Abstract 3968: Conophylline-target ARL6ip1 regulates Ran-mediated cellular migration and invasion in human colorectal cancer cells

Abstract Introduction: Conophylline is an alkaloid isolated from the leaves of Ervatamia microphylla. We isolated conophylline as an inhibitor of K-Ras functions. It is known to ameliorate various disease models in animals, including cancer, diabetes mellitus, NASH, and hepatic cirrhosis. On the other hand, its molecular target was determined to be ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) using a conophylline-biotin conjugate. ARL6ip1 is located in the endoplasmic reticulum (ER) membrane, and the conophylline-binding domain was determined by deletion mutation analysis. Known functions of ARL6ip1 include inhibition of apoptosis, inhibition of glutamate transporter, and modulation of the ER structure. However, whether ARL6ip1 is involved in the mechanisms of various biological activities of conophylline has not been proven, since knockdown of ARL6ip1 has often failed to change the cellular phenotypes. Therefore, in the present research, we knocked out ARL6ip1 in human colon carcinoma cells by CRISPR-Cas9 and studied the involvement and mechanism of ARL6ip1 in the anticancer activity of conophylline. Ran activity was measured by a pull-down assay with Ran-GTP antibody. Materials and Methods: Conophylline was isolated from the leaves of Ervatamia microphylla. We employed HCT116 and DLD1 cells as human colorectal cancer cells. Tumorigenicity was measured by soft agar colony formation. Cellular migration was measured by a wound healing assay and cell tracking analysis. Cellular invasion was measured by a Matrigel chamber assay. Results: Conophylline inhibited soft agar colony formation in HCT116 and DLD1 cells. It inhibited migration and invasion in HCT116 and DLD1 cells at nontoxic concentrations. Knockout of ARL6ip1 also decreased tumorigenicity, migration, and invasion in HCT116 and DLD1 cells. The mechanistic study was carried out with HCT116 cells. ARL6 and Ran are both G-proteins and are reported to interact with ARL6ip1. Although knockdown of ARL6 did not change the cell migratory activity, knockdown of Ran inhibited the migration. Conophylline inhibited the interaction of ARL6ip1 to Ran in the proximity ligation assay. Moreover, conophylline inhibited the Ran activity. Conclusion: CRISPR-Cas9 knockout of ARL6ip1 showed a similar anticancer activity as treatment with conophylline. It is likely that the anticancer activity of conophylline would be mediated by the ARL6ip1-Ran system. Thus, ARL6ip1 would be a useful molecular target for the treatment of cancer. Citation Format: Yinzhi Lin, Sivasundaram Karnan, Hideaki Ito, Kazuo Umezawa. Conophylline-target ARL6ip1 regulates Ran-mediated cellular migration and invasion in human colorectal cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3968.