, depleted of CCDC6, are quite resistant to cisplatinum, and the PARP inhibitor olaparib reestablishes cisplatinum sensitivity, by acting in synergy with cisplatinum (CI < 1).…”