1. ¹Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan
2. ²Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
3. ³Pathology Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan

Correspondence: H Esumi, Cancer Physiology Project, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. E-mail: hesumi@east.ncc.go.jp

⁴Current address: Cancer Physiology Project, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan

Received 17 March 2004; Revised 4 June 2004; Accepted 8 June 2004; Published online 26 July 2004.

Abstract

Colorectal cancer cells are unique in that they escape Fas-mediated cell death in the presence of Fas ligand, and we recently reported that AMP-activated protein kinase-related kinase 5 (ARK5) suppresses cell death signaling mediated by cell death receptor in Akt-dependent manner. In the current study, therefore, we examined whether ARK5 is involved in the escape from Fas-mediated cell death of colorectal cancer cells. Among 10 cell lines, ARK5 mRNA expression was observed in LoVo, SW480, and SW1116 cell lines. Interestingly, SW480 and SW1116 cell lines, but not LoVo cell line, showed expressions of both Fas ligand (FasL) and Fas mRNAs. SW620 cell line also showed FasL mRNA; however, Fas and ARK5 mRNAs were not detected. Furthermore, well-coincided expression among ARK5, FasL, and Fas mRNAs was observed in tumor tissues from patients with colorectal cancer, suggesting the suppression of FasL/Fas system-induced cell death by ARK5 in colorectal cancer cell lines. Intensive cell death, which was dependent on the FasL/Fas system was encountered when ARK5 antisense RNA (ARK5/AS) was introduced into SW480 cells. FLIP was expressed in only ARK5 mRNA-expressing cell lines, and ARK5/AS induced FLIP cleavage in a caspase-6-dependent manner. Amino-acid sequence analysis of caspase-6 revealed two putative sites of phosphorylation by ARK5 at Ser⁸⁰ and Ser²⁵⁷. Although active caspase-6 overexpression induced cell death in SW480 and DLD-1 cell lines, SW480 cells, but not DLD-1 cells, exhibited strong resistance to procaspase-6 overexpression. Moreover, mutant caspase-6, in which the Ser²⁵⁷ was substituted by Ala (caspase-6/SA), induced cell death and FLIP degradation, even in SW480 cells. Active ARK5 was found to phosphorylate wild-type caspase-6 in vitro, but not caspase-6/SA, and the prevented activation of caspase-6 was promoted due to its phosphorylation by active ARK5 in vitro. On the basis of the results of this study, we propose that ARK5 negatively regulates procaspase-6 by phosphorylation at Ser²⁵⁷, leading to resistance to the FasL/Fas system.