Salvatore Delogu

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)

Functional crosstalk between AKT/mTOR and Ras/MAPK pathways in hepatocarcinogenesis: Implications for the treatment of human liver cancer

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, with limited treatment options. AKT/mTOR and Ras/MAPK pathways are frequently deregulated in human hepatocarcinogenesis. Recently, we generated an animal model characterized by the co-expression of activated forms of AKT and Ras in the mouse liver. We found that concomitant activation of AKT/mTOR and Ras/MAPK cascades leads to rapid liver tumor development in AKT/Ras mice, mainly through mTORC1 induction. To further define the role of mTORC1 cascade in AKT/Ras induced HCC development, the mTORC1 inhibitor Rapamycin was administered to AKT/Ras mice at the time when small tumors started to emerge in the liver. Of note, Rapamycin treatment significantly delayed hepatocarcinogenesis in AKT/Ras mice. However, some microscopic lesions persisted in the livers of AKT/Ras mice despite the treatment and rapidly gave rise to HCC following Rapamycin withdrawal. Mechanistically, Rapamycin inhibited mTORC1 and mTORC2 pathways, lipogenesis and glycolysis, resulting in inhibition of proliferation in the treated livers. However, activated ERK and its downstream effectors, Mnk1 and eIF4E, were strongly upregulated in the residual lesions. Concomitant suppression of AKT/mTOR and Ras/MAPK pathways was highly detrimental for the growth of AKT/Ras cells in vitro. The study indicates the existence of a complex interplay between AKT/mTOR and Ras/MAPK pathways during hepatocarcinogenesis, with important implications for the understanding of HCC pathogenesis as well as for its prevention and treatment.

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;)

Inactivation of Spry2 accelerates AKT-driven hepatocarcinogenesis via activation of MAPK and PKM2 pathways

BACKGROUND & AIMS Aberrant activation of the AKT oncogenic pathway and downregulation of the Sprouty 2 (Spry2) tumor suppressor gene are frequently observed molecular events in human hepatocarcinogenesis. The goal of the present study was to investigate the eventual biochemical and genetic crosstalk between activated AKT and inactivation of Spry2 during liver cancer development by using in vivo and in vitro approaches. METHODS Activated AKT and/or Spry2Y55F, a dominant negative form of Spry2, were overexpressed in the mouse liver via hydrodynamic gene delivery. Histological and biochemical assays were applied to characterize the molecular features of AKT and AKT/Spry2Y55F liver tumors. The human HLE hepatocellular carcinoma (HCC) cell line, stably overexpressing AKT, was transfected with Spry2Y55F to study the molecular mechanisms underlying hepatocarcinogenesis driven by Spry2 loss. RESULTS Spry2Y55F overexpression significantly accelerated AKT-induced hepatocarcinogenesis in the mouse. AKT/Spry2Y55F liver lesions had increased proliferation and glycolysis and decreased lipogenesis when compared with AKT corresponding lesions. At the molecular level, AKT/Spry2Y55F HCCs exhibited a significantly stronger induction of activated mitogen-activated protein kinase (MAPK) and pyruvate kinase M2 (PKM2) pathways than in AKT corresponding lesions. This phenotype was reproduced in HLE cells overexpressing AKT following transfection with Spry2Y55F. Furthermore, we found that concomitant suppression of the MAPK cascade and PKM2 strongly inhibited the growth induced by Spry2Y55F in AKT-overexpressing cells. CONCLUSIONS Inactivation of Spry2 accelerates AKT-induced hepatocarcinogenesis via activation of MAPK and PKM2 pathways.

Abstract 984: High-level expression of YAP induces protumorigenic Notch signalling in human hepatocarcinogenesis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The evolutionary conserved Hippo-pathway negatively regulates organ size control by phosphorylation and cytoplasmic retention of the transcriptional co-activator yes-associated protein (YAP). Recent studies demonstrated that deletion of essential Hippo-pathway constituents (e.g., Mst-1/2 and WW45) or overexpression of YAP lead to the development of liver cancer. However, the underlying molecular tumor-supporting mechanisms in carcinogenesis have not been defined so far. Overexpression and nuclear accumulation of YAP in nearly 70% of all human hepatocellular carcinomas (HCC) significantly correlated with tumor cell proliferation and dedifferentiation. In human HCC cell lines, siRNA-mediated inhibition of YAP significantly reduced tumor cell viability, and migration/invasion. Based on transcriptomic profiling approaches, the Notch ligand Jagged-1 (Jag-1) was identified as YAP-dependent target gene in HCC cells and in primary murine hepatocytes of transgenic animals expressing constitutively active YAPS127A. Inhibition of YAP reduced the protein levels of Jag-1, cleaved Notch receptor (NICD), and Hes-1, while YAP overexpression increased the amounts of all factors. As detected for YAP knock down, transfection of gene-specific siRNA targeting Jag-1 diminished HCC cell viability and migration. Overexpression and concomitant inhibition of Jag-1 abolished Hes-1 expression and YAP-induced HCC cell viability. By applying different mutant isoforms of YAP (e.g., YAPS127A and YAP5SA-delta-C - dominant negative isoform), TEAD4 but not TEAD1 was identified as the transcription factor required for YAP-dependent regulation of Jag-1 and Hes-1. Furthermore, the WNT/beta-catenin pathway, a putative inducer of Jag-1, did not influence the YAP-dependent modulation of Jag-1. Knock down experiments revealed Mst-2 and Lats-2 as negative regulators of YAP activity and Jag-1/Hes-1 expression. The amounts of YAP, Jag-1, and Hes-1 transcripts as well as proteins significantly correlated with each other in human HCC tissues. Most importantly, increased concentrations of all factors significantly associated with poor prognosis of HCC patients. These data demonstrate that high-level expression of YAP in HCC cells induces tumor growth and tumor cell dissemination in part through activation of the Jag-1/Notch pathway in a TEAD4-dependent and beta-catenin-independent manner. This regulatory cross-talk between Hippo- and Notch-signalling defines a group of HCC patients with poor overall survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 984. doi:1538-7445.AM2012-984