FOXO1 Expression In Endometrioid Carcinoma And The Influence And Mechanisum Of FOXO1 On The Biological Behaviorr Of Endometrial Cancer Cells

BackgroundEndometrial carcinoma(EC)is one of the commonest malignancies in the female reproductive system.In recent years,its incidence has shown an increasing tendency.Younger demographic susceptibility to endometrial carcinoma is on the rise.It is the second commonest malignant tumor in female reproductive system,following cervical carcinoma(CC),whereas based on mortality rate it takes the third rank.In the United States,Europe and other countries,its incidence is far higher than CC and ovarian cancer(OC),and is the commonest malignant tumor of female genital system.It has various histopathological types,endometrioid cancinoma being the most common type,accouting for 70-80%.Patients with endometrial carcinoma are always accompanied with abnormal vaginal bleeding,which can be detected early,and treated in the early stage.Compared with other malignant tumors of female reproductive system better prognosis is observed in endometrial carcinoma.In addition,early endometrial cancer has a 5-year survival rate of about 90%.But the 5-year survival rate in patients with advanced disease is not so satisfactory,which is around 50%.5-year survival rate in case of distant metastasis even drops to 20%or less.Therefore,it is of great clinical significance to seek out new targets for the diagnosis and treatment of endometrial carcinoma.Forkhead box O(FOXO)belongs to the Forkhead(FOX)transcription factor family,which was first discovered in the study of fruit flies,and in 2000 was named the Forkhead transcription factor family.FOXO1 was found in the study of vesicular rhabdomyosarcoma,the first member detected in this subfamily.It is widely expressed in various normal human tissues,has a variety of biological activities and is involved in hepatic glycogen production,adipocyte differentiation,cell cycle regulation,apoptosis,cell differentiation,wound healing and other physiological and pathological processes.FOXO1 is a downstream gene of PI3K/Akt pathway,which is a very vital signal transduction pathway in tumorigenesis.Many genes play very important role in cancer though targeting PI3K/Akt pathway.Some studies found that FOXO1 also plays a key role in a variety of tumor progression.Previous studies have reported that the expression of FOXO1 in breast cancer,cervical cancer,gastric cancer,prostate cancer and other tumors was significantly downregulated.Other studies suggested that re-expression of FOXO1 and restoration of its transcriptional activity can inhibit tumor growth,which might provide new ideas for targeted therapy of tumors.Sterol regulatory element-binding protein(SREBP)is a very important transcription factor for lipid biosynthesis.In the past,the study of SREBP1 mainly focused on diabetes,fat synthesis,fatty liver and so on.In recent years,its role in the progression of cancer has been paid more and more attention.Studies have shown that the expression of SREBP 1 in breast cancer,prostate cancer,ovarian cancer and other tumors was remarkably upregulated.FOXO1 functions as an oncogene and was closely related to tumor progression and metastasis.Studies have shown that FOXO1 can inhibit the growth of endometrial cancer cells,which indicates FOXO1 may play a role in cancer therapy as a tumor suppressor,but its specific mechanism is still not clear.Previous study of our team found that expression of SREBP1 in endometrial carcinoma was significantly higher than in normal endometrial tissues,In vitro experiments suggested that it might promote growth,invasion and metastasis of endometrial cancer cells.Additionally,in vivo experiments also confirmed that SREBP 1 has a promoting effect on growth of endometrial cells.FOXO1 and SREBP1 both play important role in lipid synthesis and endometrial cancer,but the mechanism is still unknown.Studies have showed that FOXO1 can inhibit SREBP transcription by reducing activity of Spl and SREBP-1c.Whether there is any link between the two genes is still unclear and the relationship between FOXO1 and SREBP1 in endometrial cancer has not been reported so far.In addition,more and more studies is focus on the relationship between tumors and FOXO1.Therefore,this study was conducted to investigate the role and mechanism of FOXO1 in carcinogenesis and development of endometrial cancer,and we would carry out it in three parts.Part One FOXO1 expression in EC tissues and its correlation with EC clinicopathological characteristicsObjective:The study was conducted to investigate the expression of FOXO1 mRNA and protein in normal endometrial tissue,endometrial tissue with atypical hyperplasia and endometrioid carcinoma,to explore the expression of FOXO1-and the clinicopathological features of endometrioid carcinoma(patient age,tumor differentiation,clinicopathological stages,lymph node metastasis)in order to confirm the role of FOXO1 in the development and progression of endometrioid carcinoma.Methods:In this study,differential expression analysis of FOXO1 mRNA in endometrial carcinoma and normal endometrium was perfomed based on TCGA database.Real-time quantitative PCR was used to analyze the expression of FOXO1 mRNA in endometrial carcinoma and normal endometrium.Immunohistochemistry was used to analyze the expression of FOXO1 protein level in normal endometrial tissue,atypically hyperplastic endometrial tissue and endometrioid carcinoma.SPSS statistical software was used to analyze the correlation between expression of FOXO1 and endometrioid cancer and to compare their clinicopathological features(patient age,tumor tissue differentiation,clinical pathological stage,lymph node metastasis).Results:1.TCGA data showed that the expression of FOXO1 mRNA in endometrial carcinoma tissue was significantly lower than that in normal tissues(P<0.01).2.RT-PCR showed that the expression of FOXO1 mRNA in endometrioid carcinoma was significantly lower than that in normal tissues(P<0.01).3.Immunohistochemistry showed that the positive expression rates of FOXO1 in normal endometrium,endometrial atypical hyperplasia and endometrioid carcinoma were 90.00%,85.00%and 45.07%,respectively.Compared with the normal endometrium and endometrial atypical hyperplasia,the positive expression rate of FOXO1 in endometrioid carcinoma was significantly lower(P<0.01).There was no difference between the normal endometrial group and atypical hyperplasia group(P>0.05).4.In 71 cases of endometrioid carcinoma the positive expression rate of FOXO1 in poorly differentiated group(38.18%)was significantly lower than highly differentiated group(68.75%)(P<0.05).The positive expression rate of FOXO1 in stage III and IV(16.67%)was significantly lower than that in stage Ⅰ and Ⅱ(50.85%)(P<0.05).The positive expression rate of FOXO1 in lymph node metastasis group(11.11%)was significantly lower than that in non-lymph node metastasis group(50.00%)(P<0.05).There was no significant difference between expression of FOXO1 in the age group of<60 years and>60 years old group(P>0.05).Conclusion:1.Expression of FOXO1 in endometrioid carcinoma was low,FOXO1 might act as a tumor suppressor gene in endometrial cancer.2.Expression of FOXO1 was closely related to the degree of differentiation of endometrioid carcinoma,clinicopathological staging and lymph node metastasis.Part Two The influence of FOXO1 on biological behavior of endometrial cancer cellsObjective:Previous studies have shown that FOXO1 plays an important role in the development and progression of various tumors,but its specific role and mechanism in endometrial carcinoma is still not clear.The study was conducted to further explore the role of FOXO1 in the pathogenesis and development of endometrial carcinoma.Protein expression of FOXO1 in endometrial carcinoma cell lines:HEC-1-A,RL95-2,AN3-CA,SPEC-2,Ishikawa and KLE were detected.Lentiviral vectors were used to overexpress FOXO1 and the influence of FOXO1 overexpression on proliferation,growth,migration and in vivo tumorigenesis of endometrial carcinoma cells were detected and the correlation between FOXO1 and SREBP1 were investigated.Methods:Expression of FOXO1 in six endometrial carcinoma cell lines was detected by Western blotting.FOXO1 overexpressing lentiviral vector with GFP was constructed and stably transfected endometrial cancer cell lines were screened.The effect of FOXO1 overexpression on the cytological behavior of endometrial carcinoma was detected by MTT assay,clone formation assay,transwell assay and in vivo tumorigenesis test.The effect of FOXO1 overexpression on the expression of SREBP1 was detected by Western blotting.Results:1.Relative values of FOXO1 protein expression in HEC-1-A,RL95-2,AN3-CA,SPEC-2,Ishikawa,KLE cell lines were detected using WB.The expression level of FOXO1 protein in AN3-CA,SPEC-2,Ishikawa and KLE endometrial carcinoma cell lines was lower.AN3-CA and Ishikawa were screened out for later experiments.2.Transduction efficiency observed by inverted fluorescence microscope of FOXO1 overexpressing lentiviral vector in AN3-CA and Ishikawa cell lines was 91.42%and 87.94%respectively,lentiviral vectors were successed transducted.3.Results of MTT assay showed that the cell proliferation rate of FOXO1 overexpression lentiviral vector group was significantly lower than that of blank vector group(P<0.05).4.Results of clone formation assay showed that the number of clones was significantly fewer in the overexpression lentiviral vector group than in the blank vector group(P<0.05).5.Results showed that the number of cell migration and cell invasion in FOXO1 overexpression lentiviral vector group was significantly less than that in blank vector group(P<0.05).6.Results of immunoblotting showed that the expression of SREBP1 protein in FOXO1 overexpression lentivirus group was significantly lower than that in blank vector group(P<0.05).7.Results of nude mice experiment showed that the formation and growth rate of transplanted tumor in FOXO1 overexpression lentiviral vector group were significantly lower than those in blank vector group(P<0.01).After 28 days of injection,transplanted tumor weight of FOXO1 overexpression lentiviral vector group was significantly lower than that of the blank vector group(P<0.01).Conclusion:1.FOXO1 overexpression can inhibit the proliferation,growth,migration and invasion of cells in endometrial carcinoma.2.Overexpression of FOXO1 inhibits the in vivo tumorigenesis of endometrial cancer cells.Part Three The study on mechanism of FOXO1 on endometrial carcinomaObjective:Our previous studies showed that FOXO1 and SREBP1 both play important role in the development of endometrial cancer and also studies have showed that FOXO1 can inhibit SREBP transcription by reducing activity of Spl and SREBP-1c.Whether there is any link and the relationship between the two genes is still unclear.FOXO1 is a downstream gene of PI3K/Akt pathway,which is a very vital signal transduction pathway in tumorigenesis and many genes play very important role in cancer though targeting this pathway.To preliminarily explore the molecular mechanism by which FOXO1 exerts its anti-cancer function,we further analyzed the interaction between FOXO1 and SREBP1,and examined the effects of FOXO1 overexpression on functional pathways and PI3K/AKT signaling pathway in endometrial cancer cells.Methods:Co-immunoprecipitation technology was used to test the interaction between FOXO1 and SREBP1.Western blot analysis was applied to examine the effects of FOXO1 overexpression on functional pathway-associated proteins and PI3K/AKT signaling transduction pathways.Results:1.Co-immunoprecipitation analysis showed that SREBP1 was one of the interaction partners of FOXO1,which indicated that FOXO1 might exert its anti-cancer function via interacting with SREBP1.2.Western blot analysis demonstrated that FOXO1 overexpression significantly reduced the phosphorylation levels of PI3K and AKT in AN3-CA and Ishikawa cells in comparison with control group(P<0.05),which suggested that FOXO1 overexpression inhibited PI3K/AKT signaling transduction pathway.Conclusion:1.FOXO1 may exert its anti-cancer function through interacting with SREBP1.2.FOXO1 may have an inhibitory effect on endometrial carcinoma via PI3K/AKT signal pathway.3.FOXO1,a tumor suppressor gene,may be a novel therapeutic target for endometrial carcinoma,and the current study provides new ideas and clues of gene therapies for endometrial carcinoma.