The Role And Molecular Mechanism Of EIF3a In The Vemurafenib Resistance Of Melanoma

Background: The serine/threonine kinase BRAF is a key regulator of the mitogen-activated protein kinase(MAPK)cascade,which modulates intracellular signal transduction and plays an important role in tumor growth,development and prognosis.BRAF mutation is the main oncogene in melanoma,among all identified BRAF missense mutations,BRAF V600 E with a single nucleotide transversion from valine to glutamate at position 600 is the most clinically prevalent mutation.The mutational activation of BRAF V600 E increases BRAF kinase activity extremely higher than the wild-type BRAF,leading to a subsequent persistent activation of MAPK signaling cascade,and promotes cell proliferation and apoptosis escape.Approximately 50 % of melanomas harbor activating BRAF mutations,the BRAF V600 E inhibitor vemurafenib is the first-line drug for the treatment of malignant melanoma.However,patients often develop resistance to vemurafenib after a few months of treatment,which severely limits the therapeutic effects of vemurafenib and restricted the prognosis of melanoma patients.Nowadays,the mechanism of vemurafenib resistance has not been fully elucidated and there is still no effective strategy to overcome resistance clinically.Therefore,it is urgent to identify the molecular mechanisms of vemurafenib resistance and develop novel drug targets.Eukaryotic translation initiation factors 3a(eIF3a)is associated with physiological and pathological processes and elevated eIF3 a expression favors the maintenance of the tumor malignant phenotype.Many studies have suggested that eIF3 a affects patient prognosis and treatment responses.In recent years,eIF3 a has received extensive attention and research,and in-depth exploration of the biological function of eIF3 a may provide new therapeutic strategies for the treatment of tumors.Objective: This study aims to explore the mechanism of vemurafenib resistance in melanoma and reveal new biomarkers that affect the sensitivity of vemurafenib.Based on molecular biology experiment to explore the role and molecular mechanism of eIF3 a regulating melanoma vemurafenib resistance,and clarify eIF3 a as a new molecular marker affecting the sensitivity of vemurafenib in vitro and in vivo.In addition,this study intends to develop novel drug combinations to reverse vemurafenib resistance and provide a new strategy and theoretical basis for the treatment of melanoma.Methods: Gene Set Enrichment Analysis(GSEA)was used to explore the changes of pathways after silencing eIF3 a.CCK-8,EDU and plate cloning assay were used to detect the effect of eIF3 a on the sensitivity of vemurafenib.Bioinformatics analysis was used to reveal the correlation between the eIF3 a expression level and the sensitivity of skin tumor cells to vemurafenib via the tumor Dependency Map Portal database.Western blotting,and immunofluorescence experiment proved the effect of eIF3 a on ERK activity.Protein spectrum analysis combined with Western blotting were performed to reveal the possible phosphatase regulated by eIF3 a.The intracellular proteins synthesis efficiency and the regulation of eIF3 a on PPP2R1 B translation were analyzed by metabolic labeling with puromycin,luciferase,and RNA binding protein immunoprecipitation(RIP)assay.The correlation between eIF3 a and PPP2R1 B expression was further verified by immunohistochemistry in animal tissues,TCGA,and Human Protein Atlas database.Next,a phospho-tyrosine kinase array was utilized to assess whether dysregulation of tyrosine kinases caused by eIF3 a downregulation may contribute to vemurafenib resistance.CCK-8 assay was used to evaluate the inhibitory effect of these tyrosine kinase inhibitors on vemurafenib resistant cells.The inhibitory effect of BMX inhibitor CHMFL-BMX-078 on vemurafenib resistant cells was further detected by CCK-8,EDU and clone formation experiment,sphere formation assay and flow cytometry.CCK-8,EDU,and flow cytometry assay was used to detect the reversal effect of BMX inhibitor CHMFL-BMX-078 on vemurafenib resistance.Compu Syn software was used to calculate the Combination Index(CI)of vemurafenib and CHMFL-BMX-078.Western blotting was used to demonstrate the molecular mechanism of CHMFL-BMX-078 reversing the drug resistance of melanoma.Lastly,a xenograft model of A375 R cells were established to test whether CHMFL-BMX-078 could significantly enhance vemurafenib efficacy in vivo,and H&E staining and blood biochemical analysis were used to detect the liver and kidney toxicity in nude mice.Results: Gene enrichment analysis showed that silencing eIF3 a activated the KRAS pathway,which suggested that eIF3 a may be involved in the vemurafenib resistance.eIF3 a was significantly down-regulated in vemurafenib resistant melanoma cells,and the level of eIF3 a was correlated with the sensitivity of vemurafenib.Meanwhile,the expression of eIF3 a also affected the sensitivity of another BRAF inhibitor dabrafenib.Through bioinformatics website analysis,it was found that the IC50 value of skin tumor for vemurafenib was negatively correlated with eIF3 a expression level in Dep Map Portal database.Mechanistically,we found that silencing eIF3 a activated the ERK signaling pathway,the downstream of KRAS.After silencing eIF3 a,the inhibitory effect of vemurafenib on ERK was weakened,and combination of ERK inhibitors increased the cytotoxicity of vemurafenib against cancer cell with silencing eIF3 a.Proteomics showed that silencing eIF3 a down-regulated the expression of phosphatase PPP2R1 B.Silencing PPP2R1 B could activate ERK,and eIF3 a could affect ERK phosphorylation through PPP2R1 B,thus determining the sensitivity of melanoma cells to vemurafenib.Further studies demonstrated that eIF3 a could regulate the expression of PPP2R1 B at the translation level.Immunohistochemistry experiments in animal tissues and bioinformatics analysis via TCGA database and Human Protein Atlas database further showed a positive correlation between eIF3 a and PPP2R1 B.The phosphorylation expression levels of four tyrosine kinases,including EGFR,ACK1,Eph A2,and BMX,were increased after eIF3 a knockdown in A375 cells via a phospho-tyrosine kinase array,indicating that these kinases may contribute to vemurafenib resistance.Furthermore,the results in vitro showed that the BMX inhibitor CHMFL-BMX-078 selectively reduced cell viability,clone formation,and the number of EDU positive cells of vemurafenib-resistant melanoma cells and caused cell cycle arrest in G0/G1 phase.A combination therapy of vemurafenib and BMX inhibitor CHMFL-BMX-078 could synergistically inhibit the proliferation of vemurafenib-resistant melanoma strains and induce cell cycle arrest,and their combination index(CI)is < 0.3,indicating a strong synergism.Furthermore,our findings showed that CHMFL-BMX-078 could exert anti-tumor effect by inhibiting the activity of Akt.In addition,the combination treatment of vemurafenib and CHMFL-BMX-078 suppressed the Akt and ERK signaling pathways,which are both important in vemurafenib resistance.In vivo xenograft tumor model of nude mice further demonstrated that the combination of vemurafenib and CHMFL-BMX-078 could reverse the vemurafenib resistance without causing significant toxicity to liver and kidney.Conclusions:1.The downregulation of eIF3 a contributes to vemurafenib resistance in melanoma;2.eIF3 a modulates vemurafenib resistance by regulating the translation of phosphatase PPP2R1 B,thereby affecting the phosphorylation of ERK.Our study revealed a new pathway in cancer cell,eIF3a-PPP2R1B-ERK,that affects vemurafenib resistance;3.The combination treatment with CHMFL-BMX-078 and vemurafenib overcame vemurafenib resistance by suppressing both Akt and ERK signaling pathways in vitro and in vivo,which might serve as a new adjuvant agent for treating patients with vemurafenib resistance.