Digestive Diseases and Sciences https://doi.org/10.1007/s10620-018-5307-x
ORIGINAL ARTICLE
Clinical Significance of the Thioredoxin System and Thioredoxin‑Domain‑Containing Protein Family in Hepatocellular Carcinoma Sang Yeon Cho1 · Sungha Kim2 · Mi‑Ju Son2 · Woo Sun Rou3,4 · Seok Hyun Kim3,4 · Hyuk Soo Eun3,4 · Byung Seok Lee3,4 Received: 5 July 2018 / Accepted: 25 September 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Background Oxidative stress occurs due to the excessive generation of cellular reactive oxygen species and antioxidant system dysfunction. The thioredoxin (TXN) system and TXN-domain-containing protein (TXNDC) family form networks maintaining the cellular reducing environment. Recently, the importance of these genes in the tumor environment has been emphasized. Aim To investigate the clinical significance of TXNs and TXNDC family members in HCC. Methods Genomic data from 367 hepatocellular carcinoma (HCC) patients who underwent hepatic resections were analyzed to determine genetic alterations in mRNA and protein levels between patients and healthy controls. In addition, functional enrichment and survival analyses were performed. Results HCC patients were shown to have enhanced expression of TXN, TXNRD1, and TXNDC7/9/14 mRNA and protein compared with controls. In accordance with the survival analyses, strong associations were found that patients with TXN, TXNRD1, and TXNDC1/7/9 alterations were proven to have poor prognosis in overall survival. Moreover, gene set enrichment analysis and network analyses revealed that positive correlations were found in mRNA expression of TXN, TXNRD1, and TXNDC7/9 genes with upregulation of the tumor-promoting genes, specifically mTORC1, E2F targets, and Myc targets. On the other hand, elevated expressions of TXNIP and TXNDC11 genes were correlated with suppression of the above tumor-promoting genes. Conclusions TXN system and TXNDC family gene panel obtained from the resected tissue of the HCC patients could be used to predict survival prognosis of HCC, and these genes could be considered as potential therapeutic targets for improving HCC survival. Keywords Thioredoxin · TXN-domain-containing protein · Hepatocellular carcinoma · Overall survival
Sang Yeon Cho and Sungha Kim have contributed equally to this work. * Hyuk Soo Eun
[email protected] * Byung Seok Lee
[email protected] 1
School of Medicine, Chungnam National University, 266, Munwha‑ro, Jung‑gu, Daejeon 35015, Republic of Korea
Department of Clinical Research, Korea Institute of Oriental Medicine, 1672 Yuseong‑daero, Yuseong‑gu, Daejeon 34054, Republic of Korea
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3
Department of Internal Medicine, Chungnam National University Hospital, 282, Munwha‑ro, Jung‑gu, Daejeon 34952, Republic of Korea
4
Department of Internal Medicine, School of Medicine, Chungnam National University, 266, Munwha‑ro, Jung‑gu, Daejeon 35015, Republic of Korea
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Introduction Liver is a principal functional organ that detoxifies several substances that produce reactive oxygen species (ROS); therefore, disruption of the hepatic reduction–oxidation (redox) system results in oxidative stress and deleterious processes associated with liver cancer [1]. Accumulating evidences have revealed that tumor initiation and progression are associated with oxidative stress, and specifically in the liver, oxidative stress can promote and exacerbate tumorigenesis [2, 3]. Hepatocellular carcinoma (HCC) represents the most common form of liver cancer, with its associated mortality rate having increased in the previous decades. Paradoxically, in the liver, oxidative stress promotes HCC and also HCC induces redox capacity [4]. Because cancer cells can also be damaged by high ROS levels, these cells self-protect by regulating ROS metabolism via multiple signaling pathways linked to tumorigenesis [5, 6]. The thioredoxin system is indispensable for retaining harmony and regulation of the redox status in cells [7]. This system comprises cytoplasmic thioredoxin (TXN), mitochondrial TXN2, cytoplasmic TXN reductase 1 (TXNRD1), mitochondrial TXNRD2, and the TXN inhibitor TXN-interacting protein (TXNIP). Interestingly, previous studies report that elevated TXN expression in various malignancies or aggressive tumors related to poor survival of cancer patients [8–10]. Moreover, TXNRD overexpression has been reported in HCC, as well as breast, prostate, and colorectal cancers, and is closely related to aggressive tumor growth and poor patient survival [11–14]. On the other hand, TXNIP expression is suppressed in many cancers, and several studies report that blockage of cell cycle progression suppresses TXNIP overexpression related to the inhibition of tumor growth [15–17]. As another regulator of redox status, the TXN-domaincontaining protein (TXNDC) family includes 17 members that participate in controlling redox status at distinct cellular locations, including the plasma membrane, cytosol, endoplasmic reticulum, and nucleus [18]. However, few studies have investigated TXNDC members for their roles in cancer progression, especially in that of HCC. A previous study reported upregulation of TXNDC5, which is involved in protein folding and regulation of chaperone activity, in cervical, colon, stomach, liver, and lung cancers [19]. Additionally, overexpression of TXNDC9, involved in chaperon-assisted protein folding, is associated with poor prognosis in colorectal cancer, and TXNDC17, involved in tumor necrosis factor signaling, promotes chemotherapy resistance in ovarian cancer [20, 21]. However, the cancer-specific roles of other TXNDC family members have not been fully elucidated.
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Digestive Diseases and Sciences
Despite their involvement in maintaining the cellular redox environment, the clinical significance of TXNs and TXNDC family members in HCC has not been comprehensively examined. This study investigated their roles in HCC using data from The Cancer Genome Atlas (TCGA) and bioinformatics analyses.
Methods Gene‑Expression Profiles RNAseqV2-RSEM_genes and clinical data from 377 liver HCC samples were obtained from Firebrowse (http://fireb rowse.org/) for gene-expression analysis. Seven datasets for hepatocholangiocarcinoma and three for fibrolamellar carcinoma were excluded to ensure utilization of only 367 HCC samples for mRNA-expression profiling. Table 1 summarizes the clinicopathologic data of the patients with HCC enrolled in this study.
Analysis of mRNA and Protein Expression The data from TCGA portal were analyzed using R software (v.3.2.5; http://www.r-project.org). The Rank Normalize module in GenePattern (http://broadinstitute.org/cancer/ software/genepattern) was used to normalize the chip data. The data for the fold change of thioredoxin system and TXNDC family genes in HCC compared to normal controls were derived from Firebrowse (http://firebrowse.org/viewG ene.html?gene=thioredoxin), where the gene name in the web address for Firebrowse could be substituted by the name of the gene of interest. Protein expression and immunohistochemical staining were obtained from the Human Protein Atlas portal (http://www.proteinatlas.org).
Integrative Analysis of Genomic Data Integrative genomic analyses, including gene alterations, mRNA expression along with gene alterations, and survival analysis along with gene alterations were performed in cBioportal (http://www.cbioportal.org/).
Functional Enrichment Analysis Gene set enrichment analysis (GSEA) was performed to enrich genes predicted to correlate with pathways in the hallmark and curated gene sets using Kyoto Encyclopedia of Genes and Genomes information. GSEA was performed between two groups exhibiting high (top 10%) and low
Digestive Diseases and Sciences Table 1 Clinicopathologic information of hepatocellular carcinoma patients Feature
Total (%)
Number Sex Female Male Age (years) ≤ 60 > 60 NA TNM stage Stage I Stage II Stage III Stage IV NA Histological grade Grade 1 Grade 2 Grade 3 Grade 4 NA Vital status Alive Dead Child–Pugh classification A B C NA Ishak fibrosis score 0—no fibrosis 1, 2—portal fibrosis 3, 4—fibrous septa 5—nodular formation and incomplete cirrhosis 6—established cirrhosis NA
367 (100.0) 367 (100.0) 118 249 367 (100.0) 176 190 1 367 (100.0) 171 83 85 4 24 367 (100.0) 53 174 123 12 5 367 (100.0) 236 131 367 (100.0) 218 21 1 127 367 (100.0) 74 31 30 9 72 151
NA not applicable
(bottom 10%) levels of gene expression. Enrichment maps were visualized using Cytoscape (v.3.5.1; www.cytoscape. org). Relationships were considered significant at p
