The Role Of AKT And TAOK1 In Esophageal Squamous Cell Carcinoma Growth And Its Inhibitor Screening

Background and PurposeEsophageal cancer(EC)is one of the most common malignant tumors in China,accounting for 53%of the incidence of esophageal cancer in the world.EC ranks the fourth in frequently diagnosed cancer types and leading cause of cancer death in China.Esophageal squamous cell carcinoma(ESCC),which acts as the main histological type of esophageal cancer,is highly prevalent in developing regions and accounting for 90%of the total incidence rate of esophageal cancer annually.In China,the incidence of ESCC has significant regional characteristics and concentrated in the North Central of China and Taihang Mountain,such as Linzhou(Henan)and Shexian(Hebei).Currently,surgery combined with chemoradiotherapy is a common method for clinical treatment of ESCC.5-Fluorouracil,cisplatin and paclitaxel are frequently used chemotherapeutic agents for ESCC.However,the incidence rate of ESCC is still continue rising,adverse effect are constantly emerging,and the 5 year survival rate is only 15%to 25%.Therefore,it is of great value to find and evaluate the new therapeutic method of esophageal cancer which will help to improve the 5-year survival rate and reduce the pain of esophageal cancer patients.PI3-K/AKT/mTOR pathway was reported to involved in carcinogenesis and exerted different functions.AKT,as a direct downstream target of PI3-K,is one of the key nodes of PI3-K pathway,which is considered as a very attractive therapeutic target.AKT plays an important role in the occurrence and development of various cancers.AKT is an important signal center with more than 100 downstream substrates,it affects cell metabolism,growth,survival and proliferation and has found to act as an oncogene in colon cancer,breast cancer and other cancers.However,there is few publications focused on the function of AKT in esophageal cancer.Mitogen activated protein kinases(MAPK)pathway is an important signaling pathway in carcinogenesis.Tao kinases belong to MAP3K family in p38 MAPK cascade.It has 1001 amino acid residues,which regulate the stability of cytoskeleton,G-protein-coupled receptor family signaling pathway,cell viability and activate p38 in DNA damage mechanism.Inhibition of TAOK1 enhanced the depolymerization of centrosome and destroy mitosis of centrosome enriched breast cancer cells,revealing the possibility of TAOK1 becoming a potential target of breast cancer.At present,there are few reports about the function of TAOK1 in cancer,and its function in esophageal cancer is still waiting to be explored.Natural molecular inhibitors are a kind of bioactive components in plants,such as carotenoids,flavonoids,anthocyanins,etc.Some natural compounds have the ability to regulate cell cycle,cell differentiation and apoptosis through different signaling pathways.Compared with chemical synthetic small molecular compounds,natural small molecular compounds are more safety and have lower cytotoxicity in human body which making it to have a better effect to deal with the unexpected situation in cancer treatment.Therefore,screening and studying natural small molecular inhibitors of AKT and TAOK1 will bring huge significance to the clinical prevention and treatment of esophageal cancer.In this study,we explored the underlying function of AKT and TAOK1,and identified their onco-protein role in ESCC.Furthermore,we screened the inhibitor of AKT and TAOK1 and found that oridonin and resveratrol specific target AKT and TAOK1 kinase in ESCC,separately.Finally,we evaluated and verified the effects of this two compounds in ESCC cells and PDXs,the results indicated that oridonin and resveratrol inhibited ESCC in vitro and in vivo by directly targeting AKT or TAOK1.Methods1.Detection the expression of p-AKT and AKT(pan)in ESCC tissue array by IHC;Detection the expression of TAOK1 in ESCC cells and tissues by IHC and western blot;2.Exploration the function of AKT1/2 in ESCC via shRNA and overexpression;Evaluation the related mechanism of TAOK1 in ESCC by shRNA,overexpression,MTT,soft agar and flow cytometry;3.Evaluation the effect of MK-2206 in ESCC by MTT assay;4.Verification the function of TAOK1 in ESCC through CDX and PDX animal experiment;5.Measurement of the binding between oridonin and AKT 1/2 by binding assay and ATP competitive binding experiment;Detection the activity of oridonin on AKT1/2 kinase by in vitro kinase assay;Docking model was used to simulate the combination of oridonin with AKT 1 and AKT2 protein;6.Screening high affinity natural small molecular inhibitors of TAOK1 through computational docking model;Evaluation the IC50 dose of those compounds via MTT assay;Verification the action between inhibitor and TAOK1 by binding assay,kinase assay,KINOME ScanTM profiling and SPR methods;7.Measurement of ESCC cell proliferation after the treatment of oridonin and resveratrol by MTT and soft agar assay,separately;8.Detection cell cycle and cell apoptosis by flow cytometry combined with western blotting;9.Evaluation the effect of oridonin on PI3-K/AKT signal pathway and resveratrol on p38 MAPK signal pathway by western blot or luciferase assay;10.Verification the inhibitory effect of oridonin and resveratrol in vivo by PDX animal experiment,separately.Results1.Both of p-AKT(ser473)and AKT were highly expressed in ESCC tissue samples;Overexpression of AKT promoted cell proliferation and knock-down of AKT inhibited cell growth in vitro;2.MK-2206,a specific synthetic inhibitor of AKT,significantly reduced cell growth in ESCC cells;3.Oridonin directly bond with AKT1/2 and decreased AKT1/2 kinase activity with the IC50 value 8.4±1.34 ?M(AKT1)and 8.9±0.29?M(AKT2),respectively;4.Oridonin combined with AKT1 at Asp292 in the ATP pocket and Asp293 and Lys277 in the ATP pocket of AKT2;5.Oridonin significantly inhibited ESCC cell proliferation,arrested cell cycle at G2/M phase,induced cell apoptosis,suppressed PI3-K/AKT signaling pathway and dramatically decreased ESCC PDX growth;6.TAOK1 was significantly overexpressed in ESCC tissues and cells;7.Knock-down of TAOK1 in ESCC inhibited cell proliferation and colony formation,arrested cell cycle at G1 phase,down-regulated p38 MAPK signaling pathway and suppressed the growth of ESCC CDX and PDX;Overexpression of TAOK1 promoted cell growth and colony formation,increased cell cycle progression,up-regulated p38 MAPK signaling pathway and promoted ESCC CDX tumor growth;8.20 natural small molecular inhibitors were predicted by computational docking software,among them,resveratrol showed the strongest IC50 dose toward ESCC cells;9.Resveratrol had a high affinity to TAOK1 kinase,it bond with TAOK1 directly in the ATP-binding pocket and significantly decreased TAOK1 kinase activity;10.Resveratrol significantly inhibited ESCC cell proliferation,induced cell apoptosis,down-regulated p38 MAPK signaling pathway and dramatically suppressed ESCC PDX tumor growth in vitro and in vivo.Conclusion1.AKT is overexpressed in ESCC,knock-down of AKT decrease ESCC proliferation,on the contrary,AKT overexpression promote cell growth.2.Oridonin is a specific inhibitor of AKT in ESCC with directly binding with AKT1/2 in a ATP-competitive way.3.Oridonin inhibits cell proliferation,cell cycle progression,induces apoptosis,attenuates the growth of ESCC PDX and interrupts PI3-K/AKT signal in ESCC by targeting AKT kinase.4.TAOK1 is overexpressed in both of ESCC tissues and cells,suggesting the potential of TAOK1 as an oncogene.Knock-down of TAOK1 suppress ESCC cell proliferation,arrest cell cycle progression,interrupt p38 MAPK signal pathway and inhibit ESCC CDX and PDX tumor growth.Overexpression of TAOK1 promote ESCC cell proliferation and cell cycle progression,up-regulate p38 MAPK signal pathway and increase ESCC CDX tumor growth.5.Resveratrol specifically targets TAOK1 in ESCC and decreases TAOK1 kinase activity.6.Resveratrol inhibits ESCC cell proliferation,colony formation,induces apoptosis and suppresses ESCC PDX tumor growth via directly targeting TAOK1.