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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 Pathol ; 253(1): 55-67, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32918742

RESUMO

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet- or drug-treated mice, in vitro primary cells, and in human tissue. TM4SF5-overexpressing mice exhibited nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory-element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3-mediated ECM production for NASH progression. A TM4SF5-associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high-fat diet- or CCl4 -treated mice and human patients exhibited TM4SF5-dependent steatotic or steatohepatitic livers with links between TM4SF5-mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5-mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl4 -mediated mouse liver damage. TM4SF5-mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Matriz Extracelular/enzimologia , Metabolismo dos Lipídeos , Cirrose Hepática Experimental/enzimologia , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Sirtuína 1/metabolismo , Animais , Tetracloreto de Carbono , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Dieta Hiperlipídica , Progressão da Doença , Matriz Extracelular/patologia , Humanos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais
3.
Blood ; 136(16): 1837-1850, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32845007

RESUMO

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells, from the thymic tumor microenvironment support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in 2 distinct mouse models of T-ALL and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of antitumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid-depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.


Assuntos
Comunicação Celular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Microambiente Tumoral , Biomarcadores , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais
4.
Cell Death Dis ; 10(9): 645, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501417

RESUMO

Reactive oxygen species (ROS) regulate cell fate, although signaling molecules that regulate ROS hormesis remain unclear. Here we show that transmembrane 4 L six family member 5 (TM4SF5) in lung epithelial cells induced the alternatively spliced CD44v8-10 variant via an inverse ZEB2/epithelial splicing regulatory proteins (ESRPs) linkage. TM4SF5 formed complexes with the cystine/glutamate antiporter system via TM4SF5- and CD44v8-10-dependent CD98hc plasma-membrane enrichment. Dynamic TM4SF5 binding to CD98hc required CD44v8-10 under ROS-generating inflammatory conditions. TM4SF5 and CD44v8-10 upregulated cystine/glutamate antiporter activity and intracellular glutathione levels, leading to ROS modulation for cell survival. Tm4sf5-null mice exhibited attenuated bleomycin-induced pulmonary fibrosis with lower CD44v8-10 and ESRPs levels than wild-type mice. Primary mouse alveolar epithelial cells (AECs) revealed type II AECs (AECII), but not type I, to adapt the TM4SF5-mediated characteristics, suggesting TM4SF5-mediated AECII survival following AECI injury during idiopathic pulmonary fibrosis (IPF). Thus, the TM4SF5-mediated CD44v8-10 splice variant could be targeted against IPF.


Assuntos
Células Epiteliais Alveolares/metabolismo , Receptores de Hialuronatos/metabolismo , Proteínas de Membrana/metabolismo , Fibrose Pulmonar/metabolismo , Células A549 , Células Epiteliais Alveolares/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Receptores de Hialuronatos/genética , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Splicing de RNA , Espécies Reativas de Oxigênio/metabolismo
5.
Cell Metab ; 29(6): 1306-1319.e7, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30956113

RESUMO

The mechanistic target of rapamycin complex (mTORC1) is a signaling hub on the lysosome surface, responding to lysosomal amino acids. Although arginine is metabolically important, the physiological arginine sensor that activates mTOR remains unclear. Here, we show that transmembrane 4 L six family member 5 (TM4SF5) translocates from plasma membrane to lysosome upon arginine sufficiency and senses arginine, culminating in mTORC1/S6K1 activation. TM4SF5 bound active mTOR upon arginine sufficiency and constitutively bound amino acid transporter SLC38A9. TM4SF5 binding to the cytosolic arginine sensor Castor1 decreased upon arginine sufficiency, thus allowing TM4SF5-mediated sensing of metabolic amino acids. TM4SF5 directly bound free L-arginine via its extracellular loop possibly for the efflux, being supported by mutant study and homology and molecular docking modeling. Therefore, we propose that lysosomal TM4SF5 senses and enables arginine efflux for mTORC1/S6K1 activation, and arginine-auxotroph in hepatocellular carcinoma may be targeted by blocking the arginine sensing using anti-TM4SF5 reagents.


Assuntos
Arginina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/fisiologia , Animais , Arginina/química , Transporte Biológico , Células Cultivadas , Células HEK293 , Células Hep G2 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , Transdução de Sinais/genética
6.
FASEB J ; 33(3): 4341-4354, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30592630

RESUMO

Fibrosis is characterized by the increased accumulation of extracellular matrix (ECM), which drives abnormal cell proliferation and progressive organ dysfunction in many inflammatory and metabolic diseases. Studies have shown that halofuginone, a racemic halogenated derivative, inhibits glutamyl-prolyl-transfer RNA-synthetase (EPRS)-mediated fibrosis. However, the mechanism by which this occurs is unclear. We explored the mechanistic aspects of how EPRS could develop liver fibrotic phenotypes in cells and animal models. Treatment with TGF-ß1 up-regulated fibronectin and collagen I levels in LX2 hepatic stellate cells. This effect was inhibited in prolyl-transfer RNA synthetase (PRS)-suppressed LX2 cells. Using the promoter luciferase assay, TGF-ß1-mediated collagen I, α1 chain transcription and γ2 basal laminin transcription in LX2 cells were down-regulated by EPRS suppression, suggesting that EPRS may play roles in ECM production at transcriptional levels. Furthermore, signal transducer and activator of transcription (STAT) signaling activation was involved in the effects of TGF-ß1 on ECM expression in a PRS-dependent manner. This was mediated via a protein-protein complex formation consisting of TGF-ß1 receptor, EPRS, Janus kinases, and STAT6. Additionally, ECM expression in fibrotic livers overlapped with EPRS expression along fibrotic septa regions and was positively correlated with STAT6 activation in carbon tetrachloride-treated mice. This was less obvious in livers of Eprs-/+ mice. These findings suggest that, during fibrosis development, EPRS plays roles in nontranslational processes of ECM expression via intracellular signaling regulation upon TGF-ß1 stimulation.-Song, D.-G., Kim, D., Jung, J. W., Nam, S. H., Kim, J. E., Kim, H.-J., Kim, J. H., Lee, S.-J., Pan, C.-H., Kim, S., Lee, J. W. Glutamyl-prolyl-tRNA synthetase induces fibrotic extracellular matrix via both transcriptional and translational mechanisms.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Matriz Extracelular/metabolismo , Biossíntese de Proteínas/genética , Transcrição Genética/genética , Aminoacil-tRNA Sintetases/genética , Animais , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Regulação para Baixo/genética , Matriz Extracelular/genética , Fibrose/genética , Fibrose/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/genética
7.
Front Pharmacol ; 9: 1337, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524284

RESUMO

Idiopathic pulmonary fibrosis (IPF), a chronic disease of unknown cause, is characterized by abnormal accumulation of extracellular matrix (ECM) in fibrotic foci in the lung. Previous studies have shown that the transforming growth factor ß1 (TGFß1) and signal transducers and activators of transcription (STAT) pathways play roles in IPF pathogenesis. Glutamyl-prolyl-tRNA-synthetase (EPRS) has been identified as a target for anti-fibrosis therapy, but the link between EPRS and TGFß1-mediated IPF pathogenesis remains unknown. Here, we studied the role of EPRS in the development of fibrotic phenotypes in A549 alveolar epithelial cells and bleomycin-treated animal models. We found that EPRS knockdown inhibited the TGFß1-mediated upregulation of fibronectin and collagen I and the mesenchymal proteins α-smooth muscle actin (α-SMA) and snail 1. TGFß1-mediated transcription of collagen I-α1 and laminin γ2 in A549 cells was also down-regulated by EPRS suppression, indicating that EPRS is required for ECM protein transcriptions. Activation of STAT signaling in TGFß1-induced ECM expression was dependent on EPRS. TGFß1 treatment resulted in EPRS-dependent in vitro formation of a multi-protein complex consisting of the TGFß1 receptor, EPRS, Janus tyrosine kinases (JAKs), and STATs. In vivo lung tissue from bleomycin-treated mice showed EPRS-dependent STAT6 phosphorylation and ECM production. Our results suggest that epithelial EPRS regulates the expression of mesenchymal markers and ECM proteins via the TGFß1/STAT signaling pathway. Therefore, epithelial EPRS can be used as a potential target to develop anti-IPF treatments.

8.
J Clin Invest ; 128(11): 5034-5055, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30188867

RESUMO

Lysyl-tRNA synthetase (KRS) functions canonically in cytosolic translational processes. However, KRS is highly expressed in colon cancer, and localizes to distinct cellular compartments upon phosphorylations (i.e., the plasma membranes after T52 phosphorylation and the nucleus after S207 phosphorylation), leading to probably alternative noncanonical functions. It is unknown how other subcellular KRSs crosstalk with environmental cues during cancer progression. Here, we demonstrate that the KRS-dependent metastatic behavior of colon cancer spheroids within 3D gels requires communication between cellular molecules and extracellular soluble factors and neighboring cells. Membranous KRS and nuclear KRS were found to participate in invasive cell dissemination of colon cancer spheroids in 3D gels. Cancer spheroids secreted GAS6 via a KRS-dependent mechanism and caused the M2 polarization of macrophages, which activated the neighboring cells via secretion of FGF2/GROα/M-CSF to promote cancer dissemination under environmental remodeling via fibroblast-mediated laminin production. Analyses of tissues from clinical colon cancer patients and Krs-/+ animal models for cancer metastasis supported the roles of KRS, GAS6, and M2 macrophages in KRS-dependent positive feedback between tumors and environmental factors. Altogether, KRS in colon cancer cells remodels the microenvironment to promote metastasis, which can thus be therapeutically targeted at these bidirectional KRS-dependent communications of cancer spheroids with environmental cues.


Assuntos
Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Lisina-tRNA Ligase/biossíntese , Macrófagos/enzimologia , Proteínas de Neoplasias/biossíntese , Esferoides Celulares/enzimologia , Microambiente Tumoral , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lisina-tRNA Ligase/genética , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Metástase Neoplásica , Proteínas de Neoplasias/genética , Esferoides Celulares/patologia
9.
Cancer Lett ; 438: 219-231, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30217560

RESUMO

CD133 is a surface marker of liver cancer stem cells. Transmembrane 4 L six family member 5 (TM4SF5) promotes sphere growth and circulation. However, it is unknown how CD133 and TM4SF5 cross-talk with each other for cancer stem cell properties. Here, we investigated the significance of inter-relationships between CD133, TM4SF5, CD44, and protein tyrosine phosphatase receptor type F (PTPRF) in a three-dimensional (3D) sphere growth system. We found that CD133 upregulated TM4SF5 and CD44, whereas TM4SF5 and CD44 did not affect CD133 expression. Signaling activity following CD133 phosphorylation caused TM4SF5 expression and sphere growth. TM4SF5 bound to CD133 and promoted c-Src activity for CD133 phosphorylation as a positive feedback loop, leading to CD133-mediated sphere growth that was inhibited by TM4SF5 inhibition or suppression. TM4SF5 also bound PTPRF and promoted paxillin phosphorylation. Decreased sphere growth upon CD133 suppression was recovered by TM4SF5 expression and partially by PTPRF suppression. TM4SF5 inhibition enhanced PTPRF levels and abolished PTPRF suppression-mediated sphere growth. Altogether, CD133-induced TM4SF5 expression and function were important for liver cancer sphere growth and may be a promising target to block metastasis.


Assuntos
Antígeno AC133/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Esferoides Celulares/metabolismo , Antígeno AC133/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Mutação , Fosforilação , Interferência de RNA , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Transdução de Sinais/genética
10.
Oncotarget ; 8(48): 83480-83494, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-29137358

RESUMO

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells.

11.
FASEB J ; 31(4): 1461-1481, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28073834

RESUMO

Membrane proteins sense extracellular cues and transduce intracellular signaling to coordinate directionality and speed during cellular migration. They are often localized to specific regions, as with lipid rafts or tetraspanin-enriched microdomains; however, the dynamic interactions of tetraspanins with diverse receptors within tetraspanin-enriched microdomains on cellular surfaces remain largely unexplored. Here, we investigated effects of tetraspan(in) TM4SF5 (transmembrane 4 L6 family member 5)-enriched microdomains (T5ERMs) on the directionality of cell migration. Physical association of TM4SF5 with epidermal growth factor receptor (EGFR) and integrin α5 was visualized by live fluorescence cross-correlation spectroscopy and higher-resolution microscopy at the leading edge of migratory cells, presumably forming TM4SF5-enriched microdomains. Whereas TM4SF5 and EGFR colocalized at the migrating leading region more than at the rear, TM4SF5 and integrin α5 colocalized evenly throughout cells. Cholesterol depletion and disruption in TM4SF5 post-translational modifications, including N-glycosylation and palmitoylation, altered TM4SF5 interactions and cellular localization, which led to less cellular migration speed and directionality in 2- or 3-dimensional conditions. TM4SF5 controlled directional cell migration and invasion, and importantly, these TM4SF5 functions were dependent on cholesterol, TM4SF5 post-translational modifications, and EGFR and integrin α5 activity. Altogether, we showed that TM4SF5 dynamically interacted with EGFR and integrin α5 in migratory cells to control directionality and invasion.-Kim, H.-J., Kwon, S., Nam, S. H., Jung, J. W., Kang, M., Ryu, J., Kim, J. E., Cheong, J.-G., Cho, C. Y., Kim, S., Song, D.-G., Kim, Y.-N., Kim, T. Y., Jung, M.-K., Lee, K.-M., Pack, C.-G., Lee, J. W. Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.


Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colesterol/metabolismo , Receptores ErbB/metabolismo , Glicosilação , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Hepatócitos/ultraestrutura , Humanos , Integrina alfa5/metabolismo , Lipoilação , Microdomínios da Membrana/ultraestrutura , Ligação Proteica , Processamento de Proteína Pós-Traducional
12.
Oncotarget ; 8(8): 13277-13292, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28129652

RESUMO

The transmembrane 4 L six family proteins TM4SF1, TM4SF4, and TM4SF5 share 40-50% overall sequence identity, but their C-terminus identity is limited. It may be likely that the C-termini of the members are important and unique for own regulatory functions. We thus examined how the TM4SF5 C-terminus affected cellular functions differentially from other family members. Using colon cancer cells expressing wildtype (WT), C-terminus-deleted, or chimeric mutants, diverse cellular functions were explored in 2-dimensional (2D) and 3-dimensional (3D) condition. The C-termini of the proteins were relatively comparable with respect to 2D cell proliferation, although each C-terminal-deletion mutant exhibited increased proliferation relative to the WT. Using chimeric constructs, we found that the TM4SF5 C-terminus was critical for regulating the diverse metastatic functions of TM4SF5, and could positively replace the C-termini of other family members. Replacement of the TM4SF1 or TM4SF4 C-terminus with that of TM4SF5 increased spheroids growth, transwell migration, and invasive dissemination from spheroids in 3D collagen gels. TM4SF5-mediated effects required its extracellular loop 2 linked to the C-terminus via the transmembrane domain 4, with causing c-Src activation. Altogether, the C-terminus of TM4SF5 appears to mediate pro-migratory roles, depending on a structural relay from the second extracellular loop to the C-terminus.


Assuntos
Antígenos de Superfície/genética , Proliferação de Células/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Animais , Antígenos de Superfície/metabolismo , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HCT116 , Células HT29 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/metabolismo , Transplante Heterólogo
14.
Int J Oncol ; 48(4): 1553-60, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26891990

RESUMO

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6ß1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.


Assuntos
Transição Epitelial-Mesenquimal , Matriz Extracelular/genética , Lisina-tRNA Ligase/genética , Neoplasias/patologia , Adesão Celular , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/genética , Células HCT116 , Humanos , Lisina-tRNA Ligase/metabolismo , Neoplasias/genética , Transdução de Sinais
15.
Lung Cancer ; 90(1): 22-31, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26190015

RESUMO

OBJECTIVES: The membrane glycoprotein TM4SF5 (transmembrane 4 L6 family member 5), which is similar to the tetraspanins, is highly expressed in different cancers and causes epithelial-mesenchymal transition (EMT). TM4SF5 interacts with other membrane proteins during its pro-tumorigenic roles, presumably at tetraspanin-enriched microdomains (TEMs/TERMs). Here, we explored TM4SF5-mediated resistance against the clinically important EGFR kinase inhibitors, with regards to cooperation with other membrane proteins, particularly the insulin-like growth factor 1 receptor (IGF1R). MATERIALS AND METHODS: Using cancer cells including NSCLC with TM4SF5 overexpression or IGF1R suppression in either normal 2 dimensional (2D), 3D aqueous spheroids, or 3D collagen I gels systems, the sensitivity to tyrosine kinase inhibitors (TKIs) were evaluated. RESULTS AND CONCLUSION: We found that TM4SF5 and IGF1R transcriptionally modulated one another, with each protein promoting the expressions of the other. Expression of TM4SF5 in gefitinib-sensitive HCC827 cells caused resistance to erlotinib and gefitinib, but not to sorafenib [a platelet derived growth factor receptor (PDGFR) inhibitor]; whereas suppression of IGF1R from gefitinib-resistant NCI-H1299 cells caused enhanced sensitization to the inhibitors. Expression of TM4SF5 and IGF1R in the drug-sensitive cells promoted signaling activities of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and S6 kinase (S6K), and resulted in a higher residual EGFR activity, even after EGFR kinase inhibitor treatment. Complex formation between TM4SF5 and IGF1R was observed, and also included EGFR, dependent on TM4SF5 expression. The TM4SF5-mediated drug resistance was further confirmed in an aqueous 3D spheroid system or upon being embedded in 3D extracellular matrix (ECM)-surrounded gel systems. Collectively, these data suggest that anti-TM4SF5 reagents may be combined with the EGFR kinase inhibitors to enhance the efficacy of chemotherapies against NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptores de Somatomedina/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Receptor IGF Tipo 1 , Receptores de Somatomedina/biossíntese , Receptores de Somatomedina/genética , Transdução de Sinais , Esferoides Celulares
16.
Oncotarget ; 6(25): 21655-74, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26091349

RESUMO

The adhesion properties of cells are involved in tumor metastasis. Although KRS at the plasma membrane is shown important for cancer metastasis, additionally to canonical roles of cytosolic KRS in protein translation, how KRS and its downstream effectors promote the metastatic migration remains unexplored. Disseminative behaviors (an earlier metastatic process) of colon cancer cell spheroids embedded in 3D collagen gels were studied with regards to cell adhesion properties, and relevance in KRS(-/+) knocked-down animal and clinical colon cancer tissues. Time-lapse imaging revealed KRS-dependent cell dissemination from the spheroids, whereas KRS-suppressed spheroids remained static due to the absence of outbound movements supported by cell-extracellular matrix (ECM) adhesion. While keeping E-cadherin at the outward disseminative cells, KRS caused integrin-involved intracellular signaling for ERK/c-Jun, paxillin, and cell-ECM adhesion-mediated signaling to modulate traction force for crawling movement. KRS-suppressed spheroids became disseminative following ERK or paxillin re-expression. The KRS-dependent intracellular signaling activities correlated with the invasiveness in clinical colon tumor tissues and in KRS(-/+) knocked-down mice tissues. Collectively, these observations indicate that KRS at the plasma membrane plays new roles in metastatic migration as a signaling inducer, and causes intracellular signaling for cancer dissemination, involving cell-cell and cell-ECM adhesion, during KRS-mediated metastasis.


Assuntos
Colágeno Tipo I/metabolismo , Neoplasias do Colo/enzimologia , Lisina-tRNA Ligase/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Citosol/metabolismo , Matriz Extracelular/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Técnica Indireta de Fluorescência para Anticorpo , Células HCT116 , Humanos , Camundongos , Metástase Neoplásica , Paxilina/metabolismo , Fosforilação , Biossíntese de Proteínas , Transdução de Sinais
17.
Hepatology ; 61(6): 1978-97, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25627085

RESUMO

UNLABELLED: Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Epithelial-mesenchymal transition (EMT) is related to self-renewal capacity and circulating tumor cell (CTC) characteristics for tumor metastasis. Although tumor metastasis is a life-threatening, complicated process that occurs through circulation of tumor cells, mechanistic aspects of self-renewal and circulating capacities have been largely unknown. Hepatic transmembrane 4 L six family member 5 (TM4SF5) promotes EMT for malignant growth and migration, so it was rationalized that TM4SF5, as a hepatocellular carcinoma (HCC) biomarker, might be important for metastatic potential. Here, self-renewal capacity by TM4SF5 was mechanistically explored using hepatocarcinoma cells with or without TM4SF5 expression, and we explored whether they became CTCs using mouse liver-orthotopic model systems. We found that TM4SF5-dependent sphere growth correlated with CD24(-) , aldehyde dehydrogenase (ALDH) activity, as well as a physical association between CD44 and TM4SF5. Interaction between TM4SF5 and CD44 was through their extracellular domains with N-glycosylation modifications. TM4SF5/CD44 interaction activated proto-oncogene tyrosine-protein kinase Src (c-Src)/signal transducer and activator of transcription 3 (STAT3)/Twist-related protein 1 (Twist1)/B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) signaling for spheroid formation, whereas disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts using 200∼5,000 cells per injection, TM4SF5-positive tumors exhibited subpopulations with locally increased CD44 expressions, supporting for tumor cell differentiation. TM4SF5-positive, but not TM4SF5- or CD44-knocked-down, cells were identified circulating in blood 4-6 weeks after orthotopic liver injection using in vivo laser scanning endomicroscopy. Anti-TM4SF5 reagent blocked their metastasis to distal intestinal organs. CONCLUSION: TM4SF5 promotes self-renewal and CTC properties supported by TM4SF5(+) /CD44(+(TM4SF5-bound)) /ALDH(+) /CD24(-) markers during HCC metastasis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas de Membrana/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Esferoides Celulares , Proteína 1 Relacionada a Twist/metabolismo , Quinases da Família src/metabolismo
18.
PLoS One ; 9(7): e102817, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25033048

RESUMO

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl4-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFß1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.


Assuntos
Movimento Celular/fisiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Proteínas de Membrana/metabolismo , Invasividade Neoplásica/patologia , Tetraspanina 24/metabolismo , Tetraspanina 30/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fenótipo , Tetraspaninas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
19.
Biochem J ; 462(1): 89-101, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24897542

RESUMO

TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling.


Assuntos
Integrina alfa5/fisiologia , Proteínas de Membrana/genética , Desenvolvimento Muscular/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Transição Epitelial-Mesenquimal , Integrina alfa5/biossíntese , Proteínas de Membrana/biossíntese , Camundongos , Transdução de Sinais/fisiologia , Somitos/metabolismo , Peixe-Zebra/embriologia
20.
Mol Cell Biol ; 34(16): 2946-60, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24912675

RESUMO

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Interleucina-6/metabolismo , Neoplasias Hepáticas/imunologia , Proteínas de Membrana/genética , Fator de Transcrição STAT3/metabolismo , Evasão Tumoral , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Adesões Focais/genética , Hepatócitos/citologia , Humanos , Interleucina-6/biossíntese , Interleucina-6/farmacologia , Neoplasias Hepáticas/genética , Proteínas de Membrana/biossíntese , Metástase Neoplásica , Fosforilação , Receptores de Interleucina-6/imunologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia
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