Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.

Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines.

Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear. Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays. Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level. Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.

Polymorphic variants of IGF2BP3 and SENCR have an impact on predisposition and/or progression of Ewing sarcoma.

Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient's risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis.

A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response.

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 µg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.

ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis.

PURPOSE: The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. METHODS: The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. RESULTS: We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. CONCLUSIONS: Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.

Bone Turnover Marker (BTM) Changes after Denosumab in Giant Cell Tumors of Bone (GCTB): A Phase II Trial Correlative Study.

Background: Giant cell tumors of bone (GCTB) are osteolytic tumors. Denosumab, a RANK-L inhibitor, is approved for GCTB. Data on serum bone turnover marker (sBTM) changes are lacking. We present a phase II correlative study on sBTMs in GCTB patients treated with denosumab. Methods: All GCTB patients receiving denosumab within a multicentre, open-label, phase 2 study were enrolled. Serum levels of carboxyterminal-crosslinked-telopeptide of type I collagen (s-CTX), alkaline phosphatase (ALP), bone-alkaline phosphatase (bALP), parathyroid hormone (sPTH), and osteocalcin (OCN) were prospectively assessed (baseline, T0, 3 months, T1, 6 months, T2). The primary endpoint was assessment of sBTM changes after denosumab; the secondary endpoints were disease-free survival (DFS) and sBTM correlation. Results: In 54 cases, sBTMs decreased during denosumab treatment except for sPTH. With a median follow-up of 59 months, 3-year DFS was 65% (%CI 52−79), with a significantly worse outcome for patients with high (≥500 UI/mL) s-CTX at baseline, as compared to low s-CTX (<500 UI/mL) (3-year DFS for high CTX 45% (95%CI 23−67) vs. 75% (95%CI 59−91) for low s-CTX. Higher median ALP and s-CTX were found for patients with tumor size ≥ 5 cm (p = 0.0512; p = 0.0589). Conclusion: Denosumab induces ALP/OCN and s-CTX reduction. High baseline s-CTX identifies a group of patients at higher risk of progression of the disease.

miR-214-3p Is Commonly Downregulated by EWS-FLI1 and by CD99 and Its Restoration Limits Ewing Sarcoma Aggressiveness.

Ewing's sarcoma (EWS), an aggressive pediatric bone and soft-tissue sarcoma, has a very stable genome with very few genetic alterations. Unlike in most cancers, the progression of EWS appears to depend on epigenetic alterations. EWS-FLI1 and CD99, the two hallmarks of EWS, are reported to severely impact the malignancy of EWS cells, at least partly by regulating the expression of several types of non-coding RNAs. Here, we identify miR-214-3p as a common mediator of either EWS-FLI1 or CD99 by in silico analysis. MiR-214-3p expression was lower in EWS cells and in clinical samples than in bone marrow mesenchymal stem cells, and this miRNA was barely expressed in metastatic lesions. Silencing of EWS-FLI1 or CD99 restored the expression of miR-214-3p, leading to a reduced cell growth and migration. Mechanistically, miR-214-3p restoration inhibits the expression of the high-mobility group AT-hook 1 (HMGA1) protein, a validated target of miR-214-3p and a major regulator of the transcriptional machinery. The decrease in HMGA1 expression reduced the growth and the migration of EWS cells. Taken together, our results support that the miR-214-3p is constitutively repressed by both EWS-FLI1 and CD99 because it acts as an oncosuppressor limiting the dissemination of EWS cells.

Targeting CD99 Compromises the Oncogenic Effects of the Chimera EWS-FLI1 by Inducing Reexpression of Zyxin and Inhibition of GLI1 Activity.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

Integrated Molecular Characterization of Patient-Derived Models Reveals Therapeutic Strategies for Treating CIC-DUX4 Sarcoma.

Capicua-double homeobox 4 (CIC-DUX4)-rearranged sarcomas (CDS) are extremely rare, highly aggressive primary sarcomas that represent a major therapeutic challenge. Patients are treated according to Ewing sarcoma protocols, but CDS-specific therapies are strongly needed. In this study, RNA sequencing was performed on patient samples to identify a selective signature that differentiates CDS from Ewing sarcoma and other fusion-driven sarcomas. This signature was used to validate the representativeness of newly generated CDS experimental models-patient-derived xenografts (PDX) and PDX-derived cell lines-and to identify specific therapeutic vulnerabilities. Annotation analysis of differentially expressed genes and molecular gene validation highlighted an HMGA2/IGF2BP/IGF2/IGF1R/AKT/mTOR axis that characterizes CDS and renders the tumors particularly sensitive to combined treatments with trabectedin and PI3K/mTOR inhibitors. Trabectedin inhibited IGF2BP/IGF2/IGF1R activity, but dual inhibition of the PI3K and mTOR pathways was required to completely dampen downstream signaling mediators. Proof-of-principle efficacy for the combination of the dual AKT/mTOR inhibitor NVP-BEZ235 (dactolisib) with trabectedin was obtained in vitro and in vivo using CDS PDX-derived cell lines, demonstrating a strong inhibition of local tumor growth and multiorgan metastasis. Overall, the development of representative experimental models (PDXs and PDX-derived cell lines) has helped to identify the unique sensitivity of the CDS to AKT/mTOR inhibitors and trabectedin, revealing a mechanism-based therapeutic strategy to fight this lethal cancer. SIGNIFICANCE: This study identifies altered HMGA2/IGF2BP/IGF2 signaling in CIC-DUX4 sarcomas and provides proof of principle for combination therapy with trabectedin and AKT/mTOR dual inhibitors to specifically combat the disease.

Impact of ABC Transporters in Osteosarcoma and Ewing's Sarcoma: Which Are Involved in Chemoresistance and Which Are Not?

The ATP-binding cassette (ABC) transporter superfamily consists of several proteins with a wide repertoire of functions. Under physiological conditions, ABC transporters are involved in cellular trafficking of hormones, lipids, ions, xenobiotics, and several other molecules, including a broad spectrum of chemical substrates and chemotherapeutic drugs. In cancers, ABC transporters have been intensely studied over the past decades, mostly for their involvement in the multidrug resistance (MDR) phenotype. This review provides an overview of ABC transporters, both related and unrelated to MDR, which have been studied in osteosarcoma and Ewing's sarcoma. Since different backbone drugs used in first-line or rescue chemotherapy for these two rare bone sarcomas are substrates of ABC transporters, this review particularly focused on studies that have provided findings that have been either translated to clinical practice or have indicated new candidate therapeutic targets; however, findings obtained from ABC transporters that were not directly involved in drug resistance were also discussed, in order to provide a more complete overview of the biological impacts of these molecules in osteosarcoma and Ewing's sarcoma. Finally, therapeutic strategies and agents aimed to circumvent ABC-mediated chemoresistance were discussed to provide future perspectives about possible treatment improvements of these neoplasms.

The ABC subfamily A transporters: Multifaceted players with incipient potentialities in cancer.

Overexpression of ATP-binding cassette (ABC) transporters is a cause of drug resistance in a plethora of tumors. More recent evidence indicates additional contribution of these transporters to other processes, such as tumor cell dissemination and metastasis, thereby extending their possible roles in tumor progression. While the role of some ABC transporters, such as ABCB1, ABCC1 and ABCG2, in multidrug resistance is well documented, the mechanisms by which ABC transporters affect the proliferation, differentiation, migration and invasion of cancer cells are still poorly defined and are frequently controversial. This review, summarizes recent advances that highlight the role of subfamily A members in cancer. Emerging evidence highlights the potential value of ABCA members as biomarkers of risk and response in different tumors, but information is disperse and very little is known about their possible mechanisms of action. The only clear evidence is that ABCA members are involved in lipid metabolism and homeostasis. In particular, the relationship between ABCA1 and cholesterol is becoming evident in different fields of biology, including cancer. In parallel, emerging findings indicate that cholesterol, the main component of cell membranes, can influence many physiological and pathological processes, including cell migration, cancer progression and metastasis. This review aims to link the dispersed knowledge regarding the relationship of ABCA members with lipid metabolism and cancer in an effort to stimulate and guide readers to areas that the writers consider to have significant impact and relevant potentialities.

ROCK2 deprivation leads to the inhibition of tumor growth and metastatic potential in osteosarcoma cells through the modulation of YAP activity.

BACKGROUND: The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling. METHODS: The effects of ROCK2 depletion on metastasis were studied in NOD Scid gamma (NSG) mice injected with U-2OS cells in which ROCK2 expression had been stably silenced. Functional studies were performed in vitro in human U-2OS cells and in three novel cell lines derived from patient-derived xenografts (PDXs) by using standard methods to evaluate malignancy parameters and signaling transduction. The nuclear immunostaining of YAP and the evaluation of its downstream targets Cysteine Rich Angiogenic Inducer 6, Connective Tissue Growth Factor and Cyclin D1 by quantitative PCR were performed to analyze YAP activity. The effect of the expression and activity of ROCK2 and YAP on tumor progression was analyzed in 175 OS primary tumors. RESULTS: The silencing of ROCK2 markedly reduced tumor growth and completely abolished the metastatic ability of U-2OS cells. The depletion of ROCK2, either by pharmacological inhibition or silencing, induced a dose- and time-dependent reduction in the nuclear expression and transcriptional activity of YAP. The nuclear expression of YAP was observed in 80/175 (46%) tumor samples and was significantly correlated with worse patient prognosis and a higher likelihood of metastasis and death. The use of verteporfin, a molecule that specifically inhibits the TEAD-YAP association, remarkably impaired the growth and migration of OS cells in vitro. Moreover to inhibiting YAP activity, our findings indicate that verteporfin also affects the ROCK2 protein and its functions. CONCLUSIONS: We describe the functional connection between ROCK2 and YAP in the regulation of OS cell migration and metastasis formation. These data provide support for the use of verteporfin as a possible therapeutic option to prevent OS cell dissemination.

Bone sarcoma patient-derived xenografts are faithful and stable preclinical models for molecular and therapeutic investigations.

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.

Construction of Human Naïve Antibody Gene Libraries.

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naïve antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naïve library for use in isolating single-chain fragment variables against specific targets.

A Quinoline-Based DNA Methyltransferase Inhibitor as a Possible Adjuvant in Osteosarcoma Therapy.

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2-M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881-92. ©2018 AACR.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Influences Sensitivity to Anti-IGF System Agents Through the Translational Regulation of IGF1R.

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.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 is a Novel Post-Transcriptional Regulator of Ewing Sarcoma Malignancy.

Purpose: Large-scale sequencing studies have indicated that besides genomic alterations, the posttranscriptional regulation of gene expression or epigenetic mechanisms largely influences the clinical behavior of Ewing sarcoma. We investigated the significance of the RNA-binding protein IGF2BP3 in the regulation of Ewing sarcoma aggressiveness.Experimental Design: Explorative study was performed in 14 patients with localized Ewing sarcoma using RNA sequencing. Next, 128 patients with localized Ewing sarcoma were divided into two cohorts. In the training set, 29 Ewing sarcoma samples were analyzed using Affymetrix GeneChip arrays. In the validation set, 99 Ewing sarcoma samples were examined using qRT-PCR. Patient-derived cell lines and experimental models were used for functional studies.Results:Univariate and multivariate analyses indicated IGF2BP3 as a potent indicator of poor prognosis. Furthermore, ABCF1 mRNA was identified as a novel partner of IGF2BP3. Functional studies indicated IGF2BP3 as an oncogenic driver and ABCF1 mRNA as a sponge that by binding IGF2BP3, partly repressed its functions. The combined evaluation of IGF2BP3 and ABCF1 could identify different patient outcomes-high IGF2BP3 and low ABCF1 levels indicated poor survival (25%), whereas low IGF2BP3 and high ABCF1 levels indicated favorable survival (85.5%). The bromodomain and extraterminal domain inhibitor (BETi) JQ1 decreased IGF2BP3 expression, modified the expression of its validated targets and inhibited the capability of Ewing sarcoma cells to grow under anchorage-independent conditions.Conclusions: The combined assessment of IGF2BP3 and ABCF1 predicts recurrence in Ewing sarcoma patients. Thus, for patients with high expression of IGF2BP3 and poor probability of survival, the use of BETis should be clinically evaluated. Clin Cancer Res; 24(15); 3704-16. ©2018 AACR.

CD99: A Cell Surface Protein with an Oncojanus Role in Tumors.

The cell surface molecule CD99 has gained interest because of its involvement in regulating cell differentiation and adhesion/migration of immune and tumor cells. However, the molecule plays an intriguing and dual role in different cell types. In particular, it acts as a requirement for cell malignancy or as an oncosuppressor in tumors. In addition, the gene encodes for two different isoforms, which also act in opposition inside the same cell. This review highlights key studies focusing on the dual role of CD99 and its isoforms and discusses major critical issues, challenges, and strategies for overcoming those challenges. The review specifically underscores the properties that make the molecule an attractive therapeutic target and identifies new relationships and areas of study that may be exploited. The elucidation of the spatial and temporal control of the expression of CD99 in normal and tumor cells is required to obtain a full appreciation of this molecule and its signaling.