Xihuang Pill ameliorates the immune microenvironment of TNBC by activation of AMPKα-ACC signaling-mediated lipid metabolic reprogramming

Background: Triple-negative breast Cancer (TNBC) is a specific subtype of breast Cancer with limited treatment options and a generally poorer prognosis compared to Other subtypes, making the exploration of effective therapeutic strategies for TNBC particularly important. Recorded in the traditional Chinese medicine (TCM) monograph of Qing Dynasty, the classical TCM formula Xihuang Pill (XHP) has been used to treat "mammary rock" (breast Cancer). XHP is now widely employed in clinical practice for breast Cancer treatment in China. Nonetheless, the efficacy of XHP in treating TNBC and its associated mechanisms remain unclear.

Purpose: We aimed to investigate the anti-TNBC effects of XHP and its underlying mechanisms.

Methods: The chemical compositions of XHP and the absorbed components within blood were analyzed by using LC-IT-TOF-MS, UPLC-Q-Orbitrap MS, and UPLC-MS/MS. The anti-TNBC efficacy of XHP in vitro and in vivo were evaluated using orthotopic 4T1 mouse TNBC model and TNBC cell lines. Transcriptome Sequencing, untargeted metabolomics, and broad-spectrum targeted lipidomics were used to investigate XHP's regulation of lipid metabolism in TNBC. Propargylcholine (Pro-Cho) was synthesized to label Phosphatidylcholine (PC). Flow cytometry, QRT-PCR, western blotting, and tyramide signal amplification (TSA) multiplex immunofluorescence staining were conducted to explore the specific mechanisms related to XHP's anti-TNBC efficacy. Mouse CD8⁺ T cell or macrophage depletion experiments were employed to investigate the roles of CD8⁺ T cells and macrophages in the anti-TNBC effects of XHP. Co-culture systems were utilized to explore the effects of XHP on lipid crosstalk between TNBC cells and M2-type macrophages. Additionally, molecular docking, molecular dynamics simulations, and cellular thermal shift assay (CETSA) were performed to assess whether Acetyl-11-keto-β-boswellic acid (AKBA), 11-Keto-β-boswellic acid (KBA), β-Boswellic acid (β-BA), and 3-Acetyl-β-boswellic acid (ABA) directly interact with AMPKα.

Results: XHP exhibited potent anti-TNBC effects in vitro and in vivo. Moreover, XHP reduced PC levels in TNBC cells by activating the AMPKα-ACC signaling pathway. Additionally, XHP increased the proportion of CD8⁺ T cells within mouse tumor tissues, and CD8⁺ T cells depletion attenuated the in vivo anti-TNBC efficacy of XHP. Furthermore, XHP inhibited M2-type macrophage polarization in TNBC and induced the repolarization of M2-type macrophages toward the M1 phenotype. Macrophage depletion markedly diminished the anti-TNBC efficacy of XHP. ‌Mechanistically‌, XHP disrupted the lipid crosstalk between TNBC cells and M2-type macrophages by inhibiting lipid metabolism in TNBC cells, thereby reducing lipid transfer to M2-type macrophages. Furthermore, XHP inhibited M2-type macrophage polarization by activating the MyD88/NFκB signaling pathway and downregulating CD36-mediated fatty acid uptake. In addition, we identified 31 prototype components of XHP in rat drug-containing serum. Among these, the compounds AKBA, KBA, β-BA, and ABA exhibited anti-TNBC activity through activation of AMPKα-ACC signaling-mediated the reduction of PC levels and inhibition of M2-type macrophage polarization driven by the disrupted lipid crosstalk.

Conclusions: XHP induced lipid metabolic reprogramming mediated by activation of AMPKα-ACC signaling pathway, inhibited lipid crosstalk between TNBC cells and macrophages, interfered with M2-type macrophage polarization, and ameliorated the tumor immunosuppressive microenvironment, thereby impeding TNBC progression. This study offers a scientific basis for applying XHP in clinical TNBC treatment, provides new candidate drug or lead compound for the treatment of TNBC, and establishes a new theoretical foundation for synergistically regulating lipid metabolic reprogramming and the tumor immune microenvironment to effectively treat TNBC.