The Isotopic Tracing Based Study Of HSP60 Knockdown Mediated Tumor Progression In Clear Cell Renal Cell Carcinoma

Heat shock protein 60?HSP60?is a major mitochondrial chaperone for maintaining mitochondrial proteostasis.Our previous studies have demonstrated that HSP60 is downregulated in clear cell renal cell carcinoma?cc RCC?tissues.In the present study,we analyzed datasets in The Cancer Genome Atlas and revealed that higher HSP60expression correlated with better overall survival in cc RCC patients.We stably knocked down or overexpressed HSP60 in cc RCC cells and confirmed that HSP60 knockdown increased cell proliferation,whereas its overexpression decreased cell growth.Herein,the isotope tracing assisted metabolomics and proteomics were employed to investigate the effects of HSP60 knockdown on metabolic reprogramming in cc RCC cells.First we established a high-resolution mass spectrometry based workflow for high throughput mapping of isotope labeled metabolites.This approach incorporates an in-house MS/MS library for metabolite identification and ID-based quantitation.An“Isotopic”software was developed to generate potential labeled isotopomers.The platform was tested in 293T cells.The method was applied to 786-O-HSP60-KD,and¹³C₆-glucose tracing showed that the metabolic flow from glycolysis to TCA was reduced.Consistently,proteomics and metabolomics revealed that HSP60 knockdown promoted Warburg-like phenotypes.¹³C₅-glutamine tracing showed that HSP60 silencing enhanced mitochondrial functions in glutamine-directed biosynthesis with increased flow in two parts of the TCA cycle:Gln??-KG?OAA?Asp and Gln??-KG?ISO?acetyl-Co A,resulting in elevated de novo nucleotide synthesis and lipid synthesis.Glutamate generated from glutamine increased glutathione synthesis for quenching excessive reactive oxygen species?ROS?produced upon elevated cell growth.We further found that HSP60 silencing activated the MEK/ERK/c-Myc axis to promote glutamine addiction,and confirmed that cc RCC cells were susceptible to oxidative stress and glutaminase inhibition.Collectively,our data show that HSP60 knockdown drives metabolic reprogramming in cc RCC to promote tumor progression and enhances mitochondrial-dependent biosynthesis.