EGFR-PI3K-AKT Pathway Is Involved In ATP5B-inhibited Pathogenesis In Colon Cancer

Cancer cells undergo metabolic changes during oncogenic transformation to meet the higher metabolic and biosynthetic needs.Energy supplement mainly depends on oxidation phosphorylation(OXPHOS)in normal cells;however,an increased glycolytic rate is occurred in many cancers,leading to downregulation of OXPHOS In OXPHOS,the complexes of the electron transport chain(ETC)transfers electrons to molecular oxygen,generating proton gradient conferring ATP synthesis via H+-ATP synthase.H+transporting F1 complex beta subunit of mitochondrial ATP synthase(ATP5B)is involved in the regulation of mitochondrial bioenergy and death signal transduction.Studies have shown that PI3K-AKT pathway is involved in cancer cell metabolism,and the pathway is overactivated in various tumors.PI3K on cell membrane could be activated by the receptor of tyrosine kinases to activate signal transduction,further regulating the proliferation,invasion,and apoptosis of cancer cellsColorectal cancer(CRC)is the third most common cancer in the world.The expression level of ATP5B in CRC has been controversial in previous studies;furthermore,the biological role of ATP5B in CRC has not been fully elucidated.In this study,the association between ATP5B and CRC was verified by three levels,including population,tissue,and cell.Bioinformatics analysis was used to investigate the differential expression of ATP5B in cancers,and meta-analysis was utilized to investigate the association between ATP5B expression and prognosis in CRC patients We also examined the expression of ATP5B in CRC tissues,and investigated its association with clinicopathological parameters and prognosis in CRC patients Furthermore,we focused on human colon cancer cell lines(SW1116 and Lovo)to investigate the effects of ATP5B on cell biological behaviors(proliferation,migration,invasion,and apoptosis)and the association in ATP5B and EGFR-PI3K-AKT pathway.Methods:1.Oncomine and Timer databases were used to analyze the differential expression of ATP5B between CRC and normal tissues.Cytoscape and cBioPortal were used to analyze co-expression genes and related pathways of ATP5B in CRC.PrognoScan database was used to analyze the association between ATP5B expression and prognosis in CRC patients.2.Electronic databases were comprehensively searched to collect relevant studies about the association between ATP5B expression and overall survival in patients with solid tumor.A meta-analysis was performed to investigate the association between ATP5B expression and the prognosis of solid tumors.Combined with the survival statistic data in PrognoScan database,the association between ATP5B expression and prognosis of CRC was also investigated by meta-analysis.3.The expression of ATP5B in CRC and adjacent normal tissues was detected in 10 pairs of tissues by RT-qPCR and 41 pairs of tissues by immunohistochemistry.The correlation between ATP5B expression level and clinicopathological parameters was also analyzed.Kaplan-Meier survival analysis and cox multivariate analysis were used to investigate the association between ATP5B expression and prognosis in CRC patients.4.Overexpression and knockdown of ATP5B were constructed in colon cancer cell lines(SW1116 and Lovo)by transient liposome transfection,and the transfection efficiency of ATP5B overexpression was observed by fluorescence microscopy.RT-qPCR and western blot were used to detect the transfection effect at mRNA level and protein level,respectively.Cell proliferation was detected by CCK8 assay.Wound healing assay and transwell assay were applied to investigate migration and invasion of CRC cells.Hoechst 33258 staining was used to observe the morphology of nucleus in apoptotic cells.Furthermore,the effects of ATP5B expression level on reactive oxygen species(ROS)generation and cell apoptosis were determined by flow cytometry.Also,cells were disposed with 740Y-P,an activator of PI3K,to investigate the association in ATP5B and PI3K-AKT pathway.5.Apoptosis-related protein(Bcl2 and Bax)and protein related to EGFR-PI3K-AKT pathway(EGFR,pEGFR,PI3K,pPI3K,AKT,pAKT,and PTEN)were detected by western blot in cells with overexpression and knockdown of ATP5B.Results:1.Oncomine and Timer database both showed that ATP5B was down-regulated in CRC tissues compared with that in normal tissues.The functional analysis of GO and KEGG showed that ATP5B was associated with xenobiotic transport,monocarboxylic acid transport,and phospholipids translocation.PrognoScan database revealed that there was not significant association between ATP5B expression and overall survival in CRC patients,but patients with higher ATP5B expression had better prognosis in disease-free survival.2.A total of 1,458 patients with solid tumor from four published studies including six cohorts were included in the meta-analysis of solid tumors.The combined hazard ratio identified that there was not significant association between ATP5B expression and overall survival(HR=1.85,95%Cl:0.85-4.02).In the meta-analysis of CRC,high ATP5B expression had a favorable influence on disease-free survival(HR=0.42,95%Cl:0.22-0.82),but had no significant association with overall survival(HR=0.80,95%Cl:0.54-1.19).3.The results of RT-qPCR showed that ATP5B expression was down-regulated in CRC tissues compared with that in adjacent normal tissues.The results of immunohistochemistry showed that ATP5B was mainly expressed in cytoplasm,and ATP5B expression in CRC tissues was significantly lower than that in adjacent normal tissues.As for the association between ATP5B and clinicopathological parameters,we found the correlation of ATP5B with TNM stage and lymph node metastasis,but there was not significant correlation with gender,age,tumor size,and tumor differentiation.Kaplan-Meier survival analysis suggested that ATP5B expression was associated with poor prognosis.However,cox regression analysis revealed that ATP5B expression had no significant association with overall survival.4.Overexpression and knockdown of ATP5B were successfully constructed by liposome transfection in SW1116 and Lovo cells.Further study revealed that overexpression of ATP5B inhibited proliferation,migration,and invasion,but promoted ROS generation and apoptosis in CRC cells.On the contrary,knockdown of ATP5B exhibited the opposite effect.Furthermore,we treated ATP5B-overexpressed cells with a PI3K activator(740Y-P).We found that ATP5B-induced inhibition of cell proliferation and migration,as well as the promotion of cell apoptosis were recovered.5.The expression of apoptosis-related protein was detected by western blot.The results showed that Bcl2/Bax ratio was significantly decreased in ATP5B-overexpressed cells,but the downregulation was reversed by 740Y-P.However,knockdown of ATP5B induced converse results.Furthermore,western blot analysis showed that overexpression of ATP5B inhibited the expression of pEGFR,pPI3K,and pAKT,but improved the expression of PTEN.However,ATP5B had no significant effect on the expression level of EGFR,PI3K,and AKT.Administration of 740Y-P reversed the regulation of pPI3K,pAKT,and PTEN by ATP5B.However,knockdown of ATP5B exhibited converse results.Conclusions:1.ATP5B is down-regulated in CRC tissues,and negatively correlated with TNM stage and lymph node metastasis.2.Low expression of ATP5B is a risk factor for the prognosis in CRC patients,and ATP5B may be used as a prognostic marker for CRC.3.ATP5B inhibits proliferation,migration,and invasion,but promotes ROS generation and apoptosis in CRC cells.4.EGFR-PI3K-AKT pathway is involved in ATP5B-inhibited progression in CRC.