Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: Wnt signaling pathway activates S6K1 and induces its translocation into the nucleus. ( a ) Wnt-3a and LiCl induce S6K1 phosphorylation. Cells (293T) were treated with Wnt-3a (300 ng/mL) and LiCl (25 mM) for the indicated time points. Immunoblotting was performed using specific antibodies as indicated. ( b , c ) LiCl promotes S6K1 nuclear translocation. Immunoblotting was performed using cytoplasmic and nuclear extracts from 293T cells treated with LiCl for the indicated time points ( b ). Localization of S6K1 and β-catenin was detected by immunostaining 293T cells treated with LiCl for the indicated time points ( c ). ( d ) Rapamycin inhibits Wnt-induced S6K1 nuclear translocation. Immunoblotting was performed using cytoplasmic and nuclear extracts from 293T cells treated with LiCl in the absence or presence of rapamycin (25 nM). Cells were pretreated with specific inhibitors for 1 h before LiCl treatment.

Article Snippet: Rapamycin, the mTOR specific inhibitor (Calbiochem, 553210, Darmstadt, Germany); PF-4708671, the S6K1 specific inhibitor (Tocris, 4032, Bristol, UK); Wnt-3a, Wnt activator that binds to Frizzled (R&D Systems, 5036-WN, Minneapolis, MN, USA); lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator (Sigma Aldrich, L9650, St. Louis, MO, USA); and active p70 S6K1 protein for in vitro kinase assay (SignalChem, R21-10H, Richmond, BC, Canada).

Techniques: Translocation Assay, Phospho-proteomics, Western Blot, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: S6K1 binds directly to β-catenin. ( a , b ) Endogenous S6K1 binds to β-catenin. Co-immunoprecipitation assays were performed in DLD-1 cells using S6K1 ( a ) and β-catenin ( b ) antibodies. Each binding partner was detected by immunoblot performed with the indicated antibodies. ( c ) Exogenous S6K1 binds to β-catenin. Co-immunoprecipitation assay was performed using Flag-antibody and 293T cells that were transiently transfected with exogenous Flag-β-catenin and Myc-S6K1. Immunoblot was performed with anti-Flag and anti-Myc antibodies. ( d ) S6K1 binds to β-catenin in vitro. In vitro GST pulldown assay was performed using recombinant GST or recombinant GST-β-catenin with 293T cell extracts. Presence of the GST and GST-fusion proteins were confirmed by Ponceau staining ( top ). Eluted S6K1 and GSTs were detected by immunoblot performed with anti-S6K1 and anti-GST antibodies ( bottom ). ( e ) S6K1 binds to β-catenin in a kinase activity-dependent manner. Co-immunoprecipitation assay was performed using Flag-antibody and 293T cells stably expressing Flag-tagged WT, CA, and DN forms of S6K1 treated with LiCl (25 mM) for 8 h. ( f ) The interaction between nuclear S6K1 and β-catenin was increased in a Wnt activation-dependent manner. Co-immunoprecipitation assay was performed using S6K1 antibody and cytoplasmic and nuclear extracts from 293T cells treated or untreated with LiCl (25 mM, 8 h).

Article Snippet: Rapamycin, the mTOR specific inhibitor (Calbiochem, 553210, Darmstadt, Germany); PF-4708671, the S6K1 specific inhibitor (Tocris, 4032, Bristol, UK); Wnt-3a, Wnt activator that binds to Frizzled (R&D Systems, 5036-WN, Minneapolis, MN, USA); lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator (Sigma Aldrich, L9650, St. Louis, MO, USA); and active p70 S6K1 protein for in vitro kinase assay (SignalChem, R21-10H, Richmond, BC, Canada).

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Transfection, In Vitro, GST Pulldown Assay, Recombinant, Staining, Activity Assay, Stable Transfection, Expressing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: S6K1 regulates Wnt/β-catenin-mediated transcription. ( a ) Reduced S6K1 affects cyclin D1 protein expression. Immunoblotting was performed using 293T cells with stable S6K1 knockdown (293T-S6K1 KD) treated with LiCl (25 mM) for 8 h. ( b ) Reduced S6K1 suppresses TOPFlash luciferase reporter activity. The TOPFlash luciferase reporter assay was performed using 293T cells treated with LiCl for the indicated time points after transient transfection with S6K1 siRNA (48 h). ( c ) Reduced S6K1 suppresses β-catenin transcriptional activity. qRT-PCR was performed using 293T cells treated with LiCl for 8 h after transient transfection with S6K1 siRNA (48 h). ( d ) S6K1 inhibition suppresses TOPFlash luciferase reporter activity. The TOPFlash luciferase reporter assay was performed using 293T cells treated with LiCl for the indicated time points after pretreatment for 1 h with rapamycin (25 nM) and PF-4708671 (10 µM). ( e ) S6K1 inhibition suppresses β-catenin transcriptional activity. qRT-PCR was performed using 293T cells treated with LiCl for 8 h after pretreatment for 1 h with rapamycin (25 nM) and PF-4708671 (10 µM). ( f ) TOPFlash luciferase reporter activity is regulated by S6K1 in a kinase activity-dependent manner. The TOPFlash luciferase reporter assay was performed using 293T cells stably expressing Flag-tagged WT, CA, and DN forms of S6K1 treated with LiCl (25 mM) for the indicated time points. ( g ) Kinase activity of S6K1 affects β-catenin transcriptional activity. qRT-PCR was performed using 293T cells stably expressing Flag-tagged WT, CA, and DN forms of S6K1 treated with LiCl (25 mM) for 8 h. Data are presented as the mean ± SEM for n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Rapamycin, the mTOR specific inhibitor (Calbiochem, 553210, Darmstadt, Germany); PF-4708671, the S6K1 specific inhibitor (Tocris, 4032, Bristol, UK); Wnt-3a, Wnt activator that binds to Frizzled (R&D Systems, 5036-WN, Minneapolis, MN, USA); lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator (Sigma Aldrich, L9650, St. Louis, MO, USA); and active p70 S6K1 protein for in vitro kinase assay (SignalChem, R21-10H, Richmond, BC, Canada).

Techniques: Expressing, Western Blot, Knockdown, Luciferase, Activity Assay, Reporter Assay, Transfection, Quantitative RT-PCR, Inhibition, Stable Transfection

Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: S6K1 does not affect β-catenin. ( a ) β-catenin is not a kinase substrate of S6K1. In vitro kinase assays were performed using GST, GST-β-catenin, and recombinant histone H2B as substrates and recombinant active-S6K1 as the kinase in the presence of 32P-ATP. The presence of substrates was confirmed by Coomassie blue staining ( left ), and 32P-mediated phosphorylation was detected by photosensitization with X-ray film ( right ). ( b ) Inhibition of S6K1 did not influence LiCl-induced β-catenin accumulation. Immunoblotting was performed using 293T cells pretreated with rapamycin (25 nM, 1 h) followed by LiCl (25 mM) for the indicated time points. ( c ) S6K1 inhibition did not affect the nucleocytoplasmic distribution of β-catenin. Immunoblotting was performed using cytoplasmic and nuclear extracts from 293T cells pretreated with or without rapamycin (25 nM) and PF-4708671 (10 µM) for 1 h and then treated with LiCl for 8 h.) ( d ) S6K1 does not affect the acetylation of K49 residue of β-catenin in a kinase activity-dependent manner. Immunoblotting was performed using 293T cells stably expressing Flag-tagged WT, CA, and DN forms of S6K1 treated with LiCl (25 mM) for 8 h. ( e ) β-catenin knockdown did not influence LiCl-induced phosphorylation of S6K1. Immunoblotting was performed using 293T cells treated with LiCl for the indicated time points after transient transfection with β-catenin siRNA (48 h).

Article Snippet: Rapamycin, the mTOR specific inhibitor (Calbiochem, 553210, Darmstadt, Germany); PF-4708671, the S6K1 specific inhibitor (Tocris, 4032, Bristol, UK); Wnt-3a, Wnt activator that binds to Frizzled (R&D Systems, 5036-WN, Minneapolis, MN, USA); lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator (Sigma Aldrich, L9650, St. Louis, MO, USA); and active p70 S6K1 protein for in vitro kinase assay (SignalChem, R21-10H, Richmond, BC, Canada).

Techniques: In Vitro, Recombinant, Staining, Phospho-proteomics, Inhibition, Western Blot, Residue, Activity Assay, Stable Transfection, Expressing, Knockdown, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: S6K1 affects the formation of the Wnt/β-catenin transcriptional complex. ( a , b ) S6K1 knockdown reduces the interaction between β-catenin and TCF4. Co-immunoprecipitation assay was performed using TCF4 ( a ) and β-catenin ( b ) antibodies and 293T cells treated with LiCl (25 mM) for 8 h after transient transfection with S6K1 siRNA (48 h). ( c , d ) Inhibition of S6K1 disrupts formation of Wnt/β-catenin transcriptional complex. Each co-immunoprecipitation assay was performed with nuclear extracts from 293T cells treated with DMSO or PF-4708671 (10 µM) followed by LiCl treatment for 8 h, using Pygo2 ( c ) and p300 ( d ) antibody, respectively.

Article Snippet: Rapamycin, the mTOR specific inhibitor (Calbiochem, 553210, Darmstadt, Germany); PF-4708671, the S6K1 specific inhibitor (Tocris, 4032, Bristol, UK); Wnt-3a, Wnt activator that binds to Frizzled (R&D Systems, 5036-WN, Minneapolis, MN, USA); lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator (Sigma Aldrich, L9650, St. Louis, MO, USA); and active p70 S6K1 protein for in vitro kinase assay (SignalChem, R21-10H, Richmond, BC, Canada).

Techniques: Knockdown, Co-Immunoprecipitation Assay, Transfection, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Nuclear S6K1 Enhances Oncogenic Wnt Signaling by Inducing Wnt/β-Catenin Transcriptional Complex Formation

doi: 10.3390/ijms232416143

Figure Lengend Snippet: Effects of S6K1 deficiency in colon cancer cell lines. ( a ) S6K1 is aberrantly activated in colon cancer cell lines. Immunoblotting was performed using total protein extracts from CCD-18Co (normal colon cells), LoVo, DLD-1, and HT-29 (colon cancer cells) and specific antibodies as indicated. ( b ) HT-29 and DLD-1 S6K1-knockdown cell lines. Immunoblotting was performed using these S6K1-knockdown two colorectal cancer cell lines and antibodies as indicated. ( c ) Reduced S6K1 levels suppressed TOPFlash luciferase reporter activity. TOPFlash luciferase reporter assay was performed in HT-29 and DLD-1 S6K1-knockdown cell lines. ( d ) S6K1 knockdown suppresses the expression of Wnt target genes in colon cancer cells. qRT-PCR was performed in HT-29 and DLD-1 S6K1-knockdown cell lines. ( e ) Loss of S6K1 inhibits cell proliferation in HT-29 and DLD-1 colon cancer cells. Cell proliferation assay was performed in HT-29 and DLD-1 S6K1-knockdown cell lines using an automated cell counter as indicated time points. ( f ) Loss of S6K1 inhibits cell migration in HT-29 and DLD-1 colon cancer cells. Wound-healing assay was performed using HT-29 and DLD-1 S6K1-knockdown cells. Data are represented as the mean ± SEM for n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Rapamycin, the mTOR specific inhibitor (Calbiochem, 553210, Darmstadt, Germany); PF-4708671, the S6K1 specific inhibitor (Tocris, 4032, Bristol, UK); Wnt-3a, Wnt activator that binds to Frizzled (R&D Systems, 5036-WN, Minneapolis, MN, USA); lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator (Sigma Aldrich, L9650, St. Louis, MO, USA); and active p70 S6K1 protein for in vitro kinase assay (SignalChem, R21-10H, Richmond, BC, Canada).

Techniques: Western Blot, Knockdown, Luciferase, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Proliferation Assay, Migration, Wound Healing Assay

Journal: Frontiers in Oncology

Article Title: Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Tumor Growth Through Inhibiting the mTOR Signaling Pathway

doi: 10.3389/fonc.2021.780011

Figure Lengend Snippet: Ethyl ferulate strongly inhibits the mammalian target of rapamycin (mTOR) signaling pathway. (A) The effect of ethyl ferulate on epidermal growth factor (EGF)-induced kinase signaling in JB6 cells. Serum-starved [without fetal bovine serum (FBS); 24 h] cells were treated with different concentrations of ethyl ferulate for 6 h followed by treatment with EGF for 30 min. (B) The effect of ethyl ferulate on various kinase signaling proteins in esophageal squamous cell carcinoma (ESCC) cells. Cells were treated with ethyl ferulate for 24 h, and then various signaling proteins were examined by Western blotting. (C) Ethyl ferulate directly binds to recombinant mTOR protein and mTOR present in KYSE510 cell lysates. The recombinant mTOR protein or cell lysate was incubated with Sepharose 4B beads or ethyl ferulate-conjugated Sepharose 4B beads. Pulled down proteins were examined by Western blotting. (D) The effect of ethyl ferulate on mTOR kinase activity. mTOR kinase activity was assessed by in vitro kinase assay using active mTOR and inactive p70S6K proteins. For all experiments, similar results were shown as mean values ± SD for 3 independent experiments and band density is shown as a bar graph. One-way analysis of variance (ANOVA) was used to analyze the data. The asterisk (*) indicates a significant ( P < 0.05) inhibitory effect of ethyl ferulate.

Article Snippet: Active mTOR recombinant protein used for in vitro kinase assay or in vitro pull-down assay was purchased from Thermo Fisher (Shanghai, China), and inactive p70S6K recombinant protein used for in vitro kinase assay was purchased from SignalChem (Richmond, BC, Canada).

Techniques: Western Blot, Recombinant, Incubation, Activity Assay, In Vitro, Kinase Assay

Journal: Frontiers in Oncology

Article Title: Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Tumor Growth Through Inhibiting the mTOR Signaling Pathway

doi: 10.3389/fonc.2021.780011

Figure Lengend Snippet: Ethyl ferulate reduces esophageal squamous cell carcinoma (ESCC) patient-derived xenograft tumor growth in vivo. Mice were divided into two groups to assess the effect of ethyl ferulate on ESCC patient-derived xenograft (PDX) tumor growth. Groups are as follows: 1) vehicle group or 2) group treated with 100 mg/kg of ethyl ferulate. Tumor-bearing mice were orally treated (by gavage) with ethyl ferulate or vehicle for 32 (LEG110; n = 10) or 53 (LEG45; n = 7) days. Tumor volumes were measured on the days indicated. The effect of ethyl ferulate on ESCC tumor growth in (A) LEG110 or (B) LEG45 ESCC PDX tissues. For panels (A, B) , data are shown as mean ± SE from each group. The effect of ethyl ferulate on Ki-67 expression in (C) LEG110 or (D) LEG45 ESCC PDX tissues (n = 5). Three slices per PDX tissue were analyzed. Treated or untreated groups of tumor tissues were stained with antibodies to detect Ki-67 (×100; scale bar: 100 μm; ×20; scale bar: 50 μm). (E, F) Ethyl ferulate inhibits phosphorylated mammalian target of rapamycin (mTOR), p70S6K, and AKT protein expression in esophageal tumor tissues. Tumor tissues from each group were immunoblotted with antibodies to detect mTOR, p70S6K, and AKT and phosphorylated mTOR, p70S6K, AKT, and β-actin. β-Actin was used to verify equal protein loading. Band density was measured using the ImageJ (NIH) software program, and the results are shown as a bar graph. One-way ANOVA was used to compare significant differences. The asterisk (*) indicates a significant (* P < 0.05; ** P < 0.01) inhibitory effect of ethyl ferulate treatment.

Article Snippet: Active mTOR recombinant protein used for in vitro kinase assay or in vitro pull-down assay was purchased from Thermo Fisher (Shanghai, China), and inactive p70S6K recombinant protein used for in vitro kinase assay was purchased from SignalChem (Richmond, BC, Canada).

Techniques: Derivative Assay, In Vivo, Expressing, Staining, Software

Journal: Frontiers in Oncology

Article Title: Ipriflavone Suppresses Growth of Esophageal Squamous Cell Carcinoma Through Inhibiting mTOR In Vitro and In Vivo

doi: 10.3389/fonc.2021.648809

Figure Lengend Snippet: Ipriflavone is a novel mTOR inhibitor. (A) Effect of Ipriflavone on EGF‐induced kinase signaling molecules in JB6 cells. After serum starvation for 24 h, cells were treated with different doses of Ipriflavone for 6 h followed by EGF treatment for 30 min. various signaling molecules were analyzed by Western blotting. (B) Effect of Ipriflavone on various signaling molecules in KYSE450 ESCC cells. Cells were treated with Ipriflavone for 24 h and signaling molecule proteins were examined by Western blotting. (C) Effect of Ipriflavone on mTOR kinase activity was assessed by an in vitro kinase assay using active mTOR and inactive p70S6K proteins. The activity of mTOR was determined by Western blotting using a phosphorylated p70S6K antibody. AZD8055 (mTOR inhibitor) was used as a positive control. For all data, similar results were observed from three independent experiments and band density was measured using the Image J (NIH) software program. The asterisk (*) indicates a significant ( p < 0.05) difference. (D) Modeling of Ipriflavone binding with mTOR. ATPγS ( D , upper left panel ) and Ipriflavone ( D , upper right panel ) binding with mTOR at the ATP binding pocket. ( D , lower panel ) Ligand Interaction Diagram (LID) of the binding. The Ipriflavone is shown as stick. LID legend is shown below.

Article Snippet: Active mTOR and p70S6K human recombinant proteins for kinase assays were purchased from SignalChem (Richmond, BC, Canada).

Techniques: Western Blot, Activity Assay, In Vitro, Kinase Assay, Positive Control, Software, Binding Assay

Journal: Frontiers in Oncology

Article Title: Ipriflavone Suppresses Growth of Esophageal Squamous Cell Carcinoma Through Inhibiting mTOR In Vitro and In Vivo

doi: 10.3389/fonc.2021.648809

Figure Lengend Snippet: Ipriflavone is a novel mTOR inhibitor. (A) Effect of Ipriflavone on EGF‐induced kinase signaling molecules in JB6 cells. After serum starvation for 24 h, cells were treated with different doses of Ipriflavone for 6 h followed by EGF treatment for 30 min. various signaling molecules were analyzed by Western blotting. (B) Effect of Ipriflavone on various signaling molecules in KYSE450 ESCC cells. Cells were treated with Ipriflavone for 24 h and signaling molecule proteins were examined by Western blotting. (C) Effect of Ipriflavone on mTOR kinase activity was assessed by an in vitro kinase assay using active mTOR and inactive p70S6K proteins. The activity of mTOR was determined by Western blotting using a phosphorylated p70S6K antibody. AZD8055 (mTOR inhibitor) was used as a positive control. For all data, similar results were observed from three independent experiments and band density was measured using the Image J (NIH) software program. The asterisk (*) indicates a significant ( p < 0.05) difference. (D) Modeling of Ipriflavone binding with mTOR. ATPγS ( D , upper left panel ) and Ipriflavone ( D , upper right panel ) binding with mTOR at the ATP binding pocket. ( D , lower panel ) Ligand Interaction Diagram (LID) of the binding. The Ipriflavone is shown as stick. LID legend is shown below.

Article Snippet: The inactive p70S6K recombinant protein (100 ng, Signalchem) and ATP (Cell Signaling Technology) were added and the mixtures were incubated at 30°C for 30 min.

Techniques: Western Blot, Activity Assay, In Vitro, Kinase Assay, Positive Control, Software, Binding Assay