Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene

Abstract

Hippo-Lats-Yorkie signaling regulates tissue overgrowth and tumorigenesis in Drosophila. We show that the Mst1 and Mst2 protein kinases, the mammalian Hippo orthologs, are cleaved and constitutively activated in the mouse liver. Combined Mst1/2 deficiency in the liver results in loss of inhibitory Ser127 phosphorylation of the Yorkie ortholog, Yap1, massive overgrowth, and hepatocellular carcinoma (HCC). Reexpression of Mst1 in HCC-derived cell lines promotes Yap1 Ser127 phosphorylation and inactivation and abrogates their tumorigenicity. Notably, Mst1/2 inactivates Yap1 in liver through an intermediary kinase distinct from Lats1/2. Approximately 30% of human HCCs show low Yap1(Ser127) phosphorylation and a majority exhibit loss of cleaved, activated Mst1. Mst1/2 inhibition of Yap1 is an important pathway for tumor suppression in liver relevant to human HCC.

Figure 1

Phenotypes of Mst1/2 mutant mice. A) E8.5 Mst1−/−Mst2−/− embryos (a) are smaller than littermate controls with one or more functional Mst1 or Mst2 alleles (b). By E9.5 Mst1−/−Mst2−/− embryos (c, d) show various degrees of developmental abnormalities [compare with (e)] including stunted growth, failure to turn and close the neural tube (d). Also, embryos and yolk sacs (ys) appear pale and undervascularized and abnormally large pericardial cavities are observed (arrowheads in c, d). Scale bars 500μm. (B) Tumor-free survival curve of mice of the indicated genotypes. All deaths in the Mst1−/− Mst2+/− and Mst1+/−Mst2−/− cohorts were due to HCC. Statistical significance by the log-rank test is indicated. (C) Gross appearance of HCC arising in an Mst1−/−Mst2+/− mouse. (D) H&E stained sections of murine HCC. Scale bars: left panel 500 μm, right panel 125 μm. (E) PCR analysis of the wild type and null Mst1 (upper) and Mst2 (lower) alleles in normal liver tissue from the indicated genotypes and of HCC from Mst1−/− Mst2+/− mice (T1 and T2). Note that the HCCs lack the wild type Mst1 and Mst2 alleles. (F) Left: Western blots of liver from a wild type mouse (N) and the normal liver from an Mst1−/−Mst2+/− mouse (N) that bore an HCC (T). Note the absence of total Mst1 and Mst2 and phospho-Mst1/2 in the HCC. In the wild type liver, the majority of Mst1 and a significant proportion of Mst2 is present as an N-terminal truncated form (NT); Mst1/2 activity (as reflected by p-Mst1/2 levels) is restricted to the truncated Mst1/2 forms. Right: an Mst1 immunoprecipitate from wildtype and Mst1−/− liver, demonstrating immunoprecipitation of the 34kDa polypeptide and the pMst1(Ser183) 34kDa polypeptide. (G) Western blot showing that Mst1 is exclusively full length in the spleen.

Figure 2

Acute inactivation of Mst2 in Mst1 null liver initiates hepatocyte proliferation and HCC development. (A) Western blot showing expression of Mst1 and Mst2 in the liver of mice of the indicated genotypes before (day 0) or at intervals after Adeno-Cre injection in the tail vein. An HCC is shown, far right. (B) Gross images of livers 8 days after Adeno-Cre administration. (C) Liver weight to body weight ratios at various time points after Adeno-Cre delivery. Statistical significance (unpaired T test) is indicated. (D) Ki-67 staining at day 8; scale bar = 500 μm. (E) Quantitation of Ki-67 staining. Statistical significance (unpaired T test) is indicated; error bars show standard deviation. (F) HCC-free survival curve of mice of the indicated genotypes treated at 6 weeks with Adeno-Cre. (G) Gross and H&E images of an HCC from an Adeno-Cre treated Mst1−/−Mst2F/− mouse; scale bar = 500 μm. (H) Western blot analysis of a series of cell cycle regulators in liver tissue from WT and Mst1−/−Mst2F/− mice before, or at the indicated number of days after Adeno-Cre administration and in an HCC. (I) Western blot analysis of liver extracts from wild type and Mst1−/−Mst2F/− mice 8 days after AdenoCre, and of a set of 10 HCC’s arising after AdenoCre treatment. β-catenin*= β-catenin dephosphorylated at Ser37 or Thr41.

Figure 3

Analysis of the Hippo pathway in Mst1−/−Mst2−/− MEFs. (A) Western blot of immortalized MEFs grown to low (L) and high (H) density. Note that high density induces pYap1 regardless of Mst1/2 status. In contrast, pMob1 strictly requires Mst1/2. The MEFs were generated from wildtype or Mst1−/−Mst2F/− mice and infected with a retrovirus expressing Cre recombinase. The cells were then immortalized by infection with a retrovirus expressing SV 40 T-Antigen. (B) Western blot showing expression of full length (FL), N-terminal truncated (NT), and activated phosphorylated forms of Mst1 and Mst2 in liver and MEFs. (C) Western blot showing that the induction of Lats1/2 activation loop (AL) phosphorylation in MEFs at high cell density does not require Mst1 or Mst2. (D) Immunofluorescence showing that Yap1 (red) translocates out of the nucleus to the cytoplasm upon cellular confluence both in wild type and Mst1/2 null MEFs; scale bar = 30 μm.

Figure 4

Mst1/2 regulate Yap1 phosphorylation and cytoplasmic retention in the liver. (A) Western blot of liver lysates prior to (day 0) or at 3 and 8 days after Adeno-Cre injection of mice with the indicated parental genotypes; an HCC is shown at the far right. Note that elimination of Mst1/2 leads to immediate loss of pYap1 and pMob1. The Mst1/2 site at the C-terminus (CT) of pLats1/2 is minimally affected whereas there is a delayed and transient decrease of Lats1/2 activation loop (AL) phosphorylation, however both Lats1/2 sites in the HCC are phosphorylated at WT levels. Mst1 and Mst2 are shown in Fig. 2A. (B) Western blot analysis of WT liver as well as normal liver (N) and HCC (T1, T2) from an Alb-Cre; Mst1−/−Mst2F/F mouse. (C) Western blots of cytoplasmic [C] and nuclear [N] fractions from WT and Mst1−/−Mst2F/− livers 8 days after Adeno-Cre injection (left) and from a murine HCC (right). (D). IHC analysis of Yap1 expression in WT liver and in HCC; scale bar = 250 μm. (E) qPCR analysis of CTGF and AFP mRNA expression in WT and Mst1−/−Mst2F/− livers 8 days after Adeno-Cre delivery, and in murine HCC (data is mean for 3 specimens per group; standard error of the mean is shown). (F) Hepatic Yap1(Ser127) kinases. An extract of normal mouse liver was separated by anion exchange chromatography and each fraction was assayed for the ability to catalyze the phosphorylation of a prokaryotic recombinant GST-Yap1 at Ser127 (arrow) as detected by immunoblot (upper panel). (*) Endogenous hepatic Yap1. The middle and lowest panels show the elution of the endogenous Lats1 and Lats2. Note that two peaks of Yap1(Ser127) kinase activity are present, eluting in fractions 13–15 and 20–23; Lats1 co-elutes with the latter peak of Yap1(Ser127) kinase whereas Lat2 elutes in the interval between the Yap1 kinase peaks. (G) Yap1(Ser127) kinase activity in fractions from livers from adeno-Cre treated wild type and Mst1−/−Mst2F/− mice. Recombinant Yap1 (arrows) and endogenous Yap1 (*) are indicated. Note that the earlier eluting peak of Yap1(Ser127) kinase activity (fractions 13–15) is absent in the Mst1/2-deficient liver whereas the later eluting activity (fractions 20–22) is unaffected by Mst1/2 loss.

Figure 5

Mst1/2 deletion protects against Fas-induced apoptosis. (A) WT and Mst1−/− Mst2F/− mice were injected with Adeno-Cre and 8 days later were treated with either vehicle or anti-Fas antibody (Jo-2). Livers were harvested 3 hrs after treatment and analyzed by TUNEL staining. The Mst1/2 deficient livers (Mst1/2−/−) were resistant to Fas-induced apoptosis. Scale bar = 125 μm. (B) Quantitation of TUNEL staining, n=3 (error bars show standard deviation). (C) Western blot analysis of liver lysates shows the upregulation of cleaved caspase-3 (CL) in anti-Fas-treated mice but not Mst1/2-deficient mice.

Figure 6

Mst1 restoration inactivates Yap1 and reverts tumorigenicity of Mst1/2 deficient HCC cells. (A) The HCC-1 cell line, derived from an HCC arising in an Mst1−/−Mst2F/− mouse, was infected with lentiviruses that express GFP alone or in combination with Mst1. HCC-1 cells were either FACS sorted for GFP (+) or were unsorted (−). The western blot of cells harvested 96 hours post-lentiviral infection shows that Mst1 induces Yap1 and Mob1 phosphorylation, whereas Lats1/2 carboxyterminal (CT) and activation loop (AL) phosphorylation are unaffected. (B) Western blot of sorted HCC-1 cells that were either untreated (−) or exposed to H₂O₂ (+), 0.5mM. H2O2 induces strong Lats1/2 phosphorylation independent of Mst1/2 whereas Yap1 phosphorylation is increased more by Mst1 than by H₂O₂ in the absence of Mst1/2. (C) Western blots of cytoplasmic (C) and nuclear (N) fractions of unsorted HCC-1 cells showing that Mst1 promotes increased phosphorylation and cytoplasmic retention of Yap1. (D) BrdU/DAPI staining and flow cytometry of HCC cells 72 hours post-infection. Mst1 induces an increase in G1 content (lower left quadrant) and a decrease in S phase (upper quadrant) cells. (E) Annexin V/7- AAD staining and flow cytometry of HCC-1 cells 48 hours post-infection. Mst1 induces early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant). (F) Western blot expression of unsorted HCC-1 cells analyzed 96 hours post-infection showing that Mst1 induces cleavage of caspase-3 (CL) and a reduction in expression of c-Myc, cyclin D3 and cyclin E. (G) Mst1 reexpression suppresses HCC1 colony formation (left) and growth in soft agarose (right). Error bars show standard deviation.

Figure 7

Yap1 is required for tumorigenicity of Mst1/2 mutant HCC cells. (A) Western blots of lysates from HCC-1 cells infected with lentiviruses expressing scrambled or Yap1 shRNAs and analyzed at day 6 post-infection. Yap1 shRNA silences Yap1 expression and results in caspase-3 cleavage (CL) and reduction in full length (FL) caspase-3. (B) BrdU/DAPI staining and flow cytometry. Yap1 shRNA increases the proportion of HCC-1 cells in G1 (lower left quandrant) and decreases cells in S phase (upper quandrant). (C) Annexin V/7-AAD staining and flow cytometry. Yap1 shRNA induces early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant). (D) HCC-1 cells expressing Yap1 shRNA are unable to form colonies in clonogenic assays. (E and F) Yap1 knockdown prevents the proliferation of HCC-1 cells as determined by MTT assay (E) and suppresses growth in soft agarose (F).

Figure 8

Immunoblot analysis of Yap1, Mob1, and Mst1/2 in human HCC. Western blot analysis of 21 paired samples of non-tumorous liver (N) and HCC (T) from the same patient. Patients 2,3,5,10,11,13 and 15 exhibit substantially lower pYap1 in the tumor relative to the non-tumorous liver. Patients 4,6,12, and 15 exhibit Yap1 polypeptide overexpression in the tumor. The majority of HCCs exhibit much lower pMob1 relative to the paired non-tumorous liver. The Mst1 34kDa polypeptide fragment is lacking in the tumors while present in the non-tumorous livers of patients 3,4,5,7,8,9,11,13,15,17,19,20,21 whereas only patients 6 and 18 exhibit detectable 34kDa Mst1 in their tumors but little or none in their non-tumorous livers. The associated clinical information is presented in Supplementary Table 1.