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1.
Oncogene ; 34(33): 4320-32, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25381824

RESUMO

The acquisition of cell motility is an early step in melanoma metastasis. Here we use intravital imaging of signalling reporter cell-lines combined with genome-wide transcriptional analysis to define signalling pathways and genes associated with melanoma metastasis. Intravital imaging revealed heterogeneous cell behaviour in vivo: <10% of cells were motile and both singly moving cells and streams of cells were observed. Motile melanoma cells had increased Notch- and SRF-dependent transcription. Subsequent genome-wide analysis identified an overlapping set of genes associated with high Notch and SRF activity. We identified EZH2, a histone methyltransferase in the Polycomb repressive complex 2, as a regulator of these genes. Heterogeneity of EZH2 levels is observed in melanoma models, and co-ordinated upregulation of genes positively regulated by EZH2 is associated with melanoma metastasis. EZH2 was also identified as regulating the amelanotic phenotype of motile cells in vivo by suppressing expression of the P-glycoprotein Oca2. Analysis of patient samples confirmed an inverse relationship between EZH2 levels and pigment. EZH2 targeting with siRNA and chemical inhibition reduced invasion in mouse and human melanoma cell lines. The EZH2-regulated genes KIF2C and KIF22 are required for melanoma cell invasion and important for lung colonization. We propose that heterogeneity in EZH2 levels leads to heterogeneous expression of a cohort of genes associated with motile behaviour including KIF2C and KIF22. EZH2-dependent increased expression of these genes promotes melanoma cell motility and early steps in metastasis.


Assuntos
Movimento Celular/genética , Melanoma/genética , Complexo Repressor Polycomb 2/genética , Receptores Notch/genética , Fator de Resposta Sérica/genética , Transdução de Sinais/genética , Albinismo Oculocutâneo/genética , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Cinesinas/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Transcrição Gênica/genética , Regulação para Cima/genética
2.
Oncogene ; 32(6): 747-58, 2013 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-22450746

RESUMO

The invasion of squamous cell carcinoma (SCC) is a significant cause of morbidity and mortality. Here, we identify an E3 ligase, Traf6 and a de-ubiquitinating enzyme, Cezanne/ZA20D1, as important regulators of this process in organotypic models. Traf6 can promote the formation of Cdc42-dependent F-actin microspikes. Furthermore, Traf6 has a key role in autocrine interleukin-1ß signalling in SCC cells, which in turn is required to drive the expression of tumour necrosis factor α (TNFα). TNFα acts in a paracrine manner to increase the invasion-promoting potential of carcinoma-associated fibroblasts (CAFs). Exogenous TNFα signalling can restore invasion in cells depleted of Traf6. In conclusion, Traf6 has two important roles in SCC invasion: it promotes cell intrinsic Cdc42-dependent regulation of the actin cytoskeleton and enables production of the paracrine signal, TNFα, that enhances the activity of CAFs.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Endopeptidases/metabolismo , Fibroblastos/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Comunicação Autócrina , Humanos , Interleucina-1beta/metabolismo , Invasividade Neoplásica , Comunicação Parácrina , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
3.
Br J Cancer ; 102(2): 392-402, 2010 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-19953096

RESUMO

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) can promote the progression of tumours in many ways. They can remodel the extracellular matrix to generate an environment that enables the invasion of cancer cells. We hypothesised that compounds that prevent matrix remodelling by CAFs would block their ability to promote carcinoma cell invasion. METHODS: We designed a screen for compounds that interfere with CAF-promoted matrix remodelling. Hits from this screen were investigated in organotypic invasion models of squamous cell carcinoma (SCC). RESULTS: We find that lovastatin and simvastatin reduce matrix remodelling by fibroblasts and thereby reduce SCC invasion. This class of compounds exert their effects partly through disrupting the function of Rab proteins, and we show a new role for Rab21 in promoting cancer cell invasion promoted by CAFs. CONCLUSIONS: Rab21 is required for CAFs to promote the invasion of cancer cells. It enables the accumulation of integrin alpha5 at the plasma membrane and subsequent force-mediated matrix remodelling.


Assuntos
Actomiosina/metabolismo , Fibroblastos/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Invasividade Neoplásica/fisiopatologia , Proteínas rab de Ligação ao GTP/metabolismo , Carcinoma de Células Escamosas/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Integrina alfa5/metabolismo , Lovastatina/farmacologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Sinvastatina/farmacologia
4.
J Microsc ; 231(3): 441-5, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18754999

RESUMO

The availability of multi-photon intravital microscopy has recently allowed researchers to start to visualise the dynamic behaviour of cancer cells in vivo. This imaging has revealed that many cancer cells ranging from carcinoma to melanoma move in an amoeboid manner in order to invade surrounding tissue and escape from the primary tumour. This mode on cell motility is extremely rapid and does not require the activity of proteases to degrade the extra-cellular matrix (ECM). This review details the techniques that are available to study cell motility in vivo and discusses the current knowledge about the mechanisms of amoeboid cell motility.


Assuntos
Fenômenos Fisiológicos Celulares , Locomoção , Microscopia/métodos , Neoplasias
5.
Oncogene ; 26(36): 5214-28, 2007 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-17334396

RESUMO

Loss of E-cadherin-mediated cell-cell junctions has been correlated with cancer cell invasion and poor patient survival. p120-catenin has emerged as a key player in promoting E-cadherin stability and adherens junction integrity and has been proposed as a potential invasion suppressor by preventing release of cells from the constraints imposed by cadherin-mediated cell-cell adhesion. However, it has been proposed that tyrosine phosphorylation of p120 may contribute to cadherin-dependent junction disassembly during invasion. Here, we use small interfering RNA (siRNA) in A431 cells to show that knockdown of p120 promotes two-dimensional migration of cells. In contrast, p120 knockdown impairs epidermal growth factor-induced A431 invasion into three-dimensional matrix gels or in organotypic culture, whereas re-expression of siRNA-resistant p120, or a p120 isoform that cannot be phosphorylated on tyrosine, restores the collective mode of invasion employed by A431 cells in vitro. Thus, p120 promotes A431 cell invasion in a phosphorylation-independent manner. We show that the collective invasion of A431 cells depends on the presence of cadherin-mediated (P- and E-cadherin) cell-cell contacts, which are lost in cells where p120 expression is knocked down. Furthermore, membranous p120 is maintained in invasive squamous cell carcinomas in tumours suggesting that p120 may be important for the collective invasion of tumours cells in vivo.


Assuntos
Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/fisiologia , Invasividade Neoplásica , Fosfoproteínas/fisiologia , Sequência de Bases , Cateninas , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Primers do DNA , Fator de Crescimento Epidérmico/metabolismo , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , RNA Interferente Pequeno , Proteínas Recombinantes/metabolismo , Tirosina/metabolismo , delta Catenina
6.
Br J Cancer ; 92(1): 102-12, 2005 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-15597106

RESUMO

Integrin beta1 is both overexpressed and in an 'active' conformation in vulval squamous cell carcinomas (VSCCs) compared to matched normal skin. To investigate the significance of integrin beta1 deregulation we stably knocked-down integrin beta1 expression in the VSCC cell line A431. In vitro analysis revealed that integrin beta1 is required for cell adhesion, cell spreading and invasion. However, integrin beta1 is not required for cell growth or activation of FAK and ERK signalling in vitro or in vivo. Strikingly, while control tumours were able to invade the dermis, integrin beta1 knockdown tumours were significantly more encapsulated and less invasive.


Assuntos
Carcinoma de Células Escamosas/patologia , Integrina beta1/fisiologia , Neoplasias Vulvares/patologia , Carcinoma de Células Escamosas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Integrina beta1/genética , Invasividade Neoplásica , RNA Interferente Pequeno , Transdução de Sinais , Transfecção , Neoplasias Vulvares/metabolismo
7.
Nat Cell Biol ; 3(4): 339-45, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11283606

RESUMO

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin-myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin-myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin-myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Animais , Inibidores de Caspase , Membrana Celular/patologia , DNA/metabolismo , Fragmentação do DNA , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Células Tumorais Cultivadas , Quinases Associadas a rho
8.
EMBO J ; 20(4): 755-66, 2001 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11179220

RESUMO

Transformation by oncogenic Ras requires the function of the Rho family GTPases. We find that Ras-transformed cells have elevated levels of RhoA-GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho-GTP are not a direct consequence of Ras signalling but are selected for in response to sustained ERK-MAP kinase signalling. While the elevated levels of Rho-GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibres in transformed cells. We show that the sustained ERK-MAP kinase signalling resulting from transformation by oncogenic Ras down-regulates ROCK1 and Rho-kinase, two Rho effectors required for actin stress fibre formation. The repression of Rho- dependent stress fibre formation by ERK-MAP kinase signalling contributes to the increased motility of Ras-transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibres and inhibits the motility of Ras-transformed fibroblasts. We propose a model in which Ras and Rho signalling pathways cross-talk to promote signalling pathways favouring transformation.


Assuntos
Divisão Celular , Movimento Celular , Proteínas de Ligação ao GTP/metabolismo , Receptor Cross-Talk , Proteínas ras/metabolismo , Animais , Linhagem Celular , Linhagem Celular Transformada , Fibroblastos/enzimologia , Imunofluorescência , Sistema de Sinalização das MAP Quinases , Camundongos , Microinjeções
9.
Mol Cell Biol ; 21(4): 1173-84, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11158304

RESUMO

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.


Assuntos
MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Fator Natriurético Atrial/genética , Cardiomegalia/etiologia , Células Cultivadas , Endotelina-1/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Humanos , Miocárdio/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Transfecção , Proteína rhoA de Ligação ao GTP/metabolismo
10.
Mol Cell ; 5(1): 13-25, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10678165

RESUMO

We have examined the role of the mouse Diaphanous-related formin (DRF) Rho GTPase binding proteins, mDia1 and mDia2, in cell regulation. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperated with Rho-kinase or ROCK to form stress fibers but independently activated SRF. Src tyrosine kinase associated and co-localized with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocked cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK. Our results show that the DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fetais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células 3T3 , Animais , Divisão Celular , Endossomos/metabolismo , Forminas , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Transfecção , Quinases Associadas a rho
11.
Curr Biol ; 9(3): 136-45, 1999 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-10021386

RESUMO

BACKGROUND: The Ras-related GTPase RhoA controls signalling processes required for cytoskeletal reorganisation, transcriptional regulation, and transformation. The ability of RhoA mutants to transform cells correlates not with transcription but with their ability to bind ROCK-I, an effector kinase involved in cytoskeletal reorganisation. We used a recently developed specific ROCK inhibitor, Y-27632, and ROCK truncation mutants to investigate the role of ROCK kinases in transcriptional activation and transformation. RESULTS: In NIH3T3 cells, Y-27632 did not prevent the activation of serum response factor, transcription of c-fos or cell cycle re-entry following serum stimulation. Repeated treatment of NIH3T3 cells with Y-27632, however, substantially disrupted their actin fibre network but did not affect their growth rate. Y-27632 blocked focus formation by RhoA and its guanine-nucleotide exchange factors Dbl and mNET1. It did not affect the growth rate of cells transformed by Dbl and mNET1, but restored normal growth control at confluence and prevented their growth in soft agar. Y-27632 also significantly inhibited focus formation by Ras, but had no effect on the establishment or maintenance of transformation by Src. Furthermore, it significantly inhibited anchorage-independent growth of two out of four colorectal tumour cell lines. Consistent with these data, a truncated ROCK derivative exhibited weak ability to cooperate with activated Raf in focus formation assays. CONCLUSIONS: ROCK signalling is required for both the establishment and maintenance of transformation by constitutive activation of RhoA, and contributes to the Ras-transformed phenotype. These observations provide a potential explanation for the requirement for Rho in Ras-mediated transformation. Moreover, the inhibition of ROCK kinases may be of therapeutic use.


Assuntos
Amidas/farmacologia , Transformação Celular Neoplásica , GTP Fosfo-Hidrolases/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Piridinas/farmacologia , Transdução de Sinais/fisiologia , Células 3T3/enzimologia , Células 3T3/ultraestrutura , Actinas/análise , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas de Neoplasias/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Deleção de Sequência , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/ultraestrutura , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP
12.
EMBO J ; 17(5): 1350-61, 1998 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-9482732

RESUMO

The RhoA GTPase regulates diverse cellular processes including cytoskeletal reorganization, transcription and transformation. Although many different potential RhoA effectors have been identified, including two families of protein kinases, their roles in RhoA-regulated events remain unclear. We used a genetic screen to identify mutations at positions 37-42 in the RhoA effector loop that selectively disrupt effector binding, and used these to investigate the role of RhoA effectors in the formation of actin stress fibres, activation of transcription by serum response factor (SRF) and transformation. Interaction with the ROCK kinase and at least one other unidentified effector is required for stress fibre formation. Signalling to SRF by RhoA can occur in the absence of RhoA-induced cytoskeletal changes, and did not correlate with binding to any of the effectors tested, indicating that it may be mediated by an unknown effector. Binding to ROCK-I, but not activation of SRF, correlated with the activity of RhoA in transformation. The effector mutants should provide novel approaches for the functional study of RhoA and isolation of effector molecules involved in specific signalling processes.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Mutação/fisiologia , Proteínas Nucleares/fisiologia , Ativação Transcricional/fisiologia , Células 3T3 , Animais , Transformação Celular Neoplásica , Proteínas de Ligação ao GTP/genética , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Proteínas Recombinantes de Fusão , Fator de Resposta Sérica , Transdução de Sinais/fisiologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP
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