Investigation of tumourigenic and metastasising CD44 properties in vitro and in vivo

• Applicant: Monika Hartmann, Ph.D.
• Subject Area: Cell Biology
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 290419794

Osteosarcoma (OS) is the most common type of primary malignant bone tumour with high prevalence in childhood and adolescence. Despite intensive efforts to improve both chemotherapeutics and surgical management, 40% of all OS patients succumb to the disease. To study the molecular events leading to OS malignant transformation, the applicant generated a spontaneous tumour mouse model by applying mutation in the tumour suppressor gene Nf2. These mice were prone to develop aggressive OSs, fibrosarcomas and hepatocellular carcinomas (HCCs). The main focus of the applicant is on the role of the cell adhesion molecule CD44. It has been shown that the NF2 gene product, Merlin needs to interact with CD44 in order to perform its tumour suppressor function. Consistently, numerous NF2 mutations in cancer patients are predicted to disturb the interaction of Merlin with CD44. In order to study the effect of Cd44 gene depletion on tumour development and progression, Nf2¬-mutant mice were crossed to Cd44-knockout mice. Importantly, while the absence of Cd44 gene had no effect on frequency of OSs, it virtually diminished metastasis formation of OSs in the Nf2-mutant mice. Moreover, in contrast to Cd44-positive Nf2+/− mice, none of the analysed Cd44-negative mice developed HCC. Because of the prevalence of OS in Nf2+/− mice, further studies of the applicant concentrated on this tumour subtype. In order to enable detailed and reproducible functional studies, tumour cells were isolated from Nf2-deficient OSs and Cd44 expression was depleted using CRISPR/Cas9. Compared to Cd44-positive OS cells, Cd44-negative cells were more susceptible to TNFα-induced cell death. Moreover, CD44 facilitated transendothelial migration of OS cells in vitro and contributed to formation of lung metastases in xenotransplantation assay in vivo. Altogether, these results of the initial DFG grant suggest that CD44 contributes to aggressive behaviour of OS cells to promote their metastatic spread. The goal of the current proposal is to study the detailed molecular mechanism underlying CD44 function in OS progression. In particular, the applicant will investigate whether the interaction of CD44 with its major ligand hyaluronic acid, its co-receptor function or its proteolytic processing are required for tumour-relevant phenotypes. In the past the applicant generated multiple constructs for overexpression of CD44 isoforms and mutants that allow to distinguish between different molecular functions of CD44. OS cells with reconstituted Cd44 expression will be subjected to in vitro functional assays as well as to xenotransplantation assays in order to study tumourigenic and metastatic potential in vivo. Transcriptome sequencing will be evaluated to model the functional networks induced by the CD44 isoform/mutant. The results will further be applied to authentic human patient’s samples and functionally validated in human OS cells to confirm the translational significance of the study.

• DFG Programme: Research Grants