The Investigation Of IATL Combined With MAPK Pathway Inhibitor In The Treatment Of BRAF Mutant Melanoma

Background: Clinically,BRAF and MEK inhibitors significantly prolonged the progression free survival(PFS)of BRAF mutated melanoma,but drug resistance limits its long-term efficacy.Previous studies have shown that targeted inhibition of the mitogen-activated protein kinase(MAPK)pathway in BRAF mutated melanoma cells promotes activation of the JAK2/STAT3 pathway.SOX2 protein is one of the factors involved in the reprogramming of cancer cells to IPS cells,and IPS cells have been proved to have strong resistance to targeted treatment of BRAF mutant melanoma.IATL is reported to exert anti-cancer effects in a different of tumors.However,the underlying effects is unclear in melanoma.Objective: The aim of this experiment was to investigate whether the combination of IATL and MAPK pathway inhibitors could improve the therapeutic effect of melanoma and delay the development of drug resistance through dual inhibition of MAPK and JAK2/STAT3 pathways,which in turn could down-regulate SOX2 and cancer cell proliferation and apoptosis-related protein expression.Methods: 1.The molecular docking method in network pharmacology was used to verify whether IATL could target STAT3 protein.2.Human skin melanoma cell lines A375,A375 R,A2058 and normal renal tubular epithelial cell line HK-2 were cultured and used for experiments when they grew to logarithmic cycle.3.In A375 cells,the experiment was divided into three parts.To verify IATL combined with BRAF inhibitor on the expression of JAK2/STAT3 and MAPK pathway related proteins,growth status and cell viability in A375 cells,the first part was divided into:(1)control group:DMSO solvent treatment;(2)vemurafenib or dabrafenib group;(3)IATL group with different concentration;(4)vemurafenib or dabrafenib+IATL with different concentrations.To verify the effect of IATL combined with BRAF and MEK inhibitors on the expression of JAK2/STAT3 and MAPK pathway related proteins and cell viability in A375 cells,the second part was divided into:(1)control group: DMSO solvent treatment;(2)vemurafenib or dabrafenib group;(3)cobimetinib or trametinib group;(4)vemurafenib+cobimetinib or dabrafenib+trametinib group;(5)IATL group;(6)vemurafenib or dabrafenib+IATL group;(7)cobimetinib or trametinib +IATL group;(8)vemurafenib+cobimetinib+IATL or dabrafenib+trametinib+IATL group.The morphological and growth changes A375 cells were detected by the reverse microscopy after the treatment of different drugs for 6h.Western blot were used to examine protein expression with MAPK and JAK2/STAT3 pathways,SOX2,c-Myc,Cyclin D1 and Cleaved PARP after 6 and 24 hours of drug treatment.Cell counting Kit-8 assays were used to assess the effects of different drug on A375 cells proliferation after 72 h.To verified the effects of IATL combined with BRAF or BRAF and MEK inhibitors on cell proliferation and apoptosis,the third part was divided into:(1)control group: DMSO solvent treatment;(2)vemurafenib group;(3)vemurafenib+cobimetinib group;(4)IATL group;(5)vemurafenib+IATL group;(6)vemurafenib+cobimetinib+IATL group.Colony formation assays were used to observe the ability of cell colonies to form,flow cytometry was used to evaluate the cell apoptosis.4.In order to verified the toxic and side effects of the combined drug on normal cells,HK-2 cells were treated as normal cells and grouped with A375 cells:(1)control group: DMSO solvent treatment;(2)vemurafenib group;(3)cobimetinib group;(4)vemurafenib+cobimetinib group;(5)IATL group;(6)vemurafenib+IATL group;(7)cobimetinib+IATL group;(8)vemurafenib+cobimetinib+IATL group.Protein expression levels of MAPK and JAK2/STAT3 pathways,SOX2,c-Myc,Cyclin D1 and cleaved PARP were detected by western blots after 24 h treatment.5.In order to further verify whether the combination of IATL and MAPK pathway inhibitor has the same inhibitory effect on drug-resistant cells,in A375 R and A2058 cells,the experimental group was divided into three parts as in A375 cells,where only vemurafenib and cobimetinib were used for BRAF and MEK inhibitors.Protein expression levels of MAPK and JAK2/STAT3 pathways,SOX2,c-Myc,Cyclin D1,and Cleaved PARP were detected by western blot after 24 h treatment.Clone formation rates of each drug group in A375 R cells were observed by plate cloning assay.Results: 1.IATL can target the SH2 domain of STAT3.2.The A375 cells without drug treatment were polygonal or spindle shape and proliferated more vigorously.The number of cells with treatment of vemurafenib and IATL was significantly reduced compared with that with vemurafenib alone,irregular morphology was more serious,and the number of desquamated suspension cells was significantly increased.3.After A375 cells were treated with drugs in each group for 6 hours,the expressions of p-STAT3/STAT3,SOX2,c-Myc and Cyclin D1 in vemurafenib or dabrafenib+IATL groups were significantly lower than those in vemurafenib or dabrafenib groups(P<0.05),increased Cleaved PARP protein expression(P<0.05).After 24 hours of treatment,the expression of p-STAT3/STAT3 and SOX2 in the vemurafenib or dabrafenib+IATL group with different concentrations was still lower than that in the vemurafenib or dabrafenib group(P<0.05),the expression of Cleaved PARP was still increased(P<0.05),and there was no statistically significant difference in expression of other proteins(P>0.05).Similarly,the p-STAT3/STAT3 and SOX2 expressions in vemurafenib+cobimetinib+IATL or dabrafenib+trametinib+IATL groups were also lower than those in vemurafenib+cobimetinib or dabrafenib+trametinib groups(P<0.05).4.In A375 cells,the survival rate of vemurafenib or dabrafenib+IATL groups was higher than that of either drug(P<0.05).Similarly,compared with any single or combination of drugs,the survival rate of cells in the vemurafenib+cobimetinib+IATL or dabrafenib+trametinib+IATL group was also higher(P<0.05).5.In A375 cells,the percentage of apoptosis was significantly increased in the vemurafenib+IATL group or vemurafenib+cobimetinib+IATL group compared with any of the drugs(P<0.05).6.Compared with normal HK-2 cells,vemurafenib+IATL selectively inhibited MAPK and JAK2/STAT3/SOX2 pathways in A375 cells,and increased Cleaved PARP expression(P<0.05).However,both pathways in HK-2 cells and A375 cells were inhibited after the addition of cobimetinib,but the expression of Cleaved PARP was still more significant in A375 cells(P<0.05).7.In A375 and A375 R cells,the clone formation rate in vemurafenib+IATL or vemurafenib+cobimetinib+IATL groups was significantly lower than that in either of them(P<0.05).8.In A375 R and A2058 cells,the expression of p-STAT3/STAT3,SOX2 and Cyclin D1 in the vemurafenib+IATL group was significantly lower than that in the vemurafenib group(P<0.05).Similarly,the expression of p-STAT3/STAT3,SOX2 and Cyclin D1 in the vemurafenib+cobimetinib+IATL group was also lower than that in the vemurafenib+cobimetinib group(P<0.05),c-Myc and p-ERK1/2/ERK1/2 were not activated(P>0.05),and the expression of Cleaved PARP was increased(P<0.05).Conclusion: 1.The JAK2/STAT3/SOX2 pathway plays an important role in resistance to MAPK pathway inhibitors in the treatment of BRAF mutated melanoma.2.IATL combined with MAPK pathway inhibitors may down regulate the expression of SOX2 and c-Myc by jointly inhibiting JAK2/STAT3 and MAPK pathways,thus promoting the death of BRAF mutated melanoma cells,and the toxic side effects may be low.