Generation and investigation of resistance mechanisms to AZD5363 in breast cancer

The PI3K/AKT pathway is a key regulator of proliferation, growth and survival in mammalian cells and aberrations in the components of this pathway are often implicated in malignancy. In particular, mutations in this pathway are frequently responsible for the development of breast, prostate, and ovarian cancers. The high frequency of deregulation in this pathway means it represents a good therapeutic target, and there are now several AKT inhibitors in Phase II clinical trials for breast cancer, including the ATP-competitive AKT inhibitor AZD5363. Despite the initial success of many molecularly targeted drugs, acquired resistance is an emerging issue, and resistance mechanisms to AKT inhibitors are still relatively unknown. Research into resistance to CCT129254 (a precursor of AZD5363) in the A2780 ovarian cancer cell line has been previously undertaken (Akan, Jakubowski, Garrett; unpublished), revealing a reduction in 4EBP1 and increased phosphorylation of p70S6K. The aim of this project was therefore to develop breast cancer cell line models of acquired resistance to AZD5363, and investigate how these resistance mechanisms compare in the two different disease types.

Breast cancer cell lines with mutations causing upregulation in the PI3K/AKT pathway were selected for analysis and characterised for their response to AZD5363. Resistant clones were generated through limiting dilution and chronic exposure to twice the GI50 of AZD5363. Characterisation of parental cell lines T47D, MCF-7, and ZR-75-1 showed IC50 values of 0.92, 1.34, and 0.05 µM respectively for AZD5363. Sub-clones B9 and D2 were generated from ZR-75-1, with 7.3 and 5.8-fold resistance to AZD5363, respectively. However, there was no significant cross-resistance to other AKT inhibitors GDC0068 and MK2206, and Western blotting revealed no significant changes in PI3K/AKT signalling. Taken together, this suggests that the resistance mechanism is not proximal to AKT, and another pathway may be responsible for the resistant phenotype. Additionally, a new and innovative cell-based ELISA was developed to investigate cellular signalling of GSK3? and S6RP as an alternative to Western blotting.

In conclusion, characterisation of AKT inhibitor-sensitive breast cancer cell lines has led to generation of resistant cell line models for AZD5363. However, these novel findings showing a lack of AKT inhibitor cross-resistance and no significant changes in PI3K/AKT signalling in these models suggests the resistance mechanism is not proximal to AKT. Therefore, the novel resistance mechanisms observed in these breast cancer cell line models are not the same as that observed in the ovarian carcinoma model. Further investigation will be required to determine the pathway responsible for acquired resistance to AZD5363 in breast cancer cells.