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. 2010 May 4;107(18):8248-53.
doi: 10.1073/pnas.0912203107. Epub 2010 Apr 19.

The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis

Kwang-Pyo Lee  1 , Joo-Hyeon Lee, Tae-Shin Kim, Tack-Hoon Kim, Hee-Dong Park, Jin-Seok Byun, Min-Chul Kim, Won-Il Jeong, Diego F Calvisi, Jin-Man Kim, Dae-Sik Lim
Affiliations

The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis

Kwang-Pyo Lee et al. Proc Natl Acad Sci U S A. .

Abstract

Loss of Hippo signaling in Drosophila leads to tissue overgrowth as a result of increased cell proliferation and decreased cell death. YAP (a homolog of Drosophila Yorkie and target of the Hippo pathway) was recently implicated in control of organ size, epithelial tissue development, and tumorigenesis in mammals. However, the role of the mammalian Hippo pathway in such regulation has remained unclear. We now show that mice with liver-specific ablation of WW45 (a homolog of Drosophila Salvador and adaptor for the Hippo kinase) manifest increased liver size and expansion of hepatic progenitor cells (oval cells) and eventually develop hepatomas. Moreover, ablation of WW45 increased the abundance of YAP and induced its localization to the nucleus in oval cells, likely accounting for their increased proliferative capacity, but not in hepatocytes. Liver tumors that developed in mice heterozygous for WW45 deletion or with liver-specific WW45 ablation showed a mixed pathology combining characteristics of hepatocellular carcinoma and cholangiocarcinoma and seemed to originate from oval cells. Together, our results suggest that the mammalian Hippo-Salvador pathway restricts the proliferation of hepatic oval cells and thereby controls liver size and prevents the development of oval cell-derived tumors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Abnormal expansion of A6-positive oval cells in the liver of WW45 Liv-cKO mice. (A) Representative livers of 6-month-old WW45 Liv-cKO and control (Ctrl) mice (Upper). Liver weight as a percentage of body weight was also measured for mice (n ≥ 7) at the indicated ages (Lower). **P < 0.01; ***P < 0.001. (B) Liver sections from 6-month-old control and WW45 Liv-cKO mice were stained with H&E (Upper), revealing an increased number of immature progenitor cells (arrowheads) in the mutant mice. Such sections were also stained with antibodies to A6 (green) and to pan-CK (red), revealing colocalization of both antigens in oval cells. PT, portal tract; CV, central vein. (C) Evaluation of cell proliferation by immunofluorescence analysis with antibodies to PCNA (red) and to pan-CK (green) in liver sections of WW45 Liv-cKO and control mice at the indicated ages (Upper). White dotted lines, portal tract; yellow dotted lines, central vein. The percentages of pan-CK–negative parenchymal cells or pan-CK–positive oval cells that were positive for PCNA were determined (Lower). **P < 0.01; ***P < 0.001 (n = 4). (Scale bars, 100 μm in B; 200 μm in C.)
Fig. 2.
Fig. 2.
Increased proliferative response of A6-positive oval cells to DDC treatment in WW45 Liv-cKO mice. (A) Five-week-old WW45 Liv-cKO or control mice (n = 5) were fed a diet containing 0.1% DDC for the indicated times, after which liver weight as a percentage of body weight was measured. *P < 0.05. (B) Livers from control and WW45 Liv-cKO mice fed a 0.1% DDC diet for 6 weeks. (C) Liver sections from control or WW45 Liv-cKO mice fed a 0.1% DDC diet for 4 weeks were subjected to H&E staining or to immunohistochemical staining for A6, as indicated. Asterisks demarcate porphyrin accumulation. (D) The area of A6 staining in liver sections was quantitated with the use of ImageJ software for mutant and control mice fed a 0.1% DDC diet for the indicated times. Data are expressed as a percentage of thresholded areas. *P < 0.05 (n ≥ 3). (E) Oval cells isolated from DDC-treated control or WW45 Liv-cKO mice were incubated in growth medium containing 10 μM BrdU for 5 h. The cells were then subjected to immunofluorescence analysis (Upper) with antibodies to A6 (green) and to BrdU (red), and nuclei were stained with DAPI (blue). The percentage of A6-positive cells that were also positive for BrdU was determined for individual colonies (Lower). *P < 0.05 (n = 26 colonies, control; n = 22 colonies, mutant). (F) Oval cells isolated from DDC-treated control or WW45 Liv-cKO mice were cultured in growth medium for 1 week and then subjected to immunofluorescence analysis (Upper) with antibodies to A6 (green) and to Ki67 (red); nuclei were stained with DAPI (blue). DIC images are shown in the top four panels. The number of DAPI-stained nuclei per colony was also determined (Lower). Data are presented as a box-and-whisker plot for 33 (control) or 28 (mutant) colonies. ***P < 0.001. (Scale bars, 200 μm in C; 50 μm in E and F.)
Fig. 3.
Fig. 3.
Deregulation of YAP as a result of liver-specific ablation of WW45. (A) Liver lysates prepared from control (C) and WW45 Liv-cKO (cKO) mice either at the indicated ages (Left) or after maintenance on a 0.1% DDC diet for the indicated times (Right) were subjected to immunoblot analysis with antibodies to the indicated proteins, with GAPDH examined as a loading control. FL and NT, full-length and NH2-terminal fragment of MST1, respectively. (B) Nuclear (N) and cytosolic (C) fractions of the liver of 6-month-old (Upper) or DDC-treated (Lower) control and WW45 Liv-cKO mice were subjected to immunoblot analysis. Lamin B and α-tubulin were examined as nuclear and cytosolic marker proteins, respectively. (C) Liver sections of control and WW45 Liv-cKO mice either at 6 months of age or maintained on a 0.1% DDC diet for 4 weeks were stained with antibodies to YAP and to A6. Arrowheads, periductal oval cells; asterisks demarcate porphyrin accumulation. (D) Excision PCR analysis of hepatocytes and an oval cell–enriched fraction (OEF) that were derived from WT or WW45flox/flox (f/f) mice and infected with the Adeno-Cre virus (Upper). Arrow indicates the position of products (165 bp) derived from the excised WW45 allele. Lysates of such infected cells were also subjected to immunoblot analysis (Lower). Asterisks indicate nonspecific bands. (E) Oval cells isolated from the liver of DDC-treated control or WW45 Liv-cKO mice were subjected to immunofluorescence staining for A6 (green) and YAP (red) as well as to staining with DAPI (blue). (F) Oval cells isolated from the liver of DDC-treated WT or WW45flox/flox mice were infected with Adeno-Cre and then subjected to immunofluorescence staining for A6 (green) and YAP (red) as well as to staining with DAPI (blue). (G) Oval cells isolated and infected as in F were incubated in growth medium containing 10 μM BrdU for 5 h. The cells were then stained with antibodies to A6 and to BrdU, and nuclei were stained with DAPI. The percentage of A6-positive cells that were also positive for BrdU was determined for individual colonies (WT, n = 21; WW45 flox/flox, n = 19). *P < 0.05. (Scale bars, 100 μm in C; 50 μm in E; 20 μm in F.)
Fig. 4.
Fig. 4.
Mixed-type liver tumor development in WW45 mutant mice. (A) Liver or hepatoma sections of WW45+/− mice at 8 or at 12 or 18 months of age, respectively, were subjected to H&E staining and to immunohistochemical staining for A6. (Insets) Small A6-positive oval cells in hepatic cords at a magnification twice that for the main panels. (B) Hepatoma sections from WW45+/− or WW45 Liv-cKO mice at 14 months of age were subjected to H&E staining and to immunohistochemical staining for A6. Strands or trabeculae of small A6-positive oval cells were distributed within the hepatomas (a, a′, c, and c′). A6-positive oval cells were also organized in glandlike structures within the tumors (b, b′, d, and d′). (C) Sections of normal liver or hepatoma tissue from WW45+/+ or WW45+/− mice, respectively, were stained with antibodies to YAP. (D) Immunoblot analysis of tumors (T) at initiation, early, or advanced (Adv) stages and of corresponding nontumor (N) regions in the liver of WW45+/− mice as well as of tumor regions (T1, T2) and a nontumorous region (N) from the same liver of a WW45 Liv-cKO mouse. (Scale bars, 200 μm in A; 100 μm in B; 50 μm in C.)
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