USP43 stabilizes c-Myc to promote glycolysis and metastasis in bladder cancer

Abstract

A hallmark of tumor cells, including bladder cancer (BLCA) cells, is metabolic reprogramming toward aerobic glycolysis (Warburg effect). The classical oncogene MYC, which is crucial in regulating glycolysis, is amplified and activated in BLCA. However, direct targeting of the c-Myc oncoprotein, which regulates glycolytic metabolism, presents great challenges and necessitates the discovery of a more clarified regulatory mechanism to develop selective targeted therapy. In this study, a siRNA library targeting deubiquitinases identified a candidate enzyme named USP43, which may regulate glycolytic metabolism and c-Myc transcriptional activity. Further investigation using functional assays and molecular studies revealed a USP43/c-Myc positive feedback loop that contributes to the progression of BLCA. Moreover, USP43 stabilizes c-Myc by deubiquitinating c-Myc at K148 and K289 primarily through deubiquitinase activity. Additionally, upregulation of USP43 protein in BLCA increased the chance of interaction with c-Myc and interfered with FBXW7 access and degradation of c-Myc. These findings suggest that USP43 is a potential therapeutic target for indirectly targeting glycolytic metabolism and the c-Myc oncoprotein consequently enhancing the efficacy of bladder cancer treatment.

Fig. 1. siRNA screening reveals USP43 as a key deubiquitinase for glycolysis and c-Myc transcriptional activity.

A 293 T cells were transfected with siRNA pools targeting seven deubiquitinase candidates for 24 h, transfected with 5× E-box luciferase reporter for 36 h, and finally subjected to a dual-luciferase reporter assay. NC was the negative control, and MYC RNAi was the positive control (n = 5, one-way ANOVA followed by Tukey’s correction). B USP43 expression levels in bladder cancer and normal tissue at GEPIA (http://gepia.cancer-pku.cn/index.html). |Log₂FC| Cutoff: 0.5, p-value Cutoff: 0.01. C Immunohistochemical detection of USP43 in carcinoma samples paired with adjacent normal tissues in the BLCA tissue microarray. Average optical density values were calculated using ImageJ software (tumor n = 16, adjacent n = 16, paired two-tailed Student’s t-test). D Representative immunohistochemical image of BLCA tissue microarray showing USP43 content in normal adjacent tissues and low-grade and high-grade bladder cancer tissues. E, F GSEA enrichment of TCGA-BLCA samples shows that USP43 is positively related to the glycolysis pathway (E) and MYC target pathway (F). G Dual-luciferase reporter assay in 293 T cells cotransfected with LDHA promoter plasmid and empty vector, c-Myc overexpression plasmid, or c-Myc overexpression plasmid plus USP43 overexpression plasmid (n = 6, one-way ANOVA followed by Tukey’s correction). H, I The media from T24 cells was collected for the analysis of glucose consumption (H) and lactate production (I) (H, I, n = 3, one-way ANOVA followed by Tukey’s correction). J T24 cells were transfected with two different siRNAs against USP43 or with a control siRNA for 48 h. The mRNA levels of USP43, GLUT, HK2, PKM2, and LDHA were detected using qRT-PCR (n = 3, two-way ANOVA test followed by Tukey’s correction). K Dual-luciferase reporter assay in 293 T cells transfected as indicated, and LDHA promoter activity was measured (n = 6, one-way ANOVA test followed by Tukey’s correction). The n number represents n biologically independent experiments in each group. The data are presented as the mean ± SD (bar plots).

Fig. 2. Knockdown of USP43 suppresses BLCA cell metastasis.

A Wound healing assay demonstrated that knockdown of USP43 inhibited the migration ability of T24 cells (n = 4, one-way ANOVA test followed by Tukey’s correction). Migration rate = (wound area (0 h) – wound area (24 h))/wound area (0 h). B Transwell migration assays demonstrated that knockdown of USP43 inhibited the migration ability of T24 cells (n = 5, one-way ANOVA followed by Tukey’s correction). The scale bar is 200 μm. C, D c-Myc and epithelial-mesenchymal transition-related proteins were detected by immunoblot assays following USP43 knockdown (C) and overexpression (D). E The knockdown efficiency of USP43 in T24 stable cell lines was verified by qRT-PCR (left) (n = 3, unpaired two-tailed Student’s t-test) and Western blot analysis (right). F Representative image of the popliteal lymph node metastasis model. G, H Images of dissected popliteal lymph nodes (G) and lymph node volumes (H) in each group (H, n = 5 for BALB/c nude mice, unpaired two-tailed Student’s t-test). I Representative images of the popliteal lymph nodes analyzed by H&E staining and IHC staining using an anti-GFP antibody. The scale bar is 200 μm. J, K Images of lung fluorescence after T24-shNC/T24-shUSP43 cells were injected into the tail veins of NOD/SCID mice for six weeks (J) and quantitation of the fluorescence intensity of lung metastases (K) (J, K, n = 5 for BALB/c nude mice, unpaired two-tailed Student’s t-test). L, M Images of dissected whole lungs (L) and representative H&E-stained lung tissue sections (M). The scale bar is 200 μm. N Kaplan-Meier survival curves were generated for NOD/SCID mice within 60 days after tail vein injection of T24-shNC/T24-shUSP43 cells (n = 5 for BALB/c nude mice, log-rank test). The n number represents n biologically independent experiments in each group. The data are presented as the mean ± SD (bar plots).

Fig. 3. USP43 regulates c-Myc stability.

A After knocking down USP43 in T24 cells, the mRNA level was detected by qRT-PCR (n = 3, two-way ANOVA followed by Tukey’s correction). B 293 T cells were transfected with empty vector or with increasing concentrations of USP43 and c-Myc was detected by subsequent immunoblot analysis. C–F 48 h after USP43 knockdown, UM-UC-3 (C) and 5637 (D) cells were treated with 50 μg/mL CHX and then harvested at the indicated time points. The statistical plot (E, F) represents the intensity of c-Myc bands detected by Western blot analysis (C, D, n = 3). G, H After 48 h of transfection, T24 (G) and 5637 (H) cells were treated with 10 μM MG132 for 6 h before harvest, and then c-Myc was detected by Western blot analysis. The n number represents n biologically independent experiments in each group. The data are presented as the mean ± SD (bar plots).

Fig. 4. USP43 interacts with c-Myc.

A, B Co-IP assay showed that exogenous USP43 interacted with c-Myc in 293 T cells. USP43 and c-Myc were precipitated with the corresponding anti-Flag (A) and anti-HA antibodies (B), respectively. C Recombinant purified GST-tagged c-Myc protein and His-tagged USP43 protein from E. coli were subjected to a GST pull-down assay in vitro. D, E Schematic diagram of c-Myc (D) and USP43 (E) truncations. F, H Flag-USP43 was cotransfected with c-Myc truncation mutants. Interactions were analyzed using the Co-IP assay. G HA-c-Myc was cotransfected with USP43 truncation mutants. Interactions were analyzed using the Co-IP assay. I c-Myc in T24 cell lysates was precipitated by c-Myc antibody. The interaction between endogenous USP43 and c-Myc was examined by Western blot analysis. J USP43 and c-Myc were transfected into UM-UC-3 cells for immunofluorescence assays. Colocalization analysis showed that USP43 and c-Myc colocalized in the nucleus. The scale bar is 10 μm.

Fig. 5. USP43 deubiquitinates c-Myc at K148 and K289.

A 293 T cells were transfected with the indicated siRNAs and plasmids. Cells were treated with 10 μM MG132 for 6 h before harvest, and then ubiquitination experiments were performed to analyze polyubiquitination of c-Myc. B 5637 cells transfected with the indicated siRNA were treated with 10 μM MG132 for 6 h before collection. c-Myc was immunoprecipitated with anti-c-Myc and immunoblotted with anti-ubiquitin. C 293 T cells were transfected with HA-c-Myc, Myc-Ubiquitin and Flag-USP43 (wild-type or C110S). Cells were treated with 10 μM MG132 for 6 h before harvest, and then ubiquitination experiments were performed to analyze polyubiquitination of c-Myc. D Sequence alignment was used to determine the enzyme inactivating mutation site of USP43. E HA-c-Myc was cotransfected with wild-type USP43 or an enzyme inactivating mutant USP43 (C110S) into 293 T cells and subsequent immunoblot analysis. F Schematic diagram of the in vitro ubiquitination experimental procedure. G Deubiquitination of c-Myc in vitro by GFP-USP43. Polyubiquitinated c-Myc was lysed from 293 T cells transfected with HA-c-Myc and Myc-Ubiquitin plasmids and precipitated with anti-HA antibodies. 293 T cells transfected with GFP-USP43 plasmid were lysed and precipitated with anti-GFP antibodies followed by nondenaturing elution to obtain GFP-USP43. Polyubiquitinated c-Myc was incubated with or without GFP-USP43 and then analyzed using IB with anti-Myc antibodies. H 293 T cells were transfected with HA-c-Myc, Myc-Ubiquitin (K48O or K63O) and Flag-USP43. Cells were treated with 10 μM MG132 for 6 h before harvest, and then ubiquitination experiments were performed to analyze polyubiquitination of c-Myc. I HA-tagged wild-type c-Myc and three HA-tagged c-Myc mutants were cotransfected with Flag-USP43 into 293 T cells, and the expression of wild-type c-Myc and c-Myc mutants was detected by an anti-HA antibody. J The effect of USP43 on the ubiquitination level of wild-type c-Myc and three c-Myc mutants was analyzed by deubiquitination assay.

Fig. 6. USP43 antagonizes c-Myc degradation by FBXW7.

A HA-tagged wild-type or mutant c-Myc was cotransfected with Flag-tagged FBXW7 into 293 T cells, and HA-c-Myc was detected using Western blot analysis. B HA-tagged wild-type or mutant c-Myc was cotransfected with Flag-tagged wild-type or mutant USP43 into 293 T cells, and HA-c-Myc was detected using Western blot. C T24 and 5637 cells were transfected with siRNA against USP43 or FBXW7 as indicated. The protein levels were detected by Western blot analysis. D 293 T cells cotransfected with HA-c-Myc and Flag-FBXW7 or Flag-USP43 were lysed, and then the protein level was detected by Western blot analysis. E 293 T cells were transfected as indicated and treated with 10 μM MG132 for 6 h before harvest. The effect of FBXW7 or USP43 on the ubiquitination level of c-Myc was analyzed by ubiquitination assay. F The effect of FBXW7 on HA-tagged wild-type and three mutant c-Myc proteins was examined by Western blot. G The effect of FBXW7 on the ubiquitination level of wild-type c-Myc and three c-Myc mutants was analyzed by ubiquitination assay. H A co-IP assay was used to detect the interaction between the MBI domain of c-Myc and USP43. I Co-IP assay to detect the interaction between c-Myc and FBXW7 in the presence of USP43. 293 T cells were transfected with plasmids expressing c-Myc and FBXW7 and increasing amounts of USP43. J, K 5637 cells were treated with siRNA targeting USP43 or FBXW7 as indicated. Wound healing (J) (n = 3) and transwell migration assays (K) (n = 5) were performed to detect changes in cell migration ability (J, K, one-way ANOVA followed by Tukey’s correction). The scale bar is 200 μm. Migration rate = (wound area (0 h) – wound area (24 h)) / wound area (0 h). The n number represents n biologically independent experiments in each group. The data are presented as the mean ± SD (bar plots).

Fig. 7. USP43 is a direct target of c-Myc.

A The binding sites of c-Myc on promoter sequences obtained from the JASPAR database. B Schematic diagram of the potential sequence of c-Myc at the USP43 promoter binding site and the constructed luciferase plasmids containing wild-type or mutant USP43 promoter sequences. C Dual-luciferase reporter assay in 293 T cells cotransfected with USP43 promoter plasmid (wild-type or mutant) and empty vector or c-Myc overexpression plasmid (n = 5, two-way ANOVA followed by Tukey’s correction). D Schematic diagram of primers designed for ChIP-qPCR on the USP43 promoter sequence. E ChIP-qPCR analysis showed the enrichment degree of c-Myc in different regions of the USP43 promoter. IgG indicates the negative control (n = 3, two-way ANOVA followed by Tukey’s correction). F The c-Myc plasmid was transfected into 293 T cells, and then the mRNA levels were detected by qRT-PCR (n = 3, unpaired two-tailed Student’s t-test). G 5637 cells were transfected with siRNA targeting MYC, and mRNA levels were detected by qRT-PCR (n = 3, two-way ANOVA test followed by Tukey’s correction). H–I 5637 cells were treated with siRNA targeting USP43 3’ UTR followed by reconstitution with wild-type USP43 and a USP43 deubiquitinase inactivating mutant as indicated. Wound healing assays (H) (n = 3) and Transwell migration assays (I) (n = 3) were performed to detect changes in cell migration ability (H, I, one-way ANOVA followed by Tukey’s correction). The scale bar is 200 μm. J, K 5637 cells were treated with siRNA targeting USP43 or c-Myc overexpression plasmid as indicated. Wound healing assays (J) (n = 3) and Transwell migration assays (K) (n = 5) were performed to detect changes in cell migration ability (H, I, one-way ANOVA followed by Tukey’s correction). The scale bar is 200 μm. The n number represents n biologically independent experiments in each group. The data are presented as the mean ± SD (bar plots).

Fig. 8. A simplified schematic diagram showing that the USP43/c-Myc positive feedback loop promoted glycolysis and metastasis in bladder cancer.

USP43 stabilizes c-Myc by deubiquitinating c-Myc at K148 and K289 primarily through deubiquitinase activity. Moreover, upregulation of USP43 protein in BLCA increased the chance of interaction with c-Myc and interfered with FBXW7 access and degradation of c-Myc. In turn, stable c-Myc can function as a transcription factor that activates USP43 transcription. The dysregulation of the USP43/c-Myc positive feedback loop leads to abnormal glycolysis and the accumulation of c-Myc protein, both of which contribute to the malignant behaviors of BLCA.