Cystatin A suppresses tumor cell growth through inhibiting epithelial to mesenchymal transition in human lung cancer.

Yunxia Ma,Yong Li,Iver Petersen,Katja Grün,Alexander Berndt,Zhongwei Zhou,Yuan Chen

doi:10.18632/oncotarget.23505

Abstract

Cystatin A (CSTA), belonging to type 1 cystatin super-family, is expressed primarily in epithelial and lymphoid tissues for protecting cells from proteolysis of cytoplasmic and cytoskeletal proteins by cathepsins B, H and L. CSTA acts as a tumor suppressor in esophageal cancer, however, its role in lung cancer has not yet been elucidated. Here we found that CSTA was down-regulated in all lung cancer cell lines compared to normal lung epithelial cells. CSTA was restored in most lung cancer cell lines after treatment with demethylation agent 5-aza-2-deoxycytidine and deacetylation agent Trichostatin. Bisulfite sequencing revealed that CSTA was partially methylated in the promoter and exon 1. In primary lung tumors, squamous cell carcinoma (SCC) significantly expressed more CSTA compared to adenocarcinoma (p<0.00001), and higher expression of CSTA was significantly associated with lower tumor grade (p<0.01). CSTA stable transfection reduced the activity of cathepsin B and inhibited the ability of colony formation, migration and invasion, and enhanced gemcitabine-induced apoptosis. CSTA overexpression resulted in reduced activity of ERK, p-38, and AKT. Additionally, CSTA overexpression led to a mesenchymal to epithelial transition (MET) and prevented the TGF-β1-induced epithelial to mesenchymal transition (EMT) through inhibiting the ERK/MAPK pathway. In conclusion, our date indicate 1) epigenetic regulation is associated with CSTA gene silencing; 2) CSTA exerts tumor suppressive function through inhibiting MAPK and AKT pathways; 3) Overexpression of CSTA leads to MET and prevents TGF-β1-induced EMT by modulating the MAPK pathway; 4) CSTA may be a potential biomarker for lung SCC and tumor differentiation.

Highlights

Lung cancer is the first leading cause of cancerrelated death in the United States with 222,500 new cases being estimated to occur in 2017 [1]
Northern blot (NB), real-time RT-PCR and western blot (WB) showed that CSTA was not detected in all lung cancer cell lines at both mRNA (Figure 1A and Supplementary Figure 1A) and protein (Figure 1B) levels except H1650 compared to human bronchial epithelial cells (HBEC) and Small airway epithelial cells (SAEC)
Both increased cysteine protease inhibition and cystatin A expression were associated with better survival in non-small cell lung cancer (NSCLC) [25], and enforced expression of CSTA reduced metastasis in breast cancer [26]

Summary

Introduction

Lung cancer is the first leading cause of cancerrelated death in the United States with 222,500 new cases being estimated to occur in 2017 [1]. Through the development of advanced technologies and methods in lung cancer treatment, the 5-year survival rate was just slightly increased from 13% to 18% in the United States [2]. A cancer therapy strategy based on cystatin functions was proposed in recent years [3]. Cystatin A (CSTA, Stefin A), one of the type 1 cystatin super-family members, is mainly www.impactjournals.com/oncotarget intracellularly located and it can be found in body fluids [5]. CSTA is expressed primarily in epithelial and lymphoid tissues for protecting cells from proteolysis of cytoplasmic and cytoskeletal proteins by inhibiting cathepsin B, H and L [4, 5]. Mutations in CSTA are associated with skin fragility phenotype including exfoliative ichthyosis and acral peeling skin syndrome [6, 7]

Methods

Results

Conclusion