RESUMO
Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-ß (TGF-ß) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-ß comprises three isoforms, TGF-ß1, -ß2, and -ß3, and transduces intracellular signals via TGF-ß type I receptor (TßRI) and TGF-ß type II receptor (TßRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TßRII (TßRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-ß ligands, but several lines of evidence indicate that TßRII-Fc more effectively traps TGF-ß1 and TGF-ß3 than TGF-ß2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-ß receptor containing both TßRI and TßRII (TßRI-TßRII-Fc) and found that TßRI-TßRII-Fc trapped all TGF-ß isoforms, leading to inhibition of both the TGF-ß signal and TGF-ß-induced EMT of oral cancer cells, whereas TßRII-Fc failed to trap TGF-ß2. Furthermore, we found that TßRI-TßRII-Fc suppresses tumor growth and angiogenesis more effectively than TßRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-ß expression. These results suggest that TßRI-TßRII-Fc may be a promising tool for targeting all TGF-ß isoforms in the TME.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Receptores Fc/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Carcinoma de Células Escamosas/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Receptores Fc/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Microambiente TumoralRESUMO
Clinical development of anti-angiogenic agents has been a major landmark in cancer therapy for several types of cancers. Signals mediated by both vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-9 and 10 have been implicated in tumor angiogenesis. However, previous studies have shown that targeting the individual signals was not sufficiently effective in retarding tumor growth in certain preclinical and clinical conditions. In the present study, we developed a novel decoy chimeric receptor that traps both VEGF and BMP-9/10. Single targeting of either VEGF or BMP-9/10 signals significantly reduced the formation of tumor vessels in a mouse xenograft model of human pancreatic cancer; however, it did not show significant therapeutic effects on tumor growth. In contrast, dual targeting of the angiogenic signals resulted in more significant inhibition of tumor angiogenesis, leading to delay of tumor growth. Our findings suggest that simultaneous blockade of VEGF and BMP-9/10 signals is a promising therapeutic strategy for the cancers that are resistant to anti-VEGF and BMP-9/10 therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/farmacologia , Receptores de Activinas Tipo II/uso terapêutico , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Feminino , Fator 2 de Diferenciação de Crescimento/antagonistas & inibidores , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/irrigação sanguínea , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lymphatic vessels (LVs) play critical roles in the maintenance of fluid homeostasis and in pathological conditions, including cancer metastasis. Although mutations in ALK1, a member of the transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) receptor family, have been linked to hereditary hemorrhagic telangiectasia, a human vascular disease, the roles of activin receptor-like kinase 1 (ALK-1) signals in LV formation largely remain to be elucidated. We show that ALK-1 signals inhibit LV formation, and LVs were enlarged in multiple organs in Alk1-depleted mice. These inhibitory effects of ALK-1 signaling were mediated by BMP-9, which decreased the number of cultured lymphatic endothelial cells. Bmp9-deficient mouse embryos consistently exhibited enlarged dermal LVs. BMP-9 also inhibited LV formation during inflammation and tumorigenesis. BMP-9 downregulated the expression of the transcription factor prospero-related homeobox 1, which is necessary to maintain lymphatic endothelial cell identity. Furthermore, silencing prospero-related homeobox 1 expression inhibited lymphatic endothelial cell proliferation. Our findings reveal a unique molecular basis for the physiological and pathological roles of BMP-9/ALK-1 signals in LV formation.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Vasos Linfáticos/fisiologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Peritonite/fisiopatologia , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Primers do DNA/genética , Diafragma/patologia , Perfilação da Expressão Gênica , Células HEK293 , Técnicas Histológicas , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The lymphatic system plays important roles not only in the physiological processes, such as maintenance of tissue fluid homeostasis, but also in pathological processes including the lymph node metastasis of tumor cells. Therefore, understanding of the molecular mechanisms underlying lymphatic vessel formation is crucial. Previous studies have shown that proliferation and migration of lymphatic endothelial cells (LECs) are activated by multiple types of signals mediated by tyrosine kinase receptors such as vascular endothelial growth factor receptor (VEGFR) 3. Although signals mediated by platelet-derived growth factor receptor ß (PDGFRß) have been implicated in lymphangiogenesis, the mechanisms explaining how PDGFRß expression is maintained in LECs remain to be fully elucidated. In the present study, we show that PDGFRß expression in LECs is maintained by Prox1 transcription factor. Knockdown of Prox1 expression in human dermal LECs decreased the expression of PDGFRß, leading to the lowered migration of human dermal LECs towards PDGF-BB. Furthermore, we found that PDGF signals play important roles in inflammatory lymphangiogenesis in a chronic aseptic peritonitis model. Intraperitoneal administration of imatinib, a potent inhibitor of PDGFRß, and transduction of PDGFRß/Fc chimeric protein by an adenoviral system both reduced inflammatory lymphangiogenesis induced by thioglycollate in mice. We also found that the expression of PDGFRß/Fc reduced tumor lymphangiogenesis in a BxPC3 human pancreatic cancer xenograft model. These findings suggest that PDGFRß is one of the key mediators of lymphatic vessel formation acting downstream of Prox1.
Assuntos
Proteínas de Homeodomínio/fisiologia , Vasos Linfáticos/fisiopatologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Antineoplásicos/farmacologia , Becaplermina , Benzamidas/farmacologia , Linhagem Celular Tumoral , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Mesilato de Imatinib , Linfangiogênese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-sis/fisiologia , Pirimidinas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de SinaisRESUMO
The tumor microenvironment contains various components, including cancer cells, tumor vessels, and cancer-associated fibroblasts, the latter of which are comprised of tumor-promoting myofibroblasts and tumor-suppressing fibroblasts. Multiple lines of evidence indicate that transforming growth factor-ß (TGF-ß) induces the formation of myofibroblasts and other types of mesenchymal (non-myofibroblastic) cells from endothelial cells. Recent reports show that fibroblast growth factor 2 (FGF2) modulates TGF-ß-induced mesenchymal transition of endothelial cells, but the molecular mechanisms behind the signals that control transcriptional networks during the formation of different groups of fibroblasts remain largely unclear. Here, we studied the roles of FGF2 during the regulation of TGF-ß-induced mesenchymal transition of tumor endothelial cells (TECs). We demonstrated that auto/paracrine FGF signals in TECs inhibit TGF-ß-induced endothelial-to-myofibroblast transition (End-MyoT), leading to suppressed formation of contractile myofibroblast cells, but on the other hand can also collaborate with TGF-ß in promoting the formation of active fibroblastic cells which have migratory and proliferative properties. FGF2 modulated TGF-ß-induced formation of myofibroblastic and non-myofibroblastic cells from TECs via transcriptional regulation of various mesenchymal markers and growth factors. Furthermore, we observed that TECs treated with TGF-ß were more competent in promoting in vivo tumor growth than TECs treated with TGF-ß and FGF2. Mechanistically, we showed that Elk1 mediated FGF2-induced inhibition of End-MyoT via inhibition of TGF-ß-induced transcriptional activation of α-smooth muscle actin promoter by myocardin-related transcription factor-A. Our data suggest that TGF-ß and FGF2 oppose and cooperate with each other during the formation of myofibroblastic and non-myofibroblastic cells from TECs, which in turn determines the characteristics of mesenchymal cells in the tumor microenvironment.
Assuntos
Células Endoteliais/patologia , Fatores de Crescimento de Fibroblastos/metabolismo , Miofibroblastos/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Antiestrogen agents are commonly used to treat patients with estrogen receptor (ER)-positive breast cancer. Tamoxifen has been the mainstay of endocrine treatment for patients with early and advanced breast cancer for many years. Following tamoxifen treatment failure, however, there are still limited options for subsequent hormonal therapy. We discovered a novel compound, NK150460, that inhibits 17ß-estradiol (E2)-dependent transcription without affecting binding of E2 to ER. Against our expectations, NK150460 inhibited growth of not only most ER-positive, but also some ER-negative breast cancer cell lines, while never inhibiting growth of non-breast cancer cell lines. Cell-based screening using a random shRNA library, identified aryl hydrocarbon receptor nuclear translocator (ARNT) as a key gene involved in NK150460's antitumor mechanism. siRNAs against not only ARNT but also its counterpart aryl hydrocarbon receptor (AhR) and their target protein, CYP1A1, dramatically abrogated NK150460's growth-inhibitory activity. This suggests that the molecular cascade of AhR/ARNT plays an essential role in NK150460's antitumor mechanism. Expression of ERα was decreased by NK150460 treatment, and this was inhibited by an AhR antagonist. Unlike two other AhR agonists now undergoing clinical developmental stage, NK150460 did not induce histone H2AX phosphorylation or p53 expression, suggesting that it did not induce a DNA damage response in treated cells. Cell lines expressing epithelial markers were more sensitive to NK150460 than mesenchymal marker-expressing cells. These data indicate that NK150460 is a novel AhR agonist with selective antitumor activity against breast cancer cell lines, and its features differ from those of the other two AhR agonists.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Quinolinas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Estrogênios/farmacologia , Feminino , Compostos Heterocíclicos com 2 Anéis/química , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Camundongos Nus , Quinolinas/química , Ratos Nus , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Endothelial-mesenchymal transition (EndMT) plays important roles in various physiological and pathological processes. While signals mediated by transforming growth factor (TGF)-ß have been implicated in EndMT, the molecular mechanisms underlying it remain to be fully elucidated. Here, we examined the effects of TGF-ß signals on the EndMT of mouse pancreatic microvascular endothelial cells (MS-1). By addition of TGF-ß2, MS-1 cells underwent mesenchymal transition characterized by re-organization of actin stress fibre and increased expression of various mesenchymal markers such as α-smooth muscle actin (α-SMA) through activation of Rho signals. Whereas activation of Rho signals via TGF-ß-induced non-Smad signals has been implicated in epithelial-mesenchymal transition (EMT), we found that Arhgef5, a guanine nucleotide exchange factor, is induced by Smad signals and contributes to the TGF-ß2-induced α-SMA expression in MS-1 cells. We also found that TGF-ß2 induces the expression of myocardin-related transcription factor-A (MRTF-A) in a Smad-dependent fashion and its nuclear accumulation in MS-1 cells and that MRTF-A is required and sufficient for TGF-ß2-induced α-SMA expression. These results indicate that activation of Smad signals by TGF-ß2 have dual effects on the activation of Rho signals and MRTF-A leading to the mesenchymal transition of MS-1 endothelial cells.