RESUMO
BACKGROUND & AIMS: Acinar to ductal metaplasia is the prerequisite for the initiation of Kras-driven pancreatic ductal adenocarcinoma (PDAC), and candidate genes regulating this process are emerging from genome-wide association studies. The adaptor protein p130Cas emerged as a potential PDAC susceptibility gene and a Kras-synthetic lethal interactor in pancreatic cell lines; however, its role in PDAC development has remained largely unknown. METHODS: Human PDAC samples and murine KrasG12D-dependent pancreatic cancer models of increasing aggressiveness were used. p130Cas was conditionally ablated in pancreatic cancer models to investigate its role during Kras-induced tumorigenesis. RESULTS: We found that high expression of p130Cas is frequently detected in PDAC and correlates with higher histologic grade and poor prognosis. In a model of Kras-driven PDAC, loss of p130Cas inhibits tumor development and potently extends median survival. Deletion of p130Cas suppresses acinar-derived tumorigenesis and progression by means of repressing PI3K-AKT signaling, even in the presence of a worsening condition like pancreatitis. CONCLUSIONS: Our observations finally demonstrated that p130Cas acts downstream of Kras to boost the PI3K activity required for acinar to ductal metaplasia and subsequent tumor initiation. This demonstrates an unexpected driving role of p130Cas downstream of Kras through PI3K/AKT, thus indicating a rational therapeutic strategy of targeting the PI3K pathway in tumors with high expression of p130Cas.
Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Proteína Substrato Associada a Crk , Neoplasias Pancreáticas , Células Acinares/patologia , Adenocarcinoma/patologia , Animais , Carcinogênese , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/patologia , Proteína Substrato Associada a Crk/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Metaplasia/patologia , Camundongos , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMO
Following publication of the original article [1], the authors reported an error in the name of the 11th author. The author's name was incorrectly published as "Vincenzo Calautti", instead of "Enzo Calautti".
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BACKGROUND: p130 Crk-associated substrate (p130CAS; also known as BCAR1) is a scaffold protein that modulates many essential cellular processes such as cell adhesion, proliferation, survival, cell migration, and intracellular signaling. p130Cas has been shown to be highly expressed in a variety of human cancers of epithelial origin. However, few data are available regarding the role of p130Cas during normal epithelial development and homeostasis. METHODS: To this end, we have generated a genetically modified mouse in which p130Cas protein was specifically ablated in the epidermal tissue. RESULTS: By using this murine model, we show that p130Cas loss results in increased cell proliferation and reduction of cell adhesion to extracellular matrix. In addition, epidermal deletion of p130Cas protein leads to premature expression of "late" epidermal differentiation markers, altered membrane E-cadherin/catenin proteins localization and aberrant tyrosine phosphorylation of E-cadherin/catenin complexes. Interestingly, these alterations in adhesive properties in absence of p130Cas correlate with abnormalities in progenitor cells balance resulting in the amplification of a more committed cell population. CONCLUSION: Altogether, these results provide evidence that p130Cas is an important regulator of epidermal cell fate and homeostasis.
Assuntos
Adesão Celular , Diferenciação Celular , Proteína Substrato Associada a Crk/deficiência , Proteína Substrato Associada a Crk/genética , Epiderme/metabolismo , Deleção de Genes , Homeostase/genética , Animais , Proliferação de Células , Matriz Extracelular/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
BACKGROUND: Most metastatic gastrointestinal stromal tumors (GISTs) develop resistance to the first-line imatinib treatment. Recently, increased vessel density and angiogenic markers were reported in GISTs with a poor prognosis, suggesting that angiogenesis is implicated in GIST tumor progression and resistance. The purpose of this study was to investigate the relationship between tumor vasculature and imatinib resistance in different GIST mouse models using a noninvasive magnetic resonance imaging (MRI) functional approach. METHODS: Immunodeficient mice (n = 8 for each cell line) were grafted with imatinib-sensitive (GIST882 and GIST-T1) and imatinib-resistant (GIST430) human cell lines. Dynamic contrast-enhanced MRI (DCE-MRI) was performed on GIST xenografts to quantify tumor vessel permeability (K trans) and vascular volume fraction (v p). Microvessel density (MVD), permeability (mean dextran density, MDD), and angiogenic markers were evaluated by immunofluorescence and western blot assays. RESULTS: Dynamic contrast-enhanced magnetic resonance imaging showed significantly increased vessel density (P < 0.0001) and permeability (P = 0.0002) in imatinib-resistant tumors compared to imatinib-sensitive ones. Strong positive correlations were observed between MRI estimates, K trans and v p, and their related ex vivo values, MVD (r = 0.78 for K trans and r = 0.82 for v p) and MDD (r = 0.77 for K trans and r = 0.94 for v p). In addition, higher expression of vascular endothelial growth factor receptors (VEGFR2 and VEFGR3) was seen in GIST430. CONCLUSIONS: Dynamic contrast-enhanced magnetic resonance imaging highlighted marked differences in tumor vasculature and microenvironment properties between imatinib-resistant and imatinib-sensitive GISTs, as also confirmed by ex vivo assays. These results provide new insights into the role that DCE-MRI could play in GIST characterization and response to GIST treatment. Validation studies are needed to confirm these findings.
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Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal/diagnóstico por imagem , Tumores do Estroma Gastrointestinal/patologia , Neovascularização Patológica/diagnóstico por imagem , Animais , Antineoplásicos , Linhagem Celular Tumoral , Meios de Contraste , Xenoenxertos , Humanos , Mesilato de Imatinib , Imageamento por Ressonância Magnética/métodos , Masculino , CamundongosRESUMO
Signals downstream of Akt can either favor or oppose stem cell (SC) maintenance, but how this dual role can be achieved is still undefined. Using human limbal keratinocyte stem cells (LKSCs), a SC type used in transplantation therapies for corneal regeneration, we show that Akt signaling is prominent in SC populations both in vivo and in vitro, and that Akt1 promotes while Akt2 opposes SC self-renewal. Noteworthy, loss of Akt2 signaling enhances LKSC maintenance ex vivo, whereas Akt1 depletion anticipates SC exhaustion. Mechanistically, the antagonistic functions of Akt1 and Akt2 in SC control are mainly dictated by their differential subcellular distribution, being nuclear Akt2 selectively implicated in FOXO inhibition. Akt2 downregulation favors LKSC maintenance as a result of a gain of FOXO functions, which attenuates the mechanistic target of rapamycin complex one signaling via tuberous sclerosis one gene induction, and promotes growth factor signaling through Akt1. Consistently, Akt2 deficiency also enhances limbal SCs in vivo. Thus, our findings reveal distinct roles for nuclear versus cytosolic Akt signaling in normal epithelial SC control and suggest that the selective Akt2 inhibition may provide novel pharmacological strategies for human LKSC expansion in therapeutic settings and mechanistic research.
Assuntos
Núcleo Celular/enzimologia , Fatores de Transcrição Forkhead/metabolismo , Queratinócitos/citologia , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco/citologia , Serina-Treonina Quinases TOR/metabolismo , Células 3T3 , Adulto , Animais , Proliferação de Células , Células Clonais , Ativação Enzimática , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Humanos , Isoenzimas/metabolismo , Limbo da Córnea/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Repressoras/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , Transcrição GênicaRESUMO
Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy.
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Neoplasias da Mama/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Animais , Neoplasias da Mama/patologia , Centrossomo/patologia , Regulação para Baixo/fisiologia , Feminino , Humanos , Camundongos , Chaperonas Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The members of the Cas protein family (p130Cas/BCAR1, Nedd9/HEF1, EFS and CASS4) are scaffold proteins required for the assembly of signal transduction complexes in response to several stimuli, such as growth factors, hormones and extracellular matrix components. Given their ability to integrate and coordinate multiple signalling events, Cas proteins have emerged as crucial players in the control of mammary cell proliferation, survival and differentiation. More importantly, it has been found that alterations of their expression levels result in aberrant signalling cascades, which promote initiation and progression of breast cancer. Based on the increasing data from in vitro, mouse model and clinical studies, in this review we will focus on two Cas proteins, p130Cas/BCAR1 and Nedd9, and their coupled signalling pathways, to examine their role in mammary cell transformation and in the acquirement of invasiveness and drug resistance of breast cancer cells.
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Neoplasias da Mama/patologia , Proteína Substrato Associada a Crk/fisiologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Glândulas Mamárias Humanas/patologia , Invasividade Neoplásica , Transdução de SinaisRESUMO
It has recently been proposed that defective differentiation of mammary luminal progenitors predisposes to basal-like breast cancer. However, the molecular and cellular mechanisms involved are still unclear. Here, we describe that the adaptor protein p130Cas is a crucial regulator of mouse mammary epithelial cell (MMEC) differentiation. Using a transgenic mouse model, we show that forced p130Cas overexpression in the luminal progenitor cell compartment results in the expansion of luminal cells, which aberrantly display basal cell features and reduced differentiation in response to lactogenic stimuli. Interestingly, MMECs overexpressing p130Cas exhibit hyperactivation of the tyrosine kinase receptor c-Kit. In addition, we demonstrate that the constitutive c-Kit activation alone mimics p130Cas overexpression, whereas c-Kit downregulation is sufficient to re-establish proper differentiation of p130Cas overexpressing cells. Overall, our data indicate that high levels of p130Cas, via abnormal c-Kit activation, promote mammary luminal cell plasticity, thus providing the conditions for the development of basal-like breast cancer. Consistently, p130Cas is overexpressed in human triple-negative breast cancer, further suggesting that p130Cas upregulation may be a priming event for the onset of basal-like breast cancer.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/patologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Proteína Substrato Associada a Crk/biossíntese , Proteína Substrato Associada a Crk/genética , Feminino , Humanos , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Gravidez , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
INTRODUCTION: Intrinsic plasticity of breast carcinoma cells allows them to undergo a transient and reversible conversion into mesenchymal cells to disseminate into distant organs, where they can re-differentiate to an epithelial-like status to form a cohesive secondary mass. The p130Cas scaffold protein is overexpressed in human ER+ and HER2+ breast cancer where it contributes to cancer progression, invasion and resistance to therapy. However, its role in regulating mesenchymal aggressive breast cancer cells remains to be determined. The aim of this study was to investigate the molecular and functional involvement of this adaptor protein in breast cancer cell plasticity. METHODS: We used silencing strategies and rescue experiments to evaluate phenotypic and biochemical changes from mesenchymal to epithelial traits in breast tumor cell lines. In the mouse A17 cell model previously related to mesenchymal cancer stem cells and basal-like breast cancer, we biochemically dissected the signaling pathways involved and performed functional in vivo tumor growth ability assays. The significance of the signaling platform was assessed in a human setting through the use of specific inhibitors in aggressive MDA-MB-231 subpopulation LM2-4175 cells. To evaluate the clinical relevance of the results, we analyzed publicly available microarray data from the Netherlands Cancer Institute and from the Koo Foundation Sun Yat-Sen Cancer Center. RESULTS: We show that p130Cas silencing induces loss of mesenchymal features, by downregulating Vimentin, Snail, Slug and Twist transcriptional factors, resulting in the acquirement of epithelial-like traits. Mechanistically, p130Cas controls Cyclooxygenase-2 transcriptional expression, which in turn contributes to p130Cas-dependent maintenance of mesenchymal phenotype. This cascade of events also compromises in vivo tumor growth through inhibition of cell signaling controlling cell cycle progression. c-Src and JNK kinases are sequential players in p130Cas/ Cyclooxygenase-2 axis and their pharmacological inhibition is sufficient to downregulate Cyclooxygenase-2 leading to an epithelial phenotype. Finally, in silico microarray data analysis indicates that p130Cas and Cyclooxygenase-2 concomitant overexpression predicts poor survival and high probability of breast tumor recurrence. CONCLUSIONS: Overall, these data identify a new p130Cas/Cyclooxygenase-2 axis as a crucial element in the control of breast tumor plasticity, opening new therapeutic strategies leading to inhibition of these pathways in aggressive breast carcinoma.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Substrato Associada a Crk/metabolismo , Ciclo-Oxigenase 2/metabolismo , Animais , Neoplasias da Mama/genética , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Ciclo-Oxigenase 2/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Modelos Biológicos , Fenótipo , Característica Quantitativa Herdável , Quinases da Família src/metabolismoRESUMO
The Cas family of multiadaptor and scaffold molecules has an essential role in intracellular signaling events. Although these proteins do not have enzymatic or transcriptional activity, they spatially and temporally control signaling events through their ability to undergo changes in phosphorylation and to associate with effectors proteins in multimolecular complexes. The involvement of p130Cas in cell motility as a component of the integrin signaling machinery is well established. Here, we discuss recent developments that highlight a fundamental role in cell transformation and microbial pathogenesis and the implications of these developments on p130Cas function under normal and pathological conditions.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína Substrato Associada a Crk/fisiologia , Neoplasias/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Humanos , Neoplasias/patologia , Fosforilação , Transdução de Sinais/fisiologiaRESUMO
The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.
Assuntos
Transformação Celular Neoplásica , Proteína Substrato Associada a Crk/fisiologia , Genes erbB-2 , Neoplasias Mamárias Experimentais/patologia , Animais , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Feminino , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , RNA Interferente Pequeno , Transdução de SinaisRESUMO
p130Cas/BCAR1 is an adaptor protein devoid of any enzymatic or transcriptional activity, whose modular structure with various binding motifs, allows the formation of multi-protein signaling complexes. This results in the induction and/or maintenance of signaling pathways with pleiotropic effects on cell motility, cell adhesion, cytoskeleton remodeling, invasion, survival, and proliferation. Deregulation of p130Cas/BCAR1 adaptor protein has been extensively demonstrated in a variety of human cancers in which overexpression of p130Cas/BCAR1 correlates with increased malignancy. p140Cap (p130Cas associated protein), encoded by the SRCIN1 gene, has been discovered by affinity chromatography and mass spectrometry analysis of putative interactors of p130Cas. It came out that p140Cap associates with p130Cas not directly but through its interaction with the Src Kinase. p140Cap is highly expressed in neurons and to a lesser extent in epithelial tissues such as the mammary gland. Strikingly, in vivo and in vitro analysis identified its tumor suppressive role in breast cancer and in neuroblastoma, showing an inverse correlation between p140Cap expression in tumors and tumor progression. In this review, a synopsis of 15 years of research on the role of p130Cas/BCAR1 and p140Cap/SRCIN1 in breast cancer will be presented.
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Two recent studies show that epigenetics and inflammation play a relevant role in the regulation of transformation and cancer cell self-renewal in breast tumours, opening up the possibility that cancer progression can be controlled by interfering with inflammation cascades. Struhl's group showed that transient activation of the Src oncoprotein induces transformation and self-renewal of immortal cells via an epigenetic switch involving NF-kappaB, Lin28, Let-7 microRNA and IL-6. Concomitantly, Wicha's laboratory developed a strategy to selectively target cancer stem cells, retarding tumour growth and reducing metastasis by blocking the IL-8 receptor CXCR1 using either an inhibitor, repertaxin or a specific blocking antibody.
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Neoplasias da Mama/patologia , Transformação Celular Neoplásica/patologia , Inflamação/patologia , Células-Tronco Neoplásicas/patologia , Anti-Inflamatórios/uso terapêutico , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Progressão da Doença , Epigênese Genética , Genes de Troca/genética , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteína Oncogênica pp60(v-src)/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/imunologia , Sulfonamidas/uso terapêuticoRESUMO
Integrin signaling has a critical function in organizing cells in tissues during both embryonic development and tissue repair. Following their binding to the extracellular ligands, the intracellular signaling pathways triggered by integrins are directed to two major functions: organization of the actin cytoskeleton and regulation of cell behaviour including survival, differentiation and growth. Basic research conducted in the past twelve years has lead to remarkable breakthroughs in this field. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factor and cytokine receptors. The purpose of this chapter is to highlight recent experimental and conceptual advances in integrin signaling with particular emphasis on the ability of integrins to regulate Fak/Src family kinases (SFKs) activation and the cross-talk with soluble growth factors receptors and cytokines.
Assuntos
Integrinas/metabolismo , Transdução de Sinais/fisiologia , Actinas/metabolismo , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Citoesqueleto/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Citocinas/metabolismo , Quinases da Família src/metabolismoRESUMO
Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Pulmonares/secundário , Neuroblastoma/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Biomarcadores Tumorais/genética , Proliferação de Células , Sobrevivência Celular , Humanos , Lactente , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neuroblastoma/diagnóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The early gene early growth response (Egr-1), a broadly expressed member of the zing-finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr-1 expression is mainly regulated by integrin-mediated adhesion. Integrin-dependent adhesion plays a dual role in Egr-1 regulation, either being sufficient "per se" to induce Egr-1, or required for EGF-dependent expression of Egr-1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin-dependent Egr-1 regulation, we show by FLIM-based FRET that in living cells beta1-integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr-1 induction depends on integrin-dependent PI3K/Akt activation, as indicated by the decrease in Egr-1 levels in presence of the pharmacological inhibitor LY294002, the kinase-defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr-1-increased levels, indicating that FoxO1 behaves as a negative regulator of Egr-1 expression. These data demonstrate that integrin/EGFR cross-talk is required for expression of Egr-1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Receptores ErbB/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Integrina beta1/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteína Forkhead Box O1 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptor Cross-Talk/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The ErbB2 receptor tyrosine kinase is overexpressed in approximately 15-20% of breast tumors and associated with aggressive disease and poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration and proliferation in normal and pathological contexts. p130Cas overexpression in ErbB2 human breast cancer correlates with poor prognosis and metastasis formation. Recent data indicate that p130Cas association to ErbB2 protects ErbB2 from degradation, thus enhancing tumorigenesis. Therefore, inhibiting p130Cas/ErbB2 interaction might represent a new therapeutic strategy to target breast cancer. Here we demonstrate by performing Molecular Modeling, Molecular Dynamics, dot blot, ELISA and fluorescence quenching experiments, that p130Cas binds directly to ErbB2. Then, by structure-based virtual screening, we identified two potential inhibitors of p130Cas/ErbB2 interaction. Their experimental validation was performed in vitro and in ErbB2-positive breast cancer cellular models. The results highlight that both compounds interfere with p130Cas/ErbB2 binding and significantly affect cell proliferation and sensitivity to Trastuzumab. Overall, this study identifies p130Cas/ErbB2 complex as a potential breast cancer target revealing new therapeutic perspectives for protein-protein interaction (PPI).
Assuntos
Antineoplásicos , Neoplasias da Mama , Proteína Substrato Associada a Crk/metabolismo , Descoberta de Drogas , Ligação Proteica/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Escherichia coli/genética , Feminino , Células HEK293 , Humanos , Trastuzumab/uso terapêuticoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
To investigate the mechanisms through which p130Cas adaptor protein is linked to tumorigenesis, we generated mouse mammary tumor virus (MMTV)-p130Cas mice overexpressing p130Cas in the mammary gland. MMTVp130Cas transgenic mice are characterized by extensive mammary epithelial hyperplasia during development and pregnancy and by delayed involution at the end of lactation. These phenotypes are associated with activation of Src kinase, extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Akt pathways, leading to an increased rate of proliferation and a decreased apoptosis. A double-transgenic line derived from crossing MMTV-p130Cas with MMTV-HER2-Neu mice expressing the activated form of the HER2-Neu oncogene develops multifocal mammary tumors with a significantly shorter latency than the HER2-Neu parental strain alone. Mammary epithelial cells isolated from tumors of double-transgenic mice display increased tyrosine phosphorylation, c-Src, and Akt activation compared with cells derived from HER2-Neu tumors. In addition, p130Cas down-regulation by RNA interference increases apoptosis in HER2-Neu-expressing cells, indicating that p130Cas regulates cell survival. Consistently with the double-transgenic mice model, p130Cas is overexpressed in a significant subset of human breast cancers and high levels of p130Cas in association with HER2 expression correlate with elevated proliferation. These findings provide evidences for a role of p130Cas as a positive regulator of both proliferation and survival in normal and transformed mammary epithelial cells. Its overexpression contributes to HER2-Neu-induced breast tumorigenesis, thus identifying this protein as a putative target for clinical therapy.