Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma

Abstract

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

Keywords: HILPDA; MYC; clear cell renal cell carcinoma; glutamine; lipid droplets.

Fig. 1.

HIF stabilization is required for LD accumulation upon MYC inhibition in ccRCC cells. (A) Lipid droplet staining and (B) western blot of the indicated proteins in 786-O VHL+ and VHL− cells after MYC inhibition. (C) Lipid droplet staining in 786-O VHL+ EV, 786-O VHL+ Omomyc, 786-O VHL- EV, or 786-O VHL- Omomyc cells after vehicle (−Omomyc, H₂O) or DOX treatment (+Omomyc). (D) Western blot of the indicated proteins in RCC4 VHL+ and VHL− cells after treatment with MYCis in 1% O₂ (hypoxia). (E) Lipid droplet staining in RCC4 VHL+ and VHL− cells after treatment with MYCis in 1% O₂ (hypoxia). (F) Lipid droplet staining in RCC4 VHL+ and RCC4 VHL− cells after treatment with MYCis in combination with DMSO (−chetomin) or chetomin. For western blots, β-actin was used as loading control. Molecular weight markers are shown to the Right. For fluorescence images: blue, DAPI; green, LD-BTD1 (LDs). (Scale bars: 20 μm.) All results are representative of three independent replicates.

Fig. 2.

Low glutamine levels or inhibition of glutaminase prevent LD formation. (A) Lipid droplet staining in RCC4 VHL− cells after culture in complete, low glucose (Low Glc) or low glutamine (Low Gln) medium treated with MYCis. (B) Lipid droplet staining in RCC4 VHL− cells after treatment with DMSO or MYCi alone or in combination with BPTES. Blue: DAPI; green: LD-BTD1 (LDs). (C) Bright-field images of live 786-O VHL- EV cells treated with vehicle (-DOX), DOX, or/and BPTES, and 786-O VHL- Omomyc cells treated with vehicle (-DOX), DOX (+Omomyc), or/and BPTES. RFP (red) upon DOX induction. (Scale bars: 20 or 100 μm.) Images are representative of three independent replicates.

Fig. 3.

Lipidomic analysis reveals different lipid species content in RCC4 VHL+ versus VHL- cells. (A) Mass isotopologue distribution of palmitate using U-¹³C₅-glutamine in RCC4 VHL+ and VHL− cells after treatment with JQ1. Data are presented as mean ± SD. Statistical analysis: two-way ANOVA with *, **, and **** indicating P < 0.05, <0.01, and <0.0001, respectively. (B) Heatmap with different lipid species in RCC4 VHL+ or VHL− cells after DMSO or JQ1 treatment, with red up-regulated and blue down-regulated lipid species. DG/DAG: diacylglycerides, PC: phosphatidylcholine, PC-O: phosphatidylcholine-O, PE: phosphatidylethanolamine, PE-O: phosphatidylethanolamine-O, LPG: lysophosphatidylglycerol, PG: phosphatidylglycerol, LPI: lysophosphatidylinositol, PI: phosphatidylinositol, LPS: lipopolysaccharide, PS: phosphatidylserine, TG: triglycerides, LPC: lysophosphatidylcholine, LPE: lysophosphatidylethanolamine, SL: saccharolipids, PA: phosphatidic acid. (C) Graphical representation of the different lipid species accumulated and the key enzymes in lipid metabolism upon JQ1 treatment in RCC4 VHL+ (purple) and VHL− (green). Image created with Biorender.com. G3P: glyceraldehyde 3-phosphate, FA-CoA: fatty acyl-CoA, LPAT: lysophosphatidyl acyltransferase, LPA: lysophosphatidic acid, A/GPAT: acyl/glycerol-3-phosphate acyltransferase, PAP: phosphatidic acid phosphatase, CDP-DAG: cytidine diphosphate-diacylglycerol, CDS: CDP-DAG synthase, PIS: phosphatidylinositol synthase, IP: inositol phosphate, DGAT: diacylglycerol acyltransferase. Lipidomic experiments were performed in three independent replicates.

Fig. 4.

Combined inhibition of MYC signaling and glutamine metabolism results in reduced tumor growth. (A) Tumor index (volume each day/initial volume) in mice with tumors from 786-O VHL− Omomyc cells divided into four groups: −Omomyc −BPTES (n = 6), −Omomyc +BPTES (n = 6), +Omomyc −BPTES (n = 8), or +Omomyc +BPTES (n = 7). Data are presented as mean ± SD. Statistical analysis: two-way ANOVA with **, and **** indicating P < 0.01 and <0.0001, respectively. (B) Representative images of tumors at end point. (C) Immunohistochemistry analysis of tumor sections stained with anti-Ki67, anti-c-MYC, or anti-Omomyc. (Scale bar: 50 μm.) (D) Tumor sections stained with LD-BTD1 (green) for LD visualization. Blue: DAPI. (Scale bar: 50 μm.) Results in C and D are representative of at least three tumor sections.

Fig. 5.

The HILPDA gene is up-regulated in RCC4 VHL− cells after MYC inhibition. (A) Visualization of functional enrichment maps in JQ1− versus DMSO-treated RCC4 VHL− cells (n = 3). The nodes represent significantly enriched genes participating in indicated processes. (B) Bar plot of the most significantly changed lipid metabolic processes in JQ1− versus DMSO-treated RCC4 VHL− cells. (C) Volcano plot (P < 0.05, horizontal dotted line) of the log₂ fold change (<−2, >+2, vertical dotted lines) of expression of genes between JQ1− and DMSO-treated RCC4 VHL+ cells (Left plot) and RCC4 VHL− cells (Right plot). The five most down-regulated (blue) and up-regulated (red) genes in each cell line are boxed. Not significantly changed genes are depicted in gray. (D) RT-qPCR of MYC and HILPDA in RCC4 VHL+ and VHL− cells. B2M was used as housekeeping gene. Data are presented as mean ± SD (n = 3). Statistical analysis: one-way ANOVA with *, **, ***, and **** indicating P < 0.05, <0.01, <0.001, and <0.0001, respectively. (E) Immunohistochemistry analysis of tumor sections stained with anti-HILPDA. (Scale bar: 50 μm.) (F) Immunofluorescence staining of HILPDA and (G) lipid droplet staining in RCC4 VHL− negative control (NC) and siHILPDA cells after treatment with DMSO or JQ1. Green: LD-BTD1; red: HILPDA; blue: DAPI. (Scale bar: 20 μm.) (H) Bright-field images of lipid droplet staining with Oil-red O (red) in 786-O VHL− Omomyc shSCR and shHILPDA cells upon Omomyc induction. (Scale bar: 20 μm.) Images in E–H are representative of three independent replicates/sections.

Fig. 6.

Identification of HILPDA as a biomarker for ccRCC. (A) RT-qPCR of HILPDA in RCC4 and 786-O VHL+ and VHL− control or HILPDA overexpressing cells. B2M was used as housekeeping gene. Data are presented as mean ± SD (n = 3). Statistical analysis: t test with **** indicating P < 0.0001. (B) Lipid droplet staining in RCC4 VHL+ and VHL− control and HILPDA overexpressing cells. Green: LD-BTD1, blue: DAPI. (Scale bar: 20 μm.) Images are representative of three independent replicates. (C) Tumor index (volume each day/initial volume) in mice with tumors derived from 786-O VHL− Omomyc shSCR or shHILPDA cells divided into two groups: −Omomyc (n = 8 and n = 7) or +Omomyc (n = 6 for both cell lines). Data are presented as mean ± SD. Statistical analysis: two-way ANOVA with ** and **** indicating P < 0.01 or <0.0001, respectively. (D) Immunohistochemistry analysis of tumor sections stained with anti-Ki67, anti-c-MYC, anti-Omomyc and anti-HILPDA. (Scale bar: 50 μm.) (E) Tumor sections stained with LD-BTD1 (green) for LD visualization. Blue: DAPI. (Scale bar: 50 μm.) Images in D and E are representative of at least three independent tumors per condition. (F) Relative expression of HILPDA across different tumors, including the three renal cell carcinomas: clear cell (KIRC) blue, chromophobe (KICH) green, and papillary (KIRP) orange. (G) Single-cell RNA-seq analysis of normal (Left panel) or tumor (Right panel) renal tissue. The upper graphs represent dimensional reduction plots (UMAP). The lower graphs indicate the levels of different genes of interest in the cell entities found within the samples. (H) Graphical abstract, created with Biorender.com.