Study On The Effect And Mechanism Of KLF4 In Ovarian Cancer

Ovarian cancer is one of the most common malignant type of cancer among all gynecological tumors.The incidence rate is third in gynecological malignancy,and the mortality rate is first in female reproductive system.In 2012 there were 529,800 new cases of ovarian cancer in the world.The incidence of new cases of ovarian cancer is about 1,315,000 yearly in China,which is six times more than the developed countries.Ovarian cancer presents therapeutic challenges due to its cryptogenic,without early signs of clinical symptoms and specific molecular markers,and about 70% of patients are typically diagnosed late.The disease is easily invaded adjoining parts of the body and spread to other organs.It is too difficult to eradicate by surgery.The standard treatment for ovarian cancer is resection of carcinoma and postoperative with chemotherapy using cisplatin combined with paclitaxel.But chemotherapy is prone not only to therapeutic resistance,but also to bring severe side effects.Moreover,it leads to poor effect in clinical treatment and poor prognosis of patients.However,it is not clear the occurrence and development mechanism of ovarian cancer and how ovarian cancer cells spread to distant organs.Therefore,how to inhibit tumor cell proliferation,infiltration and metastasis.How to enhance the sensitivity of tumor cells to chemotherapy drugs and improve the effect of treatment of ovarian cancer.How to improve survival and quality of life.Kruppel-like factor 4(KLF4)is a transcription factor in the zinc finger proteinfamily of transcription factors,called one of the pluripotent genes encoding stem cells.It plays an important role in the regulation of cell proliferation,differentiation,apoptosis and embryonic development.KLF4 has been shown to function as a tumor suppressor or oncogene in cancer depending on its cellular context.KLF4 has been shown to function as an oncogene in colon and prostate cancers but as a tumor suppressor in lung cancer,lymp Homa,cervical cancer,gastric cancer,and hepatocellular carcinoma.In breast cancer,KLF4 function is controversial with reports of both oncogenic and tumor suppressive properties.The role of KLF4 in ovarian cancer is still unclear at present.Recent studies have shown that KLF4 functions as a tumor suppressor by inhibiting the epithelial to mesenchymal transition(EMT).EMT refers to the morphological transformation of epithelial cells into mesenchymal cells and the process of invasion and migration ability.This is a fundamental process in tumor metastasis.During the process of EMT it showed the phenotype and polarity has changed from epithelial cell to interstitial cell with the anti-aging and apoptosis.It got the ability of migration and invasion.It could be infiltrate adjacent tissue or invade the lymp Hatic system and the blood circulation system,transfer and colonization in other parts.During the process cell surface markers changed:such as down-regulation of the expression of E-cadherin as epithelial cell biomarkers,up-regulation of the expression of β-catenin,up-regulation of the expression of Vimentin,N-cadherin,and Snail(Snail1 and Snail2)as interstitial cell markers.KLF4 overexpression inhibited TGF-β induced EMT in ovarian cancer cells.Therefore,we hypothesize that low KLF4 expression may contribute to ovarian cancer chemoresistance and metastasis,and inducing KLF4 expression may be a novel strategy in treating ovarian cancer.It is well known that EMT is associated with tumor resistance and metastasis in different human cancers.EMT is a prominent feature of ovarian cancer.It plays a very important role in invasion and metastasis and other malignant progress of the ovarian cancer.Experiments show that KLF4 overexpression can reduce cell proliferation,migration and invasion.A small molecule inducer of KLF4(APTO-253)was recently approved by FDA for p Hase I trial in treating several other tumors.Previous studies have shown that APTO-253 can induce KLF4 expression in human leukemia,kidneycancer cell lines and other cancer cell lines,which can inhibit cell cycle and promote apoptosis.Therefore,we hypothesize that the small molecule inducer APTO-253 in ovarian cancer could induce the expression of KLF4.It can increase the sensitivity of ovarian cancer cells to chemotherapy drugs,such as paclitaxel and cisplatin.It can regulate cell cycle and gene expression leading to cell cycle arrest and promote chemotherapeutic drug-induced apoptosis and enhances the efficacy of chemotherapy drugs in ovarian cancer cells.The expression of KLF4 can inhibit EMT.It can inhibit cell proliferation,invasion and metastasis.Part I KLF4 expression enhances the efficacy of chemotherapy drugs in ovarian cancer cellsObjective Examine the KLF4 expression in ovarian serous carcinoma and determine whether KLF4 expression mediated by lentiviral vector or induced by a small molecule inducer APTO-253 sensitizes ovarian cancer cell response to chemotherapy drug paclitaxel and cisplatin.Material and methods1 Material Ovarian cancer cell lines:The ovarian cancer SKOV3,OVCAR3 cell lines and HEK293 FT cell were obtained from ATCC.Tissue specimens:16 cases of pathologically confirmed human ovarian cancer specimens and 5 cases of normal ovarian tissue specimens were come from Memp His Southwestern Cancer Hospital,Tennessee.2 Methods(1)The expression level of KLF4 in human epithelial ovarian cancer and normal ovarian tissue was detected by Real Time-PCR,and the difference between them was analyzed.(2)The expression of KLF4 and PCNA in human ovarian cancer tissues and normal ovarian tissues were detected by immunofluorescence staining.(3)Exploring the correlation between KLF4 expression and survival by Kaplan-Meier analysis of ovarian cancer database.(4)Construction of lentiviral vector,Stable cell lines were generated by transducing the SKOV3 and OVCAR3 cells with the lentiviral KLF4 expression or EV and selected with puromycin.The established KLF4 overexpression both in SKOV3 and OVCAR3 cell were treated with different concentrations and different chemotherapeutic drugs of paclitaxel(20n M,40 n M)and cisplatin(2,4μg/ml),theapoptosis was detected by Caspase 3/7 kit.(5)The apoptosis of SKOV3 and OVCAR3 cells treated by cisplatin or paclitaxel which has induced by APTO-253 was measured by Caspase3/7 kit.(6)Different concentrations of cisplatin or paclitaxel were used to treat the KLF4 overexpression SKOV3 and OVCAR3 cell which has induced by 5μM APTO-253,the expression of Cleaved-PARP and Cleaved-Caspase-3 protein was detected by Western blot.(7)The cell cycle was analyzed by flow cytometry in the KLF4 overexpression SKOV3 and OVCAR3 cells.SKOV3 and OVCAR3 cells were induced with APTO-253.(8)The expression of cell cycle associated genes CDK6,c Myc and p21 in SKOV3 and OVCAR3 cells were examined by Western blot following APTO-253 treatment at different time points(0h,1h,3h,6h,12 h,24h).3 Statistical analysis The experimental data were analyzed by SPSS21.0 statistical software.The measurement data were expressed as mean±standard deviation(x ±s).The two groups were analyzed by independent t test.Difference between multiple sets of samples using single factor analysis of variance(one-way ANOVA),two comparison use of LSD-t,α=0.05 was used as the test level.Results1.KLF4 expression was downregulated in ovarian cancer and low expression was correlated with high risk ovarian carcinoma(1)KLF4 expression was significantly downregulated in ovarian cancer compared with control tissue.(2)We further examined KLF4 expression by immunofluorescent staining in sections of human ovarian carcinoma.KLF4 staining was strong in cell nuclei of adjacent normal tissue but weak in tumor tissue.In contrast,the cell proliferation marker PCNA strongly stained tumor cell nuclei and weakly stained normal tissue.(3)To define the correlation of KLF4 expression with patient survival,we analyzed several ovarian cancer databases.Among 133 ovarian cancer patients in the Duke-OVC dataset,KLF4 was highly expressed in 53 patients but expressed at relatively low levels in 80 patients.Most importantly,overall survival was significantly longer in patients with high KLF4 expression as compared to patients with low KLF4expression(P<0.05).We also found that patients with high KLF4 expression had better overall survival than low expressing patients among 415 ovarian cancer patients in the GSE13876 dataset that included 208 with high KLF4 expression and 207 with low KLF4 expression.KLF4 expression is significantly lower in high-risk ovarian cancer patients(P<0.05).In addition,KLF4 expression was significantly reduced in high-risk ovarian cancer patients.Our data indicated that low KLF4 expression is correlated with poor prognosis and reduced overall survival in high-risk ovarian cancer patients(P<0.001).2.Lentiviral vector mediated KLF4 expression sensitized cells to chemotherapy drug treatment(1)Compared with the control group,overexpression of KLF4 in SKOV3 cells led to approximately 2-fold increase in basal apoptosis.Moreover,KLF4 expression promoted apoptosis induced by chemotherapy drug paclitaxel,which also showed a2-fold increase in apoptosis induced by 20 n M and 40 n M paclitaxel.Similarly,KLF4 expression in OVCAR3 cells led to approximately 3-fold increase in basal apoptosis,and less than two-fold increase in apoptosis induced by 20 n M or 40 n M paclitaxel.By Western blot for cleaved-PARP and cleaved-caspase-3 as a measure of apoptosis,KLF4 expression was found to promote paclitaxel induced cell apoptosis in both SKOV3 and OVCAR3 cells.(2)we examined cell apoptosis induced by cisplatin in both KLF4 expressing SKOV3 and OVCAR3 cells.Overexpression of KLF4 in SKOV3 cells led to 2-fold increase in apoptosis in cells treated with 0,2μg/ml,and 4μg/ml cisplatin.An 3-fold increase in apoptosis was also induced by these doses of cisplatin in KLF4 expressing OVCAR3 cells.Cell apoptosis was verified by determining cleaved-PARP and cleaved-caspase-3 in KLF4 expressing and control OVCAR3 cells following cisplatintreatment.3.Inducing KLF4 expression using APTO-253 enhanced the efficacy of chemotherapy drug treatment APTO-253 significantly enhanced paclitaxel-induced or cisplatin-induced cell apoptosis in both SKOV3 and OVCAR3 cells.Apoptosis induced by APTO-253 and paclitaxel was also evaluated by determining PARP and caspase3 cleavage using Western blot in both SKOV3 and OVCAR3 cells.APTO-253 not only induced cleaved-PARP and cleaved-caspase-3,but also enhanced the induction of apoptosis as shown by cleaved-PARP and cleaved-caspase-3 in both SKOV3 and OVCAR3 cells.We also tested whether APTO-253 enhanced the efficacy of cisplatin in both SKOV3 and OVCAR3 cells.APTO-253 enhanced apoptosis induced by cisplatin in both SKOV3 and OVCAR3 cells.The apoptosis induced by APTO-253 with cisplatin was examined by cleaved-PARP and cleaved-caspase-3,indicating that APTO-253 enhanced the efficacy of the chemotherapy drug cisplatin in both cell lines.4.KLF4 expression resulted in cell cycle arrest in ovarian cancer cells Overexpression of KLF4 in both SKOV3 and OVCAR3 cells led to arrest at G1 p Hase.Cell cycle associated genes CDK6 and c Myc were downregulated,while p21 was upregulated in KLF4 expressing compared to control group.We found that APTO-253 treatment led to G1 p Hase arrest and in a significant reduction in S and G2/M phase cells in both SKOV3 and OVCAR3 cells.APTO-253 treatment induced KLF4 expression in both SKOV3 and OVCAR3 cells,cell cycle associated genes CDK6 and c Myc were downregulated,and p21 was upregulated in a time dependent manner.Our data demonstrated that increased KLF4 expression mediated by lentiviral vector or induced by APTO-253 led to G1 p Hase arrest in ovarian cancer cells.Conclusions1.KLF4 was downregulated in ovarian cancer patients,and the expression of KLF4 was well-correlated with the survival and poor prognosis of ovarian cancer patients.2.For the first time we showed that APTO-253 can induce the expression of KLF4 and improve the sensitivity of ovarian cancer cells to chemotherapeutic drugs and enhance the efficacy of paclitaxel or cisplatin in SKOV3 and OVCAR3 cells.3.For the first time we demonstrated that KLF4 overexpression can also promote apoptosis and improve the sensitivity of chemotherapeutic drugs in SKOV3 and OVCAR3 cells.Inducing KLF4 expression may be a novel strategy to treat ovarian cancer.4.KLF4 is a critical regulator of cell apoptosis by arresting cell cycle in ovarian cancer cells.Part II The role of APTO-253-induced KLF4 expression on the fine biological behavior of ovarian cancer and its molecular mechanismObjectives To study the effect and the related mechanism of APTO-253 on the proliferation,invasion and metastasis of SKOV3 and OVCAR3 cells and to provide a new theoretical basis and experimental data for the targeted therapy of ovarian cancer.Material and methods1 Object of study The ovarian cancer SKOV3,OVCAR3 and HEK293 FT cell lines were obtained from ATCC.2 Methods(1)MTT assay The proliferation of SKOV3 and OVCAR3 cells induced by APTO-253 were detected after 24 h,48h and 76 h.(2)The expression of KLF4 and PCNA in SKOV3 and OVCAR3 cells were examined by Western blot following APTO-253 treatment at different time points(0h,1h,3h,6h,12 h,24h).(3)Cell colony formation assay Measure the number and size of clone colony formation of SKOV3 and OVCAR3 cells induced by APTO-253.(4)Cell Transwell assay was used to test the ability ofmigrationwhich induced by APTO-253 in SKOV3 and OVCAR3 cells.(5)Cell invasion assay was used to test the ability of invasion which induced by APTO-253 in SKOV3 and OVCAR3 cells.(6)The expression of KLF4,E-cad,N-cad,Snail(slug),β-catenin and Vimentin were detected in SKOV3 and OVCAR3 cells which induced by APTO-253 using Western blot.(7)SKOV3 and OVCAR3 cells were treated with APTO-253 for 24 h,then TGF-β was added to the experimental and control groups.The expression of P-Smad2 and Total-Smad2 in SKOV3 and OVCAR3 cells were detected by Western blot after30 min and 60 min.(8)The expression of P-Smad2 and Total-Smad2 was detected by Western blot after add TGF-β30 min and 60 min in lentiviral vector-mediated KLF4 overexpression SKOV3 and OVCAR3 cells.3 Statistical analysis The experimental data were analyzed by SPSS21.0 statistical software.The measurement data were expressed as mean±standard deviation(x ±s).The two groups were analyzed by independent t test.α=0.05 was used as the test level.Results(1)The proliferation of SKOV3 and OVCAR3 ovarian cancer cells induced by APTO-253 were detected after 24 h,48h and 76 h.It was found that the MTT absorbance value of ovarian cancer cells decreased significantly after induced by APTO-253.Compared to control group at each time point(24h,48 h,72h),there was statistically significant(P<0.001).It was indicated that the expression of KLF4 inhibited the proliferation of ovarian cancer cells.APTO-253 treatment induced KLF4 expression in both SKOV3 and OVCAR3 cells,the expression of PCNA gradually decreased with the increasing of KLF4 expression in a time dependent manner.(2)In both SKOV3 and OVCAR3 ovarian cancer cells,it was found that the number of colonies formed by APTO-253 induced KLF4 expression were significantly lower than that of control group.The number of colonies in SKOV3 ovarian cancer cells were 43.333±7.638,and the number of colonies in the control group were 86.667±11.930,which was statistically significant(P<0.01).The number of colonies in OVCAR3 ovarian cancer cells were 82.333±7.506,and the number of colonies in the control group were 163.667±5.508,which was statistically significant(P<0.001).The size of the colony-forming group was significantly smaller compare to control group in SKOV3 and OVCAR3 ovarian cancer cells.(3)The ability of migration in the experimental group was significantly decreased after induced by APTO-253 in SKOV3 and OVCAR3 ovarian cancer cells.Compared to control group,there was significant statistical difference(P<0.001;P<0.01).(4)The ability of invasion was significantly decreased after induced by APTO-253 in SKOV3 and OVCAR3 ovarian cancer cells.Compared to the control group,there was significant statistical difference(P<0.05).(5)KLF4 can inhibited epithelial to mesenchymal transition.The expression of E-cad,N-cad,Snail(slug),β-catenin and Vimentin were detected by Western blot.The results show that the expression of E-cad as the marker of surface of epithelial cells was up-regulated and the expression of N-cad and Vimentin as the marker of the surface of stromal cells was down-regulated.The expression of proto-oncogeneβ-catenin and slug was down-regulated.(6)The expression of KLF4 can inhibit TGF-β induced epithelial translocation by inhibiting the Smad pathway of TGF-β signaling.It was found that the expression of P-Smad2 in SKOV3 and OVCAR3 which induced by APTO-253 or lentiviral vector mediated KLF4 overexpression was lower than that in control group at different time points(30 min and 60 min)induced by TGF-β.There were statistically significant difference.Conclusions1.For the first time we showed that APTO-253 can induce the expression ofKLF4 in SKOV3 and OVCAR3 cells.2.As a tumor suppressor in ovarian cancer cells KLF4 inhibits the proliferation of ovarian cancer cells.It significantly reduce the ability of migration and invasion in ovarian cancer cells.3.KLF4 can inhibit TGF-β induced epithelial translocation by inhibiting the Smad pathway of TGF-β signaling.