EGFR-AS1/HIF2A regulates the expression of FOXP3 to impact the cancer stemness of smoking-related non-small cell lung cancer

Abstract

Background: Early data showed that FOXP3 could induce epithelial-mesenchymal transition by stimulating the Wnt/β-catenin signaling pathway in non-small cell lung cancer (NSCLC). However, how the expression of FOXP3 is regulated in NSCLC remains unknown. We thus explored the impacts of the long noncoding RNA EGFR antisense RNA 1 (EGFR-AS1) and hypoxia-inducible factor-2A (HIF2A) on FOXP3 expression and the cancer stemness of NSCLC.

Methods: Lung tissues samples from 87 patients with NSCLC and two NSCLC cell lines were used in this study. The regulation of FOXP3 and lung cancer cell stemness by EGFR-AS1 and HIF2A was determined at molecular levels in NSCLC tissue samples and cultured cells in the presence/absence of the smoking carcinogen, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (also known as nicotine-derived nitrosamine ketone). The results were confirmed in tumor xenograft models.

Results: We found that NNK decreased the expression of EGFR-AS1 in the long term, but increased the expression of HIF2A and FOXP3 to stimulate lung cancer cell stemness. EGFR-AS1 significantly inhibited FOXP3 expression and NSCLC cell stemness, whereas HIF2A obviously promoted both. The enhancement of lung cancer stemness by FOXP3 was, at least partially, via stimulating Notch1, as the inhibition of Notch1 could markedly diminish the effect of FOXP3.

Conclusions: FOXP3, the expression of which is under the fine control of EGFR-AS1, is a critical molecule that promotes NSCLC cancer cell stemness through stimulating the Notch1 pathway.

Keywords: EGFR antisense RNA 1; FOXP3; cancer cell stemness; hypoxia-inducible factor-2 alpha; non-small cell lung cancer.

Figure 1.

Effects of smoking on the expression of EGFR-AS1, HIF2A, and FOXP3, and lung cancer cell stemness. (a) Two NSCLC cell lines were treated with NNK for different periods. The expression of EGFR-AS1 was increased significantly at first (p < 0.01), and then reduced from the elevated level (p < 0.01). (b) Two NSCLC cell lines were treated with NNK for different periods. The expression of FOXP3, HIF2A, and two lung cancer stem-cell markers (ALDH1A1 and OCT4A) was evaluated by western blot. (c) and (d) The expression of HIF2A in patient tissues was evaluated by immunohistochemistry (IHC). IRS was applied to quantify the expression of HIF2A in adjacent normal tissues (N) and tumor tissues (T), **p < 0.01. (e) and (f) Real-time PCR was used to evaluate the expression of long noncoding RNA EGFR-AS1 in samples from patients with NSCLC, *p < 0.05. (f) The correlation analysis showed that the expression of EGFR-AS1 was negatively associated with smoking status, **p < 0.01. The experiments were performed in triplicate at least. EGFR-ASI, EGFR antisense RNA 1; HIF2A, hypoxia-inducible factor-2A; IRS, immunoreactive score; NSCLC, non-small cell lung cancer; NNK, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; PCR, polymerase chain reaction.

Figure 2.

The influence of EGFR-AS1 on FOXP3 expression and NSCLC cells stemness in vitro. (a) EGFR-AS1 plasmid was transfected into the NSCLC cells, and the expression of EGFR-AS1 was evaluated by qRT-PCR assay, *p < 0.05. (b) EGFR-AS1 plasmid was transfected into the NSCLC cells, and then the levels of HIF2A, FOXP3, and two CSC markers were evaluated by western blot. (c) EGFR-AS1 expression plasmid was transfected into NSCLC cells to construct stable expression cell lines, and then tumor-sphere formation was evaluated (left panel). Compared with scramble, EGFR-AS1 decreased the efficiency of tumor-sphere formation (right panel), **p < 0.01. (d) Colony-formation assay was conducted to evaluate the proliferative ability of the two cell lines **p < 0.01. (e) Transwell assay was conducted to evaluate the invasive ability of the two cell lines, **p < 0.01. (f) MTT assay was used to evaluate the proliferative ability of the two cell lines, *p < 0.05. The experiments were performed in triplicate at least. CSC, cancer stem cell; EGFR-ASI, EGFR antisense RNA 1; HIF2A, hypoxia-inducible factor-2A; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcriptase-polymerase chain reaction.

Figure 3.

Effects of HIF2A on FOXP3 expression and NSCLC cell stemness in vitro. (a) After HIF2A plasmid was transfected into the NSCLC cell lines, the expression of HIF2A was evaluated by qRT-PCR assay, **p < 0.01. (b) After HIF2A plasmid was transfected into the NSCLC cell lines, the expression of FOXP3 and two CSC markers, ALDH1A1 and OCT4A, was evaluated by western blot. (c) After the stable expression of HIF2A in H23 and H460 cells, tumor-sphere formation was evaluated (left panel). Compared with scramble, HIF2A increased the efficiency of tumor-sphere formation (right panel) **p < 0.01, *p < 0.05. (d) Colony-formation assay was conducted to evaluate the proliferative ability of the four cell lines, **p < 0.01. (e) Transwell assay was conducted to evaluate the invasive ability of the two cell lines, **p < 0.01. (f) MTT assay was used to evaluate the proliferative ability of the four cell lines, **p < 0.01, *p < 0.05. The experiments were performed in triplicate at least. CSC, cancer stem cell; HIF2A, hypoxia-inducible factor-2A; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcriptase-polymerase chain reaction.

Figure 4.

Effects of shHIF2A on FOXP3 expression and NSCLC cell stemness in vitro. (a) After HIF2A shRNA was transfected into the NSCLC cell lines, the expression of HIF2A was evaluated by qRT-PCR assay **p < 0.01. (b) After HIF2A shRNA plasmid was transfected into the NSCLC cell lines, the expression of FOXP3 and two CSC markers, ALDH1A1 and OCT4A, was evaluated by western blot. (c) After the stable expression of HIF2A shRNA in H23 and H460 cells, tumor-sphere formation was evaluated (left panel). Compared with scramble, shHIF2A decreased the efficiency of tumor-sphere formation (right panel) **p < 0.01, *p < 0.05. (d) Colony-formation assay was conducted to evaluate the proliferative ability of the two cell lines, **p < 0.01. (e) Transwell assay was conducted to evaluate the invasive ability of the two cell lines, *p < 0.01. (f) MTT assay was used to evaluate the proliferative ability of the four cell lines, ** p < 0.05, *p < 0.01. The experiments were performed in triplicate at least. CSC, cancer stem cell; HIF2A, hypoxia-inducible factor-2A; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcriptase-polymerase chain reaction.

Figure 5.

The regulation of FOXP3 expression in NSCLC cells by EGFR-AS1/HIF2A and the effects of FOXP3 on lung cancer stemness through the Notch pathway. (a) After FOXP3 plasmid was transfected into the NSCLC cell lines, the expression of FOXP3 was evaluated by qRT-PCR assay, **p < 0.01. (b) After FOXP3 plasmid was transfected into the NSCLC cell lines, the expression of FOXP3 and two CSC markers, ALDH1A1 and OCT4A, was evaluated by western blot. (c) After the stable expression of FOXP3 in H23 and H460 cells, tumor-sphere formation was evaluated (left panel). Compared with scramble, FOXP3 increased the efficiency of tumor-sphere formation (right panel) **p < 0.01, *p < 0.05. (d) The immunoprecipitation experiment results demonstrated that HIF2A could bind with FOXP3. (e) After FOXP3 shRNA was transfected into the NSCLC cell lines, the expression of FOXP3 was evaluated by qRT-PCR assay, **p < 0.01. (f) After FOXP3 shRNA plasmid was transfected into the NSCLC cell lines, the expression of FOXP3 and two CSC markers, ALDH1A1 and OCT4A, was evaluated by western blot. (g) After the stable expression of FOXP3 shRNA in H23 and H460 cells, tumor-sphere formation was evaluated (left panel). Compared with scramble, shFOXP3 decreased the efficiency of tumor-sphere formation (right panel) **p < 0.01, *p < 0.05. (h) Luciferase report assay was conducted to evaluate the influence of EGFR-AS1 and HIF2A on the activity of HIF2A promoter, **p < 0.01. (i) Luciferase report assay was conducted to evaluate the influence of EGFR-AS1 and HIF2A on the activity of the FOXP3 promoter, **p < 0.01, *p < 0.05. (j) Chromatin immunoprecipitation assays were conducted to evaluate the influence of HIF2A on the FOXP3 promoter, *p < 0.05. The experiments were performed in triplicate at least. CSC, cancer stem cell; EGFR-ASI, EGFR antisense RNA 1; HIF2A, hypoxia-inducible factor-2A; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcriptase-polymerase chain reaction.

Figure 6.

The critical role of the Notch pathway in FOXP3-mediated NSCLC cell stemness. (a) Colony- formation assay was conducted to evaluate the impact of the Notch inhibitor DAPT on the proliferative ability of the NSCLC cell lines, **p < 0.01, *p < 0.05. (b) After the treatment of DAPT, tumor-sphere formation was assayed (left panel). Compared with DMSO, DAPT decreased tumor- sphere formation (right panel) **p < 0.01, *p < 0.05. (c) The expression of cancer stem markers was evaluated by western blot after DAPT treatment. (d) After NNK treatment, the expression of Notch1 and HES1 increased with the treatment time extended. (e) Western blot analysis was used to evaluate the influence of EGFR-AS1, HIF2A, and FOXP3 on the Notch pathway. The experiments were performed in triplicate at least. DAPT, N-[2S-(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine; DMSO, dimethyl sulfoxide; EGFR-ASI, EGFR antisense RNA 1; HIF2A, hypoxia-inducible factor-2A; NNK, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; NSCLC, non-small cell lung cancer.

Figure 7.

The potential mechanism of FOXP3-mediated regulation of cancer cell stemness. NNK could decrease EGFR-AS1 but increase HIF2A and FOXP3. The transcription factor HIF2A may bind to the FOXP3 promoter to increase the expression of FOXP3, promoting lung cancer cell stemness through the activation of the Notch pathway. In contract to HIF2A, the long noncoding RNA EGFR-AS1 may interact with the FOXP3 promoter to suppress the expression of FOXP3, thus inhibiting lung CSCs. CSC, cancer stem cells; EGFR-ASI, EGFR antisense RNA 1; HIF2A, hypoxia-inducible factor-2A; NNK, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone.