Research Article
miR-19 targeting of GSK3β mediates sulforaphane suppression of lung cancer stem cells

https://doi.org/10.1016/j.jnutbio.2017.02.020Get rights and content

Abstract

Cancer stem cells (CSCs) play a central role in the development of cancer. The canonical Wnt/β-catenin pathway is critical for maintaining stemness of CSCs. Phytochemicals from dietary compounds possess anti-CSCs properties and have been characterized as promising therapeutic agents for the prevention and treatment of many cancers. To date, the involvement and function of miR-19, a key oncogenic miRNA, in regulating Wnt/β-catenin pathway and lung CSCs has not been defined. Meanwhile, the effect of sulforaphane (SFN) on lung CSCs also remains to be elucidated. Here, we reported that lung CSCs up-regulated miR-19a and miR-19b expression. Overexpression of miR-19a/19b enhanced the ability of tumorsphere formation, up-regulated the expression of lung CSCs markers, increased Wnt/β-catenin pathway activation and β-catenin/TCF transcriptional activity in lung CSCs. In contrary, down-regulation of miR-19 suppressed lung CSCs activity and Wnt/β-catenin activation. We further revealed that miR-19 activated Wnt/β-catenin pathway by directly targeting GSK3β, the key negative modulator of this pathway. Moreover, we showed that SFN exhibited inhibitory effect on lung CSCs through suppressing miR-19 and Wnt/β-catenin pathway. Taken together, these data illustrate the role of miR-19 in regulating lung CSCs traits and miR-19/GSK3β/β-catenin axis in SFN intervention of lung CSCs. Findings from this study could provide important new insights into the molecular mechanisms of lung CSCs regulation as well as its target intervention.

Introduction

Lung cancer is the leading cause of cancer mortality worldwide, accounting for about 13% of total cancer deaths [1], [2]. Despite significant improvement being made in cancer therapy, the 5-year relative survival rate of lung cancer still remains low [1]. Accumulating evidence has shown that cancer stem cells (CSCs), a rare subpopulation of cancer cells, possess the capacity of self-renewal and multipotent differentiation [3], [4]. CSCs express distinct and specific cell markers. For example, CD133, CD44 and aldehyde dehydrogenase (ALDH) are widely used to identify lung CSCs [5], [6]. It is well acknowledged that CSCs are responsible for the heterogeneity, metastasis and relapse of tumors [6]. Thus, the CSCs concept provides more effective intervention strategies in the prevention and treatment of cancers.

Several signal pathways are crucial in orchestrating CSCs activity. Dysregulation of these pathways has been implicated in the maintenance and function of CSCs. Aberrant Wnt/β-catenin signal pathway has been reported in several types of CSCs [7], [8], [9], [10]. The activation of Wnt/β-catenin pathway depends on the key regulator β-catenin, the intracellular level of whose is mainly modulated by glycogen synthase kinase-3β (GSK3β). In the absence of Wnt, the cytoplasmic β-catenin is phosphorylated by the destruction complex consisting of Axin, adenomatous polyposis coli (APC), casein kinase 1α (CK1α) and GSK3β and subsequently undergoes ubiquitin-proteasome degradation. Upon the Wnt ligand binding to the Frizzled receptor, β-catenin is dissociated from the destruction complex and accumulates in cytoplasm. The stabilized β-catenin then translocates into the nucleus where it forms a transcriptional complex with the T-cell factor (TCF)/lymphocyte enhancer factor (LEF), leading to the activation of downstream genes such as c-Myc, Cyclin D1, CD44, ALDH and others [11], [12], [13].

miRNAs are 21–25 nucleotides in length, small noncoding RNAs that regulate target genes by binding to the 3′-untranslated regions (3’UTRs), which leads to either degradation of mRNA or inhibition of protein translation [14]. Mounting evidence has indicated the pivotal role of miRNA in the development and progression of cancers by regulating CSCs activity [15], [16]. Some oncogenic miRNAs have been characterized, for example, the miR-17-92 cluster [17], [18], [19]. Among the miR-17-92 cluster, miR-19 is the key miRNA which exerts oncogenic function by targeting several critical tumor suppressor genes such as PTEN [20]. Previous studies revealed that miR-19 triggers the epithelial–mesenchymal transition (EMT) and is correlated with metastasis of lung cancer [21]. In gastric cancer, miR-19 promotes multidrug resistance (MDR) and regulates the self-renewal and proliferation of gastric CSCs [22], [23]. Moreover, a recent study has elucidated the role of miR-17-92 in lung cancer stem cells through regulation of Wnt signaling [24].

Numerous epidemiological studies have substantiated the efficient anticancer properties of dietary components in vegetables and fruits [25]. These bioactive and non/low-toxic phytochemicals have been proved to be promising candidates for cancer intervention [25], [26]. Sulforaphane (SFN), a major isothiocynate (ITC) abundant in broccoli or broccoli sprouts, has been shown to possess anticarcinogenic potential in vitro and in vivo [27]. Recently, it has been documented that SFN exhibits its anticancer property by targeting CSCs in various cancer types [28], [29], [30], [31]. In addition, by combining with other chemotherapeutic agents, SFN effectively abolishes CSCs characteristics [32]. To date, however, no studies have been conducted yet to examine the inhibitory effect of SFN on lung CSCs. Therefore, the present study aimed to investigate the regulatory role of the oncogenic miR-19 in lung CSCs and SFN modulation of lung CSCs.

Section snippets

Cell culture

Human lung cancer cell lines A549 and H1299 were purchased from Chinese Academy of Typical Culture Collection Cell Bank. Both cell lines were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum and antibiotics (100-units/mL penicillin and 100-μg/mL streptomycin) in a humidified atmosphere of 5% CO2 at 37 °C.

Tumorsphere formation assay

One major feature of CSCs is their ability to form three-dimensional spheres in serum-free medium (SFM) culture condition. In SFM with some

miR-19a/19b is up-regulated in lung CSCs

CSCs are characterized by their capacity to form three-dimensional structures or spheres, and tumorsphere formation assay via serum-free medium (SFM) culturing is widely used in the isolation and enrichment of CSCs in vitro. These sphere-forming cells are considered to have CSCs characteristics like self-renewal and unlimited differentiation. In our study, turmorsphere formation assay was utilized to isolate and enrich lung CSCs from two human lung cancer cell lines A549 and H1299. Along with

Discussion

Emerging evidence has suggested that CSCs play essential role in the progress, metastasis and recurrence of human cancers, through their capacity for self-renewal, production of heterogeneous progeny, resistance to chemotherapy and unlimited proliferation. As important posttranscriptional regulators of gene expression, miRNAs are critically implicated in tumorigenic process. In the present study, we illustrated the pivotal role of miR-19 in regulating lung CSC traits and miR-19/GSK3β/β-catenin

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81573139), the National Basic Research Program of China (973 Program) (No. 2013CB910303), the Science and Technology Planning Project of Guangdong Province of China (No. 2013B022000041), and by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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    Conflict of interest: The authors declare no conflict of interest.

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