Abstract
Aim: Overexpression of stathmin (STMN1) has been reported for several tumor entities. STMN1 expression correlated with the detection of mutant p53, also suggesting loss-of-function-dependent mechanisms for its accumulation in hepatocellular carcinoma (HCC) cells. On the other hand, miR-223 has been identified as one of the most down-regulated miRNAs in HCC, and its expression was shown to be negatively correlated with STMN1 expression. The aim of this study was to investigate the clinical significance of STMN1 and miRNA-223 expression. Patients and Methods: Fifty-six consecutive patients with HCC who underwent curative hepatectomy as initial treatment were enrolled. They were divided into two groups based on the STMN1 expression level: high (n=36) and low (n=20) gene-expression groups. We compared the clinicopathological factors between the groups with high and low expression in non-tumor tissues. Thirty out of 56 patients were also divided into groups with high (n=15) and low (n=15) miR-223 expression and compared the clinicopathological factors between the two groups. Results: There were no significant differences in patient background between high and low STMN1 expression groups. The incidence of multicentric (MC) recurrence in the high-expression group was significantly higher than in the low-expression group and high STMN1 expression was shown to be an independent risk factor for MC recurrence. There were also no significant differences in patient background between high and low miR-223 expression groups; however, the disease-free survival rate in the group with low expression was significantly worse. Furthermore, MC-related miRNAs identified by miRNA microarray clearly clustered patients into MC recurrence and non-recurrence groups. Conclusion: Both gene and miRNA expression profiles in non-tumor liver tissues could predict the risk for MC recurrence. Such molecular information may be useful in enabling better decision making for patients with HCC.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality (1). Although the mechanisms of liver carcinogenesis associated with chronic liver damage are still unclear, the increased turnover of hepatocytes and inflammatory cell infiltrate seen in chronic hepatitis and cirrhosis may lead to an accumulation of genetic alterations, which ultimately result in the development of HCC (2-4). Despite some progress in the treatment of cancer, existing therapies are limited in their ability to cure malignancies and to prevent metastasis and relapse. The long-term results of hepatic resection for HCC in cirrhotic patients have been disappointing because of the high rate of intrahepatic recurrence as a result of either multicentric (MC) recurrence or intrahepatic metastasis of HCC (5, 6). The development of an effective prognostic predictive model for HCC recurrence after curative treatment would, therefore, help identify the patients who would most benefit from such treatment, and could also lead to the development of new therapeutic strategies.
Several studies have shown that the specific gene expression patterns in cancerous tissues of HCC can accurately predict early intrahepatic recurrence, possibly due to the intrahepatic metastasis of HCC (7-17). It is necessary to investigate the noncancerous portion of liver tissue specimens to examine the molecular mechanisms occurring during the process of liver carcinogenesis based on the idea of ‘field cancerization’ because MC recurrence of HCC is mainly associated with underlying chronic liver damage rather than adverse tumor factors (13, 18).
STMN1 encodes for the cytosolic phosphoprotein stathmin (synonym: oncoprotein-18/Op18, metablastin). Stathmin belongs to a family of proteins that also includes stathmin-like 2 (STMN2), STMN3, and STMN4, which are involved in the regulation of microtubule (MT) dynamics (19). Although all family members share a highly conserved STMN1 domain, differences in the number of phosphorylation sites and sequence variations may facilitate diverse biofunctionality in different cell types (20).
Stathmin modulates MT dynamics through two different mechanisms. Firstly, it sequesters α/β-tubulin-heterodimers, preventing MT polymerization. Secondly, it directly binds to MTs, subsequently promoting the so-called ‘MT catastrophe’, which describes the transition from MT growth to MT shrinkage. In response to a number of cellular stimuli (e.g. leading to proliferation), stathmin is phosphorylated on up to four serine residues (Ser16, Ser25, Ser38, and Ser63) by various kinases (e.g. cyclin-dependent kinases), which inactivates it and facilitates mitotic spindle assembly (20). Accordingly, the dephosphorylation of stathmin (e.g. by protein phosphatases type 1, 2A, and 2B; PP1, PP2A, PP2B) mediates proper disassembly of the spindle apparatus and the subsequent exit from mitosis (21). Besides its role in cell-cycle progression, emerging evidence indicates that stathmin affects cell migration, because cell motility depends on MT cytoskeleton reorganization in distinct subcellular regions (22).
Overexpression of stathmin has been reported for several tumor entities such as leukemia and breast cancer (23, 24). Because the tumor-suppressor gene TP53 negatively regulates stathmin expression in various cell types (25), and expression of mutant TP53 is associated with elevated stathmin levels in breast cancer cells (26, 27), functional p53 deficiency (loss-of-function) is discussed as the most probable reason for increased stathmin expression in cancer cells. Recently, immunohistochemical analyses of HCCs showed that elevated stathmin levels correlated with clinical parameters such as tumor size and reduced 5-year survival. Furthermore, stathmin expression correlated with the detection of mutant TP53, also suggesting loss-of-function-dependent mechanisms for its accumulation in HCC cells (28).
The patients with HCC often have chronic liver damage, which may be associated with a risk of MC recurrence after curative hepatectomy. However, some patients are at low risk of MC recurrence, while the other are at high risk. Therefore, there should be specific differences in molecular features between these two liver tissues. In fact, it has been shown that even when histological degrees of liver fibrosis are comparable, specific molecular signatures in non-tumor liver tissues may predict the risk of intrahepatic recurrence and patient prognosis (29). Using a DNA microarray analysis, we also found there was a cluster for patients with a high-risk of MC recurrence (30). We therefore consider specific molecular alterations associated with hepatocarcinogenesis already exist in non-tumor liver tissues. To date, we have identified some candidate genes related to hepatocarcinogenesis. These include up-regulated genes, such as an STMN1 (31). On the other hand, miR-223 has been identified as one of the most down-regulated miRNA in HCC, and its expression was shown to be negatively correlated with STMN1 expression.
In this study, to investigate whether molecular characterization of non-tumor liver tissues is helpful in determining therapeutic strategies for patients with HCC, we examined the clinical significance of STMN1 and miR-223 expression, and the relationship between STMN1 expression and MC recurrence after hepatectomy.
Patients and Methods
Patients. Fifty-six consecutive patients with HCC who underwent hepatic resection at Tokushima University Hospital were enrolled in this study. The eligibility criteria were as follows: no treatment history before hepatectomy, without macroscopic vascular invasion, curatively resected, and non-tumor liver tissue available for examination. The clinicopathological characteristics, including age, gender, pathology, and differentiation, were available for all patients. This study was approved by the Institutional Review Board at Tokushima University, and all patients provided written informed consent to participation in the study.
Quantitative reverse transcription polymerase chain reaction (RT-PCR). The mRNA expression levels of STMN1 in non-tumor tissues were determined by TaqMan quantitative RT-PCR. The technical methods are described elsewhere (32).
The following assays (assay identification number) were used: STMN1 (Hs00217794_m1) and TaqMan Human GAPDH Endogeneous Control (4326317E) as the housekeeping gene. The mRNA expression levels of STMN1 were calculated as a ratio to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA) were used to quantify the expression level of miR-223. Total RNA was reverse-transcribed by MultiScribe (Applied Biosystems) in a reaction mixture containing miRNA-specific stem-loop RT primer. Quantitative PCR was performed in triplicate reactions containing cDNA preparation and TaqMan primers in Universal Master Mix without AmpErase UNG (Applied Biosystems). The quantitative RT-PCR was conducted at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds.
MC recurrence. MC recurrence was determined according to the Liver Cancer Study Group of Japan (32) and similar concept of image patterns (33). Lesions which were difficult to classify were not included in this study. ‘Nodule-in-nodule’ appearance is one of the typical findings of MC recurrence, which describes a well-differentiated HCC surrounding a moderately differentiated HCC. This is based on the concept of de novo and multi-step hepatocarcinogenesis.
Patient follow-up. All patients were followed-up according to a standard protocol in the outpatient clinic (34). Briefly, patients were followed up every 2 months during the first postoperative year and at least every 3-4 months thereafter. Both alpha-fetoprotein and des-gamma-carboxy prothrombin were checked during each visit. Abdominal dynamic computed tomography was performed every 6 months. A diagnosis of recurrence was based on the typical imaging appearance in computed tomography or magnetic resonance imaging (35-37). Briefly, intrahepatic recurrence of HCC was diagnosed if two criteria were met: the lesion was enhanced during the arterial phase and the lesion was hypoattenuating or hypointense relative to the surrounding liver during the venous or delayed phases. Additional imaging findings regarded as suggestive but not diagnostic of intrahepatic recurrence of HCC were a lesion that: (a) had arterial enhancement or was hypoattenuating or hypointense relative to the surrounding liver during the venous or delayed phases, (b) had peripheral rim enhancement (suggesting capsule or pseudocapsule) during the delayed phase, (c) showed decreased signal intensity (defect) during the liver-specific hepatobiliary phase, or (d) showed moderately increased signal intensity on T2-weighted MRI images. An elevated level of alpha-fetoprotein or des-gamma-carboxy prothrombin was also taken into consideration for confirmation of recurrence.
Patient and tumor characteristics according to stathmin (STMN1) expression.
Statistical analysis. All results are expressed as the mean±standard error. The Mann–Whitney test was used to compare continuous variables. The chi-squared test was applied for categorical data. The patient survival rate was calculated by the product limit method of Kaplan–Meier. Differences in survival between groups were compared using the log-rank test. Prognostic factors were examined using univariate and multivariate analyses (Cox proportional hazards regression model). Continuous variables were generally classified into two groups according to the median value of each variable. All statistical analyses were performed using JMP 8.0.1 statistical software (SAS Campus Drive, Cary, NC, USA). A value of p<0.05 was considered statistically significant.
Stathmin 1 (STMN1) expression in the non-tumor tissue and multicentric (MC) recurrence rate of patients with hepatocellular carcinoma. The incidence of MC recurrence in the high expression group was significantly higher than that in the low expression group.
Results
Clinical significance of STMN1 mRNA expression. According to STMN1 mRNA expression level in non-tumor tissues, the patients were divided into two groups: high expression (n=36) and low expression (n=20). There were no significant differences in the patient background, such as age, gender or liver function, between the two groups (Table I). There were also no significant differences in the tumor factors, such as tumor number, size, microvascular invasion, or histological grade between the two groups.
STMN1 mRNA expression and MC recurrence rate. In the analysis of STMN1 expression in non-tumor tissues, the incidence of MC recurrence in the group with high expression was significantly higher than that in the with low expression (1 year: 10% vs. 0%; 2 years: 32% vs. 6%, 3 years: 56% vs. 6%; p<0.05) (Figure 1).
Risk factors for tumor recurrence. In the univariate analysis of risk factors for MC recurrence, a low platelet count, and high STMN1 expression were determined to be significant risk factors (Table II). In the multivariate analysis, high STMN1 expression was found to be an independent risk factor of MC recurrence.
Clinical significance of miR-223 expression. According to miR-223 expression level in non-tumor tissues, the patients were again divided into two groups: high expression (n=15) and low expression (n=15). There were no significant differences in the patient background, such as age, gender or liver function, between high and low miR-223 expression groups (Table III). There were also no significant differences in the tumor factors, such as tumor size, microvascular invasion, or histological grade, although multiple tumors tended to be more frequent in the group with low miR-223 expression. However, the disease-free survival rate in the group with low expression was significantly worse than for those with high expression (1 year: 43% vs. 92%; 2 years: 36% vs. 92%, 3 years: 36% vs. 92%; p<0.05) (Figure 2). In the present study, contrary to our expectation, no significant correlation between STMN1 expression and miR-223 expression was observed (data not shown).
miR-223 expression and disease-free survival rate of patients with hepatocellular carcinoma. The disease-free survival rate in the low-expression group was significantly worse than that in the high-expression group.
Prediction of MC recurrence by molecular features. Ten patients who did not have recurrence more than 3 years after hepatectomy were defined as the non-MC group, and another 10 patients with MC recurrence were defined as the MC group. Using a miRNA microarray, we identified MC-related miRNAs. As shown in Figure 3, good separation of clusters was obtained, except for two cases (case 1 and 5). Furthermore, when STMN1 expression was taken into consideration, case 5 could be considered a low-risk patient. Finally, MC recurrence was predictable based on the molecular features of non-tumor liver tissues in 19 out of 20 patients.
Discussion
Patients with HCC often have chronic liver damage, which may be associated with a risk of MC recurrence after hepatectomy. However, some patients are at low risk of MC recurrence, while the others are at high risk. Therefore, there should be specific differences in molecular features between these two liver tissue types. In fact, it has been shown that even when histological degrees of liver fibrosis are comparable, specific molecular signatures in non-tumor liver tissues may predict a risk of intrahepatic recurrence and patient prognosis (29). We also found there were some clusters accumulating patients with a high-risk of MC recurrence, from a DNA microarray analysis (30). We therefore consider specific molecular alterations associated with hepatocarcinogenesis already exist in non-tumor liver tissues.
Risk factors of MC recurrence.
Patient and tumor characteristics according to miRNA-223 expression.
miRNA expression pattern in non-tumor tissues and a risk of multicentric (MC) recurrence in patients with HCC. Good separation of clusters with low and high risk of MC recurrence of HCC was obtained except for 2 cases. Furthermore, when the stathmin 1 (STMN1) expression was taken into consideration, case 5 could be considered as a low-risk patient. Finally, the grouping of 19 out of 20 patients might be predicted based on the molecular features of non-tumor liver tissues. H: High expression, L: low expression, N: no-MC recurrence.
In the previous study, we identified some candidate genes related to hepatocarcinogenesis and these include up-regulated genes, such as STMN1. In this study, we examined STMN1 expression in non-tumor liver tissue and revealed that the incidence of MC recurrence in the high STMN1 expression group was significantly higher than that in the group with low STMN1 expression. In addition, high STMN1 expression in non-tumor liver tissues was identified as an independent risk factor of MC recurrence.
miR-223 has been identified as one of the most down-regulated miRNAs in HCC. In the present study, low miR-223 expression was also determined to be a risk factor of recurrence. Wong et al. conducted an array-based miRNA profiling of HCC cells, and revealed a strong inverse correlation between STMN1 and miR-223 expression (38). They also demonstrated the down-regulation of miR-223 during the early carcinogenic changes of HCC development. These findings suggest that a molecular analysis using not only a DNA microarray but also miRNA microarray of noncancerous liver tissue specimens may have value in the identification of potential clinical biomarkers and therapeutic molecular targets. We also examined correlation between STMN1 and miR-223 expressions in non-tumor liver tissue in this study, however, significant correlation was not found.
In this study, MC-related miRNAs identified by miRNA microarray clearly clustered patients into MC recurrence and non-recurrence groups. Furthermore, when STMN1 expression was also taken into consideration, the grouping of almost all patients was predicted based on the molecular features of non-tumor liver tissues.
Our proposed therapeutic strategy based on the risk of MC recurrence is shown in Figure 4. In patients having low-risk miRNA expression profile and low STMN1 expression, the risk of MC recurrence may be low. Therefore, no aggressive treatment of background liver is required, and follow-up can be less frequent; even if MC recurrence occurs, local therapy can be applied. On the other hand, in the case of the high-risk group based on the miRNA and STMN1 expression profile, frequent follow-up is required, and for this type of MC recurrence, liver transplantation may be recommended. For the remaining patients, at this time, we can only say they form an intermediate group.
Our proposed therapeutic strategy based the risk of multicentric (MC) recurrence. In patients having low-risk miRNA expression profile and low stathmin 1 (STMN1) expression, the risk of MC recurrence may be low. On the other hand, in cases with a high risk of MC recurrence, frequent follow-up would be required.
In conclusion, consideration of both STMN1 gene and miRNA expression profiles in non-tumor liver tissues were predictive of the risk for MC recurrence. Such molecular information may be useful in enabling better decision making for patients with HCC.
Acknowledgements
This study was supported in part by Grants-in-Aid for Scientific Research (B) (20390359) and (C) (22591506), and a Grant-in-Aid for Challenging Exploratory Research (22659233 and 22659243) from the Japan Society for the Promotion of Science.
Footnotes
Conflicts of Interest
The Authors declare no conflict of commercial interest in regard to this study.
- Received August 23, 2017.
- Revision received September 19, 2017.
- Accepted September 21, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
