How these cascades contribute to hepatocarcinogenesis remains unknown. signal of mTORC1 in AKT-induced lymphomagenesis in mouse hepatocytes inhibits cell proliferation after partial hepatectomy.22,23 Also, when all the five phosphorylatable serine residues of RPS6 are substituted by unphosphorylatable alanine, knock-in mice display cell growth defect.24 Using mice, a previous study showed that loss of phosphorylation of RPS6 is dispensable for AKT-mediated lymphomagenesis.18 Rapamycin, an allosteric partial inhibitor of mTORC1, and its analogues (Rapalogs) have been tested clinically as anti-cancer agent in multiple tumor types.25,26 However, Rapalogs only showed modest clinical effectiveness, presumably because of the capacity to control phosphorylation of RPS6 but not 4EBP1.27,28 Concomitant activation of AKT/mTOR and Ras/MAPK cascades is frequently observed in human being HCC.13,29 To 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 PI3k-delta inhibitor 1 and N-Ras.30 In the current study, using genetic and 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 hepatocarcinogenesis mice were purchased from your Jackson Laboratory (Stock: 013188), and intercrossed to generate mice.14 Hydrodynamic injections were performed as explained previously.11, 30C32 To delete while 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 communicate Cre which led to deletion of the targeted floxed alleles, we injected pT3-Cre (20g) with Rabbit Polyclonal to NPY2R HA tagged AKT (4g) into mice. In the manifestation.33 Subsequent double immunofluorescence staining showed that all HA positive AKT expressing cells were also EYFP positive (Supplementary Fig. 1), supporting the notion that people were able to simultaneously delete the targeted floxed alleles while expressing the oncogene in the same set of hepatocytes. Rapamycin (6mg/kg/day time) or vehicle was intraperitoneally given for 7 or 11 weeks immediately after hydrodynamic injection. To block the 4EBP1/eIF4E cascade, high doses of 4EBP1A4 (20g) with low doses of AKT (4g) and/or Ras (4g) were injected. To generate eIF4E and Ras/eIF4E mice, pT3-EF1-HA-eIF4E (10g) alone or with pT2CAGGS-NRasV12 (10 g) was hydrodynamically injected into FVB/N mice. Wild-type (not injected) and mice injected with pT3-EF1 vacant plasmid were used as controls; since no difference in any parameter analyzed between the two control groups was detected, the data were merged. Mice were housed, fed, and monitored in accordance with protocols approved by the committee for animal research at the University of California, San Francisco. Detailed description of Materials and Methods is usually provided as Supplementary Material. Results Blocking of RPS6 Pathway via Rapamycin Effectively Inhibits AKT/Ras Hepatocarcinogenesis We previously showed that Rapamycin administration for 3 weeks suppresses the activation of RPS6 but not p-4EBP1 in AKT/Ras mice.34 To thoroughly investigate the contribution PI3k-delta inhibitor 1 of p70S6K/RPS6 along AKT/Ras induced hepatocarcinogenesis, Rapamycin was administered daily for 7 weeks in AKT/Ras mice immediately after hydrodynamic injection (Supplementary Fig. 2A). In accordance with previous findings,34 Rapamycin treatment efficiently blocked p-RPS6 expression in the liver of AKT/Ras mice without affecting p-4EBP1 levels (Fig. 6A). Suppression of p-RPS6 was paralleled by prevention of hepatocarcinogenesis in Rapamycin treated AKT/Ras mice. Indeed, none of the Rapamycin treated AKT/Ras mice developed palpable liver tumors 7 weeks post injection, whereas all vehicle treated AKT/Ras mice developed lethal burden of liver tumors (Fig. 1A, B). Open in a separate window Fig. 1 Rapamycin treatment effectively inhibits AKT/Ras induced hepatocarcinogenesis. (A) Gross images of livers, (B) liver weight and liver to body weight ratio of AKT/Ras mice treated with vehicle (Veh) or Rapamycin (Rapa) for 7 weeks. Data are presented as mean SEM. ****P<0.0001. (C) Hematoxylin & eosin (HE) and Ki67, (D) HA-tag, (E) p-AKT, p-ERK and p-mTOR staining in Rapamycin treated ATK/Ras mouse liver tissues. Magnifications: 100 (C&D); 400 (E and insets). 148190mm (300 PI3k-delta inhibitor 1 300 DPI) Open in a separate windows Fig. 6 Representative immunoblotting in wild-type (WT), AKT/Ras (AR), AKT/Ras/Rapa (AR/Rapa) and AKT/Ras/4EBP1A4 (AR/4EA4) liver tissues. Five to 8 samples per group were used. Arrows at the left.
How these cascades contribute to hepatocarcinogenesis remains unknown
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