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.
419 Background: A predictive biomarker for resistance to trastuzumab in HER2 positive oesophago-gastric cancer would refine patient selection. Our preclinical studies suggest that patients with HIGH baseline plasma miR-148a-3p levels will experience shorter Overall Survival (OS), Progression Free Survival (PFS), and worse Progression Free Rates (PFR) than those with LOW plasma miR-148a-3p levels. Methods: This sub-study is a prospective biomarker analysis of baseline plasma samples for HER2 positive advanced oesophago-gastric cancer patients registered within Arm B1 of the Phase 2 open label, multicentre, randomised PLATFORM trial (NCT02678182). All HER2 positive patients assessable for clinical response and miR signatures were included. Copies of miR-148a-3p and miR-16 per ml of plasma were quantified using Digital Droplet Polymerase Chain Reaction (ddPCR). OS as primary endpoint for miR-148a-3p LOW (≤median) versus HIGH (>median) was analysed using Kaplan-Meier curves and Cox model regression. Secondary endpoints were PFS and PFR. OS and PFS are from start of 1st line therapy; PFR are from start of maintenance trastuzumab. Sensitivity analysis normalised miR-148a-3p to miR-16. Results: Of 63 patients with analysable lab samples and available survival data, normalisation was possible for 41 patients. Median follow-up was 38 months. There was no statistically significant relationship between OS and miR-148-3p copies/μl of ddPCR reaction LOW versus HIGH (n= 62, Hazard Ratio (HR) 0.98, p=0.933), PFS (n=62, HR 1.08, p=0.759) or PFR (n=31, Odds Radio (OR) 0.67, p=0.577). Normalised miR-148a-3p (NmiR-148a-3p) LOW versus HIGH demonstrated a statistically significant difference in PFR at 3 months (n=23, OR=0.11, p=0.027) but no difference in OS or PFS. A model adjusting for primary tumour site, metastatic disease and number of sites demonstrated a statistically significant difference in PFR at 3 months (aOR=0.03, p=0.029). Conclusions: Patients with HIGH NmiR-148-3p have 0.03 times the odds of being progression-free at 3 months than patients with LOW NmiR-148a-3p in this population, when adjusted for key factors. Limitations include small sample size and normalisation. Clinical trial information: NCT02678182 . [Table: see text]
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