Drug-induced Epithelial-mesenchymal Transition Research Articles

Timosaponin BII inhibits TGF-β mediated epithelial-mesenchymal transition through Smad-dependent pathway during pulmonary fibrosis.

Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disease with limited therapeutic options at present, and epithelial-mesenchymal transition (EMT) is recognized as a major cause of lung fibrosis. Our previous work has confirmed that total extract of Anemarrhena asphodeloides Bunge [Asparagaceae] exerted the effect of anti-PF. As a main constituent of Anemarrhena asphodeloides Bunge [Asparagaceae], the effect of timosaponin BII (TS BII) on drug-induced EMT process in PF animals and alveolar epithelial cells remains unknown. In this study, we evaluated the effect of TS BII on bleomycin (BLM)-induced PF. The results showed that TS BII could restore the structure of lung architecture and MMP-9/TIMP-1 balance in fibrotic rat lung and inhibit collagen deposition. Moreover, we found that TS BII could reverse the abnormal expression of TGF-β1 and EMT-related marker proteins including E-cadherin, vimentin, and α-SMA. Besides, aberrant TGF-β1 expression and phosphorylation of Smad2 and Smad3 in BLM-induced animal model and TGF-β1-induced cell model were downregulated by TS BII treatment, indicating that EMT in fibrosis was suppressed by inhibition of TGF-β/Smad pathway both in vivo and in vitro. In summary, our study suggested that TS BII could be a promising candidate for PF treatment.

MiR-484: A Possible Indicator of Drug-Induced Pulmonary Fibrosis.

Drug-induced lung injury leads to serious lung diseases, such as pulmonary fibrosis. We demonstrated in an alveolar epithelial cell line A549/ABCA3 that certain microRNAs were associated with bleomycin induced epithelial-mesenchymal transition (EMT) which is closely related to pulmonary fibrosis. In this study, we focused on the role of miR-484 in drug-induced EMT using A549/ABCA3 cells and a mouse lung injury model. The expression of EMT-related genes and miR-484 was detected by real-time polymerase chain reaction. miR-484-targeted proteins were analyzed by Western blot. Pulmonary fibrosis mouse model was prepared by the intratracheal administration of BLM. As miR-484 is known to target SMAD2 and zinc finger E-box binding homeobox 1 (ZEB1), which are the well-known EMT-related transcription factors, we assessed the effects of a miR-484 inhibitor or mimic on the mRNA/protein expression of both the factors. We found that bleomycin significantly suppressed the intracellular expression and extracellular release of miR-484 in A549/ABCA3 cells. Moreover, the miR-484 mimic and inhibitor showed no drastic effects on the expression of the EMT-related transcription factors. In addition, the miR-484 mimic had no effect on the bleomycin-induced altered mRNA expression of the α-smooth muscle actin, a representative EMT marker. This suggested that miR-484 did not directly contribute to bleomycin-induced EMT in A549/ABCA3 cells. In contrast, the significant decrease in miR-484 expression in the lung tissue or plasma of bleomycin-administered mice suggested that miR-484 expression was closely correlated with bleomycin-induced lung injury. These findings indicate that miR-484 could be a novel diagnostic indicator for drug-induced pulmonary fibrosis.

Suppressive effect of quercetin against bleomycin-induced epithelial-mesenchymal transition in alveolar epithelial cells

Quercetin is a flavonol that is known to have numerous beneficial biological effects such as an anti-fibrotic effect. Epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells is one of major causes of pulmonary fibrosis. However, the effect of quercetin on drug-induced EMT in alveolar type II cells is not known. In this study, we examined the effect of quercetin on bleomycin (BLM)-induced EMT using RLE/Abca3 cells having alveolar type II cell-like phenotype. BLM induced EMT-like morphological changes, downregulation of an epithelial marker E-cadherin, and upregulation of a mesenchymal marker α-smooth muscle actin in RLE/Abca3 cells. In addition, BLM increased the levels of phosphorylated Smad2 and Slug mRNA expression, and enhanced nuclear translocation of β-catenin, suggesting that BLM induced EMT in RLE/Abca3 cells via Smad and β-catenin signaling pathways. However, when the cells were co-treated with quercetin, quercetin suppressed all of these EMT-related changes induced by BLM. Furthermore, BLM increased the intracellular level of reactive oxygen species, which was also suppressed by quercetin. These results suggest that quercetin may be a possible candidate for preventing pulmonary fibrosis caused by drugs.

P88 - Role of micrornas in drug-induced epithelial-mesenchymal transition in human lung-derived alveolar type II epithelial cell model A549/ABCA3

P88 - Role of micrornas in drug-induced epithelial-mesenchymal transition in human lung-derived alveolar type II epithelial cell model A549/ABCA3

Investigation of Drug-induced Lung Injury for the Development of a Novel Therapeutic Approach

The development of serious lung diseases, such as pulmonary fibrosis, is associated with several drugs. A recent study has shown that the epithelial-mesenchymal transition (EMT) plays an essential role in the development of pulmonary fibrosis. However, the mechanisms underlying drug-induced EMT in alveolar epithelial cells have not been characterized. The present study showed that methotrexate (MTX), a drug known to cause lung injury, induced EMT-like phenotypic changes in an A549 cell model of the alveolar epithelium. We also found that the transforming growth factor (TGF)-β1-mediated signaling pathway was associated with MTX-induced EMT. In addition, our results showed that certain secreted factors and microRNAs, a class of small noncoding RNAs, may be involved in MTX-induced EMT. The effects of COA-Cl, a novel synthetic small molecule, on TGF-β1-induced EMT were evaluated to determine the therapeutic potential of COA-Cl against drug-induced lung injury. COA-Cl significantly suppressed TGF-β1-induced EMT-like phenotypic changes, as evidenced by the inhibition of EMT-related transcription factors. Furthermore, MTX-induced EMT was completely suppressed by co-treatment with folic acid. Thus, these compounds may be promising therapeutic agents against drug-induced lung injury. In conclusion, the present study elucidated mechanisms underlying drug-induced EMT and highlighted a potential novel therapeutic approach to drug-induced lung diseases.

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells.

Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model. Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively. BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53. These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells.

Association of cell cycle arrest with anticancer drug-induced epithelial-mesenchymal transition in alveolar epithelial cells

Many drugs exert serious cytotoxic effects on pulmonary tissues. Although several reports have shown an association of epithelial-mesenchymal transition (EMT) with anticancer drug-induced lung injury, mechanisms of these effects are poorly understood. In the present study, we evaluated mechanisms of anticancer drug-induced EMT, with a focus on involvement of cell cycle arrest. We found that methotrexate (MTX) altered mRNA expression levels of many genes as determined by microarray analysis. Gene set enrichment analysis revealed that cell cycle arrest pathways may be associated with MTX-induced EMT. In addition, thymidine (THY) and nocodazole (NOC), which induce cell cycle arrest at S-phase and G2/M-phase, increased mRNA expression levels of α-smooth muscle actin (SMA), an EMT marker. Furthermore, α-SMA protein expression in cells arrested at S- and G2/M-phases by MTX and paclitaxel (PTX) was significantly higher than that in cells at G1. Notably, co-treatment of cells with THY or NOC and EMT-inducing anticancer drugs did not result in additional upregulation of α-SMA mRNA expression. These findings suggested that cell cycle arrest may be closely associated with anticancer drug-induced EMT in alveolar epithelial cells.

Abstract A002: Evidence of pazopanib-induced epithelial-mesenchymal transition (EMT) in human tumors

Abstract Background: VEGFR inhibition by pazopanib slows tumor growth through vascular pruning, although compensatory factors associated with angiogenic inhibitors (e.g., induced HIF1-α from increased hypoxia) can lead to enhanced tumor invasion and metastasis by triggering EMT in some preclinical models (Sennino et al., 2012, Cancer Discovery 2(3);270-87). The development of a clinically suitable, validated assay for EMT has made possible the analysis of EMT in a clinical trial of the VEGFR inhibitor pazopanib. Methods: Paired tumor biopsies were obtained at baseline and on cycle 1, day 7 during a phase I trial (NCT01468922) of single-agent pazopanib (800 mg/day, PO). Biopsies were formalin fixed and paraffin embedded, and tissue sections were evaluated for EMT using a multiplex immunofluorescence assay (EMT-IFA) to quantify E-cadherin (E) and vimentin (V) expression and co-localization in tumor-segmented β-catenin+ regions of interests (ROIs) using Definiens software (Navas et al., NCI-EORTC 2015). EMT-IFA quantitative data were calculated from a minimum of 8 ROIs and 2 tissue sections per biopsy. Results: Paired biopsies from 7 patients (pts) with advanced cancer were evaluable for EMT based on H&E pathologist evaluation. As expected, the mesenchymal phenotype of a patient with chondrosarcoma was stable during treatment, but 4 of 6 carcinomas exhibited significant levels of drug-associated EMT towards more transitional (E+V+) or mesenchymal (V+) phenotypes (increased ratio of V+ to E+ area, log10 V:E; P = 0.0030 to < 0.0001). Two of 4 pts with drug-induced EMT additionally displayed significant increases in the area of V+E+ co-localization (um2/cell) in individual tumor cells (P <0.0001), while the other 2 pts did not show evidence of active transitional cells, but only showed increased E+ or decreased V+ tissue areas (um2/cell) within tumor regions. These results indicate that EMT occurs in some carcinoma of pts treated with pazopanib, consistent with results from preclinical models (Navas et al., 2017, AACR). Conclusions: We report for the first time pazopanib-induced EMT and direct evidence of tumor adaptation to anti-angiogenic therapy in clinical samples. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Apurva K. Srivastava, Katherine Ferry-Galow, Hala Makhlouf, Rodrigo Chuaqui, Robert J. Kinders, Donald P. Bottaro, Alice P. Chen, Ralph E. Parchment, Shivaani Kummar, James H. Doroshow. Evidence of pazopanib-induced epithelial-mesenchymal transition (EMT) in human tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A002.

Role of miR-34a in TGF-β1- and Drug-Induced Epithelial-Mesenchymal Transition in Alveolar Type II Epithelial Cells

Epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells may play an important role in the pulmonary fibrosis induced by drugs such as bleomycin (BLM) and methotrexate (MTX). In this study, we examined the role of microRNAs (miRNAs) in drug-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Based on the screening using miRNA microarray analysis, it was found that the expression of some miRNAs, such as miR-34a, was increased by transforming growth factor (TGF)-β1 and BLM. An increase in miR-34a expression due to TGF-β1, BLM, and MTX was also observed in real-time PCR analysis. Therefore, miR-34a was focused upon in further studies. The expression of nectin-1 mRNA and protein, a possible target of miR-34a, was decreased by the treatment with TGF-β1, BLM, and MTX. In addition, when RLE/Abca3 cells were transfected with miR-34a mimic, the expression of nectin-1 mRNA and Abca3 mRNA, another target of miR34a, decreased significantly. Furthermore, the mRNA expression of cytokeratin 19, an epithelial marker, decreased, whereas that of α-smooth muscle actin, a mesenchymal marker, increased in the cells transfected with miR-34a mimic. These results suggest that miR-34a is involved in drug-induced EMT in alveolar epithelial cells, and possibly in lung fibrosis.

Abstract B47: Drug-induced EMT is fundamentally distinct from pathologic EMT

Abstract Background: Receptor tyrosine kinase ErbB2 (Her2) is overexpressed in an estimated 25% of the breast cancer patients. A therapeutic regime of Her2 targeted therapies, consists of Trastuzumab followed by the dual EGFR/Her2 tyrosine kinase inhibitor, Lapatinib. These therapies continue to show success in blocking the progression of this breast cancer subtype. Of clinical concern, however, cancer cells eventually develop pathways to evade Lapatinib-mediated tumor inhibition and acquire drug resistance. In this study, we sought to understand the molecular mechanism of resistance towards Lapatinib. Methods: We used Her2 overexpressing human mammary epithelial cells to generate Lapatinib resistant cell lines (LapR) in vitro. The LapR cells acquired growth persistence within the continued presence of Lapatinib for four weeks. We analyzed the resultant cell populations via cell viability assays, immunoblotting, flow cytometry, 3D cell culture and Nano-string gene expression analysis. Results: Generated LapR cell lines showed robust epithelial-mesenchymal transition (EMT) phenotypes that included upregulated FGFR1 and preservation of their Her2 expression. We tested the viability of LapR cells using dose response assays with Afatinib, a second-generation covalent ErbB kinase inhibitor, and Trastuzumab emtansine (T-DM1), an anti-Her2 antibody drug conjugate. We also utilized recently developed covalent inhibitors of FGFR. LapR cells were highly resistant to Afatinib but did show sensitivity to T-DM1 and FGFR kinase inhibition. Importantly, resistance to ErbB inhibition was maintained in the LapR cells for several weeks following removal of Lapatinib. This stable phenotype was consistent with a maintained yet distinct regulation of the cancer stem cell markers (CD44/CD24) compared to a transient EMT induced by transforming growth factor β (TGF-β). Strikingly, we observed that unlike their parental counterparts LapR cells were unable to grow in a compliant 3D matrix. Furthermore, unlike TGF-β-treated cells, LapR cells were unable to metastasize in vivo. By using Nano-string pan cancer progression panel analysis, we characterized key differences in the LapR EMT phenotype as compared to a TGF-β induced EMT. Future Directions: Currently, we are analyzing the expression data of LapR and TGF-β-induced EMT to identify key targets required for EMT-induced tumor progression. Overall, our studies seek to define what aspects of EMT are involved in cellular persistence in response to anti-cancer drugs versus those utilized by cells during metastatic progression. Citation Format: Saeed Salehin Akhand, Michael Wendt. Drug-induced EMT is fundamentally distinct from pathologic EMT. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B47.

Analysis of TGF-β1- and drug-induced epithelial–mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3

In this study, we examined the induction of epithelial–mesenchymal transition (EMT) by transforming growth factor (TGF)-β1 and drugs in genetically engineered type II alveolar epithelial cell line RLE/Abca3. Treatment of RLE/Abca3 cells with TGF-β1 induced marked changes in cell morphology from epithelial-like to elongated fibroblast-like morphology. With these morphological changes, mRNA expression of epithelial markers such as cytokeratin 19 (CK19) decreased, while that of mesenchymal markers such as α-smooth muscle actin (α-SMA) increased. TGF-β1 treatment also decreased the mRNA expression of Abca3, a type II cell marker, and formation of lamellar body structures. Interestingly, the effect of TGF-β1 on Abca3 mRNA expression was observed in RLE/Abca3 cells, but not in wild-type RLE-6TN, A549, and H441 cells. Treatment of RLE/Abca3 cells with bleomycin (BLM) and methotrexate (MTX) induced similar morphological and mRNA expression changes. In addition, the increase in α-SMA and the decrease in Abca3 mRNA expression by these drugs were observed only in RLE/Abca3 cells. These findings suggest that, like TGF-β1, BLM and MTX induce EMT in RLE/Abca3 cells, and RLE/Abca3 cells would be a good model to study drug-induced EMT. The effect of pirfenidone, an antifibrotic and anti-inflammatory drug, on EMT induced by TGF-β1 was also discussed.

Involvement of NF-κB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial–mesenchymal transition of breast cancer cells

The epithelial-mesenchymal transition (EMT) induced by chemotherapeutic agents promotes malignant tumor progression; however, the mechanism underlying the drug-induced EMT remains unclear. In this study, we reported that miR-448 is the most downregulated microRNA following chemotherapy. Suppression of miR-448 correlated with EMT induction in breast cancer in vitro and in vivo. With the use of chromatin immunoprecipitation-seq analysis, we demonstrated that miR-448 suppression induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, leading to elevated levels of amphiregulin and thereby, increasing epidermal growth factor receptor (EGFR)-mediated Twist1 expression, as well as nuclear factor κB (NF-κB) activation. On the other hand, we also found that the adriamycin-activated NF-κB directly binds the promoter of miR-448 suppressing its transcription, suggesting a positive feedback loop between NF-κB and miR-448. Furthermore, all patients who received cyclophosphamide (CP), epirubicin plus taxotere/CP, epirubicin plus 5-fluorouracil chemotherapy showed miR-448 suppression, an increased SATB1, Twist1 expression and acquisition of mesenchymal phenotypes. These findings reveal an underlying regulatory pathway, in which the autoregulation between NF-κB and miR-448 is important for restrain miR-448 suppression upon chemotherapy and may have a role in the regulation of chemotherapy-induced EMT. Disruption of the NF-κB-miR-448 feedback loop during clinical treatment may improve the chemotherapy response of human breast cancers in which EMT is a critical component.