Hepatocellular carcinoma (HCC) is the third most common type of cancer worldwide (But et al., 2008). Hepatocarcinogenesis is a multistep process with a multifactorial etiology. Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) and cirrhosis of any etiology are the major risk factors for HCC (Kremsdorf et al., 2006; Suruki et al., 2006). Chronic HBV/HCV infection induces an inflammatory response that often leads to chronic liver injury (Schwabe and Brenner, 2006). Persistent immune-mediated hepatic injury can initiate the process of fibrosis, cirrhosis, and eventually HCC (Choi and Ou, 2006). HCC displays gross genomic alterations, including DNA rearrangements, loss of heterozygosity, chromosomal amplification, and loss of imprinting. In the last few years it became obvious that active Wnt signaling as characterized by the presence of nuclear/cytosolic β-catenin correlates highly with the occurrence of HCC in animal models (Brui et al., 2006).
Wnt family genes encode a group of secretory glycoproteins that play important roles in embryogenesis, cell proliferation, and specification of cell fate (Peifer and Polakis, 2000; Hartmann and Tabin, 2001; Huelsken and Behrens, 2002). There are 19 human Wnt genes, several of which encode additional alternatively spliced isoforms (Miller, 2001; Kawano and Kypta, 2003). Wnt signaling is transduced through β-catenin, which is regulated by the adenomatous polyposis coli (APC)/axin/glycogen synthase kinase (GSK-3β) complex (Bienz and Clevers 2000; Woodgett, 2001; Polakis, 2002).
In the presence of Wnt stimulation, the frizzled receptors and low-density lipoprotein receptor stabilize β-catenin through multiple mechanisms, resulting in accumulation of free cytosolic β-catenin (Pinson et al., 2000; Tamai et al., 2000; Liu et al., 2002). Thereafter, elevated β-catenin translocates into the nucleus where it forms a complex with T-cell factor to stimulate the expression of Wnt-target genes (Barker et al., 2000).
In differentiated nonproliferating cells, β-catenin is associated with membrane-bound E-cadherin, and nonbound molecules are quickly removed from the cytosol in the absence of Wnt signaling, thereby preventing its translocation to the nucleus. This is accomplished by a multienzyme complex that binds cytosolic β-catenin. When bound to the complex, β-catenin is phosphorylated by active GSK-3β, then labeled by ubiquitin, and finally degraded by proteosome. The complex in addition to GSK-3β contains other proteins like axin, casine kinase Iα, and APC. Although mutations in each component of the complex may cause abnormal cytosolic stabilization of β-catenin, mutation of β-catenin itself appears to be the most common cause for stabilization in a pathological situation. Activation of β-catenin finally leads to the activation of a variety of genes (Reya et al., 2003). C-myc overexpression or amplification is reported to be associated with a variety of human cancers. Interestingly, it is identified as a transcriptional target of the APC/β-catenin/T-cell factor pathway in colorectal cancer and HCC. It has been observed that c-myc overexpression in HCC is also associated with ‘a second hit’ mutation in the β-catenin gene. C-myc is also an established inducer of apoptosis. Therefore, oncogenic transformation mediated by c-myc must require a survival signal to overcome its proapoptotic activity (Dang, 1999; Gandori et al., 2000; Reya and Clevers, 2005).
Accordingly, the present study was planned to assess the contribution of β-catenin, c-myc, and APC genes in the Wnt/β-catenin pathway and their contribution to HCV-associated hepatocarcinogenesis. Prognostic value of these genes in HCC was also evaluated.
Materials and methods
This retrospective study included 35 well-specified HCC cases that were obtained from patients who attended the clinics of the National Cancer Institute, Cairo University, between January 2007 and December 2009. Also included are 20 cases of chronic hepatitis (CH) obtained from Kasr El-Aini Hospital, Cairo University. Formalin-fixed paraffin-embedded tissue blocks were obtained from the archives of the Pathology Department at the National Cancer Institute and Kasr El-Aini Hospital. Cases were selected according to the presence of enough representative material on the paraffin blocks and the availability of clinicopathological data. All chosen patients were positive for HCV infection and negative for HBV infection by serological tests and PCR.
Immunohistochemical analysis was performed to assess the expression of β-catenin and c-myc genes, whereas methylation-specific PCR was performed to detect DNA methylation of the APC gene.
The techniques mentioned below were used.
Hematoxylin and eosin-stained slide preparation
Paraffin-embedded tissue sections of 5 μm thickness were cut and stained with hematoxylin and eosin to review the pathologic diagnosis and select blocks in which the neoplastic cells constituted more than 75% of the examined section in order to avoid the neutralizing effect of normal cells in molecular study (Bancroft and Gamble, 2006).
Tissue microarray paraffin block preparation
Tissue microarray paraffin blocks were prepared as described by Kononen et al. (1998) as follows: localization of a representative tumor tissue in the donor’s paraffin block, followed by extraction of three tissue cores with a diameter of 1.5 mm from the original blocks of each case using a Beecher Instrument tissue extractor. Tissue cores were re-embedded on a recipient paraffin block (a recipient block was constructed containing 105 tissue cores, arranged in 10×11 sectors for the HCC block, and 60 tissue cores for the CH block, arranged in 10×6 sectors). Sections of 5 μm thickness from this tissue array block were stained by hematoxylin and eosin to identify tumor tissue on the array slide. Two other 5-μm-thick sections were placed on positively charged slides for immunohistochemical staining.
Tissue sections were deparaffinized in xylene twice for 10 min and rehydrated through descending series of graded ethanol (100, 95, 85, 70%) to distilled water. Immunohistochemical staining was performed using monoclonal mouse anti-human c-myc (clone gE10.3; Thermo Fisher Scientific, USA; ready-to-use dilution for 15 min) and polyclonal rabbit anti-human β-catenin (Thermo Fisher Scientific; ready-to-use dilution). Staining was performed according to the manufacturer’s instructions. Immunohistochemical staining was performed as described by Shan-Rong Shi et al. (1999) and Petrosyan et al. (2002).
The intensity and extent of staining (% of tumor cells stained) were evaluated for each applied antibody. Staining intensity was graded as follows: weak, 1+; moderate, 2+; or strong, 3+. The extent of staining was scored as follows: 1+, if the percentage of tumor cells stained was less than 10%; 2+, if 10–50% of tumor cells were stained; and 3+, if more than 50% of tumor cells were stained. Overall, reactivity was defined as follows: negative if less than 10% of tumor cells were stained regardless of staining intensity; low if 1+ staining was observed in 10–50% of tumor cells; intermediate if 2+ or 3+ staining occurred in 10–50% of tumor cells or 1+ staining occurred in more than 50% of tumor cells; and high if 2+ or 3+ staining was present in more than 50% of tumor cells. Finally, cases were classified as negative or positive (those that scored as low, intermediate, or high were considered positive).
High-molecular-weight genomic DNA was extracted from 10-μm-thick paraffin-embedded tissue sections (five sections) of each case. DNA extraction was performed according to Shan-Rong et al. (2002). DNA concentration and purity were measured using a spectrophotometer (nano drop 2000; Thermoscientific, USA) through assessment of absorbance at wavelengths of 260 and 280. DNA is considered to be of high purity when the ratio of A260/A280 ranges from 1.8 to 2.
Assessment of APC gene methylation
DNA methylation was performed using the ‘EZ WayTM DNA methylation detection kit’ according to the manufacturer’s protocol. PCR amplification of Na bisulfate-modified DNA was performed. The PCR mixture was prepared as follows: 2.5 μl of 10× PCR buffer, 2 μl of 2.5 mmol/l dNTP mix, 1 μl of control primer, 2 μl of modified DNA template, and 0.1 μl of hot-start Taq DNA polymerase (30 U/μl). The volume was adjusted to 25 μl by adding 17.4 μl of distilled water. After initial denaturation at 94°C for 4 min for one cycle, 40 cycles of denaturation at 94°C for 15 s, annealing at 62°C for 20 s, and extention at 72°C for 40 s were performed.
Ten microliters of PCR products were electrophoresed in ethidium bromide-stained 2% agarose gel in 1× Tris-acetate-EDTA buffer. The products were visualized by an ultraviolet light transilluminator and photographed using a photodocumentation system (Biometra, Germany). The amplified methylated band was detected at 100 bp and the unmethylated band at 108 bp.
Statistical package for social science (SPSS) version 12 was used for data management and analysis. Descriptive analysis was carried out to examine the frequencies and distribution of all variables. Mean and SD were used to describe the quantitative variables, whereas proportion and percentage were used to describe qualitative variables. To test the significance of differences among the quantitative data between cases and controls the t-test was used. The χ2-test was used to compare qualitative variables between cases and controls. P-values less than or equal to 0.05 were considered significant.
Clinicopathological features of the patients
All clinicopathological features of the studied CH and HCC patients are illustrated in Table 1.
HCV-associated CH cases
The clinicopathological features of the studied CH cases (20) were collected from the patients’ records. The age of the patients ranged from 18 to 55 years with a mean of 39.25 years. Fourteen cases (70%) were men and six cases (30%) were women, with a male-to-female ratio of 2.3 : 1. Grading of CH was performed using a modified Ishack grading system. Seven cases (35%) were classified as grade I (G1), 10 cases (50%) were grade II (G2), and three cases (15%) were grade III (G3). Two cases (10%) were classified as stage I, eight cases (40%) were stage II, three cases (15%) were stage III, and seven cases (35%) were stage IV (cirrhotic).
According to patients’ records, the age of the studied HCC cases ranged from 40 to 82 years with a mean of 61.37 years. Men represented 27 cases (77.1%), whereas eight cases (22.9%) were women; the male-to-female ratio was 3.4 : 1. The 35 HCC cases were classified as follows: seven cases (20%) were classified as G1, 18 cases (51.4%) were G2, and 10 cases (28.6%) were classified as G3. Associated cirrhosis was detected in 23 cases of HCC (65.7%).
HCV-associated CH cases
Cytoplasmic expression of β-catenin was detected in 85% (17/20) of the studied CH cases. Positive cases included 6/7 cases in G1, 8/10 cases in G2, and 3/3 cases in G3 representing 85.7, 80, and 100%, respectively (Table 2, Fig. 1a and b). The distribution of positive cases in different stages was as follows: 1/2 in stage 1, 7/8 cases in stage 2, 3/3 cases in stage 3, and 6/7 cases in stage 4, representing 50, 87.5, 100, and 85.7%, respectively. There was no nuclear immunostaining in any of the CH cases (Table 3).
Positive nuclear expression of c-myc was detected in 60% (12/20) of CH cases, classified as follows: 5/7 cases in G1, 5/10 cases in G2, and 2/3 cases in G3, representing 71.4, 50, and 66.7, respectively (Table 2, Fig. 2a). In contrast, distribution of positive cases in different stages was as follows: 2/2 cases in stage 1, 5/8 cases in stage 2, 2/3 cases in stage 3, and 3/7 cases in stage 4, representing 100, 62.5, 66.7, and 42.9%, respectively (Table 3).
With regard to grading, CH cases were grouped into two groups as follows: low grade (G1) and high grade (G2, G3). In addition, cases were categorized into two staging groups: early stage (stages 1 and 2) and late stage (stages 3 and 4) for optimal statistical evaluation.
All HCC cases (35/35; 100%) showed positive cytoplasmic and membranous expression of β-catenin (Table 4, Fig. 1c and d).
Positive nuclear c-myc expression was detected in 82.9% (29/35) of HCC cases classified as follows: 3/7 cases in G1, 17/18 cases in G2, and 9/10 cases in G3, representing 42.9, 94.4, and 90%, respectively (Table 4, Fig. 2b). Twenty positive cases were cirrhotic, representing 86.95% (23 cases) of all cirrhotic HCC cases. The remaining nine positive cases were noncirrhotic, representing 75% of all noncirrhotic HCC cases (12 cases).
HCC cases were grouped into two groups in terms of grading: low grade (G1) and high grade (G2 and G3) for optimal statistical evaluation.
APC hypermethylation detection
HCV-associated CH cases
APC hypermethylation was detected in 25% (5/20) of CH cases. Positive cases were classified as follows: G1, 1/7 cases; G2, 3/10 cases; and G3, 1/3 cases, representing 14.3, 30, and 33.3%, respectively (Fig. 3). With regard to the stage of CH cases, no positive cases were detected in stage 1, one case (1/8, 12.5%) was detected in stage 2, one case (1/3, 33.3%) in stage 3, and three cases (3/7, 42.9%) were detected in stage 4 (Tables 2 and 3).
APC hypermethylation was detected in 62.9% (22/35) of HCC cases, whereas 37.1% (13/35) were negative (Fig. 3). On the basis of grade, cases were classified as follows: six cases in G1 (6/7), 10 cases in G2 (10/18), and six cases in G3 (6/10), representing 85.7, 55.6, and 60%, respectively (Table 4). Out of the 23 HCC cases associated with cirrhosis, 16 showed positive promoter hypermethylation of APC, whereas out of the 12 noncirrhotic HCC cases six showed positive promoter hypermethylation of APC.
As regards the age of cases in both CH and HCC groups, there was a statistically significant difference between the two studied groups (P=0.001). In addition, there was a statistically significant difference between the studied groups regarding the presence of cirrhosis (P=0.028).
β-Catenin overexpression: A significant difference in β-catenin expression between HCC cases and HCV-associated CH cases was observed with a P-value of 0.026 (Table 5). However, there was no significant correlation between β-catenin expression and different grades or stages of HCV-induced CH cases (P-value=1.00) (Tables 2 and 3). Moreover, there was no significant correlation between the grade of HCC and β-catenin expression as P-value was 0.5 (Table 4). Further, the correlation between the presence of cirrhosis in HCC cases and β-catenin expression was not statistically significant (P-value=0.69).
C-myc overexpression: There was a significant difference in c-myc overexpression between the CH and HCC groups (P-value=0.05) (Table 5). As regards CH grade and stage, there was no statistically significant correlation between the different grades or stages of CH and c-myc expression (P-value=0.64 and 0.65, respectively) (Tables 2 and 3). In contrast, a highly statistically significant correlation was detected between the different grades of HCC and c-myc overexpression with a P-value of 0.009 (Table 4). Correlation between cirrhosis in HCC cases and c-myc overexpression showed no statistical significance as P-value was 0.39.
APC hypermethylation correlation
A highly statistically significant difference in APC hypermethylation was calculated between HCC and HCV-induced CH groups (P-value=0.007) (Table 5). However, there was no statistically significant correlation between APC hypermethylation and different grades or stages of CH (P-value=0.61 and 0.31, respectively) (Tables 2 and 3). Further, there was no statistically significant correlation between APC hypermethylation and the different grades of HCC (Table 4) or the presence of cirrhosis (P=0.2 and 0.3, respectively).
HCC is the third most common type of cancer worldwide and the fifth most commonly diagnosed solid tumor (Alacaciaglu et al., 2008; Abdel Aziz et al., 2011). In Egypt the incidence of HCC has doubled in the past 10 years (Akiyama et al., 2011). Although the viral and environmental risk factors for HCC development have been established; the molecular pathways leading to malignant transformation of hepatocytes remain elusive (Kim et al., 2008). Some recent studies have shown that the Wnt/β-catenin pathway plays an important role in hepatocarcinogenesis (Brui et al., 2006; Austinat et al., 2008; Lai et al., 2009; Fatima et al., 2012). Overexpression of the c-myc gene and mutations in the β-catenin gene have been reported by Cavard et al. (2006) in HCC; Wang et al. (2006) mentioned that the activation of the Wnt-signaling pathway by an inactivating mutation is critical for the β-catenin degradation system, which consists of axin, APC, and GSK-3 β, and contributes to liver carcinogenesis. Therefore, the aim of the present study was to assess the contribution of β-catenin, APC, and c-myc genes in the Wnt/β-catenin pathway and their contribution to HCV-associated hepatocarcinogenesis, as well as to assess the prognostic value of these genes in HCC.
Our results showed significant difference in age (P=0.001) between HCC cases (61.37 years) and HCV-associated CH cases (39.25 years), suggesting that there is a long sequential process between the incidence of infection and transformation of the tumor. Male predominance in our Egyptian cases (77.1%) is comparable to that of other reports (Kim et al., 2008), where male presence was 88.9% of 36 studied cases.
Our frequency of β-catenin expression in HCC cases is comparable to that of Austinat et al. (2008), who reported positive cytoplasmic expression of β-catenin in 84% of their HCC cases, and to that of Kim et al. (2008), who found that more than half of their cases (55.6%) exhibited cytoplasmic and membranous expression of β-catenin. However, our results are slightly lower than those reported by Suzuki et al. (2002) and Tien et al. (2005), who mentioned that 50% of their cases express β-catenin in the cytoplasm of neoplastic cells and 41.7% in neoplastic cells in HCC cases, respectively. The difference in the frequencies reported could be attributed to differences in the genetic profile of Egyptian patients.
In this study, no significant correlation was noticed in the expression level of β-catenin among different grades of HCC (P=0.5), indicating that β-catenin does not affect tumor differentiation, which is in agreement with Tien et al. (2005) who reported that well and moderately differentiated HCC cases express the same level of cytoplasmic and membranous β-catenin.
The role of c-myc in human HCC remains unclear, although its overexpression was reported to be common (Nishida et al., 1994; Kawate et al., 1999; Liu et al., 2011). In this study, c-myc was overexpressed in 29 cases of HCC (82.9%) and in 12 cases of HCV-associated CH (60%), with a borderline significance between both groups (P=0.05). These results are comparable to those of Fu et al. (2006), who reported c-myc overexpression in 55.3% of HCC cases and in 40.4% of CH cases.
In contrast, our results regarding the frequency of c-myc overexpression differ from those of Wu et al. (2007), who reported c-myc overexpression in 85.7% of HCC cases and in 92.9% of CH cases. This difference between our results and those of Wu and colleagues regarding the expression level of c-myc could be attributed to the differences in the sensitivity of the detection techniques used, as Wu and colleagues used dot hybridization and in-situ hybridization.
Highly significant correlation between different grades of HCC and c-myc overexpression (P=0.009) indicates that the c-myc gene might play an important role in tumor differentiation and aggression.
Abnormal methylation of tumor suppressor genes has received considerable attention as it is involved in the development and progression of several tumors. It has been shown that genes with promoter hypermethylation often lack transcriptional activity, which could result in gene silencing (Zhang et al., 2007; Liu et al., 2012).
The APC gene is among the genes that usually show promoter hypermethylation in several solid and hematological malignancies Iyer et al., 2009; Kiran et al., 2009). We decided to assess the frequency of APC methylation in Egyptian cases of HCV-associated CH and HCC to determine its possible role in HCV-associated liver disease. Our results showed that 22 HCC cases (62.9%) harbored promoter hypermethylation of the APC gene compared with only five cases of HCV-associated CH (25%). The highly significant difference between the two groups (P=0.007) indicates that the APC gene is involved at a late stage of the genetic cascade, contributing to hepatocarcinogenesis.
In agreement with our results, Lee et al. (2003), Csepregi et al. (2007), and Iyer et al. (2009) reported APC promoter methylation in 63.1% of HCC cases compared with only 25.1% of HCV-associated CH cases and in 85% of their HCC patients compared with only 27% of HCV-associated CH patients. This difference may be explained by the presence of other co-carcinogenes, for example, chemical carcinogenes, or by the differences in etiological factors. In addition, the difficulty in implementing the techniques, especially the bisulfate conversion of DNA extracted from paraffin, and the high possibility of DNA loss during the procedure, which further complicates the problem of the small cell number in the tested material, especially in the CH group, which usually contains a lot of inflammatory cells and fibrosis, should also be considered.
Still obscure mechanisms, but may be involving the Wnt signaling and twist proteins, would allow presenescent hepatocytes to bypass senescence, acquire immortality by telomerase reactivation, and obtain the last genetic/epigenetic hits necessary for cancerous transformation. Among some of the oncogenic pathways that might play key driving roles in hepatocarcinogenesis, c-myc signaling and Wnt/β-catenin signaling seem to be of particular interest. Finally, antiproliferative and apoptosis deficiencies involving the TGF-β, Akt/PTEN, and IGF2 pathways, for instance, are prerequisites for cancerous transformation (Merle and Trepo, 2009).
From this study we concluded that β-catenin, c-myc, and APC gene aberrations seem to play an important role in the development and progression of HCV-associated liver disease to a varied extent. In addition, β-catenin and c-myc could be used as biomarkers for early detection of HCC and hence can be used to follow up CH patients, in addition to other currently used laboratory tests. Moreover, c-myc is significantly associated with poorly differentiated tumors as it could be used as a prognostic marker for HCC patients. Thus, it should be added to the routine prognostic panel for HCC patients. Also, APC promoter hypermethylation could be used as a marker for early detection of HCC in the high-risk groups, especially if these results are verified in larger, more expansive studies including testing for methylation in the blood of patients.
Conflicts of interest
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