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Identifying and characterizing novel genetic risk factors for BRCA1/2 negative breast cancers is highly relevant for early diagnosis and development of a management plan. Mutations in a number of DNA repair genes have been associated with genomic instability and development of breast and various other cancers. Whole exome sequencing efforts by 2 groups have led to the discovery in distinct populations of multiple breast cancer susceptibility mutations in RECQL, a gene that encodes a DNA helicase involved in homologous recombination repair and response to replication stress. RECQL pathogenic mutations were identified that truncated or disrupted the RECQL protein or introduced missense mutations in its helicase domain. RECQL mutations may serve as a useful biomarker for breast cancer. Targeting RECQL associated tumors with novel DNA repair inhibitors may provide a new strategy for anti-cancer therapy.
Breast cancer is one of the most prevalent cancers in women, irrespective of their ethnicity or race. Identification of pre-disposing mutations in both women and men having a family history of breast cancer can significantly affect early diagnosis and follow-up recommendations in these high-risk individuals. Only ~27% of familial breast cancers have been designated to genes with rare mutations that have high or moderate penetrance,1 leaving more than 70% of cases in which the novel cancer-causing genetic mutation is yet to be identified. Mutations and deletions in BRCA1 and BRCA2 are the most common in breast cancers and exhibit very high penetrance. Other genes associated with high penetrance for familial breast cancer are PTEN, TP53, CDH1, and STK11, whereas genes such as CHEK2, BRIP1, ATM, and PALB2 display only moderate penetrance. Identification of canonical genes with high penetrance is of great translational importance because their inheritance increases a woman's risk to develop breast cancer in her lifetime by 80%.2,3
Increasing efforts are being made to identify novel genetic mutations that predispose individuals to malignant tumors, including breast cancer. Candidate genes, such as those that encode proteins involved in the DNA damage response or DNA repair, have been identified by high throughput next generation sequencing and are being evaluated to determine their importance in cancer risk. Within the last month, Cybulski et al.4 and Sun et al.5 independently performed whole exome sequencing in breast cancer patients from distinct population groups and provided convincing evidence that mutations in the gene encoding the RECQL DNA helicase, a member of the conserved RecQ family of DNA helicases implicated in the maintenance of genomic stability, are associated with breast cancer susceptibility.
In a screen of 144 Polish and 51 French-Canadian women with early onset of familial breast cancer, 2.6% carried truncating mutations in RECQL.4 Analysis of an expanded group of 950 Polish and French Canadian breast cancer patients negative for BRCA1/BRCA2 founder mutations revealed 19 individuals with RECQL mutations. For validation, a recurrent RECQL breast cancer associated mutation (c.1667_1667+3delAGTA) was screened in over 13,000 breast cancer patients and 4,702 cancer-free individuals of Polish descent. The RECQL mutation appeared in 30 patients (0.23%) and only 2 controls (0.04%). Sequencing the RECQ1 mRNA from patients with this mutation revealed a 27 nucleotide net addition to the mRNA resulting in deletion of a lysine and addition of 10 amino acids that displaced a conserved β-hairpin, previously shown to be essential for RECQL helicase activity.6 The RECQL truncating mutation c.634C>T;p.Arg215* that appeared twice in the French population was further screened in 538 patients and 7,136 newborn controls of French-Canadian descent and it was detected in 5 patients and one control, a 50-fold elevated frequency in affected versus unaffected individuals.
In an independent study of residents from the northern region of China, RECQL was narrowed down as a potential breast cancer associated gene from whole exome sequencing in 9 patients with familial breast cancer but without the canonical BRCA1/2 mutations.5 A sequencing analysis of an additional 439 breast cancer patients revealed 9 RECQL germline mutations predicted to be pathogenic which included 3 nonsense mutations, one helicase domain-disruptive splice-site mutation, and 5 missense mutations in which the corresponding purified recombinant RECQL proteins were defective in helicase activity. Altogether, the pathogenic RECQL mutation frequency in the 448 familial breast cancer patients was 2.0% compared to 0.06% in 1,588 controls. In five cases analyzed, no loss of heterozygosity was observed in the RECQL mutation carriers, suggesting that RECQL haploinsufficiency contributes to familial breast cancer development.
RECQL joins a growing list of genes encoding DNA damage response and DNA repair proteins in which mutation predisposes individuals to familial breast cancer (Table 1).7 Many of these proteins are implicated in homologous recombination (HR) repair of double strand breaks (DSB) that can result directly from either ionizing radiation (IR) or clastogenic chemicals, or are a consequence of broken replication forks that arise when the fork stalls due to bulky or helix-distorting DNA damage lesions or nucleotide starvation. Several functional phases of the DNA damage response and HR repair pathway (that are not mutually exclusive of one another) stand out from inspection of the proteins encoded by genes mutated in breast cancer: 1) DSB recognition (BRCA18); 2) DNA end processing (MRE11, RAD50, NBS19,10); 3) strand invasion (BRCA1, BRCA2,8 PALB211,12); 4) ATP-dependent DNA unwinding (FANCJ,13 RECQL14,15); 5) signal transduction (ATM,16 CHK217); 6) regulation of gene expression and HR branch-migration (p5318,19). Recessive allelic mutations of genes in which monoallelic germline mutations predispose individuals to breast cancer are genetically linked to chromosomal instability disorders including Fanconi Anemia (FA), Ataxia-Telangiectasia, Nijmegan Breakage syndrome, and Li-Fraumeni syndrome (Table 1). Interestingly, mutations in a number of genes suspected to increase susceptibility to breast cancer encode DNA repair proteins: RAD51 paralogs,20 FANCC which is linked to FA,21 or the RecQ DNA helicases RECQL522 and BLM,21 the latter defective in Bloom's syndrome.23 Thus, it is evident that breast-cancer predisposing mutations are commonly found in genes whose protein products are responsible for maintenance of genomic stability.
Previously, RECQL polymorphisms were associated with the survival of patients with pancreatic cancer.24,25 Upon discovery that RECQL mutations predispose individuals to breast cancer,4,5 it is an even greater priority to determine the precise molecular roles of RECQL. It is highly probable that RECQL functions with some of the proteins listed in Table 1 and other HR proteins to prevent the accumulation of DNA damage or repair DSBs that are incurred at stalled replication forks. Interestingly, RAD51-mediated replication fork reversal induced by sublethal genotoxic treatment with agents that interfere with the normal progression of replication is antagonized by poly (ADP-ribose) polymerase (PARP)/RECQL-regulated restart,26 leading us to speculate that the direct interaction between RECQL and RAD5127 may affect their cross-talk at stalled replication forks. RECQL was also found to physically and functionally interact with mismatch repair factors that are implicated in cancer suppression,28 which is likely to be important for the mechanism(s) whereby RECQL regulates genetic recombination to suppress DNA damage and chromosomal instability.27,29
The clinical spectrum of mutations predisposing individuals to cancer can be informative. Indeed, a number of missense mutations in DNA helicase genes are genetically linked to hereditary disorders or associated with various types of cancer.30 Five breast cancer associated missense mutations identified in RECQL from the ethnic Chinese Han were found to reside at highly conserved residues in the helicase core or RecQ C-terminal (RQC) domains of RECQ1 that are implicated in strand separation, ATP hydrolysis, dimer formation, or protein stability.5 Of these, the researchers determined that 4 RECQL missense mutations completely abolished helicase activity and the other greatly reduced helicase activity on a 19-bp forked duplex DNA substrate when incubated with the corresponding purified recombinant RECQL proteins under multi-turnover conditions in vitro. Three RECQL missense mutations identified in the breast cancer patients did not apparently affect helicase activity; however, it is conceivable that under more stringent conditions there may be detectable effects on DNA unwinding, other RECQL catalytic functions (strand annealing, branch-migration), or RECQL protein interactions. It will be informative to determine the precise molecular defects of the RECQL missense mutant proteins by state-of-the-art technologies. A powerful single-molecule approach that combines optical tweezers and fluorescence microscopy31 allows a physical analysis of catalytic unwinding function that would be informative for the characterization of clinically relevant helicase missense mutations in the gene encoding RECQL, an enzyme that is capable of assuming multiple ATP-dependent conformational states.32,33 Understanding how the breast cancer associated RECQL mutations affect the protein's strand annealing and branch-migration activities,33,34 as well as its DNA unwinding, possibly by altering its oligomerization or ligand binding properties,35 will help to identify the critical functions of RECQL necessary for its biological activity in vivo. For example, the dynamic interplay of RECQL duplex separation and rezipping of unwound strands is likely to aid in the remodeling of stalled forks in human cancer cells exposed to the topoisomerase inhibitor camptothecin.36
In an insightful commentary by Ellis and Offit,37 the question is raised: how much risk in cancer susceptibility is increased by rare alleles in DNA repair genes that are thought a priori to have a function in pathway(s) related to cancer? Statistical significance was achieved in the 2 RECQL breast cancer association studies;4,5 however, translation of this information to make useful predictions for families at risk for hereditary breast cancer will require additional epidemiological studies. The degree of penetrance of breast cancer associated RECQL mutations should be addressed as well as the requirement for additional mutated alleles in order for certain RECQL alleles to be clinically relevant. Clinical heterogeneity is likely attributed to genetic background and environmental factors, as well as the nature of the RECQL mutant allele (e.g., truncating vs. missense, or degree of inactivation by the missense allele). Further studies to characterize genotype-phenotype relationships for RECQL mutations are warranted.
Although a number of reports have suggested a causal relationship between a polymorphism in a given DNA repair gene and its effect on DNA repair capacity and cancer risk, this field remains controversial, as articulated in a commentary by Clarkson and Wood.38 In this regard, a combination of RECQL genetic complementation assays with isogenic cell lines expressing breast cancer associated RECQL mutants versus normal unmutated RECQL, in combination with detailed biochemical characterization of the corresponding purified recombinant RECQL proteins, will be informative. Potential effects of RECQL mutations on RECQL protein stability as well as partial loss of function effects should be thoroughly characterized.
RECQL mutations may serve as a biomarker for cancer of the breast or other tissues and their sensitivity to anticancer therapies, which would provide a window for personalized medicine to eradicate tumor cells. The principal of synthetic lethality may apply in which a pre-existing DNA repair deficiency due to RECQL mutation provides the opportunity to treat a tumor with an inhibitor of a compensatory DNA repair pathway. The synthetic lethality approach was pioneered by the discovery that BRCA1/2-deficient tumors defective in HR are hypersensitive to PARP inhibitors.39,40 Advances in this area continue as recently evidenced by the demonstration that HR-deficient tumors are hypersensitive to inhibitors of DNA polymerase POLQ implicated in an error-prone microhomology end-joining pathway.41 Assessment of novel DNA repair inhibitors for their effects on RECQL-associated tumors may provide new avenues for the treatment of human cancer.
No potential conflicts of interest were disclosed.
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging.