Background
Hepatocellular carcinoma (HCC) is the fifth most common malignancy as well as the second leading cause of cancer-related deaths worldwide, and its incidence continues to rise [
1,
2]. Although in the past decade, there have been improvements in surgical and medical treatments, the outcome of patients with HCC remains unsatisfactory, mainly because of frequent postsurgical recurrence and metastasis [
3,
4]. Cancer classification based on biomarkers may effectively define the risk of recurrence, allowing the use of appropriate treatment for a better prognosis [
5]. However, molecular markers that effectively define the risk of recurrence have not been completely explored in patients with HCC, and the underlying mechanisms responsible for metastasis remain largely unknown [
4]. Therefore, more reliable biomarkers for predicting relapse and understanding the mechanisms underlying cancer metastasis need to be developed urgently [
5,
6].
Aldolase B (ALDOB), also known as fructose-bisphosphate aldolase, is an important enzyme for glucose and fructose metabolism. It cleaves fructose1-phosphate to yield glyceraldehyde and dihydroxyacetonephosphate. Accumulation of fructose 1-phosphate in tissues due to a defect in ALDOB may result in hereditary fructose intolerance [
7,
8]. Serum levels of ALDOB appear to be a useful measure of liver cell necrosis in both benign and malignant liver diseases [
9]. Results from studies using different technologies but small sample sizes have shown that both ALDOB mRNA and protein are dramatically downregulated in HCC as compared to adjacent non-cancerous liver tissue [
10‐
14]. However, the clinical significance of ALDOB and its role in the pathogenesis of HCC remain largely unknown.
In the current study, we found that ALDOB expression was further reduced in patients with metastasis-inclined HCC (MIH), in those with primary HCC and venous metastases (e.g., portal vein tumor thrombus) or in those with confirmed extrahepatic metastases at follow-up, compared with metastasis-averse HCC (MAH) and in those with HCC without detectable metastases. Using semi-quantitative immunohistochemistry, we explored the clinico-pathological significance of ALDOB protein in a cohort of 313 patients with HCC. Downregulation of ALDOB was associated with multiple malignant characteristics of HCC. Multiple analyses revealed that the downregulation of ALDOB was an independent risk factor for recurrence-free survival (RFS) and overall survival (OS). Moreover, ALDOB downregulation was consistently indicative of a shorter RFS and OS in BCLC 0-A and TNM I stage patients. Furthermore, we generated stable ALDOB expressing cell lines to explore the role of ALDOB in HCC. We found that overexpression of ALDOB could inhibit cell migration and reduce metastasis in vitro and in vivo. A gene microarray study on an ALDOB stably expressing cell line and its control showed that overexpression of ALDOB induced a high level of Ten–Eleven Translocation 1 (TET1) expression. We also found that ALDOB stably expressing cells had a high level of methylglyoxal (MG), which contributes to TET1expression.
Discussion
Despite recent improvements in surgical and medical treatment strategies, prognosis of patients with HCC still remains unsatisfactory due to frequent recurrence and metastasis [
3,
4]. However, the mechanisms underlying metastasis remain largely unknown, and the molecular markers that effectively define the risk of recurrence in patients with HCC have not been greatly explored. Therefore, reliable biomarkers are needed to predict relapse and outcome of patients for their optimal medical management. Moreover, additional biomarkers that play a role in HCC are needed to explore novel therapeutic targets.
Increased glycolysis, a hallmark of malignant cancers, correlates with invasive potential and poor prognosis [
22]. Many glycolysis-related genes are abnormally expressed in HCC tissues, and some of these genes are thought to be involved in carcinogenesis of hepatocytes; moreover, they influence the prognosis of patients with HCC [
23,
24]. ALDOB, an important glycolytic enzyme, is a well-known specific marker of hepatic differentiation and is expressed abundantly in the adult liver [
25,
26]. ALDOB expression is frequently decreased in liver cancer [
10‐
14,
24]. However, its clinical significance and role in HCC pathogenesis are largely unknown. Our previous microarray data demonstrated the dramatic downregulation of this gene in HCC tissues compared with paired non-tumor tissues. More interestingly, the expression of ALDOB is further reduced in MIH. This suggests that ALDOB could be used as a biomarker for aggressive HCC and that it might be involved in the progression of HCC.
Hence, in this study, we explored the clinical significance and role of ALDOB in HCC pathogenesis. We measured the ALDOB protein levels in a large cohort of 313 patients and correlated them with the clinico-pathological features of the patients. ALDOB downregulation correlated with female gender (P = 0.016), tumor size >5 cm (P = 0.006), absence of encapsulation (P = 0.002) and early recurrence (P = 0.002). A Kaplan–Meier analysis showed that patients with lower ALDOB expression had higher recurrence rates (P = 0.0031) and shorter survival times after curative resection (P = 0.0023). Moreover, a multivariate analysis revealed that the ALDOB level was an independent risk factor for both RFS and OS.
Prognostic algorithms (the BCLC and the TNM staging systems) based on multiple clinico-pathological characteristics have better prognostic functions than algorithms based on a single clinico-pathologic factor. However, even patients with tumors of the same stage can have different outcomes [
27]. Based on the current staging systems, patients with early stage tumors are thought to be at a lower risk for recurrence. However, some of these patients may still show poor prognosis in clinical practice. Therefore, predicting the prognosis of these patients is a major challenge for attending clinicians. Considering the downregulation of ALDOB in patients with poor prognosis, we explored its prognostic value in tumors showing different stages according to the BCLC and the TNM staging systems. We first stratified all 313 patients according to the BCLC and TNM staging systems. The patient distribution and 3 year survival rates in this series were similar to those of previous reports [
28]. Intriguingly, ALDOB downregulation was consistently indicative of a poor RFS and a poor OS in patients with BCLC 0-A and TNM I stage tumors. Thus, the findings of the present study suggest that measurement of ALDOB protein levels could identify a worse prognosis among patients in early stages of HCC. The prognostic value of ALDOB may be valuable to clinicians for distinguishing early stage patients with a high risk of recurrence. In such cases, close follow-up and appropriate adjuvant therapies should be recommended to prolong survival.
ALDOB overexpression also inhibited the motility and metastatic potential of HCC cells both
in vitro and
in vivo. In addition, we explored the underlying molecular mechanism of action of ALDOB. As no significant aberration was observed in the expression of glycolysis-related genes and initial and final metabolic products of glycolysis, we speculated that ALDOB might not affect glycolysis. This might due to: First, ALDOA, another enzyme has similar function, predominantly expressing in cell lines than ALDOB (Additional file
3: Figure S2 A and B). Second, ALDOB has a 10 fold lower Km for glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) than ALDOA [
29], which means ALDOB having the less ability of conversion fructose-1,6-bisphosphate F-1,6-BP to G3P and DHAP in glycolysis. Third, the ALDs are not the key enzyme in controlling glycolysis rate. It is reported that ALDOA knocking down does not affect the glycolytic flux and intracellular ATP concentration [
30]. However, we found that the MG level was elevated in ALDOB-expressing cells, which is primarily generated from G3P and DHAP through non-enzymatic fragmentation of triosephosphates [
31]. This might rely on the ability of ALDOB in cleaving the fructose-1-phosphate (F-1-P) into G3P and DHAP [
32], and this might be the reason why is ALDOB but not ALDOA affect the MG level in endothelial cells [
18,
19]. We validated the relationship of MG level and ALDOB expression in HCC cells. However, the influences of ALDOB in metabolism still need further exploration. MG has been reported to inhibit cell growth in several types of tumors. In HCC cell lines, MG is thought to influence cell adhesion and inhibit cell migration. However, since it is difficult to eliminate MG, whether ALDOB inhibits tumor migration mainly through MG is still unclear.
We therefore analyzed the resultant expression profile upon ALDOB overexpression. We observed that increased levels of ALDOB elevated the expression levels of TET1. TET1 belongs to the ten-eleven translocation (TET) family that promotes DNA demethylation by catalyzing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and to a lesser extent, 5-formylcytosine or 5-carboxylcytosine [
33]. TET1 is suppressed in several solid tumors including HCC and is thought to influence cell adhesion and inhibit cell growth and metastasis [
15‐
17]. In this study, we observed that ALDOB promotes TET1 expression. Knock down of TET1 by siRNA promoted HCC migration and reversed the inhibition of metastasis induced by ALDOB. All of these results suggest that ALDOB inhibits cell metastasis, partly through its regulation of TET1. Moreover, some reports have shown that addition of MG to cultures can elevate TET1 expression in lens epithelial cells [
21]. We validated this observation by adding MG to HCC cells. This experiment indicated that elevation of TET1 by ALDOB might occur at least partly through MG. However, as described above, since we lack the means to eliminate MG under experimental conditions, the relationship between ALDOB/MG/TET1is still unclear and requires further investigation.
In conclusion, this study investigated the expression of ALDOB and its value as a prognostic biomarker in HCC. It also explored the biological function of ALDOB in HCC. We observed that the downregulation of ALDOB is a predictor of high-risk metastasis and recurrence in HCC patients. These patients should be closely monitored and should receive appropriate adjuvant therapies for a better prognosis. We also found that ALDOB inhibits HCC metastasis by elevating TET1, a process that may involve MG. However, the detailed mechanism of this process is yet to be elucidated.
Materials and methods
Patients and specimens
The study protocol was approved by the clinical research ethics committee of the Eastern Hepatobiliary Surgery Hospital. Written informed consent was obtained from all patients according to the policies of the committee. Any information that could identify the patients was not included in this article.
This study included 29 HCC tissues and paired non-tumorous tissues (including 14 MIH and 15 MAH) for microarray analysis. Another 50 HCC specimens (including 20 MIH with and 30 MAH) were used for the validation of microarray data by qRT-PCR analysis. The MIH and MAH status was defined as described previously [
34,
35]. The MIH group was composed of patients with solitary HCC that was accompanied either by portal vein metastasis or by venous metastases at follow-up, or those who had two nodules present at the time of sample collection. The MAH group was restricted to only those patients with solitary HCC and no recurrence at follow-up. Samples from 25 patients with HCC and portal vein tumor thrombosis (PVTT) were used to test the expression level of ALDOB in paired HCC/non-tumor tissues/PVTT by qRT-PCR and IHC. Another 288 HCC cases and paired non-tumor tissues were used to validate ALDOB protein level by IHC. Moreover, a large HCC cohort of 313 patients with HCC (randomly collected from January 2006 to September 2010) who received a high-quality follow-up, were used to examine ALDOB protein levels and the clinical significance of ALDOB by tissue microarray (TMA) and IHC. Normal liver tissues were collected from patients who underwent resection of hepatic hemangiomas. All liver specimens were randomly selected from patients who underwent curative hepatectomy, and all specimens were pathologically confirmed at the Eastern Hepatobiliary Surgery Hospital between 2005 and 2010 [
36].
The preoperative diagnosis was obtained as described previously, and surgical procedures were performed on the patients with HCC as described previously [
37]. The clinical characteristics of the HCC cohort are listed in Additional file
2: Table S1. The differentiation of HCC was defined according to the criteria proposed by Edmondson and Steiner (I, well-differentiated; II, moderately differentiated; III, poorly differentiated; IV, undifferentiated). Micro-metastases were defined as tumors that were located adjacent to the border of the primary tumor and that could only be observed under the microscope. Tumor staging was conducted according to the BCLC staging system and the sixth edition of the Tumor-Node-Metastasis (TNM) classification system published by the International Union Against Cancer.
All HCC specimens were obtained immediately after hepatectomy. Tissues were then fixed in 10 % buffered formalin and embedded in paraffin. Fresh specimens used in this study were snap-frozen from tissues prior to formalin fixation and were then transferred to liquid nitrogen and stored at −80 °C until use.
Follow-up
All patients with HCC received check-ups every 2–3 months during the first 2 years and every 3–6 months there after until the follow-up period ended in January 2013. Physicians who were blinded to the study performed the follow-up examinations. Serum AFP levels and abdominal ultrasound examinations were performed every month during the first year after surgery and every 3–6 months thereafter. Computed tomography and/or magnetic resonance imaging were performed every 3–6 months or when a recurrence was suspected. A diagnosis of recurrence was based on preoperative diagnosis criteria. Once recurrence was confirmed, further treatment was implemented based on the tumor diameter, the number of tumors, the location of the tumor, and the extent of vessel invasion as well as liver function and performance statuses. The recurrence-free survival (RFS) was calculated from the date of tumor resection until the detection of tumor recurrence, death from a cause other than HCC, or the last follow-up visit. The overall survival (OS) was defined as the length of time between surgery and either the death of the patient or the last follow-up visit.
Cell lines
The SMMC7721, LM3, LM6, HepG2, Huh7, and Hep3B cells were obtained from the Chinese Academy of Sciences Cell Bank and were authenticated by the providers by DNA-Fingerprinting analysis or isoenzyme analysis. All cell lines were used at early passages and no morphological changes or altered growth rates were observed during maintenance of the cultures. Cells were grown in Dulbecco’s modified Eagle’s medium with 10 % fetal bovine serum (Gibco BRL). Cells were maintained in an atmosphere of 5 % CO2 in a humidified 37 °C incubator.
Total RNA was extracted from snap-frozen tissues with Trizol reagent (Takara, Dalian, China) according to the manufacturer’s instructions. The quality of the total RNA was assessed by a Nanodrop 2000 and agarose gel electrophoresis. First-strand cDNA was synthesized from 1–2 μg of total RNA using random primers and M-MLV Reverse Transcriptase (Invitrogen, CA). Real-time PCR was performed according to the Taqman probe assay protocol or the SYBR Green protocol in a StepOne Plus system (Applied Biosystems, Foster City, CA) with β-actin as the endogenous control. The relative mRNA levels were calculated based on the Ct values and were according to the β-actin expression. The primer and probe sequences are listed in Additional file
2: Table S3.
Western blot analysis
Total protein was extracted from snap-frozen tissues with RIPA Lysis Buffer and PMSF (Beyotime Co., China) according to the manufacturer’s instructions. Western blotting was performed as described previously [
38]. Antibody dilutions were 1:1500 for the ALDOB polyclonal antibody (Proteintech Group, Inc., Chicago, USA) and 1:5000 for β-actin (Sigma-Aldrich, USA). Antibody binding was detected with an Odyssey infrared scanner (Li-Cor Biosciences Inc).
Tissue microarray construction and immunohistochemical analysis
The tissue microarray (TMA) was constructed as described previously [
39]. Briefly, all samples from patients in the HCC cohort were reviewed histologically by hematoxylin and eosin staining. Representative areas were premarked on the paraffin blocks, away from necrotic and hemorrhagic tissue. Two 1.0-mmcores were extracted from each tumor, were paired with non-tumor tissue and were mounted on a new recipient block with a semi-automated arraying device (TMArrayer, Pathology Devices, Westminster, MD, USA).
Immunohistochemistry was performed on the TMA with a two-step immunoperoxidase technique [
40]. The ALDOB polyclonal antibody (Proteintech Group, Inc, Chicago, USA) diluted 1:60 was used as the primary antibody. Briefly, after heating the sections in 10 mmol/L citrate buffer for antigen retrieval, the sections were incubated first with the primary antibody and then with the secondary antibody for an hour at room temperature. Finally, the sections were developed in diaminobenzidine solution under a microscope and counter-stained with hematoxylin.
The immunohistochemical stains were assessed by three separate observers who had no knowledge of patient characteristics. ALDOB staining was abundant in the cytoplasm and nucleus, which was similar to previous studies [
41,
42]. The expression was recorded after an evaluation of the staining intensity of positive cells (0 = none; 1 = +weak; 2 = ++ intermediate, 3 = +++ strong). Scoring differences were discussed until a consensus was reached. A low level of ALDOB in the HCC specimens was defined as staining intensity that was designated as either “none” or “weak.”
Wound healing migration assay
Wound healing migration assays were performed as described previously [
38]. Briefly, 1 × 10
5 cells were plated in each well of 6-well plates. Once cells were attached, they were scraped to form a wound in the middle of the plates and the medium was replaced with serum-free medium. We measured the area of healing across the line after an incubation of 36 h.
Transwell assays
Millicell 24-well culture insert plates (Millipore, USA) and polycarbonate membranes with a pore size of 8 μm were used in Transwell assays as described previously [
38]. First, The insert plates were equilibrated with 0.5 ml of DMEM for1 h at 37 °C in 5 % CO
2, which was then replaced with 0.5 ml of DMEM supplemented with 10 % FBS in the lower chambers. In all, 50,000 cells in 400 μl of serum-free DMEM were loaded into the upper chambers. After 24 h of incubation, the insert plates were rinsed with PBS and the upper surfaces of the membranes were scraped toremove the cells. The cells on the underside of the membrane were stained with Giemsa stain and counted under a microscope. Cells from each culture condition were examined in quadruplicate.
Animal studies
The animal studies were approved by the Institutional Animal Care and Use Committee of the Second Military Medical University, Shanghai, China. To explore the effects of ALDOB on tumor growth
in vivo, 1 × 10
7SMMC7721 cells thatstablyexpressedALDOB (SMMC-ALDOB) and control cells (SMMC-GFP) were subcutaneously implanted into the bilateral armpit of nine BALB/C nude mice. The tumor volume was measured every week after implantation (the volume V = length × width × length × 1/2). All mice were sacrificed five weeks later, and sections of tumor tissues were transplanted to establish an orthotopic model as described previously [
37,
43]. A tail vein injection model was also used to evaluate the potential of the cells to metastasize to the lungs. The metastases were monitored by the IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA) for 10 min after intraperitoneal injection of 4.0 mg of luciferin (Gold Biotech) in 50 μl of saline.
Statistical analysis
All statistical analyses were performed with SPSS version 18.0 software. The χ
2 test or Fisher’s exact test were used to compare qualitative variables, and the Student’s t-test or the Mann–Whitney test were used to compare continuous variables. Survival curves were calculated according to the Kaplan–Meier method and were compared by a log-rank test. A Cox proportional hazards model was used to determine the independent factors of survival and recurrence based on variables selected after the univariate analysis. A difference was defined as significant at P < 0.05.
Acknowledgement
The authors thank Feng Liu, Jin-zhao Ma, Tian-tian Wang, and Feng-rui Bi for their technical assistance. This work was supported by National Key Basic Research Program of China (Grant Numbers: 2014CB542102); State key infection disease project of China (2012ZX10002010); Science Fund for Creative Research Groups,NSFC, China,(81221061); the National High Technology Research and Development Program of China 2013AA032202; National Natural Science Foundation of China (Grant No. 81372207, 81201940, 81472691, 81330037 and 81301831); Shanghai Rising-Star Program (13QA1404500).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Q-F T, S-X Y, and FY designed the study, designed experiments, analyzed results, and wrote the manuscript. Q-F T, S-X Y, SY, Z-G W, X-L T, K-Y L, JC, C-C Z, W-L H, Q-G X performed most of the experiments and analyzed results. YY, FW, S-H S, W-P Z designed the study, provided reagents and analyzed the data. All authors read and approved the final manuscript.