Cancer Letters

Cancer Letters

Volume 402, 28 August 2017, Pages 190-202
Cancer Letters

Original Article
Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C

https://doi.org/10.1016/j.canlet.2017.06.002Get rights and content

Highlights

  • miR-137 is a Slug-induced miRNA in lung cancer cells.

  • Knockdown of miR-137 abolishes Slug-induced cancer cell invasion and migration.

  • miR-137 functions as an oncogene in lung cancer.

  • Slug-induced miR-137 enhances lung cancer progression by direct targeting TFAP2C.

  • Our findings add new components to the Slug regulatory network in lung cancer.

Abstract

The epithelial-mesenchymal transition (EMT) regulator, Slug, plays multifaceted roles in controlling lung cancer progression, but its downstream targets and mechanisms in promoting lung cancer progression have not been well defined. In particular, the miRNAs downstream of Slug in non-small cell lung cancer (NSCLC) remain undetermined. Here, we report that miR-137 is downstream of the EMT regulator, Slug, in lung cancer cells. Slug binds directly to the E-box of the miR-137 promoter and up-regulates its expression in lung cancer cells. Knockdown of miR-137 abolished Slug-induced cancer invasion and migration, whereas upregulation of miR-137 was found to trigger lung cancer cell invasion and progression by direct suppressing TFAP2C (transcription factor AP-2 gamma). Clinical data showed that lung adenocarcinoma patients with low-level expression of Slug and miR-137 but high-level expression of TFAP2C experienced significantly better survival. miR-137 is a Slug-induced miRNA that relays the pro-metastatic effects of Slug by targeting TFAP2C. Our findings add new components to the Slug-mediated regulatory network in lung cancer, and suggest that Slug, miR-137, and TFAP2C may be useful prognostic markers in lung adenocarcinoma.

Introduction

The epithelial-mesenchymal transition (EMT) is an essential developmental process whereby epithelial cells lose their epithelial characteristics and acquire a migratory, mesenchymal-like phenotype [1]. Accumulating evidence shows that EMT plays important roles not only in embryonic development, but also in tissue regeneration and cancer progression [1], [2], [3]. The EMT phenotype is tightly regulated by EMT transcriptional regulators, such as Slug (also known as Snail2), Snail, Twist, Zeb1, and Zeb2 [4], most of which have been suggested as potential diagnostic markers and therapeutic targets for human cancers [5], [6], [7], [8], [9].

Slug is a member of the SNAIL family of zinc finger transcription factors. It was initially identified as mediating the formation of the mesoderm during gastrulation and the migration of neural crest cells during embryonic development [10]. Subsequently identified as an EMT regulator, Slug has recently been shown to be associated with tumor metastasis and recurrence in a variety of cancers [5], [11], [12], [13]. In lung cancer cells, elevated expression of Slug has been reported to enhance angiogenesis and cause the cells to acquire traits reminiscent of those expressed by stem cells [5], [14], [15], [16]. Slug also contributes to the ability of lung cancer cells to resist chemotherapeutic drugs and tyrosine kinase inhibitors [17], [18]. However, the downstream targets and action mechanisms through which Slug promotes lung cancer progression remain largely unknown.

MicroRNAs (miRNAs) are small noncoding single-stranded RNAs (∼22 nt) that can function as an endogenous means of RNA interference [19]. These actions play critical roles in various essential biological processes, including metabolism, development, proliferation, differentiation, and apoptosis [20], [21]. Accumulating evidence indicates that miRNAs are grossly dysregulated in lung cancer, and may serve as oncogenes or tumor suppressors. miRNAs can be used to sub-classify non-small cell lung cancers (NSCLCs), and specific miRNA profiles may predict the prognosis and/or recurrence of the disease [22], [23], [24], [25]. Similar to the case of protein-encoding genes, the transcription of miRNAs can be activated or repressed by transcription factors, which interact with their conventional binding sites in polymerase II- or III-dependent manners [26]. However, although Slug is a transcription factor, and it is known to have multiple functions in lung cancer progression, no previous study has examined a possible the link between Slug and downstream miRNAs in lung cancer cells.

In the present study, we used Illumina bead arrays and TaqMan low-density arrays (TLDAs) to identify miRNAs downstream of Slug in lung cancer cells. We demonstrate that miR-137 is a Slug-induced miRNA, and that the Slug-induced upregulation of miR-137 plays important roles in controlling lung cancer progression by targeting and downregulating TFAP2C (transcription factor AP-2 gamma). Our novel findings suggest that the Slug-miR-137-TFAP2C axis may offer new candidate target molecules for lung cancer therapeutics.

Section snippets

Cell lines

Human lung cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the Center of Genomic Medicine, National Taiwan University (Supplementary Methods). Cell lines authentication, cell culture and stable transfections were performed as described in the Supplementary Methods.

Luciferase reporter assays

Luciferase activity was measured using a dual-luciferase assay (Promega, Madison, WI, USA), according to the manufacturer's instructions. Refer to the Supplementary Methods.

Chromatin immunoprecipitation (ChIP)

ChIP

miRNAs downstream of Slug in lung cancer cells

We first used Illumina bead arrays and TLDAs to identify miRNAs that exhibited differential expression in Slug-overexpressing versus control lung cancer cells (Supplementary Methods, Table S1 and Table S2). The Illumina bead array data are available from Gene Expression Omnibus (GEO) under accession number GSE84040. These analyses identified 14 well-annotated miRNAs that were significantly altered (13 upregulated and 1 downregulated) in Slug-overexpressing cells (Supplementary Fig. S1). Of

Discussion

We previously reported that elevated mRNA expression of the transcription factor and EMT regulator, Slug, in NSCLC tumor specimens was significantly associated with early post-operative relapse and shorter survival [5]. Consistent with our report, another investigation found poorer survival among lung adenocarcinoma patients whose tumors showed positive IHC staining for Slug [27]. In addition to regulating EMT and cancer metastasis, Slug is also temporally regulated during the cell cycle, where

Funding

This work was financially supported by grant from National Taiwan University (103R7601-3) and the Far Eastern Memorial Hospital-National Taiwan University Hospital Joint Research Program (105-FTN02).

Acknowledgments

The authors thank the NTU Microarray Core Facility of the National Research Program for Genomic Medicine of Taiwan for technical assistance, and the Department of Medical Research in National Taiwan University Hospital for facility support.

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