Mahroo Rastegari

PhD Student

Verena Keitel-Anselmino

Project Leader

Biliary tract cancer, also known as cholangiocarcinoma (CCA9, accounts for about 2% of all malignancies, but incidences of intrahepatic CCA are rising and prognosis of CCA is dismal with surgical resection or liver transplantation as only curative treatment options. Chronic inflammation of the bile ducts and surrounding portal fields are a hallmark of CCA pathogenesis. Chronic diseases of the intra- and extrahepatic bile ducts, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) predispose to CCA development. Bile acids (BAs) play a fundamental role in both liver damage and regeneration. BA effects are mediated through different receptor molecules, comprising nuclear BA receptors (BARs) and G protein-coupled receptors (GPCRs), such as the Takeda G protein-coupled receptor-5 (TGR5, GPBAR1) or the sphingosine 1-phosphate receptor-2 (S1PR2). While secondary BAs have been shown to impair epithelial barrier function in different epithelial cells through disruption of tight junction integrity, BARs such as TGR5 can protect epithelial cells at the interface with bile through increased bicarbonate secretion and phosphorylation and stabilization of tight junction and adhesional molecules.
TGR5 is expressed in biliary epithelial cells (BECs, cholangiocytes) throughout the biliary tree including the extrahepatic duct as well as the gallbladder both in humans and rodents. TGR5 is localized both in the apical membrane and in the primary cilium which represent the interface of BECs with bile. Previously, we identified an essential role of TGR5 and TGR5-dependent signaling for BA-induced BEC proliferation. Besides proliferation, TGR5 protects BECs from BA toxicity and preserves integrity of the epithelial barrier. The mechanisms contributing to this protective effect comprise anti-apoptotic signaling, strengthening of tight junction integrity and increased chloride and bicarbonate secretion. The latter contributes to the formation of the so-called „bicarbonate umbrella”, which shields the biliary epithelial barrier from the detergent activity of biliary BAs. Absence of TGR5 in mice was associated with a maladaptation to injury resulting in aggravated biliary injury, reduced compensatory biliary proliferation, increased portal pressure, rapid development of liver fibrosis. In line with these findings, we observed a disease and cell-type specific downregulation of TGR5 in BECs from patients with PSC, a rare cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of the intra- and extrahepatic bile ducts. Mice lacking the hepatocyte phospholipid floppase Abcb4 recapitulate the progressive fibrosing cholangitis aspect of human PSC and also develop intrahepatic malignancy. We recently described that reduction of TGR5 in BECs of Abcb4^-/- mice triggers development of the inflammatory, reactive BEC phenotype typical for sclerosing cholangitis as demonstrated by single-cell RNA-sequencing. In contrast, overexpression of TGR5 abolished the reactive BEC phenotype, which could also be achieved by norUDCA feeding. The latter was associated with normalization of TGR5 expression in BECs of Abcb4^-/- mice. We thus identified a hitherto unknown mode of action of norUDCA, which is currently tested in a phase III clinical trial for PSC. In addition to TGR5, BECs and immune cells also express S1PR2. While liver injury was aggravated in TGR5^-/-, knockdown of S1PR2 ameliorated liver damage. The extent to which the functions of these two BARs overlap and in which aspects their role differs in BECs is yet unclear. Moreover, the interplay of TGR5 and S1PR2 in different liver cells during cholestasis is incompletely understood. Both TGR5 and S1PR2 are druggable targets with a variety of potential applications in liver diseases. A better understanding of the regulation, function and interaction of TGR5 and S1PR2 in BECs and adjacent immune and matrix cells under physiological and biliary disease conditions is the central aim of the project. We hypothesize that both TGR5 and S1PR2 contribute to BEC barrier function and that maladaptation of the crosstalk and the mutual interdependence of these BARs in BECs and surrounding immune cells and myofibroblasts in response to injury results in biliary disease.

Mechanisms of interplay of TGR5 and S1PR2 for BEC barrier function under physiological and disease conditions. TGR5 and S1PR2 are both highly expressed in BECs and exert overlapping synergistic but also distinct function. Whether both receptors contribute to BEC barrier function is unknown. Furthermore, their role for maladaptive processes and inter-cellular communication (e.g. with myofibroblasts and immune cells) following biliary injury have not been elucidated.

Shivani Singh

PhD Student

Verena Keitel-Anselmino

Project Leader

Coming soon!

Niklas Heucke

Clinician Scientist

Verena Keitel-Anselmino

Project Leader

Cholangiocellular carcinoma (CCA) remains asymptomatic for a long time and is therefore often only diagnosed at a locally advanced or metastatic stage. The low response rates to conventional chemotherapy combined with pronounced side effects highlight the need for new therapeutic approaches in CCA. Although several risk factors have already been identified, the pathogenesis of CCA remains largely unexplained. Bile acids may play a key role in the development of CCA. Besides being involved in fat digestion and absorption, they are important signalling molecules. They exert their effects by binding to nuclear as well as membrane-bound G-protein-coupled receptors. Important receptors include the farnesoid X receptor (FXR), the Takeda G protein-coupled receptor 5 (TGR5, GPBAR1) and the sphingosine-1-phosphate receptor 2 (S1PR2). While TGR5 and S1PR2 are increasingly expressed in CCA, FXR is downregulated. It has already been shown that bile acids can induce hepatic inflammation and biliary proliferation via their specific receptors and thus indirectly contribute to the development of CCA. Interestingly, bile acid receptors can be addressed pharmacologically, making them a potential target for specific tumour therapy. In human intrahepatic CCA cells, the FXR agonist obeticholic acid, already approved for the treatment of primary biliary cholangitis, showed an antiproliferative effect on tumour cells. Nevertheless, the spatial expression pattern of the different bile acid receptors in human CCA and adjacent non-tumour tissue is unknown, especially regarding the different cell types present in the tumour such as cholangiocytes, immune cells or fibroblasts. However, this knowledge could be the cornerstone of potential new bile acid receptor-associated therapeutic strategies in CCA.

In our research project, we will investigate the expression of bile acid receptors in human tissue samples of intrahepatic CCA (iCCA) and adjacent non-tumour tissue by immunostaining. A special staining technique called multiplex immunostaining will be used. This technique allows the simultaneous staining of several antigens on a single tissue section, thus enabling not only the quantification of target structures but also the investigation of their localisation and spatial relationships. By means of specific co-staining cell types can be distinguished and thus the expression of the antigens investigated can be recorded at the cellular level. Therefore, it will be possible to shed light on the tumour microenvironment of iCCA, consisting of immune cells such as macrophages and stromal cells, the so-called cancer-associated fibroblasts (CAFs), as well as adjacent non-tumour tissue, with a particular focus on the expression of bile acid receptors. A major advantage of the multiplex immunostaining protocol is the use of formalin-fixed paraffin embedded tissue samples as starting material. A large registry of CCA patients together with histopathological material is available. Therefore, the experimental results will be correlated with clinical parameters such as response to therapy or survival.

A better understanding of the cellular expression pattern of bile acid receptors in iCCA is the central aim of this project. The knowledge gained will then be used to further study the role of bile acid receptors in carcinogenesis mechanistically using in vitro approaches (cell co-culture, biliary spheroids/organoids, iCCA mouse models). The overriding goal is always to apply this knowledge to the development of new therapeutic strategies.

Somayeh Alinaghi Arjas

PhD Student

Verena Keitel-Anselmino

Project Leader

Intrahepatic cholestasis describes defective bile formation within hepatocytes and comprises a heterogeneous group of liver diseases ranging from rare inherited chronic forms of disease to more common acquired and sometimes even transient forms. The underlying molecular mechanisms comprise defects in bile acid synthesis, nuclear signaling, vesicular trafficking, canalicular transport of bile acids and bile constituents across the epithelial barrier, tight junction-mediated impaired barrier function, and maintenance of cell polarity resulting in impaired bile secretion from hepatocytes into the canalicular lumen.
The main purpose of this project is the identification and characterization of genetic variants underlying hereditary forms of intrahepatic cholestasis, which are characterized by defective barrier function either in the hepatocyte or cholangiocyte using next generation sequencing (NGS) approaches. Therefore, the focus lies on the six best-known cholestasis-associated genes ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, MYO5B, and other recently identified genes as SEMA7A, KIF12, ABCC12, USP53, ZFYVE19, FGFR4. In patients without relevant variants in these genes, other possibly related genes may be identified to be responsible for genetically determined cholestasis. Moreover, this project represents the basis for choosing variants or patients for further analysis in silico and in vitro analysis.
Another part of the project is to determine whether poly- or oligogenic variant combinations may induce cholestatic phenotypes. Genetic variants associated with other chronic hepatopathies (in HFE, SERPINA1 or genes related to metabolic liver disease) may contribute to manifestation and progression of liver disease in patients with hereditary cholestasis. Thus, another aim is to compile genetic information of the known relevant cholestasis-related genes including other specific variants for each patient to define a mutational burden using HiChol registry data.
The achieved results will lead to an improved patient cohort characterization, to the detection of novel variants in the known as well as novel cholestasis-related genes and a better understanding of the contribution of different genes to the overall phenotype. The obtained knowledge will improve health care for this rare disease with respect to diagnosis through establishment of quick and reliable genetic testing, disease monitoring (HCC risk of specific mutation subclasses) and more directed therapeutic approaches.

Workflow for genetic analysis in hereditary intrahepatic cholestasis. Variants in genes involved in bile formation are related to hereditary intrahepatic cholestasis. Modifier variants may contribute to disease severity and progression. A) Whole exome sequencing (WES) is performed to detect potentially relevant genetic variants in the protein-coding and adjacent regions. B) TaqMan genotyping assay is used to identify risk-modifying single nucleotide polymorphisms (SNPs) that may influence disease manifestation. C) Bioinformatic analysis of WES results is carried out for alignment, annotation, and variant prioritization.