RESUMEN
Hyperactivation of the Wingless-type (Wnt)/ß-catenin pathway promotes tumor initiation, tumor growth and metastasis in various tissues. Although there is evidence for the involvement of Wnt/ß-catenin pathway activation in salivary gland tumors, the precise mechanisms are unknown. Here we report for the first time that downregulation of the Wnt inhibitory factor 1 (WIF1) is a widespread event in salivary gland carcinoma ex-pleomorphic adenoma (CaExPA). We also show that WIF1 downregulation occurs in the CaExPA precursor lesion pleomorphic adenoma (PA) and indicates a higher risk of progression from benign to malignant tumor. Our results demonstrate that diverse mechanisms including WIF1 promoter hypermethylation and loss of heterozygosity contribute to WIF1 downregulation in human salivary gland tumors. In accordance with a crucial role in suppressing salivary gland tumor progression, WIF1 re-expression in salivary gland tumor cells inhibited cell proliferation, induced more differentiated phenotype and promoted cellular senescence, possibly through upregulation of tumor-suppressor genes, such as p53 and p21. Most importantly, WIF1 significantly diminished the number of salivary gland cancer stem cells and the anchorage-independent cell growth. Consistent with this observation, WIF1 caused a reduction in the expression of pluripotency and stemness markers (OCT4 and c-MYC), as well as adult stem cell self-renewal and multi-lineage differentiation markers, such as WNT3A, TCF4, c-KIT and MYB. Furthermore, WIF1 significantly increased the expression of microRNAs pri-let-7a and pri-miR-200c, negative regulators of stemness and cancer progression. In addition, we show that WIF1 functions as a positive regulator of miR-200c, leading to downregulation of BMI1, ZEB1 and ZEB2, with a consequent increase in downstream targets such as E-cadherin. Our study emphasizes the prognostic and therapeutic potential of WIF1 in human salivary gland CaExPA. Moreover, our findings demonstrate a novel mechanism by which WIF1 regulates cancer stemness and senescence, which might have major implications in the field of cancer biology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenoma Pleomórfico/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma/metabolismo , Senescencia Celular , Células Madre Neoplásicas/metabolismo , Proteínas Represoras/metabolismo , Neoplasias de las Glándulas Salivales/metabolismo , Vía de Señalización Wnt , Proteínas Adaptadoras Transductoras de Señales/genética , Adenoma Pleomórfico/genética , Adenoma Pleomórfico/patología , Biomarcadores de Tumor/genética , Carcinoma/genética , Carcinoma/patología , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Progresión de la Enfermedad , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , MicroARNs/metabolismo , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/patología , Factores de Tiempo , TransfecciónRESUMEN
RNA-binding proteins play a key role in post-transcriptional regulation of mRNA stability and translation. We have identified that RBM3, a translation regulatory protein, is significantly upregulated in human tumors, including a stage-dependent increase in colorectal tumors. Forced RBM3 overexpression in NIH3T3 mouse fibroblasts and SW480 human colon epithelial cells increases cell proliferation and development of compact multicellular spheroids in soft agar suggesting the ability to induce anchorage-independent growth. In contrast, downregulating RBM3 in HCT116 colon cancer cells with specific siRNA decreases cell growth in culture, which was partially overcome when treated with prostaglandin E(2), a product of cyclooxygenase (COX)-2 enzyme activity. Knockdown also resulted in the growth arrest of tumor xenografts. We have also identified that RBM3 knockdown increases caspase-mediated apoptosis coupled with nuclear cyclin B1, and phosphorylated Cdc25c, Chk1 and Chk2 kinases, implying that under conditions of RBM3 downregulation, cells undergo mitotic catastrophe. RBM3 enhances COX-2, IL-8 and VEGF mRNA stability and translation. Conversely, RBM3 knockdown results in loss in the translation of these transcripts. These data demonstrate that the RNA stabilizing and translation regulatory protein RBM3 is a novel proto-oncogene that induces transformation when overexpressed and is essential for cells to progress through mitosis.