Multi-omics Study Of Clear Cell Renal Cell Carcinoma

BackgroundRenal cell carcinoma is among the top 10 most common cancers.In 2020,430,000 new cases of kidney cancer were estimated to occur worldwide.Clear cell renal cell carcinoma(cc RCC)is the most common subtype of renal cancer.Localized cc RCC can be managed with partial or radical nephrectomy.However,～30% of patients with localized cc RCC eventually develop metastases.Targeted therapy and immunotherapy have been used in clinics for metastatic cc RCC.However,a large proportion of treated patients do not achieve remission.Therefore,a thorough study of the molecular characteristics of cc RCC is crucial to develop novel therapeutic strategies and to improve the survival of cc RCC patients.Large-scale genomics studies have provided us with a comprehensive understanding of the genetic landscape of cc RCC.However,proteins function as the ultimate executors of cellular processes and the direct targets of anti-cancer drugs.Proteomics study can provide important clues to further understand the mechanisms of cc RCC and to develop novel treatments.Objective and significanceFirst,to explore the regulatory mechanisms and potential precision medicine strategies of cc RCC,we conducted an integrated study of transcriptomics,proteomics and phosphoproteomics on clinical samples from 50 cc RCC patients.This is the first multi-omics study for a Chinese cc RCC cohort.Our findings provide a large data resource for future study of cc RCC.Second,the multi-omics study revealed that mTOR signaling pathway was aberrantly active in cc RCC.Involving in the mTOR signaling pathway,protein kinase mammalian target of rapamycin complex 2(mTORC2)is one the targets of mTOR inhibitors for clinical targeted therapy of renal cancer.To further explore the regulatory role of mTORC2,we studied the functions of mTORC2 in the process of DNA doublestrand break damage and explored the underlying molecular mechanisms.Method1.Proteomics and phosphoproteomics studies: we optimized sample processing procedures for mass spectrometric analysis,such as fractionation and phosphopeptide enrichment.Samples were analyzed by a Fusion Lumos mass spectrometer.Next,proteins and phosphopeptides were identified and label-free quantified by Max Quant.2.Transcriptomics study: RNA was extracted from tumors and adjacent tissues.RNA sequencing was commissioned to a company.After data collection,we performed quantitative differential analysis and subsequent integrative multi-omics analysis.3.Bioinformatics study: we used R to perform bioinformatic analysis of the multiomics data,including differential analysis,integrative analysis,biofunctional analysis and molecular subtyping.4.Mechanism study: we constructed stable transfected ACHN cell line by knocking down the expression of Rictor,a unique subunit of mTORC2.Next,we performed quantitative phosphoproteomic analysis using ionizing irradiated cells at different recovery time.Finally,we studied the regulatory function and underlying mechanism of Rictor in the process of DNA damage repair.Results and conclusions1.High-quality multi-omics data were obtained from 50 cc RCC tumors and 18 adjacent control tissues.Of which,we quantified 9379 proteins and 29957 phosphorylated peptides of 5831 proteins.2.Differential analysis revealed systemic changes of cc RCC tumors at RNA,protein and protein phosphorylation levels.The differently expressed genes were involved in a variety of biological processes,including metabolism,signal transduction,cell proliferation and immune response.For example,cc RCC tumors exhibited upregulated synthesis and downregulated catabolism of fatty acid,and this metabolic abnormality may provide materials for lipid droplet formation.Upregulation of proteins involved in glycogen synthesis and breakdown indicated that glycogen metabolism in cc RCC was abnormally active.3.We constructed a regulatory network of transcription factors and target genes by integrating multi-omics data.Transcription factors such as FOXK1,ELF1 and STAT3 showed a wide range of regulating effects on gene transcription in cc RCC.4.Abnormal phosphorylation of the endocytosis pathway was observed and may contribute to the upregulated expression of growth factor receptors,and eventually promote cell proliferation.Kinase enrichment analysis of differential protein phosphorylation sites revealed key protein kinases,such as GSK3 B,which may play an important role in the phosphorylation regulation in cc RCC.5.Based on the quantitative proteomics and phosphoproteomics data,cc RCC was classified into three subtypes,including cell proliferation,metabolism,and extracellular matrix and immune.Patients in extracellular matrix and immune subtype may have the worst clinical prognosis.Protein expression analysis of tumor drug targets across cc RCC subtypes suggested that patients in different subtypes may respond to different drugs,which provided clues for precision medicine of cc RCC.6.Mechanistic studies of mTORC2 identified that this kinase complex regulates the processes of DNA damage repair through Akt/CDK1/53BP1,and inhibition of mTORC2 activity enhances the sensitivity of renal cancer cells to radiotherapy and chemotherapeutic agents.