89 (2015) 320–328

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Lung Cancer

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Testicular orphan 4 (TR4) is a marker for metastasis and poor prognosis in non-small cell lung cancer that drives the EMT phenotype

Liyi Zhang a,1, Jianzhi Zhang a,1, Yuanyuan Ma a,1, Jinfeng Chen a, Bin Dong b, Wei Zhao c, Xing Wang a, Qinfeng Zheng a, Fang Fang a, Yue Yang a,∗ a Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Thoracic Surgery II, Beijing, People’s Republic of China b Central Laboratory, Peking University Cancer Hospital & Institute, Beijing 100142, People’s Republic of China c Department of Cell Biology, Peking University Cancer Hospital & Institute, Beijing 100142, People’s Republic of China article info abstract

Article history: Objectives: Aberrant expression of testicular orphan receptor 4 (TR4) has been shown to regulate biological Received 11 December 2014 processes near solid tumors. However, the role of TR4 in non-small cell lung cancer (NSCLC) patient Received in revised form 30 May 2015 prognosis and the development of NSCLC cancer cells are unclear. Accepted 11 June 2015 Methods: Immunohistochemical analysis was used to evaluate the correlation between TR4 expression and clinicopathological characteristics in 291 cases of NSCLC specimens. A knockdown and overex- Keywords: pression of TR4 was performed to assess the role of TR4. Transwell and colony formation assays were Testicular orphan receptor 4 completed to investigate the metastatic and proliferative abilities. Quantitative real-time PCR, Western NSCLC Prognosis blotting and immunofluorescence staining were carried out to analyze the epithelial-to-mesenchymal Metastasis transition (EMT) phenotype. EMT Results: Immunohistochemical evaluation of clinical samples revealed that most of the lung cancer tissues Immunohistochemistry were strongly positive for TR4, whereas the tissues that stained weakly positive or negative for TR4 expression were shown in the paired normal tissues. Moreover, higher levels of TR4 expression were significantly associated with higher lymph node metastases, TNM stages, tumor thrombus in vena and poor prognosis. We observed that downregulation and up-regulation of TR4 with stable cell transfection significantly influence the proliferation, invasive and metastatic abilities of NSCLC lines. In addition, aberrant TR4 expression could modulate the expression levels of several EMT related markers. Conclusions: Collectively, our results show TR4 expression in NSCLC samples is significantly associated with poor clinicopathological features, and TR4 plays an important role in the metastatic capacity of NSCLC cells by EMT regulation. © 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Testicular orphan receptor 4 (TR4; subfamily 2, group C, member 2) belongs to the nuclear receptor super family Lung cancer is the leading cause of cancer-related deaths world- and encodes a 67-kDa [4–6]. TR4 is a transcriptional factor wide and ranks among the most commonly occurring and fatal that regulates many signal pathways in normal spermatogenesis, malignancies [1]. The primary tumor, lymph node (LN) and the cerebellum development, glucose metabolism and insulin resis- metastasis (TNM) staging system remain the most important deter- tance [7–11]. TR4 heterodimerizes with testicular orphan receptor minant of outcome, and poor survival in NSCLC is partly due 2 (TR2, nuclear receptor subfamily 2, group C, member 1) and the to development of metastasis [2]. Non-small cell lung cancer complex binds to direct repeats with one nucleotide of the DR1 ele- (NSCLC) accounts for approximately 80% of cases. Of the patients ment that inhibits target [12–15]. This nuclear who exhibit NSCLC, approximately 30% to 50% will present with protein, TR4, could suppress various factors, such as ER, andro- metastatic disease [3]. gen receptor (AR) and the steroid 21-hydroxylase [16–18]. Several studies have shown that TR4 is widely expressed in human tissues, including testis, prostate, ovary, brain and lung [4,5,19,20]. In addi- tion, TR4 has been demonstrated to activate target ∗ in liver carcinoma HepG2 cells [21]. Recently, Yang et al. found Corresponding author. Tel.: +86 1088196568; fax: +86 1088196568. E-mail address: [email protected] (Y. Yang). that TR4 was located in prostate tumor cells and suggested that 1 These authors contributed equally to this work. TR4 may function through Oct4 and IL1Ra to modulate prostate http://dx.doi.org/10.1016/j.lungcan.2015.06.007 0169-5002/© 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/). L. Zhang et al. / Lung Cancer 89 (2015) 320–328 321 cancer progression and chemo-resistance [22]. Furthermore, Du 2.3. Plasmid construction and cell transfection et al. indicated that TR4 could promote pituitary corticotroph tumor progression [16]. pGPH1/GFP/Neo-shTR4 was constructed (Suzhou GenePharma Epithelial-to-mesenchymal transition (EMT) is a process dur- Co., China) and transfected into two lung cancer cell lines, A549 ing which epithelial cells acquire more migratory and invasive and PC-9. As a negative control, control vector shNC (Suzhou properties [23,24]. Several transcriptional factors are known to be GenePharma Co., China) was transfected in the A549 and PC-9 involved in the regulation of EMT, including Snail, Slug and Twist, cells. The ORF of human TR4 gene was amplified with fol- which simultaneously induce the expression of required for lowing primers (forward primer 5-CTCTCGGCCGGAATCTCCA-3 the mesenchymal phenotype, such as Vimentin, and repress the and reverse primer 5-GGCAGGTGTGTGGTTTGC-3) from A549 expression of genes required for maintaining epithelial phenotype, cells, and subcloned into the XhoI/BamHI sites of GV144 vector such as E-cadherin [25,26]. (Genechem Co., China), and transfected into H1299 and GLC82 cells NSCLC are particularly aggressive tumors, with high invasive by lipofectamine 2000 (Invitrogen) following the manufacturer’s properties and metastatic potential. About 40% stage I and 60% stage protocol. CON107 (Genechem Co., China) was used as control. Sta- II NSCLC patients are dying of distant metastases within 5 years ble single colony cells of TR4 knockdown and overexpress were after curative tumor resection [27]. Therefore, we aimed to explore screened by 500 ␮g/ml of G418 (Sigma-Aldrich) for 2 weeks and the crucial role of TR4 in 291 cases of I-IIIA NSCLC with or without LN then the cells were expanded and harvested for Quantitative real- metastasis. We demonstrated that TR4 expression was associated time PCR (QPCR) or Western blot analysis (Fig. 2). with the occurrence of LN metastasis and the postoperative survival period for NSCLC patients. TR4 induced cell metastatic and invasive 2.4. Boyden chamber cell migration and cell invasion assay capacities in lung cancer cells, at least in part, through the activation of the EMT signaling pathway. BD BioCoat Matrigel Invasion Chambers, with 8.0 ␮m PET mem- brane 24-well cell culture inserts packaged ready-to-use in BD Falcon Companion Plates and Growth Factor Reduced BD Matrigel 2. Materials and methods Matrix, a solubilized basement membrane (BD Biosciences, San Jose, CA, USA) were used in Matrigel invasion assays. Assay was 2.1. Tissue specimens and immunohistochemistry (IHC) performed according to the manufacturer’s protocol. A total of 1.0 × 105 cells per well in serum-free RPMI were placed in the upper NSCLC tumor specimens were collected with informed con- chamber. RPMI plus 10% FBS was placed in the lower chamber sent from 291 patients who underwent surgical resection at as a source of chemo attractant. Transfected A549 and PC-9 cells the Department of Thoracic Surgery of Beijing Cancer Hospital were allowed to migrate through pores for 24 h at 37 ◦C. The total between December 1995 and December 2009. Written consent numbers of cells migrated or invaded through the matrigel were statements were obtained from all patients before operation. counted after crystal violet staining and photographed. Formalin-fixed and paraffin-embedded primary lung cancer sam- ples were acquired from the Department of Pathology, Peking 2.5. Colony formation University, under approval from the Ethical Committee. The clini- cal and pathological features of all cases were summarized in Table Approximately 1000 transfected A549 and PC-9 cells were A1. replated into 10 cm dishes. Two weeks later, visible colonies were The primary antibodies used in this study included anti-TR4 fixed and stained with crystal violet, and the colony formation ratio antibody (rabbit monoclonal 1:200 dilution; Abcam, UK), anti- was calculated with the following formula: (visible colony num- Vimentin antibody (rabbit monoclonal 1:500 dilution; Abcam, bers/original number of cells) × 100%. Data are presented as the UK), anti-E-cadherin antibody (rabbit monoclonal 1:500 dilu- mean ± SD and experiments were performed in triplicate. * P < 0.05. tion; Abcam, UK). Sections (4 ␮m) were routinely processed and stained with rabbit monoclonal antibodies followed by incubation 2.6. Reverse transcription and QPCR with HRP-conjugated goat anti-rabbit secondary antibody (Sigma- Aldrich, Poole, Dorset, UK). For determination of immunoreactivity RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) for the previously mentioned markers, cytosolic staining of yellow- followed by manufacturer’s protocol. Two micrograms of total RNA ish or brownish granules was graded as follows: (A) for background was added and reverse transcribed with Moloney murine leukemia staining; (B) for negative staining; (C) for moderate staining and (D) virus (MMLV, Invitrogen). PCR reactions were performed by The for strong staining. In addition, positive staining areas in the entire LightCycler®480 SYBR Green I Master on Light-Cycler®480 Real- tissue section were graded as follows: 0, for <5%; 1, for 5–25%; 2, for Time PCR System (Roche, USA). Sequences of all primers are listed 26–50% and 3, >50%. When combining these two parameters, 0–1 in Table A2. GADPH was used as a loading control. Cycling con- and >1 were considered negative and positive staining, respectively ditions were as follows: 5 min at 95 ◦C, followed by 45 cycles each (Fig. 1A). In the case of TR4 and Vimentin, samples were scored as consisting of 10 s at 95 ◦C, 20 s at 60 ◦C and 30 s at 72 ◦C. The relative positive when greater than 5% of tumor cells showed positive stain- amount of genes was normalized to GADPH. Data was calculated ing in the nucleus or cytoplasm. In the case of E-cadherin, tumors based on 2−Ct where Ct = Ct (Target)—Ct (Reference). Fold change were scored as 0 to 3 according to different staining. Tumors scored was calculated by the 2−Ct method. as 0 or 1 were considered “negative”, and those scored as 2 or 3 were classified as “positive”. 2.7. Western blot analysis

2.2. Cell lines Protein was extracted from cells using RIPA buffer con- taining complete protease inhibitor cocktail (Roche, Mannheim, Lung cancer cell lines (A549, PC-9, H1299, GLC82) were main- Germany). were separated on SDS-PAGE and transferred tained under standard cell culture conditions in RPMI-1640 media to polyvinylidene fluoride membranes. Membranes were blocked supplemented with 10% fetal bovine serum (FBS; Gibco, Australia), with 5% nonfat dried milk in TBST (15 mM Tris–HCl, pH 7.4, 0.9% 100 U/ml penicillin and 100 ␮g/ml streptomycin (Invitrogen, Grand NaCl, and 0.05% Tween-20, pH 7.4) followed by blot analysis with ◦ Island, NY) in a humidified atmosphere of 5% CO2 at 37 C incubator. the following specified antibodies: rabbit monoclonal anti-human 322 L. Zhang et al. / Lung Cancer 89 (2015) 320–328

Fig. 1. Representative IHC staining patterns of TR4 positive and TR4 negative cells in paired specimens. (A) Left: NSCLC tissue with positive TR4 expression. Right: Matched adjacent normal tissue with negative TR4 expression. (B) The levels of TR4 in 35 NSCLC specimens were significantly higher than matched adjacent normal tissues. (C) Representative IHC staining of TR4 positive and TR4 negative cells. Left: NSCLC with the expression of TR4 interpreted as negative. Right: NSCLC with the expression of TR4 interpreted as positive. (D) TR4 expression in NSCLC tissues with lymph node metastases was found significantly higher than tissues without lymph node metastases. (E and F) Kaplan–Meier survival curves demonstrated that high levels of TR4 were associated with poor OS and DFS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

TR4 antibody (1:1000 dilution; Abcam, UK); rabbit polyclonal cells were incubated for 1–2 h with anti-vimentin (1:200) or anti-E- anti-human Twist antibody (1:400 dilution; Abcam, UK); rabbit cadherin (1:200). Cells were then washed with PBS and incubated polyclonal anti-human Snail antibody (1:1000 dilution; Abcam, with secondary antibody (Rhodamine TRITC-Conjugated Affinity UK); rabbit monoclonal anti-human Vimentin (1:1000 dilution; Pure Goat Anti-Rabbit IgG, ZSBIO, Beijing) for 45 min. Nuclei were Cell Signaling Technology, USA); rabbit monoclonal anti-human E- counterstained with 4,6-diamidino-2-phenylindole (1:1000) for cadherin (1:1000 dilution; Cell Signaling Technology, USA); rabbit 10 min at room temperature followed by three washes (5–10 min anti-GAPDH (1:10,000 dilution; Cell Signaling Technology, USA) each). The cells were then mounted with polymount antifade solu- and goat anti-rabbit secondary antibody (1:2000). Signals were tion (Sigma) and observed under a TCS-SP5 confocal laser scanning visualized by using chemiluminescence (Millipore). microscope (LEICA, Germany).

2.8. Immunofluorescence staining 2.9. Statistical analysis

Stable transfected cells were cultured in 60 mm culture plates The immunohistochemical expression and clinicopathologic and trypsinized then plated in chamber slides (Millicell EZ Slide: data were summarized using standard frequency tabulations. Asso- Millipore, EMD Millipore Corporation, Billerica, MA, USA). Cells ciations between the markers expression and patients’ clinical were then fixed with 4.0% paraformaldehyde in phosphate- variables were assessed using the 2 test and the Fisher exact test. buffered saline for 30 min and then permeabilized with 0.2% Triton The Spearman rank r was used to estimate the correlation between X-100 for 15 min at room temperature. After rinsing twice with PBS, immunohistochemical markers. Survival rates were estimated with L. Zhang et al. / Lung Cancer 89 (2015) 320–328 323

Fig. 2. (A) TR4 expression in lung cancer cell lines. (B) Knock-down of TR4 was evaluated by QPCR and Western blotting in PC-9 and A549 cells. (C) Overexpression of TR4 was verified by QPCR and Western blotting in GLC82 and H1299 cells. (D–G) Effects of TR4 on NSCLC cell migration and invasion, and effects of TR4 on NSCLC cell proliferation. (D) Transwell assays were used to investigate the changes in migratory and invasive abilities of shTR4-transfected PC-9 and A549 cells. (E) Transwell assays were used to investigate the changes in migratory and invasive abilities of TR4-overexpressing GLC82 and H1299 cells. (F) Colony-formation assays were conducted to determine the proliferation of shTR4-transfected A549 cells. (G) Colony-formation assays were conducted to determine the proliferation of shTR4-transfected PC-9 cells. (H) Analysis of E-cadherin, N-cadherin, Vimentin, Snail-1 and Twist expression in A549 cells treated with shTR4 by QPCR. (I) Analysis of E-cadherin, Vimentin, Snail-1 and Twist expression in A549 cells treated with shTR4 by western blot. All experiments were performed in triplicate independently. (J) Analysis of E-cadherin, N-cadherin, and Vimentin mRNA expression in TR4-overexpressing H1299 cells by QPCR. (K) Analysis of E-cadherin, Vimentin and N-cadherin expression in TR4-overexpressing H1299 cells by western blot. All experiments were performed in triplicate independently. 324 L. Zhang et al. / Lung Cancer 89 (2015) 320–328

Table 1 ably in advanced NSCLC TNM stages (P = 0.003) and tumor venous Correlation between the clinicopathological features and expression of TR4 (n = 291). thrombus (P = 0.025). Variables No. of patients % TR4 expression P-value We examined whether variables of TR4 expression could predict overall survival (OS) and disease-free survival (DFS) in the cohort Negative Positive of patients. Compared to the TR4-negative expression group, the Age (years) 0.801 patients with TR4 positive staining showed significantly shorter ≤60 120 (41.2) 90 30 >60 171 (58.8) 126 45 OS (69 months versus 35 months; P = 0.006) and DFS (53 months Gender 0.410 versus 21 months; P = 0.005) by Kaplan–Meier analyses (Fig. 1F). Male 167 (57.4) 127 40 These results suggest that the increased expression of TR4 may play Female 124 (42.6) 89 35 an important role in the development and progression of NSCLC. Histology 0.997 Furthermore, a Cox proportional hazards model was applied Squamous cell carcinoma 117 (39.5) 85 30 Adenocarcinoma 152 (52.2) 113 39 to estimate the effect of TR4 expression on OS and DFS. The Others 23 (7.9) 17 6 results showed that TR4 remained significantly associated with DFS Unknown 1 (0.3) 1 0 (Table 2, P = 0.022), and exhibited a borderline significance in OS Histologic grading 0.102 (Table 2, P = 0.057). The crude hazard ratio (HR) of TR4 positive Well 17 (5.8) 11 6 Moderate 145 (49.8) 115 30 expression compared with TR4 negative expression was 1.407 of Poorly 122 (42.0) 84 38 OS (95% CI, 0.989–2.001) and 1.478 of DFS (95% CI, 1.057–2.067), Unknown 7 (2.4) 6 1 respectively. Taken together, our data predicted that the expres- TNM stage 0.003* sion of TR4 demonstrated a significantly worse prognosis in NSCLC I, II 193 (66.4) 154 39 patients. Table 2 further showed the results of the univariate and IIIA 98 (33.7) 62 36 Tumor stage 0.163 multivariate analyses. T1 and T2 197 (67.7) 151 46 T3 and 95 (32.0) 65 30 Unknown 1 (0.3) 0 1 3.3. TR4 induced cell migratory and invasive ability in NSCLC cell Lymph node metastasis <0.0001* Yes 134 (51.5) 84 50 lines No 150 (46.0) 125 25 Unknown 7 (2.4) 7 0 Our data mentioned above suggests that the increased expres- Tumor thrombus in vena 0.025* sion of TR4 in NSCLC specimens correlated with lymph node No 244 (83.8) 187 57 metastasis and poor outcome in NSCLC patients. Previously, it Yes 46 (15.8) 28 18 Unknown 1 (0.3) 1 0 has been reported that TR4 promoted prostate cancer cell migra- tion/invasion in vitro and in vivo mice studies [28]. Therefore, we * Values with statistical significance (P < 0.05) were indicated. further evaluated the roles of TR4 in the metastatic ability of NSCLC cell lines. First, expression of TR4 was detected in lung cancer cell the use of the Kaplan–Meier method. The prognostic value of TR4 lines (A549, H1299, GLC82 and PC-9) by Western blotting. TR4 was studied with the use of a Cox model, which was adjusted for mRNA and protein were highly expressed in A549 and PC-9 cell age, gender, stage and histology. P < 0.05 was considered significant. lines (Fig. 2A). The knockdown assay was performed in A549 and All statistical analyses were performed using SPSS 17.0 statistical PC-9 cells transfected by the knockdown agent of GPH1/GFP/Neo- software. shTR4 (named as shTR4) and the pGPH1/GFP/Neo-shNC was used as control (named as control). H1299 and GLC82 cells that lack 3. Results endogenous TR4 expression were used in this study to establish two stable cell lines that constitutively overexpress the TR4 pro- 3.1. TR4 expression is upregulated in NSCLC tein via transfected by the GV144 vector (named as TR4) and the control vector (named as control). The transfection efficiency was To determine the clinical relevance of TR4, we first detected verified by using QPCR and Western blotting (Fig. 2B and C). Next, this protein expression level in 35 paired NSCLC tumor and nor- we used the Transwell migration and invasion assays to assess the mal tissues by IHC. The IHC data revealed that TR4 was strongly effects of TR4 on the migration and invasion in both TR4 gain-of- expressed in tumor tissues compared with normal counterpart tis- function and loss-of-function cell lines. As shown in Fig. 2D, stable sues (Fig. 1A). In addition, our results showed that the TR4 positive TR4 knockdown resulted in a 44.87% reduction in migration rates frequency was significantly higher in tumor tissues compared with (P = 0.0185) and a 45.15% reduction in invasion rates (P = 0.0037) normal counterpart tissues (Fig. 1B, P < 0.05). compared with the control cells. In contrast, overexpression of TR4 augmented the migratory and invasive capabilities of H1299 and 3.2. A high level of TR4 expression is associated with poor GLC82 cells. TR4 overexpression in H1299 cells resulted in a 60.60% prognosis in NSCLC patients increase in migration rates (P = 0.0035) and a 68.75% increase in invasion rates (P = 0.05) compared with the control cells (Fig. 2E). Next, we investigated TR4 expression in 291 clinical NSCLC These results were consistent with clinical sample analyses, indi- specimens with clinicopathological characteristics shown in cating that TR4 was associated with metastatic potentials of NSCLC Appendices A and B Table A1. According to IHC staining of TR4, cells. weak/negative staining for TR4 expression was considered to exhibit negative expression and the tumor samples with higher staining were scored as positive expression (Fig. 1C). As shown in 3.4. TR4 enhanced colony formation ability in NSCLC cell lines Table 1, there was no significant correlation between TR4 levels and gender, age at surgery, tumor status (T), degree of cell differentia- We further examined whether TR4 influenced the proliferation tion or histological subtype. Importantly, the expression of TR4 in ability of NSCLC cells. The A549-shTR4 cells and the A549-shNC cells the NSCLC tissues with lymph node metastases was significantly were seeded in plates for colony formation analysis. Knockdown of higher than tissues without lymph node metastases (P < 0.0001; TR4 significantly diminished the colony formation rates of A549 Fig. 1D). Additionally, the expression of TR4 increased remark- cells compared with the control group (P = 0.0002) (Fig. 2F). We L. Zhang et al. / Lung Cancer 89 (2015) 320–328 325

Table 2 Univariate and multivariate Cox regression analyses for disease free survival (DFS) or overall survival (OS) of patients (n = 291).

DFS OS

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

Univariate analysis Age (>60 vs. 60 years) 0.812(0.590–1.116) 0.199 1.208(0.879–1.661) 0.243 Gender (male vs. female) 0.900(0.657–1.233) 0.511 0.808(0.591–1.106) 0.183 Histological classification (Squamous cell carcinoma vs. Adenocarcinoma/Others) 1.066(0.768–1.478) 0.703 1.050(0.811–1.359) 0.7102 TNM stage (I vs. II/IIIA) 1.829(1.224–2.732) 0.000 1.403(1.192–1.653) 0.000 Tumor stage (T1 vs. T2/T3) 1.274(0.820–1.980) 0.281 1.228(1.002–1.504) 0.047 Lymph node metastasis (negative vs. positive) 2.171(1.581–2.982) 0.000 2.034(1.481–2.793) 0.000 Tumorthrombus in vana (negative vs. positive) 1.365(0.902–2.067) 0.141 1.379(0.916–2.076) 0.124 Histologic grading (well vs. moderate/poorly) 1.517(1.154–1.995) 0.003 1.544(1.171–2.035) 0.002 TR4 expression (negative vs. positive) 1.631(1.159–2.296) 0.005 1.616(1.151–2.269) 0.006 Multivariate analysis Age (>60 vs. 60years) NA NA Gender (male vs. female) NA NA Histological classification (Squamous cell carcinoma vs. Adenocarcinoma/others) NA NA TNM stage (I vs. II/IIIA) 1.610(1.311–1.977) 0.000 1.486(1.225–1.804) 0.000 Tumor stage (T1 vs. T2/T3) 0.821(0.655–1.031) 0.089 NA Lymph node metastasis (negative vs. positive) NA NA Tumorthrombus in vana (negative vs. positive) NA NA Histologic grading (well vs. moderate/poorly) 1.259(0.972–1.631) 0.081 1.365(1.030–1.809) 0.030 TR4 expression (negative vs. positive) 1.478(1.057–2.067) 0.022 1.407(0.989–2.001) 0.057 also observed that TR4 knockdown induced colony formation rates in TR4-positive tumors and TR4-negative tumors was 20% (6/30) decreased in PC-9 cells (P = 0.0029) (Fig. 2G). and 33.3% (10/30), respectively. Subsequently, we analyzed the cor- relations between TR4, Vimentin and E-cadherin expression levels 3.5. Down-regulation of TR4 suppressed the EMT phenotype and showed that TR4 expression had positive correlations with the expression of Vimentin (r = 0.336, P = 0.009). However, no correla- To further explore mechanisms associated with increased tion was found between TR4 and E-cadherin expression (r = −0.151, metastasis potential, we tested essential EMT-related factors, P = 0.243). including E-cadherin, Vimentin, Twist and Snail by conducting QPCR and Western blotting assays in the cells. We found that 4. Discussion A549-shTR4 cells expressed lower mRNA levels of mesenchymal markers, such as Vimentin (P = 0.0001), Snail (P = 0.025), N-cadherin It has been established that TR4 plays a critical role in embryonic (P = 0.026), and Twist, whereas higher mRNA levels of the epithe- development, lipid metabolism [29] and the progression of prostate lial marker of E-cadherin were observed. In addition, we observed cancer as well as pituitary corticotroph tumor development [16,22]. increased expression of the epithelial marker E-cadherin and A number of studies have clearly shown that TR4 is aberrantly decreased expression of mesenchymal markers Vimentin, Twist expressed in many types of human cancer, such as HepG2 cells and Snail in A549-shTR4 cells by immunoblotting (Fig. 2H and I). of hepatocellular carcinoma, prostate cancer and pituitary cor- We then examined both epithelial and mesenchymal markers in ticotroph tumors [16,21,22]. However, little is known about its TR4-expressing H1299 cells. As can be seen in Fig. 2J and K, TR4- expression and significance in NSCLC. Thus, we have explored this expressing H1299 cells exhibited a significant loss of E-cadherin research to better understand the effects of TR4 in NSCLC. (P = 0.026), meanwhile the mesenchymal markers Vimentin, N- In our present study, we revealed that TR4 is strongly expressed cadherin were upregulated. However, the expression of two known in NSCLC specimens with 42.9% positive frequency in tumor tis- EMT inducers, Twist and Snail, were not detectable in H1299 and sues and 20% in corresponding normal tissues. Furthermore, we GLC82 cells. QPCR and Western blotting assays were confirmed explored TR4 expression in 291 cases of NSCLC and high level of by confocal microscopy (Fig. 3A–D). We found that a significant TR4 expression was significantly associated with poor DFS and increase of E-cadherin was concurrent with a loss of Vimentin OS. All those results strongly suggested that TR4 overexpression expression in A549-shTR4 cells. Similar results were obtained by might represent a biologically and clinically distinct type of cancer immunofluorescence in PC-9-shTR4 cells. Together, these observa- exhibiting highly-malignant clinical behavior. tions show that induction of metastasis might be through the EMT It is well known that lung cancer is a disease of uncontrolled phenotype in NSCLC cells. cell growth in lung tissues that may lead to metastasis, including invasion of neighboring tissues and infiltration beyond the primary 3.6. Relationship between TR4 and EMT markers in NSCLC tumor. We observed that high expression of TR4 was related to lymph node metastasis in NSCLC, indicating TR4 could be used as We used immunohistochemical methods to analyze the fre- an independent prognostic and metastatic factor for NSCLC. Next, quency of two EMT-related markers (Vimentin and E-cadherin) we investigated the mechanism of metastasis mediated by TR4. in an investigation cohort, including 30 TR4-positive tumors and As expected, the inhibition or over-expression of TR4 was shown 30 TR4-negative tumors. Representative microphotographs of the to successfully reduce or promote the proliferation, migration and expression of these two biomarkers are shown in Fig. 3E–H. As invasion of NSCLC cells, similar to its function in prostate cancer the presence of Vimentin in tumor cells rather than its inten- [37]. EMT has been implicated to be a potential marker of invasion sity is examined as a marker of EMT, the percentage of Vimentin and metastasis in lung cancer [30,31]. We assessed these various in TR4-positive tumors and TR4-negative tumors was 20% (6/30) EMT-related markers in TR4-inhibited or over-expressed lung and 0 (0/30), respectively. The loss of E-cadherin expression has cancer cells and observed down-regulation of Snail, Twist, previously been implicated in the progression and metastasis of Vimentin and N-cadherin expressions [25] and up-regulation of E- several malignancies. In this cohort, the percentage of E-cadherin cadherin, CK19 and EpCAM in the lung cancer cells with knockdown 326 L. Zhang et al. / Lung Cancer 89 (2015) 320–328

Fig. 3. (A–D) Fluorescence staining for E-cadherin and Vimentin in A549 cells. Representative confocal pictures are shown. Rhodamine TRITC-red is used for E-cadherin and Vimentin, whereas DAPI-blue stains the nuclei. (E–H) Representative IHC staining for Vimentin and E-cadherin. (E) NSCLC with the expression of E-cadherin interpreted as positive. (F) NSCLC with the expression of E-cadherin interpreted as negative. (G) NSCLC with the expression of vimentin interpreted as positive. (H) NSCLC with the expression of vimentin interpreted as negative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of TR4 compared with the control cells and opposite manner in There are still many details to be explored regarding the mech- those with over-expressed TR4. But the limited number of spec- anisms of TR4 in NSCLC. TR4 has been reported to belong to the imens used to explore the relationship of TR4 and E-cadherin, (ER)/thyroid subfamily that Vimentin by IHC makes it difficult to get a clear conclusion. regulates several genes, including ER and AR [32,33]. It has been L. Zhang et al. / Lung Cancer 89 (2015) 320–328 327 recently reported that ER1 is an independent positive progno- protected against obesity-linked inflammation, hepatic steatosis, and insulin stic factor for survival in early-stage NSCLC patients as well as in resistance. Diabetes 2011;60:177–88. [9] Liu NC, Lin WJ, Kim E, Collins LL, Lin HY, Yu IC, Sparks JD, Chen LM, Lee YF, metastatic NSCLC [34,35]. Subsequently, the interaction between Chang C. Loss of TR4 orphan nuclear receptor reduces phosphoenolpyruvate TR4 and ER is required for further study. carboxykinase-mediated gluconeogenesis. Diabetes 2007;56:2901–9. TR4 has been found to be involved in fundamental biological [10] Chen YT, Collins LL, Uno H, Chang C. Deficits in motor coordination with aber- rant cerebellar development in mice lacking testicular orphan nuclear receptor process, such as RNA metabolism, protein translation, ubiquitin 4. Mol Cell Biol 2005;25:2722–32. cycle, metabolic processes, lipid recruitment and gluconeogene- [11] Collins LL, Lee YF, Heinlein CA, Liu NC, Chen YT, Shyr CR, Meshul CK, Uno sis [9,29]. It has previously been proven that TR4 can regulate H, Platt KA, Chang C. Growth retardation and abnormal maternal behavior several signal pathways in cancer cells of liver, skin and prostate in mice lacking testicular orphan nuclear receptor 4. Proc Natl Acad Sci USA 2004;101:15058–63. [16,22,29,38]. The studies from TR4 gene knock-out mice (TR4−/−) [12] Omori A, Tanabe O, Engel JD, Fukamizu A, Tanimoto K. Adult stage gamma- provide valuable clue to intensive research on TR4 [10,11,36–38]. globin silencing is mediated by a promoter direct repeat element. Mol Cell Biol These results indicate TR4 might not only affect the metastasis 2005;25:3443–51. [13] Tanabe O, Katsuoka F, Campbell AD, Song W, Yamamoto M, Tanimoto K, Engel of lung cancer, but also play some role in drug resistance and JD. An embryonic/fetal beta-type globin gene repressor contains a nuclear metabolism. receptor TR2/TR4 heterodimer. EMBO J 2002;21:3434–42. In conclusion, the present study showed that TR4 is frequently [14] Tanabe O, McPhee D, Kobayashi S, Shen Y, Brandt W, Jiang X, Campbell AD, Chen YT, Chang C, Yamamoto M, Tanimoto K, Engel JD. Embryonic and fetal upregulated in NSCLCs, leading to poor prognosis, an increase in beta-globin gene repression by the orphan nuclear receptors, TR2 and TR4. lymph node metastasis and advanced NSCLC TNM stages. Our data EMBO J 2007;26:2295–306. further demonstrated that TR4 could induce migratory and invasive [15] Tanabe O, Shen Y, Liu Q, Campbell AD, Kuroha T, Yamamoto M, Engel JD, The TR2 TR4 orphan nuclear receptors repress Gata1 transcription. Genes Dev properties in NSCLC cells, and that down-regulation of TR4 could 2007;21:2832–44. reduce the EMT phenotype. The evidence may lead to the applica- [16] Du L, Bergsneider M, Mirsadraei L, Young SH, Jonker JW, Downes M, Yong WH, tion of TR4 in the development of new diagnostic and therapeutic Evans RM, Heaney AP. Evidence for orphan nuclear receptor TR4 in the etiology of Cushing disease. Proc Natl Acad Sci USA 2013;110:8555–60. approaches for the treatment of NSCLC. [17] Fang F, Zheng Q, Zhang J, Dong B, Zhu S, Huang X, Wang Y, Zhao B, Li S, Xiong H, Chen J, Wu N, Song SW, Chang C, Yang Y. Testicular orphan nuclear receptor Conflict of interest statement 4-associated protein 16 promotes non-small cell lung carcinoma by activating and blocking testicular orphan nuclear receptor 2. Oncol Rep 2013;29:297–305. The authors declare no conflict of interest. [18] Yang Y, Wang X, Dong T, Kim E, Lin WJ, Chang C. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. J Biol Chem 2003;278: Acknowledgements 7709–17. [19] van Schaick HS, Rosmalen JG, Lopes da Silva S, Chang C, Burbach JP. Expression This work was supported partially by Beijing Natural Sci- of the orphan receptor TR4 during brain development of the rat. Brain Res Mol ence Foundation (Grant no. 7112025), Peking University (PKU) Brain Res 2000;77:104–10. [20] Young WJ, Smith SM, Chang C. Induction of the intronic enhancer of the 985 Special Funding for Collaborative Research with PKU Hospi- human ciliary neurotrophic factor receptor (CNTFRalpha) gene by the TR4 tals, the National Natural Science Foundation of China (Grant no. orphan receptor. A member of steroid receptor superfamily. J Biol Chem 81350028), the National High Technology Research and Devel- 1997;272:3109–16. [21] Lee YF, Pan HJ, Burbach JP, Morkin E, Chang C. Identification of direct repeat opment Program of China (863 Program, no. 2012AA02A502), 4 as a positive regulatory element for the human TR4 orphan receptor. A and the Beijing Municipal Science & Technology Commission (no. modulator for the thyroid hormone target genes. J Biol Chem 1997;272: Z111107067311018). 12215–20. [22] Yang DR, Ding XF, Luo J, Shan YX, Wang R, Lin SJ, Li G, Huang CK, Zhu J, Chen Y, Lee SO, Chang C. Increased chemosensitivity via targeting testicular nuclear Appendix A. Supplementary data receptor 4 (TR4)-Oct4-interleukin 1 receptor antagonist (IL1Ra) axis in prostate cancer CD133+ stem/progenitor cells to battle prostate cancer. J Biol Chem 2013;288:16476–83. Supplementary data associated with this article can be found, in [23] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial–mesenchymal transitions the online version, at http://dx.doi.org/10.1016/j.lungcan.2015.06. in development and disease. Cell 2009;139:871–90. 007 [24] Gavert N, Ben-Ze’ev A. Epithelial–mesenchymal transition and the invasive potential of tumors. Trends Mol Med 2008;14:199–209. [25] Zeisberg M, Neilson EG. Biomarkers for epithelial–mesenchymal transitions. J References Clin Invest 2009;119:1429–37. [26] Zhang HJ, Wang HY, Zhang HT, Su JM, Zhu J, Wang HB, Zhou WY, Zhang H, Zhao [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: Cancer J Clin MC, Zhang L, Chen XF. Transforming growth factor-beta1 promotes lung adeno- 2014;64:9–29. carcinoma invasion and metastasis by epithelial-to-mesenchymal transition. [2] Saji H, Tsuboi M, Yoshida K, Kato Y, Nomura M, Matsubayashi J, Nagao T, Kak- Mol Cell Biochem 2011;355:309–14. ihana M, Usuda J, Kajiwara N, Ohira T, Ikeda N. Prognostic impact of number [27] Strauss GM. Adjuvant chemotherapy of lung cancer: methodologic issues and of resected and involved lymph nodes at complete resection on survival in therapeutic advances. Hematol Oncol Clin North Am 2005;19:263–81, vi. non-small cell lung cancer. J Thorac Oncol 2011;6:1865–71. [28] Lin SJ, Zhang Y, Liu NC, Yang DR, Li G, Chang C. Minireview Pathophysiological [3] Tonnies M, Pfannschmidt J, Bauer TT, Kollmeier J, Tonnies S, Kaiser D. Metasta- roles of the TR4 nuclear receptor: lessons learned from mice lacking TR4. Mol sectomy for synchronous solitary non-small cell lung cancer metastases. Ann Endocrinol 2014;28(6):805–21 (me20131422). Thorac Surg 2014;98(1):249–56. [29] O’Geen H, Lin YH, Xu X, Echipare L, Komashko VM, He D, Frietze S, Tanabe O, Shi [4] Hirose T, Fujimoto W, Tamaai T, Kim KH, Matsuura H, Jetten AM. TAK1: molec- L, Sartor MA, Engel JD, Farnham PJ. Genome-wide binding of the orphan nuclear ular cloning and characterization of a new member of the nuclear receptor receptor TR4 suggests its general role in fundamental biological processes. BMC superfamily. Mol Endocrinol 1994;8:1667–80. Genomics 2010;11:689. [5] Blumenthal GM, Karuri SW, Zhang H, Zhang L, Khozin S, Kazandjian D, Tang [30] Yang Y, Ahn YH, Chen Y, Tan X, Guo L, Gibbons DL, Ungewiss C, Peng DH, S, Sridhara R, Keegan P, Pazdur R. Overall response rate, progression-free sur- Liu X, Lin SH, Thilaganathan N, Wistuba II, Rodriguez-Canales J, McLendon vival, and overall survival with targeted and standard therapies in advanced G, Creighton CJ, Kurie JM. ZEB1 sensitizes lung adenocarcinoma to metastasis non-small-cell lung cancer: US food and drug administration trial-level and suppression by PI3K antagonism. J Clin Invest 2014;124:2696–708. patient-level analyses. J Clin Oncol 2015;33:1008–14. [31] Yue D, Li H, Che J, Zhang Y, Tseng HH, Jin JQ, Luh TM, Giroux-Leprieur E, Mo M, [6] Murphy SJ, Wigle DA, Lima JF, Harris FR, Johnson SH, Halling G, Asiedu MK, Seto Zheng Q, Shi H, Zhang H, Hao X, Wang C, Jablons DM, He B. Hedgehog/Gli pro- CT, Terra S, Kosari F, Peikert T, Yang P, Aubry MC, Vasmatzis G. Genomic rear- motes epithelial–mesenchymal transition in lung squamous cell carcinomas. J rangements define lineage relationships between adjacent lepidic and invasive Exp Clin Cancer Res 2014;33:34. components in lung adenocarcinoma. Cancer Res 2014;74:3157–67. [32] Lee YF, Young WJ, Lin WJ, Shyr CR, Chang C. Differential regulation of direct [7] Kim E, Liu NC, Yu IC, Lin HY, Lee YF, Sparks JD, Chen LM, Chang C. Metformin repeat 3 vitamin D3 and direct repeat 4 thyroid hormone signaling pathways inhibits nuclear receptor TR4-mediated hepatic stearoyl-CoA desaturase 1 gene by the human TR4 orphan receptor. J Biol Chem 1999;274:16198–205. expression with altered insulin sensitivity. Diabetes 2011;60:1493–503. [33] Shyr CR, Hu YC, Kim E, Chang C. Modulation of estrogen receptor- [8] Kang HS, Okamoto K, Kim YS, Takeda Y, Bortner CD, Dang H, Wada T, Xie W, Yang mediated transactivation by orphan receptor TR4 in MCF-7 cells. J Biol Chem XP, Liao G, Jetten AM. Nuclear orphan receptor TAK1/TR4-deficient mice are 2002;277:14622–8. 328 L. Zhang et al. / Lung Cancer 89 (2015) 320–328

[34] Atmaca A, Al-Batran SE, Wirtz RM, Werner D, Zirlik S, Wiest G, Eschbach [36] Chen LM, Wang RS, Lee YF, Liu NC, Chang YJ, Wu CC, Xie S, Hung YC, Chang C, Claas S, Hartmann A, Ficker JH, Jager E, Brueckl WM. The validation C. Subfertility with defective folliculogenesis in female mice lacking testicular of estrogen receptor 1 mRNA expression as a predictor of outcome in orphan nuclear receptor 4. Mol Endocrinol 2008;22:858–67. patients with metastatic non-small cell lung cancer. Int J Cancer 2014;134: [37] Chen YT, Collins LL, Chang SS, Chang C. The roles of testicular orphan nuclear 2314–21. receptor 4 (TR4) in cerebellar development. Cerebellum 2008;7:9–17. [35] Brueckl WM, Al-Batran SE, Ficker JH, Claas S, Atmaca A, Hartmann A, [38] Collins LL, Lee YF, Ting HJ, Lin WJ, Liu NC, Meshul CK, Uno H, Bao BY, Rieker RJ, Wirtz RM. Prognostic and predictive value of estrogen receptor 1 Chen YT, Chang C. The roles of testicular nuclear receptor 4 (TR4) in male expression in completely resected non-small cell lung cancer. Int J Cancer fertility–priapism and sexual behavior defects in TR4 knockout mice. Reprod 2013;133:1825–31. Biol Endocrinol 2011;9:138.