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Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

ABSTRACT: Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma.The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential.Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors.The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy-resistant cancer.

SUBMITTER: Zecena H

PROVIDER: S-EPMC5883534 | biostudies-other | 2018 Apr

REPOSITORIES: biostudies-other

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Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

Zecena Helma H Tveit Daniel D Wang Zi Z Farhat Ahmed A Panchal Parvita P Liu Jing J Singh Simar J SJ Sanghera Amandeep A Bainiwal Ajay A Teo Shuan Y SY Meyskens Frank L FL Liu-Smith Feng F Filipp Fabian V FV

BMC systems biology 20180404 1

<h4>Background</h4>Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma.<h4>Methods</h4>The cellular model evolved i ...[more]

PMID: 29615030

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Project description:BackgroundPatients with malignant melanoma often relapse after treatment with BRAF and/or mitogen-activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance.ObjectivesTo establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy.MethodsImmunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib-induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib-resistant melanoma cell survival and invasion, respectively.ResultsCD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF-mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271-expressing subpopulations. Trametinib-induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271-expressing melanoma subpopulations with RNA interference and small-molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9-tetrahydrocannabinol and Vps34 - reduced survival of MEKi-resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi-resistant cells in an in vivo zebrafish xenograft.ConclusionsThese results highlight a novel mechanism of MEKi-induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug-resistant melanoma cells to the cytotoxic effects of MEKi.

Project description: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 [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.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.

Project description:Skin cancer is one of the most common cancers worldwide. Melanoma accounts for ~5% of skin cancers but causes the large majority of skin cancer‑related deaths. Recent discoveries have shown that the mitogen‑activated protein kinase (MAPK) signaling pathway is critical for melanoma development and progression. Many oncogenic pathways that cause melanoma tumorigenesis have been identified, most of which are due to RAF/MEK/ERK (MAPK) pathway activation. However, the precise role of p38 remains unclear. Using specific short hairpin (sh) RNA to silence p38α and p38β, the present findings demonstrated that p38α was a crucial factor in regulating cell migration in the A375 melanoma cell line. Silencing p38α downregulated the expression of epithelial‑mesenchymal transition markers, such as matrix metallopeptidase (MMP) 2, MMP9, twist family bHLH transcription factor 1, snail family transcriptional repressor 1 and vimentin, while mesenchymal‑epithelial transition markers, such as E‑cadherin, were upregulated. Of note, the results also demonstrated that p38α silencing impaired vascular endothelial growth factor expression, which regulates tumor angiogenesis. Furthermore, p38α knockdown inhibited cell proliferation in melanoma cells. In addition, silencing p38α induced senescence‑like features, but not cell cycle arrest. Expression of the senescence markers p16, p21, p53 and β‑galactosidase was upregulated, and an increase in the number of senescence‑associated β‑galactosidase‑positive cells was observed in a p38α knockdown stable clone. However, no significant difference was found between control and p38β stable knockdown cells. Taken together, the present results suggested that p38α knockdown impaired migration and proliferation, and increased senescence, in A375 melanoma cells. However, p38β may not be involved in melanoma tumorigenesis. Therefore, targeting p38α may be a valuable approach towards inhibiting tumor growth and metastasis in patients with melanoma.

Dataset Information

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

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Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

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