Wwox–Brca1 interaction: role in DNA repair pathway choice

Abstract

In this study, loss of expression of the fragile site encoded Wwox protein, was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. WWOX gene deletions occur in a variety of human cancer types and reduced Wwox protein expression can be detected early during cancer development. We find that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). Cancer cells that survive radiation recur more rapidly in a xenograft model of irradiated breast cancer cells; Wwox-deficient cells exhibited significantly shorter tumor latencies vs Wwox-expressing cells. This Wwox effect has important consequences in human disease: in a cohort of cancer patients treated with radiation, Wwox-deficiency significantly correlated with shorter overall survival times. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR), and decreased non-homologous end-joining repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. Upon silencing of Rad51, a protein critical for HDR, Wwox-deficient cells were resensitized to radiation. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for Wwox, in which Brca1-Wwox interaction supports non-homologous end-joining as the dominant DSB repair pathway in Wwox-sufficient cells. Together, the experimental results suggest that reduced Wwox expression, a common occurrence in cancers, dysregulates DSB repair, enhancing efficiency of likely mutagenic repair, and enabling radiation and cisplatin treatment resistance.

WWOX, the FRA16D gene: A target of and a contributor to genomic instability

“…As illustrated in Figure A, normal breast epithelial cells (MCF10A) silenced for WWOX expression by means of shRNA (short hairpin RNA) showed significant resistance to the DNA damaging effects of bleomycin surviving 25‐fold better at 1 and 1.5 μg/mL and 100‐fold better at 3 μg/mL when compared with WWOX expressing controls (shScr). Increased survival of MCF10A WWOX silenced cells was also observed when exposed to IR at 7.7 Gy and above compared to WWOX sufficient cells (Figure B) . We observed similar results in Wwox KO mouse embryonic fibroblasts (MEFs) when compared with wild‐type (WT) counterparts, with Wwox KO cells surviving 10‐fold better than WT cells when exposed to IR doses of 7.7 Gy and above, and such IR resistant phenotype in Wwox KO MEFs was reverted upon ectopic Wwox expression .…”

“…Failure of proper DNA damage response is, therefore, the most common cause of genomic instability in eukaryotic cells . Recently we provided evidence that WWOX is not only a common target affected by genomic instability, as summarized in previous sections, but it also exhibits a genome caretaker function influencing DNA DSBs repair pathway choice . Interestingly, we observed that human and mouse cells deficient in WWOX expression displayed survival advantage following treatment with agents that induce DNA DSBs, such as radiomimetic bleomycin and ionizing radiation (IR).…”

“…Evidence from multiple studies has accumulated suggesting that indeed WWOX behaves as a low‐penetrance tumor suppressor with far more significant roles for affecting tumor progression rather than initiation in specific tumor types . Interestingly, loss of WWOX function has recently been associated with abnormalities in DNA damage repair (DDR), increased genomic instability, and resistance to chemoradiotherapy . In conjunction, the described observations place WWOX both as a target of and a contributor to genomic instability.…”

WWOX Tumor Suppressor Gene in Breast Cancer, a Historical Perspective and Future Directions

“…It was also suggested that WWOX might serve as a genome caretaker, with its function based on Brca1-Wwox interaction in turn supporting NHEJ as the principal DSB repair pathway in Wwox-expressing cells (64). …”

Mechanisms shaping the mutational landscape of the FRA3B/FHIT‐deficient cancer genome

“…It seems more logical that evolution would select against an unstable locus encoding a genome caretaker function, yet the evidence in mice and human cells indicate it has been conserved in mammals. Moreover, another putative DNA caretaker, WWOX, is encoded at the other most fragile CFS, FRA16D . Thus, for unknown reasons, evolution may have allowed on more than one occasion an unstable locus to encode a genome‐stabilizing function.…”