A neomorphic cancer cell-specific role of MAGE-A4 in trans-lesion synthesis

Abstract

Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape.

Figure 1. MAGE-A4 is a novel component of the RAD18 complex in cancer cells.

(a) Domain organization of full-length RAD18 and RAD18 Δ402–444 (which harbours an internal deletion removing the Polη-binding domain). (b) Spectral counts and estimated probability of true interaction by SAINT analysis for selected proteins identified in HA–RAD18-WT and Control (HA) APMS experiments. (c) Total protein signal intensity versus relative abundance between HA–RAD18-WT and HA–RAD18 Δ402–444 APMS. Signal intensity was normalized to the corresponding experiment's bait intensity (x axis). (d) H1299 cells were infected with adenoviruses encoding WT HA–RAD18, HA–RAD18 Δ402–444 or with an ‘empty' control adenovirus. Infected cells were treated with CPT (2 μM) or UVC (20 J m⁻²). Two hours (h) later, cell extracts were prepared and immunoprecipitated with anti-HA antibody-conjugated magnetic beads. The resulting immune complexes and input fractions were analysed by immunoblotting with anti-HA and anti-MAGE-A4 antibodies. (e) Expression vectors encoding MYC–RAD18, MYC–TRIM69 or green fluorescent protein (GFP) (for control plasmid) were transiently transfected into H1299 cells. Extracts from the resulting cells were immunoprecipitated with an anti-MYC antibody and the resulting immune complexes (or input fractions) were analysed by immunoblotting with antibodies against MAGE-A4 and MYC.

Figure 2. MAGE-A4 associates with the RAD6-binding domain of RAD18.

(a) The indicated RAD18 fragments were expressed as GST fusions in E. coli. The RAD6-binding domain spanning residues 267–402 is highlighted in red. (b) GST–RAD18 fragments were incubated with H1299 cell extracts. After ‘pull-down' with GSH-sepharose beads, the recovered GST–RAD18 fusions and 5% of ‘input' H1299 cell lysate were analysed by immunoblotting with antibodies against GST, MAGE-A4 and RAD6. (c) GST–MAGE-A4 or GST was incubated with extracts from H1299 or 293T cells. After pulldown with GSH-sepharose beads, the recovered GST proteins (and 5% of input cell extract) were analysed by immunoblotting with antibodies against GST and RAD18. (d) Domain organization of full-length RAD18 and the RAD18 Δ340–395 (ΔR6BD) mutant harbouring an internal deletion that removes the RAD6-binding domain. (e) H1299 cells were transiently transfected with expression plasmids encoding HA–RAD18 and HA–RAD18 Δ340–395 (ΔR6BD) or with an empty vector control. Lysates from the resulting cells were immunoprecipitated with anti-HA antibodies. Anti-HA immune complexes and inputs (20 μg) were analysed by immunoblotting with antibodies against RAD18, MAGE-A4 and RAD6. (f) Recombinant GST, GST–RAD18 267–402 or GST–RAD6 were incubated with H1299 cell extracts then pulled down with GSH-sepharose beads. The recovered GST proteins were analysed by immunoblotting with antibodies against MAGE-A4 and GST. (g) Recombinant GST and GST–RAD18 267–402 were incubated with full-length recombinant Hexa-histidine-tagged MAGE-A4 (His-MAGE-A4). GST proteins were recovered using GSH-sepharose beads. Recovered GST proteins (and 5% of input) were analysed by immunoblotting with antibodies against GST and MAGE-A4.

Figure 3. MAGE-A4 promotes RAD18 stability.

(a) Recombinant RAD18–RAD6 complex (0, 0.27, 0.54 and 0.82 μM) was incubated with E1, ubiquitin and purified PCNA. Reaction products were analysed by immunoblotting with antibodies against the indicated proteins. (b) Soluble and chromatin fractions from H1299 cells were analysed by SDS–PAGE (20 μg per lane) and immunoblotting with antibodies against the indicated proteins. (c) H1299 cells were transiently transfected with an expression plasmid encoding CFP-RAD18 (or empty vector for control), ultraviolet irradiated (20 J m⁻²) and processed for immunofluorescence microscopy after 6 h. Scale bar, 10 μm. (d) H1299 and 293T cells were transfected with two independent siRNAs targeting MAGE-A4 or with control non-targeting siRNA oligonucleotides. After 72 h, extracts from the siRNA-transfected cells were analysed by immunoblotting with antibodies against the indicated proteins. (e,f) H1299 cells were transfected with siRNA oligonucleotides against MAGE-A4, RAD18 or control non-targeting siRNA as indicated. Forty eight hours later, cells were treated with cycloheximide (CHX, 100 μg ml⁻¹) and collected at different time points for immunoblot analysis.

Figure 4. MAGE-A4 protects RAD18 from ubiquitin-mediated proteolysis.

(a) Replicate plates of H1299 cells were transfected with siRNA against MAGE-A4, USP7 or with non-targeting control siRNA. After 48 h, one plate of each replicate was treated with 10 μM MG132 for 16 h. Extracts from control and MG132-treated cells were analysed by immunoblotting with antibodies against the indicated proteins. (b) 293T cells were co-transfected with an HA–RAD18 expression vector in combination with a CMV-MAGE-A4 plasmid or an empty vector for control. After 48 h, RAD18 complexes were immunoprecipitated with anti-HA antibodies. The resulting immune complexes were incubated in a rabbit reticulocyte lysate (RRL) to reconstitute ubiquitin-coupled proteolysis in vitro. Relative levels of RAD18 and MAGE-A4 were determined by immunoblotting and quantified using densitometry. (c) H1299 cells were transiently co-transfected with WT or mutant HA–RAD18 expression plasmids in combination with a MAGE-A4 expression vector (or empty vector control). Forty eight hours later, cells were harvested for immunoblot analysis of RAD18 and MAGE-A4. The white arrowhead indicates the RAD18Δ340–395 mutant protein band that is insensitive to MAGE-A4. (d) Replicate cultures of H1299 cells were transfected with an expression vector encoding MYC–TRIM69 or with an empty vector plasmid for control. Sixteen hours later, the cells were transfected with siRNA against MAGE-A4 or with a scrambled control siRNA and incubated for an additional 48 h before immunoblot analysis.

Figure 5. Mutational analyses to define structural requirements for MAGE-A4-induced RAD18 stabilization.

(a) Domain structure of full-length MAGE-A4 and MAGE-A4 mutants used in this study. The MAGE-homology domain (MHD) is conserved between MAGE family members and comprises juxtaposed WH-A and WH-B regions. (b) 293T cells were transiently transfected with expression vectors encoding the MAGE-A4 mutants shown in a or with an empty vector (EV). After 48 h, extracts from the resulting cells were analysed by immunoblotting with anti-Pan-MAGE-A (which recognizes an epitope in the WH-B domain) or with anti-MAGE-A4 (which recognizes a C-terminal epitope of MAGE-A4 in residues 275–317). (c) Replicate plates of 293T cells were transiently transfected with expression vectors encoding WT or mutant forms of MAGE-A4. Forty-eight hours post transfection, cells were treated with cycloheximide (CHX) and then harvested at different times post CHX. Cell extracts were analysed by immunoblotting with antibodies against RAD18, MAGE-A4 and actin. (d) RAD18 levels in each lane of immunoblots in c were quantified by densitometry with ImageJ software. The graph indicates the levels of RAD18 remaining at each time point following CHX treatment in control and MAGE-A4-expressing cells.

Figure 6. Effect of MAGE family members on RAD18 stability.

(a) 293T cells were transfected with expression vectors encoding FLAG-tagged forms of WT MAGE-A4, MAGE-A4 AB (see Fig. 5a), MAGE-A12, MAGE-B10 and MAGE-A1. After 48 h, extracts were prepared from the transfected cells and analysed by immunoblotting with anti-Pan-MAGE and anti-FLAG antibodies. (b) Replicate plates of 293T cells were transiently transfected with expression vectors encoding WT MAGE-A4, MAGE-A4 AB, MAGE-A12, MAGE-B10 and MAGE-A1. Forty-eight hours post transfection, cells were treated with cycloheximide (CHX) and then harvested at different times post CHX. Cell extracts were analysed by immunoblotting with antibodies against RAD18, FLAG and actin. (c) RAD18 levels in each lane of immunoblots in b were quantified by densitometry with ImageJ software. The graph indicates the levels of RAD18 remaining at each time point following CHX treatment in control and MAGE-expressing cells.

Figure 7. MAGE-A4 promotes TLS and DNA-damage tolerance.

(a) H1299 cells were transiently transfected with MAGE-A4 or non-targeting siRNAs. After 16 h, cells were transfected with a siRNA-resistant MAGE-A4 expression plasmid (or empty vector control). Forty-eight hours later, cells were sham or ultraviolet irradiated (20 J m⁻²) and harvested for immunoblot analysis after 2 h. (b) H1299 cells were transfected with siRNA against RAD18, MAGE-A4 or non-targeting siRNA. Forty-eight hours later, cells were pulsed labelled with BrdU (10 μM) for 1 h and collected for flow cytometry. (c) H1299, A549 or mouse embryonic fibroblast (MEF) cells were transfected with a MAGE-A4 expression plasmid or empty vector. After 48 h, cells were sham or ultraviolet irradiated (20 J m⁻²) and extracted 2 h later for immunoblotting. Human dermal fibroblasts (HDFs) stably transduced with a pINDUCER-MAGE-A4 were treated with indicated doxycycline concentrations for 48 h and then collected for immunoblotting. (d) H1299 cells were transfected with siRNA against RAD18 and MAGE-A4 (or with non-targeting oligonucleotides). Twenty-four hours post transfection, cells were re-plated in 24-well dishes and ultraviolet irradiated (5 J m⁻²) 48 h later. DNA synthesis rates were measured immediately before and at different times after ultraviolet treatment. (e) H1299 cells were transfected with siRNA against MAGE-A4 or with non-targeting siRNA. Seventy-two hours post transfection, cells were sham or ultraviolet irradiated (5 J m⁻²) and harvested at different times for immunoblotting. (f) 293T cells were co-transfected with ultraviolet-damaged pSP189 reporter plasmid and MAGE-A4 or RAD18 expression vectors. Forty-eight hours later, 293T cell extracts were collected for immunoblot analysis of MAGE-A4 and RAD18 (right). Recovered pSP189 plasmid was transformed into electro-competent MBM7070 bacteria and pSP189 mutation rates were determined by enumerating blue and white bacterial colonies. Data represent means±s.e.m. of four independent experiments each performed in triplicate. P-values were calculated using a two-tailed Student's t-test. Baseline mutation rates for the experiments ranged from 5.6 to 9.6%.