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.

UNLABELLED: 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.

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.

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.

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

UNLABELLED: 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.

Integration of genomic analysis and in vivo transfection to identify sprouty 2 as a candidate tumor suppressor in liver cancer.

UNLABELLED: Hepatocellular carcinoma (HCC) is 1 of the leading causes of cancer-related deaths worldwide, yet the molecular genetics underlying this malignancy are still poorly understood. In our study, we applied statistical methods to correlate human HCC gene expression data obtained from complementary DNA (cDNA) microarrays and corresponding DNA copy number variation data obtained from array-based comparative genomic hybridization. We have thus identified 76 genes that are up-regulated and show frequent DNA copy number gain, and 37 genes that are down-regulated and show frequent DNA copy loss in human HCC samples. Among these down-regulated genes is Sprouty2 (Spry2), a known inhibitor of receptor tyrosine kinases. We investigated the potential role of Spry2 in HCC by expressing dominant negative Spry2 (Spry2Y55F) and activated beta-catenin (DeltaN90-beta-catenin) in the mouse liver through hydrodynamic injection and sleeping beauty-mediated somatic integration. When stably expressed in mouse hepatocytes, Spry2Y55F cooperates with DeltaN90-beta-catenin to confer a neoplastic phenotype in mice. Tumor cells show high levels of expression of phospho-extracellular signal-regulated kinase (ERK), as well as deregulation of genes involved in cell proliferation, apoptosis, and angiogenesis. CONCLUSION: We identified a set of candidate oncogenes and tumor suppressor genes for human HCC. Our study provides evidence that inhibition of Spry activity cooperates with other oncogenes to promote liver cancer in mouse models, and Spry2 may function as a candidate tumor suppressor for HCC development in vivo. In addition, we demonstrate that the integration of genomic analysis and in vivo transfection is a powerful tool to identify genes that are important during hepatic carcinogenesis.

RNA interference as therapeutics for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a major form of primary liver cancer, is one of the leading causes of cancer related deaths worldwide. Hepatitis B and C infections are major risk factors for the development of HCC. Currently, the treatment options are rather limited, and the prognosis for this malignancy is poor for most of these patients. RNA interference has emerged as an innovative technology for gene silencing and as a potential therapeutic for various diseases, including cancer. HCC has been widely chosen as a model system for the development of RNAi therapy due to the convenience and availability of effective delivery of RNA molecules into liver tissues. Targets for HCC treatment include HBV and HCV viruses, oncogenes, as well as cellular genes mediating angiogenesis, tumor growth and metastasis. Here, we summarized the progress of RNAi therapeutics in HCC treatment, relevant patents, potential challenges and prospects in the future.

Role of cyclin D1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis.

Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.

The zebrafish forkhead transcription factor Foxi1 specifies epibranchial placode-derived sensory neurons.

Vertebrate epibranchial placodes give rise to visceral sensory neurons that transmit vital information such as heart rate, blood pressure and visceral distension. Despite the pivotal roles they play, the molecular program underlying their development is not well understood. Here we report that the zebrafish mutation no soul, in which epibranchial placodes are defective, disrupts the fork headrelated, winged helix domain-containing protein Foxi1. Foxi1 is expressed in lateral placodal progenitor cells. In the absence of foxi1 activity, progenitor cells fail to express the basic helix-loop-helix gene neurogenin that is essential for the formation of neuronal precursors, and the paired homeodomain containing gene phox2a that is essential for neuronal differentiation and maintenance. Consequently, increased cell death is detected indicating that the placodal progenitor cells take on an apoptotic pathway. Furthermore, ectopic expression of foxi1 is sufficient to induce phox2a-positive and neurogenin-positive cells. Taken together, these findings suggest that Foxi1 is an important determination factor for epibranchial placodal progenitor cells to acquire both neuronal fate and subtype visceral sensory identity.