Abstract
Background:
Focal Adhesion Kinase (FAK) is a key tyrosine kinase often overexpressed in tumors, making it a promising target for cancer therapy. This study aims to screen and investigate the activity and mechanisms of Traditional Chinese Medicines (TCMs) targeting FAK within breast cancer.
Materials and methods:
Using xCELLigence system, we identified TCMs affecting FAK signaling and validated its anti-tumor effects. The 4T1-Luc breast cancer models and MMTV-PyMT mice were used to evaluate Moutan Cortex Radicis (MCR) on tumor growth and lung metastasis, with flow cytometry assessing immune cell changes. We utilized CRISPR-Cas9 to investigate the anti-tumor mechanisms of MCR by specifically targeting the FAK signaling pathway. To elucidate the underlying molecular mechanisms, RNA sequencing was performed, and the results were subsequently validated through quantitative reverse transcription PCR (qRT-PCR) and Western blot analysis.
Results:
We have successfully developed the EGF-induced Time-dependent Cell Response Profilings (FAK-TCRPs) and discovered that MCR effectively attenuated EGF-mediated responses in a dose-dependent manner. Additionally, MCR significantly inhibited tumor growth and lung metastasis by partly targeting FAK. MCR increased CD8+ T cell infiltration and the proportion of CD44HiCD62LHi central memory and CD44HiCD62LLow effector memory T cells, while reducing regulatory T cells (Tregs) and CTLA-4 expression. MCR converted M2 tumor-associated macrophages (TAMs) to M1 within the tumor microenvironment (TME). In FAK-deficient mice, MCR did not affect on CD8+ cells, TAMs, or CTLA-4 expression. MCR modulated chemokine signaling and ECM-receptor interactions, decreasing CCL1, CCL5, TGF, IL-4, IL-10, TNF-α, and IL-6, while increasing CCL7, CXCL10, and IL-24. It significantly inhibited FAK mRNA in tumors. Cellular experiments demonstrated that MCR suppressed P-FAK and P-Erk activation and reduced MMP-2, MMP-9, Laminin, and Fibronectin levels.
Conclusion:
Collectively, MCR is identified as a FAK-targeting agent through FAK-TCRPs. MCR inhibits TAMs, CTLA-4, and chemokine transcription via the FAK/MMPs pathway, showing antitumor effects in breast cancer.