STAT3 inhibitor Stattic Exhibits the Synergistic Effect with FGFRs Inhibitor Erdafitinib in FGFR1-positive Lung Squamous Cell Carcinoma

Abstract

Lung squamous cell carcinoma (LUSC), a subset of non-small cell lung cancer (NSCLC), accounts for about 30% of all lung cancers (LC) and exhibits a dismal response to current therapeutic protocols. Existed studies have indicated that aberrations in fibroblast growth factor receptors (FGFRs) play a pivotal role in the progression of LUSC, rendering them as attractive targets for therapeutic intervention in this cancer type. This study found that Erdafitinib (Erda), a novel pan-FGF receptor tyrosine kinase inhibitor (TKI), exerted a cytotoxic effect on LUSC cells. However, STAT3, the downstream target of FGFRs, remained still activated despite Erdafitinib treatment. Then, a STAT3 inhibitor, Stattic (Sta), was concurrently used with Erdafitinib, and the combined treatment demonstrated a synergistic efficacy in both in vitro and in vivo models of LUSC when compared to that of the treatment of the Erdafitinib or Stattic alone. Further molecular studies showed that such an effect of Erdafitinib and Stattic was associated with their concurrently inhibitory effect on FGFR1 and STAT3 signaling in LUSC cells. Therefore, the findings of this study indicated that the concurrent use of Erdafitinib and Stattic is a promising therapeutic approach for the treatment of FGFR1-positive LUSC.

Keywords: Erdafitinib; FGFR1; Lung Squamous Cell Carcinoma; STAT3; Stattic.

Figure 1

The effects of Erdafitinib on FGFRs, AKT, MAPK, STAT3 signaling. A: The chemical structure of Erdafitinib. B: The expressions of FGFRs in LUSC H520 cells from THE HUMANS PROTEIN ATLAS. C: H520 cells were treated with Erdafitinib (10 µM) for 24 h, and the expressions of FGFRs in H520 cells were assessed by Western blot analysis. D: H520 cells were treated with Erdafitinib (10 µM) for 24 h, and the expressions of molecules involved in AKT, MAPK, STAT3 signaling were assessed by Western blot analysis. Data was expressed as mean ± SD of three experiments and each experiment included triplicate repeats. ^**p<0.01 vs. Erda (0 µM).

Figure 2

The synergistic cytotoxic effects of Erdafitinib and Stattic on LUSC cells. A: The chemical structure of Stattic. B: The effect of Erdafitinib on H520 and BEAS-2B cells was assessed by MTT assay. C: The effect of Stattic on H520 and BEAS-2B cells was assessed by MTT assay. D: The effects of Erdafitinib and Stattic on H520 cells were assessed by MTT assay, and the combination index (CI) was calculated. E: The concurrent effects of Erdafitinib and Stattic on H520 cells were assessed by colony formation assay. F: The concurrent pro-apoptosis effects of Erdafitinib and Stattic on H520 cells were assessed by flow cytometry analysis. The Annexin V+/PI- and Annexin V+/PI+ cells were considered as early and late apoptotic cells, respectively, and the sum of the above two was calculated as apoptotic cells. Data was expressed as mean ± SD of three experiments and each experiment included triplicate repeats. ^**p<0.01 vs. Control, ^##p<0.01 vs. Erda alone, ^&&p<0.01 vs. Sta alone.

Figure 3

The synergistic anti-metastatic effects of Erdafitinib and Stattic on H520 cells. A: The concurrent anti-metastatic effects of Erdafitinib and Stattic on H520 cells was assessed by wound healing assay. B: The concurrent anti-invasive effects of Erdafitinib and Stattic on H520 cells was assessed by Transwell assay. C: The effects of Erdafitinib and Stattic co-treatment on the expressions of E-Cadherin and Vimentin in H520 cells. Data was expressed as mean ± SD of three experiments and each experiment included triplicate repeats. ^**p<0.01 vs. Control, ^##p<0.01 vs. Erda alone, ^&&p<0.01 vs. Sta alone.

Figure 4

The molecular mechanism responsible for the synergistic effects of Erdafitinib and Stattic on H520 cells. A: Analysis of Volcano plots, GO pathway and KEGG pathway between the Erdafitinib (Erda,10 µM, 24 h) group and Control group. B: Analysis of Volcano plots, GO pathway and KEGG pathway between the Erdafitinib (Erda,10 µM, 24 h)+Stattic (Sta, 2.5 µM, 24 h) group and Control group. C: The concurrent effects of Erdafitinib (Erda,10 µM, 24 h) and Stattic (Sta, 2.5 µM, 24 h) on FGFR1/STAT3 signaling in H520 cells. D: The concurrent effects of FGFR1 knockdown and Stattic (Sta, 2.5 µM) on FGFR1/STAT3 signaling in H520 cells. Data was expressed as mean ± SD of three experiments and each experiment included triplicate repeats. ^**p<0.01 vs. Control, ^##p<0.01 vs. Erda alone, ^&&p<0.01 vs. Sta alone.

Figure 5

The synergistic effects of Erdafitinib and Stattic against tumorigenesis in vivo. H520 xenograft model was constructed and treated with Erdafitinib (Erda, 10 mg/kg/day), Stattic (Sta, 5 mg/kg/day), or their combination (10 mg/kg/day Erdafitinib, 5 mg/kg/day Stattic) for 2 weeks. A: Tumor volumes were measured every other day. B: Body weights were measured every other day. C&D: MicroPET was conducted using ¹⁸F-FDG. E: Representative images of hematoxylin-eosin (H&E), TUNEL, Ki67 and FGFR1/pSTAT3 staining in H520 xenografts. Data was expressed as mean ± SD of three experiments and each experiment included triplicate repeats. ^**p<0.01 vs. Control, ^##p<0.01 vs. Erda alone, ^&&p<0.01 vs. Sta alone.