Genomic Instability and DNA Damage Response in Soft Tissue Sarcoma

Abstract

Soft tissue sarcomas (STSs) originate from mesenchymal stem cells (MSCs) and, although rare, represent a diverse group of tumours. Making up less than 1% of all adult malignancies, these uncommon tumours are significant despite their rarity. Previous research in our laboratory has shown that STS sarcoma cell lines have high levels of genomic instability. Additional studies have identified a deletion in the ATM gene, notably in gastrointestinal stromal tumours and leiomyosarcomas. Despite this deletion, ATM protein expression persists. It has been demonstrated that sarcoma samples maintain functional ATM, enabling these cancers to repair DNA even in the presence of ATM inhibition. This PhD thesis examined genomic instability in STS subtypes through chromosome counting, assessing DNA damage responses under spontaneous conditions and following ATM inhibitor (ATMi) treatment. It also evaluated the effects of mitomycin C (MMC), with and without ATMi. Techniques such as sister chromatid exchange (SCE), immunofluorescence, and Annexin V experiments were employed to assess DNA exchange levels, repair mechanisms, and apoptosis, respectively. Additionally, comet and clonogenic assays measured DNA damage and cytotoxic effects at various concentrations. STS cell lines exhibit a higher level of endogenous DNA repair compared to other tested cancer cell lines. Even following ATMi and MMC, STS cells demonstrated the ability to repair double-strand breaks (DSBs), evidenced by the continued formation of DSB repair proteins (γH2AX, RAD51, and DNA-PKcs). This was further indicated by the varying levels of SCE in response to treatment. These observations suggest that alternative pathways may be utilised for DNA repair in response to ATMi and MMC, or that mutations in the ATM gene could render it unresponsive to such treatments. The combined ATMi and MMC treatment had no effect, indicating further research is needed to understand DNA damage response pathways in STS.