Sulforaphane suppresses EMT and metastasis in human lung cancer through miR-616-5p-mediated GSK3β/β-catenin signaling pathways.

Da-Xuan Wang,Yu-Jiao Zou,Zhi-Qiang Lin,Yong Lin,Jun-Jin Lin,Xi-Bin Zhuang,Shu-Xing Chen,Wen-Lan Li

doi:10.1038/aps.2016.122

Abstract

Sulforaphane is a common antioxidant selectively abundant in cruciferous plants, which exhibits effective anti-cancer actions in control of tumorigenesis or progression of various cancers. A recent study has shown that sulforaphane attenuates the EGFR signaling pathway in non-small cell lung cancer (NSCLC), suggesting its potential anti-metastatic effects. In this study we assessed the involvement of sulforaphane and miR-616-5p in epithelial-mesenchymal transition (EMT) and NSCLC metastasis. Sulforaphane suppressed the cell proliferation in human NSCLC cell lines H1299, 95C and 95D with IC50 values of 9.52±1.23, 9.04±1.90 and 17.35±2.03 μmol/L, respectively. At low concentrations (1-5 μmol/L), sulforaphane dose-dependently inhibited the migration and invasion of 95D and H1299 cells with relatively high metastatic potential. The anti-metastatic action of sulforaphane was confirmed in 95D and H1299 cell xenografts in vivo. In fresh NSCLC tissue samples from 179 patients, miR-616-5p levels were upregulated in late-stage NSCLCs, and strongly correlated with risk of NSCLC recurrence and metastasis. Consistent with the clinic observation, miR-616-5p levels in the 3 NSCLC cell lines were correlated with their metastatic ability, and were decreased by sulforaphane treatment. Silencing miR-616-5p markedly suppressed the migration and invasion of 95D cells in vitro and NSCLC metastasis in vivo. Further studies revealed that miR-616-5p directly targeted GSK3β and decreased its expression, whereas sulforaphane decreased miR-616-5p levels by histone modification, and followed by inactivation of the GSK3β/β-catenin signaling pathway and inhibition of EMT, which was characterized by loss of epithelial markers and acquisition of a mesenchymal phenotype in NSCLC cells. Our findings suggest that sulforaphane is a potential adjuvant chemotherapeutic agent for the prevention of NSCLC recurrence and metastasis, and miR-616-5p can be clinically utilized as a biomarker or therapeutic target to inhibit metastasis.

Highlights

Sulforaphane is one of the most powerful and effective bioactive substances derived from plants in cancer research and is a common antioxidant that is selectively abundant in cruciferous plants
The anti-proliferative effects of sulforaphane were evaluated in the human non-small cell lung cancer (NSCLC) cell lines H1299, 95C and 95D by MTT assays
Differential expression analysis based on the TCGA data set identified miR-616 as a microRNA candidate that is upregulated in stage III/IV NSCLCs versus stage I/II NSCLCs (P=0.01) (Figure 2C)

Summary

Introduction

Sulforaphane is one of the most powerful and effective bioactive substances derived from plants in cancer research and is a common antioxidant that is selectively abundant in cruciferous plants. Many studies have examined the anti-cancer effects of sulforaphane in control of tumorigenesis or progression of MicroRNAs (miRNAs), which function as negative regulator of gene expression, are involved in various biological functions, including development and differentiation, metabolism, immune response, proliferation, apoptosis and metastasis. In www.nature.com/aps recent years, many studies have revealed that miRNAs play an important physiological and pathological role in cancers; therapies targeting miRNAs may be an effective strategy to block cancer oncogenesis and progression. Several miRNAs have previously been shown to be regulated by sulforaphane[8,9,10]. Sulforaphane can modify the activity of miR-200c/ ZEB1 and the snail pathway to affect epithelial-mesenchymal transition (EMT)[11], suggesting that sulforaphane may target specific miRNAs to promote its anti-metastatic effects in cancers. In EMT, the Wnt/β-catenin signal pathway plays an important role. Β-catenin is prevented from being phosphorylated, ubiquitylated and degraded and translocates into the nucleus to bind transcription factors of the T cell factor/lymphoid enhancer binding factor (TCF/LEF) family; this process enables β-catenin to regulate gene expression[12]

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