Abstract
BackgroundMAP kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9–12 months. Understanding mechanisms of resistance and identifying effective therapeutic alternatives is one of the most important scientific challenges in melanoma. Using proteomics, we want to specifically gain insight into the pathophysiological process of cerebral metastases.MethodsCerebral metastases from melanoma patients were initially analyzed by a LC–MS shotgun approach performed on a QExactive HF hybrid quadrupole-orbitrap mass spectrometer. For further validation steps after bioinformatics analysis, a targeted LC-QQQ-MS approach, as well as Western blot, immunohistochemistry and immunocytochemistry was performed.ResultsIn this pilot study, we were able to identify 5977 proteins by LC–MS analysis (data are available via ProteomeXchange with identifier PXD007592). Based on PFS, samples were classified into good responders (PFS ≥ 6 months) and poor responders (PFS le 3 months). By evaluating these proteomic profiles according to gene ontology (GO) terms, KEGG pathways and gene set enrichment analysis (GSEA), we could characterize differences between the two distinct groups. We detected an EMT feature (up-regulation of N-cadherin) as classifier between the two groups, V-type proton ATPases, cell adhesion proteins and several transporter and exchanger proteins to be significantly up-regulated in poor responding patients, whereas good responders showed an immune activation, among other features. We identified class-discriminating proteins based on nearest shrunken centroids, validated and quantified this signature by a targeted approach and could correlate parts of this signature with resistance using the CPL/MUW proteome database and survival of patients by TCGA analysis. We further validated an EMT-like signature as a major discriminator between good and poor responders on primary melanoma cells derived from cerebral metastases. Higher immune activity is demonstrated in patients with good response to MAPKi by immunohistochemical staining of biopsy samples of cerebral melanoma metastases.ConclusionsEmploying proteomic analysis, we confirmed known extra-cerebral resistance mechanisms in the cerebral metastases and further discovered possible brain specific mechanisms of drug efflux, which might serve as treatment targets or as predictive markers for these kinds of metastasis.
Highlights
mitogen activated protein (MAP) kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9-12 months
By evaluating these proteomic profiles according to gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene set enrichment analysis (GSEA), we could characterize differences between the two distinct groups
We further validated an epithelial to mesenchymal transition (EMT)-like signature as a major discriminator between good and poor responders on primary melanoma cells derived from cerebral metastases
Summary
MAP kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9-12 months. Resistance to therapy is still challenging and especially the high mortality of brain metastases poses a huge ongoing clinical problem It is questionable whether there are brain specific efflux transporters contributing to the resistance of intracranial metastases and where differences or similarities between visceral and cerebral metastases can be found. Proliferative melanoma cells have shown to change their phenotype from proliferative to invasive state in response to long-term treatment with targeted therapy (BRAFi and MEKi), which is associated with drug resistance. The down-regulation of E-cadherin, balanced by the increased expression of mesenchymal neural cadherin (N-cadherin), this, so called cadherin switch, alters cell adhesion [26, 27] and is considered to be a fundamental event in EMT, leading to a loss of cell–cell contact, increased invasive properties and typical morphological changes [21, 28, 29]
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