Abstract A76: Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma
Authors: Hameiri-Grossman Michal , Porat-Klein Adi , Cohen Ian J. , Ash Shifra , Kloog Yoel , Haklai Ronit , Elad-Sfadia Galit , Weizman Avraham , Yaniv Isaac , Avigad Smadar 
Published: May 2015

Aim: Ewing sarcoma (ES) is the second most common and aggressive primary malignant bone tumor in children and adolescents. The hallmarks of the tumor are specific translocations; the most frequent involves the EWS gene on chromosome 22 and the FLI-1 gene on chromosome 11. Our aim was to identify microRNAs (miRs) and genes involved in the aggressiveness of ES. Out of the miRs located on these chromosomes, 3 belong to the let-7 family.

Methods: We evaluated the expression levels of hsa-let-7a and hsa-let-7b by RQ-PCR in 57 primary ES tumors. We measured the protein levels of p-ERK, HIF-1a and EWS-FLI-1 following transfection with let-7a, let-7b or HIF-1a . The effect of a Ras inhibitor was evaluated in vitro and in vivo.

Results: In the group of patients with localized disease, a significant correlation with outcome was observed. Progression free survival (PFS) at ten years for patients with high hsa-let-7b expression was 62% versus 29% for those with low hsa-let-7b expression level (p=0.029). Multivariate Cox regression analysis identified expression of let-7b as an independent prognostic factor in ES, with a 4.4 fold increased risk of relapse (p=0.021).

hsa-let-7 is known to be a negative regulator of the RAS oncogene. Positive p-ERK immuno-staining was detected in 31 out of the 45 (69%) tumors analyzed, and a significant inverse correlation was identified between hsa-let-7a and p-ERK (p=0.037).

One of the target genes of RAS is Hypoxia inducible factor 1 (HIF-1). We detected a high level of HIF-1α protein in ES cell lines, under normoxic conditions, which did not change following induced hypoxia. This implies for a mechanism that mimics hypoxia and probably contributes to the high frequency of metastasis and thereby aggressiveness of this tumor.

Following transfection with hsa-let-7a and hsa-let-7b mimics (overexpression) the levels of RAS, p-ERK, HIF-1α and EWS-FLI-1 proteins were significantly reduced (60%, p=0.0019, 52%, p=0.003, 45%, p=0.0028 and 38%, p=0.006, respectively).

Silencing of HIF-1α resulted in a decrease of 61% of EWS-FLI-1, suggesting that HIF-1α regulates EWS-FLI-1, and we were able to show that HIF-1α directly binds EWS promoter. This was accompanied by a reduction of 32% (p=0.009) in cell proliferation rate and by a significant decrease of 36% (p=0.0013) in cell migration ability. Using a RAS inhibitor, Salirasib, we observed a significant decrease of HIF-1α and EWS-FLI-1 protein levels in both in-vitro and in-vivo settings. In addition, a significant inhibition of tumor growth was demonstrated in the treated animals.

Conclusions: These results imply that Ras and HIF-1α might contribute to the high frequency of metastasis and thereby aggressiveness of ES tumors. The reduction in tumor burden in a mouse model of ES following Salirasib treatment, demonstrates the therapeutic potential of this RAS inhibitor in ES.

Citation Format: Michal Hameiri-Grossman, Adi Porat-Klein, Ian J. Cohen, Shifra Ash, Yoel Kloog, Ronit Haklai, Galit Elad-Sfadia, Avraham Weizman, Isaac Yaniv, Smadar Avigad. Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A76.