Investigating Protein Expression of AKT/mTOR Pathway in HR+/HER2- Breast Cancer Tumors using Laser Capture Microdissection and Reverse Phase Protein Arrays

Tumors classified as HR+ and HER2- are most likely to develop resistance to endocrine therapy through over-activation of the AKT/mTOR signaling pathway. Immunohistochemistry staining (IHC) is typically used to identify and quantify protein expression; however, similar to using whole tissue lysates in protein analysis, loss of sensitivity is a critical issue. This study used laser capture microdissection (LCM) coupled with reverse phase protein arrays (RPPA) to isolate tumor cells from twelve biopsies from high-risk breast cancer patients and investigate the expression of AKT/mTOR related proteins compared to whole tissue (WT) lysates from the same biopsies. Using a t-test, the protein expression of both mTOR S2448 (p= 0.042) and Estrogen Receptor alpha (ERa) (p= 0.005) were found to be significantly higher in the microdissected tumor population compared to WT lysates. The mean intensity value of mTOR S2448 expression in WT lysates was only of the microdissected tumor cells and in the same trend the mean intensity value of ERa was only 9% of the microdissected. These results indicate that LCM together with RPPA is a far more sensitive option for the detection and quantification of total and phospho-proteins providing a more accurate read out of their activation state.