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Functional in vivo analysis of cholangiocarcinoma development, progression and metastasis.

Periodic Reporting for period 4 - CholangioConcept (Functional in vivo analysis of cholangiocarcinoma development, progression and metastasis.)

Reporting period: 2020-03-01 to 2020-08-31

Primary liver cancer comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which are distinct with respect to their morphology, metastatic capacity and their response to cancer therapy. HCC mostly show a local invasive growth restricted to the liver, whereas ICC tend to metastasize early. ICC can be seen as a prototype of a therapy-resistant tumor. It represents the second most frequent primary liver cancer (PLC) and constitutes an emerging health problem with sharply increasing incidence rates. The reason for the steadily increasing ICC incidence, in particular in patients without defined classical risk factors (e.g. liver flukes, biliary stones) remains unclear, but an influence of western lifestyle and altered hepatic metabolism have been discussed. Interestingly, recent work has suggested a high degree of cellular plasticity in the hepatobiliary system and it has been suggested that intrahepatic cholangiocarcinomas can be derived from de- or transdifferentiated hepatocytes.
It is thought that the GI tract is inhabited by approximately 1000 microbial species from relatively few phyla. Besides beneficial contributions of the gut microbiome to the host, it was described to impact disease onset and progression. For example, it seems that the intestinal microbiome may be involved in the development of obesity-associated cancers. Obesity dependent alterations in intestinal flora alter bile acid metabolism to generate cancer promoters, which together with inflammatory cytokines secreted from senescent cells may increase both the incidence and the rate of progression of HCC (Yoshimoto S et al., Nature 2013). Interestingly, other studies showed that commensal bacteria are important for the modulation of cancer therapy responses (Iida N et al., Science 2013). CholangioConcept was dedicated to mechanistically dissect the initiation, progression and metastasis of intrahepatic cholangiocarcinomas (ICC). A particular focus was to elucidate mechanisms of hepatocyte-cholangiocyte transdifferentiation and the functional genetic identification of novel therapeutic targets in ICC. Furthermore, CholangioConcept aimed to characterize the role of the gut microbiome for cholangiocarcinoma development, progression and metastasis.
One key aim of CholangioConcept was a functional and mechanistic dissection of cellular plasticity and lineage commitment in the hepatobiliary system. All aims in this regard could be fully accomplished and the conducted work was published as an article in Nature (Seehawer M et al., Nature 2018). Specifically, we showed that the hepatic microenvironment epigenetically shapes lineage commitment in liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment was found to determine ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors. Our study provides fundamental insights into how lineage commitment in liver tumorigenesis is regulated and explains how common risk factors such as a western lifestyle and fatty liver disease can lead to the development of either HCC or ICC. The study was presented at 22 international scientific conferences (see table 1) and published as an article in Nature.
To study the impact of the gut microbiome on cholangiocarcinogenesis we took advantage of mice harboring different defined and stable gut microbiomes. We identified dysbiotic microbiomes, which markedly impacted ICC initiation, progression and metastasis. We found that long-term gut dysbiosis is induced by even short-term antibiotics treatment and that antibiotics-induced gut dysbiosis fueled cholangiocarcinoma growth and metastasis. To identify microbial products which might mediate the protumorigenic effects, we undertook a comprehensive omics-profiling approach. Portal vein blood and stool from eubiotic and dysbiotic mice was subjected to metabolomics and lipidomics analyses. Dysbiotic microbiomes were found to be associated with profound changes in the lipidome of portal venous blood and also with a significantly altered hepatic metabolism, e.g bile acid metabolism and glycolysis. Therapeutic interventions directed against secondary bile acids and hepatic glucose metabolism resulted in an attenuated cholangiocarcinoma development.
Work conducted within CholangioConcept was highly successful and resulted in novel and innovative data with a high translational potential. For example, our work on cell plasticity and lineage commitment in liver tumorigenesis led to the identification of a novel mechanism on how lineage commitment in liver tumorigenesis is regulated.
Our study was the first to demonstrate that the hepatic microenvironment plays a key role in determining lineage commitment in liver tumorigenesis via epigenetic regulation. Using mosaic mouse models, we demonstrated that a necroptosis-associated hepatic cytokine microenvironment switches HCC to ICC development, independently of the oncogenic drivers. Pharmacological or genetic suppression of necroptosis revert the necroptosis-dependent cytokine microenvironment and switches ICC to HCC. Epigenome and transcriptome profiling of mouse HCC and ICC identified Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, which were validated in a cohort of 199 cases of human HCC and ICC. Together, our results provides insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC. Owing to the groundbreaking character of our results, we were able to disseminate our results via a publication in Nature (Seehawer et al., Nature 2018).
Similarly, we believe that our results on the interconnection of gut dysbiosis and cholangiocarcinogenesis will have a major impact. This subproject of CholagioConcept shows for the first time that even short term antibiotic treatment exerts a long-term impact on cholangiocarcinoma development. Our results represent a major contribution to the field, as they relativize recent studies wherein sustained antibiotic treatment attenuated hepatocellular carcinoma progression in several mouse models. In contrast to these data, our study pinpoints that antibiotics can result in the formation of dysbiotic microbiomes which harbor tumor-promoting potential.
: Schematic representation of the proposed model of microenvironment and epigenetically shaped devel