Prognostic significance of epidermal growth factor-like domain 7 in pancreatic cancer

Beijing,China

Introduction

Pancreatic cancer (PC),has long been recognized as a lethal malignancy with extremely poor prognosis.[1,2]In addition,the long-term survival of PC has not been improved within two decades,despite widely-performed surgical resections.[3]Therefore,factors predictive for poor prognosis of the disease are of interest.Among these factors,conventional clinical and pathological parameters such as lymph node metastasis,neural/perineural invasion and resection margin were previously identified as significant variables.[4-8]Recently,much attention has been paid to many tumor initiation/development-associated molecules that were associated with PC prognosis.[9,10]However,further clues and evidences are required.

Epidermal growth factor-like domain 7 (EGFL7),a secreted protein containing two EGF-like domains,was initially identified as a modulator of smooth muscle cell migration.[11]The biological roles of EGFL7 in the vascular system have been extensively investigated.[12-15]In human cancer cells,EGFL7 accelerates migration through the focal adhesion kinase (FAK)-associated pathway,[16]and promotes tumor progression by reducing the expression of endothelial molecules that mediate immune cell infiltration.[17]A study[18]on human tissues revealed that EGFL7 is overexpressed in ten human epithelial tumor types,including hepatocellular carcinoma,lung cancer,breast cancer,prostate cancer,colorectal cancer,gastric cancer,esophageal cancer,malignant glioma,ovarian cancer and renal cancer.In addition,correlations between EGFL7 expression and unfavorable biological behaviors as well as poor prognosis were also suggested in many malignancies.[16,18-21]However,controversy still remains because EGFL7 expression was associated with better prognosis and with the absence of lymph node invasion in human breast cancer.[22]So far,there is no study on the expression and prognostic role of EGFL7 in PC.

The present study was to explore the expression of EGFL7 in PC,its association with clinicopathological variables,and the role of EGFL7 in the prognosis of PC.

Methods

Cell culture

Nine human PC cell lines (AsPC-1,BxPC-3,Capan-1,Colo357,MIAPaCa-2,PANC-1,Su86.86,SW1990 and T3M4),gifts from Professor Helmut Friess,Heidelberg University,Germany,were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 medium (Hyclone,Thermo Fisher Scientific Inc.,Waltham,MA,USA),supplemented with 10% fetal bovine serum (FBS,Hyclone),as previously reported.[23]

Western blotting analysis

Western blotting detection of EGFL7 in PC cell lines was performed according to the method of Liu et al,[24]using a rabbit anti-human EGFL7 antibody (Santa Cruz Biotechnology,Inc.,Santa Cruz,CA,USA).

Patients

Eighty-three patients with PC after radical surgical resection (R0) were recruited,53 males and 30 females,with a mean age of 61.8±10.6 years (mean±SD).The clinicopathological features of the patients are shown in Table 1.Standard lymphadenectomy (including resection of nodes of No.12,13,17,8 and 14 groups) was performed.Post-surgical gemcitabine-based adjuvant chemotherapy/chemoradiotherapy was recommended for all patients.The study was approved by the Institutional Ethics Committee.

Construction of tissue microarray (TMA)

A tissue microarray was constructed using formalinfixed paraffin-embedded blocks of PC.Diagnosis was proven by hematoxylin-eosin staining-based routine pathologic examination.After careful review and screening of representative tumor and non-tumor regions,two cores of corresponding tissues for each patient were sampled from typical areas using a 1.5-mm punch.The TMA was constructed with a manual tissuearrayer (Beecher Instruments,Sun Prairie,WI,USA).

Table 1.EGFL7 expression and clinicopathological features of PC

Immunohistochemical staining

TMA-based immunohistochemical staining for EGFL7 was performed in tissues from 83 patients with PC.A rabbit anti-human EGFL7 polyclonal antibody(Santa Cruz) and a two-step staining kit (EnVision+kit,Dako,Glostrup,Denmark) were used.The tissues were cross-sectioned in 4 μm thickness and mounted,deparaffinized and rehydrated.An autoclave was used to retrieve antigen.Slides were subsequently incubated with 3% hydrogen peroxide to block the endogenous peroxidase.Then,the slides were incubated with the primary antibody (dilution:1:20) overnight at 4 ℃.Following washing in PBS,the slides were incubated with horseradish peroxidase (HRP)-labeled secondary antibody,and after washing,the slides were stained with diaminobenzidine and then counterstained with hematoxylin.Non-immune rabbit serum at the same dilution was applied as the negative control.

Evaluation of staining

Two pathologists with no prior information of clinicopathological and survival data (CQC and ZWX)performed staining evaluation,reaching a consensus after discussion when there was a controversy.The brown color located in the cytoplasm was defined as the positive signal.The initial scoring was given using previously introduced criteria.[20]Then,afinal decision for EGFL7 expression was made according to the standard (low:scores 0-4; high:scores 5-12).

Follow-up

Post-operative follow-up,with a median term of 15 months (range 2-87),was given to all patients.Among the patients,54 died and 29 were alive at the time of follow-up.

Statistical analysis

EGFL7 staining between tumor and non-tumor tissues was compared using the Mann-Whitney U test.The Chi-square test was used to show the association between EGFL7 expression and clinicopathological variables.Overall survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test.Cox regression (proportional hazard model) was used for multivariate analysis of prognostic factors.Statistical software package SPSS11.5 (SPSS Inc.,Chicago,IL,USA)was employed for all the analyses.A P value of less than 0.05 was defined as statistical significance.

Results

Expression of EGFL7 in PC cell lines

Western blotting analysis showed that EGFL7 was widely expressed in all PC cell lines tested (Fig.1).The expression was relatively higher in SW1990 and T3M4 and lower in PANC-1 cells.

Expression of EGFL7 in PC tissues and its association with clinicopathological features

Fig.1.Expression of EGFL7 in pancreatic cancer cell lines.

Positive EGFL7 staining was present in both tumor and non-tumor tissues (Fig.2A,2B,respectively).The Mann-Whitney U test showed that the staining score in tumor tissues was statistically higher than that in non-tumor samples (Fig.2C,P=0.040).No significant association between tumoral EGFL7 expression and estimated clinicopathological variables was observed(Table 1,P＞0.05).

Prognostic significance of EGFL7 in PC after resection

Univariate analysis showed that patients with high EGFL7 expression in tumor tissues carried poor overall survival (Fig.3,Table 2,P=0.032).In addition,other conventional clinical and pathological parameters including gender,histological grade and lymph node metastasis were also predictive for prognosis (Table 2,P＜0.05).In a multivariate Cox regression test,EGFL7 expression in tumor tissues was identified as an independent prognostic marker (Table 2,P=0.001),in addition to the aforementioned significant univariate variables (Table 2,P＜0.05).

Fig.2.Expression of EGFL7 in pancreatic cancer.A:EGFL7 expression in tumor cells (original magnification ×200).B:EGFL7 expression in non-tumor cells (original magnification ×200); C:Comparison of the EGFL7 expression staining scores between tumor and non-tumor tissues (Mann-Whitney U test; P=0.040).EGFL7:epidermal growth factor-like domain 7.

Fig.3.Overall survival of patients with PC after surgical resection according to EGFL7 expression in tumor tissues (Log-rank test;P=0.032).EGFL7:epidermal growth factor-like domain 7.

Discussion

It was shown that EGFL7 affects smooth muscle cell migration- and vessel-associated phenotypes,such as vascular tubulogenesis,elastogenesis,endothelial integrity and cell death.[11-15]In 2009,Wu et al[16]found that EGFL7 accelerated migration through the EGFL7-EGFR-FAK axis in hepatocellular carcinoma cells,thus providing the first evidence of the effect of EGFL7 on cancer cell behavior.An observation focusing on EGFL7 and immune cells,based on experiments in breast and lung carcinoma cells,indicated that the protein contributes to tumor escape from immunity.[17]Therefore,EGFL7 might play important roles in cancer development.Recently,EGFL7 was found to be overexpressed in ten human epithelial tumor tissues.[18]In addition,EGFL7 expression was also associated with unfavorable clinicopathological features and poorer prognosis in the majority of malignant tumors tested,[16,18-21]although the adverse clue could also be seen.[22]Thus,EGFL7 might be a cancer promoter.However,the expression of EGFL7 in PC remains unknown.In the present study,EGFL7 was widely expressed in all of the 9 PC cell lines,suggesting its possible role in PC.Furthermore,determination of the baseline level of EGFL7 expression lays a foundation for further investigations.Immunohistochemical staining showed that the expression of EGFL7 in PC tissues was significantly higher than that in non-tumor samples.The data were consistent with those in most cancers,[16,18-21]and suggested that EGFL7 might be involved in pancreatic carcinogenesis.However,unlike previous reports,[16,18-21]no positive associations existed between EGFL7 and clinicopathological variables in patients with PC.Our findings were consistent with those of studies on other proteins such as cyclin D1 Survivin and HUGL-1,all of which were associated with the survival in different cancers but none of them was correlated with clinical variables.[25-27]The variations in sample size and patient characteristics as summarized by Knutsen et al[26]might account,at least in part,for the observed phenomenon.Future prospective studies with more specimens might be necessary.

Nevertheless,EGFL7 was previously found to be associated with unfavorable outcome in some cancers.[16,18,21]However,its prognostic significance in PC has not been clarified.Univariate analysis in our study showed that EGFL7 expression in tumor tissues was predictive for poor survival.Besides EGFL7,we also found the conventional clinicopathological variables,such as gender,differentiation and N status,were predictors of survival in patients with PC.Our results were consistent with other studies.[4-6,28,29]More importantly,multivariate Cox regression test concluded that high tumor expression of EGFL7 was an independent prognostic factor of PC.This finding strengthened the potential of the protein as a novel biomarker of poor prognosis of PC,and might add a new promising candidate to the lists published.[9,10]Previously,some proteins that are mainly related to metastatic potential such as CD44v6,c-Met and EpCAM were found to have prognostic value in PC.[30-35]In the future,combined estimation of EGFL7 and other clinicopathological or the aforementioned molecular factors,especially those associated with invasion/metastasis,could provide stronger predictive power and might be of interest to promote further investigations.

In conclusion,our findings suggest that EGFL7 is extensively expressed in PC and that EGFL7 may have prognostic implications in this malignancy.

Contributors:ZL proposed the study and wrote the first draft.ZL and LJ performed the experiments.CQC and ZWX evaluated staining results.ZL,LJ,ZYP and GJC analyzed the data.All authors contributed to the design and interpretation of the study and to further drafts.ZL and LJ contributed equally to this work.ZYP is the guarantor.

Funding:This study was supported by a grant from the Research Special Fund for Public Welfare Industry of Health (201202007).

Ethical approval:This study has been approved by the Institutional Ethics Committee.

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

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