RESUMEN
Cancer cells universally increase glucose and glutamine consumption, leading to the altered metabolic state known as the Warburg effect; one metabolic pathway, highly dependent on glucose and glutamine, is the hexosamine biosynthetic pathway. Increased flux through the hexosamine biosynthetic pathway leads to increases in the post-translational addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to various nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer, and recently, O-GlcNAcylation was shown to play a role in breast cancer; however, O-GlcNAcylation in other cancers remains poorly defined. Here, we show that O-GlcNAc transferase (OGT) is overexpressed in prostate cancer compared with normal prostate epithelium and that OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines. Reducing O-GlcNAcylation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF, resulting in inhibition of invasion and angiogenesis. OGT-mediated regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, a key regulator of invasion and angiogenesis, as reducing OGT expression led to increased FoxM1 protein degradation. Conversely, overexpression of a degradation-resistant FoxM1 mutant abrogated OGT RNAi-mediated effects on invasion, MMP levels, angiogenesis, and VEGF expression. Using a mouse model of metastasis, we found that reduction of OGT expression blocked bone metastasis. Altogether, these data suggest that as prostate cancer cells alter glucose and glutamine levels, O-GlcNAc modifications and OGT levels become elevated and are required for regulation of malignant properties, implicating OGT as a novel therapeutic target in the treatment of cancer.
Asunto(s)
N-Acetilglucosaminiltransferasas/metabolismo , Neovascularización Patológica/enzimología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Acetilglucosamina/metabolismo , Animales , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Mutación , N-Acetilglucosaminiltransferasas/deficiencia , N-Acetilglucosaminiltransferasas/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteolisis , Interferencia de ARN , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Proper adhesion to extracellular matrix is critical for epithelial cell survival. Detachment from matrix signals results in apoptosis, referred to as anoikis. Selective apoptosis of cells that become detached from matrix is associated with the formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro. Because early breast cancer lesions such as carcinoma in situ, characterized by ducts exhibiting lumens filled with cells, are often associated with hypoxic markers, we sought to examine the role of hypoxia in anoikis and lumen formation in mammary epithelial cells. Here, we show that hypoxic conditions inhibit anoikis and block expression of proapoptotic BH3-only family members Bim and Bmf in epithelial cells. Hypoxia-mediated anoikis protection is associated with increased activation of the epidermal growth factor receptor-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (Erk) kinase pathway and requires the hypoxia-activated transcription factor. Consistent with these data, hypoxic conditions inhibit luminal clearing during morphogenesis in human mammary epithelial acini when grown in three-dimensional cultures and are associated with decreased expression of Bim and Bmf as well as Erk activation. We show that hypoxia regulates specific cell survival pathways that disrupt tissue architecture related to clearing of luminal space during mammary morphogenesis and suggest that hypoxia-mediated anoikis resistance may contribute to cancer progression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anoicis , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Butadienos/farmacología , Técnicas de Cultivo de Célula , Hipoxia de la Célula , Línea Celular , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Immunoblotting , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/metabolismo , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfogénesis , Nitrilos/farmacología , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
ErbB2, a receptor tyrosine kinase highly expressed in many tumors, is known to inhibit apoptotic signals. Overexpression of ErbB2 causes anoikis resistance that contributes to luminal filling in three-dimensional mammary epithelial acinar structures in vitro. Given that integrins and growth factor receptors are highly interdependent for function, we examined the role of integrin subunits in ErbB2-mediated survival signaling. Here, we show that MCF-10A cells overexpressing ErbB2 upregulate integrin alpha5 via the MAP-kinase pathway in three-dimensional acini and found elevated integrin alpha5 levels associated with ErbB2 status in human breast cancer. Integrin alpha5 is required for ErbB2-mediated anoikis resistance and for optimal ErbB2 signaling to the Mek-Erk-Bim axis as depletion of integrin alpha5 reverses anoikis resistance and Bim inhibition. Integrin alpha5 is required for full activation of ErbB2 tyrosine phosphorylation on Y877 and ErbB2 phosphorylation is associated with increased activity of Src in the absence of adhesion. Indeed, we show that blocking elevated Src activity during cell detachment reverses ErbB2-mediated survival and Bim repression. Thus, integrin alpha5 serves as a key mediator of Src and ErbB2-survival signaling in low adhesion states, which are necessary to block the pro-anoikis mediator Bim, and we suggest that this pathway represents a potential novel therapeutic target in ErbB2-positive tumors.