TLE1 promotes EMT in A549 lung cancer cells through suppression of E-cadherin

https://doi.org/10.1016/j.bbrc.2014.11.007Get rights and content

Highlights

  • Exogenous TLE1 in A549 cells enhances cell migration and suppresses E-cadherin.

  • Knockdown of TLE1 in A549 cells reduces cell motility and upregulates E-cadherin.

  • TLE1 suppresses E-cadherin expression at the transcriptional level.

  • TLE1 recruits HDAC1 to the E-cadherin promoter.

  • HDAC inhibition attenuates TLE1-induced E-cadherin downregulation and motility.

Abstract

The Groucho transcriptional corepressor TLE1 protein has recently been shown to be a putative lung specific oncogene, but its underlying oncogenic activity in lung cancer has not been fully elucidated. In this report, we investigated whether TLE1 regulates lung cancer aggressiveness using the human lung adenocarcinoma cell line A549 as a model system. Through a combination of genetic approaches, we found that TLE1 potentiates epithelial-to-mesenchymal transition (EMT) in A549 cells in part through suppression of the tumor suppressor gene E-cadherin. Exogenous expression of TLE1 in A549 cells resulted in heightened EMT phenotypes (enhanced fibroblastoid morphology and increased cell migratory potential) and in molecular alterations characteristic of EMT (downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal marker Vimentin). Conversely, downregulation of endogenous TLE1 expression in these cells resulted in reversal of basal EMT characterized by a cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Mechanistic studies showed that TLE1 suppresses E-cadherin expression at the transcriptional level in part by recruiting histone deacetylase (HDAC) activity to the E-cadherin promoter. Consistently, the HDAC inhibitor TSA partially reversed the TLE1-induced E-cadherin downregulation and cell migration, suggesting a role for HDACs in TLE1-mediated transcriptional repression of E-cadherin and EMT function. These findings uncover a novel role of TLE1 in regulating EMT in A549 cells through its repressive effect on E-cadherin and provide a mechanism for TLE1 oncogenic activity in lung cancer.

Introduction

TLE1 is a member of the Groucho (Gro)/TLE family of transcriptional co-repressors that regulate the transcriptional activity of a wide range of genes [1]. As a co-repressor, the TLE1 protein does not bind to DNA directly but is recruited to target gene(s) by direct interaction with DNA-binding transcription factors to form large multi-protein complexes [2]. Alternatively, it has also been shown that TLE1 may interact with chromatin through its interactions with the amino-terminal tail of histone H3 [3]. The exact mechanism underlying the transcriptional repression function of TLE1 is yet to be fully elucidated. Numerous data indicate that the TLE1 gene silencing function appears to depend in part on recruitment of the histone deacetylase (HDAC) protein to target genes and the subsequent removal of acetyl groups from nearby DNA bound histones. Indeed, the interaction of TLE1 with HDAC1 in particular has been demonstrated using genetic and biochemical approaches [4], [5]. Importantly, inhibition of HDAC activity via chemical inhibitors partially blocks TLE1 repressive activity, suggesting the importance of HDAC activity on TLE1 repressive function [4]. Based on the inability of HDAC inhibitors to completely block the TLE1 repressive function, it is likely that TLE1 may depend on other chromatin remodeling proteins to effect transcriptional repression [4].

As transcriptional corepressors, the Groucho/TLE proteins regulate diverse cellular functions including neurogenesis and a number of developmental processes [6], [7]. Recently, a series of independent findings indicate a survival promoting role of Groucho proteins, TLE1 in particular, in several cellular models. Exogenous TLE1 expression stimulated anchorage-independent growth in chicken embryo fibroblast [8]. As a negative regulator of apoptosis, TLE1 inhibited low potassium-induced apoptosis in neurons [9] and protected synovial sarcoma cells from doxorubicin-mediated apoptosis [10]. In line with its anti-apoptotic function, we have found that TLE1 specifically inhibited the caspase-independent cell death induced by Bit1 (Bcl2-inhibitor of transcription 1) in several types of malignant cells [11]. Recently, we showed that TLE1 effectively abrogates Bit1-induced anoikis in lung cancer cells [12]. Based on the latest report indicating that TLE1 is a putative lung specific oncogene [13], it is interesting to speculate whether the anti-apoptotic function of TLE1 represents as an oncogenic stimulus. In line with its function as a regulator of a survival-promoting gene transcription program, TLE1 was shown to positively regulates Bcl2 expression [11] and ErbB1 and ErbB2 signaling [13], two survival pathways that impact cancer progression.

As a putative lung specific oncogene, TLE1 was found to be overexpressed in a subset of aggressive and advanced human lung tumors [13]. In light of this finding, we examined the possibility that TLE1 may regulate lung cancer aggressiveness. Here, we have uncovered a novel function of TLE1 in regulating EMT in the human lung adenocarcinoma cell line A549. Importantly, our findings indicate that TLE1 mediates transcriptional silencing of the invasive suppressor E-cadherin gene in part through recruitment of HDAC activity, and further suggest TLE1 as a novel in vivo repressor of E-cadherin in lung cancer.

Section snippets

Cell culture and transfection assays

The human lung adenocarcinoma cell line A549 from American Type Culture Collection (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) with glutamine containing 10% fetal bovine serum, penicillin, and streptomycin. Stable A549 control GFP and GFP-TLE1 pool of cells were generated by transduction with the lentiviral GFP-TLE1 or the empty control GFP construct (Open Biosystems, Huntsville, AL) according to the manufacturer’s protocol. 48 h post-transduction, cells were cultured in the

TLE1 expression induces EMT in A549 cells

To address the hypothesis that TLE1 may play a role in aggressiveness of lung cancer cells, we generated stable TLE1 expressing clones by infecting the human adenocarcinoma A549 cell line with lentiviral expression vectors containing either the GFP-tagged TLE1 or control GFP alone construct. Several GFP-TLE1 and control GFP clones were pooled together to generate the GFP-TLE1 pool and control GFP pool, respectively. Exogenous TLE1 expression in the GFP-TLE1 pool was confirmed by immunoblotting

Conclusion

In summary, we have uncovered a novel function of the corepressor TLE1 in EMT by suppressing the expression of the tumor suppressor E-cadherin. Although the exact mechanism(s) by which TLE1 is recruited to the E-cadherin promoter remains to be examined (likely through known EMT regulatory DNA binding transcriptional factors), the present findings indicate that TLE1 is a functional component of the E-cadherin corepressor complex in part to recruit HDAC1 (Fig. 4D). With numerous evidence

Acknowledgments

This work was supported by Louisiana Cancer Research Consortium (LCRC) Start up Grant (HB), NIH RCMI G12RR026250-03 Grant (to Xavier University of Louisiana), and NIH 1R15CA158677-01A1 Grant (HB).

References (19)

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