Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma

Abstract

Dysregulation of growth factors and their receptors is central to human hepatocellular carcinoma (HCC). We previously demonstrated that the Frizzled-7 membrane receptor mediating the Wnt signalling can activate the beta-catenin pathway and promotes malignancy in human hepatitis B virus-related HCCs. Expression patterns of all the 10 Frizzled receptors, and their extracellular soluble autoparacrine regulators (19 Wnt activators and 4 sFRP inhibitors) were assessed by real-time RT-PCR in 62 human HCC of different etiologies and their matched peritumorous areas. Immunostaining was performed to localise Frizzled on cell types in liver tissues. Regulation of three known Frizzled-dependent pathways (beta-catenin, protein kinase C, and C-Jun NH(2)-terminal kinase) was measured in tissues by western blot. We found that eight Frizzled-potentially activating events were pleiotropically dysregulated in 95% HCC and 68% peritumours as compared to normal livers (upregulations of Frizzled-3/6/7 and Wnt3/4/5a, or downregulation of sFRP1/5), accumulating gradually with severity of fibrosis in peritumours and loss of differentiation status in tumours. The hepatocytes supported the Wnt/Frizzled signalling since specifically overexpressing Frizzled receptors in liver tissues. Dysregulation of the eight Frizzled-potentially activating events was associated with differential activation of the three known Frizzled-dependent pathways. This study provides an extensive analysis of the Wnt/Frizzled receptor elements and reveals that the dysregulation may be one of the most common and earliest events described thus far during hepatocarcinogenesis.

Figure 1

Expression patterns of FZD/LRP/WNT/sFRP genes in HCC tissues in T (A) and pT (B), by comparison to cutoff values obtained from NL. Each line represents a different HCC tissue depending on the aetiologic factor (lines 1–18 for HBV, lines 19–38 for HCV, and lines 39–42 for NBNC). Upregulation of genes are indicated in coloured boxes as light blue (one- to five-fold), medium dark blue (five- to 10-fold), or dark blue (>10-fold). Downregulation of genes are indicated in pink (five-fold), orange (five- to 10-fold), or brown (<10-fold).

Figure 2

Evidence of accumulating WNT/FZD events with occurrence of cirrhosis in peritumorous tissues, with development of tumours and subsequently with severity of tumours in terms of dedifferentiation status. Bars (±s.d.) represent the average number of events – that is, upregulation of FZD3, FZD6, FZD7, WNT3, WNT4, and WNT5A or downregulation of sFRP1, sFRP5 – per tissue sample. Comparisons were statistically assessed by the Mann–Whitney U-test : ^*P<0.01 between noncirrhotic and cirrhotic peritumours; ^**P<0.05 between cirrhotic peritumours and well-differentiated tumours; ^***P<0.05 between well-differentiated and moderately/poorly differentiated tumours.

Figure 3

Representative examples of immunostaining for Frizzled receptors (FZD) in liver sections involving both T and pT: FZD3 (A), FZD6 (C) and FZD7 (E). Staining of NL is presented for comparison for (B, D, and F). FZD3 was detected mainly in HCC cells as compared to adjacent nontumorous hepatocytes (A). In the normal liver, only a faint labelling was observed in the perivenous area of the lobule (B). FZD6 was strongly expressed mainly in HCC cells as compared to adjacent nontumorous hepatocytes (C). In the normal liver, no expression was detected either in hepatocytes or normal biliary epithelial cells (D). FZD7 was highly expressed by HCC cells in a well-differentiated HCC (E). In the normal liver, faint labelling was observed in hepatocytes located in the perivenous region of the lobule (F). Size bars=500 μm.

Figure 4

Western blot analysis of FZD-dependent intracellular pathways (β-catenin, PKC, and JNK). Activity was assessed in a panel of HCC (T) and their matched nontumorous liver parenchyma (pT). The β-catenin activity was assessed as the ratio between total-β-catenin and phsopho-β-catenin, whereas the PKC and JNK activities were assessed as the ratio between the phospho-PKC or phosphor-JNK and the total PKC or total JNK. All of the blots were standardized for equal protein loading by β-actin.