Inhibition of Ras-like Protooncogenes A and B, RALA & RALB, Sensitize Soft Tissue Sarcomas to Radiation Therapy
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Date
2023-05
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The Ohio State University
Abstract
Treatment options for patients with soft tissue sarcomas (STS) have changed very little over the past 50 years. While radiation treatment (RT) improves outcome for patients with many types of cancer, STS are notoriously RT resistant. Understanding the mechanism(s) of this resistance and how to overcome it will improve outcomes for STS patients. Recent approaches to understand radiation resistance in cancers have placed an emphasis on uncovering key genetic alterations that are helping to confer this radiation resistance. RALA and RALB are small GTPases that are differentially expressed in a variety of cancer and have been shown to play a role in conferring radiation resistance. In STS the RALs are also altered in expression making them promising targets to study the issue of radiation resistance in STS. The role of RALA and RALB in STS radiation resistance were investigated through in vitro assays studying cellular response to radiation with variable expression of RALs. Radiation responses analysis included cell survival and sustained DNA damage. RALA and RALB expression was experimentally decreased in STS cell lines to study the roles of the RALs during RT. Colony forming assays were performed to understand how the survival and growth of STS cells were altered with and without the expression of RALA and/or RALB. Additionally, DNA damage, one of the major responses to RT, was analyzed post-radiation in STS cells in which RAL expression was once again experimentally decreased. Assays involved immunofluorescence methods measuring the presence of yH2AX, a biomarker representative of DNA damage. Knockout or depletion of the RAL small GTPases inhibited the radiation resistance of STS cancer cells. Clonogenics assays showed that in vitro when the RALs were depleted there was a decrease in the surviving fraction of STS cells after radiation, particularly when RALB was depleted. This showed that increase in RAL could be responsible for radiation resistance in STS and inhibiting RAL expression could re-sensitize cells to RT. DNA damage post-radiation showed similar results with RAL depletion resulting in increased sustained DNA damage after radiation exposure. This once again shows that when RAL expression was experimentally decreased there was a more effective radiation response as the cells sustained a greater amount of DNA damage. Together these data show the significant role that RALs play in conferring radiation resistance to STS and perhaps a possibility in inhibiting RAL expression to sensitize STS cells to RT. Further investigation will be necessary to understand the mechanisms by which RALA and RALB are conferring this resistance for the eventual use of RALs as targets for more effective RT.
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Keywords
Soft Tissue Sarcomas, Radiation Resistance, RalA and RalB