GAL3ST1 Deficiency Reduces Epithelial-Mesenchymal Transition and Tumorigenic Capacity in a Cholangiocarcinoma Cell Line.

Cholangiocarcinoma (CCA), or bile duct cancer, is the second most common liver malignancy, with an increasing incidence in Western countries. The lack of effective treatments associated with the absence of early symptoms highlights the need to search for new therapeutic targets for CCA. Sulfatides (STs), a type of sulfoglycosphingolipids, have been found in the biliary tract, with increased levels in CCA and other types of cancer. STs are involved in protein trafficking and cell adhesion as part of the lipid rafts of the plasma membrane. We aimed to study the role of STs in CCA by the genetic targeting of GAL3ST1, an enzyme involved in ST synthesis. We used the CRISPR-Cas9 system to generate GAL3ST1-deficient TFK1 cells. GAL3ST1 KO cells showed lower proliferation and clonogenic activity and reduced glycolytic activity compared to TFK1 cells. Polarized TFK1 GAL3ST1 KO cells displayed increased transepithelial resistance and reduced permeability compared to TFK1 wt cells. The loss of GAL3ST1 showed a negative effect on growth in 30 out of 34 biliary tract cancer cell lines from the DepMap database. GAL3ST1 deficiency partially restored epithelial identity and barrier function and reduced proliferative activity in CCA cells. Sulfatide synthesis may provide a novel therapeutic target for CCA.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins.

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.

The Epidermal Growth Factor Receptor Ligand Amphiregulin Protects From Cholestatic Liver Injury and Regulates Bile Acids Synthesis.

Intrahepatic accumulation of bile acids (BAs) causes hepatocellular injury. Upon liver damage, a potent protective response is mounted to restore the organ's function. Epidermal growth factor receptor (EGFR) signaling is essential for regeneration after most types of liver damage, including cholestatic injury. However, EGFR can be activated by a family of growth factors induced during liver injury and regeneration. We evaluated the role of the EGFR ligand, amphiregulin (AREG), during cholestatic liver injury and regulation of AREG expression by BAs. First, we demonstrated increased AREG levels in livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In two murine models of cholestatic liver injury, bile duct ligation (BDL) and alpha-naphthyl-isothiocyanate (ANIT) gavage, hepatic AREG expression was markedly up-regulated. Importantly, Areg-/- mice showed aggravated liver injury after BDL and ANIT administration compared to Areg+/+ mice. Recombinant AREG protected from ANIT and BDL-induced liver injury and reduced BA-triggered apoptosis in liver cells. Oral BA administration induced ileal and hepatic Areg expression, and, interestingly, cholestyramine feeding reduced postprandial Areg up-regulation in both tissues. Most interestingly, Areg-/- mice displayed high hepatic cholesterol 7 α-hydroxylase (CYP7A1) expression, reduced serum cholesterol, and high BA levels. Postprandial repression of Cyp7a1 was impaired in Areg-/- mice, and recombinant AREG down-regulated Cyp7a1 mRNA in hepatocytes. On the other hand, BAs promoted AREG gene expression and protein shedding in hepatocytes. This effect was mediated through the farnesoid X receptor (FXR), as demonstrated in Fxr-/- mice, and involved EGFR transactivation. Finally, we show that hepatic EGFR expression is indirectly induced by BA-FXR through activation of suppressor of cytokine signaling-3 (SOC3). Conclusion: AREG-EGFR signaling protects from cholestatic injury and participates in the physiological regulation of BA synthesis.

Dual Targeting of Histone Methyltransferase G9a and DNA-Methyltransferase 1 for the Treatment of Experimental Hepatocellular Carcinoma.

Epigenetic modifications such as DNA and histone methylation functionally cooperate in fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n = 268), peritumoral tissues (n = 154), and HCC cell lines (n = 32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cell growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1, and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. Conclusion: Combined targeting of G9a/DNMT1 with compounds such as CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC.

Bile acids, FGF15/19 and liver regeneration: From mechanisms to clinical applications.

The liver has an extraordinary regenerative capacity rapidly triggered upon injury or resection. This response is intrinsically adjusted in its initiation and termination, a property termed the "hepatostat". Several molecules have been involved in liver regeneration, and among them bile acids may play a central role. Intrahepatic levels of bile acids rapidly increase after resection. Through the activation of farnesoid X receptor (FXR), bile acids regulate their hepatic metabolism and also promote hepatocellular proliferation. FXR is also expressed in enterocytes, where bile acids stimulate the expression of fibroblast growth factor 15/19 (FGF15/19), which is released to the portal blood. Through the activation of FGFR4 on hepatocytes FGF15/19 regulates bile acids synthesis and finely tunes liver regeneration as part of the "hepatostat". Here we review the experimental evidences supporting the relevance of the FXR-FGF15/19-FGFR4 axis in liver regeneration and discuss potential therapeutic applications of FGF15/19 in the prevention of liver failure. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration.

OBJECTIVE: Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. DESIGN: Fgf15-/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. RESULTS: Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15-/- mice. Hepatic expression of Pparγ2 was elevated in Fgf15-/- mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. CONCLUSIONS: FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.

Fibroblast Growth Factor 15/19 in Hepatocarcinogenesis.

BACKGROUND: Advanced hepatocellular carcinoma (HCC) is a neoplastic disease with a very bad prognosis and increasing worldwide incidence. HCCs are resistant to conventional chemotherapy and the multikinase inhibitor sorafenib is the only agent that has shown some clinical efficacy. It is therefore important to identify key molecular mechanisms driving hepatocarcinogenesis for the development of more efficacious therapies. However, HCCs are heterogeneous tumors and different molecular subclasses have been characterized. This heterogeneity may underlie the poor performance of most of the targeted therapies so far tested in HCC patients. The fibroblast growth factor 15/19 (FGF15/19), FGF receptor 4 (FGFR4) and beta-Klotho (KLB) correceptor signaling system, a key regulator of bile acids (BA) synthesis and intermediary metabolism, is emerging as an important player in hepatocarcinogenesis. Key Messages: Aberrant signaling through the FGF15/19-FGFR4 pathway participates in the neoplastic behavior of HCC cells, promotes HCC development in mice and its overexpression has been characterized in a subset of HCC tumors from patients with poorer prognosis. Pharmacological interference with FGF15/19-FGFR4 signaling inhibits experimental hepatocarcinogenesis, and specific FGFR4 inhibitors are currently being tested in selected HCC patients with tumoral FGF19-FGFR4/KLB expression. CONCLUSIONS: Interference with FGF19-FGFR4 signaling represents a novel strategy in HCC therapy. Selection of candidate patients based on tumoral FGF19-FGFR4/KLB levels as biomarkers may result in increased efficacy of FGFR4-targeted drugs. Nevertheless, attention should be paid to the potential on target toxic effects of FGFR4 inhibitors due to the key role of this signaling system in BA metabolism.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Bile Processing Protocol for Improved Proteomic Analysis.

One of the critical issues to warrant the success of a proteome-wide analysis is sample preparation. Efficient protein extraction in the absence of interferent material is mandatory to achieve an ample proteome coverage by mass spectrometry. The study of biological fluids is always challenging due to their specific biochemical composition. However, there is increasing interest in their characterization as it will provide proteins that may advice disease setting, state, and progression. In particular, bile is proximal to liver and pancreas, and its study is especially attractive since it might provide valuable information for the clinical management of severe diseases afflicting these organs, which are at an urgent need of new biomarkers. Though previous efforts have been made to optimize protocols to analyze bile proteome, only partial descriptions were achieved due to its complex composition, where proteins represent less than 5% of the organic components. Here we describe a new method that significantly increases the bile proteome coverage while reducing by a factor of six the amount of sample required for the proteomic analysis.