Diego F. Calvisi

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

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.

Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma.

BACKGROUND & AIMS De novo lipogenesis is believed to be involved in oncogenesis. We investigated the role of aberrant lipid biosynthesis in the pathogenesis of human hepatocellular carcinoma (HCC). METHODS We evaluated expression of enzymes that regulate lipogenesis in human normal liver tissues and HCC and surrounding, nontumor, liver tissues from patients using real-time reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and biochemical assays. Effects of lipogenic enzymes on human HCC cell lines were evaluated using inhibitors and overexpression experiments. The lipogenic role of the proto-oncogene AKT was assessed in vitro and in vivo. RESULTS In human liver samples, de novo lipogenesis was progressively induced from nontumorous liver tissue toward the HCC. Extent of aberrant lipogenesis correlated with clinical aggressiveness, activation of the AKT-mammalian target of rapamycin signaling pathway, and suppression of adenosine monophosphate-activated protein kinases. In HCC cell lines, the AKT-mammalian target of rapamycin complex 1-ribosomal protein S6 pathway promoted lipogenesis via transcriptional and post-transcriptional mechanisms that included inhibition of fatty acid synthase ubiquitination by the USP2a de-ubiquitinase and disruption of the SREBP1 and SREBP2 degradation complexes. Suppression of the genes adenosine triphosphate citrate lyase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, or sterol regulatory element-binding protein 1, which are involved in lipogenesis, reduced proliferation, and survival of HCC cell lines and AKT-dependent cell proliferation. Overexpression of an activated form of AKT in livers of mice induced lipogenesis and tumor development. CONCLUSIONS De novo lipogenesis has pathogenic and prognostic significance for HCC. Inhibitors of lipogenic signaling, including those that inhibit the AKT pathway, might be useful as therapeutics for patients with liver cancer.

Cholangiocarcinomas can originate from hepatocytes in mice.

Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.

Loss of the glycine N‐methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice

Glycine N‐methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S‐adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor‐susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT‐knockout (GNMT‐KO) mice. GNMT‐KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT‐KO mice coincidently with the suppression of the Ras inhibitors Ras‐association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1–3 and cytokine‐inducible SH2‐protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT‐KO mice. Conclusion: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice. (HEPATOLOGY 2008.)

Yes-Associated Protein Up-regulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma

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 activity are unclear. METHODS By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a 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 (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated 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 TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS The transcriptional regulator YAP up-regulates 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. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

The RASSF1A tumor suppressor activates Bax via MOAP-1.

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. RASSF1A may serve as a node in the integration of signaling pathways controlling a range of critical cellular functions including cell cycle, genomic instability, and apoptosis. The mechanism of action of RASSF1A remains under investigation. We now identify a novel pathway connecting RASSF1A to Bax via the Bax binding protein MOAP-1. RASSF1A and MOAP-1 interact directly, and this interaction is enhanced by the presence of activated K-Ras. RASSF1A can activate Bax via MOAP-1. Moreover, activated K-Ras, RASSF1A, and MOAP-1 synergize to induce Bax activation and cell death. Analysis of a tumor-derived point mutant of RASSF1A showed that the mutant was defective for the MOAP-1 interaction and for Bax activation. Moreover, inhibition of RASSF1A by shRNA impaired the ability of K-Ras to activate Bax. Thus, we identify a novel pro-apoptotic pathway linking K-Ras, RASSF1A and Bax that is specifically impaired in some human tumors.

Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the “European Network for the Study of Cholangiocarcinoma” (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.

AKT (v-akt murine thymoma viral oncogene homolog 1) and N-Ras (neuroblastoma ras viral oncogene homolog) coactivation in the mouse liver promotes rapid carcinogenesis by way of mTOR (mammalian target of rapamycin complex 1), FOXM1 (forkhead box M1)/SKP2, and c-Myc pathways.

Activation of v‐akt murine thymoma viral oncogene homolog (AKT) and Ras pathways is often implicated in carcinogenesis. However, the oncogenic cooperation between these two cascades in relationship to hepatocellular carcinoma (HCC) development remains undetermined. To investigate this issue, we generated a mouse model characterized by combined overexpression of activated forms of AKT and neuroblastoma Ras viral oncogene homolog (N‐Ras) protooncogenes in the liver by way of hydrodynamic gene transfer. The molecular mechanisms underlying crosstalk between AKT and N‐Ras were assessed in the mouse model and further evaluated in human and murine HCC cell lines. We found that coexpression of AKT and N‐Ras resulted in a dramatic acceleration of liver tumor development when compared with mice overexpressing AKT alone, whereas N‐Ras alone did not lead to tumor formation. At the cellular level, concomitant up‐regulation of AKT and N‐Ras resulted in increased proliferation and microvascularization when compared with AKT‐injected mice. Mechanistic studies suggested that accelerated hepatocarcinogenesis driven by AKT and N‐Ras resulted from a strong activation of mammalian target of rapamycin complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/SKP2 and c‐Myc also contributed to rapid tumor growth in AKT/Ras mice, yet by way of mTORC1‐independent mechanisms. The biological effects of coactivation of AKT and N‐Ras were then recapitulated in vitro using HCC cell lines, which supports the functional significance of mTORC1, FOXM1/SKP2, and c‐Myc signaling cascades in mediating AKT and N‐Ras‐induced liver tumor development. Conclusion: Our data demonstrate the in vivo crosstalk between the AKT and Ras pathways in promoting liver tumor development, and the pivotal role of mTORC1‐dependent and independent pathways in mediating AKT and Ras induced hepatocarcinogenesis. (HEPATOLOGY 2011)

SKP2 and CKS1 Promote Degradation of Cell Cycle Regulators and Are Associated With Hepatocellular Carcinoma Prognosis

BACKGROUND & AIMS The cell cycle regulators P21(WAF1), P27(KIP1), P57(KIP2), P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. METHODS Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, <3 years survival) and HCC cell lines were analyzed. RESULTS The promoters of P21(WAF1), P27(KIP1), and P57(KIP2) were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. CONCLUSIONS Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.

Activation of β-Catenin and Yap1 in Human Hepatoblastoma and Induction of Hepatocarcinogenesis in Mice

BACKGROUND & AIMS Aberrant activation of β-catenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with the development of multiple tumor types. Yap functions as a transcriptional coactivator by interacting with TEA domain DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. METHODS We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the United States, consisting of 104 hepatocellular carcinoma, 62 intrahepatic cholangiocarcinoma, and 94 hepatoblastoma samples. We assessed β-catenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering RNAs, and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (ΔN90/β-catenin) and Yap1 (YapS127A) in livers of mice; tissues were collected, and histological and immunohistochemical analyses were performed. RESULTS We observed nuclear localization of β-catenin and Yap1 in 79% of hepatoblastoma samples but not in most hepatocellular carcinoma or intrahepatic cholangiocarcinoma samples. Yap1 and β-catenin coprecipitated in hepatoblastoma but not hepatocellular carcinoma cells. Small interfering RNA-mediated knockdown of Yap1 or β-catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to coactivate transcription with β-catenin; β-catenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and β-catenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumor cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEA domain factors was required for tumorigenesis in mice. CONCLUSIONS β-catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets.