| Background and Objects:Patients with metastatic hepatocellular carcinoma(HCC)usually have a poor prognosis,and invadopodium formation is a crucial early event of invasion and metastasis of this disease.However,the molecular mechanisms underlying regulating invadopodia remain elusive.Tyrosine kinase substrate with five SH3 domains(Tks5,five SH3 domains,FISH,or SH3 multiple domains 1),a substrate of Src,is the master scaffold for invadopodium formation and function.Src-dependent phosphorylation of Tks5 at Y557/Y619 is important for recruiting adaptor proteins to assemble an F-actin network in Tks5-riched invadopodium precursors,and for invadopodium-associated matrix degradation in melanoma and prostate cancer cells.Discs large homolog 5(Dlg5)is a member of the membrane-associated guanylate kinase family.Dlg5 is required for the maintenance of adherens junctions and epithelial cell polarity in mammalian brain,kidneys,and lungs.Recent studies suggest that Dlg5 may inhibit epithelial-mesenchymal transition(EMT)in renal epithelial cells and prostate cancer cells by attenuating TGF-β1 signaling.Indeed,down-regulated Dlg5 expression is detected in aggressive malignancies.However,little is known about how Dlg5 regulates cancer cell invasion and metastasis.Notably,Dlg5 can interact with girders of actin filament(Girdin)and inhibit Akt-mediated phosphorylation of Girdin at Ser-1416 in prostate cancer PC3 cells.Recently Leyme et a121 found that Girdin localized in invadopodia of NIH3T3-Src-Y527F cells though they did not determine its potential role in invadopodia.Our preliminary studies observed that Girdin interacted with SH3 multiple domains 2,a protein with Tks5 in the same family(Wang L,unpublished data).Moreover,Girdin is necessary for FAK Tyr397 and Src Tyr418 phosphorylation,both are involved in Tks5 activation and invadopodium development.Accordingly,we hypothesize that Dlg5 may regulate the formation and function of invadopodia through a mechanism that requires Girdin and Tks5.This study aimed to(1)investigate the difference of Dlg5 expression profile in HCC and the adjacent non-tumor liver tissues,and the correlation of its expression in HCC with clinicopathological features,(2)find out the potential role of Dlg5 in invadopodia formation and function of HCC cells and the molecular mechanism underlying the regulation(we will focus on the possible participation of Girdin and Tks5 in the mechanistic aspect),(3)determine Dlg5 effect on cellular invasion in vitro and tumor number and size in vivo.Methods:At a tissue level:HCC specimens and corresponding adjacent liver tissues(≥ 2 cm from the tumors)from 100 patients were collected from Department of Hepatobiliary Surgery,the Second Affiliated Hospital of Kunming Medical University from January 2008 to December 2009.Patients were followed-up until December 2015,and their demographic and clinical data were collected.(1)The levels of Dlg5 expression in HCC tissues were analyzed by immunohistochemistry.(2)The correlation of Dlg5 expression in HCC with clinicopathological features was further explored.Cellular phenotype assays:(1)The expression of Dlg5 protein was detected using Western blot in human hepatoma HepG2,SMMC-7721,SK-Hep1,and MHCC97-L cells,and non-tumor HL-7702 liver cells.(2)HepG2 cells were infected with lentivirus for expression of Dlg5-specific shRNAs or control shRNA.The relative levels of Dlg5,E-cadherin,Fibronectin,andα-SMA to β-tubulin in different groups of cells were determined by Western blot,and the bright field images of different groups of cells were taken using a phase contrast microscope.Invadopodium formation in HCC cells was determined by immunofluorescent assays.Briefly,different groups of cells were stained with phalloidin(red),DAPI(blue),and anti-Tks5 or anti-Cortactin(green).Co-localization of F-actin with the unique adaptor protein Tks5 or the nucleation promoting factor Cortactin(specified where used including in the context of Tks5 knockdown)was used to determine invadopodium formation.For monitoring the gelatin degradation by invadopodia,Different groups of cells were plated on FITC-gelatin(green),and stained with phalloidin(red)and DAPI(blue).Invadopodium-associated gelatin degradation was defined as F-actin puncta co-localizing with gelatin degradation.Furthermore,transwell Matrigel assays were used to determine invasion of different groups of cells.(3)SK-Hepl cells were transfected with control pcDNA3.1(+)or Dlg5-expressing H302 pcDNA3.1(+)using lipofectamine 3000 and the positively transduced cells were selected by treatment with Neomycin.The relative levels of Dlg5,E-cadherin,Fibronectin,α-SMA to(β-tubulin expression in different groups of SK-Hepl cells were tested by Western blot assay.The invadopodium formation,function,and cellular invasion of different groups of cells were determined as the above.Furthermore,the different groups of cells were cultured for four days and the numbers of cells were determined at indicated times.(4)Control shRNA-expressing and Dlg5-silenced HepG2 cells were infected with lentiviruses expressing control or Tks5-specific shRNAs and these cells were infected with control adenoviruses(vector)alone,or adenoviruses expressing shRNA-resistant Tks5 WT or Tks5-Y557F/Y619F(or Girdin).The levels of Dlg5,Tks5,and β-tulin expression in cell lysates and Tks5 Tyr phosphorylation in immunocomplex from different groups of cells were determined by Western blot.Control and Dlg5-silenced HepG2 cells were infected with lentiviruses encoding control or Girdin shRNAs.These cells were infected with control adenoviruses(vector),or adenoviruses expressing shRNA-resistant Girdin WT.The levels of Girdin,Dlg5,and β-tubulin expression were determined by Western blot.The invadopodium formation,function,and cellular invasion of different groups of cells were determined as the above.Molecular mechanism studies:(1)HepG2 cell lysates were probed with anti-Dlg5 or anti-Girdin and the levels of Dlg5,Girdin,and Tks5 in immunocomplex or cell lysates were determined by Western blot.Control or Dlg5-silenced HepG2 cells that had been infected with control adenoviruses(vector)or adenoviruses expressing shRNA-resistant Dlg5 were harvested.The levels of Dlg5,Girdin,and Tks5 in immunocomplex or cell lysates were determined by Western blot.The distribution of Girdin and Tks5 in different groups of cells was determined by immunofluorescent assays.(2)Control and Dlg5-silenced HepG2 cells were infected with control lentiviruses or lentiviruses expressing Girdin-specific shRNAs.These cells were infected with control adenoviruses(vector)or adenoviruses expressing shRNA-resistant Girdin.The relative levels of Dlg5,Girdin,Tks5,FAK,and Src toβ-tubulin in different groups of cells were determined by Western blot.The different groups of cell lysates were immunoprecipitated with anti-Girdin,anti-Tks5,anti-FAK,or anti-Src and the levels of Girdin Ser1416 and Tyr1764,Tks5 Tyr,FAK Tyr397,and Src Tyr416 phosphorylation were determined by Western blot.Control and Dlg5-silenced HepG2 cells were treated with vehicle DMSO or PF-573228(the FAK-specific inhibitor)or SU-6656(the Src-specific inhibitor)for 8 hours and the levels of Dlg5,Tks5,FAK,Src,and β-tubulin in cell lysates and Tks5 Tyr,FAK Tyr397,Src Tyr416 phosphorylation in immunocomplex from different groups of cells were determined by Western blot.(3)Control and Dlg5-overexpressing HepG2 cells were treated with the increasing concentrations of TGF-β1(0,5,or 10 ng/ml)for three days.The relative levels of Dlg5,Girdin,and Tks5 were determined by Western blot.Their cell lysates were immunoprecipitated with anti-Girdin and the levels of Tks5 in the immunocomplex were determined by Western blot.Control and Dlg5-overexpressing HepG2 cells were treated with 15 ng/ml TGF-β1 or DMSO for three days.The relative levels of Dlg5,Girdin,Tks5,FAK,and Src to β-tubulin in different groups of cells were determined by Western blot.The different groups of cell lysates were immunoprecipitated with anti-Girdin,anti-Tks5,anti-FAK,or anti-Src and the levels of Girdin Ser1416 and Tyr1764,Tks5 Tyr,FAK Tyr397,and Src Tyr416 phosphorylation were determined by Western blot.Furthermore,the invadopodium formation and function of different groups of cells were determined as the above.In vivo assays:Male BALB/c nude mice were implanted orthotopically with control or Dlg5 over-expressing SK-Hep1 cells(n = 10 mice/group),(A)Five weeks later,the metastatic lung tumors in individual mice were measured by microPET/microCT imaging.Subsequently,the mice were sacrificed and the tumor nodules in the liver and lungs of individual mice were examined by histology.Results:At a tissue level:(1)A moderate-strong Dlg5 expression was detected in 70%of non-tumor liver tissues,but only in 28%of HCC tissues(P<0.05).(2)Stratification analysis revealed that the Dlg5 expression was inversely associated with tumor nodules(Multiple nodules/single nodule:high expression 6/22 vs.low expression 32/40,P = 0.033),vascular invasion(Yes/No:high expression 5/23 vs.low expression 29/43,P=0.034),cellular differentiation grades(EdmonsonⅢ-Ⅳ/Ⅰ-Ⅱ:high expression 4/24 vs.low expression 31/41,P = 0.007),and TNM stages(TNM Ⅲ-Ⅳ/Ⅰ-Ⅱ:high expression 2/26 vs.low expression 25/47,P = 0.005).Furthermore,low Dlg5 expression was associated significantly with poor overall survival(median OS of 20 monthsvs.69 months;P = 0.010),and disease-free survival(median DFS of 16 months vs.48 months;P =0.017)of HCC patients.Cellular phenotype assays:(1)High levels of Dlg5 expression were detected in HL-7702 cells,its expression was associated negatively with invasiveness of different HCC cell lines(low invasive:HepG2,SMMC-7721,high invasive:SK-Hepl,and MHCC97-L cells).(2)Infection with lentivirus significantly reduced Dlg5 expression by 90%-95%.Second,Dlg5-silencing significantly decreased the levels of E-cadherin,but increased a-SMA and fibronectin expression in HepG2 cells.Bright field images indicated that Dlg5-silencing changed HepG2 cells from a typically cobblestone-like epithelial to fibroblast-like morphology.Dlg5-silencing significantly promoted the formation of invadopodia in HepG2 cells.Evidentially,20%-25%of Dlg5-silenced HepG2 cells exhibited Tks5 and F-actin co-localization,and was significantly higher than 5%of control cells(P<0.05).Furthermore,the numbers of invadopodia per Dlg5-silenced HepG2 cell were significantly greater than the control(1.5-1.8 vs.0.3,P<0.05).Dlg5-silencing also enhanced the function of invadopodia in HepG2 cells.The numbers of cells degrading gelatin(30%-35%vs.7%,P<0.05)and the relative areas of degraded gelatin per Dlg5-silenced HepG2 cell(5-6 vs.1,P<0.05)were significantly greater than that in the control.Furthermore,Dlg5-silencing also increased cellular invasion of HepG2 cells by～2 times(P<0.05).(3)Induction of DIg5 over-expression significantly inhibited EMT process(the levels of E-cadherin went up,while the levels of a-SMA and fibronectin went down),invadopodium formation(the numbers of cells exhibiting Tks5 and F-actin co-localization:20%vs.control 5%,P<0.05),function(the numbers of cells degrading gelatin:30%vs.control 5%,P<0.05),and cellular invasion(decreased by～66%vs.control,P<0.05)in naturally Dlg5-deficient SK-Hepl cells.In contrast,Dlg5 over-expression did not affect the dynamic growth of SK-Hepl cells.(4)Tks5 silencing dramatically inhibited the enhanced invadopodium formation by～90%(P<0.05),gelatin degradation by>90%(P<0.05),and invasion by-40%(P<0.05)in the Dlg5-silenced HepG2 cells.Induction of shRNA-resistant Tks5 wild-type(WT),but not the Tks5 non-phosphorylatable mutant(Tks5-Y557F/Y619F),expression not only restored Tks5’s expression and phosphorylation,but also abrogated defective invadopodium formation and function and cell invasion in endogenous Dlg5 and Tks5-silenced HepG2 cells.Girdin silencing almost completely demolished the increased invadopodium formation and function as well as invasion in Dlg5-silenced HepG2 cells(P<0.05),which were rescued by ectopic expression of shRNA-resistant Girdin.Molecular mechanism studies:(1)Co-immunoprecipitation with anti-Dlg5 detected Girdin and vice versa in HepG2 cells.However,anti-Dlg5 failed to immunoprecipitate Tks5.Anti-Girdin immunoprecipitated a little endogenous Tks5 in the control HepG2 cells but a comparable amount of Girdin co-precipitated much more Tks5,which was mitigated by induction of shRNA-resistant Dlg5 in the Dlg5-silenced HepG2 cells.Immunofluorescence revealed that co-localization of Girdin and Tks5 signals was in the cytoplasm of HepG2 cells,and significantly increased at invadopodium-like puncta of Dlg5-silenced HepG2 cells(Pearson’s coefficient of～0.2 vs.～0.5,P<0.05),which was significantly reduced in the Dlg5-rescued cells(Pearson’s coefficient of-0.6 vs.-0.2,P<0.05).(2)Low levels of Girdin and Tks5 phosphorylation were detected in the control cells and Dlg5 and/or Girdin silencing did not affect the relative levels of FAK,Src,and Tks5 expression in HepG2 cells.Dlg5-silencing increased the levels of Girdin Ser-1416 and Tyr-1764 phosphorylation,which was abrogated by Girdin silencing and rescued by inducing shRNA-resistant Girdin expression.Furthermore,obvious Tks5 tyrosine-phosphorylation was found in Dlg5-silenced HepG2 cells,but not in the Dlg5 and Girdin co-silenced HepG2 cells,which was rescued by ectopic expression of shRNA-resistant Girdin.Dlg5-silencing enhanced FAK Tyr397 and Src Tyr416 phosphorylation in HepG2 cells,but not in the Dlg5 and Girdin co-silenced HepG2 cells,which was rescued by induction of shRNA-resistant Girdin.Furthermore,treatment with PF-573228 or SU-6656,a pharmacological FAK-or Src-specific inhibitor,significantly reduced Tks5 phosphorylation in the Dlg5-silenced HepG2 cells.(3)Treatment with TGF-β1 for 3 days inhibited Dlg5 expression in a concentration-dependent manner in control HepG2 cells,which was almost completely abrogated by overexpressing Dlg5.Similarly,treatment with TGF-β1 enhanced the interaction between Tks5 and Girdin in control HepG2 cells,which was almost completely abrogated by overexpressing Dlg5.Furthermore,TGF-β1 treatment resulted in a significant increase in the levels of Girdin,FAK,Src,and Tks5 phosphorylation in control HepG2 cells,which were obviously demolished in Dlg5-overexpressing cells.Furthermore,TGF-β1 treatment significantly increased invadopodium formation(the numbers of cells exhibiting Tks5 and F-actin co-localization:5%vs.35%,P<0.05)and function(the numbers of cells degrading gelatin:5%vs.45%,P<0.05),which were obviously demolished by Dlg5 overexpression.In vivo assays:(1)The radioactive signals in the lungs of the control mice were significantly higher than that in the Dlg5 group of mice(mean Standard uptake value,SUV:1.8 vs.0.7,P<0.05).(2)Histological examination revealed that the numbers(median numbers of metastatic foci/lung:4 vs.1,P<0.05;median numbers of intrahepatic foci/liver;15 vs.5,P<0.05)and sizes(median numbers of metastatic foci>0.5mm/lung:3 vs.0,P<0.05;median numbers of intrahepatic foci/liver:9 vs.3,<0.05)of intrahepatic and lung metastatic tumor nodules in the control mice were significantly greater than that in the Dlg5 group of mice.Conclusions:Taken together,our data indicate that(1)Dlg5 was significantly underexpressed in HCC tissues compared with adjacent non-tumor tissues,and its underexpression was significantly associated with tumor progression and poorer survival outcomes of HCC patients.(2)Dlg5 knockdown increases invasion of HCC cells,at least partly through promoting invadopodium formation and function.(3)Dlg5 acts as a novel regulator of invadopodia-associated invasion through a mechanism that requires Giridn and Tks5.(4)Dlg5 can interfere with the interaction between Girdin and Tks5,which may be important for Tks5 phosphorylation in HCC cells.(5)TGF-β1 could serve as a potential physiological regulator of Dlg5 expression,thus regulating EMT and invadopodia.(6)Dlg5 overexpression significantly inhibited HCC intrahepatic and lung metastasis in vivo.These findings identified a new cellular function of the well-known EMT-related Dlg5 and provided new insights into the molecular mechanisms of invadopodium formation and cancer cell invasion.Furthermore,Dlg5 may act as a new biomarker for prognosis of HCC patients. |
