(PTFS03-03-25) n-3 Polyunsaturated Fatty Acid Exposure Influences Notch 3 Protein Expression in Mouse Mammary Epithelial Cells

Objectives: Over 50% of breast cancers (BC) exhibit aberrant activation of Notch signalling; however, dysregulation of the Notch 3 protein that causes cell cycle arrest, accelerated mammary epithelial cell proliferation, and tumour initiation is less studied. Notch 3 is uniquely expressed in luminal epithelial cells and exhibits a tumour-suppressive role by inhibiting the proliferation of luminal progenitor (LP) cells. Long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) derived from marine oils reduce the risk of BC and delay tumour onset in vivo. We explored the dynamic effects of lifelong n-3 exposure on Notch 3-expressing cells and cell cycle regulation in mammary epithelial cells (MECs) throughout the estrous cycle.

Methods: Transgenic fat-1 female mice capable of de novo n-3 PUFA synthesis from n-6 PUFA and wild-type mice were fed a 10% (w/w) safflower oil diet until 11 weeks of age. At ten weeks, the estrous cycle phases: proestrus, estrus, metestrus, and diestrus were examined by vaginal flush. Five cycle-progressing mice from each estrous cycle phase per genotype were euthanized for MEC isolation from the inguinal glands. MEC were stained for (1) Notch 3 and Ki-67 and (2) propidium iodide and Ki-67. Separated populations by flow cytometry included (i) total Notch 3 and Notch 3+ Ki67- (LP) and (ii) G1 and G2/M, and hyperproliferative cells. Results were processed in FlowJo, and data were analyzed by analysis of variance (p < 0.05).

Results: There were significant interactions between genotype and estrous cycle on populations of interest (p < 0.01). Lifelong n-3 PUFA was shown to prevent a decrease in Notch 3 protein as compared to WT in proestrus and metestrus within MEC (p < 0.01). Also, n-3 PUFA decreased G1 phase arrest in MEC between metestrus (p=0.018) and diestrus (p < 0.01), and the percentage of LP cells increased by 4.3-6.6% (p < 0.001), and the percentage of hyperproliferating cells decreased by 11.2-25% in diestrus (p < 0.001).

Conclusions: Our findings indicate that n-3 PUFA influences the regulation of Notch 3 protein expression in the mouse mammary gland according to the estrous cycle. These observations enhance our basic understanding of potential linkages between diet and cancer in support of prevention strategies.

Funding Sources: Canadian Institutes of Health Research and Ontario Graduate Scholarship.