Enhancing the antitumor efficacy using a combination of FGFR4 Inhibitor (H3B-6527) and oxaliplatin in gastric cancer

Abstract

Oxaliplatin, a platinum-based anticancer drug, is commonly used to treat gastrointestinal cancers, including gastric cancer. However, resistance to platinum-based therapies often leads to poor clinical outcomes for gastric cancer patients. Overexpression and activation of FGFR4 signaling have been identified as drivers of tumorigenesis in several types of cancer, including gastric cancer. In this study, we investigated the therapeutic efficacy of combining the FGFR4 inhibitor H3B-6527 with oxaliplatin using in vitro and in vivo gastric cancer models. Using gastric cancer cell lines, cell viability and clonogenic cell survival assays revealed that the combination treatment significantly reduced cancer cell viability and colony formation, compared to either agent alone (p < 0.01). Interestingly, treatment with oxaliplatin alone increased FGFR4 expression in the resistant cancer cell population. Western blot analysis confirmed the heightened DNA damage (γH2AX, cleaved PARP) alongside suppressed pro-survival signals (phospho-STAT3 and BCL2 family). Apoptosis was markedly enhanced, as demonstrated by Caspase-3/7 and TUNEL assays (p < 0.01). In human gastric cancer-derived tumoroids, the combination therapy significantly reduced both the size and number of tumoroids. In patient-derived xenograft (PDX) models, the combined treatment approach outperformed single-agent treatments in reducing tumor growth and improving survival. Immunofluorescence and immunohistochemistry analyses of PDX tumors showed an increase in DNA damage (γH2AX) and apoptosis (cleaved caspase-3) along with a reduction in cell proliferation (KI67). These findings indicate that H3B-6527 enhances gastric cancer sensitivity to oxaliplatin by amplifying DNA damage and disrupting cell survival pathways. This study provides a rationale for clinical trials targeting FGFR4 in gastric cancer.

Keywords: FGFR4; Gastric cancer; H3B-6527; Oxaliplatin.

Fig. 1

H3B-6527 and oxaliplatin combination treatment significantly reduces gastric cancer cell survival. A-B) HGC-27 and MKN28 cells were treated with H3B-6527, Oxaliplatin and their combination. Cell viability was measured using the CellTiter-Glo assay, and the IC50 values were determined from nonlinear regression from at least three independent experiments. C-D) Representative images of clonogenic cell survival of HGC-27 (C) and MKN28 (D) cells following treatment with H3B-6527 (3 µM and 2 µM in HGC-27 and MKN28 respectively), Oxaliplatin (1 µM and 2 µM in HGC-27 and MKN28 respectively) and combination of both drugs. E-F) Colony formation quantification from three independent experiments. * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Fig. 2

FGFR4 inhibition enhances apoptotic markers with oxaliplatin. A-B) Western blot analysis showing increase of Cleaved-PARP and ƴH2AX proteins and decrease levels of phospho-STAT3, BCL2 and BCL-XL after combination of Oxaliplatin and H3B-6527 in HGC-27 (A) and MKN28 (B) gastric cancer cell lines. C-D) Western blot analysis showing increase of Cleaved-PARP and ƴH2AX proteins and decrease of phospho-STAT3, BCL2 and BCL-XL after FGFR4 siRNA knock down and treatment with Oxaliplatin in HGC-27 (C) and MKN28 (D) gastric cancer cell lines. These data are representative of three independent experiments.

Fig. 3

Apoptosis induction and organoid growth suppression by combined treatment. (A-B) Caspase-3/7 activity assay showed significant increases in HGC-27 (A) and MKN28 (B) gastric cancer cell lines treated with H3B-6527 (5 µM HGC-27 or 8 µM MKN28), Oxaliplatin (Oxp) (2.5 µM HGC-27 or 8 µM MKN28), or the combination (Comb) for 48 hours. Data are represented as mean ± SD (n = 3). ****P < 0.0001. (C-D) Representative images of TUNEL staining in HGC-27 (C) and MKN28 (D) cells following treatment with Ctrl, H3B-6527, Oxp, or Comb for 48 hours. Green fluorescence indicates apoptotic cells. Quantifications are shown on the right of each panel, and data are represented as mean ± SD (n = 3). ****P < 0.0001. (E) Human gastric adenocarcinoma organoids were treated for 10 days with Ctrl, H3B-6527, Oxaliplatin, or the Combination. With Representative images taken on Day 0 and Day 10 demonstrate morphological changes. Quantification of mean organoid area size in µm² from at least 50 organoids per condition and the graph showing the counts of organoids per well are shown on the right panel. Data are represented as mean ± SD (n = 3 wells per condition). ****P < 0.0001. (F) Representative images of ƴH2AX (red) and DAPI (blue) immunofluorescent staining in human gastric adenocarcinoma organoids treated for 10 days with H3B-6527, Oxaliplatin, or the Combination as compared to control. The percentage of ƴH2AX positive cells are shown on the right panel and are presented as mean ± SD. ^⁎⁎P < 0.01.

Fig. 4

Characterization of oxaliplatin resistance cell lines. A) IC50 curves for oxaliplatin in AGS parental cell line and three Oxaliplatin resistant AGS variants (AGS Oxp-R_C1, AGS Oxp-R_C2, and AGS Oxp-R_C3). B) Western blot analysis showing increased of FGFR4, Phospho-STAT3 (Y705) and total STAT3 levels in Oxaliplatin resistant AGS cell lines compared to parental controls. β-Actin was used as a loading control. C) Dose response curves of AGS single treated with H3B-6527(IC50= 3.1 µM), Oxaliplatin (IC50= 1.3 µM) and combination (IC50= 0.2 µM+0.2 µM). (D) Dose-response curves of AGS Oxp-R cells treated with oxaliplatin alone (IC50 >20 µM), H3B-6527 alone (IC50= 3.1 µM), and the combination of H3B-6527 with oxaliplatin (2 µM + 2 µM). (E) Western blot analysis of FGFR4, PARP, cleaved PARP (Cl-PARP), γH2AX, H2AX, p-STAT3 (Y705), STAT3, BCL2, BCL-XL, and β-Actin expression in AGS Oxp-R cells treated with 5 µM of oxaliplatin or H3B-6527, or combination. Data represent mean ± SD from triplicate experiments.

Fig. 5

Increased DNA damage and apoptosis in PDX models after combined treatment. A) Representative images of ƴH2AX (red) and Ki67 (Green) immunofluorescent staining showing higher ƴH2AX levels and low levels of Ki67 in PDX498 tumor tissues after treatment with the combination of H3B-6527 and oxaliplatin as compared to single treatment and control. Scale bar 50 µm. B-C) Quantification of immunostaining of Ki67 and ƴH2AX respectively. D) Representative immunohistochemistry images in the upper panels show high nuclear Ki67 staining in control and single treatments but low in the combination treatment. The lower panels demonstrate high expression of Cleaved Caspase 3 in the combined treatment and low in single or no treatment group. Scale bar 100 µm. E) Quantification nuclear Ki67 staining in at least four fields as a percentage in the right panel. Data is graphed with mean ± SEM. F) Intensity densitometry of cleaved caspase 3 staining in at least four different fields. Data are graphed with mean ± SEM.

Fig. 6

Tumor Growth and Survival Enhancement in PDX Models with Combined Treatment. (A-B) Volume growth rate of tumors in xenografted animals using PDX498 and FJ0155 models. Each line represents the average tumor volume (mm³) for treatment groups: H3B-6527 (300mg/kg daily, Oral gavage), oxaliplatin (2.5 mg/kg i.p. thrice a week) or their combination. C-D) Kaplan–Meier survival plot showing significant difference in overall survival among treatment groups for PDX498 (P = 0.0105) and FJ0155 (P < 0.0001). E-F) Mice body weight data show no toxic effect from the treatments compared to controls.