Abstract 4071: Angiogenin enhances proliferation, progression and angiogenesis in human bladder cancer.

Introduction: Angiogenin (ANG) is a member of RNase A family and regulates synthesis of various proteins. ANG is reported to act on both vascular endothelial cells and cancer cells. Although it has been demonstrated that upregulated ANG plays important roles on cellular proliferation, migration and angiogenesis in several malignancies, there are very few reports addressing the relationship between ANG expression and bladder cancer. The aim of this study is to investigate the biological function of ANG in bladder cancer. Methods: UROtsa (benign urothelial cell line), RT4 (urothelial papilloma cell line), KU7 (non-invasive bladder cancer) and T24 (invasive bladder cancer) were used in this study. ANG-overexpressing stable clones in UROtsa were created using plasmid DNA transfection and ANG down-regulation in KU7 and T24 were created using siRNA transfection or short-hairpin RNA (shRNA) transfection. Proliferation assay, migration/invasion assay, anchorage-independent colony formation assay in soft agar and in-vitro Matrigel tube formation assay using HUVEC were carried out with conditioned media. ANG overexpression clone (UROtsa) and ANG shRNA expressing clones (KU7 and T24) were subcutaneously injected into nude mice to investigate whether ANG expression increase tumorigenicity and tumor growth in vivo. Results: Higher level of ANG was expressed in cancer cell lines, KU7 and T24, compared to non-cancer cell lines, UROtsa and RT4. Proliferation assay and soft agar assay revealed that ANG expression was associated with cell growth and anchorage independent growth in the mediation of dephophorylation of ERK1/2 and JNK/SAPK. Migration was inhibited significantly by knocking down ANG in KU7 (non-invasive cancer), while invasion was inhibited in T24 (invasive cancer). An in vitro Matrigel angiogenesis assay demonstrated that conditioned media collected from ANG overexpressing stable clones resulted in increased tube formation as compared to the control clone in UROtsa, while the tube formation ability was decreased by knocking down ANG in cancer cells. In tumorigenicity testing in nude mice, tumor growth was correlated with higher expression of ANG. Four weeks after inoculation of cells in nude mice, there was a significant difference in tumor size in all the cell lines (UROtsa control vs ANG overexpression, KU7 control shRNA vs ANG shRNA, T24 control shRNA vs ANG shRNA), suggesting that ANG expression supported tumorigenicity and tumor growth. Conclusions: These results suggest that ANG overexpression enhances tumor growth and progression. ANG secreted from cancer cells promotes angiogenesis in bladder cancer. Therefore, ANG may be a potential therapeutic target in bladder cancer. Citation Format: Makito Miyake, Steve Goodison, Evan Gomes Giacosia, Jeongsoon Kim, Shanti Ross, Ge Zhang, Charles J. Rosser. Angiogenin enhances proliferation, progression and angiogenesis in human bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2013-4071