Abstract Background and Aim: Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with high metastatic capacity and dismal prognosis. Accumulating evidence suggests that Traditional Chinese Medicine (TCM) is a promising strategy for cancer treatment. Our team previously reported that a TCM formula consisting of H. diffusa, A. paniculata, A. senticosus, and C. sinensis exhibited a potent antitumor effect in a metastatic breast cancer mouse model. The formula was shown to reduce metastatic lesions in the lungs and livers of experimental mice. Recently, we modified this herbal formula by substituting C. sinensis with G. lucidum (a potent immunostimulatory herb) with an aim to improve the antitumor and antimetastatic efficacy. This study aimed to (i) investigate this modified formula (hereafter called GHAA) systematically both in vitro and in vivo, and (ii) elucidate its mechanism of action by bioinformatics analysis. Method: Morphological and chemical authentications of the herbs were accomplished in accordance with the Chinese Pharmacopoeia. The anticancer effect of GHAA or its 4 individual herb extracts was evaluated in the TNBC cell line by MTT assay. The synergistic anticancer effect was analyzed by the SynergyFinder software. Anti-migration and -invasion effects were evaluated by wound healing and invasion assays, as well as in the zebrafish xenograft model. RNA sequencing was conducted in TNBC cells treated with GHAA or its individual herbs to elucidate the differentially expressed genes and altered pathways following treatment. The transcriptomic signatures induced by GHAA or its individual herbs were queried against the Connectivity Map database to elucidate their possible mechanism of action. The antitumor and anti-metastatic efficacy of GHAA was also investigated in nude mice bearing TNBC tumor xenografts in vivo. Results: GHAA inhibited TNBC proliferation in a concentration-dependent manner in vitro (IC50 ∼ 1 mg/mL). Simultaneous combination of GHAA and doxorubicin was mildly synergistic. GHAA induced apoptosis, but it had no effect on cell cycle regulation. Also, GHAA was shown to significantly inhibit TNBC migration and invasion, and metastasis in the zebrafish model. RNA-seq analysis revealed that ribosomal biogenesis is the top altered biological process by GHAA. Using the connectivity mapping approach, the most potent herb (A. paniculata; IC50 ∼ 0.2 mg/mL) within GHAA was found to share a highly similar transcriptomic signature as cathepsin inhibitors, implying its mechanism of action. Finally, GHAA also displayed significant antitumor and anti-metastatic efficacy in TNBC-inoculated nude mice without inducing notable toxicity. Conclusion: The findings advocate clinical evaluation of GHAA for the treatment of metastatic TNBC. Citation Format: Longling Wang, Julia K.M. Lee, Judy Y.W. Chan, Zhong Zuo, Kenneth K.W. To, . A novel 4-herb traditional Chinese medicine formula inhibits metastasis of triple-negative breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3664.
Wang et al. (Fri,) studied this question.