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Suppressor PAQR3 associated with the clinical significance and prognosis in esophageal squamous cell carcinoma

Progestin and adipoQ receptor family member 3 (PAQR3) is a novel tumor suppressor; however, its function in esophageal cancer is not well understood. The present study explored the association between PAQR3, and the survival and clinical phenotype in patients with esophageal squamous cell carcinoma (ESCC). The expression of PAQR3 in 80 cases of ESCC and its corresponding adjacent tissues was detected by reverse transcription-quantitative polymerase chain reaction. The results demonstrated that PAQR3 expression in cancer tissues was significantly lower compared with that in adjacent tissues. Clinicopathological analysis indicated that PAQR3 expression was significantly correlated with ethnicity (P=0.032), tumor length (P=0.019), lymph node metastasis (P=0.011) and local recurrence (P=0.009). Notably, the Kaplan-Meier survival curve demonstrated that a decrease in PAQR3 expression was associated with poor prognosis in patients with ESCC. Multivariate analysis indicated that PAQR3 expression was an independent prognostic indicator for patients with ESCC. PAQR3 may serve an important role in the progress of ESCC and become a potential candidate for ESCC targeted therapy.


Introduction
Esophageal cancer (EC) is one of the most common types of malignancy globally, including esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. In China, >90% of ECs are ESCC (1). Currently, the morbidity of EC is increasing annually and the rate of metastasis remains high with a poor prognosis (2). The diagnosis of EC usually occurs in the middle and late stages due to the lack of symptoms in the early stage, and as a result, the 5-year survival of patients with EC is 18-30% (3). The RAS/rapidly accelerated fibrosarcoma (RAF)/extracellular signal-regulated kinase (ERK) signaling pathway is critical for the malignant biological behavior of ESCC (4). This pathway can be activated in multiple ways, and has an important role in the proliferation, metastasis and infiltration of ESCC (5). Therefore, the identification of molecules that specifically inhibit activation of the RAS/RAF/ERK signaling pathway may enable the development of novel treatments for ESCC.
Raf kinase trapping to Golgi apparatus [also known as progestin and adipoQ receptor family member 3 (PAQR3)] is a type III seven-transmembrane protein with an extracellular N-terminus and a cytosolic C-terminus determined in the membrane of the Golgi apparatus (6). Binding of PAQR3 to B-Raf and C-Raf kinases anchors cytosolic RAF to the Golgi apparatus, and prevents the binding of RAF to RAS (upstream) and MEK (downstream) (7). Blocking signal transduction from RAS inhibits the activation of the Ras/Raf/MEK/ERK signaling pathway, and consequently affects the proliferation and malignant transformation of cells, as well as the development and progression of cancer (8,9). Therefore, PAQR3 is considered a negative regulator that suppresses RAS/RAF/ERK pathway activation-dependent tumors (10,11). A study by Wang et al (12) determined that kinases in the Raf/MEK/ERK signaling pathway were abnormally activated in various tissues of PAQR3 gene knockout mice. These knockout mice had significantly increased proliferation of epidermal keratinocytes, and PAQR3 gene deletion promoted the development and progression of dimethylbutylamine/12-Otetradecanoyl-phorbol-12-acetate-induced skin cancer. Other studies have reported downregulated expression of PAQR3 in liver (10), gastric (13,14), colon (15) and breast cancer (11), and determined that this downregulation was associated with tumor progression, metastasis and prognosis. However, the role and mechanism underlying the action of PAQR3 in ESCC has not been reported. Therefore, the purpose of the present study was to examine the association between PAQR3 and the prognosis and clinicopathological characteristics of patients with ESCC, and to increase the understanding of the molecular functions of PAQR3.

Materials and methods
Ethical statement. All selected patients agreed to participate in the present study and signed informed consent forms. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ürümqi, China), and was implemented in accordance with ʻThe Declaration of Helsinkiʼ.
Tissue specimens. A total of 80 histopathological specimens from patients surgically diagnosed with ESCC between January and March 2012 were collected from the First Affiliated Hospital and Affiliated Tumor Hospital of Xinjiang Medical University. All patients had undergone radical surgery and had not received prior treatments. The clinicopathological data of these patients, including age, sex, ethnic group, tumor location, tumor length, tumor type, degree of differentiation, number of lymph node metastases and T staging, were obtained from the hospitals. A total of 40 male and 40 female patients (mean age, 54.7±12.6 years) underwent tumor resection. Tumor and paracancerous histological normal tissue (PCHNT) specimens were obtained at the same time during surgery, and were stored at -80˚C. PCHNT is defined as esophageal tissue that is located ≥5 cm away from the tumor margin and is confirmed to be normal esophageal tissue by postoperative histopathology. Patient follow-up was conducted from the day following surgery, and anastomotic or regional lymph node recurrence was diagnosed by clinical or pathological examination at 3 months post-operation. Local recurrence within 3 months following surgery was considered a part of the initial treatment process. Distant metastasis was defined as distant metastatic disease diagnosed by clinical examination or imaging. Disease-free survival (DFS) time was defined as the time from the day following surgery to the first recurrence or metastasis. Overall survival (OS) time was defined as the time from day 1 post-operation to last follow-up or mortality. Complete follow-up data included the time and location of recurrence or metastasis, and condition and time of survival. All patients were followed up until March 2017 and the duration of follow-up was 9-62 months (median, 28 months).
Cell culture. Human ESCC cell lines (ECA-109 and TE-1) were obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). They were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37˚C and in a 5% CO 2 atmosphere.
Plasmid and transfection. Full-length human PAQR3 cDNA was amplified using a polymerase chain reaction (PCR), as previously described (2). PCR products were purified and inserted into a pcDNA3.1(+) vector. The PAQR3-expressing plasmid (pcDNA3.1/PAQR3) or an empty vector were transfected into ECA-109 and TE-1 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. A total of 24 h after transfection, cells were examined for gene expression, proliferation and cell cycle distribution. For stable transfection, 1x10 5 ECA-109 cells were transfected with the pcDNA3.1/PAQR3 plasmid (0.5 µg) and 24 h later. Subsequent to culturing for another 24 h, the cells were incubated at 37˚C in DMEM with 15% FBS containing 600 µg/ml G418 (Sigma-Aldrich; Merck KGaA) for 3 weeks. The G418-resistant colonies were pooled together and used for colony formation and tumorigenic studies.
RNA extraction and reverse transcriptase-quantitative PCR (RT-qPCR) analysis of tumor specimens. Total RNA was extracted using the TRIzol ® (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's one-step instructions. cDNA was synthesized from 3 µg total RNA using the M-MLV reverse transcriptase: 4 µl 5X buffer, 2 µl 10 mM dNTPs, 1 µl RNA inhibitor and 1 µl Reverse transcriptase from the QIAGEN74903-RNeasy Plantmini kit 74903 (cat. no. M1705; Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. GAPDH was used as the reference gene for RT-qPCR. The forward and reverse primers for PAQR3 were: 5'-TGT CGA AGA TGG ATG GCA TTA GA-3' and 5'-ACC TGA CGC CAG TAG TTA TTA CA-3', respectively. The forward and reverse primers for GAPDH were: 5'-TGT TGC CAT CAA TGA CCC CTT-3' and 5'-CTC CAC GAC GTA CTC AGC G-3' , respectively. Gene-specific amplifications of the 20 µl PCR mixture were performed using the ABI 7500HT real-time PCR system (Thermo Fisher Scientific, Inc.). The PCR mixture contained the following: 1 µl DNA, 2X SYBR ® Select Master mix (cat. no. AT311; Beijing TransGen Biotech Co., Ltd., Beijing, China), and 10 µl each of forward and reverse primers (10 µM). The PCR amplification conditions were as follows: 95˚C for 30 sec, 95˚C for 60 sec and 72˚C for 60 sec for a total of 45 cycles, followed by 72˚C for 7 min. DNA concentration was approximately doubled following each cycle of denaturation, annealing and elongation. The cycle quantification (Cq) of each sample was calculated, and the relative expression of the gene was calculated using the 2 -ΔΔCq (16) method.
Immunohistochemistry. Formalin-fixed paraffin-embedded ESCC tissues (as aforementioned) were sectioned (4-µm) for immunohistochemical analysis. For antigen retrieval, the slides were immersed in EDTA [1 mmol/l, (pH 8.0)] and boiled 60˚C for 20 min in a microwave oven. Following rinsing with PBS 3 times, endogenous peroxidase was blocked using 0.3% hydrogen peroxide for 15 min at room temperature. The slides were incubated with the PAQR3 primary antibody (1:100; cat. no. ab174327; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), in a humidified chamber at 4˚C overnight. Following additional washing with PBS three times, the sections were sequentially incubated with horseradish peroxidase-conjugated the anti-rabbit secondary antibody PV-6001 (1:50; Envision™ detection kit; cat. no. GK500705; Gene Tech Co., Ltd., Hong Kong, China) at 37˚C for 30 min, and then washed three times with PBS. Finally, 3,3'-diaminobenzidine tetrahydrochloride was used for signal development and then the sections were lightly counterstained with 20% hematoxylin at room temperature for 20 min. The paraffin slides were dehydrated (using 95% ethanol every 2 h) and mounted on coverslips. For the negative controls, PBS was used in place of the primary antibody. A light microscope was used at a magnification of x200. Immunohistochemistry scores of 0-4 were considered to be negative/low expression and 5-11 was considered to be normal/high expression. Anti-PAQR3 (ab174327; Abcam, Cambridge, UK).
Statistical analysis. All statistical analyses were performed using the SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Count data were analyzed using the χ 2 test and Fisher's exact test. Paired-samples Student's t-test was used to compare mRNA/protein expression of PAQR3 in ESCC tissues with paired adjacent non-tumor tissue samples. Spearman's correlation analysis was used to compare the mRNA/protein expression of PAQR3 in ESCC tissues. Survival analysis was performed using the Kaplan-Meier estimator method, and compared using the log rank test. Prognostic analysis was performed using the Cox proportional hazards regression model. P<0.05 was considered to indicate a statistically significant difference

PAQR3 mRNA expression is downregulated in ESCC tissue.
The levels of PAQR3 mRNA expression in the 80 ESCC and corresponding PCHNT specimens were measured using RT-qPCR. The mRNA expression of PAQR3 was significantly lower in ESCC tissues compared with that in the corresponding PCHNT tissues (P<0.0001; Fig. 1A). A total of 71.25% of subjects (57/80) had significantly lowered PAQR3 mRNA expression in their ESCC tissues compared with the PCHNT tissues (Fig. 1B). Low PAQR3 mRNA expression was defined as ESCC/PCHNT <0.5. All 80 pairs of specimens were independently measured twice.
PAQR3 protein expression is downregulated in ESCC tissue. PAQR3 protein was detected by immunohistochemical staining. The normal and adjacent tissues stained positive, in contrast with the staining of PAQR3 in cancer tissues, which was mostly negative (Fig. 2A). PAQR3 protein expression scores were also significantly reduced in EC tissues compared with adjacent normal tissues (P<0.0001; Fig. 2B). The protein levels of PAQR3 were significantly correlated with its mRNA levels (r=0.783; P=0.014; Fig. 2C).
Association between PAQR3 expression and clinicopathological characteristics of ESCC. The association between

PAQR3 suppresses epithelial-mesenchymal transition (EMT) features in human ESCC cells.
Previous studies have revealed that PAQR3 is able to inhibit Ras/Raf/MEK/ERK signaling cascades. Whether PI3K/AKT signaling pathways were also affected by PAQR3 in ESCC cells was explored (15). Western blot analysis of p-Akt and Akt proteins in ECA-109 and TE-1 cells transfected with the PAQR3-expressing plasmid or vector was performed. However, the activation of Akt was not altered by PAQR3 overexpression (Fig. 4A). The effect of PAQR3 on EMT was also analyzed, a critical step for tumor migration and metastasis (10). In ECA-109 and TE-1 cells, overexpression of PAQR3 suppressed EMT features as demonstrated by the marked increase in epithelial marker, E-cadherin (Fig. 4B).

Discussion
PAQR3 is a member of the PAQR family (17). A search of this gene in the world's largest cancer microarray database, Oncomine (https://www.oncomine.org/resource/login.html), demonstrated that PAQR3 is associated with multiple cancer types, including breast cancer, colon cancer, gastric cancer, leukemia and lymphoma. In addition, the expression of PAQR3 mRNA is downregulated in numerous cancer types, including gastric cancer, but upregulated in a number of cancer types of the circulatory system, including leukemia and lymphoma (18). However, there are no relevant data on the expression of PAQR3 in EC. In the present study, the levels of PAQR3 mRNA expression in 80 ESCC and corresponding PCHNT tissue specimens were measured. In addition, a complete follow-up database was constructed to examine the association between PAQR3 and the prognosis and clinicopathological characteristics of patients with ESCC, and to increase the understanding of the diagnosis and prognosis of EC, a highly prevalent disease in Xinjiang, China (19).
RT-qPCR analysis demonstrated that PAQR3 mRNA expression was significantly lower in ESCC tissues compared with in PCHNT tissues, which was consistent with the data in other cancer types, including breast cancer (11), colon cancer (15) and gastric cancer (13), demonstrating that PAQR3 may be an ESCC suppressor gene. The tumor suppressive function of PAQR3 is primarily achieved by inhibiting activation of the ERK signaling pathway (9). The broad anticancer activity of PAQR3 is mediated through the alteration of multiple different signaling pathways (20). PAQR3 overexpression decreased the phosphorylation of ERK1/2 in ESCC cells, without affecting the activation of Akt (21). PAQR3 has demonstrated the ability to interfere with multiple aspects of tumor biology, including proliferation, migration, invasion, and EMT (22,23). In the present study, it was revealed that the overexpression of PAQR3 suppressed EMT features as demonstrated by the marked increase in the epithelial marker E-cadherin.
In the present study, convincing evidence that PAQR3 is a novel genetic marker associated with the progression, proliferation, infiltration and prognosis of EC has been indicated. Analysis of the clinicopathological characteristics of 80 patients with EC indicated that the level of PAQR3 mRNA expression was associated with ethnic group, status of lymph node metastasis and tumor length. In particular, the level of PAQR3 mRNA expression was significantly higher among Han patients with ESCC than among those of the Kazakh and Uygur ethnic groups. Although current studies have indicated improved short-term efficacy of treatment in Kazakh and Uygur patients with ESCC, their prognosis and long-term survival were lower compared with those of Han patients. Whether this characteristic is associated with the difference in PAQR3 mRNA expression between ethnic groups will require further studies to confirm. In the present study, it was demonstrated that an association between patients with low PAQR3 expression, and lymph node metastasis and poor prognosis, indicating that PAQR3 may be a potential tumor suppressor. The total of 80 histopathological specimens was a limiting factor for this study as the number of samples after grouping was too small, reducing the statistical reliability. In the future study, the sample size should be increased to confirm the credibility of the findings of the present study.
The present study demonstrated that low PAQR3 expression was associated with lymph node metastasis and survival. Multivariate prognostic analysis of the association between PAQR3 expression and the status of lymph node metastasis indicated reduced DFS in patients with low PAQR3 expression and lymph node metastasis. Thus, lymph node metastasis or low PAQR3 expression may be adverse prognostic factors for EC. The combined evaluation of PAQR3 expression and lymph node status may be helpful in the prognosis of patients, and may provide insights into postoperative adjuvant therapy strategies. Notably, a previous study have demonstrated that PAQR3 expression may be associated with the susceptibility of breast cancer to epirubicin, whereby PAQR3 overexpression enhances the susceptibility of breast cancer cells to epirubicin via caspase-associated signaling pathways and epirubicin inhibition-induced ERK activation (24). This data indicated that PAQR3 may be a novel cancer treatment target that can be used to evaluate radiation therapy and chemotherapy sensitivity. However, further studies are required to determine the effect of PAQR3 on the sensitivity of ESCC to treatment.
In conclusion, the present study demonstrated that PAQR3 downregulation is associated with the progression of tumor recurrence and increased lymph node metastasis in EC, and is significantly associated with reduced survival of patients with EC. This data demonstrated that PAQR3 is a tumor suppressor gene associated with the survival and prognosis of esophageal malignancies. PARQR3 may be a potential valuable prognostic factor for ESCC, and a novel predictor of radiation and chemotherapy sensitivity. To the best of our knowledge, the present study is the first to examine the association between PAQR3 and ESCC. However, further in vitro and in vivo studies are required to elucidate the molecular mechanism underlying PAQR3 in the regulation of ESCC.