GPR1 and CMKLR1 Control Lipid Metabolism to Support the Development of Clear Cell Renal Cell Carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer, is largely incurable in the metastatic setting. ccRCC is characterized by excessive lipid accumulation that protects cells from stress and promotes tumor growth, suggesting that the underlying regulators of lipid storage could represent potential therapeutic targets. Here, we evaluated the regulatory roles of GPR1 and CMKLR1, two G protein-coupled receptors of the protumorigenic adipokine chemerin that is involved in ccRCC lipid metabolism. Both genetic and pharmacologic suppression of either receptor suppressed lipid formation and induced multiple forms of cell death, including apoptosis, ferroptosis, and autophagy, thereby significantly impeding ccRCC growth in cell lines and patient-derived xenograft models. Comprehensive lipidomic and transcriptomic profiling of receptor competent and depleted cells revealed overlapping and unique signaling of the receptors granting control over triglyceride synthesis, ceramide production, and fatty acid saturation and class production. Mechanistically, both receptors enforced suppression of adipose triglyceride lipase, but each receptor also demonstrated distinct functions, such as the unique ability of CMKLR1 to control lipid uptake through regulation of sterol regulatory element-binding protein 1c and the CD36 scavenger receptor. Treating patient-derived xenograft models with the CMKLR1-targeting small molecule 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) led to a dramatic reduction in tumor growth, lipid storage, and clear-cell morphology. Together, these findings provide mechanistic insights into lipid regulation in ccRCC and identify a targetable axis at the core of the histologic definition of this tumor that could be exploited therapeutically. Significance: Extracellular control of lipid accumulation via G protein receptor-mediated cell signaling is a metabolic vulnerability in clear cell renal cell carcinoma, which depends on lipid storage to avoid oxidative toxicity.

Figure 1.. GPR1 and CMKLR1 are critical for ccRCC growth.

A/B: Western blot analysis showing GPR1 or CMKLR1 knockout and relevant changes in cell growth. C/D: In vitro proliferation assay of ccRCC cells with GPR1 or CMKRL1 knockout. E/F: In vitro colony formation of ccRCC cells with GPR1 or CMKRL1 knockout. G: EdU incorporation flow diagram of ccRCC cells with GPR1 or CMKRL1 knockout. H/I: Quantification of EdU incorporation. J/K: Quantification of C11 Bodipy staining.

Figure 2.. GPR1 and CMKLR1 regulate lipid metabolism in ccRCC cells.

A/C: qRT-PCR of lipid metabolism-related genes due to GPR1/CMKLR1 knockout. B/D: Quantification of ORO staining of GPR1/CMKLR1 knockout cell. E/F: Mito-stress assay of GPR1/CMKLR1 knockout cell. G/H: Quantification of oxygen consumption rate of ccRCC cells with GPR1 or CMKRL1 knockout.

Figure 3.. GPR1 and CMKRL1 modulate ccRCC growth and metabolic shift in vivo.

A/B: In vivo tumor assay of ccRCC cells with GPR1 and CMKRL1 knockout. C: tumor weight of UOK101 with GPR1 and CMKRL1 knockout at terminal point D: qRT-PCR of key lipid metabolism-related genes of RNAs extracted from UOK101 tumor E: ORO quantification of UOK101 tumor with receptor knockouts. F: Hematoxylin and eosin staining of UOK101 tumors. G: ORO staining of UOK101 tumors.

Figure 4.. Multiomic analysis revealing the global impact of GPR1 and CMKRL1 on ccRCC.

A: Global heatmap of differentially regulated lipids in 769-P cells. B: PCA analysis of samples for lipidomic analysis. C: Enrichment plot of unsaturated lipid species in GPR1/CMKLR1/RARRES2 knockout compared to the control. D: Bar graph showing ceramide lipid species enrichment in four sample groups. E: Bar graph showing Phosphatidylethanolamine (PE) lipid species enrichment in four sample groups. F: Western blot of LC3B in 769-P cells with GPR1 knockout. G: Western blot of LC3B in 769-P cells with CMKLR1 knockout. H: Western blot of LC3B in 769-P cells with RARRES2 knockout. I: Venn diagram showing the number of differentially regulated genes in RARRES2 KO, GPR1 KO and CMKLR1 KO from RNA-seq.

Figure 5.. Lipolysis is commonly regulated by GPR1 and CMKLR1 via suppression of ATGL, while CMKLR1 regulates SREBP1c-mediated lipid uptake.

A/B: Western blot of 769-P/UOK101 with GPR1 or CMKLR1 knockout. C/D: Western blot of 769-P/UOK101 rescued with 2 μM of Atglistatin. E/F: ORO quantification of 769-P/UOK101 with or without GPR1/CMKLR1 knockout treated with Atglistatin. G/H: C11 BODIPY quantification of 769-P/UOK101 with or without GPR1/CMKLR1 knockout treated with Atglistatin. I: ORO quantification of knockout cells treated with external fatty acids. J: BODIPY (493/503) quantification of knockout cells treated with external fatty acids. K/L: Western blot of CD36 in ccRCC cells with GPR1 or CMKLR1 knockout. M/N: Western blot of SREBP1c in ccRCC cells with GPR1 or CMKLR1 knockout.

Figure 6.. CMKLR1-targeting α-NETA induces lipid oxidation and induces a shift in lipid metabolism.

A/B: Western blot probing for ATGL and precursor/active SREBP1c on ccRCC cells treated with $\mathrm{\alpha }$-NETA. C: qRT-PCR of key CMKLR1 regulatory genes on ccRCC cells treated with $\mathrm{\alpha }$-NETA. D: Oil Red-O staining of ccRCC cells treated with $\mathrm{\alpha }$-NETA. E/F: ORO quantification of ccRCC cells treated with $\mathrm{\alpha }$-NETA.

Figure 7.. α-NETA suppresses ccRCC growth in both cell line and patient derived xenograft models.

A: UOK101 tumor growth curve with or without the treatment of $\mathrm{\alpha }$-NETA. B: UOK101 tumor weight at the terminal point of $\mathrm{\alpha }$-NETA treatment. C: Normalized ORO staining quantification of UOK101 tumor treated with $\mathrm{\alpha }$-NETA. D: Human ccRCC PDX (XP296) tumor subcutaneous growth curve with or without the treatment of $\mathrm{\alpha }$-NETA and the tumor weight at the terminal point E: XP296 orthotopic tumor weight at the terminal point with or without $\mathrm{\alpha }$-NETA. F: Normalized ORO staining quantification of XP296 subcutaneous tumor treated with $\mathrm{\alpha }$-NETA. G: Oil Red-O staining of XP296 orthotopic tumor with or without the treatment of $\mathrm{\alpha }$-NETA. H: ORO staining quantification of XP296 tumor treated with $\mathrm{\alpha }$-NETA. I: Pathway diagram showing the regulatory role of both GPR1 and CMKRL1 on lipid metabolism in ccRCC.