Studies On Hepatocellular Carcinoma Based Multi-omics Data Integration And Application

Part IStudies on the Signal Network of ALDH2 in HCC Progression and the Clinical SignificanceBACKGROUND & AIMS:As one of the most common digestive system cancers,Hepatocellular carcinoma(HCC)greatly threatens human health and becomes the third most frequent cause of cancer-related deaths all over the world.Treatments for HCC have continued to develop during the last decades,and carefully selected early-stage patients can be effectively managed by hepatic resection,ablation,liver transplantation,locoregional therapies and so on.In addition,cancer classifications which are based on molecular biomarkers can be used to effectively predict recurrence and metastasis,which improves the possibility of practitioners and patients to choose appropriate treatment methods for better prognoses.However,current situation continues to be dismal in HCC because of high rates of postsurgical recurrence and metastasis.Therefore,identifying more efficient biomarkers which can be used for predicting HCC progression and increasing our understanding of the mechanisms underlying its progress will contribute to the development of better tumor treatment methods,then prolong survival and improve life quality in HCC patients.In recent years,metabolic alterations have been reported in most tumor tissues.Several studies of HCC have found that metabolic dysfunctions contribute to hepatocarcinogenesis and its progression.In the current study,we combined gene expression and proteomic profiling analyses to identify aldehyde dehydrogenase 2(ALDH2)associated with outcomes of patients with hepatocellular carcinoma(HCC).Nonetheless,the role of ALDH2 in the pathogenesis of HCC remains controversial,and little is known about the effects of ALDH2 during HCC progression.METHODS:We compared gene and protein expression patterns between paired tumor and nontumor tissues,and different metastatic potential tissues from 50 or 27 patients with HCC who underwent hepatectomy in Shanghai,China,from January 2007 to September 2010.ALDH2 were identified for further study.And then,m RNA and protein levels of ALDH2 were measured by quantitative reverse transcription polymerase chain reaction,western blot and immunohistochemical analyses.Statistical analysis was used to associate levels of ALDH2 with tumor features and patient outcomes.Moreover,ALDH2 was overexpressed from a lentiviral vector in HCC-LM3 and SMMC-7721 cells,or knocked down by small interfering RNAs against ALDH2 in PLC/PRF/5 cells which were analyzed in migration and invasion assays.Lung and intrahepatic metastatic tumors were grown from these cells in nude mice.We used microarray analysis to compare expression patterns between ALDH2-overexpressed and control HCC-LM3 cells.We studied the effects of ALDH2 mediated aldehyde metabolism in metastasis in vitro,and the mechanistic target of AMPK in different ALDH2-overexpressed cell lines.To investigate whether it is the level of ALDH2 or the activity of ALDH2 that is governing the HCC risk,we extensively examined the activity of ALDH2 in both ALDH2-overexpressing cells(HCCLM3-ALDH2,SMMC7721-ALDH2)and their counterpart control cells.RESULTS:ALDH2 was significantly downregulated in a good deal of HCC samples than normal controls on both m RNA and protein levels.Western blot assays and RT-PCR results also showed that ALDH2 expression levels were lower in patients with metastasis-inclined HCC(MIH)than in those with metastasis-averse HCC(MAH).Further studies found that the m RNA and protein levels of ALDH2 were significantly lower in PVTT tissues than in the corresponding matched tumor or peritumoral tissues.And patients whose tumors had reduced ALDH2 levels were more likely to have aggressive traits(eg,high levels of extrahepatic metastasis,microvascular invasion,and embolus)than those with low levels.Patients with reduced levels of ALDH2 had shorter overall survival times and time to recurrence.Stable overexpression of ALDH2 in HCC cell lines(HCC-LM3 and SMMC-7721)suppressed their migration and invasion traits in vitro.Lung and intrahepatic metastatic tumors grown from ALDH2-overexpressed cells formed more slowly and fewer metastases than control cells in mice.And ALDH2 could activate AMPK signaling through modulating the ALDH2-acetaldehyde-redox-AMPK(AMP-activated protein kinase)axis,which was required for ALDH2-mediated cell metastasis.And further analysis suggests that it is the enhanced ALDH2 activity but not protein level in both HCCLM3-ALDH2 and SMMC7721-ALDH2 that contributes largely for ALDH2-manipulated cell migration and invasion.CONCLUSIONS:ALDH2 is frequently expressed at more reduced levels in clinical HCC samples,even when the tissues bear metastatic HCC,and in invasive HCC cell lines.And decreased levels of ALDH2 could indicate a poor prognosis for HCC patients,and forced expression of ALDH2 in HCC cells inhibits their aggressive behavior in vitro and in mice by regulating AMPK signaling through modulating the ALDH2-acetaldehyde-redox-AMPK(AMP-activated protein kinase)axis.Additionally,it is the enhanced ALDH2 activity but not protein level that contributes largely for ALDH2-manipulated cell migration and invasion.Therefore,identifying ALDH2 expression levels of HCC might provide a strategy for classification of patients and potential therapy target of metastatic HCC.Part IIDimethylglycine Dehydrogenase(DMGDH)Suppresses Hepatocellular Carcinoma Progression and Metastasis via Akt Signaling PathwayBACKGROUND & AIMS:Primary liver cancer is the fourth most frequently seen malignancy with poor prognosis and high mortality all around the world.In the year of 2015,there were about 466,000 new cases and up to 422,000 deaths from liver cancer in China.Therefore,identifying efficient molecular biomarkers of HCC progression and achieving a better understanding of the molecular mechanisms underlying HCC progression and post-surgical recurrence will contribute to tumor inhibition,improve life quality and extend the life expectancy of HCC patients.Several reports have indicated that metabolic dysfunction contributes to hepatocellular carcinogenesis and progression.However,current metabolic biomarkers are still insufficient for diagnosis and prognosis in HCC.In the vein,we combined gene expression analyses to identify metabolism-related gene DMGDH associated with outcomes of patients with hepatocellular carcinoma(HCC).Nonetheless,little is known about the role of DMGDH in the pathogenesis of HCC.METHODS:We compared HCC samples and matched corresponding non-tumor liver tissues collected from 47 patients who underwent hepatectomy in Shanghai,China,from January 2007 through September 2010.Levels of m RNA and protein were determined by quantitative reverse transcription polymerase chain reaction,western blot and immunohistochemical analyses.Statistical analyses were used to associate levels of DMGDH with tumor features and patient outcomes.Moreover,DMGDH was overexpressed from a lentiviral vector in HCC-LM3 and SMMC-7721 cells,or knocked down by small interfering RNAs against DMGDH in PLC/PRF/5 cells which were analyzed in migration and invasion assays.Lung metastatic tumors were grown from these cells in nude mice.We used microarray analysis to compare expression patterns between DMGDH-overexpressed and control HCC-LM3 cells.We studied the mechanistic target of Akt in different DMGDH-overexpressed cell lines.RESULTS:DMGDH was significantly reduced in a greater percentage of HCC samples than control tissues on both m RNA and protein levels.Tumors with reduced levels of DMGDH were more likely to have aggressive characteristics(e.g.,high levels of AFP,embolus,recurrence)than those with low levels.Patients whose tumors had reduced levels of DMGDH had shorter overall survival times.Forced expression of DMGDH in HCC cell lines(SMMC-7721 and HCC-LM3)reduced their invasive features in vitro,and ability to form metastatic tumors in nude mice.And DMGDH has a better prognostic and diagnostic effect in HCC patients.Further study shows DMGDH suppresses metastasis partly through inhibiting the Akt signaling pathway.CONCLUSIONS:Reduced levels of DMGDH in HCCs are associated with shorter survival times of patients,high levels of AFP,embolus,recurrence.Expression of DMGDH in HCC cell lines reduces their aggressive behavior in vitro and in mice partly by inhibiting the Akt signaling pathway.Therefore,identifying DMGDH expression levels of HCC could provide a novel strategy for classification of patients and potential therapy target of metastatic HCC.