EWS/FLI1 suppresses retinoblastoma protein function and senescence in Ewing's sarcoma cells

Abstract

Ewing's Family Tumors (EFTs) most commonly harbor a specific t(11;22) translocation that generates the EWS/FLI1 fusion protein responsible for malignant transformation. Many potential downstream targets of EWS/FLI1 have been identified but a detailed mechanism by which the fusion protein brings about transformation remains unknown. In this report, we show that depletion of EWS/FLI1 in Ewing's cell lines results in a senescence phenotype, a marked increase in expression of the G1/S regulatory proteins p27 kip1 and p57 kip2 , and a significant decrease in cyclin D1 and CDK2. We also demonstrate for the first time, to our knowledge, that knockdown of EWS/FLI1 leads to hypophosphorylation and functional activation of the retinoblastoma (pRb) family of proteins. Consistent with activation of the pRb proteins, E2F-responsive genes such as cyclin A are repressed in EWS/FLI1-depleted cells. In over 85% of Ewing's Family Tumors (EFTs), a specific t(11;22) chromosomal translocation results in a chimeric oncogenic product fusing the N-terminal domain of the RNA-binding protein EWS with the C-terminal DNAbinding domain of the ETS family transcription factor FLI1. 1 EWS/FLI1 and other related fusion proteins have been shown to be critical for transformation in vivo and in vitro. 2,3 A number of downstream biological pathways have been proposed for EWS/FLI1 but a detailed mechanism of transformation remains elusive. [4][5][6][7] Most of these downstream targets have been identified by ectopic expression of EWS/FLI1 in nonEwing's cells. However, recent studies demonstrate that the genetic constitution of these cells may not be a valid representation of Ewing's cells. 8 To circumvent this problem, we and others have studied the effects of transient knockdown of EWS/FLI1 by synthetic siRNA in actual Ewing's cell lines. [9][10][11] We found that knockdown of EWS/FLI1 in Ewing's cells leads to growth arrest and reduced invasiveness. More recently, it was shown that EWS/FLI1 accelerates p27 kip1 protein degradation and appears to inhibit cellular senescence. 12 These findings demonstrate that EWS/FLI1 is essential for tumor growth and its downregulation triggers signaling pathways leading to terminal growth arrest.Tumorigenesis involves accumulation of genetic alterations to promote cellular proliferation and dedifferentiation. Emerging evidence suggests that these alterations also interfere with the cellular senescence in response to oncogene activation, DNA damage, and oxidative stress. 13,14 Senescence limits the proliferative capacity of cells and has been shown to be a barrier to cell immortalization and development of tumors. 15 Bypassing senescence is therefore a critical step in tumorigenesis. For instance, full transformation by oncogenic Ras requires additional genetic mutations that bypass Ras-induced senescence. These mutations include inactivation of tumor-suppressor genes such as pRb and p53. 16,17 Similar to EWS/FLI1, evasion of senescence has also been implicated in transformation by the oncogenic fusion p...

Epidermal Growth Factor Receptor Regulation of Ewing Sarcoma Cell Function

“…Previous evidence has indicated that direct depletion of EWS/FLI1 in ES cell lines induces a senescence phenotype [38]. Knockdown of EWS/FLI1 was also coupled to decreases in cyclin D1 and promotion of p53 expression [38, 39] which were observed after EGFR inhibition in our experiments. Thus, activities displayed by an EGFR inhibitor are consistent with other experimental agents that induce antiproliferative effects in ES associated with stimulation of p53 expression [40-42].…”

“…Also, the EGFR inhibitor increased the fraction of cells in senescence, i.e., in a stable state of cell cycle arrest [37]. Previous evidence has indicated that direct depletion of EWS/FLI1 in ES cell lines induces a senescence phenotype [38]. Knockdown of EWS/FLI1 was also coupled to decreases in cyclin D1 and promotion of p53 expression [38, 39] which were observed after EGFR inhibition in our experiments.…”

Novel peptide binds EWS-FLI1 and reduces the oncogenic potential in Ewing tumors

“…26 Also, reduction of EWS-FLI1 protein levels can lead to G 1 arrest by increasing the cyclin-dependent kinase inhibitors (p27 Kip1 and p57 Kip2 ) expression and increasing pRB protein. 27 EWS-FLI1 affects the transcription of a large number of proteins that function in the regulation of the cell cycle, 28 supporting our findings that ESAP1 interrupts cell cycle kinetics at important downstream pathways from EWS-FLI1.…”

RNA-binding proteins as molecular links between cancer and neurodegeneration

“…EWSR1-null spermatocytes are deficient in XY bivalent formation and have reduced meiotic recombination, resulting in massive apoptosis and complete arrest in gamete maturation. Notably, the homozygous EWSR1-null mice also show premature cellular senescence in embryonic fibroblasts (Li et al 2007) and hematopoietic stem cells (Cho et al 2011), an effect that seems to be mediated by hypophosphorylation and functional activation of the retinoblastoma (pRb) family of proteins (Hu et al 2008a).…”