Focal adhesion kinase confers pro‐migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma

Abstract

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1-ETS fusion oncogenes, most often EWSR1-FLI1 (E/ F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F-dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate-independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho-dependent cell migration, and impaired caspase-3-mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) enhanced caspase-mediated apoptosis and EwS cell migration, independent from the respective EWSR1-ETS fusion type, mimicking an anoikis-like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.Abbreviations CAM, chorioallantoic membrane; E/F, EWSR1-FLI1 fusion oncogene; EwS, Ewing sarcoma; FACS, fluorescence-activated cell sorting; FAK, focal adhesion kinase; TMA, tissue microarray; Y15, FAK inhibitor 15 (1,2,4,5-benzenetetraamine tetrahydrochloride.

Ewing Sarcoma—Diagnosis, Treatment, Clinical Challenges and Future Perspectives

“…One study focused on the contribution of focal adhesion kinase to EwS cell migration via its effects on focal adhesion formation and Rho-dependent cell migration. These investigators employed an avian chorioallantoic membrane model for in vivo testing of the effect of FAK1 inhibitors on EwS cell growth [ 385 ]. Another approach is the use of zebrafish embryos and larvae as a host for human EwS cell xenografts.…”

Sarcoma treatment in the era of molecular medicine

“…In vivo , the chick chorioallantoic membrane (CAM) assay is a valuable option due to its low costs and relatively easy implementation. CAM assays have been employed to study sarcoma angiogenesis, fibroblast infiltration, tumorigenesis, tumor invasion, and metastasis in CHS, EwS, fibrosarcoma, LPS, and OS (Sys et al , 2013; Patil et al , 2014; Manjunathan & Ragunathan, 2015; Cimpean et al , 2018; Kunz et al , 2019; Perut et al , 2019; Steinestel et al , 2020). Numerous additional in vivo models of inducible or spontaneous sarcomas have been described in non‐mammalian vertebrates (e.g., zebrafish; Leacock et al , 2012; Mohseny et al , 2012; Brown et al , 2017b; Hayes & Langenau, 2017; Ignatius et al , 2018; Fleming et al , 2019) and in mammalians (e.g., mouse, rat, and dog; Cannon, 2015; Jacques et al , 2018; Castillo‐Tandazo et al , 2019; Pomella & Rota, 2020).…”

Focal adhesion kinase inhibitors, a heavy punch to cancer

“…Y15 did not target homologous Pyk-2, c-Src, c-RAF, EGFR, IGFR, PDGFR, PI3K, VEGFR-3, and c-Met [ 76 ]. From in vitro studies, Y15 significantly inhibited cancer cell viability in six cancer cell lines, including breast cancer, thyroid cancer, colon cancer [ 77 ] glioblastoma tumor [ 78 ], Lung cancer [ 79 ] and Ewing’s sarcoma [ 80 ]. Further evidence indicated that Y15 promoted the pancreatic cancer cells apoptosis and inhibited the cell adhesion in a dose-dependent manner [ 81 ].…”