Alvaro Santos-Laso

Background & Aims: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD‐causative genes, encoding for endoplasmic reticulum (ER)‐resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target.Methods: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4‐phenylbutyric acid (4‐PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro.Results: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4‐PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4‐PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis.Conclusions: Restoration of proteostasis in cystic cholangiocytes with 4‐PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

: Cholangiocarcinoma (CCA) comprises a group of heterogeneous biliary cancers with dismal prognosis. The etiologies of most CCAs are unknown, but primary sclerosing cholangitis (PSC) is a risk factor. Non-invasive diagnosis of CCA is challenging and accurate biomarkers are lacking. We aimed to characterize the transcriptomic profile of serum and urine extracellular vesicles (EVs) from patients with CCA, PSC, ulcerative colitis (UC), and healthy individuals. Serum and urine EVs were isolated by serial ultracentrifugations and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. EVs transcriptome was determined by Illumina gene expression array [messenger RNAs (mRNA) and non-coding RNAs (ncRNAs)]. Differential RNA profiles were found in serum and urine EVs from patients with CCA compared to control groups (disease and healthy), showing high diagnostic capacity. The comparison of the mRNA profiles of serum or urine EVs from patients with CCA with the transcriptome of tumor tissues from two cohorts of patients, CCA cells in vitro, and CCA cells-derived EVs, identified 105 and 39 commonly-altered transcripts, respectively. Gene ontology analysis indicated that most commonly-altered mRNAs participate in carcinogenic steps. Overall, patients with CCA present specific RNA profiles in EVs mirroring the tumor, and constituting novel promising liquid biopsy biomarkers.

Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of symptomatic biliary cysts. Current surgical and pharmacological approaches are ineffective, and liver transplantation represents the only curative option. Ursodeoxycholic acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6i) have arisen as promising therapeutic strategies but with partial benefits. Here, we tested a novel approach based on the design, synthesis and validation of a family of UDCA synthetic conjugates with selective HDAC6i capacity (UDCA‐HDAC6i). Four UDCA‐HDAC6i conjugates presented selective HDAC6i activity, UDCA‐HDAC6i #1 being the most promising candidate. UDCA orientation within the UDCA‐HDAC6i structure was determinant for the HDAC6i activity and selectivity. Treatment of polycystic rats with UDCA‐HDAC6i #1 reduced their hepatomegaly and cystogenesis, increased UDCA concentration and inhibited HDAC6 activity in liver. In cystic cholangiocytes in vitro, UDCA‐HDAC6i #1 restored the primary cilium length and exhibited potent anti‐proliferative activity. UDCA‐HDAC6i #1 was actively transported into cells through bile acid and organic cation transporters. Conclusion these novel UDCA‐HDAC6i conjugates open a new therapeutic avenue for PLDs.