An inflammatory-CCRK circuitry drives mTORC1-dependent metabolic and immunosuppressive reprogramming in obesity-associated hepatocellular carcinoma

Abstract

Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men, but the molecular mechanism remains obscure. Here, we show that an androgen receptor (AR)-driven oncogene, cell cycle-related kinase (CCRK), collaborates with obesity-induced pro-inflammatory signaling to promote non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Lentivirus-mediated Ccrk ablation in liver of male mice fed with high-fat high-carbohydrate diet abrogates not only obesity-associated lipid accumulation, glucose intolerance and insulin resistance, but also HCC development. Mechanistically, CCRK fuels a feedforward loop by inducing STAT3-AR promoter co-occupancy and transcriptional up-regulation, which in turn activates mTORC1/4E-BP1/S6K/SREBP1 cascades via GSK3β phosphorylation. Moreover, hepatic CCRK induction in transgenic mice stimulates mTORC1-dependent G^-csf expression to enhance polymorphonuclear myeloid-derived suppressor cell recruitment and tumorigenicity. Finally, the STAT3-AR-CCRK-mTORC1 pathway components are concordantly over-expressed in human NASH-associated HCCs. These findings unveil the dual roles of an inflammatory-CCRK circuitry in driving metabolic and immunosuppressive reprogramming through mTORC1 activation, thereby establishing a pro-tumorigenic microenvironment for HCC development.

Fig. 1

Dietary obesity-induced CCRK over-expression promotes lipid accumulation, glucose intolerance, and liver damages in male mice. a Schematic diagram of NASH mouse model with different diets (CD chow diet, HFHC high-fat high-carbohydrate) and lentivirus-mediated Ccrk knockdown (CD+shCtrl, n=8; HFHC+shCtrl, n = 15; HFHC+shCcrk, n = 15). b Body weight, c blood triglyceride and non-esterified fatty acid (NEFA) levels in mice were increased by HFHC at 28 weeks, which could be reduced by Ccrk knockdown. d, e CCRK impaired insulin sensitivity in mice. d Intraperitoneal glucose tolerance test (IPGTT) and e intraperitoneal insulin tolerance test (IPITT) were performed on CD-fed and HFHC-fed mice, blood glucose was measured at indicated time points after glucose or insulin injection (left), and area under the curve (AUC) is shown in a bar chart (right). f CCRK protein expression was induced by HFHC in mouse livers, which could be reduced by shRNA-mediated knockdown. Quantification of CCRK protein levels (relative to β-actin) is shown in a bar chart (bottom). g Representative pictures of Oil Red O, ballooning degeneration, and spotty necrosis of liver tissues in different groups (image magnification = ×200 or ×400, scale bar = 20 μm). h, i Quantifications of Oil Red O, ballooning degeneration and j scoring of spotty necrosis showed increased lipid accumulation, ballooning and spotty necrosis in HFHC-fed mice, which were reduced by Ccrk knockdown. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; and ***p < 0.001 as calculated by unpaired two-tailed Student’s t-test (b), one-way ANOVA followed by Bonferroni post-hoc test (c–f, h, j), two-way ANOVA followed by Bonferroni post-hoc tests (d, e), and Chi-square test (i)

Fig. 2

Knockdown of Ccrk improves glucose sensitivity, and reduces hepatic lipid accumulation and hepatocarcinogenesis. a Schematic diagram of NASH-HCC mouse model with carcinogen induction (DEN diethylnitrosamine), different diets, and lentivirus-mediated Ccrk knockdown (CD+shCtrl, n = 8; HFHC+shCtrl, n = 15; HFHC+shCcrk, n = 15). b Body weight of mice at 28 weeks. c CCRK protein expression in mouse livers were increased by HFHC at 28 weeks, which could be reduced by shRNA-mediated knockdown. Quantification of CCRK protein levels (relative to β-actin) is shown in a bar chart (right). d Representative pictures of dissected livers (scale bar = 1 cm), and Oil Red O and Ki67 staining of liver tissues in different groups (image magnification = ×200 or ×400, scale bar = 20 μm). e Tumor multiplicity and f tumor sizes were higher in the livers of HFHC-fed mice, which could be reduced by Ccrk knockdown. g, h Dietary obesity-induced CCRK increased lipid accumulation and cell proliferation, which could be abrogated by Ccrk knockdown as shown by Oil Red O staining and Ki67 staining in the livers. i, j Blood triglyceride and NEFA levels were elevated by dietary obesity-induced CCRK at 28 weeks, which could be reduced by Ccrk knockdown. k CCRK over-expression impaired insulin sensitivity in mice. IPGTT was performed on CD-fed and HFHC-fed mice, blood glucose was measured at indicated time points after glucose injection (left), and AUC is shown in a bar chart (right). Data are presented as mean ± SD. *p < 0.05; **p < 0.01; and ***p < 0.001 as calculated by unpaired two-tailed Student’s t-test (b), one-way ANOVA followed by Bonferroni post-hoc test (c, e–k), and two-way ANOVA followed by Bonferroni post hoc test (k)

Fig. 3

IL-6 activates STAT3 and AR signaling to stimulate CCRK expression. a Western blot and b qRT-PCR analysis of HepG2 and Huh7 cells treated with or without IL-6, combined with siRNA-mediated knockdown of STAT3 or AR. c Luciferase reporter assay was used to measure CCRK promotor activity in HepG2 and Huh7 cells expressing either WT or AR response element (ARE)-deleted mutant CCRK promoter, treated with or without IL-6, combined with siRNA-mediated knockdown of STAT3 or AR. d CCRK expression was blocked by knockdown of STAT3, but rescued by over-expression of AR. e STAT3 phosphorylation suppressed by AR knockdown could be rescued by over-expression of CCRK. f WT but not kinase-defective (KD) CCRK-induced STAT3 phosphorylation and AR expression. g CCRK induces its own promoter activity, which was abolished by deletion of ARE or knockdown of either STAT3 or AR in CCRK-expressing cells. h Co-immunoprecipitation of p-STAT3^Tyr705 and AR in LO2 cells transfected with WT CCRK, but not in those transfected with empty vector or KD CCRK. IgG is a control for non-specific immunoprecipitation. i ChIP-re-ChIP assay demonstrated an increased co-occupancy of STAT3 and AR at the ARE of CCRK promotor in LO2 cells transfected with WT CCRK relative to those transfected with vector only. IgG is a control for non-specific immunoprecipitation. The antibodies used in the 1st and 2nd ChIP are as indicated. j Schematic representation of the IL-6/STAT3/AR/CCRK circuitry in hepatocarcinogenesis. Data are presented as mean ± SD. *p < 0.05 and **p < 0.01 as calculated by one-way ANOVA followed by Bonferroni post hoc test (i)

Fig. 4

CCRK activates mTORC1 signaling through GSK3β/TSC2 cascade. a Ectopic CCRK expression in LO2 and CCRK KO Huh7 cells activates mTORC1 signaling, which was abrogated by suppression of GSK3β phosphorylation at Ser9 (p-GSK3β^Ser9) via over-expression of the constitutively-active S9A-GSK3β mutant. Western blot analysis was used to detect the protein expression of mTORC1 downstream molecules. b Knockdown of CCRK in HepG2 and Huh7 cells impaired the activation of mTORC1 signaling, which was rescued by the silencing of TSC2. c, d Dietary obesity-induced CCRK expression is responsible for the activation of mTORC1 signaling cascades in both c NASH and d NASH-HCC models, and such CCRK-dependent effects were abolished following knockdown of Ccrk. The establishment of NASH and NASH-HCC models are as described in Figs. 1a and 2a

Fig. 5

Hepatic CCRK promotes lipid accumulation, glucose uptake, insulin resistance, and tumorigenicity through mTORC1 activation. a Doxycycline (Dox)-induced expression of CCRK-activated mTORC1 signaling pathway in LO2-CCRK cells, which was abolished by inhibition of mTOR via Raptor knockdown or treatment with Rapamycin (Rapa). b, c Lipid accumulation (image magnification =×400, scale bar = 20 μm) as well as d glucose uptake were increased by CCRK-mediated mTOR activation, but were reduced by mTORC1 inhibition. e mTORC1 signaling was suppressed in CCRK KO Huh7 cell line. f CCRK impaired insulin sensitivity in Huh7 cells, which was restored by CCRK KO. Insulin sensitivity was assessed by p-Akt^Ser473 expression via Western blot analysis in cells treated with high dose of insulin followed by low dose of insulin stimulation. g The CCRK-induced insulin intolerance was restored by inhibition of mTORC1 using shRaptor or Rapamycin. h, i Mice injected with Dox-induced LO2-CCRK cells developed larger tumors (scale bar = 1 cm) compared to control mice and those treated with shRaptor or Rapamycin (n = 5 per group). j, k CCRK promoted tumorigenicity through mTORC1 activation. j The cell proliferation was assessed by Ki67 staining (scale bar = 20 μm). k The CCRK-activated mTORC1 signaling was detected by Western blot analysis. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; and ***p < 0.001 as calculated by one-way ANOVA followed by Bonferroni post-hoc test (b, d, i, j), and two-way ANOVA followed by Bonferroni post-hoc test (h)

Fig. 6

Hepatic CCRK-mTORC1 signaling recruits MDSCs to form a tumor-prone liver microenvironment. a mTORC1 signaling was activated in the livers of CCRK TG mice. The downstream molecules of mTORC1 signaling were detected by Western blot. b mTORC1 signaling components were activated in the primary hepatocytes isolated from CCRK TG mice. c Schematic diagram of CCRK TG mouse model with intrahepatic injection of Ccrk KO Hepa1–6 cells by CRISPR/Cas9, and lentiviral-mediated Raptor knockdown. d CCRK TG mice (n = 8) developed significantly larger tumors compared to controls (n = 11), which was abolished by down-regulation of Raptor (shRaptor; n = 5) (scale bar = 1 cm). e PMN-MDSCs were induced in CCRK TG mice, but were reduced after Raptor knockdown, and f PMN-MDSC levels positively correlated with tumor weight. g PMN-MDSCs were induced by HFHC diet, but were reduced by Ccrk knockdown in NASH-HCC model (CD+shCtrl, n = 8; HFHC+shCtrl, n = 15; HFHC+shCcrk, n = 15), wherein h PMN-MDSC levels positively correlated with tumor multiplicity. i G-csf mRNA levels were induced by HFHC diet, but were reduced by Ccrk knockdown in NASH-HCC model. j G-CSF mRNA level was augmented by CCRK over-expression in LO2 cells, which could be attenuated by Raptor knockdown. k G-CSF mRNA expression was down-regulated in CCRK KO Huh7 cells. Data are presented as mean ± SD. *p < 0.05 and **p < 0.01 as calculated by one-way ANOVA followed by Bonferroni post hoc test (d, e, g, i, j), Pearson correlation (f, h), and unpaired two-tailed Student’s t-test (k)

Fig. 7

CCRK over-expression correlates with mTOR signaling activation in patients with NASH-associated HCCs. a Western blots showed the induced expressions of CCRK/mTOR signaling proteins in HCC tissues relative to matched non-tumor tissues (23 pairs), and representative results from three patients are shown. b The Western blot results of all patients from a were quantified and shown in dot plots, wherein the fold enrichments of target proteins relative to their corresponding controls are shown. c The positive correlations of STAT3/AR/CCRK/GSK3β/mTORC1 signaling components were confirmed in the patient specimens. d Schematic diagram showing the IL-6-triggered self-reinforcing circuitry of STAT3/AR/CCRK (highlighted in the yellow panel), which activates mTORC1 signaling (highlighted in the green panel) through GSK3β/TSC2 to promote NASH and HCC development. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 as calculated by unpaired two-tailed Student’s t-test (b), and Pearson correlation (c)