The Expression and Clinical Significance of Aldo-Keto Reductase 1 Member B1 in Gastric Carcinoma

Abstract

To study the expression of aldo-keto reductase 1 member B1 (AKR1B1) in gastric carcinoma (GC), the correlation between AKR1B1 and the clinicopathological characteristics of GC patients, and provide reference for the diagnosis and prognosis of GC patients. One hundred thirty-six patients with GC were collected, and the expression level of AKR1B1 in GC and adjacent tissues was detected by immunohistochemistry assays. The clinicopathological features and prognosis of GC patients were collected to analyze the relationship with AKR1B1 expression. The positive expression of AKR1B1 in GC tissues was significantly higher than that of adjacent nontumor tissues. The difference of AKR1B1 expression between GC tissues and paired adjacent nontumor tissues was statistically significant (p < 0.001). AKR1B1 was closely related to tumor size, regional lymph node (N), metastases (M), and tumor–node–metastasis (TNM) stage (p < 0.05). The overall survival of patients with low expression of AKR1B1 was significantly better than that of patients with high expression of AKR1B1 by Kaplan–Meier survival analysis (p < 0.001). AKR1B1 plays an important role in the occurrence and development of GC, and it has a certain reference value for the prognosis of GC patients.

Introduction

Gastric carcinoma (GC) is one of the most common gastrointestinal cancers in the world. According to the latest statistics, East Asia is the region with the highest 5-year survival rate of GC. The 5-year survival rate of GC in South Korea is as high as 68.9% (Allemani et al., 2018), while the 5-year survival rate of GC in China is only 35.9% (Chen et al., 2016). The current treatments for GC include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. The prognosis of GC is closely related to the timing of diagnosis and treatment. The treatment of early GC is surgical resection as much as possible, and the 5-year survival rate after surgery can exceed 90%, and even achieve the curative effect (Sumiyama, 2017). However, most of the GC patients in China are already in the advanced stage. The 5-year survival rate of advanced GC patients is still less than 30% even after receiving comprehensive treatment mainly based on surgery (Katai et al., 2018). Postoperative recurrence and metastasis are the main causes of death for GC patients. The development of GC is a multifactor process. The abnormal expression of genes plays an important role, but the specific mechanism is still unclear.

Aldo-keto reductases (AKRs) are oxidoreductases dependent on nicotinamide adenine dinucleotide phosphate (NADPH), which are the most glycoreductive enzymes. The increase of AKR expression is related to the lung, breast, prostate, cervix, ovary, and colon tumors, which have an important significance in the pathophysiology of tumors (Taskoparan et al., 2017). Aldo-keto reductase 1 member B1 (AKR1B1) is a member of the AKR family. It is mainly involved in the following metabolic pathways: (1) production of sorbitol by catalyzing glucose; (2) removal of aldehyde group of glutathione to participate in lipid metabolism; and (3) catalyzes prostaglandin H2 to produce prostaglandin F2α (Fujimori et al., 2010; Ramana, 2011). The relationship between the expression of AKR1B1 in GC and its clinical characteristics is still unclear. Therefore, for the first time, we detected the expression of AKR1B1 in tumor tissues and adjacent tissues of GC patients, and analyzed its relationship with the clinical characteristics and prognosis of patients.

Materials and Methods

Tissue samples

A total of 136 paired GC tissues and adjacent tissues were collected from patients who were diagnosed with GC by both pathological and clinical methods. The distance between GC tissues and adjacent nontumor tissues was >3 cm. All the patients did not receive any radiotherapy or chemotherapy before surgery. All pathological samples in this study were taken from GC tissues removed during surgery and made into tissue chips. The tissues and clinical information were obtained with the consent of patients, and approved by the Ethics Committee of the First Affiliated Hospital of Naval Military Medical University (ID: CHEC2014–098).

Reagents

The AKR1B1 antibody (ARG57863) for immunohistochemistry (IHC) was purchased from Arigo Bio Company (Taiwan, China). Kit for IHC was from Maixin Biotechnologies (Fuzhou, China). The sheep-derived serum, phosphate-buffered saline, and hydrogen peroxide were provided by ZSGB-BIO (Beijing, China).

Immunohistochemistry

The IHC assay was performed to detect the expression of AKR1B1 in GC and adjacent tissues. The specific steps are carried out according to the instructions of the immunohistochemistry kit. First, we put the tissue chip in the 63° baking sheet for 1 h. It is then dewaxed and hydrated in a fully automatic dyeing machine. Second, we used high-pressure heat repair method for antigen retrieval, added a primary antibody for 1 h at 37°C, and then added the goat anti-rabbit secondary antibody for 30 min at 37°C. Finally, DAB was developed for 1 min, hematoxylin was counterstained for 10 min, and the film was sealed.

Five fields in each specimen were selected randomly for analysis. More than 500 cells were counted to determine the mean percentage of immunostained cells relative to the total number of cells. AKR1B1 was scored by staining intensity: no coloring was 0 (negative), light yellow was 1 point (1+), brownish yellow was 2 points (2+), and dark brown was 3 points (3+). The AKR1B1 staining positive rate score was as follows: 0 points (negative), 1 point (1–25%), 2 points (26–50%), 3 points (51–75%), and 4 points (76–100%). The product of the staining intensity score and the staining positive rate score are the total score. Scores >4 were defined as AKR1B1-positive expression and scores ≤4 were defined as AKR1B1-negative expression (Zhu et al., 2019).

Statistical analysis

The expression difference of AKR1B1 between GC and adjacent tissues was compared by the chi-square test. The correlation between AKR1B1 expression and clinicopathological parameters of GC patients was analyzed by chi-square test and multivariate logistic regression analysis. The Kaplan–Meier method was used for survival analysis, and difference in survival was estimated by using the log-rank test, p < 0.05 was defined as statistically significant. Multivariate Cox regression analysis was performed to determine survival trends adjusted for clinical and demographic factors. The statistical software SPSS 19.0 (SPSS, Chicago, IL) was used for all data analysis.

Results

Clinicopathological characteristics of GC patients

This study enrolled altogether 136 GC cases. Among the selected patients, there were 100 males and 36 females, aged 28 to 77 years, with an average of 67.5 ± 12.4 years; 30 patients with tumors <3 cm in diameter, 76 patients with tumors 3–6 cm in diameter, and 30 patients with tumors of diameter >6 cm. The tumor–node–metastasis (TNM) stage of patients with I, II, III, and IV was 21 (15.4%), 33 (24.3%), 64 (47.1%), and 18 (13.2%); and the high, middle, and low differentiation of tumors was 14 (10.3%), 68 (50.0%), and 54 (39.7%), according to the AJCC criteria staging system (Liu et al., 2018). There were 9 cases of vascular cancer thrombus and 127 cases without vascular cancer thrombus (Supplementary Table S1).

Expression of AKR1B1 in GC tissues and adjacent tissues

We detected the expression of AKR1B1 in 136 paired GC and adjacent tissues by IHC assay. As shown in Figure 1, AKR1B1 located mainly at the cytoplasm. The positive expression of AKR1B1 in GC and adjacent tissues was 52.9% (72/136) and 33.1% (45/136), respectively. After statistical analysis, we found that AKR1B1 was upregulated in GC tissues (p < 0.001, Table 1 and Fig. 2).

FIG. 1.

Expression of AKR1B1 in GC tissues and adjacent tissues. Representative positive expression of AKR1B1 in GC tissues, negative expression of AKR1B1 in paratumor stromal cells (image above), and negative expression of AKR1B1 in adjacent tissues (image below) were detected by IHC. AKR1B1, aldo-keto reductase 1 member B1; GC, gastric carcinoma; IHC, immunohistochemistry. Color images are available online.

FIG. 2.

Comparison of AKR1B1 expression in GC and its adjacent tissues by IHC. p-Value is based on the McNemar chi-square test.

Table 1.

Expression of Aldo-Keto Reductase 1 Member B1 in Paired Gastric Carcinoma and Adjacent Tissues

GC tissues	Adjacent tissues		Total
GC tissues	Positive	Negative	Total
Positive	43	29	72
Negative	2	62	64
Total	45	91	136

p < 0.001, p-value is based on the McNemar chi-square test.

GC, gastric carcinoma.

Relationship between AKR1B1 expression and clinicopathological features of GC patients

The 136 cases of GC patients were divided into two groups according to the expression of AKR1B1. We collected these clinicopathological features of GC patients, such as gender, age, tumor site, tumor size, tumor invasion depth (T), regional lymph node (N), metastases (M), TNM stage, and tumor differentiation. As shown in Table 2, AKR1B1 expression was significantly correlated with tumor size, regional lymph node (N), metastases (M), and TNM stage (p < 0.05). There was no relationship between AKR1B1 expression and other clinicopathological features of GC patients (p > 0.05). Multivariate logistic regression analysis showed that AKR1B1 expression was related to the TNM stage (Table 3).

Table 2.

Relationship Between Aldo-Keto Reductase 1 Member B1 Expression and Clinicopathological Characteristics in Gastric Carcinoma

Variables	Total	Expression of AKR1B1		p
Variables	Total	Positive	Negative	p
Gender
Male	100	57	43	0.114
Female	36	15	21	0.114
Age, year
<30	11	4	7	0.337
30–59	45	22	23
>60	80	46	34
Tumor site
Cardia	13	9	4	0.387
Gastric body	50	23	27
Gastric antrum	64	34	30
Whole stomach	9	6	3
Tumor size
<3 cm	30	9	21	0.013
3–6 cm	76	47	29
>6 cm	30	16	14
Tumor invasion depth (T)
T1	12	4	8	0.440
T2	25	12	13
T3	84	47	37
T4	15	9	6
Regional lymph node (N)
N0	48	18	30	0.011
N1	54	30	24
N2	34	23	10
Metastases (M)
M0	128	65	63	0.023
M1	18	14	4	0.023
TNM stage
I	21	5	16	0.008
II	33	18	15
III	64	35	29
IV	18	14	4
Tumor differentiation
High differentiation	14	4	10	0.156
Middle differentiation	68	38	30
Low differentiation	54	30	24

Bold values indicate significance. p-Value is based on the chi-square test.

AKR1B1, aldo-keto reductase 1 member B1; TNM, tumor–node–metastasis.

Table 3.

The Multivariate Logistic Regression Analysis Between the Clinically Related Risk Factors and Aldo-Keto Reductase 1 Member B1 Expression in Gastric Carcinoma Patients

Variables	OR (95% CI)	p
TNM stage		0.016
I	Reference
II	3.840 (1.139–12.951)	0.030
III	3.862 (1.262–11.817)	0.018
IV	11.200 (2.505–50.080)	0.002

CI, confidence interval; OR, odds ratio.

Relationship between AKR1B1 expression and prognosis of GC patients

The mean survival time of GC patients with negative AKR1B1 expression and positive AKR1B1 expression was 85.86 ± 3.57 months (95% confidence interval [CI]: 78.85–92.87) and 48.91 ± 4.01 months (95% CI: 41.05–56.77) by the Kaplan–Meier method, respectively (Table 4). After the log-rank test, the survival time of GC patients with AKR1B1-negative expression was significantly longer than that of patients with AKR1B1-positive expression (p < 0.001), indicating that positive expression of AKR1B1 was associated with poor prognosis of GC patients (Table 4 and Fig. 3). Tumor size, tumor invasion depth (T), regional lymph node (N), metastases (M), TNM stage, and AKR1B1 expression were included in COX regression analysis. We found that TNM stage and AKR1B1 expression were independent protective factors for GC patients (Table 5).We then sought to elucidate its clinical relevance by examining its correlation with patient survival in the GEPIA database, and the data of GEPIA database came from TCGA and GTEx, indicating that the high expression of AKR1B1 was closely related to shorter overall survival and disease-free survival (Fig. 3).

FIG. 3.

Effect of AKR1B1 expression on the survival of GC patients. The survival rates were determined by Kaplan–Meier analysis (Left). AKR1B1 (+) indicates AKR1B1-positive expression and AKR1B1 (−) indicates AKR1B1-negative expression. Relationship between AKR1B1 expression and overall survival in 137 gastric cancer patients (Middle). Kaplan–Meier survival analysis of published database (GEPIA) for the relationship between AKR1B1 expression and overall survival (Right). Kaplan–Meier survival analysis of published database (GEPIA) for the relationship between AKR1B1 expression and disease-free survival. Color images are available online.

Table 4.

Relationship Between Aldo-Keto Reductase 1 Member B1 Expression and Overall Survival of Gastric Carcinoma Patients

Expression of AKR1B1	Total	Overall survival (month)		p
Expression of AKR1B1	Total	Mean survival time (x ± s)	95% CI	p
Positive	72	48.91 ± 4.01	41.05–56.77	0.000
Negative	64	85.86 ± 3.57	78.85–92.87	0.000

Table 5.

The Multivariate Cox Proportional Hazard Regression Analyses Between the Clinically Related Factors and Survival in Gastric Carcinoma Patients

Variables	HR (95% CI)	p
AKR1B1	3.317 (1.667–6.600)	0.001
TNM stage		0.028
I	Reference
II	2.063 (0.433–9.829)	0.363
III	4.290 (1.011–18.205)	0.048
IV	4.753 (1.005–22.483)	0.049

CI, confidence interval; HR, hazard ratio.

Discussion

Gastric adenocarcinoma is the fifth-most common and the third-most lethal cancer worldwide (Johnston and Beckman, 2019). Approximately half of the world's GC cases and deaths occur in China (Nie et al., 2017). Early screening plays an important role in early diagnosis of GC (Ponzetto and Figura, 2019). Early GC can even be cured by surgery, while advanced GC cannot achieve satisfactory results even through radiotherapy, chemotherapy, and targeted immunotherapy. GC presents challenges in early diagnosis and effective therapy due to a lack of understanding of the underlying molecular biology (Nie et al., 2017). Therefore, using sufficient clinical samples to screen and identify more new tumor markers is of great significance for the in-depth exploration of the pathogenesis of cancer.

The AKR protein superfamily contains >190 members that fall into 16 families and are found in all phyla. These enzymes reduce carbonyl substrates such as sugar aldehydes, keto-steroids, keto-prostaglandins, retinals, quinones, and lipid peroxidation by-products (Penning, 2015). Study uncovers the active function of AKR1C1 as a key component of the STAT3 pathway, which promotes lung cancer metastasis (Zhu et al., 2018). Researchers found that AKR1C3 overexpression promotes the growth of both androgen-dependent prostate cancer and castration-resistant prostate cancer xenografts, with concomitant reactivation of androgen signaling (Yepuru et al., 2013). Recent researches showed that combination of AKR1B10 with alpha-fetoprotein (AFP) increased diagnostic accuracy for hepatocellular carcinoma (HCC) compared with AKR1B10 or AFP alone; this suggests that AKR1B10 is a new potent serum marker for detection of HCC (Ye et al., 2018). Another study confirmed that AKR1B10 was overexpressed in breast cancer and promoted the migration and invasion of MCF-7 and BT-20 cells by activating the extracellular signal-regulated kinase (ERK) signaling pathway (Li et al., 2017).

AKR1B1 is an NADPH-dependent multifunctional enzyme. The spatial structure of the protein is a single-chain polypeptide containing sulfhydryl groups, which generally exists as monomer in organisms. AKR1B1 can be reversibly combined with NADPH to reduce aldehydes to corresponding alcohols by using its nicotinamide ring C4 hydride (Anil and Bhanuprakash, 2006). AKR1B1 is a rate-limiting enzyme of polyol pathway in vivo, which is closely related to glucose metabolism. Abnormal glucose metabolism is the main way of energy metabolism reprogramming in cancer. At present, the molecular regulatory mechanism between energy metabolism and tumor is the forefront of research in oncology. Studies have shown that impaired glucose metabolism can lead to tumorigenesis and development (Wulaningsih et al., 2013). In recent years, it has been found that AKR1B1 can promote the migration and cell cycle progression in colorectal cancer cells, and patients with high expression of AKR1B1 are significantly correlated with shorter disease-free survival (Taskoparan et al., 2017). In breast cancer, immunoblotting and IHC data indicated overexpression of AKR1B1 in all grades of tumors compared with their corresponding nontumor samples (Reddy et al., 2016). In basal-like breast cancer, Twist2 transcriptionally induces AKR1B1 expression, which leads to activation of the nuclear factor kappa B (NF-κB) signaling pathway. In turn, NF-κB upregulates the expression of Twist2, thereby fulfilling a positive feedback loop, activating the epithelial–mesenchymal transition program, and enhancing cancer stem cell-like properties in basal-like breast cancer (Wu et al., 2017). In pancreatic cancer, AKR1B1 is colocated with β2-adrenergic receptor (β2-AR), a high-risk factor of pancreatic cancer, and overexpression of AKR1B1 can activate extracellular signal-regulated kinase 1/2 (p-ERK1/2) signal pathway, thus inhibiting apoptosis and promoting proliferation of pancreatic cancer cells (Xiao et al., 2018). AKR1B1 overexpression was found to be associated with shortened patient survival in acute myelogenous leukemias and multiple myelomas (Laffin and Petrash, 2012). It has been found that nuclear factor E2-related factor 2 (NRF2) may inhibit the occurrence of tumor ferroptosis and promote tumor progression through transcriptional regulation of AKR1B1 expression (Matthew et al., 2019). These studies indicate that AKR1B1 may be an oncogene during tumorigenesis. Until now, some scholars have found a kind of AKR1B1 inhibitor, which was an old drug used in clinical treatment of diabetic peripheral neuropathy—epalrestat. This suggests that AKR1B1 may become a new target of tumor therapy and provide a valuable treatment strategy.

Similar to these studies, in our study we found that AKR1B1 expression was significantly upregulated in tumor tissues of patients with GC. AKR1B1 expression was significantly correlated with tumor size, regional lymph node (N), metastases (M), and TNM stage. The overall survival of patients with low expression of AKR1B1 was significantly better than those with high expression. Therefore, AKR1B1 has certain value for the prognosis of GC patients. Since the clinical sample size of this study is not large enough, it is necessary to further expand the clinical sample size in the follow-up and explore the specific molecular mechanism of AKR1B1 in the process of tumorigenesis and development at the cell level and animal model by means of molecular biology.

Conclusion

This study used IHC to detect AKR1B1 expression, and so, it has its own limitations, such as positive criteria, definition of cutoff values, and specificity and reproducibility of antibodies used. Despite all data being preliminary and numerous questions to be further studied, AKR1B1 may be used as a specific therapeutic target for cancer.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the Budget internal medicine research project of Shanghai municipal education commission (2019LK003); Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Dragon Medical Scholars (Nursery Program) of National Clinical Research Base of Traditional Chinese Medicine (LYTD-82), and Research project of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine affiliated to Shanghai University of Traditional Chinese Medicine (2019YYQ25).

Supplementary Material

Supplementary Table S1

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