Amplification and/or activation of the c-Myc protooncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of the mTORC1 complex, strongly inhibits c-Myc liver tumor formation. Also, p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E (4EBP1/eIF4E) signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed the upregulation of multiple amino acid transporters, including SLC1A5 and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1, as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human HCC specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. Conclusion Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis. Thus, targeting mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of HCC with activated c-Myc signaling.

Activation of the AKT/mTOR cascade and overexpression of c-Met have been implicated in the development of human hepatocellular carcinoma (HCC). To elucidate the functional crosstalk between the two pathways, we generated a model characterized by the combined expression of activated AKT and c-Met in the mouse liver. Co-expression of AKT and c-Met triggered rapid liver tumor development and mice required to be euthanized within 8 weeks after hydrodynamic injection. At the molecular level, liver tumors induced by AKT/c-Met display activation of AKT/mTOR and Ras/MAPK cascades as well as increased lipogenesis and glycolysis. Since a remarkable lipogenic phenotype characterizes liver lesions from AKT/c-Met mice, we determined the requirement of lipogenesis in AKT/c-Met driven hepatocarcinogenesis using conditional Fatty Acid Synthase (FASN) knockout mice. Of note, hepatocarcinogenesis induced by AKT/c-Met was fully inhibited by FASN ablation. In human HCC samples, coordinated expression of FASN, activated AKT, and c-Met proteins was detected in a subgroup of biologically aggressive tumors. Altogether, our study demonstrates that co-activation of AKT and c-Met induces HCC development that depends on the mTORC1/FASN pathway. Suppression of mTORC1 and/or FASN might be highly detrimental for the growth of human HCC subsets characterized by concomitant induction of the AKT and c-Met cascades.

ObjectiveIncreased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).DesignWe investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies.ResultsAblation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture.ConclusionOur study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

Background & Aims Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second-generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. Methods We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/Oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their antitumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for characterization and mechanistic study. Results Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/Oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. Conclusions Altogether, our study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics such as gemcitabine/Oxaliplatin based regimen, especially in the subset exhibiting activated AKT/mTOR cascade. Lay Summary We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 versus Gemcitabine/Oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to Gemcitabine/Oxaliplatin based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most prevalent types of primary liver cancer. These malignancies have limited treatment options resulting in poor patient outcomes. Metabolism reprogramming, including increased de novo lipogenesis, is one of the hallmarks of cancer. Fatty Acid Synthase (FASN) catalyzes the de novo synthesis of long-chain fatty acids from acetyl-CoA and malonyl-CoA. Increased FASN expression has been reported in multiple tumor types, and inhibition of FASN expression has been shown to have tumor-suppressing activity. Intriguingly, we found that while FASN is upregulated in human HCC samples, its expression is frequently low in human ICC specimens. Similar results were observed in mouse ICC models induced by different oncogenes. Ablating FASN in the mouse liver did not affect activated AKT and Notch (AKT/NICD) induced ICC formation in vivo. Furthermore, while both HCC and ICC lesions develop in mice following hydrodynamic injection of AKT and NRas oncogenes (AKT/Ras), deletion of FASN in AKT/Ras mice triggered the development almost exclusively of ICCs. In the absence of FASN, ICC cells might receive lipids for membrane synthesis through exogenous fatty acid uptake. In accordance with the latter hypothesis, ICC cells displayed high expression of fatty acid uptake related proteins and robust long-chain fatty acid uptake. Conclusion, our data demonstrate that FASN dependence is not a universal feature of liver tumors: while HCC development is highly dependent of FASN and its mediated lipogenesis, ICC tumorigenesis can be insensitive to FASN deprivation. Our study supports novel therapeutic approaches to treat this pernicious tumor type with the inhibition of exogenous fatty acid uptake.

Background & Aims Although it is well established that fatty acids (FA) are indispensable for the proliferation and survival of cancer cells in hepatocellular carcinoma (HCC), inhibition of Fatty Acid Synthase (FASN) cannot completely repress HCC cell growth in culture. Thus, we hypothesized that uptake of exogenous FA by cancer cells might play an important role in the development and progression of HCC. Lipoprotein lipase (LPL) is the enzyme that catalyzes the hydrolysis of triglycerides into free fatty acids (FFA) and increases the cellular uptake of FA. Methods We used immunohistochemistry and quantitative reverse transcription real-time polymerase chain reaction to evaluate LPL expression in human and mouse HCC samples. Using lipoprotein-deficient medium as well as siRNAs against LPL and/or FASN, we investigated whether human HCC cells depend on both endogenous and exogenous fatty acids for survival in vitro. Results We found that LPL is upregulated in mouse and human HCC samples. High expression of LPL in human HCC samples is associated with poor prognosis. In HCC cell lines, silencing of FASN or LPL or culturing the cells in lipoprotein-deficient medium significantly decreased cell proliferation. Importantly, when FASN suppression was coupled to concomitant LPL depletion, the growth restraint of cell lines was further augmented. Conclusions The present study strongly suggests that both de novo synthetized and exogenous FA play a major role along hepatocarcinogenesis. Thus, combined suppression of LPL and FASN inhibitors might be highly beneficial for the treatment of human HCC.

Concomitant expression of activated forms of AKT and Ras in the mouse liver (AKT/Ras) leads to rapid tumor development via strong activation of the mTORC1 pathway. mTORC1 functions via regulating p70S6K/RPS6 and 4EBP1/eIF4E cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of RPS6 pathway via Rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras induced hepatocarcinogenesis. Combined treatment with Rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong anti-neoplastic effect was successfully recapitulated by ablating Raptor, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the protooncogene whose activity is specifically inhibited by 4EBP1, resulted in HCC development in cooperation with activated Ras. Mechanistically, we identified the ENTPD5/AK1/CMPK1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues. Conclusions Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras protooncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the most frequent primary tumor of the liver, is an aggressive cancer type with limited treatment options. Cumulating evidence underlines a crucial role of aberrant lipid biosynthesis (a process known as de novo lipogenesis) along carcinogenesis. Previous studies showed that suppression of fatty acid synthase (FASN), the major enzyme responsible for de novo lipogenesis, is highly detrimental for the in vitro growth of HCC cell lines. To assess whether de novo lipogenesis is required for liver carcinogenesis, we have generated various mouse models of liver cancer by stably overexpressing candidate oncogenes in the mouse liver via hydrodynamic gene delivery. We found that overexpression of FASN in the mouse liver is unable to malignantly transform hepatocytes. However, genetic deletion of FASN totally suppresses hepatocarcinogenesis driven by AKT and AKT/c-Met protooncogenes in mice. On the other hand, liver tumor development is completely unaffected by FASN depletion in mice coexpressing b-catenin and c-Met. Our data indicate that tumors might be either addicted to or independent from de novo lipogenesis for their growth depending on the oncogenes involved. Additional investigation is required to unravel the molecular mechanisms whereby some oncogenes render cancer cells resistant to inhibition of de novo lipogenesis.