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.
Severe congenital neutropenia (SCN) is characterized by a near absence of neutrophils, rendering individuals with this disorder vulnerable to recurrent life-threatening infections. The majority of SCN cases arise because of germline mutations in the gene elastase, neutrophil-expressed (ELANE) encoding the neutrophil granule serine protease neutrophil elastase. Treatment with a high dose of granulocyte colony-stimulating factor increases neutrophil production and reduces infection risk. How ELANE mutations produce SCN remains unknown. The currently proposed mechanism is that ELANE mutations promote protein misfolding, resulting in endoplasmic reticulum stress and activation of the unfolded protein response (UPR), triggering death of neutrophil precursors and resulting in neutropenia. Here we studied the ELANE mutation p.G185R, often associated with greater clinical severity (e.g. decreased responsiveness to granulocyte colony-stimulating factor and increased leukemogenesis). Using an inducible expression system, we observed that this ELANE mutation diminishes enzymatic activity and granulocytic differentiation without significantly affecting cell proliferation, cell death, or UPR induction in murine myeloblast 32D and human promyelocytic NB4 cells. Impaired differentiation was associated with decreased expression of genes encoding critical hematopoietic transcription factors (Gfi1, Cebpd, Cebpe, and Spi1), cell surface proteins (Csf3r and Gr1), and neutrophil granule proteins (Mpo and Elane). Together, these findings challenge the currently prevailing model that SCN results from mutant ELANE, which triggers endoplasmic reticulum stress, UPR, and apoptosis.
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.
CRM1-mediated nucleocytoplasmic transport has been shown as a potential anti-cancer target in various malignancies. However, the role of the most characterized CRM1 cofactor RanBP3 in cancer cell biology has never been investigated. Utilizing a loss-of-function experimental setting in a vast collection of genetically varied melanoma cell lines, we witness the requirement of RanBP3 in melanoma cell proliferation and survival. Mechanistically, we suggest the reinstatement of TGF-β-Smad2/3-p21 tumor-suppressor axis as part of the RanBP3 silencing-associated anti-proliferative program. Employing extensive Nuclear Export Sequence (NES) analyses and immunofluorescence-based protein localization studies, we further present evidence suggesting the requirement of RanBP3 function for the nuclear exit of the weak NES-harboring ERK protein, while being dispensable for general CRM1-mediated nuclear export of strong NES-harboring cargoes. Rendering mechanistic support to RanBP3 silencing-mediated apoptosis, consequent to ERK nuclear entrapment, we observed increased levels of cytoplasmically-restricted non-phosphorylated/active pro-apoptotic Bad protein. Lastly, we present evidence suggesting the frequently activated MAPK signaling in melanoma as a potential founding basis for a deregulated post-translational control of RanBP3 activity. Collectively, the presented data suggests RanBP3 as a potential target for therapeutic intervention in human melanoma.Journal of Investigative Dermatology accepted article preview online, 08 October 2015. doi:10.1038/jid.2015.401.
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