Thus, we conclude that Hippo-Yap signaling suppresses cell polyploidy and oncogenesis through Skp2

Thus, we conclude that Hippo-Yap signaling suppresses cell polyploidy and oncogenesis through Skp2. transgenic (Yap Tg) mice exhibited markedly enlarged nuclear size and increased cell polyploidy, whereas allele deletion in Mst1/2 DKO liver (Mst1/2 DKO Yap+/?) was sufficient to reduce the level of the p27 protein to the level in normal WT hepatocytes (Figures 2E and S2C). p27 protein to the level in normal WT hepatocytes (Figures 2E and S2C). These data indicated that Hippo signaling controls the protein level of p27. Open in a separate window Figure 2 Loss of Hippo signaling resulted in the accumulation of p27 leading to polyploidy(A) The quantification of the relative protein expression levels of cell cycle related proteins p27, p21, CDK2, CDK4, CDK6, Cyclin A1, Cyclin D1 and Cyclin E1 in livers from the indicated mouse strains with a liver-specific mutation of the Hippo signaling components. (B) Quantitative PCR analysis of the mRNA expression in hepatocytes from the indicated liver-specific mutant mice. (C) Immunoblot analysis of p27, p-Yap, Yap, Lats1, Lats2 and GAPDH in WT or Lats1/2 DKO MEFs. (D) Immunoblot analysis of p27, Yap and GAPDH in WT, Yap Tg or Yap KO control liver tissues. (E) Immunoblot analysis of p27, Yap, Mst1, Mst2 and GAPDH in WT, Mst1/2 DKO, allele deletion (Mst1/2 DKO p27+/?) (Figures S2ICS2N). We further observed much lower incidences of abnormal anaphase cells in the livers of Mst1/2 DKO p27+/? mice than in Mst1/2 DKO livers, indicating that p27 downregulation restored cellular cytokinesis to normal levels in Mst1/2 DKO livers (Figures S2KCS2L). As the mitosis of polyploid cells leads to genomic instability and a higher incidence of cancer formation, it is not surprising that we observed that the loss of p27 resulted in a lower incidence and delayed tumor formation in Hippo signal-deficient livers by reducing cell polyploidy in the context of a much higher fraction of polyploid cells in Mst1/2 DKO liver tissues, although p27 downregulation increased the cell mitosis and proliferation of diploid cells (Figures 2LC2M and S2MCS2N). These results indicated that the Hippo signaling pathway limits polyploidy formation and prevents tumor formation, at least in part, through the downregulation of p27 (Figure 2N). Hippo signaling deficiency enhances the cytoplasmic retention of Skp2 Previous studies showed that S-phase kinase-associated protein 2 (Skp2) in the nuclear compartment is required for ubiquitin-mediated p27 degradation. We measured the levels of Skp2 and p27 in whole cell lysates and the cytoplasmic and nuclear fractions from WT, Mst1/2 DKO or Yap Tg hepatocytes and found that the protein levels of Skp2 and p27 were increased in whole-cell lysates of Mst1/2 DKO or Yap Tg hepatocytes compared with those in WT cells (Figures S3A and S3B). Dynemicin A However, these proteins were present in distinct subcellular locations (Figures 3A and 3B). The cytoplasmic retention of Skp2 in Mst1/2 DKO or Yap Tg livers was further confirmed by IHC staining (Figures 3C and 3D) and was observed in primary MEFs isolated from Lats1/2 DKO or Yap Tg mice (Figure 3E) and a HepG2 cell line overexpressing Yap (Figure S3C). Furthermore, the loss of one allele of Yap in Mst1/2 DKO hepatocytes restored the nuclear localization of Skp2 and thereby reducing the p27 levels (Figures 3C and 3D). These data suggested that loss of Hippo signaling resulted in the Dynemicin A cytoplasmic retention of Skp2, leading to the nuclear accumulation of p27. Previous studies showed that the acetylation of Skp2 promotes its translocation from the nuclei to the cytosol (Inuzuka et al., 2012). In line with its sub-cellular localization, Skp2 acetylation levels were greatly increased in Mst1/2 DKO and Yap Dynemicin A Tg hepatocytes, and attenuated in Yap KO hepatocytes (Figures 3FC3H). In addition, p27 ubiquitination was remarkably attenuated in cells overexpressing Yap or an acetylation-mimetic mutant Skp2 (KLKL) which was mainly located in the cytosol (Figures 3I and 3J). Consistently, the p27 levels were greatly reduced in Skp2 KO livers infected with adenoviruses expressing wild-type Skp2 (WT), but only slightly reduced in Skp2 KO liver infected with acetylation-mimetic mutant Skp2 (Ad-Skp2 (KLKL)) (Figure 3K). These results indicated that Hippo signaling regulates p27 stability through modulating Skp2 acetylation and sub-cellular localization (Figure 3L). Open in Spp1 a separate window Figure 3 Loss of Hippo signaling enhances the cytoplasmic retention of Skp2(A and B) Immunoblot analysis of p27, Skp2, -tubulin or PARP in the cytoplasmic (c) and nuclear (n) fractions of WT, Mst1/2 DKO (A) or Yap.