Mechanism Of Fox M1 Promoting Paclitaxel Resistance By Regulating PHB1

Background Pancreatic cancer is a highly malignant cancer with a five-year survival rate of less than 5%.One of the standard treatments for pancreatic cancer is the combination of gemcitabine and paclitaxel.Paclitaxel is an effective stabilizer of cellular microtubule.It has been used to treat a variety of human cancers,including ovarian cancer,breast cancer,pancreatic cancer,non-small cell lung cancer and other malignancies.Paclitaxel binds to tubulin and promotes the tubulin polymerization and stabilization.The formation of spindle is inhibited and the cell cycle is blocked in the G2 and M phase.Although,initially effective to paclitaxel,many patients treated with paclitaxel eventually develop innate or progressive chemoresistance.Several potential mechanisms and modifications contributed to drug resistance.These included the overexpression of the ABC transporters,alterations in microtubule dynamics and so on,in addition,the activation of extracellular signal-regulated kinase(ERK1/2)and oncogenes that interacted with microtubules also promoted paclitaxel resistance.Nowadays,the emergence of drug resistance is one of the main obstacles to clinical drug treatment.The assessment and establishment of a biomarker in diagnosis and treatment of cancer are necessary.The transcriptional factor Forkhead Box M1(FoxM1)is a member of the Forkhead family with a highly conserved and winged-helix DNA-binding domain.FoxM1 gene consists of 10 exons.There are mainly three FoxM1 isoforms: FoxM1 a,FoxM1 b,FoxM1 c,which are generated by the alternative splicing.Only the FoxM1 b and FoxM1 c are the active isoforms.FoxM1 is the downstream of PI3K-AKT,RAF-MEK-ERK signaling pathways and it plays an important roles in cell cycle,the repair of DNA damage and drug resistance.Recent studies have shown that higher expression of FoxM1 in breast cancer cells are resistant to cisplatin,trastuzumab and paclitaxel.However,the mechanism of paclitaxel resistance is still not clear.Prohibitin1(PHB1)is an evolutionarily conserved protein and resides at the inner mitochondrial membrane,nuclear and cell membrane.It has multifunction in cellular biology,including cellular proliferation,apoptosis and mitochondrial energy metabolism.Prohibitin1(PHB1)is capable of recruiting C-RAF to Caveolin-1-rich lipid rafts and is essential to activation of the RAS-RAF-MEK-ERK signaling pathway.The activation of the FoxM1-Cav-1 signaling pathway promotes the invasion and metastasis of pancreatic cancer cells.Caveolae are lipid rafts enriched in cholesterol and are 50–100 nm flask-shaped invaginations of the plasma membrane,which are involved in molecular transportation,signal transduction and the sensitivity of drugs.Cav-1 is critical for albumin uptake in cells and determines sensitivity of cells when are confronted with albumin related drug.PHB1 and Caveolin-1 are lipid rafts with the Stomatin/Prohibitin/Flotillin/Hfl K/C(SPFH)domains.However,the molecular mechanism of PHB1 underlying paclitaxel resistance is not clear.FoxM1 regulates gene expression activity by binding to the forkhead transcription factor response element in promoter region.It is an important transcription factor involved in tumorigenesis and development.FoxM1 exhibits a stable and high level of expression in paclitaxel resistant cells and carcinoma.It confers drug resistance by regulating the microtubule or microtubule-associated protein,which impacts the stability of microtubule and inhibits the apoptosis pathway induced by paclitaxel.Thiostrepton(THR)belongs to thiazole-peptide and it is a polypeptide natural product of microbial ribosome.It is proved that thiostrepton interacts directly with FoxM1 and inhibits the binding of FoxM1 to the genomic targets.Purpose To demonstrate that the signaling pathway mediated by FoxM1 could confer paclitaxel resistance.To analyze the expression content of FoxM1 and PHB1 in paclitaxel-resistant cells and the molecular mechanism of FoxM1 regulats PHB1.To identify the mechanism of FoxM1/PHB1/RAF-MEK-ERK signaling pathway to confer cell resistance to paclitaxel.To evaluate whether the inhibitor of FoxM1 combined with paclitaxel cloud reverse the paclitaxel resistance in vitro and in vivo,which provides a molecular therapy for the treatment of paclitaxel resistance.Methods 1.The gene MANIA database was used to predict possible targets of FoxM1.We identified the relationship between FoxM1 and PHB1.The expression and correlations between the FoxM1 and PHB1 were detected by western blotting and immunohistochemistry in clinical cancer tissue.Moreover,the correlations of m RNA levels between the FoxM1 and PHB1 in pancreatic cancer were evaluated by GEO database.2.FoxM1 bound to the promoter region of PHB1,the differneces between the isforms of FoxM1 to enhance the transcriptional activity of PHB1 were verified by using the chromatin immunoprecipitation.Cycloheximide was used to detect the protein stability when HEK293 T cells were transfected with FoxM1 b,FoxM1 c or THR at the indicated time points.Western blot was used to detect the effect of FoxM1 on protein stability of PHB1.Co-Immunoprecipitation was adopted to evaluate whether the protein of FoxM1 could bind to PHB1.3.We cultivated the paclitaxel resistant cells.The expression of FoxM1 and PHB1 in protein and m RNA levels were detected by q PCR and WB in paclitaxel resistant and sensitive cell lines with increasing dosage of paclitaxel.The correlations between the expression levels of protien and drug resistance were determined.Western blot identified whether the FoxM1/PHB1 signaling pathway had an affect on the phosphorylation of ERK1/2 and whether the signaling pathway was mediated by RAF-MEK-ERK.Immunostaining was used to detect the localization of PHB1 after it cells were transfected with FoxM1 b or FoxM1 c.Western blot was adopted to compare the localization of PHB1 in sensitive and resistant cell lines.The ability of PHB1 bound to C-RAF after transfected with FoxM1 b or FoxM1 c was analyzed in cell membrane fractions.4.The IC50 was detected to confirm that FoxM1-PHB1 signaling pathway promoted the paclitaxel resistance.The feedback loop of FoxM1/PHB1/RAF-MEK-ERK was evaluated when sensitive and resistant cell lines were confronted with paclitaxel and/or THR.The effects of activation or inhibition of the pathway were verified by flow cytometry and confocal microscopy when cells were treated with Oregon Green? 488 paclitaxel.Mice treated with paclitaxel and/or THR were to detect the tumor growth and overall survival.Results 1 FoxM1 positive regulated the transcriptional activity of PHB1 The gene MANIA database was assighed to predict the possible target of FoxM1.It was found that FoxM1 and Prohibitin1(PHB1)were co-expressed in cells.We analyzed the information of GEO database from patients with pancreatic cancer and found that FoxM1 and PHB1 were correlated significantly between the m RNA levels.FoxM1 can be recruited to the PHB1 promoter via Forkhead response element and it enhanced the transcriptional activity of PHB1.Chromatin immunoprecipitation confirmed that FoxM1 was enriched in the PHB1 promoter and the enrichment levels were different between FoxM1 b and FoxM1 c.FoxM1 b could enhance the enrichment in a dose depentent manner.However,FoxM1 c did not.It may be due to the different structure between FoxM1 b and FoxM1 c.After administration of the THR,an inhibitor of FoxM1,the levels of enrichment in the promoter region were significantly decreased when compared with the control group.2 The interactions between FoxM1 and PHB1 and it significantly enhanced the stability of PHB1 protein Co-immunoprecipitation showed that FoxM1 directly bound to PHB1.The molecular weight of 30-32 KD,double and triple molecular weight were blotted with PHB1,which may indicate that FoxM1 interacted with PHB1 in the form of single molecule or homologous polymer.After administration of FoxM1 inhibitor THR,the binding capacity between the PHB1 and FoxM1 was significantly decreased when compared with the control group.It showed that FoxM1 b or FoxM1 c significantly promoted the stability of PHB1,while FoxM1 inhibitor THR decreased the stability of PHB1.3 FoxM1-PHB1 pathway promoted paclitaxel resistance The m RNA levels of PHB1 were positively correlated with the IC50 value when they were confronted with paclitaxel in 11 kinds of cancer cell lines.Western blot showed that the expression level of PHB1 was significantly increased when the sensitive cell(Aspc-1)was transfected with FoxM1 b or FoxM1 c.At the same time,the overexpression of FoxM1 b or FoxM1 c conferred Panc-02 resistance to paclitaxel when compared with the control group.Silencing of PHB1 in Panc-02 cells attenuated FoxM1 induced paclitaxel resistance.Aspc-1,SW1990,Panc-02 and Panc-02-PTX cells treated with paclitaxel showed that the expression levels of FoxM1 and PHB1 in paclitaxel-resistant cell lines decreased less than that of the sensitive ones.4 FoxM1-induced phosphorylation of ERK1/2 was required the involvement of PHB1 and FoxM1/PHB1/RAF–MEK–ERK formed a positive feedback loop to promote paclitaxel resistance FoxM1 b and FoxM1 c promoted the expression of PHB1 with an increase of p-ERK1/2 levels.When cells were co-transfected with FoxM1 b and H1,FoxM1 c and H1,the protein levels of FoxM1,PHB1 and p-ERK1/2 were decreased to the control level,suggesting that PHB1 may affect FoxM1 level by RAF–MEK–ERK signaling pathway.Studies had shown that FoxM1 was a downstream target of ERK1/2.We hypothesized that FoxM1 regulated PHB1 to form a positive feedback loop and sustain activation of FoxM1/PHB1/RAF–MEK –ERK.Western blot showed that a significant increase of p-ERK1/2 in resistant cell lines compared to the sensitive ones.THR,an inhibitor of FoxM1,decreased the abundance of FoxM1,PHB1 and p-ERK1/2 in SW1990,but had no effect on total ERK1/2 level.Paclitaxel combined with THR further significantly reduced p-ERK1/2 level.We concluded that FoxM1 induced p-ERK1/2 by sustaining FoxM1/PHB1/RAF-MEK-ERK feedback loop and activated genes involved in paclitaxel resistance.5 FoxM1 regulated the expression of ABCA2 After overexpressing of FoxM1 b or FoxM1 c,the m RNA levels of ABC transporters were detected in the HEK293 T.The m RNA analysis was performed on 47 ABC transporters.ABCA2 and ABCD2 were increased significantly when cells were transfected with FoxM1.However,when cells were transfected with FoxM1 b and H1,FoxM1 c and H1,the m RNA levels of ABCA2 and ABCD2 were decreased,but ABCD2 m RNA level was not affected.Sensitive and resistant cell lines treated with paclitaxel showed that the m RNA of ABCA2 was decreased in sensitive cell lines while it was increased in the drug resistant cells,but ABCD2 did not change.The same results were observed in the administration of THR in HEK293 T cells.We concluded that FoxM1 up-regulated ABCA2 and it was also regulated by the FoxM1/PHB1/RAF-MEK-ERK feedback loop,which may further contribute to paclitaxel resistance.6 FoxM1 inhibitor significantly enhanced drug sensitivity We validated the effect of the FoxM1/PHB1/RAF-MEK-ERK feedback loop on paclitaxel resistance in vitro and in vivo.After administration of Oregon Green? 488 paclitaxel,the cells treated with THR showed an increase of the apoptosis.Overexpression of FoxM1 b or FoxM1 c in the sensitive cell line(Panc-02)significantly decreased the average of the fluorescent intensity.While silencing of PHB1,the average of fluorescent intensity in the cell was significantly increased.In the animal model,paclitaxel combined with THR,an inhibitor of FoxM1,significantly reduced tumor growth and prolonged the overall survival time.Conclusion Collectively,we have predicted and identified that FoxM1 regulates PHB1 at both transcriptional and post-transcriptional level,which promotes the activation of RAF-MEK-ERK pathway.On the other hand,p-ERK1/2 directly targets and regulates the expression of FoxM1,thereby forming a positive feedback loop to increase the cell proliferation and paclitaxel resistance.At the same time,the FoxM1 directly up-regulates the expression of ABCA2 and it is also regulated by the FoxM1/PHB1/RAF-MEK-ERK feedback loop,which collectively confers the cell resistance to paclitaxel.Paclitaxel with the combination of THR,an inhibitor of FoxM1,significantly inhibits the activation of FoxM1/PHB1/RAF-MEK-ERK feedback loop and the expression of ABCA2,which reverses paclitaxel resistance and induces the apoptosis of drug-resistant cells.