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. 2020 Oct 14;12(10):2976.
doi: 10.3390/cancers12102976.

FGFR4 Inhibitor BLU9931 Attenuates Pancreatic Cancer Cell Proliferation and Invasion While Inducing Senescence: Evidence for Senolytic Therapy Potential in Pancreatic Cancer

Norihiko Sasaki  1 , Fujiya Gomi  2 , Hisashi Yoshimura  3 , Masami Yamamoto  3 , Yoko Matsuda  4 , Masaki Michishita  5 , Hitoshi Hatakeyama  6 , Yoichi Kawano  7 , Masashi Toyoda  1 , Murray Korc  8 , Toshiyuki Ishiwata  2
Affiliations

FGFR4 Inhibitor BLU9931 Attenuates Pancreatic Cancer Cell Proliferation and Invasion While Inducing Senescence: Evidence for Senolytic Therapy Potential in Pancreatic Cancer

Norihiko Sasaki et al. Cancers (Basel). .

Abstract

Fibroblast growth factor receptor 4 (FGFR4), one of four tyrosine kinase receptors for FGFs, is involved in diverse cellular processes. Activation of FGF19/FGFR4 signaling is closely associated with cancer development and progression. In this study, we examined the expression and roles of FGF19/FGFR4 signaling in human pancreatic ductal adenocarcinoma (PDAC). In human PDAC cases, FGFR4 expression positively correlated with larger primary tumors and more advanced stages. Among eight PDAC cell lines, FGFR4 was expressed at the highest levels in PK-1 cells, in which single-nucleotide polymorphism G388R in FGFR4 was detected. For inhibition of autocrine/paracrine FGF19/FGFR4 signaling, we used BLU9931, a highly selective FGFR4 inhibitor. Inhibition of signal transduction through ERK, AKT, and STAT3 pathways by BLU9931 reduced proliferation in FGF19/FGFR4 signaling-activated PDAC cells. By contrast, BLU9931 did not alter stemness features, including stemness marker expression, anticancer drug resistance, and sphere-forming ability. However, BLU9931 inhibited cell invasion, in part, by downregulating membrane-type matrix metalloproteinase-1 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. Thus, we propose that BLU9931 is a promising therapeutic agent in FGFR4-positive PDAC, especially when combined with senolysis (195/200).

Keywords: FGFR4; FGFR4 inhibitor; growth; invasion; pancreatic cancer; senescence; senolytic therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
FGFR4 expression in human pancreatic tissues and PDAC cell lines. (A) Representative photographs of immunohistochemistry for FGFR4. In normal human pancreatic tissues, weak FGFR4 immunoreactivity was present in the normal exocrine and endocrine pancreas. AI: Arrowheads indicate endocrine islets, whereas arrow indicates ductal cells. AII: Strong FGFR4 immunoreactivity was present in the cytoplasm and cell membrane of human PDAC cells. Scale bar: 200 μm; n = 136 PDAC cases. Inset: strong membranous FGFR4 immunoreactivity is readily evident. Scale bar: 200 μm (B) Real-time qPCR analysis of FGFR4 in PDAC cell lines. Representative results from triplicate measurements are shown. Results shown were normalized to values obtained for PK-45P cells (value = 1). (C) Western blot analysis of FGFR4 was performed in PDAC cell lines. The expression of each band is shown under or above the blot. (D) FACS analysis of FGFR4 expression in PDAC cell lines. Controls are indicated by thin lines with gray color. (E) Cell surface levels of FGFR4 expression in PDAC cell lines. Mean fluorescence intensities (MFIs) from FACS analysis are shown. Results are presented as means ± SD from three independent experiments. (F) Real-time qPCR analysis of FGF19 in PDAC cell lines. Representative results from triplicate measurements are shown. Control PK-45P cells were assigned a value of 1, and all other values in this series of experiments were calculated in relation to this reference control value. (value = 1).
Figure 2
Figure 2
Oncoprints visualizing the FGF19 amplicon and analysis of related genes in human PDAC. (A) TCGA analysis of the 11q13.3 amplicon in PDAC revealed the presence of the following amplified genes: FGF19, FGF4, FGF3, CCND1, RAD9A, RPS6KB2, GAB2, and to a lesser extent PAK1. (B) TCGA data for FGFR4 encoding the receptor that is activated by FGF19, KLB encoding β-klotho, and FGFR1, FGFR2, and FGFR3 genes. (C) Real-time qPCR analysis of RNA from indicated cell lines was performed for FGF4, FGF3, CCND1, RAD9A, RPS6KB2, GAB2, PAK1, and KLB. Representative results from triplicate measurements are shown. Results shown were normalized to values obtained for PK-45P cells (value = 1).
Figure 3
Figure 3
Effects of BLU9931 on PDAC cells. (A) PDAC cells were incubated with or without BLU9931 for 3 days at the indicated concentrations and growth rates were determined. Results are presented as means ± SD from three independent experiments. (B) Quantification of apoptotic, necrotic, and live cells by flow cytometry in PK-1 cells incubated with or without 2 μM BLU9931 for 3 days. (C) Cell cycle analysis in PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 days. (D) Real-time qPCR analysis of cell cycle-related genes in PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 days. Results shown are normalized to values obtained for control cells (value = 1). Results are presented as means ± SD from three independent experiments. (E) Western blot analysis for FGF19/FGFR4 signaling was performed in PK-1 or PK-45P cells that were incubated with or without 2 μM BLU9931 for 3 days. The expression of each band is shown under the blot. The histograms show mean densitometric readings ± SD for the phosphorylated proteins normalized to those of the loading controls. Results are the means ± SD from three independent experiments. (F) Real-time qPCR analysis of FGFR4 or FGF19 or KLB in PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 days. Results are normalized to values obtained for control cells (value = 1), and are the as means ± SD from three independent experiments. * p < 0.05, ** p < 0.01. Control (Ctr): Control cells were incubated with dimethyl sulfoxide.
Figure 4
Figure 4
Effects of BLU9931 on stemness features of PDAC cells. (A) Real-time qPCR analysis of stemness markers in PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 days. Results shown are normalized to values obtained for control cells (value = 1). (B) Anticancer drug resistance assay in PK-1 cells that were incubated with or without 2 μM BLU9931. The dose response (10 or 100 μM) of PK-1 cells to gemcitabine, 5-FU, and abraxane was determined using the ATP assay. (C) Real-time qPCR analysis of transporters in PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 days. Results shown are normalized to values obtained for control cells (value = 1). (D) Sphere-forming assays performed in PK-1 cells that were incubated with or without 2 μM BLU9931. (E) Real-time qPCR analysis of FGFR4 or FGF19 in PK-1 cells cultured under adherent or 3-D conditions. Results shown are normalized to values obtained for adherent-cultured cells (value = 1). All histograms are presented as means ± SD from three independent experiments. * p < 0.05, ** p < 0.01. Control (Ctr): Control cells were incubated with dimethyl sulfoxide.
Figure 5
Figure 5
Effects of BLU9931 on migration and invasion of PDAC cells. (A) Wound healing/cell scratch assay was performed in PK-1 cells incubated with or without 2 μM BLU9931 for 3 days. Images show the times (0, 6, and 24 h) after the ibidi 2-well culture-insert was removed. The histograms show the average normalization of the wound migration rate in three experiments. (B) Migration assays were performed in PK-1 cells incubated with or without 2 μM BLU9931 for 3 days. Representative results from measurements of 12 fields are shown. (C) Matrigel invasion assays performed in PK-1 cells incubated with or without 2 μM BLU9931 for 3 days. Representative results from measurements of 12 fields are shown. (D) Real-time qPCR analysis of MMPs in PK-1 cells incubated with or without 2 μM BLU9931 for 3 days. Results shown are normalized to values obtained for control cells (value = 1). Results are the means ± SD from three independent experiments. (E) Western blot analysis of MT1-MMP was performed in PK-1 that were incubated with or without 2 μM BLU9931 for 3 days. The expression of each band is shown under the blot. The histograms show mean densitometric readings ± SD for MT1-MMP normalized to those of the loading controls. Results are the as means ± SD from three independent experiments. (F) Gelatin zymography was performed using culture supernatants from PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 days. Relative band intensity is shown. * p < 0.05, ** p < 0.01. Control (Ctr): Control cells were incubated with dimethyl sulfoxide.
Figure 6
Figure 6
Induction of senescence in PDAC cells by long-term incubation with BLU9931. (A) PK-1 cells that were incubated with or without 2 μM BLU9931 for 7 days were stained for SA-β-Gal activity. Representative images of staining for SA-β-Gal and DAPI are shown. Regions surrounded by dotted lines in the lower panel show representative senescent cells that are highlighted by arrows. (B) SA-β-Gal-positive cells in (A) were quantitated as a percentage of total cell numbers. Representative results from measurements of 4 fields are shown. (C) Immunocytochemical staining performed in PK-1 cells that were incubated with or without 2 μM BLU9931 for 3 or 7 days. Representative images are shown (γH2A.X, green; DAPI, blue). The associated histogram shows the percentage of γH2A.X-positive cells (mean ± SD) from four fields. (D) Telomere length assay by real-time PCR in PK-1 cells incubated with or without 2 μM BLU9931 for 7 days. Results are the means ± SD from three independent experiments. (E) Real-time qPCR analysis of SASP factors in PK-1 cells that were incubated with or without 2 μM BLU9931 for 7 days. Results shown are normalized to values obtained for control cells (value = 1). Results are the means ± SD from three independent experiments. (F) Real-time qPCR analysis of SIRT1 and SIRT6 in PK-1 cells that were incubated with or without 2 μM BLU9931 for 7 days. Results shown are normalized to values obtained for control cells (value = 1). Results are the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01. Control (Ctr): Control cells were incubated with dimethyl sulfoxide.
Figure 7
Figure 7
Effects of senolytic drugs on BLU9931-induced senescent PDAC cells. (A) All cells were incubated for 7 days in the presence or absence of 2 μM BLU9931. PK-1 cells were then incubated for 4 days with quercetin (12.5 or 25 μM) or dasatinib (31.25 or 62.5 nM) whereas PK-45P cells were incubated for 4 days with quercetin (12.5 or 25 μM) or dasatinib (3.9 or 7.8 nM). Cell viability was then measured by ATP assays. Results are the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01. Control (Ctr): Control cells were incubated with DMSO. (B) Schematic representation of autocrine/paracrine FGF19/FGFR4 signaling in PDAC cells. Klotho-β (KLB) binding to FGFR4 enables FGF19 binding to the FGFR4-KLB heterodimer and promotes FGFR4 dimerization, thereby activating downstream signaling, such as ERK, AKT, and STAT3. These pathways promote cell proliferation, upregulate membrane-type matrix metalloproteinase-1 (MT1-MMP) that contributes to enhanced cell invasion, and upregulate sirtuin1 (SIRT1) and SIRT6 to help suppress senescence. Additionally, FGF19 auto-induces FGF19 expression, thereby promoting an autocrine/paracrine loop for persistent FGF19-mediated activation of FGFR4. BLU9931, a highly specific and irreversible FGFR4 inhibitor, inhibits the tyrosine kinase activity of FGFR4 by interacting with cysteine residue 552 near FGFR4’s ATP binding region, thereby suppressing downstream signaling and impeding upregulation of the above genes.
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References

    1. Siegel R.L., Miller K.D., Jemal A. Cancer statistics, 2020. CA Cancer J. Clin. 2019;70:7–30. doi: 10.3322/caac.21590. - DOI - PubMed
    1. Korc M., Jeon C.Y., Edderkaoui M., Pandol S.J., Petrov M.S. Consortium for the study of chronic pancreatitis, diabetes, and pancreatic cancer (CPDPC). Tobacco and alcohol as risk factors for pancreatic cancer. Best Pract. Res. Clin. Gastroenterol. 2017;31:529–536. doi: 10.1016/j.bpg.2017.09.001. - DOI - PMC - PubMed
    1. Ahrendt S.A., Pitt H.A. Surgical management of pancreatic cancer. Oncology. 2002;16:725–734. - PubMed
    1. Kleeff J., Korc M., Apte M., La Vecchia C., Johnson C.D., Biankin A.V., Neale R.E., Tempero M., Tuveson D.A., Hruban R.H., et al. Pancreatic cancer. Nat. Rev. Dis Primers. 2016;21:16022. doi: 10.1038/nrdp.2016.22. - DOI - PubMed
    1. Ornitz D.M., Itoh N. The fibroblast growth factor signaling pathway. Wiley Interdiscip. Rev. Dev. Biol. 2015;4:215–266. - PMC - PubMed

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