Extracellular Interactors of the IGF System: Impact on Cancer Hallmarks and Therapeutic Approaches.

Dysregulation of the insulin-like growth factor (IGF) system determines the onset of various pathological conditions, including cancer. Accordingly, therapeutic strategies have been developed to block this system in tumor cells, but the results of clinical trials have been disappointing. After decades of research in the field, it is safe to say that one of the major reasons underlying the poor efficacy of anti-IGF-targeting agents is derived from an underestimation of the molecular complexity of this axis. Genetic, transcriptional, post-transcriptional and functional interactors interfere with the activity of canonical components of this axis, supporting the need for combinatorial approaches to effectively block this system. In addition, cancer cells interface with a multiplicity of factors from the extracellular compartment, which strongly affect cell destiny. In this review, we will cover novel extracellular mechanisms contributing to IGF system dysregulation and the implications of such dangerous liaisons for cancer hallmarks and responses to known and new anti-IGF drugs. A deeper understanding of both the intracellular and extracellular microenvironments might provide new impetus to better decipher the complexity of the IGF axis in cancer and provide new clues for designing novel therapeutic approaches.

CD99 Modulates the Proteomic Landscape of Ewing Sarcoma Cells and Related Extracellular Vesicles.

Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.

PROTAC-Based Protein Degradation as a Promising Strategy for Targeted Therapy in Sarcomas.

Sarcomas are heterogeneous bone and soft tissue cancers representing the second most common tumor type in children and adolescents. Histology and genetic profiling discovered more than 100 subtypes, which are characterized by peculiar molecular vulnerabilities. However, limited therapeutic options exist beyond standard therapy and clinical benefits from targeted therapies were observed only in a minority of patients with sarcomas. The rarity of these tumors, paucity of actionable mutations, and limitations in the chemical composition of current targeted therapies hindered the use of these approaches in sarcomas. Targeted protein degradation (TPD) is an innovative pharmacological modality to directly alter protein abundance with promising clinical potential in cancer, even for undruggable proteins. TPD is based on the use of small molecules called degraders or proteolysis-targeting chimeras (PROTACs), which trigger ubiquitin-dependent degradation of protein of interest. In this review, we will discuss major features of PROTAC and PROTAC-derived genetic systems for target validation and cancer treatment and focus on the potential of these approaches to overcome major issues connected to targeted therapies in sarcomas, including drug resistance, target specificity, and undruggable targets. A deeper understanding of these strategies might provide new fuel to drive molecular and personalized medicine to sarcomas.

Extracellular vesicle-associated IGF2BP3 tunes Ewing sarcoma cell migration and affects PI3K/Akt pathway in neighboring cells.

Ewing sarcoma (EWS) is a challenging pediatric cancer characterized by vast intra-tumor heterogeneity. We evaluated the RNA-binding protein IGF2BP3, whose high expression correlates with a poor prognosis and an elevated tendency of metastases, as a possible soluble mediator of inter-cellular communication in EWS. Our data demonstrate that (i) IGF2BP3 is detected in cell supernatants, and it is released inside extracellular vesicles (EVs); (ii) EVs from IGF2BP3-positive or IGF2BP3-negative EWS cells reciprocally affect cell migration but not the proliferation of EWS recipient cells; (iii) EVs derived from IGF2BP3-silenced cells have a distinct miRNA cargo profile and inhibit the PI3K/Akt pathway in recipient cells; (iv) the 11 common differentially expressed miRNAs associated with IGF2BP3-positive and IGF2BP3-negative EVs correctly group IGF2BP3-positive and IGF2BP3-negative clinical tissue specimens. Overall, our data suggest that IGF2BP3 can participate in the modulation of phenotypic heterogeneity.

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.

CIC-Rearranged Sarcomas: An Intriguing Entity That May Lead the Way to the Comprehension of More Common Cancers.

Capicua transcriptional repressor (CIC)-rearranged sarcoma, belonging to the undifferentiated round cells sarcoma family, is characterized by high metastatic rate and poor chemo response. CIC sarcoma represents a new entity harboring the recurrent chromosomal translocation between CIC and, in most of the cases, DUX4. CIC-DUX4 imposes a CIC-specific transcriptional signature, which drives cell transformation, proliferation, and migration. While the discovery of the fusion represented the first evidence of a role of CIC in cancer, a complete comprehension of CIC-rearranged activity is still required before providing new potential avenues for therapy. To date, a specific and effective treatment for CIC sarcoma has yet to be defined. In this review, we initially highlight the clinical features and pathogenesis of CIC-rearranged sarcomas along with current therapeutic approaches and then focus on the specific oncogenic mechanisms driven by the CIC-rearrangement. We discuss novel therapeutic options evoked by the aberrant relations of CIC-DUX4 with the IGF system, DUSP6, P300/CBP, and CCNE1. We also discuss how different mutations involving CIC might converge on a common upregulation of CIC-target genes across human cancers. A deeper understanding of the oncogenic mechanisms driven by the chimera CIC-DUX4 might provide novel therapeutic opportunities with a general impact in cancer.

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.

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.

Unraveling the IGF System Interactome in Sarcomas Exploits Novel Therapeutic Options.

Aberrant bioactivity of the insulin-like growth factor (IGF) system results in the development and progression of several pathologic conditions including cancer. Preclinical studies have shown promising anti-cancer therapeutic potentials for anti-IGF targeted therapies. However, a clear but limited clinical benefit was observed only in a minority of patients with sarcomas. The molecular complexity of the IGF system, which comprises multiple regulators and interactions with other cancer-related pathways, poses a major limitation in the use of anti-IGF agents and supports the need of combinatorial therapeutic strategies to better tackle this axis. In this review, we will initially highlight multiple mechanisms underlying IGF dysregulation in cancer and then focus on the impact of the IGF system and its complexity in sarcoma development and progression as well as response to anti-IGF therapies. We will also discuss the role of Ephrin receptors, Hippo pathway, BET proteins and CXCR4 signaling, as mediators of sarcoma malignancy and relevant interactors with the IGF system in tumor cells. A deeper understanding of these molecular interactions might provide the rationale for novel and more effective therapeutic combinations to treat sarcomas.

Novel Regulators of the IGF System in Cancer.

The insulin-like growth factor (IGF) system is a dynamic network of proteins, which includes cognate ligands, membrane receptors, ligand binding proteins and functional downstream effectors. It plays a critical role in regulating several important physiological processes including cell growth, metabolism and differentiation. Importantly, alterations in expression levels or activation of components of the IGF network are implicated in many pathological conditions including diabetes, obesity and cancer initiation and progression. In this review we will initially cover some general aspects of IGF action and regulation in cancer and then focus in particular on the role of transcriptional regulators and novel interacting proteins, which functionally contribute in fine tuning IGF1R signaling in several cancer models. A deeper understanding of the biological relevance of this network of IGF1R modulators might provide novel therapeutic opportunities to block this system in neoplasia.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Modulates Aggressiveness of Ewing Sarcoma by Regulating the CD164-CXCR4 Axis.

Ewing sarcoma (EWS) is the second most common bone and soft tissue-associated malignancy in children and young adults. It is driven by the fusion oncogene EWS/FLI1 and characterized by rapid growth and early metastasis. We have previously discovered that the mRNA binding protein IGF2BP3 constitutes an important biomarker for EWS as high expression of IGF2BP3 in primary tumors predicts poor prognosis of EWS patients. We additionally demonstrated that IGF2BP3 enhances anchorage-independent growth and migration of EWS cells suggesting that IGF2BP3 might work as molecular driver and predictor of EWS progression. The aim of this study was to further define the role of IGF2BP3 in EWS progression. We demonstrated that high IGF2BP3 mRNA expression levels correlated with EWS metastasis and disease progression in well-characterized EWS tumor specimens. EWS tumors with high IGF2BP3 levels were characterized by a specific gene signature enriched in chemokine-mediated signaling pathways. We also discovered that IGF2BP3 regulated the expression of CXCR4 through CD164. Significantly, CD164 and CXCR4 colocalized at the plasma membrane of EWS cells upon CXCL12 stimulation. We further demonstrated that IGF2BP3, CD164, and CXCR4 expression levels correlated in clinical samples and the IGF2BP3/CD164/CXCR4 signaling pathway promoted motility of EWS cells in response to CXCL12 and under hypoxia conditions. The data presented identified CD164 and CXCR4 as novel IGF2BP3 downstream functional effectors indicating that the IGF2BP3/CD164/CXCR4 oncogenic axis may work as critical modulator of EWS aggressiveness. In addition, IGF2BP3, CD164, and CXCR4 expression levels may constitute a novel biomarker panel predictive of EWS progression.

IGF2BP3 From Physiology to Cancer: Novel Discoveries, Unsolved Issues, and Future Perspectives.

RNA network control is a key aspect of proper cellular homeostasis. In this context, RNA-binding proteins (RBPs) play a major role as regulators of the RNA life cycle due to their capability to bind to RNA sequences and precisely direct nuclear export, translation/degradation rates, and the intracellular localization of their target transcripts. Alterations in RBP expression or functions result in aberrant RNA translation and may drive the emergence and progression of several pathological conditions, including cancer. Among the RBPs, insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is of particular interest in tumorigenesis and tumor progression. This review highlights the molecular mechanisms underlying the oncogenic functions of IGF2BP3, summarizes the therapeutic potential related to its inhibition and notes the fundamental issues that remain unanswered. To fully exploit IGF2BP3 for tumor diagnosis and therapy, it is crucial to dissect the mechanisms governing IGF2BP3 re-expression and to elucidate the complex interactions between IGF2BP3 and its target mRNAs as normal cells become tumor cells.

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.

IGF system in sarcomas: a crucial pathway with many unknowns to exploit for therapy.

The insulin-like growth factor (IGF) system has gained substantial interest due to its involvement in regulating cell proliferation, differentiation and survival during anoikis and after conventional and targeted therapies. However, results from clinical trials have been largely disappointing, with only a few but notable exceptions, such as trials targeting sarcomas, especially Ewing sarcoma. This review highlights key studies focusing on IGF signaling in sarcomas, specifically studies underscoring the properties that make this system an attractive therapeutic target and identifies new relationships that may be exploited. This review discusses the potential roles of IGF2 mRNA-binding proteins (IGF2BPs), discoidin domain receptors (DDRs) and metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) in regulating the IGF system. Deeper investigation of these novel regulators of the IGF system may help us to further elucidate the spatial and temporal control of the IGF axis, as understanding the control of this axis is essential for future clinical studies.

Insulin-like growth factor 1 receptor affects the survival of primary prostate cancer patients depending on TMPRSS2-ERG status.

BACKGROUND: Prostate cancer (PCa) is characterized by clinical and biological heterogeneity and has differential outcomes and mortality rates. Therefore, it is necessary to identify molecular alterations to define new therapeutic strategies based on the risk of progression. In this study, the prognostic relevance of the insulin-like growth factor (IGF) system was examined in molecular subtypes defined by TMPRSS2-ERG (T2E) gene fusion within a series of patients with primary localized PCa. METHODS: A cohort of 270 formalin-fixed and paraffin-embedded (FFPE) primary PCa samples from patients with more than 5 years' follow-up was collected. IGF-1R, IGF-1, IGFBP-3 and INSR expression was analyzed using quantitative RT-PCR. The T2E status and immunohistochemical ERG findings were considered in the analyses. The association with both biochemical and clinical progression-free survival (BPFS and PFS, respectively) was evaluated for the different molecular subtypes using the Kaplan-Meier proportional risk log-rank test and the Cox proportional hazards model. RESULTS: An association between IGF-1R overexpression and better BPFS was found in T2E-negative patients (35.3% BPFS, p-value = 0.016). Multivariate analysis demonstrated that IGF-1R expression constitutes an independent variable in T2E-negative patients [HR: 0.41. CI 95% (0.2-0.82), p = 0.013]. These data were confirmed using immunohistochemistry of ERG as subrogate of T2E. High IGF-1 expression correlated with prolonged BPFS and PFS independent of the T2E status. CONCLUSIONS: IGF-1R, a reported target of T2E, constitutes an independent factor for good prognosis in T2E-negative PCa. Quantitative evaluation of IGF-1/IGF-1R expression combined with molecular assessment of T2E status or ERG protein expression represents a useful marker for tumor progression in localized PCa.

CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling.

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.