Background:Fascin is crucial for cancer cell filopodium formation and tumor metastasis,and is functionally regulated by post-translational modifications.However,whether and how Fascin is regulated by acetylation remai...Background:Fascin is crucial for cancer cell filopodium formation and tumor metastasis,and is functionally regulated by post-translational modifications.However,whether and how Fascin is regulated by acetylation remains unclear.This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis.Methods:Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor(PCAF),and immunofluorescence was used to investigate their colocalization.An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry.A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma(ESCC)cells using Western blotting by overexpressing and knocking down PCAF expression.An in vitro cell migration assay was performed,and a xenograft model was established to study in vivo tumor metastasis.Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation.The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry.Results:Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471(K471)by PCAF.Using the specific antiAcK471-Fascin antibody,Fascin was found to be acetylated in ESCC cells,and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown.Functionally,Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis,whereas Fascin-K471 deacetylation exhibited a potent oncogenic function.Moreover,Fascin-K471 acetylation reduced filopodial length and density,and lifespan of ESCC cells,while its deacetylation produced the opposite effect.In the filipodium shaft,K471-acetylated Fascin displayed rapid dynamic exchange,suggesting that it remained in its monomeric form owing to its weakened actinbundling activity.Clinically,high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients.Conclusions:Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells.Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.展开更多
Dear Editor,Fascin actin-bundling protein 1(Fascin,gene name:FSCN1)is the primary actin-bundling protein that clusters filaments into stable and bundled filopodia[1].In previous studies,elevated levels of Fascin promo...Dear Editor,Fascin actin-bundling protein 1(Fascin,gene name:FSCN1)is the primary actin-bundling protein that clusters filaments into stable and bundled filopodia[1].In previous studies,elevated levels of Fascin promoted filopodia formation and enhanced the invasive ability of tumor cells,suggesting that Fascin is a target for therapeutic intervention[2,3].Therefore,it is necessary to elucidate the detailed molecular mechanism by which Fascin bundles F-actin filaments.展开更多
基金National Natural Science Foundation of China,Grant/Award Numbers:81872372,81902469Natural Science Foundation of China-Guangdong Joint Fund,Grant/Award Number:U0932001+2 种基金National Cohort of Esophageal Cancer of China,Grant/Award Number:2016YFC0901400China Postdoctoral Science Foundation,Grant/Award Number:2018M6431342020 Li Ka Shing Foundation Cross-Disciplinary Research Grant,Grant/Award Number:2020LKSFG07B。
文摘Background:Fascin is crucial for cancer cell filopodium formation and tumor metastasis,and is functionally regulated by post-translational modifications.However,whether and how Fascin is regulated by acetylation remains unclear.This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis.Methods:Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor(PCAF),and immunofluorescence was used to investigate their colocalization.An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry.A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma(ESCC)cells using Western blotting by overexpressing and knocking down PCAF expression.An in vitro cell migration assay was performed,and a xenograft model was established to study in vivo tumor metastasis.Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation.The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry.Results:Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471(K471)by PCAF.Using the specific antiAcK471-Fascin antibody,Fascin was found to be acetylated in ESCC cells,and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown.Functionally,Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis,whereas Fascin-K471 deacetylation exhibited a potent oncogenic function.Moreover,Fascin-K471 acetylation reduced filopodial length and density,and lifespan of ESCC cells,while its deacetylation produced the opposite effect.In the filipodium shaft,K471-acetylated Fascin displayed rapid dynamic exchange,suggesting that it remained in its monomeric form owing to its weakened actinbundling activity.Clinically,high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients.Conclusions:Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells.Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.
基金supported by the grants from the National Natural Science Foundation of China(81872372)the Guangdong Basic and Applied Basic Research Foundation(2020A1515011532)+3 种基金the Natural Science Foundation of China-Guangdong Joint Fund(U0932001)the National Cohort of Esophageal Cancer of China(2016YFC0901400)the China Postdoctoral Science Foundation(2018M643134)2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant(2020LKSFG07B).
文摘Dear Editor,Fascin actin-bundling protein 1(Fascin,gene name:FSCN1)is the primary actin-bundling protein that clusters filaments into stable and bundled filopodia[1].In previous studies,elevated levels of Fascin promoted filopodia formation and enhanced the invasive ability of tumor cells,suggesting that Fascin is a target for therapeutic intervention[2,3].Therefore,it is necessary to elucidate the detailed molecular mechanism by which Fascin bundles F-actin filaments.
