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. 2022 May 26;41(1):183.
doi: 10.1186/s13046-022-02386-2.

New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming

Leticia Colyn #  1 , Gloria Alvarez-Sola #  1   2 , M Ujue Latasa #  1   3 , Iker Uriarte  1   2 , Jose M Herranz  1   2 , Maria Arechederra  1   2   3 , George Vlachogiannis  4 , Colin Rae  5 , Antonio Pineda-Lucena  6 , Andrea Casadei-Gardini  7 , Federica Pedica  8 , Luca Aldrighetti  9 , Angeles López-López  10 , Angeles López-Gonzálvez  10 , Coral Barbas  10 , Sergio Ciordia  11 , Sebastiaan M Van Liempd  12 , Juan M Falcón-Pérez  2   12   13 , Jesus Urman  3   14 , Bruno Sangro  2   3   15 , Silve Vicent  3   16   17 , Maria J Iraburu  18 , Felipe Prosper  19 , Leonard J Nelson  20 , Jesus M Banales  2   18   21 , Maria Luz Martinez-Chantar  2   22 , Jose J G Marin  2   23 , Chiara Braconi  5   24 , Christian Trautwein  25 , Fernando J Corrales  2   11 , F Javier Cubero  2   26 , Carmen Berasain #  1   2   3 , Maite G Fernandez-Barrena #  1   2   3 , Matias A Avila #  27   28   29
Affiliations

New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming

Leticia Colyn et al. J Exp Clin Cancer Res. .

Abstract

Background: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA.

Methods: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model.

Results: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression.

Conclusions: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

Keywords: Bile; Cholangiocarcinoma; G9a histone methyl-transferase; Inflammation; Interleukin-6; KRAS; Metabolic reprogramming; Serine-glycine pathway.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Proteomic analysis of bile in the rat TAA model of CCA development. a Schematic representation of the rat TAA model of CAA implemented in this study. c Pie chart showing the classification of proteins identified as differentially represented in bile from control and CCA bearing rats (30 weeks) according to their cellular localization. b Volcano plot (−log10 [p-value] and log2 [fold-change]) of the proteins found in bile from rats with CCA compared with control animals. d Principal component analysis (PCA) of bile proteomic data from control (Veh-1-4) and TTA (TAA-1-4) treated rats. e Ingenuity pathway analysis (IPA) of the differentially represented proteins between control and CCA bile samples identifying the top enriched categories of canonical pathways. Created with BioRender.com
Fig. 2
Fig. 2
Most relevant proteins and metabolites differentially represented in bile from control and CCA bearing rats. a Identity of proteins showing significantly increased concentrations (Fold change > 0.9) in bile from TAA-treated rats vs controls (Vehicle). The expression of the corresponding genes as reported in the TCGA database is shown. AC: accession number. b Metabolites showing significantly altered concentrations in bile from TAA-treated rats vs controls (Vehicle). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 3
Fig. 3
Inflammatory and growth factor-related signaling activation in the TAA rat model of CCA. a Quantification of bacterial DNA levels in the livers of control (Veh) and TAA treated rats. *p < 0.05. b mRNA levels of Il6 and the EGFR ligands heparin-binding EGF (Hbegf), amphiregulin (Areg) and epiregulin (Ereg) in liver tissue samples from control rats (Vehicle), peritumour liver tissues and tumour tissues. *p < 0.05, **p < 0.01, ***p < 0.001. d Immunohistochemical analysis of p-ERK1/2 in liver tissue samples from control rats (Vehicle), peritumour liver tissues and tumour tissues. Representative images are shown. c Immunohistochemical analysis of p-STAT3 in liver tissue samples from control rats (Vehicle), peritumour liver tissues and tumour tissues. Representative images are shown
Fig. 4
Fig. 4
EGFR-KRAS signaling triggers IL6 expression in CCA cells. a Effect of HB-EGF on IL6 mRNA expression (12 h treatment) and IL6 protein release (24 h treatment) in HuCCT-1 and Huh28 cells. *p < 0.05, **p < 0.01. b Characterization of control and KRASG12D (MutKRAS) Huh28 cells. Images show representative western blot analyses of KRASG12D, p-MEK1/2, p-ERK1/2, p-STAT3, STAT3 levels, as well as GAPDH levels, as loading control, in both cell lines. c Expression levels of IL6 mRNA and IL6 protein concentrations in the conditioned media (24 h culture) of control and MutKRAS Huh28 cells. **p < 0.01. d Expression levels of IL6 mRNA in MutKRAS cells treated with PI3K (LY294002) or MEK (PD98059) inhibitors for 6 h. *p < 0.05
Fig. 5
Fig. 5
Metabolic reprogramming in experimental CAA (rat TAA model) and human iCCA. a Expression of glucose metabolism-related genes in liver tissue samples from control rats (Vehicle), peritumour liver tissues and tumour tissues. *p < 0.05, **p < 0.01, ***p < 0.001. b Immunohistochemical analysis of PHGDH in liver tissue samples from control rats (Vehicle), peritumoural liver tissues and tumoural tissues. Representative images are shown. c Immunohistochemical analysis of PHGDH in human iCCA tissue samples. Representative images of tumours with 9, 6–4 and 0 PHGDH immunostaining scores are shown. Graphs show the distribution of PHGDH scores among all iCCA tissue samples and according to tumor grade (G1-G3)
Fig. 6
Fig. 6
Expression of PHGDH in wild type (control) and KRASG12D expressing (MutKRAS) Huh28 cells, response to L-serine availability. a PHGDH mRNA levels in control and MutKRAS Huh28 cells grown in complete medium (t = 0) and at the indicated time-points after L-serine depletion. *p < 0.05 vs control. b PHGDH protein levels were analyzed by western blotting in same samples described in A. Representative blots, including HSP90 analysis as loading control, are shown. c Growth of control and MutKRAS Huh28 cells in L-serine depleted medium referenced to cell growth in complete medium. *p < 0.05, **p < 0.01
Fig. 7
Fig. 7
G9a inhibition blunts the adaptive upregulation of PHGDH expression to L-serine availability in CCA cells. a Control and KRASG12D expressing (MutKRAS) Huh28 cells were grown in complete medium for 60 h with or without CM-272 (200 nM) and then maintained in complete medium or without L-serine for another 20 h. At this point PHGDH mRNA expression was analyzed. *p < 0.05. b PHGDH protein levels were analyzed by western blotting in same samples described in a. Representative blots, including HSP90 analysis as loading control, are shown. c UPLC-ToF-MS analysis of [U-13C] glucose flux into serine in control and MutKRAS Huh28 cells treated of not with CM-272 ( 200 nM, 66 h). *p < 0.05. d ATF4 protein levels were analyzed by western blotting in same samples described in a. Representative blots, including HSP90 analysis as loading control, are shown
Fig. 8
Fig. 8
G9a inhibition reduces PHGDH expression in a mouse model of CCA. a Diagram showing the experimental model and the treatments applied (n = 6 mice per group). b Phgdh mRNA levels in the liver of wild type mice, JnkΔhepa mice, JnkΔhepa mice treated with CCl4 and diethylnitrosamine (DEN) (JnkΔhepa + CCl4 + DEN mice) and JnkΔhepa + CCl4 + DEN mice treated with CM-272 as indicated. **p < 0.01. c Immunohistochemical detection of PHGDH in liver tissue sections from mice treated as described in b. Representative images are shown. Created with BioRender.com
Fig. 9
Fig. 9
G9a targeting inhibits KRASG12D induced malignant traits in CCA cells: identification of G9a as a therapeutically relevant vulnerability in KRASG12D expressing CCA cells. a Anchorage-independent growth of control and KRASG12D expressing (MutKRAS) Huh28 cells treated or not with CM-272 (200 nM). Representative images of colonies formed at the end of experiments (3 weeks) and quantification of the area occupied by colonies are shown. ***p < 0.001. b Colony formation assay in control and MutKRAS Huh28 cells treated with CM-272 as indicated. Representative images of crystal violet-stained colonies and the corresponding quantification are shown. *p < 0.05, **p < 0.01
Fig. 10
Fig. 10
Pharmacological targeting of G9a activity in CCA cells. a Western blot analysis of the distribution of G9a between nuclear chromatin faction (CF) and soluble nuclear fraction (SF) in control and KRASG12D expressing (MutKRAS) Huh28 cells. Representative blots, including C23 (nucleolin) analysis as loading control, are shown. b Effect of G9a inhibition on the distribution of G9a between CF and SF in control and MutKRAS Huh28 cells. Cells were treated with CM-272 (200 nM) for 72 h before fractionation and western blot analyses. Representative blots, including C23 (nucleolin) analysis as loading control, are shown. c Effect of CM-272 on G9a methylation status and interaction with HP1γ in control and MutKRAS Huh28 cells. Cells were treated with CM-272 (200 nM) for 72 h before immunoprecipitations with an anti-G9a antibody, an anti-pan-methyllysine antibody (Methyl-K) or with an anti-HP1γ antibody, and subsequent western blot analyses to detect G9a. Corresponding immunoprecipitation controls using normal rabbit IgG are included. Representative blots are shown. d Effect of CM-272 on G9a methylation and interaction with HP1γ in HuCCT-1 cells. Cells were treated or not with CM272 (200 nM) for 72 h before immunoprecipitations were carried out as described in c. Corresponding immunoprecipitation controls using normal rabbit IgG are included. Representative blots, including levels of HP1γ in total cell lysates are shown
Fig. 11
Fig. 11
Schematic diagram of the most relevant findings in this study. Grey boxes indicate observations from other works. Created with BioRender.com
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