USP22Promotes Wnt/β-catenin Signaling Pathway-mediated Foxm1Expression And Induces Epithelial-mesenchymal Transition In Pancreatic Ductal Adenocarcinoma

I. ObjectivePancreatic ductal adenocarcinoma (PDA) that originates in glandular epitheliumaccounts for more than90%of all malignant pancreatic tumors and exhibits a highdegree of malignancy with a poor prognosis. Lymphatic, vascular, and distant organmetastases can occur at an early stage; therefore, when pancreatic cancer is confirmed,most patients are at intermediate or advanced stages and have passed the optimal timefor surgical treatment. To date, reliable biomarkers for the early diagnosis and effectivetreatment of PDA remain lacking.Ubiquitin-specific protease22(USP22) is an ubiquitin-specific peptidase that belongsto the deubiquitinating enzyme (DUB) family and serves as a subunit of the hSAGAcomplex. In addition, USP22can activate BMI-1-, c-Myc-, and FBP-1-mediated targetgene transcription and plays a key role in cell cycle regulation, embryonic development,and telomere homeostasis. Recently, it has been shown that USP22is closely associatedwith the metastatic potential and prognosis of many solid tumors. However, themechanism underlying the role of abnormal USP22expression in the occurrence anddevelopment of tumors and its regulatory aspects remain poorly defined; moreover, theassociation between USP22and pancreatic cancer has not been reported.The invasion and metastasis are the primary factors leading to treatment failure andpoor prognosis in cancer patients. Recent studies have shown that the epithelial-mesenchymal transition (EMT) is closely associated with the invasion andmetastasis of pancreatic cancer. The occurrence of EMT enables tumor cells to acquirethe ability to infiltrate surrounding tissues and invade adjacent vasculature; as a result,these cells can metastasize to other tissues or organs to form metastatic foci.Furthermore, immunohistochemical analysis of pancreatic cancer showed that EMToccurrence was significantly correlated with poor prognosis. However, recent studies onUSP22have mainly focused on its role in cell cycle regulation, while the potentialmechanism underlying tumor invasion and metastasis by abnormal USP22expressionhas not been reported.This study aims to reveal the relationship between USP22and the prognosis ofpancreatic ductal adenocarcinoma and investigate the role of USP22and its possibleunderlying mechanism in PDA tumorigenesis which may provide a new approach fortargeted therapy of pancreatic cancer.II. Methods1. Immunohistochemistry SP method was used to detect the expression of USP22andFoxM1in136cases of PDA paraffin histopathological tissues and42cases normalcarcinoma-adjacent tissues. SPSS17.0statistical software was used for statisticalanalysis, and p-value less than0.05were considered significant. The correlationbetween the two indicators and the major clinical characteristics of PDA were analyzedby chi-square test. Postoperative survival period analysis were computed by theKaplan-Meier method and compared by the Log-rank test. The prognostic relevance ofthe four indicators and conventional prognostic factors were analyzed by COXregression model.2. The recombinant vector was transfected into cells. Stable transfectants were selectedwith800μg/ml G418, and individual clones were isolated.3. The expression of USP22and FoxM1mRNA were detected by the methods ofRT-PCR.4. The expression of USP22、FoxM1、Wnt/β-catenin signalling pathway marker proteinsand the EMT marker proteins were detected by the method of Western blot. 5. Indirect immunofluorescence staining was used to detect the expression of USP22、FoxM1、β-catenin、F-actin、E-Cadherin and Ezrin.6. Using scratch and transwell assays, we showed the migration and invasion abilities ofPANC-1cells.III. Results(I) Overexpression of USP22and FoxM1predicts poor prognosis in non-small-cell lungcancer1. Compared with the normal tissues, PDA tissues expresses significantly high USP22and FoxM1(P<0.001). Additionally, A statistical correlation is observed betweenUSP22and FoxM1expression (r=0.450, P<0.001).2. USP22expression is significantly associated with lymph node metastasis (P=0.009)and TNM stage (P=0.031). FoxM1expression is significantly associated withhistological differentiation (P=0.020), lymph node metastasis (P=0.015) and TNM stage(P=0.018). Furthermore, a co-positive expression of USP22/FoxM1is significantlyassociated with lymph node metastasis (P=0.011) and TNM stage (P=0.041).3. Kaplan–Meier analysis demonstrates that PDA patients with USP22, FoxM1andco-expression USP22/FoxM1have a significantly poorer OS compared to the patientswith negative expression (log-rank P=0.033; log-rank P＜0.001; log-rank P＜0.001).4. In univariate analysis, USP22（HR:1.944；95%CI：0.895–2.996；P=0.034）, FoxM1（HR：1.823；95%CI：1.369–3.881；P＜0.001）, co-expression USP22/FoxM1（HR：2.895；95%CI：1.346–4.237；P＜0.001）, histological differentiation, lymph nodemetastasis and TNM stage are significant prognostic factors of PDA.5. In multivariate analysis of model A, USP22（adjusted HR：1.675；95%CI：1.008-2.146；P=0.027）, FoxM1（adjusted HR：1.732；95%CI：1.262-2.947；P=0.032）expression, TNM stage are independent prognostic factors of survival. Model B showesthat positive co-expression of USP22/FoxM1（adjusted HR：1.328；95%CI：1.175–1.923；P=0.029） and TNM stage are determined as independent prognosticfactors.(II) USP22promotes the G1/S phase transition by upregulating FoxM1expression via β-catenin nuclear localization.1. The USP22downregulation in the PANC-1cell line resulted in reductions in theexpression of2Wnt/β-catenin signalling pathway marker proteins, Axin2and lymphoidenhancer-binding factor-1(LEF-1), as well as the downstream target protein c-Myc.Using a luciferase reporter plasmid, we also confirmed a decrease in Wnt/β-cateninsignalling activity. In the CFPAC-1cell line, the USP22upregulation led to increases inthe expression of Axin2, LEF-1and the downstream target protein c-Myc. Theluciferase reporterassay also confirmed a increase in Wnt/β-catenin signalling activity.2. The USP22downregulation in PANC-1cells led to a reduction in nuclear β-cateninexpression, as well as a elevation in cytoplasmic β-catenin expression. Additionally,reductions were observed in both nuclear and cytoplasmic FoxM1expression. In theCFPAC-1cell line, the USP22upregulation led to an increase in nuclear β-cateninexpression and a reduction in cytoplasmic β-catenin expression. Concurrently, increaseswere observed in both nuclear and cytoplasmic FoxM1expression. In additionalexperiments on the PANC-1cell line, we found that when β-catenin expression wasupregulated with the β-catenin overexpression plasmid, USP22knockdown withUSP22-siRNA failed to induce significant changes in either nuclear or cytoplasmicFoxM1expression. In the CFPAC-1cell line, when β-catenin expression was disruptedwith β-catenin-siRNA, USP22overexpression failed to trigger significant changes ineither nuclear or cytoplasmic FoxM1expression.3. The CCK-8proliferation assay showed that USP22overexpression significantlystimulated the proliferation of both cell lines and the interference of FoxM1expressionproduced significant inhibition of the proliferation of both cell lines. However, thedepletion of FoxM1expression, caused by FoxM1-siRNA interference, in theUSP22-overexpressing PANC-1and CFPAC-1cell lines abolished the USP22overexpression-stimulated cell proliferation. Additionally, flow cytometry revealed thatUSP22overexpression increased the G1/S transition rate, resulting in a significantdecrease in the proportion of G1-phase cells and an increase in the proportions of S-phase. However, there was no significant changes in the G2/M-phase. Upon FoxM1 expression downregulation by RNAi, USP22overexpression no longer elicitedsignificant changes in cell cycle progression in either cell line4. Further studies showed that USP22overexpression resulted in decreased p21and p27expression in both cell lines, which were accompanied by the increased Cyclin D1levels. Similarly, the expression levels of Cdk2, Cdk4and Cdk6were also increased.FoxM1depletion abolished the USP22-induced changes in G1phase-related proteinexpression in both cell lines.(III) USP22promotes epithelial-mesenchymal transition via the FAK pathway inpancreatic cancer cells1. To examine whether USP22could promote EMT in PANC-1cells, detection ofF-actin expression in Panc-usp and Panc-uspsh cells was performed usinglaser-scanning confocalimmunofluorescence. The results showed that there were a largeamount of longitudinally oriented actin filaments in the cytoplasm of Panc-usp cells,whereas in Panc-uspsh cells, actin showed punctate expression without actin filamentsin the cytoplasm. Next, the expression of Ezrin protein, an important regulatory proteinand cell adhesion protein for connections to the cell membrane and cytoskeleton, wasdetermined. The results showed that Ezrin expression in Panc-uspshcells was mainlyconcentrated in the cytoplasm. Interestingly, in USP22stably expressing Panc-usp cells,Ezrin expression was significantly closer to the cell membrane and almost absent in thecytoplasm. The subsequent Western blot results showed that although the expressionlevels of Ezrin in PANC-1, Panc-uspsh, and Panc-usp cells did not change, the levels ofEzrin phosphorylation (P-Ezrin) were significantly altered. For example, the expressionlevel of P-Ezrin in Panc-usp cells was6.4-fold greater than that observed in Panc-uspshcells.2. Western blot results showed that the expression level of P-FAK in Panc-usp cells was3.6-fold greater than that in Panc-uspsh cells. To further confirm the function of theFAK signaling pathway, control-siRNA and FAK-siRNA were separately transfectedinto Panc-usp cells. The results showed that with the decrease in FAK expression,P-PAK and P-Ezrin expression also decreasedsignificantly. Furthermore, transfection of an empty plasmid or an FAK over-expression plasmid into Panc-uspsh cells showed thatwith the increase in FAK expression, P-FAK and P-Ezrin expression also increased.3. Western blot results for Panc-usp cells showed that the expression levels of themesenchymal markers N-cadherin, vimentin, and fibronectin were increased5.8-,4.2-,and2.7-fold compared to those in Panc-uspsh cells, while the expression level of theepithelial marker E-cadherin was decreased by75.3%compared to that in Panc-uspshcells. The evaluation of EMT-associated transcription factors showed that the expressionlevels of ZEB1and Snail in Panc-usp cells were5.6-and5.3-fold greater than those inPanc-uspsh cells.4. The results showed that with the downregulation of P-FAK, the expression levels ofthe mesenchymal markers N-cadherin, vimentin, and fibronectin decreased by63.8%,81.3%, and64.5%, respectively, while the expression level of the epithelial markerE-cadherin was increased4.2-fold. With the downregulation of P-FAK, the expressionlevels of theEMT-associated transcription factors ZEB1and Snail were decreased to86.2%and78.4%, respectively. Next, Panc-uspsh cells were transfected with anFAK-overexpressing plasmid, and with the upregulation of P-FAK, the expressionlevels of the mesenchymal markers N-cadherin, vimentin, and fibronectin wereincreased5.2-fold,1.4-fold, and3.6-fold, respectively, while the expression level of theepithelial marker E-cadherin was decreased by87.5%. In addition, the expression levelsof the EMT-associated transcription factors ZEB1and Snail were increased2.8-fold and3.6-fold, respectively.5. Using scratch and transwell assays, we showed that the migration and invasionabilities of Panc-usp cells were significantly greater than those of Panc-uspsh cells. Inaddition, downregulation of FAK in Panc-usp cells significantly decreased the migrationand invasion rates, while overexpression of FAK in Panc-uspsh cells significantlyincreased the migration and invasion rates.IV. Conclusions(I) Overexpression of USP22and FoxM1predicts poor prognosis in non-small-cell lungcancer 1. Compared with the normal tissues, PDA tissues expresses significantly high USP22and FoxM1.2. USP22expression is significantly associated with lymph node metastasis. FoxM1expression is significantly associated with histological differentiation, lymph nodemetastasis and TNM stage. Furthermore, a co-positive expression of USP22/FoxM1issignificantly associated with lymph node metastasis and TNM stage.3. Overexpression of USP22/FoxM1is an independent and significant prognostic factorin PDA.(II) USP22promotes the G1/S phase transition by upregulating FoxM1expression viaβ-catenin nuclear localization.1. USP22expression affects Wnt/β-catenin pathway activation.2. USP22expression affects FoxM1expression via Wnt/β-catenin pathway.3. USP22regulates the G1/S phase transition through FoxM1in PDA cell lines.4. USP22regulates the expression of G1phase-related proteins through FoxM1in PDAcell lines(III) USP22promotes epithelial-mesenchymal transition via the FAK pathway inpancreatic cancer cells1. USP22promotes the redistribution and phosphorylation of Ezrin and cytoskeletalremodeling in PANC-1cells.2. Overexpression of USP22induces phenotypic changes and leads to the acquisition ofmesenchymal markers and reduction of epithelial markers in PANC-1cells.3. USP22promotes PANC-1cell invasion and migration through the FAK signalingpathway.4. USP22promotes cytoskeletal remodeling and epithelial-mesenchymal transition viathe FAK pathway in PANC-1cells...