Simultaneous Targeting The PI3K And PERK Pathways In Esophageal Squamous Carcinoma

ObjectiveEsophageal cancer is a malignant tumor with high mortality and morbidity in the worldwide,and its incidence is still increasing year by year.China is known to be the highest-incidence country of esophageal cancer in the world,and at least 90%of esophageal cancers are squamous cell carcinoma.Due to late diagnosis,poor treatment and adverse effects and other shortcomings,esophageal cancer has become the fourth leading cause of cancer death in China.Chemotherapy is the most important and effective treatment strategies of patients with advanced esophageal cancer.However,drug resistance is the major reason for failure in esophageal cancer chemotherapy.Searching for novel therapeutic targets and drugs,and exploring new drug regimens are core areas of anti-esophageal cancer drug research and development.Molecular targeted therapy is a major advance in the field of cancer therapy,signal pathway abnormalities are mainly biological characteristics of tumor cells.PI3K signaling pathway is a critical anti-tumor pathway,in inhibition of apoptosis,promoting cell proliferation,promote tumor angiogenesis and tumor cell invasion and migration and so play an important role.And PI3K is usually activated abnormally in many human tumors,so PI3K is a great potential target for cancer treatment.LY294002 is the first synthetic small molecule inhibitor known to inhibit PI3K pathway,after which other PI3K inhibitors emerge in an endless stream.Studies of PI3K pathway in head and neck cancer,lung cancer and other tumors are more common.However,the relevant research of PI3K pathway in esophageal cancer is not much,and its clinical value and regulatory mechanisms are not clear.The aim of this study was to investigate the effects and mechanisms of PI3K pathway and its inhibitor LY294002 on the growth of esophageal squamous cell carcinoma.Methods(1)Gene expression and survival analysis of PI3Ks in esophageal cancer patients was carried out by using TCGA assay of the esophageal cancer database.(2)CCK8 assay was done to test the efficacy of PI3K inhibitor LY294002 in ESCC cells(Eca-109,EC9706,KYSE140,KYSE450)and normal vascular endothelial cells(HUVEC).(3)Western Blot assay was used to observe the changes of phosphorylated AKT,S6,eIF2α,and their background levels after LY294002 treatment in Eca-109 and EC9706 cells.(4)CCK8 assay was done to test the efficacy of PERK inhibitor GSK2656157 in ESCC cells(Eca-109,EC9706,KYSE140,KYSE450)and normal vascular endothelial cells(HUVEC).(5)siRNAs interfered with PERK or ATF4 were used to reduce the mRNA levels which were tested by qPCR assay.CCK8 assay was done to test the efficacy of siRNAs.(6)CCK8,Western Blot,Annexin V-FITC/PI,and clone formation assay were used to test the effects and mechanisms of LY combined with GSK2656157.Results(1)The mRNA levels of PI3Ks were expressed higher in esophageal cancer tissue compared to normal adjacent tissue.PI3Ks(especially PIK3CB,P = 0.007)alterations affected ESCC patients’ survival more significantly than that with EAC.These results implicated that PI3Ks expression may be prognostic indicators for ESCC specificity(2)LY294002 could dose dependently inhibit the growth of ESCC(Eca-109,EC9706,KYSE140,KYSE450),which had lower effect in HUVEC.It significantly reduced the number and the diameter of colony in a dose dependent manner.It effectively reduced the phosphorylation of AKT,S6 and eIF2a of in a time dependent manner,which indicated that PI3K inhibitors could affect the canonical mTOR and noncanonical PERK/eIF2α/ATF4 pathways.(3)The siRNAs targeting PERK caused a 20-40%decrease of their growth rate in Eca-109 and EC9706 cells.Using the siRNAs of ATF4 had a similar phenotype in both ESCC cell lines.The novel PERK inhibitor GSK2656157 could significantly inhibit the phosphorylation of eIF2a,and dose-dependently inhibit the growth of ESCC cells(Eca-109,EC9706,KYSE140,KYSE450)and have relative smaller effect to HUVEC.Clone formation experiments showed that GSK2656157 can significantly reduce the number and the diameter of colony in a dose dependent manner.(4)The inhibition rate of drug combination in Eca-109 cell and EC9706growth was significantly higher than that of LY294002 and GSK2656157.Combination therapy completely reduced the number and the diameter of colony.Combination could significantly enhance the PARP cleavage bands and reduce p-eIF2a level.Compared with single drug,the apoptosis rate of combination therapy was greatly increased.Dual upregulation of the key genes of PI3K(PIK3CB)and PERK pathways(EIF2AK3 and ATF4)had more significant predict value(PIK3CB&EIF2AK3,P = 0.001;Fig.4G,PIK3CB&ATF4,P = 0.0002)than that of using PIK3CB individually(P = 0.004).ConclusionWe initially provided a new strategy to evaluate the effect of clinical prognosis and a potential combination therapy in ESCC.We believe that co-analysis the mRNA expression of key genes in inhibition activity of PI3K and PERK pathways could better evaluate the clinical prognosis of the tumor patients.We developed a pharmaceutical composition of treating ESCC at which the effect of combination of LY294002 with GSK2656157 was obviously better than individual LY294002 and better clinical outcomes are expected even for other types of squamous carcinoma(such as lung cancer,head and neck cancer,etc).