Original contributionThe role of Reg IV gene and its encoding product in gastric carcinogenesis☆
Introduction
Despite a worldwide decline in incidence and mortality beginning the second half of the 20th century, gastric cancer still ranks as the fourth most common cancer and the second most lethal cancer, accounting for 10.4% of cancer deaths worldwide. It continues to be a major health concern because of the slow decrease in incidence in Asia and high mortality from diagnosed gastric carcinomas in the West, although sophisticated diagnostic and operative techniques are widely applied in clinical practice [1], [2]. Tumorigenesis and progression of gastric carcinoma is a multistage process, involving a multifactorial etiology, which mainly results from gene-environmental interactions. Generally, it is believed that cancer develops as a result of multiple genetic and epigenetic alterations. Therefore, increased understanding of the changes in gene expression that occur during carcinogenesis, particularly identification of novel biomarkers for cancer diagnosis and novel targets for treatment, may lead to improvements in cancer diagnosis, treatment and prevention.
Regenerating (Reg) gene family belongs to the calcium depending lectin gene super family and encodes 4 multi-functional small-secreted proteins, which can function as acute phase reactants, lectins, or antiapoptotic or growth agents [3]. Reg IV, a novel member of the family, was identified by high-throughput sequencing of a cDNA library from ulcerative colitis (UC) tissues, implying its important role in initiating the multistep process of colorectal carcinogenesis. It is located on human chromosome 1q12-q21, whose cDNA contains an open reading frame of 477 bp encoding a peptide of 158 amino acids with a predicted molecular mass of 18 kd [4]. Although the biologic function of Reg IV is poorly understood, it has been reported that Reg IV is a potent activator of the epidermal growth factor receptor/Akt/activator protein 1 signaling pathway in colon cancer cells and increases the expression of Bcl-2, Bcl-xl, and surviving proteins, associated with the inhibition of apoptosis [5]. These findings indicate that Reg IV might function as a tissue mitogen or play a role in the cell growth. The colocalization of Reg IV and Ki-67 expression suggested that Reg IV might be associated with proliferative behavior of epithelial cells in line with the speculation mentioned above [6]. Reg IV also induces the expression of matrix metalloproteinase 7 (also known as matrilysin). The expression of Reg IV may contribute to liver metastasis through induction of matrix metalloproteinase 7 [5].
Recently, Nanakin et al [6] found that Reg IV mRNA was strongly expressed in inflamed epithelium, dysplasia, and cancerous lesions of UC tissues, and correlated with basic fibroblast growth factor and hepatocyte growth factor mRNA expression in UC tissues. In colon cancer cell line, Reg IV expression was enhanced by stimulation with transforming growth factor α, epidermal growth factor, basic fibroblast growth factor, and hepatocyte growth factor [7]. Taken together, it is concluded that Reg IV is inducible by growth factors and may function as a growth promoting and/or an antiapoptotic factor in the pathophysiologic process of UC. In addition to gastric, colorectal, and pancreatic, hepatocellular cancers, Reg IV mRNA overexpression has been reported in prostate carcinoma (PCa) [5], [8], [9], [10], [11], [12], [13]. Most primary PCa tumors expressed a low level of Reg IV mRNA, whereas most metastatic PCa tumors expressed its high level [13]. It was reported that 29% of colorectal cancer (CRC) cases were positive for Reg IV, and CRC cases with metastatic recurrence to the liver showed more frequently Reg IV staining. Patients with Reg IV-positive CRC had a significantly worse survival than those without Reg IV staining [7]. At the protein level, Reg IV protein is expressed in a few epithelial cells showing neuroendocrine and mucin-producing features [14], [15]. However, Reg IV expression is abundantly enhanced, and the distributions of Reg IV and chromogranin A were apparently distinct in UC tissues [6]. Recently, it has been reported that forced expression of Reg IV induces phosphorylation of the epidermal growth factor receptor and inhibits 5-fluorouracil–induced apoptosis in gastric cancer cells [16].
Immunohistochemical analysis revealed that Reg IV is expressed in gastric, colorectal, and pancreatic cancer, but not in lung and breast cancer [15]. Therefore, Reg IV may serve as a marker of digestive organ cancer. In the present study, Reg IV expression was for the first time examined in a large number of gastric samples including carcinoma, adjacent nonneoplastic mucosa (NNM), adenoma, intestinal metaplasia (IM), or gastritis in combination of immunohistochemistry (IHC), in situ hybridization (ISH), tissue microarray (TMA) and compared with the clinicopathologic parameters of carcinomas, as well as prognosis to explore the clinicopathologic significance and molecular roles of Reg IV expression in stepwise development of gastric carcinoma. In addition, gastric carcinoma cell lines and frozen carcinoma tissues were subjected to Western blot or reverse transcriptase–polymerase chain reaction (RT-PCR). We also assessed the serum Reg IV level in patients with gastric carcinoma by enzyme-linked immunosorbent assay (ELISA) to determine its potential diagnostic utility.
Section snippets
Subjects
Gastric adenocarcinomas (n = 372) and adjacent nonneoplastic mucosa (NNM; n = 44) were collected from surgical resection and gastric intestinal metaplasia (n = 63), adenoma (n = 42), and gastritis (n = 93) from endoscopic biopsy or polypectomy in Takaoka Citizen Hospital, Kouserein Takaoka Hospital, or the Affiliated Hospital, University of Toyama, between 1993 and 2007. The IM or gastritis was sometime present in the NNM adjacent to carcinoma and included in ISH analysis but not in IHC. The
Reg IV expression in gastric carcinoma cell lines or samples and its serum level in carcinoma patients
As shown in Fig. 1A, Reg IV protein strongly existed in MKN45, AGS, and HGC-27 but weakly in MKN28 and KATO-III. In contrast, no positive immunoreactivity to Reg IV was observed in 5 carcinoma cell lines by IHC (data not shown). To check its mRNA expression, we performed RT-PCR and observed a little weak expression of Reg IV mRNA in MKN28 and HGC-27 cell lines in comparison with the others (Fig. 2B). In addition, all the amplicons were subjected to direct DNA sequencing and proved correct (Fig.
Discussion
In the present study, we found that Reg IV expression underwent up-regulation and then down-regulation from gastritis to carcinoma through precancerous lesions like IM and adenoma in line with other reports [9], [15]. Our ISH results also showed strong Reg IV mRNA expression in adjacent IM in comparison with adjacent NNM or carcinoma. In contrast, there was no significant difference in Reg IV protein and mRNA expression between gastric carcinoma and adjacent NNM according to the results of ISH
Acknowledgments
The authors thank Tokimasa Kumada and Hideki Hatta for their technical help and Xing-hua Luan for her manuscript's revision.
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This study was supported by Shenyang Outstanding Talent Foundation of China, Liaoning BaiQianWan Talents Program; Scientific and Technological Projects for Overseas Returning Persons; Grant-in aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology of Japan (20659109; 21790624); and Smoking Research Foundation.