Differential impact of metformin based prevention and therapeutic strategies on aberrant metabolic signals/ molecular signatures associated with triple negative breast cancer (TNBC) during high fat/ high fructose diet feeding

Metformin is routinely used in the management of type-2 diabetes (T2D) and is known for its potential to alter mitochondrial respiration-driven energetic stress. Interestingly, metformin has also been the focus of cancer research because various study outcomes have associated its intake with anti-cancer benefits. In the present study, we determined the comparative anti-cancer effects of preventive versus therapeutic regimen of oral metformin against TNBC tumorigenesis. Given that the anti-cancer benefits of metformin are observed mostly in cases associated with metabolic aberrations such as obesity/T2D, accordingly, the in vivo preclinical study was performed in diet-induced obese mice model (DIO mice) wherein four week old C57Bl/6 female mice were fed either a high fat diet (HFD, 60 kcal% fats) or high fructose diet (HFR, 60 kcal% fructose ) throughout the course of the study so as to induce obesity-associated changes in both systemic and local mammary gland microenvironment that could impact breast tumorigenesis. After 10 weeks of respective DIO feeding (at 14 weeks of mice age), orthotopic mammary tumors were induced by subcutaneous injection of 2x10 6 PY230 breast cancer cells in the 4 th mammary fat pad (either left or right). In the preventive regimen metformin (pre) treatment (daily oral gavage: 300mg/kg body wt.) was started 4 weeks before the cell inoculation and continued till 2 weeks after the cell inoculations followed by a wash out period of 6 weeks till study end (8 weeks following cell inoculations). On the other hand, in the therapeutic regimen, metformin (post) treatment (daily oral gavage: 300mg/kg body wt.) was started 2 weeks after the cell inoculation and continued till 6 weeks after the cell inoculations (till study end). Overall, the results indicated that while both preventive and therapeutic regimens of metformin had an anti-tumorigenic effect on TNBC growth and progression, the preventive regimen even after the washout period (no drug administration) had long lasting anti-cancer effect which was comparatively better than therapeutic treatment of TNBC tumors. In untargeted metabolomic profiling, the observed metabolic alterations in both the HFD and HFR groups suggest that metformin has the potential to restore metabolic homeostasis in the tumor microenvironment. In untargeted lipidomics profiling, metformin's preventive and therapeutic effects were evident in the modulation of multiple lipid classes. Specifically, the preventive regimen appeared to yield more pronounced lipid alterations, indicating the potential benefits of early intervention in modulating cancer progression.