HCC recurrence after liver transplantation remains a significant cause of poor survival in HCC recipients. In fact, expansion of donor pools in solving the problem of organ donor shortage, for instances using partial living donor grafts and marginal grafts, potentially increases the risk of tumor recurrence after liver transplantation (3, 33). Despite of employing selection strategies to minimize the potential high-risk HCC patients, deciphering its underlying risk factors becomes a critical task in developing effective strategies to predict and prevent HCC recurrence (34, 35). Increasing evidences from clinical and experimental research have demonstrated a positive correlation between post-transplant early phase I/R injury and late phase tumor recurrence (7, 36, 37). Studies have pointed out that elevated systemic and regional inflammatory responses during the early phase can increase the likelihood of late phase tumor recurrence (7, 10, 37, 38). Studies have also demonstrated that reduction of I/R injury is a conceivable strategy to diminish the risk of late phase tumor recurrence (37, 39). Still, there are many uncovered aspects of why and how does early phase I/R injury affect the happening of HCC recurrence after liver transplantation. This study aimed to identify novel HCC-recurrence-associated molecular targets from post-LT early phase, that could lead to development of novel early predictive and preventive strategies to combat HCC recurrence after liver transplantation.
In the present study, by profiling the transcriptomic of the liver biopsies obtained at the early phase after liver transplantation and comparing the difference between HCC recipients with and without late phase HCC recurrence, a panel of differential genes were identified. GSTA2 gene was identified to be the most upregulated gene in HCC recipients who developed recurred HCC at late phase after liver transplantation compared to HCC recipients without developing HCC recurrence. The expression level of hepatic GSTA2 gene was significantly correlated with the expression level of genes associated with hepatic injury including TNFa and TGFb genes, suggesting GSTA2 may be a hepatic injury-responding gene during early phase after liver transplantation. GSTA2 is a secretary protein. The EPCGSTA2 protein was detected to be significantly upregulated after reperfusion of the liver. There was no significantly association between EPCGSTA2 protein and pre-transplantation clinicopathological factors, indicating a systemic upregulation of GSTA2 resulted from I/R injury during early phase of liver transplantation. Study in mice model reported that GSTA2 is one of the upregulated gene in responding to sulfasalazine-induced liver injury (40). Our data provided several evidences to demonstrate a close relationship of EPCGSTA2 with hepatic and systemic injury at early phase after liver transplantation including warm ischemic time, post-transplant ROS level, AST level, ALT level, IL-10 level and IL-8 level (Table 1). The EPCGSTA2 was also significantly correlated with our previously identified microRNA miR-1246 (Table 1) whose level at early phase is also significantly associated with hepatic injury after liver transplantation (13). Serum aminotransferases including ALT and AST are commonly recognized markers for diagnosis and monitoring of liver injury, however the indication for liver injury by aminotransferases cannot fulfil the purpose of different clinical applications (41). New biomarkers such as microRNAs have been emerged to compensate the indications of aminotransferases on liver injury under surgical operations including liver transplantation (42). Therefore, our present findings indicated that GSTA2 is a novel biomarker to indicate hepatic and systemic injury at early phase of liver transplantation.
We further determined that this EPCGSTA2 protein exhibited significant correlation with late phase HCC recurrence of HCC recipients after liver transplantation. The accuracy of EPCGSTA2 protein in predicting overall HCC recurrence was about 70% (Table 2). Although the accuracy of EPCGSTA2 protein was slightly lower than Milan criteria (AUC = 0.752) and UCSF criteria (AUC = 0.790), the specificity of EPCGSTA2 in predicting HCC recurrence was higher than these criteria. Moreover, EPCGSTA2 protein was found to be an independent predictor with USCF in predicting HCC recurrence after liver transplantation (Table 2), demonstrating the independent risks from pre-transplant and post-transplant factors in affecting HCC recurrence after liver transplantation. Apart from predicting HCC recurrence, high level of EPCGSTA2 protein could significantly predict disease-free survival of HCC recipients after liver transplantation (Fig. 2e and Table 3). Multivariate Cox Regression analysis of the significant predictors of disease-free survival showed that there was no a significantly independent predictor defined (Table 3), suggesting that these predictors dependently influenced the disease-free survival of HCC recipients. These above findings agreed with our previous study that early phase injury-induced molecules play influential roles in late phase HCC recurrence after liver transplantation (14). Altogether, our results indicated that EPCGSTA2 protein is a potential non-invasive biomarker not only for indication of post-LT early phase hepatic and systemic injury but also for prediction of late phase HCC recurrence and survival of HCC recipients. It has been pointed out that a single biomarker is difficult to achieve perfect prediction on HCC recurrence after liver transplantation due to highly diverse tumor behaviours among patients (5). Further investigation on integrating GSTA2 with clinicopathological parameters is necessary for constructing effective prediction models on HCC recurrence after liver transplantation.
RNA sequencing analysis indicated several nucleotide polymorphisms within the GSTA2 transcript of the liver from early phase between recurrent and non-recurrent recipients. Sanger sequencing study of GSTA2 coding nucleotide sequence illustrated that the frequency of nucleotide G335C SNP of GSTA2 coding sequencing, a GSTA2 protein residue S112T substitution, was significantly different between HCC-recurrence recipients and non-HCC-recurrence recipients (Table 4). The percentage of heterozygous G/C alleles of G335C SNP in recurrent group was 5-fold greater than in non-recurrent group, indicating this heterozygous SNP to be a potential risk factor of post-transplantation HCC recurrence. In addition to the association with HCC recurrence, GSTA2 heterozygous G/C alleles of G335C SNP in the donor livers was significantly associated with the poorest overall and disease-free survival (Fig. 2h). Unlike our finding, the GSTA2 S112T serine homozygosity has been reported to be an independent prognostic factor of poor survival in acute leukemia patients received allogenic stem cell transplantation (43). We postulated that the effect of GSTA2 S112T polymorphism is dependent on different clinical situations. Owing that GSTA2 S112T polymorphism might affect the survival outcome of different diseases, assessment on GSTA2 polymorphism in the liver tissues of the donors may provide important clinical information for stratification of high-risk recipients for closely monitoring after liver transplantation. In addition, studies has demonstrated that the S112T change does not cause the change of protein structure and enzymatic activity of GSTA2 protein, but affects the level and thermostability of GSTA2 protein in the liver (44–46). A study has also suggested that GSTA2 S112T polymorphism affects bilirubin metabolism after busulfan-conditioned allogenic transplants (43). However, we did not detect a significant correlation between GSTA2 S112T polymorphism and total bilirubin in HCC recipients after liver transplantation. We postulated that bilirubin metabolism after liver transplantation may be influenced by multifactorial factors rather than a single factor.
To delineate the possible reasons of why upregulation of GSTA2 would lead to an increased risk of HCC recurrence after liver transplantation, we further investigated the expression level and functions of GSTA2 in HCC. We found that the expression level of GSTA2 mRNA in tumor tissues of HCC patients was significantly higher than the matched non-tumor tissues and the healthy donors, indicating that GSTA2 is commonly upregulated in human HCCs. The expression level of GSTA2 mRNA was significantly correlated with tumor size and presence of venous infiltration, suggesting that the expression level of GSTA2 is positively correlated with HCC malignancy. The metastatic HCC cell line expressed the highest level of GSTA2 mRNA and protein over other non-metastatic HCC cell lines, implying that GSTA2 may play important role in HCC metastasis. In addition, by analyzing the HCC tissues developed from rat liver transplantation model, we found that the expression level of GSTA2 mRNA in the more aggressive tumor tissues was significantly higher than in the less aggressive tumor tissues. So far, the expression patterns of GSTA2 has not yet to determine. Our study revealed for the first time that GSTA2 is commonly upregulated in human HCCs and positively correlated to HCC malignancy.
Functionally, we found that overexpression of endogenous GSTA2 in HCC cells could promote their proliferation rate, migration rate and invasion ability. Interestingly, administration of human recombinant GSTA2 protein could also exhibit the similar effect on HCC cells. The increased level of either endogenous or exogenous GSTA2 protein could activate the expression of several epithelial-mesenchymal-transition (EMT) - promoting proteins including N–cadherin, vimentin and claudin-1. Suppression of GSTA2 in the metastatic HCC cell line could inhibit its in vitro and in vivo metastatic ability. A recent study has demonstrated that targeted inhibition of glutathione and thioredoxin antioxidant pathways can synergistically kill cancer cells, suggesting that antioxidant systems are important for the initiation and progression of cancer cells (47). Therefore, our results indicated that GSTA2 plays essential role in metastasis of HCCs and targeted inhibition of GST2 may be a potential therapeutic method for treatment of HCC.
GSTA2 is mainly produced from the liver and functions in detoxifying electrophilic compounds such as the products of oxidative stress (19). Our results showed that the expression of hepatic GSTA2 mRNA at early phase after liver transplantation was significantly correlated with the expression level of several ROS-regulatory genes such as NRF2, SOD3, GPX2 and GPX3. The level of early phase circulating GSTA2 (EPCGSTA2) protein was also significantly correlated with the ROS level at early phase after liver transplantation. These results indicated a close relationship between GSTA2 and the hepatic and systemic ROS metabolism at the early phase after liver transplantation. Further experimental results demonstrated that elevated level of exogenous GSTA2 protein could help the normal liver cells and HCC cells to compensate their ROS level in responding to H2O2-induced oxidative stress, that subsequently protected them against the H2O2-induced apoptosis and death. Meanwhile, overexpression of GSTA2 in HCC cells could also provide protective effect for HCC cells against high ROS-induced cell death. These above findings agreed with other study that GSTA2 can contribute a protective effect on cells against ROS cytotoxicity (19). Moreover, our experiments demonstrated that suppression of GSTA2 expression in HCC cells could lead to a further elevated level of ROS in HCC cells under high H2O2 condition, increasing the numbers of died cells. We further characterized that alteration of the expression of GSTA2 could influence the activation of ROS-associated signalling pathways such as JNK and AKT pathways in responding to the change of ROS environment. In addition, downregulation of GSTA2 expression in HCC cells led to alteration of the expression of several ROS-associated genes under the H2O2 condition, suggesting that GSTA2 is important in maintaining the ROS metabolism of HCC cells in responding to the changes of ROS condition. Cancer cells exhibit a phenomenon of a relatively high intracellular level of oxidative stress because of aberrant metabolism for abnormal proliferation and progression (48, 49). However, high ROS level is harmful for cancer cells, making the role of ROS in cancer remain controversial (17, 18). Several lines of evidences have indicated that cancer cells establish counteracting effect to high ROS by increasing the level antioxidant enzymes to modulate the ROS level to be favorable for themselves (17, 50, 51). Therefore, our results indicated that upregulation of GSTA2 at early phase provides protective advantages for HCC cells to survival against high ROS-induced damage during early phase after liver transplantation and subsequently increases the likelihood of HCC recurrence.
Altogether, our results demonstrated the possible mechanism of GSTA2 in HCC recurrence after liver transplantation (Fig. 6): Elevated early phase hepatic I/R injury after liver transplantation contributes to high ROS environment which would cause harmful effects on HCC cells. GSTA2 is induced by I/R injury and upregulated in the liver and in the circulation subsequently provides protective advantages for HCC cells to survival against high ROS-induced damage. When ROS level decreased along with the recovery of hepatic I/R injury during later time point of liver transplantation, GSTA2 provides anti-oxidative benefit for HCC cells to initiate the growth and progression in an earlier period. Therefore, our study suggested the possible mechanism that upregulation of GSTA2 at early phase after liver transplantation increases the risk of HCC recurrence through maintaining a favorable ROS environment for HCC cells to survival and progression.