The Effects of Omega-3 Polyunsaturated Fatty Acids on Breast Cancer as a Preventive Measure or as an Adjunct to Conventional Treatments

In order to understand how omega-3 polyunsaturated fatty acid (ω-3 PUFA) supplements affect breast cancer prevention and treatment, a systematic review of articles published in the last 5 years in two databases was performed. Of the 679 articles identified, only 27 were included and examined based on five topics, taking into account: the induction type of the breast cancer used in animal models; the characteristics of the induction model by cell transplantation; the experimental design of the ω-3 supplementation—combined or not with a treatment antitumor drug; the fatty acids (FAs) composition used; the analysis of the studies’ outcomes. There are diverse and well-established animal models of breast cancer in the literature, with very relevant histological and molecular similarities depending on the specific objective of the study, such as whether the method of tumor induction was transgenic, by cell transplantation, or by oncogenic drugs. The analyses of outcomes were mainly focused on monitoring tumor growth, body/tumor weight, and molecular, genetic, or histological analyses, and few studies evaluated latency, survival, or metastases. The best results occurred when supplementation with ω-3 PUFA was associated with antitumor drugs, especially in the analysis of metastases and volume/weight of tumors or when the supplementation was started early and maintained for a long time. However, the beneficial effect of ω-3 PUFA supplementation when not associated with an antitumor agent remains unclear.


Introduction
Breast cancer (BC) is the second most common neoplasm in the world, with 2.3 million new cases diagnosed in 2020. By 2040, due to population growth and aging, the worldwide incidence of breast cancer is expected to rise to more than three million cases with one million deaths each year [1]. BC normally affects young and mainly Black women, who represent 10% to 20% of BC cases. The risk of developing BC is influenced by factors such as lifestyle, physical activity, eating habits, genetic predisposition, sex, and age [2][3][4][5].
BC is widely characterized into four major subtypes, based on invasiveness, proliferation, and gene expression [6][7][8], known as (1) Luminal A, (2) Luminal B, (3) HER2+, and (4) Triple Negative. This classification of BC depends on the presence or absence of some biomarkers such as the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2), which are used to diagnose and guide the adjuvant treatment options for BC.

Inclusion Criteria
The review included only original full-text articles written in English, published within the last 5 years, and that had used different inductions of BC in animal models (transgenic or induction by tumor cells or drugs) to evaluate the effect of dietary enrichment with ω-3 PUFAs or the use of ω-3 PUFAs in the treatment or prevention of BC, combined or not with other antitumor treatments. From the perspective of the patient, and the intervention, comparison, and outcome (PICO) criterion, the study problem was the unclear effect of ω-3 PUFAs on BC. Intervention: FAs supplementation before and after the induction of BC; Comparison: to assess the real benefits of the use of ω-3 PUFAs as a coadjuvant in the prevention or treatment of BC; Outcome: alternative treatment of BC.

Exclusion Criteria
We excluded studies based on the following criteria: (i) reviews, (ii) publications written in languages other than English, (iii) indexed articles published in more than one database (duplicates), (iv) only in vitro studies, (v) only clinical data analyses, and (vi) diets without ω-3 PUFAs.

Data Extraction
The papers under evaluation were analyzed using five topics, which were represented in tables that addressed the following characteristics: (1) characteristics of breast tumor experimental models; (2) characteristics of the tumor cell transplantation model; (3) design of the experiment based on the FAs dietary supplementation with or without antitumor treatment; (4) main outcomes; (5) dietary supplements based on the types of omega-3-6-9 FAs utilized and their components.

Risk of Bias Assessment
The selection of articles was performed in pairs and a third independent author decided if the articles should be included. The data selected in the tables were divided by the authors into the groups already described above, and the checking of the data was carried out by the respective group. The final inclusion of studies into the systematic review was by agreement between all reviewers.

Data Analysis
To highlight the key traits, peculiarities, and exceptions, according to the applicability, the data gathered in each of the tables were evaluated in percentages and ranges of distribution. The frequency and quantity distribution of each type of the omega-3-6-9 FAs were also examined in the chosen studies. All data are considered to be the changes in the treated or experimental group (ω-3 PUFA, drug, or a combined treatment) compared to the control group (vehicle or FAs) data for each study.

Overview of the Screening Process of the Included Studies
Following the inclusion and exclusion criteria described above, we found 679 papers in the last five years throughout the PubMed and Scopus databases, with 216 from PubMed and 463 from Scopus. Of the 216 articles identified from PubMed, 174 were excluded after screening because 112 studies were with humans, 39 were reviews, 18 were clinical trials, 2 were meta-analyses, 1 study was written in a language other than English, and 2 were editorials. After the eligibility assessment, a further 23 of the 42 studies were excluded, in which 13 showed in vitro studies only, 4 did not use a BC model for the ω-3 PUFAs' dietary supplementation, and in 6 studies the diet did not include ω-3 PUFAs. Of the 463 articles found in Scopus, 450 were excluded after the screening, 183 studies were with humans, 155 were reviews, 27 were clinical trials, 14 were meta-analyses, 2 studies were in a language other than English, 13 were editorials, 14 were book chapters, 6 were communications, and 36 were duplicates of articles from the PubMed database. The eligibility analysis excluded a further five articles-three studies reported only in vitro results and two did not use the BC model. Thus, only 27 unduplicated full-text articles [17][18][19] were included in this systematic review, 19 from PubMed and 8 from Scopus, as shown in Figure 1. and two did not use the BC model. Thus, only 27 unduplicated full-text articles [17][18][19] were included in this systematic review, 19 from PubMed and 8 from Scopus, as shown in Figure 1.

Characteristics of the Breast Cancer Experimental Animal Model
The influence of an omega-3-6-9 FAs-enriched diet, combined or not with another antitumor treatment, on the prevention and treatment of BC was evaluated in this systematic review; the evaluation specifically focused on the omega FAs, combined or not with another antitumor treatment, evaluating their influence as the coadjuvant in conventional tumor treatment. The last five years has shown increasing attention to the real benefits of ω-3 PUFAs and their types and a more in-depth search of their actions against BC. In this first analysis, we describe the characteristics of BC induction in animal models used in the selected studies, as shown in Table 1, detailing the type of model, source of breast tumor, and the animal characteristics.

Characteristics of the Breast Cancer Experimental Animal Model
The influence of an omega-3-6-9 FAs-enriched diet, combined or not with another antitumor treatment, on the prevention and treatment of BC was evaluated in this systematic review; the evaluation specifically focused on the omega FAs, combined or not with another antitumor treatment, evaluating their influence as the coadjuvant in conventional tumor treatment. The last five years has shown increasing attention to the real benefits of ω-3 PUFAs and their types and a more in-depth search of their actions against BC. In this first analysis, we describe the characteristics of BC induction in animal models used in the selected studies, as shown in Table 1, detailing the type of model, source of breast tumor, and the animal characteristics. The breast tumor experimental model most used in the selected studies was the conventional tumor cell transplant in specific tissue regions of animal models (69%) [17][18][19]39,42,43,45,46,[49][50][51]53,55,[57][58][59][60][61][62], followed by the model of spontaneous tumor generation using transgenic animals (17%) [40,41,47,52,55,56], and lastly, 14% of studies induced BC in the animal model by carcinogenic drug transplant [44,48,54].
We analyzed the percentage of animal weight change in the selected studies according to the results on the effect of using an ω-3 PUFA-enriched diet as an antitumor treatment, or only an antitumor drug treatment, and the combined treatment (ω-3 PUFAs plus an antitumor drug). A diet enriched more with ω-3 PUFAs than ω-6 PUFAs showed a mean reduction of 46% in animal weight, varying from 1% to 151%, in which the last was a 51% weight gain (median 18%). In the experimental groups that analyzed only the drug effect, when compared with the control group (vehicle), the majority of animals showed a weight gain up to 10%, and in the combined treatment versus the control group, animal weight increased in the majority of studies (median 103%, with 3% of gain). There was a reduction between 2 and 30% (mean 73%) in only a few cases. The tumor weight was also analyzed under the three different experimental conditions: the combined ω-3 PUFAs and antitumor drug treatments versus the control was more effective, reaching 86% of tumor weight reduction (mean 57%), with the use of only an FAs-enriched diet (the amount of ω-3 PUFAs was higher than ω-6 PUFAs) the mean was 43%, and with the drugs-only versus the control, the tumor weight reduction was 32%.
The tumor growth was reported at different times of tumor evolution; therefore, we grouped the results in 15-day periods for this analysis and considered the mean results of each study for each condition. In the early stage, the highest reduction of tumor growth (a mean of 77% and a maximum value of 87%) occurred with the combined ω-3 PUFAs and drug treatment versus the control group (until 15 days), from 30 to 45 days the mean reduction stayed the same (53%), but in the late stage the mean reduction for this comparison decreased to 47%, being more effective in the groups that used the ω-3 PUFAenriched diet only, compared with a diet enriched with a higher amount of ω-3 PUFAs than ω-6 PUFAs (mean 49% and maximum value 79%). Thus, the ω-3 PUFA-enriched diet was more effective in studies that followed the animal for a longer period (140 and 300 days). In the experimental groups that analyzed only the antitumor drug effect versus the control, the tumor size reduction was less effective (mean of 17%, 24%, and 7% until 15, 30, and 45 days, respectively).
The tumor metastasis was analyzed mainly in the studies that used only a ω-3 PUFAenriched diet as the tumor treatment, showing a mean reduction of 37%, varying from 14% to 70% when the diet was predominantly ω-3 PUFAs versus ω-6 PUFAs; however, the combined treatments (ω-3 PUFA plus drug) versus the control was more effective, reducing the metastasis mean to 70%, ranging from 61 to 84%. In the groups that used drugs only versus the control, the results were less effective with a reduction of 17% and 60%, and an increase of 26% (in the graphic of Figure 2 this shows as 126%). The survival analysis of the animals in the studies is reported by the Kaplan-Meier curves at different times; the common time of most studies was 50% (T50 of box plot of Figure 2), and only one study reported the analysis of 80% onwards (T80). These percentages (T50 or T80; 50% or 80%) represent the fraction of animals surviving divided by the number of animals at risk for a certain amount of time after treatment. In the studies that used the FAs-only comparison, the increased survival was 20 days (T50) and 7 days (T80) when the diet was predominantly ω-3 PUFAs versus ω-6 PUFAs. For the combined treatment the survival increase was more remarkable with a mean of 23 days ranging from 5 to 45 days, compared to their respective control groups.

Dietary Compounds
Analyzing the main types of each PUFA (ω-3 and ω-6) and ω-9 MUFA, we observed that some types were more frequently cited than others and with this detailed analysis, we tried to verify if there was a trend for the most representative or important types of FAs, as shown in Table 4 and Figure 3A-D.
A current review showed that ω-3 PUFA supplements combined with chemotherapy and radiotherapy for BC patients can reduce the pain symptoms, prevent cachexia-anorexia syndrome, increase the weight of cancer patients, decrease the process of mitosis and cancer cell proliferation, as well as reduce the inflammatory response and support chemotherapy treatment, and improve the cancer patient's overall survival rate [63]. Moreover, according to a clinical review, women with high intakes of total ω-3 PUFAs in comparison to ω-6 PUFAs have been found to have a lower risk of BC [24]. In addition, by analyzing eating habits, a clinical trial showed a good association between total MUFAs, OA, and palmitic acid (C16:0) and an increase in BC risk [64]. Nonetheless, the effects of ω-3 PUFAs on tumor treatment are controversial [65]. Some studies suggest that ω-3 PUFAs may prevent or slow cancer development by reducing inflammation, slowing cancer cell growth and division, and preventing the formation of new blood vessels [66].
Due to the significant differences in the experimental model between studies, the ω-3 PUFA source (fish oil, menhaden oil, and chia oil), ω-3 PUFA type (ALA, EPA, and DHA), total dietary amount administered, route of administration (diet or gavage), and time of use, as well as accounting for the other FAs, it was difficult to determine the appropriate dose of ω-3 PUFAs for BC prevention or treatment in the selected studies. However, according to the global standard for EPA and DHA intake, the dose of supplemental ω-3 PUFAs known to be beneficial for some treatments or aging processes for humans is already well-established, such as cardiovascular diseases (at least 500 mg/day of EPA+DHA), pregnant/lactating women (D-A-300 mg/day), and general adults (300-400 mg EPA+DHA/day). In Brazil, the standard was reported with a minor alteration, for coronary artery disease (1 g/day EPA+DHA) and pregnant/lactating women (D-A-200 mg/day). The recommended daily dose in France for reducing the risk of breast and colon cancer was 500 mg of EPA+DHA [67]. The dose that was advised in 2017 for cardiovascular diseases (greater than 1 g/day) and pregnant/lactating women (D-A-700 mg/day) was slightly raised [68]. A current review study showed that the dose of DHA of 2 g per day shows evidence for safety, good absorption, and saturating plasma levels according to distribution, interconversion, and dose response in humans [69].
ALA, DHA, and EPA were the main ω-3 PUFAs (44 to 63%) reported by the selected studies, and their antitumoral effect was analyzed as either combined with antitumor drugs or compared with a diet with a higher intake of the LA ω-6 PUFA type (70.4%), or the ARA and GLA ω-6 PUFAs (about 20%) and the OA ω-9 MUFA (63%). EPA and DHA carried by apo B-containing lipoproteins (Non-High-Density Lipoprotein (HDL)) showed a protective effect on tumor cell proliferation only in tumors negative for ER and PgR, comparing different lipoproteins and the degree of severity of BC in women [70]. Furthermore, in a Swedish cohort study, postmenopausal women who consumed more ω-6 PUFAs and fewer heterocyclic amines had an increased risk of developing BC [71].
The age of the animals was another crucial factor in how ω-3 PUFA-enriched diets affect BC incidence; younger animals showed a stronger protective effect than older animals [72]. Puppies as young as 3 weeks old were employed in the evaluation of the ω-3 PUFA effects in four of the chosen studies [41,44,52,56].
The tumor growth and tumor weight were the measures mostly used in the studies about disease progress, with 92.6% and 70.4% of studies reflecting this, respectively. For both measures, the analyses showed that ω-3 PUFAs combined with antitumor drugs were more effective in decreasing tumor weight and size in comparison to the controls, mainly in the early stage of tumor growth (until 15 days), but the continuous use of ω-3 PUFAs over time showed a higher reduction in tumor growth (mean of 11, 36, 46, and 49% until 15, 30, 45, and more than 140 days, respectively). The only group that used a longer timeframe for the analyses (140 and 300 days) showed that this was more effective than the combined treatment. The use of antitumor drugs only was not as effective.
Weight loss is frequently the first visible sign of cancer and may be easily controlled in both clinical and preclinical studies. However, as the disease progresses, cachexia can develop, which can aggravate the condition and increase mortality. This parameter was reported in 77.8% of studies; the antitumor drug groups in comparison to the control groups were more effective in avoiding weight loss and allowing weight gain than the combined treatment groups (ω-3 PUFAs and drug) or the FAs groups, in relation to their controls. All conditions had a weight gain percentage, highlighting the study of Monk [40] and Liu [46], which used obese animals in the FAs groups and showed the highest gain; however, this group also had a smaller mean reduction (46%) in comparison to the drug and combined treatment groups.
The metastasis, the incidence and latency of the tumor, and the survival rate, were analyzed in some studies (33.3, 25, and 22.2%, respectively) on the ω-3 PUFA effect, normally without antitumor drugs and mainly in the transgenic BC model and in BC chemically induced models. A higher metastasis reduction occurred during the use of ω-3 PUFAs combined with antitumor drugs (61 to 84%), followed by using only ω-3 PUFAs giving a 14% to 70% reduction. The effect of the antitumor drug compared to the vehicle (control) showed controversial results between different antitumor drugs, and either a decrease in metastasis of 17 and 60%, or an increase of 26% [42]. The role of ω-3 PUFA supplementation in the prevention of cancer metastasis was demonstrated in clinical studies in BC patients using ω-3 PUFAs in conjunction with antitumor drugs and mastectomy, and showed decreases in the presence of the proliferative marker Ki67 and the angiogenesis marker VEGF [73]. In advanced pancreatic adenocarcinoma patients there were benefits in terms of quality of life, reducing the metastatic burden, and a significant reduction in serum levels of pro-angiogenic markers [73,74]. A higher reduction in tumor incidence occurred when the ω-3 PUFA supplementation was higher than the ω-6 PUFAs without any antitumor drugs [41,44,47,52,54,56,57,60], with a mean of a 20% incidence of reduction in the evaluation of half of the tumor-free animals (50%), and a 12% reduction considering all animals analyzed (100%). The distribution of latency delay was similar for T50 and T100, with a median of 15 days; however, the follow-up of this analysis had a wide range from 20 to 300 days. Clinical studies reported the prevention effect of ω-3 PUFA intake in cancer and cardiovascular disease in adults more than 50 years old [24,64,75], and decreased latency was reported in preclinical studies [24].
In general, survival analysis aims to determine the proportion of a population that will survive through to a specific point in time. Different percentual times were used to evaluate the Kaplan-Meier, with the most common time in the studies being 50% (T50 of the box plot of Figure 2), and in only one study 80% and onwards (T80) was reported. A higher ω-3 PUFA-enriched diet (more ω-3 PUFAs than ω-6 PUFAs) showed a survival increase of 20 days, while the combined treatments (ω-3 PUFAs plus drugs) showed a mean increase of 23 days (ranging from 5 to 45 days of increase, as shown in the orange boxplot of Figure 2). Patients with locally advanced BC had dramatically extended overall survival and progression-free survival when combining the usage of an ω-3 PUFAs-enriched diet with chemotherapy and mastectomy [23].
Molecular evaluation was used for understanding the pathophysiology of BC in the selected studies through the protein expression analysis, investigating the inhibitory potential of ω-3 PUFAs in oncogenic pathways such as nuclear factor-kappa B, phosphatidylinositol-3kinase/Protein kinase B/Mammalian target of rapamycin, and Wnt/β-catenin, as well as in the cell cycles (Cyclin B1, Wee1, and Poly (ADP-ribose) polymerase), energy homeostasis/metabolism (Glyceraldehyde-3-phosphate dehydrogenase, estrogen, insulin, resistin, and leptin), fatty acid synthesis (SREBP), oxidative stress (reactive oxygen species and hypoxia-inducible factors), DNA repair mechanisms (p53), and integrity and cell death through the processes of necrosis, apoptosis, and necroptosis (caspases, cytochrome C, tumor necrosis factor, receptor-interacting protein kinase, and mixed lineage kinase domainlike pseudokinase). The immunological analyses were focused on the immunophenotype characterization of tumors (through CD-type markers), the analysis of immune and inflammatory response mediators' (interleukins and tumor necrosis factor-a) interaction capacity, and the activation of immune checkpoint receptors (Programmed death-1 and Programmed death-ligand 1). Furthermore, investigations into angiogenesis processes (signaling VEGF) have been observed, which have a significant impact on the occurrence of metastases [76]. Patients treated with chemotherapy and mastectomy for locally advanced BC, and supplemented with ω-3 PUFAs, showed significantly decreased expression levels of Ki-67 and VEGF, compared to the placebo [23].

Animal Models Used in the BC Induction
The human tumor cell transplantation model (xenograft transplantation) was the most common BC model used (67%), mainly through the MDA-MB-231 cell transplantation in immunodeficient mice. These TNBC cells have more aggressive growth and an intermediate response to chemotherapy [77]. This model is more advantageous in having short cycles, low costs, a minimal variation, and high rates of tumor growth in comparison to the induced models (biological, physical, and chemical) and the genetically engineered mouse models [78]. The local cell implantation ranged between studies, in orthotopic transplantation, such as in the mammary gland and the inguinal mammary fat pad, the number and volume of cells implanted were smaller than in ectopic transplantations such as in the flank and armpit, among others. An intraductal method of transplantation is thought to be preferable to the mammary fat-pad transplantation for orthotopic transplantation, due to the production of a better pathological microenvironment for BC cells; however, this method is technically more difficult and only allows for the injection of a limited number of cancer cells [78].
The biological induction model of BC mainly relies on lentiviruses to overexpress oncogenes or silence tumor suppressor genes in experimental animals. This model was reported in some studies (19%) using MMTV-Neundl and MMTV-PyVT. The replicationcompetent ALV-LTR splice acceptor (RCAS), the Avian Leukosis Virus (ALV) and the Long terminal repeat (LTR), which contains genes of interest, can be directly injected into the glands of MMTV transgenic mice to induce breast tumors. These mice develop tumors within three weeks, induced by RCAS viral particles encoding the T-antigen in the mouse Mammary Tumor virus-polyomavirus (PyMT) gene in mammary ducts. Other oncogenes such as Neu and Wnt-1 take about seven months [78]. The chemically induced BC model using DMBA [44,52,54], or MNU drugs [48], was also reported in some studies (15%). The animals that received DMBA administration showed PIK3CA gene mutations detected in all tumors, positively correlating with the activation of protein kinase B (AKT), and these genetic changes in the induced mammary tumors were similar to those that exist in natural mammary cancers [78].
The present study's limitation was the wide range of experimental designs used in the studies that were selected, which made it difficult to make a more accurate comparison to determine the optimal dose of ω-3 PUFAs for the prevention of BC over the short and long-term and in conjunction with conventional antitumor therapies.

Conclusions
Omega-3 PUFA supplementation is an important coadjuvant to chemotherapy or other traditional antitumor therapies and shows remarkable results in combination with these other treatments, reducing tumor growth and weight (during the first fifteen days after tumor induction) compared to the isolated use of drugs or ω-3 PUFAs alone. Furthermore, the survival rate is increased. The effect of isolated ω-3 PUFAs was mainly analyzed in terms of tumor incidence and latency, as well as tumor metastasis in the transgenic BC model and chemically induced BC model, showing significant results in terms of animal weight (improving weight gain and preventing weight loss) and tumor growth reduction after prolonged use. However, the optimal ω-3 PUFA dose for pharmaconutrition and antitumor effects or the prevention of BC is unclear from the preclinical research.