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Proteomics-Based Insights Into Mitogen-Activated Protein Kinase

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Proteomics-Based Insights Into Mitogen-Activated Protein Kinase Zila et al. Clin Proteom (2018) 15:13 https://doi.org/10.1186/s12014-018-9189-x Clinical Proteomics RESEARCH Open Access Proteomics‑based insights into mitogen‑activated protein kinase inhibitor resistance of cerebral melanoma metastases Nina Zila1,2,3, Andrea Bileck2, Besnik Muqaku2, Lukas Janker2, Ossia M. Eichhof4, Phil F. Cheng4, Reinhard Dummer4, Mitchell P. Levesque4, Christopher Gerner2 and Verena Paulitschke1,4* Abstract Background: 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. Under- standing mechanisms of resistance and identifying efective therapeutic alternatives is one of the most important scientifc challenges in melanoma. Using proteomics, we want to specifcally gain insight into the pathophysiological process of cerebral metastases. Methods: Cerebral metastases from melanoma patients were initially analyzed by a LC–MS shotgun approach per- formed on a QExactive HF hybrid quadrupole-orbitrap mass spectrometer. For further validation steps after bioinfor- matics analysis, a targeted LC-QQQ-MS approach, as well as Western blot, immunohistochemistry and immunocyto- chemistry was performed. Results: In this pilot study, we were able to identify 5977 proteins by LC–MS analysis (data are available via ProteomeXchange with identifer PXD007592). Based on PFS, samples were classifed into good responders (PFS 6 months) and poor responders (PFS ≤ 3 months). By evaluating these proteomic profles according to gene ontology≥ (GO) terms, KEGG pathways and gene set enrichment analysis (GSEA), we could characterize diferences between the two distinct groups. We detected an EMT feature (up-regulation of N-cadherin) as classifer between the two groups, V-type proton ATPases, cell adhesion proteins and several transporter and exchanger proteins to be signifcantly up-regulated in poor responding patients, whereas good responders showed an immune activation, among other features. We identifed class-discriminating proteins based on nearest shrunken centroids, validated and quantifed 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 metas- tases. 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 confrmed known extra-cerebral resistance mechanisms in the cerebral metastases and further discovered possible brain specifc mechanisms of drug efux, which might serve as treatment targets or as predictive markers for these kinds of metastasis. Keywords: BRAF mutation, Cerebral melanoma metastases, Drug resistance, Melanoma, MAP kinase inhibitor, Proteomics *Correspondence: [email protected] 1 Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18–20, 1090 Vienna, Austria Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zila et al. Clin Proteom (2018) 15:13 Page 2 of 22 Background of BRAF and MEK inhibitors (MEKi) was implemented Te incidence of melanoma is increasing rapidly world- and improved the efectiveness of targeted therapy even wide, including countries with historically low rates. further by improving median PFS [17, 18]. Unfortunately, Tis increase is occurring at a faster rate than with resistance to therapy is still challenging and especially the any other neoplasm [1]. Up to one-ffth of melanoma high mortality of brain metastases poses a huge ongo- patients develop metastatic disease, which has mainly ing clinical problem. It is questionable whether there been treated by chemotherapy, achieving response are brain specifc efux transporters contributing to the rates between 10 and 30% [2]. After cancers of the lung resistance of intracranial metastases and where difer- and breast, melanoma is the third most common cause ences or similarities between visceral and cerebral metas- of central nervous system (CNS) metastases [3, 4]. In tases can be found. Te phenotype-switching model was patients with newly diagnosed stage IV disease, brain frst described in melanoma by Hoek et al. [19, 20] and metastases are present in 20% of cases [5] and among characterizes two transcriptional distinct melanoma cell patients with documented brain involvement, these populations, a proliferative and an invasive type. Mela- lesions contribute to death in up to 95% [6]. In 40–60% noma cells are able to switch back and forth between of all melanoma patients, mutational activation of BRAF these two phenotypes and therefore explain the hetero- V600E can be found, resulting in constitutive activa- geneous nature of melanoma cells [21]. It accounts for tion of the serine-threonine protein kinase BRAF and disease progression and tumor heterogeneity, as well as the Ras-RAF-MEK-ERK signaling pathway, also known aspects of resistance [22]. Proliferative melanoma cells as mitogen activated protein (MAP) kinase pathway [7]. have been shown to be more responsive to MAPK path- Until 2011, standard treatment for patients with inopera- way inhibition than invasive phenotype cells, indepen- ble metastatic melanoma was dacarbazine (DTIC) [8]. In dently of their mutation status [23, 24]. On the other August 2011, the US Agency of Food and Drug Admin- hand, proliferative melanoma cells have shown to change istration (FDA) approved Vemurafenib, a BRAF inhibitor their phenotype from proliferative to invasive state in (BRAFi), for the treatment of metastatic or unresectable response to long-term treatment with targeted therapy melanoma with BRAF V600E mutation. Brain metasta- (BRAFi and MEKi), which is associated with drug resist- ses pose special challenges because of the poor associ- ance. Te major role in phenotype switching and the ated prognosis [9]. Tey are a major cause of mortality transition from a proliferative to an invasive cell type in patients with advanced melanoma. However, relatively and ultimately leading to metastasis is played by EMT- little was known about the intracranial efectiveness of like mechanisms [25]. Identifying critical switches in selective inhibitors because patients with brain metas- EMT processes and fnding a way to block these, might tases have historically been excluded from clinical trials serve as a strategy to prevent metastasis and to decrease [10]. Recently a few studies investigated BRAFi treatment therapeutic resistance. Especially, the down-regulation of for melanoma patients with brain metastasis [11–13]. An E-cadherin, balanced by the increased expression of mes- open-label pilot study assessed Vemurafenib therapy in enchymal neural cadherin (N-cadherin), this, so called patients with BRAF V600 mutation positive metastatic cadherin switch, alters cell adhesion [26, 27] and is con- melanoma with non-resectable, previously treated brain sidered to be a fundamental event in EMT, leading to a metastases and concluded that Vemurafenib can safely be loss of cell–cell contact, increased invasive properties used for the therapy of advanced symptomatic melanoma and typical morphological changes [21, 28, 29]. with metastasis to the brain and can result in meaning- In this study we showed, by analyzing brain tissue ful tumor regression [11]. In more than 50% of these samples of patients with low and high PFS in response cases clinical response, up to a complete remission, were to MAPKi treatment, distinct diferences between the achieved by Vemurafenib. Systemic therapy revolution- two groups, using proteome profling by shotgun MS. ized melanoma treatment but unfortunately high initial We analyzed the data according to gene ontology (GO) responses are followed by acquired drug resistance after terms, KEGG pathways and gene set enrichment analy- a median time of only 6–8 months [14, 15]. About 15% sis (GSEA). We were further able to identify an EMT of the patients treated with BRAFi do not achieve tumor mechanism (up-regulation of N-cadherin), V-type proton regression, because of intrinsic (primary) mechanisms ATPases, calcium ion binding proteins, eukaryotic trans- of resistance and most patients who respond to therapy lation initiation factors, cell adhesion proteins, several ultimately develop mechanisms of acquired (secondary) transporter and exchanger proteins in poor responding resistance, leading to progressive disease [16]. Initial suc- patients, whereas good responders showed an immune cess was made by BRAFi therapy, but due to the reacti- activation and involvement of extracellular matrix struc- vation of the MAPK signaling pathway, patients showed tural constituents, among other features. Based
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