Ewing sarcoma (ES) is the most common soft tissue and bone tumor in adolescents and young adults. The survival rate of patients with metastatic disease is poor suggesting an urgent need for the development of more effective therapeutic options. Our aim was to investigate novel targets and identify less toxic agents to improve therapeutic efficacy in ES patients. Specificity protein 1 (Sp1) transcription factor regulates critical genes involved in cell proliferation. Sp1 also mediates the expression of survivin, an inhibitor of apoptosis protein. Overexpression of Sp1 and survivin is linked to aggressiveness and poor prognosis in several cancers; however, their precise association is not adequately evaluated in ES. Previously, we showed that tolfenamic acid (TA) inhibits leukemia, medulloblastoma and neuroblastoma cell proliferation by targeting Sp1. In this study, we investigated the anti-cancer activity of this small molecule using human ES cell lines. CHLA-9 and TC-32 cells were treated with vehicle (DMSO) or TA (2.5-20 µg/ml) and cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter Glo kit. ES cells were treated with vehicle or TA (15 µg/ml) for 48 h. The mRNA and protein expression of Sp1 and survivin was determined by quantitative polymerase chain reaction (qPCR) and Western blot analysis respectively. Flow cytometry was used to measure apoptotic cell population using Annexin-V staining and cell cycle phase distribution with propidium iodide. Markers of apoptosis (caspase 3/7 activity using caspase Glo kit and cleaved PARP by Western blot) and cell cycle arrest (Cyclin D1 and p21 expression by Western blot) were also determined. Sp1 DNA-binding was evaluated by gel shift assay. TA treatment inhibited ES cell viability in a dose and time-dependent manner which is accompanied by the inhibition of Sp1 and survivin protein expression and a 2-3 fold increase in apoptotic markers, caspase 3/7 activity, Annexin-V staining, and c-PARP expression. Interestingly, TA inhibited mRNA expression of survivin but not Sp1, suggesting it has post-translational effects, perhaps proteasome-dependent degradation of Sp1. Gel shift assay revealed a decrease in Sp1 DNA-binding, indicating that TA could directly disrupt the binding of Sp1 with its consensus binding site in the promoter of target genes. Cyclin D1 is crucial for moving the cells from G0/G1 to next phases and induction of p21 is known to inhibit Cyclin D1. In this study, TA up-regulated p21, reduced Cyclin D1 expression and accumulated cells in G0/G1 phase. These results demonstrate that TA induces apoptosis and causes cell cycle arrest in ES cells by targeting Sp1 and survivin.

Citation Format: Sagar Shelake, Umesh T. Sankpal, W. Paul Bowman, Matthew Wise, Anish Ray, Riyaz Basha. Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2017-696