Abstract PR013: Novel mechanisms of ezrin function in osteosarcoma metastasis

Abstract

Abstract Ezrin, a member of the ezrin/radixin/moesin protein family, functions as a cytoskeletal linker to mediate cell adhesion, motility, proliferation, and programmed death in different tissues. Ezrin plays a critical role in osteosarcoma (OS) metastasis to the lungs. Even though ezrin has no function in proliferation of primary tumor cells, OS cells lacking ezrin do not metastasize to the lungs. Even the OS cells that extravasate in lung capillaries fail to grow metastatic foci in the absence of ezrin. The protein is believed to be in an inactive (closed) conformational state in the cytoplasm. Phosphorylated ezrin acquired an active (open) conformation and relocates to the plasma membrane. The exact molecular mechanism of how ezrin in the membrane contributes to OS metastasis remains elusive. In this study, we propose an alternative mechanism for ezrin in the cytoplasm through direct interaction with mRNA and new protein partners that are involved in stress granules. We discovered the first in class ezrin inhibitor, NSC305787, though a surface plasmon resonance screening experiment and validated its biological activities. We demonstrated that NSC305787 directly binds to ezrin protein and inhibits ezrin mediated OS metastasis in three different mouse models including a transgenic mouse with spontaneous lung metastasis and primary xenografts tumors that were removed by amputation for metastasis development. We then used the small molecule inhibitor ezrin as molecular probe to discover novel interaction partners of ezrin in the cytoplasm. Affinity column capture of ezrin and NSC305787 competition followed by mass spec analysis identified a number of novel ezrin binding proteins whose interaction was disrupted by NSC305787 in OS cell lysates. At least seven of these novel interactors were RNA binding proteins (DDX3, Caprin-1, FAK, FXR1, PABP1, EWS, FUS) that were implicated in translation regulation and found in stress granules, which are membrane-less cytoplasmic granules where particular mRNA species are stored during stress. Supporting ezrin’s role in stress granule formation, we demonstrated that ezrin knockdown or inhibition by NSC305787 affects the expression levels of a set of stress response genes. In addition to interacting with RNA binding proteins, we also identified several mRNAs preferentially associated with ezrin in OS cells by RIP-Seq (Ribonuclear protein Immuno-precipitation). Expression of ezrin binding target mRNAs correlated with presence of ezrin and can be modulated by the ezrin inhibitor. Therefore, we propose that ezrin may regulate target gene expression by directly binding to mRNA and interacting with a number of stress granule proteins. Our data challenges the conventional molecular mechanism in the field that required ezrin phosphorylation and membrane localization and proposes a novel mechanism of action in the cytoplasm. Citation Format: Aykut Uren, Emre Deniz, Taryn Shaw, Jeffrey Toretsky, Luiz O. Penalva. Novel mechanisms of ezrin function in osteosarcoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR013.