RESUMEN
Notch 1/2 genes play tumor-suppressing functions in squamous cell carcinoma (SCC), a very common malignancy in skin and internal organs. In contrast with Notch, we show that the transcription factor CSL (also known as RBP-Jκ), a key effector of canonical Notch signaling endowed with intrinsic transcription-repressive functions, plays a tumor-promoting function in SCC development. Expression of this gene decreased in upper epidermal layers and human keratinocytes (HKCs) undergoing differentiation, while it increased in premalignant and malignant SCC lesions from skin, head/neck, and lung. Increased CSL levels enhanced the proliferative potential of HKCs and SCC cells, while silencing of CSL induced growth arrest and apoptosis. In vivo, SCC cells with increased CSL levels gave rise to rapidly expanding tumors, while cells with silenced CSL formed smaller and more differentiated tumors with enhanced inflammatory infiltrate. Global transcriptomic analysis of HKCs and SCC cells with silenced CSL revealed major modulation of apoptotic, cell-cycle, and proinflammatory genes. We also show that the histone demethylase KDM6B is a direct CSL-negative target, with inverse roles of CSL in HKC and SCC proliferative capacity, tumorigenesis, and tumor-associated inflammatory reaction. CSL/KDM6B protein expression could be used as a biomarker of SCC development and indicator of cancer treatment.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/metabolismo , Transducción de Señal , Animales , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Histona Demetilasas con Dominio de Jumonji/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation-stromal co-evolution model under convergent CSL-p53 control.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Fibroblastos/fisiología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Neoplasias Cutáneas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Senescencia Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Ratones , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of ß-catenin, α-catenin and ZO-2, it decreased nuclear levels of ß-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, ß-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, ß-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering ß-catenin away from nucleus.