TRPC3 Induces Metabolic Reprogramming And Ferroptosis Resistance To Promote Hepatocellular Carcinoma Growth

Background:Hepatocellular carcinoma（HCC）is the most frequent cause of cancer death worldwide.Despite intensive investigations,the underlying mechanisms of HCC are still unclear.The present study aims to clarify the characteristics of HCC to provide novel approaches for diagnosis,prognostication,and treatment.Transient receptor potential（TRP）superfamily proteins are permeable cation channels that are regulated by a series of chemical and physical stimuli and play roles in various physiological and pathological processes.TRPC3 is involved in multiple biological processes and diseases.TRPC3 was recently found to be overexpressed and promote cancer cell progression in gastric,colon,breast,and ovarian cancers.However,the expression and mechanism of TRPC3 in HCC remain largely unknown.Objective:Part I:To explore the significance of TRPC3 protein expression in HCC;Part II:To investigate the effect of TRPC3 on the process of HCC in vitro and in vivo;Part III:To prove the mechanisms of TRPC3 on mitochondrial metabolic reprogramming in HCC;Part IV:To identify the regulatory mechanism and downstream of TRPC3 in HCC;Part V:To investigate the regulatory mechanism of TRPC3 on ferroptosis tolerance in HCC.Methods:Part I:The expression of TRPC3 in 122 HCC patients was assessed by immunohistochemistry.Cox proportional hazard regression and Kaplan–Meier curves were used to evaluating the prognostic value of TRPC3;Part II:HCC cells with stable TRPC3 protein overexpression（CRISPR/SAM）or knockout（CRISPR/CAS9）were estabilished using lentiviral transduction.Phenotypes were detected to confirm the regulation of TRPC3 in HCC under loss-and-gain-of-function assay in vitro and in vivo;Part III:Trpc3 gene knockout HCC mouse model was established and used to study the regulation of TRPC3 on mitochondrial metabolic reprogramming in HCC;Part IV:RNA-seq,ATAC-seq and histone ChIP-seq were used to examine chromatin accessibility and transcriptomic landscape of HCC cells under TRPC3 overexpressed or knocked-down;Part V:Patient-derived HCC xenograft was established,and transmission electron microscopy,metabolomics detection,flow cytometry were used under TRPC3 overexpression or knockout.TRPC3 specific inhibitor Pyr3were used to prove the regulation on ferroptosis.Results:Part I:TRPC3 was upregulated in HCC and correlated with poor prognosis;Part II:TRPC3 enhanced the proliferation,migration,invasion and cell cycle of HCC cells,and inhibited apoptosis;TRPC3 also promoted tumor growth in vivo;Part III:TRPC3 promoted HCC by promoting mitochondrial metabolic reprogramming;Part IV:TRPC3 activated the MAPK signaling pathway by promoting MEK phosphorylation and p-ERK1/2 nuclear import to regulate the transcription of HCC cells,leading to the decrease of LIPIN1 and SCARA5 expression and the increase of oncogenes c-Jun,c-Myc,and Cyclin D1 expression,finally promoted the progression of HCC.Part V:TRPC3 inhibited SCARA5-mediated Fe²⁺uptake by increasing Ca²⁺uptake,thus inhibiting ferroptosis in HCC cells.Pyr3,TRPC3 inhibitor,promoted ferroptosis by increasing iron metabolism in HCC cells.Conclusion:TRPC3 is a novel biomarker for HCC.Its high expression indicates a poor prognosis for patients.TRPC3 promotes the proliferation,migration,invasion,and cell cycle progression and inhibits apoptosis of HCC cells by regulating mitochondrial metabolism and HCC transcription both in vivo and in vitro.TRPC3 inhibits SCARA5-mediated Fe²⁺uptake by increasing Ca²⁺uptake in hepatocellular carcinoma cells and inhibits ferroptosis.