Targeting UBC9-mediated protein hyper-SUMOylation in cystic cholangiocytes halts polycystic liver disease in experimental models

Background & Aims: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. Methods: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. Results: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdhl-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUM01-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. Conclusions: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy.


RNA isolation and gene expression
RNA isolation was performed in human and rat liver samples and cell culture using TRI Reagent (Sigma). Subsequently, 1 µg of RNA isolated from human liver samples were reverse transcribed using the SuperScript VILO cDNA  Table and Supplementary Table 2) was determined by real-time quantitative polymerase chain reaction (qPCR) using iQ SYBR Green Supermix (Bio-Rad) in a CFX96 Touch Real-Time PCR Detection System as previously described. [1]

Animals and treatment
The well-defined PCK rat (commercially available by Charles River Laboratory) carries a spontaneous splicing mutation (IVS35-2AT) in the polycystic kidney and hepatic disease 1 (PKHD1) orthologous gene, responsible for an ARPKD/congenital hepatic fibrosis phenotype. [2] The PCK rat represents an accurate and useful animal model of slowly progressive PLD phenotype. [3] The liver phenotype in PCK rats is characterized by extensive cyst formation, as the majority disconnect from the bile ducts throughout time, and hepatomegaly with unaltered liver parenchyma. [4] Hepato-renal cystogenesis and fibrosis gradually develop after 8 weeks of age in both male and female PCK rats, and concurrently elevated serum levels of blood urea nitrogen, creatinine, bilirubin, cholesterol, triglycerides and alkaline phosphatase are observed. [3] Although the pathologies become evident after 25 weeks of age, there is considerable variability.
In this study, PCK rats (8 weeks of age) were grouped in: 1) non-treated (n=14) and 2) treated (n=12) with S-adenosyl-l-methionine disulfate ptoluenesulfonate (SAMe, kindly provided by Gnosis S.p.A., Derio, Italy). Every group had 50% male and 50% female PCK rats, as published studies demonstrated no significant differences between both sexes (Supplementary Figure 1). [5][6][7][8] SAMe dosage was set at 20 mg/kg/day, as published clinical trials in liver diseases showed no severe adverse events. [9,10] SAMe dissolved easily in drinking water of the animals, thus based on daily weight monitoring, SAMe was administered by oral gavage with an orogastric canula for 5 months (on a 5day-on/2-day-off schedule). As negative control of the disease, eight-weeks old wild-type (Sprague-Dawley, Charles River Laboratory) rats (n=8) were maintained in parallel. During the entire study, all animals were weighed weekly, and had ad libitum access to food (standard diet) and water.
At the beginning, and after 6 weeks, 12 weeks and at the sacrifice, blood samples were collected from every animal. Levels of serum biochemical markers such as alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, blood urea and total protein were measured.
For every blood collection, the animals were anesthetized with isoflurane (2.5% in inhaling oxygen, at a flow of 0.3 L/min). Non-treated and treated rats were sacrificed after 5 months, and blood, bile, liver, kidneys and spleen were collected. The harvested tissues were stained with hematoxylin/eosin or picrosirius red, for measuring hepatorenal cysts and liver fibrosis, respectively.
Hepatorenal cystogenesis and liver fibrosis were quantitated using ImageJ software (National Institutes of Health, USA). [11] All animal experimental procedures were approved by the Animal

Mass spectrometry and proteomic analysis
Samples of control shRNA and shRNA against UBE2I, as well as SUMO1immunoprecipitation (IP) lysates, were extracted or eluted using 7 M urea, 2 M thiourea, 4% CHAPS. Samples were incubated for 30 min at room temperature under agitation and digested following the Filter-Aided Sample Preparation protocol [14] with minor modifications. Trypsin was added to a trypsin:protein ratio of 1:10, and the mixture was incubated overnight at 37 o C, dried out in a RVC2 25 speedvac concentrator (Christ), and resuspended in 0.1% formic acid (FA).
Samples were analyzed in a novel hybrid trapped ion mobility spectrometry -quadrupole time of flight mass spectrometer parallel accumulation serial fragmentation (tims TOF Pro with PASEF, Bruker Daltonics) coupled online to a nanoElute liquid chromatograph (Bruker). This mass spectrometer takes advantage of a novel scan mode termed parallel accumulation -serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity [15] and has been proven to provide outstanding analytical speed and sensibility for proteomics analyses. [