Ratio of n-3/n-6 PUFAs and risk of breast cancer: a meta-analysis of 274135 adult females from 11 independent prospective studies

Although breast cancer (BC) is the most common cancer occurring among women worldwide, international variation of BC incidence show there is a higher incidence in North America and Western Europe, but lower incidence in Asia [1, 2]. The large geographic heterogeneity of incidence among women globally could be explained by variation of dietary patterns, especially with relation to dietary fat as a potential dietary factor that is closely correlated with increased incidence of BC [3–6]. Polyunsaturated fatty acids (PUFAs) as dietary fat subtypes consist of two families: n-3 PUFAs and n-6 PUFAs. Serum phospholipids (PL) ratio of n-3/n-6 PUFA can directly reflect dietary intake ratio of n-3/n-6 PUFA, due to the lack of interconversion between n-3 and n-6 PUFAs in humans. N-3 and n-6 PUFAs in vivo can influence breast tumor cell growth by simultaneously competing for the same metabolic pathway (COX and LOX pathway) to change the balance of tissue eicosanoids, the transcription mediated by nuclear factor κB (NF-κB), and signal transduction mediated by the mammalian target of rapamycin (mTOR) etc. [7–9]. Therefore, ratio of n-3/n-6 PUFAs in diet and serum PL probably plays an important role in the risk of BC.

The studies from cell lines and animals have shown promising results of down regulating BC tumor growth by n-3 PUFAs as a nutrient to compete with n-6 PUFAs [10, 11]. Most of the case–control studies also support that dietary or serum PL n-3/n-6 ratio is inversely associated with risk of BC [4, 5, 12–14]. However, there are some inconsistent conclusions in prospective studies [15–20], and the optimal intake ratio of n-3/n-6 PUFAs has not yet been well defined. Therefore, it is necessary to quantitatively ascertain the association between intake ratio of n-3/n-6 PUFAs and the risk of BC by means of meta-analysis. Available data from prospective studies of adult females (premenopausal, postmenopausal, or combined) across different countries were pooled to summarize the relationship between intake ratio of n-3/n-6 PUFAs and the risk of BC for highest vs. lowest quantile, to estimate the potential dose–response trend and to conduct the stratified analysis for exploring the probable source of heterogeneity.

The present study, involving 8,331 BC events from 274,135 participants, indicated that higher ratio of n-3/n-6 PUFAs is associated with lower risk of BC, and per 1/10 increment of n-3/n-6 ratio in diet is associated with a 6% reduction of BC risk. However, subgroup analysis showed there was no significant relationship between serum PL biomarker and BC risk, but the significant difference between association of dietary n-3/n-6 ratio and serum PL with BC risk was not observed with metaregression (P for metaregression = 0.87; P for a permutation test = 0.99). Fatty acid profile in serum phospholipids may be an untypical representative of sensitive biomarkers indicating post-absorptive amounts and change at the target tissue, which did not provide the same information as the dietary questionnaire tools. Therefore, the strength of the relationship between the ratio in serum PL and BC risk might be lower compared with the relationship between the ratio in diet and the risk of BC.

Although the was no significant difference between most subgroups with metaregression, study heterogeneity (I ² = 21.00%) in this meta-analysis indicated the subtotal variation was attributable to between individual studies. There are some possible reasons for explaining the potential heterogeneity in present study. Firstly, the heterogeneity from geographic regions could be partially explained by different dietary patterns. The n-3/n-6 ratio in Japanese diet is about 1 to 4, whereas that in western diet (USA and Europe) is about 1 to 15–20 [37]. Imbalance of n-3/n-6 PUFAs ratio in diet across different countries is the consequence of excessive n-6 PUFAs consumption largely from corn, sunflower or safflower oils in the Western diet, but higher n-3 PUFAs intake largely from marine foods in the Asian diet, particularly in the Japanese population, which partially explains the inconsistency between Asia and Western studies in this meta-analysis. Subsequently, lower n-3/n-6 ratio in the diet will lead to lower ratio of n-3/n-6 PUFAs in serum PL. One randomized clinical trial showed that plasma n-3/n-6 ratio significantly increased among subjects after lovaza intervention (a prescription-strength pill, 4 g/day, EPA + DHA = 3.36 g, duration for 2 years) compared with subjects without intervention [38]. In vivo, AA (20:4n-6, arachidonic acid) and EPA (20:5n-3, eicosapentaenoic acid) can simultaneously compete for the same cyclooxygenases (COX) and lipoxygenases (LOX) metabolism pathways, leading to the production of n-6 family derived 2-series PG and 4-series LT with promoting tumor growth effects [39, 40], and n-3 family derived 3-series PG and 5-series LT with suppressive effects [41, 42]. Thus, the higher ratio of n-3/n-6 PUFA entering the cellular pool from dietary sources could be involved in BC carcinogenesis by changing the balance of tissue eicosanoids. Finally, there is evidence that change of estrogen metabolism is probably involved in mammary carcinogenesis among post-menopausal females[43]. EPA/AA ratio present in cell membrane lipids could influence the balance of prostaglandin E ₃ (PGE₃)/prostaglandin E₂ (PGE₂) to inactivate the activity of adipose aromatase P 450 which catalyzes the conversion of 19-carbon steroids to estrogens, and thus reduce estrogen-stimulated cell growth action [44, 45].

This meta-analysis had several strengths. Firstly, the quantitative assessment was based on data from prospective cohort studies. This minimizes the possibility that overall analysis will be influenced due to recall bias, which could be of more common concern in retrospective case–control studies. Also, we had higher statistical power to estimate the relationship between intake ratio of n-3/n-6 PUFA and risk of BC by analyzing summary data from 274,135 participants in 11 prospective studies. Subsequently, serum PL as a biomarker directly reflecting intake ratio of n-3/n-6 fatty acids has the advantage of providing more objective ratio of n-3/n-6 PUFA in vivo, independent of subjective recall bias and probable measurable errors owing to dietary questionnaire tools used. Finally, more than 50% of the studies were high quality studies (NOS stars ≥ 8), and exclusion of any individual study did not significantly change the negative relationship between intake ratio of n-3/n-6 PUFAs and BC risk. Although publication bias could be of concern because small studies with null results tend not to be published, there was no evidence of potential publication bias in the present meta-analysis.

Meta-analyses of observational studies are susceptible to methodological and confounding biases inherent in the original studies. Therefore, there are also several limitations considered in our study. Firstly, although prospective studies are more superior to retrospective case–control studies with regards to elucidating causal relationships, selection bias from study populations might be still unavoidable. Volunteers recruited from occupational exposure populations (e.g., teacher, workers and nurse) might not be an unbiased representative of study population. Subsequently, although the fatty acid profile in serum PL, implying absorption and circulation transportation mechanisms, reflects acute dietary intakes over the past few days, it may be a untypical representation of sensitive biomarkers indicating post-absorptive amounts and change at the target tissue. Moreover, any measurement error and resulting misclassification would most likely lead to an attenuation of the true association. Intake of fatty acids was evaluated using dietary questionnaire tools, which could potentially have measurable errors, due to inaccurate fatty acids database and diet reporting bias. Similarly, fatty acids in serum PL,, especially long chain PUFAs, are probably undergoing change during the long follow-up time and storage time [46], due to double bonds being easily oxidized. Consequently, potential measurement bias might lead to underestimation or overestimation of risk of BC [47]. Although only original studies with adjusting for at least three covariates were included in the present study, the possibility of altering summary results from residual confounding factors still cannot be excluded. Data extraction and analyses were not blinded to the authors and publication agencies, and the references screening and data extraction were conducted independently and in duplicate by 2 investigators, therefore data selection bias was unlikely. Although reporting bias might be present in our study, due to English language bias from exclusion of non-English language articles, our eligible studies covered a wide range of non-English countries, such as countries across Europe and Asia, which partially eliminated the possible effects of report bias on overall risk estimates. Finally, the limited numbers of studies included in each subgroup might diminish the statistical power to detect the association between ratio of n-3/n-6 PUFA and BC risk. Although we separately made a stratified analysis for association of serum PL ratio with BC risk and dose–response trend, it is difficult to find firm evidences among study populations within subgroup analysis, particular among USA females (P for metaregression = 0.103; P for a permutation test = 0.100).

The present meta-analysis has also important medical implications of balanced n-3/n-6 fatty acid intake ratio for BC prevention, clinical diagnosis and treatment. Firstly, although many sensitive tumor biomarkers were used for the clinical diagnosis of BC, several potential limitations were still unavoidable. In addition, the BC inflammatory microenvironment plays a major role in growth, invasiveness and resistance to therapy. However, modulation ratio of n-3/n-6 fatty acids in food, as a potent inducer of metabolic responses, could provide an effective means to alter ratio of n-3/n-6 fatty acid in tumor tissue and thereby possibly affect tumor growth, by sensitizing cancer cells to chemotherapy and increasing resistance of normal cells to the toxic effects. Finally, diet intervention could represent a clinically relevant adjuvant therapy in patients with BC, based on the general tenet of clinical nutrition.

In summary, findings from this meta-analysis of prospective studies provide a conclusive evidence to support increase intake ratio of n-3/n-6 PUFAs for BC prevention among study females. Quantitative conclusions from the present study showed that per 1/10 increment of n-3/n-6 ratio in diet, there was a 6% reduction of BC risk among study populations (USA, Europe and Asia). This important evidence highlights the need to promote nutritional education programs, stressing the need to increase the consumption of food rich in n-3 PUFAs (marine foods), decrease the consumption of food rich in n-6 PUFAs (vegetable oils and processed foods), to ultimately improve intake ratio of n-3/n-6 PUFAs. However, no conclusive evidence from USA populations can be obtained and this needs to be,further confirmed by prospective population-based study on tissue biomarker of n-3/n-6 PUFAs ratio, and larger randomized controlled trials are required to determine whether higher intake ratio of n-3/n-6 PUFAs will have beneficial effects on BC risk, or improve the prognosis of patients with BC.