FoxM1,Dually Regulated By Both HIF-1α And HIF-2α,Promotes Gastrointestinal Stromal Tumor Progression

Background and Objective:Gastrointestinal stromal tumor(GIST)is the most common mesenchymal neoplasm of the gastrointestinal tract,and more than 80%of GISTs harbor activating mutations in KIT or PDGFRA.There is increasing evidence that KIT/PDGFRA mutation is not the only mechanism involved in malignant transformation of GISTs.Many other factors also account for the growth and malignant properties of GIST cells.FoxM1 signaling plays important roles in maintaining the balance between cell proliferation,differentiation and genomic stability.The FoxM1 protein is abnormally elevated in many human malignancies,and levels of FoxM1 expression increase with tumor grade and are inversely correlated with patient survival.However,there are no related studies focusing on the correlation between clinicopathological significance and FoxMl expression in the GISTs,and the effect of FoxM1 on the proliferation,migration and invasion of GIST cells.Therefore,the object of our study is to evaluate the functional role of FoxMl in GIST progression,emphasizing molecular interactions among hypoxia inducible factor(HIF)-1α,HIF-2a and FoxMl.Methods:The present study was divided into four sections.1.The clinicopathological significance of FoxM1 expression in gastrointestinal stromal tumor.A total 83 cases of primary GIST were obtained from Shanghai Changhai Hospital.Each GIST was evaluated for clinicopathologic and histologic features by using Hematoxylin and Eosin(HE).Immunohistochemistry was used to determine the expression of FoxM1,CD117,DOG1 and CD34.Genomic DNA was extracted from paraffin-embedded specimens,and KIT/PDGFRA gene mutations were analyzed by direct sequencing analysis.A statistical analysis of the relationships between FoxM1 expression and clinicopathological factors was analyzed.2.Effect of FoxMl on proliferation,migration and invasion of gastrointestinal stromal tumor cells.Quantitative PCR(qPCR)and western blot were used to detect FoxMl expression under normoxic(21%O2)or hypoxic conditions(1%O2).Human FoxMl expression plasmid(pcFoxMl)and short hairpin RNA specific to human FoxMl(shFoxMl)were constructed and were transfected into GIST-T1 and GIST 882 cells by using Lipofectamine 2000.GIST cell growth,cell cycle distribution,migration and invasion were evaluated by using CCK-8,flow cytometry,wound-healing and transwell cell matrigel and invasion assay.Western blot was used to examine the expression of proliferating cell nuclear antigen(PCNA),cyclinA-cyclin-dependent kinase2(Cdk2)and matrix metalloproteinase 2(MMP2).3.Regulation mechanism of FoxMl by HIF-la and HIF-2α.Western blot were used to detect the expression of HIF-la and HIF-2a under normoxic(21%O2)or hypoxic conditions(1%O2).Short hairpin RNA specific to human HIF-la(shHIF-la)and HIF-2a(shHIF-2a)were constructed and transfected into GIST-T1 and GIST 882 cells,using Lipofectamine 2000.Western blot and qPCR were used to detecte expression the expression of HIF-1α,HIF-2a and FoxMl.Bioinformatic analysis was performed to identify transcription start site and promoter regions,and uncover putative HRE.Chromatin immunoprecipitation(ChIP)assay was used to examine the specific association of HIF-la and HIF-2a with FoxMl promoter in the context of living GIST-T1 cells.Luciferase reporter genes were constructed and transfected into GIST-T1 cells.The luciferase activity was detected with the Luciferase Assay.4.The clinicopathological significance of HIF-la and HIF-2α expressions in gastrointestinal stromal tumor,and their correlation with FoxMl expression.Immunohistochemistry was used to determine the expression of HIF-1α and HIF-2a.A statistical analysis of the relationships between HIF-la/HIF-2a expression and clinicopathological factors,as well as correlation between HIF-la/HIF-2a expression and FoxMl was analyzed.Athymic nude mice(n=20)were inoculated subcutaneously with 2×107 GIST-T1 cells in 0.2 ml phosphate-buffered saline(PBS)and 0.1 ml Matrigel.Following injection,the mice were randomly divided into four groups,and fed PX-478 2HCl(PX),PT-2385(PT),PX-478 2HCl combined with PT-2385(PX+PT),and PBS,respectively.After two weeks,the mice were sacrificed and their tumors were excised and processed for FoxMl and ki-67 immunohistochemical staining.Results:1.The clinicopathological significance of FoxMl expression in gastrointestinal stromal tumor.The positive rates of CD 117,CD34 and DOG1 in GISTs were 97.59%,67.47%and 100%.All 83 GISTs were positive for FoxMl expression,and showed positive staining in the nucleus of the tumor cells.These 83 patients were divided into two groups based on the immunohistochemical results:high FoxMl expression group(n=21)and low FoxMl expression group(n=62).We found that the increased expression of FoxMl was significantly correlated with larger tumor size,increased mitosis,higher ki-67 index,the presence of invasion and metastasis,advanced NIH risk grade and advanced WHO risk grade(All P<0.001).However,no significant difference in FoxM1 expression was observed with respect to other factors such as sex(P=0.2307),age(P=0.4114),and tumor location(P=0.7555),histological morphology(P=0.8835),KIT/PDGFRA gene mutation(P=0.4060).2.Effect of FoxM1 on proliferation,migration and invasion of gastrointestinal stromal tumor cells.GIST-T1、GIST 882 cells were transfected with pcFoxMl and pcEGFP and were cultured under normoxic conditions.The CCK-8 results showed that pcFoxMl transfected cells had a higher growth rate than pcEGFP transfected cells at 2,3 and 4 days of the cultivation.Flow cytometry analysis for S-phase of GIST-T1 cells showed 49.80±0.34%in pcFoxMl transfected cells、32.90±0.94%in pcEGFP transfected cells,respectively.Flow cytometry analysis for S-phase of GIST 882 cells showed 45.00±2.04%in pcFoxMl transfected cells、30.49± 1.76%in pcEGFP transfected cells,respectively.There was significant difference of cell cycle distribution between pcFoxMl and pcEGFP transfected cells.Wound healing assay showed that the closure of pcFoxMl transfected cells was significantly faster than that of pcEGFP transfected cells(P<0.05).Transwell assay showed that FoxM1 increased the number of invading cells by 2.68-fold(GIST-T1)and 2.73-fold(GIST 882)compared with vector control.Western blot confirmed that FoxMl increased the expression of PCNA、Cdk2 and MMP2.In addition,the expression of FoxMl was found to increase under hypoxic conditions.Therefore,GIST-T1、GIST882 cells were transfected with shFoxMl and shCon and were cultured under hypoxic conditions.The CCK-8 results showed that shFoxM1 transfected cells had a lower growth rate than shCon transfected cells at 2,3 and 4 days of the cultivation.Flow cytometry analysis for S-phase of GIST-T1 cells showed 31.98±1.0%in shFoxMl transfected cells,48.19±0.65%in shCon transfected cells,respectively.Flow cytometry analysis for S-phase of GIST 882 cells showed 30.05 ± 1.22%in shFoxMl transfected cells,42.05±2.23%in shCon transfected cells,respectively.There was significant difference of cell cycle distribution between shFoxMl and shCon transfected cells.Wound healing assay showed that the closure of shFoxMl transfected cells was significantly slower than that of shCon transfected cells(P<0.05).Transwell assay showed that the FoxMl downregulation decreased the number of invading cells by 0.62-fold(GIST-T1)and 0.52-fold(GIST 882)compared with vector control.Western blot also confirmed that FoxM1 downregulation decreased the expression of PCNA、Cdk2 and MMP2.3.Regulation mechanism of FoxMl by HIF-1α and HIF-2a.The results of western blot showed that the expression of HIF-1α and HIF-2a was significantly increased in GIST cells under hypoxic conditions.Under normoxic conditions,protein expression of HIF-2α and FoxM1 was observed in GIST cell lines,although HIF-1α expression was absent.Both qPCR and western blot showed that the downregulation of HIF-1α or HIF-2α could decrease the expression of FoxM1 of hypoxic GIST cells.Furthermore,HIF-2α suppression could partially downregulated FoxM1 expression under normoxia.Bioinformatic analysis uncovered five pure HRE(5’-RCGTG-3’),located at-25(HRE1),-297(HRE2),-309(HRE3),-761(HRE4)and-2324(HRE5)base pairs(bp)relative to the transcriptional start site of FoxM1.Under hypoxic conditions,ChIP assay demonstrated that HIF-1α directly bond to HRE1 site,rather than other HREs.By contrast,PCR analysis did not detect any PCR signal under normoxic conditions.ChIP assay also demonstrated that HIF-2α could directly bond to HRE1,HRE2 and HRE3 sites under normoxic conditions.In addition to these three HRE sites,HIF-2α also directly bond to HRE5 site.The luciferase reporter assay showed that the HRE1 site-directed mutagenesis significantly reduced the promoter activity of FoxMl induced by HIF-1α.The luciferase reporter assay also showed that HRE2,HRE3 and HRE5 site-directly mutagenesis reduced the activation of FoxM1 promoter induced by HIF-2α.4.The clinicopathological significance of HIF-1αand HIF-2α expressions in gastrointestinal stromal tumor,and their correlation with FoxM1 expression.All 83 GISTs were positive for HIF-1α and HIF-2αexpression,and showed positive staining in the nucleus and/or cytoplasm of the tumor cells.These 83 patients were divided into two groups based on the immunohistochemical results:high HIF-1α expression group(n=35)and low HIF-1αexpression group(n=48).The increased expression of HIF-1α was correlated with higher FoxM1 expression(P=0.0341),larger tumor size(P=0.0499),advanced NIH risk grade(P=0.0401)and advanced WHO risk grade(P=0.0478).These 83 patients were also divided into two groups based on the immunohistochemical results:high HIF-2α expression group(n=46)and low HIF-2α expression group(n=37).The increased expression of HIF-2α was correlated with FoxM1 expression(P=0.0065),increased mitosis(P=0.0162),higher ki-67 index(P=0.0060),advanced NIH risk grade(P=0.0004)and advanced WHO risk grade(P=0.0025).The different higher expression of HIF-1α and HIF-2α was correlated with FoxMl expression,larger tumor size,increased mitosis,higher ki-67 index,advanced NIH risk grade and advanced WHO risk grade(All P<0.01).On the contrary,there was no obvious relationship between high expression of HIF-1α and HIF-2α simultaneously and FoxM1 expression as well as other clinicopathological factors.In addition,the tumor sizes of PBS,PX,PT,PX+PT were 80.70±32.14mm3,39.02±5.46mm3,38.90±3.87 mm3,15.14±9.30 mm3,respectively.FoxMl positive cell rates were 66.74±15.12%,50.16± 10.28%,44.66±4.96%,28.50±9.07%,respectively.ki-67 index were 79.34±11.38%,58.22±9.11%,56.86±8.90%,40.50± 12.87%,respectively.The tumor size,FoxMl expression and ki-67 index in PX+PT group were significantly lower than those of other three groups(All P<0.05).Conclusion:1.The level of FoxMl expression was positively correlated with GIST cell proliferation,migration and invasion,and could be used as a marker to indicate the progression of GISTs.2.FoxMl enhanced the cell cycle progression,promopted the proliferation,migration and invasion of GIST cells,and upregulated the expression of PCNA,Cdk2 and MMP2.3.FoxMl was the hypoxia-regulated protein.HRE1 was functional binding site of HIF-la on FoxMl promoter,whereas HRE2,HRE3 and HRE5 were functional binding sites of HIF-2a on FoxM1 promoter.4.HIF-la was an important regulated factor of FoxMl expression under normoxic conditions,whereas both HIF-la and HIF-2a concordantly enhanced the expression of FoxMl under hypoxic conditions.5.The expression of FoxMl positively correlated with HIF-la or HIF-2a.The Combined inhibition of HIF-la or HIF-2a could significantly decreased FoxMl expression and inhibited GIST tumor growth.