significance of nqo1 overexpression for prognostic evaluation of gastric adenocarcinoma

7/24/2019 Significance of NQO1 Overexpression for Prognostic Evaluation of Gastric Adenocarcinoma

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Signicance of NQO1 overexpression for prognostic evaluation of

gastric adenocarcinoma

Lijuan Lin a,b,1, Yunzhi Qin c,1, Tiefeng Jin a, Shuangping Liu a, Songnan Zhang d,Xionghu Shen d,, Zhenhua Lin a,a Department of Pathology, Yanbian University Medical College, Yanji 133002, Chinab Department of Medical Imaging, College of Medicine, Eastern Liaoning University, Dandong 118003, Chinac Department of Anesthesiology, Yanbian University Hospital, Yanji 133000, Chinad Department of Oncology, Yanbian University Hospital, Yanji 133000, China

a b s t r a c ta r t i c l e i n f o

Article history:

Received 19 November 2013

Available online 31 December 2013

Keywords:

NQO1

Gastric adenocarcinoma

Immunohistochemistry

Survival analysis

NQO1 (NAD(P)H: quinone oxidoreductase, also known as DT-diaphorase) plays a prominent rolein maintaining

cellular homeostasis. NQO1 is abnormally elevated in many solid cancer types, including those of the adrenal

gland,breast, colon,lung, ovary,and thyroid. However, little is known about the status of NQO1 in gastricadeno-

carcinoma (GAC). To investigate the clinicopathological signicance of NQO1 expression in GAC,and thus evalu-

ate its role as a potential prognostic marker, 203 cases of primary GAC, 31of gastric dysplasia, and 53of adjacent

non-tumor tissues were selected for immunohistochemical staining of NQO1 protein. Correlations between

NQO1 overexpression and clinicopathological characteristics were evaluated by 2 test and Fisher's exact test,

while survival rates were calculated by KaplanMeier method. The relationship between prognostic factors

and patient survival was analyzed by Cox proportional hazards model.

Through these analyses it was found that the strongly positive rate of NQO1 protein in GAC was signi cantly

higher than that in gastric dysplasia and adjacent non-tumor tissues. Analysis by qRT-PCR also conrmed that

NQO1 mRNA levels were increased in GAC compared with those detected in either adjacent non-tumor tissues

or normal gastric mucosa. Additionally, the NQO1 expression rate was positively correlated with tumor size,

serosal invasion, tumor stage, and both disease-free survival and 5-year survival rates. Further analysis showedthat although NQO1 was not an independent predictor of GAC, elevated expression of NQO1 could predict

lower disease-free survival and 5-year survival times in late-stage patients. In conclusion, NQO1plays an impor-

tant role in theprogression of GAC, and might be a potential,but not an independent,poor prognosticbiomarker

and therapeutic target of GAC.

2013 Elsevier Inc. All rights reserved.

Introduction

Gastric cancer is the fourth most common malignancy and the sec-

ond leading cause of cancer deaths worldwide (Geng et al., 2013). The

development and progression of gastric cancer is a multistage process

which involves multiple molecular pathways and abnormal genetic

changes. Despite great advances in surgical and medical management

of the disease, the prognosis of gastric cancer has not signicantly im-

proved. Therefore, identication of reliable criteria for predicting its re-

currence and prognosis attracts widespread research interest.

NAD(P)H: quinone oxidoreductase 1 (NQO1, also known as diphthe-

ria toxin diaphorase (DT-diaphorase)), was discovered by Professor

Ernster in 1958 (Siegel et al., 2000) and is located on chromosome

16q22 (Zhuet al., 2013). NQO1 is a mainly cytosolic enzyme which uses

NADH or NADPH as substrates to directly reduce quinones to hydroqui-

nones (Zhang et al., 2012). It is present in all tissue types with the excep-

tion of the liver (Siegel et al., 2000; Strassburget al., 2002) and is induced

along with a battery of defensive genes that provide protection against

different stresses to prevent organs from carcinogen-induced tumorigen-

esis. Because there is an increased incidence of disease and xenobiotic-

induced toxicity in individuals carrying a polymorphism in NQO1, it has

been suggested that it has a role in chemoprotection.

Paradoxically, in spite of this cell protectorstatus, NQO1 expres-

sion has been found to be increased during malignant transformation

in some tumor types including that of the adrenal gland, breast, colon,

lung, ovary, and thyroid (Garate et al., 2010; Lewis et al., 2005; Lyn-

Cook et al., 2006), and has also been detected following the induction

of cell cycle progression and proliferation of melanoma cells (Garate et

al., 2010). To date, the role of NQO1 in cancer progression remains

Experimental and Molecular Pathology 96 (2014) 200205

Correspondence to: Z. Lin, Department of Pathology, Yanbian University Medical

College, No. 977, Gongyuan-Rd., Yanji 133002, China. Fax: +86 433 2435104.

Correspondence to: X. Shen, Department of Oncology, Yanbian University Hospital,

No. 1829, Juzi-St., Yanji 133000, China.

E-mail addresses:[email protected](L. Lin),[email protected](Y. Qin),

[email protected](T. Jin),[email protected](S. Liu),[email protected](S. Zhang),

[email protected](X. Shen),[email protected](Z. Lin).1 These authors contributed equally to this work.

0014-4800/$ see front matter 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.yexmp.2013.12.008

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Experimental and Molecular Pathology

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controversial. Notably, its role in GAC progression has not yet been re-

ported. This study aimed to determine theNQO1 protein expression sta-

tus in GAC, dysplasia, and adjacent non-tumor tissues, and thereby

determineits potential as a prognostic biomarkerand therapeutic target

in this disease.

Materials and methods

Clinical samples

Two hundred and three GAC cases were selected randomly from the

Department of Pathology Tumor Tissue Bank, Yanbian University Med-

ical College. These specimens were collected from patients undergoing

surgical treatment between 2004 and 2008. The cohort included 135males and 68 females, ranging from 29 to 72 years old, with a mean

age of 49.7. All cases were conrmed as GAC by pathological examina-

tion. Tumor stage was determined according to the 7th edition of the

American Joint Committee on Cancer (AJCC). Of the 203 samples, 101

cases were stages IIIa while 102 cases were stages IIbIIIc. Tumor

stage was closely correlated with prognosis. In addition, 80 cases were

dened as well-differentiated while 123 cases were poor to mildly dif-

ferentiated. Fifty-three cases of normal gastric mucosa tissues obtained

from the periphery of malignant GAC tissue and 31 cases of gastric

dysplasia were also included in the study. None of the patients received

radio-chemotherapy before surgery. The 203 patients with GAC had

been followed for ve years or until death. At the end of the follow-

up, 105 patients remained alive.

Fresh samples were also collected and included 12 cases of GAC, 8cases of adjacent non-tumor tissue and 8 cases of normal gastric muco-

sa. These were used for RNA extraction and qRT-PCR analysis of NQO1

mRNA.

Immunohistochemistry

To eliminate endogenous peroxidase activity, 4 m thick tissue sec-

tions were deparafnized, rehydrated and incubated with 3% H2O2in

methanol for 15 min at room temperature (RT). The antigen was re-

trieved by placing the slides in 0.01 M sodium citrate buffer (pH 6.0)

at 95 C for 20 min. Theslides were then incubatedwith NQO1 antibody

(1:50, sc-32793, Santa Cruz Biotechnology, Inc. USA) at 4 C overnight.

After incubation with biotinylated secondary antibody at RT for

30 min, the slides were incubated with streptavidinperoxidase com-

plex at RT for 30 min. Immunostaining was developed using 3,3-diami-

nobenzidine, and Mayer's hematoxylin was used for counterstaining.

We used tonsil sections as the positive control and mouse IgG as an

isotope control. Some positive tissue sections were also processed withomission of the primary antibody (mouse anti-NQO1) as an additional

negative control.

All specimens were blind examined by two pathologists. In case

of discrepancies, a nal score was established by reassessment on a

double-headed microscope. The immunostaining for NQO1 was semi-

quantitatively scored as (negative) no or less than 5% positive

cells; +525% positive cells; ++2650% positive cells; and +++

more than 50% positive cells. Only cytoplasmic staining was considered

positive. For statistical analysis, the strongly positive group represents

the combined scores of++and +++positive cells.

RNA extraction and quantitative real-time polymerase chain reaction

(qRT-PCR)

Total RNA of fresh tissues was extracted using Trizol reagent

(Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized using

PrimeScript reverse transcriptase (TaKaRa Bio, Dalian, China) and oligo

Table 1

NQO1 protein expression in gastric lesions.

Diagnosis No. of cases NQO1 protein expression Positive rate Strongly positive rate

+ ++ +++

Gastric adenocarcinoma 203 49 29 54 71 75.86% 61.58%

Gastric dysplasia 31 14 5 6 6 54.84% 38.71%

Adjacent non-tumor tissues 53 32 7 9 5 39.62% 26.42%

Statistical analyses were performed using Pearson Chi-square test. Gastric adenocarcinoma compared with Gastric dysplasia, P b 0.05; Gastric adenocarcinoma compared with Adjacent

non-tumor tissues, Pb

0.01.

Fig. 1. Immunohistochemical staining of NQO1 in gastric lesions. A: Gastric mucosa. B: Atypical cells of gastricdysplasia. C: Gastricadenocarcinoma (GAC) with lymph node metastasis.D:

GAC without lymph node metastasis. E: Invasive cancer loci. F: Metastatic cancer cells in blood vessels (arrows). Magnication is 100 in A and 200 in BF.

201L. Lin et al. / Experimental and Molecular Pathology 96 (2014) 200205


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(dT) following the manufacturer's instructions. To examine expression,

real-time PCR was performed with a Bio-Rad sequence detection system

using double-stranded DNA-specic SYBR Premix Ex TaqTM II Kit

(TaKaRa Bio) according to the manufacturer's instructions. Double-

stranded DNAspecic expression was testedby the comparative Ct meth-

od using 2-Ct. NQO1 primers were as follows: 5-GGCAGAAGAGCACT

GATCGTA-3, and 5-TGATGGGATTGAAGTTCATGGC-3. GAPDH was used

for an internal control: 5-GGTCTCCTCTGACTTCAACA-3and 5-ATACCA

GGAAATGAGCTTGA-3

. All assays were performed in triplicate at leastthree times.

Statistical analysis

Analyses were performed using SPSS 17.0. Correlations between

NQO1 expression and clinicopathological characteristics were evalu-

ated by the Chi-square (2) and Fisher's exact tests. The survival

rates after tumor removal were calculated by the KaplanMeier

method, and differences in survival curves were analyzed by the

Log-rank test. Univariate and multivariate survival analysis were

performed on all the characteristics measured by the Cox propor-

tional hazard regression model. P b0.05 was considered statistically

signicant.

Results

Expression of NQO1 in GAC

The positive and strongly positive expression rates of NQO1 protein

were 75.86% (154/203) and 61.58% (125/203) in GAC, respectively,

which was signicantly higher than those in either gastric dysplasia tis-

sues (54.84% and 38.71%, both P b0.05) or adjacent non-tumor tissues

39.62% and 26.42%, both P b0.01. However, no signicant difference

between gastric dysplasia and adjacent non-tumor tissues was found

(P N0.05) (Fig. 1andTable 1).

Analysis by qRT-PCR also conrmed that NQO1 expressionincreased

at the mRNA level in GAC when compared with that in either adjacent

non-tumor tissue or normal gastric mucosa (Fig. 2).

Clinicopathologic and prognostic signicance of NQO1 expression in GAC

To elucidate the role of NQO1 in GAC progression, we analyzed the

correlation between NQO1 expression and clinicopathological features

of GAC. As shown inTable 2, elevated NQO1 expression was closely

associated with tumor size (P = 0.014), serosal invasion (P = 0.018),

and clinical stage (P = 0.015), but did not correlate with age, gender,

differentiation, or lymph node metastasis (all P N0.05). Moreover,

GAC patients with elevated NQO1 expression had both a shorter

disease-free survival rate (Log-rank = 11.960, P = 0.001) and

ve-year survival rate (Log-rank = 12.571, P b0.001) than those

with low NQO1 expression, as determined by KaplanMeier analysis

(Fig. 3).

Cox proportional hazard regression model analysis for independent

prognostic factors in GAC

Univariate analysis was performed for all of the variables by Coxanalysis. It was found that GAC patients with elevated NQO1 expression

had a signicantly lower overall survival rate than those withlow NQO1

expressing tumors (HR: 1.391, 95% CI: 1.0481.848, P = 0.022). Tumor

size, differentiation, lymph node metastasis, serosal invasion, and stage,

were also associated with reduced overall survival rate. However,

further multivariate analysis showed that only tumor stage proved to

be an independent prognostic factor for survival in GAC (HR: 1.807,

95% CI: 1.3532.413, P = 0.000). NQO1 expression, in contrast, did

not emerge as a signicant independent prognostic factor in GAC (HR:

1.196, 95% CI: 0.8901.607, P = 0.236) (Table 3).

NQO1 combined with tumor stage predicts prognosis in GAC

Further survival analysis showed that patients with late-stageGAC concomitant with elevated NQO1 expression had both poorer

disease-free survival and ve-year survival rates than those with

low NQO1 expression (Fig. 4CD), indicating that NQO1 might be

a useful prognostic marker for late-stage GAC. Of note, although

there was no statistical connection between patients with high-

and low expression of NQO1 in early-stage GACs, there was still

a tendency for survival in patients with high NQO1 expression to

be comparatively shorter than those with NQO1 low-expression

(Fig. 4AB).

Discussion

NQO1 provides protection for cells against free radical damage, oxi-

dative stress, and accumulation of toxic substances (Garate et al., 2010;

Fig. 2.qRT-PCR analysis of NQO1 mRNA. Normal gastric mucosa (normal), adjacent non-

tumortissues (non-tumor) andGAC tissueswere collectedand subjectedto qRT-PCR anal-

ysis of NQO1 mRNA levels. Data represents the mean of individual samples tested in trip-

licate relative to that of the normal control SD. **Pb

0.01.

Table 2

Univariate analysis of NQO1 expression and various risk factors in 203 GAC patients.

Variables NQO1 expression (%) HR (95% CI) P value

/+ ++/+++

Age 1.116 (0.6261.988) 0.710

b50 32 (40.00%) 48 (60.00%)

50 46 (37.40%) 77 (62.60%)

Gender 1.222 (0.6672.238) 0.516

Male 54 (40.00%) 81 (60.00%)

Female 24 (35.29%) 44 (64.71%)

Tumor size 2.046 (1.1413.668) 0.016

5 cm 51 (45.95) 60 (54.05%)

N5 cm 27 (29.35%) 65 (70.65%)

Differentiation 1.447 (0.8132.576) 0.209

Well 35 (43.75%) 45 (56.25%)

Moderate and Poor 43 (34.96%) 80 (65.04%)

Clinical stage 1.993 (1.1213.543) 0.018

IIIa 47 (46.53%) 54 (53.47%)

IIbIIIc 31 (30.39%) 71 (69.61%)

LN metastasis 0.715 (0.4021.272) 0.254

Positive 43 (35.25%) 79 (64.75%)

Negative 35 ( 43.21%) 46 ( 56.79%)

Serosal invasion 0.484 (0.2700.869) 0.015

Yes 41 (32.03%) 87 (67.97%)

No 37 (49.33%) 38 (50.67%)

P

b

0.05.P b 0.01.

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Zhu et al., 2013) by catalyzing two-electron reduction of quinones and

nitrogen oxides (Riley et al., 1992) and also by the direct conversion of

quinones to hydroquinones. Varying NQO1 genotypes encode different

enzymes which may affect enzyme activities. Indeed, the homozy-

gous TT genotype lacks NQO1 activity and thus might result in re-

duced detoxication of exogenous carcinogens. Therefore, many

studies have tested the hypothesis that the C609T polymorphism

might be linked to the risk of cancers, such as cervical, lung, bladder

and colorectal. Niwa and colleagues (Niwa et al., 2005) studied the

effect of NQO1 genotypes on cervical carcinogenesis in relation to

smoking behaviors and showed that the TT genotype was a risk

factor only for cervical squamous cell carcinoma while the C allele

was over-expressed in invasive cervical cancer and cervical

intraepithelial neoplasia. Fagerholm's study on breast cancer pa-

tients showed that NQO1 may predict the outcome of patients withoverabundant FANCD2 protein through its potential modication

of prognostic DNA repair markers (Fagerholm et al., 2013). More-

over,Li et al. (2011)highlights that NQO1 is more than a biomarker,

but also an exploitable, tumor-selective target, whose expression is not

cell-cycle regulated, nor affected by alterations in common tumor

suppressors, such as p53 or Rb. Beyond these reports, the function of

NQO1 in malignancies remains controversial.

Recently, it was reported that NQO1 protein and mRNA expres-

sion levels are abnormally elevated within many solid tumors.

Awadallah et al. (2008)reported that NQO1 is upregulated in pan-

creatic ductal adenocarcinoma (PDAC), and that combining NQO1

expression with cellular morphology assessment minimizes the

risk of false positivediagnosis. Lyn-Cook et al. (2006) also considered

that NQO1 may represent a useful biomarker for pancreatic cancer,

while Liang's research (Liang et al., 2013) showed that expression

of NQO1 tends to be higher (3060%) in lung cancers compared

with normal lung tissue, but is not signicantly different from that

in peri-cancer tissues. In the present study, we found that NQO1 pro-

tein expression was frequently upregulated in GAC compared with

that in either gastric dysplasia or adjacent non-tumor tissues. Thestrongly positive rate of NQO1 protein was 61.58% (125/203) in

GAC cases, which was signicantly greater than that in either gastric

dysplasia or adjacent non-tumor gastric mucosa. Analysis by qRT-

PCR also revealed increased NQO1 at the mRNA level in GAC com-

pared with that in adjacent non-tumor tissues and normal gastric

mucosa. These results indicate that NQO1 is involved in the progres-

sion of GAC, and that NQO1 protein level might be a diagnostic indi-

cator of this disease.

Furthermore, accumulating studies have demonstrated that el-

evated NQO1 expression is associated with the poor prognosis of

malignancies.Mikami et al. (1998)demonstrated that the expres-

sion and enzyme activity of NQO1 is not only upregulated in

colon cancer cell lines and colorectal tumors, but is also signicant-

ly greater in tumors with nodal metastases than those without.Awadallah et al. (2008)also suggests that NQO1 overexpression

is a clinically useful prognostic adjunct for detection of PDAC, inde-

pendent of tumor stage. Here we found that NQO1 overexpression

was closely related with increased tumor size, serosal invasion, and

late-stage tumors, raising the possibility that NQO1 expression

participates in the tumorigenesis and malignant progression of

GAC. Consequently, NQO1 might be useful as a p oor prognostic bio-

marker of GAC.

In regard to survival, we found that GAC patients with high NQO1

expression had both lower disease-free survival (P = 0.001) and over-

all survival rates (P b0.001) than those with low NQO1 expression.

Univariate survival analysis indicated that tumor size, differentiation,

clinical stage, lymph node metastasis, serosal invasion and NQO1

expression are all signicantly related with disease-free and overall

Fig. 3. KaplanMeier analysis of GAC patient survival rates in relation to NQO1 protein expression. Disease-free survival (A) and overall survival rates (ve-year survival) (B) of patients

with elevated (green, n = 125) and low (blue, n = 78) NQO1 expression.

Table 3

Univariateand multivariate survival analyses (Cox regression model) of various factors in

203 GAC patients.

Factors B SE Wald HR (95% CI) P value

Univariate

Age 0.052 0.144 0.131 1.054 (0.7941.398) 0.717

Sex 0.057 0.147 0.148 1.058 (0.794

1.411) 0.700Tumor size 0.339 0.141 5.770 1.404 (1.0641.851) 0.016

Differentiation 0.349 0.144 5.846 1.417 (1.0681.880) 0.016

Stage 0.623 0.142 19.358 1.864 (1.4132.460) 0.000

LN 0.307 0.143 4.577 1.359 (1.0261.800) 0.032

SI 0.308 0.145 4.457 1.361 (1.0221.811) 0.035

NQO1 0.331 0.216 5.215 1.392 (1.0481.848) 0.022

Multivariate

Tumor size 0.149 0.147 1.033 1.161 (0.8711.549) 0.309

Differentiation 0.270 0.149 3.294 1.310 (0.9791.753) 0.070

Stage 0.591 0.148 16.035 1.807 (1.3532.413) 0.000

LN 0.239 0.150 2.553 1.270 (0.9471.704) 0.110

SI 0.288 0.151 3.643 1.334 (0.9921.792) 0.056

NQO1 0.179 0.151 1.406 1.196 (0.8901.607) 0.236

SI: serosal invasion, LN: lymph node metastasis. P b 0.05.

P

b

0.01.

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survival rates of GAC patients (P b0.05). However, through multivari-

ate survival analysis, only clinical stage emerged as a signicant inde-

pendent hazard factor for overall survival in GAC. Notably, late-stage

GAC concomitant with high NQO1 expression was correlated with

shorter disease-free survival and ve-year survival times than those

with low NQO1 expression (P = 0.039 and P = 0.035, respectively).

A similar tendency was observed in patients with early-stage GAC.

Therefore, although NQO1 expression in GAC was not found to be an in-

dependent risk factor for patients' survival in the present study, it was

revealed that NQO1 serves as a useful biomarker for prognosis of GAC,

especially for late-stage disease.

In conclusion, NQO1 plays an important role in the tumorigenesisand progression of GAC, and is a potential effective predictor for its

poor prognosis, especially in late stage disease. Consequently, determi-

nation of NQO1 expression in GAC may aid the selection of appropriate

therapies.

Conict of interest statement

The authors declare that there are no conicts of interest.

Author's contributions

LL, QY, and JT participated in the study conception, design, case

selection and experiments. ZS, PL and SX carried out the data

collection. LL, LS, and LZ performed the data analysis and writing

of the manuscript. All the authors read and approved the nal

manuscript.

Acknowledgments

This study was supported by grants from the National Natural

Science Foundation of China (61371067 & 31301065), and the Pro-

jec ts of Rese arc h & Inno vation of Jil in Youth Leader and Team

(20130521017JH).

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Fagerholm, R.,et al.,2013. Overabundant FANCD2, aloneand combined with NQO1,is a sensitive marker of adverse prognosis in breast cancer. Ann. Oncol. 24,27802785.

Garate, M., et al., 2010.The NAD(P)H:quinone oxidoreductase 1 induces cell cycleprogression and proliferation of melanoma cells. Free Radic. Biol. Med. 48,16011609.

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Lewis, A.M., et al., 2005.Targeting NAD(P)H:quinone oxidoreductase (NQO1) in pancre-atic cancer. Mol. Carcinog. 43, 215224.

Li, L.S., et al., 2011.Modulating endogenous NQO1 levels identies key regulatory mech-anisms of action of-lapachone for pancreatic cancer therapy. Clin. Cancer Res. 17,

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285.

Fig. 4. KaplanMeier analysis of survivalratesin patients with highor lowNQO1 with early-or late-stage GAC. Disease-free survival (A) andoverall survival rates (ve-year survival) (B)

were assessed in patients with early-stage GAC concomitant with either high (green line, n = 54) or low (blue, n = 47) NQO1 expression. Disease-free survival (C) and overall survival

rates (D) were also assessed in patients with late-stage GAC concomitant with high (green, n = 71) or low (blue, n = 31) NQO1 expression.

204 L. Lin et al. / Experimental and Molecular Pathology 96 (2014) 200205
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significance of nqo1 overexpression for prognostic evaluation of gastric adenocarcinoma

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