RESUMEN
The mechanistic target of rapamycin (mTOR) protein kinase coordinates responses to nutrients and growth factors and is an anti-cancer drug target. To anticipate how cells will respond and adapt to chronic mTOR complex (mTORC)1 and mTORC2 inhibition, we have generated SW620 colon cancer cells with acquired resistance to the ATP-competitive mTOR kinase inhibitor AZD8055 (SW620:8055R). AZD8055 inhibited mTORC1 and mTORC2 signalling and caused a switch from cap-dependent to internal ribosome entry site (IRES)-dependent translation in parental SW620 cells. In contrast, SW620:8055R cells exhibited a loss of S6K signalling, an increase in expression of the eukaryotic translation initiation factor eIF4E and increased cap-dependent mRNA translation. As a result, the expression of CCND1 and MCL1, proteins encoded by eIF4E-sensitive and cap-dependent transcripts, was refractory to AZD8055 in SW620:8055R cells. RNAi-mediated knockdown of eIF4E reversed acquired resistance to AZD8055 in SW620:8055R cells; furthermore, increased expression of eIF4E was sufficient to reduce sensitivity to AZD8055 in a heterologous cell system. Finally, although the combination of MEK1/2 inhibitors with mTOR inhibitors is an attractive rational drug combination, SW620:8055R cells were actually cross-resistant to the MEK1/2 inhibitor selumetinib (AZD6244). These results exemplify the convergence of ERK1/2 and mTOR signalling at eIF4E, and the key role of eIF4E downstream of mTOR in maintaining cell proliferation. They also have important implications for therapeutic strategies based around mTOR and the MEK1/2-ERK1/2 pathway.
Asunto(s)
Antineoplásicos/farmacología , Factor 4E Eucariótico de Iniciación/genética , Morfolinas/farmacología , Biosíntesis de Proteínas , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bencimidazoles/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Factor 4E Eucariótico de Iniciación/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Amplificación de Genes , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de SeñalRESUMEN
The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signalregulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signalregulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF600E or KRAS13D, due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF600E. Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF600E or KRAS13D) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS13D leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Genes ras , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Bencimidazoles , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/genética , Amplificación de Genes , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Regulación hacia Arriba , Proteínas ras/genéticaRESUMEN
The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF(600E) or KRAS(13D), due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF(600E). Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF(600E) or KRAS(13D)) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS(13D) leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.
Asunto(s)
Bencimidazoles/farmacología , Neoplasias Colorrectales/enzimología , Resistencia a Antineoplásicos/efectos de los fármacos , Amplificación de Genes/efectos de los fármacos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas ras/biosíntesis , Sustitución de Aminoácidos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , Mutación Missense , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras/genéticaRESUMEN
Sialin is a lysosomal membrane protein encoded by the SLC17A5 gene, which is mutated in patients with sialic acid storage diseases (SASD). To further understand the role of sialin in normal CNS development and in the progressive neuronal atrophy and dysmyelination seen in SASD, we investigated its normal cellular distribution in adult and developing mice. Overall, sialin showed granular immunoreactivity, consistent with a vesicular protein. Adult mice showed widespread sialin expression, including in the brain, heart, lung, and liver. High-level immunoreactivity was seen in the neuropil of the hippocampus, striatum, and cerebral cortex, as well as in the perikarya of cerebellar Purkinje cells, globus pallidus, and certain thalamic and brainstem nuclei. In mouse embryos, the highest levels of expression were observed in the nervous system. We discuss the possible role of sialin in normal development and in SASD pathogenesis, as a framework for further investigation of its function in these contexts.