Frequent loss of heterozygosity (LOH) on human chromosome 7q31 has been reported in numerous malignancies. Suppressor of tumorigenicity 7 (ST7) has been identified as a candidate tumor suppressor gene in this region. To identify whether 7q31 and genetic alterations of ST7 were involved in human esophageal carcinogenesis, we performed LOH mapping of a 5.4 cM region at 7q31-q35 in 43 primary esophageal carcinomas, as well as mutational analyses of the ST7 gene in tumors with LOH in this region. Of 43 tumors, 12 (28%) showed LOH at 7q31–q35. These included four (22%) of 18 squamous cell carcinomas and eight (32%) of 25 adenocarcinomas. The peak LOH locus was D7S480, lying 4.2 Mb telomeric to ST7 and showing LOH in eight of 37 informative tumors, or 22%. No mutations were found in the entire coding or flanking intronic regions of the ST7 gene among 12 tumors with 7q-LOH. In addition, quantitative RT–PCR analyses of ST7 mRNA expression levels in 11/13 normal-tumor pairs failed to show more than a 50% decrease in tumor ST7 mRNA relative to matched normal tissues. These data suggest that LOH at 7q31–q35 is involved in the origin or progression of at least a subset of esophageal carcinomas, but that ST7 is not the target gene of this somatic event.
Esophageal carcinoma is one of the most common cancers worldwide, with a very poor prognosis and a marked increase in prevalence in developed countries (Miller et al., 1996). To make inroads against this deadly disease, it will be critical to clarify carcinogenic pathways underlying it, including the inactivation of tumor suppressor genes (TSGs). Several abnormalities have been reported in esophageal cancer, notably including frequent p53 mutation and loss of heterozygosity (LOH) involving numerous chromosomal arms (Hollstein et al., 1990; Shibagaki et al., 1994; Riegman et al., 2001; Roth et al., 2001).
Chromosomal arm 7q31 has been implicated as a TSG site because of frequent LOH at this locus in a variety of tumors of epithelial origin (Liang et al., 1998; van Dekken et al., 1999, Zenklusen et al., 1994b,1995,1999). Furthermore, introduction of a single chromosome 7 inhibited the tumorigenicity of a mouse squamous cancer cell line (Zenklusen et al., 1994a). Lately, Zenklusen et al. reported that suppressor of tumorigenicity 7 (ST7) is a candidate TSG at 7q31, showing frequent mutation in colon and breast cancers. They also demonstrated that the introduction of exogenous wild-type ST7 cDNA suppresses the in vivo tumorigenicity of PC3, a human prostate cancer cell line with LOH at 7q31 (Zenklusen et al., 2001). Since ST7 is also expressed in the normal esophagus (Zenklusen et al., 2001), we assumed that this gene might be involved in the development of esophageal cancer. Thus, we conducted LOH mapping of 7q31–q35 in esophageal cancers, tested LOH-positive tumors for somatic sequence alterations of the ST7 gene, and analysed ST7 mRNA expression levels in a subset of primary tumors.
LOH mapping was performed on 43 paired normal and tumorous esophageal tissues, consisting of 18 squamous cell carcinomas (SCCAs) and 25 adenocarcinomas (ADCAs). Four microsatellite markers localized within a 5.4 cM region at 7q31–q35 including the ST7 gene were used, i.e., D7S523, D7S480, D7S486, and D7S490 ( Table1). The ST7 gene is located within a l.9 cM region between D7S486 and D7S480. LOH was diagnosed when a more than 50% diminution in peak area was observed in tumor compared with its corresponding normal tissue. Of 43 tumors, 12 (28%) showed LOH at one or more of the four loci analysed (Figure1). These consisted of four (22%) of 18 SCCAs and eight (32%) of 25 ADCAs. D7S480 was the locus with the highest LOH rate (8/37 informative cases, or 22%). There was no significant difference in LOH frequency between SCCAs and ADCAs at any locus. The most commonly deleted region was between D7S480 and D7S486, within which the ST7 gene localizes.
Next, we sequenced the ST7 coding and intronic regions in genomic DNAs from 12 tumors with LOH at 7q31–35, in order to test whether ST7 is a target of LOH occurring at this locus. Mutation within the entire coding region as well as flanking intronic regions was examined by direct DNA sequencing. Breast cancer cell line MDA-MB231, which was previously reported to carry a frameshift mutation in the ST7 gene (insertion of a T at nucleotide 1368; Zenklusen et al., 2001), and normal esophageal mucosa were included as positive and negative controls, respectively. All sequencing analyses were performed on both sense and antisense strands at least twice for data verification. No mutations were found in either the entire coding region or flanking intronic regions of ST7 among any of the 12 tumors studied or in MDA-MB231 cells.
In order to evaluate whether LOH was associated with diminished expression of ST7 RNA, we performed quantitative real-time RT–PCR. A total of 13 esophageal cancers were examined, including eight SCCAs and five ADCAs, along with corresponding normal esophageal mucosal tissues. Relative expression levels of ST7 mRNA were measured and normalized by 18S ribosomal RNA. Figure2 displays relative ST7 mRNA expression levels after normalization. ST7 mRNA expression was detected in all normal esophageal tissues, although levels were highly variable. Furthermore, the vast majority of tumors did not show a decrease in ST7 mRNA levels relative to their corresponding normal esophageal tissues. There was no significant difference between SCCAs and ADCAs at the relative ST7 mRNA expression level. Three tumors showed greater than 50% upregulated expression compared to their corresponding normal tissues, while only two tumors exhibited more than 50% downregulation of ST7 mRNA expression.
In this study, we conducted LOH mapping of 7q31–q35, a region found to be deleted frequently in malignancies of multiple organs (Zenklusen et al., 1994b,1995,1999; Liang et al., 1998), in primary human esophageal cancers, and found LOH in 28% of cancers tested. The deleted region examined in our study included a recently described candidate TSG, ST7 (Zenklusen et al., 2001). Since it was already known that ST7 mRNA expression was detectable in normal esophagus (Zenklusen et al., 2001), we hypothesized that ST7 might also be a TSG at this locus in esophageal carcinogenesis, and examined somatic alterations of this gene in esophageal cancers. However, we did not find mutations or diminished expression of ST7 in these primary cancers. Tumors in our study were selected for mutational studies based on LOH at the ST7 flanking regions, which should have ensured a higher prevalence of mutation if ST7 was a target of LOH events in these tumors. Quantitative real-time measurement of mRNA expression levels also suggested that ST7 is not likely to be inactivated by epigenetic events such as methylation or haploinsufficiency, since these events would have been expected to result in diminished or absent ST7 expression. Altogether, these data imply that ST7 does not play a dominant role in esophageal carcinogenesis.
To our knowledge, two additional mutational analyses of the ST7 gene have been published (Hughes et al., 2001; Thomas et al., 2001,). Both of these studies failed to find any mutations in primary tumors, suggesting that ST7 is not the responsible TSG at this locus, at least in ovarian, breast, and colorectal cancers. Moreover, in agreement with our results, these two studies did not find mutations in the cell lines reported by Zenklusen et al. to carry mutations. The reason for these contradictory findings of ST7 mutations in the cell lines remains unclear. As stated by Thomas and Hughes, the mutation described by Zenklusen et al. may have been acquired during cell culturing passages.
The existence of a TSG at 7q31 is supported by the LOH rate that we found in esophageal cancers, although its identity was not determined in this study. There are potential TSGs other than ST7 that localize in this region including testis-derived transcript (TES) and inhibitor of growth family member three (ING3). Loss of expression and methylation at the 5′ CpG island of TES are found frequently in various human cancers (Tatarelli et al., 2000; Tobias et al., 2001). ING3 belongs to a TSG family, and decreased mRNA expression of ING3 is frequent among head and neck cancers (Gunduz et al., 2002). Further investigation is required to clarify the identity of any mutative target gene at 7q31.
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This work was supported by USPHS grants CA95323, CA85069, CA77057, DK (to SJM), and the Medical Research Office, Department of Veterans Affairs (to SJM).
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