Identification and characterization of novel oncogenes located in a homogeneously staining region (HSR) of human esophageal squamous cell carcinoma (ESCC)

Abstract

Esophageal squamous cell carcinoma (ESCC) has been reported to be caused by multiple genomic factors, such as amplification and overexpression of oncogenes, which lead to the malignant transformation of normal cells. In this study, a homogeneously staining region (HSR) was identified from an ESCC cell line SLMT-1 and was microdissected for FISH analysis to determine its chromosomal origin. The HSR was confirmed to be originated from chromosome 17p13.3 region with amplification and inversion. Further FISH analysis using the 17p BACs and array CGH analysis for chromosome 17p on SLMT-1 revealed that the amplicon was located within a 900kb region. According to the genomic data available at Ensembl release 46 (http://www.ensembl.org), mutliplex RT-PCR analysis for gene overexpression was performed for the eight non-tumor suppressor genes located within the amplicon on 14 ESCC cell lines compared to the immortalized non-tumor esophageal epithelial cell line. Among the genes studied, JC-1 showed the highest percentage of overexpression in 14 ESCC cell lines (78.6%; 11/14), and the translocase of inner mitochondrial membrane 22 (Timm22) showed the second highest overexpression percentage of 71.4% (10/14). Thus Timm22 and JC-1 were further investigated for their oncogenic properties. Overexpression of Timm22 and JC-1 in NIH-3T3 cells could enhance the proliferation rates; enhance the anchorage dependent and independent growth and the migration rates of transfected NIH 3T3 cells. Most importantly, the overexpression of Timm22 and JC-1 could also induce the formation of subcutaneous sarcomas in athymic nude mice. Immunohistochemical staining was performed for Timm22 in seven ESCC cell lines compared with the immortalized non-tumor esophageal epithelial cell lines. Three out of seven (42.9%) ESCC cell lines showed Timm22 protein overexpression. Further multiplex RT-PCR analysis on the primary ESCC patient samples revealed that 16 out of 29 samples (55.2%) showed overexpression of Timm22, which was most commonly detected in Stage IIa (8/16; 50%); Stage III (6/16; 37.5%) and moderately differentiated tumors (9/16; 56.3%), but only one moderately differentiated Stage IIa tumor showed JC-1 overexpression. The present study is thus the first study to demonstrate the oncogenic properties of Timm22 and JC-1 in ESCC with overexpression and the overall results suggest that Timm22 and JC-1 may play important roles in the molecular pathogenesis of ESCC.