Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress

Abstract

Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.

Defective ribosomal products challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin

“…Nuclear proteostasis maintenance is fundamental for the preservation of genome integrity (Shibata & Morimoto, 2014 In agreement with the literature, proliferating HeLa cells contained nuclear 53BP1 foci that colocalized with ubiquitinated H2A (H2A-Ub; Fig EV5B; Harrigan et al, 2011). As previously shown by independent groups, we also found that the number of these 53BP1 foci strongly decreased upon proteasome inhibition with MG132, alone or combined with OP-puro ( Fig 7A and B), and this, in turn, correlated with a reduction of H2A-Ub levels and an accumulation of polyUb substrates ( Fig EV5C; Mimnaugh et al , 1997;Jacquemont & Taniguchi, 2007;Mailand et al, 2007;Chroma et al, 2016). The number of 53BP1 foci returned to basal levels when proteasomal activity was restored during the recovery phase, indicating that nuclei recovered their ability to recognize and protect fragile chromosomal sites from erosion (Fig 7A and B).…”

“…Our data also identify nuclear protein quality control as a weak link in the proteostasis network, because of the continuous targeting of newly synthesized aberrant proteins to the nucleus for proteasomal degradation. Upon proteasome inhibition, a link between decreased levels of free ubiquitin and defective formation of DNA repair compartments at fragile chromosomal sites was previously reported by independent groups (Mailand et al, 2007;Chroma et al, 2016). Our data identify newly synthesized proteins, including DRiPs, as the main species that upon proteasome inhibition deplete free ubiquitin, thereby compromising DNA damage sensing/repair.…”

RNF168 facilitates proliferation and invasion of esophageal carcinoma, possibly via stabilizing STAT1

“…In several DDR process, RNF168 locates at DNA repair sites and promotes mono‐ubiquitination of H2A/H2AX at K13‐15 to drive DNA repair complex formation . Previous studies have shown that RNF168 could participate in chemotherapy resistance in a group of malignancies . In our analysis, the public cancer database shows RNF168 has gene amplification in oesophageal cancer patients, which tends to correlate with poor overall survival.…”

“…In several DNA damage process, RNF168 is recruited to DNA damage foci and promotes mono‐ubiquitination of H2A/H2AX at K13‐15 to facilitate the formation of DNA repair complex . Previous studies showed that RNF168 could participate in chemotherapy resistance in various type of cancers . Interestingly, RNF168 is highly amplified in oesophageal cancer patients, which lead our interests to dig into the mechanisms.…”

Targeting the unfolded protein response in cancer

“…Altogether, the above studies reflect the emerging evidence that variable expression of the components of the UPR may have important roles in human cancer. These observations can be paired with emerging preclinical studies indicate that UPR is intricately linked to transformation (16), genomic instability (59), tumorigenesis (60), metastasis (61), dormancy and resistance to anti-cancer therapy (62). Further studies are needed in both spheres to determine the context dependent roles of the UPR in such a manner that measurements of UPR components can one day serve as biomarkers for UPR targeted therapy.…”