Extracellular Signal-regulated Kinase-dependent Proliferation Is Mediated through the Protein Kinase A/B-Raf Pathway in Human Uveal Melanoma Cells

Armelle Calipel,Frédéric Mouriaux,Anne-Lise Glotin,François Malecaze,Anne-Marie Faussat,Frédéric Mascarelli

doi:10.1074/jbc.m600228200

Abstract

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.

Highlights

Activating point mutations in B-RAF were detected in a large scale screen for genes mutated in human cancer [1]
It has been suggested that a gain-of-function mutation in BRAF (V600EB-Raf) is primarily responsible for the constitutive activation of ERK1/2 and for the acquisition by cutaneous melanoma cells of self-sufficiency in the
No RAS mutations and rare B-RAF mutations have been detected, constitutive activation of ERK1/2 has been detected in primary uveal melanoma tumors, suggesting that wild-type B-Raf (WTB-Raf) may be involved in the activation of the ERK1/2 signaling pathway (10 –18)

Summary

Introduction

Activating point mutations in B-RAF were detected in a large scale screen for genes mutated in human cancer [1]. Reconstitution experiments have shown that this mutation increases the activity of ectopically expressed B-Raf kinase and induces cell transformation [1, 2], whereas siRNA-mediated suppression of V600EB-Raf abolishes cell transformation [3] This gain of function mutation in B-RAF (V600EBRaf) is largely responsible for the constitutive activation of ERK1/2 and is for the acquisition of self-sufficiency in the growth signal in human cutaneous melanoma cells [1, 3, 4]. Constitutive activation of ERK1/2 in the absence of mutations in RAS and B-RAF has been shown in primary tumors and cell lines of uveal melanoma, demonstrating a large difference between cutaneous and uveal melanomas [18, 19]. We identified PKA as a main upstream activator of WTB-Raf for ERK1/2 activation and cell proliferation, suggesting that the PKA/B-Raf pathway may be a potential target for therapeutic strategies against WTB-Raf expressing uveal melanoma

Objectives

Results

Conclusion