ABSTRACT
BACKGROUND: Activity of both c-Jun and cyclin D1 is deemed critical for melanoma cell proliferation. This functionality is corroborated by frequently elevated expression and activity of these proteins in human melanomas. Correspondingly, alleviating c-Jun and cyclin D1 function is vital to the success of antimelanoma therapeutics. OBJECTIVES: To understand the role of the c-Jun N-terminal kinase (JNK) signalling pathway in melanoma cell proliferation and survival. METHODS: The effect of JNK inhibitors SP600125 and JNK-IN-8 on the proliferation and survival of genetically highly representative human melanoma cell lines was studied in assays of proliferation and apoptosis. Changes in c-Jun and cyclin D1 protein and mRNA levels in response to JNK and mitogen-activated protein kinase kinase (MEK) inhibition were investigated through immunoblotting and quantitative reverse-transcription polymerase chain reaction. The effects of JNK and MEK inhibitors on cell-cycle distribution were assessed by flow cytometry. RESULTS: We demonstrate the requirement of JNK signalling in melanoma cell proliferation and survival. While JNK inhibition suppressed the expression and activity of c-Jun, it failed to suppress cyclin D1 levels. Consistently with its inability to downregulate cyclin D1, JNK inhibition failed to induce G1 arrest. In contrast, the blockade of MEK-extracellular signal-regulated kinase (ERK) signalling, although unable to suppress c-Jun activity and expression, paradoxically abated cyclin D1 levels and triggered G1 arrest. This previously unreported dual disconnect between JNK-cyclin D1 and ERK-c-Jun levels was confirmed by concomitant JNK and BRAF inhibition, which suppressed both c-Jun and cyclin D1 levels and exhibited a heightened antiproliferative response. CONCLUSIONS: Dual disjunction between JNK-cyclin D1 and ERK-c-Jun signalling forms the basis for further investigation of combined JNK and MAPK signalling blockade as a more effective therapeutic approach in human melanoma.
Subject(s)
JNK Mitogen-Activated Protein Kinases/metabolism , Melanoma/mortality , Skin Neoplasms/mortality , Anthracenes/pharmacology , Cell Proliferation , Cyclin D1/metabolism , Humans , Melanoma/pathology , Protein Kinase Inhibitors/pharmacology , Signal Transduction/physiology , Skin Neoplasms/pathology , Tumor Cells, CulturedABSTRACT
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.