RESUMEN
Liver kinase B1 (LKB1) is mutationally inactivated in Peutz-Jeghers syndrome and in a variety of cancers including human papillomavirus (HPV)-caused cervical cancer. However, the significance of LKB1 mutations in cervical cancer initiation and progress has not been examined. Herein, we demonstrated that, in mouse embryonic fibroblasts, loss of LKB1 and transduction of HPV16 E6/E7 had an additive effect on constraining cell senescence while promoting cell proliferation and increasing glucose consumption, lactate production and ATP generation. Knockdown of LKB1 increased and ectopic expression of LKB1 decreased glycolysis, anchorage-independent cell growth, and cell migration and invasion in HPV-transformed cells. In the tumorigenesis and lung metastasis model in syngeneic mice, depletion of LKB1 markedly increased tumor metastatic colonies in lungs without affecting subcutaneous tumor growth. We showed that HPV16 E6/E7 enhanced the expression of hexokinase-ll (HK-II) in the glycolytic pathway through elevated c-MYC. Ectopic LKB1 reduced HK-II along with glycolysis. The inverse relationship between HK-II and LKB1 was also observed in normal and HPV-associated cervical lesions. We propose that LKB1 acts as a safeguard against HPV-stimulated aerobic glycolysis and tumor progression. These findings may eventually aid in the development of therapeutic strategy for HPV-associated malignancies by targeting cell metabolism.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Glucosa/metabolismo , Glucólisis/fisiología , Infecciones por Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Transformación Celular Neoplásica/patología , Femenino , Estudios de Seguimiento , Hexoquinasa/genética , Hexoquinasa/metabolismo , Papillomavirus Humano 16/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Estadificación de Neoplasias , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/virologíaRESUMEN
Liver kinase B1 (LKB1, also known as serine/threonine kinase 11, STK11) is a tumor suppressor mutated in Peutz-Jeghers syndrome and in a variety of sporadic cancers. Herein, we demonstrate that LKB1 controls the levels of intracellular reactive oxygen species (ROS) and protects the genome from oxidative damage. Cells lacking LKB1 exhibit markedly increased intracellular ROS levels, excessive oxidation of DNA, increased mutation rates and accumulation of DNA damage, which are effectively prevented by ectopic expression of LKB1 and by incubation with antioxidant N-acetylcysteine. The role of LKB1 in suppressing ROS is independent of AMP-activated protein kinase, a canonical substrate of LKB1. Instead, under the elevated ROS, LKB1 binds to and maintains the activity of the cdc42-PAK1 (p21-activated kinase 1) complex, which triggers the activation of p38 and its downstream signaling targets, such as ATF-2, thereby enhancing the activity of superoxide dismutase-2 and catalase, two antioxidant enzymes that protect the cells from ROS accumulation, DNA damage and loss of viability. Our results provide a new paradigm for a non-canonical tumor suppressor function of LKB1 and highlight the importance of targeting ROS signaling as a potential therapeutic strategy for cancer cells lacking LKB1.
Asunto(s)
Daño del ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Acetilcisteína/farmacología , Factor de Transcripción Activador 2/metabolismo , Animales , Western Blotting , Catalasa/metabolismo , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Fibroblastos/metabolismo , Depuradores de Radicales Libres/farmacología , Células HeLa , Humanos , Ratones Noqueados , Microscopía Fluorescente , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Superóxido Dismutasa/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death.