RESUMO
Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , DNA Metiltransferase 3A , Epigênese Genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismoRESUMO
Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma.
Assuntos
Receptores ErbB/genética , Glioblastoma/genética , Proteína HMGA2/genética , Proteínas de Fusão Oncogênica/genética , Idoso , Animais , Linhagem Celular , Linhagem Celular Tumoral , Éxons/genética , Feminino , Amplificação de Genes/genética , Deleção de Genes , Glioma/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação/genética , Transdução de Sinais/genéticaRESUMO
Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses αvß5 integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma de Células Escamosas/tratamento farmacológico , Cisplatino/administração & dosagem , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Peptídeos Cíclicos/química , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Micelas , Nanopartículas , Células-Tronco Neoplásicas/metabolismo , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Angiogenesis is a multi-step process that culminates in vascular maturation whereby nascent vessels stabilize to become functional, and mural cells play an essential role in this process. Recent studies have shown that mural cells in tumors also promote and maintain vascular integrity, with wide-reaching clinical implications including the regulation of tumor growth, metastases, and drug delivery. Various regulatory signaling pathways have been hitherto implicated, but whether regulation of Fas-dependent apoptotic mechanisms is involved has not yet been fully investigated. We first compared endothelial FAS staining in human pancreatic ductal adenocarcinomas and colon carcinomas and show that the latter, characterized by lower mural cell coverage of tumor vasculature, demonstrated higher expression of FAS than the former. Next, in an in vitro coculture system of MS-1 and 10T1/2 cells as endothelial and mural cells respectively, we show that mural cells decreased endothelial Fas expression. Then, in an in vivo model in which C26 colon carcinoma cells were inoculated together with MS-1 cells alone or with the further addition of 10T1/2 cells, we demonstrate that mural cells prevented hemorrhage. Finally, knockdown of endothelial Fas sufficiently recapitulated the protection against hemorrhage seen with the addition of mural cells. These results together suggest that regulation of endothelial Fas signaling is involved in the promotion of vascular integrity by mural cells in tumors.
Assuntos
Células Endoteliais/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Receptor fas/metabolismo , Animais , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/fisiologiaRESUMO
Pancreatic cancer is notorious for its poor prognosis. The histopathologic characteristic of pancreatic ductal adenocarcinoma (PDAC), which is the most common type of pancreatic cancer, is fibrosis within tumor tissue. Although fibrosis within tumor tissue is thought to impede drug therapy by interfering with the intratumoral accumulation of anti-tumor drugs, this hypothesis has yet to be proven directly in preclinical models. Here, we evaluated the effect of enhanced fibrosis on intratumoral accumulation of macromolecular drugs by increasing fibrosis in a murine tumor model of subcutaneously xenografted BxPC-3, a human PDAC cell line. When fibroblast growth factor-2 (FGF-2) was co-administered upon BxPC-3 inoculation, stromal fibrotic area was increased and was characterized by augmented murine collagen accumulation compared to inoculation of BxPC-3 alone, which correlated with increased monocyte/macrophage contents in the tumor tissues. We further discovered that the intratumoral accumulation of intravenously administrated fluorescein isothiocyanate-dextran of 2,000,000Da (2MDa) was significantly reduced in the FGF-2 co-administered tumors despite unaltered hyaluronan accumulation and pericyte coverage of the tumor neovasculature and increased lymphangiogenesis. Finally, we found that FGF-2 co-administered tumors are more refractory to macromolecular drug therapy using nab-paclitaxel (Abraxane). The model established and analyzed in this study, characterized by increased fibrotic component, provides a preclinical animal model suited to predict the intratumoral accumulation of macromolecular drugs and to evaluate the efficacy of drugs targeting the tumor stroma.
Assuntos
Paclitaxel Ligado a Albumina/administração & dosagem , Antineoplásicos/administração & dosagem , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Paclitaxel Ligado a Albumina/farmacocinética , Paclitaxel Ligado a Albumina/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Colágeno Tipo I/genética , Dextranos/farmacocinética , Modelos Animais de Doenças , Fibrose , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Corantes Fluorescentes/farmacocinética , Humanos , Hidroxiprolina/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Overexpression of periostin (POSTN), an extracellular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II-IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn(-/-) mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn(-/-) mice. These findings suggest that POSTN produced by cancer-associated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias Gástricas/patologia , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Crescimento Transformador beta/metabolismo , Vimentina/metabolismoRESUMO
Drug release rate is an important factor in determining efficacy and toxicity of nanoscale drug delivery systems. However, optimization of the release rate in polymeric micellar nanoscale drug delivery systems has not been fully investigated. In this study NC-6301, a poly(ethylene glycol)-poly(aspartate) block copolymer with docetaxel (DTX) covalently bound via ester link, was synthesized with various numbers of DTX molecules bound to the polymer backbone. The number of DTX molecules was determined up to 14 to achieve an optimal release rate, based upon the authors' own pharmacokinetic model using known patient data. Efficacy and toxicity of the formulation was then tested in animals. When administered three times at 4-day intervals, the maximum tolerated doses of NC-6301 and native DTX were 50 and 10 mg/kg, respectively, in nude mice. Tissue distribution studies of NC-6301 in mice at 50 mg/kg revealed prolonged release of free DTX in the tumor for at least 120 hours, thus supporting its effectiveness. Furthermore, in cynomolgus monkeys, NC-6301 at 6 mg/kg three times at 2-week intervals showed marginal toxicity, whereas native DTX, at 3 mg/kg with the same schedule, induced significant decrease of food consumption and neutrophil count. NC-6301 at 50 mg/kg in mice also regressed a xenografted tumor of MDA-MB-231 human breast cancer. Native DTX, on the other hand, produced only transient and slight regression of the same tumor xenograft. NC-6301 also significantly inhibited growth of OCUM-2MLN human scirrhous gastric carcinoma in an orthotopic mouse model. Total weight of metastatic lymph nodes was also reduced. In conclusion, NC-6301 with an optimized release rate improved the potency of DTX while reducing its toxicity.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Micelas , Peptídeos/administração & dosagem , Peptídeos/química , Taxoides/administração & dosagem , Taxoides/química , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Docetaxel , Sistemas de Liberação de Medicamentos , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Nus , Análise Multivariada , Nanopartículas , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Tamanho da Partícula , Polietilenoglicóis , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. Although transforming growth factor-ß (TGF-ß) and pro-inflammatory cytokines have been implicated in EMT, the underlying molecular mechanisms remain to be elucidated. Here, we studied the effects of proinflammatory cytokines derived from the mouse macrophage cell line RAW 264.7 on TGF-ß-induced EMT in A549 lung cancer cells. Co-culture and treatment with conditioned medium of RAW 264.7 cells enhanced a subset of TGF-ß-induced EMT phenotypes in A549 cells, including changes in cell morphology and induction of mesenchymal marker expression. These effects were increased by the treatment of RAW 264.7 cells with lipopolysaccharide, which also induced the expression of various proinflammatory cytokines, including TNF-α and IL-1ß. The effects of conditioned medium of RAW 264.7 cells were partially inhibited by a TNF-α neutralizing antibody. Dehydroxy methyl epoxyquinomicin, a selective inhibitor of NFκB, partially inhibited the enhancement of fibronectin expression by TGF-ß, TNF-α, and IL-1ß, but not of N-cadherin expression. Effects of other pharmacological inhibitors also suggested complex regulatory mechanisms of the TGF-ß-induced EMT phenotype by TNF-α stimulation. These findings provide direct evidence of the effects of RAW 264.7-derived TNF-α on TGF-ß-induced EMT in A549 cells, which is transduced in part by NFκB signalling.
Assuntos
Citocinas/metabolismo , Transição Epitelial-Mesenquimal , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Humanos , Inflamação/metabolismo , Neoplasias Pulmonares , Macrófagos/imunologia , Camundongos , Transdução de SinaisRESUMO
Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.
Assuntos
Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Peritonite/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-ets-2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Células Endoteliais/imunologia , Células Endoteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Inflamação , Linfangiogênese/genética , Linfangiogênese/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Peritonite/induzido quimicamente , Peritonite/genética , Peritonite/fisiopatologia , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-2/genética , RNA Interferente Pequeno/genética , Tioglicolatos/administração & dosagem , Proteínas Supressoras de Tumor/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lysophosphatidic acid (LPA) is a potent lipid mediator with a wide variety of biological actions mediated through G protein-coupled receptors (LPA(1-6)). LPA(4) has been identified as a G(13) protein-coupled receptor, but its physiological role is unknown. Here we show that a subset of LPA(4)-deficient embryos did not survive gestation and displayed hemorrhages and/or edema in many organs at multiple embryonic stages. The blood vessels of bleeding LPA(4)-deficient embryos were often dilated. The recruitment of mural cells, namely smooth muscle cells and pericytes, was impaired. Consistently, Matrigel plug assays showed decreased mural cell coverage of endothelial cells in the neovessels of LPA(4)-deficient adult mice. In situ hybridization detected Lpa4 mRNA in the endothelium of some vasculatures. Similarly, the lymphatic vessels of edematous embryos were dilated. These results suggest that LPA(4) regulates establishment of the structure and function of blood and lymphatic vessels during mouse embryogenesis. Considering the critical role of autotaxin (an enzyme involved in LPA production) and Gα(13) in vascular development, we suggest that LPA(4) provides a link between these 2 molecules.
Assuntos
Vasos Sanguíneos/embriologia , Desenvolvimento Embrionário/fisiologia , Vasos Linfáticos/embriologia , Receptores Purinérgicos/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Northern Blotting , Southern Blotting , Feminino , Imuno-Histoquímica , Hibridização In Situ , Vasos Linfáticos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Diffuse-type gastric carcinoma is characterized by rapid progression and poor prognosis. High expression of transforming growth factor (TGF)-ß and thick stromal fibrosis are observed in this type of gastric carcinoma. We have previously shown that disruption of TGF-ß signaling via introduction of a dominant negative form of the TGF-ß type II receptor (dnTßRII) into diffuse-type gastric cancer cell lines, including OCUM-2MLN, caused accelerated tumor growth through induction of tumor angiogenesis in vivo. In the present study, we show that TGF-ß induces upregulation of expression of tissue inhibitor of metalloproteinase 2 (TIMP2) in the OCUM-2MLN cell line in vitro, and that expression of TIMP2 is repressed by dnTßRII expression in vivo. Transplantation of the OCUM-2MLN cells to nude mice exhibited accelerated tumor growth in response to dnTßRII expression, which was completely abolished when TIMP2 was coexpressed with dnTßRII. Although the blood vessel density of TIMP2-expressing tumors was only slightly decreased, the degree of hypoxia in tumor tissues was significantly increased and pericytes covering tumor vasculature were decreased by TIMP2 expression in OCUM-2MLN cells, suggesting that the function of tumor vasculatures was repressed by TIMP2 and consequently tumor growth was reduced. These findings provide evidence that one of the mechanisms of the increase in angiogenesis in diffuse-type gastric carcinoma is the downregulation of the anti-angiogenic protein TIMP2.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Neoplasias Gástricas/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipóxia , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Inibidor Tecidual de Metaloproteinase-2/genética , Fator de Crescimento Transformador beta/farmacologia , Transplante HeterólogoRESUMO
The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-kappaB) family; however, the role of NF-kappaB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kappaB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-kappaB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kappaB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kappaB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kappaB pathway by inflammatory stimuli activates Prox1, and both NF-kappaB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.
Assuntos
Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Neovascularização Fisiológica , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Feminino , Proteínas de Homeodomínio/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Subunidade p50 de NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Ratos , Fatores de Tempo , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.
Assuntos
Angiopoietina-2/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/metabolismo , Linfangiogênese , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Angiopoietina-2/genética , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Endotélio Linfático/citologia , Endotélio Linfático/embriologia , Endotélio Linfático/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Fatores de Transcrição Forkhead/genética , Proteínas de Homeodomínio/genética , Humanos , Vasos Linfáticos/citologia , Vasos Linfáticos/embriologia , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
c-Ski, originally identified as a proto-oncogene product, is an important negative regulator of transforming growth factor (TGF)-beta family signaling through interaction with Smad2, Smad3, and Smad4. High expression of c-Ski has been found in some cancers, including gastric cancer. We previously showed that disruption of TGF-beta signaling by dominant-negative TGF-beta type II receptor in a diffuse-type gastric carcinoma model accelerated tumor growth through induction of tumor angiogenesis by decreased expression of the anti-angiogenic factor thrombospondin (TSP)-1. Here, we examined the function of c-Ski in human diffuse-type gastric carcinoma OCUM-2MLN cells. Overexpression of c-Ski inhibited TGF-beta signaling in OCUM-2MLN cells. Interestingly, c-Ski overexpression resulted in extensive acceleration of the growth of subcutaneous xenografts in BALB/c nu/nu female mice (6 weeks of age). Similar to tumors expressing dominant-negative TGF-beta type II receptor, histochemical studies revealed less fibrosis and increased angiogenesis in xenografted tumors expressing c-Ski compared to control tumors. Induction of TSP-1 mRNA by TGF-beta was attenuated by c-Ski in vitro, and expression of TSP-1 mRNA was decreased in tumors expressing c-Ski in vivo. These findings suggest that c-Ski overexpression promotes the growth of diffuse-type gastric carcinoma through induction of angiogenesis.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Ligação a DNA/biossíntese , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Transdução de Sinais/fisiologia , Neoplasias Gástricas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Trombospondina 1/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Diffuse-type gastric carcinoma is a cancer with poor prognosis that has high levels of transforming growth factor beta (TGF-beta) expression and thick stromal fibrosis. However, the association of TGF-beta signaling with diffuse-type gastric carcinoma has not been investigated in detail. METHODS: We used a lentiviral infection system to express a dominant-negative TGF-beta type II receptor (dnTbetaRII) or green fluorescent protein (GFP) as a control in the diffuse-type gastric carcinoma cell lines, OCUM-2MLN and OCUM-12. These infected cells and the corresponding parental control cells were subcutaneously or orthotopically injected into nude mice. Angiogenesis was inhibited by infecting cells with a lentivirus carrying the gene for angiogenic inhibitor thrombospondin-1 or by injecting mice intraperitoneally with the small-molecule angiogenic inhibitor sorafenib or with anti-vascular endothelial growth factor (VEGF) neutralizing antibody (six or eight mice per group). Expression of phospho-Smad2 and thrombospondin-1 was investigated immunologically in human gastric carcinoma tissues from 102 patients. All statistical tests were two-sided. RESULTS: Expression of dnTbetaRII into OCUM-2MLN cells did not affect their proliferation in vitro, but it accelerated the growth of subcutaneously or orthotopically transplanted tumors in vivo (eg, for mean volume of subcutaneous tumors on day 10 relative to that on day 0: dnTbetaRII tumors = 3.49 and GFP tumors = 2.46, difference = 1.02, 95% confidence interval [CI] = 0.21 to 1.84; P = .003). The tumors expressing dnTbetaRII had higher levels of angiogenesis than those expressing GFP because of decreased thrombospondin-1 production. Similar results were obtained with OCUM-12 cells. Expression of thrombospondin-1 in the dnTbetaRII tumor or treatment with sorafenib or anti-VEGF antibody reduced tumor growth, whereas knockdown of thrombospondin-1 expression resulted in more accelerated growth of OCUM-2MLN tumors than of GFP tumors (eg, mean tumor volumes on day 14 relative to those on day 0: thrombospondin-1-knockdown tumors = 4.91 and GFP tumors = 3.79, difference = 1.12, 95% CI = 0.80 to 1.44; P < .001). Positive association between phosphorylated Smad2 and thrombospondin-1 immunostaining was observed in human gastric carcinoma tissues. CONCLUSIONS: Disruption of TGF-beta signaling in diffuse-type gastric carcinoma models appeared to accelerate tumor growth, apparently through increased tumor angiogenesis that was induced by decreased expression of thrombospondin-1.
Assuntos
Biomarcadores Tumorais/metabolismo , Neovascularização Patológica/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Infecções por Lentivirus , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Niacinamida/análogos & derivados , Análise de Sequência com Séries de Oligonucleotídeos , Compostos de Fenilureia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , RNA Neoplásico/isolamento & purificação , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Neoplasias Gástricas/irrigação sanguínea , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
There are a number of kinase inhibitors that regulate components of the neovasculature. We previously reported the use of transforming growth factor (TGF)-beta inhibitor on neovasculature in stroma-rich tumor models to increase the intratumoral distribution of nanoparticles. Here, we compared the effects of two other kinase inhibitors, imatinib and sorafenib, with TGF-beta inhibitor (LY364947) on extravasation of a modeled nanoparticle, 2 MDa dextran. We first used a mouse model of neoangiogenesis, the Matrigel plug assay, to compare neovasculature formed inside of and around Matrigel plugs (intraplug and periplug regions, respectively). Intraplug vasculature was more strongly pericyte covered, whereas periplug vasculature was less covered. In this model, TGF-beta inhibitor exhibited the most potent effect on intraplug vasculature in increasing the extravasation of dextran, whereas sorafenib had the strongest effect on periplug vasculature. Although imatinib and TGF-beta inhibitor each reduced pericyte coverage, imatinib also reduced the density of endothelium, resulting in a decrease in overall delivery of nanoparticles. These findings were confirmed in two tumor models, the CT26 colon cancer model and the BxPC3 pancreatic cancer model. The vasculature phenotype in the CT26 model resembled that in the periplug region, whereas the latter resembled that in the intraplug region. Consistent with this, sorafenib most potently enhanced the accumulation of nanoparticles in the CT26 model, whereas TGF-beta inhibitor did in the BxPC3 model. In conclusion, the appropriate strategy for optimization of tumor vasculature for nanoparticles may differ depending on tumor type, and in particular on the degree of pericyte coverage around the vasculature.
Assuntos
Benzenossulfonatos/farmacologia , Nanopartículas , Neoplasias/irrigação sanguínea , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Benzamidas , Linhagem Celular Tumoral , Extravasamento de Materiais Terapêuticos e Diagnósticos , Humanos , Mesilato de Imatinib , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/análogos & derivados , Compostos de Fenilureia , Transdução de Sinais , Sorafenibe , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Lymphangiogenesis is induced by various growth factors, including VEGF-C. Although TGF-beta plays crucial roles in angiogenesis, the roles of TGF-beta signaling in lymphangiogenesis are unknown. We show here that TGF-beta transduced signals in human dermal lymphatic microvascular endothelial cells (HDLECs) and inhibited the proliferation, cord formation, and migration toward VEGF-C of HDLECs. Expression of lymphatic endothelial cell (LEC) markers, including LYVE-1 and Prox1 in HDLECs, as well as early lymph vessel development in mouse embryonic stem cells in the presence of VEGF-A and C, were repressed by TGF-beta but were induced by TGF-beta type I receptor (TbetaR-I) inhibitor. Moreover, inhibition of endogenous TGF-beta signaling by TbetaR-I inhibitor accelerated lymphangiogenesis in a mouse model of chronic peritonitis. Lymphangiogenesis was also induced by TbetaR-I inhibitor in the presence of VEGF-C in pancreatic adenocarcinoma xenograft models inoculated in nude mice. These findings suggest that TGF-beta transduces signals in LECs and plays an important role in the regulation of lymphangiogenesis in vivo.
Assuntos
Linfangiogênese , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Linhagem Celular , Modelos Animais de Doenças , Camundongos , Camundongos Nus , Neoplasias Pancreáticas , Peritonite , Receptor do Fator de Crescimento Transformador beta Tipo I , Fator C de Crescimento do Endotélio VascularRESUMO
Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Etodolac/farmacologia , Linfangiogênese/efeitos dos fármacos , Metástase Linfática/prevenção & controle , Animais , Ciclo-Oxigenase 2/análise , Humanos , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Peritonite/fisiopatologia , Neoplasias Gástricas/patologia , Fator C de Crescimento do Endotélio Vascular/análise , Fator D de Crescimento do Endotélio Vascular/análiseRESUMO
Transforming growth factor (TGF)-beta plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-beta inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-beta signaling inhibition, including the induction of cancers by the repression of TGF-beta-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-beta type I receptor (TbetaR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TbetaR-I inhibitor altered neither TGF-beta signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TbetaR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TbetaR-I inhibitor. The use of TbetaR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Endotélio Vascular/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta , Fator de Crescimento Transformador beta/metabolismoRESUMO
Combined stimulation with VEGF-A, FGF-2, or PDGF-BB has emerged as a potent strategy for therapeutic angiogenesis, although the mechanisms underlying the synergism of these factors are not well understood. In the present study, we investigated the mechanism of synergism between VEGF-A and FGF-2 by using Matrigel plug assay in vivo and embryonic stem cell (ESC)-derived VEGF receptor 2 (VEGFR2)-positive cells in vitro. Experiments in vitro revealed that, in addition to having direct mitogenic effects, these molecules enhance intercellular PDGF-B signaling in a cell-type specific manner: VEGF-A enhances endothelial PDGF-B expression, whereas FGF-2 enhances mural PDGF receptor beta (PDGFRbeta) expression. Co-stimulation with VEGF-A and FGF-2 caused significant mural cell recruitment in vitro and formation of functional neovasculature in vivo, compared with single-agent stimulation. These effects were abrogated not only by anti-PDGFRbeta neutralizing antibody, but also by exogenous PDGF-BB, which could overwhelm the endogenous PDGF-BB distribution. These findings indicated the importance of preservation of the periendothelial PDGF-BB gradient. Thus, we demonstrated that the directional enhancement of endogenous PDGF-B-PDGFRbeta signaling is indispensable for the synergistic effect of VEGF-A and FGF-2 on neoangiogenesis in adults. The findings provide insights into the mechanisms underlying the effects of co-stimulation by growth factors, which could lead to rational design of therapeutic angiogenic strategies.