RESUMEN
To identify regulators of intracellular signaling, we targeted 541 kinases and kinase-related molecules with small interfering RNAs (siRNAs), and determined their effects on signaling with a functional proteomics reverse-phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome-wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and mitogen-activated protein kinase (MAPK) with 115 genes that, when targeted by siRNAs, demonstrated opposite effects on MAPK and AKT phosphorylation. Network-based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on tuberous sclerosis complex 2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 (Glycogen synthase kinase 3) interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.
Asunto(s)
Redes y Vías Metabólicas/fisiología , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Humanos , Redes y Vías Metabólicas/genética , Fosfoproteínas/genética , Fosforilación , Proteómica , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 'druggable' targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top 'hits' from the screen were found to result from off-target gene silencing. Interestingly, the three off-target siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3' untranslated region (3' UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.
Asunto(s)
Compuestos de Bifenilo/farmacología , Proteínas de Neoplasias/fisiología , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , ARN Interferente Pequeño/genética , Sulfonamidas/farmacología , Regiones no Traducidas 3'/genética , Antineoplásicos/farmacología , Secuencia de Bases , Bencenosulfonatos/farmacología , Western Blotting , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinas/farmacología , Piridinas/farmacología , Interferencia de ARN , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Roscovitina , Sorafenib , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Dedos de Zinc/genética , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
Tumors comprise genetically heterogeneous cell populations, whose growth and survival depend on multiple signaling pathways. This has spurred the development of multitargeted therapies, including small molecules that can inhibit multiple kinases. A major challenge in designing such molecules is to determine which kinases to inhibit in each cancer to maximize efficacy and therapeutic index. We describe an approach to this problem implementing RNA interference technology. In order to identify Akt-cooperating kinases, we screened a library of kinase-directed small interfering RNAs (siRNAs) for enhanced cancer cell killing in the presence of Akt inhibitor A-443654. siRNAs targeting casein kinase I gamma 3 (CSNK1G3) or the inositol polyphosphate multikinase (IPMK) significantly enhanced A-443654-mediated cell killing, and caused decreases in Akt Ser-473 and ribosomal protein S6 phosphorylation. Small molecules targeting CSNK1G3 and/or IPMK in addition to Akt may thus exhibit increased efficacy and have the potential for improved therapeutic index.