Novel Crosstalk Between KLF4 And ZEB1 Regulates Gemcitabine Resistance In Pancreatic Ductal Adenocarcinoma

Background and objectives:Pancreatic ductal adenocarcinoma(PDAC)is one of the most malignant tumors with an estimated 5-year survival rate less than 5%.The lack of accurate early diagnosis is one of the major factors that cause poor prognosis.Most patients are diagnosed at a later stage when tumor metastasis has occurred.In this stage,surgical resection can not improve the survival rate despite adjuvant chemotherapy,which is mainly due to that PDAC gains a wide range of chemotherapy drug resistance.Gemcitabine is a pyrimidine drug for the treatment of advanced or metastatic pancreatic cancer,and is administered alone or in combination with other chemotherapeutic agents.Gemcitabine resistance of PDAC is another important reason for poor survival rates in patients with PDAC.Therefore,it is important to understand the molecular mechanism of PDAC gemcitabine resistance and to identify novel biomarkers of PDAC gemcitabine resistance to overcome the poor prognosis of PDAC.Kriippel-like factor 4(KLF4)is a transcription factor that contains zinc finger structures,and is widely expressed in a variety of tissues.KLF4 plays a key role in maintaining cell cycle and genomic integrity.Downregulation or loss of KLF4 has been found in different types of tumors,such as colorectal cancer,bladder cancer and gastric cancer.Overexpression of KLF4 significantly inhibits the tumorigenicity of colon and gastric cancer cells in vivo.However,inhibition of KLF4 in breast cancer cells promotes p53 expression and induces p53-dependent apoptosis.A high level of KLF4 is detected in about 70%of breast cancer.In pancreatic cancer,KLF4 positively regulates the expression of p27Kipl,and inhibits the growth and metastasis of pancreatic cancer in vitro and in vivo,indicating that KLF4 is a tumor suppressor in pancreatic cancer.However,KLF4a upregulation has been demonstrated to promote pancreatic cancer cell cycle.and reduces the survival time of patients with pancreatic cancer.These results suggest that the exact role of KLF4 in PDAC is unclear,and it is necessary to clarify whether KLF4 is implicated in PDAC gemcitabine resistance.Epithelial stromal transformation(EMT)phenotype has been closely linked to chemotherapy or radiation resistance.EMT phenotypes could be observed in oxaliplatin-resistant colorectal cancer cells,paclitaxel-resistant ovarian cancer cells,tamoxifen-resistant breast cancer cells,and gefitinib-resistant lung cancer cells,such as decreased expression of E-cadherin,and increased expression of mesenchymal markers including vimentin,a-SMA and fibronectin.These findings fully link chemotherapeutic drug resistance to EMT.Gemcitabine-resistant pancreatic cancer cells can also generate EMT phenotypes,in which Notch signaling pathways play a key role.ZEB1 as EMT activator,can trigger phenotype transformation from epithelial cells to mesenchymal cells,which assists cancer cells to obtain the invasive phenotype.However,recent studies have shown that inhibition of EMT in PD AC does not alter the production of invasive PDAC,but promotes the proliferation of cancer cells and the expression of nucleoside transporters,which enhances gemcitabine sensitivity of PDAC and increases the overall survival of mice.In contrast,ZEB1-mediated EMT enhances the multidrug resistance of PDAC cells.Targeting inhibition of ZEB1 partially attenuated the sensitivity of PDAC cells to gemcitabine,5-fluorouracil and cisplatin.These results suggest that the role of ZEB1 and EMT in drugs resistance of tumors remains controversial.Therefore,it is of great clinical significance to study the role of ZEB1 in PDAC gemcitabine resistance,and it is necessary to clarify whether ZEB1 expression is positively correlated with PDAC gemcitabine resistance.MicroRNA(miRNA)is a non-coding RNAs with a length of 20 to 22 nucleotides.The miRNAs inhibit target genes by binding to the 3 ’untranslated region(3’UTR)of the target gene.The expression of miRNAs has been implicated in many normal biological processes,such as cell proliferation,differentiation and apoptosis.The dysregulated expression of miRNA is closely related to the occurrence and development of cancer.In liver cancer,the expression of KLF4 was positively correlated with the expression of miR-153,miR-506 and miR-200b.MiR-153,miR-506 and miR-200b inhibit the expression of snail,Slug and ZEB1 by targeting their 3’UTR,respectively.MiR-200 family members(miR-141 and miR-200c)inhibit EMT by targeting 3’UTR of ZEB1 and ZEB2 in different types of tumor cells,such as breast cancer cells,colon cancer cells and PDAC cells.ZEB1 as a transcription factor in turn inhibits the transcription of miR-141 and miR-200c,suggesting a feedback loop between miR-200 family members and ZEB1.ZEB1 also inhibited the expression of miR-183.MiR-183 is a member of the miR-182-96-183 gene cluster located at human chromosome 7q31-34 locus,and is up-regulated in human colorectal cancer,breast cancer and prostate cancer,but is down-regulated in retinoblastoma and lung cancer.These results indicate that the role of miR-183 is cell-type dependent.MiR-183 is down-regulated in PDAC tissues and cells,which was significantly associated with tumor grade,metastasis and TNM stage.The expression of miR-183 is closely related to the expression of miR-200 family members.MiR-183 and miR-200 family members cooperate to affect stemness properties in PDAC cells by suppressing Bmil,function as EMT inhibitors and accelerate epithelial differentiation by targeting Wnt/β-catenin signal pathway.In addition,the expression of miR-200 family members was down-regulated in gemcitabine-resistant PDAC cells,but whether gemcitabine affected the expression of miR-183 remains unknown.In this study,the effect of gemcitabine on the expression of KLF4 and ZEB1 is analyzed by exposing PDAC cells to gemcitabine.The effect of KLF4 and ZEB1 on the gemcitabine sensitivity of PDAC is analyzed by silencing or overexpressing KLF4 and ZEB1.The effect of KLF4 and ZEB1 on the expression of miR-200b and miR-183 is examined by silencing or overexpressing KLF4 and ZEB1.The effect of KLF4 overexpression on PDAC gemcitabine sensitivity and ZEB1 expression is determined by constructing PDAC nude mouse model overexpressing KLF4.PDAC gemcitabine sensitivity is determined in PDAC tissues.Differential expression of KLF4 and ZEB1 in the gemcitabine sensitive and resistant PDAC tissues are determined,which contributes to confirm the early warning function of KLF4 and ZEB1 in PDAC gemcitabine resistance.Methods:1.Experiments in vitro:(1)The PDAC cells BxPC-3,Panc-1 and MIApaca-2 are treated with gemcitabine.The inhibitory effect of gemcitabine on PDAC cells was detected by CCK8 cell proliferation assay and flow cytometry.The effects of gemcitabine on the expression of KLF4 and ZEB1 are determined by western blot and real time PCR.(2)The PDAC cells are transfected with shRNA against KLF4 or KLF4 cDNA.The effect of KLF4 expression on the ZEB1 expression is detected by western blot analysis.The effect of KLF4 on the expression of miR-200b and miR-183 are determined by norther blot and real time PCR.The effect of KLF4 on PDAC gemcitabine sensitivity is determined by CCK8 cell proliferation assay.(3)On the sensitivity of gemcitabine in PDAC cells.The binding ability of KLF4 to miR-200b and miR-183 promoters are determined by chromatin immunoprecipitation(ChIP).The transcriptional activity of KLF4 on miR-200b and miR-183 are detected by luciferase reporter assay.(4)The interaction of miR-200b and miR-183 with 3’UTR of ZEB1 are detected by luciferase reporter assay.(5)The PDAC cells are transferred with shRNA against ZEB1.The activation of Caspase 3 is analyzed by western blot.The effect of ZEB1 inhibition on the PDAC gemcitabine sensitivity is detected by CCK8 cell proliferation assay.2.Experiments in vivo:4-to 6-week-old BALB/c nude mice of both sexes were obtained from Weitonglihua Animal Center(Beijing,China)and maintained under specific pathogen-free conditions in the animal facility.All procedures were approved by the Institutional Laboratory Animal Care and Use Committee at Shandong provincial hospital.1×107 Panc-1 cells in 100 μl PBS/matrigel(1:1,v/v,BD Biosciences,USA)transfected with control lentivirus or lentivirus carrying KLF4 cDNA were injected into both flanks of the mice.One week after injection,gemcitabine(80 mg/kg;q3 days)was administered via intraperitoneal injection for six weeks.The long(L)and short axes(S)of the tumors were measured weekly using Vernier calipers,and the tumor volumes were calculated as follows:V=(L×S2)×π/6.The mice were sacrificed twenty-four hours after the last injection,and the tumors were harvested for subsequent analysis.The expression of KLF4 and ZEB1 was analyzed by immunohistochemistry and western blot.3.Clinical specimens:Primary PD AC cells are isolated from fresh PDAC tissues.LDH assay is used to determine cell gemcitabine ensitivity,the PDAC tissues are classified as gemcitabine sensitive,partially sensitive and resistant.The differential expression of KLF4 and ZEB1 in gemcitabine sensitive and resistant PDAC are determined by real time PCR,western blot and immunohistochemistry.Results:1.Experiments in vitrol:The results showed that exposure of PDAC cells to gemcitabine inhibits the expression of KLF4,miR-200b and miR-183,but promotes ZEB1 expression.KLF4 knockdown inhibits the expression of miR-200b and miR-183,but promotes ZEB1 expression.Conversely,KLF4 overexpression promoted the expression of miR-200b and miR-183,but inhibits the ZEB1 expression.These results indicate that KLF4 positively regulates the expression of miR-200b and miR-183,but negatively regulates ZEB1 expression.In addition,KLF4 knockdown enhances gemcitabine resistance,whereas KLF4 overexpresses attenuates gemcitabine resistance.ChIP assay confirmed that KLF4 positively regulates the transcription of miR-200b and miR-183 by directly binding to their promoters.MiR-200b and miR-183 directly inhibited ZEB1 expression by targeting the 3’UTR region of ZEB1.ZEB1 knockdown increased the gemcitabine sensitivity of PDAC cells.These results suggest that the KLF4/miR-200b/miR-183/ZEB1 signaling pathway regulates gemcitabine resistance in PDAC cells.2.Experiments in vivo:PDAC cells overexpressing KLF4 are inoculated into the armpits of BALB/c nude mice.After one week,the nodules are palpable at the inoculation site,but the growth is slower in one week to two weeks after inoculation,followed by rapid growth.Compared with the control group,KLF4 overexpression does not significantly affect the tumor growth,but significantly increases the gemcitabine sensitivity.Immunohistochemistry and western blot analysis confirms that in vivo overexpression of KLF4 inhibits ZEB1 expression.Linear regression analysis shows that KLF4 expression is negatively correlated with ZEB1 expression.These results suggest that the negative regulatory between KLF4 and ZEB1 attenuates gemcitabine resistance of PDAC in vivo.3.Clinical specimens:41 cases of PDAC tissues are collected,among which 13 cases are identified as gemcitabine sensitive,7 cases are gemcitabine resistance and 21 cases were sensitive by LDH method.We selected 7 cases of gemcitabine resistance and 7 cases of gemcitabine-sensitive PDAC for subsequent analysis.Western blot analysis and immunohistochemistry confirmed that the expression of KLF4 in gemcitabine resistant PDAC is significantly lower than that in gemcitabine sensitive PDAC,while ZEB1 expression in gemcitabine resistant PDAC was significantly higher than that in gemcitabine sensitive PDAC.These results indicate that the expression of KLF4 and ZEB1 may serve as new markers of PDAC gemcitabine resistance.Conclusion:1.KLF4 promotes the expression of miR-200b and miR-183 by binding to their promoters.MiR-200b and miR-183 inhibit the expression of ZEB1 by directly targeting 3’UTR of ZEB1.ZEB1 Inhibition increased the gemcitabine sensitivity in PDAC cells.2.KLF4 overexpression inhibits the expression of ZEB1 and increases the gemcitabine sensitivity in PDAC cells.3.The expression of KLF4 in gemcitabine resistant PDAC is significantly lower than that in gemcitabine sensitive PDAC,whereas ZEB1 expression is significantly higher in gemcitabine resistant PDAC than that in gemcitabine sensitive PDAC.The expression of KLF4 and ZEB1 could be novel markers of PDAC gemcitabine resistance.