ETV4 Promotes PDAC Metastasis Through Activation Of The CXCL13/CXCR5 Signaling Axis

Background:Pancreatic ductal adenocarcinoma(PDAC)is a relatively uncommon aggressive malignancy in the digestive tract.According to the latest global cancer statistics,pancreatic cancer has become the seventh-leading cause of cancer death worldwide.With a rapid increase in the mortality rate of 0.5% to 1.0% per year,PDAC is projected to become the second leading cause of cancer-related deaths in the USA by the end of the decade.Dramatically characterized by early metastasis,more than 50% of PDAC patients present with distant metastases at the time of diagnosis,and the majority of patients will develop metastasis within 4 years after tumor resection.At presentation,a considerable number of studies have provided insights into the pathological signaling pathways and genetic alterations of PDAC metastasis.Nevertheless,the molecular mechanisms underlying PDAC metastasis remain unclear.Thus,it is urgent to elucidate the molecular mechanisms underlying PDAC metastasis and to develop novel strategies for therapeutic interventions.Besides classical genetic alterations,including KRAS,CDKN2 A,TP53,and SMAD4,the aberrant expression or activation of transcription factors also has crucial role in the initiation,metastasis,angiogenesis and chemo-resistance of PDAC.ETS variant 4(ETV4)is a member of the polyoma enhancer activator protein(PEA3)subgroup,which in turn belongs to the E26 transformation-specific(ETS)transcription factor family.Since it was discovered three decades ago,ETV4,along with ETV1 and ETV5,has been reported to play a key role in the diverse physiological processes,including embryonic development,hormonal metabolism,and motor coordination.Recent studies showed that ETV4 was highly expressed in several tumors,and could promote tumor invasion and metastasis by regulating the migration of tumor cells.In pancreatic cancer,a study indicated that ETV4 was elevated in tumor tissues and cells.But the oncogenic function of ETV4 in PDAC metastases has not been systematically reported,and the underlying molecular mechanisms must still be further investigated.Aims:To explore the expression of ETV4 in PDAC tissues and elucidate its underlying molecular mechanism of promoting tumor metastasis,thus providing theoretical value and potential therapeutic target for clinical intervention of PDAC.1.To detect the expression of ETV4 in PDAC tissues and evaluate its correlation with tumor metastasis and patient prognosis.2.To clarify the role of ETV4 in PDAC invasion and metastasis.3.To identify the downstream target of ETV4 in PDAC cells.4.To elucidate the mechanism of CXCL13-induced ETV4 expression and tumor metastasis.5.To explore the potential strategy of targeting CXCL13/ETV4/CXCR5 positive feedback loop to inhibit tumor metastasis.6.To verify the correlation of ETV4,CXCL13 and CXCR5 expression in clinical PDAC samples.Methods:1.Immunohistochemistry(IHC)staining was used to detect the expression of ETV4 in PDAC tissue microarray and its adjacent non-tumor tissues.And the correlation between ETV4 expression and patient’s clinicopathological parameters was analyzed.TCGA database was used to evaluate the ETV4 m RNA levels in PDAC and normal tissues.Finally,univariate and multivariate Cox analysis were used to analyze the correlation of ETV4 expression and patient’s prognosis.2.Lentivirus was utilized to establish stably ETV4 overexpression or knockdown PDAC cell lines.Transwell and invasion assay,wound healing assay and tumor metastasis model in nude mice were used to clarify the function of ETV4 in PDAC metastasis.The change of EMT markers was determined by western blotting,immunofluorescence,and immunohistochemistry after ETV4 silencing or overexpression.The effect of ETV4 expression on tumor proliferation was subsequently evaluated by CCK-8 assay,apoptosis test and subcutaneous tumorigenesis experiment.3.Whole-gene transcriptome sequencing was used to identify the direct target of ETV4 in PDAC cells.Western blot,q RT-PCR,immunofluorescence,dual-luciferase reporter assay and chromatin immunoprecipitation were used to determine the transcriptional activation of ETV4 on downstream target molecules.The essential role of downstream target molecular for ETV4-mediated tumor metastasis was then determined by Transwell and invasion assay and tumor metastasis model in nude mice.4.The protein levels of ETV4,CXCR5 and downstream molecular were detected by western blotting after recombinant human CXCL13 protein treatment.Signal pathway specific inhibitors,Transwell and invasion assays were used to elucidate the mechanism of CXCL13-induced ETV4 expression and tumor metastasis.5.The migratory and invasive abilities of indicated cells were examined by Transwell and invasion assays after ETV4 silencing in CXCL13-overexpressing cells to determine the crucial role of ETV4 for CXCL13/CXCR5-induced PDAC metastasis.6.The protein levels of ETV4,CXCR5 and p-ERK1/2,along with the migratory and invasive abilities of indicated cells,were evaluated to explore the blockade effect of CXCR5 neutralizing antibody on CXCL13/CXCR5/ETV4 positive feedback loop.Results:1.The expression of ETV4 was significantly upregulated in PDAC tissues compared to adjacent normal tissues.And ETV4 expression was positively associated with tumor metastasis and shorter survival in PDAC patients.Univariate and multivariate Cox analysis revealed that ETV4 overexpression was an independent risk factor for survival.2.Overexpression of ETV4 promoted the proliferation and invasion of PDAC cells.In turn,downregulation of ETV4 accounts for the opposite effect.Additionally,ETV4 significantly promoted epithelial to mesenchymal transition in PDAC cells.3.The results of RNA-sequencing,dual-luciferase reporter assay and chromatin immunoprecipitation showed that ETV4 could transcriptionally activate CXCR5.Sitedirected mutagenesis experiment further illustrated that ETV4 directly bound to the CXCR5 promoter region from-894 to-885 nt.Rescue experiments validated that the essential role of CXCR5 for ETV4-induced PDAC migration and invasion.4.Recombinant human CXCL13,the specific ligand of CXCR5,promoted the expression of ETV4 and the migration and invasion of PDAC cell lines though activation of ERK1/2 pathway.The expression of ETV4 and the enhanced migratory and invasive abilities of indicated cells dramatically inhibited after ERK1/2 inhibitor treatment or CXCR5 silencing.5.The enhanced migratory and invasive abilities of CXCL13-overexpressiong cells were significantly abrogated by ETV4 knockdown both in vitro and in vivo,thus demonstrating that ETV4 was critical for CXCL13/CXCR5-mediated PDAC migration and metastasis.6.The protein levels of ETV4 and p-ERK1/2 significantly decreased after CXCR5 neutralizing antibody treatment in the presence or CXCL13,indicating that CXCR5 blocking antibody could inhibit CXCL13/CXCR5/ERK signaling transduction.Additionally,the expression of p-ERK1/2 and the migration and invasion of PANC-1-ETV4 cells were also significantly reduced upon CXCR5 blockade in vitro.Finally,we found that ETV4 m RNA levels were positive correlated with CXCL13 and CXCR5 expression in 20 cases of fresh freezing PDAC samples.Conclusion:In this study,we found that ETV4 expression was significantly upregulated in PDAC tissues and correlated with poor survival.For the first time,we have demonstrated that ETV4 promotes tumor metastasis and EMT by transactivating CXCR5.In addition,a novel CXCL13/ETV4/CXCR5 positive feedback loop was identified to be involved in PDAC metastasis,and interrupting this loop with a CXCR5 neutralizing antibody inhibited ETV4-mediated PDAC invasion and metastasis.This study provides new insights into the molecular mechanisms underlying PDAC metastasis and has implications for the development of novel strategies for therapeutic interventions.