Abstract PS17-28: Abrupt involution of lactating mammary gland induces metabolic reprogramming conducive to pro-tumorigenic changes
Authors: Shinde Neelam , Kaul Kirti , Zhang Allen , Mehra Saba , Mawalkar Resham , Kim Hee Kyung , Ganju Ramesh , Majumder Sarmila , Ramaswamy Bhuvaneswari 
Published: February 2021

Abstract
Background: Epidemiological studies indicate that prolonged breast feeding reduces the risk of triple negative breast cancer (TNBC), which carries the worst prognosis. Prolonged breastfeeding allows gradual involution (GI) of the breast while lack of or short-term breast feeding leads to abrupt involution (AI). We developed a novel murine model mimicking AI and GI of breast, and found that GI offers better protection to mammary glands from tissue remodeling associated injuries. Our data showed that AI leads to the development of pro-tumorigenic microenvironment and ductal hyperplasia1. Tissue remodeling involves orchestrated cell death and repopulation, and is closely associated with metabolic reprogramming from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Such metabolic alterations can contribute to cellular changes aiding malignant transformation2. We used our murine model to evaluate whether AI affects cellular metabolism differentially when compared to GI. Methods: Wild-type mice of FVB background were used in all our experiments. Twelve to fourteen week old uniparous mice were allowed to nurse (6 pups/dam) for 7 days postpartum. All pups were removed on day7 postpartum from the dams in AI cohort and three each on day28 and day31 from dams in GI cohort. Whole mammary glands and sorted luminal progenitor (LP) cells harvested on postpartum day28 were subjected to Affymetrix microarray analysis. Gene Set Enrichment Analysis (GSEA) was used to compute pathway enrichments in AI vs. GI glands. Differentially expressed genes were validated using qRT-PCR. Mammary glands harvested on day28 postpartum were subjected to mass spectrometry based untargeted metabolic profiling using Agilent QTOF. Raw data were analyzed using XCMS to assess key metabolic networks altered in AI vs GI glands. Targeted analysis for lactate, pyruvate, succinate and palmitic acid were performed using C13 labelled internal standards to compare OXPHOS vs glycolysis reliance in the AI and GI glands. Results: We observed enrichment of mitochondrial OXPHOS pathway, fatty acid metabolism and Myc target genes in both whole mammary gland and LP cells of AI vs. GI mice. Adipogenesis and hypoxia related genes were enriched in AI-glands. We observed significant upregulation of genes involved in glucose transport and fatty acid synthesis in AI glands, namely, Glut-5, Cidea, Acss2, Acsm3, Acly, Atp6v0d2, Acot11, and Elvol3. Several factors indicating a higher reliance on OXPHOS vs glycolysis, such as, Ppar-γ, Pgc1α, Cpt-2, Srebp1c and Chrebp were upregulated in AI glands. Upregulation of Cpt-2 and Srebp1c in the AI glands indicate higher flux through fatty acid oxidation and reliance on cholesterol synthesis. Metabolomic profiling revealed significant alteration in L-carnitine, GMP and XMP in AI glands which reflect mitochondrial fatty acid transport and nucleotide biosynthesis via guanine-guanosine salvage pathway. Pyruvate and lactate associated with glycolysis were increased in GI vs. AI glands. Conclusion: We show for the first time that in the abruptly involuting (AI) mammary glands following short-term breast feeding, there is a significant shift in the metabolic pathways towards mitochondrial OXPHOS and fatty acid oxidation compared to GI glands. Studies are underway to determine the effect of this metabolic shift on cellular transformation and tumorigenesis and the potential to target these pathways to reverse the detrimental effects of AI. 1. Basree MM, Shinde N, Koivisto C, et al. Breast Cancer Res. 2019; 21(1):80. 2. Ward PS, Thompson CB. Cancer Cell. 2012; 21(3):297.
Citation Format: Neelam Shinde, Kirti Kaul, Allen Zhang, Saba Mehra, Resham Mawalkar, Hee Kyung Kim, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt involution of lactating mammary gland induces metabolic reprogramming conducive to pro-tumorigenic changes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-28.