FOXP3 is highly expressed in NSCLC and correlated with poor prognosis

To determine whether FOXP3 was expressed in NSCLC and correlated with patient prognosis, we performed immunohistochemistry on NSCLC tissues using two types of monoclonal anti-FOXP3 antibodies. FOXP3 immunostaining was seen in NSCLC cells and Treg cells (Additional file 2: Figure S1). Tumor FOXP3 expression mainly exhibited a mixture of diffuse staining in the cytoplasm, the nucleus or in both of them. The FOXP3 expression levels in NSCLC were scored 1 to 12 according to the IRS method. Specimens scored 1–4 were classified as ‘Tumor FOXP3-Low’ and 6–12 as ‘Tumor FOXP3-High’. In 106 NSCLC specimens, 41 cancer tissues were detected high FOXP3 expression and 65 cancer tissues showed low FOXP3 expression. Statitically, the overall expression of FOXP3 was much higher in cancer tissues than in normal adjacent tissues (p < 0.001) (Fig. 1a). To confirm this finding, western blotting was performed to detect their protein levels in 5 randomly selected paired NSCLC specimens. As shown in Fig. 1b, 3 out of 5 showed higher level of FOXP3 protein in tumor tissues than that in adjacent normal tissues.

Fig. 1 FOXP3 is highly expressed in NSCLC and correlated with poor prognosis. a The levels of FOXP3 in 106 paired NSCLC tissues and adjacent normal tissues were scored by IRS method (5) and analyzed by paired t-test. Mean ± SD is shown in the figure. For images of the tissue staining, please refer to Additional file 2: Figure S1. b FOXP3 protein levels were higher in tumor than in peritumoral tissues, as determined by western blot. c Kaplan–Meier representation of the overall survival and recurrence-free survival of the two groups of patients with high (n = 41, solid line) or low (n = 65, dotted line) FOXP3 expression in NSCLC tissues. Statistical analysis was performed with the log-rank test. d Kaplan–Meier representation of the overall survival of the two groups of patients with Treg cell counts >25 (n = 42, solid line) or ≤25 (n = 64, dotted line) in NSCLC tissues. Statistical analysis was performed with the log-rank test Full size image

We analyzed the correlation between clinicopathologic characteristics and tumor FOXP3 expression, the results showed that there was no significant association of FOXP3 expression levels with clinicopathologic characteristics. However, we found a correlation between tumor FOXP3 expression and Treg cell counts (Additional file 1: Table S3).

The correlation of FOXP3 expression with the long-term survival rate after surgery operation was analyzed. Kaplan–Meier analysis showed that patients with high levels of FOXP3 expression in tumor had shorter overall survival time than those with low levels of FOXP3 expression (P < 0.05, Fig. 1c). The mean overall survival time of patients with high FOXP3 expression in tumor tissues was 44.3 ± 6.54 months, while that with low FOXP3 expression level was 78.1 ± 7.35 months (P = 0.007).

Kaplan–Meier analysis also showed that patients with high levels of FOXP3 expression in tumor had shorter recurrence-free survival time than those with low levels of FOXP3 expression (P < 0.05, Fig. 1c). The mean recurrence-free survival time of the patients with high FOXP3 expression in tumor tissues was 30.5 ± 5.47 months, while those with low FOXP3 expression was 63.8 ± 8.16 months (P = 0.019).

To further explore whther FOXP3 had any prognostic value, we conducted Cox proportional Hazard regression analysis of patients’ overall survival and recurrence-free survival. In multivariate analysis of overall survival, when other risk factors such as histology grade, pathology stage, pathology tuberculosis and nodal status were taken in consideration, the HR of high FOXP3 expression was improved from 1.86 (95% CI: 1.18–1.92, P = 0.01) to 2.09 (95% CI: 1.27–3.44, P = 0.00) (Additional file 1: Table S4). In multivariate analysis of recurrence-free survival, when other risk factors were taken in consideration, the HR of high FOXP3 expression was improved from 1.71, (95% CI: 1.09–2.70, P = 0.02) to 1.83 (95% CI: 1.11–3.02, P = 0.02) (Additional file 1: Table S5). Both univariate analyses and multivariate analyses indicate that FOXP3 is an independent predictor for overall survival and recurrence-free survival of NSCLC patients.We also analyzed the Treg cells in NSCLC. Patients were divided into two groups according Treg cell numbers, we found that Treg cells alone did not have a prognostic value in NSCLC (P > 0.05, Fig. 1d).

Ectopic expression of FOXP3 promotes tumourigenic properties of NSCLC cells

We examined the effect of ectopic expression of FOXP3 on the several tumorigentic features of cancer cells using NSCLC cell lines (A549 and H460). FOXP3 lentivirus was used to improve transfection efficiency, and the expression of FOXP3 was validated by western blot (Fig. 2a). Ectopic expression of FOXP3 caused a significant increase in cell viability in both A549 (p < 0.001) and H460 (p < 0.001) cells (Fig. 2a). This promoting effect was further confirmed by the colony formation assay, in which FOXP3 significantly increased the colony formation in A549 (p < 0.001) and H460 (p < 0.001) when compared with control (Fig. 2b).

Fig. 2 FOXP3 promotes tumor growth in NSCLC. a Effect of FOXP3 on cell viability was evaluated by MTT assay in A549 and H460 cells. b Effect of FOXP3 on colony formation in A549 and H460 cells. c Subcutaneous tumor growth curve of A549-FOXP3 cells in nude mice was compared with control cells. The FOXP3 group showed an enhanced tumor growth compared with the control group (P < 0.05). d Effects of FOXP3 knockdown on cell viability and colony formation in A549 cells Full size image

Since FOXP3 promotes cell growth in vitro, we next tested whether FOXP3 could promote the growth of NSCLC cells in nude mice. 18 days after inoculation we found that the mean volume of tumors infected with FOXP3 was 2.6-fold larger than that of infected with control virus, which formed either no tumor or much smaller tumors. The tumor growth curves of A549-FOXP3 and A549-Control in nude mice were shown in Fig. 2c and Additional file 2: Figure S2. In contrast to A549-control, A549-FOXP3 exhibited markedly increased growth rate in the xenograft model (p < 0.05). Taken together, these findings have demonstrated that FOXP3 can exhibit obvious tumorigenic functions in NSCLC.

To further validate the oncogenic effect of FOXP3 on the growth of NSCLC, shFOXP3 lentivirus was generated and the silence effect was validated by qPCR (Fig. 2d). MTT assay showed that the knockdown of FOXP3 in A549 could inhibit cell growth (p < 0.05) (Fig. 2d). The colony formed by shFOXP3 lentivirus-infected cells was fewer than the control lentivirus-infected cells (Fig. 2d). These results further support that FOXP3 is an oncogenic molecule in NSCLC.

Ectopic expression of FOXP3 induces EMT in NSCLC cells

When we first infected A549 cells by FOXP3 lentivirus, we found a significant spindle shape change in A549 cells (Fig. 3a) compared to control cells, which is further confirmed in H460 cells infected with FOXP3 lentivirus (Fig. 3a). When we seeded A549-FOXP3 cells in 6-well plate to form colony for about 1–2 weeks, it was found that cells lost cell-cell contact and formed a scattered phenomenon (Fig. 3a). A similar finding was also observed in H460-FOXP3 cells after two-week growth (Fig. 3a). Spindle shape change and lose of cell-cell contact are the characteristics of cell morphology change during Epithelial-Mesenchymal Transition [21]. E-Cadherin is the most remarkable markers of EMT, immunofluorescent staining demonstrated that E-Cadherin was markedly reduced in A549-FOXP3 cells compared with control cells (Additional file 2: Figure S3), which is consistent with the cell morphology change that FOXP3 induced the EMT. Western blot was used to analyze EMT markers induced by FOXP3 in A549 and H460 cells (Fig. 3b). E-Cadherin was downregulated in both A549-FOXP3 and H460-FOXP3 cells compared to control cells, which is consistent with our immunofluorescent staining assays. An upregulation of N-Cadherin, vimentin, Snail, Slug and MMP9 was also induced by FOXP3 in both cells. All these changes in biomarkers suggest a transition of epithelial cells to mesenchymal cells.

Fig. 3 FOXP3 induces EMT in NSCLC cells. a FOXP3 induces mesenchymal morphology changes in A549 and H460 cells: spindle shape and loss of cell-cell contact. b The effect of FOXP3 overexpression on EMT marker expression was assessed by western blot. c Effect of FOXP3 knockdown on EMT marker expression in A549 cells Full size image

Then, we knocked down FOXP3 to check whether it could reverse the change of EMT markers induced. A549-shFOXP3 cells which was generated early was used for analysis. Western blot results (Fig. 3c) showed that E-Cadherin was upregulated, N-Cadherin, Vimentin, snail, slug and MMP9 were downregulated compared to control cells. These changes were opposite to FOXP3 overexpression experiment, and further validated the role FOXP3 on EMT induction.

Ectopic expression of FOXP3 promotes cancer metastasis in vitro and in vivo

First, the result of the wound healing assay showed that cells infected with FOXP3 lentivirus had a significant healing ability than control cells in wound formation, which is about 1.35 fold faster than control cells (p < 0.01) (Fig. 4a, Additional file 2: Figure S4A), suggesting A549-FOXP3 has an stronger ability in migration than A549-control. Second, we did Matrigel invasion assays to test the ability of invasion. Photomicrographs and histograms (Fig. 4b) indicated that the number of A549-FOXP3 cells migrated to lower chambers through Matrigel is more than that of control cells migrated to the lower chambers (2.4 fold, p < 0.01). Third, soft agar assays showed that A549-FOX3 and H460-FOXP3 cells formed more and larger colonies (p < 0.01) compared to control cells (Fig. 4c). Finally, to test whether FOXP3 could promote metastasis in vivo, A549-FOXP3 and control cells were inoculated into nude mice via tail vein injection. The mice were sacrificed 2 months later to examine metastatic nodules in lung, liver and spleen. The results of H&E staining showed that ectopic expression of FOXP3 in A549 cells led to a significant increase in the total number of metastatic nodules in the lung of nude mice (Fig. 4b-d), although we didn’t find metastatic nodules in the liver and spleen of nude mice (data not shown). All these findings suggest that FOXP3 is able to promote metastasis in NSCLC cells.

Fig. 4 FOXP3 promotes tumor metastasis in NSCLC. a Effect of FOXP3 on cell migration was evaluated by wound healing assay in A549 cells. FOXP3-expressing A549 cells had a much stronger healing ability than control cells (p < 0.01). b Effect of FOXP3 on cell invasion was evaluated in A549 cells by Matrigel invasion assay. FOXP3-expressing A549 cells showed higher penetration rate through the Matrigel-coated membrane compared with control cells (p < 0.001). c Soft agar growth analysis demonstrated that FOXP3-expressing A549 and H460 cells had a stronger tumorigenicity than control cells (p < 0.01). d FOXP3 promotes tumor metastasis in vivo. The total number of metastatic nodules was quantified in lungs of nude mice 8 weeks after tail vein injection of control and FOXP3-expressing A549 cells. Values for individual mouse were dotted in the plot and values by group were also denoted. Data are mean ± s.d. Please refer to Additional file 2: Figure S4 for representative images for a, b and d Full size image

FOXP3 enhances Wnt/β-catenin pathway in NSCLC

To gain insights into the mechanism of FOXP3-mediated tumorigenicity and EMT, microarray gene expression profiling was conducted. Hierarchical clustering analysis showed that FOXP3 overexpression led to a clear and consistent difference in the gene expression profile (Additional file 2: Figure S5). We identified 1811 upregulated genes, and 2292 downregulated genes (data not shown). We conducted GO analysis to reveal general functional features implemented by FOXP3 in A549 cells using upregulated genes. The results of both Biological process analysis (Additional file 2: Figure S6A) and cellular component analysis (Additional file 2: Figure S6B) coincided with the characteristics of EMT process.

Pathway analysis suggests multiple pathways might be involved in A549 cells after FOXP3 stimulation (Additional file 2: Figure S7). Wnt/β–catenin pathway was selected for further studies as it is broadly involved in the process of promoting tumorigenicity, cell stemness and EMT induction [21, 22]. Data analysis of Wnt/β–catenin pathway (data not shown) suggested that 4 out of 6 direct downstream genes including c-Myc, Cyclin D1, c-Jun, fra-1 were upregulated, the finding of which is another evidence to support an activated Wnt/β–catenin pathway in A549-FOXP3 cells. To determine whether Wnt/β–catenin pathway was activated by FOXP3 in NSCLC cells, we first performed a luciferase experiment with Top-Luc Flash reporter and pRL-TK (as an internal control). Our results showed that FOXP3 increased luciferase activity dramatically in both A549 and H460 cells (Fig. 5a). And we observed a significant reduction of the luciferase activity in A549 cells when FOXP3 was silenced by lentivirus (Fig. 5b). These results suggest that the overexpression of FOXP3 stimulates the Wnt/β–catenin pathway in NSCLC cells.

Fig. 5 The oncogenic effect of FOXP3 is mediated by activating Wnt/β-catenin signaling pathway. a The effect of FOXP3 on Wnt/β-catenin signaling pathway was assessed by dual-luciferase reporter assays in A549 and H460 cells. b Knockdown of FOXP3 impaired Topflash reporter activities in A549 cells. c FOXP3 upregulated protein expression of c-Myc and Cyclin D1 in A549 and H460 cells. d FOXP3 upregulated mRNA expression of c-Myc and Cyclin D1 in A549 and H460 cells Full size image

We also analyzed the expression of c-Myc and Cylin D1, two well-known target genes of the Wnt/β–catenin pathway [23,24,25], in A549-FOXP3 and H460-FOXP3 cells. Both qPCR (Fig. 5d) and western blot (Fig. 5c) results showed the elevated expression of c-Myc and Cylin D1.

FOXP3 acts as a co-activator of Wnt/β-catenin signaling in NSCLC cells

In most cases, the elevated expression of β-catenin is the cause of activating canonical pathway. However, our western blot results did not show the significant changes in β-catenin levels in A549-FOXP3 and H460-FOXP3 cells compared with corresponding control cells. Since our immunofluorescent staining results (Additional file 2: Figure S8) which showed the localization of overexpressed FOXP3 protein was mainly in the nucleus of A549-FOXP3 and H460-FOXP3 cells, it is likely that the oncogenic function of FOXP3 is correlated with the LEF/TCF complex, which is the potent Wnt/β-catenin transcriptional factor in the nucleus [23, 26]. We thus examined whether FOXP3 interacted with the nuclear components of the Wnt/β-catenin signaling pathway. Reciprocal immunoprecipitation experiments indicated that FOXP3 and TCF4 interacted with each other in HEK-293T cells (Fig. 6a), suggesting that FOXP3 could form a complex with TCF4 in the nucleus. In the canonical pathway, β-catenin combined with LEF/TCF4 complex to activate Wnt signaling [19, 20, 27, 28]. We thus questioned whether FOXP3 could also form a complex with β-catenin. The Co-IP test demonstrated that β-catenin and FOXP3 could precipitate down each other in the nucleus (Fig. 6b), suggesting that FOXP3 could also associate with β-catenin to form a complex. To this end, we speculated FOXP3 might enhance the formation of β-catenin·TCF4 complex and work as a co-activator of Wnt/β-catenin pathway. Immunoprecipitation results showed that the interaction of FLAG-β-catenin with Myc-TCF4 increased 1.45 fold in the nucleus when FOXP3 was co-expressed (Fig. 6c). Furthermore, an immunoprecipitation experiment using endogenous proteins demonstrated that the interaction of endogenous β-catenin and endogenous TCF4 was significantly increased 1.79 fold in the nucleus by FOXP3 overexpression (Fig. 6d). Taken together, these results suggest that FOXP3 is a co-activator to enhance the formation of the β-catenin and TCF4 complex in the nucleus of NSCLC cells.