Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target

Abstract

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS -FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS -FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS -FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS -FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS -FLI1 inhibition. We showed that TOPK is a new target gene of EWS -FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

Overexpression of PBK/TOPK Contributes to Tumor Development and Poor Outcome of Esophageal Squamous Cell Carcinoma

“…The PBK/TOPK gene is up-regulated in various types of cancer and tumors such as bladder cancer, brain tumor, breast cancer, liver cancer, lung cancer and sarcoma (7)(8)(9)(23)(24)(25)(26). PBK/TOPK was initially identified as a mitotic protein kinase (27), assisting the recruitment of cylcin-dependent kinase 1 (CDK1)/cyclin B to mitotic spindles, where PBK/TOPK plays a role in the formation of the spindle midzone and cytokinesis (10).…”

CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis

“…ChIP analysis was carried out in TC71 and IOR/BRZ cells as described in Supplemental Methods. The negative control promoter primers used for ChIP were previously described (72,73).…”

PBK/TOPK interacts with the DBD domain of tumor suppressor p53 and modulates expression of transcriptional targets including p21

“…Indeed PBK/TOPK is overexpressed in breast cancer, and siRNA-mediated reduction of PBK/ TOPK expression suppresses breast cancer cell growth and clonogenicity (Park et al, 2006;Ayllo´n and O'Connor, 2007). In addition to its role in the MAPK oncogenic signaling pathway, PBK/TOPK has recently been found to be a downstream target of the EWS-FLI1 chimeric fusion protein that is present in 90% of cases of Ewing sarcoma (Herrero-Martin et al, 2009). We have previously shown that forced expression of a phosphomimetic mutant of PBK/TOPK resulted in loss of DNA damage checkpoint control leading to aneuploidy and suggested that this observation may be due to attenuation of p53 function (Nandi et al, 2007).…”