Acylglycerol Kinase Promotes Paclitaxel Resistance In Nasopharyngeal Carcinoma Cells By Regulating FOXM1 Via The JAK2/STAT3 Pathway

ObjectivesNasopharyngeal carcinoma(NPC),a malignant tumor of nasopharyngeal epithelial tissue,is more likely to occur in many provinces in southern China,especially in Guangdong and Guangxi areas,which is commonly known as "Guangdong tumor".Its morbidity and mortality rank in the forefront of head and neck tumors.Currently,paclitaxel is one of the most widely used chemotherapeutics in clinical practice.It has also achieved certain therapeutic effects in the clinical treatment of a variety cancers such as gastric cancer,ovarian cancer and NPC.However,the subsequent emergence of drug resistance greatly limits its therapeutic effects,which has become an important cause of treatment failure in middle and advanced patients.Therefore,in-depth exploration of the molecular mechanism of paclitaxel resistance in NPC and finding its potential targets is also a major strategic task that needs to be solved in clinical practice.AGK(Acylglycerol kinase),located on chromosome 7q34,is originally thought to be a mitochondria-localized lipid kinase that catalyzed the formation of lysophosphatidic acid(LPA)and phosphatidic acid(PA)by monosylglycerol and diglycerol.In recent years,increasing studies have found that AGK is abnormally up-regulated in multiple cancers such as cervical cancer,liver cancer,breast cancer,NPC,and it can be functioned as an oncogene to participate in many malignant biological behaviors of tumor cells.However,Whether AGK is involved in the mechanism of paclitaxel resistance remains to be studied.The objectives of this study were to investigate the expression of AGK in both NPC and its paclitaxel-resistant cell lines,and its effect on paclitaxel drug sensitivity of NPC.To determine whether AGK and FOXM1 have an association in expressive regulation,paclitaxel resistance,apoptosis and tumor growth in NPC paclitaxel-resistant cell lines.To elucidate the molecular mechanism of "AGK promotes FOXM1 gene transcription through the JAK2/STAT3 pathway".Methods(1)To investigate the expression and biological roles of AGK in the paclitaxel resistance of NPC,we first established a paclitaxel-resistant NPC cell line,and AGK was silenced by transfection sh-RNA.Then,the expression patterns of AGK,MDR1 and P-gp were elevated by qRT-PCR and western blot assays.The IC50 value and cell apoptosis of paclitaxel-resistant NPC cells were detected by MTT,Flow cytometry and Western blot assays.(2)To clarify the expressive and functional correlation between AGK and FOXM1,we co-transfected sh-AGK and FOXM1 overexpressed plasmids in NPC paclitaxel-resistant cell lines.Then,the expression patterns of FOXM1,MDR1 and P-gp were qRT-PCR and western blot assays.The IC50 value and cell apoptosis of paclitaxel-resistant NPC cells were detected by MTT,Flow cytometry and Western blot assays.(3)Western blot was used to detect the expression changes of JAK2/STAT3 pathway in NPC paclitaxel-resistant cells and the relationship between AGK and JAK2/STAT3 pathway by knocking down AGK,knocking down or overexpressing STAT3.Double luciferase reporter gene and chromatin immunoprecipitation(ChIP)assay were used to detect the molecular interaction between STAT3 and FOXM1.(4)To validate the effects of AGK mediated JAK2/STAT3-FOXM1 axis on tumor growth and paclitaxel resistance of NPC,we transfected sh-AGK or cotransfected sh-AGK and FOXM1 vector into CNE1-TR cells and then transplanted into nude mice to establish xenograft tumor model.Then,the tumor size,volume,and weight were measured.The expression levels of JAK2/STAT3-FOXM1 axis were tested by western blot.Results(1)The expression of AGK in CNE1-TR and CNE2-TR cells was higher than that in parental cells CNE1 and CNE2,and AGK knockdown significantly promoted the apoptosis of CNE1-TR and CNE2-TR cells,decreased the IC50 value of paclitaxel,and enhanced the sensitivity of NPC cells to paclitaxel.(2)Compared with CNE1 and CNE2 cells,the expression of FOXM1 was increased in CNE1-TR and CNE2-TR cells,but decreased after AGK silencing.Overexpression of FOXM1 significantly reversed the effect of AGK silencing on apoptosis and paclitaxel resistance of CNE1-TR and CNE2-TR cells.(3)AGK silencing inhibited the activation of JAK2/STAT3 pathway in CNE1-TR and CNE2-TR cells.Dual luciferase and Ch IP assay showed that STAT3 could directly bind FOXM1 promoter and enhance its transcriptional activity.(4)The expressions of AGK,JAK2/STAT3 and FOXM1 were significantly increased in the xenograft tumor volume in nude mice,while the expressions of JAK2/STAT3 and FOXM1 were decreased after knockdown of AGK.Knocking down AGK inhibited tumor growth,reduced tumor size,and inhibited paclitaxel resistance of nasopharyngeal carcinoma,while overexpression of FOXM1 significantly reversed the biological function of AGK silencing.ConclusionOur study illustrates a new molecular mechanism that AGK inhibit the apoptosis and promote the paclitaxel resistance of nasopharyngeal carcinoma cells by enhancing FOXM1 expression via activating JAK2 / STAT3 signaling pathway,suggesting AGK may be improved clinical paclitaxel drug resistance status of nasopharyngeal carcinoma a potential target.These data would provide new insights for the treatment of locally advanced nasopharyngeal carcinoma patients.