RESUMEN
Hepatocyte growth factor (HGF) activator inhibitor type-1 (HAI-1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane-anchored serine proteases, particularly matriptase. Here, we explored the role of HAI-1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI-1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI-1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI-1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI-1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase-type plasminogen activator, which induced activation of HGF/MET signaling in the co-culture with pro-HGF-expressing fibroblasts and plasminogen-dependent plasmin generation, respectively. The enhanced plasminogen-dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI-1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor-C (VEGF-C), a lymphangiogenesis factor, into the mature form in a plasminogen-dependent manner. Furthermore, HGF significantly stimulated VEGF-C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI-1KO TSCC cells compared to control cells. Our results suggest that HAI-1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease-dependent growth factors, such as HGF and VEGF-C, in a tumor microenvironment to contribute to TSCC progression.
Asunto(s)
Carcinoma de Células Escamosas , Proteínas Inhibidoras de Proteinasas Secretoras , Neoplasias de la Lengua , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Fibrinolisina/genética , Homocigoto , Humanos , Ratones , Plasminógeno/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Eliminación de Secuencia , Serina Endopeptidasas , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/patología , Microambiente Tumoral , Factor C de Crecimiento Endotelial Vascular/genéticaRESUMEN
Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the SPINT1 gene, is a membrane-bound protease inhibitor expressed on the surface of epithelial cells. Hepatocyte growth factor activator inhibitor-1 regulates type II transmembrane serine proteases that activate protease-activated receptor-2 (PAR-2). We previously reported that deletion of Spint1 in ApcMin/+ mice resulted in accelerated formation of intestinal tumors, possibly through enhanced nuclear factor-κB signaling. In this study, we examined the role of PAR-2 in accelerating tumor formation in the ApcMin/+ model in the presence or absence of Spint1. We observed that knockout of the F2rl1 gene, encoding PAR-2, not only eliminated the enhanced formation of intestinal tumors caused by Spint1 deletion, but also reduced tumor formation in the presence of Spint1. Exacerbation of anemia and weight loss associated with HAI-1 deficiency was also normalized by compound deficiency of PAR-2. Mechanistically, signaling triggered by deregulated protease activities increased nuclear translocation of RelA/p65, vascular endothelial growth factor expression, and vascular density in ApcMin/+ -induced intestinal tumors. These results suggest that serine proteases promote intestinal carcinogenesis through activation of PAR-2, and that HAI-1 plays a critical tumor suppressor role as an inhibitor of matriptase, kallikreins, and other PAR-2 activating proteases.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias Intestinales/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Receptor PAR-2/genética , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Células Epiteliales/patología , Humanos , Neoplasias Intestinales/patología , Calicreínas/genética , Ratones , FN-kappa B/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Transducción de Señal/genética , Factor de Transcripción ReIA/genéticaRESUMEN
We had previously reported that in addition to p53 inactivation, overexpression of the DNA sensor protein-absent in melanoma 2 (AIM2)-contributes to tumorigenesis of oral squamous cell carcinoma (OSCC). Given that AIM2 is highly expressed in the OSCC tumors from patients with metastasis, we investigated whether AIM2 expression contributes to the progression of OSCC metastasis. In in vitro assays using OSCC cell lines, the high migration and invasion capacity of OSCC cells were dependent on the increased expression of AIM2, resulting in enhanced epithelial-mesenchymal transition (EMT), with EMT-related gene expression. Moreover, the in vivo short-term metastasis assay using orthotopic implantation into immunodeficient mice demonstrated that OSCC cells with high levels of AIM2 expression exhibited enhanced tumor growth in the tongue, resulting in decreased survival of the mice. Further, the cells overexpressing AIM2 dominantly invaded into the tumor lymphatic vessels, unlike OSCC cells with low AIM2 expression. Thus, the high expression of AIM2 in OSCC enhances progression of tumor growth.
Asunto(s)
Carcinoma de Células Escamosas/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca/genética , Regulación hacia Arriba , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Neoplasias de la Boca/patología , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/secundarioRESUMEN
Hepatocyte growth factor activator inhibitor type 2 (HAI-2), encoded by the SPINT2 gene, is a membrane-anchored protein that inhibits proteases involved in the activation of hepatocyte growth factor (HGF), a ligand of MET receptor. Epigenetic silencing of the SPINT2 gene has been reported in a human glioblastoma cell line (U87) and glioblastoma-derived cancer stem cells. However, the incidence of SPINT2 methylation in tumor tissues obtained from glioma patients is unknown. In this study, we analyzed the methylation status of the SPINT2 gene of eight human glioblastoma cell lines and surgically resected glioma tissues of different grades (II, III, and IV) by bisulfite sequence analysis and methylation-specific PCR. Most glioblastoma lines (7/8) showed methylation of the SPINT2 gene with a significantly reduced level of SPINT2mRNA compared to cultured astrocytes and normal brain tissues. However, all glioblastoma lines expressed mRNA for HGF activator (HGFAC), a target protease of HAI-2/SPINT2. Forced expression of SPINT2 reduced MET phosphorylation of U87 glioblastoma cells both in vitro and in intracranial xenografts in nude mice. Methylation-specific PCR analysis of the resected glioma tissues indicated notable methylation of the SPINT2 gene in 33.3% (2/6), 71.4% (10/14), and 74.3% (26/35) of grade II, III, and IV gliomas, respectively. Analysis of RNA sequencing data in a public database indicated an increased HGFAC/SPINT2 expression ratio in high-grade compared to low-grade gliomas (P = .01). In summary, aberrant methylation of the SPINT2 gene is frequently observed in high-grade gliomas and might confer MET signaling in the glioma cells.
Asunto(s)
Neoplasias Encefálicas/genética , Metilación de ADN/genética , Glioblastoma/genética , Glicoproteínas de Membrana/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Astrocitos/patología , Encéfalo/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Silenciador del Gen , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Fosforilación , Transducción de Señal/genética , Trasplante HeterólogoRESUMEN
Prostasin is a glycosylphosphatidylinositol-anchored serine protease widely expressed in epithelial cells, with crucial epidermal barrier functions. Evidence has suggested prostasin may have served as a tumor suppressor in various cancers, but its role in oral squamous cell carcinoma (OSCC) remains unclear. Thus, herein, we conducted an immunohistochemical prostasin study in 119 resected OSCC cases. Prostasin expression was decreased in 63% (75/119) of cases. OSCC with decreased prostasin immunoreactivity (low prostasin cases) tended to show a higher histological grade (p = 0.0088) and a more infiltrative cancer cell morphology (p = 0.0024). We then explored the role of prostasin in the OSCC cell lines: SAS and HSC-4. SAS did not express detectable prostasin levels, whereas HSC-4 expressed low but distinct levels. Prostasin overexpression suppressed the proliferation and migration of both OSCC lines in vitro. Conversely, prostasin silencing significantly enhanced growth rates of HSC-4. Finally, we analyzed the impact of prostasin expression on the prognosis of patients with OSCC; decreased expression tended to correlate with shorter overall survival (p = 0.0291) after resection. This trend was supported by our analyses using a public database (Kaplan-Meier plotter) of head and neck squamous cell carcinomas. In conclusion, we showed decreased prostasin expression was associated with aggressive features and a poorer prognosis of OSCC.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Expresión Génica/genética , Genes Supresores de Tumor , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Pronóstico , Serina Endopeptidasas/fisiologíaRESUMEN
Hepatocyte growth factor activator inhibitor (HAI)-1/SPINT1 and HAI-2/SPINT2 are membrane-anchored protease inhibitors having homologous Kunitz-type inhibitor domains. They regulate membrane-anchored serine proteases, such as matriptase and prostasin. Whereas HAI-1 suppresses the neoplastic progression of keratinocytes to invasive squamous cell carcinoma (SCC) through matriptase inhibition, the role of HAI-2 in keratinocytes is poorly understood. In vitro homozygous knockout of the SPINT2 gene suppressed the proliferation of two oral SCC (OSCC) lines (SAS and HSC3) but not the growth of a non-tumorigenic keratinocyte line (HaCaT). Reversion of HAI-2 abrogated the growth suppression. Matrigel invasion of both OSCC lines was also suppressed by the loss of HAI-2. The levels of prostasin protein were markedly increased in HAI-2-deficient cells, and knockdown of prostasin alleviated the HAI-2 loss-induced suppression of OSCC cell invasion. Therefore, HAI-2 has a pro-invasive role in OSCC cells through suppression of prostasin. In surgically resected OSCC tissues, HAI-2 immunoreactivity increased along with neoplastic progression, showing intense immunoreactivities in invasive OSCC cells. In summary, HAI-2 is required for invasive growth of OSCC cells and may contribute to OSCC progression.