Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

Fig. 1. Diminished expression of UBB is observed in ccRCC and adversely impacts the survival of patients.

A Analysis of UBB mRNA expression in TCGA-KIRC and GEO (GSE53000, GSE53757) datasets. B qPCR (top) and western blot (bottom) of UBB expression in the HK2 and RCC cell lines. C IF analysis of UBB expression in HK2 and RCC cell lines. Scale bar, 50 μm. D Western blot of UBB expression in 9 pairs of ccRCC and corresponding paracarcinoma specimens. E qPCR of UBB expression in 60 pairs of ccRCC and corresponding paracarcinoma specimens. F IHC of UBB expression in ccRCC and corresponding paracarcinoma specimens. The data are presented as a representative image. Scale bar, 50 μm. G Overall survival of 30 ccRCC patients according to Kaplan–Meier analysis. Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. UBB hinders the proliferation and invasion of RCC cells and suppresses tumor growth in mice.

A RCC cells proliferation after UBB overexpression was assessed by EdU assay. Scale bar, 50 μm. B RCC cells proliferation after UBB overexpression was assessed by clonogenic assays. C Wound healing assay of RCC cells after UBB overexpression. D Transwell assays were performed to evaluate invasion ability. Scale bar, 50 μm. E Subcutaneous xenografts (n = 5 per group) were excised from nude mice in the control group and UBB overexpression group. Tumor growth is represented by a line chart (top), and mean tumor weights are shown in the histogram (bottom). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. UBB suppressed the angiogenesis of RCC cells in vitro.

A GO biological processes analysis. B Angiogenesis activity after UBB overexpression was assessed by CAM assay. C Representative capillary tubule structures were observed in HUVEC treated with culture medium from the indicated RCC cells (UBB_NC/UBB_OE). Scale bar, 100 μm. D qPCR analysis of cytokines and growth factors in the process of tumor angiogenesis. E Western blot analysis of VEGFA and PGF in RCC cells (UBB_NC/UBB_OE). F ELISA analysis of VEGFA culture medium from the indicated RCC cells (UBB_NC/UBB_OE). qPCR (G), western blot (H), and ELISA (I) of VEGFA from the indicated RCC cells (UBB_NC/UBB_OE and VEGFA_NC/VEGFA_OE). J Representative capillary tubule structures were observed in HUVEC treated with culture medium from the indicated RCC cells (UBB_NC/UBB_OE and VEGFA_NC/VEGFA_OE). Scale bar, 100 μm. Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. UBB interacts with SP1 to promote VEGFA transcription.

A Western blot analysis of the polyubiquitination level and expression level of SP1 in RCC cells (UBB_NC/UBB_OE). B IF analysis of UBB and SP1 in RCC cells (UBB_NC/UBB_OE). Scale bar, 50 μm. C Western blot analysis of KLF4 in RCC cells (UBB_NC/UBB_OE). D Western blot analysis of SP1 expression in MG132-treated RCC cells (786-O UBB_OE). E VEGFA promoter luciferase reporter plasmids and Renilla pRL-TK plasmids were transfected into empty vector-transduced and SP1-transduced RCC cells. Forty-eight hours later, the luciferase signal was examined. F Western blot analysis of SP1 and VEGFA expression in RCC cells (SP1_NC/SP1_sh). G ELISA analysis of VEGFA in culture medium from the indicated RCC cells (SP1_NC/SP1_sh). H Co-IP assay showing that endogenous UBB interacted with endogenous SP1 in 786-O cells. I Flag-tagged SP1 (full length or truncations) and Myc-tagged UBB were transfected into HEK293T cells. Co-IP assays revealed that UBB interacted with FL, F1, and F2 but not with F3. J Representative capillary tubule structures were observed in HUVEC treated with culture medium from the indicated RCC cells (UBB_NC/UBB_OE and SP1_NC/SP1_OE). Scale bar, 100 μm. K Representative results (top) and correlation (bottom) between UBB expression and SP1 expression. Scale bar, 50 μm. Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. Epigenetic silencing maintained by DNMT3A contributes to suppressed transcription of UBB in ccRCC.

A Promoter methylation level analysis of UBB in ccRCC based on data from the UALCAN-ccRCC dataset. B Correlation between UBB expression level and promoter methylation level based on data from the TCGA-KIRC database. qPCR (C) and western blot (D) analysis of UBB expression in RCC cells treated with 5-Aza. E qPCR analysis of UBB mRNA levels in RCC cells transfected with NC, DNMT1_OE, DNMT3A_OE, or DNMT3B_OE. F Western blot of DNMT3A and UBB expression in RCC cells transfected with NC and DNMT3A_OE. G The IGV browser image of H3K27me3 enrichment in the UBB promoter region in ccRCC. H ChIP-qPCR analysis of DNMT3A genomic occupancy and H3K27me3 methylation status at the UBB promoter. I IF analysis of DNMT3A and UBB expression in HK2 and RCC cell lines. Scale bar, 50 μm. Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. UBB/VEGFA ratio affects ccRCC prognosis and pazopanib resistance.

A Kaplan-Meier survival analysis showed the correlation of the UBB/VEGFA ratio with the survival rate of ccRCC patients. B The correlation of risk group with TKI resistance in ccRCC. qPCR (C) and western blot (D) analysis of UBB and VEGFA expression in TKI-resistant RCC cells. Data are presented as the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7. The mechanistic scheme of UBB in ccRCC.

Model showing the antitumor mechanism by which UBB affects angiogenesis via the SP1-VEGFA signaling pathway.