In utero exposure of rats to high-fat diets perturbs gene expression profiles and cancer susceptibility of prepubertal mammary glands

Abstract

Human studies suggest that high-fat diets (HFDs) increase the risk of breast cancer. The 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis rat model is commonly used to evaluate the effects of lifestyle factors such as HFD on mammary tumor risk. Past studies focused primarily on the effects of continuous maternal exposure on the risk of offspring at the end of puberty (PND50). We assessed the effects of prenatal HFD exposure on cancer susceptibility in prepubertal mammary glands and identified key gene networks associated with such disruption. During pregnancy, dams were fed AIN-93G-based diets with isocaloric high olive oil, butterfat or safflower oil. The control group received AIN-93G. Female offspring were treated with DMBA on PND21. However, a significant increase in tumor volume and a trend of shortened tumor latency were observed in rats with HFD exposure against the controls (P=.048 and P=.067, respectively). Large-volume tumors harbored carcinoma in situ. Transcriptome profiling identified 43 differentially expressed genes in the mammary glands of the HFBUTTER group as compared with control. Rapid hormone signaling was the most dysregulated pathway. The diet also induced aberrant expression of Dnmt3a, Mbd1 and Mbd3, consistent with potential epigenetic disruption. Collectively, these findings provide the first evidence supporting susceptibility of prepubertal mammary glands to DMBA-induced tumorigenesis that can be modulated by dietary fat that involves aberrant gene expression and likely epigenetic dysregulation.

Keywords: Breast cancer; DNA methyl transferases; DNA methylation; Developmental origin of health and disease; Epigenetics; Methylated DNA binding domain.

Fig. 1

Clustering analysis of genes with p<0.05 in control and treatment groups. Heat map shows the two signature panels of differentially expressed genes in response to an HFBUTTER (HFB) diet and a control AIN diet (CTL). Green represents low expression; red represents high expression.

Fig. 2

Pathway analysis of 43 genes differentially expressed in HFBUTTER (HFB) vs. CTL groups. The major networks involved are estradiol, progesterone, ERK/MAPK, NFk-B, VEGF, and ubiquitin (Ub) C signaling. Ingenuity pathway analysis shows the most significant gene networks of the HFB group. A, estradiol and progesterone network: 11 genes were mapped principally to this network as distinct node. B, ERK, NFk-B, and VEGF network: 16 genes were mapped to these networks as a distinct node. C, Ub network: 13 genes were mapped to a network with Ub as a central node. The intensity of the node color indicates the degree of up- or downregulation. Genes in uncolored nodes were not identified as differentially expressed in our array experiments and were incorporated into individual networks on the basis of the IPA knowledge database, indicating a relevance to this network.

Fig. 3

Real-time PCR analyses of differentially expressed genes in this study. RNA sequencing shows transcripts levels of 9 genes. Relative levels of transcript expression of rPla2g2a (upregulated), rCadm4, rCsn1s1, rDbf4, rLrrn1, rBtn1a1, rNf1, rTmem45b, and rSlc6a14 (downregulated) in day 21 rat mammary gland of HFBUTTER (HFB) groups (n=5) compared with control (CTL) (n=5). Comparison with CTL used Student's t-test. p<0.05 compared with control.

Fig. 4

Expression of rDNMT3a, rMBD1, and rMBD3 in HFBUTTER (HFB) vs. CTL. Relative levels of transcript expression of rDnmt3a, rMbd1 and Mbd3 in day 21 rat mammary gland of HFB (n=5) groups compared with control (CTL) (n=5). Comparison was performed with CTL using Student's t-test. p<0.01 compared with control.

Fig. 5

Real-time PCR analyses of differentially expressed genes in HFBUTTER (HFB), HFSAFF (HFS) and HFOLIVE (HFO) groups. Significant difference in the gene expression patterns of Lrrn1, Nf1, Dbf4, Cadm4, Tmem45b, and Btn1a1 genes among HFB, HFS and HFO groups relative to control (CTL). Comparison was performed using one way ANOVA. *p<0.05, **p<0.01, ***p<0.001 compared among three different fats relative to control.