Studies On The Role And Mechanism Of FOXK1 In The Growth And Metastasis Of Gallbladder Cancer

Background and Objective:Gallbladder cancer(GBC)is the most common malignancy of the biliary tract,and the sixth most common form of digestive tract malignancy.It accounts for about 165,000 cancer deaths annually,which is 1.7% of all global cancer deaths.Recently,with the development of national economy,the incidence rates of gallbladder cancer in China is increasing.Surgical resection that represents the only method associated with long-term survival is still the main treatment.Since GBC is an aggressive disease with early local invasion,early and widespread nodal involvement,and frequent distant metastases,most of patients was diagnosed at an advanced stage and lost the chance for radical surgery.Although chemotherapy and radiotherapy may benefit patients,these methods ccould not significantly prolong survival.To achieve early diagnosis and develop effective targeted or immunotherapy drugs is the key to the treatment of GBC.Therefore a deep understanding of the molecular mechanisms of GBC proliferation and metastasis is indispensable for novel prognostic biomarkers and effective adjuvant therapy.Forkhead box k1(FOXK1),a member of the FOX transcription factor family,has been reported to play pivotal roles in cell proliferation,cell metabolism,cell cycle control,and autophagy.Recent studies have demonstrated that the expression level of FOXK1 was elevated in various cancers and was associated with the prognosis of the cancer,which means that FOXK1 participates in the development of cancers.However,the expression pattern and effect of FOXK1 on the development,progression and prognosis of GBC have never been investigated.The objective of the present research is to explore the role and mechanisms of FOXK1 in gallbladder cancer.Materials and methods:1.Extract RNA from 42 pairs of gallbladder cancer samples and their corresponding adjacent normal tissues for synthesis of c DNA.The expression level of FOXK1 m RNA was validated by qRT-PCR analysis.2.The expression level of FOXK1 protein was detected by western blot analysis within 10 pairs of gallbladder cancer samples and adjacent tissues.3.Compare the difference of FOXK1 protein expression between gallbladder cancer tissues from 97 GBC patients and tissues from 64 cholecystitis patients by immunohistochemistry analysis.Then analyze the correlation between FOXK1 expression and the clinicopathological characteristics and prognosis of 97 GBC patients.4.FOXK1 was knocked down by lentivirus-mediated gene knockdown method or si RNA and over-expressed by plasmid overexpression means in gallbladder cancer cells,respectively.CCK-8 and colony formation assay were performed to detect the effects of FOXK1 modulation on cell proliferation.Xenograft tumor assay was performed in which FOXK1-depleted or control NOZ cells were injected into nude mice to evaluate the effect of FOXK1 on the tumor progression in vivo.The cell cycle progression and cell apoptosis of gallbladder cancer cells were analyzed by flow cytometry.The cell cycle regulatory proteins associated with the G1-S phase transition were examined by western blot analysis.5.Scratches wound healing assay and transwell assay were performed to detect the effects of FOXK1 expression on cell migration and invasion.The expression of epithelial mesenchymal transition markers,such as E-cadherin,N-cadherin and vimentin was detected by western blot analysis.6.Nude mouse xenograft model and mouse metastasis model were used to assess the effects of FOXK1 on the tumorigenic and metastatic ability of GBC cells in vivo.7.CHIP-seq data about FOXK1 from the public database Gene Expression Omnibus(GEO)were used to predict the mechanism of FOXK1 involved in the carcinogenesis and progress of gallbladder cancer.Then western blot analysis was used to verify these findings.Results:1.FOXK1 expression was upregulated in gallbladder cancer tissue and associated with the prognosis.Compared with the adjacent non-tumor tissues,the relative m RNA level of FOXK1 in tumor tissues was significantly increased.The western blot data showed that the protein level of FOXK1 was obviously increased in GBC tissues(8/10)compared with matched adjacent tissue samples.Immunohistochemistry analysis revealed that the positive staining of FOXK1 was mainly observed in the nucleus of cells and FOXK1 expression was significantly higher in tumor specimens compared with that in cholecystitis tissues.Among the 97 cases of GBC tissue samples,23%(22/97)of cases were strongly stained,on the contrary,only 6%(4/64)of the cholecystitis specimens showed strong staining of FOXK1 protein.The high level of FOXK1 expression was significantly correlated with increased liver invasion(P= 0.009),poor histology differentiation(P= 0.039)and advanced TNM stage(P= 0.013).The median survival of the 97 GBC patients was 9.5 months,while it was 7.5 months in 64 patients of high FOXK1 expression,and 15.3 months in 33 patients of low FOXK1 expression.Furthermore,Kaplan-Meier survival analysis with the log-rank test revealed that patients in the high FOXK1 group had a significantly shorter overall survival(OS)than those in the low FOXK1 group(P = 0.007).2.The effect of FOXK1 on the proliferation,cell cycle,apoptosis of GBC.Among the four GBC cell lines,the expression level of FOXK1 protein in GBC-SD and NOZ cells was higher than that in EH-GB1 and SGC-996 cells.Then GBC-SD and NOZ cell lines were chosen for stable transfection with si RNA or sh RNA lentivirus vectors toward FOXK1,and SGC-996 cell lines were chosen for stable transfection with FOXK1-expression vector.CCK-8 and colony formation assays showed that the proliferation of GBC-SD and NOZ cells was significantly suppressed by FOXK1 knockdown,while the cell proliferation was significantly enhanced by overexpression of FOXK1 in SGC-996 cells.The results of flow cytometry analysis demonstrated that FOXK1 knockdown led to a significant increase in the G0/G1 phase cells,but a reduction in the S and G2/M phase population.Furthermore,the expression levels of cell cycle regulatory proteins CDK4,CDK6,Cyclin D1 and Cyclin E1 were decreased post FOXK1 knockdown.We also found that FOXK1 had no significant effect on apoptosis of GBC cells.Xenograft tumor assay showed that depletion of FOXK1 significantly inhibited xenograft tumor formation,and growth and cell proliferation marker Ki-67 was decreased in FOXK1-depleted tumor tissues.3.The role of FOXK1 in invasion and metastasis of GBC cells.In vitro wound healing assay,transwell migration and invasion assay showed that cell mobility and invasive capability was dramatically attenuated when knockdown of FOXK1 in NOZ and GBC-SD cells,while overexpression of FOXK1 in SGC-996 cells demonstrated the opposite effect.We also found that the expression levels of E-cadherin were enhanced while N-cadherin and Vimentin were reduced in both GBC-SD and NOZ cells transfected with sh FOXK1.In contrast,overexpression of FOXK1 in SGC-996 cells could significantly reduce E-cadherin but increase N-cadherin and Vimentin expression.Moreover,the lung tumor metastasis model of nude mice showed that the incidence rate of lung metastasis and the size of micrometastatic lesions in sh FOXK1 group were lower than that in the control group.4.The cellular mechanism of FOXK1 in regulating the growth and metastasis of gallbladder cancer.We analyzed GEO datasets GSE 51673,which was about the FOXK1-Ch IP-seq data in 293 T cells.The result indicated that FOXK1 may exert the tumor-promoting functions through AKT/mTOR signaling pathway.Compared with control cells,the expression levels of both phosphorylated Akt(Ser473)and phosphorylated mTOR(Ser2448)were decreased in FOXK1 knockdown GBC cells.Moreover,overexpression of FOXK1 in SGC-996 cells increased the phosphorylation of Akt(Ser473)and mTOR(Ser 2448).In addition,AKT special inhibitor MK-2206 could abolish the proliferation and metastasis discrepancy between FOXK1 overexpression GBC cells and control cells.Conclusions:The expression level of FOXK1 is elevated in human GBC tissues and associated with liver invasion,histological differentiation,TNM stage of and overall survival of GBC patients.FOXK1 high expression is an independent risk factor of GBC prognosis.FOXK1 could promote the proliferation of GBC cells by regulating the cell cycle.And FOXK1 could influence the EMT of GBC cells,which resulted in the enhanced migrative and invasive ability of GBC cells.Moreover,the progression of GBC cells induced by FOXK1 may be partially related with the activations of Akt/mTOR axis.