Forkhead box K1 regulates the malignant behavior of gastric cancer by inhibiting autophagy

Abstract

Background: Forkhead box K1 (FOXK1) is a transcription factor that contributes to cancer development, but it is unclear how FOXK1 regulates the proliferation and migration of gastric cancer (GC) cells. The purpose of this study was to investigate the clinical significance, biological function, and molecular mechanisms of FOXK1 in GC.

Methods: We conducted bioinformatics assays and western blotting to assess FOXK1 expression. Then, we performed immunohistochemistry (IHC) with tissue microarrays (TMAs) to assess FOXK1 expression in order to identify an association between FOXK1 expression levels and clinical parameters. We used 5-ethynyl-2'-deoxyuridine (EdU), wound healing and Transwell assays to determine whether FOXK1 promotes malignant behaviors in GC. Furthermore, immunofluorescence staining, transmission electron microscopy and western blotting were used to verify an association between FOXK1 and autophagy.

Results: We observed high levels of FOXK1 expression in GC tissues, which were associated with the degree of malignancy in GC. FOXK1 promotes the malignant behavior of GC by regulating autophagy via activation of the class I phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway and inhibition of the expression of class III PI3K.

Conclusions: These findings provide a new target for the comprehensive treatment of GC by highlighting the relationship between FOXK1 and malignant behaviors in GC.

Keywords: 3-methyladenine (3-MA); Forkhead box K1 (FOXK1); autophagy; gastric cancer.

Figure 1

High FOXK1 expression predicts a poor prognosis for patients with GC. (A) Boxplot of FOXK1 expression in GC based on GEPIA; (B) a Kaplan-Meier plot was used to analyze the prognosis of patients with GC stratified by FOXK1 expression (low FOXK1 expression group, n=221; high FOXK1 expression group, n=408); (C) FOXK1 expression in normal and malignant human gastric tissues was detected using IHC in TMA. Scale bar, 250 µm; (D) TMA of the levels of the FOXK1 protein in GC tissues. Representative images of FOXK1 staining by IHC in TMA. Scale bar, 250 µm; (E) FOXK1 expression increased with increasing malignant progression in GC. *, P<0.05 and **, P<0.01.

Figure 2

FOXK1 promotes GC cell proliferation in vitro. (A) Protein levels of FOXK1 in normal GES1 cells and human GC cells were determined using western blotting; (B) transfection efficiency of FOXK1 overexpression and knockdown constructs, as measured by qRT-PCR; (C) transfection efficiency of the FOXK1 overexpression and knockdown constructs, as measured by western blotting; (D) DNA synthesis was measured by assessing EdU incorporation for 24 h, and the vector, FOXK1, siNC and siFOXK1 samples were compared. Scale bar, 200 µm. The data are presented as the means ± SD of three independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

Figure 3

FOXK1 promotes GC cell migration and invasion. (A,B) The motility of AGS and MGC803 cells was measured by examining wound closure following FOXK1 overexpression or knockdown. Scale bar, 500 µm; (C,D) Transwell assays were utilized to assess AGS and MGC803 cell migration and invasion following FOXK1 overexpression and knockdown. Scale bar, 500 µm. The data are presented as the means ± SD of three independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

Figure 4

FOXK1 suppresses autophagy in GC cells. (A,B) LC3 puncta in cells overexpressing FOXK1 were visualized under a confocal microscope after cells were subjected to immunofluorescence staining. Scale bar, 20 μm; (C) the protein levels of LC3-I, LC3-II, p62, Beclin1 and FOXK1 were analyzed in FOXK1-transfected cells using western blotting. The data are presented as the means ± SD of three independent experiments. *, P<0.05, **, P<0.01, and ****, P<0.0001.

Figure 5

FOXK1 knockdown promotes autophagy in GC cells. (A,B) LC3 puncta in cells transfected with siNC or siFOXK1 and subjected to immunofluorescence staining were visualized under a confocal microscope. Scale bar, 20 µm; (C) the protein levels of LC3-II, p62, Beclin1 and FOXK1 were examined using western blotting in cells transfected with siNC or siFOXK1; (D) autophagosomes were observed under a transmission electron microscope in cells with FOXK1 knockdown. Scale bar, 2 µm. The data are presented as the means ± SD of three independent experiments. **, P<0.01, ***, P<0.001, and ****, P<0.0001.

Figure 6

FOXK1 induces autophagy in GC cells by modulating the PI3K/AKT/mTOR signaling pathway. (A) KEGG pathway analysis of FOXK1; (B) levels of FOXK1, class I PI3K, and class III PI3K and the ratios of p-AKT/total AKT and p-mTOR/total mTOR in GC cells overexpressing FOXK1 were assessed using Western blotting; (C) the protein levels of FOXK1, class I PI3K, class III PI3K, p-AKT/total AKT and p-mTOR/total mTOR in GC cells with FOXK1 knockdown were measured using western blotting; (D) FOXK1 overexpression and knockdown of the mRNA levels of class I PI3K and class III PI3K were assessed by qRT-PCR. The data are presented as the means ± SD of three independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

Figure 7

FOXK1 knockdown increases autophagy inhibited by 3-MA. (A) After cells were transfected with siNC or siFOXK1, they were treated with 200 µM 3-MA for 48 h. The levels of LC3-II, p62 and FOXK1 were detected using Western blotting; (B) representative images and analysis of EdU assays using AGS and MGC803 cells. Scale bar, 200 µm; (C) invasion of cells treated with siNC, siFOXK1, siNC + 3-MA, and siFOXK1 + 3-MA. Scale bar, 500 µm. The data are presented as the means ± SD of three independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

Figure 8

Downregulation of FOXK1 inhibits GC growth in vivo. (A) Tumor tissues derived from xenograft tumors in CB17/SCID mice (n=6) 28 days after inoculation. Top panel, tumor derived from LV-NC-transfected cells; bottom panel, tumor derived from cells lacking FOXK1; (B) tumor growth curves for the LV-NC and LV-shFOXK1 groups. Tumor volumes were measured weekly; (C) the average tumor weights from mice in the LV-NC and LV-shFOXK1 groups. Tumors were excised at the end point of the experiment and weighed; (D) H&E staining of xenograft tumors from CB17/SCID mice; (E) levels of FOXK1, class I PI3K, class III PI3K, p-AKT, p-mTOR, LC3-II and p62 in tissues from xenograft tumors were detected using Western blotting. *, P<0.05, **P, <0.01, ***, P<0.001, and ****, P<0.0001.

Figure S1

The proposed mechanistic model of FOXK1 in gastric cancer. FOXK1 activates the PI3K/AKT/mTOR pathway in gastric cancer to inhibit autophagy and promote tumor growth, invasion and metastasis.