Repression of bone morphogenetic protein 4 by let-7i attenuates mesenchymal migration of head and neck cancer cells

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Abstract

The movement modes of epithelial cancer cells in three-dimensional (3D) environments include the mesenchymal mode, which is associated with local invasion, and the amoeboid mode, which facilitates distant metastasis. The migratory behavior of individual cancer cells is critical for tumor dissemination; however, the mechanism underlying regulation of the switch between movement modes is not clearly understood. For head and neck squamous cell carcinoma (HNSCC), local invasion is the major route of dissemination. We previously demonstrated that, in HNSCC cells, Twist1 represses let-7i expression to elicit mesenchymal-mode movement through activation of Ras-related C3 botulinum toxin substrate 1 (RAC1). In this study, we discover another important target gene of let-7i for regulating HNSCC migration. Using bioinformatic tools, we identified bone morphogenetic protein 4 (BMP4) as a candidate target of let-7i. Further experiments, including 3′-untranslated region (UTR) reporter assays, quantitative RT-PCR and western blotting, confirmed that BMP4 is a bona fide target repressed by let-7i. In the HNSCC cell line OECM-1, knockdown of BMP4 reduced mesenchymal-mode migration and invasion in 3D culture. In clinical HNSCC samples, let-7i expression was inversely correlated with BMP4 expression. Our results revealed that let-7i attenuates mesenchymal-mode migration of HNSCC cells through repression of a novel target, BMP4.

Highlights

► let-7i represses BMP4 expression through binding to 3′-UTR. ► Knockdown of BMP4 attenuates mesenchymal migration of head and neck cancer cells. ► let-7i expression inversely correlates with BMP4 in head and neck cancer samples.

Introduction

Head and neck cancer, including cancers arising from the oral cavity, oropharynx, hypopharynx and larynx, is one of the leading causes of cancer-related deaths worldwide [1]. More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC), which is associated with smoking, drinking and betel nut chewing [2]. The clinical characteristics of HNSCC are distinct from cancers originating from other tissues/organs: advanced HNSCC is often associated with severe destruction of surrounding tissues, and locoregional recurrence is the major pattern of treatment failure. When disease recurs, the response rate to treatment reduces significantly. Intriguingly, compared with other types of cancers, the frequency of distant metastasis in HNSCC is relatively low [3]. However, understanding of the unique mechanism responsible for HNSCC local invasion is limited.

The three-dimensional (3D) cultivation system represents the living microenvironment and is superior to traditional culture systems for studying the migration of cancer cells [4], [5]. In 3D environments, epithelial cancer cells migrate using multi-cellular collective movement [6] or individual cell movement, the latter of which includes mesenchymal- or amoeboid-mode movement [7]. For mesenchymal-mode movement, the cells possess an elongated shape with protrusion, whereas cells moving in an amoeboid mode often reveal a round shape with membrane blebbing [8], [9]. Recent studies have suggested that mesenchymal-mode movement is important for local invasion; by contrast, the amoeboid-mode movement directs distant metastasis [10]. However, knowledge concerning controlling the switch between mesenchymal and amoeboid movement is limited.

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that repress gene expression at the posttranscriptional level by partially base-pairing to the 3′-untranslated regions (3′-UTRs) of target mRNAs [11], [12]. The role of miRNAs in cancer metastasis has been highlighted recently [13], [14]. We recently discovered that, in HNSCC, the epithelial–mesenchymal transition (EMT) inducer Twist1 promotes mesenchymal-mode movement through repression of let-7i, a member of let-7 family microRNAs that are expressed during stem cell differentiation and that act as a tumor suppressor [15], [16], [17]. Repression of let-7i elicits mesenchymal-mode movement through activation of Ras-related C3 botulinum toxin substrate 1 (RAC1) [18]. In the present study, we further identified a novel let-7i target involved in regulating HNSCC migration in 3D environments. We found that bone morphogenetic protein 4 (BMP4), a TGF-β superfamily protein that plays a critical role in embryogenesis [19], is a target repressed by let-7i. Knockdown of BMP4 reduces mesenchymal-mode movement in HNSCC. The present study uncovers both a novel target of let-7i and a new function of BMP4 in cancer cells migration.

Section snippets

Cell lines and plasmids

The human HNSCC cell line FaDu and the human embryonic kidney cell line HEK-293T were obtained from the Bioresource Collection and Research Center of Taiwan. The HNSCC cell lines OECM-1, CAL-27, Ca-9 and HSC-3 were provided by Dr. Cheng-Chi Chang (National Taiwan University). OECM-1 and FaDu were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% heat-inactivated fetal bovine serum (FBS), and HEK-293T, Ca-9, HSC-3 and CAL-27 were cultured in Dulbecco’s Modified Eagle’s

The expression of let-7i inversely correlates with BMP4

We initially aimed to identify the novel let-7i target gene(s) that regulate(s) cellular migration because we previously found that let-7i represses mesenchymal-mode movement in HNSCC [18]. The cancer miRNA Regulatory Network (http://cmrn.systemsbiology.net/) [23] was applied to predict candidate targets of let-7i. We found that BMP4, a morphogenic protein that is critically involved in development and cancer progression [19], [24], [25], [26], [27], [28], [29], [30], [31], is a putative target

Discussion

BMP4 is a morphogenic protein that plays an important role in embryogenesis and stem cell development [19], [24]. The significance of BMP4 in cancer cells has been highlighted recently: increased expression of BMP4 has been shown in different types of cancers, including melanoma, ovarian carcinoma, gastric carcinoma, hepatocellular carcinoma, renal cell carcinoma and HNSCC [25], [26], [27], [28], [29], [30], [31], and increased BMP4 expression is associated with a worse outcome of HNSCC [29].

Acknowledgments

This work was supported by National Health Research Institutes (NHRI-EX100-10037BI to M.H.Y.), National Science Council (101-2321-B-010-007 to M.H.Y.), Taipei Veterans General Hospital (V102-E8-002; V102C-036 to M.H.Y.), a grant from Ministry of Education, Aim for the Top University Plan (to M.H.Y.), and a grant from Department of Health, Center of Excellence for Cancer Research (DOH101-TD-C-111-007 to M.H.Y).

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