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

doi:10.1016/j.canlet.2015.02.041

Cancer Letters

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

https://doi.org/10.1016/j.canlet.2015.02.041 Get 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 BRAF^V600E 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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Original ArticlesVemurafenib resistance selects for highly malignant brain and lung-metastasizing melanoma cells

Highlights

Abstract

Introduction

Section snippets

Cells

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

Discussion

Conflict of interest

Acknowledgements

The Raf/MEK/ERK pathway: new concepts of activation

Biol. Cell

Mutations of the BRAF gene in human cancer

Nature

Improved survival with vemurafenib in melanoma with BRAF V600E mutation

N. Engl. J. Med

Resistance to BRAF-targeted therapy in melanoma

Eur. J. Cancer

Systemic treatment for BRAF-mutant melanoma: where do we go next?

Lancet Oncol

The multiple facets of drug resistance: one history, different approaches

J. Exp. Clin. Cancer Res

Pharmacogenetics research on chemotherapy resistance in colorectal cancer over the last 20 years

World J. Gastroenterol

Detection of P-glycoprotein in ovarian cancer: a molecular marker associated with multidrug resistance

J. Clin. Oncol

Role of microRNAs in drug-resistant ovarian cancer cells

Gynecol. Oncol

VAV3 mediates resistance to breast cancer endocrine therapy

Breast Cancer Res

The metastatic microenvironment: brain-residing melanoma metastasis and dormant micrometastasis

Int. J. Cancer

The metastatic microenvironment: Claudin-1 suppresses the malignant phenotype of melanoma brain metastasis

Int. J. Cancer

Blood-brain barrier-specific properties of a human adult brain endothelial cell line

FASEB J.

In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMEC-ST1.6R

Microvasc. Res

Targeting endothelial CD146 attenuates neuroinflammation by limiting lymphocyte extravasation to the CNS

Sci. Rep

Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells

J. Immunol

RhoA is required for monocyte tail retraction during transendothelial migration

J. Cell Biol

High-throughput microtiter assay for Hoechst 33342 dye uptake

Cytotechnology

Exploration, normalization, and summaries of high density oligonucleotide array probe level data

Biostatistics

The role of tumor necrosis factor-alpha and interferon-gamma in regulating angiomotin-like protein 1 expression in lung microvascular endothelial cells

Allergol. Int

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

Cell. Signal

Transcriptional regulation of CCL20 expression

Microbes Infect

E-cadherin and gastric cancer: cause, consequence, and applications

Biomed Res. Int

Variable expression of GLIPR1 correlates with invasive potential in melanoma cells

Front. Oncol

Expression of high-mobility group AT-hook protein 2 and its prognostic significance in malignant gliomas

Hum. Pathol

Coexpression of HMGA2 and Oct4 predicts an unfavorable prognosis in human gastric cancer

Med. Oncol

Effects of micro-environment- and malignant cell-derived interleukin-1 in carcinogenesis, tumour invasiveness and tumour-host interactions

Eur. J. Cancer

The role IL-1 in tumor-mediated angiogenesis

Front. Physiol

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens

Int. J. Cancer

The paracrine effect of cancer-associated fibroblast-induced interleukin-33 regulates the invasiveness of head and neck squamous cell carcinoma

J. Pathol

Notch signalling in solid tumours: a little bit of everything but not all the time

Nat. Rev. Cancer

A role for the JARID1B stem cell marker for continuous melanoma growth

Pigment Cell Melanoma Res

Blocking autocrine VEGF signaling by sunitinib, an anti-cancer drug, promotes embryonic stem cell self-renewal and somatic cell reprogramming

Cell Res

LIF mediates proinvasive activation of stromal fibroblasts in cancer

Cell Rep

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