Background: The early exposure of nutrients during pubertal mammary gland development may reduce the risk of developing breast cancer later in life. Anticancer n-3 polyunsaturated fatty acids (n-3 PUFA) are shown to modulate pubertal mammary gland development; however, the mechanisms of action remain unclear. Prior work focused on effects at the whole tissue level, and little is known at the cellular level, such as at the level of mammary epithelial cells (MECs), which are implicated in cancer development. Methods: This pilot study examined the effects of lifelong n-3 PUFA exposure on the transcriptome by RNA-Seq in the isolated MECs of pubertal (6–8-week-old) female fat-1 transgenic mice capable of de novo n-3 PUFA synthesis. edgeR and DESeq2 were used separately for the differential expression analysis of RNA sequencing data followed by the Benjamani–Hochberg procedure for multiple testing correction. Results: Nine genes were found concordant and significantly different (p ≤ 0.05) by both the DESeq2 and edgeR methods. These genes were associated with multiple pathways, suggesting that n-3 PUFA stimulates estrogen-related signaling (Mlltl0, Galr3, and Nrip1) and a glycolytic profile (Soga1, Pdpr, and Uso1) while offering protective effects for immune and DNA damage responses (Glpd1, Garre1, and Rpa1) in MECs during puberty. Conclusions: This pilot study highlights the utility of RNA-Seq to better understanding the mechanistic effects of specific nutrients such as n-3 PUFA in a cell-specific manner. Thus, further studies are warranted to investigate the cell-specific mechanisms by which n-3 PUFA influences pubertal mammary gland development and breast cancer risk later in life.

Background: n-3 PUFA derived from marine sources, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit potential for breast cancer prevention. In contrast, higher dietary intakes of n-6 PUFA, such as linoleic acid (LA), have been implicated in promoting mammary tumourigenesis. However, there is a need for further exploration into how n-3 PUFA influence breast cancer development in comparison to different amounts and sources of LA. Objective: The purpose of this study was to compare the effects of n-3 PUFA-enriched diets versus n-6 PUFA diets differing in LA content, including corn oil (50% LA) and safflower oil (70% LA), on mammary tumour development in a HER2+ breast cancer model. Methods: Using the HER2+ breast cancer MMTV-neu(ndl)YD5 transgenic mouse model, this study determined the effects of: (1) 10% w/w corn oil (CO, n-6 PUFA, n = 14), (2) 10% w/w safflower oil (SO, n-6 PUFA, n = 14), (3) 3% w/w menhaden oil + 7% w/w CO (3% FO 7% CO, n-3 PUFA, n = 12), and (4) 3% w/w menhaden oil + 7% w/w SO (3% FO 7% SO, n-3 PUFA, n = 14) on puberty onset, tumour incidence, tumour volume, and tumour number in utero until 20 weeks of age. Results: Mice fed the n-3 PUFA-enriched diets showed a lower trajectory of tumour development compared to the n-6 PUFA diets, although the differences for palpated tumour volume and number over time reached significance only between the 10% CO and 3% FO 7% CO groups. This suggests that high LA content in CO may represent a threshold for promoting tumour growth whereby further LA content marginally influences additional tumour development. Exposure to the CO n-6 PUFA diet further resulted in earlier onset of puberty compared to the n-3 PUFA-enriched diet containing CO. To investigate the underlying mechanisms, a qPCR analysis of mammary glands and tumour tissue revealed that the n-3 PUFA diets downregulated the expression of pro-tumourigenic immune markers, including CD206 and F4/80 in the mammary glands and the cannabinoid receptor CB2 in tumours, compared to the n-6 PUFA diets. Conclusions: These findings indicate that the presence of dietary n-3 PUFA plays a key role in modulating mammary tumour development, which may be further influenced by the underlying n-6 PUFA background. The associated changes in immune markers suggest that n-3 PUFA exert anticancer effects in part by shifting the tumour immune microenvironment toward an anti-tumour phenotype and modulating cannabinoid receptor signalling. Collectively, this work informs future human studies investigating the role of dietary fat composition in breast cancer risk.