Deregulation of Hippo kinase signalling in human hepatic malignancies - PubMed (original) (raw)

Deregulation of Hippo kinase signalling in human hepatic malignancies

Hua Li et al. Liver Int. 2012 Jan.

Abstract

Background/aims: Hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and hepatoblastoma (HB) are the main hepatic malignancies with limited treatment options and high mortality. Recent studies have implicated Hippo kinase pathway in cancer development, but detailed analysis of Hippo kinase signalling in human hepatic malignancies, especially CC and HB, is lacking.

Methods: We investigated Hippo kinase signalling in HCC, CC and HB using cells and patient samples.

Results: Increased expression of yes-associated protein (Yap), the downstream effector of the Hippo kinase pathway, was observed in HCC cells, and siRNA-mediated knockdown of Yap resulted in decreased survival of HCC cells. The density-dependent activation of Hippo kinase pathway characteristic of normal cells was not observed in HCC cells and CCLP cells, a cholangiocarcinoma cell line. Immunohistochemistry of Yap in HCC, CC and HB tissues indicated extensive nuclear localization of Yap in majority of tissues. Western blot analysis performed using total cell extracts from patient samples and normal livers showed extensive activation of Yap. Marked induction of Glypican-3, CTGF and Survivin, the three Yap target genes was observed in the tumour samples. Further analysis revealed significant decrease in expression and activity of Lats kinase, the main upstream regulator of Yap. However, no change in activation of Mst-2 kinase, the upstream regulator of Lats kinase was observed.

Conclusions: These data show that Yap induction mediated by inactivation of Lats is observed in hepatic malignancies. These studies highlight Hippo kinase pathway as a novel therapeutic target for hepatic malignancies.

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Figures

Fig. 1

Increased Yap expression in HCC and HB cells. (A) Western blot analysis of Yap in total cell extracts and nuclear protein extracts of HepG2 (a HB cell line), Hep3B (a HCC cell line) and primary human hepatocytes. (B) Western blot analysis of Yap target genes CTGF, Glypican 3 and Survivin in HepG2 cells, Hep3B cells and primary human hepatocytes. (C) Immunofluorescence staining of Yap in Hep3B cells. Top panel is Yap, middle panel is nuclear staining using DAPI and bottom panel shows the merge. Arrows point to nuclear Yap staining. All images at 400x magnification. (D) Western blot of Yap and its target gene Survivin in extracts of Hep3B cells treated with either control shRNA or anti-Yap shRNA. (E) Real Time PCR analysis of CTGF mRNA in Hep3B cells treated with either control or anti-Yap shRNA. (F) Hep3B cell viability following anti-Yap shRNA treatment determined by MTT assay.

Fig. 2

Yap expression in human HCC tissues. Tissue arrays purchased from US Biomax (Rockville, MD) were stained for Yap. The upper panel shows representative photomicrographs of normal (A) and HCC (B) tissues. Arrowheads indicate nuclear Yap staining. Staining intensity was scored in three categories including high nuclear, low nuclear and cytoplasmic (no nuclear) staining as described in the methods. All images at 400x magnification. (C) Bar graph showing percent of normal, HCC and tumor-adjacent normal tissue samples in each category. (D) The HCC staining intensity data was further stratified using TNM score.

Fig. 3

Mechanisms of increased Yap activation in HCC. Western blot analysis was used to determine levels of Hippo Kinase pathway components in total cell extracts of normal and HCC samples. (A) Western blot analysis of total and phospho-Yap (B) Lats and phospho-Lats (active), and (C) Mst, phospho-Mst (active), Mob and Sav (WW45) in normal livers and HCC samples.

Fig. 4

Increased expression of Yap target genes in HCC. Quantification of (A) Survivin (BIRC5), CTGF, and (B) Glypican 3 mRNA in normal and HCC samples using Real time PCR. Total RNA was extracted from the same samples used for Western blot analysis data shown in Fig. 3 and was used for Real Time PCR analysis. (C) Western blot analysis of Survivin, CTGF, Glypican 3 and phospho-Yap in three selected normal and HCC livers.

Fig. 5

Yap expression in human cholangiocarcinoma tissues. Tissue arrays purchased from US Biomax (Rockville, MD) were stained for Yap. The upper panel shows representative photomicrographs (400x) of (A) normal, (B) and (C) CC tissues. Staining intensity was scored using three categories including high nuclear, low nuclear and cytoplasmic (no nuclear) staining as described in methods. (D) Bar graph showing percent of CC tissue samples in each category. (E) Low magnification (200x) photomicrograph of CC tissue stained for Yap. Arrowheads indicate nuclear Yap staining.

Fig. 6

Yap expression in HB tissues. Representative photomicrographs of adjacent normal liver (A), and HB samples (B-D). All images are at 400x magnification. Arrows indicate cytoplasmic Yap staining in the hepatocytes of normal liver. Arrowheads indicate nuclear Yap staining in hepatoblastoma samples. (E) Bar graph showing percent of HB samples with high and moderate nuclear staining.

Fig. 7

Loss of cell-cell contact-induced activation of Hippo Kinase pathway in HCC and CC. (A) Western blot analysis of Yap, phospho-Yap, Lats, phospho-Lats, Mst-2, and phopho-Mst-2 in RIPA extracts obtained from HepaRG cells treated with either proliferation media (PM) or differentiation media (DM). (B) Western blot analysis of Yap, phospho-Yap, Lats, phospho-Lats and Mst-2 in nuclear and cytoplasmic extracts of Hep3B cells grown at <50% density or 100% cell density. (C) Western blot analysis of Yap and phospho-Yap and (D) Lats, phospho-Lats, Mst and phospho-Mst using RIPA extracts of CCLP cells grown at <50% cell density and 100% cell density.

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Deregulation of Hippo kinase signalling in human hepatic malignancies - PubMed (original) (raw)