TGF-β induced EMT and stemness characteristics are associated with epigenetic regulation in lung cancer

Abstract

Transforming growth factor-β (TGF-β) promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT). EMT is often related with acquisition of stemness characteristics. The objective of this study was to determine whether EMT and stemness characteristics induced by TGF-β might be associated with epigenetic regulation in lung cancer. A human normal lung epithelial cell line and four lung cancer cell lines were treated with TGF-β. Transcriptome analysis of BEAS-2B and A549 cells incubated with TGF-β were analyzed through next-generation sequencing (NGS). Western blotting was carried out to investigate expression levels of epithelial and mesenchymal markers. Wound healing and Matrigel invasion assay, sphere formation assay, and in vivo mice tumor model were performed to evaluate functional characteristics of EMT and stemness acquisition. To investigate whether activation of EMT and stem cell markers might be involved in epigenetic regulation of lung cancer, experiment using a DNA methyltransferase inhibitor (5-azacytidine, AZA), methylation-specific PCR (MSP) and bisulfite sequencing were performed. NGS revealed changes in expression levels of EMT markers (E-cadherin, N-cadherin, fibronectin, vimentin, slug and snail) and stem cell markers (CD44 and CD87) in both BEAS-2B and A549 cells. Functional analysis revealed increased migration, invasion, sphere formation, and tumor development in mice after TGF-β treatment. Expression of slug and CD87 genes was activated following treatment with AZA and TGF-β. MSP and bisulfite sequencing indicated DNA demethylation of slug and CD87 genes. These results suggest that TGF-β induced EMT and cancer stemness acquisition could be associated with activation of slug and CD87 gene by their promoter demethylation.

Figure 1

Transcriptome analysis using next-generation sequencing in BEAS-2B and A549 cells treated with TGF-β for 72 h to screen EMT and stemness genes. (A) A distinct separation of mRNA expression patterns of cells treated with or without TGF-β was indicated by heat map of hierarchical clustering. (B) mRNA expression levels of N-cadherin, fibronectin, Vimentin, slug, and snail were increased in lung cells treated with TGF-β whereas those of E-cadherin were decreased in cells treated with TGF-β. (C) Expression levels of stem cell markers (CD44 and CD87) were enhanced following TGF-β treatment.

Figure 2

Expression of TGF-β induced EMT markers. E-cadherin expression levels were decreased in lung cells (BEAS-2B, A549 and H292) following TGF-β treatment whereas those of N-cadherin, fibronectin, vimentin, slug, and snail were increased, although their protein levels differed according to lung cells. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. S1.

Figure 3

Functional analysis of EMT using wound healing and Matrigel invasion assays following TGF-β treatment. (A) Although the growth rate of lung cells treated with TGF-β was different, cell migration toward the center of scratched area was higher in lung cells induced by TGF-β. (B) Transwell invasion in BEAS-2B, A549, H292, H226, and H460 cells following TGF-β treatment was enhanced. Data are presented as mean ± SE (n = 4, *P < 0.05).

Figure 4

Acquisition of stemness in vitro and in vivo. (A) Sphere forming potentials of all lung cells following TGF-β treatment were higher than those of cells untreated with TGF-β. (B, C) Tumor formation abilities in BEAS-2B, A549, and H226 cells treated with or without TGF-β were compared following injection into BALB/c nude mice. Numbers of mice that developed tumors were higher in TGF-β treated cell groups compared to those in TGF-β untreated cell groups. Weight and size of the tumors were enhanced following injection of BEAS-2B (1 × 10⁶ and 5 × 10⁶), A549 (5 × 10⁶), and H226 (5 × 10⁶) cells treated with TGF-β compared with those in TGF-β untreated cell groups. Data are presented as mean ± SE (*P < 0.05, ^†P < 0.01).

Figure 5

TGF-β induced slug and CD87 activations by their promoter DNA demethylations. (A) After treatment with either DNA methyltransferase inhibitor (AZA) or TGF-β, real-time RT-PCR indicated significantly enhanced slug and CD87 expressions compared with controls (non-AZA + non-TGF-β) in BEAS-2B and A549 cells. Data are presented as mean ± SE (n = 3, *P < 0.05). (B, C) Methylation-specific PCR revealed increased unmethylation levels of slug and CD87 genes in TGF-β treated cell groups whereas methylation levels of these genes were decreased following TGF-β treatment. These expression levels were compared with controls (non-TGF-β). Cropped images of Figs. B and C are displayed, uncropped blots are displayed in Supplementary Figs. S2 and S3. Data are presented as mean ± SE (n = 3, *P < 0.05).

Figure 6

Representative demonstration of CpG site methylation changes of CD87 promoter region in BEAS-2B and A549 cells. Bisulfite sequencing showed six CpG sites presented in underlined letters within a 100-bp promoter region of CD87 gene. Demethylated CpG sites following TGF-β treatment are indicated by red letters. Heterozygote C/T double peaks are indicated by Y.