Background & Aim: Riboflavin (vitamin B2) deficiency has been shown to link with various types of human diseases, especially as a risk factor for esophageal squamous cell carcinoma (ESCC) in high incidence areas of China. Recently, a genome-wide association study (GWAS) identified C20orf54 as one of the susceptibility loci to ESCC. Since C20orf54 loci contain human riboflavin transporter 2 (RFT2) that can transport riboflavin into cells specifically and efficiently, these results suggested that RFT2 may play an important role in ESCC tumorigenesis. The aim of this study was to investigate the function and regulation of RFT2 involved in human esophageal carcinogenesis.

Methods: A total of 156 human ESCC patients who underwent complete resection were recruited. The mRNA expression of RFT2 in tumor tissues and paired adjacent normal epithelial tissues were determined by TaqMan Real-Time PCR assay. Two single nucleotide polymorphisms (SNPs) rs13042395 and rs6140125 identified in GWAS were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Direct sequencing was employed to determine for mutations of RFT2. Meanwhile, ESCC cell lines, KYSE30 and KYSE150, were used as model systems to study the function and regulation of RFT2 in controlling cell proliferation, cell cycle progression, colony formation, anchorage-independent growth, mitochondrial membrane potential (Δψm), ATP metabolism, apoptosis and tumorigenicity in nude mice.

Results and Conclusion: The mRNA expression of RFT2 was significantly up-regulated in tumor tissues as compared to their corresponding normal tissues in 156 ESCC patients (P<0.0001). There was, however, no significant difference for RFT2 expression among different genotypes of rs13042395 and rs6140125 in these ESCC patients. No somatic mutation or deletion was detected in RFT2 in 26 ESCC tumor samples. Knockdown of endogenous RFT2 in KYSE30 cells, which expresses high levels of RFT2 protein, markedly prohibited cell proliferation, inhibited anchorage-independent growth, reduced the cellular ATP levels and decreased mitochondrial membrane potential (Δψm), inducing cell cycle arrest at G1/S phase and apoptosis. By contrast, overexpression of RFT2 in KYSE150 cells, which expresses low levels of RFT2 protein, significantly promoted cell proliferation and anchorage-independent growth, resulting in an enhanced tumorigenicity in nude mice. Overexpression of RFT2 in KYSE150 cells also strongly suppressed cell apoptosis after cells were treated with cisplatin (DDP). Thus, our study indicates that RFT2 has a critical role in regulating cell proliferation, cellular energy metabolism, cell cycle progression, apoptosis and tumorigenicity in human ESCCs. In addition, overexpression of RFT2 appears to induce cisplatin resistance with an anti-apoptosis effect, suggesting that RFT2 may serve as a novel therapeutic target in ESCC.

Citation Format: Xingran Jiang, Xiying Yu, Jinhu Fan, Liping Guo, Wei Jiang, Shih-Hsin Lu. RFT2 is involved in human esophageal carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1293. doi:10.1158/1538-7445.AM2013-1293