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Overexpression of G protein-coupled receptor 31 as a poor prognosticator in human colorectal cancer

AIM To investigate the expression of G protein-coupled receptor 31 (GPR31) and its clinical significance in human colorectal cancer (CRC). METHODS To determine the association between the GPR31 expression and the prognosis of patients, we obtained paraffin-embedded pathological specimens from 466 CRC patients who underwent initial resection. A total of 321 patients from the First Affiliated Hospital of Sun Yat-sen University from January 1996 to December 2008 were included as a training cohort, whereas 145 patients from the Sixth Affiliated Hospital of Sun Yat-sen University from January 2007 to November 2008 were included as a validation cohort. We examined GPR31 expression levels in CRC tissues from two independent cohorts via immunohistochemical staining. All patients were categorized into either a GPR31 low expression group or a GPR31 high expression group. The clinicopathological factors and the prognosis of patients in the GPR31 low expression group and GPR31 high expression group were compared. RESULTS We compared the clinicopathological factors and the prognosis of patients in the GPR31 low expression group and GPR31 high expression group. Significant differences were observed in the number of patients in pM classification between patients in the GPR31 low expression group and GPR31 high expression group (P = 0.007). The five-year survival and tumor-free survival rates of patients were 84.3% and 82.2% in the GPR31 low expression group, respectively, and both rates were 59.7% in the GPR31 high expression group (P < 0.05). Results of the Cox proportional hazard regression model revealed that GPR31 upregulation was associated with shorter overall survival and tumor-free survival of patients with CRC (P < 0.05). Multivariate analysis identified GPR31 expression in colorectal cancer as an independent predictive factor of CRC patient survival (P < 0.05). CONCLUSION High GPR31 expression levels were found to be correlated with pM classification of CRC and to serve as an independent predictive factor of poor survival of CRC patients.


INTRODUCTION
Based on the statistics of the American Cancer Society (ACS), colorectal cancer (CRC) has become the second common cause of cancer deaths [1] . CRC causes over 600 thousand deaths each year all over the world and is the fifth common cause of cancer deaths in China [2,3] . The incidence rate of CRC is predicted to increase both in urban and rural areas in the next few years [4] . The prognosis of CRC has improved recent years because of the continued advancements in CRC diagnosis and treatment [5,6] . However, many CRC patients are incurable because of various reasons [7,8] . Nowadays, the American Joint Committee on Can cer (AJCC) staging system is still the gold standard to predict the prognosis of CRC patients. However, the AJCC staging system has certain limitations due to the heterogeneity of CRC. Other reliable biomarkers, which can provide guidance to the treatment of CRC and help predict treatment prognosis, have gradually received interest of clinicians [9,10] . G proteincoupled receptor 31 (GPR31) is a member of the G proteincoupled receptor (GPCR) superfamily and has been identified as a target receptor for 12(S)hydroxyeicosatetraenoic acid [12(S)HETE] [11] . 12(S) HETE is an eicosanoid product of arachidonate meta bolism by the enzyme 12lipoxygenase (12LOX), which was first demonstrated by Hamberg and Samuelsson [12] . 12(S)HETE plays an important role in many physiological and pathological processes, such as cell growth, adhesion, differentiation, angiogenesis, in flammation, atherosclerosis and cancer promotion [1317] .
Previous studies have demonstrated that 12(S)HETE promotes cell migration during tumor progression by eliciting a wide variety of physiological and pathological responses [1822] . It is showed that 12(S)HETE could induce changes of cancer cell cytoskeleton and result in enhanced tumor invasion and motility [23,24] , which in turn enhances tumor cell motility [16] . Exogenous addition of 12(S)HETE induces overexpression of proteinases [2527] , vascular endothelial growth factor [28] , integrins [23,29] and fibronectin [30] in cancer cells, thereby prolonging cell sur vival [21,31] . 12(S)HETE promotes adhesion of tumor cell by inducing the nondestructive retraction of monolayers in endothelial cells [32,33] . 12(S)HETE also promotes tube formation by enhancing the motility of isolated endo thelial cells [34] . The diverse biological effects of this im portant proinflammatory metabolite suggest that 12(S)-HETE potentially acts through a cognate receptor, which was identified as GPR31 [11] .
In this present study, we aimed to elucidate the asso ciation between the expression level of GPR31 and CRC progression.

Patients
To determine the association between the expression of GPR31 and the prognosis of CRC patients, we obtained tissue specimens from 466 CRC patients who underwent surgery. A total of 321 patients treated at the First Affiliated Hospital of Sun Yatsen University (SYSU) from January 1996 to December 2008 were included as a trai ning cohort, whereas 145 patients treated at the Sixth Af filiated Hospital of SYSU from January 2007 to November 2008 were included as a validation cohort. The patients who underwent initial colorectal resection were included in this study. Patients who received neoadjuvant therapy were excluded. Abdominal ultrasonography, chest Xray, magnetic resonance imaging (MRI), computed tomography (CT), bone scans or positron emission tomo graphycomputed tomography (PETCT) were performed to identify tumor recurrence and distant metastasis. Clinical data including demographics, surgical method, tumor location, differentiation, TNM status and follow up data were collected from the CRC database of each hospital. MRI and/or CT were used to evaluate the pa tients at 3, 6, 9, 12, 18 and 24 mo after surgery in the first two years, and annually after that. The primary endpoint of this study was described as the overall survival (OS) and was defined as the time interval between the first surgery to clinical death.

TMA construction and IHC analysis
Tissue microarrays (TMA) were considered as an array fashion to facilitate multiplex analysis of histology [35] . In our study, the TMAs were constructed respectively by two skilled researchers. During the process of TMA experiment, the central portion of neoplasm tissue was selected by two skilled pathologists, after which two pieces of each sample were picked out and deposited into the tissue array instrument (Beecher Instruments, Alphelys, France). TMA blocks were subsequently sliced into 5μm thick sections before immunohistochemistry (IHC) staining. TMA slides were deparaffinized, rehy drated, exposed to the antigen retrieval system, endo genous peroxidase blocked, primary antibody incubated, stained with diaminobenzidine and counterstained with hematoxylin according to the methods of our previous study [2] . The primary antibody of GPR31 (Santa Cruz Biotechnology, Inc., Dallas, TX, United States) was used in a dilution of 1:200.

Evaluation of IHC analysis
Immunoreactivity of GPR31 protein was examined accor ding to previous studies [1,36] . Spots of TMA were scored ranging from 0 to 3 according to the intensity by two separated researchers. The percentage of positive cancer cells was described as 0%, 5%, …, 95%, 100%. The H score (0 to 300) was determined by calculating the sum of the product of the intensity score and the proportion of the corresponding stained area for each intensity score. The average H score was calculated by two professional researchers finally.

Cut-off point determination
Receiver operating characteristic (ROC) curve was used to determine the cutoff score according to previous studies [1,37] . The H score which met both highest sensi tivity and highest specificity was determined as the final cutoff point. Neoplasm tissues were described as "low expression" if they had scores lower than or equal to the cutoff point, whereas neoplasm tissues with scores above the cutoff point were designated as "high expression". Clinicopathological features including differentiation, pT status, pN status, pM status, TNM stage, and survival were dichotomized for ROC curve analysis, the same as our previous study [1] .

Statistical analysis
Relationship between GPR31 protein levels and the clinico pathological characteristics were analyzed using methods according to our previous study [1] : Chisquare test for categorical variables, Student's ttest for quantitative data which meet homogeneity and normality, Kaplan Meier curves with a logrank test for the correlation of the GPR31 and patient survival, and forward stepwise method for construction of a multivariate Cox propor tional hazard regression model. SPSS (v19.0, Chicago, IL, United States) was used for our statistical analyses. a P < 0.05 was considered statistically significant in this study.

Patient clinical features
The baseline clinical features of the two cohorts were listed in Table 1. Four hundred and sixtysix patients with CRC were included for analysis. Three hundred and twentyone patients were included in the training cohort and 145 in the validation cohort. Two hundred and sixty five males and 201 females were recruited. There were for each intensity score. ROC curve analysis was used to figure out the cutoff point of the different patterns of GPR31 expression. According to the generated ROC curve, the cutoff point of GPR31 expression levels was 185. H score more than 185 was categorized as "high expression", otherwise it was categorized as "low expression". In the training cohort, 124 samples were ca tegorized as "high expression" and 197 were categorized as "low expression" based on the H scores. Thirty nine were categorized as "high expression" and 106 were categorized as "low expression" in the validation cohort. Figure 1 shows the representative IHC staining for GPR31 in CRC tissue and adjacent normal colorectal mucosa. Figure 2 shows the corresponding area under curve (AUC).

Correlation between GPR31 level and clinicopathological characteristics
Advanced correlation analyses revealed that GPR31 level was notably associated with pM classification in the 99 early stage patients (stage Ⅰ or Ⅱ) and 367 advanced stage patients (stage Ⅲ or Ⅳ). For all cases, the mean followup period was 58.4 mo (range, 0.5 to 123.5 mo). One hundred and seventynine patients died during the followup period. In the training cohort, 173 patients were male (53.9%) and 148 (46.1%) were female, with an average age of 58.7 years. Tumors were located in the colon in 156 (48.6%) patients and the rectum in 163 (50.8%) patients (two patients had no record). The mean followup period was 60.1 mo. In the validation cohort, 92 (63.4%) patients were male, and 53 (36.6%) were female, with an average age of 57.3 years. These patients included 64 (44.1%) colon cancer patients and 81 (55.9%) rectal cancer patients and had a mean followup period of 55.42 mo.

Cut-off point of GPR31 expression levels
The H score, ranging from 0 to 300, was determined by calculating the sum of the product of the intensity score and the proportion of the corresponding stained area training cohort (P = 0.007) ( Table 1). pT classification (P = 0.006) and pM classification (P = 0.018) were significantly different between high-and low-GPR31 expressing patients in the validation cohort, and results showed that strong GPR31 expression was highly cor relative with neoplasm metastasis (Table 1).

GPR31 level as a novel prognostic biomarker
In the training cohort, the survival analysis showed that a high GPR31 level was correlated with decreased OS ( b P < 0.001, Figure 3 and Table 2). In addition, the following clinical characteristics were also identified as prognostic factors: age (P = 0.010), carcinoembryonic antigen (CEA) (  Table 2).
GPR31 expression levels and the clinicopathological characteristics that were found to be significantly associated above were then examined via multivariate ana lysis. Multivariate analysis revealed that high GPR31 level was a significant independent prognostic factor for poor OS [hazard ratio (HR): 1.896; 95% confidence

DISCUSSION
Prognostic value of specific biomarkers was found to be more accurate than that of the AJCC staging system [3840] . However, cancer heterogeneity limited the use of bio markers and had produced disagreeing results [41,42] . Thus, largescale prospective studies are required to validate the specificity and prognostic value of biomarkers.
G proteincoupled receptors (GPCRs) belong to a superfamily of the cell surface signaling proteins and are found only in eukaryotes. GPCRs mediate biological effects by coupling with G proteins. Currently, GPCRs are the target of approximately 40% of all modern medicinal drugs [43,44] . As a result, the molecular mechanisms and functions of GPCRs have been a hotspot in biomedical research.
Gprotein coupled receptor 31 (GPR31), also known as 12(S)HETE receptor, is a protein encoded by the GPR31 gene that is located on chromosome 6q27 and consists of 319 amino acids [45] . GPR31 plays an im portant role in a variety of physiological and pathological processes, including inflammation and tumor progres sion [45] . GPR31 was first discovered in 1997, but its critical role as a 12(S)HETEspecific receptor was first identified in 2011 [11] . 12(S)HETE promotes tumor cell proliferation and metastasis directly or indirectly. Studies have de monstrated that 12LOX upregulation leads to increased synthesis of 12(S)HETE in cancer cells [21] . Treatment with LOX inhibitors, such as baicalin, can increase the activity of apoptotic proteases and lead to downregulation of the Bcl protein. In turn, Bcl downregulation promotes cell survival by inhibiting the expression of 12LOX, there by leading to cell cycle arrest and apoptosis. This effect can be reversed by exogenous addition of 12(S)HETE [46] . In addition to promoting tumor cell survival, 12(S) HETE can directly promote tumor metastasis by acting on vascular endothelial cells or inducing PKCdependent cytoskeleton rearrangement [16] . Furthermore, 12(S) HETE can promote the release of cathepsin B, disrupt the vascular endothelial basement membrane and promote penetration of blood vessels by tumor cells, thereby leading to tumor metastasis [25,47] . 12(S)HETE can also inhibit cadherin E expression, disrupt the lymphatic endothelial cell membrane and promote the migration of tumor cells from the lymphatic vessels. Inhibition of 12(S)-HETE can significantly suppress tumor cell lymph node metastasis [48,49] . GPR31 is a specific receptor for 12(S)-HETE. A study by Guo et al [11] of prostate cancer showed that 12(S) HETE promotes invasion by tumor cells by specifically targeting GPR31. In vitro studies have revealed that activation of the 12(S)HETE/GPR31 signaling pathway is a crucial factor that determines tumor invasion and metastasis [26,50] . Previous studies have shown that 12(S)HETE can activate the extracellular signal regulated kinase (ERK)1/2, mitogenactivated protein kinase kinase (MEK) and nuclear factor kappalightchainenhancer of activated B cells (NFκB) signaling pathways [19,22,51] by specifically binding to GPR31 [11] . GPR31 upregulation enhances the activation of ERK1/2, MEK and NF κB via 12(S)HETE, whereas GPR31 suppression can completely inhibit 12(S)HETEmediated activation of these signaling pathways. The ERK1/2, MEK and NFκB pathways are involved in most human physiological and pathological processes and serve as important regulatory factors affecting immune and inflammatory processes. Moreover, NFκB is an important tumor promoter [52] . 12(S)HETE binds to GPR31 on the cell membrane and activates NFκB by activating mitogenactivated protein kinases (MAPKs)/cJun Nterminal kinases (JNK)/ERK signaling [11] . NFκB signaling pathways influence tumor cell invasion and angiogenesis by regulating a variety of tumor metastasis or invasionrelated genes and cyto kine expression, including matrix metalloproteinases, urokinasetype plasminogen activator (UPA), interleukin (IL)-8, inflammatory mediators of intercellular adhesion molecules, monocyte chemokines and cyclooxygenase2 (COX2) [53] . Results of the present study showed that GPR31 expression in colorectal cancer tissue was significantly higher than that in normal mucosa and that GPR31 ex pression levels are closely related to distant metastasis of tumors, which are consistent with findings reported in previous studies [54] . Further univariate and multivariate analyses showed that patients with high GPR31 expres sion had a worse prognosis and decreased OS and DFS than patients that exhibited low GPR31 expression. These results indicate that GPR31 is a critical prognostic factor of OS and DFS in CRC patients and suggest that GPR31 is closely related to the occurrence, development and prognosis of CRC. And GPR31 may become a novel

Research background
G protein-coupled receptor 31 (GPR31) plays an important role in a variety of physiological and pathological processes, including inflammation and tumor progression. In this present study, we aimed to elucidate the association between the expression level of GPR31 and colorectal cancer (CRC) progression.

Research motivation
12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] plays an important role in cancer promotion. It potentially acts through GPR31. We aimed to elucidate the association between the expression level of GPR31 and CRC progression. We expect GPR 31 as one of reliable biomarkers can provide guidance to the treatment of CRC and help predict treatment prognosis.

Research objectives
GPR31 is a critical prognostic factor of overall survival and disease-free survival

ARTICLE HIGHLIGHTS
biomarker and therapeutic target for CRC. Although few studies have discussed the role of GPR31 in tumors, it is reasonable to believe that GPR31 plays a key regulatory role in tumor development and progression by mediating a specific "switch" effect by 12(S)-HETE. Further studies are warranted to elucidate the detailed mechanisms underlying GPR31 function, specifically the molecular mechanisms by which GPR31 expression affects carcino genesis process, such as tumor proliferation, differen tiation, migration and invasion in CRC.
There are some limitations in this study. In order to study the clinical value and role of GPR31 in CRC more accurately, patients were divided into a training cohort and a validation cohort for analysis. However, due to the small sample size, relatively long sample age, poor storage conditions, single research center and other factors, the results still need further verification.  Carbohydrate antigen 19-9. in CRC patients and is closely related to the occurrence, development and prognosis of CRC. GPR31 may become a novel biomarker and therapeutic target for CRC.

Research methods
We obtained paraffin-embedded pathological specimens from 466 CRC patients. And we examined GPR31 expression levels in CRC tissues from two independent cohorts via immunohistochemical staining. All patients were categorized into either the GPR31 low expression group or GPR31 high expression group. The clinicopathological factors and the prognosis of patients in the GPR31 low expression group and GPR31 high expression group were compared.

Research results
Results of the present study showed that GPR31 expression in colorectal cancer tissue was significantly higher than that in normal mucosa and that GPR31 expression levels are closely related to distant metastasis of tumors, which are consistent with findings reported in previous studies. Further univariate and multivariate analyses showed that patients with high GPR31 expression had a worse prognosis and decreased overall survival and diseasefree survival than patients that exhibited low GPR31 expression.

Research conclusions
We found that GPR31 was closely related to the occurrence, development, and prognosis of CRC. And GPR31 may become a novel biomarker and therapeutic target for CRC. Although few studies have discussed the role of GPR31 in tumors, it is reasonable to believe that GPR31 plays a key regulatory role in tumor development and progression by mediating a specific "switch" effect by 12(S)-HETE. Further studies are warranted to elucidate the detailed mechanisms underlying GPR31 function, specifically the molecular mechanisms by which GPR31 expression affects carcinogenesis process, such as tumor proliferation, differentiation, migration and invasion in CRC.

Research perspectives
High GPR31 expression levels were found to be correlated with pM classification of CRC and to serve as an independent predictive factor of poor survival of CRC patients.   affects carcinogenesis process, such as tumor proliferation, differentiation, migration and invasion in CRC.