Purpose: Fibroblast growth factor receptor 4 (FGFR4), one of the four tyrosine kinase receptors for FGFs, is involved in various cellular processes. Activation of the FGF19/FGFR4 signaling is strongly associated with cancer development and progression. BLU9931 is an irreversible and highly selective small-molecule inhibitor of FGFR4. In this study, we aimed to elucidate the role of FGF19/FGFR4 signaling using BLU9931 in human PDAC.
Experimental procedures: The expression of FGFR4 in eight human PDAC cell lines were examined using real-time PCR, immunoblotting, and FACS analysis. The effects of BLU9931 on FGFR4-positive PDAC cells were examined using growth assay, cell cycle assay, immunoblotting of downstream targets of FGF19/FGFR4 signaling, real-time PCR analysis of stemness markers, and sphere forming assays. Motility was evaluated using migration and invasion assays. Matrix metalloproteinase activity was evaluated using gelatin zymography. Induction of senescence was evaluated using senescence-associated β-galactosidase activity and real-time PCR analysis of senescence-associated, phenotype-related secretory genes. The synergistic effects of senolytic drugs on BLU9931-treated cells were evaluated using ATP assay. Expression and clinicopathological features of FGFR4 in PDAC patients were examined using immunohistochemical analysis of tissue microarrays.
Results: In human PDAC cases, FGFR4 expression was positively correlated with larger primary tumors and more advanced disease stage. Among the eight PDAC cell lines, FGFR4 was expressed at the highest level in PK-1 cell line, in which the single-nucleotide polymorphism G388R in FGFR4 was detected. Inhibition of signal transduction through the ERK, AKT, and STAT3 pathways by BLU9931 reduced proliferation of FGF19/FGFR4 signaling-activated PDAC cells. In contrast, BLU9931 did not alter stemness features, including stemness marker expression, anticancer drug resistance, and sphere-forming ability of PK-1 cells. However, BLU9931 inhibited cell invasion, in part, by downregulating membrane-type matrix metalloproteinase-1 (MT1-MMP) expression in FGF19/FGFR4 signaling-activated PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 contributed to senescence induction, priming these cells for quercetin-induced death, a process termed senolysis.
Conclusions: We demonstrated that inhibition of signal transduction pathways through the ERK, AKT, and STAT3 pathways by BLU9931 inhibited PDAC cell proliferation and invasion, in part by downregulating MT1-MMP expression in autocrine/paracrine FGF19/FGFR4 signaling-positive PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 may have contributed to senolysis in these cells. Thus, we propose that BLU9931 may be a promising drug for the treatment of FGFR4-positive PDAC.
Citation Format: Norihiko Sasaki, Fujiya Gomi, Hisashi Yoshimura, Masami Yamamoto, Yoko Matsuda, Masaki Michishita, Hitoshi Hatakeyama, Yoichi Kawano, Yoko Itakura, Masashi Toyoda, Murray Korc, Toshiyuki Ishiwata. FGFR4 inhibitor BLU9931 induces cellular senescence in pancreatic ductal adenocarcinoma cell lines promoting sensitivity to senolytic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2449.