Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression

Abstract

Epidemiological studies indicate that overweight and obesity are associated with increased cancer risk. To study how obesity augments cancer risk and development, we focused on hepatocellular carcinoma (HCC), the common form of liver cancer whose occurrence and progression are the most strongly affected by obesity among all cancers. We now demonstrate that either dietary or genetic obesity is a potent bona fide liver tumor promoter in mice. Obesity-promoted HCC development was dependent on enhanced production of the tumor-promoting cytokines IL-6 and TNF, which cause hepatic inflammation and activation of the oncogenic transcription factor STAT3. The chronic inflammatory response caused by obesity and enhanced production of IL-6 and TNF may also increase the risk of other cancers.

Figure 1. Genetic and dietary obesity promote DEN-induced hepatocarcinogenesis

(A). Livers of male WT and Lep^Ob mice kept on normal chow (LFD) or high fat diet (HFD) from week 6 to week 36 after the administration of DEN (25 mg/kg) at 2 weeks of age (protocol #1; Figure S1A). (B). Tumor multiplicity, size and incidence in livers of DEN-injected WT and Lep^Ob male mice kept on LFD □ or HFD ■ as above. Results are averages ± s.d. (n=10-12). (C). Tumor multiplicity, size and incidence in livers of WT females that were given DEN at 2 weeks of age and fed as above. Results are averages ± s.d. (n=10-12). (D). Livers of WT male mice given DEN (80 mg/kg) at 16 weeks of age and kept on LFD or HFD from week 6 to week 50 (protocol #2, Figure S1B). When indicated, phenobarbital (PB) was given as a tumor promoter in drinking water (0.05%) from 4 weeks after DEN administration until sacrifice. Arrows indicate HCCs. (E). HCC multiplicity, size and incidence in WT male mice treated as in (D). Results are means ± s.d. (n=10-13). *P<0.05, **P<0.01, ***P<0.001 denote significant differences between the groups. See also Supplemental Figures S1 and S2.

Figure 2. Obesity in HCC-bearing mice is associated with increased cancer cell proliferation and reduced apoptosis

HCC-bearing (protocol #1) male mice were subjected to histological and biochemical analyses at 9 months of age. (A). Obesity reduces cell death in HCCs. Paraffin-embedded liver sections were TUNEL labeled (left panels) and counter stained with DAPI (right panels). Note the presence of apoptotic cells in tumor (T) area in lean mice. Magnification: 20×. (B). Numbers of apoptotic cells per field in (A) was determined by Image Tool software (n=5). (C). Obesity enhanced cell proliferation in HCCs. Mice were BrdU pulsed 2 hrs prior to sacrifice. Paraffin embedded liver sections were stained with anti-BrdU antibody. (D). Numbers of BrdU-positive proliferating cells in (C) was determined with Image Tool software (n=5). (E). Expression of cyclin D1 mRNA in non-tumor liver (NT) and tumors (T) was determined by qRT-PCR (n=5-6). Values were normalized to cyclophilin mRNA. (F). Mice kept on LFD or HFD for 10 wks were injected with DEN (100 mg/kg) and their livers were isolated 48 hrs after DEN administration and 2 hrs after BrdU pulse. Paraffin embedded liver sections were subjected to TUNEL and BrdU staining and positive cells were counted as above. (n=5). All values represent means ± s.d. *P<0.05, **P<0.01, ***P<0.001 denote significant differences between the groups.

Figure 3. Obesity promotes tumorigenic growth of hepatoma cells

(A). Two months old lean mice (2 M) were subcutaneously injected with established hepatoma cells (2.5×10⁶ cells/mouse) and kept on LFD or HFD for 4 weeks after inoculation with (AG+) or without (AG-) AG490 (0.5 mg/mouse/day), a JAK inhibitor that blocks STAT3 activation. The same number of hepatoma cells was also transplanted into 8 months-old mice that were kept on HFD for the preceding 6 months or were genetically obese (Lep^Ob). Tumor growth was monitored by measurement with a caliper once a week, from week 2 to week 4. The images show representative solid tumors dissected at week 4. (B). Tumor growth measured over the course of a 2 week period in mice described in (A). The results are means for 3-6 mice per group. (C). Tumor sizes at week 4 post-inoculation are shown as a histogram. Results are means ± s.d. (n=3-6). (D). Tumors grown in mice given HFD without (AG-) or with (AG+) AG490, administered as above, were collected at week 4 post-inoculation, homogenized and STAT3 phosphorylation and expression were analyzed by immunoblotting. All values represent means ± s.d. *P<0.05, **P<0.01 denote significant differences between the groups.

Figure 4. Obese HCC-bearing mice exhibit activated STAT3 and elevated expression of inflammatory cytokines

The HCC-bearing mice from Figure 2 were subjected to additional biochemical and gene expression analyses. (A). Activation state of metabolically-regulated protein kinases. Non-tumor (NT) liver and HCC (T) were lysed, gel separated and the activation (phosphorylation) states of AKT and S6 kinase (S6K) were examined by immunoblotting. (B). Non-tumor (NT) liver and HCC (T) were analyzed for phosphorylation of JNK, ERK, p38, and STAT3 by immunoblotting as above. The bottom panel shows JNK kinase assays in non-tumor liver and HCCs of 4 different mice per dietary group. (C). The results of several immunoblots similar to the one shown in B, each representing a different mouse, were quantitated using Image J software and mean values were determined (n=3-4). (D). Serum IL-6 in HCC-bearing mice was determined by ELISA (n=10-12). (E-G). Relative amounts of IL-6 (E), TNF (F) and IL-1β (G) mRNAs in non-tumor (NT) liver and HCC (T) were determined by qRT-PCR and normalized to cyclophilin mRNA (n=6-8). (H). TNF amounts in non-tumor liver (NT) and HCC (T) were examined by immunoblotting of tissue lysates. All values represent means ± s.d. *P<0.05, **P<0.01, ***P<0.001 denote significant differences between the groups. See also Supplemental Figure S3.

Figure 5. Enhanced IL-6 production is required for obesity-induced tumor promotion

(A). Livers of WT and IL-6 deficient (IL6^-/-) male mice that were kept on LFD or HFD after the administration of DEN (25 mg/kg) at 14 days of age. (B). HCC multiplicity, size, and incidence (n=10-12) in DEN-administered male mice of the indicated genotype kept on either LFD □ or HFD ■. (C). HCC multiplicity, size, and incidence (n=10-12) in DEN-administered WT females or IL-6^-/- males of the indicated genotype kept on either LFD □ or HFD ■. (D). Body weight gain during tumor development in WT and IL-6^-/- male mice kept on LFD or HFD after DEN administration. (E). Serum and liver triglycerides (TG) in mice from (D) were determined by a colorimetric assay at 9 months of age. Serum insulin was determined by ELISA at same age (serum, n=8-12; liver, n=5). (F). HCCs (T) and non-tumor (NT) liver tissue from IL6^-/- male mice kept on either LFD or HFD were isolated, homogenized and subject to immunoblot analysis to determine the phosphorylation state of the indicated proteins. Shown are the results from two separate tumors for each condition. All values are means ± s.d. *P<0.05, **P<0.01, ***P<0.001 denote significant differences between the groups. ^†P<0.05, ^‡P<0.01 presents significant differences from WT mice. See also Supplemental Figure S4.

Figure 6. TNFR1 signaling is required for obesity-induced tumor promotion

(A). Livers of WT or TNFR1 deficient male mice kept on LFD or HFD after DEN administration (25 mg/kg) at 14 days of age. (B). Tumor multiplicity and maximal sizes in DEN-administered male mice of the indicated genotypes kept on either LFD □ or HFD ■ (n=10-13). (C). Body weight gain during tumor development in WT or TNFR1^-/- male mice kept on LFD or HFD after DEN administration. (D). Non-tumor (NT) liver and HCC (T) from the mice described in (A) were isolated, lysed, gel separated and the phosphorylation states of the indicated proteins were examined by immunoblotting. Shown are the results for two separate HCCs per condition.

Figure 7. TNFR1 signaling and IL-6 support steatohepatitis

(A and C). Serum IL-6 (A) and TNF (C) were measured by ELISA in HCC bearing WT, IL6^-/- or TNFR1^-/- mice kept on LFD □ or HFD ■. The analyses were done at 9 months of age (n=8-10). (B). IL-6 mRNA amounts in HCC-bearing livers and HCCs from WT or TNFR1^-/- mice kept on HFD or LFD were measured by qRT-PCR at 9 months of age. The bars depict the relative increase in normalized mRNA amount, with the amount in non-tumor (NT) liver of lean mice given an arbitrary value of 1.0 (n=5-6). (D). Liver fat accumulation in HCC-bearing WT and IL6^-/- mice kept on LFD or HFD was examined by oil red O (ORO) staining of liver sections collected at 9 months of age (magnification: 40×). (E). Liver TG levels in HCC-bearing WT, IL6^-/- or TNFR1^-/- mice were determined at 9 months of age. (F-G). Macrophage (F and G) and neutrophil (G) infiltration in non-tumor liver and HCCs of lean and obese 9 months old WT, IL6^-/- and TNFR1^-/- male mice. Paraffin embedded sections were stained with F4/80 and Gr-1 specific antibodies to visualize macrophages and neutrophils, respectively. Results from F4/80 staining are shown in (F). Data from several experiments (n=4-5) were quantitated with Image J software and were averaged (G). All values represent means ± s.d. *P<0.05, **P<0.01, **P<0.001 denote significant differences between the groups.