Correlation of S1P1 and ERp29 expression to progression, metastasis, and poor prognosis of gallbladder adenocarcinoma

Changsha, China

Correlation of S1P1 and ERp29 expression to progression, metastasis, and poor prognosis of gallbladder adenocarcinoma

Lian-Wen Yuan, Dong-Cai Liu and Zhu-Lin Yang

Changsha, China

BACKGROUND:Gallbladder cancer (GBC) is one of the most aggressive malignant neoplasms with an extremely poor prognosis. Early diagnosis signif i cantly increases the survival rate. The present study was undertaken to evaluate the diagnostic and prognostic value of sphingosine-1-phosphate receptor 1 (S1P1) and endoplasmic reticulum protein 29 (ERp29) in benign and malignant gallbladder lesions and to develop a possible alternative treatment for GBC.

METHODS:A total of 100 gallbladder adenocarcinoma, 46 peritumoral, 30 gallbladder adenomatous, 15 gallbladder polyp, and 35 chronic cholecystitis tissues were included. S1P1 and ERp29 expressions were evaluated by immunohistochemistry. The correlation between S1P1 and ERp29 expression and tumor pathological features and prognosis was analyzed.

RESULTS:S1P1 positive rate was signif i cantly higher in gallbladder adenocarcinomas than that in peritumoral, adenomatous, polyp, and chronic cholecystitis tissues. On the contrary, ERp29 positive rate was signif i cantly lower in adenocarcinomas than that in peritumoral, adenomatous, polyp, and chronic cholecystitis tissues. Benign lesions with positive S1P1 or negative ERp29 expression showed moderate or severe atypical hyperplasia in the gallbladder epithelium. The overexpression of S1P1 or non-expression of ERp29 was signif i cantly associated with tumor differentiation, tumor mass, lymph node metastasis, and adenocarcinoma invasion. Univariate Kaplan-Meier analysis showed that the elevated S1P1 (P=0.008) or absence of ERp29 (P=0.043) was closely associated with decreased survival rate. Multivariate Cox regression analysis showed that S1P1 positive (P=0.004) or ERp29 negative (P=0.029) was an independent predictor of poor prognosis in gallbladder adenocarcinoma.

CONCLUSION:S1P1 overexpression or ERp29 absence is related to the carcinogenesis and progression, and may be potential biomarkers for early detection of gallbladder adenocarcinoma.

(Hepatobiliary Pancreat Dis Int 2013;12:189-195)

gallbladder cancer;gallbladder polyp; chronic cholecystitis; S1P1; ERp29

Introduction

Gallbladder cancer (GBC) is one of the most aggressive malignant neoplasms with an extremely poor prognosis. GBC is the fi fth most common gastrointestinal cancer in the United States, accounting for 46% of biliary tract cancers.[1]Meanwhile, the incidence of GBC is alarmingly increasing in China[2]and North Central India.[3]Early GBC detection assures proper treatment. Gallbladder surgery with partial liver and lymph dissection is currently the most common therapeutic strategy for GBC. However, only 10% of GBC patients are diagnosed at an early stage because of its asymptomatic nature. The characteristics of the gallbladder enable GBC to grow silently and therefore, most of the GBCs present at an advanced stage and have poor prognosis.[4]Generally, palliative chemotherapy and radiation therapy are not suff i cient and benef i cial for GBC patients.[4]Therefore, it is important to fi nd specif i c biomarkers for early diagnosis and to improve prognosis of GBC patients.

Sphingosine-1-phosphate (S1P) receptor 1 (S1P1), also known as S1PR1 or EDG1 (endothelial differentiation gene 1), is a G-protein-coupled receptor that binds to the lipid-signaling molecule S1P, acting as a key regulator of cell proliferation and apoptosis.[5,6]Recently, accumulated data implicated that S1P1 is linked to cancer by regulatingcell motility and migration, angiogenesis, survival, and vascular maturation.[7-11]The relationship between S1P1 expression and malignancy is contradictory. Yoshida and colleagues[12]found that down-regulation of S1P1 expression enhances the malignancy of glioblastoma by increasing cell proliferation and is correlated with poor survival. Whereas Arikawa et al[13]demonstrated that overexpression of S1P1 enhances B16F10 mouse melanoma cell migration and invasion. Tumor vessels induce strong S1P1 expression and siRNAs targeting S1P1 in mouse results in tumor growth suppression by vascular stabilization inhibition and angiogenesis.[14]The high expression of S1P1 is associated with shorter disease-specif i c survival and relapse time in estrogen receptor-positive breast cancer patients, implicating that S1P1 causes breast cancer progression.[15]Numerous studies have demonstrated the role of S1P1 in human cancers. However, the expression of S1P1 in human GBC and its clinical signif i cance have not been reported.

Endoplasmic reticulum (ER) protein 29 (ERp29) is a newly discovered ER protein that facilitates early processing of secretory proteins. This protein is directly associated with the folding and/or secretion of thyroglobulin.[16,17]ERp29 is highly expressed in several tumors, such as basal cell carcinoma[18]and lung cancer.[19]ERp29 is a radio-resistant factor that affects nasopharyngeal carcinoma,[20]implicating its pathological role in cancer progression. Moreover, Bambang et al[21]reported that overexpression of ERp29 signif i cantly inhibits cell proliferation and prevents tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells, indicating a negative association between ERp29 and cancer cell aggression. There is a recognized role of mesenchymalepithelial transition in facilitating distant metastasis,[22]ERp29 is essential for the promotion of distant metastasis through cancer progression because ERp29 drives mesenchymal-epithelial transition in mesenchymal breast cancer cells.[21]However, the clinical signif i cance of ERp29 in GBC remains unclear.

The present study examined the expression of S1P1 and ERp29 in tissues from surgery, including gallbladder adenocarcinoma, peritumoral, adenomatous, polyp, and chronic cholecystitis tissues. The correlations of S1P1 and ERp29 expressions with the histological characteristics and prognosis of GBC, including patients' survival rate were evaluated.

Methods

Patients

Resected or biopsy specimens were collected from Second Xiangya Hospital, Central South University between 1996 and 2006. The specimens included 100 cases of gallbladder adenocarcinoma, 46 peritumoral tissues taken from 100 adenocarcinomas, 30 gallbladder adenoma tissues, 15 gallbladder polyp tissues, and 35 chronic cholecystitis tissues. One hundred patients with GBC included 70 females and 30 males, with an average age of 52.4±11.3 years. All diagnoses were based on histological study. Among the 100 GBCs, histological subtypes included 36 well-differentiated, 29 moderately-differentiated, 25 poorly-differentiated, and 10 mucinous adenocarcinomas. Invasion was evaluated using the standard criteria for T-stages.[4]Among the 100 GBCs, 13 were T1, 33 T2, 34 T3, and 20 T4 stage. Moreover, 53 of the 100 patients had regional lymph node metastasis. Radical resections were performed in 32 patients, palliative surgery in 46, and biopsy in 22. Among all GBC patients, 65 were followed up through letters and phone calls. Nineteen patients survived over a year and 46 less than a year. Among the 46 peritumoral noncancerous tissues (distance from cancer≥3 mm), 10 were normal, 10 showed mild dysplasia, 12 revealed moderate dysplasia, and 14 showed severe dysplasia. Among the 15 gallbladder polyps, 10 were from normal epithelium to mild dysplasia, 5 were moderate to severe dysplasia. Of the 30 patients with gallbladder adenomatous, 5 were not pathologically conf i rmed with dysplastic adenomatous epithelium, 10 with mild dysplasia, 9 with moderate dysplasia, and 6 with severe dysplasia. Among the 35 patients with chronic cholecystitis, 15 had chronic cholecystitis, whereas 20 had chronic cholecystitis with gallstones. Pathological examination conf i rmed that 11 gallbladders had normal mucosa, 12 had mild dysplasia, 7 had moderate dysplasia, and 5 had severe dysplasia. Benign gallbladders were used as references.[23,24]Pathological evaluation and immunohistochemical conclusions were performed by two experts in microscopy.

EnVision™ immunohistochemistry

Paraff i n-embedded tissues were sectioned at 4 µm thickness. Rabbit anti-human S1P1 polyclonal antibody and mouse anti-human ERp29 monoclonal antibody were purchased from Abgent Company (California, USA). An EnVision™ Detection Kit was purchased from Dako Laboratories (California, USA). S1P1 and ERp29 staining was performed according to the protocol of the manufacturer (ChemMate™ EnVision+/HRP/DAB). Brief l y, the sections were deparaff i nized and incubated with peroxidase inhibitor (3% H2O2) in the dark for 15 minutes, followed by EDTA-trypsin digestion for another 15 minutes. The sections were then washed three times in phosphate-buffered saline (PBS) solutionfor 5 minutes, and then incubated with mouse anti-S1P1 or anti-ERp29 for 60 minutes at room temperature. The sections were incubated with solution A (HRP-conjugated anti-rabbit or mouse second antibody) for 30 minutes at room temperature, with addition of DAB substrate, followed by hematoxylin counterstaining. The sections were dehydrated in different alcohol concentrations (70% to 100%), incubated with xylene for 5 minutes, and mounted with neutral balsam. Images were taken using an Olympus microscope. The positive controls used were the positive sections of S1P1 and ERp29 in breast cancer provided by Dako Company Agent (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China). The negative controls used 5% fetal bovine serum as a substitute for antibodies. Each section was evaluated by two pathologists independently. The fi nal conclusions were made by discussion if there were any discrepancy. The percentage of positively stained cells relative to the total number of cells (400 cells) was determined in 10 random fi elds at a magnif i cation ×200. The def i nition of positive means the positive cells were ≥25%.[25-27]

Statistical analysis

Data analysis was conducted using the SPSS 13.0. S1P1 or ERp29 expression inter-relationship was analyzed using the independent Chi-square test, considering the histological or clinical factors. Statistical association analysis between the two independent sample groups was conducted using the Fisher's exact probability test. The Kaplan-Meier method and log-rank test were used for univariate survival analysis. The Cox proportional hazards model was used for multivariate analysis and to determine the 95% conf i dence interval (CI). APvalue less than 0.05 was considered as statistically signif i cant.

Results

S1P1 and ERp29 expression in gallbladder adenocarcinoma

EnVision™ immunohistochemistry revealed that S1P1 and ERp29 positive reactions were mainly in the cytoplasm (Fig. 1). S1P1 and ERp29 expressions in benign and malignant gallbladder lesions are summarized in Table 1. S1P1 positive rate was signif i cantly higher in gallbladder adenocarcinomas than that in peritumoral (P<0.01), adenomatous (P<0.01), polyp (P<0.05), and chronic cholecystitis (P<0.01) tissues. On the contrary, ERp29 positive rate was signif i cantly lower in GBCs compared with that in peritumoral (P<0.01), adenomatous (P<0.01), polyp (P<0.01), and chronic cholecystitis (P<0.01) tissues. Benign and precancerous gallbladder epithelium, with positive S1P1 and/or negative ERp29 expression, showed moderate to severe dysplasia (Table 1), suggesting that both S1P1 and ERp29 were regarded as molecular markers in evaluating pre-malignant lesions. ERp29 was negative in 34 of the 55 S1P1 positive cases, whereas ERp29 was positive in 29 of the 45 S1P1 negative cases (χ2=6.83,P<0.05). Therefore, S1P1 positive and ERp29 negative showed high consistency in gallbladder adenocarcinoma.

Fig. 1.S1P1 and ERp29 expression in benign and malignant gallbladder lesions (EnVisionTMimmunohistochemistry, original magnification×200). S1P1 positive reaction and ERp29 positive expression localized in the cytoplasm.A: S1P1 negative expression in gallbladder polyp;B: S1P1 positive expression in poorly differentiated adenocarcinoma;C: ERp29 negative expression in gallbladder adenomatous;D: ERp29 positive expression in well differentiated adenocarcinoma.

Table 1.S1P1 and ERp29 expression in the benign and malignant lesions of the gallbladder

Association of S1P1 or ERp29 expression with histological characteristics of gallbladder adenocarcinoma

Table 2 lists S1P1 staining, tumor size, lymph node metastasis, and stages. These results showed that S1P1 positive staining is signif i cantly higher in poorly-differentiated GBCs than that in well-differentiated ones. Tumor size and lymph node metastasis also affected S1P1 staining (P<0.05). On the contrary, ERp29 expression showed an opposite pattern. S1P1 or ERp29 expression did not show any signif i cant association with mucinous adenocarcinoma and other clinicopathological characteristics, such as gender, age, and gallstone history (P>0.05).

Table 2.S1P1 and ERp29 expression associated with clinicopathological characteristics of gallbladder adenocarcinoma

S1P1 or ERp29 expression and the survival of patients with GBC

Table 3 shows the survival information of 65 followed up patients from the 100 patients with gallbladder adeno-carcinoma. Among the 65 patients, S1P1 was positive in 32 (49.2%) and ERp29 positive in 30 (46.2%). Kaplan-Meier survival analysis showed that histological type (P=0.03), tumor size (P=0.003), lymph node metastasis (P=0.005) and tumor stages (P=0.002) were signif i cantly correlated with the average survival time of the patients. Patients with S1P1 positive survived signif i cantly shorter than those with S1P1 negative (P=0.008) (Table 3 and Fig. 2A). On the contrary, patients with ERp29 positive survived signif i cantly longer than those with ERp29 negative (P=0.043) (Table 3 and Fig. 2B). Cox multivariate analysis showed that tumor size (≥2 cm), lymph node metastasis, invasion, surgical type (radical resection) and S1P1 overexpression were negatively correlated with post-operative survival and positively correlated with mortality. While ERp29 negativity favored the patient's survival (Table 4).

Table 3.Relationship between clinicopathologic characteristics and average survival of 65 gallbladder adenocarcinoma patients

Fig. 2.S1P1 and ERp29 expression and survival in patients with gallbladder adenocarcinoma.A: Kaplan-Meier plots showing the overall patient survival with gallbladder adenocarcinoma, including S1P1 positive and negative expression;B: Kaplan-Meier plots showing the overall patient survival with gallbladder adenocarcinoma, including ERp29 positive and negative expression.

Table 4.Multivariate Cox regression analysis of survival rate in 65 gallbladder adenocarcinoma patients

Discussion

GBC is a very aggressive malignancy and commonly presents at an advanced stage. In addition, GBC is not sensitive to chemo- and radiotherapy. Therefore, the prognosis is extremely poor and the median survival is less than 6 months after diagnosis. Although previous studies demonstrated that S1P1 and ERp29 are highly expressed in several human tumors,[17,22,23]their expression in GBC and benign gallbladder lesions has not been reported. The current study measured the expression of S1P1 and ERp29 in 100 GBC specimens, benign and precancerous gallbladder lesions. About 55.0% of GBCs exhibited S1P1 expression and 50.0% expressed ERp29. Furthermore, S1P1 positive or ERp29 negative was signif i cantly correlated with tumor differentiation, lymph node metastasis, and tumor stages. Both S1P1 positive and ERp29 negative were important poor prognostic markers independent of other clinicopathological factors. The current study is the fi rst to describe the association between S1P1 or ERp29 expression and clinicopathological characteristics in GBC patients.

Prognosis of GBC patients remains poor because of the lack of effective treatment except surgery. Therefore, novel cancer treatments for GBC are needed. Our results showed that S1P1 is important in tumor proliferation, migration, invasion, and metastasis.[28-30]S1P1 is essential in lymphocyte egress from lymph nodes and vascular maturation.[14,31]S1P1 silenced by S1P1 siRNA in endothelial cells inhibits the growth of neovessels into subcutaneous implants of Matrigel. Vascular stabilization and suppression of angiogenesis result in tumor growth inhibition.[16]A previous study found that S1P receptors antagonist, FTY720, inhibited tumor-associated angiogenesis, decreased tumor cell proliferation and increased apoptosis.[32]A biospecif i c anti-S1P monoclonal antibody[33]signif i cantly reduced tumor progression and, in some cases, eliminated measurable tumors in several tumor models. S1P and its receptors, especially S1P1, are currently regarded as potential targets in cancer therapy. We found a signif i cant correlation between S1P1 expression and lymph node metastasis, this implied that S1P1 is a metastasis-associated gene. This theory may apply to GBC treatment. Currently, there is no effective treatment for advanced GBC. High expression of S1P1 in gallbladder tumor cells makes it high-potential specif i c targets for gene therapy. Therefore, this result may provide a novel therapeutic approach for gallbladder adenocarcinoma through S1P1 molecular targeting.

Unlike S1P1, the role of ERp29 in tumorigenesis has not been fully elucidated. ERp29 is highly expressed in primary tumor and cell lines.[19-21]Recent fi ndings showed that ERp29 overexpression signif i cantly downregulated the modulators of cell proliferation[34]and siRNA-mediated ERp29 silencing in non-invasive MCF-7 breast cancer cells reduces tumor formation.[35]This result imply the oncogenic role of ERp29 in breast tumor. Moreover, a previous study showed that the overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells, including an inverse correlation between ERp29 expression and tumor progression.[36]Furthermore, ERp29 overexpression can indirectly up-regulate the transcriptional activation of some anti-oncogenic genes and down-regulate some carcinogenic genes. Because of these conf l icting results, the pathophysiological role of ERp29 in tumorigenesis remains elusive and needs further evaluation. The current study demonstrated that ERp29 expression was signif i cantly lower in gallbladder adenocarcinoma than in benign lesions. Low expressionlevels of ERp29 were signif i cantly correlated with tumor differentiation, invasion, and lymph node metastasis. These fi ndings strongly suggested that ERp29 affects distant metastasis during gallbladder adenocarcinoma progression. Hence, ERp29 can be a potential therapeutic target for the treatment of gallbladder adenocarcinoma.

Another important fi nding showed that the epithelium of benign gallbladder, which has highly expressed S1P1 and/or negatively expressed ERp29, showed moderate to severe dysplasia. However, negative S1P1 expression and ERp29 overexpression were exhibited in mildly dysplastic or normal epithelium. The expression of S1P1 was signif i cantly lower in moderate than in poorly differentiated adenocarcinoma. Conversely, ERp29 overexpression was signif i cantly higher in moderate than in poorly differentiated adenocarcinoma. Therefore, both markers can be used as effective predictors of metaplastic malignancy and as biomarkers for early diagnosis. Patients with S1P1 overexpression and ERp29 negativity are more prone to invasion and metastasis, and these patients need a close follow-up for clinical manifestations of relapse.

In summary, the current study conf i rms that S1P1 positive and ERp29 negative are important biomarkers for metastasis, progression, and prognosis of gallbladder adenocarcinoma, and that they are likely potential markers in detecting precancerous gallbladder lesions to GBC. However, the detailed mechanisms of S1P1 and ERp29 involvement in gallbladder adenocarcinoma remain to be elucidated. Further studies will provide a new therapeutic way for gallbladder adenocarcinoma.

Acknowledgement:We thank Dr. Ling-Hui Zeng for his critical review of the article.

Contributors:YLW proposed the study. YLW and LDC performed research, wrote the fi rst draft and analyzed the data. YZL designed the study and helped the interpretation of the study.

Funding:This study was supported by grants from the National Science Foundation of China (No. 81410292) and the Natural Science Foundation of Hunan Province (No. 09JJ3077).

Ethical approval:This study was approved by the Ethics Committee of Second Xiangya Hospital, Central South University. Signed informed consent forms were obtained from all subjects who participated in the study. The Code of Ethics of the World Medical Association (Declaration of Helsinki) was followed.

Competing interest:No benef i ts in any form have been received or will be received from a commercial party related directly or indirectly tot the subject of this article.

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Received May 8, 2012

Accepted after revision January 4, 2013

AuthorAff i liations:Department of Geriatric Surgery (Yuan LW and Liu DC); Research Laboratory of Hepatobiliary Diseases (Yang ZL), Second Xiangya Hospital, Central South University, Changsha 410011, China

Zhu-Lin Yang, MD, PhD, Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, China (Tel: 86-731-88187376; Fax: 86-731-84898168; Email: yangzhulin8@sina.com)

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(13)60030-2