Med Oncol (2013) 30:454 DOI 10.1007/s12032-012-0454-y
ORIGINAL PAPER
Reduced group IVA phospholipase A2 expression is associated with unfavorable outcome for patients with gastric cancer Xia Zhang • Qiong Wu • Lu Gan • Guan-Zhen Yu Rui Wang • Zi-Shu Wang • Jie-Jun Wang • Xi Wang
•
Received: 19 December 2012 / Accepted: 29 December 2012 Ó Springer Science+Business Media New York 2013
Abstract Arachidonic acid metabolic pathway has been implicated in the inflammation-associated tumorigenesis of gastrointestinal cancers. As the rate-limiting enzyme of arachidonic acid production, group IVA phospholipase A2 (PLA2G4A) is hypothesized to play a fundamental role in gastric tumorigenesis as well as cyclooxygenase-2 (COX-2). However, little is known about the expression and role of PLA2G4A in gastric cancer, and the association of PLA2G4A with COX-2 remains to be elucidated. In this study, the mRNA expression of PLA2G4A and COX-2 in 60 pairs of fresh gastric tumors and corresponding adjacent non-cancerous mucosa was detected by using real-time quantitative PCR and the immunostaining of the both proteins in paired samples from 866 gastric cancer patients were assessed by using immunohistochemistry method. The clinicopathological and the prognostic relevance of PLA2G4A and COX-2 expression were determined. The results revealed a significantly reduced expression of PLA2G4A in gastric tumors compared to in non-cancerous tissues, as opposite to the increased expression of COX-2. PLA2G4A was significantly associated with tumor size (P = 0.003), tumor grade (P \ 0.001), intestinal type
Xia Zhang and Qiong Wu contributed equally to this work. X. Zhang L. Gan G.-Z. Yu J.-J. Wang (&) X. Wang (&) Department of Oncology, Changzheng Hospital, Second Military Medical University, 64 Hetian Road, Shanghai 200070, China e-mail:
[email protected] X. Wang e-mail:
[email protected] Q. Wu R. Wang Z.-S. Wang Department of Medical Oncology, First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, China
(P = 0.003), T classification (P \ 0.001), N classification (P \ 0.001), and thereby TNM stage (P \ 0.001). PLA2 G4A and COX-2 expression were both identified as independent prognostic factors in multivariate Cox model analysis (P = 0.024 for PLA2G4A and P \ 0.001 for COX-2). Moreover, the reduced PLA2G4A and increased COX-2 expression was both associated with unfavorable survival for patients with gastric cancer. PLA2G4A might serve as a promising target for future therapeutic approaches to gastric cancer combined with COX-2 inhibitors. Keywords Gastric cancer Group IVA phospholipase A2 Cyclooxygenase-2 Prognosis
Introduction Chronic inflammation has been implicated in a multitude of human malignancies [1]. Among multifaceted links between chronic inflammation and cancer, cyclooxygenase-2 (COX-2)/ prostaglandin E2 (PGE2) pathway has been the one to attract the most attention [2]. PGE2 is a major prostaglandin derived from arachidonic acid, and it can promote the carcinogenesis of gastrointestinal cancers by stimulating angiogenesis and inhibiting apoptosis [3]. COX-2 is the key enzyme catalyzing the biosynthesis of PGE2. The aberrant overexpression of COX-2 has been observed in gastrointestinal cancers and recognized as a promising target for prevention and treatment [3–5]. Recently, accumulating data suggest that a wide spectrum of eicosanoids such as thromboxanes, leukotrienes, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs), all produced from arachidonic acid, may also be involved in human tumorigenesis [6]. These bioactive lipid metabolites may act as crucial
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mediators as important as prostaglandins between chronic inflammation and cancer. Thus, the key molecules in the upstream of eicosanoid cascade, for example, phospholipase A2 (PLA2) which is the rate-limiting enzyme of arachidonic acid mobilization, has been proposed as a novel therapeutic target for gastrointestinal cancers as well as COX-2 [7]. Phospholipase A2 represents a superfamily of enzymes that can catalyze the hydrolysis of the fatty acyl ester bond at the sn-2 position of phospholipids to produce free fatty acids and lysophospholipids [8]. In particular, cytosolic PLA2 (cPLA2) has a high selectivity for liberating arachidonic acid which can be metabolized by a panel of downstream enzymes for eicosanoid production mentioned above [9]. In view of the elevated COX-2 expression and prostaglandins yield in inflammation-associated gastrointestinal carcinomas [10, 11], group IVA PLA2 (PLA2G4A), the most abundant subtype of cPLA2, is hypothesized to be synchronously overexpressed and participated in tumorigenesis through producing sufficient substrate for the metabolic cascade of COX-2/PGE2 and other pathways. However, little is known about the expression and role of PLA2G4A in gastric cancer, and the association of PLA2G4A with COX-2 remains to be elucidated. Therefore, the present study was designed to examine the expression pattern of PLA2G4A and COX-2 in a large Chinese cohort of gastric cancer, and their clinicopathological and prognostic relevance were determined.
Materials and methods Patients and tissue samples A total of 1,072 gastric cancer patients underwent gastrectomy from 2000 to 2005 in Changhai Hospital, Second Military Medical University, Shanghai, China. Among these patients, those with insufficient tissue samples or incomplete information, or those diagnosed as having double primary tumors or remnant gastric cancer, or those died within 2 months of surgery, were excluded, and thus, 866 eligible patients were included. The paired cancerous and non-cancerous tissues from these patients were routinely fixed in 10 % buffered formalin and blocked in paraffin, ready to tissue microarray construction. Additionally, 60 pairs of fresh gastric tumors and corresponding adjacent non-cancerous mucosa were obtained, stabilized in RNAlaterÒ solution (Ambion, Austin, TX, USA) at 4 °C overnight and preserved at -20 °C until RNA extraction. All patients had not received any anticancer therapy before surgery, and they were followed up every 6 months until death or study end (March 30 2010), except for those lost to follow up. The tissue samples were obtained with patient informed consent, and the protocol was approved by
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Institutional Review Board of Second Military Medical University. RNA preparation and reverse transcription Total RNA was extracted from the RNAlaterÒ-stabilized samples by using an RNAqueousÒ-4PCR kit (Cat# AM1914, Ambion) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized with use of a High Capacity cDNA Reverse Transcription Kit (PN4374 966, Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. The reaction was incubated in an ABI 2720 Thermocycler (Applied Biosystems) for 10 min at 25 °C, 120 min at 37 °C, and 5 min at 85 °C. cDNA samples were stored at -20 °C before real-time PCR amplification. Real-time quantitative PCR Real-time quantitative PCR was performed with an ABI PRISMÒ 7900HT Sequence Detection System (Applied Biosystems) using a Power SYBRÒ Green PCR Master Mix kit (PN4367659, Applied Biosystems) as described by the manufacturer. The primers were synthesized by Sangon Biotech Co., Ltd., Shanghai, China, and the primer sequences were as follows: PLA2G4A 50 -AGCACC AGTATTCCCACAAGTT-30 (forward) and 50 -TCAAAGG TCTCATTCCACACAG-30 (reverse), COX-2 50 -CTCAGC CATACAGCAAATCCTT-30 (forward) and 50 -TACTGGT CAAATCCCACACTCA-30 (reverse), and b-actin 50 -TGT TACAGGAAGTCCCTTGC-30 (forward) and 50 -AAGCA ATGCTATCACCTCCC-30 (reverse). The amplification was run at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All samples were run in triplicate, and the data were analyzed by use of the Sequence Detection System (SDS) Software version 2.3 (Applied Biosystems). The specificity of amplification reaction was confirmed by analyzing the corresponding dissociation curves. The expression of mRNA for PLA2G4A and COX-2 was normalized to b-actin by using the 2-DDCt method. Tissue microarray construction and immunohistochemistry Tissue microarrays were assembled from the paraffinembedded tissue blocks using a tissue arrayer (Beecher Instruments, Silver Spring, MD) as described in our previous report [12]. For each of 866 gastric cancer patients, duplicate cancerous cylinders and at least one matched non-cancerous cylinder with a diameter of 1.5 mm were arrayed and consecutive 4-lm sections were cut. Immunohistochemistry assay for PLA2G4A and COX-2 was performed using an UltraSensitiveTM SP kit (#9710,
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Maixin, Fuzhou, China) according to the manufacturer’s instructions. Briefly, the microarray sections were deparaffinized in xylene, rehydrated with graded ethanol, and subjected to antigen retrieval in citrate buffer (pH 6.0) in a high-pressure cooker. The sections were subsequently blocked for endogenous peroxide activity with 3 % hydrogen peroxide, treated with preimmune goat serum to block nonspecific binding sites, and then incubated with the primary mouse polyclonal antibody against human PLA2G4A (H00005321-A01, Abnova; 1:100) and the primary rabbit polyclonal antibody against human COX-2 (#4842, Cell Signaling Technology; 1:150), respectively, at 4 °C overnight. The sections were washed and incubated with a secondary biotinylated anti-mouse/rabbit antibody. The immunostaining was visualized with a diaminobenzidine detection kit (DAB-0031, Maixin), and then, the sections were counterstained with hematoxylin, dehydrated, cleared, and coverslipped. Evaluation of immunostaining
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cancerous and non-cancerous tissues obtained from 60 gastric cancer patients. As shown in Fig. 1, no difference in PLA2G4A mRNA levels was found between cancerous and non-cancerous tissues (P = 0.147). However, the mRNA expression of COX-2 in gastric tumors was significantly increased compared to that in the matched noncancerous mucosa (P \ 0.001). Immunohistochemistry assay in 866 patients demonstrated that the immunostaining of PLA2G4A and COX-2 was both localized in the cytoplasm of non-cancerous epithelium and gastric cancer cells (Fig. 2). After exclusion of inevaluable cases due to tissue loss or inadequate tissue, the positive rates of PLA2G4A and COX-2 expression in non-cancerous mucosa were 84.0 % (694/826) and 25.9 % (212/819), and the positive rates of the two proteins in gastric tumors were 49.3 % (417/845) and 54.0 % (448/ 830), respectively. The immunostaining of the PLA2G4A in gastric tumors was significantly reduced than that in non-cancerous tissues (P \ 0.001), whereas COX-2 in the opposite direction (P \ 0.001).
Brown cytoplasmic staining in the gastric cancer cells or non-cancerous epitheliums was considered positive. The signal was scored on a scale representing the estimated intensity combined with proportion of positive staining cells, as previously described [13]. A score C3 was designated as positive staining, and a score of 0 or 2 was regarded as negative. The stained sections were viewed by two individuals independently using an Olympus CX31 microscope (Olympus, Japan). Statistical analysis The difference in mRNA expression was examined by Wilcoxon matched-pairs signed-rank test, and the difference in protein expression was checked by Chi square test. Correlations were computed using the Spearman rank test. The probability of survival was estimated by Kaplan– Meier method and compared by log-rank test. The prognostic relevance of PLA2G4A and COX-2 was evaluated by using univariate and multivariate Cox model. Statistical analyses were performed by the SPSS 15.0 (SPSS, Chicago, IL, USA), and a two-sided P value \0.05 was considered significant.
Results Expression of PLA2G4A and COX-2 in gastric cancer Real-time quantitative PCR showed that the mRNAs of both PLA2G4A and COX-2 were detected in all paired
Fig. 1 Scatter plots of PLA2G4A (a) and COX-2 (b) mRNA levels in gastric tumors and corresponding non-cancerous mucosa (n = 60). The line indicates the median value
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Fig. 2 Representative immunostaining of PLA2G4A and COX-2 in gastric cancerous and non-cancerous tissues. Positive staining of PLA2G4A and COX-2 in non-cancerous tissues (a, e and i, m) and well (b, f and j, n) and moderate grade (c, g and k, o) cancerous
tissues, and negative staining of PLA2G4A and COX-2 (d, h and l, p) in poor grade cancerous tissues, are shown, respectively. Original magnification, 9100 for (a–d) and (i–l); 9400 for (e–h) and (m–p)
Correlation between PLA2G4A and COX-2 expression in gastric cancer
grade (P \ 0.001), Lauren type (P = 0.003), T classification (P \ 0.001), N classification (P \ 0.001), and consequently TNM stage (P \ 0.001). Positive immunostaining of PLA2G4A was more frequently observed in patients with smaller, well-differentiated, intestinal type, and earlier tumors, and positive immunostaining of COX-2 was more frequently observed in patients with well-differentiated (P \ 0.001) and intestinal type tumors (P = 0.008).
Nonparametric Spearman analysis was performed to examine the correlation between PLA2G4A and COX-2 expression in gastric tumors. No significant correlation was found on mRNA level (r = 0.021, P = 0.875) while a significant correlation was observed on protein level (r = 0.097, P = 0.005). Associations of PLA2G4A and COX-2 expression with clinicopathological features of gastric cancer Associations of PLA2G4A and COX-2 expression with clinicopathological features for 866 gastric cancer patients are presented in Table 1. The PLA2G4A expression was significantly related to tumor size (P = 0.003), tumor
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Prognostic role of PLA2G4A and COX-2 expression in gastric cancer Table 2 presents the findings from univariate Cox analysis for 866 gastric cancer patients. Age, tumor site, tumor size, tumor grade, Lauren type, T classification, N classification, radical resection, TNM stage, PLA2G4A expression, and COX-2 expression were identified as significant prognostic
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Table 1 Associations of PLA2G4A and COX-2 expression with clinicopathological characteristics of gastric cancer Variable
Total patients (n = 866)
Evaluable patientsa PLA2G4A (n = 845) Positive no. (%)
Sex
COX-2 (n = 830) Pb 0.863
Female
261 (30.1)
126 (49.8)
Male Age, year
605 (69.9)
291 (49.2)
0.295 119 (51.1) 329 (55.1)
0.276
0.353
Median, range
60, 20–86
62, 20–83
61, 20–83
\60
412 (47.6)
189 (47.4)
206 (52.3)
C60
454 (52.4)
228 (51.1)
242 (55.5)
Upper
138 (15.9)
74 (54.0)
75 (54.7)
Middle
263 (30.4)
123 (48.2)
138 (55.0)
Lower
416 (48.0)
197 (48.6)
210 (52.9)
Diffuse
49 (5.7)
23 (47.9)
Tumor site
Pb
Positive no. (%)
c
0.696
Tumor size, cm
0.947
25 (55.6) 0.003
0.889
Median, range
4.25, 1–17
4, 1–17
4.5, 1–17
B2 cm
133 (15.4)
81 (64.3)
66 (54.1)
B3 cm
160 (18.5)
71 (46.1)
79 (51.6)
B5 cm
273 (31.5)
132 (49.1)
142 (53.6)
300 (34.6)
133 (44.9)
161 (55.5)
48 (5.5)
32 (66.7)
[5 cm Tumor grade Well
\0.001
\0.001 34 (72.3)
Moderate
286 (33.0)
169 (59.5)
170 (61.2)
Poor
532 (61.4)
216 (42.1)
244 (48.3)
Lauren type
0.003
0.008
Intestinal
535 (61.8)
283 (53.7)
301 (58.1)
Diffuse
297 (34.3)
117 (41.2)
131 (46.8)
Mixed
34 (3.9)
17 (50.0)
T classificationd
16 (50.0) \0.001
0.639
pT1
132 (15.2)
82 (63.6)
pT2
142 (16.4)
82 (61.2)
74 (56.1)
pT3
528 (61.0)
226 (43.5)
279 (54.8)
pT4
64 (7.4)
27 (42.9)
N classificationd
65 (51.6)
30 (47.6) \0.001
0.538
pN0
309 (35.7)
175 (58.9)
162 (54.7)
pN1 pN2
314 (36.3) 191 (22.1)
148 (48.2) 78 (41.1)
169 (56.1) 91 (49.5)
pN3
52 (6.0)
16 (31.4)
TNM staged
26 (53.1) \0.001
0.828
I
187 (21.6)
118 (65.6)
96 (54.2)
II
158 (18.2)
78 (52.0)
84 (56.4)
III
294 (33.9)
137 (46.9)
148 (51.9)
IV
227 (26.2)
84 (37.7)
120 (54.8)
PLA2G4A group IVA phospholipase A2, COX-2 cyclooxygenase-2 a
Total patients except for inevaluable cases due to tissue loss or inadequate tissue in immunohistochemistry assay
b
Chi square test
c
Japanese Classification of Gastric Carcinoma (3rd English edition) proposed by the Japanese Gastric Cancer Association (JGCA) The 6th TNM Classification of Malignant Tumors proposed by the AJCC/UICC
d
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Table 2 Univariate and multivariate analysis of overall survival by Cox model Variable
Univariate Cox HR
Multivariate Cox 95 % CI
P
0.906–1.374
0.302
1.323–1.968
\0.001
HR
95 % CI
P
1.217–1.846
\0.001
Sex Male
1
Female
1.116
Age (years) \60
1
C60
1.613
Tumor sitea Upper
1
1
1.499 1
Middle
0.746
0.559–0.996
0.047
0.820
0.604–1.114
0.204
Lower
0.703
0.537–0.919
0.010
0.844
0.637–1.118
0.238
Diffuse
1.765
1.191–2.615
0.005
1.398
0.914–2.139
0.122
0.448
Tumor size (cm) B2
1
1
B3
2.276
1.441–3.595
\0.001
1.250
0.755–2.071
B5
3.113
2.044–4.742
\0.001
1.141
0.703–1.852
0.593
[5
4.867
3.228–7.340
\0.001
1.244
0.767–2.017
0.377
Tumor grade Well
1
Moderate
1.673
0.943–2.970
0.079
1.063
1 0.592–1.909
0.837
Poor
2.527
1.449–4.407
0.001
1.305
0.724–2.350
0.376
Intestinal Diffuse
1 1.240
1.012–1.519
0.038
1 1.153
0.898–1.481
0.264
Mixed
1.329
0.833–2.121
0.232
1.468
0.898–2.398
0.126
Lauren type
T classification
b
pT1
1
pT2
2.895
1.575–5.321
0.001
1.901
1.014–3.563
0.045
pT3
8.267
4.837–14.130
\0.001
3.194
1.786–5.710
\0.001
10.192
5.591–18.580
\0.001
3.797
1.995–7.225
\0.001
pT4 N classification
1
b
pN0
1
pN1
3.002
2.243–4.019
\0.001
2.224
1 1.631–3.031
\0.001
pN2
6.825
5.075–9.179
\0.001
4.124
2.972–5.722
\0.001
pN3
10.236
6.941–15.096
\0.001
5.136
3.324–7.935
\0.001
2.706–4.114
\0.001
2.129
1.692–2.680
\0.001
1.032–1.555
0.024
Radical resection Yes
1
No
3.337
TNM stageb,c I
1
1
II
2.840
1.671–4.828
\0.001
III
8.143
5.123–12.945
\0.001
IV
14.793
9.292–23.549
\0.001
1.233–1.826
\0.001
PLA2G4A expression Positive
1
Negative
1.500
123
1 1.267
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Table 2 continued Variable
Univariate Cox HR
Multivariate Cox 95 % CI
P
HR
95 % CI
P
1.337–2.027
\0.001
COX-2 expression Negative
1
Positive
1.318
1 1.078–1.610
0.006
1.646
PLA2G4A group IVA phospholipase A2, COX-2 cyclooxygenase-2, HR hazard ratio, CI confidence interval a b c
Japanese Classification of Gastric Carcinoma (3rd English edition) proposed by the Japanese Gastric Cancer Association (JGCA) The 6th TNM Classification of Malignant Tumors proposed by the AJCC/UICC TNM stage was not included into multivariate Cox model to avoid repetition with T and N classifications
factors. Particularly, PLA2G4A-positive patients had a significantly better outcome than PLA2G4A-negative ones, and the latter population was associated with a hazard ratio (HR) of 1.500 (95 % CI 1.233–1.826; P \ 0.001; Fig. 3a). Conversely, patients with positive immunostaining for COX-2 showed a significant worse survival than COX-2negative ones with a HR of 1.318 (95 % CI 1.078–1.610; P = 0.006; Fig. 3b). When multivariate Cox analysis was performed, variables such as age, T classification, N classification, radical resection, PLA2G4A expression, and COX-2 expression were determined as independent prognostic factors (Table 2). The reduced PLA2G4A and increased COX-2 expression was associated with unfavorable prognosis in patients with gastric cancer (HR = 1.267; 95 % CI 1.032–1.555; P = 0.024 for PLA2G4A and HR = 1.646; 95 % CI 1.337–2.027; P \ 0.001 for COX-2). According to the expression phenotypes of both PLA2G4A and COX-2, the patients were divided into four subgroups. The 5-year survival rates for PLA2G4A-negative/ COX-2-negative, PLA2G4A-negative/COX-2-positive, PLA 2G4A-positive/COX-2-negative, and PLA2G4A-positive/ COX-2-positive subgroups were 51.7 % (95 % CI 44.8– 58.6 %), 41.7 % (95 % CI 34.8–48.6 %), 65.2 % (95 % CI 57.8–72.6 %), and 55.9 % (95 %, 49.4–62.4 %), respectively. Obviously, PLA2G4A-positive/COX-2-negative patients had the best survival, while PLA2G4A-negative/COX-2positive patients suffered the worst outcome with a HR of 2.102 (95 % CI 1.536–2.877; P \ 0.001) (Fig. 3c). Pairwise comparisons found no difference between PLA2G4Anegative/COX-2-negative and PLA2G4A-positive/COX-2positive subgroups (P = 0.474), whereas significant survival difference was noticed individually in the other pairwise comparisons (all P \ 0.05).
Discussion cPLA2 regulates the release of arachidonic acid and lysophospholipids from membrane phospholipid pools.
The arachidonic acid can be metabolized by distinct enzymatic systems initiated by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 to generate a variety of eicosanoids [7]. Meanwhile, platelet activating factor derived from lysophospholipids also is a potent inducer of cancer growth and metastasis [14]. These lipids and their derivatives are involved in the regulation of cell proliferation, differentiation, motility, vascularisation, and immune surveillance in both normal tissues and tumors [15]. Thus, the relevance of cPLA2-dependent pathway to human tumorigenesis has been recently suggested [16]. In light of the possible involvement of cPLA2, in particular PLA2G4A, in the pathogenesis of cancer, a few of studies have reported the expression and function of PLA2G4A in several types of malignancies. Specifically, an elevated PLA2G4A expression was noticed in human intestinal tumors [17–20], non-small cell lung cancer [21, 22], prostate cancer [23], and oral squamous carcinoma [24]. The knockout of cPLA2 gene dramatically reduced lung tumorigenesis in mice [25]. By contrast, a reduced pattern of PLA2G4A expression was also observed in human colon cancer [26]. cPLA2 expression appears to be reduced in mouse colonic tumors [27], and the knockout of cPLA2 gene enhanced mouse colonic tumor development [28]. Therefore, the true role of PLA2G4A in human cancer is not clearly understood and more studies are needed to clarify these contradictory findings. When the expression of PLA2G4A in gastric cancer is concerned, only a single preliminary study enrolled 17 patients showed that the amounts and activity of PLA2G4A were similar in gastric tumor and normal mucosa [29]. In the large patient cohort enrolled in this study, the hypothesized overexpression of PLA2G4A did not occur but a reduced expression was observed in gastric tumors compared to noncancerous mucosa (49.3 vs 84.0 %). Moreover, PLA2G4A expression correlated with small size, well-differentiated, intestinal type, and early stage tumors. The results indicated that PLA2G4A expression seemed to be a favorable variable for gastric cancer patients which were consistent with the findings in Barrett’s adenocarcinomas [30].
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Fig. 3 Kaplan–Meier survival curves according to expression phenotypes of PLA2G4A (a), COX-2 (b), and PLA2G4A combined with COX-2 (c) (log-rank test)
To date, the prognostic role of PLA2G4A in human cancer still remains unclear. Except that PLA2G4A expression correlated with an adverse prognosis in breast luminal tumors [31], significant prognostic vale of PLA2G4A has not yet been determined in cancer patients [30, 32]. However, in this study, PLA2G4A expression was confirmed as a favorable prognostic indicator for gastric cancer patients. Combined with the existing data, our study
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suggested that the expression and role of PLA2G4A appeared to be organ-specific, and PLA2G4A was indeed involved in gastric tumorigenesis. COX-2, the key enzyme in the downstream of eicosanoid biosynthetic pathway, has been extensively studied in gastric cancer. The increased COX-2 expression was shown to be associated with intestinal type, proximal location, large size, and advanced stage by several previous studies and might be an important predictor of poor survival [11]. In this study, our results confirmed the adverse prognostic role of COX-2 expression for patients with gastric cancer. Although the small correlation coefficients indicated that PLA2G4A and COX-2 were expressed independently in gastric cancer, the reduced PLA2G4A and increased COX-2 were both determined to be independent unfavorable factors in this study (Fig. 3a, b). Just as expected, PLA2G4A-positive/COX-2-negative patients had the best survival while PLA2G4A-negative/COX-2-positive patients suffered the worst outcome (Fig. 3c). Taken together, our results suggested that PLA2G4A combined with COX-2 could serve as important indicators in survival prediction for gastric cancer patients. Previous studies demonstrated that activated PLA2G4A could augment arachidonic acid release, triggering sphingomyelinases to generate ceramide which in turn induced G0/G1 cell cycle arrest and apoptosis [28]. Whereas COX-2 overexpression might deplete pools of free arachidonic acid, thereby limiting apoptotic signaling mediated by the downstream ceramide pathway [28]. Therefore, the elevated COX-2 concomitant with inactivated of PLA2G4A may greatly diminish arachidonic acid level through the enhanced utilization and insufficient production simultaneously. The inactivation of PLA2G4A and activation of COX-2 would lead to an imbalance in the cellular pools of arachidonic acid and the loss of several key apoptotic pathways, then facilitating tumorigenesis and progression [7]. This explanation has been verified by the findings that inhibition of PLA2G4A with an inhibitor or genetic deletion could attenuate TNF-a-mediated apoptosis during mouse colon tumorigenesis and contribute to tumor progression [27]. Thus, the inactivation of PLA2G4A and activation of COX-2 might be critical molecular events in the inflammation-associated tumorigenesis of gastric cancer. In summary, the reduced PLA2G4A expression was associated with unfavorable survival for patients with gastric cancer as well as the increased expression of COX-2. The expression phenotypes and prognostic roles of PLA2G4A and COX-2 revealed in this study may provide further insights into the mechanisms of inflammationassociated tumorigenesis of gastric cancer. PLA2G4A might serve as a promising target for future therapeutic
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approaches to gastric cancer combined with COX-2 inhibitors. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30973432 and No. 81000983). Conflict of interest
None.
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