The importance of fusion protein activity in Ewing sarcoma and the cell intrinsic and extrinsic factors that regulate it: A review

Abstract

Accumulating evidence shows that despite clonal origins tumors eventually become complex communities comprised of phenotypically distinct cell subpopulations. This heterogeneity arises from both tumor cell intrinsic programs and signals from spatially and temporally dynamic microenvironments. While pediatric cancers usually lack the mutational burden of adult cancers, they still exhibit high levels of cellular heterogeneity that are largely mediated by epigenetic mechanisms. Ewing sarcomas are aggressive bone and soft tissue malignancies with peak incidence in adolescence and the prognosis for patients with relapsed and metastatic disease is dismal. Ewing sarcomas are driven by a single pathognomonic fusion between a FET protein and an ETS family transcription factor, the most common of which is EWS::FLI1. Despite sharing a single driver mutation, Ewing sarcoma cells demonstrate a high degree of transcriptional heterogeneity both between and within tumors. Recent studies have identified differential fusion protein activity as a key source of this heterogeneity which leads to profoundly different cellular phenotypes. Paradoxically, increased invasive and metastatic potential is associated with lower EWS::FLI1 activity. Here, we review what is currently understood about EWS::FLI1 activity, the cell autonomous and tumor microenvironmental factors that regulate it, and the downstream consequences of these activity states on tumor progression. We specifically highlight how transcription factor regulation, signaling pathway modulation, and the extracellular matrix intersect to create a complex network of tumor cell phenotypes. We propose that elucidation of the mechanisms by which these essential elements interact will enable the development of novel therapeutic approaches that are designed to target this complexity and ultimately improve patient outcomes.

Keywords: Ewing sarcoma; cell phenotype; heterogeneity; oncogene; plasticity.

Figure 1

(A) Ewing sarcoma cells exist along a spectrum of EWS::FLI1 expression and activity states. Ewing tumors consist of different populations of these cells, and epigenetic plasticity allows cells to shift along this axis within the tumorigenic window. EWS::FLI1 activity above or below a permissive window leads to growth arrest and death, consistent with the “Goldilocks” principle (19). (B) EWS::FLI1 “high” cells have increased proliferation, tumorigenic potential, and exhibit more cell-cell contact. EWS::FLI1 “low” cells have increased migration/invasion, metastatic potential, and enhanced extracellular matrix association (–25). EWS::FLI1 “high” and “low” cells have an inverse expression pattern of fusion-regulated genes. EWS::FLI1 “low” activity cells have increased expression of the repressed signature, but can have either high or low expression of the activating signature. Figure created with BioRender.com.

Figure 2

(A) EWS::FLI1 expression and activity are highly regulated through many cell-intrinsic processes. 1) The EWS::FLI1 and endogenous EWSR1 promoter are positively regulated by SP1 in complex with the histone acetyltransferase, P300. HDAC6 keeps SP1 deacetylated, allowing for continued binding and expression of the fusion (58). 2) SF3B1 is a member of the spliceosome that is critical to for proper pre-mRNA splicing of EWS::FLI1. HNRNPH1 is a RNA binding protein that facilitates the splicing of EWSR1 exon 8-containing fusions (59). 3) The RNA-binding protein LIN28B can bind to and stabilize EWS::FLI1 mRNA (60). 4) In opposition, the microRNA miRNA-145 can bind the 3’ UTR of FLI1 and cause mRNA degradation of the fusion (64). EWS::FLI1 protein is degraded by the proteasome and regulated by the competing action of deubiquitinases and E3 ubiquitin ligases. 5) USP7, USP19, and OTUD7A are three deubiquitinases that have been shown to promote EWS::FLI1 expression (–69). 6) TRIM8 and SPOP are two E3 ubiquitin ligases that have been reported to promote EWS::FLI1 degradation (19, 69). (B) (i) EWS::FLI1 activity is regulated by chromatin topologies and chromatin modifying complexes. In Ewing sarcoma cells with mutant STAG2, the EWS::FLI1 transcriptional signature is disrupted by alterations in chromatin looping (20, 21). LSD1 functions to regulate fusion activity in Ewing sarcoma cells through interaction with the fusion itself and either coactivator or repressive (NuRD) complexes resulting in either gene activation or repression (, , –72). BRD4 regulates both the fusions activating and repressive activity through indirect binding with EWS::FLI1 in a large multi-subunit transcriptional complex (73). ii. EWS::FLI1 activity is also regulated by fusion-activated TFs or context-dependent TFs. These TFs then can either augment or antagonize expression of different subsets of the EWS::FLI1 target gene signature. Figure created with BioRender.com.

Figure 3

EWS::FLI1 activity is regulated by signals in the osteolytic bone tumor microenvironment. (A) Bone is constantly remodeled and homeostasis is maintained by osteoclasts (which resorb bone through protease and proton secretion), osteoblasts (which deposit mineralized, collagenous matrix), and osteoblast-derived osteocytes which form a network within bone and modulate osteoclast and osteoblast activity (110). Soluble signals exist either embedded in the bone matrix (and released upon dissolution) or generated via paracrine secretion from bone cells. These signals couple remodeling to deposition and shifts in these signals can either balance bone remodeling, favor deposition during periods of bone growth, or favor osteolysis during regression or calcium deficiency. (B) Ewing sarcoma cells can secrete numerous signals which dysregulate this balance, including osteoblast-activating factors such as RANKL, IL6, TNF-alpha, and LOX (, –113). In turn, the dissolution of bone matrix releases embedded growth factors such Wnts, TGF-beta, PDGFs, FGFs, and IGF1 which can promote tumor growth and/or metastasis (111). (C) EWS::FLI1 “low” cells may generate a positive feedback loop with the osteolytic “vicious cycle”. Recent findings suggest that bone-derived signals such as Wnt and TGF-beta partially antagonize EWS::FLI1 activity, promoting an EWS::FLI1 “low” transcriptional state that upregulates mesenchymal-identify genes including pro-metastatic ECM molecules and angiogenesis-inducing genes (23). EWS::FLI1 antagonism also induces expression of LOX and IL-6, known promoters of osteolysis (17, 35, 114). Figure created with BioRender.com.