1D), overexpression of p-AKT, and adjustable expression of p-ERK1/2 (positive cells shown) and p-mTOR (Fig. 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. == Introduction == Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the fourth leading cause of cancer death worldwide.1Treatment options for HCC are limited and generally ineffective: only a small percentage of HCC Liarozole dihydrochloride is diagnosed at an early stage and amenable to potential curative treatments such as surgical resection, liver transplantation, and radiofrequency ablation.24The only approved drug for the treatment of advanced HCC is Sorafenib, a multi-kinase inhibitor, whose overall efficacy in terms of patients survival extension is, however, rather limited.5Thus, the development of novel therapeutic strategies against advanced HCC is mandatory. For this purpose, a deeper knowledge of the molecular mechanisms underlying hepatocarcinogenesis is necessary.6,7 One of the most frequently activated pathways in HCC is the PI3K/AKT/mTOR cascade,8which has been implicated in tumor initiation, progression, and metastasis.9,10Following PI3K (phosphoinositide 3-kinase) activation by receptor tyrosine kinases or G protein coupled receptors, AKT (v-akt murine thymoma viral oncogene homolog) is recruited to the membrane and phosphorylated.9,10In mouse hepatocytes, overexpression of an activated form of AKT (Myr-AKT) induces lipogenesis and hepatocyte proliferation, eventually leading to HCC development within six months.11AKT exerts many effects on target cells through its key downstream effector, the mTOR complex 1 (mTORC1).10,12 The mTORC1 axis is also frequently activated in human HCC.13Using mice with liver-specific deletion ofTSC1andRaptor(regulatory associated protein of mTORC1, an essential mTORC1 component), a recent study showed that mTORC1 controls ketogenesis in mice in response to fasting and its modulation by ageing.14Moreover, chronic activation of mTORC1 in mice with liver-specific deletion ofTSC1leads to hepatocarcinogenesis.15 The major regulators downstream of mTORC1 are 4EBP1 and p70S6K1/2.16,174EBP1 negatively regulates eIF4E, a key rate-limiting initiation factor for cap-dependent translation. Phosphorylation by mTORC1 leads to 4EBP1 dissociation from eIF4E, allowing translation initiation complex formation at the 5 end of mRNAs. 4EBP1/eIF4E mediated translational control has been shown to be the key downstream signal of mTORC1 in AKT-induced lymphomagenesisin vivo,18and a critical mediator of mTORC1-induced cell proliferation.19Moreover, 4EBP1/eIF4E has been found to be a major effector of the oncogenic activation of AKT and ERK signaling pathways in tumor cell lines and xenograft models.20p70S6K1/2 is another downstream effector of mTORC1. Once phosphorylated Liarozole dihydrochloride and activated by mTORC1, p70S6K1/2 in turn regulates 40S ribosomal protein S6 (RPS6) as well as other regulators of translation initiation.21Ablation ofRPS6in mouse hepatocytes inhibits cell proliferation after partial hepatectomy.22,23Also, when all the five phosphorylatable serine residues of RPS6 are substituted by unphosphorylatable alanine, knock-inRPS6(P/)mice display cell growth defect.24UsingRPS6(P/)mice, a Liarozole dihydrochloride previous study showed that loss of phosphorylation of RPS6 is dispensable for AKT-mediated lymphomagenesis.18Rapamycin, an allosteric partial inhibitor of Liarozole dihydrochloride mTORC1, and its analogues (Rapalogs) have been tested clinically as anti-cancer agent in multiple tumor types.25,26However, Rapalogs only showed modest clinical efficacy, presumably due to their capacity to suppress phosphorylation of RPS6 but not 4EBP1.27,28 Concomitant activation of AKT/mTOR and Ras/MAPK cascades is frequently observed in human HCC.13,29To elucidate the molecular and biochemical crosstalk(s) between the two pathways, we generated a mouse model of liver cancer characterized by co-expression of activated forms of AKT and N-Ras.30In the current study, using genetic and Actb pharmacological approaches, we systematically investigated the requirement of each of the two main mTORC1 downstream effectors, 4EBP1/eIF4E and p70S6K/RPS6, in AKT/Ras-induced hepatocarcinogenesisin vivo. Our data demonstrate that complete inhibition of mTORC1 is required to suppress hepatocarcinogenesis driven by N-Ras and AKT oncogenes in mice. == Materials and Methods == == Hydrodynamic injection and mouse treatment == Wild-type FVB/N mice were obtained from Charles River Liarozole dihydrochloride (Wilmington, MA).Raptorfl/+mice were purchased from the Jackson Laboratory (Stock: 013188), and intercrossed to generateRaptorfl/flmice.14Hydrodynamic injections were performed as described previously.11,3032To deleteRaptorwhile co-expressing AKT and/or Ras, we injected high dose of pT3-Cre (20g) with low dose of AKT (4g) and/or Ras (4g). To ensure that all oncogene expressing cells also express Cre which led to deletion of the targeted floxed alleles, we injected pT3-Cre.
1D), overexpression of p-AKT, and adjustable expression of p-ERK1/2 (positive cells shown) and p-mTOR (Fig