Maternal fish oil consumption has a negative impact on mammary gland tumorigenesis in C3(1) Tag mice offspring

Abstract

Purpose

Omega-3 fatty acids have been shown to reduce the incidence and slow the growth of mammary gland cancer in rodent models. Since exposure to dietary components during the critical developmental times of gestation and lactation may alter risk for mammary gland cancer in females, we tested whether exposure to increased levels of long-chain omega-3 fatty acids from fish oils would be preventive or promotional to mammary gland cancer in the offspring.

Methods:

Normal SV129 female mice were fed AIN 76 diets containing either 10% corn oil (control, 50% omega 6, n-6) or 5% of an omega-3 (n-3) fatty acid concentrate (fish oil 60% n-3) + 5% canola oil (10% n-3 + 20% n-6). Females were then mated with C(3)1 TAg transgenic mice. At weaning (3 weeks), pups were randomized to either the corn (C) or fish oil (F) diet, 15–17 mice per group. Four experimental groups were generated: FF, FC, CF and CC. Tumor incidence and multiplicity were assessed at the following time points 120, 130 and 140 days of age. A panel of genes encoding signal transduction proteins were analyzed in mammary glands at 130 days.

Results:

Mice never exposed to fish oil (CC group) had a significantly higher incidence and multiplicity of mammary gland tumors than mice exposed to fish oil throughout life (FF group). Mice exposed to fish oil during a portion of life (CF or FC) had intermediate tumor incidences and multiplicities. Results also indicate that maternal consumption of fish oil increased the expression of genes associated with immune system activation (Ccl20, Cd5, Il2, Lef1, Lta).

Conclusions:

Adequate omega-3 fatty acids in the maternal diet may reduce the risk for mammary gland cancer in the offspring. If humans make dietary change by consuming more omega-3 fat instead of corn oil with 0% omega 3 fat, breast cancer may be reduced in the next generation.

Introduction

A comprehensive report published in 2018 by World Cancer Research Fund (WCRF) and American Institute for Cancer Research (AICR) under Continuous Update Project (CUP) states that over 2 million new cases of breast cancer were diagnosed in 2018 [1]. Based on the report, countries with the highest incidence rates of breast cancers are in Europe, Oceania and North America. The report stresses that overweight is an increased risk factor for breast cancer development. In this context, food intake becomes an essential player in slowing or increasing the risk for breast cancer development. A diversity of approaches involving food (e.g., nuts, veggies, fruits, spices) [2–5], food components (e.g., fat, vitamins, minerals) [6, 7] and dietary supplements (e.g., quercetin, curcumin, resveratrol) [8–10] are recognized as complementary methodologies in cancer prevention and treatment. It is recognized that the maternal diet during pregnancy impacts the mammary gland health of offspring in animal models [11–15]. The impact of in utero high-fat exposure is not limited only to the mammary glands [15, 16]. It is well known that dietary fat also affects the development of the immune system [15, 17, 18]. Human studies are coming along and supporting the positive impact of omega-3 (n-3) FA (EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid)) supplemented maternal diet on the infants’ immune system development [19]. The fish oil supplemented maternal diet (during late pregnancy and/or lactation) and feeding infant formula enriched in ARA (arachidonic acid) and DHA seems to alter the immune function markers in a direction that is beneficial for the infant’s health in the first year of life [19]. The beneficial effect of n-3 FA on the immune system development and lowering inflammation is associated with a decreased risk of breast cancer development [20–22]. Conversely, in many studies, high levels of n-6 FA are associated with inflammation, increased risk of obesity and the development of mammary tumors [11, 23, 24]. The questions are: 1. How efficient is a diet rich in n-3 FA consumed by mother and/or offspring to reduce the incidence of breast tumors when compared with a diet high in n-6 FA consumed by both mother and offspring? 2. If the diet of the mother is rich in n-6 fats and the offspring have a rich n-3 FA diet to what extend can this lower the risk of developing mammary tumors in the offspring? In this study, we chose a mixture of fish oil (to provide premade long-chain n-3 FAs) and canola oil (18C, n-3 FA and less of the essential n-6 linoleic acid than corn oil) as a source of n-3 FA. The beneficial effect of fish oil intake can rely on less active inflammatory response, production of anti-inflammatory molecules, inhibition of nuclear factor-κB and decreased signalling through growth factor receptor [21, 25, 26]. Animal models offer a handy approach to overcome the tremendous challenge of following up the effects of maternal/early life infant diet on the adult long-term health.

Methods

Animals

Forty-six female SV 129 mice, at 6 weeks of age, were obtained from Charles River Laboratories (Wilmington, MA). Mice that bear a transgene for the SV40 large T antigen with a C3(1) rat prostatic steroid binding protein promoter were obtained from Dr. Jeffrey Green for breeding. The hemizygous female transgenic mice are expected to develop mammary gland cancer due to expression of the large T antigen in the mammary gland [27]. The transgenic line was maintained in the laboratory and all experimental mice were genotyped to ensure presence of the transgene [28]. All animal work was approved by the Marshall University School of Medicine Institutional Animal Care and Use Committee.

Study design

Mice were quarantined for 2 weeks, and then moved to a study room. SV129 females were split into two groups and numbered for identification. Twenty-three female mice were placed on a diet containing 10% w/w corn oil (high n-6, control diet) [28] and 23 female mice were placed on a diet containing 5% w/w fish oil concentrate + 5% canola oil w/w (high n-3 diet, test diet). After 2 weeks, these females were bred with homozygous C3(1)/TAg male mice. The hemizygous female pups from these breedings were the experimental mice NOT the wild-type mother mice. Pups were weaned at 21 days old and placed on the two diets resulting in four experimental groups: corn/corn (CC); corn/fish (CF); fish/corn (FC) and fish/fish (FF) (the first diet is the maternal diet and the second diet is the pup’s diet). Mice that were not exposed to an n-3 diet at any time are considered the baseline controls. The offspring were housed three to four in a cage, individually numbered for identification, and weighed weekly.

Diet

Diets were prepared in the Marshall University School of Medicine animal diet prep room. Diets were based on the AIN-76A formula and only the fat content was altered to obtain a 10% by weight total fat [28]. The diets were isocaloric and isonutrient, and are relevant to human consumption. The dry ingredients of the diet, except sugar, were obtained in bulk from MP Biomedicals (Solon, Ohio). Omega-3 concentrates from fish oil (containing 60% EPA and DHA) were obtained from Zone Labs Inc. (Danvers, MA). Sugar and oil (100% canola oil and 100% corn oil) were obtained locally. Diets were prepared in batches as needed. The diet mixture was pressed into trays and cut into small squares. Individual cage sized portions (25–30 g) were stored in sealed containers at −20°C to prevent oxidation of the fat and bacterial growth in the food. Mice had free access to food and water and were fed fresh food daily. Food removed from cages was discarded.

Assessment of transgene copy number

Ear punches stored at −20°C were digested as previously described [28]. The presence of SV40 Tag transgene was assessed by real-time PCR method, using an ABI Prism 7000 instrument (Applied Biosystems, Foster City, CA).

Body weights

Body weights were recorded each week (data not shown) and terminally.

Necropsy

Mice were euthanized at 120, 130, 140 days of age. The left 4^th mammary gland was quickly removed and frozen in liquid nitrogen. All ten mammary glands were examined for the presence of a tumor 1 mm or larger. All tumors detected were measured, removed and weighed, thus total tumor weight and numbers include many tumors that were too small to be detected by palpation. The number of tumors in each gland and the number of glands with tumor were recorded for every mouse. Samples of inguinal fat and liver were removed and frozen in liquid nitrogen until further analyses.

Whole mount mammary glands of C3(1)T_AG/129 female mice: glands were flattened, fixed and defatted in xylene then stained with hematoxylin and eosin [29, 30]. The pictures were taken using a 2.5X objective on a Zeiss microscope and a zoom factor of 100 on the Kodak digital camera.

Fatty acid composition

The fatty acid composition of mammary glands, liver and fat pad at 130 days was analyzed by gas chromatography. Frozen tissues were thawed and homogenized in distilled water containing 0.1% BHT to prevent oxidation of the fatty acids. Lipids were extracted with chloroform/methanol, and the fatty acids were methylated followed by separation and identification using gas chromatography. Briefly, gas chromatography was performed using a PerkinElmer Clarus 500 Gas Chromatograph (Shelton, CT) with an Elite-WAX Polyethylene Glycol Capillary Column (Length: 30m, Inner Diameter: 0.53mm), at 220°C for 100 min with a helium carrier gas flow rate of 2ml/min. A fatty acid methyl ester standard (Nu-Chek-Prep, Elysian, MN) GLC #704, which contained ten fatty acids (methyl esters of stearate, oleate, linoleate, alpha linolenate, gamma linolenate, homogamma linolenate, arachidonate, eicosapentaenoate, docosapentaenoate, and docosahexaenoate) was used for peak identification. The fatty acid methyl esters were reported as the percent of the total methylated fatty acids (area under the curve).

Gene expression assay

Mouse Signal Transduction Pathway Finder^™ RT² Profiler^™ PCR Array, RT2 First Strand Kit and SuperArray RT2 qPCR Master Mix (SuperArray Bioscience Corporation, Frederick, MD) were used to analyze the expression of a panel of genes in mammary glands without any visible tumors at 130 days. Frozen tissue was homogenized in Tri Reagent (Sigma-Aldrich, St. Louis, Mo) following the protocol of the manufacturer to isolate the RNA. RNA quality control was performed for all samples. The gene expression assay followed the protocol provided by SuperArray. The relative fold differences in gene expression and statistical analyses were calculated on SuperArray software.

Immunohistochemistry

Expressions of proliferating cell nuclear antigen (PCNA) were assessed in mouse mammary gland at 140 days by immunohistochemistry. Paraffin sections (six per slides) were deparaffinized in xylene, hydrated in a series of graded ethanol, and rinsed in distilled water. Antigen retrieval was done in a microwave oven using Vector Antigen Unmasking solution (Vector Laboratories, Inc., Burlingame, CA). Endogenous peroxidase and Avidin/Biotin activity were blocked by 3% hydrogen peroxide and by Vector Avidin/Biotin blocking kit (Vector Laboratories, Inc., Burlingame, CA), respectively, following the manufacturers protocols. Following the blocking, the sections were probed with mouse anti-PCNA (Biogenex, San Ramon, CA). Vector M.O.M kit (Vector Laboratories, Inc., Burlingame, CA) was used to localize mouse primary antibodies. Sections were incubated with 3, 3’-diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA) and counterstained using hematoxylin. The number of PCNA positively stained nuclei per 1000 cells was compared between the four diet groups. Counting was performed within viable areas of lesions.

Results

Diet influence on body weight

The body weight was recorded terminally for each mouse. The mean weight of CF group at 120 days was significant higher compared to all other groups (Fig 1a). There was no significant difference between the four groups in body weight at 130 days. FC group at 140 days had a mean weight significant lower compared to CF and CC groups (Fig 1a).

Figure 1.

a Mean body weight at 120, 130 and 140 days. Body weight was determined for each mouse. There was significant difference between the groups at 120 and 140 days of age. At 120 days, CF* group had the highest mean weight compared to FF, FC and CC groups. At 140 days FC* group had the lowest mean weight compared to CF and CC. p<0.05 by Newman-Keuls Multiple Comparison Test, n>15. b Number of tumors per mouse. The mean number of tumors at 120 and 140 days was statistically lower in the FF, FC and CF groups than in CC group. The mean number of tumors at 130 days was statistically lower in the FF and CF groups than in CC group. (1 way ANOVA, Bonferroni’s Multiple Comparison). c Number of glands with tumor per mouse. 1 way ANOVA, Bonferroni’s Multiple Comparison Test showed that the number of glands with tumor per mouse was significantly lower in FF group than in CC group at all time points. FC and CF groups had a significantly lower number of glands with tumor than CC group at 120 and 140 days. d Tumor mass. The tumor mass at 120 days was significantly less in the FF and CF groups than in the CC group by Kruskal-Wallis test, Dunn’s Multiple Comparison. n>15, *p<0.05 for all statistical analysis. e Effect of diet on PCNA expression at 140 days. Expression of PCNA was significantly lower in FF and FC groups than in CC group. n=4, *p<0.05 by one-way Anova, Bonferroni’s Multiple Comparison Test

Diet influence on tissue lipid composition

The lipid composition of mammary glands, liver and fat pad at 130 days of age is shown in Table 1. At 130 days of age, the lipid composition of the tissues reflected the current diet of the mice. Gas chromatography was performed for FF and FC groups. The tissues of mice fed with fish oil containing diet had significantly more (p<0.05 by T-test) n-3 fatty acids (ALA, EPA, DPA, DHA) and significantly less (p<0.05 by T-test) n-6 fatty acids (LNA, GLA) than the mice switched to the corn oil-containing diet. The content of ARA (n-6) in mammary glands and fat pads was not statistically different (p>0.05 by T-test) between the groups (FF and FC). There was a statistically significant increase (p<0.05 by T-test) of ARA in the liver of mice switched to corn diet at weaning compared to fish diet fed mice.

Table 1.

Tissue lipid composition at 130 days.

	Mammary gland			Liver			Fat pad
Fatty acid	FF	FC	p value	FF	FC	p value	FF	FC	p value
LNA	14.26±0.92	42.47±1.86	0.000000	11.78±0.80	31.72±2.53	0.000006	15.97±0.35	47.77±0.30	0.000000
GLA	0.04±0.01	0.08±0.01	0.000448	0.04±0.01	0.49±0.09	0.000070	0.03±0.01	0.07±0.01	0.000014
ARA	0.19±0.04	0.26±0.05	0.065569*	7.27±0.39	13.75±1.24	0.000058	0.13±0.03	0.13±0.01	1.000000*
ALA	2.47±0.47	0.29±0.01	0.000089	1.05±0.19	0.15±0.03	0.000093	2.74±0.20	0.38±0.02	0.000000
EPA	1.13±0.37	0.05±0.01	0.001086	11.49±1.35	0.20±0.05	0.000003	1.07±0.54	0.04±0.01	0.008769
DPA	0.25±0.05	0.04±0.01	0.000183	2.70±0.05	0.26±0.06	0.000000	0.27±0.10	0.02±0.00	0.002893
DHA	0.50±0.15	0.02±0.01	0.000691	20.68±1.24	4.38±0.41	0.000000	0.64±0.27	0.02±0.00	0.00397

The percents of assayed fatty acids of mammary gland, liver and fat pad at 130 days are shown. The results of T-tests between each pair of treatments for each tissue are shown, n =4.

^*indicates a p>0.05 which is not statistically significant. LNA—linoleic acid; GLA—gamma linolenic acid; ARA—arachidonic acid; ALA—α-linolenic acid; EPA—eicosapentaenoic acid; DPA—docosapentaenoic acid; DHA—docosahexaenoic acid.

Diet influence on tumor multiplicity and weight

The total number of tumors, glands with tumors, the tumor weight per mouse (Fig 1b, c, d) and the number of tumor-free animals (Table 2) were assessed in C3(1)T_AG/129 female offspring at 120, 130, 140 days of age. The presence of the transgene was confirmed in all pups used in the experiment (data not showed). Images of fully developed mammary gland, hyperplasia and mammary tumor are presented in Supplementary Fig 1.

Table 2.

Tumor free animals at 120, 130 and 140 days

Days of age	% Tumor free animals							Tumor free				Number of animals
	FF	FC	CF	CC	%FF-%FC	%FF-%CF	%FF-%CC	FF	FC	CF	CC	FF	FC	CF	CC
120	87.5	62.5	80	33.33	25	7.5	54.17	14	10	12	5	16	16	15	15
130	87.5	60	68.75	53.33	27.5	18.75	34.17	14	9	11	8	16	15	16	15
140	64.7	38.88	33.33	13.33	25.82	31.37	51.37	11	7	5	2	17	18	15	15

Multiplicity:

Since these mice bear a tumor promoting transgene, all mice are expected to develop tumors at some point. As shown in Fig 1b, the tumor multiplicity (mean number of tumors per mouse) at 120 and 140 days of age was significantly lower (p<0.05 by one-way ANOVA, Bonferroni’s Multiple Comparison) in the FF, FC and CF groups than in the CC group. At 130 days of age, Fig 1b, the multiplicity of tumors in the FF and CF groups was significantly lower than in the CC group. The number of glands with tumor at each time point for each group is presented in Fig 1c. The one-way ANOVA, Bonferroni’s Multiple Comparison revealed that there were significant effects due to the diet. The number of glands with tumor was significantly lower in the FF group than in the CC group at all time points, p<0.05. FC and CF groups at 120 and 140 days showed significantly lower number of glands with tumours per mouse compared to CC group (Fig 1c), p<0.05. The percent of tumor-free animals was higher for FF group at all time points investigated (Table 2). The comparison of the groups showed that the percentage of tumor-free animals had the same pattern for all time points investigated. At 120 days, there were 7.5%, 25% and 54.17% more tumor-free animals in FF group than in CF, FC and CC group, respectively. At 130 days, there were 27.5%, 18.75% and 34.17% more tumor-free animals in FF group than in FC, CF and CC group. At 140 days, the difference followed the same trend: 25.82%, 31.37% and 51.33% more tumor-free animals in FF group than in FC, CF and CC group. Mice exposed to the fish oil containing diet at any time (FF, FC and CF groups) were free of tumors at a higher rate than the mice never exposed to fish oil diet (CC group). As expected, the number of tumor-free animals decreased with time in all groups (Table 2). Tumor mass: The mean tumor mass, calculated from autopsy data, indicates a difference in tumor burden due to the diet. In Fig 1d, the tumor mass in the FF and CF groups was significantly lower (p<0.004 by Kruskal-Wallis test) than that of the CC group at 120 days. At 130 and 140 days, there were not statistically differences among the groups. The comparison of the groups showed that the difference between the number of tumors/mouse, glands with tumor/mouse and tumor mass/mouse, respectively, was not statistically significant between the FF and FC or CF groups nor between FC and CF groups (Fig 1 b, c, d).

Diet influence on gene expression at 130 days of age

The diet-induced changes in genes expression associated with reduction of tumor number per mouse were assessed by RT-PCR (the raw data are available as Supplementary Table 1). The assay was performed on mammary gland tissue without any visible tumor from C3(1)T_AG/129 mice offspring at 130 days of age. The CC group was the control group, the FF, FC and CF groups were the experimental groups for analysis. For a better understanding of similarities and differences in gene expression of different diet groups, the following groups were compared too: FF versus FC; FF versus CF; FC versus CF (Table 3). Table 3 presents genes that were analyzed and found to be at least two fold different between the compared groups. Differences in gene expression were likely induced by the maternal diet: 1. FF and FC groups had a similar trend in gene expression when compared to CC group. A significant fold up regulation of Ccl2, Ccl20, Cd5, Il2, Il2ra, Il4ra, Lef1, Lta gene expression suggests that the maternal diet might be responsible for boosting the immune response of progeny. 2. The CF group did not show a significant change in any of the genes analysed when compared to CC group. 3. The only statistically significant difference between FF and FC groups was present in two of the 84 genes investigated: Csf2 (Colony stimulating factor 2 (granulocyte-macrophage)) and Fasn (Fatty acid synthase). 4. The gene expression trend was similar when FF and FC groups were compared to CF. Although the trend was similar, a statistically significant difference was only present between FC/CF groups.

Table 3.

Mammary gland gene expression at 130 days

			Fold up-or down-regulation
			FF vs CC		FC vs CC		CF vs CC		FF vs FC		FF vs CF		FC vs CF
				p value		p value		p value		p value		p value		p value
LR family, apoptosis inhibitory protein 1	Naipl	Anti-apoptotic	11.54	0.046	18.97	0.001	1.62	0.442	−1.64	0.274	11.72	0.267	11.72	0.098
Baculoviral IAP repeat-containing 3	Birc3	Apoptotic suppressor	2.31	0.011	2.37	0.008	1.22	0.482	−1.03	0.885	1.94	0.268	1.94	0.098
Bone morphogenetic protein 4	Bmp4	Growth and differentiation factor	−2.03	0.026	−2.07	0.013	−1.45	0.058	1.02	0.849	0.70	0.266	−1,43	0.054
Chemokine (C-C motif) ligand 2	Ccl2	Augments monocyte anti-tumor activity	2.39	0.062	2.56	0.056	1.34	0.590	−1.07	0.759	1.92	0.267	1.92	0,075
Chemokine (C-C motif) ligand 20	Ccl20	Chemoattractant produced by activated T cells	3.53	0.008	3.72	0.008	−1	0.991	−1.05	0.729	3.73	0.267	3.73	0.008
CD5 antigen	Cd5	Regulator of T cell activation	28.29	0.050	36.22	0.0008	3.75	0.350	−1.28	0.684	9.66	0.271	9.66	0.039
Cyclin-dependent kinase inhibitor 2A	Cdkn2a	Tumor suppressor	2.75	0.060	1.74	0.272	−1.26	0.546	1.58	0.098	2.19	0.181	2.19	0.022
Colony stimulating factor 2 (granulocyte-macrophage)	Csf2	Granulocytes and monocytes growth factor	1.00	0.626	2.13	0.473	−1.21	0.963	−2.13	0.027	2.58	0.268	2.58	0.643
Fas ligand (TNF superfamily, member 6)	Fasl	Apoptosis	3.69	0.018	4.45	0.008	1.31	0.374	−1.21	0.491	3.40	0.267	3.40	0.025
Fatty acid synthase	Fasn	Catalyzes the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH.	−2.59	0.007	−1.15	0.311	−1.08	0.728	−2.26	0.011	0.94	0.631	−1.06	0.683
Hexokinase 2	Hk2	Glucose metabolism	−2.23	0.004	−1.35	0.032	1.18	0.348	−1.65	0.065	0.62	0.350	−1.60	0.096
Interleukin 2	Il2	Regulator of immune activation	5.04	0.003	4.98	0.038	−2.36	0.263	1.01	0.893	11.72	0.267	11.72	0.024
Interleukin 2 receptor, alpha chain	Il2ra	Receptor for interleukin-2	2.44	0.028	2.44	0.059	1.07	0.726	−1.00	0.947	2.28	0.267	2.28	0.155
Interleukin 4 receptor, alpha	Il4ra	Receptor for both interleukin 4 and interleukin 13	2.04	0.076	2.06	0.063	1.29	0.532	−1.01	0.966	1.60	0.267	1.60	0.327
Lymphoid enhancer binding factor 1	Lef1	Participates in the Wnt signaling pathway; Regulates T-cell receptor alpha enhancer function	41.32	0.003	62.05	0.0007	4.72	0.353	−1.50	0.096	13.16	0.268	13.16	0.022
Leptin	Lep	Fat metabolism	−1.99	0.137	−3.06	0.026	−1.29	0.266	1.54	0.411	0.42	0.201	−2.38	0.066
Lymphotoxin A	Lta	Stimulation of B-cells; inhibition of tumor angiogenesis	11.87	0.009	23.9	0.014	2.32	0.359	−2.01	0.107	10.30	0.269	10.30	0.065
Matrix metallopeptidase10	Mmp10	Degrades proteoglycans and fibronectin	4.18	0.211	5.11	0.094	−1.15	0.530	−1.22	0.978	5.88	0.267	5.88	0.084
Nitric oxide synthase 2, inducible	Nos2	Produces nitric oxide	3.59	0.266	3.28	0.163	−1.27	0.647	1.09	0.944	4.17	0.266	4.17	0.149
Peroxisome proliferator activated receptor gamma	Pparg	Adipogenesis regulator	−1.78	0.072	−1.64	0.054	1.32	0.261	−1.08	0.850	0.46	0.273	−2.18	0.038
Transcription factor 7, T-cell specific	Tcf7	Wnt signaling pathway	9.18	0.048	10.62	0.001	1.94	0.379	−1.16	0.857	5.47	0.285	5.47	0.037
Tumor necrosis factor	Tnf	Cytokine involved in cell proliferation, differentiation, apoptosis, lipid metabolism, coagulation	2.84	0.008	2.77	0.001	1.2	0.545	1.02	0.811	2.30	0.266	2.30	0.101
WNT1 inducible signaling pathway protein 1	Wisp1	Downstream regulator in the Wnt signaling pathway. Associated with cell survival	3.79	0.034	2.03	0.030	1.11	0.808	1.60	0.400	1.12	0.267	1.12	0.565

Gene expression was analyzed in 3 mammary glands from mice at 130 days of age. Mammary tissue without macroscopic tumor was selected for assay. Most significant differences in gene expression were due to the diet of the mother. FF=fish/fish; FC=fish/corn; CF=corn/fish; CC=corn/corn; p by ttest

The diet of the mother during pregnancy and lactation can be important to ‘set up’ or imprint the offspring in such a way that cancer development during adult life might be altered.

Diet influence on PCNA expression

Maternal consumption of a high n-3 diet (FF and FC groups) was able to up regulate certain genes in female offspring (Table 3) and to reduce the expression of PCNA (Fig 1e). The diet induced changes in cell proliferation was assessed by immunohistochemistry of PCNA (Proliferating Cell Nuclear Antigen) expression in mammary gland tissue of mice at 140 days of age. FF and FC groups had a significantly lower expression of PCNA than CC group by one-way Anova, Bonferroni’s Multiple Comparison Test.

Discussion

Western diet is well known for its high n-6 fat composition and the negative impact on the human health [24, 31–33]. On the basis of this understanding, a high n-6:n-3 ratio is likely to support the development and progression of breast cancer while a low n-6:n-3 ratio has beneficial effects of slowing down the cancer progression [34]. Even more, the results presented herein point out that fish oil consumption was able to delay the breast cancer development in mice genetically modified to develop the tumours. C3(1)/SV40 TAg model used in this study is very well characterized [35]. The breast invasive carcinoma was observed in 100% of homozygous female mice at 16 weeks of age (112 days). Hemizygous female mice used in this study resulted from the breeding of SV129 female with C3(1)/SV40 TAg males. The hemizygous female mice bearing the transgene were prone to develop breast tumours. The efficiency of fish oil consumption by both mother and offspring to reduce the incidence of the tumors in this study was significant. This was translated into 87.5% tumor-free animals at the age of 17 weeks (120 days) compared to the mice on a diet rich in n-6 FA (corn oil) where the percentage of tumor-free animals at 120 days was only 33.33%. The efficiency of fish oil diet to slow down the tumor progression was shown in a longer term. At the age of 20 weeks (140 days), the percentage of tumor-free animals in the FF group was 64.7% compared to only 13.33% in the CC group. Similar pattern in percentage difference of free tumor animals was shown between the two groups (FF and CC) at 120 and 140 days. The consumption of fish oil after weaning appeared to delay the development of tumors. Exposure of mothers or/and offspring to a fish oil diet seemed to be beneficial though not a significantly reduction on tumor multipliticy. Even though the intermediate groups (FC and CF) showed a high tumor multiplicity at 140 days the mean glands with tumor and mean tumor/mouse scored less than in the CC group which never had a fish oil intake. The underlying mechanism for the efficiency of fish oil diet in slowing down breast cancer development may rely on the immune system boost. The efficiency of fish oil supplementation in breast cancer prevention and immune regulation was also shown on HER-2/ neu transgenic mouse model [36]. In our study, the fish oil immune regulation was supported by the fact that the gene expression of Cd5 (regulator of T cell activation), Lef1 (regulates T-cell receptor alpha enhancer function), Lta (responsible for B-cells stimulation) were highly up-regulated in mammary glands from mice that were on a fish oil diet (mother and/or offspring). The data showed that the most significant differences in gene expression were due to the diet of the mother. The diet of the mother appears to improve the lifelong outcome in the offspring. Il2 (regulator of immune activation), Il2ra (receptor for interleukin-2), Tcf7 (Transcription factor 7, T-cell specific) were also up-regulated in offspring whose mothers were on fish oil diet. The same diet was also responsible for Bmp4 (growth and differentiation factor) down-regulation. The marker for proliferating cells, PCNA staining additionally supported the fact that the fish oil diet of the mother highly and negatively impacted the development of tumors in offspring. The findings of this study are in line with our [12, 26, 28] and others investigations [11, 37–39] in supporting the fact that a healthier n-3:n-6 ratio is beneficial for diseases promoted by inflammation [34, 40–42]. The study supports the notion that an n-3-rich diet consumed by the mother may reduce cancer development and/or progression in offspring. In humans, the positive impact of n-3 FA on the immune system function is also supported by different clinical trials [19, 21, 26, 43]. In patients with early CLL, fish oil supplements reduced NFkB activation by altering lymphocyte mRNA expression in patients with higher initial NFkB activation [26]. A study published in 2017 by Paixão EMDS et al. showed that newly diagnosed breast cancer patients treated with fish oil supplements were able to maintain the level of CD4⁺ T cells and had less inflammatory response [21]. An extended review by Richard et al. (2016) covered the importance of DHA in postnatal/breast-feeding for the immune system development in early life [19].

In humans, it is very difficult to track how the diet of the mother (before/during pregnancy and lactation) can affect not only the early life development of the infants but the health events that occur in adult life. Epigenetic events that occur in utero might lead to changes in gene expression that persist throughout life and animal models provide a good glimpse on what and how that might be.

Supplementary Material

1691652_Sup_1

Supplementary Figure 1 Whole mount mammary glands of C3(1)T_AG/129 female mice. In pups at weaning (3 weeks old), the mammary gland was developing. In adult mice, the mammary gland was fully developed, with hyperplastic areas and tumors.

Abbreviations

n-3

Omega 3

n-6

Omega 6

C

corn oil

F

fish oil

FA

fatty acids

LNA

linoleic acid

GLA

gamma linolenic acid

ARA

arachidonic acid

ALA

α-linolenic acid

EPA

eicosapentaenoic acid

DPA

docosapentaenoic acid

DHA

docosahexaenoic acid

Naip1

NLR family, apoptosis inhibitory protein 1

Birc3

Baculoviral IAP repeat-containing 3

Bmp4

Bone morphogenetic protein 4

Ccl2

Chemokine (C-C motif) ligand 2

Ccl20

Chemokine (C-C motif) ligand 20

Cd5

CD5 antigen

Cdkn2a

Cyclin-dependent kinase inhibitor 2A

Csf2

Colony stimulating factor 2 (granulocyte-macrophage)

Fasl

Fas ligand (TNF superfamily, member 6)

Fasn

Fatty acid synthase

Hk2

Hexokinase 2

Il2

Interleukin 2

Il2ra

Interleukin 2 receptor, alpha chain

Il4ra

Interleukin 4 receptor, alpha

Lef1

Lymphoid enhancer binding factor 1

Lep

Leptin

Lta

Lymphotoxin A

Mmp10

Matrix metallopeptidase10

Nos2

Nitric oxide synthase 2, inducible

Pparg

Peroxisome proliferator activated receptor gamma

Tcf7

Transcription factor 7, T-cell specific

Tnf

Tumor necrosis factor

Wisp1

WNT1 inducible signaling pathway protein 1