The Study Of The Mechanism And Synergistic Lethal Effect On SHP2 Inhibitor SHP099 Together With AKT Inhibitor MK-2206 In Colorectal Cancer

Background:Colorectal cancer(CRC)is one of the most common malignant tumors in the world.Surgery and chemotherapy are still common strategies in clinical treatment.Although targeted therapy combined with chemotherapy had extended the average survival of patients with advanced CRC from 15 months to 30 months,it has limited to 30 months at present.SHP2,a non-receptor protein tyrosine phosphatase encoded by PTPN11 gene,mediates the regulation of RAS/MAPK and PI3K/AKT signaling pathways by receptor protein tyrosine kinase(RPTK).Hyperactivity of two signaling pathways occur frequently in many tumors including CRC.Therefore,SHP2 shows great potential as a therapeutic target for cancer.SHP099 is a novel allosteric SHP2 inhibitor and triggers less off-target effects.However,research has found that CRC cells with KRAS or BRAF mutants are insensitive to SHP099.And nearly 50%CRC patients in clinical have KRAS or BRAF mutants.Therefore,it has an important clinical significance to solve the problem that CRC cells with KRAS or BRAF mutants are insensitive to SHP099.Drug combination is an important method to solve the insensitivity of single drug in clinical.This paper aims to solve the problem that CRC cells with KRAS or BRAF mutants are insensitive to SHP099 by using drug combination.Methods:High-throughput and short-term growth inhibition assay was used to find drugs that can increase sensitivity of SHP099;CCK-8 cell proliferation assay,long-term plate colony formation assay,flow cytometry cell cycle and apoptosis analysis assay,and tumour xenograft assay were used to evaluate the antitumor effects of SHP099 combinated with MK-2206 in CRC cells in vitro and in vivo;Combination index(CI)analysis assay was used to evaluate the synergy of SHP099 combined with MK-2206;Western blot and immunohistochemistry assays were used to detect the expression or phosphorylation of target proteins such as AKT after treating with SHP099 or MK-2206 in cells or tissues;qRT-PCR was used to detect the mRNA levels of RTKs and RTK ligands in CRC cells treated with SHP099;RTK tyrosine phosphorylation antibody chips were used to detect the phosphorylation of RTKs in CRC cells treated with SHP099.Results:1.SHP099 has a stronger killing or growth inhibitory effect on SW480,RKO and Caco-2 cell lines when combined with 5-Fu,Oxaliplatin,Honokiol,MK-2206,Rapamycin,SB590885,Lapatinib and Pimasertib;2.SHP099 combined with MK-2206 has a synergistic lethal effect on CRC cells(0<CI<0.61),and significantly inhibit the cell proliferation and clony formation abilities,and triggered cell cycle arrest at GO/G1 phase,and promote apoptosis in vitro,and has no obvious side effects,inhibit tumor angiogenesis,inhibit the growth of SW620 and Colo205 cells in vivo;3.SHP099 combined with MK-2206 promotes apoptosis of CRC cells by up-regulating the protein expression levels of Cleaved-caspase-3 and Cleaved-PARP;4.The phosphorylation level of AKT(S473)was correlated with the sensitivity of SHP099.SHP099 showed dose dependent inhibition and activation effects on AKT(S473)phosphorylation levels in vitro and in vivo,respectively.MK-2206 reinforced the inhibition effect of SHP099 to the AKT/mTOR/S6K1/S6 signaling axis in vitro and blocked the activation of AKT by SHP099 in vivo,thereby it increased the sensitivity of CRC cells to SHP099;5.Low concentration FBS increases the sensitivity of SHP099 in RKO and SW620;6.SHP099 can up-regulate mRNA expression or tyrosine phosphorylation levels of multiple RTKs or RTK ligands associated with AKT activation in CRC cells.EphA2 may mediate the reactivation of AKT by SHP099 in RKO and Co1o205.Conclusion:Functionally,SHP099 combined with MK-2206 significantly inhibited the growth of CRC cells in vitro and in vivo,with a synergistic lethal effect.Mechanismly,SHP099 combined with MK-2206 induced apoptosis by up-regulated the levels of Cleaved-caspase-3 and Cleaved-PARP.Considering the two-sided feature of SHP099 on the regulation of AKT,MK-2206 can increase the sensitivity of SHP099 in CRC cells by enhancing the inhibitory effect of SHP099 on the AKT/mTOR/S6K1/S6 axis or blocking the reactivation of AKT by SHP099.SHP099 may activate AKT through different RTKs in different cells.