RESUMO
The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Técnicas Imunoenzimáticas , Camundongos , Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.
Assuntos
Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP27/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Pulmonares/secundário , Neoplasias Ovarianas/patologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Western Blotting , Proliferação de Células , Feminino , Imunofluorescência , Proteínas de Choque Térmico HSP27/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Vascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.
Assuntos
Células Endoteliais/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Contagem de Células , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Biológicos , Proteínas Tirosina Fosfatases/metabolismo , Transdução de SinaisRESUMO
Casein kinase 1 delta (CK1δ) has a tumor-promoting role in different cancers and it is genetically amplified in a portion of human epithelial ovarian cancer (EOC). CK1δ is involved in pleiotropic cellular functions such as cell proliferation, DNA damage, and migration. We specifically knocked down CK1δ by short hairpin RNA (shRNA) in human ovarian cancer cells and we performed proliferation, chemosensitivity, as well as in vitro and in vivo migration assays. CK1δ knocked-down cells displayed reduced proliferation capability both in vitro and in vivo. Nonetheless, these cells were sensitized to the first line chemotherapeutic agent carboplatin (CPT), and this observation could be associated to reduced expression levels of p21(Cip1/Waf1), involved in DNA damage response, and the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP). Moreover, CK1δ knocked-down cells were affected in their migratory and lung homing capability, even if in opposite ways, i.e., IGROV1, SKOV3 and MES-OV lost, while OVCAR3 gained motility potential. The results suggest CK1δ as a potential exploitable target for pharmacological EOC treatment, but they also advise further investigation of its role in cell migration.
RESUMO
Receptors tyrosine kinase (RTK) enable normal and tumor cells to perceive and adapt to stimuli present in the microenvironment. These stimuli, also known as growth factors, are important molecular cues actively supporting cancer stem cell (CSC) self-renewal and viability. Since in epithelial ovarian cancer (EOC) the expression of c-Kit (CD117) has been identified as a CSC hallmark, we investigated the existence of a tumor growth-promoting loop between c-Kit and its ligand Stem Cell Factor (SCF). SCF exists as a soluble or transmembrane protein and through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions. High amounts of SCF were found in the ascitic effusions collected from EOC patients. While tumor cells and CSC only expressed the membrane-associated SCF isoform, both secreted and membrane-bound isoforms were expressed by tumor-associated macrophages (TAM, here shown to be M2-like) and fibroblasts (TAF). Circulating monocytes from EOC-bearing patients and healthy donors did not express both SCF isoforms. However, monocytes isolated from healthy donors produced SCF upon in vitro differentiation into macrophages, irrespectively of M1 or M2 polarization. In vitro, both SCF isoforms were able to activate the Akt pathway in c-Kit+ cells, and this effect was counteracted by the tyrosine kinase inhibitor imatinib. In addition, our results indicated that SCF could help c-Kit+ CSC survival in selective culture conditions and promote their canonical stemness properties, thus indicating the possible existence of a juxtacrine/paracrine circuit in EOC.
Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Antineoplásicos/farmacologia , Carcinoma Epitelial do Ovário/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Fibroblastos/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/genética , Comunicação Parácrina/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Isoformas de Proteínas/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/química , Transdução de Sinais/genética , Fator de Células-Tronco/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
An epithelial to mesenchymal transition (EMT) has been correlated to malignant tumor progression and metastasis by promoting cancer cell migration and invasion and chemoresistance. Hence, finding druggable EMT effectors is critical to efficiently interfere with metastasis formation and to overcome therapy resistance. We have employed a high-content microscopy screen in combination with a kinome and phosphatome-wide siRNA library to identify signaling pathways underlying an EMT of murine mammary epithelial cells and breast cancer cells. This screen identified the MEK5-ERK5 axis as a critical player in TGFß-mediated EMT. Suppression of MEK5-ERK5 signaling completely prevented the morphological and molecular changes occurring during a TGFß-induced EMT and, conversely, forced highly metastatic breast cancer cells into a differentiated epithelial state. Inhibition of MEK5-ERK5 signaling also repressed breast cancer cell migration and invasion and substantially reduced lung metastasis without affecting primary tumor growth. The results suggest that the MEK5-ERK5 signaling axis via activation of MEF2B and other transcription factors plays an important role in the induction and maintenance of breast cancer cell migration and invasion and thus represents an exploitable target for the pharmacological inhibition of cancer cell metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , MAP Quinase Quinase 5/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Metástase Neoplásica/patologia , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFß)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFß receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias Mamárias Animais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Receptores Proteína Tirosina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: The Interferon Regulatory Factors (IRFs) are transcription factors involved in immune responses and oncogenesis and most of them are classified as tumour suppressors. The expression and activation of IRF(s) are stimulated by several cytokines and by DNA damage. Here we examine the role of the IRF-1 in the response of ovarian cancer cells to the front-line chemotherapeutic drug cisplatin (CDDP). METHODS: We evaluated the transcriptional response of three ovarian cancer cell lines to CDDP both under control conditions and after IRF-1 silencing using expression microarrays. The role played by IRF-1 in the response of these cells to CDDP was evaluated after silencing and overexpressing IRF-1. We studied cell cycle progression, colony forming ability in monolayer culture and semisolid medium, and apoptosis in the response to the drug. RESULTS: The treatment of ovarian cancer cells with CDDP boosted the expression and the nuclear translocation of IRF-1, which in turn modulated the expression of putative IRF-1 target genes. Accordingly, IRF-1 silencing re-orchestrated the expression profiles of CDDP-treated cells. In agreement with its role as a tumour suppressor, overexpressing IRF-1 suppressed the transformed phenotype of ovarian cancer cells. Nevertheless, IRF-1 silencing sensitized cells to the apoptotic death induced by CDDP. Over-expression was associated with cell G1 arrest and p21 induction irrespective of p53 proficiency, while IRF-1 silencing reduced the induction of p21 by CDDP. CONCLUSIONS: These data demonstrate that IRF-1 is up-regulated by CDDP in ovarian cancer cells and might limit the cell response to CDDP, likely by inhibiting cell proliferation. Data suggest that IRF-1 induction might interfere with the effectiveness of combination therapy with platinum drugs and cytokines.