During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.

Germ-line mutations in BRCA1 predispose to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify 53BP1 as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Importantly, loss of 53BP1 partially restores the homologous recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the retinoblastoma protein (RB). The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the CDKN2 gene on human chromosome 9p21 (refs 12-14). The frequent deletion or mutation of CDKN2 in tumour cells suggests that p16 acts as a tumour suppressor. We show that wild-type p16 arrests normal diploid cells in late G1, whereas a tumour-associated mutant of p16 does not. Significantly, the ability of p16 to induce cell-cycle arrest is lost in cells lacking functional RB, including primary fibroblasts from Rb-/- mouse embryos. Thus, loss of p16, overexpression of D-cyclins and loss of RB have similar effects on G1 progression, and may represent a common pathway to tumorigenesis.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to such unmet need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (Antabuse), an old alcohol-aversion drug effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify ditiocarb-copper complex as the metabolite of disulfiram responsible for anticancer effects, and provide methods to detect its preferential accumulation in tumours and candidate biomarkers for impact in cells and tissues. Finally, our functional and biophysical analyses reveal the long-sought molecular target of disulfiram’s tumour suppressing effects as NPL4, an adapter of p97/VCP segregase essential for protein turnover involved in multiple regulatory and stress-response cellular pathways.