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

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Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Electronic supplementary material

The online version of this article (10.1186/s12918-018-0554-1) contains supplementary material, which is available to authorized users.

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Most cited references 26

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Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.

Ramin Nazarian, Hubing Shi, Qi Wang … (2010)

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

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Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma.

Levi A Garraway, Hans R. Widlund, Mark Rubin … (2005)

Systematic analyses of cancer genomes promise to unveil patterns of genetic alterations linked to the genesis and spread of human cancers. High-density single-nucleotide polymorphism (SNP) arrays enable detailed and genome-wide identification of both loss-of-heterozygosity events and copy-number alterations in cancer. Here, by integrating SNP array-based genetic maps with gene expression signatures derived from NCI60 cell lines, we identified the melanocyte master regulator MITF (microphthalmia-associated transcription factor) as the target of a novel melanoma amplification. We found that MITF amplification was more prevalent in metastatic disease and correlated with decreased overall patient survival. BRAF mutation and p16 inactivation accompanied MITF amplification in melanoma cell lines. Ectopic MITF expression in conjunction with the BRAF(V600E) mutant transformed primary human melanocytes, and thus MITF can function as a melanoma oncogene. Reduction of MITF activity sensitizes melanoma cells to chemotherapeutic agents. Targeting MITF in combination with BRAF or cyclin-dependent kinase inhibitors may offer a rational therapeutic avenue into melanoma, a highly chemotherapy-resistant neoplasm. Together, these data suggest that MITF represents a distinct class of 'lineage survival' or 'lineage addiction' oncogenes required for both tissue-specific cancer development and tumour progression.

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Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.

Willy Hugo, Hubing Shi, Lu Sun … (2015)

Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.

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Author and article information

Contributors

Helma Zecena: hzecena1@ucmerced.edu

Daniel Tveit: dtveit1@ucmerced.edu

Zi Wang: zwang@uci.edu

Ahmed Farhat: afarhat@uci.edu

Parvita Panchal: ppanchal@uci.edu

Jing Liu: jliu@uci.edu

Simar J. Singh: ssingh@ucmerced.edu

Amandeep Sanghera: asanghera@ucmerced.edu

Ajay Bainiwal: abainiwal@ucmerced.edu

Shuan Y. Teo: steo@ucmerced.edu

Frank L. Meyskens Jr: flmeyskens@uci.edu

Feng Liu-Smith: +1-949-302-1968 , liufe@uci.edu

Fabian V. Filipp:

ORCID: http://orcid.org/0000-0001-9889-5727

+1-858-349-0349 , filipp@ucmerced.edu

Journal

Journal ID (nlm-ta): BMC Syst Biol

Journal ID (iso-abbrev): BMC Syst Biol

Title: BMC Systems Biology

Publisher: BioMed Central (London )

ISSN (Electronic): 1752-0509

Publication date (Electronic): 4 April 2018

Publication date PMC-release: 4 April 2018

Publication date Collection: 2018

Volume: 12

Electronic Location Identifier: 33

Affiliations

[1 ]ISNI 0000 0001 0049 1282, GRID grid.266096.d, Systems Biology and Cancer Metabolism, Program for Quantitative Systems Biology, , University of California Merced, ; 2500 North Lake Road, Merced, CA 95343 USA

[2 ]ISNI 0000 0001 0668 7243, GRID grid.266093.8, Department of Medicine, School of Medicine, Chao Family Comprehensive Cancer Center, , University of California Irvine, ; Irvine, CA 92697 USA

[3 ]ISNI 0000 0001 0379 7164, GRID grid.216417.7, The State Key Laboratory of Medical Genetics and School of Life Sciences, Department of Molecular Biology, , Central South University, ; Changsha, 410078 China

[4 ]ISNI 0000 0001 0668 7243, GRID grid.266093.8, Department of Epidemiology, School of Medicine, , University of California, ; Irvine, CA 92697 USA

Author information

Fabian V. Filipp http://orcid.org/0000-0001-9889-5727

Article

Publisher ID: 554

DOI: 10.1186/s12918-018-0554-1

PMC ID: 5883534

PubMed ID: 29615030

SO-VID: 28ef2cef-64af-4646-9d68-030d81453e6d

License:

Open AccessThis 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.

History

Date received : 29 October 2017

Date accepted : 21 February 2018