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BPFS and clinical PFS log-rank and Cox regression tests in primary PCa patients analyzed with IHC. (DOC 72 kb)

Association between IGF system components and clinico-pathological parameters according to Fisher’s or chi-square tests (when more than 2 categories were present) in T2E-positive cases. (DOC 38 kb)

Association between IGF system components and clinico-pathological parameters according to Fisher’s or chi-square tests (when more than 2 categories were present) in 270 cases. (DOC 39 kb)

BPFS and clinical PFS log-rank and Cox regression tests in T2E-negative PCa patients analyzed with qRT-PCR. (DOC 80 kb)

Association between IGF-1R and clinico-pathological parameters according to Fisher’s or chi-square tests (when more than 2 categories were present) in ERG-positive cases. (DOC 32 kb)

BPFS and clinical PFS log-rank and Cox regression tests in ERG-negative PCa patients analyzed with IHC. (DOC 69 kb)

Association between IGF system components and clinico-pathological parameters according to Fisher’s or chi-square tests (when more than 2 categories were present) in T2E-negative cases. (DOC 38 kb)

BPFS and clinical PFS log-rank and Cox regression tests in T2E-positive PCa patients analyzed with qRT-PCR. (DOC 83 kb)

BPFS and clinical PFS log-rank and Cox regression tests in ERG-positive PCa patients analyzed with IHC. (DOC 71 kb)

Aberrant expression of ETS transcription factors, including FLI1 and ERG, due to chromosomal translocations has been described as a driver event in initiation and progression of different tumors. In this study, the impact of prostate cancer (PCa) fusion gene TMPRSS2-ERG was evaluated on components of the insulin-like growth factor (IGF) system and the CD99 molecule, two well documented targets of EWS-FLI1, the hallmark of Ewing sarcoma (ES). The aim of this study was to identify common or distinctive ETS-related mechanisms which could be exploited at biological and clinical level. The results demonstrate that IGF-1R represents a common target of ETS rearrangements as ERG and FLI1 bind IGF-1R gene promoter and their modulation causes alteration in IGF-1R protein levels. At clinical level, this mechanism provides basis for a more rationale use of anti-IGF-1R inhibitors as PCa cells expressing the fusion gene better respond to anti-IGF-1R agents. EWS-FLI1/IGF-1R axis provides rationale for combination of anti-IGF-1R agents with trabectedin, an alkylator agent causing enhanced EWS-FLI1 occupancy on the IGF-1R promoter. TMPRSS2-ERG also influences prognosis relevance of IGF system as high IGF-1R correlates with a better biochemical progression free survival (BPFS) in PCa patients negative for the fusion gene while marginal or no association was found in the total cases or TMPRSS2-ERG-positive cases, respectively. This study indicates CD99 is differentially regulated between ETS-related tumors as CD99 is not a target of ERG. In PCa, CD99 did not show differential expression between TMPRSS2-ERG-positive and –negative cells. A direct correlation was anyway found between ERG and CD99 proteins both in vitro and in patients putatively suggesting that ERG target genes comprehend regulators of CD99. Despite a little trend suggesting a correlation between CD99 expression and a better BPFS, no clinical relevance for CD99 was found in the field of prognostic biomarkers.

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.

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.

In this article an incorrect affiliation had inadvertently been added. The original article has been updated.

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.

INTRODUCTION: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3, also known as IMP3) is an oncofetal protein which binds RNA thus influencing the transcript target fate. IGF2BP3 is de novo synthesized in malignancies where it promotes proliferation, drug resistance and metastases. Moreover, IGF2BP3 represents a promising indicator of outcome in several cancers. Ewing sarcoma (EWS) is a rare bone and soft tissue tumor where the IGF system plays a pivotal role. This study aimed to investigate the relevance of IGF2BP3 in EWS and verify its value as a prognostic biomarker. MATERIAL AND METHODS: Total RNA was extracted from two independent cohorts of 30 and 109 snap-frozen tissues from localized primary EWS samples with more than 5 years follow-up. Microarray analysis (Affymetrix) was performed in the cohort of 30 patients while IGF2BP3 expression was analysed by qRT-PCR (Applied Biosystem) in the cohort of 109 cases. Prognostic value of IGF2BP3 was evaluated both in univariate and multivariate analysis. IGF2BP3 expression was assessed by qRT-PCR and western blot in a panel of 14 EWS cell lines. Plasmids for IGF2BP3 silencing or over-expression were transfected in A673 or LAP-35 and IOR/CAR EWS cells, respectively. Soft-agar growth of cell lines with differential IGF2BP3 expression was evaluated. catRAPID express human web server was employed for prediction of novel IGF2BP3/mRNAs interactions. IGF-2 and ABCF1 expression levels were analysed in vitro and in clinical specimens. RESULTS: Microarray results evidenced that IGF2BP3 higher expression correlated with a worse outcome (p-value 0.0002). This data was confirmed in the cohort analysed by qRT-PCR both considering univariate (p-value 0.001) and multivariate analysis (HR:2.83. IC95% [1.22-6.53].p = 0.015). In vitro, higher IGF2BP3 expression correlated with an increased capability of growth in anchorage-independent conditions and IGF2BP3 knockdown significantly decreased soft-agar growth of EWS cells. Oncogenic potential of IGF2BP3 was not found to be mediated through IGF-2-dependent mechanism but ABCF1, an ABC family member with a key role in translation initiation, was predicted as a target of IGF2BP3 by in silico analysis. Accordingly, IGF2BP3 loss- or gain-of-functions approaches induced down- or up-regulation of ABCF1 protein levels, respectively. In addition, a direct correlation between IGF2BP3 and ABCF1 was found in 109 EWS patients (p-value minor than 0.0001). Functional studies on the role of ABCF1 in EWS will follow. CONCLUSIONS: IGF2BP3 represents an indicator of prognosis in EWS therefore its detection may be of help to stratify patients according to risk. We also provided evidences for IGF2BP3 in the regulation of EWS aggressiveness and identified ABCF1 as a new putative mediator of IGF2BP3 malignant effects. (Grants: FIRB RBAP11884M_005 and AIRC IG2013_14049 to KS) Citation Format: Caterina Mancarella, Linda Calzolari, Stefano Ferrari, Davide Donati, Piero Picci, Katia Scotlandi. Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3201.