RESUMEN
MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.
Asunto(s)
Apoptosis , Aurora Quinasa A/metabolismo , Resistencia a Antineoplásicos , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Transducción de Señal , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Azepinas/farmacología , Línea Celular Tumoral , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirimidinas/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-specific expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identified as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Indoles/farmacología , Espectrometría de Masas , Melanocitos/efectos de los fármacos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Oncogenes , Pirroles/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Sunitinib , TransfecciónRESUMEN
Overexpression of Ras(G12V) in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21(Waf1/Cip1) rescues from Ras(G12V)-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras(G12V)-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras(G12V)-induced senescence by prevention of Ras(G12V)-induced upregulation of p21(Waf1/Cip1). Our results establish an important role for the cell cycle inhibitor p21(Waf1/Cip1) in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor.