AIM: To detect the expression of sal-like protein 4 (SALL4) and to explore its relationship with clinicopathological characteristics and prognosis of hepatocellular carcinoma (HCC).

METHODS: One hundred and twenty-six samples of HCC tissue, 44 of adjacent noncancerous cirrhotic tissue and 10 of liver hemangioma tissue, were obtained from patients who underwent hepatectomy for HCC at the Fourth Hospital of Hebei Medical University. None of the patients had received any form of treatment before the operation. After resection, all the tissues were fixed in 10% neutral formaldehyde and embedded in paraffin. Expression of SALL4 was detected by immunohistochemistry. Patients were followed up for postoperative survival until February 2014. The relationships between SALL4 expression level and clinicopathological data and prognosis of HCC were analyzed.

RESULTS: SALL4 expression was negative in the 10 samples of tissue from liver hemangioma, was weakly positive in the two samples from adjacent noncancerous cirrhotic tissue, and positive in 58 samples of HCC tissues. The differences were statistically significant (P < 0.05). Expression of SALL4 was higher in patients with higher α-fetoprotein (AFP) levels, portal vein tumor thrombus, and later clinical stage based on the Barcelona Clinic Liver Cancer classification (P < 0.05). Among patients with negative expression, weakly positive expression, positive expression, and strongly positive expression of SALL4, the median survival time was 39, 25, 23, and 9 mo, respectively (P < 0.001). When both AFP and SALL4 were detected, patients who were negative for both AFP and SALL4, SALL4-positive only, AFP-positive only, and positive for both AFP and SALL4, had a median survival time of 41, 38, 31, and 12 mo, respectively (P < 0.001).

CONCLUSION: Expression of SALL4 is relevant to the prognosis of HCC patients. Patients with higher expression levels of SALL4 and AFP have worse prognosis.

Hepatocellular carcinoma (HCC) is a primary liver cancer and accounts for > 90% of cases. As HCC patients have no specific symptoms in the early stage, most patients have lost the best opportunity for treatment (radical surgery) when diagnosed. Effects to improve the survival rate of HCC patients are limited by the drug intervention, radiotherapy, chemotherapy, and molecular targeted therapy. Thus far, the pathogenesis of HCC is still not clear. However, with the rapid development of genetics and molecular biology, researchers have begun to explore the pathogenesis of HCC at the molecular genetic level.

The occurrence of HCC is relevant to a variety of signaling pathways, including the Notch, Wnt, mitogen-activated protein kinase (MAPK), and hedgehog signaling pathways[1-4]. Sal-like protein 4 (SALL4) is a newly discovered proto-oncogene that is expressed in embryonic stem cells and liver stem cells but is not expressed in normal adult liver cells. Recent studies have found that expression of SALL4 can be detected in HCC specimens.

SALL4 is located on human chromosome 20q13 and is a zinc finger transcription factor that includes two subtypes: SALL4A and SALL4B. Mutation of SALL4 causes Okihiro syndrome and familial IVIC syndrome, which are autosomal dominant genetic diseases that manifest as multiple organ dysfunction[5,6]. SALL4 is highly expressed in embryonic stem cells and plays an important role in early embryogenesis, organogenesis, embryonic stem cell proliferation, and maintenance of pluripotency, interacting with octamer-binding transcription factor 4 (OCT4), SRY-box containing gene 2 (SOX2), and NANOG[7-9]. SALL4 is expressed in normal hematopoietic stem cells, and its expression decreases gradually with cell differentiation and maturation. High expression levels of SALL4 are related to certain hematopoietic malignancies and can lead to acute leukemia in transgenic mice[10]. In addition, acute and chronic myeloid leukemia, along with acute lymphocytic leukemia, is also associated with high levels of SALL4 expression[11], which suggests that SALL4 is a key regulator of leukemic cells[12]. SALL4 is expressed as an oncogene in a variety of tumors, including germ cell tumors, breast cancer, and α-fetoprotein (AFP)-producing gastric cancer[13-15], which indicates that SALL4 may be involved in the development of tumors[16]. Recent studies have found that SALL4 is actively expressed in HCC with stem cell characteristics and plays an important role in predicting the prognosis of HCC patients. Therefore, further studies of SALL4 may provide new therapeutic targets for HCC patients.

Some researchers have pointed out that SALL4 is not expressed in HCC tissues, but is highly expressed in AFP-producing gastric carcinoma tissues, and this was used as a method for distinguishing the two types of tissues[15,16]. However, the present study showed that SALL4 was detectable in HCC tissues. The reason for the different results is that SALL4 is not expressed in all HCC tissues. The HCC tissues in which SALL4 was positively expressed had features in common with stem cells and were characterized by a highly invasive nature and were also resistant to chemotherapy and radiotherapy[17]. Some reports showed that SALL4 is expressed in the nucleus and cytoplasm. The immunohistochemical results of Oikawa et al[18] indicated that SALL4 is expressed in both the nucleus and cytoplasm. Zeng et al[17], who evaluated the effect of SALL4 overexpression, found little expression of SALL4 in HuH1 cells. They transfected plasmid constructs encoding SALL4 (pCMV6-SALL4) and identified the expression of two isoforms using these constructs. Evaluation of the subcellular localization of GFP-tagged SALL4 (pCMV6-SALL4-GFP) showed that it could be detected in both the cytoplasm and the nucleus. The overexpression of SALL4 in HuH1 cells resulted in the activation of spheroid formation and invasive capacities and upregulated the level of hepatic stem cell markers cytokeratin (CK)19, epithelial cell adhesion molecule, and CD44. However, knockdown of SALL4 with SALL4 short hairpin RNA resulted in a decrease in spheroid formation capacity and invasive capacity, which suggests that SALL4 has the potential to enhance tumor invasiveness.

A recent study suggested that SALL4 forms a nucleosome remodeling and deacetylase (NuRD) complex with histone deacetylases (HDACs) and potentially regulates the activity of HDAC[19]. Knockdown of SALL4 decreased HDAC activity, while overexpression of SALL4 increased the activity of HDAC and enhanced the chemosensitivity to HDAC inhibitor. That study also found that the SALL4-NuRD complex had the same start sites as phosphatase and tensin homolog protein (PTEN) and, through competitive binding with the PTEN binding site, the expression of PTEN was decreased. As a tumor suppressor gene, PTEN plays an important role in the development of a variety of tumors, and the PTEN/phosphoinositide 3 kinase (PI3K)/Akt signaling pathway is closely related to HCC.

A recent report[20] described a SALL4 peptide, containing 12 amino acids, that competitively inhibited the binding of SALL4-NuRD complex, thereby blocking the interaction between SALL4 and NuRD. In addition, SALL4 peptides decreased the viability of HCC cells and increased expression of PTEN, but had no interaction with SALL4-negative cells, which suggests that SALL4 peptides have no toxic effect on normal cells. In a mouse xenograft model, tumor sizes were significantly smaller in mice that received the SALL4 peptide compared with mice that did not receive it. These results suggest that the SALL4 peptide is expected to be a new breakthrough therapy for SALL4-positive HCC patients.

It has been reported that SALL4 is not detected in chronic hepatitis but is faintly detected in bile ductules and in hepatocytes at the interface of parenchymal and stromal cells in liver cirrhosis[18]. Overexpression of SALL4 results in increased expression of CK19, which is a specific gene of the bile duct, and knockdown of SALL4 inhibited expression of CK19. These results suggest that SALL4 plays an important role in promoting differentiation of hepatoblasts into bile duct cells[21]. The observed expression of SALL4 in bile duct cells suggests that the hepatoblasts are activated and differentiated into bile duct cells. In our study, SALL4 expression was detected at weak levels in adjacent noncancerous cirrhotic tissues, suggesting that cirrhosis is the early stage of liver cancer.

In this study, we report for the first time the combined detection of SALL4 and AFP, which enabled evaluation of the prognosis of HCC patients. Nowadays, AFP is used as a diagnostic marker. However, the diagnostic value of AFP is obscured by its low specificity and high sensitivity[22-24], and some researchers believe that AFP has no significant prognostic value for HCC patients. According to our results, joint detection of SALL4 and AFP allowed for evaluation of the prognostic value. The results showed that patients who were both SALL4 and AFP positive had the worst prognosis (Figure 2B and Table 5). Among the SALL4-positive patients, the mean survival time decreased with increasing AFP levels (Table 6). Gene expression analysis has demonstrated that genes that are upregulated during proliferation and metastasis are significantly enriched when SALL4 is highly expressed in HCC[19], which suggests that SALL4-positive HCC patients have greater malignant potential and worse prognosis. In our study, we confirmed that AFP is positively correlated with SALL4 (Table 2). SALL4 was positively expressed in patients with increased AFP levels. The higher the level of SALL4 expression, the higher the grade of malignancy of the tumor, which resulted in shorter survival time and worse prognosis. Therefore, AFP had a prognostic value for HCC patients who were SALL4 positive. However, due to the small sample size, we were unable to analyze the influence of different AFP levels when SALL4 expression was weakly positive, moderately positive, and strongly positive.

In conclusion, expression of SALL4 in HCC tissues was stronger than in liver hemangioma tissues and adjacent noncancerous cirrhotic tissues. The expression level of SALL4 was related to the AFP level, clinical stage, BCLC classification, and portal vein tumor thrombosis. SALL4 as an independent predictor affected the prognosis of HCC patients. Patients with higher expression levels of SALL4 and AFP have worse prognosis.