Peripubertal Nutritional Prevention of Cancer-Associated Gene Expression and Phenotypes

Abstract

Breast cancer (BC) is a nearly ubiquitous malignancy that effects the lives of millions worldwide. Recently, nutritional prevention of BC has received increased attention due to its efficacy and ease of application. Chief among chemopreventive compounds are plant-based substances known as dietary phytochemicals. Sulforaphane (SFN), an epigenetically active phytochemical found in cruciferous vegetables, has shown promise in BC prevention. In addition, observational studies suggest that the life stage of phytochemical consumption may influence its anticancer properties. These life stages, called critical periods (CPs), are associated with rapid development and increased susceptibility to cellular damage. Puberty, a CP in which female breast tissue undergoes proliferation and differentiation, is of particular interest for later-life BC development. However, little is known about the importance of nutritional chemoprevention to CPs. We sought to address this by utilizing two estrogen receptor-negative [ER(-)] transgenic mouse models fed SFN-containing broccoli sprout extract during the critical period of puberty. We found that this treatment resulted in a significant decrease in tumor incidence and weight, as well as an increase in tumor latency. Further, we found significant alterations in the long-term expression of cancer-associated genes, including p21, p53, and BRCA2. Additionally, our transcriptomic analyses identified expressional changes in many cancer-associated genes, and bisulfite sequencing revealed that the antiproliferation-associated gene Erich4 was both hypomethylated and overexpressed in our experimental group. Our study indicates that dietary interventions during the CP of puberty may be important for later-life ER(-) BC prevention and highlights potential important genetic and epigenetic targets for treatment and study of the more deadly variants of BC.

Keywords: DNA methylation; breast cancer; cancer prevention; critical periods; gene expression; nutrition; puberty.

Figure 1

Overview of experimental design. Experiments were conducted in both SV40 and HER2/neu transgenic mouse lines. Puberty in mice begins about 10 days following vaginal opening (VO). Mice in the experimental (BSp) group received chow infused with SFN-containing BSp for a total of 5 weeks, beginning on the first day of the 5th week and ending on the last day of the 9th week. Mice were monitored for tumor formation beginning at 10 weeks of age. Experiments were terminated and tumor samples were collected for downstream analysis when tumor size reached 1 cm³. For all groups n = 24.

Figure 2

Tumor incidence for SV40 (a) and HER2/neu (b) mice. Tumor formation began around 16 weeks for SV40 mice and 21 weeks for HER2/neu mice. For both controls and experimental mice in both SV40 and HER2 experiments n = 24 mice. * = p < 0.05 and ** = p < 0.01.

Figure 3

Tumor weight (a) and latency (b) of HER2/neu mice. BSp treatment significantly decreased mean tumor weight by 0.9 g and significantly increased mean tumor latency. For each group n = 24 mice. * = p < 0.05.

Figure 4

Expression of key tumor suppressor genes in HER2/neu mouse breast tumors. We observed significant increases in relative gene expression in p53 (a), p21 (b), and BRCA2 (c). We also observed significant increases in protein expression for p53 (d,g), p21 (e,h), and BRCA2 (f,i). For RT-qPCR, n = 10, and for Western blots, n = 6. * = p < 0.05. The uncropped blots are shown in File S1.

Figure 5

RNA sequencing heatmap (a), downregulated gene ontology (b), and upregulated gene ontology (c) analyses for breast tumor samples of peripubertal BSp-treated HER2/neu mice. For RNA-sequencing (a), n = 7 and genes were deemed significant if their adjusted p-value was p < 0.05. RNA sequencing results informed GO analyses in (b,c). For GO analyses, a cutoff of adjusted p < 0.05 was used to determine significant enrichment. The histograms shown here represent the GO groupings with the top 5 highest gene counts, as well as other groupings with physiological relevance to cancer biology.

Figure 6

Heatmap of differentially methylated genes within breast tumor samples of BSp-treated mice vs. controls (a), as well as subsequent Venn diagram (b) of overlap between differentially expressed (red) and differentially methylated (blue) genes. Erich4 was hypomethylated and overexpressed, and these results were verified with RT-qPCR (c). For RRBS and RNAseq, n = 7. For RT-qPCR, n = 10. * = p < 0.05.