Functional consequences of WNT3/Frizzled7-mediated signaling in non-transformed hepatic cells

Abstract

We have previously demonstrated that WNT3 and Frizzled7 (FZD7) expression levelswere upregulated in hepatocellular carcinoma (HCC) and that they directly interact to activate the canonical Wnt/β-catenin pathway in HCC cell lines. In this study, we investigated the functional consequences of WNT3 and FZD7 expression levels in non-transformed hepatic cells to address the question of whether WNT3/FZD7-mediated signal transduction could be involved in cellular transformation. After stable transfection of WNT3 and FZD7, the activation of the Wnt/β-catenin pathway was confirmed by western blot, immunostaining and quantitative real-time reverse transcriptase-PCR (qRT-PCR) analysis in two non-transformed hepatocyte-derived cell lines. In vitro characteristics of the malignant phenotype were measured, including cell proliferation, migration, invasion and anchorage-independent growth in soft agar. Stable expression of WNT3 and FZD7 in the two cell lines led to cellular accumulation of β-catenin and expression of downstream target genes activated by this pathway. In the stable WNT3/FZD7-expressing clones, hepatic cell proliferation, migration, invasion as well as soft agar colony formation were enhanced compared with the non-transformed control cells. The epithelial-mesenchymal transition (EMT) factors, Twist, Snail and Vimentin, were increased in cells expressing WNT3 and FZD7. However, the WNT3/FZD7-expressing cells did not form tumors in vivo. We conclude that activation of the WNT3/FZD7 canonical pathway has a role in the early stages of hepatocarcinogenesis by promoting the acquisition of a malignant phenotype with features of EMT.

Figure 1

Expression of WNT3-Myc and FZD7-EE in HepaRG and OUMS-29 cells. (a) Immunostaining showing protein expression of WNT3 (Myc-Tag, green) and FZD7 (EE-Tag, red) in RG-W3/F7- and OU-W3/F7-stable-expressing clones. RG-EV and OU-EV were used as negative controls ( × 400). (b) Expression of WNT3 and FZD7 mRNAs by qRT–PCR normalized to 18S rRNA expression. Bar graphs depict a ratio to the controls OU-EV or RG-EV. Stable clones expressing single WNT3 (W3) or FZD7 (F7) were used as positive controls. *P<0.01 compared with the negative controls OU-EV or RG-EV.

Figure 2

Activation of the β-catenin pathway in cells expressing WNT3 and FZD7. (a) Top: Western blot analysis of β-catenin levels after subcellular fractionation in HepaRG and OUMS-29 clones. Bottom: β-catenin expression was quantified using ImageJ (NIH) and normalized to HSP90 expression (cytoplasmic fraction) or Lamin A/C (nuclear fraction). Bar graphs represent percentage to RG-EV or OU-EV control cells. (b) Immunostaining showing the subcellular localization of β-catenin (red) in HepaRG (top) and OUMS-29 (bottom) stable clones expressing WNT3 and FZD7 ( × 400). (c) Top: Expression of Cyclin D1 by western blot analysis. Bottom: Cyclin D1 expression level was normalized to Actin expression. Bar graphs represent percentage to RG-EV or OU-EV control cells. (d) Analysis of WISP2 expression by qRT–PCR, normalized to 18S rRNA expression. Bar graphs depict percentage to RG-EV or OU-EV control cells. *P<0.01 compared with the negative controls OU-EV or RG-EV.

Figure 3

WNT3/FZD7-mediated signaling promotes hepatic cell proliferation. (a) The proliferation rate of OUMS-29 clones was measured by MTS assay over 7 days. (b) Left: western blot analysis of PCNA expression as proliferation marker in HepaRG clones using nuclear extracts. Right: PCNA levels were quantified and normalized to Lamin A/C expression. Bar graphs represent percentage to RG-EV control cells. *P< 0.01 compared with the negative controls OU-EV.

Figure 4

Activation of the WNT3/FZD7-mediated signaling promotes cell migration. (a, b) Analysis of cell migration in HepaRG (a) and OUMS-29 (b) expressing both WNT3 and FZD7 as measured by a wound-healing assay. Top: Representative photographs taken at 0 h, 12 h and 24 h post-wound ( × 40). Bottom: The wound closure was quantified at 8 h, 12 h and 24 h post-wound by measuring the remaining unmigrated area using ImageJ. (c) Transwell migration assay. Left: Quantification of the number of migratory cells using the transwell assay. Migratory cells were counted in 10 non-overlapping frames of the membrane using Stereologer (Dissector, Newton, MA, USA). Right: Representative photographs of stained cells attached to the bottom membrane of a transwell ( × 100). *P<0.01 compared with the negative controls OU-EV or RG-EV.

Figure 5

WNT3/FZD7-mediated signaling promotes invasion and anchorage-independent growth. (a, b) Analysis of invasion using a matrigel-coated transwell assay in HepaRG (a) and OUMS-29 (b). Left: Representative photographs of stained cells ( × 100). Right: Quantification of invasive cells using Stereologer. (c) Results of soft agar colony formation assay with OUMS-29-derived clones. Top: Representative microscopic photographs of colony size and number ( × 40). Bottom: The number of colonies was calculated from macroscopic photographs of the wells. *P<0.01 and **P<0.05 compared with the negative controls OU-EV or RG-EV.

Figure 6

WNT3/FZD7-mediated signaling induces epithelial–mesenchymal transition. (a) Phase-contrast photographs ( × 200) demonstrating the morphological appearance of HepaRG (top) and OUMS-29 (bottom) following phalloidin–rhodamin staining of F-Actin filaments as visualized by confocal microscopy (red, 630X). (b) Top: Expression of EMT markers Twist, Snail and Vimentin in HepaRG and OUMS-29 cells by western blot analysis. Middle and bottom: Protein expression level was normalized to Actin expression. Bar graphs represent percentage to EV cells. (c and d) Analysis of E-cadherin and K19 expression in HepaRG (c) and OPN, BMP4 and MMP7 expression in OUMS-29 (d) by qRT–PCR, and normalized to 18S rRNA expression. Data are expressed as percentage to the control cells RG-EV (c) or OU-EV (d). *P<0.01 and **P<0.05 compared with the negative controls OU-EV or RG-EV.