Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the seventh in women. Many HCC patients have progressed to advanced stages at time of diagnosis, resulting in poor prognoses and high mortality. Despite various treatment options, survival of HCC is poor due to late diagnosis and resistance to chemotherapy. The current gold standard and most commonly used diagnostic marker for HCC is plasmic alpha-fetoprotein (AFP) along with ultrasound every 6 to 12 months, but this is far from satisfactory. Only serum AFP levels of more than 400 ng/mL are considered truly diagnostic, but such high values are observed only in a small percentage of patients with HCC at advanced stages [
1]. Although many molecules have been considered to associate with diagnosis and prognosis, their presumed merits are controversial [
2,
3]. Accordingly, new diagnostic and prognostic factors are needed to improve HCC diagnosis and treatment.
Evidence is accumulating that perturbed cellular metabolism predisposes human to tumor development. Metabolic diseases such as obesity and diabetes are associated with increased risk to develop various cancers [
4‐
6]. In addition, many human tumors display a high rate of aerobic glycolysis, de novo fatty acid synthesis and nucleotide biosynthesis [
7,
8]. It has been proposed that increased glucose metabolism promotes lipogenesis and nucleotide biosynthesis, and enhances tumor cell growth and proliferation by providing essential synthetic and bioenergetic molecules [
9,
10].
Cancer cells acquire energy from glucose to satisfy their high metabolic demands. Glucose and related hexoses are transported into cells via glucose transporter (GLUT) family proteins (Solute carrier, SLC2A family) [
11]. To date, 14 GLUT family members have been identified, each of them with different affinities for glucose and their expression patterns are tissue dependent [
12,
13]. Many studies have demonstrated that the expression of glucose transporters, especially GLUT1, is increased in a variety of tumors, including pancreatic, breast, esophageal, and brain cancers [
14,
15]. GLUT1 overexpression is associated with tumor progression and poor overall survival in various malignant tumors [
16,
17]. GLUT2 is expressed in liver, pancreatic islet cells and retina and it is the most abundant glucose transporter in liver [
18]. GLUT2 mRNA expression was shown to be increased in gastric tumors [
19] but decreased in insulinomas when compared to normal tissues [
20]. However, data on these glucose transporter expression in different stages of HCC have been lacking.
One of the master regulators of intracellular glucose metabolism is carbohydrate responsive element binding protein (ChREBP), a basic helix–loop–helix leucine zipper (bHLH-LZ) transcription factor expressed in liver, white and brown adipose tissues, intestine, muscle, and pancreatic β-cells [
21,
22]. Elevated insulin, in response to increased glucose levels, promotes ChREBP dephosphorylation and its translocation from the cytoplasm to the nucleus where, in association with its binding partner MLX (Max-like interacting protein), it binds to carbohydrate response elements, present in the promoters of target genes. Glucose metabolites F6P and acetyl-CoA can increase ChREBP activity via O-linked glycosylation and acetylation, respectively [
23‐
25]. The ChREBP/Mlx heterodimer controls glucose and lipid metabolism through regulating glycolytic (
Pklr, Fk, Glut2, Glut4), gluconeogenic (
G6pc), and lipogenic (
Fasn, Acc1, Scd1, Elovl6) gene expressions [
22,
26,
27], suggesting that ChREBP may have an important role in the pathogenesis of metabolic diseases and cancer. So far, most of the work on ChREBP has focused on its function as a hepatic transcription factor, its activation by glucose metabolites and its role in the regulation of lipogenesis. Little is known about the role of ChREBP in cancer cells. Genomic analysis of ChREBP target gene expression in human hepatocellular carcinoma cell line HEPG2 by ChIP-sequencing showed that ChREBP regulates genes associated with a tumor metabolic phenotype and malignant progression, such as tumor differentiation and motility [
28]. Suppression of ChREBP in hepatocellular carcinoma and colorectal cancer cells led to reduced lipogenesis and nucleotide synthesis and decreased proliferative and tumorigenic potential in mice [
29]. In breast cancer, the level of ChREBP protein expression is positively correlated with tumor progression [
30]. However, hepatic ChREBP expression has not been examined in human HCC and the relationship between ChREBP expression and the degree of liver tumor malignancy has yet to be investigated.
In this study, we analyzed GLUT family members GLUT2 and GLUT1, and ChREBP protein expression levels in human liver tissue array composed of normal, HCC and adjacent liver tissue. We found that the expression of ChREBP showed a tendency to increase with liver malignancy, but unexpectedly, that GLUT2 protein expression was decreased in cancer cells compared to normal hepatocytes and its expression was negatively associated with advanced stages of HCC. GLUT1 was increased in cancer tissues and its expression was significantly correlated with clinical stage. Moreover, ChREBP and GLUT1 expressions were positively correlated to each other but GLUT1 and GLUT2 expressions were negatively correlated. Therefore, a combined evaluation of ChREBP, GLUT1 and GLUT2 protein expression profile may provide a new diagnostic and prognostic marker for HCC, that might be useful in improving patient treatment and survival.
Materials and Methods
Human hepatic carcinoma and normal tissue microarray was purchased from AURAGENE (TC0145, Changsha, China). The tissue array contains 40 cases of hepatocellular carcinoma, 17 cases of normal tissue and 13 cases of adjacent normal tissue. In addition, 5 pairs of cancer and adjacent normal tissues were collected from the Second Affiliated Hospital of Shantou University Medical College, Shantou, China. This study has been approved by Shantou University medical college Ethic Committee. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all participants and/or their legal guardians.
Immunohistochemistry
Immunohistochemistry was carried out following an established protocol [
31]. Briefly, liver tissue microarray or paraffin tissue sections were dewaxed, rehydrated through graded ethanol and incubated with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 mol/l citrate buffer (pH 6.0) for 15 min. Slides were then washed in PBS for 15 min and treated with 10% normal horse serum for 30 min and incubated with primary antibody at 4 °C overnight. The reaction products were detected with 3-amino-9-ethylcarbozole (AEC) substrate-chromogen kit after incubating with the secondary antibody of Dako REAL EnVision Detection Kit (Dako, Carpinteria, CA) for 30 min and washing in 0.1 M PBS at room temperature. Staining with AEC resulted in red signals. The primary antibody for GLUT2 (Novus Biologicals, NBP2-22218SS, USA), GLUT1 (Abcam, ab115730, USA), were used at a 1:500 dilution and anti-ChREBP (Novus Biologicals, NB400–135, USA) was used at a 1:200 dilution. The antibodies against GLUT2 and ChREBP were rabbit-derived polyclonal and the antibody against GLUT1 was rabbit-derived monoclonal. For negative controls, the primary antibodies were replaced with PBS.
Stain-Decolorize-Stain (SDS) Method for Liver Microarray
The stain-decolorize-stain method was performed to show whether GLUT2 and ChREBP are expressed in the same hepatocytes/pattern according to an established procedure [
32]. Briefly, immunohistochemistry of ChREBP was first performed on the liver microarray. After visualization and taking photos, the slides were decolorized with 80% alcohol for half an hour at room temperature and then heated in a microwave oven for 10 min to remove the bound antibodies. The microarray and slides were then incubated with GLUT2 antibody at 4 °C overnight. After visualization with the AEC kit, photos were taken at the same fields as for the first immunostaining. Appropriate controls were carried out and the specificity of this technique has been reported previously [
32].
Semiquantition of Stainings of ChREBP and Glucose Transporters
Semiquantitative scoring was carried out as described in a previous study [
33]. Briefly, tissue microarray sample ‘spots’ were viewed at 400× magnification and an overall score was assigned according to intensity and area of positive immunostaining. Sample scorings are as follows: 0, no red staining at all (negative); 1, pink staining in the minority of tissue (mild); 2, pink staining in the majority of the tissue (middle); 3, red staining in the majority of the tissue (strong) and 4, dark-red staining in all the tissue (very strong) following a well established protocol [
33]. To reduce the variation of scoring, all slides, including microarray, were scored by two observers independently (Y. L. and Q. H.).
Statistical Analysis
The effect of HCC malignancy on protein expression was analyzed with one-way ANOVA. The correlation between 2 protein expression levels were analyzed with Pearson Correlation test. If protein expression levels were normally distributed and had equal variance, Student’s t test was used. If the scores were not normally distributed, we used the Mann-Whitney U test. p < 0.05 was considered statistically significant. All figures and statistics were made with Graphpad Prism 7 (CA, USA).
Discussion
It has been proposed that the glucose-sensitive transcription factor ChREBP plays a role in cancer pathology. It has been reported that suppression of ChREBP mRNA inhibited cell proliferation in vitro and reduced tumor growth in vivo [
29]. ChREBP protein expression was positively correlated to breast cancer malignancy, i.e., more malignant cancers express more ChREBP protein [
30]. However, in hepatocellular carcinoma, the relationship between ChREBP expression and cancer malignancy has not been established. In this study, we found that ChREBP protein expression tended to increase with cancer progression although, with the limited number of samples, could not reach statistical significance in compassion to normal liver tissue. In normal liver tissue, there was no or weak ChREBP protein expression, but in liver cancers the expression was abundant. In stage III HCC patients, 32% of the samples showed strong staining of ChREBP. The increased ChREBP expression may serve to support increased glycolysis and lipogenesis in cancerous hepatocytes. Although ChREBP protein levels can be used as a novel diagnostic and prognostic marker in breast cancer [
30], variation in HCC appears to be too large for this purpose. Nevertheless, ChREBP may contribute to diagnosis of cancer grading in combination with other markers.
Most cancer cells have increased aerobic glycolysis under both anaerobic and aerobic conditions [
37]. This increased glycolysis, accompanied by accelerated glucose uptake, is known as the Warburg effect, after the German biochemist Otto Warburg who first described the phenomenon in 1920s [
38]. Increased glucose uptake is contributed by transmembrane transport mediated by specific glucose transporters [
39,
40] and increased activity of hexokinases [
41,
42]. Many studies have demonstrated that the expression of glucose transporters, especially GLUT1, was increased in a variety of malignancies and GLUT1 overexpression was associated with invasiveness and poor overall survival of various malignant tumors [
43‐
47]. GLUT2 mRNA expression was inversely associated with overall survival in HCC [
18] and invasiveness in insulinomas [
20]. Thus our data confirm that GLUT2 can be potentially used for HCC diagnosis and prognostic prediction [
18]. It has been reported that GLUT2 is a target of ChREBP [
22], but its expression had an opposite pattern compared to ChREBP in our study. The possible reason could be that GLUT2 is not only regulated by ChREBP but also that other regulators could have stronger influences on GLUT2 expression. This notion, however, requires further investigation.
It has been reported that the increased glucose transport was due, at least in part, to increased expression of GLUT1 in cancers compared to normal tissues [
36] and that GLUT2 was replaced by GLUT1 in human hepatocellular carcinoma cell line HepG2 [
48]. These results suggest that the increased expression of GLUT1 gene may closely relate to cellular transformation. In our study, we found that GLUT1 was significantly increased in HCC compared to adjacent normal hepatocytes and its expression was positively associated with HCC progression. Therefore, GLUT1 might serve as a diagnostic marker for HCC. Moreover, we found that ChREBP-expressing hepatocytes do not express GLUT2 protein but express GLUT1, and that ChREBP-negative hepatocytes express GLUT2 but not GLUT1. This indicates that ChREBP and GLUT1 have a similar expression profile. Upon statistical analysis, we found that ChREBP and GLUT1 protein levels were significantly positively correlated to each other and GLUT1 and GLUT2 levels were significantly reversely associated. Different glucose transporters have different binding affinities for glucose. GLUT1/HKIV has high affinity for glucose [
46] but GLUT2/GCK has relatively low affinity for glucose, mannose and galactose [
47]. Therefore, a transition from GLUT2/GCK to GLUT1/HKIV mediated glucose metabolism could contribute to cancer differentiation because of losing sensitivity of the liver to circulating glucose levels.
Taken together, this is to our best knowledge the first to report of ChREBP expression in HCC and its relationship with glucose transporters GLUT1 and GLUT2. Analysis of combined profile of ChREBP, GLUT1 and GLUT2 expression should be helpful for HCC diagnosis and shed light on improvement of HCC treatment and patient survival.
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