AKT/mTOR Pathway Protein Expression in the Tumor Microenvironment of High-Risk, HR+, and HER2- Breast Cancer using Laser-Capture Microdissection (LCM) and Reverse Phase Protein Arrays (RPPA)

Abstract

Over-activation of the AKT/mTOR signaling pathway is one of the main contributors of tumor proliferation and endocrine therapy resistance in breast cancer patients. The tumor microenvironment participates in tumor maintenance and metastasis and may be a key to identify targetable biomarkers and developing personalized treatments. However, current treatments focus mainly on targeting cancer cells and there is still much to learn about how the tumor microenvironment contributes to tumor progression. In this study, pure populations of stroma cells were isolated from twelve HR+/HER2- breast tumor specimens using laser capture microdissection (LCM) and the activation of AKT/mTOR signaling pathway related proteins was quantified and compared to matched whole stroma/tissue lysates utilizing reverse-phase protein arrays (RPPA). Our findings revealed significant differences in 4E-BP1 (S65) (p=0.0003), HER2 (p=0.00001), and elF4E (S209) (p=0.003) in the isolated stroma population compared to the whole tissue lysates where intensity values were higher by 61.6% in 4E-BP1 (S65), 73.7% in HER2, and 86.5% in elF4E (S209), indicating whole tissue lysates were not suitable for a true representation of protein activation. Interestingly, elF4E (S209) was moderately expressed in three of the twelve microdissected biopsies, while all whole tissue lysates showed increased activation. In addition, expression of AKT (S473), AKT (T308) and p70 S6 Kinase (T389) was not detected in isolated stroma or whole tissue lysates. Such differences in protein expression support the rationale that utilizing pure cell populations produces more sensitive results representing the true activation of proteins, which can guide our understanding on how the tumor microenvironment can support development of targeted therapies for high-risk breast cancer patients.