Forkhead Box q1 promotes invasion and metastasis in colorectal cancer by activating the epidermal growth factor receptor pathway

Figure 1 Expression and prognosis of forkhead Box q1 and heparin binding epidermal growth factor in colorectal cancer and normal colorectal tissues. A and B: Forkhead Box q1 (FOXQ1) (left) and heparin binding epidermal growth factor (HB-EGF) (right) expression was analyzed in human colorectal cancer (CRC) tissues and normal tissues using the Gene Expression Profiling Interactive Analysis online database. ^aP < 0.05; C and D: Overall survival rate of FOXQ1 (left) and HB-EGF (right) in CRC patients. FOXQ1: Forkhead Box q1; HB-EGF: Heparin binding epidermal growth factor; GEPIA: Gene Expression Profiling Interactive Analysis.

Figure 2 Forkhead Box q1 knockdown induces heparin binding epidermal growth factor suppression and epidermal growth factor/platelet-derived growth factor signaling pathway blockade in vitro according to transcriptional analysis of the generated forkhead Box q1 knockdown cell line. A: Quantitative real-time polymerase chain reaction was performed to evaluate mRNA expression in the knockdown of forkhead Box q1 in DLD1 cells (DLD1-shFOXQ1) and SW480-shFOXQ1 cells (two-tailed, unpaired Student’s t test). Bars represent the mean ± SE of three independent experiments; B: Western blotting was performed to evaluate FOXQ1 protein expression in DLD1-shFOXQ1 and SW480-shFOXQ1 cells; C: Volcano plot illustrating differentially expressed genes using the epidermal growth factor (EGF)/platelet-derived growth factor (PDGF) array to compare DLD1-shFOXQ1 and DLD1-shControl cells. The vertical blue points in the plot represent 84 genes related to the EGF/PDGF signaling pathways. The vertical green line indicates a fold-change in gene expression of 1. The vertical pink lines indicate the threshold for fold-change (≥ 2.0 and ≤ -2.0). The horizontal blue line indicates the threshold for the P value of the t test. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. DLD1-shFOXQ1: The knockdown of forkhead Box q1 in DLD1 cells; SW480-shFOXQ1: The knockdown of forkhead Box q1 in SW480 cells.

Figure 3 Effect of forkhead Box q1 expression on heparin binding epidermal growth factor expression and extracellular release ability in colorectal cancer cells. A: Quantitative analysis of western blots was performed to detect the expression of heparin binding epidermal growth factor (HB-EGF) with forkhead Box q1 (FOXQ1) inhibition; B: Flow cytometry was performed to detect the effect of changes in FOXQ1 expression on the expression of proHB-EGF in DLD1 and SW480 cells; C: ELISA verified the effect of FOXQ1 knockdown on sHB-EGF expression in DLD1 and SW480 cells; D: ELISA confirmed the effect of FOXQ1 on the expression of proHB-EGF extracellular release proteins (ADAM9, ADAM10, ADAM12 and MMP-7) in DLD1 and SW480 cells. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. FOXQ1: Forkhead Box q1; HB-EGF: Heparin binding epidermal growth factor; ProHB-EGF: Membrane-bound HB-EGF; SHB-EGF: Souble HB-EGF; ADAM9: A disintegrin and a metalloprotease 9; ADAM10: A disintegrin and a metalloprotease 10; ADAM12: A disintegrin and a metalloprotease 12; MMP7: Matrix metallopeptidase 7.

Figure 4 Forkhead Box q1 regulates epidermal growth factor receptor and downstream signaling pathways by regulating heparin binding epidermal growth factor. A: Quantitative real-time polymerase chain reaction (qRT–PCR) was performed to analyze the levels of forkhead Box q1 (FOXQ1), heparin binding epidermal growth factor (HB-EGF), epidermal growth factor receptor (EGFR), Akt, kRas and Raf in DLD1-shFOXQ1 and DLD1-shControl cells; B: qRT–PCR was performed to analyze the levels of FOXQ1, HB-EGF, EGFR, AKT, RAF and KRAS in SW480-shFOXQ1 and SW480-shControl cells; C: Western blot analyses were performed to analyze the protein levels of FOXQ1, HB-EGF, EGFR, RAF and KRAS as well as the phosphorylation levels of PI3K, Akt, and MAPK in DLD1-shFOXQ1, SW480-shFOXQ1, DLD1-shControl and SW480-shControl cells. β-actin was used as the loading control; D: Exogenous rhHB-EGF (50 ng/mL) was added to the culture media of DLD1-shFOXQ1 and SW480-shFOXQ1 cells for 1.5 h and 2 h, respectively. Western blotting was performed to detect the expression of proteins in the HB-EGF/EGFR signaling pathway. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001 indicate significant differences compared to the control group (two-tailed, unpaired Student’s t test). Bars represent the mean ± S.E. of three independent experiments. rhHB-EGF: Recombinant human HB-EGF; FOXQ1: Forkhead Box q1; HB-EGF: Heparin binding epidermal growth factor.

Figure 5 Recombinant human heparin binding epidermal growth factor reverses the forkhead Box q1-induced suppression of colorectal cancer cell proliferation and migration in vitro. A: A cell counting Kit-8 (CCK-8) assay was performed to detect the effects of recombinant human heparin binding epidermal growth factor (rhHB-EGF) on the proliferation of forkhead Box q1 (FOXQ1)-deficient DLD1 and SW480 cells; B: The Transwell migration assay was performed to evaluate the migration ability after knocking out FOXQ1 and after adding rhHB-EGF; C: A statistical analysis of the results of the Transwell migration assay was performed. ^bP < 0.01; D: After 0 h and 24 h, the scratch experiment evaluated the migration ability of DLD1 and SW480 cells with downregulated FOXQ1 and rhHB-EGF; E: Statistical analysis of the above scratch experiment results. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; F: Quantitative analysis of Western blot to detect the expression of E-cadherin, N-cadherin, vimentin and Snail protein levels in DLD-shFOXQ1 and SW480-shFOXQ1 cells compared to DLD1-shControl and SW480-shControl cells. β-actin was used as the loading control. CCK-8: Cell Counting Kit-8.

Figure 6 Prognostic value of the combination of forkhead Box q1 and heparin binding epidermal growth factor. A: Representative immunohistochemical images of forkhead Box q1 (FOXQ1) and heparin binding epidermal growth factor (HB-EGF) positive coexpression and negative coexpression in human colorectal cancer tissues and adjacent nontumorous tissues. Scale bars represent 200 μm; B: Kaplan-Meier survival analysis was used to evaluate the overall survival rate in the following patients: FOXQ1-positive and FOXQ1-negative patients; HB-EGF-positive and HB-EGF-negative patients; and FOXQ1-positive and FOXQ1-negative coexpressing HB-EGF-positive and HBEGF-negative patients. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001 indicate a significant difference between the indicated groups (log-rank test). FOXQ1: Forkhead Box q1; HB-EGF: Heparin binding epidermal growth factor.