Introduction: Lipid accumulation is pathognomonic for clear cell renal cell carcinoma (ccRCC) and occurs due to VHL loss and accumulation of HIF. General Control of Amino Acid Synthesis 5-like 1 (GCN5L1) augments mitochondrial protein acetylation and inhibits beta oxidation leading to lipid accumulation. Furthermore, VHL reconstitution lowers GCN5L1 transcript levels in VHL -/- ccRCC. We hypothesized that GCN5L1 regulates ccRCC lipid metabolism in a VHL-dependent manner.
Methods: GCN5L1 protein levels in various RCC subtypes were assessed. To determine if GCN5L1 expression is dependent on VHL or HIF, we reintroduced wtVHL in VHL -/- ccRCC, knocked down (KD) VHL, HIF1 or HIF2 in RPTEC cells or grew cells in hypoxia to assess GCN5L1 protein levels. GCN5L1 ubiquitination was determined by immunoprecipitation after VHL reconstitution. Mitochondria acetylation was assessed using purified mitochondria. Oil red O staining and cell proliferation were assessed after GCN5L1 KD. Beta oxidation was assessed via Seahorse. Finally, acetylation and activity levels of key beta oxidation enzymes were assessed after GCN5L1 KD.
Results: GCN5L1 protein level and mitochondrial acetylation were augmented in ccRCC compared to other RCC subtypes and was dependent on GCN5L1 expression. Re-expression of wtVHL in ccRCC attenuated, while VHL KD, but not HIF1 & 2 KD, in RPTEC augmented levels of GCN5L1. GCN5L1 was not induced in RPTEC under hypoxia. Further, GCN5L1 ubiquitination in ccRCC was elevated with wtVHL reconstitution. Thus, VHL, and not HIF, appears to directly regulate GCN5L1 levels. Functionally, GCN5L1 KD in ccRCC led to increased levels of CPT1a and lower lipid levels as seen with Oil-red O stain. Furthermore, GCN5L1 KD selectively inhibited growth of ccRCC but not other RCC subtypes/RPTEC. Finally, GCN5L1 KD attenuated acetylation levels of HADHA, LCAD and SCAD and augmented beta oxidation.
Conclusions: VHL loss augments GCN5L1 levels through lost ubiquitin mediated degradation. Thus, GCN5L1 and mitochondrial acetylation levels are selectively elevated in VHL-deficient ccRCC. GCN5L1 KD also reverses ccRCC repression of CPT1a, attenuates acetylation (augmenting activity) of key beta oxidation enzymes leading to decreased lipid accumulation. Finally, cell proliferation is selectively attenuated in ccRCC with GCN5L1 KD. GCN5L1 appears to play a unique role in ccRCC vs. non-ccRCC subtypes. Future work will examine GCN5L1’s role in mouse xenografts and patient tumors further highlighting the selective importance of GCN5L1 in ccRCC tumor biology.
.jpg)
.jpg)