The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

Abstract

Renal cell carcinoma (RCC) is a kind of malignant tumor originating from the renal tubular epithelium. Approximately 30% of patients with renal cancer are found to have metastasis when first diagnosed. Exploring other effective treatment methods in addition to surgery is an urgent need in the research field of renal cell carcinoma. Polybromo 1 (PBRM1) is the second most mutated gene in RCC, with a mutation rate of ~40%. Notably, the posttranscriptional modification of PBRM1 in RCC is unclear. In this study, we performed unbiased mass spectrometry of PBRM1 and identified ubiquitin-protein ligase E3A (UBE3A), an extensively studied E3 ligase that can bind with PBRM1 and regulate the stability of PBRM1 in renal cancer cells. We further found that RBPJ/DAPK3 modulated the E3 ligase activity of UBE3A by interfering with the PKA phosphorylation of UBE3A. Finally, we demonstrated that the RBPJ/DAPK3/UBE3A/PBRM1/p21 axis contributed to the sensitivity of renal cancer cells to CDK4/6 inhibitors. In addition, in combination with RBPJ inhibitors, CDK4/6 inhibitors showed synergistically enhanced effects on renal cancer cells. In summary, we not only revealed a novel RBPJ/DAPK3/UBE3A/PBRM1/p21 signaling axis but also identified a combination strategy for overcoming the resistance of renal cancer cells to CDK4/6 inhibitors.

Fig. 1. UBE3A binds PBRM1 to decrease the PBRM1 protein level in renal cancer cells.

a–c Using the PBRM1 and UBE3A antibodies to performed the IP assay. Western blotting analysis the whole-cell lysates (WCL) of 293T (a), 786-O (b), and ACHN (c) cells. d Western blotting analysis of UBE3A proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-PBRM1 recombinant proteins. Asterisks indicated the corresponding protein band of GST-EV and GST-PBRM1. e A schematic diagram depicting a set of GST-UBE3A recombinant protein constructs. f Western blotting analysis of PBRM1 proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-UBE3A recombinant proteins. Asterisks indicated the corresponding protein band of GST-EV and GST-UBE3A recombinant proteins. g, h 786-O and ACHN cells were infected with indicates shRNAs for 72 h. Cells were harvested for western blotting analysis (g) and RT-qPCR assay (h). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as mean ± SEM with three replicates (n = 3). ns not significant. i–k IHC analysis of the tissue microarray with a cohort of patients with renal cell carcinoma by using the UBE3A and PBRM1 antibodies. The typical images of IHC were shown in (i). Heatmap showing the IHC score of PBRM1 and UBE3A in (j). Correlation analysis of the IHC score of PBRM1 and UBE3A proteins in (k).

Fig. 2. UBE3A promotes PBRM1 degradation in renal cancer cells.

a 786-O cells were transfected with indicated plasmids for 24 h. Before harvested for western blotting analysis, cells were treated with or without 20 µM MG132 for 6 h. b 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. c 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. d 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with cycloheximide (CHX), and cells were collected for western blot analysis at different timepoints. e 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. f 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h. g 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for Western blotting after treatment with MG132 for 8 h. h 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Fig. 3. DAPK3 as a binding partner regulates PBRM1 stability in renal cancer cells.

a A schematic diagram depicted that UBE3A contained a consensus DAPK phosphorylation motif which was adjacent to the PKA phosphorylation site. b Western blotting analysis the whole-cell lysates (WCL) of 293T cells. c Western blotting analysis the WCL 786-O and ACHN cells. d Western blotting analysis of UBE3A proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-DAPK3 recombinant proteins. e Western blotting analysis of DAPK3 proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-UBE3A recombinant proteins. f, g 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for Western blotting analysis (f) and RT-qPCR analysis (g). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). ns not significant. h, i 786-O cells were transfected with indicated shRNAs. Seventy-two hours post infection, cells were harvested for western blotting analysis (h) and RT-qPCR analysis (i). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). ns not significant. j 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. k 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. The GAPDH was recognized as the loading control. The protein level of PBRM1 was first normalized to loading control. The normalized values were further normalized to the values in 0 h group. Immunoblots (IB) are representative of results from two independent experiments (n = 2). Statistical significance was determined by multiple student’s t-test at the time point of 5, 10, 15 h. Data presented as Mean ± SEM with two replicates. Ns not significant; ***P < 0.001. Data presented as Mean ± SEM with two replicates. Ns not significant; ***P < 0.001. l 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Fig. 4. DAPK3 competes with PKA to regulate the E3 ligase activity of UBE3A.

a 786-O and ACHN cells were infected with indicated shRNAs. Seventy-two hours post infection, cells were harvested for western blotting analysis. b 786-O and ACHN cells were infected with shControl and shUBE3A for 48 h. Then, 786-O and ACHN cells were transfected with indicated plasmids for another 24 h. Cells were harvested for western blotting analysis. c 786-O cells were transfected with indicated plasmids for 24 h. The WCL of cells were subjected to western blotting analysis. d 786-O cells were transfected with indicated plasmids for 24 h. The WCL of cells were subjected to western blotting analysis. e 786-O cells were treated with 0, 1 µM, 5 µM, and 10 µM HS38 (the DAPK3 inhibitor) for 24 h. The WCL of cells were subjected to western blotting analysis. f 786-O cells were transfected with indicated plasmids for 24 h. The WCL of cells were subjected to western blotting analysis. g 786-O cells were infected with shControl and shDAPK3 for 48 h. Then, 786-O cells were transfected with indicated plasmids for another 24 h. Cells were harvested for western blotting analysis. h 786-O cells were transfected with indicated plasmids for 24 h. The WCL of cells were subjected to western blotting analysis. i 786-O cells were transfected with indicated plasmids for 24 h. The WCL of cells were subjected to western blotting analysis. j 786-O were infected with shControl and shDAPK3 for 72 h. The WCL of cells were subjected to western blotting analysis. k 786-O cells were transfected with indicated plasmids for 24 h. The WCL of cells were subjected to western blotting analysis. l Western blotting analysis of in vitro expressed DAPK3 and PKA GST-pulled down by the C-terminus of UBE3A.

Fig. 5. RBPJ transcriptionally increases DAPK3 expression in renal cancer cells.

a Bioinformatic analysis of the potential binding proteins of the promoter of DAPK3 via analysis the ChIP-seq datasets. b, c 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were harvested for western blotting analysis (b) and RT-qPCR analysis (c). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). Ns not significant; **P < 0.01; ***P < 0.001. d, e 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting analysis (d) and RT-qPCR analysis (e). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01. f, g 786-O cells were treated with 0, 1 µM, 5 µM, and 10 µM the RBPJ inhibitors (the DAPK3 inhibitor) for 24 h. The WCL of cells were subjected to western blotting analysis (f) and RT-qPCR analysis (g). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). Ns not significant; ***P < 0.001. h the ChIP-seq of RBPJ on the promoter region of DAPK3. i 786-O cells were infected with indicated shRNAs for 72 h. Cells were harvested for ChIP-qPCR analysis by using the IgG or RBPJ antibodies. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. j 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for ChIP-qPCR analysis by using the IgG or RBPJ antibodies. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. k The correlation between RBPJ and DAPK3 were analyzed by the GEPIA web tool (http://gepia.cancer-pku.cn/) in different types of cancer. l 786-O cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting analysis. m 786-O cells were infected with indicated shRNAs for 48 h. Then, cells were treated with or without the 5 µM RBPJ inhibitor for another 24 h. Cells were harvested for western blotting analysis.

Fig. 6. RBPJ/DAPK3/UBE3A/PBRM1/p21 contributes to the resistance of renal cancer cells to CDK4/6 inhibitors.

a analysis of the protein levels of UBE3A in the TMA of patients with renal cell carcinoma. N = 40; ***P < 0.001. b, c 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for RT-qPCR analysis (b) and MTS assay (c). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. d, e 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were harvested for RT-qPCR analysis (d) and MTS assay (e). Statistical significance was determined by Student’s t-test. Data presented as Mean ± SEM with three replicates (n = 3). ***P < 0.001. f The bioinformatics analysis indicated that UBE3A is positively correlate with cell cycle in renal cancer specimens through analyzing the TCGA dataset. g, h 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for the MTS assay (g) and colony formation assay (h) treated with or without palbociclib (20 µM). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. i–k 786-O cells were infected with indicated shRNAs for 72 h. After puromycin selection, cells were harvested and injected subcutaneously into nude mice for xenografts assay and treated with or without palbociclib. The image of tumor was shown in panel i. The tumor mass was demonstrated in panel j. The tumor growth curve was indicated in panel k. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with five replicates (n = 5). **P < 0.01; ***P < 0.001. l 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were collected for the MTS assay after treated with indicated concentration of palbociclib for another 24 h. Statistical significance was determined by Student’s t-test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. m 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were collected for the MTS assay after treated with indicated concentration of palbociclib for another 24 h. Statistical significance was determined by Student’s t-test. Data presented as Mean ± SEM with three replicates (n = 3). Ns not significant; *P < 0.05; ***P < 0.001. n–p 786-O cells were treated with indicated small inhibitors. Cells were subjected to MTS assay (n) and Xenografts assay (o, p). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For MTS assay, data presented as Mean ± SEM with three replicates (n = 3). For xenografts assay, data presented as Mean ± SEM with five replicates (n = 5). **P < 0.01; ***P < 0.001.

Fig. 7. A hypothesis model depicted that PKA phosphorylated UBE3A to prevent UBE3A degrading PBRM1.

DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.