Understanding the molecular processes characterizing Ewing sarcoma (EWS) cell migration is crucial to highlight novel therapies for patients with disseminated disease. In this study we analyzed the role of ROCK kinases in the regulation of cell migration, growth and differentiation of EWS cells. Overexpression of ROCK promotes invasion and metastasis in many solid tumors. However, the effect of ROCK in EWS has not been extensively investigated. Expression of ROCK1 and ROCK2 was analyzed by western blotting in a representative panel of human EWS cell lines, in comparison with the parameters of in vitro malignancy. We investigated the effects of a ROCK2 specific inhibitor toward those of a pan-ROCK inhibitor on the growth, migration and differentiation of two EWS cell lines. ROCK2 but not ROCK1 expression was found to be associated with in vitro cell migration and anchorage‑independent growth capabilities. Exposure of EWS cells to ROCK inhibitors significantly reduced migration and growth, while favoring morphology changes and neural differentiation. These effects were more striking when cells were specifically deprived of ROCK2 activity. Our findings lead to consider ROCK2, rather than ROCK1, as a possible molecular target for the treatment of EWS.

Circulating tumor cells (CTCs) analysis is a promising new diagnostic field to estimate risk and monitor treatment efficacy, metastatic relapse, and progression in cancer patients. The study aim was to isolate and characterize CTCs in blood samples of Ewing sarcoma (ES) patients exploiting two main characteristics: CD99 expression and presence of chromosomal translocations.

Ewing sarcoma (EWS) is a pediatric cancer characterized by the EWSR1-FLI1 fusion. We performed a genome-wide association study of 733 EWS cases and 1346 unaffected individuals of European ancestry. Our study replicates previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1, and identifies new loci at 6p25.1, 20p11.22 and 20p11.23. Effect estimates exhibit odds ratios in excess of 1.7, which is high for cancer GWAS, and striking in light of the rarity of EWS cases in familial cancer syndromes. Expression quantitative trait locus (eQTL) analyses identify candidate genes at 6p25.1 (RREB1) and 20p11.23 (KIZ). The 20p11.22 locus is near NKX2-2, a highly overexpressed gene in EWS. Interestingly, most loci reside near GGAA repeat sequences and may disrupt binding of the EWSR1-FLI1 fusion protein. The high locus to case discovery ratio from 733 EWS cases suggests a genetic architecture in which moderate risk SNPs constitute a significant fraction of risk.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.

Ewing sarcoma (EWS), the second most common primary bone tumor in pediatric age, is known for its paucity of recurrent somatic abnormalities. Apart from the chimeric oncoprotein that derives from the fusion of EWS and FLI genes, recent genome-wide association studies have identified susceptibility variants near the EGR2 gene that regulate DNA binding of EWS-FLI. However, to induce transformation, EWS-FLI requires the presence of additional molecular events, including the expression of CD99, a cell surface molecule with critical relevance for the pathogenesis of EWS. High expression of CD99 is a common and distinctive feature of EWS cells, and it has largely been used for the differential diagnosis of the disease. The present study first links CD99 germline genetic variants to the susceptibility of EWS development and its progression. In particular, a panel of 25 single nucleotide polymorphisms has been genotyped in a case-control study. The CD99 rs311059 T variant was found to be significantly associated [P value = 0.0029; ORhet = 3.9 (95% CI 1.5-9.8) and ORhom = 5.3 (95% CI 1.2-23.7)] with EWS onset in patients less than 14 years old, while the CD99 rs312257-T was observed to be associated [P value = 0.0265; ORhet = 3.5 (95% CI 1.3-9.9)] with a reduced risk of relapse. Besides confirming the importance of CD99, our findings indicate that polymorphic variations in this gene may affect either development or progression of EWS, leading to further understanding of this cancer and development of better diagnostics/prognostics for children and adolescents with this devastating disease.

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 104 features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.

Ewing sarcoma (EWS) is an aggressive mesenchymal tumor with unmet clinical need and significant social impacts on children, adolescents, and young adults. CD99, a hallmark surface molecule of EWS, participates in crucial biological processes including cell migration, differentiation, and death. EWS cells can release CD99 through exosomes (EXOs), specialized extracellular vesicles with major cell communication roles. Here we show that, as a consequence of CD99 silencing, EWS cells deliver exosomes with oncosuppressive functions that significantly reduce tumor aggressiveness. These CD99-lacking microvesicles modulate gene expression of the EWS-recipient cells, reduce proliferation and migration, in turn inducing a more-differentiated less-malignant phenotype. The most relevant effects were detected on the activator protein-1 signaling pathway whose regulation was found to be dependent on the specific cargo loaded in vesicles after CD99 shutdown. Investigation of the miRNA content of CD99-deprived EXOs identified miR-199a-3p as a key driver able to reverse EWS malignancy in experimental models as well as in clinical specimens. All together, our data provide evidence that the abrogation of CD99 in EWS tumor cells leads to produce and release EXOs capable to transfer their antineoplastic effects into the nearby tumor cells, suggesting a novel atypical role for these microvesicles in reversion of malignancy rather than in priming the soil for progression and metastatic seeding. This conceptually innovative approach might offer a new therapeutic opportunity to treat a tumor still refractory to most treatments.

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.

Metformin, a well-known insulin-sensitizer commonly used for type 2 diabetes therapy, has recently emerged as potentially very attractive drug also in oncology. It is cheap, it is relatively safe and many reports have indicated effects in cancer prevention and therapy. These desirable features are particularly interesting for pediatric sarcomas, a group of rare tumors that have been shown to be dependent on IGF and insulin system for pathogenesis and progression. Metformin exerts anti-mitogenic activity in several cancer histotypes through several molecular mechanisms. In this paper, we analyzed its effects against osteosarcoma, Ewing sarcoma and rhabdomyosarcoma, the three most common pediatric sarcomas. Despite in vitro metformin gave remarkable antiproliferative and chemosensitizing effects both in sensitive and chemoresistant cells, its efficacy was not confirmed against Ewing sarcoma xenografts neither as single agent nor in combination with vincristine. This discrepancy between in vitro and in vivo effects may be due to hypoxia, a common feature of solid tumors. We provide evidences that in hypoxia conditions metformin was not able to activate AMPK and inhibit mTOR signaling, which likely prevents the inhibitory effects of metformin on tumor growth. Thus, although metformin may be considered a useful complement of conventional chemotherapy in normoxia, its therapeutic value in highly hypoxic tumors may be more limited. The impact of hypoxia should be considered when novel therapies are planned for pediatric sarcomas.

Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones.

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3) is an oncofetal protein that binds RNA, thereby influencing the fate of target transcripts. IGF2BP3 is synthesized de novo in cancer, where it promotes proliferation, drug resistance, and metastasis via both IGF2-dependent and IGF2-independent mechanisms. Ewing sarcoma (ES) is a rare bone and soft tissue tumor in which the IGF system plays a pivotal role. This study aimed to investigate the effect of IGF2BP3 on the regulation of the IGF system in ES. Among the components of the IGF axis, a direct significant correlation was identified between IGF2BP3 and IGF1R at mRNA and protein levels in two independent series of clinical specimens from patients with localized ES. After the formal demonstration of a direct association between IGF2BP3 and IGF1R mRNA using ribo-immunoprecipitation assay, we performed in vitro studies using A673 and TC-71 ES cell lines to demonstrate that IGF2BP3 loss promotes the downregulation of IGF1R and a decreased biological response to IGF1, represented by reduced migration and cell growth. Additionally, the compensatory activation of insulin receptor (IR) and its mitogenic ligand IGF2 is triggered in some but not all cell lines in response to IGF2BP3-mediated IGF1R loss. These findings have therapeutic implications because cells with a decreased expression of IGF2BP3/IGF1R axis but an increased expression of the IR/IGF2 loop display higher sensitivity to the dual inhibitor OSI-906 than do control cells. Therefore, studies on IGF2BP3, which was confirmed as a post-transcriptional regulator of IGF1R, provide a step forward in the identification of new mechanisms regulating the IGF system. In addition, our results demonstrate that the detection of IGF2BP3 expression should be combined with the assessment of the IGF1R/IR ratio to predict cell responses to anti-IGF1R/IR agents.