P53.08 Analysis of Mitochondria-Mediated OCIAD2 Oncogenic Function in Lung Adenocarcinoma

Abstract

Ovarian carcinoma immune-reactive antigen domain 2 (OCIAD2) has been reported as a novel tumor-specific protein (Cancer Sci, 2007). Overexpression of OCIAD2 is closely associated with the stepwise progression of early-stage lung adenocarcinoma and significantly associated with poorer outcomes in patients with ovarian and lung cancer (Pathol Int, 2012 and 2018). OCIAD2 is localized predominantly in the mitochondrial membrane in cancer cells. However, the oncogenic functions mediated by OCIAD2 are poorly understood. In this study, we explored the potential role of OCIAD2 in mitochondria and identified the OCIAD2 binding partner in lung adenocarcinoma cells. We performed most experiments after 72 hours of siRNA (scramble or siOCIAD2) transfection using lipofectamine RNAiMAX or after 24 hours of Flag-OCIAD2 plasmid transfection using Fugene HD. Alterations to mitochondrial morphology in the siRNA-transfected lung adenocarcinoma cells (A549 or HCC827) were observed using transmission electron microscopy (TEM). The siRNA-transfected cells were stained with MitoPT TMRM fluorescent dye and healthy cells were detected with electrical potential gradient by flow cytometry. To identify the OCIAD2-binding partners in lung adenocarcinoma cells, the membrane fraction including the mitochondria membrane was isolated from 3'-Flag-OCIAD2- or 5'-Flag-OCIAD2-transfected cells, followed by pull-down assay and LC-MS/MS analysis. We found that suppression of OCIAD2 led to marked destruction of morphologic features of mitochondria such as cristae in lung adenocarcinoma cells. The number of mitochondria per cell and the number of cristae per mitochondrion were significantly decreased, and there was a loss of mitochondrial electrical potential gradient in comparison with siCON. By LC-MS/MS analysis, we identified several OCIAD2-binding partner proteins that are significantly associated with apoptosis, crista formation, metabolic processes, membrane permeability, and transport. OCIAD2 is abnormally overexpressed in lung adenocarcinoma and may be associated with mitochondria-mediated oncogenic signaling such as blocking of apoptosis, active mitochondrial fitness, and maintenance of the mitochondrial electrical potential gradient through interaction with mitochondrial proteins. The fact that tumor cells are controlled by OCIAD2 and depend on its oncogenic function is of considerable interest.