Wnt/β-catenin signaling in malignant mammary tumor progression and metastasis formation & Mechanisms of evasive resistance to sorafenib in hepatocellular carcinoma

Büchel, David Martin. Wnt/β-catenin signaling in malignant mammary tumor progression and metastasis formation & Mechanisms of evasive resistance to sorafenib in hepatocellular carcinoma. 2019, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_13234

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β-catenin exerts a dual role as the key nuclear effector of the canonical Wnt signaling pathway and as a crucial component of the E-cadherin-mediated cell-cell adhesion complex. In breast cancer, aberrations in the canonical Wnt signaling pathway have been found in correlation with poor prognosis. To provide a better understanding of β-catenin’s function in breast tumor progression and metastasis formation, we have performed functional in vivo analysis in the MMTV-PyMT mouse model. We first analyzed breast cancer progression and metastasis formation in the complete absence of β-catenin by conditional knockout. We found that the knockout of β–catenin and hence the loss of its signaling and adhesion function leads to apoptosis in vitro and in vivo. To specifically dissect its transcriptional function without affecting its role in cell adhesion, we have used mice expressing mutant forms of β-catenin which retained their function in cell adhesion but lacked either the N- or C-terminal transcriptional output or both, resulting in the abrogation of β-catenin’s transcriptional activity and thus the canonical Wnt signaling output. Introducing the N-terminal mutation D164A abrogates the interaction of β-catenin with its coactivators BCL9 and BCL9L, deletion of the C-terminus (ΔC) on the other hand prevents binding of multiple coactivators to the C-terminus. The double mutant (dm) combines both, the D164A mutation and the C-terminal truncation and thereby completely abrogates Wnt signaling. From the different mouse tumor genotypes, cell lines have been established to further characterize and examine the effects of an altered canonical Wnt signaling output. As compared to the β-catenin knockout cells, these cells were viable, suggesting a key role of β-catenin’s adhesion function in cell survival. Here, we demonstrate that selective abrogation of the N- and/or C-terminal transcriptional output of β-catenin affects tumor progression and tumor cell proliferation. Lack of the N-terminal transcriptional output only or the complete abrogation of Wnt signaling activity in the dm form of β-catenin also affects metastasis formation in vivo and epithelial-to-mesenchymal transition (EMT) in vitro. Furthermore, the different mutant forms exert a dominant-negative effect. RNA sequencing analysis to examine global changes in transcription and signaling pathways affected by the expression of the different β-catenin mutant forms upon activation of the Wnt pathway and during EMT by Wnt3a or TGFβ treatment, respectively, has been performed. With this approach we were able to identify candidate genes, which seem to be either regulated by N- and C-terminal or specifically by N- or C-terminal coactivators binding to β-catenin. Furthermore, preventing the binding of coactivators to β-catenin in the β-catenindm/- cell line resulted in the almost complete abrogation of the canonical Wnt pathway showing only minor expression changes in some known Wnt target genes as well as pathway components. Moreover, also upon TGFβ treatment we detected target genes that seem to be specifically regulated by either the N- or the C-terminal coactivators during EMT. The mutant forms of β-catenin were able to alter and partially ablate the EMT response as compared to wild-type β-catenin, however, the cell lines are still able to upregulate most of the mesenchymal markers examined so far. In summary this study provides new insight of the involvement of the canonical Wnt signaling pathway in breast tumor progression and metastasis formation and allows the identification of β-catenin target genes important for EMT and tumor progression. These data may help identifying new therapeutic targets in breast cancer tumor progression and metastasis formation.
The second project presented in this thesis aimed to examine mechanisms of evasive resistance to sorafenib in hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is the second most common cause for cancer-related death worldwide. There is need for more effective medical therapies, so far the standard treatment for patients with advanced HCC is sorafenib, a multikinase inhibitor which prolongs the time of tumor progression and improves the overall survival of the patients by two to three months. However, the use of Sorafenib is hampered by the occurrence of drug resistance leading to progression of the tumor after initial response to chemotherapy. To examine the escape of such therapies by the activation of compensatory pathways or via other mechanisms was therefore the aim of this study. We aimed to investigate the molecular mechanisms involved in evasive resistance to sorafenib in HCC. Therefore, we determined the IC50 for sorafenib of a number of HCC cell lines. This enabled us to differentiate between more sensitive cell lines and intrinsically drug resistant ones. We established sorafenib-resistant HCC cell lines out of the most susceptible ones, using two different approaches and investigated their changes in morphology, gene expression and migratory capacity. The resistant cells seemed to undergo an EMT since they became more mesenchymal as well as more migratory. Furthermore, they decreased the expression of diagnostic HCC markers and increased the expression of stem cell markers. However, those changes seemed to be epigenetic, since sorafenib withdrawal partially reverted the observed effects. Therefore, treatment with HDAC inhibitors was tested on intrinsically resistant cell lines with a mesenchymal morphology. Initial tests suggested a reversion of the mesenchymal to an epithelial morphology of those cells and a synergistic effect on cell growth of the co-treatment of pan-HDAC inhibitors together with sorafenib. To compare the differences in gene expression between the parental- and the established resistant cell lines and to able to identify pathways or genes contributing to evasive resistance to sorafenib, RNA sequencing has been performed. The results revealed changes in pathways involved in ECM organization and cell surface interactions which are also associated with EMT as well as pathways associated with epigenetic alterations and cell cycle control. Taken together, this study provides a model to examine evasive resistance and preliminary results show overlapping mechanisms between different cell lines. Sorafenib-resistant cell lines seem to undergo an EMT and become more migratory. This cell model was further used in preliminary experiments to investigate the restoration of the morphology and sensitivity to sorafenib using HDAC inhibitors.
Advisors:Christofori, Gerhard M. and Heim, Markus
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Former Units at DBM > Tumor Biology (Christofori)
05 Faculty of Science
UniBasel Contributors:Christofori, Gerhard M.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13234
Thesis status:Complete
Number of Pages:1 Online-Ressource (145 Blätter)
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edoc DOI:
Last Modified:23 Aug 2019 04:30
Deposited On:22 Aug 2019 08:17

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