Molecular cloning and characterization of a novel esophageal cancer related gene

We previously identified four novel cDNA fragments related to human esophageal cancer. One of the fragments was named esophageal cancer related gene 2 (ECRG2). We report here the molecular cloning, sequencing, and expression of the ECRG2 gene. The ECRG2 cDNA comprises a 258 bp nucleotide sequence which encodes for 85 amino acids with a predicted molecular weight of 9.2 kDa. Analysis of the protein sequence reveals the presence at the N terminus of a signal peptide followed by 56 amino acids with a significant degree of sequence similarity with the conserved Kazal domain which characterizes the serine protease inhibitor family. Pulse-chase experiments showed that ECRG2 protein was detected in both cell lysates and culture medium, indicating that the ECRG2 protein was extracellularly secreted after the post-translational cleavage. In vitro uPA/plasmin activity analysis showed the secreted ECRG2 protein inhibited the uPA/plasmin activity, indicating that ECRG2 may be a novel serine protease inhibitor. Northern blot analysis revealed the presence of the major band corresponding to a size of 569 kb throughout the fetal skin, thymus, esophagus, brain, lung, heart, stomach, liver, spleen, colon, kidney, testis, muscle, cholecyst tissues and adult esophageal mucosa, brain, thyroid tissue and mouth epithelia. However, ECRG2 gene was significantly down-regulated in primary esophageal cancer tissues. Taken together, these results indicate that ECRG2 is a novel member of the Kazal-type serine protease inhibitor family and may function as a tumor suppressor gene regulating the protease cascades during carcinogenesis and migration/invasion of esophageal cancer.