Nuclear beta-catenin induces an early liver progenitor phenotype in hepatocellular carcinoma and promotes tumor recurrence

Abstract

Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.

Figure 1

TGF-β-dependent EMT of hepatocytes associates with dissociation of E-cadherin/β-catenin complexes. Ras-transformed epithelial MIM-R hepatocytes, the same after EMT induced by TGF-β (MIM-RT cells), and MIM-R-S7 cells overexpressing Smad7, which disrupts TGF-β signaling, were transplanted into the spleen of immunocompromised SCID mice to generate orthotopic experimental HCC. The resulting liver tumors (Tu) and neighboring tissue from MIM-R tumors (adjacent) were collected after 20 days and processed for standard histology by H&E staining and for immunohistochemical staining by using antibodies against E-cadherin and total β-catenin (β-catenin). Black lines in upper panels indicate tumor-host borders. Scale bar = 25 μm.

Figure 2

Accumulation of nuclear β-catenin is blocked by interference with TGF-β signaling. A: Experimental HCC generated by MIM-R, MIM-RT or MIM-R-S7 hepatocytes (Figure 1) were analyzed by immunohistochemical staining using an antibody against active β-catenin. “Adjacent” designates peritumoral tissue of MIM-R tumors. Insets: Part of panels at 10-fold higher magnification. Scale bar = 100 μm. B: Detailed analysis of MIM-R-S7-generated tumors stained as in A. Black lines indicate tumor-stroma border. Black boxes in left panels show location of fivefold magnified regions (middle panels). Boxed regions in upper middle panel show further fourfold magnifications of the stroma and the tumor (upper and lower right panels, respectively). Boxed regions in lower middle panel show low nuclear β-catenin levels in central tumor regions (upper small panel), whereas regions at the host-tumor border show intense staining for nuclear β-catenin (lower small panel). Tu, tumor. Scale bar = 500 μm.

Figure 3

Activation of nuclear β-catenin in liver tumors by TGF-β or constitutive active β-catenin abolishes expression of the epithelial differentiation markers CK8 and CK18. The same cell types as in Figure 1 (MIM-R, MIM-RT) plus MIM-R cells with blocked (MIM-R-Axin) or constitutively active β-catenin signaling (MIM-R-ABC) were orthotopically transplanted into immunocompromised SCID mice. Green fluorescent protein-positive liver tumors were collected after 20 days and processed for immunohistochemistry using anti-CK8 or anti-CK18 antibodies. Scale bar = 25 μm.

Figure 4

Nuclear β-catenin accumulation in liver tumor cells correlates with an immature hepatocyte progenitor phenotype. Orthotopic liver tumors generated by the same cell types as in Figure 3 were collected after 20 days and immunohistochemically stained with anti-M2-PK and anti-CK19 antibodies. Sections in middle panels show an overview of CK19 staining at lower magnification. Adjacent liver: Tissue surrounding MIM-R tumors. Arrow indicates bile duct in normal tissue. Black lines show tumor-host borders. Tu, tumor. Scale bar = 25 μm.

Figure 5

Poorly differentiated human HCC show cytoplasmic dislocation or loss of E-cadherin expression at plasma membrane, suggesting EMT. Human HCC samples were stained with H&E or anti-E-cadherin (E-cadherin) antibody. Two representative examples from both well-differentiated HCC (histological grading G1) (A) and poorly differentiated HCC tissues (histological grading G3) (B) are shown. The left panels show H&E staining. Black boxes in middle panels are regions depicted at fourfold magnification in right panels. E-cadherin is expressed at cell borders in HCC with G1 grade, whereas poorly differentiated HCC with G3 grade show either cytoplasmic delocalization or loss of E-cadherin. Hepatocytes of peritumoral tissues (adjacent) show plasma membrane-localized E-cadherin (right panels). Scale bar = 50 μm.

Figure 6

Nuclear β-catenin correlates with vascular invasion and tumor relapse in human HCC after OLT. Immunolocalization and staining intensity of samples from a human HCC tissue array (133 patients) was grouped into four arbitrary classes after staining with anti-activate β-catenin antibody. A: Representative examples for no (cytoplasmic β-catenin only), low (weak nuclear staining), medium (majority of nuclei strongly stained) or high nuclear β-catenin staining (all nuclei strongly stained) are shown. Insets: Selected regions at fivefold higher magnification. Scale bar = 100 μm. B and C: Nuclear β-catenin accumulation evaluated as shown in A associates with fostered recurrence of HCC (ROD) after OLT (B) and increased vascular invasion (C). Black circles show the percentages of patients positive for ROD (B) or vessel invasion (C) calculated from raw numbers of the different classes of nuclear β-catenin expression. Error bars = 95% confidence interval.