PTEN Gene Status Affects Melanoma Resistance Mechanism To BRAF Inhibitor

BackgroundMelanoma,is a malignant tumor that develops from melanocytes and primarily involves the skin.Cutaneous melanoma is the most potentially dangerous form of skin cancer.Until recently,metastatic melanoma was a poor prognostic disease with very limited treatments and low curative effect.The therapeutic landscape for metastatic melanomais rapidly changing with the development of immunity and targeted therapy,which have demonstrated better efficacy than traditional chemotherapy.Mutations of BRAF are present in 40-60%of the melanomas,and 90%of them are BRAF V600E substitution.Selective BRAF inhibitors(BRAFi)Vemurafenib(PLX-4032)is one of BRAFi,which had been approved for clinical application in 2011.PLX-4032 directly targets the MAPK pathway and significantly improve the overall and progression-free survival of patients with BRAF-mutant melanomas.Unfortunately,despite the initial success of BRAF inhibitor therapy,acquired resistance of BRAF-mutant melanoma to B-Rafis develops within one year and almost invariably.A thorough understanding of the causes of resistance to BRAFi is necessary to develop more effective treatment strategies.Numerous mechanisms of resistance have been predicted and detected based on in vitro and in vivo models and many of them have been confirmed on pre-and post-treatment tumor samples.Resistance mechanisms are associated mainly with the reactivation of the MAP kinase pathway and the PI3K-AKT pathway.Proposed mechanisms include:Overexpression or hyperactivation of RTKs,such as PDGFRs,EGFR,FGFRs,IGF1R;Activating NRAS and MEK mutations.PTEN plays an important role insignal transduction and identified as a tumor suppressor frequently altered in melanomas.Loss of PTEN,which is observed in 30%of melanoma specimens and often occurred simultaneously with the BRAF mutation,may contribute to intrinsic resistance to BRAFi via increased PI3K/AKT signaling and suppression of apoptosis.There is,however,evidence that some BRAF mutant/PTEN-melanoma cells still respond to BRAFi monotherapy.Thus,the relationship between PTEN gene status and BRAF inhibitor resistance has not been reported.Therefore,this study focused on whether the PTEN gene status affects the acquired resistance of melanoma to BRAF inhibitors and its specific mechanisms.Methods and Contents1.We generated BRAF inhibitor-resistant human melanoma cells through increase stepwise exposure to PLX4032 in BRAFV600E-positive melanoma cells(4 strains of PTEN wild type 101,103,111 and A375sm,2 strains of PTEN mutations 105 and M28).2.Western blot was used to detect the activation of MAPK and PI3K/AKT pathway of resistant melanoma cells in PLX4032 at different concentrations or times.3.The inhibitory effects of BRAF,AKT and MEK inhibitors alone or in combination on the growth of drug-resistant cells were detected by CCK8 assay.CalcuSyn 2.11 software was used to analyze the synergistic effect of the two drugs.4.The phosphorylated RTK antibody array was used to detect the relative phosphorylation levels of 42 receptor tyrosine kinases(RTKs)in parental and drug resistant cells.The susceptibility of BRAF inhibitor to drug resistant cells was detected by AXL inhibitor R428.5.Statistical analyses:One-way ANOVA and Student’s t test were respectively used to compare the datas between the multiple groups and two groups.Results1.Six strains of BRAF inhibitor-resistant human melanoma cell lines 101R,103R,105R,111R,A375smR and M28R were successfully induced and the resistance index(RI)were 35.1,89.4,1.9,3.8,22.9 and 19.0,respectively.2.MAPK signaling pathway is activated in PTEN mutant and wild-type resistant cells;the PI3K/AKT pathway is only activated in PTEN wild-type resistant cells and is not activated or down-regulated in PTEN mutant resistant cells.3.In the PTEN wild-type resistant cells,the BRAF inhibitor had a synergistic inhibitory effect with the AKT inhibitor and had no synergistic inhibitory effect with the MEK inhibitor;In the PTEN mutant resistant cells,BRAF inhibitors have synergistic inhibitory effects with AKT inhibitors or MEK inhibitors,with better synergistic effects with MEK inhibitors;The combination of the three inhibitors showed a better effect than the two drugs only in PTEN mutant resistant cells.4.The phosphorylation level of the receptor tyrosine kinase AXL protein was only significantly elevated in PTEN wild type resistant cells.By inhibiting the activation of AXL protein,the sensitivity of drug-resistant cells to BRAF inhibitors was restored and the expression level of p-AKT was down-regulated.Conclusions1.Different status of PTEN gene melanoma have different mechanisms of resistance to BRAF inhibitors.2.Activation of PI3K/AKT signaling pathway is one of the major mechanisms leading to the resistance of PTEN wild-type melanoma to BRAF inhibitors.MAPK signaling pathway plays a leading role in the resistance of PTEN mutation melanoma to BRAF inhibitors.3.Receptor tyrosine kinase AXL mediates the resistance of PTEN wild-type melanoma to BRAF inhibitors by activating the PI3K/AKT signaling pathway and may be a new target for reversing melanoma resistance to BRAF inhibitor.