STATISTICAL ANALYSIS

All experiments were performed in triplicate unless otherwise stated. Statistical analyses were performed with the use of SPSS software, versions 15.0 and 16.0 (SPSS). To examine the association between levels of SALL4 expression and clinicopathological characteristics, Fisher’s exact test was used for analysis of the Singapore specimens of primary hepatocellular carcinomas and the chi-square test was used for analysis of the Hong Kong specimens. In the Hong Kong specimens, levels of SALL4 expression were divided into two groups. The group with a high level of SALL4 expression (high-SALL4 group) had SALL4 expression intensity that was equal to or exceeded the median level of expression detected on microarray analysis, whereas the group with a low level of SALL4 expression (low-SALL4 group) had SALL4 expression intensity that was lower than the median level of expression. Univariate analyses of cumulative overall survival and tumor recurrence were performed with the use of a Cox proportional-hazards regression model in the Singapore cohort and with the use of the Kaplan–Meier method followed by the log-rank test in the Hong Kong cohort. Factors with prognostic significance were included in the subsequent multivariate analyses with the use of the Cox proportional-hazards regression model in both cohorts. For statistical analysis of the Singapore specimens of primary hepatocellular carcinomas, 79 of the 179 specimens for which full clinicopathological data were available were analyzed. The correlation significance was determined by means of Spearman and Pearson correlation analyses. The chi-square test and Student’s t-test were used for comparisons between the high-SALL4 and low-SALL4 groups. A P value of 0.05 or less was considered to indicate statistical significance.

TARGETING SALL4 BY A NOVEL PEPTIDE

Our loss-of-function studies suggested that SALL4 is a potential therapeutic target for hepatocellular carcinoma and that down-regulation of SALL4 might reverse the aggressive phenotype of hepatocellular carcinomas. We next sought to develop targeted therapy to antagonize the oncogenic role of SALL4. It is known that SALL4 functions as a transcription repressor by recruiting a histone deacetylase (HDAC)–containing nucleosome remodeling and HDAC (NuRD) complex, and that the tumor suppressor PTEN (phosphatase and tensin homologue) is among the target genes repressed by SALL4.¹⁹ Our SALL4 loss-of-function microarray data provided direct evidence that in hepatocellular carcinoma, the PTEN–AKT pathway is involved in SALL4-induced hepatocarcinogenesis, since the class I phosphatidylinositol 3–kinase (PI3K) signaling events mediated by AKT were significantly affected by SALL4 down-regulation (P = 0.02).

We recently described a SALL4 12-AA peptide as a competitive inhibitor that blocks the interaction between SALL4 and NuRD and as a result blocks the NuRD-mediated SALL4-repression function.²⁰ We tested whether this 12-AA SALL4 peptide was effective in targeting the SALL4–PTEN–AKT pathway and resulted in decreased viability of hepatocellular carcinoma cells. When 5 µM or 20 µM of nonmutant SALL4 peptide was added to SNU-398 cells, the number of viable cells was reduced, as compared with SNU-398 cells treated with control mutant peptide and scrambled peptide. Trichostatin A, a general HDAC inhibitor, was used as a positive control drug.^21,22 When the PTEN inhibitor SF1670 was added to the cells treated with SALL4 peptide, it rescued the phenotype, as evidenced by the maintenance of cell viability (Fig. 4A). These results suggest that PTEN plays an important role in the SALL4 peptide–induced loss of tumor-cell viability in hepatocellular carcinoma. In contrast, SALL4 peptide had no effect on SNU-387 cells with undetectable endogenous SALL4 expression (Fig. 4B). This finding suggests that SALL4 peptide is a specific agent inhibiting SALL4-overexpressing hepatocellular carcinoma cells, with minimal toxic effects on SALL4-negative cells, as compared with the HDAC inhibitor trichostatin A.

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Figure 4

Specific Targeting of SALL4 by SALL4 Peptide in Hepatocellular Carcinoma Cells with High SALL4 Expression

A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell-viability assay shows the effects of SALL4 peptide on SNU-398 cells with high endogenous SALL4 expression (Panel A) and SNU-387 cells with undetectable SALL4 expression (Panel B). Cell viability was determined 72 hours after peptide treatments. Absorbance at 490 nm was directly correlated with cell viability. Nonmutant functional SALL4 peptide, mutant nonfunctional peptide, trichostatin A (TSA), scrambled control peptide, and nonmutant peptide plus PTEN (phosphatase and tensin homologue) inhibitor were added to the cells at the indicated concentration. The PTEN inhibitor concentration was 400 nM (Panels A through D). In Panels A and B, treatment 1 was a no-treatment control, treatment 2 was treatment of cells with 5 µM of peptides or 50 nM of TSA, and treatment 3 was treatment of cells with 20 µM of peptides or 100 nM of TSA. Western blot analyses show the effects of peptides on PTEN, AKT, and phosphorylated AKT (pAKT) expression on SNU-398 cells (Panel C) and SNU-387 cells (Panel D) 72 hours after peptide treatments; α-tubulin was analyzed as a loading control. Twenty µM of nonmutant or scrambled peptide or 100 nM of TSA was used. The PTEN inhibitor concentration was 400 nM. Panel E shows nonobese diabetic mice with severe combined immunodeficiency that underwent transplantation with 3 million SNU-398 cells at the left flank and were treated intraperitoneally for 5 consecutive days with 56 mg of TAT-mutant peptide per kilogram of body weight or 60 mg of nonmutant peptide per kilogram (top panel). The dissected subcutaneous tumors are shown below. Panel F shows the mean weight of tumors dissected at day 18 after transplantation and treatment in five mice that received TAT fusion peptides intraperitoneally. T bars indicate standard deviations.

To confirm the involvement of the tumor suppressor PTEN in the SALL4 peptide–induced reduction of hepatocellular carcinoma cell viability, we analyzed PTEN expression by means of Western blotting. SALL4 peptide treatment induced an extensive increase in levels of PTEN protein in SNU-398 cells (Fig. 4C). In contrast, SALL4 peptide had a negligible effect on PTEN expression in SNU-387 cells (Fig. 4D). We next examined whether the increase in PTEN expression had an effect on PI3K signaling. PTEN functions as a phosphatase to dephosphorylate AKT. Western blot analysis showed a marked reduction of phosphorylated AKT protein levels with SALL4 peptide treatment in SNU-398 cells (Fig. 4C), but not in low-SALL4 SNU-387 cells (Fig. 4D); this suggests that the increase in the level of PTEN expression had a functional role in blocking the PI3K survival signaling by dephosphorylating AKT. Furthermore, this effect could be rescued by the PTEN inhibitor, which is consistent with the role of SALL4 in regulating this pathway.

To further test the therapeutic effect of this peptide for in vivo treatment, we conjugated mutant and nonmutant peptides with the transactivator of transcription (TAT) protein transduction domain and administered the peptides intraperitoneally in nonobese diabetic mice with severe combined immunodeficiency, in which SNU-398 cells had been transplanted subcutaneously. TAT fusion nonmutant peptides reduced the tumorigenicity of SNU-398 hepatocellular carcinoma cells (Fig. 4E). The tumor loads at 18 days after treatment in mice that received TAT-nonmutant peptides were significantly smaller than tumor loads in mice that received TAT–mutant peptides (Fig. 4F). These experiments confirm that the SALL4 peptide had biologic activity both in vitro and in vivo in a xenotransplant model.

Our in vitro and murine data suggest that the SALL4 peptide is a potential targeted therapy for a subgroup of hepatocellular carcinomas, since it effectively and specifically reduced viable SALL4-positive hepatocellular carcinoma cells.