Liver damage, inflammation, and enhanced tumorigenesis after persistent mTORC1 inhibition

Abstract

Obesity can result in insulin resistance, hepatosteatosis, and nonalcoholic steatohepatitis (NASH) and increases liver cancer risk. Obesity-induced insulin resistance depends, in part, on chronic activation of mammalian target of rapamycin complex 1 (mTORC1), which also occurs in human and mouse hepatocellular carcinoma (HCC), a frequently fatal liver cancer. Correspondingly, mTORC1 inhibitors have been considered as potential NASH and HCC treatments. Using a mouse model in which high-fat diet enhances HCC induction by the hepatic carcinogen DEN, we examined whether mTORC1 inhibition attenuates liver inflammation and tumorigenesis. Notably, rapamycin treatment or hepatocyte-specific ablation of the specific mTORC1 subunit Raptor resulted in elevated interleukin-6 (IL-6) production, activation of signal transducer and activator of transcription 3 (STAT3), and enhanced HCC development, despite a transient reduction in hepatosteatosis. These results suggest that long-term rapamycin treatment, which also increases IL-6 production in humans, is unsuitable for prevention or treatment of obesity-promoted liver cancer.

Figure 1. Rapamycin treatment enhances IL-6 expression and STAT3 activation

Eight weeks old male mice kept on normal chow (LFD) or high fat diet (HFD) for 3 months were treated with vehicle or rapamycin (5 mg/kg/d, ip) for 2 weeks. During rapamycin treatment the same feeding regimens were continued. Livers were collected and analyzed. (A) Frozen liver sections were stained for lipid droplets and ROS accumulation with oil red O (ORO) and dihydroethidium (DHE), respectively. (Magnification: 200x). (B) Data from mice in (A) were quantitated with Image J software and averaged (n=3-4). (C) Serum ALT and IL-6 were measured by Infinity reagent and ELISA, respectively (n= 3-4). (D) Livers were homogenized and STAT3 and p70S6K expression and phosphorylation were analyzed by immunoblotting. All the bar graphs in Figure 1 represent means +/− S.D. See also Figure S1.

Figure 2. Characterization of liver specific Raptor knockout mice

(A) (Left panel) Raptor PCR on genomic DNA extracted from tail, whole liver and isolated hepatocytes of WT, Raptor^F/+ (F/+), Raptor^F/F (F/F or FF), and Raptor^Δhep (KO) mice. (Right panel) Immunoblot analysis of isolated hepatocytes from F/F and KO mice with raptor, p70S6K, and its phosphorylation antibody. (B) Serum ALT and liver weight as percentage of body weight (LW/BW) in 8 weeks old FF and KO mice (n= 4). (C, D) H&E, Sirius red, and immune cell staining of liver sections from FF and KO mice (scale bar: 100 μm). Sirius red positive areas were quantified with Image J software and shown as bar graphs (n= 4). Immune cell infiltration was analyzed by immunostaining of liver sections with indicated antibodies. Bar graphs indicate frequencies of positive cells (n= 4). (E, F) TUNEL, cleaved-caspase 3 (clv-casp 3), and phospho-γH2AX staining were performed on liver sections, positive cells were quantified and results are shown in the bar graphs (n= 4). All the bar graphs in Figure 2 represent means +/− S.D. See also Figure S2.

Figure 3. Loss of Raptor inhibits mitosis and results in enhanced liver damage after partial hepatectomy

(A) Serum ALT and liver weight/body weight ratio (LW/BW) in 8 weeks old Raptor^F/F (FF) and Raptor^Δhep (KO) mice 48 hrs after partial hepatectomy (PH) (n= 3). (B) Liver sections from FF and KO mice 48 hrs after PH were evaluated by H&E staining (Magnification bar: 100 μm). (C) BrdU labeling and immunostaining with antibodies to Ki67 and cyclin D1 at 48 hrs after PH. Bar graphs indicate frequencies of BrdU, Ki67, and cyclin D1 positive cells (n= 3). (D) Phospho-Histone H3 and cyclin B1 staining of liver sections from FF and KO mice after PH and their quantitative analysis (n= 3). (E) Immunoblot analysis of liver extracts prepared 48 hrs after PH. All the bar graphs in Figure 3 represent means +/− S.D. See also Figure S3.

Figure 4. Loss of Raptor enhances hepatocyte death and inflammation after DEN challenge

Raptor^F/F (FF) and Raptor^Δhep (KO) 8 weeks old mice were i.p. injected with 100 mg/Kg DEN and their sera and livers analyzed 24-72 hrs later. (A) Serum ALT after DEN injection (n= 3). (B,C) H&E, Ki67, p-ERK, and cyclin D1 staining of liver sections from FF and KO mice 48 hrs after DEN injection (n= 3). Bar graphs indicate frequencies of Ki67, p-ERK, and cyclin D1 positive cells. (D) Cell death in FF and KO livers was analyzed 48 hrs after DEN injection by TUNEL and immunostaining with the antibody against cleaved-caspase 3 (clv-casp 3). Quantitative analysis is shown in the bar graphs (n= 3). (E) Immune cell infiltration was analyzed 48 hrs after DEN injection by immunostaining with the indicated antibodies. F4/80 positive areas were quantified with Image J software. B220 and CD3 positive cells were counted (n= 3). (F) Immunoblot analysis of liver lysates collected after DEN injection. All the bar graphs in Figure 4 represent means +/− S.D. See also Figure S4.

Figure 5. Increased DEN-induced hepatocarcinogenesis in Raptor^Δhep mice

Raptor^F/F (FF) and Raptor^Δhep (KO) 2 weeks old males were injected with 25 mg/Kg DEN. Tumor development was analyzed 7 months later. (A) Gross morphology of livers and typical HCC histology (magnification bar; 100 μm) in mice of the indicated genotypes. (B) Tumor numbers and maximal tumor sizes. Results are means +/− S.E.M. (C) Raptor protein expression in tumor and non-tumor liver tissue was analyzed by immunoblotting. See also Figure S5.

Figure 6. Hepatocyte Raptor ablation does not reduce hepatosteatosis and augments liver fibrosis in HFD fed mice

Raptor^F/F (FF) and Raptor^Δhep (KO) mice were kept on HFD or LFD for 6 months. (A) Liver histology, lipid accumulation and fibrosis were analyzed by staining liver sections with H&E, oil red O, Sirius Red and α smooth muscle actin (SMA) antibody. The positive areas were quantified with Image J software and shown as bar graphs. (B) Liver triglyceride (TG) content in 7 months old mice kept on LFD or HFD. (C) The mRNA amounts of fibrogenic markers, collagen α1(I) (Col1A1), collagen α1(IV) (Col4A1), actin α (Acta), and TIMP1 (Timp1) in livers of 7 months old FF and KO mice were determined by real time qPCR. (D) Glucose tolerance tests were performed on above mice kept on LFD and HFD (n= 4). All the bar graphs in Figure 6 represent means +/− S.D. See also Figure S6.