| Background:Pancreatic cancer(PC)is a highly lethal malignancy,in which mortality closely parallels incidence with a 5-year overall survival rate of approximately 5%[1].The poor clinical outcome of PC is attributed to this tumor exhibits rapid progression with no obvious symptoms,extensive local tumor invasion and distant metastases[2].Thus,it is great importance to investigate genes and molecular pathways related with PC growth and metastasis and further elucidate the potential mechanism underlying PC progression,which will be helpful to find effective diagnostic and therapeutic targets for improving the prognosis of PC patients with selecting most suitable personalized therapies.Sparc/osteonectin,cwcv,and kazal-like domains proteoglycan 1(SPOCK1)belongs to a matricellular protein family named secreted protein,acidic,cysteine-rich(SPARC)[3].SPARC appeared to function as an oncogene in a variety of cancer types including gastric cancer[4]and breast cancer[5],due to its important roles in regulating multiple cellular processes including proliferation,apoptosis,cell cycle progression,adhesion,and cell-matrix interaction.In recent years,many researchers have focused on the study of SPOCK1 in various malignancies owing to its structure is very similar to SPARC.Study by Yang et al.showed that overexpression of SPOCK1 promoted the proliferation and inhibited apoptosis in glioma cells[6].Ma et al.have showed that the expression levels of SPOCK1 were significantly associated with the advanced tumor stage and the presence of distal metastasis in urothelial carcinoma[7].In lung cancer[8],SPOCK1 had been reported as a transforming growth factor-? target gene that regulated cell migration and invasion by inducing epithelial-mesenchymal transition(EMT).Additionally,Yang et al.[6]and Shu et al.[9]have reported that the overexpression of SPOCK1 could activate multiple intracellular signaling pathways,such as PI3K/AKT,mTOR-S6K and Wnt/?-catenin pathway.These findings suggest that SPOCK1 is a novel oncogene that contributes to rapid tumor progression in numerous types of cancers.Recently,Veenstra et al.has reported that SPOCK1 played an important role in invasive PC growth[10].However,few researches ever specified the expression or the underlying mechanism of SPOCK1 in PC cells.Therefore,it is urgent need to study the biological function and molecular mechanism of SPOCK1 in PC,which provides reliable theoretical and experimental foundations for finding an effective therapeutic target for PC treatment.Objectives:This study aims to investigate the expression and clinicopathological significance of SPOCK1 in PC and understand the underlying molecular mechanism of SPOCK1 implicated in tumor growth and development of metastasis in PC.Methods:1)Tissue specimens and database analysis:Oncomine and cBioPortal database were performed to analyze the mRNA expression of SPOCK1 in PC;OncoLnc database was used to analyze the prognostic significance of SPOCK1 in PC;immunohistochemistry was used to evaluate SPOCK1 and phospho-NF-?B expression in PC tissues and normal tissues.2)Experiments in vitro:Knockdown or overexpression of SPOCK1 expression in PC cells was constructed with lentivirus-mediated transduction and knockdown of ZEB2 expression in PC cells was constructed with siRNA-mediated transduction;the effects of SPOCK1 on PC cell proliferation were measured by MTT,colony formation,EdU incorporation assay;the effects of SPOCK1 on cell cycle distribution were measured by flow cytometry assay;the effects of SPOCK1 on PC cell migration and invasion were measured by wound healing and transwell assay;immunofluorescence staining was used to detect the localization and expression of E-cadherin,Vimentin and phospho-NF-?B in cells;western blot was used to detect the expression of EMT-related proteins,G2/M phase-related proteins and NF-?B signaling pathway-related proteins;luciferase reporter assay was used to detect the transcriptional activity of NF-?B.3)Experiments in vivo:For the in vivo proliferation assay,PC cells were inoculated subcutaneously into left axilla of nude mice;for the in vivo lung metastasis assay,PC cells were intravenously injected into the tail vein of nude mice;immunohistochemistry was used to detect the expression of Ki67,SPOCK1,phospho-NF-?B and E-cadherin in collected tumor tissues;haematoxylin and eosin staining was performed to confirm the tumor colonies formed in lung tissues.Results:1)SPOCK1 expression is increased and associated with metastasis and poor prognosis in PC:Oncomine and cBioPortal database analysis showed that SPOCK1 was overexpressed in PC tissues,which was further validated by immunohistochemistry in our cohort of PC samples and normal pancreatic tissues.Meanwhile,the overexpression of SPOCK1 was positively correlated with lymph node metastasis and poor prognosis of PC.2)SPOCK1 accelerates PC cell proliferation:We found that ectopic expression of SPOCK1 promoted PC cell proliferation and colony formation by MTT and colony formation assay.Furthermore,through the EdU retention assay,we observed that SPOCK1 could increase the ability of DNA replication of PC cells.3)SPOCK1 affects PC cell cycle progression:Cell cycle analysis showed that SPOCK1 had an effect on the G2/M phase transition with up-regulating the expression of G2/M associated proteins including CDK1,CyclinB1 and Cdc25c and down-regulating the expression of p-Cdc2,p21WAF1/CIPI and p27KIPI.4)SPOCK1 regulates the PC cell migration and invasion via inducing EMT:We found that overexpression of SPOCK1 facilitated the capacities of cell migration and invasion by wound healing and Transwell assay.In addition,overexpression of SPOCK1 down-regulated the expression of EMT epithelial-related marker E-cadherin and up-regulated the expression of EMT mesenchymal-related marker Vimentin and EMT-related transcriptional factors such as Snail,Slug,ZEB1 and ZEB2.These results suggest that SPOCK1 may promote PC cell migration and invasion via the induction of EMT.5)SPOCK1 activates NF-?B pathway:The analysis between SPOCK1 and phospho-NF-?B expression showed that the expression of them had a similar pattern in PC tissues and PC cell lines.More importantly,SPOCK1 controlled the NF-?B by enhancing NF-?B nuclear translocation and increasing the transcriptional activity of NF-?B,indicating that the overexpression of SPOCK1 is related to the activation of NF-?B pathway.6)SPOCK1 contributes to tumor metastasis via NF-?B/ZEB2 axis-mediated EMT in PC:Inhibiting the activity of NF-?B with an IKK inhibitor BAY11-7082 successfully reversed the EMT process and PC cell motilities induced by SPOCK1;among the several transcriptional factors,ZEB2 was remarkably abrogated when we inhibited the activity of NF-?B with BAY11-7082 in both SPOCK1 overexpressed cells;siRNA-mediated knock-down of ZEB2 in SPOCK1 overexpressed PC cells could effectively prevent the SPOCK1 triggered EMT process,indicating that the NF-?B/ZEB2 axis plays a critical role in SPOCK1-mediated PC cell metastasis.7)SPOCK1 facilitates PC cell growth and metastasis in vivo:SPOCK1 dramatically facilitated tumor growth and metastasis in our animal models and the up-regulated expression of Ki67,and phospho-NF-?B,down-regulated expression of E-cadherin was also observed by immunohistochemical stainingConclusions:1)SPOCK1 is overexpressed in PC tissues than in matched normal tissues and is significantly associated with aggressive behaviors of PC.2)SPOCK1 promotes the PC cell proliferation via controlling cell cycle transition from G2/M phase and regulating the expression of SPOCK1 on the G2/M-associated proteins such as CDK1,CyclinB1,Cdc25c,p-Cdc2,p21WAF1/CIPI and p27KIPI.3)SPOCK1 contributes to metastasis of PC cells by enhancing NF-?B nuclear translocation and increasing the transcriptional activity NF-?B.4)The activation of NF-?B plays a critical role in SPOCK1-mediated metastasis by targeting the EMT transcriptional factor ZEB2,suggesting that SPOCK1/NF-?B/ZEB2/EMT axis may serve as a novel diagnostic and therapeutic approach for managing PC patients. |
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