Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin

Abstract

Evasion of apoptosis allows many cancers to resist chemotherapy. Apoptosis is mediated by the serial activation of caspase family proteins. These proteases are often activated upon the release of cytochrome c from the mitochondria, which is promoted by the proapoptotic Bcl-2 family protein, Bax. This function of Bax is enhanced by the MOAP-1 (modulator of apoptosis protein 1) protein in response to DNA damage. Previously, we reported that MOAP-1 is targeted for ubiquitylation and degradation by the APC/C^Cdh1 ubiquitin ligase. In this study, we identify the HECT (homologous to the E6-AP carboxyl terminus) family E3 ubiquitin ligase, UBR5, as a novel ubiquitin ligase for MOAP-1. We demonstrate that UBR5 interacts physically with MOAP-1, ubiquitylates MOAP-1 in vitro and inhibits MOAP-1 stability in cultured cells. In addition, we show that Dyrk2 kinase, a reported UBR5 interactor, cooperates with UBR5 in mediating MOAP-1 ubiquitylation. Importantly, we found that cisplatin-resistant ovarian cancer cell lines exhibit lower levels of MOAP-1 accumulation than their sensitive counterparts upon cisplatin treatment, consistent with the previously reported role of MOAP-1 in modulating cisplatin-induced apoptosis. Accordingly, UBR5 knockdown increased MOAP-1 expression, enhanced Bax activation and sensitized otherwise resistant cells to cisplatin-induced apoptosis. Furthermore, UBR5 expression was higher in ovarian cancers from cisplatin-resistant patients than from cisplatin-responsive patients. These results show that UBR5 downregulates proapoptotic MOAP-1 and suggest that UBR5 can confer cisplatin resistance in ovarian cancer. Thus UBR5 may be an attractive therapeutic target for ovarian cancer treatment.

Figure 1

UBR5 is identified as a novel interacting factor of MOAP-1. (a) Flag-MOAP-1 was transfected into 293T cells, treated with or without 100  μM of etoposide (ETP) for 18 or 24 h and lysates were prepared for co-immunoprecipitation (Co-IP) with Flag M2 agarose beads. Co-IP samples were applied for SDS–PAGE and proteins were visualized by silver staining (top). Whole-cell lysates were immunoblotted with Flag antibody for Flag-MOAP-1 (bottom). n=2 independent experiments. (b) Transfection was performed as in panel (a), and Co-IP samples with Flag beads were immunoblotted as indicated. n=3 independent experiments. (c) GST-MOAP-1 recombinant protein was incubated with or without His-UBR5 recombinant protein on ice for 4 h, and nickel beads were added and incubated for 45 min. Beads were washed with 0.5% TritonX-100 wash buffer for five times. The proteins were immunoblotted with UBR5 or MOAP-1 antibody. n=3 independent experiments. Molecular weight markers are in kDa.

Figure 2

UBR5 regulates MOAP-1 protein stability by ubiquitylation. (a) 293T cells were transfected with control or UBR5-targeting siRNA (siCtrl, siUBR5_A or siUBR5_B). Forty-eight hours posttransfection, lysates were prepared and immunoblotted as indicated. n=3 independent experiments. (b) 293T cells transfected with siCtrl or siUBR5_A were treated with cycloheximide (CHX) 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (top). MOAP-1 protein level was quantified and plotted (bottom). The MOAP-1 abundance at 0 time point was set at 100%. n=3 independent experiments (means±s.e.m.). (c) siCtrl, siUBR5_A or siUBR5_B was transfected into 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h posttransfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n=3 independent experiments. (d) MBP-MOAP-1 recombinant protein was incubated with or without UBR5 recombinant protein in the presence of E1, E2 (UbcH5b), ubiquitin and ATP at 30 °C for 1 h. Active wild-type (wt) or catalytically inactive mutant (C2768A) of UBR5 recombinant protein was used. Reaction was immunoblotted with MOAP-1 antibody. n=3 independent experiments. Original uncropped image is shown in Supplementary Figure S6. (e) HeLa cells transfected with siCtrl or siUBR5_A were synchronized with double thymidine and released from G1/S phase. Cells were collected at the indicated time points after the release and lysates were immunoblotted with antibodies as indicated. n=2 independent experiments. Cell cycle data from the same experiment is shown in Supplementary Figure S2B. Molecular weight markers are in kDa.

Figure 3

UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. (a) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n=3 independent experiments. (b) In vitro ubiquitylation assay was performed as Figure 2d in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n=3 independent experiments. Original uncropped image is shown in Supplementary Figure S6. (c) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n=3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in Supplementary Figure S3B. (d) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n=4 independent experiments (means±s.e.m.). Molecular weight markers are in kDa.

Figure 4

UBR5 knockdown enhances apoptosis dependent on MOAP-1. (a) A2780 cells were transfected with siCtrl, siUBR5, siMOAP-1 or siUBR5 and siMOAP-1. Cell lysates were prepared 48 h posttransfection and immunoblotted as indicated. n=3 independent experiments. (b) A2780 cells were prepared as described in panel (a) and treated with or without 10 μM of cisplatin for 48 h and stained with Alexa Fluor 488-conjugated Annexin V for flow cytometric analysis. Percentage of apoptotic cells is indicated as Annexin V-positive cells. n=4 independent experiments (means+s.e.m.). *P<0.05 by unpaired two-tailed t-test. (c) A2780 cells were transfected as in panel (a) and treated with or without 10 μM of cisplatin for 12 h. Cell lysates were used for IP with active Bax-specific antibody (6A7). IP samples were immunoblotted with total Bax antibody. n=3 independent experiments.

Figure 5

UBR5–MOAP-1 axis contributes to cisplatin resistance in ovarian cancer. (a) A2780 or A2780CIS cells were treated for 24 h with the indicated concentration of cisplatin (cisPt). Tyknu or Tyknu cisR cells were treated for 6 h with 10 μM of cisplatin. Cell lysates were prepared and immunoblotted as indicated. n=3 independent experiments. (b) Pairs of sensitive and resistant of A2780 and Tyknu cells were transfected with siCtrl or siUBR5; lysates were prepared 48 h posttransfection and immunoblotted as indicated. n=2 independent experiments. (c) Cells prepared as described in panel (b) were treated with 10 μM of cisplatin for 48 h. Cells were stained with Alexa Fluor 488-conjugated Annexin V and analyzed by flow cytometry. n=4 independent experiments (means+s.e.m.). *P<0.005 (A2780 pair) and P<0.05 (Tyknu pair) by unpaired two-tailed t-test. (d) Ovarian cancer tissue extracts from treatment-naive patients with high-grade serious ovarian cancer (from 11 or 9 patients, cisplatin-responsive or cisplatin-resistant group, respectively) were prepared as described in Materials and Methods section and immunoblotted as indicated. Additional information of ovarian cancer patients is shown in Supplementary Figure S5B. (e) Quantification results of panel (d). Band intensities were calculated by the ImageJ software and normalized based on Coomassie Brilliant Blue (CBB). The band intensities were plotted as means±s.d. *P-values by unpaired two-tailed t-test. (f) Representative images of immunohistochemical staining of UBR5 and MOAP-1 in tumor samples. Formalin-fixed, paraffin-embedded tissues were stained as described in Materials and Methods section.