The role of mTOR signalling in mediating T-DM1 resistance

Abstract

HER2-positive breast cancer possesses an invasive phenotype and a notably aggressive clinical course, where patients typically face a poor prognosis in the absence of clinical intervention. Trastuzumab emtansine (T-DM1) is a HER2-targeted therapy that has significantly improved the overall survival in patients. However, its clinical use is limited by intrinsic and acquired resistance. In an attempt to identify the potential determinants of T-DM1 sensitivity, CRISPR/Cas9 functional genomic screens were conducted and revealed that knockout of TSC1 and TSC2, both of which are negative regulators of mTOR signalling, promoted T-DM1 resistance in HER2-positive breast cancer cell lines. The aim of this Masters project was to determine the role of TSC1 and TSC2 in mediating T-DM1 resistance and evaluate if mTOR inhibitors can enhance T-DM1 efficacy, in order to address the role of mTOR signalling in the sensitivity and resistance to T-DM1 in HER2-positive breast cancer. This study utilised MDA-MB-453 TSC1 and TSC2 knockout clones generated outside of this project. The efficiency of gene knockout was evaluated by Western immunoblotting. Sensitivity of TSC1 and TSC2 knockout clones to T-DM1 were analysed by sulforhodamine B (SRB) and competitive cell growth assays. The efficacy of T-DM1 in combination with three mTOR inhibitors (KU-0063794, everolimus and rapamycin) was assessed in MDA-MB-361 and MDA-MB-453 cells. Fixed ratio combinations of T-DM1 and mTOR inhibitors were tested using SRB and/or 3[H] thymidine incorporation assays, after which isobolograms and combination indices were determined for each combination to evaluate drug interactions. Partial knockout of TSC1 was achieved across the clones, one isoform of TSC1 (124 kDa) was knocked out and the other isoform (130 kDa) remained. Consequently, these clones showed reduced sensitivity to T-DM1 relative to the wild type cells in both SRB and competitive cell growth assays, which suggests that TSC1 may be implicated in T-DM1 sensitivity. In contrast, TSC2 did not seem to be knocked out across the clones and despite showing a reduced TSC2 protein expression, these clones appeared to be sensitive to T-DM1, particularly in the competitive cell growth assay. Combinations of T-DM1 and mTOR inhibitors were effective at inhibiting cell viability or proliferation in both cell lines tested. Most combinations showed nearly additive effects, while some showed slight to moderate antagonism, with the lack of synergy possibly reflecting that both cell lines were highly sensitive to T-DM1. The findings from this study necessitate the generation of complete TSC1 and TSC2 knockout clones and a repeat of the combination studies in T-DM1-resistant HER2-positive breast cancer cell lines, in order to further evaluate the role of mTOR signalling in T-DM1 resistance in HER2-positive breast cancer.