Maternal intake of high n-6 polyunsaturated fatty acid diet during pregnancy causes transgenerational increase in mammary cancer risk in mice

Abstract

Background: Maternal and paternal high-fat (HF) diet intake before and/or during pregnancy increases mammary cancer risk in several preclinical models. We studied if maternal consumption of a HF diet that began at a time when the fetal primordial germ cells travel to the genital ridge and start differentiating into germ cells would result in a transgenerational inheritance of increased mammary cancer risk.

Methods: Pregnant C57BL/6NTac mouse dams were fed either a control AIN93G or isocaloric HF diet composed of corn oil high in n-6 polyunsaturated fatty acids between gestational days 10 and 20. Offspring in subsequent F1-F3 generations were fed only the control diet.

Results: Mammary tumor incidence induced by 7,12-dimethylbenz[a]anthracene was significantly higher in F1 (p < 0.016) and F3 generation offspring of HF diet-fed dams (p < 0.040) than in the control offspring. Further, tumor latency was significantly shorter (p < 0.028) and burden higher (p < 0.027) in F1 generation HF offspring, and similar trends were seen in F3 generation HF offspring. RNA sequencing was done on normal mammary glands to identify signaling differences that may predispose to increased breast cancer risk by maternal HF intake. Analysis revealed 1587 and 4423 differentially expressed genes between HF and control offspring in F1 and F3 generations, respectively, of which 48 genes were similarly altered in both generations. Quantitative real-time polymerase chain reaction analysis validated 13 chosen up- and downregulated genes in F3 HF offspring, but only downregulated genes in F1 HF offspring. Ingenuity Pathway Analysis identified upregulation of Notch signaling as a key alteration in HF offspring. Further, knowledge-fused differential dependency network analysis identified ten node genes that in the HF offspring were uniquely connected to genes linked to increased cancer risk (ANKEF1, IGFBP6, SEMA5B), increased resistance to cancer treatments (SLC26A3), poor prognosis (ID4, JAM3, TBX2), and impaired anticancer immunity (EGR3, ZBP1).

Conclusions: We conclude that maternal HF diet intake during pregnancy induces a transgenerational increase in offspring mammary cancer risk in mice. The mechanisms of inheritance in the F3 generation may be different from the F1 generation because significantly more changes were seen in the transcriptome.

Keywords: Breast cancer; Maternal diet; Primordial germ cells; Transgenerational; n-6 Polyunsaturated fatty acids.

Fig. 1

Transgenerational study design. a Pregnant C57BL/6NTac mice (F0) were fed either a high-fat (HF; n = 10) or control (CON; n = 10) diet. The HF diet was fed to dams from gestational day (GD) 10 to GD 20. All offspring were fed the CON diet after birth for the remainder of the study, including during pregnancies of F1 and F2 generation offspring. b All pups were cross-fostered at birth (postnatal day [PND] 1) to a CON mother to eliminate litter bias. Pups were weighed on PNDs 2 and 3 and weaned on PND 21. Tumorigenesis was initiated on PND 42 by priming female mice with medroxyprogesterone acetate (MPA; 15 mg/kg), followed by oral gavage of 7,12-dimethylbenz[a]anthracene (DMBA; 1 mg/dose/week) for 3 weeks. Tumorigenesis was monitored by palpation once per week starting 3 weeks after final DMBA administration up to 20 weeks post-DMBA. Mammary glands (MGs) and tumors were collected and processed from F1 and F3 offspring unexposed to DMBA on PND 50 for whole mounts and on PND 100 to perform RNA sequencing analysis

Fig. 2

Transgenerational effect of maternal control (CON) or high-fat (HF) diet on offspring mammary tumorigenesis. Differences in mammary tumor incidence in (a) F1 (p < 0.016; CON, n = 30 mice; HF, n = 28 mice) and (b) F3 (p < 0.040; CON, n = 19 mice; HF, n = 24 mice) generation female offspring of dams fed either CON or HF diet during pregnancy. Differences in mammary tumor burden in (c) F1 (p < 0.027) and (d) F3 (p < 0.242) generation female offspring. Differences in mammary tumor latency in (e) F1 (p < 0.028) and (f) F3 (p < 0.110) generation female offspring. Mean ± SEM data are shown in c–f. TEB Terminal end bud

Fig. 3

Effect of maternal control (CON) or high-fat (HF) diet exposure on offspring mammary gland development. a The left fourth mammary glands were obtained on postnatal day 50 for whole mounts. Terminal end buds, structures in the enlarged image indicated by the arrows, were counted for (b) F1 (p < 0.035; n = 8 for HF and n = 6 for CON) and (c) F3 (p < 0.023; n = 5 for HF and n = 4 for CON). Mean ± SEM data are shown. DMBA 7,12-Dimethylbenz[a]anthracene

Fig. 4

Differentially expressed genes (DEGs) in mammary glands of F1 and F3 generation offspring of dams fed either control (CON) or high-fat (HF) diet during pregnancy. a RNA-sequencing analysis identified 1587 DEGs in F1 and 4423 DEGs in F3 generation mammary glands obtained on postnatal day 100 from mice not exposed to 7,12-dimethylbenz[a]anthracene (n = 5 CON and n = 3 HF offspring in F1 generation, and n = 4 CON and n = 5 HF offspring in F3 generation). A total of 390 common DEGs were found in the F1 and F3 generations, with 48 regulated in the same direction in both generations. Heat map of common 48 DEGs in (b) F1 mammary glands and (c) F3 mammary glands. Knowledge-fused differential dependency networks cluster map of nodes uniquely connected to different sets of genes in HF or CON offspring in (d) F1 or (e) F3 generation. Yellow ovals indicate nodes. Single-lined green connections indicate gene interactions in HF offspring. Double-lined red connections indicate gene interaction in CON offspring

Fig. 5

Verification of differential gene expression. Validation by quantitative real-time polymerase chain reaction of the following 13 differentially expressed genes identified in RNA-sequencing analysis: (a) Akt2, (b) Egr3, (c) Hes1, (d) Id4, (e) Jam3, (f) Pcdhga8, (g) Slc26a10, (h) Tbx2, (i) Igfbp6, (j) Oas3a, (k) p21, (l) Slfn1, and (m) Zbp1 (p < 0.05, a different from control diet [CON], b different from F1 high-fat (HF) diet, c different from F3 HF; p < 0.06, d marginally different from CON). We used fourth mammary glands obtained on postnatal day 100 from six CON and six HF offspring in F1 generation, as well as from six control and six HF offspring in F3 generation for the analysis. Mean ± SEM data are shown. Akt2 Serine/threonine kinase 2, CON Control diet, Egr3 Early growth response 3, Hes1 Hairy and enhancer of split-1, HF High fat, Id4 DNA-binding protein inhibitor ID-4, Igfbp6 Insulin-like growth factor binding protein 6, Jam3 Junctional adhesion molecule 3, Oas3 2′-5′-Oligoadenylate synthetase 3, Pcdhga8 Protocadherin gamma subfamily A, 8, Slc26a10 Solute carrier family 26 member 10, Slfn1 Schlafen 1, Zbp1, Z-DNA binding protein 1