Targeting Sp1 in Ewing Sarcoma: A multi-approach method for the utilization of Mithramycin

Purpose: Ewing Sarcoma (ES) is a bone and soft tissue cancer affecting young adults and children. ES mostly occurs in the bones or soft tissue of the arms, legs, and pelvis. Localized ES presents with 5-year survival rate of 70%, but metastatic 5-year survival rate is between 15% and 30%. Our laboratory is interested in combination treatments using less toxic agents to induce sensitization to chemotherapy in ES. The anti-cancer activity of an antineoplastic antibiotic, Mithramycin, against ES cells has been shown. Mithramycin inhibits Specificity protein 1 (Sp1) a marker associated with aggressive cancer cell growth and resistance to chemo/radiation therapies. However, its mechanistic effects on other oncogenic proteins have yet to be elucidated in ES. The purpose of this study is to evaluate the effectiveness of Mithramycin and various combinations with other chemotherapeutic agents, Etoposide and Vincristine, to inhibit ES cell growth and assess the effect on key cancer related proteins regulated by Sp1. Future studies will include expanding upon Mithramycin’s mechanism of action in Ewing Sarcoma utilizing RNA sequencing and various computational methods.

Methods: Cell lines were obtained from Children’s Oncology Group (COG). Anti-proliferative activity of Mithramycin and/or Vincristine and Etoposide against ES cell lines, TC205 and CHLA10, was evaluated using CellTiterGlo kit. Dose curves were plotted and IC50 values were determined by Sigma-Plot software. The expression of Sp1 and survivin was determined by Western blot analysis. The specific type of effect (additive/antagonistic/ synergistic) of the combination treatments were determined by analyzing the combination index obtained via Calcusyn software. Nude mice were injected with TC205 cells and treated over two weeks with either Mithramycin (1mg/kg per week) and/or Etoposide (5mg/kg per week) and tumor volume was compared. Protein models were obtained from RCSB PDB and homology tests were performed using the Swiss-model workspace.

Results: Mithramycin, etoposide, and vincristine decreased ES cell line viability in TC205 and CHLA10 cells as monotherapies, but more effectively in combination. Tumor volume was greatly attenuated upon Mithramycin and/or etoposide introduction, but more significantly when used in combination. Mithramycin showed the ability to reduce the expression of Sp1 and offer differing effects on survivin expression, indicative of anti-apoptotic mechanisms being implemented in the ES cell lines. Decreases in viability upon chemotherapeutic and Mithramycin introduction were drastically increased when used in combination and this effect was mirrored in further decreases in Sp1 expression. Synergistic drug responses were shown in the combination of Mithramycin with both Vincristine and Etoposide (CI <1). Sp1 and survivin protein models were established and homology verification using Ramachandran plots and QMEAN Z-scores indicated quality protein models for further computational studies.

Conclusions: Mithramycin may effectively sensitize ES cells and improve the response of chemotherapy while lowering necessary effective dosages. Studies to understand the mechanism of action of Mithramycin on Sp1, survivin, and other proteins involved in Ewing Sarcoma are underway.