NUCB2 – SEREX-identified antigen in gastric adenocarcinoma – possible reasons for immunogenicity

Z Slucka MSc^a, A Line PhD^a, J Miltins BSc^a, K Silina^a, A Stengrevics MD^b, R Bruvere MD^a, N Grabuseva MSc^a

^aBiomedical Research and Study Centre, University of Latvia, Riga, Latvia, ^bLatvian Oncology Center, Riga, Latvia

AIM. Identification and characterization of new cancer-associated antigens is of great importance for diagnostic and therapeutic purposes. In our study we applied a serological screening method SEREX to gastric cancer, which resulted in isolation of fourteen distinct serum-reactive cDNA clones. The aim of this study was to characterize one of the identified antigens NUCB2/NEFA, a calcium binding protein, in order to assess the nature of its immunogenicity and possible role in cancer etiology. METHODS. cDNA clone encoding NUCB2 was isolated by screening gastric cancer cDNA library with autologous patient sera. The frequency of antibody responses against NUCB2 was analysed in the set of healthy donor and cancer patient sera. In order to search for genetic alterations underlying the immunogenicity we used a range of analyses, including full-length sequencing of cDNA and 5’RLM-RACE analysis. NUCB2 mRNA and protein expression in normal and cancerous tissues was analyzed by comparative RT-PCR, Western blot and immunohistochemistry. RESULTS. Anti-NUCB2 antibody responses were found exclusively in gastric cancer patient sera but not in 35 healthy donor sera. NUCB2 mRNA expression analysis showed differential tissue distribution being highest in testis, spleen and stomach. Surprisingly we found that NUCB2 mRNA and protein levels are consistently decreased in more than 50% of gastric tumors when compared with the adjacent normal tissue. Although several DNA alterations in the protein-coding region of the gene were found, including three-nucleotide insertion/deletion, none of them turned out to be a somatic mutation, which might be responsible for the immunogenicity of NUCB2. Western blot analysis showed that NUCB2 is phosphorylated in cancerous and non-cancerous gastric tissues and demonstrated existence of yet unidentified posttranslational modifications in cancer tissues. 5’RLM-RACE analysis revealed a novel transcript variant of NUCB2 differing in its 5’ UTR, which is predominantly expressed in gastric cancer tissues and might be responsible for alterations in posttranslational modifications that we observed. Immunohistochemical analysis revealed NUCB2 expression in parietal cells of normal gastric mucosa and in infiltrative plasma cells in cancerous and non-cancerous stomach tissue. CONCLUSIONS. It has been proposed by Taniguchi et al that NUCB2 might have role in the regulation of cell death and survival by controlling calcium homeostasis in cytoplasm. Downregulation of NUCB2 expression in tumors may suggest its putative role in cancer progression but this would require determination of NUCB2 status in gastric gland progenitor cells in various stages of differentiation. The reasons of NUCB2 immunogenicity still remain unclear – one of the possibilities is that antibody production is elicited by altered posttranslational modifications in tumor tissues; alternatively it might be associated with autoimmune responses to parietal cells in gastritis or inflammatory processes.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 797 (Manifestations of cancer).