Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 43
Filtrar
1.
Expert Rev Proteomics ; 20(12): 357-370, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37874121

RESUMO

INTRODUCTION: Tissue-based proteomic studies of colorectal cancer (CRC) metastasis have delivered fragmented results, with very few therapeutic targets and prognostic biomarkers moving beyond the discovery phase. This situation is likely due to the difficulties in obtaining and analyzing large numbers of patient-derived metastatic samples, the own heterogeneity of CRC, and technical limitations in proteomics discovery. As an alternative, metastatic CRC cell lines provide a flexible framework to investigate the underlying mechanisms and network biology of metastasis for target discovery. AREAS COVERED: In this perspective, we comment on different in-depth proteomic studies of metastatic versus non-metastatic CRC cell lines. Identified metastasis-related proteins are introduced and discussed according to the spatial location in different cellular fractions, with special emphasis on membrane/adhesion proteins, secreted proteins, and nuclear factors, including miRNAs associated with liver metastasis. Moreover, we analyze the biological significance and potential therapeutic applications of the identified liver metastasis-related proteins. EXPERT OPINION: The combination of protein discovery and functional analysis is the only way to accelerate the progress to clinical translation of the proteomic-derived findings in a relatively fast pace. Patient-derived organoids represent a promising alternative to patient tissues and cell lines, but further optimizations are still required for achieving solid and reproducible results.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , MicroRNAs , Humanos , Proteômica/métodos , Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias , Metástase Neoplásica
2.
Br J Cancer ; 126(11): 1604-1615, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35347323

RESUMO

BACKGROUND: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells. METHODS: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments. RESULTS: A significant association of high AIP expression with poor CRC patients' survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver. CONCLUSIONS: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Hepáticas , Neoplasias Retais , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Hidrocarbonetos , Imuno-Histoquímica , Neoplasias Hepáticas/secundário , Metástase Neoplásica
3.
Biochim Biophys Acta Rev Cancer ; 1869(2): 321-332, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29673969

RESUMO

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2ß1 integrin, these cadherins promote integrin ß1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2ß1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2ß1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2ß1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.


Assuntos
Caderinas/metabolismo , Movimento Celular , Integrina alfa2beta1/metabolismo , Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Sítios de Ligação , Caderinas/antagonistas & inibidores , Caderinas/imunologia , Adesão Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Humanos , Integrina alfa2beta1/antagonistas & inibidores , Integrina alfa2beta1/imunologia , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/imunologia , Transdução de Sinais
4.
Carcinogenesis ; 41(2): 203-213, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-31095674

RESUMO

Pancreatic adenocarcinoma upregulated factor (PAUF), also known as ZG16B, was previously found in the secretome of metastatic colorectal cancer cells. Here, we demonstrated the presence of PAUF at the intracellular level and its multiple effects on cancer progression. An initial decline of PAUF expression was observed at early stages of colorectal cancer followed by an increase at the metastatic site. PAUF was located at different cellular compartments: membrane-associated vesicles, endosomes, microtubule-associated vesicles, cell growth cones and the cell nucleus. PAUF loss in two colorectal cancer cell lines caused severe alterations in the cell phenotype and cell cycle, including tetraploidy, extensive genomic alterations, micronuclei and increased apoptosis. An exhaustive analysis of the PAUF interactome using different proteomic approaches revealed the presence of multiple components of the cell cycle, mitotic checkpoint, Wnt pathway and intracellular transport. Among the interacting proteins we found ZW10, a moonlighting protein with a dual function in membrane trafficking and mitosis. In addition, PAUF silencing was associated to APC loss and increased ß-catenin nuclear expression. Altogether, our results suggest that PAUF depletion increases aneuploidy, promotes apoptosis and activates the Wnt/ß-catenin pathway in colorectal cancer cells facilitating cancer progression. In summary, PAUF behaves as a multifunctional protein, with different roles in cancer progression according to the extra- or intracellular expression, suggesting a therapeutic value for colorectal cancer.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Neoplasias Colorretais/patologia , Lectinas/metabolismo , Neoplasias Hepáticas/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Via de Sinalização Wnt , Proteína da Polipose Adenomatosa do Colo/metabolismo , Aneuploidia , Linhagem Celular Tumoral , Colo/patologia , Neoplasias Colorretais/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Lectinas/genética , Fígado/patologia , Neoplasias Hepáticas/secundário , Mapeamento de Interação de Proteínas , Proteômica , Reto/patologia , Regulação para Cima
5.
Int J Mol Sci ; 20(13)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31324051

RESUMO

Cell-cell adhesion molecules (cadherins) and cell-extracellular matrix adhesion proteins (integrins) play a critical role in the regulation of cancer invasion and metastasis. Although significant progress has been made in the characterization of multiple members of the cadherin superfamily, most of the published work continues to focus in the switch E-/N-cadherin and its role in the epithelial-mesenchymal transition. Here, we will discuss the structural and functional properties of a subset of cadherins (cadherin 17, cadherin 5 and cadherin 6) that have an RGD motif in the extracellular domains. This RGD motif is critical for the interaction with α2ß1 integrin and posterior integrin pathway activation in cancer metastatic cells. However, other signaling pathways seem to be affected by RGD cadherin interactions, as will be discussed. The range of solid tumors with overexpression or "de novo" expression of one or more of these three cadherins is very wide (gastrointestinal, gynaecological and melanoma, among others), underscoring the relevance of these cadherins in cancer metastasis. Finally, we will discuss different evidences that support the therapeutic use of these cadherins by blocking their capacity to work as integrin ligands in order to develop new cures for metastatic patients.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Neoplasias/metabolismo , Humanos , Transdução de Sinais/fisiologia
6.
J Proteome Res ; 17(1): 252-264, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29131639

RESUMO

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly metastatic KM12SM cells relative to nonmetastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. In both cell lines there were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1, or SRSF3. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4, and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration, and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated with cancer progression. Loss of SRSF3 expression was significantly associated with poor survival and shorter disease-free survival, particularly in early stages, in colorectal cancer.


Assuntos
Neoplasias Colorretais/química , Metástase Neoplásica , Proteômica/métodos , Fatores de Processamento de RNA/análise , Fatores de Transcrição/análise , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Prognóstico , Fatores de Processamento de Serina-Arginina/análise
7.
Br J Cancer ; 119(8): 940-949, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30318506

RESUMO

BACKGROUND: Interleukin 13 receptor α2 (IL13Rα2) is overexpressed in metastatic colorectal cancer. Here, we have developed novel strategies to block IL-13 binding to IL13Rα2 in order to reduce metastatic spread. METHODS: Synthetic IL13Rα2 D1 peptide (GSETWKTIITKN) was tested for the inhibition of IL-13 binding to IL13Rα2 using ELISA and different cellular assays. Peptide blocking effects on different cell signalling mediators were determined by western blot. An enantiomer version of the peptide (D-D1) was prepared to avoid proteolytic digestion. Nude mice were used for tumour growth and survival analysis after treatment with IL13Rα2 peptides. RESULTS: IL13Rα2 D1 peptide inhibited migration, invasion, and proliferation in metastatic colorectal and glioblastoma cancer cells treated with IL-13. Residues 82K, 83T, 85I and 86T were essential for blocking IL-13. IL13Rα2 peptide abolished ligand-mediated receptor internalisation and degradation, and substantially decreased IL-13 signalling capacity through IL13Rα2 to activate the FAK, PI3K/AKT and Src pathways as well as MT1-MMP expression. In addition, D1 significantly inhibited IL-13-mediated STAT6 activation through IL13Rα1. Nude mice treated with the enantiomer D-D1 peptide showed a remarkable survival increase. CONCLUSIONS: We propose that the D-D1 peptide from IL13Rα2 represents a promising therapeutic agent to inhibit metastatic progression in colorectal cancer and, likely, other solid tumours.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/terapia , Glioblastoma/terapia , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/uso terapêutico , Interleucina-13/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Quinase 1 de Adesão Focal/metabolismo , Glioblastoma/patologia , Células HT29 , Humanos , Subunidade alfa2 de Receptor de Interleucina-13/genética , Metaloproteinase 14 da Matriz/biossíntese , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Pathol ; 242(1): 39-51, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28054337

RESUMO

The process of liver colonization in colorectal cancer remains poorly characterized. Here, we addressed the role of microRNA (miRNA) dysregulation in metastasis. We first compared miRNA expression profiles between colorectal cancer cell lines with different metastatic properties and then identified target proteins of the dysregulated miRNAs to establish their functions and prognostic value. We found that 38 miRNAs were differentially expressed between highly metastatic (KM12SM/SW620) and poorly metastatic (KM12C/SW480) cancer cell lines. After initial validation, we determined that three miRNAs (miR-424-3p, -503, and -1292) were overexpressed in metastatic colorectal cancer cell lines and human samples. Stable transduction of non-metastatic cells with each of the three miRNAs promoted metastatic properties in culture and increased liver colonization in vivo. Moreover, miR-424-3p and miR-1292 were associated with poor prognosis in human patients. A quantitative proteomic analysis of colorectal cancer cells transfected with miR-424-3p, miR-503, or miR-1292 identified alterations in 149, 129, or 121 proteins, respectively, with an extensive overlap of the target proteins of the three miRNAs. Importantly, down-regulation of two of these shared target proteins, CKB and UBA2, increased cell adhesion and proliferation in colorectal cancer cells. The capacity of distinct miRNAs to regulate the same mRNAs boosts the capacity of miRNAs to regulate cancer metastasis and underscores the necessity of targeting multiple miRNAs for effective cancer therapy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neoplasias Colorretais/genética , MicroRNAs/genética , Metástase Neoplásica/genética , RNA Neoplásico/genética , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Creatina Quinase Forma BB/biossíntese , Creatina Quinase Forma BB/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Prognóstico , Proteômica/métodos , Enzimas Ativadoras de Ubiquitina/biossíntese , Enzimas Ativadoras de Ubiquitina/genética
9.
Proteomics ; 17(19)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28861940

RESUMO

Colorectal cancer (CRC) is the second deadliest cancer worldwide. Here, we aimed to study metastasis mechanisms using spatial proteomics in the KM12 cell model. Cells were SILAC-labeled and fractionated into five subcellular fractions corresponding to: cytoplasm, plasma, mitochondria and ER/golgi membranes, nuclear, chromatin-bound and cytoskeletal proteins and analyzed with high resolution mass spectrometry. We provide localization data of 4863 quantified proteins in the different subcellular fractions. A total of 1318 proteins with at least 1.5-fold change were deregulated in highly metastatic KM12SM cells respect to KM12C cells. The protein network organization, protein complexes and functional pathways associated to CRC metastasis was revealed with spatial resolution. Although 92% of the differentially expressed proteins showed the same deregulation in all subcellular compartments, a subset of 117 proteins (8%) showed opposite changes in different subcellular localizations. The chaperonin CCT, the Eif2 and Eif3 initiation of translation and the oxidative phosphorylation complexes together with an important number of guanine nucleotide-binding proteins, were deregulated in abundance and localization within the metastatic cells. Particularly relevant was the relationship of deregulated protein complexes with exosome secretion. The knowledge of the spatial proteome alterations at subcellular level contributes to clarify the molecular mechanisms underlying colorectal cancer metastasis and to identify potential targets of therapeutic intervention.


Assuntos
Núcleo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citoplasma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteoma/análise , Humanos , Metástase Neoplásica , Proteômica , Células Tumorais Cultivadas
10.
Mol Cell Proteomics ; 14(2): 303-15, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25505127

RESUMO

Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Fatores de Transcrição COUP/metabolismo , Diferenciação Celular , Interleucina-17/metabolismo , Proteômica/métodos , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Adipogenia , Animais , Extratos Celulares , Núcleo Celular/metabolismo , Regulação para Baixo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Biológicos , Proteínas Repressoras , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de Transcrição da Família Snail , Transfecção
11.
J Biol Chem ; 289(50): 34801-14, 2014 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-25336636

RESUMO

Little is known about the mechanism of integrin activation by cadherin 17 (CDH17). Here we observed the presence of a tri-peptide motif, RGD, in domain 6 of the human CDH17 sequence and other cadherins such as cadherin 5 and cadherin 6. The use of CDH17 RAD mutants demonstrated a considerable decrease of proliferation and adhesion in RKO and KM12SM colon cancer cells. Furthermore, RGD peptides inhibited the adhesion of both cell lines to recombinant CDH17 domain 6. The RGD motif added exogenously to the cells provoked a change in ß1 integrin to an active, high-affinity conformation and an increase in focal adhesion kinase and ERK1/2 activation. In vivo experiments with Swiss nude mice demonstrated that cancer cells expressing the CDH17 RAD mutant showed a considerable delay in tumor growth and liver homing. CDH17 RGD effects were also active in pancreatic cancer cells. Our results suggest that α2ß1 integrin interacts with two different ligands, collagen IV and CDH17, using two different binding sites. In summary, the RGD binding motif constitutes a switch for integrin pathway activation and shows a novel capacity of CDH17 as an integrin ligand. This motif could be targeted to avoid metastatic dissemination in tumors overexpressing CDH17 and other RGD-containing cadherins.


Assuntos
Caderinas/química , Caderinas/metabolismo , Integrina alfa2beta1/metabolismo , Oligopeptídeos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caderinas/genética , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Humanos , Integrinas/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Estrutura Terciária de Proteína , Transdução de Sinais
12.
Mol Cell Proteomics ; 12(6): 1602-20, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23443137

RESUMO

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/isolamento & purificação , Proteoma/isolamento & purificação , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica , Humanos , Marcação por Isótopo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Espectrometria de Massas , Invasividade Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Prognóstico , Mapeamento de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
13.
J Cell Sci ; 125(Pt 22): 5338-52, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22946047

RESUMO

Cell migration and invasion require regulated turnover of integrin-dependent adhesion complexes. Rap1-GTP-interacting adaptor molecule (RIAM) is an adaptor protein that mediates talin recruitment to the cell membrane, and whose depletion leads to defective melanoma cell migration and invasion. In this study, we investigated the potential involvement of RIAM in focal adhesion (FA) dynamics. RIAM-depleted melanoma and breast carcinoma cells displayed an increased number, size and stability of FAs, which accumulated centrally at the ventral cell surface, a phenotype caused by defective FA disassembly. Impairment in FA disassembly resulting from RIAM knockdown correlated with deficient integrin-dependent mitogen-activated protein kinase kinase (MEK)-Erk1/2 activation and, importantly, overexpression of constitutively active MEK resulted in rescue of FA disassembly and recovery of cell invasion. Furthermore, RIAM-promoted Ras homologue gene family, member A (RhoA) activation following integrin engagement was needed for subsequent Erk1/2 activation. In addition, RhoA overexpression partially rescued the FA phenotype in RIAM-depleted cells, also suggesting a functional role for RhoA downstream of RIAM, but upstream of Erk1/2. RIAM knockdown also led to enhanced phosphorylation of paxillin Tyr118 and Tyr31. However, expression of phosphomimetic and nonphosphorylatable mutants at these paxillin residues indicated that paxillin hyperphosphorylation is a subsequent consequence of the blockade of FA disassembly, but does not cause the FA phenotype. RIAM depletion also weakened the association between FA proteins, suggesting that it has important adaptor roles in the correct assembly of adhesion complexes. Our data suggest that integrin-triggered, RIAM-dependent MEK activation represents a key feedback event required for efficient FA disassembly, which could help explain the role of RIAM in cell migration and invasion.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adesões Focais/metabolismo , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Modelos Biológicos , Paxilina/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 12/metabolismo , Regulação para Cima , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
14.
J Exp Clin Cancer Res ; 43(1): 31, 2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38263178

RESUMO

BACKGROUND: Cadherin-17 (CDH17), a marker of differentiation in intestinal cells, binds and activates α2ß1 integrin to promote cell adhesion and proliferation in colorectal cancer (CRC) metastasis. Furthermore, CDH17 associates with p120- and ß-catenin in a manner yet to be fully elucidated. In this report, we explored the molecular mediators involved in this association, their contribution to CRC dissemination and potential therapeutic implications. METHODS: Proteomic and confocal analyses were employed to identify and validate CDH17 interactors. Functional characterization involved the study of proliferation, migration, and invasion in cell lines representative of various phenotypes. Immunohistochemistry was conducted on CRC tissue microarrays (TMA). In vivo animal experiments were carried out for metastatic studies. RESULTS: We found that desmocollin-1 (DSC1), a desmosomal cadherin, interacts with CDH17 via its extracellular domain. DSC1 depletion led to increased or decreased invasion in CRC cells displaying epithelial or mesenchymal phenotype, respectively, in a process mediated by the association with p120-catenin. Down-regulation of DSC1 resulted in an increased expression of p120-catenin isoform 1 in epithelial cells or a shift in cellular location in mesenchymal cells. Opposite results were observed after forced expression of CDH17. DSC1 is highly expressed in budding cells at the leading edge of the tumor and associates with poor prognosis in the stem-like, mesenchymal CRC subtypes, while correlates with a more favorable prognosis in the less-aggressive subtypes. In vivo experiments demonstrated that DSC1 silencing reduced tumor growth, liver homing, and metastasis in CRC mesenchymal cells. Furthermore, a synthetic peptide derived from CDH17, containing the NLV motif, effectively inhibited invasion and liver homing in vivo, opening up new possibilities for the development of novel therapies focused on desmosomal cadherins. CONCLUSIONS: These findings shed light on the multifaceted roles of CDH17, DSC1, and p120-catenin in CRC metastasis, offering insights into potential therapeutic interventions for targeting desmosomal cadherins in poorly-differentiated carcinomas.


Assuntos
Neoplasias Colorretais , Desmocolinas , Animais , delta Catenina , Proteômica , Caderinas
15.
Nucleic Acids Res ; 39(1): 347-58, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20817927

RESUMO

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/química , Códon sem Sentido , Células HeLa , Humanos , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo
16.
Cell Death Dis ; 14(11): 742, 2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-37963919

RESUMO

Interleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/ß-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.


Assuntos
Subunidade alfa2 de Receptor de Interleucina-13 , Neoplasias , Animais , Camundongos , Interleucina-13 , Subunidade alfa2 de Receptor de Interleucina-13/genética , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias/genética , NF-kappa B/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteômica
17.
Cells ; 12(21)2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37947626

RESUMO

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7's role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Fenótipo , Proteômica/métodos
18.
J Biol Chem ; 286(21): 18492-504, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21454517

RESUMO

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces ß1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Inativação Gênica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transplante Heterólogo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
19.
Biochim Biophys Acta Rev Cancer ; 1877(5): 188802, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36152905

RESUMO

Interleukin 13 receptor alpha 2 (IL13Rα2) is increasingly recognized as a relevant player in cancer invasion and metastasis. Despite being initially considered a decoy receptor for dampening the levels of interleukin 13 (IL-13) in diverse inflammatory conditions, accumulating evidences in the last decades indicate the capacity of IL13Rα2 for mediating IL-13 signaling in cancer cells. The biological reasons behind the expression of this receptor with such extremely high affinity for IL-13 in cancer cells remain unclear. Elevated expression of IL13Rα2 is commonly associated with invasion, late stage and cancer metastasis that results in poor prognosis for glioblastoma, colorectal or breast cancer, among others. The discovery of new mediators and effectors of IL13Rα2 signaling has been critical for deciphering its underlying molecular mechanisms in cancer progression. Still, many questions about the effects of inflammation, the cancer type and the tumor degree in the expression of IL13Rα2 remain largely uncharacterized. Here, we review and discuss the current status of the IL13Rα2 biology in cancer, with particular emphasis in the role of inflammation-driven expression and the regulation of different signaling pathways. As IL13Rα2 implications in cancer continue to grow exponentially, we highlight new targeted therapies recently developed for glioblastoma, colorectal cancer and other IL13Rα2-positive tumors.


Assuntos
Glioblastoma , Subunidade alfa2 de Receptor de Interleucina-13 , Glioblastoma/patologia , Humanos , Inflamação , Interleucina-13/uso terapêutico , Subunidade alfa2 de Receptor de Interleucina-13/genética , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/uso terapêutico , Transdução de Sinais
20.
J Pathol Clin Res ; 8(6): 495-508, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36134447

RESUMO

The necessity to accurately predict recurrence and clinical outcome in early stage colorectal cancer (CRC) is critical to identify those patients who may benefit from adjuvant chemotherapy. Here, we developed and validated a gene-based risk-score algorithm for patient stratification and personalised treatment in early stage disease based on alterations in the secretion of metastasis-related proteins. A quantitative label-free proteomic analysis of the secretome of highly and poorly metastatic CRC cell lines with different genetic backgrounds revealed 153 differentially secreted proteins (fold-change >5). These changes in the secretome were validated at the transcriptomic level. Starting from 119 up-regulated proteins, a six-gene/protein-based prognostic signature composed of IGFBP3, CD109, LTBP1, PSAP, BMP1, and NPC2 was identified after sequential discovery, training, and validation in four different cohorts. This signature was used to develop a risk-score algorithm, named SEC6, for patient stratification. SEC6 risk-score components showed higher expression in the poor prognosis CRC subtypes: consensus molecular subtype 4 (CMS4), CRIS-B, and stem-like. High expression of the signature was also associated with patients showing dMMR, CIMP+ status, and BRAF mutations. In addition, the SEC6 signature was associated with lower overall survival, progression-free interval, and disease-specific survival in stage II and III patients. SEC6-based risk stratification indicated that 5-FU treatment was beneficial for low-risk patients, whereas only aggressive treatments (FOLFOX and FOLFIRI) provided benefits to high-risk patients in stages II and III. In summary, this novel risk-score demonstrates the value of the secretome compartment as a reliable source for the retrieval of biomarkers with high prognostic and chemotherapy-predictive capacity, providing a potential new tool for tailoring decision-making in patient care.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Biomarcadores Tumorais/análise , Neoplasias do Colo/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fluoruracila/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Prognóstico , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/uso terapêutico , Secretoma , Transcriptoma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA