RESUMEN
Deregulation of matriptase is a consistent feature of human epithelial cancers and correlates with poor disease outcome. We have previously shown that matriptase promotes multi-stage squamous cell carcinogenesis in transgenic mice through dual activation of pro-hepatocyte growth factor-cMet-Akt-mTor proliferation/survival signaling and PAR-2-Gαi-NFκB inflammatory signaling. Matriptase was congenitally and constitutively deregulated in our prior studies, and therefore it was unclear if aberrant matriptase signaling supports only initiation of tumor formation or if it is also critical for the progression of established tumors. To determine this, we here have generated triple-transgenic mice with constitutive deregulation of matriptase and simultaneous inducible expression of the cognate matriptase inhibitor, hepatocyte growth factor inhibitor (HAI)-2. As expected, constitutive expression of HAI-2 suppressed the formation of matriptase-dependent tumors in 7,12-Dimethylbenz(a)anthracene-treated mouse skin. Interestingly, however, the induction of HAI-2 expression in already established tumors markedly impaired malignant progression and caused regression of individual tumors. Tumor regression correlated with reduced accumulation of tumor-associated inflammatory cells, likely caused by diminished expression of pro-tumorigenic inflammatory cytokines. The data suggest that matriptase-dependent signaling may be a therapeutic target for both squamous cell carcinoma chemoprevention and for the treatment of established tumors.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Serina Endopeptidasas/fisiología , Neoplasias Cutáneas/enzimología , Animales , Carcinogénesis/inmunología , Carcinogénesis/metabolismo , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patologíaRESUMEN
The membrane-anchored serine protease, matriptase, is consistently dysregulated in a range of human carcinomas, and high matriptase activity correlates with poor prognosis. Furthermore, matriptase is unique among tumor-associated proteases in that epithelial stem cell expression of the protease suffices to induce malignant transformation. Here, we use genetic epistasis analysis to identify proteinase-activated receptor (PAR)-2-dependent inflammatory signaling as an essential component of matriptase-mediated oncogenesis. In cell-based assays, matriptase was a potent activator of PAR-2, and PAR-2 activation by matriptase caused robust induction of nuclear factor (NF)κB through Gαi. Importantly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced pre-malignant progression, including inflammatory cytokine production, inflammatory cell recruitment, epidermal hyperplasia and dermal fibrosis. Selective ablation of PAR-2 from bone marrow-derived cells did not prevent matriptase-driven pre-malignant progression, indicating that matriptase activates keratinocyte stem cell PAR-2 to elicit its pro-inflammatory and pro-tumorigenic effects. When combined with previous studies, our data suggest that dual induction of PAR-2-NFκB inflammatory signaling and PI3K-Akt-mTor survival/proliferative signaling underlies the transforming potential of matriptase and may contribute to pro-tumorigenic signaling in human epithelial carcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas ras/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular , Transformación Celular Neoplásica/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Progresión de la Enfermedad , Células Epiteliales/patología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Receptor PAR-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Proteínas ras/genéticaRESUMEN
OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) progression and metastasis have previously been associated with the activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) and Wnt signalling pathways, which lead to the activation of pro-proliferative genes, such as cyclin D1. The current study aims to investigate whether there is a crosstalk between these pathways in HNSCC and which pathway is more likely to regulate cyclin D1. MATERIAL AND METHODS: Two HNSCC and a control keratinocyte cell lines were treated with EGF and wortmannin to respectively activate and block the PI3K-Akt and Wnt pathways. Partial and total levels of cyclin D1, beta-catenin and Akt were evaluated by Western blotting and immunofluorescence. Twenty-four paraffin-embedded samples of human HNSCC, as well as normal oral mucosa biopsies, were also immunohistochemically evaluated for beta-catenin and cyclin D1 expression. RESULTS: Following both treatments, change in cyclin D1 protein was correlated with Akt levels only. Cytoplasmic staining for beta-catenin and loss of its membranous expression in the HNSCC invasive areas were found in 92% of the HNSCC biopsies. CONCLUSION: Taken together, we show that the change in cyclin D1 levels is more likely to be due to the EGFR-Akt pathway activation than due to beta-catenin nuclear translocation.
