NR4A2, an orphan nuclear receptor essential in the generation of dopaminergic neurons, has been recently linked to inflammation and cancer. This study sought to identify the role of NR4A2 on chemoresistance and postoperative prognosis of gastric cancer (GC).
NR4A2 was transfected into GC cells to investigate its effects on chemoresistance to 5-fluorouracil and the tumorigenicity in nude mice. This study also investigated prostaglandin E2 (PGE2)-induced NR4A2 expression and its effect on chemoresistance. Surgical specimens from patients with stage I through III GC were examined immunohistochemically for NR4A2 expression. Median follow-up time was 76 months for 245 patients.
Ectopic expression of NR4A2 significantly increased the chemoresistance and attenuated 5-fluorouracil–induced apoptosis. Transient treatment of GC cells with PGE2 significantly upregulated NR4A2 expression via the protein kinase A pathway and increased the chemoresistance. Ectopic expression of NR4A2 significantly increased the tumorigenicity. In clinical samples, NR4A2 was preferentially expressed in lymphocytes and epithelial cytoplasm in adjacent mucosa. High expression of NR4A2 (immunoreactive score ≥ 3) in cancer cells significantly predicted an unfavorable postoperative disease-specific survival of patients with stage I to III GC (P = .011), especially for those who received 5-fluorouracil–based chemotherapy (P = .016). This effect was not found in those without the chemotherapy. In multivariate Cox analyses, age, TNM (tumor/node/metastasis) stage, and high NR4A2 expression significantly predicted an unfavorable postoperative survival.
Gastric cancer (GC) is the fourth most common cancer in men and the fifth in women worldwide, and more than 70% of new cases and deaths occur in developing countries. Surgery in combination with adjuvant chemotherapy remains the mainstay of curative treatment. However, a subset of patients will relapse and/or develop metachronous metastases. To characterize this heterogeneity, it is necessary to develop prognostic and/or predictive markers useful in identifying patients with GC who will benefit from adjuvant therapies after surgery, thus leading to an improved prognosis.
Convincing epidemiological and experimental evidences have demonstrated that chronic infection with Helicobacter pylori, environmental insults, and host immune response work synergistically to maintain inflammation, driving the initiation and progression of mucosal atrophy, metaplasia, and dysplasia toward GC. Bacterial infection and prostaglandin E2 (PGE2) signaling are required for gastric carcinogenesis. Chronic inflammation contributes not only to carcinogenesis, but also to poor postoperative prognosis of GC. Daily aspirin significantly reduces death risk from cancers, including GC. Selective cyclooxygenase-2 (COX-2) inhibitors have beneficial effects on preventing inflammation-related carcinogenesis and on the regression of advanced GC.[6, 7] COX-2 induces the production of PGE2, which upregulates NR4A2 expression via sequential activation of the Rho, protein kinase A (PKA), and nuclear factor-κB signaling pathways.
NR4A2 (also termed as Nurr1), a transcription factor belonging to orphan nuclear receptor family 4 subgroup A (NR4A) superfamily, is essential in the generation of dopaminergic neurons and is involved in neurological diseases such as Parkinson's disease.[8, 9] NR4A2 is pivotal for the induction and function of regulatory T cell and implicated in autoimmune diseases such as rheumatoid arthritis.[10, 11] NR4A2 is an immediate early gene induced by PGE2 and promotes cancer cell survival via inhibiting apoptosis. NR4A2 activates the promoter of osteopontin (OPN), an important molecule promoting tumor progression and angiogenesis in many cancer types. Selective COX-2 inhibitors downregulate OPN, probably via blockade of NR4A2 and Wnt/β-catenin signaling. Downregulation of NR4A2 reduced anchorage-independent growth of cancer cells and promoted intrinsic apoptosis. Our previous study using a complementary DNA (cDNA) microarray found that NR4A2 was upregulated in adjacent gastric mucosa tissues compared to that in GC tissues. Wnt/β-catenin signaling and β-catenin target molecule cyclin D1 are important in promoting GC progression and chemoresistance.[16-18] We hypothesize that NR4A2 is activated in an inflammatory microenvironment, participates in an evolutionary process from inflammation to cancer via affecting PKA and/or OPN–β-catenin–cyclin D1 pathways, and promotes the progression of GC. However, the role of NR4A2 in the pathogenesis of GC remains unknown.
Here, we report that NR4A2 expression conferred chemoresistance and predicted an unfavorable postoperative prognosis of patients who have stage I through III GC, especially for those who received chemotherapy. These novel findings may shed light on the role of NR4A2 in chemoresistance and provide a predictive and prognostic factor for GC prognosis.
MATERIALS AND METHODS
Two representative human GC cell lines—AGS, which is derived from an intestinal-type moderately differentiated gastric adenocarcinoma, and HGC-27, which is derived from a lymph node metastasis of a potential diffuse-type undifferentiated gastric carcinoma—were purchased from ATCC (Manassas, Va) and Cell Bank, Chinese Academy of Science (Shanghai, China), respectively, 6 weeks before our in vitro study. These cell lines have been characterized by karyotyping in the cell banks and were maintained in Dulbecco's modified Eagle medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified atmosphere containing 5% CO2 at 37°C.
A full-length human NR4A2 cDNA (GenBank:BC009288) in pOTB7 vector was purchased from Open Biosystems (Waltham, Mass) and released by BamHI/XhoI digestion and then inserted into pCDNA 3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, Calif) to construct a NR4A2 expression vector TOPO-NR4A2. A total of 105 cells were transfected by TOPO-NR4A2 or empty TOPO-vector using Lipofectamine 2000 (Invitrogen) and were selected in the presence of 1 mg/mL G418 (Sigma-Aldrich, St Louis, Mo). The G418-resistant cells were not subcloned. Cell invasive ability and anchorage-independent growth of the transfected cells were examined as described.
Real-Time Quantitative Reverse-Transcription PCR and Western Blot
Total RNA was isolated and subjected for quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) as described, and primers for NR4A2 were described. Primers used for OPN were (forward) 5′-TTCCAAGTAAGTCCAACGAAAG-3′, (reverse) 5′-GTGACCAGTTCATCAGATTCAT-3′. Primers used for β-catenin were (forward) 5′-GGCTACTCAAGCTGATTTGATGGA-3′, (reverse) 5′-TAAGACTGTTGCTGCCAGTGACTAA-3′. Primers used for cyclin D1 were (forward) 5′-GCCGAGAAGCTGTGCATCTA-3′, (reverse) 5′-CTGGCATTTTGGAGAGGAAG-3′. Primers used for GAPDH were (forward) 5′-TCCTCTGACTTCAACAGCGACAC-3′, (reverse) 5′-TCTCTCTTCCTCTTGTGCTCTTGC-3′. Protein was extracted, quantified, and subjected to western blot with antibodies against NR4A2 (sc-81345, 1:100; Santa Cruz Biotechnology, Delaware, Calif), OPN (sc-73631, 1:100; Santa Cruz Biotechnology), β-catenin (1247-1, 1:1000; Abcam, Cambridge, UK), cyclin D1 (2261-1, 1:1000; Abcam), and β-actin (3700, 1:1000; Cell Signaling Technology), according to our previous protocol.
The NR4A2-transfected cells, empty TOPO-vector transfected cells, and parental cells were seeded in 96-well plates (5 × 103 per well) and allowed to grow for 24 hours, then treated with 5-fluorouracil (5-FU) (Sigma) at various concentrations for 24 hours. Number of viable cells was assayed using WST tetrazolium salt (CCK-8; Dojindo, Shanghai, China) as described.
AGS and HGC-27 cells were allowed to grow in FBS-free media for 24 hours. The starved cells were treated with 10 μM H-89 (a selective inhibitor of cyclic adenosine monophosphate [cAMP]/PKA) (Sigma), 10 μM NS398 (a selective inhibitor of COX-2) (Sigma), or dimethyl sulfoxide (control) for 1 hour, and then treated with 10 μM PGE2 (Sigma) for 4 hours. The expression of NR4A2 was measured using qRT-PCR and western blot.
The TOPO-NR4A2–transfected cells, 10 μM PGE2-induced cells, and TOPO-vector tranfected cells (5 × 103 per well) were incubated with 5-FU at the median inhibitory concentrations (IC50) of TOPO-vector transfected cells for 24 hours. The cells were then fixed with methanol–acetic acid followed by staining with 100 μL Hoechst 33258 staining reagent (Beyotime, Haimen, China) in the dark for 30 minutes. After being washed twice with phosphate-buffered saline, cells were immediately photographed with excitation wavelength of 340 to 360 nm to determine ratios of apoptotic cells.
Three- to 4-week-old male BALB/c nude mice were purchased from the Chinese Academy of Science (Shanghai, China) and maintained under pathogen-free conditions. After 1 week adaptable feeding, approximately 1 × 106 TOPO-NR4A2–transfected HGC-27 cells and TOPO–vector-transfected counterparts were subcutaneously implanted into the nape region. Tumor sizes were measured once a week and calculated as described. This experiment was performed in accordance with recommendations for the proper care and use of laboratory animals and approved by the ethics committee of this university.
GC Patients and Postoperative Follow-Up
Consecutive patients with stage I through III GC who received curative surgery at Dr. Liye Ma's surgery group, Department of General Surgery, Changhai Hospital (Shanghai, China) from March 2001 to December 2005 were enrolled in this study. The diagnoses were pathologically confirmed. Clinicopathological variables before surgery and chemotherapy were documented. We excluded such patients as those with concurrent malignant diseases and those who received radiotherapy, lost contact information, died of other causes, and refused to join the study. Follow-up was performed every 6 months by in-person interview at our outpatient department and/or by telephone calls according to our standard epidemiologic procedure. Death from GC relapse was defined as an event, and patients were censored at the last follow-up. Median follow-up time was 76 months (interquartile range, 25-89 months). The final date of follow-up was November 30, 2012. A total of 245 patients with stage I through III GC who had intact follow-up information and intact NR4A2 immunohistochemistry data were involved in survival analysis. We also acquired freshly frozen and formalin-fixed paraffin-embedded (FFPE) tumor specimens and their paired adjacent mucosa specimens from 28 patients with GC who received surgery at the same surgery group for comparative analysis of NR4A2 expression. This protocol conformed to the 1975 Declaration of Helsinki and was approved by the ethics committee of the Second Military Medical University, Shanghai, China. Informed consent was obtained from the subject(s) and/or guardian(s).
The FFPE specimens of primary GCs were examined by immunohistochemistry for NR4A2 expression. The specimens were processed basically according to a previous protocol. Polyclonal antibody against NR4A2 (ab60149, 1:50; Abcam) was used for immunostaining. Immunostains were independently examined by 4 researchers (Y.H., H.C., Y.Y., and G.C.) who were blind to clinical information. Immunostaining intensity was rated as 0 (negative), 1 (weak), 2 (moderate), and 3 (intense). The extent of positive tumor cells was graded as 0 (0%-4%), 1 (5%-24%), 2 (25%-49%), 3 (50%-74%), and 4 (75%-100%). The intensity and the extent were multiplied for each score. Because the expression of NR4A2 occurred in a patchy pattern in some specimens, we randomly selected 3 visual fields in each section and scored each visual field. A mean score of the 3 visual fields was given to each specimen. Specimens of the first 150 patients were applied to optimize a cutoff of immunoreactive score ranging from 0 to 12 by Kaplan–Meier analysis with log-rank test. The score of 3 was set as the cutoff point that best dichotomized patients into high-risk and low-risk groups.
Differences in categorical variables were examined using the chi-square test. Differences in continual variables were tested by Student t test. IC50 value was determined by probit analysis. The correlation of immunoreactive score with each clinical factor was determined by Spearman rank test. Kaplan–Meier analysis with log-rank test was used to estimate the survival. Univariate Cox regression analysis was used to obtain hazard ratio and 95% confidence interval of factors that were significantly associated with disease-specific survival. Multivariate Cox hazard model was employed to determine factors contributing significantly to the survival. All statistical tests were 2-sided and conducted using SPSS version 16.0 for Windows (SPSS, Chicago, Ill). P < .05 was defined as statistically significant.
Ectopic Overexpression of NR4A2 Increased the Tumorigenicity of GC Cells
Following the transfection with TOPO-NR4A2, NR4A2 expression was significantly upregulated in AGS and HGC-27 cells at both messenger RNA (mRNA) and protein levels, whereas expression of OPN was significantly upregulated in AGS cells at mRNA and protein levels, compared with the empty-vector controls (Fig. 1). Ectopic expression of NR4A2 did not significantly affect invasion ability and anchorage-independent growth of these cells in vitro (data not shown). HGC-27 cells stably formed aggressive tumors in nude mice. Ectopic expression of NR4A2 significantly increased the tumorigenicity (Fig. 2).
Ectopic Expression of NR4A2 Increased Chemoresistance and Conferred Resistance to 5-FU–Induced Apoptosis
NR4A2-transfected AGS, empty-vector-transfected AGS, and parental cells were examined for their sensitivity to 5-FU, resulting in IC50 concentrations of 250.2 μg/mL, 181.9 μg/mL, and 125.0 μg/mL, respectively. The IC50 concentrations of 5-FU in NR4A2-transfected HGC-27, empty-vector-transfected HGC-27, and parental cells were 322.6 μg/mL, 146.5 μg/mL, and 127.4 μg/mL, respectively. Ectopic expression of NR4A2 significantly increased the chemoresistance of both cell lines to 5-FU (Fig. 3A). The ratio of apoptotic cells in empty-vector–transfected AGS cells was significantly higher than that in NR4A2-transfected AGS cells, after being exposed to 5-FU at the IC50 concentration of empty-vector–transfected cells (53.6% ± 12.1% versus 21.2% ± 6.9%, P = .016). The same was true in HGC-27 cells (23.4% ± 2.7% versus 7.6% ± 1.7%, P = .010) (Fig. 3B).
PGE2 Induced NR4A2 Expression via PKA Pathway and Conferred Chemoresistance
The expression of NR4A2 in both GC cells was significantly up-regulated by transient PGE2 treatment, moreover, the expression of NR4A2 was significantly blocked by H-89 at both mRNA and protein levels (Fig. 4A). Interestingly, PGE2-induced NR4A2 expression was significantly associated with an increased chemoresistance to 5-FU at all concentrations in AGS cells but only at high concentration in HGC-27 cells (Fig. 4B).
Expression Pattern of NR4A2 in GCs and Adjacent Mucosa
In the paired freshly frozen GC and adjacent mucosa tissues of 28 patients, NR4A2 expression was higher in adjacent mucosa than in GCs as measured by qRT-PCR (P = .002). This was consistent with our previous finding. Immunohistochemistry of the 28 paired FFPE specimens indicated that the ratio of positive NR4A2 immunostaining in adjacent mucosa was 64.3% in lymphocyte nuclei, 39.3% in epithelial cytoplasm, and 25.0% in epithelial nuclei; the ratio in tumors was 14.3% in lymphocyte nuclei, 0% in cancer cytoplasm, and 53.6% in cancer cell nuclei.
In all FFPE specimens included in this study, the immunoreactive score of NR4A2 in cancer cells did not significantly differ among stage I, stage II, and stage III GC (Spearman correlation coefficient [rs] = .071, P = .268) and did not significantly differ among well-differentiated, moderately differentiated, poorly differentiated, and Signet-ring cell GC (rs = .105, P = .101). Figure 5 shows the representative positive immunostains of NR4A2 in adjacent mucosa and tissues from stages I through III GC.
NR4A2 Expression in Tumors Predicted Unfavorable Prognosis
Demographic and clinicopathogical variables of the patients enrolled in survival analysis are presented in Table 1. High expression of NR4A2 (immunoreactive score ≥ 3) in cancer cells was significantly associated with an unfavorable postoperative disease-specific survival (Fig. 6A). Further stratification analyses showed that this effect was only found in the patients who received 5-FU–based chemotherapy after curative surgery (Fig. 6B), but not in those without the chemotherapy (Fig. 6C). Table 2 depicts the contribution of NR4A2 expression in cancer cells and clinicopathological variables to disease-specific survival in multivariate Cox regression analyses. High expression of NR4A2 in cancer cells significantly predicted an unfavorable prognosis, and this effect was independent of age and advanced TNM stage.
Table 1. Demographics and Clinical Features of the Patients With Gastric Cancer Involved in Survival Analysis
In this study, we presented a line of evidence to elucidate the effect of NR4A2 in GC. The major findings of this study were that NR4A2 expression in cancer cells conferred chemoresistance to 5-FU, attenuated 5-FU–induced apoptosis, promoted the tumorigenicity in nude mice, and contributed to an unfavorable prognosis in GC patients, especially for those who received postoperative chemotherapy. To our knowledge, this is the first study to elucidate the role of NR4A2 in GC.
NR4A2 expression was increased up to 5-fold in AGS cells and approximately 2.5-fold in HGC-27 cells at the mRNA levels under the same transfection condition (Fig. 1). The NR4A2-transfected AGS cells exhibited a stronger chemoresistance to 5-FU, compared to that of NR4A2-transfected HGC-27 cells (Fig. 3). The same was true for the correlation between PGE2-induced NR4A2 expression and the chemoresistance (Fig. 4). These “dose-response” effects reflex an actual role of NR4A2 in chemoresistance. OPN is a direct target of NR4A2 in AGS cells rather than in HGC-27 cells with a high background level of OPN (Fig. 1). It is understandable that HGC-27 derived from metastatic GC has a high level of OPN that facilitates GC metastasis. Wnt/β-catenin–cyclin D1 might be important in the development of chemoresistance. Ectopic expression of NR4A2 did not affect the expression of β-catenin and cyclin D1 in both GC cell lines (Fig. 1), indicating that NR4A2 and Wnt/β-catenin might represent distinct pathways involved in chemoresistance.
To comprehend the complex process of GC progression, attention should be paid to effective accommodation of cancer cells against inflammatory microenvironment. Ectopic expression of NR4A2 did not affect the growth of GC cells in vitro but significantly increased the tumorigenicity in vivo (Fig. 2), indicating the importance of the microenvironment in GC progression. The COX-2/PGE2 pathway links cancer cells with the inflammatory microenvironment. Transient treatment with PGE2 significantly induced NR4A2 expression in GC cells, and the NR4A2 induction was blocked by a selective inhibitor of PKA (Fig. 4A), indicating that exogenous PGE2 promotes NR4A2 expression in a cAMP/PKA-dependent manner. The cAMP/PKA signaling pathway also links chronic inflammation and cancers. A selective inhibitor of COX-2 did not block NR4A2 expression induced by PGE2, indicating that COX-2 is an upstream element of the PGE2–PKA–NR4A2 axis. Our immunohistochemistry results showed that expression patterns of NR4A2 in adjacent mucosa and in tumors were different, and NR4A2 was preferentially expressed in lymphocytes and epithelial cytoplasm in adjacent mucosa tissues (Fig. 5). NR4A2 regulates CD4+ T cells by inducing Foxp3 and strongly repressing Th1 cytokine production. This may contribute to immunosuppression and chronic inflammation, the latter of which promotes PGE2 production. Ectopic expression of NR4A2 promotes the production of interleukin-8, an important inflammation-promoting Th2 cytokine. The feedback loop of PGE2-NR4A2 may play an active role in maintaining inflammation and promoting GC progression. Interestingly, PGE2-treated GC cells exhibited a significant resistance to 5-FU–induced apoptosis (Fig. 4). PGE2-mediated protection from apoptosis can be completely inhibited by a dominant-negative NR4A2 construct; moreover, downregulation of NR4A2 can promote intrinsic apoptosis in several cancer cell lines.[12, 14, 28] This evidence, together with our data (Fig. 3), support that NR4A2 participates in an evolutionary process from inflammation to cancer and confers a resistance of cancer cells to chemotherapeutic-induced apoptosis.
Importantly, NR4A2 expression in cancer cells was significantly associated with unfavorable postoperative survival, especially for those who received postoperative chemotherapy (Fig. 6). This effect was independent of TNM stage (Table 2). NR4A2 expression was mostly nuclear in cancer cells (Fig. 5). However, cytoplasmic mislocalization of NR4A2 has been related to a poor prognosis of bladder cancer. The function of NR4A2 in different subcellular localization should be further elucidated. Because NR4A2 expression confers chemoresistance, the chemotherapy may select a subset of cancer cells with high NR4A2 expression, and these cancer cells might be major populations of cancer recurrence. Targeting NR4A2 pathway might be an alternative to improve the effect of postoperative chemotherapy. Thus, this study provides a new predictive and prognostic biomarker for postoperative outcome of patients with GC and also a candidate therapeutic target to improve GC prognosis.
Our study has limitations. First, the mechanism by which NR4A2 confers chemoresistance remains to be clarified. Constitutive activation of the epidermal growth factor receptor (EGFR) family is associated with PGE2-induced NR4A2 expression and chemoresistance in oral squamous cell carcinoma. EGFR is able to predict GC prognosis. The role of EGFR and its related somatic mutations in PGE2-induced NR4A2 expression and chemoresistance should be investigated. Second, we did not find a difference in invasion and anchorage-independent growth among the GC cells transfected with NR4A2, parental cells, and those with NR4A2 downregulation by short hairpin RNA in our preliminary study (data not shown). However, ectopic expression of NR4A2 significantly increased the tumorigenicity in nude mice (Fig. 2). Potential interaction of cancer cells with mesenchyma as a source of this inconsistency should be elucidated. Third, postoperative 5-FU–based chemotherapy is effective in improving the prognosis of GC. However, this study gave different data in univariate Cox analysis (Table 2), possibly because the chemotherapy had been selectively applied to the patients at an advanced stage (Table 1). In addition, the survival study was retrospectively designed, which might have introduced a selection bias.
In conclusion, the expression of NR4A2, an orphan nuclear factor linking neurological abnormalities, inflammation, and cancers, confers the chemoresistance to 5-FU, attenuates 5-FU–induced apoptosis, increases the tumorigenicity of human GC cells, and predicts an unfavorable postoperative prognosis of patients with GC, especially for those who received postoperative chemotherapy. Although the mechanisms by which NR4A2 expression in GC confers chemoresistance remains to be further clarified, this study provides a new predictive and prognostic factor for postoperative prognosis of GC. Targeting NR4A2 might be an alternative to improve the survival of patients with GC.
No specific funding was disclosed.
CONFLICT OF INTEREST DISCLOSURE
This study was supported by the National Natural Scientific Foundation of China (grants 81025015 and 91129301 to Dr. Cao). The other authors made no disclosure.