Radiosensitising efficacy of histone deacetylase inhibitors CUDC-101 and SAHA in breast cell lines

Abstract

Introduction: Photon-based radiation therapy remains to be an important treatment modality in the management of breast cancers. However, proton therapy has attracted a lot of interest due to the ability to reduce dose to healthy tissues. It is rapidly evolving as a radiation therapy tool for women with left-sided breast cancer in order to limit unnecessary dose to the heart and reduce the risk of long-term cardiac morbidity. In addition to the evolution in radiation therapy modalities, increasing evidence shows that histone deacetylase inhibitors (HDACi) are able to sensitise cancer cells to ionising radiation with little effect on healthy cells. Studies on radiosensitising capabilities of HDACi in combination with proton irradiation remain limited. To date, the published manuscripts presented in this thesis were the first to be published on radiosensitising capabilities of HDACi in combination with proton therapy in breast cell lines. Aim and hypothesis: The study aimed to compare in vitro radiosensitising capacities of two different HDACi, suberoylanilide hydroxamic acid (SAHA) and CUDC-101, to photon and proton irradiation in multiple breast cell lines. CUDC-101 is an inhibitor of histone deacetylases (HDACs), epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and was therefore expected to be a more effective radiosensitiser than SAHA. Research design: The study followed a factorial experimental design with three main effects (HDACi, cell types, radiation types) with three repeats and 180 observations. Methods: MCF-7, MCF-10A and MDA-MB-231 human breast cell lines were utilised to conduct cell proliferation, clonogenic survival, gamma-Histone subtype H2A isoform X (γ-H2AX), cell migration, apoptosis and cell cycle analysis assays. Cells were pre-treated with HDACi and each sample irradiated at room temperature with varying doses of 184 MeV proton irradiation or 250Kvp X-rays. Data analysis was conducted using Graphpad Prism software. For statistical analysis, unpaired Student t-tests were used to test for significance. Testing was done at the 0.05 level of significance. Significance of the study: The study contributes new knowledge on HDACi-mediated sensitisation of breast cell lines to proton irradiation. The results highlighted the role of CUDC-101 as a potent radiosensitizer and a promising strategy to mitigate breast cancer metastasis. The results also add to the ongoing efforts to clarify the mechanisms that underlie the radiosensitising effects of HDACi particularly to proton irradiations.

Results: Both HDACi, SAHA and CUDC-101 are able to enhance the radiation-induced cytotoxicity in the MCF-7 and MDA-MB-231 breast cancer cell lines when combined with either X-rays or protons. In the triple negative MDA-MB-231 cell line, increased DNA DSB induction and retention following treatment with CUDC-101 or SAHA monotherapy and in combination treatments with 2Gy protons or X-rays. An unexpected finding in the study was the increase in migration and invasion after SAHA treatment in the MCF-7 cell line, whereas both SAHA and CUDC-101 reduced migration and invasion in the triple negative MDA-MB-231 cells.