| Background: Pancreatic ductal adenocarcinoma is regarded as the most lethal solid tumor worldwide.The incidence of PAAD has been steadily increasing,but the five-year survival rate of PAAD has not been significantly improved,mainly because of its special anatomical location which makes it difficult to be detected at an early stage,approximately 75% of the patients have lost the opportunity for surgery at the time of diagnosis.In addition,the pancreatic cancer tumor microenvironment,increased stromal components and lack of vascularity led to limited effect of gemcitabine-based chemotherapy.Immune checkpoint inhibitors like PD-1/PD-L1 monoclonal antibodies have been proven to be ineffective in pancreatic cancer.Therefore,there is an urgent need to find new therapeutic targets for pancreatic cancer.It is also important to explore the molecular biological mechanisms of pancreatic cancer development to find new therapeutic targets.Recently,FYVE,Rho GEF and PH domain-containing protein 3(FGD3)have been found to be prognostically protective in many tumors,clinical and epidemiological datas show that patients with high expression of FGD3 in breast cancer usually have longer survival,lower lymph node metastases,that is mean FGD3 is a strongly protective factors for prognosis.This phenomenon can also be observed in lung cancer,hepatocellular carcinoma,head and neck squamous cell carcinoma.However,It remains unknown what role the FGD3 may play in pancreatic cancer,and how FGD3 exerts its oncogenic effect is not reported yet.Methods: In order to investigate whether there is any difference in the expression of FGD3 in PAAD and normal pancreatic tissues and whether it is related to prognosis,we analyzed the expression levels of FGD3 in PAAD and adjacent normal tissues in TCGA database.Clinical data of patients with high or low FGD3 expression groups were also analyzed the to assess whether FGD3 expression correlates with prognosis of PAAD.Subsequently,the changes of FGD3 expression and its relationship with prognosis were verified with clinical patient specimens and tissue microarray(TMA)data.The biological functions of FGD3 in pancreatic cancer cells were evaluated by knockdown,overexpression,and knockdown followed by overexpression of FGD3 in different pancreatic cancer cell lines,Subsequently,CCK-8 assay,plate clone formation,Transwell invasion assay,scratch assay,mouse xenograft model,and mouse lung metastasis model were used to assess the biological function of FGD3 in pancreatic cancer cells.After knocking down FGD3 in PANC-1 cell line,we sent transcriptome sequencing(RNA-seq)and combined with bioinformatics analysis to speculate the mechanism of FGD3’s cancer suppression;subsequently,FGD3 immunoprecipitation were sent to mass spectrometryproteomics analysis to find the interactions of FGD3.The molecular interactions between FGD3 and HSF4 were assessed using immunoprecipitation(CO-IP),glutathione Stransferase pull-down assay(GST-pulldown),immunofluorescence(IFC)staining;Chromatin immunoprecipitation-quantitative reverse transcription PCR(Ch IP-q PCR)assays were used to assess the regulation of p65 by HSF4.Results: The expression level of FGD3 was significantly downregulated in pancreatic cancer compared with normal pancreatic tissue,which was observed in multiple datasets including TCGA.Clinical data showed that pancreatic cancer patients in the low FGD3 expression group had higher distant metastasis rates,later pathological stages,poorer overall survival time;Knockdown of FGD3 promote the proliferation,invasive and migration ability of pancreatic cancer cell while overexpression of FGD3 inhibited the proliferation,invasive and migration ability of pancreatic cancer cell.The animal experiments also confirmed that FGD3 has an inhibitory effect on pancreatic cancer proliferation and lung metastasis;RNA-seq combined with bioinformatics analysis revealed that knockdown of FGD3 significantly upregulated the NF-κB pathway,the protein and m RNA levels of p65 were significantly upregulated.The presence of significant NF-κB pathway upregulation in patients with low FGD3 expression was also observed in clinical data.A subsequent decrease/increase in p65 was observed after overexpression/knockdown of FGD3.FGD3 promotes the expression of epithelial markers ZEB1,E-cadherin and inhibits the expression of mesenchymal marker Vimentin.Mass spectrometry-proteomics analysis identified that FGD3 could bind with 101 proteins in PANC-1 and the only transcription factor was HSF4.However,the expression of HSF4 was not regulated by FGD3;CO-IP and GST-pulldown experiments confirmed that the binding of FGD3 and HSF4 occurrs at the C-terminus of the proteins.Nucleoplasmic separation assay and IFC confirmed that FGD3 binding to HSF4 and inhibited HSF4 entry into the nucleus;Knockdown/overexpression of HSF4 was followed by a decrease/increase in p65 expression,Ch IP-seq data suggested that HSF4 enriched in the promoter region of p65,and Ch IP-q PCR confirmed that HSF4 could bind to the p65 promoter region;knockdown of HSF4 in pancreatic cancer cell lines reversed the increase in p65 expression caused by knockdown of FGD3.Similarly,the decrease in p65 expression caused by overexpression of FGD3 could be reversed by Overexpression of HSF4.These results confirmed that the FGD3-HSF4-p65 regulatory axis exists in pancreatic cancer and regulates the biological behavior of pancreatic cancer.Conclusion: This study confirmed that FGD3 was lowly expressed in pancreatic cancer and that low FGD3 expression was associated with poorer prognosis.FGD3 inhibits the proliferation,invasion,migration and EMT of pancreatic cancer cells.FGD3 suppresses pancreatic cancer progression by inhibiting the expression of p65 through binding to HSF4 and inhibiting HSF4 entry into the nucleus.HSF4 acts as a transcription factor to directly promote the transcriptional level of p65. |
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