The role of the receptor tyrosine kinase-like orphan receptors (RORs) in human breast cancer
von Saskia Heinrichs
Datum der mündl. Prüfung:2022-01-21
Betreuer:Prof. Dr. Annalen Bleckmann
Gutachter:Prof. Dr. Annalen Bleckmann
Gutachter:PD Dr. Laura Zelarayan-Behrend
EnglischBreast and colorectal cancers are among the most common tumor types worldwide and the occurrence of metastases is often associated with a shortened lifespan. One of the signaling pathways that has been frequently associated with metastasis in these tumor entities is the Wnt signaling pathway. Wnt signaling can be either β-catenin dependent (canonical) or βcatenin independent (non-canonical). In breast and colorectal cancer, tumor-promoting properties could be attributed to members of the non-canonical Wnt signaling pathway. Preliminary results showed that overexpression of ROR2 as a non-canonical Wnt receptor, mediated an aggressive phenotype in breast cancer cells and could significantly increase invasion. Accordingly, the first aim of this work was to investigate which ligand binds ROR2 and thus triggers the invasive behavior of MCF-7 cells. RNA-seq analysis revealed increased expression levels of the non-canonical ligand Wnt11 in ROR2 overexpressing cells. Hence, Wnt11 was further investigated as a potential ROR2-ligand. Using co-immunoprecipitation experiments, this work demonstrated the interaction of ROR2 with Wnt11 in human breast cancer cells. To determine which domain facilitates the ROR2-mediated invasion, sequential deletions of the different ROR2 domains were induced. This demonstrated that the cysteine-rich and the tyrosine kinase domain mediate this effect. The next step was to determine whether Wnt11 binds other receptors that trigger the invasive behavior of MCF-7 cells since ROR2 is not expressed in these cells. Furthermore, a cell line screening of different breast and colorectal cancer cell lines identified FZD4 and FZD6 as highly expressed non-canonical Wnt receptors. An interaction between Wnt11 and FZD6 was validated by using Co-IP, in which PTK7 appears to act as a co-receptor. To investigate functional implications of Wnt11-mediated signaling in breast cancer, MCF-7 cells were stimulated with recombinant Wnt11, which caused increased invasion and migration rates, whereas loss of FZD6 resulted in a significant decrease of the elevated invasion rates. In line with the collected data, a signature with non-canonical Wnt pathway members including FZD receptors, ROR receptors, Wnt ligands and PI3K signaling members was defined and was analyzed for its DMFS prognostic values in breast and colorectal cancer patients. Therefore, the signature was applied to gene expression data of primary breast and colorectal cancer patients. For the primary breast cancer patients, the signature clustered the data set into two patient groups, among which the group with high Wnt11 expression was associated with poor DMFS. Considering FZD4 and FZD6 individually, breast cancer patients with high FZD6 gene expression showed worse DMFS, while high FZD4 levels were associated with favorable DMFS. Interestingly, the defined signature clustered the data set of the colorectal cancer patients into four groups and the cohort with a high FZD6 expression exhibited a poor DMFS compared to the other groups. Another important aspect of tumor progression is the reprogramming of the tumor microenvironment. It has been shown that tumor-derived extracellular vesicles can influence the tumor microenvironment in different ways. Preliminary data demonstrated that Wnt proteins can be transported via extracellular vesicles to target cells and induce Wnt signaling responses there. It was shown that RORs are transported on microvesicles and exosomes. Modulation of ROR1 and ROR2 expression resulted in altered protein compositions for both vesicle populations. However, no major impact on vesicle size or concentration was evident. Further analysis addressed functional consequences of ROR1 and ROR2 expression on extracellular vesicles. Tumor-derived EVs isolated from the aggressive breast cancer cell line MDA-MB231 induced invasiveness in MCF-7 cells, which was shown to be dependent on vesicular ROR1 expression. To determine whether ROR1/2 can function as a biomarker in breast cancer patients, plasma-derived microvesicles were analyzed for their ROR1 and ROR2 expression by flow cytometry, which is currently ongoing. In conclusion, this work demonstrated the importance of non-canonical Wnt signaling in breast cancer progression. Wnt11 has been identified as a novel ligand for ROR2 and FZD6 by co-immunoprecipitation experiments, thereby mediating tumor-promoting properties in breast cancer. In particular, it has been shown that ROR proteins not only play an important role in breast cancer cells themselves, but also appear to be additionally involved in vesicle biogenesis and can furthermore be transferred to target cells. A clinical applicability of both ROR proteins regarding their usage as tumor biomarkers for breast cancer is still under investigation.
Keywords: Breast cancer; Metastasis; Wnt signaling