JNK2 downregulation promotes tumorigenesis and chemoresistance by decreasing p53 stability in bladder cancer

Abstract

Bladder cancer is one of the most common malignancies of the urinary system, and the 5-year survival rate remains low. A comprehensive understanding of the carcinogenesis and progression of bladder cancer is urgently needed to advance treatment. c-Jun N-terminal kinase-2 (JNK2) exhibits both tumor promoter and tumor suppressor actions, depending on tumor type. Here, we analyzed the JNK2 function in bladder cancer. Using gene expression microarrays, we demonstrated that JNK2 mRNA is downregulated in an orthotopic rat model of bladder cancer. JNK2 protein levels were lower in rat and human bladder cancer tissues than in normal tissues, and the levels correlated with those of p53. Moreover, JNK2 phosphorylated p53 at Thr-81, thus protecting p53 from MDM2-induced proteasome degradation. Decreased expression of JNK2 in T24 cells conferred resistance to cell death induced by mitomycin C. Furthermore, lower JNK2 expression was associated with poorer overall survival among patients who underwent radical cystectomy. These results indicate that JNK2 acts as a tumor suppressor in bladder cancer, and that decreased JNK2 expression promotes bladder cancer tumorigenesis.

Keywords: JNK2; bladder cancer; chemoresistance; p53; tumorigenesis.

Figure 1. Both mRNA and protein levels of JNK2 decrease in bladder cancer

a. Representative images of macrography, H&E staining and IHC of anti-JNK2 antibody on tissues of orthotopic rat bladder cancer model. Scale bar represents 100μm. b. Left panel shows changed genes in rat bladder cancer tissues (n=3) compared with normal rat bladder tissues (n=3) analyzed by gene expression microarray (GEM). Right panel shows details of MAPK9 gene and genes around. c. Heat map shows IHC staining index of JNK2 of human bladder tissues. See a detailed scoring method in Material and Methods section. Left panel comes from tissue microarray (TMA) of 44 patients underwent RC (Radical cystectomy). Right panel comes from 16 patients underwent TURBT (Transurethral resection of bladder tumor). ANT: adjacent normal tissue. d. Representative images of H&E staining and IHC of anti-JNK2 antibody on TMA of 44 RC patients. Areas indicated by arrows are blood vessels and connective tissues, not tumor area. Scale bar represents 100μm. e. Box-plot shows IHC staining index of JNK2 on tissues from 44 RC patients and 16 TURBT patients respectively. f. Western blotting analysis of protein from bladder tissues of 7 MIBC patients. Histogram shows ratios of relative quantity of indicated proteins from ANT to tumor tissues. T: tumor tissues. A: adjacent normal tissues.

Figure 2. JNK2 promotes p53 stability and apoptosis activity through phosphorylation of p53 at Thr81

a. The p53 has consensus binding motif with JNK in different species. b. Western blotting detection of co-immunoprecipitated endogenous JNK2 and p53 proteins in T24 cells. c. Histogram shows changes of p53 mRNA between normal and tumor tissues, which are from human and rat bladder tissues respectively. d. Scatter plot of relative grayscale values of JNK2 and p53. Western blotting detected JNK2 and p53 proteins of tumor and paired adjacent normal tissues (ANTs) from 28 MIBC patients, then the grayscale values were measured. R² in tumors and ANTs are 0.163 (p=0.033, ANOVA) and 0.412 (p=0.000, ANOVA) respectively. e. Western Blotting shows JNK2, p53, phosphor-p53 and related apoptotic proteins in T24 cells. Cells were infected with lentivirus expressing indicated shRNAs for 48h, then transfected with indicated plasmids for 24h before harvest. f. Western blot analysis of indicated proteins in T24 cells 24 h after treated with or without 10 μM SP600125.

Figure 3. JNK2 prevents p53 from mdm2 mediated degradation

a. Western blot analysis of whole cell lysate (WCL) and co-IP samples of IgG or anti-MDM2 antibody obtained from T24 cells 24 h after transfected with indicated plasmids. b. Western blot analysis of WCL and co-IP samples of IgG or anti-MDM2 antibody obtained from T24 cells 24 h after treated with or without 10 μM SP600125. c. Western blotting analysis of ubiquitin co-IP samples of anti-p53 antibody in T24 cells 24h after transfected with indicated plasmids. d. Western blotting analysis of ubiquitin co-IP samples of anti-p53 antibody in T24 cells 48h after transfected with indicated siRNAs. e. Western blotting analysis of ubiquitin co-IP samples of anti-p53 antibody in T24 cells 24h after treated with or without 10 μM SP600125. f. Western blot analysis of WCL and co-IP samples of IgG or anti-Flag antibody obtained from T24 cells 24 h after transfected with indicated plasmids. The cells were treated with 20μM MG132 for 6 hours before they were harvested. g. Western blotting analysis of ubiquitin co-IP samples of anti-Flag antibody in T24 cells 24h after transfected with indicated plasmids. The cells were treated with 20μM MG132 for 6 hours before they were harvested.

Figure 4. Decreased expression of JNK2 confers resistance to cell death induced by MMC

(a. and b.) Flow cytometry (FCM) analysis of apoptotic and necrotic T24 cells. Cells were transfected with indicated plasmids (24h) or siRNAs (48h), then treated with or without 100ug/ml mitomycin C (MMC) for 12h before dual-stained by Annexin V-Alexa Fluor 488 and PI solution. Stained cell suspension was analyzed by FCM. *: p<0.001 vs. control group; #: p<0.001 vs. EV+MMC group. (c and d) MTS assay analysis of T24 cells proliferation. Cells were transfected with indicated plasmids (24h) or siRNAs (48h) before treated with or without 100ug/ml MMC for 12h followed by MTS assay. *: p<0.05 vs. Group 1; #: p<0.05 vs. Group 2; &: p<0.05 vs. Group 3. e. MTS assay analysis of 5637 cells proliferation. Cells were transfected with indicated plasmids (24h) or siRNAs (48h) before treated with 100ug/ml MMC for 12h followed by MTS assay. *: p<0.05 vs. JNK2+si-P53+MMC group.

Figure 5. Survival analysis for patients with different expression levels of JNK2

a. Kaplan-Meier analysis of overall survival of patients underwent RC (Radical cystectomy) in different JNK2 conditions. b. Kaplan-Meier analysis of recurrence-free survival of patients underwent TURBT (Transurethral resection of bladder tumor) in different JNK2 conditions. HR=0.516 (95%CI: 0.303-0.930). c. A hypothetical model depicts that JNK2 serves as a tumor suppressor in bladder cancer, and that decreased JNK2 expression promotes bladder tumorigenesis.