The Length of Lactation and Model of Weaning Modulate Key Regulatory Nodes of Murine Mammary Gland Involution

Reduced lactation after forced weaning induces a tumor-promoting environment in the mammary gland, triggering key regulatory nodes common to both post-lactation involution and breast cancer, including calpains and miRs. We investigated how lactation duration modulates these nodes using two murine models: short lactation (forced weaning) and prolonged lactation (spontaneous-weaning). Additionally, the role of inflammation in calpain regulation was assessed in forced-weaned NOS-2 knockout mice. Morphological and molecular analysis in mammary tissue included histochemical staining, qPCR, enzymatic activity, Western blot and miRNA-seq. Mammary gland involution after prolonged lactation resulted in milder inflammation, reduced cell death and tissue remodeling, and lower collagen deposition compared to short lactation. The expression, activity and function of calpain-2 was found to be more sensitive to the model of lactation and inflammatory environment compared to calpain-1. Even after full regression (28 days postpartum), prolonged lactation maintained lower calpain-2 levels and higher expression of tumor suppressors miR-10b and miR-143/145. These persistent molecular differences suggest spontaneous weaning as the optimal model for healthy mammary gland regression, whereas forced weaning sustains alterations in calpain-2 and regulatory miRs that may increase post-partum breast cancer risk. The potential long-term influence of lactation duration on breast cancer development warrants further consideration in both clinical and public health contexts.