Osteosarcoma (OS) is the malignant tumor of bone that is most common in children and adolescents. OS is characterized by a high propensity for metastasis, especially to lungs, which is the main cause of death. The membrane-cytoskeleton cross-linker ezrin plays a key role in the metastatic progression of OS. In our earlier studies, we discovered that a small molecule, NSC305787, directly binds to ezrin and inhibits its function related to metastatic phenotype. In order to gain a better insight into the molecular mechanisms of NSC30787 action, an affinity pull-down coupled mass spectrometry was conducted to find the putative ezrin-interacting proteins whose interaction with ezrin is decreased or lost in the presence of NSC305787. Detailed mass spectrometry analysis identified 78 proteins after filtering for the potential background contaminants. Out of 78 proteins, 9 proteins were known ezrin-binding proteins including ezrin, radixin, moesin, polyadenylate-binding protein 1 (PABP-1), Na(+)/H(+) exchange regulatory cofactor (NHERF1/EPB-50), calpains, epidermal growth factor receptor kinase substrate 8 (EPS8), protein kinase C ioto (PKC-ioto) and focal adhesion kinase (FAK). For the remaining 69 proteins, we immunoprecipitated ezrin from K7M2 OS cells, and then western blotted for 8 selected functionally relevant proteins in order to test the validity of our approach. Of these 8 candidate proteins, Co-IP experiments verified binding of ezrin to 5 novel binding partners including DDX3 RNA helicase, caprin-1, AEG-1/MTDH/LYRIC, Rho guanine nucleotide exchange factor 2 (GEFH1) and catenin delta-1 (p120-catenin). NSC305787 effectively reduced the binding of DDX3 RNA helicase, AEG-1/MTDH/LYRIC and GEFH1 to ezrin in Co-IP experiments. Direct binding of ezrin to DDX3 RNA helicase was also demonstrated by surface plasmon resonance analysis using Biacore instrument. In conclusion, our results suggest that NSC305787 can modulate ezrin function through blocking its interaction with crucial proteins involved in adhesion, migration, invasion and survival. We also identified novel ezrin interactors that are involved in different levels of mRNA metabolism suggesting a completely new molecular function of ezrin that is independent from its classic role as a membrane cytoskeletal cross-linker at the plasma membrane. Thus, these results highlight novel molecular mechanisms for ezrin in cancer metastasis.

Citation Format: Haydar Celik, Jenny Han, Sung-Hyeok Hong, Gulay Bulut, Jeffrey Toretsky, Aykut Uren. NSC305787 inhibits specific protein–protein interactions involving ezrin in osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2014-3979