High VHL Expression Reverses Warburg Phenotype and Enhances Immunogenicity in Kidney Tumor Cells

Abstract

Clear cell renal cell carcinoma (ccRCC) is a frequently occurring renal cancer. The Von Hippel-Lindau disease tumor suppressor VHL, a known tumor suppressor gene, is frequently mutated in about 50% of patients with ccRCC. However, it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL. In the present study, we found that higher expression of VHL was correlated with the better disease-free survival (DFS) in ccRCC patients using The Cancer Genome Atlas (TCGA) datasets. We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition (EMT), sterol regulatory element-binding protein 1 (SREBP1)-regulated triglyceride synthesis, and cell proliferation. Proteomic analysis provided us a global view that VHL regulated four biological processes, including metabolism, immune regulation, apoptosis, and cell movement. Importantly, we found that VHL overexpression led to up-regulated expression of proteins associated with antigen processing and interferon-responsive proteins, thus rendering ccRCC cells more sensitive to interferon treatment. We defined an interferon-responsive signature (IRS) composed of ten interferon-responsive proteins, whose mRNA expression levels were positively correlated with DFS in ccRCC patients. Taken together, our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.

Keywords: Immunogenicity; Interferon; Multi-omics; Renal cancer; VHL.

Figure 1

High VHL expression was correlated with better DFS in ccRCC A. Analysis of the mutation frequency of VHL from four different clinical datasets “China 2012” , “Japan 2013” , “USA 2013” , and “Europe 2014” . B. Correlation analysis between the DFS and VHL expression levels of different cancer patients using datasets from TCGA with GEPIA (http://gepia2.cancer-pku.cn/#index). Blue-coded rectangle represents that high VHL expression is positively correlated with better DFS, whereas red-coded rectangle represents that high VHL expression is correlated with poor DFS. C. Higher VHL mRNA expression levels correlated with longer DFS of ccRCC patients using the KIRC datasets. n = 129, each in low (blue) or high (red) group. Log-rank test was used to determine the survival differences (P = 0.043). D. RT-PCR analysis of VHL expression in the control and VHL-OE cells. E. Western blotting analysis of HA-tagged VHL expression in the control and VHL-OE cells. F.VHL overexpression decreased cell proliferation in ccRCC cells. G.VHL overexpression decreased the colony formation in ccRCC cells. H. Representative images of the scratch assay of the control and VHL-OE cells. I. Quantitative result of the scratch assay shown in (H) by using ImageJ software. J.VHL overexpression decreased cellular ROS level in ccRCC cells. VHL, Von Hippel-Lindau disease tumor suppressor; DFS, disease-free survival; TCGA, The Cancer Genome Atlas; ccRCC, clear cell renal cell carcinoma; ROS, reactive oxygen species; OE, overexpression; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Figure 2

VHL overexpression reversed the Warburg phenotype and decreased cellular TAG by inhibiting SREBP1 maturation A. and B.VHL overexpression decreased basal glycolysis (A) but increased basal respiration (B) in ccRCC cells. C. Diagram showed the sequence of sgRNA targeting VHL. D. Sequence alignment of the exon 2 in VHL in control and VHL-KO cells. E. Western blotting analysis of VHL and HIF1A expression in control and VHL-KO cells. F. and G.VHL knockout increased the basal glycolysis (F) but decreased the basal respiration (G) in 293T cells. H.VHL overexpression decreased the cellular TAG levels in ccRCC cells. I.VHL knockout increased the cellular TAG levels in 293T cells. J. RT-PCR analysis of SREBP1, SREBP2, SCD, and ACACA in VHL-OE and control cells. K. Western blotting analysis showing the expression of flSREBP1, nSREBP1, FAS, and VHL in VHL-OE and control cells. TAG, triglyceride; flSREBP1, full-length SREBP1; nSREBP1, nuclear SREBP1; KO, knockout.

Figure 3

VHL overexpression altered protein expression profiling in ccRCC cells A. The correlation of two biological replicates from proteomes exhibited a good repeatability. B. A volcano diagram of up-regulated (red) and down-regulated (blue) proteins between the VHL-OE and control cells based on fold change (x-axis) and adjusted P value (y-axis). Each point represents a single quantified protein. The significantly changed proteins were labeled with the gene symbols. C. A heatmap showing the changes of the proteins involved in glycolysis and oxidative phosphorylation. D. RT-PCR analysis of GLUT1, PGK, ENO2, and HK2 in VHL-OE and control cells. E. Western blotting analysis confirmed VHL overexpression-induced changes in GAPDH, NDUFV2, and NDUFA4 in VHL-OE cells. F. The top 20 canonical pathways can be grouped into four biological processes analyzed by IPA software. G. RT-PCR analysis revealed that VHL overexpression significantly up-regulated HLA-related genes but down-regulated PD-L1. H. Western blotting analysis confirmed VHL overexpression-induced changes in HLA-A, HLA-DR, and PD-L1 in VHL-OE cells. I. Antigen processing and presentation (red) and antigen processing and presentation via MHC class I (blue) were all highly up-regulated by GSEA (using “C5” signature from the MSigDB). The plots indicated a significant (FDR adjusted P value < 0.05) enrichment after VHL overexpression. IPA, Ingenuity Pathway Analysis; GSEA, gene set enrichment analysis; NES, normalized enrichment score.

Figure 4

VHL overexpression up-regulated interferon-responsive proteins via STAT1-mediated signalling A. Responses to interferon-β (red) and interferon-γ (blue) genes were highly up-regulated by GSEA. Plots indicated a significant (FDR adjusted P value < 0.05) enrichment after VHL overexpression. B. RT-PCR analysis showing the significantly up-regulated mRNA levels of interferon and interferon-responsive genes in VHL-OE cells. C. Western blotting analysis showing the up-regulated protein levels of ISG15, UBE1L, UBE2L6, HERC5, and VHL in VHL-OE cells. D. RT-PCR analysis showing the significantly up-regulated mRNA levels of the IRS genes in VHL-OE cells. E. RT-PCR analysis showing the down-regulated expression of interferon-responsive genes in VHL-OE cells treated with 2.5 µM or 5 µM fludarabine. IRS, interferon-responsive signature.

Figure 5

VHL overexpression rendered ccRCC cells sensitive to interferon treatment A. and B.VHL overexpression sensitized ccRCC cells to interferon treatment. VHL-OE cells and control cells were treated with different concentrations of interferon-β (A) or interferon-γ (B) for 24 h. C.VHL expression was positively correlated with IRS expression (R = 0.78, P < 1E−5). Data were from the KIRC datasets from TCGA, and normalized by GAPDH. D. IRS expression was positively correlated with DFS in ccRCC patients using the KIRC datasets. n = 129, each in low (blue) or high (red) group. Log-rank test was used for comparing the survival curves (P = 0.0025).