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. 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.
METHODS: Cerebral
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.
RESULTS: In 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 [Formula: see text] 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.
CONCLUSIONS: Employing 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.