TGF-β-mediated epithelial-mesenchymal transition and tumor-promoting effects in CMT64 cells are reflected in the transcriptomic signature of human lung adenocarcinoma

Abstract

Epithelial-mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal phenotypes. Cancer cells undergo EMT to acquire malignant features and TGF-β is a key regulator of EMT. Here, we demonstrate for the first time that TGF-β could elicit EMT in a mouse lung adenocarcinoma cell line. TGF-β signaling activation led to cell morphological changes corresponding to EMT and enhanced the expression of mesenchymal markers and EMT-associated transcription factors in CMT64 lung cancer cells. RNA-sequencing analyses revealed that TGF-β increases expression of Tead transcription factors and an array of Tead2 target genes. TGF-β stimulation also resulted in alternative splicing of several genes including Cd44, tight junction protein 1 (Tjp1), and Cortactin (Cttn). In parallel with EMT, TGF-β enhanced cell growth of CMT64 cells and promoted tumor formation in a syngeneic transplantation model. Of clinical importance, the expression of TGF-β-induced genes identified in CMT64 cells correlated with EMT gene signatures in human lung adenocarcinoma tissue samples. Furthermore, TGF-β-induced gene enrichment was related to poor prognosis, underscoring the tumor-promoting role of TGF-β signaling in lung adenocarcinoma. Our cellular and syngeneic transplantation model would provide a simple and useful experimental tool to study the significance of TGF-β signaling and EMT.

Figure 1

TGF-β induces EMT in CMT64 lung adenocarcinoma cells. (A) CMT64 cells were pretreated with 3 μM of LY364947, a TGF-β type I kinase inhibitor, or control DMSO, and were further stimulated with 5 ng/ml of TGF-β for 1 h. LY364947 or DMSO were added 1 h before TGF-β treatment. Immunoblotting was performed for phosphorylated Smad2 (p-Smad2; C-terminal region, Ser 465/467), total Smad2, phosphorylated Smad3 (p-Smad3; C-terminal region, Ser 423/425), and total Smad3. (B) CMT64 cells were cultured with or without 5 ng/ml TGF-β for 72 h. Upper: Cells were analyzed by phase-contrast microscopy (bar: 100 µm). Lower: Actin reorganization was visualized by phalloidin staining (bar: 20 µm). Green: phalloidin. Blue: DAPI (nuclei). (C) CMT64 cells were incubated with or without 5 ng/ml of TGF-β for 72 h. Immunocytochemistry for E-cadherin or fibronectin (green) was performed. Blue: DAPI (nuclei).

Figure 2

Gene expression profiling following TGF-β and/or TNF-α stimulation. (A) Venn diagram illustrating overlaps between genes upregulated by TGF-β alone and TGF-β/TNF-α costimulation in CMT64 cells. The threshold was set as fold change > 2 and RPKM > 2 in the group treated with TGF-β alone or both TGF-β and TNF-α. Numbers of identified genes are indicated. (B) Heatmap indicates relative expression levels of EMT markers (Cdh1, Cdh2, and Fn1), EMT-associated transcription factors (Snai1, Snai2, Zeb1, Zeb2, and Hmga2), and TGF-β target genes (Smad7, Serpine1, Thbs1, Pdgfa, Pdgfb, Jun, Junb, Wnt7a, and Wnt7b). CMT64 cells were treated with 5 ng/ml of TGF-β and/or 10 ng/ml TNF-α for 24 h. For the indicated genes, z-scores were calculated from RPKM values obtained by RNA-seq analysis. (C) Quantitative RT-PCR was performed for Cdh1 and EMT-associated transcription factors (Snai1, Snai2, Zeb1, Zeb2, and Hmga2) in CMT64 cells treated with 5 ng/ml of TGF-β for 24 h in the presence or absence of 3 μM of LY364947. Expression levels were normalized to that of Gapdh. Experiments were performed in triplicate. Error bars: SE.

Figure 3

TGF-β-mediated EMT is accompanied by enhanced activity of Tead transcription factors. (A) Three known recognition sequences for TEAD transcription factors were among top 10 enriched motifs at the promoters of genes induced by TGF-β. Genes without changes upon TGF-β treatment were used as backgrounds. Percentage of genes that have the indicated motif is shown. (B) Heatmap indicates relative expression levels of Yap/Taz signaling components (Yap1, Wwtr1, Tead1, Tead2, Tead3, and Tead4) and Yap/Taz target genes (Amotl1, Amotl2, Crim1, Fstl1, Ccnd1, Ctgf, and Cyr61). CMT64 cells were treated with 5 ng/ml of TGF-β and/or 10 ng/ml TNF-α for 24 h. For the indicated genes, z-scores were calculated from RPKM values obtained by RNA-seq analysis. (C) Gene set enrichment analysis was performed to examine the enrichment of Tead2 target gene signature in TGF-β-induced genes in CMT64 cells.

Figure 4

Differential splicing is induced by TGF-β in CMT64 cells. (A) Alternative transcripts for genes involved in ECM-cell interactions (Cd44), cell junction (Tjp1), and cytoskeletal organization (Cttn). Dotted square indicates the exons of alternative splicing by TGF-β treatment. Expression levels determined by RNA-seq in CMT64 cells cultured with or without 5 ng/ml of TGF-β for 24 h were shown by Integrative Genomics Viewer. (B) Quantitative RT-PCR was performed for Cd44 exon 13, Tjp1 exon 20, and Cttn exon 11 in CMT64 cells treated with 5 ng/ml of TGF-β for 24 h in the presence or absence of 3 μM of LY364947. LY364947 or DMSO was added 1 h before TGF-β treatment. Expression levels were normalized to that of Gapdh. Error bars: SE.

Figure 5

TGF-β enhances cell growth and tumorigenesis of CMT64 cells. (A) Cell count of CMT64 cells on days 2, 4, and 6 after 5 ng/ml of TGF-β stimulation are shown. (B) Hematoxylin & eosin staining image of tumors derived from CMT64 cells. CMT64 cells treated with or without 5 ng/ml of TGF-β for 96 h were injected subcutaneously into C57BL/6 mice. CMT64 cells were exposed to TGF-β only at the pre-transplant stage. The tumor was harvested after 26 d after subcutaneous injection of 1 × 10⁷ CMT64 cells. Bar: 50 μm. (C) CMT64 cells treated with or without 5 ng/ml of TGF-β for 96 h were injected subcutaneously into C57BL/6 mice. Tumor volume and tumor weight were measured after 17 days. Eight mice were used in both groups. P < 0.05 for each measurement; Student’s t-test.

Figure 6

Clinical relevance of TGF-β-mediated gene expression changes. (A) Correlation between TGF-β-induced gene signature scores and those of EMT or ECM in lung adenocarcinoma tissue samples derived from the GSE81089 dataset. (B) Box plot of TGF-β scores were compared between two subgroups of staining scores for Ki67. (C) Kaplan–Meier analysis of lung adenocarcinoma cases in the GSE81089 dataset by stratification of TGF-β-induced gene signature scores.