Whole-exome Mutational Landscape Of Metastasis In Patient-derived Hepatocellular Carcinoma Cells

Objective: Liver cancer was the sixth most common cancer in the world and the fourth leading cause of cancer death.China account for more than 50% of new cases and deaths of liver cancer worldwide,most liver cancer patients were diagnosed with distant metastasis.Due to the heterogeneity of liver cancer,it is easy to develop drug resistance in chemotherapy,targeted therapy,immunotherapy and other treatments,so it is particularly important to explore the heterogeneity of liver cancer.Methods: Whole-exome sequencing(WES)was performed on patient-derived hepatocellular carcinoma(HCC)cell lines(MHCC97-L?MHCC97-H ? HCC97LM3)with differential metastatic potentials and analyzed their clonal evolution relationships.WES data was annotated in Oncotator,and non-silent mutations were found.An evolutionary tree based on genomic single nucleotide polymorphism(SNP)was constructed in Mega X software.According to the mutation sites and mutation genes of the three HCC cell lines,Venn diagrams were drawn and heterogeneities were calculated.The base changes of the three HCC cell lines,known liver cancer driving genes and gene mutations on 10 classical carcinogenic signal pathways were counted in Excel.The protein interaction network was constructed by STRING,and hub genes were found in the shared trunk mutation genes and the heterogeneous private mutations respectively.The relationship between the mutations of hub genes and the overall survival time(OS)of hepatocellular carcinoma(HCC)was searched in c Bio Portal database.The expression of hub genes in hepatocellular carcinoma and its effect on overall survival time of liver cancer were explored in ULCAN database.The mutated genes of interest were selected and primers were designed according to the mutation sites,and then verified by PCR.Results: The Venn diagram of the mutated genes of the three HCC cell lines showed that there were 4691 mutated genes shared by the three HCC cell lines,and the private mutation genes of MHCC97-L,MHCC97-H and HCC97LM3 were 243,315 and 289,respectively.The WES data showed that the average percentage of heterogeneous mutations in each HCC cell lines was 16.55%(range,15.38%-18.17%).C: G > T: A and T: A > C: G somatic transitions were the two most frequent substitutions.In these metastatic HCC cell lines,non-silent gene mutations were found in 7 known driver genes(including ARID1A?CDKN1A?P53?KEAP1?APOB?XPO1?HIST1H1E)and 10 classical signaling pathways(such as ALK?RET?EGFR?ROS1?KRAS?HRAS?APC,et al).In c Bio Portal database,some of the selected hub genes(including SDC1?ITGB4?ITGA4?BPTF?COL4A1?MYOD1?AR?EFTUD2)were found to be associated with poor overall survival(OS)of HCC patients.In ULCAN database,the high expression of hub genes(including CDC27?RELA?EIF4E?POXO3?EZH2?POLR2G?WDTC1?DLG?EPRS?EFTUD2)in liver cancer is related to the poor prognosis of liver cancer patients.Among the mutated HCC driver genes,a novel KEAP1 mutation(1334del C,p.P445fs)with a homozygous frameshift truncation at the c-terminal Nrf2 binding region was detected and verified in MHCC97-H and HCC97LM3 cells.Conclusions: WES data demonstrate that HCC cell lines from tumor biopsy specimens of the same patient have obtained different metastatic potentials through repeated selection in rodents in vivo,and they do indeed have a genetic relationship at the genomic level.Mutations in liver cancer driver genes and classical signaling pathways,as well as hub genes screened from trunk mutations and private mutations,may play an important role in the occurrence and development of liver cancer.