Abstract P5-01-08: Mechanistic differences between abrupt and gradual involution of mouse mammary gland

Abstract

Abstract Objective: Epidemiological studies indicate a direct relation between length of breast feeding and protection against risk of triple negative breast cancer (TNBC), an aggressive subtype. While prolonged breastfeeding allows gradual involution (GI) of the breast, short-term or no breast feeding leads to abrupt involution (AI). We modelled AI and GI of breast in mice, and showed that mice subjected to GI have better protection from tissue remodeling associated injuries in the mammary gland. Our data revealed late development of ductal hyperplasia aided by pro-tumorigenic microenvironment in the AI glands1. Detailed mechanism of mammary gland involution immediately following cessation of lactation has been studied in the past, but only in the AI setting, that is after abrupt removal of pups at the peak of lactation2,3. The goal of this study is to conduct stepwise comparison of the mechanism of GI vs. AI early on to understand the protective effect of GI against breast cancer risk.Methods: Wild-type FVB mice were used in all our studies. Females were mated at 8 weeks of age and uniparous mice were allowed to nurse (6 pups/dam) for 7 days. Females were then assigned randomly to AI or GI cohort. All pups were removed from the AI dams on postnatal day7 (PND7). Three pups each were removed from GI dams on day28 and 31. Mammary glands were harvested intermittently between PND7 and PND35. H&E stained sections were used for histological studies. Unstained FFPE sections were used for immunohistochemistry and TUNEL assay. Total RNA and protein from whole mammary gland was used for qPCR and western blot respectively. Results: GI glands transitioned from fully active lactating to near involuted glands over a period of 8 days (PND17- PND25), while for AI glands it took < 4 days (PND8.5-PND12). The shrinkage and flattening of tall epithelia and loss of acini was gradual in GI glands as opposed to rapid breakdown of acini and adipocyte repopulation in AI gland. Apoptotic cell count peaked on PND11 (5%) in AI vs. PND25 (3%) in GI glands. The pStat3Y705+ cells were highest on PND8.5 (25%) in AI vs. on PND25 (11%) in GI glands. Macrophage infiltration (F4/80+) peaked on PND11 (35%) and remained elevated at ~24% till PND25, while in GI glands increase was gradual from PND17 through PND25 (27%). Expression of key genes identified in AI mice2,3 have markedly different expression pattern in GI mice (Table). While some peaked at a later time point in GI vs. AI coinciding with maximum cell death, expression of some are significantly low or undetectable in GI glands at both RNA and protein level. Conclusions: We show for the first time that kinetics of cell death, adipocyte repopulation, immune cell infiltration and inflammatory state of glands undergoing abrupt vs. gradual involution are markedly different. Several genes known to play a key role during AI are either not expressed or barely detectable in the GI glands at any time point during involution. These data suggests that not only the kinetics, but mechanism of GI and AI are not identical. We conclude that orchestrated cell death in GI protects from drastic lysosomal, and immune cell activities that predisposes mammary glands to higher risk of neoplastic changes.Significance: Epidemiological data highlights the benefits of prolonged breastfeeding in protecting against breast cancer, particularly, TNBC, an aggressive subtype prevalent in the African American women. Our study highlights the mechanism underlying the benefits of gradual involution of breast. GeneFold Change compared to PND7 (GI vs. AI) Peaked on (GI vs. AI)Stat34.0 vs. 5.9PND25 vs. PND8.5Ctsb2.8 vs. 3.4PND25 vs. PND8.5CD143.4 vs. 20PND25 vs. PND8.5Orm11.7 vs. 10PND25 vs. PND12Lrg130 vs. 216PND25 vs. PND12MMP215.8 vs. 27PND25 vs. PND12Chi3L11350 vs. 572, 859PND28 vs. PND11, 28Cebpδnone vs. 4.4None vs. PND8.5CtsLnone vs. 5.7None vs. PND8.5Orm2none vs. 370None vs. PND12Slpinone vs. 92None vs. PND8.5 Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Morgan Bauer, Maria Cuitino, Saba Mehra, Resham Mawalkar, Mustafa Basree, Allen Zhang, Kirti Kaul, Xiaoli Zhang, Ramesh Ganju, Sarmila Majumder. Mechanistic differences between abrupt and gradual involution of mouse mammary gland [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-08.