Abstract B17: Traf2- and NCK-interacting kinase (TNIK) inhibitor down-regulates Wnt signaling pathway in cancer cells

Abstract

Abstract Introduction: Aberrant activation of the Wnt signaling pathway has been implicated as the key driver of carcinogenesis, particularly in colorectal cancers. Because nearly 90% of colorectal cancers carry mutations either APC or β-catenin, downstream signaling molecules of the β-catenin destructive complex are considered as more attractive therapeutic targets in drug discovery research for the treatment of colorectal cancers. Recently, Traf2- and NCK-interacting kinase (TNIK) has been identified as one of components of the T-cell factor-4 (TCF4) transcriptional complex and essential for the expression of Wnt target genes. Knockdown of TNIK gene expression suppressed the transcriptional activity of the Wnt signaling pathway, and decreased in proliferation of colorectal cancer cells. Therefore inhibitors of TNIK may represent a promising therapeutic approach for treating Wnt-signal activated cancers. Previously we reported a thiazol-based TNIK inhibitor, N5355 have single digit nM IC50 value and the compound significantly down-regulated the Wnt target genes such as AXIN2 and cMYC in colorectal cancer cell line, HCT116. Further evaluations of N5355 have been implemented to characterize the effects of TNIK inhibition on cancer cell lines. Methods: To study the effects of N5355 on the Wnt signaling pathway, the expression levels of Wnt target proteins were analyzed by Western blotting using Wnt-signal activated cancer cell lines. The ability of N5355 for blocking of nuclear translocation of phosphorylated TNIK was also analyzed by Western blotting using HCT116 cells. Anti-proliferative activity profile of N5355 was studied using a panel of 44 cancer cell lines. The pharmacokinetic profile of TNIK inhibitor was also analyzed. Results: The Wnt signal inhibition was validated by significant suppression of Wnt target protein expressions by treating with N5355 in several cancer cells. In addition, Wnt co-receptor proteins LRP6 and LRP5 were found to be down-regulated by N5355 treatment. N5355 exhibited strong anti-proliferation activities against Wnt-driven cancer cell lines. Interestingly in DLD1 colorectal cancer cell lines, N5355 showed no anti-proliferative effect in conventional 2D cell culture assay, but displayed strong inhibitory activity in 3D cell culture assay using soft agar, which is biologically relevant assay system. The fact that N5355 inhibited the autophosphorylation of TNIK in the cytosol and subsequent nuclear translocation of TNIK in HCT116 cells suggested the depletion of TNIK proteins in nucleus resulted in the observed Wnt signal inhibition effect. Detailed results will be presented in the conference. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Mari Masuda, Masaaki Sawa, Tesshi Yamada. Traf2- and NCK-interacting kinase (TNIK) inhibitor down-regulates Wnt signaling pathway in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B17.