Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin

Abstract

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin‐CD44 complex through loss of Merlin, unleashes putative tumor‐ or metastasis‐promoting functions of CD44. To evaluate the relevance of the Merlin‐CD44 interaction in vivo, we compared tumor growth and progression in Cd44‐positive and Cd44‐negative Nf2‐mutant mice. Heterozygous Nf2‐mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2‐mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4β1 (very late antigen‐4, VLA‐4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA‐4 β1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis‐promoting role in the absence of Merlin.

CD44 Contributes to the Regulation of MDR1 Protein and Doxorubicin Chemoresistance in Osteosarcoma

“…We also showed that the shortest isoforms of CD44, so called CD44s, which does not contain any sequences encoded by alternatively spliced variant exons, was sufficient to confer resistance to doxorubicin in osteosarcoma cells ( Figure 2 ). Nevertheless, as we have shown previously, the Nf2 -deficient osteosarcomas express additional CD44 splice variants [ 38 ]. Thus, we cannot exclude that splice variants of CD44 might regulate additional processes involved in osteosarcoma chemoresistance.…”

“…In order to study the relevance of CD44 in mediating chemoresistance, we previously isolated osteosarcoma cells from mice that developed endogenous osteosarcoma, due to a mutation of the tumor suppressor gene Nf2 [ 38 ]. To enable the analysis of CD44-dependent processes in genetically comparable tumor backgrounds, Cd44 was deleted in Nf2 -deficient osteosarcoma cells, using the CRISPR/Cas9 approach [ 38 ]. Our results show that Cd44 -positive cells were more resistant to doxorubicin treatment than the Cd44 -negative cells (IC50 of 1.224 versus 0.7523 µM, respectively, p = 0.000793) ( Figure 1 A).…”

“…Altogether, our results suggest that CD44 contributes to doxorubicin chemoresistance in osteosarcoma cells by regulating MDR1 expression and function. To test if CD44 regulates additional processes that may be involved in chemoresistance regulation, RNA sequencing was performed on Cd44 -positive and Cd44 -negative primary osteosarcoma tissues isolated from Nf2 -mutant mice described previously [ 38 ]. Three samples of each group were analyzed.…”

“…NF2 downregulation could be due to either mutations or transcriptional inactivation [ 40 ]. Absence of the Cd44 gene prevents osteosarcoma metastasis in mice with a mutation of the tumor suppressor gene Tp53 [ 42 ] and, as we have shown recently, Nf2 [ 38 ]. In the current study, we applied osteosarcoma tissues and cells isolated from mice with a mutation of the Nf2 gene, to evaluate the function of CD44 in the resistance to chemotherapy.…”

“…The interaction partner of CD44, Merlin, was also implicated in osteosarcoma. Deletion of the Nf2 gene, encoding Merlin, leads to the generation of highly metastatic osteosarcoma tumors in mice [ 38 ]. The role of NF2 in the pathogenesis of human osteosarcoma is less well described.…”