Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.
Purpose: Ewing tumor cell survival and proliferation depends on several autocrine loops. Targeting these loops is a promising therapeutic approach. We recently showed the cytostatic role of imatinib, an inhibitor of the SCF-KIT loop, on Ewing tumor cells, and in this study, we intend to analyze the inhibition of the insulin-like growth factor I receptor (IGF1R) loop. Experimental Design: We analyzed IGF1R blockade by ADW742, a small molecule specific for this receptor, alone and in combination with imatinib, vincristine, and doxorubicin on Ewing tumor cell lines. We studied the effect on proliferation, apoptosis, cell cycle, pathway phosphorylation, soft-agar growth, motility, and vascular endothelial growth factor expression levels. Results: Treatment with ADW742 induced down-regulation of IGF1R/AKT/mammalian target of rapamycin (mTOR) phosphorylation, which was deeper in cell lines having higher IGF1R activation levels. Treatment also induced dose-dependent inhibition of cell proliferation (IC 50 = 0.55-1.4 Amol/L), inducing a G 1 phase blockage and apoptosis. Addition of imatinib to ADW742 synergistically augmented these effects and was especially effective in inhibiting AKT/mTOR phosphorylation and reducing vascular endothelial growth factor expression in cell lines having high IGF1R activation levels. Combination with usual chemotherapeutic agents vincristine and doxorubicin showed synergistic interactions. Conclusions: Inhibition of Ewing tumor cell proliferation byADW742 is mediated through blockade of IGF1R signaling. Combination of ADW742 with imatinib, vincristine, and doxorubicin induces a significant reduction of tumor cell growth, mainly by the increase in apoptosis with a pattern depending on IGF1R activation levels.This study supports a potential role forADW742 in the treatment of Ewing tumor and AKT/mTOR as a possible surrogate marker of response to therapy.
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.
Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES. We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n ¼ 37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.
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