Molecular Mechanism Of Combination Therapy To Overcome Primary And Acquired Resistance To Trametinib In Colorectal Cancer Cells

Background:Tumor targeted therapy blocks the growth of cancer cells by interfering with specific molecules which are needed for carcinogenesis and tumor development.Compared with traditional chemotherapeutic drugs,targeted therapeutic drugs have the advantages of high efficacy and less toxic side effects.However,in clinical practice,primary and acquired resistance limits the effectiveness of targeted therapy,which impede development of precision medicine and the personalized therapy.Trametinib is an inhibitor of MEK1/2,and pervious report showed that trametinib resistance was induced after therapy of CRC patients.Therefore,it is critical to investigate the mechanism of trametinib resistance and establish strategies to overcome resistance.One of the strategy that can be applied in clinic is combination therapy,which can partly or completely overcome the resistance to targeted drugs.In our previous work,we identified that two colorectal cancer cells with KRAS mutations SW480 and CW2 were resistant to trametinib.Therefore,our decision is to set up a synergistic combination of drugs or inhibitors that can reverse the primary and acquired resistance to trametinib in CRC cells,and analyze the molecular mechanism of trametinib resistance.This study will provide a reference for the selection of clinical targeted therapeutic drugs in CRC.Objective:To screen synergistic drug combinations with trametinib in colorectal cancer cells,to analyze the effects of drug combinations which have primary and acquired resistance to trametinib,and to explore the molecular mechanisms of trametinib resistance and the mechanism of resistance overcome of drug combination.Methods:1.Chou-Talalay method for screening synergistic drug combinationThe MTT assay was used to screen synergistic combination of chemotherapy drugs,and the “CompuSyn” software was used to calculate the combination index(CI)to quantitatively describe the synergistic(CI<1),additive(CI=1)and antagonistic(CI>1)effects.2.Establishment of an acquired trametinib resistant cell model RKO-RThe acquired trametinib resistant cell model was established by concentration gradient increment method.The parental cell RKO were sequentially grown in medium containing 0.01,0.05,and 0.1 ?M for at least three months.MTT assay,colony formation assay and flow cytometry were used to detect the difference between the acquired resistant cell and the parental cell line,and to determine whether the acquired resistant cell model was successfully established.3.The effect of combination therapy on CRC cellsCells were divided into four group including the blank control group,two single drug groups and combination group.The effect of drugs on the proliferation of SW480,CW2 and RKO-R cells was evaluated by colony formation assay.The expression level of Cleaved-PARP was detected by western blot.4.Molecular mechanism of trametinib resistance and combination therapyWestern blot was used to detect trametinib treatment at different time point on the expression of MAPK and PI3K-AKT signaling pathway proteins.Then,the expression of the above pathway proteins was detected after the combined treatment of trametinib and GSK2126458.Results: 1.The synergistic drug combination of trametinib and GSK2126458 was obtained,and the CI values were less than 1 in the primary resistant cell lines SW480,CW2 and the acquired drug-resistant cell line RKO-R after combination treatment.2.Compared with the control group and the monotherapy group,the drug combination could reduce cell clone numbers and increase the inhibition rates of colony formation.The expression level of Cleaved-PARP was also up-regulated.3.The phosphorylation levels of IGF1 R and AKT were up-regulated after SW480 was treated with trametinib.After treatment of CW2 cells with trametinib alone,the phosphorylated ERK was down,but the phosphorylated AKT was up.While the phosphorylated of ERK and AKT were not significantly changed after trametinib treatment to RKO-R cells.4.In SW480,CW2 and RKO-R cells,combination therapy reduced the phosphorylation levels of both ERK and AKT,and the combination therapy not only inhibited the MAPK pathway,but also blocked the PI3K-AKT pathway.Conclusion:While inhibiting the MAPK pathway,trametinib can activate the bypass signaling PI3K-AKT pathway,which may be one of molecular mechanisms of primary and acquired resistance to trametinib in colorectal cancer cells.The combination of trametinib and GSK2126458 can overcome the drug resistance through simultaneously block MAPK and PI3K-AKT pathways,and thereby inhibit the proliferation of colorectal cancer cells and promote apoptosis.