The Role And Mechanism Of Ribosomal Protein S6 Kinase 2 Inhibitor In Melanoma

Background: Cutaneous melanoma is a highly life-threatening tumor causing the majority of the cutaneous cancer related deaths,and one of the most difficult tumors to treat.Over the past years,several novel systematic therapies including immune-checkpoint inhibitors,targeted therapies,or the combine of both,have contributed to improve survival rate of melanoma patients.However,long-term clinical efficacy is often compromised because of innate or acquired resistance and dose-limiting toxicity.Therefore,it is necessary to clarify the disease pathogenesis and find new therapeutic drugs with low toxicity for melanoma treatment.There are numerous genomic mutations in melanoma patients.The mainly mutation genes include BRAF,NRAS,KIT and are associated with the treatment and prognosis.Mutations of these genes usually lead to abnormal activation of RAS/RAF/MEK signaling pathway.Therefore,targeting the signaling pathways is promising treatment for advanced melanoma patients.RSK2 is an important downstream effector of the MAPK pathway that controls proliferation,transformation,cell cycle progression,and apoptosis.RSK2 phosphorylates various nuclear proteins including c-Fos,P53,cyclic AMP responsive element-binding(CREB),activating transcription factor4(ATF4),nuclear factor of activated T cells 3(NFAT3).RSK2 is also dysregulated in several malignancies,such as breast,lung,and prostate cancers,and malignant melanoma.Previously,we found that knockdown of RSK2 expression in melanoma cells induces autophagy and inhibits its proliferation,migration and invasion.More importantly,RSK2 inhibition significantly attenuated BRAF inhibitor resistant melanoma cells malignant phenotype.These findings highlighted the significance of targeting RSK2 in melanoma.To study the effects of AE007 on the proliferation and invasion of melanoma cells,and to explore the effect and mechanism of AE007 on melanoma through in vivo and in vitro experiments.Methods:1.Combining virtual screening method and CCK-8 experiment to screen out AE007 with significant inhibitory effect on melanoma cells;2.CCK-8 experiment to detect the effect of AE007 on the proliferation of melanoma cells and non-tumor cells,and to detect the IC50 value;3.Flow cytometry and western blot assays to detect melanoma cell cycle and apoptosis;4.Pull down experiments to study the direct binding of AE007 to RSK2;5.In vitro phosphorylation experiments to study the phosphorylation of the downstream substrate IκBα by the kinase RSK2;6.The effect of AE007 on the expression of RSK2 and its downstream p-CREB was studied by western blot;7.The changes in the gene transcription level of AE007 were verified by sequencing and RT-PCR experiments;8.Detect the effect of AE007 on tumor proliferation and metastasis in mice by skin melanoma xenograft model and lung metastasis mouse model;9.Construct stable RSK2 knockout by plasmid extraction experiment,lentivirus packaging experiment and lentivirus infection experiment The effect of AE007 on the proliferation of knockdown RSK2 melanoma cells was examined.Results:1.Through computer simulation docking,28 small-molecule compounds targeting RSK2 were screened from the database,among which AE007 had the best inhibitory effect on melanoma cells,and it is 3-nitro-N’-(5,6,7,8-tetrahydrobenzo [4,5] thieno [2,3-d] pyrimidin-4-yl)benzohydrazide,with a molecular weight of 369.4;2.AE007 directly binds to RSK2 and inhibits the protein kinase activity of its downstream IκBα,and reduces the expression of p-CREB;Objective:3.AE007 inhibited the proliferation of melanoma cells SK-MEL-5and SK-MEL-28 in a concentration-and time-dependent manner,but had no significant effect on the proliferation of non-tumor cells PIG1 and Ha Ca T;4.The IC50 value of AE007 in inhibiting melanoma cells was significantly lower than that of the commercial RSK2 inhibitor Carnosol.After 48 hours of AE007 treatment,the IC50 values of A375,SK-MEL-5and SK-MEL-28 cells were 7.152 μM,7.079 μM and 9.050 μM,respectively.while the IC50 values of A375,SK-MEL-5 and SK-MEL-28 were 36.40 μM,38.20 μM and 40.41 μM after Carnosol treatment,respectively;5.AE007 inhibited the invasion and migration of melanoma cells SK-MEL-5 and SK-MEL-28 in a concentration-and time-dependent manner;6.AE007 can induce cycle arrest and apoptosis of melanoma cells SK-MEL-5 and SK-MEL-28,up-regulate the cleavage of pro-apoptosis-related protein Parp,down-regulate the expression level of anti-apoptotic protein Bcl-2,and down-regulate at the same time Expression levels of cycle-related proteins Cyclin B1,Cyclin D1 and CDK2;7.AE007 can effectively inhibit the growth of melanoma cell SK-MEL-5 subcutaneously transplanted tumor in nude mice and the expression of Ki67,and can also significantly reduce the number of lung metastatic nodules of melanoma cell B16F10 in vivo;8.RNA sequencing analysis of SK-MEL-5 and SK-MEL-28 in melanoma cells after AE007 treatment,bioinformatics analysis and PCR results showed that the expression of CCNB2 involved in the cell cycle was significantly reduced;Apoptosis-related P21 and GADD45 A expressions were significantly increased,suggesting that the mechanism by which AE007 inhibits melanoma cells is related to P21,DNA replication and cell cycle.Conclusion:1.AE007 can inhibit the proliferation of melanoma cells in vivo and in vitro,and has low toxicity to non-tumor cells,Compared with the commercial RSK2 inhibitor Carnosol,AE007 has a lower IC50 on melanoma cells,indicating that AE007 has a better research value;2.AE007 directly binds to RSK2 and inhibits the expression of RSK2,while inhibiting its downstream protein kinases,and reducing the expression of p-CREB,causing cell cycle arrest and apoptosis,thereby significantly inhibiting the proliferation,migration and proliferation of melanoma cells.There are totally 20 figures,13 tables and 84 references.