Sara Ladu

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.

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).

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.

Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC

Background and aim: Previous studies indicate unrestrained cell cycle progression in liver lesions from hepatocarcinogenesis-susceptible Fisher 344 (F344) rats and a block of G1–S transition in corresponding lesions from resistant Brown Norway (BN) rats. Here, the role of the Forkhead box M1B (FOXM1) gene during hepatocarcinogenesis in both rat models and human hepatocellular carcinoma (HCC) was assessed. Methods and results: Levels of FOXM1 and its targets were determined by immunoprecipitation and real-time PCR analyses in rat and human samples. FOXM1 function was investigated by either FOXM1 silencing or overexpression in human HCC cell lines. Activation of FOXM1 and its targets (Aurora Kinose A, Cdc2, cyclin B1, Nek2) occurred earlier and was most pronounced in liver lesions from F344 than BN rats, leading to the highest number of Cdc2–cyclin B1 complexes (implying the highest G2–M transition) in F344 rats. In human HCC, the level of FOXM1 progressively increased from surrounding non-tumorous livers to HCC, reaching the highest levels in tumours with poorer prognosis (as defined by patients’ length of survival). Furthermore, expression levels of FOXM1 directly correlated with the proliferation index, genomic instability rate and microvessel density, and inversely with apoptosis. FOXM1 upregulation was due to extracellular signal-regulated kinase (ERK) and glioblastoma-associated oncogene 1 (GLI1) combined activity, and its overexpression resulted in increased proliferation and angiogenesis and reduced apoptosis in human HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of human HCC cell lines. Conclusions: FOXM1 upregulation is associated with the acquisition of a susceptible phenotype in rats and influences human HCC development and prognosis.

Altered methionine metabolism and global DNA methylation in liver cancer: relationship with genomic instability and prognosis

Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c‐Myc and c‐Myc/Tgf‐α transgenic mice and human HCCs. S‐adenosyl‐L‐methionine/S‐adenosylhomocysteine ratio and liver‐specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c‐Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patients survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c‐Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c‐Myc/Tgf‐α lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c‐Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c‐Myc/Tgf‐α HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c‐Myc/Tgf‐α tumors, may develop in the absence of alterations in DNA methylation.

Disregulation of E-cadherin in transgenic mouse models of liver cancer

E-cadherin is a cell–cell adhesion molecule that plays a pivotal role in the development and maintenance of cell polarity. Disruption of E-cadherin-mediated adhesion represents a key step toward the invasive phenotype in a variety of solid tumors, including hepatocellular carcinoma (HCC). Here, we investigate whether deregulation of E-cadherin occurs along the multistep process of hepatocarcinogenesis in transgenic mouse models, including c-Myc, E2F1, c-Myc/TGF-α and c-Myc/E2F1 mice. Liver tumors from the transgenic mouse lines could be divided into two categories based on E-cadherin levels. Of 28, 20 (71.4%) c-Myc HCCs showed marked reduction of E-cadherin expression when compared with wild-type livers. In contrast, all of c-Myc/TGF-α and the majority of E2F1 and c-myc/E2F1 preneoplastic and neoplastic lesions exhibited overexpression of E-cadherin. Downregulation of E-cadherin was associated with promoter hypermethylation in seven of 20 c-Myc HCCs (35%), while no loss of heterozygosity at the E-cadherin locus was detected. Nuclear accumulation of β-catenin did not correlate with E-cadherin downregulation. Furthermore, c-Myc HCCs with reduced E-cadherin displayed upregulation of hypoxia-inducible factor-1α and vascular endothelial growth factor proteins. Importantly, loss of E-cadherin was associated with increased cell proliferation and higher microvessel density in c-Myc tumors. Taken together, these data suggest that loss of E-cadherin might favor tumor progression in relatively more benign HCC from c-Myc transgenic mice by stimulating neoplastic proliferation and angiogenesis under hypoxic conditions.

Synergistic role of Sprouty2 inactivation and c-Met up-regulation in mouse and human hepatocarcinogenesis.

Sprouty2 (Spry2), a negative feedback regulator of the Ras/mitogen‐activated protein kinase (MAPK) pathway, is frequently down‐regulated in human hepatocellular carcinoma (HCC). We tested the hypothesis that loss of Spry2 cooperates with unconstrained activation of the c‐Met protooncogene to induce hepatocarcinogenesis via in vitro and in vivo approaches. We found coordinated down‐regulation of Spry2 protein expression and activation of c‐Met as well as its downstream effectors extracellular signal‐regulated kinase (ERK) and v‐akt murine thymoma viral oncogene homolog (AKT) in a subset of human HCC samples with poor outcome. Mechanistic studies revealed that Spry2 function is disrupted in human HCC via multiple mechanisms at both transcriptional and post‐transcriptional level, including promoter hypermethylation, loss of heterozygosity, and proteosomal degradation by neural precursor cell expressed, developmentally down‐regulated 4 (NEDD4). In HCC cell lines, Spry2 overexpression inhibits c‐Met–induced cell proliferation as well as ERK and AKT activation, whereas loss of Spry2 potentiates c‐Met signaling. Most importantly, we show that blocking Spry2 activity via a dominant negative form of Spry2 cooperates with c‐Met to promote hepatocarcinogenesis in the mouse liver by sustaining proliferation and angiogenesis. The tumors exhibited high levels of activated ERK and AKT, recapitulating the subgroup of human HCC with a clinically aggressive phenotype. Conclusion: The occurrence of frequent genetic, epigenetic, and biochemical events leading to Spry2 inactivation provides solid evidence that Spry2 functions as a tumor suppressor gene in liver cancer. Coordinated deregulation of Spry2 and c‐Met signaling may be a pivotal oncogenic mechanism responsible for unrestrained activation of ERK and AKT pathways in human hepatocarcinogenesis. (HEPATOLOGY 2010)

EEF1A2 inactivates p53 by way of PI3K/AKT/mTOR‐dependent stabilization of MDM4 in hepatocellular carcinoma

Mouse Double Minute homolog 4 (MDM4) gene up‐regulation often occurs in human hepatocellular carcinoma (HCC), but the molecular mechanisms responsible for its induction remain poorly understood. Here we investigated the role of the phosphoinositide‐3‐kinase/v‐akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/AKT/mTOR) axis in the regulation of MDM4 levels in HCC. The activity of MDM4 and the PI3K/AKT/mTOR pathway was modulated in human HCC cell lines by way of silencing and overexpression experiments. Expression of main pathway components was analyzed in an AKT mouse model and human HCCs. MDM4 inhibition resulted in growth restraint of HCC cell lines both in vitro and in vivo. Inhibition of the PI3K‐AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells and reactivated p53‐dependent transcription. Deubiquitination by ubiquitin‐specific protease 2a and AKT‐mediated phosphorylation protected MDM4 from proteasomal degradation and increased its protein stability. The eukaryotic elongation factor 1A2 (EEF1A2) was identified as an upstream inducer of PI3K supporting MDM4 stabilization. Also, we detected MDM4 protein up‐regulation in an AKT mouse model and a strong correlation between the expression of EEF1A2, activated/phosphorylated AKT, and MDM4 in human HCC (each rho > 0.8, P < 0.001). Noticeably, a strong activation of this cascade was associated with shorter patient survival. Conclusion: The EEF1A2/PI3K/AKT/mTOR axis promotes the protumorigenic stabilization of the MDM4 protooncogene in human HCC by way of a posttranscriptional mechanism. The activation level of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis significantly influences the survival probability of HCC patients in vivo and may thus represent a promising molecular target. (Hepatology 2014;59:1886–1899)

V-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin activation induces a module of metabolic changes contributing to growth in insulin-induced hepatocarcinogenesis†‡

Mounting epidemiological evidence supports a role for insulin‐signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v‐akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin‐induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP‐BEZ235, both in vitro and in vivo. Conclusions: The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin‐induced hepatocarcinogenesis. (Hepatology 2012;)