Published OnlineFirst August 4, 2020; DOI: 10.1158/1541-7786.MCR-20-0180 MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS Wnt/b-Catenin Signaling Axis Is Required for TFEB-Mediated Gastric Cancer Metastasis and Epithelial– Mesenchymal Transition Shuxuan Li1, Fenglin Liu2, Ling Xu1, Can Li1, Xu Yang1, Bao Guo1, Jianxin Gu1, and Lan Wang1 ABSTRACT ◥ Gastric cancer remains the third leading cause of cancer-related b-catenin signaling pathway members in TFEB high-expression death, and tumor metastasis is the main risk factor for poor group, and the TOP/FOPflash assay verified the effect of TFEB on prognosis of patients with gastric cancer. Transcription factor EB b-catenin transcription activity. Besides, we found that TFEB could (TFEB) is a MiT family member and has been found to drive trigger the aggregation of b-catenin in nucleus and activate its tumorigenesis in a number of tissues, whereas few studies were transcription, as well as facilitate the expression of Wnt/b-catenin focused on investigating its prometastasis role and mechanism in target genes and EMT-related markers, which could be reversed by gastric cancer. Here, we found TFEB was upregulated in gastric the Wnt/b-catenin inhibitor XAV-939. Collectively, TFEB cancer tissues compared with adjacent normal gastric epithelial enhances gastric cancer metastatic potential by activating Wnt/ tissues. IHC analysis from gastric cancer tissue microarray revealed b-catenin signaling pathway and may become a promising thera- that TFEB in gastric cancer was correlated with depth of tumor peutic target for gastric cancer metastasis. invasion, lymph node or distant metastasis, tumor tumor–node– metastasis stage, and overall survival. Gastric cancer cells with TFEB Implications: Overexpressed TFEB predicts a higher rate of metas- overexpression presented an increased cell migration or invasion, tasis and worse survival in patients with gastric cancer. Mechanis- and epithelial–mesenchymal transition (EMT). Furthermore, gene tically, TFEB activates Wnt/b-catenin signaling to fuel migratory correlation analysis and gene set enrichment analysis enriched Wnt/ and invasive activities of gastric cancer cells, as well as EMT. Introduction version of cytokeratin to vimentin-based cytoskeleton, and an increase in transcription factors (Snail, Twist, ZEB1, ZEB2; refs. 6, 7). In 2018, there were more than one million new cases of gastric In addition, EMT involves a variety of signaling pathways, including cancer with a third death rate in the world despite early diagnoses and TGFb,NF-kB, Notch, RTK/Ras, and Wnt/b-catenin pathways (8). evolving therapeutic methods (1). More than 70% of patients with Transcription factor EB (TFEB) is a member of the basic helix-loop- gastric cancer are advanced and metastasized at the time of diagnosis, helix leucine zipper (bHLH-Zip) transcription factor MITF/TFE fam- and the 5-year survival rate of gastric cancer is only 20% to 50% (2, 3). ily, which contains four different transcription factors, TFEB, TFE3, At present, the treatment of gastric cancer is mainly chemotherapy MITF, and TFEC (9, 10). TFEB coordinates lysosomal biogenesis and drugs, lacking targeted drugs and personalized treatment. More and autophagy induction to keep cell homeostasis, including immune more research has committed to exploring the mechanism of gastric response, mitochondrial biogenesis, unfolded protein response, cancer metastasis, and hope to develop new solutions for the treatment lysosomal exocytosis. In vivo studies have highlighted the role of TFEB of gastric cancer. in lipid catabolism, which is associated with a variety of diseases such as Epithelial–mesenchymal transition (EMT) is an important bio- neurodegenerative diseases, cancer, and lysosomal storage dis- logical process for epithelial-derived tumor cells to acquire the ease (11, 12). Lectin-like oxidized low density lipoprotein receptor-1 ability of migration and invasion. Epithelial cells are transformed (LOX-1) is first identified as a receptor of oxidized low density into mesenchymal-like cells with significant genetic and phenotypic lipoprotein (LDL) in endothelial cells and has been found to be changes, further manifested by loss of polarity, tight junctions, involved in the activation of autophagy (13, 14), which contributes and adhesions (4, 5). The main features of EMT are decreased to the development and progression of digestive system tumors, expression of cell adhesion molecules (such as E-cadherin), con- including gastric cancer (15, 16). As a major regulator of autophagy, many studies on TFEB were about tumor chemical resistance, and it was 1NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry reported that TFEB could abnormally expressed or mutated in papillary and Molecular Biology, School of Basic Medical Sciences, Fudan University, renal cell carcinoma, lung cancer, pancreatic cancer, and colorectal Shanghai, China. 2Department of General Surgery, Zhongshan Hospital, Fudan cancer (17–20), whereas few studies were about gastric cancer. University, Shanghai, China. Here we present a comprehensive characterization of the expres- Note: Supplementary data for this article are available at Molecular Cancer sional pattern, clinical value, and functional mechanism of TFEB in Research Online (http://mcr.aacrjournals.org/). gastric cancer, and expected to explore a new method for the diagnosis S. Li and F. Liu contributed equally to this article. and treatment of gastric cancer. Corresponding Author: Lan Wang, Fudan University, Shanghai 200032, China. Phone: 86-21-54237361; E-mail: [email protected] Materials and Methods – Mol Cancer Res 2020;XX:XX XX Patient samples and tissue microarray construction doi: 10.1158/1541-7786.MCR-20-0180 A human tissue microarray (TMA) of 80 pairs of gastric tumors/ Ó2020 American Association for Cancer Research. nontumor specimens collected from patients with gastric cancer AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst August 4, 2020; DOI: 10.1158/1541-7786.MCR-20-0180 Li et al. undergoing surgery at Zhongshan Hospital were paraffin-embedded 30), si2# (50-GACGAAGGUUCAACAUCAATT-30), si3# (50-GCGG- and stained with hematoxylin and eosin by Shanghai Superbiotek CAGAAGAAAGACAAUTT-30). Transfection was performed with Pharmaceutical Technology Co. Ltd. Tumor staging was graded Lipofectamine 3000 (Invitrogen) according to the manufacturer's according to the eighth edition of the American Joint Committee on instructions. Tumor TNM Classification (21). In addition, 26 pairs of gastric cancer and adjacent gastric tissue samples were obtained from patients Immunoblot analysis undergoing surgery at Zhongshan Hospital and stored at 80C until The protein samples extracted from tissue or cell lysates were use. The study was authorized by the Ethics Committee of Basic separated by SDS-PAGE and transferred to polyvinylidene fluoride Medical College of Fudan University. All patients gave written (PVDF) membrane. The membrane was incubated with the first informed consent before participation in this study, and the IRB antibody (1:1,000) for 4C overnight, including anti-TFEB (Pro- number is 2018-Y007. teintech), anti-Myc-tag (Cell Signaling Technology), anti-ZO-1 (CST), anti-E-cadherin (Proteintech), anti-N-cadherin (Protein- IHC analysis tech), anti-Vimentin (CST), anti-Snail (CST), anti-Twist1 (Protein- IHC analysis was applied on TMA specimens according to standard tech), anti-b-catenin (CST), anti-TCF4 (Proteintech), anti-c-Myc protocols of Rabbit-specific HRP/DAB (ABC) Detection IHC Kit (CST). The second antibody (1:2,000; Santa Cruz Biotechnology) (Abcam), followed by anti-TFEB antibodies (1:200; Proteintech) and b-actin (1: 4,000; Proteintech) coupled with HRP was incubated incubation, and hematoxylin was used for counterstaining. The IHC at room temperature for 1.5 hours. The bands were detected by result was assessed by an experienced independent pathologist and enhanced chemiluminescence (ECL). were unaware of the patient's condition. The staining intensity score was 0 to 3 points. The heterogeneity of staining varies from 0 to 4 Real-time PCR (RT-qPCR) assay depending on the percentage of positive tumor cells. To obtain an IHC Total RNA was purified from stomach tissues or cancer cells score that considers IHC signal intensity and positive cell frequency, using TRizol (Invitrogen) and reversed transcription to cDNA using we generated a complete range of composite expression scores (CES) PrimeScript RT reagent Kit (Takara). Real-time PCR were per- from 0 to 12. The best cutoff value (CES ¼ 6) is determined using ROC formed using SYBR Premix Ex Taq (Takara) Taq (Takara) on ABI curve analysis. CES > 6 represents a high TFEB expression and CES ≤ 6 StepOne Plus (Applied Biosystems) according to the manufacturer's represents a low TFEB expression. instructions. TCGA and GEO datasets Transwell assay The databases applied in this study are publicly available from the Transwell migration and invasion was assessed using a 12-well Cancer Genome Atlas of Stomach Adenocarcinoma (TCGA-STAD) Transwell plate (8 mm pore size) according to the manufacturer's and Gene Expression Omnibus (GEO) database (GSE62254, instructions (US Microwell). In the migration assay, 20,000 to 30,000 GSE66229, GSE35809). For the TCGA dataset, all data was down- cells in serum-free medium were inoculated into the upper chamber, loaded using the TCGA assembler software, and mRNA level of was whereas