Norcantharidin Overcomes Vemurafenib Resistance In BRAF Mutant Melanoma

BackgroundMelanoma is derived from malignant melanocytes,which promote migration and metastasis due to neural crest origin.Despite accounting for only 1% of skin cancer,it constitutes about 80% of deaths from all,with a 5-year survival rate of between 5% and19%.BRAF mutation can make BRAF activity abnormally enhanced,MAPK pathway over-activated,and promote tumor cell proliferation,invasion and metastasis.BRAF mutated melanoma accounts for over 50% of melanoma in global data,and about 25% of melanoma in China,of which V600 E mutant represents 70%–90%.Vemurafenib(Vem)is an FDA-approved drug for the treatment of late-stage melanoma in 2011.By specifically targeting V600E-mutated BRAF,Vem has shown appreciable antitumor activity,and improved survival rates in patients with BRAF V600E-mutant melanoma.Unfortunately,most of these patients treated with Vem will develop acquired resistance,which provides a great challenge for follow-up treatment.Despite Vemurafenib,Dabrafenib,and Trametinib are approved for melanoma by FDA in2015,but drug resistant is inevitable.At present,the known mechanisms of drug resistance of vemurafenib resistance melanoma include reactivation of MAPK pathway and PI3K/AKT/m TOR pathway,secondary mutation of MAPK and phenotypic changes.Thus,uncovering the mechanism underlying resistance development and discovering effective therapies to resolve resistance is still the primary concern in clinics.Cantharidin(CTD)is a natural toxin extracted from it.Its chemical structure is 7-oxabicycloheptane-2,3-dicarboxylic acid derivative.Norcantharidin(NCTD)is a novel self-dependent anti-cancer drug in our country.It is a demethylated analogue of CTD.It is a water-soluble synthetic small molecule with significant anti-tumor activity,small toxic side effects and unique characteristics of raising white blood cells.It has been used in the treatment of liver cancer,gastric cancer,and esophagus cancer,but it is not currently used for the treatment of melanoma.NCTD can inhibit cell proliferation,induce cell cycle arrest,induce apoptosis,block invasion/metastasis,and enhance immunity.However,the targets of antitumor action are unclear,which limit their clinical application.In my study,we had established a drug resistant melanoma cell line(A375R),and found NCTD can significantly reverse the drug resistance of melanoma cell line.Using the integration technology of muti-omics,combined with the cell experiments,it revealed that NCTD can inhibit the pentose phosphonate pathway and lipogenesis in drug resistant melanoma cells by downregulating the m TOR pathway.m TOR pathway plays a key role in the mechanism of drug resistance of Vem.This study provides experimental basis and new strategies for the treatment of vemurafenib-resistant melanoma.Meanwhile,the integration of multi-omics and interdisciplinary provides a new way for the research of molecular mechanisms of vemurafenib-resistant melanoma.Methods 1、A drug resistant cell line A375 R was established by the parental melanoma cell line A375.2、The proliferation of A375 and A375 R cells were detected by CCK-8,and the changes of drug resistance related proteins including p-ERK,p-AKT and p-Glycoprotein in melanoma were observed by Western blot analysis.3、CCK-8 was used to screen the anti-tumor drug,which was sensitive to A375 R cell line.And we found NCTD more significantly inhibit the proliferation of A375 R cells than A375 cells.4、Western blot was used to analyze changes of proteins related to melanoma cells and drug-resistant melanoma cells separately treated with different concentrations of NCTD(0,25,50 and 75 μ M).The changes of cell cycle and apoptosis in A375 and A375 R cells treated with NCTD were observed by Flow cytometry.The crystal violet staining was used to investigate the proliferation of A375 and A375 R cells treated with different concentrations of NCTD(0,25,50 and 75 μ M)for 7 days.5、Nude mice subcutaneous tumorigenesis test was used to detect the inhibition of NCTD on the growth of vemurafenib-resistant melanoma cells in mice.6、The alterations of metabolites and lipids in A375 and A375 R cells treated with NCTD were analyzed by capillary electrophoresis-mass spectrometary(CE-MS)and liquid chromatography-mass spectrometry(LC-MS).7、The changes of gene in A375 and A375 R cells treated with NCTD were analyzed by transcriptome analysis.8、The molecular mechanisms of NCTD against vemurafenib-resistant melanoma were analyzed based on the contents of literature reports and the results of metabonomic and transcriptomic bioinformatics analysis.The proteins were verified by Western blot analysis.9、All experimental data were expressed as the mean±standard deviation of three repeated tests.Chart using Excel and Graph Pad Prism 5.The data were not normally distributed an compared between groups were analyzed by the t test and one-way ANOVA test.Grayscale analysis uses Image J software web version(https://cnij.imjoy.io).p<0.05 indicated statistical difference.Results Section 1 Establishment of vemurafenib-resistant melanoma cell line A375 R and the inhibitory effect of norcantharidin on vemurafenib-resistant cells.1.The IC50 of vemurafenib(0,0.01,0.02,0.04,0.08,0.16,0.32,0.64,1.25 and 2.5 μM)on A375 cells was 0.157μM after 48 hours.The IC50 of vemurafenib(0,0.3125,0.625,1.25,2.5,5,10,20,40 and 80 μM)on A375 R cells was 21.5μM.2.p-ERK,p-AKT and p-Glycoprotein related to melanoma drug resistance were significantly up-related in drug resistant cells.3.NCTD can significantly inhibit the proliferation of A375 R by CCK-8 method.4.NCTD caused death by inducing G2/M phase arrest in A375 R cells by Flow cytometry.5.NCTD showed the effect of overcoming the drug resistance of vemurafenib in nude mice subcutaneous tumorigenesis test.Section 2 Exploring the molecular mechanism of NCTD against vemurafenib resistant melanoma based on multi-omics analysis.1.The levels of lauric acid,ethanolamine phosphate,phenylacetylglycine,Sedum heptanose 7-phosphate,glucose 1-phosphate,and 6-phosphate glucuronic acid in A375 R cells increased significantly,but decreased significantly after NCTD treatment by CEMS.Pathway enrichment analysis showed that NCTD down-regulated the pentose phosphate pathway of drug resistant cell A375 R.2.LC-MS showed most triacylglycerol,sphingomyelin,phosphatidylglycerol,hexoceramide and cholesterol ester significantly upregulated in A375 R cells,while A375 R significantly downregulated after NCTD treatment.It is suggested that NCTD can down-regulate the lipogenesis of A375 R cells.3.In A375 R vs A375 group,965 genes were upregulated and 521 genes were downregulated;In A375 R vs A375R+NCTD group,140 genes were upregulated and 630 genes were downregulated;In A375 vs A375+NCTD group,314 genes were upregulated and 986 genes were downregulated.4.Based on the contents of literature reports and the results of metabonomic and transcriptomic bioinformatics analysis,NCTD can significantly downregulated the genes related to pentose phosphate pathway and lipid synthesis,which was consistent with the results of metabonomics.The mechanisms of vemurafenib resistant melanoma may be closely related to downregulation of m TOR pathway.At the same time,NCTD can also down-regulate genes related to G2/M phase arrest,which is consistent with flow cytometry.5.RNA-seq analysis showed BTN family genes BTNL8 and BTNL9,which played an important role in immune signaling pathway,were significantly upregulated in NCTD treated drug resistant cells.6.At the protein level,NCTD can down-regulate the phosphorylation level of m TOR in A375 R cells in a concentration-dependent manner,but has no significant effect on A375 cells.Conclusion 1.Vemurafenib-resistant melanoma cell line A375 R was successfully constructed.2.NCTD significantly inhibited the proliferation of vemurafenib-resistant melanoma cell A375 R.3.NCTD could induce G2/M arrest of A375 R cells,but did not induce apoptosis.4.NCTD significantly inhibited the proliferation of vemurafenib-resistant melanoma cells in nude mice.5.NCTD specifically down-regulated pentose phosphate pathway and lipogenesis in drug resistant cell A375 R via inhibiting m TOR pathway.