PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

Azathioprine promotes intestinal epithelial cell differentiation into Paneth cells and alleviates ileal Crohn's disease severity.

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn's disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

Unique Metabolomic and Lipidomic Profile in Serum From Patients With Crohn's Disease and Ulcerative Colitis Compared With Healthy Control Individuals.

BACKGROUND: Accurate biomarkers for disease activity and progression in patients with inflammatory bowel disease (IBD) are a prerequisite for individual disease characterization and personalized therapy. We show that metabolic profiling of serum from IBD patients is a promising approach to establish biomarkers. The aim of this work was to characterize metabolomic and lipidomic serum profiles of IBD patients in order to identify metabolic fingerprints unique to the disease. METHODS: Serum samples were obtained from 55 patients with Crohn's disease (CD), 34 patients with ulcerative colitis (UC), and 40 healthy control (HC) individuals and analyzed using proton nuclear magnetic resonance spectroscopy. Classification of patients and HC individuals was achieved by orthogonal partial least squares discriminant analysis and univariate analysis approaches. Disease activity was assessed using the Gastrointestinal Symptom Rating Scale. RESULTS: Serum metabolome significantly differed between CD patients, UC patients, and HC individuals. The metabolomic differences of UC and CD patients compared with HC individuals were more pronounced than the differences between UC and CD patients. Differences in serum levels of pyruvic acid, histidine, and the branched-chain amino acids leucine and valine were detected. The size of low-density lipoprotein particles shifted from large to small dense particles in patients with CD. Of note, apolipoprotein A1 and A2 serum levels were decreased in CD and UC patients with higher fecal calprotectin levels. The Gastrointestinal Symptom Rating Scale is negatively associated with the concentration of apolipoprotein A2. CONCLUSIONS: Metabolomic assessment of serum samples facilitated the differentiation of IBD patients and HC individuals. These differences were constituted by changes in amino acid and lipoprotein levels. Furthermore, disease activity in IBD patients was associated with decreased levels of the atheroprotective apolipoproteins A1 and A2.

The metabolic and lipidomic serum profile of patients with inflammatory bowel disease was analyzed using proton nuclear magnetic resonance spectroscopy. A significantly altered profile in comparison with healthy control individuals was identified, characterized by more atherogenic properties.