Targeting FGFRs by pemigatinib induces G1 phase cell cycle arrest, cellular stress and upregulation of tumor suppressor microRNAs

Abstract

Background: Fibroblast growth factor receptor (FGFR) gene family alterations are found in several cancers, indicating their importance as potential therapeutic targets. The FGFR-tyrosine kinase inhibitor (TKI) pemigatinib has been introduced in the treatment of advanced cholangiocarcinoma and more recently for relapsed or refractory myeloid/lymphoid neoplasms with FGFR2 and FGFR1 rearrangements, respectively. Several clinical trials are currently investigating the possible combination of pemigatinib with immunotherapy. In this study, we analyzed the biological and molecular effects of pemigatinib on different cancer cell models (lung, bladder, and gastric), which are currently objective of clinical trial investigations.

Methods: NCI-H1581 lung, KATO III gastric and RT-112 bladder cancer cell lines were evaluated for FGFR expression by qRT-PCR and Western blot. Cell lines were treated with Pem and then characterized for cell proliferation, apoptosis, production of intracellular reactive oxygen species (ROS), and induction of senescence. The expression of microRNAs with tumor suppressor functions was analyzed by qRT-PCR, while modulation of the proteins coded by their target genes was evaluated by Western blot and mRNA. Descriptive statistics was used to analyze the various data and student's t test to compare the analysis of two groups.

Results: Pemigatinib exposure triggered distinct signaling pathways and reduced the proliferative ability of all cancer cells, inducing G1 phase cell cycle arrest and strong intracellular stress resulting in ROS production, senescence and apoptosis. Pemigatinib treatment also caused the upregulation of microRNAs (miR-133b, miR-139, miR-186, miR-195) with tumor suppressor functions, along with the downregulation of validated protein targets with oncogenic roles (c-Myc, c-MET, CDK6, EGFR).

Conclusions: These results contribute to clarifying the biological effects and molecular mechanisms mediated by the anti-FGFR TKI pemigatinib in distinct tumor settings and support its exploitation for combined therapies.

Keywords: Apoptosis; Calreticulin; Cell cycle arrest; Cellular stress; FGFR; Pemigatinib; ROS; Senescence; TKI; miRNA.

Fig. 1

FGFR expression profile in cancer cell lines and downstream signaling pathways. A Expression of FGFR1 and 2 in H1581 lung cancer cells by Western blot. GAPDH was employed as reference marker for relative protein expression and histograms represent the relative band intensity calculated as mean ± SEM of three independent experiments. B Expression of FGFR2 in KATO III gastric cancer cells by Western blot. β-actin was employed as reference marker for relative protein expression and histograms represents the relative band intensity calculated as mean ± SEM of three independent experiments. C Expression of FGFR3 and FGFR3-TACC3 fusion in RT-112 bladder cancer cell line by Western blot analysis. The FGFR3 western blot (left panel) recognizes both native FGFR3 and the FGFR3-TACC3 fusion protein with slightly higher molecular weight. The TACC3 western blot (panel below) recognizes both native TACC3 and the FGFR3-TACC3 fusion protein with slightly higher molecular weight. β-actin was employed as reference marker for relative protein expression and histograms represent the relative band intensity calculated as mean ± SEM of three independent experiments. D Western blot analysis of c-RAF/p–c-RAF, AKT/p-AKT, ERK1/2/p-ERK1/2 in untreated (NT) and treated (Pem) H1581 (upper panels), KATO III (middle panels) and RT-112 (bottom panels) cells. GAPDH and β-actin were employed as reference markers. **p < 0.01; ns, not significative

Fig. 2

Pemigatinib treatment modulates proliferation and cell cycle arrest in cancer cell lines. A Effect of Pem (100 nM) on the proliferation of H1581, KATO III and RT-112 cells after 24 h and 48 h; B The flow cytometry histogram plots and bar-plots represent the mean ± SEM of Ki67-BV421 geometric mean of fluorescence in untreated (NT) and Pem-treated (100 nM) H1581, KATO III and RT-112 cells. C Effect of Pem (100 nM) on the G1 phase-cell cycle. Cell cycle analysis was performed by propidium iodide (PI) staining by flow cytometry. The flow cytometry histogram plots are representative of the cell cycle of one experiment at 24 h and 48 h and the histogram bar-plots represent the count of PI-positive cells found in G1 phase in untreated (NT) and Pem-treated (100 nM) H1581, KATO III and RT-112 cells; All experiments are represented as the mean of three independent experiments ± SEM. *p < 0.05; **p < 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Student’s t test

Fig. 3

Effect of pemigatinib on apoptosis and calreticulin exposure in cancer cell lines. A Apoptosis of cancer cells exposed to Pem (100 nM) at 24 h and 48 h. Untreated cells (NT) were employed as control. Apoptosis was detected as Annexin V-7-AAD staining by flow cytometry. Histograms correspond to the average percentage of apoptotic cells of three independent experiments ± SEM. *p < 0.05; **p < 0.01; ***p ≤ 0.001. B Dot-plot graphs are representative of the expression of Annexin V and 7-AAD nuclear intercalant of one experiment for each cancer cell line untreated (NT) and treated with Pem. C Calreticulin cell surface expression after Pem exposure (100 nM) was evaluated by flow cytometry. The results are plotted as the ratio between the percentages of calreticulin-positive pemigatinib-treated cells and calreticulin-positive Pem-untreated cells. D Dot-plot graphs are representative of the expression of Calreticulin-PE and 7-AAD nuclear intercalant of vital cells of one experiment for each cancer cell line untreated (NT) and treated with Pem. Each histogram represents the average of values of 3 independent experiments. Variability is expressed as the SEM. *p < 0.05; **p < 0.01; ***p ≤ 0.001

Fig. 4

Effect of pemigatinib on ROS and senescence in cancer cell lines. A Flow cytometry plots (left panel) and immunofluorescence staining (40 × magnification) with DCFH-DA probe (right panel) of intracellular ROS in cancer cells untreated and treated with Pem (100 nM). The histogram results are plotted as the fold change in the MFI of the treated (light blue) vs. the untreated samples (pink) of three independent experiments. *p < 0.05; **p < 0.01; ***p ≤ 0.001. B Cellular senescence. Western blot of lamin B, p21 and γ-H2A.X protein expression in untreated (NT) and treated (Pem) H1581, RT-112 and KATO III cells at 48 h. GAPDH and β-actin were used as reference markers (left panel). β-Galactosidase staining of untreated and Pem-treated H1581, KATO III and RT-112 cells at 48 h was assessed with a magnification of 40X under a Leyca microscope (right panel). The β-Galactosidase staining assay was performed in three independent experiments

Fig. 5

Expression levels of selected miRNA and protein targets in cancer cell upon pemigatinib treatment. A Left: histograms show the expression of miR-186 and miR-195 and right: Western blots of CDK6, target of miR-195, and c-Myc, target of miR-186 and miR-195 in H1581 cells treated with Pem for 48 h. B Left: histogram showing the expression of miR-133b and right: Western blots of MET, a target of miR-133b, in KATO III cells treated with Pem for 48 h. C Left: histogram showing the expression of miR-139 and miR-186 and right: Western blots of EGFR, target of miR-139, and c-Myc, target of miR-186, in RT-112 cells treated with Pem for 48 h. Histograms represent the mean ± SEM of three experiments, and the dashed line indicates the control (NT). *p < 0.05; ***p ≤ 0.001; Student’s t-test