Identification Of HCC Subtypes With Different Prognosis And Metabolic Patterns Based On Mitophagy

BackgroundAs the third leading cause of cancer-related death worldwide,the 5-year overall survival rate for hepatocellular carcinoma（HCC）is only 18%.Different from alcoholic liver disease abroad,cirrhosis is the most common cause of HCC in China due to underlying liver diseases such as hepatitis CAUSED by HBV or HCV viruses.The prevalence of HCC is increasing year by year worldwide,and the new cases of HCC in China account for about 55% of the global total,which greatly increases the social medical burden.HCC is characterized by rapid proliferation,easy metastasis,high recurrence,and insidious onset.Most patients are already in advanced stage when they are found,which contribute to the high mortality rate of HCC.Moreover,there are few effective treatment measures for advanced HCC,poor sensitivity of chemotherapy,and serious drug resistance of targeted drugs such as sorafenib.Therefore,the exploration of HCC heterogeneity and potential therapeutic targets became a research hotspots.Mitochondrial autophagy can recognize some damaged,defective mitochondria and degrade them in lysosomes.Each liver cell contains about 1000 mitochondria,accounting for about 18% of the cell volume.In order to maintain the normal functioning of mitochondria,appropriate and moderate mitochondrial autophagy is essential.Meanwhile,it has been reported that mitochondria are the main and important source of reactive oxygen species（ROS）.Excessive ROS can lead to damage to DNA,proteins and lipids,which are closely related to the development of cancer.It is well known that more than 90% of HCC is closely related to liver damage and inflammation.In this case,mitochondrial dysfunction can release ROS and mitochondrial DNA（mt DNA）into the cytoplasm,thereby activating the main innate immune response,which is one of the important reasons for the occurrence and development of HCC.At the same time,we learned that the uncontrolled proliferation of tumor cells requires mitochondrial autophagy to ensure normal mitochondrial homeostasis,and the dysfunction of mitochondrial autophagy can disrupt metabolism,increase oxidative stress and induce apoptosis of tumor cells.In addition,mitochondrial autophagy mediated by PINK1 leads to inactivation of the tumor suppressor p53,which is thought to play an important role in maintaining the number of HCC stem cells（CSCs）.In conclusion,mitochondrial autophagy prevents tumorigenesis of HCC by inhibiting dysfunctional mitochondrial accumulation,cellular oxidative stress,genomic instability,and inflammatory formation.On the contrary,when tumor masses have formed,the activation of mitochondrial autophagy can fully meet the metabolic needs of cancer cells and promote the progress of HCC.Therefore,such a paradoxical role of cellular mitochondrial autophagy may be a promising though challenging therapeutic direction for HCC in the future,and it is of great significance to explore the heterogeneity of mitochondrial autophagy in HCC for the prevention and treatment of HCC.ObjectiveTo comprehensively identify the expression pattern of mitochondrial autophagy-related regulatory factors in HCC and systematically explore the heterogeneity of HCC related to mitochondrial autophagy through bioinformatics analysis and high-throughput sequencing data of HCC from the public Cancer Genome Atlas Database（TCGA）and the International Cancer Genome Consortium Database（ICGC）.Specifically,differences in prognosis and tumor metabolism.Meanwhile,prognostic risk models were constructed to accurately predict the prognosis of different patients.Methods（1）Gene sets related to mitochondrial autophagy,glycolysis and cholesterol biosynthesis were obtained from the Reactome database.The relative expression levels of different mitochondrial autophagy regulatory factors in normal and tumor samples were determined by TCGA and ICGC databases.（2）Consensus Cluster Plus algorithm was used to identify tumor subtypes related to mitochondrial autophagy and metabolism.（3）Univariate Cox regression analysis was performed to determine the prognostic regulatory factors of mitochondrial autophagy.Principal component analysis（PCA）was performed for prognostic mitochondrial autophagy regulatory factors.The sum of PC1 and PC2 obtained by PCA analysis was used as the prognostic risk score of HCC patients,and the corresponding prognostic model,mitochondrial autophagy score,was established.Patients with a mitophagy score above or below the median were categorized as the high-or low-risk group.（4）Kaplan-Meier survival analysis was utilized to estimate differences in overall survival between high-risk and low-risk groups.ROC curve analysis was used to evaluate the prognostic ability of mitochondrial autophagy score.Use the "RMS" R package to draw the linogram and calibration curve.（5）R package "Limma" is used to explore differentially expressed genes.（6）In the CMap database,differentially expressed genes（DEGs）between high-risk and low-risk groups were used as input files to explore the corresponding small molecule drugs with potential for treatment.A literature search was conducted to obtain potential anti HCC drugs with experimental validation,and R package "Prophytic" and cancer genomics drug sensitivity（GDSC）databases were used to determine the sensitivity of different anti-HCC drugs to different mitochondrial autophagy subtypes.Results（1）By searching the Reactome database,we obtained 28 regulatory factors of mitochondrial autophagy,including 26 genes that could be retrieved in TCGA and ICGC databases.Only PINK1,MAPILC3 A and UBB were significantly down-regulated in tumor samples,while other genes were significantly up-regulated in tumor samples.（2）Consensus Cluster Plus algorithm has identified three hepatocellular carcinoma subtypes（low,medium and high mitochondrial autophagy subtypes）with different amounts of mitochondrial autophagy accumulation.The high mitochondrial autophagy subtype had the worst prognosis and the highest glycolysis level.Low mitochondrial autophagy subtype had the lowest degree of hypoxia and the highest cholesterol synthesis level.The intermediate type had the lowest expression dryness index.（3）PCA analysis of mitochondrial autophagy score can be used as a new accurate prognostic index of HCC.Patients in the high-score group had a worse prognosis（log-rank test,P < 0.001）.The AUC of the 1-year survival rate of mitochondrial autophagy score in the TCGA set was 0.77,and that in the ICGC set was 0.75.（4）from the CMap database identified nine candidates for small molecules that could be used in the treatment of liver cancer drugs（DL-thiorphan,Sanguinarine,Chrysin,Blebbistatin,Metyrapone,Medrysone,Verteporfin,Apigen In meticrane）.（5）According to the GDSC database,we obtained 21 different anti-HCC drugs,and found that the sensitivity of 21 anti-HCC drugs decreased in low-risk patients.Moreover,we also found a new key gene SPP1,which is highly correlated with different mitochondrial autophagy subtypes of HCC species,and this association has not been studied by previous scholars.ConclusionsBased on bioinformatics analysis,we systematically examined the heterogeneity of HCC associated with mitochondrial autophagy and observed that three different HCC subtypes had different prognostic and metabolic patterns...