Polysaccharides of Huaier Suppress Metastasis and Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Inducing Autophagy to Degrade Snail

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and lacks targeted therapies. Trametes robiniophila Murr (Huaier) has been widely used in clinical anti-cancer treatments in China. Previously, we found that Huaier can effectively improve 5-year overall survival and disease free survival in stage III TNBC patients. The Polysaccharides of Huaier (PS-T) were identified as the major components of Huaier. Further study will be needed to elucidate the efficacy of PS-T as potential anti-tumor adjuvant strategy in TNBC. Methods: Transwell assay, scratch assay and mice lung metastasis model were used to observe cell invasion and migration in vitro and in vivo. Western blotting was used to determine the expression of epithelial-mesenchymal transition (EMT) and autophagy related proteins. Immunofluorescence analysis was used to detect autophagosome-lysosome fusion and protein expression of LC3 and Snail. A cell line MDA-MB-231-siATG5 in which autophagy key protein ATG5 was stably interfered was constructed. An E-cadherin promoter reporter gene plasmid and a Snail overexpression plasmid were sequentially introduced into MDA-MB-231 cells. Immunohistochemistry and Database analysis online(the Human Protein Atlas, UALCAN, Kaplan-Meier plotter) were used to explore the distinct prognostic and potential therapeutic value of Snail in breast cancer patients. Findings: PS-T inhibited cell invasion and migration in vitro and in vivo, and reversed the EMT while induced autophagy. Utilization of autophagy inhibitor LY294002 or knockdown of ATG5 by lentivirus infection suppressed the inhibitory effects of PS-T. Snail, a key transcription factor that controls EMT initiation was found to be degraded by PS-T induced autophagy. Overexpression of Snail reversed the inhibitory effects of PS-T. IHC for breast cancer tissues of 33 cases of TNBC patients and Database analysis online verified that high expression of Snail was associated with poor prognostics. Interpretation: We demonstrate that PS-T can inhibit EMT in breast cancer cell by the induction of autophagy to degrade Snail protein which contributes to both the EMT and metastasis. This study provides a new insight into the mechanism by which PS-T reduce the recurrence of TNBC and new ideas for comprehensive treatment strategies for TNBC patients. Funding Statement: National Natural Science foundation of China (No. 81802664). Declaration of Interests: All authors declare no competing interest. Ethics Approval Statement: Animals were treated according to protocols established by the ethics committee of Army Medical University and the experiments in vivo were conducted according to the approved guidelines and approved by the ethics committee of Army Medical University (AMUWEC2019199).