Abstract PS3-12-17: Baicalein Reverses CAFs Activation in Triple-Negative Breast Cancer by Inhibiting the PI3K/Akt/mTOR Pathway

Abstract Background and purpose: Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastatic potential and the poorest prognosis. Cancer-associated fibroblasts (CAFs) promote tumor progression and treatment resistance. Baicalein (BAI), the primary active constituent of the traditional Chinese herb Scutellaria baicalensis, exhibits broad-spectrum antitumor activity and multi-target effects. Studies on baicalein-mediated regulation of CAFs remain limited, so this study aims to analyze the correlation between tumor-stroma ratio (TSR), CAFs activation markers, clinicopathological features, and prognosis in TNBC; explore novel mechanisms and key molecules underlying CAFs activation; elucidate the role and molecular mechanism of baicalein in reversing CAFs activation, thereby providing a scientific foundation for developing novel TNBC combination therapies. Methods: Clinicopathological data from TNBC patients and the TCGA database were analyzed to evaluate the association of TSR and CAFs activation markers with clinicopathological characteristics and prognosis. An ex vivo CAFs activation model was established to investigate the effects of activated CAFs on 4T1 cell migration, pathway alterations during CAFs activation, and underlying mechanisms. The reversal effect of baicalein on CAFs activation and its molecular mechanism were systematically evaluated. Molecular docking and dynamics simulations were performed to assess the binding affinity and stability of baicalein with key pathway proteins. A murine orthotopic TNBC co-implantation model was established to validate the in vivo effects of baicalein on CAFs activation reversal and TME remodeling. Results: Stroma-rich tumors were significantly associated with high histological grade, rapid proliferation, and PIK3CA mutations in TNBC patients. Both CAFs activation and high PI3K expression correlated with poor prognosis. Stroma-rich TNBC tissues exhibited higher CAFs activation and elevated stromal PI3K expression. Conditioned medium (CM) from 4T1 cells induced a CAFs-like morphology in 3T3 cells, with significantly upregulated protein levels of CAFs activation markers (α-SMA, FAP, S100A4, and Vimentin), upregulated CAFs activation-related genes (eg.Acta2) and extracellular matrix-related genes (eg.Col1a1). KEGG enrichment analysis highlighted significant alterations in the PI3K-Akt pathway. Activated CAFs showed increased ratios of p-PI3K/total PI3K, p-Akt/total Akt, and p-mTOR/total mTOR. Activated CAFs markedly promoted 4T1 cell migration. Baicalein downregulated the CAFs activation and extracellular matrix-related genes. KEGG analysis revealed the most pronounced enrichment in the PI3K-Akt pathway. The binding energy of baicalein and p110α is −8.4 kcal/mol, forming a thermodynamically stable complex. In the orthotopic TNBC model, the baicalein plus nab-paclitaxel exhibited the smallest tumor volume/weight, fewest lung metastases, and prolonged survival. Baicalein significantly reduced α-SMA, Vimentin, and Collagen I expression, decreased collagen deposition, and attenuated TME fibrosis. Tumor tissues from the baicalein group showed reduced p-PI3K/total PI3K, p-Akt/total Akt, and p-mTOR/total mTOR ratios. Conclusion: In TNBC patients, CAFs activation, stroma-rich TME, and aberrant PI3K activation are interrelated and associated with poor prognosis. PI3K/Akt/mTOR pathway activation is a novel mechanism driving CAFs activation in TNBC. Baicalein reverses CAFs activation by inhibiting this pathway, suppresses TNBC cell migration, and synergizes with taxanes to enhance antitumor efficacy. Citation Format: W. Lv, F. Wu, C. Du, S. Zhang. Baicalein Reverses CAFs Activation in Triple-Negative Breast Cancer by Inhibiting the PI3K/Akt/mTOR Pathway [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-17.