IFN-γ induces ZEB1 expression in lung adenocarcinoma.
Previously, we performed a global transcriptome study (microarray analysis) and compared tumor tissues with high versus low IFNG expression level. ZEB1 mRNA levels were significantly higher in tumors with high IFNG expression (GSE99995). To determine whether IFN-γ has the ability to induce ZEB1 transcription in lung cancer cells, five lung adenocarcinoma cell lines and the lung carcinoid cell line UMC-11 were cultured with IFN-γ, and ZEB1 mRNA levels were evaluated. Significantly higher ZEB1 mRNA levels were observed in all five lung adenocarcinoma cell lines cocultured with IFN-γ, whereas IFN-γ appeared to have no effect on ZEB1 expression in lung carcinoid cells (Fig. 1a). Immunoblotting analysis revealed that ZEB1 protein levels increased within 12 h upon IFN-γ stimulation and sustained for at least 72 h after a single treatment (Fig. 1b). Strikingly, prolonged exposure of A549 and H1975 cells to IFN-γ led to sustained ZEB1 expression even after removal of IFN-γ (Fig. 1c).
Furthermore, we cultured human-derived lung adenocarcinoma cells with IFN-γ ex vivo. IFN-γ was able to induce ZEB1 transcription (Fig. 1d). Interestingly, exogenous IFN-γ did not significantly increase ZEB1 mRNA levels in distant non-tumor lung tissues from patients with lung cancer (Fig. 1e). Our data demonstrate that IFN-γ induces ZEB1 expression at both mRNA and protein levels in lung adenocarcinoma cells.
IFN-γ induces EMT in lung adenocarcinoma cells.
E-cadherin and Vimentin are the most commonly used EMT markers, as their expression patterns undergo dramatic changes during EMT. In A549, HCC827, and H2228 lung cancer cells, IFN-γ stimulation downregulated E-cadherin expression and upregulated Vimentin expression, as determined by immunoblot analysis (Fig. 2a). The altered expression patterns of E-cadherin and Vimentin in lung cancer cells upon IFN-γ treatment were further validated by immunofluorescence analysis (Fig. 2b). Real-time PCT (RT-PCR) analysis revealed that CDH1 transcription was decreased at 24 h and maintained at a low level in A549 and H2228 cells, whereas VIM transcription was significantly increased to various degrees in the three cell lines in response to IFN-γ stimulation (Fig. 2c).
Treatment with low amounts of IFN-γ (10 IU/ml) for 3 days did not significantly affect E-cadherin levels in A549 and HCC827 (Supplementary Fig. 1a). While treatment with IFN-γ at 25 IU/ml significantly downregulated E-cadherin levels in A549 and HCC827 cells (Supplementary Fig. 1b). Interestingly, prolonged exposure to IFN-γ induced morphological changes in A549 and HCC827 cells, which acquired a fibroblast-like appearance (Fig. 2c). The fibroblast-like appearance of A549 cells became even more evident after treatment with IFN-γ for 21 days (Supplementary Fig. 1c), indicating that IFN-γ−treated cells enter a stable mesenchymal-associated state. Substantial evidence has shown that EMT is associated with increased cell migration in vitro. As shown in Fig. 2e, the migratory capability of A549 and HCC827 cells was indeed significantly increased after IFN-γ treatment. IFN-γ treatment led to increases in migration of 57% and 45% in A549 and HCC827 cells, respectively. Collectively, these data demonstrate that IFN-γ induces EMT in lung adenocarcinoma cells and promotes cell migration in vitro.
IFN-γ-induced ZEB1 expression stimulates EMT.
EMT involves a robust reprogramming of gene expression. We analyzed the transcriptome alternations by RNA-seq analysis during EMT following IFN-γ treatment. It has been reported that A549 cells have mesenchymal characteristics, whereas HCC827 cells have epithelial features . For these reasons, we selected A549 and HCC827 cells to investigate the reprogramming of gene transcription by IFN-γ. An EMT signature consisting of 130 genes was analyzed, including 67 upregulated mesenchymal-associated genes and 63 downregulated epithelial-associated genes (Supplementary Table 3) . Of the 63 epithelial-associated genes, 35 genes including CDH1, were highly expressed in HCC827 cells compared with A549 cells; of the 67 mesenchymal-associated genes, 35 genes, including VIM, were expressed at lower levels in HCC827 cells than in A549 cells (Fig. 3a). These results indicate that relative to A549 cells, HCC827 cells have more epithelial-associated features, which was consistent with the reports by others . EMT is a dynamic process with intermediary states that is not easily identified in cultured cells. We performed transcriptome analysis after the cells were treated with IFN-γ for 8 or 24 h. The number of differentially expressed genes was dramatically increased after 24 h of stimulation with IFN-γ compared with 8 h of stimulation (Supplementary Fig. 2A).
In A549 cells, 58 genes were differentially expressed after 24 h of IFN-γ treatment, including 20 upregulated and 38 downregulated genes (Log2FC ≥ 0.5, P < 0.05) (Fig. 3b and Supplementary Table 4). Among these differentially expressed genes, of 67 genes that are upregulated in EMT, 13 genes were upregulated after IFN-γ treatment, and of 63 genes that are downregulated in EMT, 23 genes were downregulated after IFN-γ treatment. In HCC827 cells, 58 genes were differentially expressed after 24 h of IFN-γ treatment, including 32 upregulated and 26 downregulated genes (Log2FC ≥ 0.5, P < 0.05) (Fig. 3c and Supplementary Table 5); among these differentially expressed genes, of 67 genes that upregulated in EMT, 21 genes were upregulated after IFN-γ treatment, and of 63 genes that are downregulated in EMT, 12 genes were downregulated after IFN-γ treatment. Detailed analysis revealed that IFN-γ stimulation of the mesenchymal-like A549 cells led to downregulation of more epithelial-associated genes (genes downregulated during EMT) whereas IFN-γ stimulation of the epithelial-like HCC827 cells led to upregulation of more mesenchymal-associated genes (genes upregulated during EMT). Although IFN-γ altered the expression patterns of E-cadherin and Vimentin in both A549 and HCC827 cells, transcriptome analysis of EMT-associated genes in IFN-γ-treated cells revealed that IFN-γ induced EMT in lung cancer cells is not a unified state. The characteristics of IFN-γ-induced EMT could be associated with the intrinsic state of untreated lung cancer cells.
Although IFN-γ differentially alters the expression of EMT-associated genes in A549 and HCC827 cells, RNA-seq analysis showed that the expression of 15 genes was equally affected in both cell lines upon IFN-γ treatment, including 7 upregulated and 8 downregulated genes. Among the upregulated genes, CTGF, which encodes connective tissue growth factor (CTGF), is able to induce EMT and its expression levels are highly correlated with EMT markers . FGF2, which encodes basic fibroblast growth factor, and MAP1B, which encodes a protein belonging to the microtubule-associated protein family, are both upregulated during EMT . FGF2 promotes EMT and metastasis through the FGFR1–ERK1/2–SOX2 axis in FGFR1-amplified lung cancer . Actin binding LIM protein 1, encoded by ABL1M1 gene, plays multiple roles in establishing and maintaining cellular structure through mediating interactions between actin filaments and cytoplasmic LIM binding partners . ABLIM1 is downregulated during EMT . These differentially expressed genes in response to IFN-γ stimulation in both A549 and HCC827 cells were confirmed by quantitative RT-PCR (Fig. 3d).
RNA-seq analysis revealed that ZEB1 but not Snail and Slug was significantly upregulated in both A549 and HCC827 cells as early as 8 h upon IFN-γ stimulation (Supplementary Fig. 2A). The effect of IFN-g on the expression of Snail and Slug was confirmed by quantitative RT-PCR (Supplementary Fig. 2B). To determine whether ZEB1 is involved in IFN-γ induced EMT, we knocked down ZEB1 using small interfering RNA (siRNA) in lung cancer cells (Supplementary Fig. 3a). Knockdown of ZEB1 abrogated the IFN-γ-induced upregulation of VIM transcription (Fig. 3e). IFN-γ-mediated alterations in the expression patterns of E-cadherin and Vimentin in A549 and HCC827 cells were reversed upon ZEB1 knockdown (Fig. 3f).
ZEB1 is required for IFN-γ-promoted cell migration and metastasis.
To determine whether ZEB1 is required for IFN-γ-promoted cell migration, we used siRNA-ZEB1 to knock down ZEB1 (Supplementary Fig. 3a). The migratory capability of A549 cells promoted by IFN-γ was significantly compromised by the downregulation of ZEB1 expression (Fig. 4a). To examine the in vivo effects of IFN-γ on lung cancer cell metastasis, we established an in vivo metastasis model by intravenous injection of A549 cells that had been treated with IFN-γ in vitro for 4 days into NCG mice (2 × 106 cells/mouse) (Fig. 4b). The mice were given recombinant human IFN-γ (2000 IU/mouse) intraperitoneally (i.p.) every other day for total three times. Seven days after injection of A549 cells, lung tissues were collected and the presence of metastatic foci and the size of metastases were analyzed microscopically. Control mice were given untreated A549 cells and the mice were not given IFN-γ. As shown in Fig. 4c, IFN-γ treatment significantly increased the number and the size of metastatic nodules in lung tissues.
To determine the role of ZEB1 in this event, we transfected A549 cells with short hairpin RNA against ZEB1 (shRNA-ZEB1) and obtained a stable ZEB1-depleted cell line (Supplementary Fig. 3b). The IFN-γ-promoted increase of A549 cell migration in vitro was diminished in shRNA-ZEB1 A549 cells (Fig. 4d). Our in vivo metastasis model showed that the number and the size of metastatic nodules were reduced in mice injected with IFN-γ-treated shRNA-ZEB1 A549 cells compared with mice injected with IFN-γ-treated control A549 cells, indicating that loss of ZEB1 dramatically reduces IFN-γ-promoted metastasis of A549 cells (Fig. 4e). Our data demonstrate that ZEB1 is responsible for IFN-γ-induced cell migration in vitro and metastasis in vivo.
JAK2-STAT1-dependent upregulation of JMJD3 enhances ZEB1 transcription via demethylation of H3K27.
We next evaluated whether IFN-γ-induced activation of the JAK2–STAT1 pathway is involved in regulation of ZEB1 expression. IFN-γ–treated JAK2–deficient H1573 cells did not exhibit altered expression levels of E-cadherin or ZEB1 (Supplementary Figs. 3c and d), suggesting that IFN-γ–induced EMT and upregulation of ZEB1 might be dependent on JAK2–mediated signaling. To confirm this, we knocked down JAK2 by siRNA-JAK2 in A549 and HCC827 cells (Supplementary Fig. 3e). These cells were subsequently stimulated with IFN-γ. Upregulation of ZEB1 by IFN-γ was no longer observed (Fig. 5a). We used the same approach to determine whether STAT1 is required for IFN-γ–induced ZEB1 expression (Supplementary Fig. 3f). As shown in Fig. 5b, IFN-γ–induced ZEB1 expression was diminished in cells transfected with siRNA-STAT1, indicating that IFN-γ–induced ZEB1 expression requires JAK2–STAT1.
ZEB1 transcription is regulated by the modulation of the chromatin environment at gene regulatory elements [25, 26]. H3K27me3 is often associated with transcriptional repression. The relative absence of H3K27me3 in the chromatin at the ZEB1 promoter signals active transcription . Interestingly, the expression of H3K27 trimethylation and the ratio of H3K27me3 to H3 were rapidly reduced in A549 cells after exposure to IFN-γ (Figs. 5c and d). JMJD3, a direct transcriptional target of STAT1, catalyzes the demethylation of H3K27me3 . For these reasons, we examined whether IFN-γ induces JMJD3 expression. JMJD3 transcription was rapidly upregulated in A549 and HCC827 cells upon exposure to IFN-γ (Fig. 5e). Immunoblot analysis revealed increased JMJD3 expression after 3 h of IFN-γ treatment (Fig. 5f). To determine whether JMJD3 is associated with IFN-γ-induced ZEB1 expression, the JMJD3 specific inhibitor GSK-J4 was used . GSK-J4 significantly reduced IFN-γ stimulated increasing in ZEB1 mRNA levels (Fig. 5g) and protein levels (Fig. 5h). Knockdown experiments with siRNA-JMJD3 further confirmed that IFN-γ-induced ZEB1 expression requires JMJD3 (Supplementary Fig. 3g and Fig. 5i). Downregulation of STAT1 expression led to abrogation of IFN-γ-induced JMJD3 expression (Fig. 5j), confirming that IFN-γ-induced JMJD3 expression is STAT1–dependent.
Since H3K27 demethylation is linked to transcriptional activation, we examined whether H3K27 demethylation is associated with the transcription of ZEB1. The ZEB1 promoter exhibits a bivalent chromatin configuration . H3K4me3 is associated with transcriptional initiation , whereas the H3K27me3 is associated with transcriptional repression. We performed a chromatin immunoprecipitation (ChIP) assay at the ZEB1 promoter to compare the levels of histone modifications in control versus IFN-γ–treated cells. IFN-γ treatment led to a significant reduction in H3K27me3 levels at the ZEB1 promoter in A549 cells, whereas IFN-γ did not significantly affect H3K4me3 levels at the ZEB1 promoter (Fig. 5k). These data demonstrate that IFN-γ enables the ZEB1 promoter to transition from the bivalent to the active chromatin state, at least in part through the demethylation of H3K27me3.
IFN-γ regulates PD-L1 expression via miR-200–ZEB1 axis.
ZEB1 transcription is tightly regulated by microRNA. Recent studies have revealed that IFN-γ promotes ZEB1 expression through IFIT5-mediated suppression of miR-363 in prostate cancer and renal cancer . We did not observe downregulation of miR-363 expression by IFN-γ in lung cancer cells (Supplementary Fig. 4). It is well known that the miR-200 family inhibits ZEB1 expression and plays a major role in preventing ZEB1 from triggering EMT. In turn, ZEB1 can directly repress the transcription of miR-200 loci . As shown in Fig. 6a, miR-200c expression was significantly reduced in A549 and HCC827 cells after 12 h of IFN-γ treatment, and the reduction of miR-200c expression was even more dramatic after 24 and 48 h of IFN-γ treatment. We wondered whether IFN-γ–promoted ZEB1 expression in lung cancer cells is related to the downregulation of miR-200 expression. IFN-γ-induced upregulation of ZEB1 transcription was observed after 4 h in both A549 and HCC827 cells, whereas miR-200c expression was not affected even after 6 h of IFN-γ stimulation (Figs. 6b and c). Our data suggest that IFN-γ first promotes ZEB1 transcription, and subsequently suppresses miR-200c expression.
PD-L1 expression has been reported to be directly regulated by miR-200 family members . We examined whether IFN-γ–induced PD-L1 expression can be regulated by ZEB1. Knockdown of ZEB1 by siRNA did indeed lead to a significant reduction of IFN-γ-induced PD-L1 expression in A549 and HCC827 cells (Fig. 6d). Further analysis revealed that downregulation of ZEB1 enhanced miR-200 expression (Fig. 6e). Our data indicate that IFN-γ stimulation rapidly induces ZEB1 expression and consequently downregulates miR-200c, which at least partially contributes to the upregulation of PD-L1 expression.
IFN-γ–mediated anti–proliferation and induction of CXCL9 and CXCL10 expression are not affected by ZEB1 knockdown.
Previous studies by us and others have shown that IFN-γ suppresses the proliferation of lung cancer cells [2, 32]. We wondered whether ZEB1 is involved in IFN-γ-mediated suppression of cell proliferation. The JMJID3 inhibitor GSK-J4 revered IFN-γ-induced ZEB1 expression (Figs. 5g and h). However, GSK-J4 did not affect IFN-γ-mediated suppression of A549 cell proliferation (Fig. 7a). Knockdown of ZEB1 by siRNA did not alter the anti-proliferative effects of IFN-γ in both A549 and HCC827 cells (Fig. 7b). ZEB1 knockdown had no significant effect on IFN-γ-mediated suppression of colony formation (Fig. 7c). Cyclin E1, which is encoded by the CCNE1 gene, plays a critical role in the control of cell cycle progression by allowing G1 to S phase transition . IFN-γ-treated A549 cells exhibited significantly lower CCNE1 mRNA levels than untreated A549 cells (Fig. 7d). Knockdown of ZEB1 had no effect on the IFN-γ-mediated reduction of CCNE1 expression (Fig. 7d). A previous study by us has shown that IFN-γ-mediated suppression of cell proliferation requires STAT1 and IRF1 . Downregulation of ZEB1 had no effect on IFN-γ-induced STAT1 and IRF1 expression at mRNA and protein levels (Figs. 7e and f). ZEB1 knockdown did not affect IFN-γ-induced phosphorylation of STAT1 (Fig. 7g). Additionally, CXCL9 and CXCL10, which encodes two chemokines that are important for the recruitment of activated lymphocytes to tumors, are also target genes of STAT1-IRF1. As shown in Fig. 7h, knockdown of ZEB1 did not alter the IFN-γ-induced expression pattern of CXCL9 and CXCL10.
Based on our results, we proposed a functional mechanism as shown in Fig. 7i. IFN-γ stimulation results in the rapid upregulation of the expression of the STAT1 target gene JMJD3, which consequently leads to the demethylation of H3K27me3 near the ZEB1 promoter, enabling the transition from the bivalent to an active chromatin state. This leads to increased ZEB1 transcription and protein levels. Elevated ZEB1 expression mediates IFN-γ-induced EMT and cell migration in vitro and promotes metastasis in vivo. ZEB1 knockdown abrogates the pro-tumor effects of IFN-γ, including the induction of EMT, enhanced cell migration and upregulated PD-L1 expression, while retaining IFN-γ−activated STAT1−IRF1−mediated anti-tumor functions.