Stem Cell Gene SALL4 in Aggressive Hepatocellular Carcinoma: A Cancer Stem Cell-Specific Target?

Abstract

Background

Hepatocellular carcinoma is the third leading cause of cancer-related deaths worldwide. In the heterogeneous group of hepatocellular carcinomas, those with characteristics of embryonic stem-cell and progenitor-cell gene expression are associated with the worst prognosis. The oncofetal gene SALL4, a marker of a subtype of hepatocellular carcinoma with progenitor-like features, is associated with a poor prognosis and is a potential target for treatment.

Methods

We screened specimens obtained from patients with primary hepatocellular carcinoma for the expression of SALL4 and carried out a clinicopathological analysis. Loss-of-function studies were then performed to evaluate the role of SALL4 in hepatocarcinogenesis and its potential as a molecular target for therapy. To assess the therapeutic effects of a peptide that targets SALL4, we used in vitro functional and in vivo xenograft assays.

Results

SALL4 is an oncofetal protein that is expressed in the human fetal liver and silenced in the adult liver, but it is reexpressed in a subgroup of patients who have hepatocellular carcinoma and an unfavorable prognosis. Gene-expression analysis showed the enrichment of progenitor-like gene signatures with overexpression of proliferative and metastatic genes in SALL4-positive hepatocellular carcinomas. Loss-of-function studies confirmed the critical role of SALL4 in cell survival and tumorigenicity. Blocking SALL4-corepressor interactions released suppression of PTEN (the phosphatase and tensin homologue protein) and inhibited tumor formation in xenograft models in vivo.

Conclusions

SALL4 is a marker for a progenitor subclass of hepatocellular carcinoma with an aggressive phenotype. The absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in hepatocellular carcinoma.

Comment

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer and is a leading cause of liver-associated morbidity worldwide. Underlying etiology includes alcohol abuse, hepatitis B or C, and other inflammatory processes, including autoimmune disorders. Liver cirrhosis is the major risk factor for HCC.¹

The current treatment standard for patients with advanced HCC is largely confined to sorafenib, a small molecule receptor tyrosine kinase inhibitor that reduces tumor cell proliferation and angiogenesis.² The heterogeneous nature of HCC has been the main obstacle to developing a broad-spectrum alternative therapy, with no reproducible survival benefits in over 100 randomized trials in the past 30 years with anti-cancer agents ranging from chemotherapy, hormonal therapies, and immunotherapy.

Targeting cancer stem cells (CSCs) could lead to more effective therapies for HCC.³ The stem cell theory of cancer was first established in hematopoietic malignancies and recently extended to solid tumors including HCC. In this hierarchical model, self-renewing CSCs are at the apex and ultimately give rise to all other tumor cells.⁴ CSCs are chemoresistant and have a unique gene expression signature.^5,6 Upregulation of this signature in subgroups of HCC, i.e., increased “stemness,” correlates with poor clinical outcome. Therefore, stem genes and associated pathways have been identified as potential targets for new therapeutics in HCC.

SALL4, the human homolog of the Drosophila spalt homeotic gene, is a zinc finger transcription factor that regulates pluripotency and self-renewal in embryonic stem cells. SALL4 is essential for maintaining stemness properties of embryonic stem cells through both transcriptional and epigenetic controls including direct interaction with Oct4 and Nanog.⁷ To that effect, SALL4 has been used to reprogram fibroblasts to induced pluripotent stem (iPS) cells.^8,9

SALL4 stem gene is involved in a variety of CSCs since both cancer and adult stem cells share functional properties in self-renewal. SALL4 was first shown to be a key regulator in leukemogenesis, with constitutive expression in acute myeloid leukemia.¹⁰ SALL4 expression was also detected in lymphoma, plasma cell myeloma, and acute lymphoblastic leukemia,¹¹ as well as in solid tumors including breast,¹² lung,¹³ and colorectal cancer.¹⁴

Recently, we have shown that SALL4 is also an important transcriptional regulator in normal adult hematopoiesis.¹⁵ Our previous studies have found that SALL4 mediates increased human hematopoietic stem and progenitor cell (HSC/HPC) expansion, likely by increasing self-renewal activity and inhibiting differentiation processes.¹⁶ Based on these findings, SALL4 was proposed for use in human HSC expansion.^17,18

The authors of this article provide convincing evidence that high levels of SALL4 expression by hepatocytes correlate with a more aggressive subtype of HCC and poor prognosis. In a case-control study with two independent cohorts, immunohistochemistry showed SALL4 expression to be significantly higher in HCC specimens than in matched nonneoplastic specimens. Analysis of pooled global gene expression data from public databases further supported this finding. It is important to note that in these studies the authors did not find SALL4 expression to be entirely absent in nonneoplastic tissue, but rather, significantly less when compared to that of neoplastic tissue. Additionally, within HCC, SALL4 expression levels varied, further supporting the potential for SALL4 expression-based prognostic subgroups. Subsequently, Cox regression modeling was used to demonstrate that within HCC cases from both study cohorts, high expression of SALL4 was a predictor of decreased overall survival. Furthermore, in the Hong Kong cohort, high SALL4 expression was shown to be a predictor of early recurrence of HCC after treatment. Accordingly, hierarchical cluster analysis of global gene expression data showed that high SALL4-expressing HCC samples clustered with fetal liver samples, and low SALL4-expressing HCC samples clustered with nonneoplastic hepatocytes, indicating that high SALL4-expressing HCCs were more likely to be poorly differentiated and more aggressive than low SALL4-expressing HCCs. The potential prognostic value of HCC expression was further supported by gene-enrichment analysis that showed that high SALL4-expressing HCC cells had increased expression of genes associated with embryonic stem-cell signatures, metastasis, and poor survival.

Knockdown of SALL4 in HCC cells using short hairpin RNA (shRNA) resulted in decreased tumorigenicity, increased apoptosis, and a more hepatocyte-like gene expression signature. Specific inhibition of SALL4 using a 12-AA peptide directed against its nucleosome remodeling and histone deacetylation activity affected downstream phosphatase and tensin homolog protein (PTEN) and AKT signaling. Treatment with the peptide was shown to reduce viability in HCC cells, and it was shown that these cells could be rescued with a PTEN inhibitor, strongly supporting that the PTEN-AKT pathway is involved in SALL4-induced hepatocarcinogenesis. Finally, the authors were able to confirm a potential therapeutic role of the peptide in SALL4-expressing tumors by demonstrating its biological activity in vivo. Treatment with peptide correlated with decreased tumor burden in NOD-SCID mice transplanted with high SALL4-expressing HCC cells (Fig. 1).

Fig. 1.

Proposed action of SALL4 inhibiting peptide.

Prognostic subgroups based on levels of SALL4 expression would allow for multitiered treatment protocols. Furthermore, specific targeting of high SALL4-expressing HCCs with a SALL4 peptide would allow for personalized medicine in an area of unmet therapeutic need. The authors argue that SALL4 is present in the fetal liver, and then silenced in the adult liver only to be reactivated in a subset of HCCs.

Since SALL4 is a known inhibitor of PTEN, the authors tested the SALL4 targeting peptide by looking at AKT, phosphorylated AKT (pAKT), and PTEN in both high and low SALL4-expressing cell lines. PTEN acts as a phosphatase to dephosphorylate pAKT, yielding AKT, which in turn blocks PI3K survival signaling. In a high SALL4-expressing cell line, SALL4 peptide treatment markedly reduced pAKT levels, but this was not observed for a low SALL4-expressing cell line. Therefore, in the high SALL4-expressing cell line, SALL4 promotes PI3K survival signaling and inhibits apoptotic pathways by way of a higher level of pAKT—a process that can be downregulated by the peptide and then rescued by a PTEN inhibitor. However, SALL4 peptide increased PTEN expression in both cell lines. Predictably, the increase in PTEN expression was most pronounced in the high SALL4 line; however, the effect was also present in the low SALL4 line by way of visual observation of the western blot. Therefore, even when SALL4 expression is undetectable relative to high SALL4-expressing HCCs, inhibition of SALL4 with the peptide still leads to an increase in PTEN. Consequently, the western blot data suggest that a functional baseline of SALL4 expression cannot be ruled out.

A baseline level of SALL4 could appear to be low or close to zero relative to a high SALL4-expressing HCC. It could also be subthreshold for detection if at a tissue-wide low level of expression. Additionally, if specific cells have enhanced SALL4 expression and the cell population is a small fraction of liver cells, the overall SALL4 expression will be measured as low. This possibility is especially salient in light of the liver’s unique regenerative abilities. SALL4 has been shown to play an important role in adult hematopoiesis, and consequently, functional significance in adult hepatocytes is a possibility that should not be discounted.

Overall, this study holds great expansive potential for SALL4-expressing cancers. Nonetheless, there is still the need for clinical data to evaluate toxicities that may result from effects on low SALL4-expressing non-neoplastic cells. Additionally, there is a need to clinically evaluate whether such a targeted therapeutic can safely and effectively be given in combination with the standard locoregional and systemic regimens for HCC. It also remains to be determined how we can effectively target SALL4 expressing cancer cells but spare their normal counterparts in the adult tissue.