Ainhoa Lapitz

Background and Aim: Polycystic liver disease (PLD) is a hereditary disorder characterized by the progressive development and enlargement of intrahepatic biliary cysts, which can lead to significant morbidity. Liver transplantation remains the only curative treatment. Mutations in endoplasmic reticulum (ER)-related genes contribute to ER stress in cystic cholangiocytes, promoting disease progression. Given the functional interplay between ER and mitochondria, we investigated mitochondrial dynamics and metabolism in cystic cholangiocytes from both PLD patients and Pkhd1 ^mut rats to identify novel therapeutic targets. Approach and Results: Cystic cholangiocytes exhibited increased mitochondrial mass, membrane potential, and superoxide levels, along with enhanced bioenergetic capacity and ATP production compared with normal cholangiocytes. These alterations were linked to the upregulation of electron transport chain protein complexes. Metabolic reprogramming involved enhanced oxidation of glucose, glutamine, and/or fatty acids, as well as increased de novo cholesterol synthesis and accumulation. Chronic treatment with pravastatin, a cholesterol synthesis inhibitor, significantly reduced hepatomegaly, cyst volume, and fibrosis in Pkhd1 ^mut rats. It also normalized mitochondrial hyperactivity and reduced the proliferation of cystic cholangiocytes in culture, effects that were similarly observed with other statins such as atorvastatin and simvastatin. Importantly, a case-control study in PLD patients showed that statin use was associated with reduced liver growth, further supporting its potential therapeutic role. Conclusions: Mitochondrial and metabolic dysregulation are central to the pathogenesis of PLD. Targeting cholesterol metabolism with statins represents a promising therapeutic strategy to slow disease progression and reduce cyst burden.

Background: Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are immune-mediated cholestatic disorders characterized by progressive biliary inflammation and fibrosis, for which treatment options remain limited, underscoring the need for novel therapeutic targets. The leukemia inhibitory factor (LIF) is an IL-6-related cytokine that dysregulates the communication between epithelial cells and extracellular matrices by binding a heterodimeric complex formed by LIF receptor (LIFR) and gp130. The role of the LIF/LIFR system in PSC and PBC and its potential as a therapeutic target remain unclear. Methods: We investigated LIF/LIFR system alteration in PSC and PBC and assessed the therapeutic potential of LIFR antagonism in a genetic mouse model of PSC (Abcb4-/- mice). Single-cell transcriptomics analyses were performed to evaluate LIF and LIFR expression in human liver samples. Whole liver RNA-seq and immunostaining were used to assess LIF/LIFR levels and correlation with fibrotic and immune markers. The effects of LIFR antagonism were evaluated in vitro using LRI-310, a steroidal LIFR antagonist, on human cholangiocytes, HSCs, endothelial cells, and macrophages. In vivo, LRI-310 was administered to Abcb4–/– mice, and effects on liver injury, cholestasis, fibrosis, leukocyte infiltration, and gene expression were assessed. Results: LIF expression s enriched in human cholangiocytes, while LIFR is predominantly expressed by HSCs, endothelial cells, and macrophages. Whole liver RNAseq analysis and liver sections immunostaining demonstarted that increased LIF expression correlates with expression of markers of hepatic fibrosis and immune activation in PSC and PBC patients. LRI-310, a steroidal LIFR antagonist, attenuated human cholangiocytes activation and expression of inflammatory mediators, as well as the activation of liver sinusoidal cells and hepatic fibroblasts. In Abcb4 −/− mice administration of LRI-310 mitigated liver injury, cholestasis, liver leukocytes infiltration and reduced the expression of biomarkers associated with fibrosis, inflammation, and bile acid dismetabolism. Conclusion: Cholangiocyte-derived LIF promotes the formation of a pro-inflammatory and pro-fibrotic niche centred on damaged cholangiocytes. LIFR antagonism reverses fibrosis and immune dysregulation in Abcb4 ^−/− mice, supporting the development of anti-LIFR therapies in human cholangiopathies.

Background and Aims: Cholangiocarcinoma (CCA) includes a diverse group of biliary malignancies with poor prognosis. Alterations in post-translational modifications contribute to disrupted protein dynamics, cellular disturbances, and disease. This study investigates the role of protein SUMOylation in cholangiocarcinogenesis and its potential as a therapeutic target. Approach and Results: Analysis of CCA tumors from 4 patient cohorts and CCA cell lines, revealed increased expression of the SUMOylation machinery genes SUMO activating enzyme 1 ( SAE1 ) and the SUMO conjugating enzyme UBE2I , regardless of the tumor’s molecular profile, resulting in elevated levels of SUMO1-conjugated proteins. Higher SAE1 and UBE2I levels were both indicative of unfavorable clinical outcomes. Deregulated SUMOylated proteins in CCA, mostly linked to cell proliferation, survival, and homeostasis, were identified through immunoprecipitation and mass spectrometry. Genetic ( UBE2I -knockdown) and pharmacological (ML792 and S-adenosyl-methionine) inhibition of SUMOylation effectively suppressed tumorigenesis in subcutaneous and oncogene-driven CCA models, reducing the presence of cancer-associated fibroblasts and increasing the recruitment of antitumor immune cells. In vitro, targeting SUMOylation induced CCA cell death and reduced cell proliferation, colony formation, and spheroid growth. Importantly, ML792 and S-adenosyl-methionine did not adversely affect normal human cholangiocytes. Moreover, co-culture of wild-type or UBE2I -knockdown CCA cells with cancer-associated fibroblasts revealed that depleting SUMOylation in CCA cells impaired cancer-associated fibroblast cell growth and altered their protein secretome, ultimately disrupting CCA growth through a regulatory feedback loop. Conclusions: Aberrant SUMOylation drives CCA progression by enhancing cell survival, proliferation, and shaping the tumor microenvironment. Targeting SUMOylation shows potential in inhibiting CCA growth, representing a promising therapeutic strategy.

The proteomic analysis of serum extracellular vesicles (EVs) could be a useful tool for studying the pathophysiology of Crohn’s disease (CD) and ulcerative colitis (UC), as well as for biomarker discovery. To characterize the proteomic composition of serum EVs in patients with CD and UC to identify biomarkers and molecular pathways associated with pathogenesis and activity. Methods: Serum EVs were enriched and analyzed in patients with quiescent CD, active CD (aCD), quiescent UC, active UC (aUC), and healthy controls (HCs) (n = 30 per group). All groups were matched for age and sex. Disease activity was assessed by ileocolonoscopy and categorized based on the SES-CD (CD) and the endoscopic sub-score of the Mayo Score (UC). EVs were enriched by ul-tracentrifugation, and their size and concentration were determined by nanoparticle tracking analysis. The expression of CD63, CD81, and CD9 was determined using West-ern blotting. Proteomic analysis was performed by label-free nano-LC MS/MS. A total of 324 proteins were identified; 60 showed differential abundance in CD-HC, 34 in UC-HC, and 21 in CD-UC. Regarding disease activity, the abundance of 58 and 32 proteins was altered in aCD-HC and aUC-HC, respectively. Functional analyses revealed that proteins associated with aCD were involved in immune regulation, whereas those linked to aUC were enriched in oxidative stress. We have identified expressed proteins between EVs from patients with CD and UC, depending on the presence of disease, the disease type, and the disease activity. These proteins are potential candidates as disease biomarkers and open new research avenues to better understand these conditions.

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Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of low‐molecular‐weight metabolites by new high‐throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19‐9 (CA 19‐9) and alpha‐fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy‐proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14‐15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases.

Primary hepatobiliary malignancies include a heterogeneous group of cancers with dismal prognosis, among which hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatoblastoma (HB) stand out. These tumors mainly arise from the malignant transformation of hepatocytes, cholangiocytes (bile duct epithelial cells) or hepatoblasts (embryonic liver progenitor cells), respectively. Early diagnosis, prognosis prediction and effective therapies are still a utopia for these diseases. Extracellular vesicles (EVs) are small membrane-enclosed spheres secreted by cells and present in biological fluids. They contain multiple types of biomolecules, such as proteins, RNA, DNA, metabolites and lipids, which make them a potential source of biomarkers as well as regulators of human pathobiology. In this review, the role of EVs in the pathogenesis of hepatobiliary cancers and their potential usefulness as disease biomarkers are highlighted. Moreover, the therapeutic value of EV regulation is discussed and future directions on basic and clinical research are indicated.