RESUMEN
AMPK is a serine threonine kinase composed of a heterotrimer of a catalytic, kinase-containing α and regulatory ß and γ subunits. Here we show that individual AMPK subunit expression and requirement for survival varies across colon cancer cell lines. While AMPKα1 expression is relatively consistent across colon cancer cell lines, AMPKα1 depletion does not induce cell death. Conversely, AMPKα2 is expressed at variable levels in colon cancer cells. In high expressing SW480 and moderate expressing HCT116 colon cancer cells, siRNA-mediated depletion induces cell death. These data suggest that AMPK kinase inhibition may be a useful component of future therapeutic strategies. We used Functional Signature Ontology (FUSION) to screen a natural product library to identify compounds that were inhibitors of AMPK to test its potential for detecting small molecules with preferential toxicity toward human colon tumor cells. FUSION identified 5'-hydroxy-staurosporine, which competitively inhibits AMPK. Human colon cancer cell lines are notably more sensitive to 5'-hydroxy-staurosporine than are non-transformed human colon epithelial cells. This study serves as proof-of-concept for unbiased FUSION-based detection of small molecule inhibitors of therapeutic targets and highlights its potential to identify novel compounds for cancer therapy development.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Ontologías Biológicas , Neoplasias del Colon/patología , Inhibidores de Proteínas Quinasas/farmacología , Productos Biológicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
Identification and characterization of survival pathways active in tumor cells but absent in normal tissues provide opportunities to develop effective anticancer therapies with reduced toxicity to the patient. We show here that, like kinase suppressor of Ras 1 (KSR1), EPH (erythropoietin-producing hepatocellular carcinoma) receptor B4 (EPHB4) is aberrantly overexpressed in human colon tumor cell lines and selectively required for their survival. KSR1 and EPHB4 support tumor cell survival by promoting the expression of downstream targets, Myc and the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß). While KSR1 promotes the aberrant expression of Myc and the PGC1ß protein via a posttranscriptional mechanism, EPHB4 has a greater effect on Myc and PGC1ß expression via its ability to elevate mRNA levels. Subsequent analysis of the posttranscriptional regulation demonstrated that KSR1 promotes the translation of Myc protein. These findings reveal novel KSR1- and EPHB4-dependent signaling pathways supporting the survival of colorectal cancer cells through regulation of Myc and PGC1ß, suggesting that inhibition of KSR1 or EPHB4 effectors may lead to selective toxicity in colorectal tumors.
Asunto(s)
Proteínas Portadoras/genética , Neoplasias del Colon/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Receptor EphB4/metabolismo , Células CACO-2 , Línea Celular Tumoral , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN , Regulación hacia ArribaRESUMEN
A major goal of cancer research is the identification of tumor-specific vulnerabilities that can be exploited for the development of therapies that are selectively toxic to the tumor. We show here that the transcriptional coactivators peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß) and estrogen-related receptor α (ERRα) are aberrantly expressed in human colon cell lines and tumors. With kinase suppressor of Ras 1 (KSR1) depletion as a reference standard, we used functional signature ontology (FUSION) analysis to identify the γ1 subunit of AMP-activated protein kinase (AMPK) as an essential contributor to PGC1ß expression and colon tumor cell survival. Subsequent analysis revealed that a subunit composition of AMPK (α2ß2γ1) is preferred for colorectal cancer cell survival, at least in part, by stabilizing the tumor-specific expression of PGC1ß. In contrast, PGC1ß and ERRα are not detectable in nontransformed human colon epithelial cells, and depletion of the AMPKγ1 subunit has no effect on their viability. These data indicate that Ras oncogenesis relies on the aberrant activation of a PGC1ß-dependent transcriptional pathway via a specific AMPK isoform.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Portadoras/genética , Colon/patología , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Colon/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptor Relacionado con Estrógeno ERRalfaAsunto(s)
Adenocarcinoma/epidemiología , Factores de Edad , Neoplasias Colorrectales/epidemiología , Adenocarcinoma/terapia , Adolescente , Adulto , Quimioradioterapia , Niño , Preescolar , Neoplasias Colorrectales/terapia , Femenino , Hospitales , Humanos , India/epidemiología , Masculino , Metástasis de la Neoplasia , Estadificación de Neoplasias , Estudios Retrospectivos , Adulto JovenRESUMEN
Kinase suppressor of ras 1 (KSR1) is a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase (ERK) cascade that enhances oncogenic Ras signaling. Here we show KSR1-dependent, but ERK-independent, regulation of metabolic capacity is mediated through the expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and estrogen-related receptor α (ERRα). This KSR1-regulated pathway is essential for the transformation of cells by oncogenic Ras. In mouse embryo fibroblasts (MEFs) expressing H-Ras(V12), ectopic PGC1α was sufficient to rescue ERRα expression, metabolic capacity, and anchorage-independent growth in the absence of KSR1. The ability of PGC1α to promote anchorage-independent growth required interaction with ERRα, and treatment with an inhibitor of ERRα impeded anchorage-independent growth. In contrast to PGC1α, the expression of constitutively active ERRα (CA-ERRα) was sufficient to enhance metabolic capacity but not anchorage-independent growth in the absence of KSR1. These data reveal KSR1-dependent control of PGC1α- and ERRα-dependent pathways that are necessary and sufficient for signaling by oncogenic H-Ras(V12) to regulate metabolism and anchorage-independent growth, providing novel targets for therapeutic intervention.