Background
Granulin-epithelin precursor (GEP) contributes in multiple vital biological processes and its alias partly indicates its function, including progranulin, proepithelin, acrogranin, and PC cell-derived growth factor. GEP is a pluripotent growth factor important in fetal development, neuronal cell survival, wound healing and tumorigenesis [
1-
4]. Over-expression of GEP has been reported in a number of human cancers including breast, prostate, and ovary cancers [
5-
7]. In addition to its classical well-known biological function on growth regulation, GEP has recently been shown to control chemo-resistance [
8-
10]. Nonetheless, the mechanism of GEP over-expression remains elusive.
The incidence of primary liver cancer, hepatocellular carcinoma (HCC), has been increasing globally in the past two decades. HCC is the second most frequent cause of cancer-related death worldwide [
11]. Major risk factors for HCC development include infections with hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic liver diseases and fatty liver diseases. In China, with endemic HBV infection, HCC is the second leading cause of cancer death [
12]. In Western countries, HCC incidence is increasing steadily [
11]. Treatment of HCC remains a challenge, as curative partial hepatectomy and liver transplantation are only applicable for early stage patients [
13]. However, with limited surveillance, and early stage HCC usually asymptomatic, the majority of HCC patients are diagnosed at advanced stages. Prognosis of advanced stage HCC is poor with overall survival rate less than 5%. Treatment options for advanced HCC are limited, systemic chemotherapy and hormone therapy have not been effective [
14]. The research effort should continue to comprehend hepatocarcinogenesis. GEP has shown over-expression in HCC with functional roles on growth, invasion, tumorigenicity and cancer stem cell properties [
8,
9,
15]. In addition, GEP has demonstrated the potential to serve as therapeutic target [
16-
18]. Noted the GEP gene locus at chromosome 17q21 and the region has been frequently reported with copy number gain in HCC [
19,
20]. In this study, we investigated the GEP DNA copy number, and analyzed the association with gene expression levels and clinico-pathological features.
Methods
Clinical specimens
Sixty patients underwent curative partial hepatectomy for hepatocellular carcinoma (HCC) between September 2002 and July 2005 at Queen Mary Hospital, Hong Kong, were recruited with informed consent. Clinico-pathological data were prospectively collected. Ten blood samples from healthy individuals were recruited and served as control with informed consent. The study protocol was approved by the Institutional Review Board of the University of Hong Kong / Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB).
Quantitative microsatellite analysis (QuMA)
Copy number of GEP gene was measured by Quantitative Microsatellite Analysis (QuMA) as described using quantitative real-time PCR amplification [
21]. Chromosome 3 consistently showed stable copy number in HCC [
19,
20,
22] and thus microsatellite D3S1609 was used as a reference locus. Copy Number Assay for GEP and D3S1609 were ready-made reagents (Applied Biosystems, Foster City, CA). The number of PCR cycles (CT) required for the signal intensities to exceed a threshold just above background was calculated for the test and reference reactions. CT values were determined for test and reference reactions in each sample, averaged, and subtracted to obtain deltaCT (dCT) [dCT = CT (test locus) – CT (reference locus)]. dCT values were measured for each unknown sample [dCT (test DNA)] and for samples from ten unrelated healthy individuals (calibrator) [average dCT (calibrator DNA)]. Relative copy number at each locus in the test sample was then calculated as described [
21]:
$$ \mathrm{Relative}\kern0.5em \mathrm{D}\mathrm{N}\mathrm{A}\kern0.5em \mathrm{copy}\kern0.5em \mathrm{number}=\mathrm{N}\times {\left(1+\mathrm{E}\right)}^{\hbox{-} \mathrm{ddCT}} $$
where ddCT = dCT (test DNA) – average dCT (calibrator DNA), and E = PCR efficiency. The primers had showed PCR efficiencies >95% (Additional file
1: Figure S1), and N = 2 for diploid normal individuals, and for simplicity, relative DNA copy number = 2 × 2
-ddCT. Tolerance interval (TI) was calculated to determine if the test sample in the QuMA measurement was significantly different from the mean of measurements made on samples from the healthy individuals [
21]:
$$ \mathrm{T}\mathrm{I}=\mathrm{N}\pm \mathrm{S}\mathrm{D}\times 3.38 $$
where SD was the standard deviation and 3.38 was the two-sided tolerance limiting factor for the measurements on healthy samples, and N = 2 for diploid status. Measurements outside this range were considered significantly different from normal.
Fluorescence in situ hybridization (FISH)
FISH analysis was performed to confirm the copy number status in paraffin-embedded HCC tissues as described [
23]. Two primary HCCs were investigated by FISH based on histology assessment among the samples with DNA copy number gain by QuMA. Two BAC clones RP11-436 J4 and RP11-52 N13 flanking GEP gene located at 17q21 were labelled with Spectrum Green (Molecular Probes, Life Technologies). The centromeric probes at chromosome 3 (pAE0.68) and chromosome 17 (pEZ17-4) were labelled with Spectrum Orange (Molecular Probes) and used as reference probes. Chromosomal locations of these probes were validated in metaphases of normal individual.
Statistical analysis
All analyses were performed using the statistical software IBM SPSS Statistics Version 21 (Armonk, NY). Continuous variables were assessed by Pearson correlation analyses. The comparison of categorical variables was examined by Pearson chi-square test with Yates continuity correction. Difference was considered statistical significant if the P value was less than 0.05.
Discussions
Increased GEP transcript and protein levels have been reported in various human cancers [
5-
7]. The enhanced GEP expressions have been demonstrated to associate with aggressive tumor features, including large tumor size [
15,
24], metastasis [
15,
25], and poor prognosis [
5,
8,
25-
27]. Biological roles have been demonstrated with cell models and xenograft systems with regulatory functions on growth [
7,
15,
25,
28,
29], invasion [
15,
30,
31], tumorigenicity [
15,
25,
30], drug resistance [
8,
28,
30,
32] and cancer stem cell properties [
8,
9]. The biological function of GEP corroborates very well with the aggressive clinical features of the tumors showing over-expression. Studies on the signaling pathways demonstrated substantial molecules associated with GEP expressions. GEP stimulated MAPK and PI3K pathways [
29]. GEP was a cofactor for toll-like receptor 9 signaling [
33]. In addition, GEP protein over-expressions were associated with accumulation of wild-type p53 protein [
34]. GEP has also been shown to be regulated by endothelin, lysophosphatidic acid and cAMP [
7]. Protein kinase C signaling has demonstrated to influence GEP protein levels [
35]. Nonetheless, the exact mechanism for GEP over-expression in the majority of human cancers warrant further investigation.
Elevated expressions of growth factors and oncogenes have been reported to associate with gene copy number gain. In the current study, the GEP gene copy number was investigated by real-time PCR based method QuMA and FISH analysis. QuMA has been commonly employed for quantitative measurement of DNA copy number [
21,
36,
37]. Nonetheless, there were technical limitations that DNA samples were extracted from tumor mass which would contain tumor cells and tumor-infiltrating cells including lymphocytes etc. Therefore, copy number assays would underestimate the tumor chromosomal aberrations and would need further computational analyses [
38,
39]. The extent of underestimation would depend on the percentage of tumor-infiltrating cells which fluctuate between specimens. In contrast, FISH analysis was performed under microscope. Thus, tumor cells could be distinguished from non-tumor components and focused to analyze for genetic alterations. FISH has been shown to be useful to facilitate the diagnosis of neoplasms [
40-
42]. Comparatively, FISH is reliable but technically demanding and expensive, while QuMA prone to underestimate genetic aberration but is economical and suitable for large scale screening by automation. The two methods should be used in complementary for investigation of cancer genetic aberrations.
Copy number gain at specific chromosomal region contributes on activation of oncogenes and growth factors. The process is important during tumor initiation and also progression along carcinogenesis. However, there always have concerns on whether high levels of amplification are necessary or if the gain of single extra copy would be able to advance cancer. Recently, there are reports that low copy number gain contributes on cancer progression [
43-
45]. Gain of single supernumerary segment encompassing Myc, Pvt1, Ccdc26 and Gsdmc has shown to promote cancer [
43]. The present study demonstrated low copy number gain at centromere 17 and GEP gene at 17q21 associated with increased GEP expressions. Therefore, the specific chromosomal region 17q21 would be the focus to examine if this segment contains the essential gene set for tumor initiation and progression.
Recurrent genomic and expression alterations have been reported on chromosomal arm 17q. Independent studies demonstrated expression gains of gene set at 17q12-21 [
46] and 17q21-25 [
47], respectively, in HCC by expression imbalance map analysis. These expression gain regions corroborated with chromosomal gain regions frequently reported in HCC [
48,
49]. Furthermore, TOP2A gene locus at 17q21-22 has reported copy gains and overexpression, and regulated chemo-resistance in HCC [
47]. Similar copy number gain status has also been revealed in HER2 with copy number gain at gene locus 17q21 and centromere 17, and showed protein over-expression in breast cancer [
50]. Elevated HER2 expression has been demonstrated in HCC tissues [
51] and blood samples [
52], and associated with poor survival [
49]. HER2 associated with hepatitis B virus infection [
52] in particular hepatitis B x (HBx) antigen [
51,
53] where HBx has been shown to promote chronic liver disease and HCC development [
54]. Further investigation would be warranted to examine the minimal gene set that drives neoplasia. Potential candidates at chromosomal segment 17q21 that demonstrated copy number gain and overexpression included GEP, TOP2A and HER2. These genes could constitute partly the essential gene set that initiate and promote HCC progression.
Acknowledgement
This study was supported in part by Hong Kong Research Grants Council (764111, 764112 and T12-401/13-R), Health and Medical Research Fund (01121566), and Sun C.Y. Research Foundation for Hepatobiliary and Pancreatic Surgery.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MKY, CWY and PFYC carried out and analyzed the QuMA data. MKY, KWL, GTYC and CYKT performed and analyzed the FISH data. TTC, RTPP, and STF provided clinical specimens and information. STF and SS advised clinical analysis. STC conceived the study, analyzed data and drafted the manuscript. All authors read and approved the final manuscript.