A20/TNFAIP3 Regulates the DNA Damage Response and Mediates Tumor Cell Resistance to DNA-Damaging Therapy

Abstract

A competent DNA damage response (DDR) helps prevent cancer, but once cancer has arisen, DDR can blunt the efficacy of chemotherapy and radiotherapy that cause lethal DNA breakage in cancer cells. Thus, blocking DDR may improve the efficacy of these modalities. Here, we report a new DDR mechanism that interfaces with inflammatory signaling and might be blocked to improve anticancer outcomes. Specifically, we report that the ubiquitin-editing enzyme A20/TNFAIP3 binds and inhibits the E3 ubiquitin ligase RNF168, which is responsible for regulating histone H2A turnover critical for proper DNA repair. A20 induced after DNA damage disrupted RNF168-H2A interaction in a manner independent of its enzymatic activity. Furthermore, it inhibited accumulation of RNF168 and downstream repair protein 53BP1 during DNA repair. A20 was also required for disassembly of RNF168 and 53BP1 from damage sites after repair. Conversely, A20 deletion increased the efficiency of error-prone nonhomologous DNA end-joining and decreased error-free DNA homologous recombination, destablizing the genome and increasing sensitivity to DNA damage. In clinical specimens of invasive breast carcinoma, A20 was widely overexpressed, consistent with its candidacy as a therapeutic target. Taken together, our findings suggest that A20 is critical for proper functioning of the DDR in cancer cells and it establishes a new link between this NFκB-regulated ubiquitin-editing enzyme and the DDR pathway. This study identifies the ubiquitin-editing enzyme A20 as a key factor in mediating cancer cell resistance to DNA-damaging therapy, with implications for blocking its function to leverage the efficacy of chemotherapy and radiotherapy. .

Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase–dependent manner and regulates the DNA damage response

“…Chromatin extraction was performed as previously described (49). The cells were lysed in 100 l of chromatin extraction buffer A (10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl 2 , 1 mM EGTA, 0.2% Triton X-100, pH 6.8) on ice.…”

“…His-tagged proteins were also expressed in E. coli BL21 cells and purified using a Ni 2ϩ -Sepharose affinity column (GE Healthcare). In vitro pulldown assays were performed as described previously (49).…”

“…The cells were first transfected with scrambled siRNA or RPL6 siRNA followed by NHEJ or HR reporters, after which the analyses were carried out following the procedures described previously (49).…”

The function and regulation of OTU deubiquitinases

“…The zinc finger domain of A20 targets TRAF2 for lysosomal degradation [98]. Yang et al found that A20 regulates the DNA damage response and mediates tumor cell resistance to DNA damage therapy [99].…”

The translational science of hodgkin lymphoma

“…TNFAIP3 mutations were also identified in this study at a higher frequency than prior reports (60% in NSHL) (Reichel et al , ). TNFAIP3 encodes an ubiquitin‐converting enzyme of the NF‐κB pathway, and loss of function mutations was recently linked to lower efficacy of chemotherapy (Yang et al , ). By whole exome sequencing, Tiacci et al () found that over 80% of patient samples carry mutations in the JAK/STAT pathway.…”