Tussilagone suppresses triple-negative breast cancer progression by inhibiting the TLR4/NF-κB pathway and enhancing anti-tumor immunity

Background: As a highly aggressive form of breast Cancer, triple-negative breast Cancer (TNBC) is characterized by significant metastatic potential, a lack of targeted therapies, and an unfavorable prognosis. Tussilagone (TUS), a bioactive sesquiterpene isolated from Tussilago farfara's medicinal herb, has shown anti-inflammatory and Anticancer properties. However, its potential role in TNBC treatment and the underlying molecular mechanisms remain unexplored.

Methods: The antitumor effects of TUS on the proliferation, epithelial-mesenchymal transition (EMT), and metastasis of TNBC cell lines MDA-MB-231 and BT549 were assessed through cell viability, invasion, and colony formation assays. Western blot analysis was performed to detect Ki67, vimentin, E-cadherin, N-Cadherin, PD-L1, LAG-3, TIM-3, and key TLR4/NF-κB regulators. Impact on the tumor immune microenvironment (TIME) was evaluated using CD8⁺ T cell co-culture, ELISA, and flow cytometry. The in vivo anti-tumor efficacy of TUS was investigated in a TNBC xenograft mouse model.

Results: TUS exhibited a dose-dependent inhibition of TNBC cell proliferation and invasion, reversed EMT by upregulating E-cadherin and downregulating N-Cadherin and vimentin expression. It showed minimal cytotoxicity toward normal breast epithelial cells. TUS suppressed the TLR4/NF-κB pathway by downregulating TLR4, MyD88, and phosphorylated NF-κB, and decreased PD-L1 expression. Furthermore, TUS enhanced CD8⁺ T cell activation, increased cytokine secretion (IFN-γ, IL-2, TNF-α), reduced LAG-3 and TIM-3 expression, and attenuated CD8⁺ T cell Apoptosis. Treatment of CD8⁺ T cells directly with TUS did not affect cytokine secretion or Apoptosis, suggesting that its immunomodulatory effects are mediated through tumor cell modulation. In vivo studies demonstrated significant tumor growth inhibition by TUS without inducing toxicity.

Conclusion: This study demonstrated that TUS inhibited TNBC progression by suppressing the TLR4/NF-κB pathway, reversing EMT, and modulating TIME. These findings support the potential of TUS as a promising therapeutic candidate for TNBC and underscore the need for further clinical investigation.

PD-L1; TLR4/NF-κB pathway; Tussilagone; triple-negative breast cancer; tumor immune microenvironment.