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1.
Theranostics ; 11(16): 8092-8111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335982

RESUMO

Active c-Src non-receptor tyrosine kinase localizes to the plasma membrane via N-terminal lipid modification. Membranous c-Src causes cancer initiation and progression. Even though transmembrane 4 L six family member 5 (TM4SF5), a tetraspan(in), can be involved in this mechanism, the molecular and structural influence of TM4SF5 on c-Src remains unknown. Methods: Here, we investigated molecular and structural details by which TM4SF5 regulated c-Src devoid of its N-terminus and how cell-penetrating peptides were able to interrupt c-Src activation via interference of c-Src-TM4SF5 interaction in hepatocellular carcinoma models. Results: The TM4SF5 C-terminus efficiently bound the c-Src SH1 kinase domain, efficiently to the inactively-closed form. The complex involved protein tyrosine phosphatase 1B able to dephosphorylate Tyr530. The c-Src SH1 domain alone, even in a closed form, bound TM4SF5 to cause c-Src Tyr419 and FAK Y861 phosphorylation. Homology modeling and molecular dynamics simulation studies predicted the directly interfacing residues, which were further validated by mutational studies. Cell penetration of TM4SF5 C-terminal peptides blocked the interaction of TM4SF5 with c-Src and prevented c-Src-dependent tumor initiation and progression in vivo. Conclusions: Collectively, these data demonstrate that binding of the TM4SF5 C-terminus to the kinase domain of inactive c-Src leads to its activation. Because this binding can be abolished by cell-penetrating peptides containing the TM4SF5 C-terminus, targeting this direct interaction may be an effective strategy for developing therapeutics that block the development and progression of hepatocellular carcinoma.


Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Tirosina Quinase CSK/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Genes src/genética , Genes src/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Tetraspaninas/genética , Tetraspaninas/metabolismo
2.
J Cell Sci ; 132(20)2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31562188

RESUMO

When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1) then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gαißγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTNVEGFR2), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes.


Assuntos
Movimento Celular/imunologia , Integrina alfa4beta1/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Receptores CXCR4/imunologia , Sindecana-1/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Vigilância Imunológica , Integrina alfa4beta1/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Fosforilação/imunologia , Receptores CXCR4/genética , Sindecana-1/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
3.
Sci Rep ; 9(1): 9126, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235839

RESUMO

Epidermal growth factor receptor (EGFR) and its downstream phosphoinositide 3-kinase (PI3K) pathway are commonly deregulated in cancer. Recently, we have shown that the IQ motif-containing GTPase-activating protein 1 (IQGAP1) provides a molecular platform to scaffold all the components of the PI3K-Akt pathway and results in the sequential generation of phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P3). In addition to the PI3K-Akt pathway, IQGAP1 also scaffolds the Ras-ERK pathway. To define the specificity of IQGAP1 for the control of PI3K signaling, we have focused on the IQ3 motif in IQGAP1 as PIPKIα and PI3K enzymes bind this region. An IQ3 deletion mutant loses interactions with the PI3K-Akt components but retains binding to ERK and EGFR. Consistently, blocking the IQ3 motif of IQGAP1 using an IQ3 motif-derived peptide mirrors the effect of IQ3 deletion mutant by reducing Akt activation but has no impact on ERK activation. Also, the peptide disrupts the binding of IQGAP1 with PI3K-Akt pathway components, while IQGAP1 interactions with ERK and EGFR are not affected. Functionally, deleting or blocking the IQ3 motif inhibits cell proliferation, invasion, and migration in a non-additive manner to a PIPKIα inhibitor, establishing the functional specificity of IQ3 motif towards the PI3K-Akt pathway. Taken together, the IQ3 motif is a specific target for suppressing activation of the PI3K-Akt but not the Ras-ERK pathway. Although EGFR stimulates the IQGAP1-PI3K and -ERK pathways, here we show that IQGAP1-PI3K controls migration, invasion, and proliferation independent of ERK. These data illustrate that the IQ3 region of IQGAP1 is a promising therapeutic target for PI3K-driven cancer.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Invasividade Neoplásica , Deleção de Sequência , Proteínas Ativadoras de ras GTPase/genética
4.
Cancer Res ; 76(17): 4981-93, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27364558

RESUMO

Syndecan-1 (Sdc1/CD138) expression is linked to disease severity in multiple myeloma, although the causal basis for this link remains unclear. Here we report that capture of the IGF1 receptor (IGF1R) by Sdc1 suppresses ASK1-dependent apoptosis in multiple myeloma cells. Sdc1 binds two different fractions of IGF1R, one that is constitutively active and a second that is activated by IGF1 ligand. Notably, IGF1R kinase activity in both fractions is blocked by synstatinIGF1R (SSTNIGF1R), a peptide that inhibits IGF1R capture by Sdc1, as well as by a truncated peptide (SSTNIGF1R-T) that appears to be specific for multiple myeloma cells. Mechanistically, we show that ASK1 is bound to active IGF1R and inhibited by Tyr and Ser83/Ser966 phosphorylation. When IGF1R engagement with Sdc1 is blocked by SSTNIGF1R, ASK1 becomes activated, and initiates JNK- and caspase-3-mediated apoptosis. In pharmacologic tests, we find SSTNIGF1R is highly stable in human plasma and displays a half-life of 27 hours in mice, wherein it significantly reduces both the size and neovascularization of CAG myeloma tumor xenografts. Taken together, our results offer a preclinical proof of concept and mechanistic rationale for the exploration of SSTNIGF1R as an experimental therapeutic to dually attack multiple myeloma tumor cell survival and tumor angiogenesis. Cancer Res; 76(17); 4981-93. ©2016 AACR.


Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/patologia , Receptor IGF Tipo 1/farmacologia , Receptores de Somatomedina/metabolismo , Sindecana-1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imunoprecipitação , MAP Quinase Quinase Quinase 5/metabolismo , Camundongos , Camundongos Nus , Mieloma Múltiplo/metabolismo , Peptídeos/farmacologia
5.
Biochim Biophys Acta ; 1833(3): 629-42, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23220047

RESUMO

Transmembrane 4 L six family member 5 (TM4SF5) enhances cell migration and invasion, although how TM4SF5 mechanistically mediates these effects remains unknown. In the study, during efforts to understand TM4SF5-mediated signal transduction, TM4SF5 was shown to bind c-Src and thus hepatoma cell lines expressing TM4SF5 were analyzed for the significance of the interaction in cell invasion. The C-terminus of TM4SF5 bound both inactive c-Src that might be sequestered to certain cellular areas and active c-Src that might form invasive protrusions. Wildtype (WT) TM4SF5 expression enhanced migration and invasive protrusion formation in a c-Src-dependent manner, compared with TM4SF5-null control hepatoma cell lines. However, tailless TM4SF5(ΔC) cells were more efficient than WT TM4SF5 cells, suggesting a negative regulatory role by the C-terminus. TM4SF5 WT- or TM4SF5(ΔC)-mediated formation of invasive protrusions was dependent or independent on serum or epidermal growth factor treatment, respectively, although they both were dependent on c-Src. The c-Src activity of TM4SF5 WT- or TM4SF5(ΔC)-expressing cells correlated with enhanced Tyr845 phosphorylation of epidermal growth factor receptor. Y845F EGFR mutation abolished the TM4SF5-mediated invasive protrusions, but not c-Src phosphorylation. Our findings demonstrate that TM4SF5 modulates c-Src activity during TM4SF5-mediated invasion through a TM4SF5/c-Src/EGFR signaling pathway, differentially along the leading protrusive edges of an invasive cancer cell.


Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular , Receptores ErbB/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Tirosina/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Adesão Celular , Proliferação de Células , Receptores ErbB/genética , Imunofluorescência , Humanos , Imunoprecipitação , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/genética , Invasividade Neoplásica , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas pp60(c-src)/genética , RNA Mensageiro/genética , Transdução de Sinais , Células Tumorais Cultivadas
6.
J Cell Sci ; 125(Pt 24): 5960-73, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23077174

RESUMO

Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Quinase 1 de Adesão Focal/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Tetraspaninas/genética , Sequência de Aminoácidos , Animais , Carcinoma Hepatocelular/enzimologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Ativação Enzimática , Feminino , Xenoenxertos , Humanos , Neoplasias Hepáticas/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Metástase Neoplásica , Fosforilação , Transdução de Sinais , Tetraspaninas/metabolismo
7.
Biochem J ; 443(3): 691-700, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22292774

RESUMO

The EMT (epithelial-mesenchymal transition) is involved in fibrosis and cancer, and is regulated by different signalling pathways mediated through soluble factors, actin reorganization and transcription factor actions. Because the tetraspan (also called tetraspanin) TM4SF5 (transmembrane 4 L6 family member 5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that TGFß1 (transforming growth factor ß1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR [EGF (epidermal growth factor) receptor] pathway. Inhibition of EGFR activity following TGFß1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFß1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFß1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in the development of liver pathologies.


Assuntos
Transição Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Humanos
8.
Cancer Lett ; 314(2): 198-205, 2012 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-22014979

RESUMO

Transmembrane 4L six family member 5 (TM4SF5) can regulate cell-cell adhesion and cellular morphology via cytoplasmic p27(Kip1)-mediated changes in RhoA activity. However, how TM4SF5 causes cytosolic p27(Kip1) stabilization remains unknown. In this study we found that TM4SF5-mediated Ser10 phosphorylation of p27(Kip1) required for cytosolic localization was not always correlated with Akt activity. Inhibition or suppression of c-Jun N-terminal kinase (JNK) in TM4SF5-expressing cells decreased Ser10 phosphorylation of p27(Kip1) and rescued expression levels and localization of adherence junction molecules to cell-cell contacts. These observations suggest involvement of JNKs in TM4SF5-mediated p27(Kip1) Ser10 phosphorylation and localization during epithelial-mesenchymal transition.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/fisiologia , Adesão Celular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Fosforilação
9.
Cancer Biol Ther ; 11(3): 330-6, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21099346

RESUMO

Two separate clinical studies of advanced hepatocarcinoma patients recently reported that the multikinase inhibitor sorafenib (nexavar) could extend survival of the patients only by 2-3 months. We also previously demonstrated that 4'-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) blocks the multilayer growth and migration mediated by TM4SF5, which is highly expressed in approximately 80% of Korean hepatocarcinoma patients. Therefore, we wondered how TSAHC might be different from sorafenib to deal with hepatocarcinoma in terms of the therapeutic characteristics including specificity for TM4SF5. TM4SF5 is previously shown to mediate tumorigenesis through cytosolic p27Kip1-mediated inactivation of RhoA, epithelial-mesenchymal transition, multilayer growth, migration, invasion, and tumor angiogenesis. In this study, TSAHC and two derivatives showed similar antagonistic activities against TM4SF5-mediated signaling and multilayer growth in vitro and anti-tumorigenic activity even in early stages of TM4SF5-mediated tumor formation in nude mice. Meanwhile, sorafenib was only effective much later in tumorigenesis in vivo and affected in vitro proliferation in a TM4SF5-independent manner. Altogether, these observations suggest that TSAHC may be a promising anti-tumorigenic reagent, especially against TM4SF5-mediated hepatocarcinoma.


Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Chalcona/análogos & derivados , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Membrana/metabolismo , Piridinas/farmacologia , Sulfonamidas/farmacologia , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Chalcona/metabolismo , Chalcona/farmacologia , Descoberta de Drogas , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Niacinamida/análogos & derivados , Compostos de Fenilureia , Transdução de Sinais , Sorafenibe , Sulfonamidas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Biol Chem ; 285(46): 36021-31, 2010 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-20829364

RESUMO

Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic ß cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process.


Assuntos
Acetilglucosamina/metabolismo , Actinas/metabolismo , Glucosamina/farmacologia , Paxilina/metabolismo , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Humanos , Imuno-Histoquímica , Insulinoma/metabolismo , Insulinoma/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microscopia de Fluorescência , Mutação , Paxilina/genética , Fosforilação/efeitos dos fármacos , Ratos , Serina/genética , Serina/metabolismo , Estreptozocina
11.
Biochim Biophys Acta ; 1803(8): 975-82, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20399237

RESUMO

Transmembrane 4 L six family member 5 (TM4SF5) causes epithelial-mesenchymal transition (EMT) for aberrant cell proliferation. However, the effects of TM4SF5 expression on cell cycle are unknown so far. In this study, using hepatocytes that either ectopically or endogenously express TM4SF5 and human hepatocarcinoma tissues, the role of TM4SF5 in G1/S phase progression was examined. We found that TM4SF5 expression accelerated G1/S phase progression with facilitated cyclin D1 and E expression and Rb phosphorylation. Furthermore, TM4SF5 enhanced trafficking of CDK4 and cyclin D1 into the nucleus and induced complex formation between them. However, TM4SF5-facilitated G1/S phase progression was blocked by silencing of p27Kip1 using siRNA or by infection of active RhoA. Pharmacological inhibition of ROCK accelerated the G1/S phase progression of control TM4SF5-unexpressing cells. Altogether, these observations suggest that TM4SF5 accelerates G1/S phase progression with facilitated CDK4/cyclin D1 entry into the nucleus, which might be supported by TM4SF5-mediated actin reorganization through cytosolic p27Kip1 expression and Rho GTPase activity.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fase G1/fisiologia , Proteínas de Membrana/metabolismo , Fase S/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas de Membrana/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética
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