Aldo-keto reductase family 1 member B10 prevents esophageal squamous cell carcinoma from reactive carbonyl species-induced cell death and promotes its progression

Abstract

Introduction: Chronic alcohol consumption and tobacco usage are major risk factors for esophageal squamous cell carcinoma (ESCC). Excessive tobacco and alcohol consumption lead to oxidative stress and the generation of reactive carbonyl species (RCS) which induce DNA damage and cell apoptosis. This phenomenon contributes to cell damage and carcinogenesis in various organs including ESCC. However, it also raises an important question on how ESCC cells evade RCS-induced apoptosis and grow rapidly under these conditions. Therefore, we hypothesize that some enzymes produced by ESCC cells are capable of catabolizing RCS, preventing ESCC neoplastic cells from undergoing RCS-induced apoptosis, potentially contributing to ESCC progression.

Methods: To identify significant gene clusters involved in the metabolism of RCS in ESCC, we used an Agilent SurePrint G3 Human V2 GE 8 × 60 K microarray kit to analyze differentially expressed genes between nine paired ESCC tissues and adjacent normal esophageal tissues taken from areas distant from the tumor site. Bioinformatics analysis using gene ontology (GO) was performed to categorize these genes. To validate the findings, immunohistochemical staining in specimens from 169 surgically resected ESCC patients was performed and then correlated with treatment outcomes. Furthermore, the identified signaling pathway and its biological effects were investigated in ESCC cell lines in vitro and 4-nitroquinoline 1-oxide (4-NQO)-induced-ESCC murine model in vivo.

Results: Interestingly, we found that one of the significantly altered 57 GO molecular function domain terms (GO:0004033 aldo-keto reductase activity; P = 0.021) between nine paired ESCC tumors and adjacent normal tissue specimens was associated with the RCS metabolism. Among significant genes within this domain, AKR1B10 (aldo-keto reductase family 1 member B10; P = 0.006) was identified as the most significantly altered gene. Immunohistochemical analysis revealed that AKR1B10 expression was higher in ESCC cells than in adjacent normal esophageal epithelium. In addition, AKR1B10 expression was independently significantly associated with a poorer prognosis in 169 ESCC patients. Enzyme-linked immunosorbent assay results further demonstrated that blood AKR1B10 concentrations were significantly higher in 72 ESCC patients than in 24 healthy controls. In vitro experiments revealed that inhibiting endogenous AKR1B10 enhanced the cytotoxicity of 4-hydroxy trans-2-nonenal, a type of RCS. In a 4-NQO-induced-ESCC murine model, oleanolic acid, an AKR1B10 inhibitor, significantly reduced the incidence of esophageal tumors.

Conclusions: Our findings suggested that AKR1B10 is an independent adverse prognosticator for patients with ESCC, and could prevent ESCC neoplastic cells from undergoing RCS-induced apoptosis, and promote ESCC progression. Therefore, AKR1B10 signaling could be a potential therapeutic strategy for ESCC.

Keywords: AKR1B10; ESCC; RCS.

Fig. 1

(A) Upper: Venn diagram analysis showed 3406 significant (> 1.5-fold) transcripts including 1785 upregulated and 1621 downregulated transcripts in nine-paired ESCC tissues in comparison with adjacent normal esophageal tissues. Bioinformatics analysis using gene ontology (GO) revealed 57 GO molecular function domain, and one domain, GO:0004033 (aldo-keto reductase activity; P = 0.021), was associated with the RCS metabolism. Among the significant genes in aldo-keto reductase activity (GO:0004033), AKR1B10 was the most significant gene identified between ESCC tumors and adjacent normal esophageal tissue specimens, followed by AKR1B15, AKR1B1, AKR1C4, AKR1C3, SPR, and AKR1C1.(B) The differentially expressed gene, AKR1B10, between nine paired ESCC tumors and adjacent normal tissue specimens identified by microarray was validated by q-RT-PCR. (C) Representative immunohistochemical staining of AKR1B10 in esophageal squamous cell carcinoma and adjacent normal esophageal epithelium. Immunohistochemistry of AKR1B10 in esophagectomy samples showed higher AKR1B10 protein expression in ESCC tissues than in adjacent normal esophageal epithelium. Positive AKR1B10 immunoreactivity was observed in the normal human colon mucosa, which served as a positive control. Original magnification: ×200. (D) AKR1B10 concentration was significantly higher in 22 ESCC patients than in 22 healthy controls. ELISA of AKR1B10 concentration of blood samples from ESCC patients and healthy controls. The results are presented as the mean ± SD of triplicate wells in three independent experiments. *statistically significant (P = 0.001). (E) The 4-HNE, a reactive carbonyl species (RCS), is recognized as a general marker of oxidative stress induced by excessive tobacco smoking or alcohol consumption. AKR1B10 silencing by small-interfering RNA (left panel) significantly enhance 4-HNE cytotoxicity in ESCC cell lines, KYSE70 and TE11, suggesting that AKR1B10 can prevent ESCC cells from undergoing RCS-induced apoptosis. The percentage (%) of cell growth inhibition = (viable cell number under 0µM 4-HNE - viable cell number under 40µM 4-HNE)/ viable cell number under 0µM 4-HNE. *Significantly different from the control (P < 0.05); ESCC, esophageal squamous cell carcinoma; RCS, reactive carbonyl species; AKR1B10, aldo-keto reductase family 1 member B10; qRT-PCR, quantitative real-time polymerase chain reaction; 4-HNE, 4-hydroxy trans-2-nonenal

Fig. 2

Kaplan–Meier curves showed that patients with AKR1B10 overexpression had significantly worse overall survival and disease-free survival than those with low AKR1B10 expression. (A) Overall survival according to AKR1B10 expression. (B) Disease-free survival according to AKR1B10 expression. AKR1B10, Aldo-keto reductase 1B10

Fig. 3

AKR1B10 inhibitor, oleanolic acid, significantly decrease the incidence of ESCC in 4-NQO-induced ESCC murine model. (A) The incidence of ESCC in mice treated with oleanolic acid was significantly lower than that in mice treated with placebo control (56% versus 88%; P = 0.013). (B) Left: Gross appearance of the esophagus from representative mice treated with placebo control showed enlargement of the esophageal tumor indicated by the arrow. Right: Gross appearance of the esophagus from representative mice treated with oleanolic acid revealed no enlargement of the esophageal tumor. (C) Hematoxylin and eosin stained sections from representative mice treated with placebo control showed ESCC with stromal invasion, whereas sections from representative mice treated with oleanolic acid displayed only esophageal dysplasia. Original magnification ×100. Immunohistochemistry revealed lower AKR1B10 expression in the oleanolic acid group than in the placebo group. Original magnification ×200. AKR1B10, Aldo-keto reductase 1B10; ESCC, esophageal squamous cell carcinoma; 4-NQO, 4-nitroquinoline 1-oxide *statistically significant