Abstract A24: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis via ectodomain shedding mediated by a disintegrin and metalloproteinase 9
Authors: Sonoda Kenzo 
Published: August 2014

Objectives: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a type-II membrane protein that can induce apoptosis in immune cells including peripheral lymphocytes and natural killer cells after converted to a secreted type by proteolytic processing referred to as “ectodomain shedding.” So far, over 150 scientific reports have been published concerning the biological functions and clinical significance of RCAS1. Because RCAS1 not only helps tumor cells to evade immune surveillance but also induces cancer stromal tissue remodeling, RCAS1 is believed to exaggerate aggressive characteristics of human malignancies. Clinically, RCAS1 expression correlates with several pathological variables including tumor size, stage, invasion depth, and lymph node metastasis and is a negative predictor of overall survival in 15 different kinds of cancer derived from brain, oral cavity, lung, pleural mesothelium, esophagus, stomach, bile duct, gallbladder, pancreas, colon, gastrointestinal mesenchyme, kidney, prostate, uterine cervix, and endometrium. RCAS1 is secreted significantly higher in serum and pleural effusion in cancer patients than non-cancer individuals and its level is changed in response to treatment. We here studied regarding ectodomain shedding that affects the biological activity of membrane proteins such as growth factors, growth factor receptors, cell-adhesion molecules, extracellular matrix proteins by altering their localization and mode of action. The objective of this study is to investigate (1) a key protease involved in RCAS1 shedding and (2) an association between tumor protease expression and serum RCAS1 concentration in uterine cancer patients.

Materials and Methods: (1) The key protease involved in RCAS1 shedding was investigated using two cell lines: a human cervical adenocarcinoma cell line SiSo and a breast adenocarcinoma cell line MCF-7. Both SiSo and MCF-7 express RCAS1, but RCAS1 secretion is undetectable in MCF-7. The protease expression was evaluated by microarray analysis and immnocytochemistry. The expression and secretion of RCAS1 were measured by using flow cytometry and ELISA after knockdown or introduction of protease expression. The induction of apoptosis in an erythroleukemia cell line K562, which express a putative RCAS1 receptor, was investigated. K562 cells were co-cultured with SiSo, MCF-7, and protease cDNA transfected MCF-7 to follow RCAS1 secretion in apoptosis initiation. (2) The association between tumor protease expression and serum RCAS1 concentration was studied in 50 cases of uterine cancer patients. The tumor protease expression was evaluated by immunohistochemistry and serum RCAS1 concentration was measured by ELISA.

Results: (1) Microarray analysis revealed that SiSo expressed MMP-28, ADAM9, and ADAM21 at significantly higher levels than did MCF-7. The association of RCAS1 and ADAM9 was detected by immunocytochemistry in SiSo. ADAM9 cDNA transfection in MCF-7 induced RCAS1 secretion and reduced its expression, on the other hand, ADAM9 siRNA transfection in SiSo suppressed RCAS1 secretion and augmented its expression. Growth inhibition and apoptosis were induced by ADAM9 cDNA tansfected MCF-7 but not parent cell. Therefore, it was revealed that ADAM9 is a key protease for RCAS1 shedding. (2) There was a significant correlation between tumor ADAM9 expression and serum RCAS1 concentration in uterine cancer.

Conclusions: Further exploration regarding the regulatory mechanisms involved in the conversion of membrane-anchored RCAS1 into its soluble form by ADAM9 should aid the development of novel therapeutic strategies against human malignancies by targeting RCAS1.

Citation Format: Kenzo Sonoda. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis via ectodomain shedding mediated by a disintegrin and metalloproteinase 9. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A24.