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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 4  |  Page : 867-872

Increased expression of epidermal growth factor-like domain-containing protein 7 is predictive of poor prognosis in patients with hepatocellular carcinoma


1 Department of Interventional Radiology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
2 Department of General Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China

Date of Web Publication27-Jun-2018

Correspondence Address:
Cai-Fang Ni
Department of Interventional Radiology, First Affiliated Hospital of Soochow University, Suzhou, No. 188, Shizi Street, Jiangsu
China
Zhi Li
Department of Interventional Radiology, First Affiliated Hospital of Soochow University, Suzhou, No. 188, Shizi Street, Jiangsu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_745_17

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 > Abstract 


Objective: Epidermal growth factor-like domain-containing protein 7 (EGFL7) is an endothelial cell-derived secreted factor that regulates vascular tube formation. In human cancer, the specificity of expression is lost as EGFL7 has been detected in tumor cells, in addition to endothelial cells. This study evaluated the intricate relationship between hypoxia-inducible factor 1-alpha (HIF-1α) and EGFL7 under both hyperoxia and hypoxia states.
Materials and Methods: In the present study, immunohistochemical staining and ELISA were applied to examine the relative level of EGFL7 in 182 cases of hepatocellular carcinoma (HCC) formalin-fixed and paraffin-embedded tissues and 110 cases of HCC serum samples. Quantitative polymerase chain reaction and Western blotting were applied to verify the correlation between serum EGFL7 level and anoxic microenvironment. Immunohistochemical staining was performed to determine the correlation between EGFL7 and HIF1-α.
Results: The correlations between EGFL7 expression and patients' age, tumor size, gender, N-stage, history of cirrhosis, M-stage, history of hepatitis C, and history of hepatitis B were statistically insignificant (P = 0.28, 0.34, 0.71, 0.15, 0.8, 0.2, 0.052, and 0.14, respectively). High level of EGFL7 was significantly correlated with overall survival as well as disease-free survival in 182 HCC patients (P = 0.0016 and P < 0.001, respectively). The correlations between serum EGFL7 and vascular invasion and extrahepatic metastasis were statistically significant (P < 0.0001). Among the 35 HIF1-α-positive HCC patients, 69% were medium positive and 31% were strong positive. EGFL7 protein expression level was oxygen dependent in HCC line (P < 0.05).
Conclusions: EGFL7 was found to be a potential predictor for HCC survival and metastasis state; EGFL7 may be a promising biomarker and therapeutic target in human HCC.

Keywords: Anoxic, hepatocellular carcinoma, metastasis, transcatheter arterial chemoembolization


How to cite this article:
Yang C, Wang YL, Sun D, Zhu XL, Li Z, Ni CF. Increased expression of epidermal growth factor-like domain-containing protein 7 is predictive of poor prognosis in patients with hepatocellular carcinoma. J Can Res Ther 2018;14:867-72

How to cite this URL:
Yang C, Wang YL, Sun D, Zhu XL, Li Z, Ni CF. Increased expression of epidermal growth factor-like domain-containing protein 7 is predictive of poor prognosis in patients with hepatocellular carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Nov 12];14:867-72. Available from: http://www.cancerjournal.net/text.asp?2018/14/4/867/235099




 > Introduction Top


Hepatocellular carcinoma (HCC) has been the second leading cause of cancer-related death worldwide.[1] The high probability of metastasis made the prognosis of HCC very dismal even after potentially curative treatment. Transcatheter arterial chemoembolization (TACE) was the current standard of care for patients with large or multinodular HCC and relatively preserved liver function, without cancer-related symptoms, and with no evidence of vascular invasion or extrahepatic spread (i.e., those classified as intermediate stage according to the Barcelona Clinic Liver Cancer staging system).[2] However, TACE was often associated with invasion and metastasis, which limited the overall survival of HCC patients.[3]

Epidermal growth factor-like domain-containing protein 7 (EGFL7), also known as vascular endothelial statin, is an endothelial cell-derived secreted factor that regulates vascular tube formation.[4] In human cancer, the specificity of expression is lost as EGFL7 has been detected in tumor cells themselves, in addition to endothelial cells.[5] Its expression levels correlate with a higher tumor grade in glioma and gastric colon cancer patients and with a poorer prognosis and higher metastatic score in HCC patients.[6],[7],[8] We previously reported that EGFL7 expression was also associated with poor overall survival in human pancreatic cancer.[9] However, few studies have been performed on the relationship between blood EGFL7 level and HCC clinical significance.

A study indicated that hypoxia was responsible for the upregulation of EGFL7 in human coronary artery endothelial cells.[10] Xu et al. reported that EGFL7 gene expression was significantly decreased in the lungs of neonatal rat after anoxic exposure.[11] These studies seem to indicate that the expression of EGFL7 was regulated by oxygen exposure.[12] TACE inevitably led to a hypoxic insult to HCC and the surrounding liver tissue.[3] We previously reported that EGFL7 expression increased in rabbit VX2 liver tumors after transcatheter arterial embolization (TAE).[13] Hence, we hypothesized that there could be a correlation between expression of EGFL7 and TACE in human.

In this study, we explored the expression level of EGFL7 in human HCC tumors as well as in the blood. The correlation between EGFL7 expression level and patient prognosis, as well as the correlation between hypoxia and EGFL7 expression in human HCC tissues, was examined. This marker may be a potential prognostic indicator of survival in patients with HCC.


 > Materials and Methods Top


Hepatocellular carcinoma patient specimens

Frozen serum samples were collected from 110 HCC patients in the Department of Interventional, Department of General Surgery, and Department of Infectious Diseases of our hospital during the period January 2015–July 2016. Formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected from 182 HCC patients in the Department of General Surgery of our hospital during the period January 2007–November 2009. All cases were reevaluated for grade and histological type by two independent pathologists. The mean age of patients at the time of surgery was 63 years (ranged 35–83 years). Other original clinical data were also collected, including gender, age, tumor size, histological type, hepatitis B history, Child–Pugh score, lesion size, lesion number, TNM stage, and 80-month follow-up survival. Both frozen serum samples and FFPE tumor samples were collected from 50 HCC patients in the Department of General Surgery of our hospital during the period January 2014–November 2015. No patients had received chemotherapy or radiation before the operation. Written informed consent and any related picture were acquired from each patient for publication of this study. This research was approved by the Local Human Research Ethics Committee of our hospital.

Immunohistochemical staining

FFPE samples were sliced to be sections with a thickness of 3 μm. The Polink-1 HRP DAB Detection System One-Step Polymer Detection Method was applied as follows: tissue sections were deparaffinized in xylene and then gradually rehydrated through ethanol to water. Endogenous peroxidase activity blocking was performed with 3% H2O2 for 15 min. Antigen retrieval was performed as follows: Sections were heated in a high-pressure cooker with 10 mmol/L citrate buffer (pH 6.0) at 1000 kW power heated by an induction cooker for 2.5 min and then left to cool naturally.

Nonspecific binding was minimized with 5% goat serum (Beyotime Biotechnology, Beijing, China) for 30 min. Prediluted primary rabbit anti-EGFL7 and anti-hypoxia-inducible factor 1-alpha (HIF-1α) polyclonal antibodies (Abs) (1:80, Abcam, Cambridge, MA, USA) were applied. Negative control slides without primary Ab were included in this step. The slides were incubated for 16 h at 4°C. After washing with phosphate-buffered saline, secondary Ab at working dilutions (rat anti-rabbit IgG, Abcam, Cambridge, MA, USA) was added to the sections, which was incubated for 1 h at room temperature. After washing, diaminobenzene was added to develop the color reaction. After the reaction ended, the sections were counterstained with hematoxylin, cleared, and mounted. Samples were scored with the immunohistochemical score (IHS) value as described by Soslow et al.[14] IHS = A × B, where A was the percentage of positive cells and B was the positive cell staining intensity grade. B was scored from 0 to 3 as follows: 0, negative; 1, low positive; 2, medium; and 3, strong positive.

Western blotting

Protein samples were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene Fluoride (PVDF) membranes. After blocking, the membranes were incubated with the indicated specific primary Abs for 2 h at room temperature followed by washing and incubation with horseradish peroxidase (HRP)-conjugated secondary Ab. The involved Abs were rabbit polyclonal anti-EGFL7 and anti-HIF-1α Abs (1:1000, Abcam, Cambridge, MA, USA) and rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000, Abcam, Cambridge, MA, USA). The secondary Ab was an HRP-conjugated anti-rabbit IgG Ab (1:4000; Zhongshan, Beijing, China). The immunoreactive proteins were developed with enhanced chemiluminescence detection kit (Tiangen Biotech) and visualized with the image analyzer Image Quant LAS 4000 (GE Healthcare).

ELISA assay

The serum samples were collected and centrifuged. These samples were stored at −80°C until being detected. The secreted EGFL7 was determined by ELISA kit (Abcam, UK). Absorbance was measured at 450 nm with a microplate reader. The concentration of EGFL7 in the samples was calculated by comparing with the standard curve.

Quantitative real-time polymerase chain reaction

For cultured cells, the TRIzol (Qiagen) was applied to extract total RNA. Reverse transcription was performed with the Maxima First Strand cDNA Synthesis Kit for real-time quantitative polymerase chain reaction (RT-qPCR, Thermo Scientific Rockford, USA). RT-PCR was run with the ViiA 7 Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) and the Fast Start Universal SYBR Green Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturers' specifications. Relative levels of EGFL7 mRNA were normalized to the expression of the housekeeping gene GAPDH. Quantification values were calculated according to a standard curve method generated from a dilution series. The amplification was performed in triplicates.

Data analysis

GraphPad Software 5 for Windows (GraphPad, San Diego, USA) and Microsoft Excel 2007 were applied for statistical analysis and graph creation. Data were expressed as mean±standard error (from three to eight independent experiments) depending on the specific assay. The nonparametric Mann–Whitney U-test was applied for analyzing the difference between the mean of two datasets. Fisher's exact probability was performed in tables. P< 0.05 was considered as statistically significant.


 > Results Top


Epidermal growth factor-like domain-containing protein 7 was a potential predictor of poor survival in hepatocellular carcinoma tissues

EGFL7 expression level was overexpressed in several tumor tissues. We first detected the protein expression level in 182 HCC tissues. As shown in [Table 1], the correlations between EGFL7 expression and patient's age, tumor size, gender, N-stage, history of cirrhosis, M-stage, history of hepatitis C, and history of hepatitis B were statistically insignificant (P = 0.28, 0.34, 0.71, 0.15, 0.8, 0.2, 0.052 and 0.14, respectively). On the other hand, high expression level of EGFL7 was significantly correlated with T-stage and grade (P< 0.001, respectively). These data indicated that EGFL7 expression was associated with malignance degree and metastasis state of HCC patients. We then hypothesized whether the expression level of EGFL7 was correlated with the poor survival of HCC patients. The results [Figure 1]a and [Figure 1]b showed that high level of EGFL7 was significantly correlated with overall survival as well as disease-free survival in 182 HCC patients (P = 0.0016 and P< 0.001, respectively).
Table 1: Clinicopathological characteristics of patient stratified with the epidermal growth factor-like domain-containing protein 7 expression levels in formalin-fixed and paraffin-embedded samples of hepatocellular carcinoma

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Figure 1: (a) Kaplan–Meier curve showing the relationship between epidermal growth factor-like domain-containing protein 7 expression and the overall survival of hepatocellular carcinoma patients (P < 0.05). (b) Kaplan–Meier curve showing the relationship between epidermal growth factor-like domain-containing protein 7 expression and the disease-free survival of hepatocellular carcinoma patients (P < 0.05)

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Epidermal growth factor-like domain-containing protein 7 was a potential predictor for malignance degree and metastasis stage in serum of hepatocellular carcinoma patient

As a secreted factor, EGFL7 was also detectable in the blood, as well as serum or plasma. However, few studies have been performed on correlation between blood level of EGFL7 and HCC clinical significance. Then, EGFL7 expression levels in serums of 110 HCC patients were examined. As shown in [Table 2], serum EGFL7 expression level was not significantly correlated with the gender and age of patients (P = 0.46 and 0.41, respectively). Among a total of 110 HCC patients, 78 patients suffered a history of hepatitis B. No significant correlation was observed between the blood EGFL7 expression level and the history of hepatitis B (P = 0.51). Interestingly, the EGFL7 expression level was gradually increased in Child–Pugh Score A, B, and C, respectively. We observed that the correlation between serum EGFL7 and vascular invasion and extrahepatic metastasis was statistically significant (P< 0.0001). However, there was no relevance between serum EGFL7 level and focus number or focus size.
Table 2: Clinicopathological characteristics of patient stratified with the epidermal growth factor-like domain-containing protein 7 expression levels in serum samples of hepatocellular carcinoma

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Epidermal growth factor-like domain-containing protein 7 expression was oxygen dependent

We have previously reported that EGFL7 expression level was upregulated after TAE in rabbit VX2 tumor. However, whether anoxic microenvironment was also necessary for EGFL7 regulation remained unknown. Anoxic marker HIF-1α expressed in 35/50 patients. Among the 35 HIF-1α-positive HCC patients, 69% were medium positive and 31% were strong positive. We then analyzed the correlation between serum EGFL7 level and anoxic microenvironment. As shown in [Table 3] and [Figure 2], EGFL7 expression level was HIF-1α dependent (P< 0.05). Our data indicated that EGFL7 expression was also regulated by anoxic microenvironment in HCC patients.
Table 3: The correlation between the levels of epidermal growth factor-like domain-containing protein 7 and hypoxia-inducible factor 1 alpha

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Figure 2: Protein level of epidermal growth factor-like domain-containing protein 7 and hypoxia-inducible factor 1-alpha in clinical tissues of hepatocellular carcinoma patients

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Then, HCC cell lines were applied to investigate the mechanism underlying anoxic-induced EGFL7 overexpression. Five HCC cell lines including Hep3B, SMMC-7721, BEL-7701, MHCC-97H, and MHCC-97L were employed. The levels of both EGFL7 mRNA and protein were highest in SMMC-7721 and lowest in MHCC-97H cells [Figure 3]a and [Figure 3]b. Then, these two lines were involved and analyzed in the subsequent experiments. Two cell lines were incubated in 1% oxygen condition for 1, 2, 4, and 8 h, respectively. Interestingly, the level of EGFL7 protein was gradually increased in two hepatoma cell lines, but there was no significant change in the level of mRNA [Figure 3]c,[Figure 3]d,[Figure 3]e,[Figure 3]f. Our data indicated that EGFL7 may be regulated by oxygen condition through posttranscriptional pathway.
Figure 3: (a) Protein level of epidermal growth factor-like domain-containing protein 7 in Hep3B, SMMC-7721, BEL-7701, MHCC-97H, and MHCC-97L cells. (b). The mRNA level of epidermal growth factor-like domain-containing protein 7 in Hep3B, SMMC-7721, BEL-7701, MHCC-97H, and MHCC-97L cells. (c and d) Protein level and mRNA level of epidermal growth factor-like domain-containing protein 7 in hypoxia environment in SMMC-7721. (e and f) Protein level and mRNA level of epidermal growth factor-like domain-containing protein 7 in hypoxia environment in MHCC-97H

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 > Discussion Top


We showed here that high level of tissue EGFL7 expression was significantly correlated to HCC vascular invasion, extrahepatic metastasis, and poor overall survival and disease-free survival. High expression of EGFL7 may be regulated by anoxic microenvironment.

EGFL7 was expressed in several tumors and many cancer cell lines.[5],[6],[7],[9],[11] EGFL7 expression was highly elevated in human tumors, including kidney tumors, malignant gliomas, HCC, and colon cancers. Because endothelial EGFL7 overexpression in mice would lead to abnormal vessel patterning and remodeling, it was possible that EGFL7 stimulated tumor angiogenesis and contributed to the irregularly shaped, tortuous, leaky vessels that were specific to tumor vasculature, which was in a similar manner to vascular endothelial growth factor (VEGF).[15],[16] We found that EGFL7 not only existed in HCC tissue but also in serum. Serum was very readily available and not invasive to the patients. Hence, EGFL7 was more suitable as a predictor for HCC prognosis and diagnosis.

Previous studies on the expression of EGFL7 in human cancers generally suggested that tumor growth and metastasis could be promoted with EGFL7.[4],[17] The elevated expression levels of EGFL7 in tumors and its possible roles in promoting tumor angiogenesis made EGFL7 also a potential target for antiangiogenesis therapy. Recent preclinical studies involving a combination of anti-VEGF therapy and humanized monoclonal Abs against EGFL7 provided additional inhibition of tumor growth compared with anti-VEGF treatment alone, in human cancer xenograft mouse models (http://ip.com/patapp/US20110200602). Currently, clinical trials have been conducted to evaluate the effects of combined anti-EGFL7 and Avastin (anti-VEGF Ab) therapy on tumor vascular function and growth (http://www.gene.com/gene/pipeline/status/oncology/anti-egfl7). It is expected that blocking EGFL7 signaling in conjunction with anti-VEGF would provide additional inhibition of angiogenesis. With respect to the roles of EGFL7 in vascular remodeling and patterning during development, anti-EGFL7 therapy may also involve normalization of the vasculature which, when used together with anti-VEGF treatment, could extend the window for improved drug delivery. Although the potential for anti-EGFL7 therapy was promising, the long-term outcome and its possible effects need to be further confirmed.

TACE is an important therapy which improved survival in cirrhotic patients with HCC.[3] The main finding of this study was that EGFL7 was regulated by anoxic conditions and was associated with higher grade and poor survival. TACE would lead to hypoxia, which stimulated angiogenesis and promoted tumor growth; thus, the current efficacy could be improved by combining TACE with antiangiogenic agents. In a tumor context, TACE caused an anoxic microenvironment in liver tissues and induced an increase expression level of EGFL7. The overexpressed EGFL7 may result in the subsequent metastasis and poor survival. Hence, small molecular compounds or Abs against EGFL7 may result in potential hypoxia in TACE for improving HCC curative effect.

Because of its potential significance to predict HCC survival, as well as promoting tumor growth and metastasis, we speculated that EGFL7 may be a promising biomarker and therapeutic target for human HCC.


 > Conclusion Top


EGFL7 may be a promising biomarker and therapeutic target for human HCC. High level of tissue EGFL7 expression was significantly correlated to HCC vascular invasion, extrahepatic metastasis, and poor overall survival and disease-free survival. EGFL7 protein expression level was oxygen dependent in HCC line. The level of EGFL7 protein was gradually increased in hypoxic condition, but there was no significant change in the level of mRNA. EGFL7 may be regulated by oxygen condition through posttranscriptional pathway.

Acknowledgment

This study was supported by a grant from National Natural Science Foundation of China (No. 81501563) and Jiangsu Provincial Medical Youth Talent (QNRC2016711) and Suzhou Science and Technology Plan (SYS201611 and KJXW2016005).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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