Abstract 1123: TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells

Abstract

Abstract [Introduction] Osteosarcoma (OS) is a rare malignant bone tumor and affects predominantly adolescents and young adults. There is no molecular targeted therapy for OS and breakthrough intervention has long been awaited for decades. Disturbances in differentiation towards mature osteoblast from mesenchymal stem cell are related to OS pathogenesis. Wnt signaling is a key component in managing this differentiation process. We previously reported that TNIK was a positive regulator of Wnt signaling. To develop the first molecular targeted therapy for OS patients, we focused on Wnt pathway-targeted therapy in OS and initiated this study to demonstrate TNIK as a novel therapeutic target for OS treatment. [Methods] Eleven different osteosarcoma cell lines were used in the present study. Twenty clinical samples were obtained at biopsy and surgical resection from ten OS patients. TNIK expression was assessed by western blotting, immunofluorescence and immunohistochemical staining. The generation of the novel TNIK inhibitor was previously described (Masuda et al., 2016). Cells were cultured with TNIK inhibitor or siTNIK. Cell viability was evaluated by real-time cell analysis system or ATP quantitation assay. The effect of TNIK inhibitor in vivo was also assessed by a mouse xenograft model. RNA-seq was performed using the RNA extracted from vehicle or TNIK inhibitor-treated OS cells. Cancer stem cell (CSC)-like properties were assessed by soft-agar colony-formation assay, limiting dilution assay, ALDH activities and the expression of CSC markers. Adipogenic phenotypes were qualitatively and quantitatively analyzed by Oil red O staining and the expression of adipogenesis markers. [Results] TNIK was identified in all cell lines and most of cases. Immunohistochemistry of OS tissue microarray also revealed that 52 out of 55 OS samples were positive for TNIK protein. TNIK inhibitor suppressed OS cell proliferation both in vitro and in vivo. SiTNIK also inhibited OS cell growth. Global gene expression analysis revealed a significant decrease in the expression of the genes relating to Wnt signaling pathway and maintenance of stem cell pluripotency. Soft-agar colony-formation assay and limiting dilution assay revealed that TNIK inhibitor decreased sphere formation activities in OS cells. Accordingly, TNIK inhibitor significantly suppressed ALDH activities and the expression of the proteins that are involved in the maintenance of CSCs, including NANOG, SOX2, OCT4A and MYC. Concomitantly, TNIK inhibitor drove adipogenic transdifferentiation of OS cells with PPARG activation. TNIK knock-down U2OS cells created by shRNAs showed nearly identical results. [Conclusion] We present that TNIK inhibition promotes adipogenic transdifferentiation of OS cells and eliminates CSCs. Our study thus suggests the feasibility of TNIK as a target for OS treatment. Citation Format: Toru Hirozane, Mari Masuda, Naoko Goto, Teppei Sugano, Naofumi Asano, Eisuke Kobayashi, Keisuke Horiuchi, Hideo Morioka, Akira Kawai, Masaaki Sawa, Morio Matsumoto, Masaya Nakamura, Tesshi Yamada. TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1123.