Mammary gland architectural modulation with a natural galactagogue Asparagus racemosus (Shatavari root extract) and its impact against triple negative breast cancer (TNBC) tumorigenesis

The roots of Asparagus Racemosus Willd. (Shatavari) are known for its galactagogue properties and often used as a traditional/ alternative way to increase milk production during lactation and for breast size augmentation. There is also supporting evidence that it has anti-cancer properties against various cancers including carcinogen-induced breast cancer in pre-clinical in vivo studies and against ER positive breast cancer cell lines. In the present study we investigated the anti-cancer potential of standard extract of roots of Asparagus Racemosus (AR RE ) against triple negative breast cancer (TNBC). We conducted orthotopic tumorigenesis studies in C57Bl/6 female mice. Five weeks old C57Bl/6 female mice (Jackson Labs), were maintained on control AIN-76A diet from Envigo diets, Inc. After 1 week of acclimatization mammary tumors were induced by orthotopic injection of 2x10 6 PY230 breast cancer cells in the 4 th mammary fat pad (either left or right) on day 0. n=7 mice per group were treated with or without AR RE as an oral intervention agent (including with and without tumor inoculation). The dose of AR RE was 250 mg/kg body weight/day (volume 200μl sterile distilled water)-prepared fresh daily. Study end point was 8 weeks following cell inoculations. At study end, tumor tissues, tumor adjacent breast fat pad was harvested from mice and processed for pathological/ molecular analysis. AR RE treatment caused an increase in latency time (time taken for appearance of palpable TNBC tumors) indicating its preventive benefits. AR RE treatment did not have any significant impact on TNBC tumorigenesis; however, tumors had the appearance of differentiated lobular/glandular formation in AR RE -fed groups compared to undifferentiated tumor cells in the controls. Proteomic profiling of mammary gland tissues indicated there were 43 differentially expressed biomarkers after AR RE intervention. Out of the 43 differentially expressed biomarkers, there were 2 molecules [Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) that were significantly decreased in the AR RE control (treated tissue). Importantly, these two molecules are reported to be overexpressed in breast cancer and implicated in the growth and progression of breast cancer. Overall, the results indicated that AR RE intervention results in modulation of signaling pathways (that are associated with breast cancer growth and progression) and had the potential to induce differentiation in TNBC tumors, which could possibly be a novel approach to target TNBC malignancy. Taken together, the study outcomes favor the use of AR RE as a preventive or therapeutic modality against TNBC but warrants further investigation.