Hepatocellular carcinomas with characteristics of embryonic stem cell and progenitor cell gene expression are associated with particularly poor prognosis. SALL4 is an oncofetal protein that is expressed in the human fetal liver and normally silenced in the adult liver, but re-expressed in a subgroup of patients with hepatocellular carcinoma. As reported by Kol Jia Yong, MD, of Cancer Science Institute of Singapore, and colleagues in The New England Journal of Medicine, a series of studies showed that the SALL4 gene is a marker of a subtype of hepatocellular carcinoma with progenitor-like features, associated with poor prognosis, and a potential therapeutic target.¹

Study Details

In these studies, specimens from patients with primary hepatocellular carcinoma were screened for the expression of SALL4, and clinicopathologic analysis was performed to assess correlations with clinical outcomes. Loss-of-function studies examined the role of SALL4 in hepatocarcinogenesis and its potential as a target for therapy, and the potential therapeutic effects of a peptide that targets SALL4 were evaluated.

Immunohistochemistry analysis of 179 hepatocellular carcinoma specimens (87% grade 1 or 2 tumors) and matched nonneoplastic specimens (Singapore cohort) showed significantly more SALL-expressing cells in the hepatocellular carcinoma specimens (P < .001), with 56% (95 of 171) exhibiting SALL4 positivity at various expression levels. A similar pattern of differential SALL4 expression (P < .001) was found in 228 matched neoplastic and nonneoplastic specimens in another cohort of patients (Hong Kong cohort) and in analysis of pooled global gene-expression data from public databases (P = .003).

Associated with Poor Survival

Clinicopathologic analysis of the Singapore and Hong Kong hepatocellular carcinoma specimens showed that patients with a high level of SALL4 expression had worse prognosis than patients with a low level of SALL4 expression.

On univariate analysis, absence of SALL4 protein conferred a significant survival advantage in the Singapore cohort (P = .02) and presence of SALL4 was associated with poor survival in the Hong Kong cohort (P = .002). On multivariate analyses, SALL4 was an independent prognostic factor for overall survival (hazard ratio [HR] = 2.87, P = .03) in the Singapore cohort and an independent predictor of both overall survival (HR = 1.52, P = .05) and early recurrence (HR = 1.67, P = .01) in the Hong Kong cohort.

Progenitor-like Characteristics

Hierarchical cluster analysis of global gene-expression data on hepatocytes, fetal liver tissue, and hepatocellular carcinomas showed that high-SALL4 hepatocellular carcinomas clustered tightly with fetal livers, whereas low-SALL4 hepatocellular carcinomas clustered with normal hepatocytes. Gene-set enrichment analysis showed that genes upregulated in hepatocellular carcinoma associated with poor survival, genes in an embryonic stem cell signature, and genes upregulated in metastasis, hepatoblastoma, and a proliferation subclass of hepatocellular carcinoma were significantly enriched in the high-SALL4 hepatocellular carcinoma subgroup.

Overall, these findings suggest that hepatocellular carcinomas that express SALL4 have a gene-expression pattern similar to that of hepatic progenitor cells and thus are poorly differentiated, aggressive, and associated with poor prognosis.

Role in Cell Survival, Tumorigenesis

Loss-of-function studies confirmed the role of SALL4 in cell survival and tumorigenicity. SALL4 knockdown studies using shRNA resulted in reduced cell viability, increased apoptosis, and reduced tumorigenicity of hepatocellular carcinoma cells.

The investigators then performed microarray analysis of gene-expression profiles of SNU-398 hepatocellular carcinoma cells treated with control scrambled shRNA or SALL4-specific shRNA and hierarchical clustering analysis of these profiles and profiles derived from normal human hepatocytes and fetal liver tissues. They found that SNU-398 cells treated with scrambled shRNA clustered closely with human fetal liver tissue (both expressing high levels of SALL4), whereas SALL4-knocked-down SNU-398 cells clustered with human hepatocytes. These findings suggest that downregulation of SALL4 can render hepatocellular carcinoma more hepatocyte-like in terms of gene expression.

Studies with Inhibitory Peptide

Studies to develop a targeted agent to inhibit the oncogenic activity of SALL4 were guided by the recognition that SALL4 functions as a transcription repressor by recruiting histone deacetylase (HDAC)-containing nucleosome remodeling and HDAC (NuRD) complex and that the tumor suppressor PTEN is one of the genes repressed by SALL4. Further, the loss-of-function microarray data provided evidence that the PTEN-AKT pathway is involved in SALL4-induced hepatocarcinogenesis, with PI3K signaling events mediated by AKT being significantly affected by SALL4 downregulation (P = .02).

The investigators had recently characterized a SALL4 12-amino acid peptide as a competitive inhibitor of the interaction between SALL4 and NuRD that acts to block the NuRD-mediated SALL4-repression function. In studies to determine the effect of the SALL4 peptide on the SALL4-PTEN-AKT pathway, the addition of nonmutant SALL4 peptide to SNU-398 cells reduced the number of viable cells. The addition of a PTEN inhibitor abrogated this effect and maintained cell viability, with these findings indicating that PTEN plays an important role in the SALL4 peptide–induced loss of tumor-cell viability in hepatocellular carcinoma.

SALL4 peptide had no effect on SNU-387 cells with undetectable endogenous SALL4 expression. Additional analysis showed that SALL4 peptide induced marked increases in PTEN protein levels in SNU-398 cells but had a negligible effect in the low-SALL4 SNU-387 cells. The increase in PTEN expression was associated with a marked reduction in phosphorylated AKT protein levels (not observed in the low-SALL4 SNU-387 cells), suggesting that the increase in PTEN expression acted to block PI3K survival signaling by dephosphorylating AKT. This effect was abrogated by the addition of a PTEN inhibitor, supporting the role of SALL4 in regulating this pathway.

The investigators then administered nonmutant and mutant SALL4 peptides to nonobese diabetic mice with severe combined immunodeficiency and transplanted SNU-398 cells. The nonmutant peptide reduced the tumorigenicity of the SNU-398 hepatocellular carcinoma cells, with tumor burden at 18 days after treatment being significantly smaller in mice receiving nonmutant peptide compared with those receiving mutant peptide.

As noted by the investigators, the absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in hepatocellular carcinoma. They concluded, “[O]ur study shows that the oncofetal protein SALL4 plays an important role in the extensive network of heterogeneous cellular pathways underlying hepatocarcinogenesis. A 12-amino acid peptide can block the oncogenic role of SALL4 and hence has therapeutic potential in SALL4-positive hepatocellular carcinoma.” ■

Disclosure: For full disclosures of the study authors, visit www.nejm.org.

Reference

1. Yong KJ, Gao C, Lim JSJ, et al: Oncofetal gene SALL4 in aggressive hepatocellular carcinoma. N Engl J Med 368:2266-2276, 2013.