SRP14AS1 in stress granules and Ewing sarcoma susceptibility

Ewing sarcoma is the second most frequent bone cancer in children and young adults of European descent, but hardly occurs in Africans. The molecular basis for this ethnic difference in disease incidence is unknown. This project tests the hypothesis that natural genetic variations in the regulation of components of intracellular bodies that form upon cellular stress predispose to Ewing sarcoma. Ewing sarcoma is characterized by a highly recurrent chromosomal rearrangement leading to the expression of an aberrant gene-regulatory protein which drives the disease. Previously variations in three genomic regions were identified that associate with Ewing sarcoma and differ between people of European and African descent. Our preliminary results indicate that two of these susceptibility loci affect the regulation of putative components of insoluble cytoplasmic foci which form during early cellular stress response, the stress granules. Previously, we interrogated the functional relevance of the Ewing sarcoma susceptibility region on chromosome 15q15. We found this locus to be bound by the Ewing sarcoma oncogene and to display properties of a gene regulatory element that controls the expression of a long non-coding RNA, SRP14- AS1. Experimental modulation of SRP14-AS1 expression or deletion of the regulatory element significantly reduced the ability of Ewing sarcoma cells to form stress granules under cellular stress, reminiscent of the consequences of loss of the stress granule component TDP-43, which is also a target of Ewing sarcoma predisposing genetic variation. This observation was paralleled by the loss of proliferative activity under non-adherent cell culture mimicking in vivo tumor growth conditions. Preliminary results revealed that SRP14-AS1 binds to SRP14, a further stress granule component, suggesting a functional link between the products of two genes affected by Ewing sarcoma predisposing variations. This study will therefore interrogate the role of stress granules in Ewing sarcoma pathogenesis and the mechanisms by which variations in SRP14-AS1 and TDP43 expression affect Ewing sarcoma susceptibility. In a comprehensive work program we will explore in detail the dynamic regulation and mechanisms controlling early stress response, the functional interaction of Ewing sarcoma susceptibility loci in stress granule formation, and the role of the long non-coding RNA SRP14- AS1 and associated proteins in this process. These goals will be achieved by the application of state-of- the-art gene editing, epigenomic and proteomic methods. Finally, we will explore SRP14-AS1 expression as a potential predictive marker for Ewing sarcoma susceptibility, and test stress granule inhibitory compounds for their anti-tumor activity. This project will therefore provide unprecedented insights into a novel mechanism of long non-coding RNA mediated tumor predisposition.

Ewing sarcoma is the second most frequent bone cancer. It affects predominantly children and adolescents in puberty. The disease is caused by a chromosomal rearrangement that perturbs cellular gene expression leading to uncontrolled cell proliferation, which is due to the aberrant activity of a fusion protein, EWS-FLI1, generated by this aberration. For unknown reasons, the tumor occurs much more frequently in Caucasians than in people of African or Southeast Asian descent. Previous studies identified three polymorphic chromosomal regions as being associated with the disease in Europeans. Two of these regions contain genes, which play a putative role in the acute protein translation response to various kinds of cellular stress. Therefore, this project was based on the supposition that Ewing sarcoma susceptibility may be caused by variable ability of the protein translational machinery to cope with different forms of cellular stress. We tested this hypothesis for oxidative and metabolic stress, however, with negative results. Among the various types of stress to which a tumor cell is naturally exposed are mechanical stimuli from the tumor microenvironment not the least during tumor cell migration. Mechanical stress elicits changes in the transcriptome through activation of the YAP/TAZ/TEAD signaling pathway. Our previous research revealed that fluctuations in EWS-FLI1 expression activates this pathway leading to migration and invasion of the tumor cells at the basis of metastatic spread. As metastasis constitutes a major risk to Ewing sarcoma patients, we investigated if inhibition of this mechanism affects Ewing sarcoma plasticity and metastatic potential. We found that EWS-FLI1 is mainly increasing the expression of TAZ. We demonstrated on the single cell level that EWS-FLI1 fluctuations lead to complex formation between the co-factors YAP and TAZ and the transcription factor TEAD in a subpopulation of tumor cells. Treatment with verteporfin, a drug that has so far been used in the therapy of age-related macula degeneration, was able to suppress this complex formation. This led to the reversion of altered gene expression associated with tumor cell plasticity and inhibition of tumor cell migration already at nanomolar concentrations of the drug. In three pilot experiments testing verteporfin treatment in a mouse xenotransplantation model, we found that the drug reduced and delayed lung metastases and also decreased the number of metastatic nodules per affected lung. The anti-migratory effect on tumor cells was reproduced with genetic TAZ downregulation but not with depletion of YAP. Our results therefore identify TAZ/TEAD signaling as a promising candidate target of future anti-metastatic therapies of Ewing sarcoma. As verteporfin is not suited for systemic application due to its light-sensitivity, we are currently testing a new generation of TEAD-binding small molecules for their potential to block YAP/TAZ/TEAD complex formation and reduce tumor cell migration.