Background & Aims Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP are unclear. Methods Using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAPS127A), and measured protein levels in HCC and colorectal and pancreatic tumor samples from patients. Results Human HCC cell lines and mouse hepatocytes that overexpress YAPS127A upregulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEAD4; TEAD4 binding required Mst1/2, but not WNT-β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and shorter survival times of patients with HCC or colorectal cancer. Conclusion The transcriptional regulator YAP upregulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer.

Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. Conclusion: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. (HEPATOLOGY 2010;52:2096-2108 P rohibitin (PHB) proteins are highly conserved and ubiquitously expressed proteins that have diverse cellular functions.1,2 Two PHB proteins, PHB1 and PHB2, encoded by genes located on different chromosomes, form a large multimeric complex (PHB complex) that is found largely in the inner mitochondrial membrane where it exerts a chaperone-like function to stabilize newly synthesized mitochondrial

Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/ CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription-PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC.

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Down-regulation of the liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and up-regulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Here we found Mat1A:Mat2A switch and low SAM levels, associated with CpG hypermethylation and histone H4 deacetylation of Mat1A promoter, and prevalent CpG hypomethylation and histone H4 acetylation in Mat2A promoter of fast-growing HCC of F344 rats, genetically susceptible to hepatocarcinogenesis. In HCC of genetically resistant BN rats, very low changes in the Mat1A:Mat2A ratio, CpG methylation, and histone H4 acetylation occurred. The highest MAT1A promoter hypermethylation and MAT2A promoter hypomethylation occurred in human HCC with poorer prognosis. Furthermore, levels of AUF1 protein, which destabilizes MAT1A messenger RNA (mRNA), Mat1A-AUF1 ribonucleoprotein, HuR protein, which stabilizes MAT2A mRNA, and Mat2A-HuR ribonucleoprotein sharply increased in F344 and human HCC, and underwent low/no increase in BN HCC. In human HCC, Mat1A:MAT2A expression and MATI/ III:MATII activity ratios correlated negatively with cell proliferation and genomic instability, and positively with apoptosis and DNA methylation. Noticeably, the MATI/III:MATII ratio strongly predicted patient survival length. Forced MAT1A overexpression in HepG2 and HuH7 cells led to a rise in the SAM level, decreased cell proliferation, increased apoptosis, down-regulation of Cyclin D1, E2F1, IKK, NF-jB, and antiapoptotic BCL2 and XIAP genes, and up-regulation of BAX and BAK proapoptotic genes. In conclusion, we found for the first time a post-transcriptional regulation of MAT1A and MAT2A by AUF1 and HuR in HCC. Low MATI/III:MATII ratio is a prognostic marker that contributes to determine a phenotype susceptible to HCC and patients' survival. Conclusion: Interference with cell cycle progression and I-kappa B kinase (IKK)/nuclear factor kappa B (NF-jB) signaling contributes to the antiproliferative and proapoptotic effect of high SAM levels in HCC. (HEPATOLOGY 2012;56:165-175)