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. 2024 Apr 22;10(1):188.
doi: 10.1038/s41420-024-01944-1.

MED15 is upregulated by HIF-2α and promotes proliferation and metastasis in clear cell renal cell carcinoma via activation of SREBP-dependent fatty acid synthesis

Xiaoliang Hua  1   2 , Shengdong Ge  2 , Li Zhang  2 , Qing Jiang  1 , Juan Chen  3   4 , Haibing Xiao  5 , Chaozhao Liang  6
Affiliations

MED15 is upregulated by HIF-2α and promotes proliferation and metastasis in clear cell renal cell carcinoma via activation of SREBP-dependent fatty acid synthesis

Xiaoliang Hua et al. Cell Death Discov. .

Abstract

Emerging evidence has highlighted that dysregulation of lipid metabolism in clear cell renal cell carcinoma (ccRCC) is associated with tumor development and progression. HIF-2α plays an oncogenic role in ccRCC and is involved in abnormal lipid accumulation. However, the underlying mechanisms between these two phenomena remain unknown. Here, MED15 was demonstrated to be a dominant factor for HIF-2α-dependent lipid accumulation and tumor progression. HIF-2α promoted MED15 transcriptional activation by directly binding the MED15 promoter region, and MED15 overexpression significantly alleviated the lipid deposition inhibition and malignant tumor behavior phenotypes induced by HIF-2α knockdown. MED15 was upregulated in ccRCC and predicted poor prognosis. MED15 promoted lipid deposition and tumor progression in ccRCC. Mechanistic investigations demonstrated that MED15 acts as SREBP coactivator directly interacting with SREBPs to promote SREBP-dependent lipid biosynthesis enzyme expression, and promotes SREBP1 and SREBP2 activation through the PLK1/AKT axis. Overall, we describe a molecular regulatory network that links MED15 to lipid metabolism induced by the SREBP pathway and the classic HIF-2α pathway in ccRCC. Efforts to target MED15 or inhibit MED15 binding to SREBPs as a novel therapeutic strategy for ccRCC may be warranted.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. MED15 was upregulated in ccRCC.
A Venn diagram of the results for four independent datasets, including lipid metabolism-related genes, survival-related genes and differentially expressed genes between normal and tumor samples from the TCGA dataset, and differentially expressed genes from transcriptome sequencing data obtained after HIF-2α knockdown. Three genes (MED15, ACLY, and TRIB3) were identified. B Heatmap showing the expression of MED15, ACLY, and TRIB3 in normal and tumor samples from the TCGA dataset. C The receiver operating characteristic (ROC) curves for MED15, ACLY, and TRIB3. Kaplan–Meier survival curves showed lower overall survival D and disease-free survival (DFS) times E for the high MED15, ACLY, and TRIB3 expression groups. F The mRNA levels of MED15 were increased in 40 ccRCC samples compared with 40 paired normal renal samples. G The protein levels of MED15 were increased in ccRCC samples compared with matched normal renal samples, as detected by western blot assay. H Immunohistochemical staining for MED15 in ccRCC samples and matched normal renal samples (scale bar: 100 μm). I The mRNA and protein levels of MED15 in five ccRCC cell lines and the HK-2 cell line (n = 3). The results are expressed as the mean ± standard deviation (SD). * indicates P < 0.05; ** indicates P < 0.01.
Fig. 2
Fig. 2. MED15 promoted the progression of ccRCC.
A Western blot assay and qRT–PCR revealed that MED15 protein and mRNA levels were efficiently knocked down in RCC cells with MED15 knockdown. B Growth curves of RCC cells with MED15 knockdown detected by CCK-8 assay (n = 5). C The colony formation assay showed a decreased proliferation rate of RCC cells with MED15 knockdown at the second week of cultivation. D, E The wound healing assay indicated decreased migration of RCC cells with MED15 knockdown. F, G Transwell assays showed decreased migration and invasion of RCC cells with MED15 knockdown. H Western blot assay and qRT–PCR revealed that MED15 protein and mRNA were efficiently increased in RCC cells with MED15 overexpression. I Growth curves for 786-O cells (n = 3) and OS-RC-2 cells (n = 6) with MED15 overexpression detected by CCK-8 assay. J The colony formation assay showed an increased proliferation rate in RCC cells with MED15 overexpression on day ten after cultivation. K, L The wound healing assay indicated increased migration of RCC cells with MED15 overexpression. M, N Transwell assays showed increased migration and invasion of RCC cells with MED15 overexpression. The results are expressed as the mean ± standard deviation (SD). ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001. RCC renal cell carcinoma, qRT–PCR quantitative reverse transcription-polymerase chain reaction.
Fig. 3
Fig. 3. MED15 promoted lipid accumulation in ccRCC.
A The results of the KEGG enrichment analysis of differentially expressed genes identified based on analysis of transcriptome sequencing data of OS-RC-2 cells with MED15 knockdown. B Enriched biological processes related to lipid metabolism with P < 0.05 according to the GO enrichment analysis. C GSEA of TCGA data showed that lipid metabolism terms were associated with the mRNA expression of MED15 according. FDR < 25% and p < 0.05 were used as criteria for determining statistical significance. D Oil Red O staining in RCC cells with MED15 knockdown. E The TG and cholesterol contents were measured in RCC cells with MED15 knockdown (n = 4). F Oil Red O staining in RCC cells overexpressing MED15. G The TG and cholesterol contents were measured in RCC cells overexpressing MED15 (n = 4). H The differentially expressed lipid components in negative ion mode were detected by LC/MS lipidomics assay in OS-RC-2 cells with or without MED15 knockdown (n = 5). The results are expressed as the mean ± standard deviation (SD). * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001. KEGG Kyoto Encyclopedia of Genes and Genomes, GO Gene Ontology, GSEA gene set enrichment analysis, TCGA The Cancer Genome Atlas, FDR false discovery rate, TG triglyceride, RCC renal cell carcinoma; LC/MS liquid chromatography/mass spectrometry.
Fig. 4
Fig. 4. HIF-2α directly regulates MED15 transcription in ccRCC.
A–C The protein and mRNA levels of HIF-2α and MED15 in RCC cells with HIF-2α knockdown. D Chromatin immunoprecipitation assays were performed on OS-RC-2 cells using anti-HIF-2α antibody followed by SYBR Green quantitative real-time polymerase chain reaction (qRT−PCR) for assessment of vascular endothelial growth factor (VEGF) and MED15 hypoxia response element (HRE) sites. E Luciferase reporter assays were performed in 293T cells cotransfected with truncated plasmids and HIF-2α expression plasmids or empty plasmid. After 24 h, Firefly luciferase and Renilla luciferase activities were measured using a dual-luciferase reporter assay, and the ratio of the Firefly/Renilla values was calculated (n = 3). The results are expressed as the mean ± standard deviation (SD). “ns” indicates P > 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.
Fig. 5
Fig. 5. MED15 promoted activation of the SREBP pathway.
A Downregulated lipid metabolism genes identified in transcriptome sequencing data of OS-RC-2 cells with MED15 knockdown. B Functional protein association networks for these downregulated lipid metabolism genes were constructed in the STRING database. C, D Western blot assays showed decreased protein levels of SREBP1, SREBP2, PLK1 and SREBP-dependent lipid biosynthesis enzymes (FASN, ACC1, ACLY, and SCD1) in RCC cells with MED15 knockdown. E, F Western blot assays showed increased protein levels of SREBP1, SREBP2, PLK1 and SREBP-dependent lipid biosynthesis enzymes (FASN, ACC1, ACLY, and SCD1) in RCC cells overexpressing MED15. G, H PLK1 knockdown eliminated the effects of MED15 overexpression on SREBP1 and SREBP2 expression. The results are expressed as the mean ± standard deviation (SD). “ns” indicates P > 0.05; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.
Fig. 6
Fig. 6. Overexpression of MED15 rescued the effects of HIF-2α knockdown on the lipid accumulation, migration, and invasion of RCC cells and tumor growth.
A, B RCC cells with stable HIF-2α knockdown and MED15 overexpression. Western blot assays were performed to detect the expression of HIF-2α, MED15, PLK1, SREBP1 and SREBP2. C, D Growth curves for 786-O cells (n = 3) and OS-RC-2 cells (n = 6) with stable HIF-2α knockdown and MED15 overexpression detected by CCK-8 assay. E Oil Red O staining in RCC cells with stable HIF-2α knockdown and MED15 overexpression. F, G The TG and cholesterol contents were measured in RCC cells with stable HIF-2α knockdown and MED15 overexpression (n = 4). H, I Transwell assays of migration and invasion were performed with the transfected cell lines. The results are expressed as the mean ± standard deviation (SD). “ns” indicates P > 0.05; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.
Fig. 7
Fig. 7. MED15 knockdown inhibited tumor progression and lipid accumulation in ccRCC in vivo.
A–C OS-RC-2 cells with stable MED15 knockdown were implanted subcutaneously into the flanks of nude mice, and tumor size was measured every three days. Both tumor weight and tumor volumes were reduced in cells with stable MED15 knockdown (n = 5). D Macroscopic view and H&E staining of lung from the tail vein metastasis model at 4 weeks and 7 weeks. E The numbers of metastatic nodules in the lung were reduced in cells with stable MED15 knockdown at 4 weeks and 7 weeks (n = 6). F, G Immunohistochemical staining was performed to detect the levels of MED15, Ki67, PLK1, and SREBP-dependent lipid biosynthesis enzymes (SREBP1, SREBP2, FASN, ACC1, ACLY, and SCD1) in the subcutaneous tumorigenesis nude mouse model (n = 5). H, I The TG and cholesterol contents were measured in the subcutaneous tumorigenesis nude mouse model (n = 5). The results are expressed as the mean ± standard deviation (SD). “ns” indicates P > 0.05; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.
Fig. 8
Fig. 8. MED15 expression was positively associated with tumor stage and predicted poor prognosis in ccRCC specimens.
A Examples of MED15 staining intensity (weak, moderate, positive, and strong). B Kaplan–Meier survival curves showing poor prognosis for ccRCC patients in the high MED15 expression group. C, D MED15 expression was increased in ccRCC specimens and was positively associated with tumor stage. E Schematic diagram depicting a proposed model for the major mechanism underlying the effects of HIF-2α dependent MED15 expression on the regulation of ccRCC progression and lipid accumulation.
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