RESUMO
Receptor tyrosine kinases (RTKs) are recognized as targets of precision medicine in human cancer upon their gene amplification or constitutive activation, resulting in increased downstream signal complexity including heterotypic crosstalk with other RTKs. The Met RTK exhibits such reciprocal crosstalk with several members of the human EGFR (HER) family of RTKs when amplified in cancer cells. We show that Met signaling converges on HER3-tyrosine phosphorylation across a panel of seven MET-amplified cancer cell lines and that HER3 is required for cancer cell expansion and oncogenic capacity in vitro and in vivo. Gene expression analysis of HER3-depleted cells identified MPZL3, encoding a single-pass transmembrane protein, as HER3-dependent effector in multiple MET-amplified cancer cell lines. MPZL3 interacts with HER3 and MPZL3 loss phenocopies HER3 loss in MET-amplified cells, while MPZL3 overexpression can partially rescue proliferation upon HER3 depletion. Together, these data support an oncogenic role for a HER3-MPZL3 axis in MET-amplified cancers.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor ErbB-3/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Instabilidade de Microssatélites , Fosforilação , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética , Transdução de Sinais/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transplante HeterólogoRESUMO
BACKGROUND & AIMS: Incidence of and mortality from pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, are almost equivalent, so better treatments are needed. We studied gene expression profiles of PDACs and the functions of genes with altered expression to identify new therapeutic targets. METHODS: We performed microarray analysis to analyze gene expression profiles of 195 PDAC and 41 non-tumor pancreatic tissue samples. We undertook an extensive analysis of the PDAC transcriptome by superimposing interaction networks of proteins encoded by aberrantly expressed genes over signaling pathways associated with PDAC development to identify factors that might alter regulation of these pathways during tumor progression. We performed tissue microarray analysis to verify changes in expression of candidate protein using an independent set of 152 samples (40 nontumor pancreatic tissues, 63 PDAC sections, and 49 chronic pancreatitis samples). We validated the functional relevance of the candidate molecule using RNA interference or pharmacologic inhibitors in pancreatic cancer cell lines and analyses of xenograft tumors in mice. RESULTS: In an analysis of 38,276 human genes and loci, we identified 1676 genes that were significantly up-regulated and 1166 genes that were significantly down-regulated in PDAC compared with nontumor pancreatic tissues. One gene that was up-regulated and associated with multiple signaling pathways that are dysregulated in PDAC was G protein subunit αi2, which has not been previously associated with PDAC. G protein subunit αi2 mediates the effects of dopamine receptor D2 (DRD2) on cyclic adenosine monophosphate signaling; PDAC tissues had a slight but significant increase in DRD2 messenger RNA. Levels of DRD2 protein were substantially increased in PDACs, compared with non-tumor tissues, in tissue microarray analyses. RNA interference knockdown of DRD2 or inhibition with pharmacologic antagonists (pimozide and haloperidol) reduced proliferation of pancreatic cancer cells, induced endoplasmic reticulum stress and apoptosis, and reduced cell migration. RNA interference knockdown of DRD2 in pancreatic tumor cells reduced growth of xenograft tumors in mice, and administration of the DRD2 inhibitor haloperidol to mice with orthotopic xenograft tumors reduced final tumor size and metastasis. CONCLUSIONS: In gene expression profile analysis of PDAC samples, we found the DRD2 signaling pathway to be activated. Inhibition of DRD2 in pancreatic cancer cells reduced proliferation and migration, and slowed growth of xenograft tumors in mice. DRD2 antagonists routinely used for management of schizophrenia might be tested in patients with pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptores de Dopamina D2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/secundário , Estudos de Casos e Controles , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Antagonistas dos Receptores de Dopamina D2/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Haloperidol/farmacologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Pimozida/farmacologia , RNA Interferente Pequeno , Receptores de Dopamina D2/metabolismo , Transdução de Sinais , Transcriptoma , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima , eIF-2 Quinase/metabolismoRESUMO
Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell-cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Cotargeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors. SIGNIFICANCE: The combination of cytostatic CDK4/6 and BRD4 inhibitors induces senescent cancer cells that are primed for activation of ferroptotic cell death by targeting GPX4, providing an effective strategy for treating cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Citostáticos , Ferroptose , Neoplasias Pulmonares , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Quinase 4 Dependente de Ciclina , Proteínas Nucleares/metabolismo , Citostáticos/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Quinase 6 Dependente de Ciclina , Inibidores de Proteínas Quinases/farmacologiaRESUMO
SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.
Assuntos
Glutamina , Neoplasias , Humanos , Transportador de Glucose Tipo 1 , Adenosina Trifosfatases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Suplementos Nutricionais , DNA Helicases/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo. METHODS: The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer. RESULTS: A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation. CONCLUSIONS: These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Animais , Neoplasias Ósseas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteína Substrato Associada a Crk/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Integrinas/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks both effective patient stratification strategies and therapeutic targets. Whilst elevated levels of the MET receptor tyrosine kinase are associated with TNBCs and predict poor clinical outcome, the functional role of MET in TNBC is still poorly understood. In this study, we utilise an established Met-dependent transgenic mouse model of TNBC, human cell lines and patient-derived xenografts to investigate the role of MET in TNBC tumorigenesis. We find that in TNBCs with mesenchymal signatures, MET participates in a compensatory interplay with FGFR1 to regulate tumour-initiating cells (TICs). We demonstrate a requirement for the scaffold protein FRS2 downstream from both Met and FGFR1 and find that dual inhibition of MET and FGFR1 signalling results in TIC depletion, hindering tumour progression. Importantly, basal breast cancers that display elevated MET and FGFR1 signatures are associated with poor relapse-free survival. Our results support a role for MET and FGFR1 as potential co-targets for anti-TIC therapies in TNBC.
RESUMO
Inactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca2+) release in these cancers. By restricting chromatin accessibility to ITPR3, encoding Ca2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca2+-dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , DNA Helicases/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias/metabolismo , Mutação , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/genética , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Subsets of breast tumors present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare histologies. Here, we developed 37 patient-derived xenografts (PDX) from these difficult-to-treat cancers to interrogate their molecular composition and functional biology. Whole-genome and transcriptome sequencing and reverse-phase protein arrays revealed that PDXs conserve the molecular landscape of their corresponding patient tumors. Metastatic potential varied between PDXs, where low-penetrance lung micrometastases were most common, though a subset of models displayed high rates of dissemination in organotropic or diffuse patterns consistent with what was observed clinically. Chemosensitivity profiling was performed in vivo with standard-of-care agents, where multi-drug chemoresistance was retained upon xenotransplantation. Consolidating chemogenomic data identified actionable features in the majority of PDXs, and marked regressions were observed in a subset that was evaluated in vivo. Together, this clinically-annotated PDX library with comprehensive molecular and phenotypic profiling serves as a resource for preclinical studies on difficult-to-treat breast tumors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Mutação , Medicina de Precisão , Prognóstico , Estudo de Prova de Conceito , Análise Serial de Proteínas/métodos , Sequenciamento Completo do GenomaRESUMO
CDK4/6 inhibitors are FDA-approved drugs for estrogen receptor-positive (ER+) breast cancer and are being evaluated to treat other tumor types, including KRAS-mutant non-small cell lung cancer (NSCLC). However, their clinical utility is often limited by drug resistance. Here, we sought to better understand the resistant mechanisms and help devise potential strategies to overcome this challenge. We show that treatment with CDK4/6 inhibitors in both ER+ breast cancer and KRAS-mutant NSCLC cells induces feedback upregulation of cyclin D1, CDK4, and cyclin E1, mediating drug resistance. We demonstrate that rocaglates, which preferentially target translation of key cell-cycle regulators, effectively suppress this feedback upregulation induced by CDK4/6 inhibition. Consequently, combination treatment of CDK4/6 inhibitor palbociclib with the eukaryotic initiation factor (eIF) 4A inhibitor, CR-1-31-B, is synergistic in suppressing the growth of these cancer cells in vitro and in vivo Furthermore, ER+ breast cancer and KRAS-mutant NSCLC cells that acquired resistance to palbociclib after chronic drug exposure are also highly sensitive to this combination treatment strategy. Our findings reveal a novel strategy using eIF4A inhibitors to suppress cell-cycle feedback response and to overcome resistance to CDK4/6 inhibition in cancer.
Assuntos
Benzofuranos/farmacologia , Neoplasias da Mama/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Benzofuranos/química , Neoplasias da Mama/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Células MCF-7 , Piperazinas/farmacologia , Purinas/farmacologia , Piridinas/farmacologiaRESUMO
Neutrophils are phenotypically heterogeneous and exert either anti- or pro-metastatic functions. We show that cancer-cell-derived G-CSF is necessary, but not sufficient, to mobilize immature low-density neutrophils (iLDNs) that promote liver metastasis. In contrast, mature high-density neutrophils inhibit the formation of liver metastases. Transcriptomic and metabolomic analyses of high- and low-density neutrophils reveal engagement of numerous metabolic pathways specifically in low-density neutrophils. iLDNs exhibit enhanced global bioenergetic capacity, through their ability to engage mitochondrial-dependent ATP production, and remain capable of executing pro-metastatic neutrophil functions, including NETosis, under nutrient-deprived conditions. We demonstrate that NETosis is an important neutrophil function that promotes breast cancer liver metastasis. iLDNs rely on the catabolism of glutamate and proline to support mitochondrial-dependent metabolism in the absence of glucose, which enables sustained NETosis. These data reveal that distinct pro-metastatic neutrophil populations exhibit a high degree of metabolic flexibility, which facilitates the formation of liver metastases.
Assuntos
Neoplasias Hepáticas/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neutrófilos/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Neutrófilos/patologiaRESUMO
Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease.
Assuntos
Neoplasias da Mama/enzimologia , Movimento Celular , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Podossomos/enzimologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Células HEK293 , Xenoenxertos , Humanos , Masculino , Camundongos Nus , Proteínas dos Microfilamentos/genética , Invasividade Neoplásica , Metástase Neoplásica , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfoproteínas/metabolismo , Podossomos/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Förster resonance energy transfer-based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell's leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers.
Assuntos
Movimento Celular , Neoplasias Epiteliais e Glandulares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Transporte Proteico/genética , Proteínas Supressoras de Tumor/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The Met receptor tyrosine kinase is activated or genetically amplified in some gastric cancers, but resistance to small-molecule inhibitors of Met often emerges in patients. We found that Met abundance correlated with a proliferation marker in patient gastric tumor sections, and gastric cancer cell lines that have MET amplifications depended on Met for proliferation and anchorage-independent growth in culture. Inhibition of Met induced temporal changes in gene expression in the cell lines, initiated by a rapid decrease in the expression of genes encoding transcription factors, followed by those encoding proteins involved in epithelial-mesenchymal transition, and finally those encoding cell cycle-related proteins. In the gastric cancer cell lines, microarray and chromatin immunoprecipitation analysis revealed considerable overlap between genes regulated in response to Met stimulation and those regulated by signal transducer and activator of transcription 3 (STAT3). The activity of STAT3, extracellular signal-regulated kinase (ERK), and the kinase Akt was decreased by Met inhibition, but only inhibitors of STAT3 were as effective as the Met inhibitor in decreasing tumor cell proliferation in culture and in xenografts, suggesting that STAT3 mediates the pro-proliferative program induced by Met. However, the phosphorylation of ERK increased after prolonged Met inhibition in culture, correlating with decreased abundance of the phosphatases DUSP4 and DUSP6, which inhibit ERK. Combined inhibition of Met and the mitogen-activated protein kinase kinase (MEK)-ERK pathway induced greater cell death in cultured gastric cancer cells than did either inhibitor alone. These findings indicate combination therapies that may counteract resistance to Met inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Sistema de Sinalização das MAP Quinases , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Células HEK293 , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.
Assuntos
Neoplasias da Mama/patologia , Claudinas/metabolismo , Hepatócitos/metabolismo , Neoplasias Hepáticas/secundário , Animais , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/patologia , Claudinas/biossíntese , Claudinas/genética , Matriz Extracelular/metabolismo , Feminino , Células HEK293 , Hepatócitos/patologia , Humanos , Integrinas/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Camundongos , Interferência de RNA , RNA Interferente Pequeno , Neoplasias Cutâneas/metabolismo , Junções Íntimas/metabolismoRESUMO
The non-receptor protein-tyrosine phosphatases (PTPs) 1B and T-cell phosphatase (TCPTP) have been implicated as negative regulators of multiple signaling pathways including receptor-tyrosine kinases. We have identified PTP1B and TCPTP as negative regulators of the hepatocyte growth factor receptor, the Met receptor-tyrosine kinase. In vivo, loss of PTP1B or TCPTP enhances hepatocyte growth factor-mediated phosphorylation of Met. Using substrate trapping mutants of PTP1B or TCPTP, we have demonstrated that both phosphatases interact with Met and that these interactions require phosphorylation of twin tyrosines (Tyr-1234/1235) in the activation loop of the Met kinase domain. Using confocal microscopy, we show that trapping mutants of both PTP1B and the endoplasmic reticulum-targeted TCPTP isoform, TC48, colocalize with Met and that activation of Met enables the nuclear-localized isoform of TCPTP, TC45, to exit the nucleus. Using small interfering RNA against PTP1B and TCPTP, we demonstrate that phosphorylation of Tyr-1234/1235 in the activation loop of the Met receptor is elevated in the absence of either PTP1B or TCPTP and further elevated upon loss of both phosphatases. This enhanced phosphorylation of Met corresponds to enhanced biological activity and cellular invasion. Our data demonstrate that PTP1B and TCPTP play distinct and non-redundant roles in the regulation of the Met receptor-tyrosine kinase.