Breast cancer is the second leading cause of cancer death in female patients after lung cancer [1
]. Thus, much research is being conducted for its prevention and treatment, including the role of diet. Because of its unique composition, flaxseed (FS) has been studied for its effect on breast cancer (reviewed in [3
Flaxseed (FS; Linum usitatissimum
) is an oilseed containing approximately 40% oil (FSO), more than half of which is the n-3 polyunsaturated fatty acids (PUFA), α-linolenic acid (ALA), the highest level of the dietary phytoestrogen lignan, secoisolariciresinol diglucoside (SDG) (~1%), and approximately 30% dietary fiber [4
]. Due to the elevated contents of ALA, FSO is considered to be an anti-inflammatory agent, while the lignan components have documented anti-oxidant and estrogenic/anti-estrogenic activities. Dietary fiber is generally recognized as having many beneficial effects, including modulation of the gut microbiota with the production of short-chain fatty acids (SCFA) [5
We previously showed that FS consumption was associated with the reduction in breast cancer risk (reviewed in [3
]). In mice injected with breast tumor cells, 10% FS significantly decreased the tumor size and growth at a late stage of carcinogenesis [6
]. The 10% FS-induced inhibition of breast cancer growth and metastasis was related to the downregulation of insulin-like growth factor 1 and epidermal growth factor [7
]. The FS inhibitory effect on breast cancer was also attributed to both FSO and SDG components.
In addition, studies from our group showed the link between early-life exposure to FS, mammary gland (MG) development, and breast cancer risk during adulthood. Notably, a 10% FS exposure in utero, during suckling, and throughout life could improve MG morphogenesis and differentiation, which in turn, helps to prevent MG carcinogenesis (reviewed in [3
]). In addition, SDG-based diet during pregnancy and lactation helped in reducing the susceptibility of mice to carcinogenesis and reduced tumorigenesis. This pattern was not observed with FSO-based dietary intervention [8
]. Taken together, these findings support the use of FS and SDG during MG development to reduce the future risk of breast cancer. However, the mechanisms involved in the protective effects of early-life exposure to FS and its components remain unclear. These may involve gene expression regulation via microRNAs (miRNAs).
MiRNAs are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level [9
]. MiRNAs are involved in several physiological processes across multiple tissues, including playing a vital role during MG development through the regulation of cell differentiation, proliferation, and involution across different stages of life [10
]. Their aberrant expression levels were associated with cancers, including breast cancer (reviewed in [12
]). In a recent systematic review, potential markers of breast cancer were identified for early detection and diagnosis, including miR-210 and miR-21 [13
Interestingly, environmental factors such as diet have been shown to alter the expression of miRNAs. Recent studies have showed that dietary phytochemicals may inhibit breast cancer through the regulation of miRNAs expression (reviewed in [14
]). We recently found that n-3 PUFA altered the expression of miR-21 in MCF-7 breast cancer cells [15
]. To date, no studies have investigated the effect of FS and its components, FSO and SDG, on the miRNA expression signature during MG development. Understanding the functions and contextual interactions between dietary components and MG miRNA signature (miRNome) during development could provide insights on the mechanism employed by bioactive agents to generate positive health outcomes and subsequently mitigate disease risk for lifelong health.
The objective of this study was to investigate the effect of exposing female C57BL/6 mice to 10% FS, as well as FSO or SDG found at the level in 10% FS, during MG development on MG miRNA expression signature and to identify potential targets to prevent breast cancer development during adulthood.
To investigate mechanisms underlying beneficial effects of FS in the context of breast health, we analyzed miRNA responses in the MG of pubertal mice to two main FS components, FSO and SDG, versus FS. We found overlap in miRNA expression across the three diets, but three diet-specific miRNA signatures could be identified. Specifically, a four-miRNA signature (higher miR-1 and miR-210 and lower miR-297c and miR-500 expression) significantly associated with the FS diet, a different four-miRNA signature (higher miR-30b, miR-324-5p and miR-423-3p and lower miR-382 expression) significantly associated with the FSO diet, and a two-miRNA signature (higher miR-142-5p and miR-1966) significantly associated with the SDG diet. This shows that the effects of FS components on MG miRNA expression are not additive. Upon ingestion, FS and its components are processed in the intestine mainly through the action of the gut microbiota. The microbiota ferments FS fiber, producing short-chain fatty acids and gases, and converts SDG to the enterolignans ED and EL, which may travel to distal organs. Here, despite being fed the same amount of SDG, the 10% FS group had relatively higher serum enterolignans concentrations compared to SDG-fed group, in line with our previously published studies, showing significantly higher urinary enterolignan levels in female mice receiving 10% FS compared to SDG-fed mice [25
]. The fiber in FS may have enhanced the conversion of SDG to enterolignans due to enhanced microbial fermentation.
In this study, we examined the MG miRNome at the end of the MG developmental period. MiRNAs are known as one of the regulators of MG development. MG miRNA profiling in C57BL/6 female mice during development showed a distinctive miRNA signature based on developmental stages [27
]. This study identified seven temporally co-expressed clusters, which were enriched in miRNAs associated with breast cancer including, miR-210, miR-30b, and miR-21. The breast cancer-associated miRNAs may play a role in cell proliferation and invasion during normal MG development, but their expression may become deregulated in breast cancer.
Taken together, this shows that miRNA expression regulation is critical throughout various stages of life in the MG development. Alterations in MG during the early stages of development have been associated with increased susceptibility to breast cancer [28
]. Exposures to environmental factors, including hormones, chemicals, and diets, could play an important role in increasing this susceptibility [28
This is the first comprehensive study of the effects of FS as a whole food compared to its isolated components, FSO and SDG, on MG miRNA signature in healthy female mice during MG development. Our key findings include the identification of (1) diet-specific MG miRNA signatures, (2) diet-dependent deregulation of selected breast cancer-associated miRNAs, and (3) enriched gene sets targeted by these miRNAs in each dietary intervention group.
Interestingly, most of the miRNAs identified in this study were shown to be deregulated in several types of cancers, including in breast cancer.
In the FS group, miR-1 was upregulated. This miRNA is known as a tumor suppressor in breast cancer, but was significantly downregulated in breast cancer compared to non-tumor tissues [30
]. Breast cancer patients with low miR-1 expression levels were found to have a poor survival time [31
]. Moreover, previous studies suggest that upregulation of miR-1 beneficially counters several types of cancers by repressing the expression of Egfr
(coding for an epidermal growth factor receptor) to inhibit cell proliferation, migration, and invasion [32
]. Diet-induced restoration of miR-1 may have a potential therapeutic significance.
Increased miR-500 serum levels have been linked to hepatocellular carcinoma [36
], while its downregulation was found to suppress lung cancer proliferation [37
]. However, it is not known if this miRNA is deregulated in breast cancer. FSO-associated miR-500 downregulation could potentially protect against mammary gland carcinogenesis and breast cancer development.
MiR-297c inhibition in human or animal subjects was found to be correlated with the efficacy of NSAIDs (non-steroidal anti-inflammatory drugs) in cancer chemoprevention treatment. This miRNA has, however, been suggested for use as a marker to assess the effectiveness of treatment [38
]. Interestingly, in our study, miR-297c expression was significantly decreased in FS-fed mice.
In the FSO group, with mice receiving the equivalent amount of 3.67% FSO as in the FS group, both miR-30b and miR-324-5p were upregulated compared to the BD and SDG groups, with no significant changes in the expression levels when compared to the FS group.
MiR-30b is a member of the miR-30 family, identified as tumor suppressors, which have frequently been found to be downregulated in different types of tumors, including breast cancers (reviewed in [30
]). This miRNA was shown to be able to alter drug resistance in breast cancer. A previous study found that a 6-day treatment of breast cancer cell lines with trastuzumab (Herceptin) resulted in a significant upregulation of miR-30b. Trastuzumab-induced miR-30b expression could, in consequence, induce cell growth inhibition by targeting cyclin E2 (CCNE2) [39
]. MiR-30b was also found to be involved in MG development; its upregulation in the tissue led to an impaired MG function and structure during lactation and involution [40
In a recent study, miRNA profiling of triple-negative breast cancer (TNBC) tissues showed high miR-324-5p expression levels. This expression was significantly correlated with decreased overall survival in breast cancer patients [41
MiR-382 was reported to be upregulated in the serum and plasma of patients with breast cancer and suggested to be used as a non-invasive marker for the diagnosis of breast cancer [42
]. This miRNA was also shown to promote cell viability, invasion, and survival in breast cancer patients [43
MiR-142-5p is one of the oncogenic miRNAs targeting cancer-related pathways. Its expression levels are significantly higher in breast cancer cell lines compared to controls [44
]. This miRNA was also identified as a negative regulator of TGF-β signaling pathway and suggested to be used as a therapeutic target in breast cancer [44
For the SDG-induced miR-1966, very limited data are available to describe the role of this miRNA in MG development and/or in cancers. A very recent study showed that miR-1966 is significantly downregulated in toosendanin-induced liver injury in mice [45
Interestingly, six of the ten miRNAs identified in this study (with the exception of miR-210 in the FS group, miR-423-3p in the FSO group, as well as the SDG-induced miR142-5p and miR-1966), were expressed in the opposite direction compared to their expression in the breast cancer tissues and/or cell lines. Therefore, it is speculated that FS and FSO dietary interventions could be used either to antagonize breast cancer-associated alterations of selected miRNAs or to prevent their deregulation in the MG during development. Note that mice used in this study were 4–5 weeks old, which corresponds to late stages of pubertal development. This supports the notion of early-life dietary intervention, using FS, to mitigate the risks of mammary gland malignancies and breast cancer.
This study brings a new concept of diet-dependent MG response that is mediated by miRNAs. Our data discern the differences between the consumption of purified FS components, SDG and FSO, and whole FS on the MG miRNA signature. However, the mechanisms behind these specific responses are not clear. This is likely due to the gut-microbiota derived metabolites resulting from the ingestion of FS as whole food and its isolated FSO (3.67%) or SDG (0.148%) components.
Overall, the gut microbiota outcomes may have a significant consequence on the amount and types of metabolites entering the systemic circulation, which could in consequence influence the miRNA-mediated mammary gland response (MG). Though, given the complexity and because the effect of FS and its components, FSO and SDG, on the gut-MG axis has not been completely elucidated yet, gut microbial profiling in response to different dietary interventions is needed for a better understanding of the mechanisms driving our diet-associated significant miRNA signatures.
To further explore the role of miRNAs in mediating MG response to different diets, we identified a regulatory network of the deregulated miRNAs with their experimentally validated targets. Although distinctive miRNA signatures were associated with different diets, the in silico analysis revealed the presence of shared gene sets between the three groups. Growth regulation and tube morphogenesis were identified among the most significantly enriched pathways. Thus, it could be postulated that deregulated miRNAs may be involved in promoting growth properties such as cell proliferation and differentiation in the MG.
It is not surprising to see that the FS-fed group had the greatest number of enriched gene sets followed by the FSO and SDG groups. Compared to isolated FSO and SDG, the FS contains soluble and insoluble dietary fiber, digestible proteins, and lignans, which may also have either a direct or an indirect effect on the MG response.
Interestingly, enrichment analysis shows that FS- and FSO- dependent miRNAs are associated with gene sets involved in MG development, including Bcl2l11, Csf1, Gja1, Hif1a, Igf1, Igf1r, Pax3, Pgr, Slc12a2, and Vdr.
Finally, constructing the miRNA-mRNA interaction network served not only to visualize the interaction between miRNAs and their targets, but also to identify potential biomarkers or novel therapeutic targets to prevent and/or reduce the risk of breast cancers. Of these, FS-induced miR-1 may be a potential candidate. In addition to its association with MG development, this miRNA also targets genes involved in breast cancer development, including Igf-1
coding for insulin-like growth factor 1 and its receptor (reviewed in [46
] and [47
]), as well as Cdk9
coding for cyclin-dependent kinase 9 (CDK9), involved in cell survival and the regulation of apoptosis. Cdk9 inhibitors are considered to be anticancer therapeutics [48