FIBP interacts with transcription factor STAT3 to induce EME1 expression and drive radioresistance in lung adenocarcinoma

Abstract

Cancer cells inevitably develop radioresistance during lung adenocarcinoma radiotherapy. However, the mechanisms are incompletely clarified. In this study, we show that FIBP protein expression in lung adenocarcinoma tissues is upregulated and associated with worse overall survival. Functionally, we find that depletion of FIBP inhibits lung adenocarcinoma progression and radioresistance in vitro and in vivo. Moreover, we uncover that FIBP interacts with STAT3 to enhance its transcriptional activity, thereby inducing the expression of the downstream target gene EME1. Importantly, we demonstrate that the biological effects of FIBP are partially dependent on EME1 in lung adenocarcinoma. Our work reveals that FIBP modulates the STAT3/EME1 axis to drive lung cancer progression and radioresistance, indicating that targeting FIBP may be a novel intervention strategy for lung adenocarcinoma radiotherapy.

Keywords: EME1; FIBP; STAT3; lung adenocarcinoma; radioresistance.

Fig 1

FIBP is upregulated in lung adenocarcinoma and associated with poor outcomes. (A-B) Analysis of FIBP mRNA expression in normal and tumor tissues from the TCGA and GEO lung adenocarcinoma datasets. (C-D) PCR and Western blot analysis of FIBP in human bronchial epithelial (HBE) cells and different lung adenocarcinoma cell lines. (E) Images of FIBP IHC staining in the lung adenocarcinoma tissue microarray. Scale bar, 25 μm. (F) Statistical analysis of FIBP IHC results. (G) High FIBP expression was associated with worse outcomes in lung adenocarcinoma patients.

Fig 2

FIBP knockdown suppresses lung adenocarcinoma cell growth and proliferation in vitro and in vivo. (A) Immunoblot analysis of FIBP protein expression in A549 and H1299 cells. (B-C) FIBP depletion inhibited the growth and proliferation of A549 and H1299 cells. ***P < 0.001. (D) FIBP was stably depleted by two different shRNAs (Sh-FIBP) in A549 cells. (E) Pictures of xenograft tumors (n = 7 mice/group). (F) FIBP silencing suppressed tumor growth in vivo. The data are presented as the mean ± SEM values. ***P < 0.001. (G) Tumor weights in the three groups. ***P < 0.001. (H) Pictures of FIBP and Ki-67 staining of xenograft tumors.

Fig 3

FIBP depletion enhances DNA damage response and inhibits DNA repair in lung adenocarcinoma. (A) Cells were transfected with indicated FIBP siRNAs for 48 h and exposed to IR. After 24 h, cells were collected and analyzed by flow cytometry. ***P < 0.001. (B) Representative images and quantification of the Olive tail moment in control cells and FIBP-depleted lung adenocarcinoma cells. ***P < 0.001. (C) Left panel: Control cells and FIBP-depleted lung adenocarcinoma cells were irradiated and were then harvested after 4 h. Representative images of γH2AX foci. Right panel: quantification of γH2AX foci. ***P < 0.001. Scale bar, 10 μm. (D) FIBP depletion impaired Rad51 foci formation. ***P < 0.001. Scale bar, 10 μm.

Fig 4

FIBP depletion contributes to enhanced radiosensitivity in lung adenocarcinoma in vitro and in vivo. (A) The cell survival rate showed that FIBP depletion sensitized lung cancer cells to radiation. ***P < 0.001. (B) FIBP overexpression led to radioresistance in lung adenocarcinoma cells. **P < 0.01. (C) Mice were irradiated with 10 Gy when the tumor volume had increased to about 130 mm³. (D-F) Images of xenograft tumors (D), tumor growth curves (E) and tumor weight curves (F) in different groups (n = 6 mice/group). *P < 0.05, ***P < 0.001.

Fig 5

Suppression of FIBP downregulates EME1 expression in lung adenocarcinoma cells. (A) Volcano plot showing up- and downregulated genes in FIBP-depleted H1299 cells. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of transcriptionally altered genes, as identified by RNA-seq, between control cells and FIBP-depleted lung adenocarcinoma cells. (C) The pathway enrichment analysis shows that FIBP is closely correlated with homologous recombination. (D) Heatmap of the transcriptional changes. (E) Real-time PCR analysis showed the changes in mRNA expression in H1299 cells. ***P < 0.001. (F-G) FIBP positively regulates EME1 protein expression in lung adenocarcinoma cells.

Fig 6

FIBP interacts with STAT3 in lung adenocarcinoma cells. (A) Exogenously expressed STAT3 bound to Exogenously expressed FIBP and vice versa in H1299 cells. (B) Exogenously expressed STAT3 formed a complex with endogenous FIBP and vice versa in H1299 cells. (C) Endogenous binding between FIBP and STAT3 in H1299 cells. (D) The GST or recombinant GST-STAT3 protein was coimmunoprecipitated with SFB-FIBP overnight. The immunoprecipitates were analyzed by western blotting. (E) Colocalization of endogenous FIBP and STAT3 in H1299 cells.

Fig 7

FIBP enhances STAT3 transcriptional activity to induce EME1 expression in lung adenocarcinoma cells. (A-B) FIBP positively regulated the phosphorylation of STAT3 at Ser727 in lung adenocarcinoma cells. (C-D) The luciferase reporter assay showed that FIBP positively regulates the transcriptional activity of STAT3 in A549 and H1299 cells. **P < 0.01, ***P < 0.001. (E-F) FIBP depletion decreased the binding affinity of STAT3 to EME1 gene promoter. ***P < 0.001. (G-H) PCR and Western blot analysis of the expression of EME1 in lung adenocarcinoma cells after transfection with the indicated plasmids or siRNAs.

Fig 8

The biological effects of FIBP inhibition are mediated partially by modulating EME1 in lung adenocarcinoma cells. (A) Immunoblot analysis of the indicated proteins in H1299 cells. (B-C) Cell growth (B) and colony formation (C) assays showed that the ability of FIBP to inhibit cell growth and proliferation was abrogated by overexpression of EME1 in H1299 cells. ***P < 0.001. (D) Representative images and quantification of the Olive tail moment indicating DNA damage induced by irradiation. ***P < 0.001. (E-F) Representative immunofluorescence images and quantification of γH2AX foci (E) and Rad51 foci (F) in H1299 cells. *P < 0.05, ***P < 0.001.