Prenylation inhibition-induced cell death in melanoma: reduced sensitivity in BRAF mutant/PTEN wild-type melanoma cells.

Tamás Garay,Viktória László,Balázs Döme,József Tímár,Walter Klepetko,Eszter Molnár,József Tóvári,István Kenessey,Anita Rózsás,Walter Berger,Andrea Réti,Judit Dobos,Éva Juhász,Balázs Hegedűs

doi:10.1371/journal.pone.0117021

Abstract

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.

Highlights

Melanoma is characterized by high mortality among solid tumors due to the very high metastatic potential of melanoma cells and their resistance to therapy especially at late stage diseases [1, 2]
Short-term effect of the treatment with different concentrations of zoledronic acid (ZA) on cell proliferation of melanoma cells was measured by Sulforhodamine B (SRB)-assay (Fig. 1A)
All four BRAF mutant/PTEN-null mutant cells were sensitive to long-term ZA treatment in contrast to three BRAF mutant and PTEN wild-type line that were essentially resistant against prenylation inhibition

Summary

Introduction

Melanoma is characterized by high mortality among solid tumors due to the very high metastatic potential of melanoma cells and their resistance to therapy especially at late stage diseases [1, 2]. The majority of melanoma cases demonstrate oncogenic activation of the KIT—NRAS—BRAF—MEK—ERK central axis [5] that is a major regulator of cell differentiation and proliferation [6, 7]. The importance of this pathway is highlighted by the finding that BRAF and NRAS mutation are the two most important oncogenic mutations in melanoma and both of these mutations result in the constitutive activation of the RAS-RAF-MEK-ERK signaling cascade. PTEN-null mutations are present in 20% of melanoma cases [17, 18] PTEN null mutation is often concurrent with BRAF mutation in melanoma [19]

Methods

Results

Conclusion