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Laura Hinze - One of the best experts on this subject based on the ideXlab platform.
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exploiting the therapeutic interaction of wnt pathway activation and asparaginase for colorectal cancer therapy
2020Co-Authors: Laura Hinze, Salmaan Karim, James Degar, Roxane Labrosse, Teng Han, Emma M Schatoff, Sabine Schreek, Connor McguckinAbstract:Colorectal cancer is driven by mutations that activate canonical WNT/β-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a β-catenin-independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or β-catenin mutations) of GSK3, thus allowing WNT/β-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or β-catenin-mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or β-catenin-mutant colorectal cancer, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy. SIGNIFICANCE: Solid tumors are thought to be asparaginase-resistant via de novo asparagine synthesis. In leukemia, GSK3α-dependent protein degradation, a catabolic amino acid source, mediates asparaginase resistance. We found that asparaginase is profoundly toxic to colorectal cancers with WNT-activating mutations that inhibit GSK3. Aberrant WNT activation can provide a therapeutic vulnerability in colorectal cancer.See related commentary by Davidsen and Sullivan, p. 1632.This article is highlighted in the In This Issue feature, p. 1611.
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synthetic lethality of wnt pathway activation and asparaginase in drug resistant acute leukemias
2019Co-Authors: Kristen E Stevenson, Laura Hinze, Maren Pfirrmann, Salmaan Karim, James Degar, Connor Mcguckin, Divya S Vinjamur, Joshua R Sacher, Donna NeubergAbstract:Summary Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits glycogen synthase kinase 3 (GSK3)-dependent protein ubiquitination and proteasomal degradation, a catabolic source of asparagine. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3α inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.
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synthetic lethality of wnt pathway activation and asparaginase in drug resistant acute leukemias
2018Co-Authors: Laura Hinze, Donna Neuberg, Kristen E Stevenson, Maren Pfirrmann, Salmaan Karim, James Degar, Connor Mcguckin, Divya S Vinjamur, Joshua R Sacher, Daniel E BauerAbstract:Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. We hypothesized, from the concept of synthetic lethality, that gain-of-fitness alterations in drug-resistant cells had conferred a survival advantage that could be exploited therapeutically. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, including T-lymphoblastic, hypodiploid B-lymphoblastic, and acute myeloid leukemias, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and degradation. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3α inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.
Maria Luisa Lavitrano - One of the best experts on this subject based on the ideXlab platform.
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GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis
2014Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Carola Missaglia, Maria Grazia Cerrito, Roberto Giovannoni, Sara Bonomo, Laura Masiero, Maria Luisa LavitranoAbstract:Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy.
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abstract b113 gsk3a is redundant with GSK3B in modulating drug resistance and chemotherapy induced necroptosis
2013Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Elena A Federzoni, Robert Narloch, Kristian Helin, Maria Luisa LavitranoAbstract:Purpose of the study. GSK3A and GSK3B isoforms perform similar functions but they are not completely redundant, depending on the cell type, biological process and differentiative status (Rayasam GV et al, Br J Pharmacol, 2009). We recently demonstrated that GSK3B inhibition enables necroptosis in response to chemotherapy in drug-resistant colon carcinoma cells (Grassilli E et al, Clin Cancer Res, 2013). Here we investigated whether GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. Materials and Methods. Cell lines stably interfered were obtained by retroviral infection and selection with the appropriate antibiotic. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cell viability was assessed by Trypan blue staining. Cell proliferation was evaluated by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Cell cycle was analysed by flow cytometry upon PI staining using Modfit Cell Cycle Analysis (Becton Dickinson). Reporter activity was analysed by Dual-Glo Luciferase Assay (Promega). IF staining was performed on 4% paraformaldehyde-fixed cells. Active caspase-3/7 was quantified by the Caspase-Glo3/7 Assay System (Promega). Western blots were performed on lysates obtained by RIPA buffer. Results and Discussion. GSK3A stable silencing in HCT116p53KO cells did not alter cell proliferation as assessed by growth curve, cell cycle distribution and β-catenin activation assays. GSK3A depletion abolished colony formation after 5FU treatment and re-sensitized drug-resistant HCT116p53KO cells to 5FU-induced cell death. Blocking GSK3A did not interfere with DNA damage sensing upon 5FU treatment, as demonstrated by H2AX foci formation, but impaired the DNA repair response by affecting RPA70 foci formation. GSK3A silencing allowed a cell death response to DNA-damaging drugs in absence of p53: 5FU-treated, GSK3A-depleted HCT116p53KO cells underwent caspase-independent PARP1- and tBid-dependent cell death, which was unaffected by RIP1 inhibition and was characterized by PARP-dependent nuclear re-localization of AIF. Our data demostrate that GSK3A, like GSK3B, contributes to cell survival upon DNA-damaging chemotherapy by suppressing RIP-independent necroptosis and pinpoint that inhibition of one isoform is sufficient to bypass drug resistance of p53-null cells. Being GSK3A and GSK3B redundant in the response to DNA-damaging drugs, it is likely that, when used together with anticancer drugs, GSK3 inhibitors should be particularly effective in overcoming drug resistance even at low doses, since inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells. We therefore propose that GSK3 inhibition in combination with DNA-damaging drugs would be an appealing strategy to induce necroptosis in p53-null drug-resistant tumor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B113. Citation Format: Emanuela Grassilli, Elena Federzoni, Robert Narloch, Leonarda Ianzano, Kristian Helin, Marialuisa Lavitrano. GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B113.
Emanuela Grassilli - One of the best experts on this subject based on the ideXlab platform.
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GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis
2014Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Carola Missaglia, Maria Grazia Cerrito, Roberto Giovannoni, Sara Bonomo, Laura Masiero, Maria Luisa LavitranoAbstract:Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy.
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abstract b113 gsk3a is redundant with GSK3B in modulating drug resistance and chemotherapy induced necroptosis
2013Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Elena A Federzoni, Robert Narloch, Kristian Helin, Maria Luisa LavitranoAbstract:Purpose of the study. GSK3A and GSK3B isoforms perform similar functions but they are not completely redundant, depending on the cell type, biological process and differentiative status (Rayasam GV et al, Br J Pharmacol, 2009). We recently demonstrated that GSK3B inhibition enables necroptosis in response to chemotherapy in drug-resistant colon carcinoma cells (Grassilli E et al, Clin Cancer Res, 2013). Here we investigated whether GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. Materials and Methods. Cell lines stably interfered were obtained by retroviral infection and selection with the appropriate antibiotic. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cell viability was assessed by Trypan blue staining. Cell proliferation was evaluated by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Cell cycle was analysed by flow cytometry upon PI staining using Modfit Cell Cycle Analysis (Becton Dickinson). Reporter activity was analysed by Dual-Glo Luciferase Assay (Promega). IF staining was performed on 4% paraformaldehyde-fixed cells. Active caspase-3/7 was quantified by the Caspase-Glo3/7 Assay System (Promega). Western blots were performed on lysates obtained by RIPA buffer. Results and Discussion. GSK3A stable silencing in HCT116p53KO cells did not alter cell proliferation as assessed by growth curve, cell cycle distribution and β-catenin activation assays. GSK3A depletion abolished colony formation after 5FU treatment and re-sensitized drug-resistant HCT116p53KO cells to 5FU-induced cell death. Blocking GSK3A did not interfere with DNA damage sensing upon 5FU treatment, as demonstrated by H2AX foci formation, but impaired the DNA repair response by affecting RPA70 foci formation. GSK3A silencing allowed a cell death response to DNA-damaging drugs in absence of p53: 5FU-treated, GSK3A-depleted HCT116p53KO cells underwent caspase-independent PARP1- and tBid-dependent cell death, which was unaffected by RIP1 inhibition and was characterized by PARP-dependent nuclear re-localization of AIF. Our data demostrate that GSK3A, like GSK3B, contributes to cell survival upon DNA-damaging chemotherapy by suppressing RIP-independent necroptosis and pinpoint that inhibition of one isoform is sufficient to bypass drug resistance of p53-null cells. Being GSK3A and GSK3B redundant in the response to DNA-damaging drugs, it is likely that, when used together with anticancer drugs, GSK3 inhibitors should be particularly effective in overcoming drug resistance even at low doses, since inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells. We therefore propose that GSK3 inhibition in combination with DNA-damaging drugs would be an appealing strategy to induce necroptosis in p53-null drug-resistant tumor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B113. Citation Format: Emanuela Grassilli, Elena Federzoni, Robert Narloch, Leonarda Ianzano, Kristian Helin, Marialuisa Lavitrano. GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B113.
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inhibition of GSK3B bypass drug resistance of p53 null colon carcinomas by enabling necroptosis in response to chemotherapy
2013Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Roberto Giovannoni, Laura Masiero, Elena A Federzoni, Robert Narloch, Fabio Pisano, Gabriele Romano, Biagio Eugenio LeoneAbstract:Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR .
Leonarda Ianzano - One of the best experts on this subject based on the ideXlab platform.
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GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis
2014Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Carola Missaglia, Maria Grazia Cerrito, Roberto Giovannoni, Sara Bonomo, Laura Masiero, Maria Luisa LavitranoAbstract:Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy.
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abstract b113 gsk3a is redundant with GSK3B in modulating drug resistance and chemotherapy induced necroptosis
2013Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Elena A Federzoni, Robert Narloch, Kristian Helin, Maria Luisa LavitranoAbstract:Purpose of the study. GSK3A and GSK3B isoforms perform similar functions but they are not completely redundant, depending on the cell type, biological process and differentiative status (Rayasam GV et al, Br J Pharmacol, 2009). We recently demonstrated that GSK3B inhibition enables necroptosis in response to chemotherapy in drug-resistant colon carcinoma cells (Grassilli E et al, Clin Cancer Res, 2013). Here we investigated whether GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. Materials and Methods. Cell lines stably interfered were obtained by retroviral infection and selection with the appropriate antibiotic. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cell viability was assessed by Trypan blue staining. Cell proliferation was evaluated by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Cell cycle was analysed by flow cytometry upon PI staining using Modfit Cell Cycle Analysis (Becton Dickinson). Reporter activity was analysed by Dual-Glo Luciferase Assay (Promega). IF staining was performed on 4% paraformaldehyde-fixed cells. Active caspase-3/7 was quantified by the Caspase-Glo3/7 Assay System (Promega). Western blots were performed on lysates obtained by RIPA buffer. Results and Discussion. GSK3A stable silencing in HCT116p53KO cells did not alter cell proliferation as assessed by growth curve, cell cycle distribution and β-catenin activation assays. GSK3A depletion abolished colony formation after 5FU treatment and re-sensitized drug-resistant HCT116p53KO cells to 5FU-induced cell death. Blocking GSK3A did not interfere with DNA damage sensing upon 5FU treatment, as demonstrated by H2AX foci formation, but impaired the DNA repair response by affecting RPA70 foci formation. GSK3A silencing allowed a cell death response to DNA-damaging drugs in absence of p53: 5FU-treated, GSK3A-depleted HCT116p53KO cells underwent caspase-independent PARP1- and tBid-dependent cell death, which was unaffected by RIP1 inhibition and was characterized by PARP-dependent nuclear re-localization of AIF. Our data demostrate that GSK3A, like GSK3B, contributes to cell survival upon DNA-damaging chemotherapy by suppressing RIP-independent necroptosis and pinpoint that inhibition of one isoform is sufficient to bypass drug resistance of p53-null cells. Being GSK3A and GSK3B redundant in the response to DNA-damaging drugs, it is likely that, when used together with anticancer drugs, GSK3 inhibitors should be particularly effective in overcoming drug resistance even at low doses, since inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells. We therefore propose that GSK3 inhibition in combination with DNA-damaging drugs would be an appealing strategy to induce necroptosis in p53-null drug-resistant tumor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B113. Citation Format: Emanuela Grassilli, Elena Federzoni, Robert Narloch, Leonarda Ianzano, Kristian Helin, Marialuisa Lavitrano. GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B113.
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inhibition of GSK3B bypass drug resistance of p53 null colon carcinomas by enabling necroptosis in response to chemotherapy
2013Co-Authors: Emanuela Grassilli, Leonarda Ianzano, Roberto Giovannoni, Laura Masiero, Elena A Federzoni, Robert Narloch, Fabio Pisano, Gabriele Romano, Biagio Eugenio LeoneAbstract:Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR .
Pengda Guo - One of the best experts on this subject based on the ideXlab platform.
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traf6 inhibits colorectal cancer metastasis through regulating selective autophagic ctnnb1 β catenin degradation and is targeted for GSK3B gsk3β mediated phosphorylation and degradation
2019Co-Authors: Jing-ru Wang, Tian-yu Yang, Wen-juan Gan, Pengda GuoAbstract:Aberrant CTNNB1 signaling is one of the fundamental processes in cancers, especially colorectal cancer (CRC). Here, we reported that TRAF6, an E3 ubiquitin ligase important for inflammatory signaling, inhibited epithelial-mesenchymal transition (EMT) and CRC metastasis through driving a selective autophagic CTNNB1 degradation machinery. Mechanistically, TRAF6 interacted with MAP1LC3B/LC3B through its LC3-interacting region 'YxxL' and catalyzed K63-linked polyubiquitination of LC3B. The K63-linked ubiquitination of LC3B promoted the formation of the LC3B-ATG7 complex and was critical to the subsequent recognition of CTNNB1 by LC3B for the selective autophagic degradation. However, TRAF6 was phosphorylated at Thr266 by GSK3B in most clinical CRC, which triggered K48-linked polyubiquitination and degradation of TRAF6 and thereby attenuated its inhibitory activity towards the autophagy-dependent CTNNB1 signaling. Clinically, decreased expression of TRAF6 was associated with elevated GSK3B protein levels and activity and reduced overall survival in CRC patients. Pharmacological inhibition of GSK3B activity stabilized the TRAF6 protein, promoted CTNNB1 degradation, and effectively suppressed EMT and CRC metastasis. Thus, targeting TRAF6 and its pathway may be meaningful for treating advanced CRC. Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; AOM: azoxymethane; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CoIP: co-immunoprecipitation; CQ: chloroquine; CRC: colorectal cancer; CTNNB1/β-catenin: catenin beta 1; DSS: dextran sodium sulfate; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; GFP: green fluorescent protein; GSK3B/GSK3β: glycogen synthase kinase 3 beta; IgG: Immunoglobulin G; IHC: immunohistochemistry; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; RFP: red fluorescent protein; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TRAF6: TNF receptor-associated factor 6; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.
