Cancer-associated overexpression of RNF168 ubiquitin ligase alters DNA repair and response to PARP inhibitors under proteotoxic stress

Introduction

During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event which orchestrates the recruitment of the downstream effectors. Under conditions of ubiquitin starvation the ubiquitin dependent accrual of DDR proteins at the DNA lesions is impaired and the DDR is attenuated. A common manifestation of the attenuation is disappearance of the 53BP1 and BRCA1 proteins from the irradiation induced foci (IRIF). However, we have identified several cancer cell lines that display sustained 53BP1 recruitment to IRIFs under proteasome inhibitor induced ubiquitin starvation. We set out to investigate mechanism and the potential relevance of the phenotype in cancer.

Materials/methods

The number and intensity of 53BP1 IRIF after 5μM MG132 treatment was quantified in the primary BJ cell line and several cancer cell lines (MDA-MB-231, MDA-MB-436, MCF7, CAL 51, U2OS, U2OS-GFPRNF168) using the ScanR system. Cellular level of selected ubiquitinproteasome and DDR pathways related proteins (53BP1, RNF168, RNF8, UBC13) was detected by standard Western blot using ImageJ for band quantification. The abundance of RNF168 was verified by quantitative PCR using a Nano LightCycler instrument. For probing of the DNA repair pathway choice the Traffic light system with subsequent flow cytometry analysis was employed.

Results and conclusions

We show that central to the proteotoxic stress resistant phenotype is elevated level of the E3 ubiquitin ligase RNF168 that enables more efficient exploitation of the scarce free ubiquitin resources. Elevated RNF168 levels harbouring cells are more resistant to combined treatment by gamma irradiation and proteasome inhibitor. Moreover, the overabundant RNF168 E3 ligase causes a boost in 53BP1 recruitment thus shifting the repair pathway balance towards the nonhomologous end-joining (NHEJ). Importantly, this imbalance might account for increased sensitivity of particular HR proficient breast cancer lines towards PARP1 and topoisomerase inhibitors. As tumors often display heterogeneity in RNF168 expression, upregulation of the RNF168/53BP1 pathway could provide a useful biomarker for assessment of tumor sensitivity to PARP1 and topoisomerase inhibitors.