1. ¹Division of Stem Cell Regulation, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi, Japan
2. ²Department of Dermatology, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi, Japan
3. ³Divison of Organ Transplantation, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi, Japan
4. ⁴Department of Nephrology, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi, Japan
5. ⁵Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan
6. ⁶Division of Medical Oncology, Tochigi Cancer Center, Yonan, Utsunomiya, Tochigi, Japan
7. ⁷Department of Dermatology, Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo, Japan

Correspondence: Dr Y Furukawa, Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail: furuyu@jichi.ac.jp

Received 4 March 2005; Revised 26 July 2005; Accepted 29 July 2005; Published online 26 September 2005.

Abstract

Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras–MAP kinase signaling in melanomas.