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

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.

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1.

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-ß interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-ß pathway and implicate this mechanism in the maintenance of genome integrity.

Heterochromatin marks HP1γ, HP1α and H3K9me3, and DNA damage response activation in human testis development and germ cell tumours.

Heterochromatinization has been implicated in fundamental biological and pathological processes including differentiation, senescence, ageing and tumourigenesis; however, little is known about its regulation and roles in human cells and tissues in vivo. Here, we show distinct cell-type- and cancer-stage-associated patterns of key heterochromatin marks: histone H3 trimethylated at lysine 9 (H3K9me3) and heterochromatic adaptor proteins HP1α and HP1γ, compared with the γH2AX marker of endogenously activated DNA damage response (DDR) and proliferation markers in normal human foetal (n=4) and adult (n=29) testes, pre-invasive carcinoma in situ (CIS; n=26) lesions and a series of overt germ cell tumours, including seminomas (n=26), embryonal carcinomas (n=18) and teratomas (n=11). Among striking findings were high levels of HP1γ in foetal gonocytes, CIS and seminomas; enhanced multimarker heterochromatinization without DDR activation in CIS; and enhanced HP1α in teratoma structures with epithelial and neuronal differentiation. Differential expression of the three heterochromatin markers suggests their partly non-overlapping roles, and separation of heterochromatinization from DDR activation highlights distinct responses of germ cells vs. somatic tissues in early tumourigenesis. Conceptually interesting findings were that subsets of human cells in vivo proliferate despite enhanced heterochromatinization, and that cells can strongly express even multiple heterochromatin features in the absence of functional retinoblastoma protein and without DDR activation. Overall, these results provide novel insights into cell-related and tumour-related diversity of heterochromatin in human tissues in vivo, relevant for andrology and intrinsic anti-tumour defence roles attributed to activated DDR and cellular senescence.