Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

Hepatocellular carcinoma (HCC) is the fifth most frequent malignancy in the world [1]. With a mortality rate of about one million per year, it ranks the third leading cause of cancer deaths worldwide [2]. Roughly, 80 % HCC patients, among all cases, live in the Asia–Pacific region and Sub-Saharan Africa area while the incidence has also increased markedly in Europe and the United States [3–5]. It has been estimated that there would be 35,660 new cases with liver cancer in America in 2015 [6]. Several environmental factors are considered to be contributed to the main causes of HCC, such as long-time exposure to aflatoxin B1, hepatitis B and C viral infections and alcohol abuse [7–9]. Despite great progress has been made in diagnosis and therapy for HCC, there are limitations in early detection, and the patients in advanced stage, when diagnosed, generally face a dismal prognosis and a high incidence of recurrence [10, 11]. At present, α-fetoprotein (AFP) is the common clinical diagnostic criteria for HCC [12], especially in Asia. However, its specificity and sensitivity are not satisfied [13]. Therefore, identifying reliable novel biomarkers for the early screening and prediction of HCC arouses our interest.

Tumor necrosis factor receptor-associated factors (TRAFs) are a group of cytoplasmic adapter proteins which link a wide range of cell surface receptors. TRAFs mediate intracellular signal transduction, including tumor necrosis factor (TNF), toll-like receptor (TLR), interleukin-1 receptor (IL-1R) and superfamily transforming growth factor-β (TGF-β) [14, 15]. As a result, TRAFs lead to the activation of mitogen-activated protein and nuclear factor kappa B (NF-κB) kinases [16]. Additionally, TRAFs also interact with varieties of proteins which regulate receptor-induced cell death and survival [16]. Therefore, TRAF-mediated signal cascades may directly cause the receptor-induced apoptosis and interfere with cell survival.

Among the six TRAF family members, TRAF6 is unique. TRAF6 is involved in a variety of physiological processes, including congenital immunity, adaptive immunity, and inflammation [17–19]. TRAF6, functioning as a signal transducer, is a key activator of NF-κB pathway which activates inhibitor of IκB kinase in response to proinflammatory cytokines [20, 21]. In recent years TRAF6 has been found to play some roles in carcinogenesis in some cancers, such as lung cancer [22], glioblastoma [23], and squamous cell carcinoma [24]. However, none of the studies have cast light upon the expression and function of TRAF6 and its association with HCC. Thus, in order to investigate the relationship between TRAF6 and clinicopathological parameters of HCC, whether TRAF6 plays a role in the proliferation and apoptosis process of HCC or not, we carried out the current study, quantifying the TRAF6 differential expression in liver tissues with continuous pathological states, which could lead to potential development of HCC using immunohistochemistry (IHC) technique, and testing the effects of silencing TRAF6 in HCC HepG2 and Hep3B cell lines with in vitro experiments.

HCC is one of the most common, aggressive and lethal cancers in the world [26]. Owing to its burden to human health, early and effective diagnosis is of vital importance. As the genomic researches develop, increasing numbers of novel molecular biomarkers have been regarded as specific diagnosis predictor and targeted therapeutic agents for HCC [27–30]. However, most of those molecular biomarkers for diagnosis and treatment have not been clearly clarified. Additionally, the carcinogenesis of HCC is a multistep process and the mechanism of tumorigenesis and deterioration of HCC has not been fully confirmed. Therefore, we devoted great efforts to look for novel biomarkers of HCC and cast light on the potential role that TRAF6 might play in the genesis and progression of HCC.

Several studies have paid close attention on the role of TRAF6. For instance, Liu et al. proposed that TRAF6 might be considered as a potential target for the treatment of Multiple myeloma (MM) as the receptor activator of nuclear factor-kappaB/receptor activator of nuclear factor-kappa B ligand/tumor necrosis factor receptor-associated factor (RANK/RANKL-TRAF6) signal pathway mediated osteolytic bone lesions [22]. Our previous study got the conclusion that TRAF6 may become a target for diagnosis and gene treatment for lung cancer patients as the TRAF6 had a higher level of expression in lung cancer tissues than in normal lung tissues [31]. Chiu et al. suggested that TRAF6 inhibition might represent a new therapeutic strategy for pancreatic cancer as the down-regulation of TRAF6 led to a remarkable increase in autophagy and apoptosis [32]. However, only Xu Z et al. focused on the relationship between TRAF6 and HCC. They indicated that Cezanne2 interacted with TRAF6 and cleaved the polyubiquitin from TRAF6 substrates whose suppression might have a key role in the HCC malignancy transformation [33]. However, the study was lack of in vivo and in vitro verification.

In the current study, different from previous experiment methods comparing only normal liver tissues and HCC tissues, we examined TRAF6 expression in continuous liver tissues, including normal liver, cirrhosis, para-cancer liver and HCC tissues, which gave us a chance to observe a relatively continuous expressional change of TRAF6 in the genesis of HCC. Positive TRAF6 expression rates were 12.1, 21.6, 36.3, 49.7 % in the normal, cirrhosis, para-cancer and HCC tissues, respectively. The results showed an increasing expression trend in the process of carcinogenesis. Moreover, we performed ROC curve to analyze its diagnostic ability. That we found the AUC of TRAF6 was 0. 595 in HCC, lower than that in lung cancer which was 0.663, indicated the diagnosis value of TRAF6 was moderate for HCC or lung cancer [31]. However, coexistent significant statistical differences were not observed in the dynamic development of liver cancer. Namely, significant differences were only found apart between normal and HCC tissues (P < 0.001), cirrhosis and HCC tissues (P = 0.002), para-cancer and HCC tissues (P = 0.012), normal and para-cancer tissues (P = 0.007). This may be due to the limitation of case number. Further larger cirrhosis and normal samples should be enrolled to investigate the differential expression in the dynamic development of HCC.

In order to explain the role of TRAF6 in the HCC angiogenesis, we quantified the expression of VEGF and MVD in this study. No correlation was found between TRAF6 and VEGF, while TRAF6 was associated with MVD closely (r = −0.249, P = 0.004). This indicated that TRAF6 might act as a repressor in the angiogenesis of HCC. However, the AUC of MVD was 0.624, lacking high accuracy in predicting the status of micro-vessels. Besides, TRAF6 was connected with metastasis (r = 0.158, P = 0.039). Although VEGF and MVD were able to predict vascular metastasis and invasion of HCC [34], the role of TRAF6 in contributing to metastasis did not lie in the aspect of angiogenesis, as indicated above.

To confirm the biological function of TRAF6 in HCC cells, we focused on the application of siRNA interference technology with in vitro experiments, using HepG2 and Hep3B cell lines. We found that cell growth was inhibited in both two cell lines after TRAF6 was knocked-down with siRNA. Since good cell viability is important for cellular material transporting, high expression of TRAF6 might promote cancer growth by keeping cancer cells alive. As is known, cell proliferation and apoptosis play important roles in cancers, we also explored the effects of TRAF6 on cell proliferation and apoptosis by its knock-down, and results showed that the ability of cell proliferation was reduced while the action of cell apoptosis and caspase activity was enhanced after TRAF6 was silenced. Peng et al. reported that knock-down of TRAF6 could suppress cell proliferation and promote cell apoptosis in the glioma cell [35], and Sun and his colleagues found that the TRAF6 level was increased in the colon cancer and its knockdown inhibited cell proliferation but did not improve the survival time [36]. In general, TRAF6 may participate in the cell growth and apoptosis in HCC as well as other cancers being reported.

As for the role that TRAF6 played in cancer invasion and metastasis, a study on esophageal squamous cell carcinoma revealed that TRAF6 promoted migration and metastasis by regulating the RAS pathway [37]. In the analysis of correlations between TRAF6 expression and clinicopathological features, we found that high expression of TRAF6 was associated with metastasis. Although there was no in vitro experiment on the metastasis in this study, based on existing literatures, HCC may be similar with esophageal squamous cell carcinoma where TRAF6 influences cell migration and metastasis, which we will perform experiments to verify.

In summary, our study revealed that the level of TRAF6 was higher in the HCC tissues compared to normal, cirrhosis and para-cancer liver tissues. Meanwhile, TRAF6 expression was related to metastasis and MVD. Most importantly, experiments in vitro showed that TRAF6 could increase the cell viability, promote cell proliferation, and inhibit cell apoptosis and caspase-3/7 activity. All these results elucidated that overexpression of TRAF6 might promote HCC progression. TRAF6 may be a key biomarker in the HCC carcinogenesis and development. It would be beneficial to pay more attention on exploring the regulation mechanisms of TRAF6 in the HCC deterioration in the future.