Cancer Letters

Cancer Letters

Volume 361, Issue 1, 28 May 2015, Pages 86-96
Cancer Letters

Original Articles
Vemurafenib resistance selects for highly malignant brain and lung-metastasizing melanoma cells

https://doi.org/10.1016/j.canlet.2015.02.041Get rights and content

Highlights

  • Vemurafenib-resistant melanoma cells express an altered gene expression profile.

  • Vemurafenib-resistant melanoma cells express higher levels of certain stem cell markers.

  • Vemurafenib-resistance of melanoma cells is associated with increased tumorigenicity.

  • Vemurafenib-resistant melanoma cells express an increased metastatic phenotype.

  • Vemurafenib-resistant and sensitive cells alter differently their microenvironment.

Abstract

V600E being the most common mutation in BRAF, leads to constitutive activation of the MAPK signaling pathway. The majority of V600E BRAF positive melanoma patients treated with the BRAF inhibitor vemurafenib showed initial good clinical responses but relapsed due to acquired resistance to the drug. The aim of the present study was to identify possible biomarkers associated with the emergence of drug resistant melanoma cells. To this end we analyzed the differential gene expression of vemurafenib-sensitive and vemurafenib resistant brain and lung metastasizing melanoma cells. The major finding of this study is that the in vitro induction of vemurafenib resistance in melanoma cells is associated with an increased malignancy phenotype of these cells. Resistant cells expressed higher levels of genes coding for cancer stem cell markers (JARID1B, CD271 and Fibronectin) as well as genes involved in drug resistance (ABCG2), cell invasion and promotion of metastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigrate more efficiently through lung endothelial cells than drug-sensitive cells. The former cells also alter their microenvironment in a different manner from that of drug-sensitive cells. Biomarkers and molecular mechanisms associated with drug resistance may serve as targets for therapy of drug-resistant cancer.

Introduction

The MAPK signaling pathway involves activation of BRAF which phosphorylates and activates MEK which in turn phosphorylates and activates ERK. These reactions result in activation of transcription factors that regulate cell survival, proliferation and differentiation [1].

BRAF mutations have been found in different malignancies including melanoma. V600E is the most common mutation in BRAF leading to constitutive activation of the MAPK signaling pathway [2]. Several small molecule inhibitors targeting the V600E BRAF mutation such as vemurafenib were developed [3]. Treatment of V600E BRAF positive metastatic melanoma with vemurafenib showed initial good clinical responses. However most of the patients relapsed due to acquired resistance [4].

Acquired drug resistance is one of the major obstacles in cancer treatment and management [5], [6]. Several approaches have been adopted to overcome drug resistance, among them attempts to detect novel markers that can be targeted on resistant cells [7], [8], [9], [10].

We have previously generated xenograft human melanoma brain metastasis models, consisting of local, cutaneous variants as well as of brain and lung-metastasizing variants yielding either dormant micrometastasis or overt metastasis. These cell lines comprise BRAFV600E mutation. All the variants originated from single melanomas thus sharing a common genetic background. Genes that are differentially expressed by these variants can, thus, be assigned to the differential malignancy phenotype of the different variants [11]. Using these models we demonstrated that brain-metastasizing melanoma variants expressed a set of genes whose expression pattern differed from that of cutaneous melanoma variants [11].

In this study we analyzed the differential gene expression of vemurafenib-sensitive brain and lung metastasizing melanoma cells and corresponding cells in which resistance to this bio-drug was induced by repeated cycles of in vitro exposure to the drug. The vemurafenib sensitive melanoma cells and their resistant counterparts originated from a single melanoma tumor having therefore a common genetic background [11]. Any difference in gene expression between these metastatic variants can therefore be attributed to the difference in the metastatic microenvironment they originated from (brain versus lungs) and their drug sensitivity/resistance status.

Section snippets

Cells

All human melanoma cells (YDFR.CB3, YDFR.SB3, YDFR.CB3CSL3) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/ml L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 12.5 units/ml nystatin and 1% Hepes (Biological Industries, Beit-Haemek, Israel). Medium of melanoma cells resistant to Vemurafenib was supplemented with 1 µM PLX-4032 (Vemurafenib) (Selleck, Houston, TX) dissolved in Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, St. Louis,

Vemurafenib resistance of melanoma cells is associated with an altered gene expression profile

In previous studies we generated variants of human melanoma cells that metastasize to the brain and lungs of xenotransplanted nude mice [11]. In this study we utilized variants that metastasize specifically and spontaneously to brain and lungs of nude mice forming micro-metastasis (YDFR.SB3 and YDFR.CB3CSL3, respectively) in these organs following an orthotopic sub-dermal inoculation. We also used a variant that generates brain macro-metastasis following an intra-cardiac inoculation (YDFR.CB3).

Discussion

A major finding of this study is that the in vitro induction of vemurafenib-resistance of melanoma cells is associated with an increased malignancy phenotype of these cells. It is not unlikely that selecting for drug resistance selects for tumor and metastasis initiating cells [51], [52]. This possibility is supported by the findings that vemurafenib-resistant cells express higher levels of certain stem cell markers and that these cells also express higher levels of ABCG2 functioning as a key

Conflict of interest

None.

Acknowledgements

This study was supported by The Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (04-7023433) (Needham, MA).

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