The Role And Mechanism Of MARK2 In Cisplatin Resistance Of Osteosarcoma Cells

BackgroundOsteosarcoma is the most common primary malignant bone tumor in children and adolescents.Primary therapy typically consists of surgery and neoadjuvant and adjuvant chemotherapy,keeping the five-year survival rate around 70%in patients with non-metastatic disease.Unfortunately,the outcome are reduced for patients with metastatic disease and have a poor response to initial treatment,with less than 25%5-year overall survival.Cisplatin(DDP)-based chemotherapy is a standard regimens for osteosarcoma.Chemotherapy resistance,especially cisplatin resistance,is a major challenge for recurrence and prognosis of osteosarcoma.There are many mechanisms that can lead to chemoresistance,including reduced drug influx,increased drug efflux,activation of detoxification systems,alteration of the targets of the drug,enhanced DNA repair,impaired apoptosis,and altered oncogene expression.Therefore,it is very necessary to find a novel biomarker that can influence the drug resistance and prognosis of chemotherapy.Microtubule-affinity regulating kinase2(MARK2),a member of the MARK family,is a serine/threonine protein kinase,playing an important role in neurodifferentiation,neurodegeneration,cell polarization,intracellular transport,and cell migration.Recent studies have found that MARK2 is closely related to malignant biological behavior and drug resistance of tumors.Overexpression of MARK2 was observed in cisplatin-resistant Hela cell lines.In cervical cancer,the sensitivity of cells to cisplatin increased after MARK2 gene silencing,but there was no significant change in the sensitivity to vincristine and paclitaxel.MARK2 is correlated with DNA damage response and cisplatin resistance in non-small cell lung cancer[8].However,the role and mechanism of MARK2 in cisplatin resistance of osteosarcoma remain unclear.Increasing drug efflux and inhibiting apoptosis are two important reasons for chemoresistance.P-glycoprotein(P-gp),a transmembrane glycoprotein,is considered as an energy-dependent drug efflux pump to reduce intracellular concentration of anticancer drugs.Increasing evidence indicates that overexpression of P-gp plays an important role in multidrug resistance of tumors.Besides,studies showed that high expression of P-gp was involved in cisplatin resistance and was associated with poorprognosis in osteosarcoma.Apoptosis is the main mode of chemotherapy-induced cell death.Furthermore,studies have showed that expression of apoptosis-related genes,including p53,BCL-2,BAX,etc.,are associated with cellular chemoresistance in osteosarcoma.Previous studies have shown that in many tumors,the expression of P-gp associated with chemoresistance is regulated by the PI3K/AKT/NF-?B pathway.Moreover,study has shown that AKT/NF-?B pathway is involved in regulating drug resistance of osteosarcoma.However,whether MARK2 promotes cisplatin resistance of osteosarcoma cells by regulating P-gp expression and cell apoptosis through PI3K/AKT/NF-?B signaling pathway still remains to be uncovered.PurposeChemotherapy resistance is a major clinical challenge in the treatment and prognosis of osteosarcoma.The purpose of this study is to investigate the role and mechanism of MARK2 in cisplatin resistance of osteosarcoma.MethodsWe first analyzed the correlation between MARK2 and chemoresistance and clinical prognosis in the public datasets.Next we analyzed the expression of MARK2 in osteosarcoma cell lines and normal osteoblast cell line using Western blotting.Then the role of MARK2 in osteosarcoma cisplatin-resistance was explored in vitro and in vivo.CCK-8 assay,Western blotting,Immunofluorescence,qRT-PCR,cell apoptosis assay,and rescue experiments were used to investigate the effect of MARK2 overexpression and silencing on cisplatin resistance,expression of P-gp,cell apoptosis,and PI3K/AKT/NF-?B signaling pathway.Results1.GEO database analysis showed that osteosarcoma patients with high expression of MARK2 had lower overall survival and poorer response to chemotherapy.Spearman rank correlation analysis found a positive correlation between MARK2 and P-gp mRNA expression in patients with osteosarcoma.MARK2 was significantly upregulated in osteosarcoma cells compared to normal osteoblast.After knocked down the MARK2 level by siRNA,the P-gp expression level decreased in MG-63 and MNNG/HOS cells.Inhibition of P-gp reduced cisplatin resistance in osteosarcoma cells.These results suggested that high expression of MARK2 was associated with poor prognosis of osteosarcoma and may be positively correlated with chemoresistance.2.Overexpression and inhibition of MARK2 promoted and suppressed,respectively,the resistance to cisplatin of osteosarcoma cells in vitro and in vivo.Overexpression of MARK2 increased the cisplatin IC50 of osteosarcoma cell lines,and inhibited the rate of cisplatin-induced tumor reduction in xenograft osteosarcoma tumors.Knockdown of MARK2 reduced the cisplatin IC50 of osteosarcoma cell lines,and promoted the rate of cisplatin-induced tumor reduction in xenograft osteosarcoma tumors.3.Overexpression of MARK2 promoted the expression of P-gp,inhibited the apoptosis rate of osteosarcoma cells,and increased the protein levels of p-PI3K p85,p-AKT,p-NF-?B/p65,and p-I?B-? in PI3K/AKT/NF-?B signaling pathway.Knockdown of MARK2 inhibited the expression of P-gp,promoted the apoptosis rate of osteosarcoma cells,and inhibit the protein levels of p-PI3K p85,p-AKT,p-NF-KB/p65,and p-I?B-? in PI3K/AKT/NF-?B signaling pathway.4.Rescue experiments showed that in osteosarcoma cells overexpressing MARK2 and treated with LY294002,the IC50 value of cisplatin was partially reduced,while the proportion of apoptotic cells was partially increased,with the expression of P-gp,p-AKT,p-NF-?B/p65,and BCL-2 inhibited,and the expression of BAX and cleaved caspase 3 increased.The expression of MARK2 is not affected.These results indicated that PI3K inhibitor rescued the effect of MARK2 overexpression on cisplatin resistance to some extent,further confirming that MARK2 overexpression enhanced P-gp expression and decreased cell apoptosis through PI3K/AKT/NF-?B signaling pathway activation,resulting in cisplatin resistance.ConclusionMARK2 overexpression is correlated with chemoresistance and poor prognosis in osteosarcoma.MARK2 promotes cisplatin resistance in osteosarcoma cells by regulating P-gp expression and cell apoptosis through the PI3K/AKT/NF-?B signaling pathway.These results indicate that MARK2 may be a new potential therapeutic target and a biomarker for predicting chemotherapeutic resistance in osteosarcoma.