Downregulation of SFRP1 is a protumorigenic event in hepatoblastoma and correlates with beta-catenin mutations

Abstract

Background: Hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are the most common malignant liver tumors in childhood. Both tumor types exhibit genetic and epigenetic alterations in the WNT/β-catenin signaling pathway, which is a key regulator of liver progenitor cells in embryonic development. The tumors demonstrate a high rate of β-catenin mutations and gene expression changes of several WNT antagonists. However, the role of the WNT inhibitory factor secreted frizzled-related protein 1 (SFRP1) has not been addressed in pediatric liver cancer so far.

Results: In our study, we investigated the gene expression level, DNA methylation status and functional relevance of SFRP1 in HB cell lines and in pediatric liver tumor patient samples. SFRP1 was downregulated due to DNA promoter methylation in all tested HB cell lines. Overexpression of SFRP1 in HB cell lines diminished tumor cell proliferation, colony formation and migration potential. In addition, the SFRP1-expressing HB cell lines showed reduced WNT/β-catenin signaling pathway activity and decreased expression of WNT target genes. To evaluate the utility of SFRP1 as a biomarker in pediatric liver cancer, we determined the gene expression level and DNA methylation status of SFRP1 in 45 pediatric liver tumor patient samples. The correlation analysis of different clinical parameters and tumor characteristics revealed a significant correlation of reduced SFRP1 expression with the presence of mutant β-catenin. The methylation status of SFRP1 was furthermore associated to a pediatric liver tumor type with HCC-like characteristics, TERT mutations and an older age at diagnosis.

Conclusion: Altogether, our data demonstrate that the epigenetic suppression of the WNT/β-catenin antagonist SFRP1 has an important impact on the malignant behavior of HB cells. Although SFRP1 methylation is a common event in HCC-like pediatric liver tumors, its potential as a prognostic or diagnostic biomarker needs to be further investigated.

Keywords: APC; DKK1; DNA methylation; Epigenetics; Hepatoblastoma; Hepatocellular carcinoma; Pediatric liver cancer; SFRP1; WIF1; WNT signaling.

Fig. 1

Promoter methylation causes SFRP1 silencing in human HB cell lines. a mRNA expression of APC, DKK1, SFRP1 and WIF1 in HuH-6, HepT1, Hep-T3, and HepG2 and normal liver (NL, n = 7) was determined by qRT-PCR (n = 3) and calculated as normalized mRNA expression (fold change) to normal liver controls. b DNA methylation status (M methylated, U unmethylated) of APC, DKK1, SFRP1 and WIF1 promoter regions in HuH-6, HepT1, Hep-T3, and HepG2 after solvent (Ø), 3 days (3d) and 5 days (5d) 5-aza treatment was conducted by MSP. c mRNA expression of APC, DKK1, SFRP1 and WIF1 in HuH-6, HepT1, Hep-T3, and HepG2 after solvent (Ø), 3 days (3d) and 5 days (5d) 5-aza treatment (n = 3) was determined by qRT-PCR and summarized as log₂ relative gene expression in heatmaps. All data are represented as mean ± SEM; p values were calculated by one-way ANOVA with Dunnett’s multiple comparisons test; *p < 0.05, ***p < 0.001, ****p < 0.0001

Fig. 2

Restored SFRP1 expression affects WNT signaling activity and HB tumor cell characteristics. a mRNA expression of SFRP1 in transient pcDNA3.1- and pSFRP1-transfected HuH-6 cells after 48 and 72 h was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to pcDNA3.1 control (n = 2). b Cell growth of transient pcDNA3.1- and pSFRP1-transfected HuH-6 cells was assessed by MTT assay at indicated time points. Values were normalized to zero hour time point and shown as mean ± SEM (n = 2). Slope difference was analyzed by linear regression, ****p < 0.0001. c mRNA expression and representative immunoblot image of SFRP1 in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cell clone 2. GAPDH served as loading control in immunoblot analysis (n = 2). Gene expression was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to pcDNA3.1 control (n = 3). d Cell growth of stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells was assessed by MTT assay at indicated time points. Values were normalized to zero hour time point and shown as mean ± SEM (n = 4). Slope difference was analyzed by linear regression, *p < 0.05. e Representative pictures and quantification of number of colonies per well of stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells (n = 3). f Representative pictures and quantification of cell migration at indicated time points were normalized to zero hour time points. Slope difference was analyzed by linear regression, ****p < 0.0001. g TOP/FOP reporter plasmid activity was assessed by a relative luciferase activity in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells 48 h after co-transfection (n = 5). h mRNA expression of MYC and CCND1 in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cell clone 2 was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to pcDNA3.1 control (n = 4). i Immunofluorescence staining of β-catenin (CTNNB1, green) and DAPI nuclear staining (blue) in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells. Scale bars: 100 µm. All data are represented as mean ± SEM; unless otherwise stated p values were calculated by two-tailed, unpaired Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

SFRP1 DNA methylation correlates with the tumor type and late onset of the disease. a mRNA expression of SFRP1 in normal liver (NL) and patient tumor samples (TU) was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to NL controls. The red line marks the median expression level. b For each tumor and normal tissue (N—number) sample, the normalized SFRP1 gene expression and promoter methylation status is shown. The embedded table displays the distribution of methylated (M) and unmethylated (U) SFRP1 in the SFRP1 low and high gene expression categories. c Representative pictures of the SFRP1 MSP reaction from selected normal and tumor tissue samples. Sss1-treated DNA serves as methylated positive control, 5-aza-treated DNA as unmethylated control. d Illustration of the correlation analysis of SFRP1 gene expression and β-catenin mutation status, see also Table 1. e Illustration of the correlation analysis of SFRP1 DNA methylation status to the tumor type, considering only HB and HCC/TLCT tumors, and to the TERT mutation status, see also Table 2. f Forest plot with a simple and cumulative generalized linear regression model considering SFRP1 methylation status, gene expression category and a combined profile in respect to the age at diagnosis