Mechanism Of FoxM1 Promoting Paclitaxel Resistance By Regulating PHB1

BackgroundPancreatic 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,which 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.Fox M1 gene consists of 10 exons.There are mainly three Fox M1 isoforms: Fox M1 a,Fox M1 b and Fox M1 c,which are generated by the alternative splicing.Only the Fox M1 b and Fox M1 c are the active isoforms.Fox M1 is the downstream of PI3K-AKT and RAF-MEK-ERK signaling pathways and plays important roles in cell cycle,DNA damage repair and drug resistance.Recent studies have shown that higher expression of Fox M1 in breast cancer cells is 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(Cav-1)-rich lipid rafts and is essential to activation of the RAS-RAF-MEK-ERK signaling pathway.The activation of the Fox M1-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 drugs sensitivity.Cav-1 is critical for albumin uptake in cells and determines sensitivity of cells when 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.Fox M1 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.Fox M1 exhibits stable and high levels of expression in paclitaxel resistant cells and carcinomas.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 Fox M1 and inhibits the binding of Fox M1 to the genomic targets.PurposeTo demonstrate that the signaling pathway mediated by FoxM1 could confer paclitaxel resistance.To analyze the expression content of Fox M1 and PHB1 in paclitaxel-resistant cells and the molecular mechanism of Fox M1 in regulating PHB1.To identify the mechanism of Fox M1/PHB1/RAF-MEK-ERK signaling pathway to confer cell resistance to paclitaxel.To evaluate whether the inhibitor of Fox M1 combined with paclitaxel cloud reverse the paclitaxel resistance in vitro and in vivo,which provides a molecular therapy for the treatment of paclitaxel resistance.Methods1.The gene MANIA database was used to predict possible targets of Fox M1.We identified the relationship between Fox M1 and PHB1.The expression and correlations between the Fox M1 and PHB1 were detected by western blotting and immunohistochemistry in clinical pancreatic cancer tissue.Moreover,the correlations of m RNA levels between the Fox M1 and PHB1 in pancreatic cancer were evaluated by GEO database.2.The chromatin immunoprecipitation was used to verified the bounding of FoxM1 to the promoter region of PHB1 and the differences of the PHB1 transcriptional activity redulated by different Fox M1 isoforms.To detect the effects of Fox M1 on protein stability of PHB1,THR(Fox M1 inhibitor)and Cycloheximide were used.HEK293 T cells were transfected with Fox M1 b,Fox M1 c or treated with THR,Cycloheximide at the indicated time,then protein stability of PHB1 was detected by western blot.Co-Immunoprecipitation was adopted to evaluate whether the protein of Fox M1 could bind to PHB1.3.We cultivated the paclitaxel resistant cells.The expression of Fox M1 and PHB1 in protein and m RNA levels were detected respectively by q PCR and WB in paclitaxel resistant and sensitive cell lines with increasing dosage of paclitaxel.Then the correlations between the expression levels of protien and drug resistance were determined.Western blot was used to identify whether the Fox M1/PHB1 signaling pathway had an effect on the phosphorylation of ERK1/2 and whether the signaling pathway was mediated by RAF-MEK-ERK.Immunofluorescence was used to detect the translocation of PHB1 in cells after transfected with Fox M1 b or Fox M1 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 Fox M1 b or Fox M1 c was analyzed in cell membrane fractions.4.The IC50 was detected to confirm that Fox M1-PHB1 signaling pathway mediated the paclitaxel resistance.The THR-sensitive and-resistant cell lines were treated with paclitaxel and/or THR,then Fox M1/PHB1/RAF-MEK-ERK pathway was evaluated by WB.The Oregon Green? 488 paclitaxel was conjugated,and the effects of activation or inhibition of the pathway were verified by flow cytometry and confocal microscopy.The tumor growth and overall survival were detected in mice treated with paclitaxel and/or THR.Results1.FoxM1 positively regulated the transcriptional activity of PHB1The gene MANIA database was assighed to predict the possible target of Fox M1.It was found that Fox M1 and PHB1 were co-expressed in cells.We analyzed the information of GEO database from patients with pancreatic cancer and found that Fox M1 and PHB1 were correlated significantly in the m RNA levels.Fox M1 can be recruited to the PHB1 promoter via Forkhead response element and enhances the transcriptional activity of PHB1.The results of chromatin immunoprecipitation confirmed that Fox M1 was enriched in the PHB1 promoter,and the enrichment levels were different between Fox M1 b and Fox M1 c.Fox M1 b could enhance the enrichment in a dose depentent manner but Fox M1 c did not.It may be due to the different structure between Fox M1 b and Fox M1 c.After administration of the THR,the levels of enrichment in the promoter region were significantly decreased when compared with the control group.2.The interactions between Fox M1 and PHB1 and it significantly enhanced the stability of PHB1 proteinCo-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 Fox M1 interacted with PHB1 in the form of single molecule or homologous polymer.After administration of Fox M1 inhibitor THR,the binding capacity between the PHB1 and Fox M1 was significantly decreased.It showed that Fox M1 b or Fox M1 c significantly promoted the stability of PHB1,while Fox M1 inhibitor THR decreased the stability of PHB1.3.Fox M1-PHB1 pathway promoted paclitaxel resistanceThe 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 results showed that the expression level of PHB1 was significantly increased when the sensitive cell(Aspc-1)was transfected with Fox M1 b or Fox M1 c.At the same time,the overexpression of Fox M1 b or Fox M1 c conferred Panc-02 resistance to paclitaxel when compared with the control group.Silencing of PHB1 in Panc-02 cells attenuated Fox M1 induced paclitaxel resistance.Aspc-1,SW1990,Panc-02 and Panc-02-PTX cells treated with paclitaxel showed that the expression levels of Fox M1 and PHB1 in paclitaxel-resistant cell lines decreased less than that of the sensitive ones.4.Fox M1-induced phosphorylation of ERK1/2 was required the involvement of PHB1 and Fox M1/PHB1/RAF–MEK–ERK formed a positive feedback loop to promote paclitaxel resistanceFoxM1b and FoxM1 c promoted the expression of PHB1 with an increase of p-ERK1/2 levels.When cells were co-transfected with Fox M1 b and H1,Fox M1 c and H1,the protein levels of Fox M1,PHB1 and p-ERK1/2 were decreased to the control level,suggesting that PHB1 may affect Fox M1 level by RAF–MEK–ERK signaling pathway.Studies had shown that Fox M1 was a downstream target of ERK1/2.We hypothesized that Fox M1 regulated PHB1 to form a positive feedback loop and sustain activation of Fox M1/PHB1/RAF–MEK–ERK.Western blot results showed that a significant increase of p-ERK1/2 in resistant cell lines compared to the sensitive ones.THR decreased the abundance of Fox M1,PHB1 and p-ERK1/2 in SW1990,but had no effect on total ERK1/2 levels.Paclitaxel combined with THR further significantly reduced p-ERK1/2 level.We concluded that Fox M1 induced p-ERK1/2 by sustaining Fox M1/PHB1/RAF-MEK-ERK feedback loop and activated genes involved in paclitaxel resistance.5.Fox M1 regulated the expression of ABCA2After overexpressing of Fox M1 b or Fox M1 c,the m RNA levels of ABC transporters were detected in the HEK293 T.The m RNA levels analyses were performed on 47 ABC transporters.ABCA2 and ABCD2 were increased significantly when cells were transfected with Fox M1.However,when cells were transfected with Fox M1 b and H1 or Fox M1 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 in sensitive cell lines was decreased 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.So,we concluded that Fox M1 up-regulated ABCA2 and it was also regulated by the Fox M1/PHB1/RAF-MEK-ERK feedback loop,which may further contribute to paclitaxel resistance.6.Fox M1 inhibitor significantly enhanced drug sensitivityWe validated the effect of the Fox M1/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 Fox M1 b or Fox M1 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 significantly reduced tumor growth and prolonged the overall survival time.ConclusionCollectively,we have predicted and identified that Fox M1 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 Fox M1,thereby forming a positive feedback loop to increase the cancer cell proliferation and paclitaxel resistance.At the same time,Fox M1 directly up-regulates the expression of ABCA2 and it is also regulated by the Fox M1/PHB1/RAF-MEK-ERK feedback loop,which collectively confers the cell resistance to paclitaxel.Paclitaxel with the combination of THR significantly inhibits the activation of Fox M1/PHB1/RAF-MEK-ERK feedback loop and the expression of ABCA2,which reverses paclitaxel resistance and induces the apoptosis of drug-resistant cells.