Tangyi Wang ¹ Yadian Lei ¹ Jingwei Sun ² Li Wang ¹ Yuxin Lin ¹ Zhijing Wu ¹ Shoude Zhang ³ Chengzhu Cao ^1,4,5 Haiyan Wang ^1,4,5*

• ¹ Qinghai University Medical College, Xining, China
• ² Qinghai Provincial People's Hospital, Xining, Qinghai Province, China
• ³ State Key Laboratory of Sanjiangyuan Ecology and Plateau Agriculture and Animal Husbandry, Qinghai University, Xining, Qinghai Province, China
• ⁴ Research Center for High Altitude Medicine, Qinghai University Medical College, Xining, Qinghai Province, China
• ⁵ Key Laboratory of the Ministry of High Altitude Medicine, Qinghai University, Xining, Qinghai, China

Introduction:Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer resistant to endocrine and targeted therapies. Immune checkpoint inhibitors (ICIs) have shown significant efficacy in various cancers. Taraxacum officinale (dandelion), traditionally used for breast-related conditions, is known for its beneficial composition and minimal side effects. FDA-approved drugs, validated for safety and efficacy, provide a basis for drug repurposing. Researchers are exploring FDA-approved drugs targeting NANOS1 for TOE (Taraxacum officinale extract) treatment to develop new strategies. Digoxin (Dig) and Algestone acetophenide (AA) have emerged as promising candidates targeting NANOS1. Methods: RNA sequencing (RNA-seq) was used to identify potential targets for triple-negative breast cancer (TNBC) from TOE. Bioinformatics tools, including bc-GenExMiner v4.8, the Human Protein Atlas, and TIMER, aided target identification. Molecular docking studies assessed interactions between FDA-approved drugs and these targets, with Dig and AA selected as candidates. The therapeutic efficacy of Dig and AA, combined with PD-1 inhibitors, was evaluated in the 4T1 mouse model. Flow cytometry assessed lymphocyte infiltration in the tumor immune microenvironment. RNA-seq following target silencing by small interfering RNA (siRNA) was performed, followed by GO and KEGG pathway analysis. Validation was done through quantitative PCR and Western blot. Results: TOE inhibited TNBC cell growth, migration, and invasion, as assessed by CCK-8 and transwell assays. RNA-seq indicated the effects may be due to NANOS1 down-regulation. Survival analysis showed lower NANOS1 expression correlated with better prognosis. Immunoinfiltration analysis indicated a negative correlation between NANOS1 levels and activated NK cells. Molecular docking identified Dig and AA as high-affinity binders of NANOS1. Animal experiments showed Dig and PD-1 inhibitor combination enhanced immunotherapy efficacy for TNBC. Discussion: This study suggests TOE as a novel therapeutic approach for TNBC by targeting NANOS1, a protein whose down-regulation correlates with improved patient outcomes. The negative correlation between NANOS1 and activated NK cells highlights the immune system's potential role in TNBC pathogenesis and treatment response. Identifying Dig as a drug targeting NANOS1 offers new opportunities for drug repurposing in TNBC. Overall, this study positions NANOS1 as a new therapeutic target for TNBC and supports a combination approach to enhance immunotherapy efficacy and improve outcomes.