Cecal microbiota and mammary gland microRNA signatures are related and modifiable by dietary flaxseed with implications for breast cancer risk.

IMPORTANCE: Breast cancer is a leading cause of cancer mortality worldwide. There is a growing interest in using dietary approaches, including flaxseed (FS) and its oil and lignan components, to mitigate breast cancer risk. Importantly, there is recognition that pubertal processes and lifestyle, including diet, are important for breast health throughout life. Mechanisms remain incompletely understood. Our research uncovers a link between mammary gland miRNA expression and the gut microbiota in young female mice. We found that this relationship is modifiable via a dietary intervention. Using data from The Cancer Genome Atlas, we also show that the expression of miRNAs involved in these relationships is altered in breast cancer in humans. These findings highlight a role for the gut microbiome as a modulator, and thus a target, of interventions aiming at reducing breast cancer risk. They also provide foundational knowledge to explore the effects of early life interventions and mechanisms programming breast health.

MicroRNAs: A Link between Mammary Gland Development and Breast Cancer.

Breast cancer is among the most common cancers in women, second to skin cancer. Mammary gland development can influence breast cancer development in later life. Processes such as proliferation, invasion, and migration during mammary gland development can often mirror processes found in breast cancer. MicroRNAs (miRNAs), small, non-coding RNAs, can repress post-transcriptional RNA expression and can regulate up to 80% of all genes. Expression of miRNAs play a key role in mammary gland development, and aberrant expression can initiate or promote breast cancer. Here, we review the role of miRNAs in mammary development and breast cancer, and potential parallel roles. A total of 32 miRNAs were found to be expressed in both mammary gland development and breast cancer. These miRNAs are involved in proliferation, metastasis, invasion, and apoptosis in both processes. Some miRNAs were found to have contradictory roles, possibly due to their ability to target many genes at once. Investigation of miRNAs and their role in mammary gland development may inform about their role in breast cancer. In particular, by studying miRNA in development, mechanisms and potential targets for breast cancer treatment may be elucidated.

Data on mammary gland microRNAs expression, their predicted gene targets and corresponding pathway analysis in female mice receiving flaxseed or its oil and secoisolariciresinol diglucoside components.

Dietary flaxseed may act via microRNAs (miRNAs) to affect the health of the mammary gland. These data are in support of the article entitled "Effects of flaxseed and its components on mammary gland miRNome: identification of potential biomarkers to prevent breast cancer development" [1]. Here, we provide miRNA expression data obtained from NanoString nCounter® profiling of mammary gland RNA from C57BL/6 female mice who received a control diet or isocaloric diets containing 10% FS, 3.67% FSO, or 0.15% SDG for 21 days. The raw miRNA data were deposited at the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193847) under the accession number GSE193847. We also identified diet-associated miRNA-gene targets and corresponding enriched pathways. These data can be found at the HARVARD Dataverse (https://doi.org/10.7910/DVN/3ZNYES). These data will be valuable as a reference to understand the effects of FS versus its components and to study responses to these ingredients in hosts of different genetic backgrounds, sex and age. These data will contribute to future investigations regarding mechanisms underlying FS effects within the mammary gland.

Data on cecal and fecal microbiota and predicted metagenomes profiles of female mice receiving whole flaxseed or its oil and secoisolariciresinol diglucoside components.

Dietary flaxseed (FS) and its components including FS oil (FSO), secoisolariciresinol diglucoside (SDG) and fiber, are processed by the gut microbiota. These data are in support of the article entitled "Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components", Journal of Nutritional Biochemistry [1]. Here we describe data generated by 16S rRNA sequencing of DNA obtained from cecum contents and feces of C57BL/6 female mice fed either a basal diet (BD, AIN93G), or isocaloric diets containing 10% FS, or 10% FS-equivalent amounts of FSO or SDG for 21 days. These include bacterial community composition and inferred KEGG pathways; the raw data are publicly available at the NCBI SRA database (BioProject ID PRJNA683934). Furthermore, this work includes detailed experimentation procedures, total bacterial counts (qPCR) in the cecum content and feces, and correlation analysis between a selected bacterial genus, Bacteroides and a predicted metabolic pathway. FS is utilized worldwide, especially for the prevention and/or treatment of diseases including cardiovascular diseases, diabetes and cancer. These data will be valuable as a reference to study different FS cultivars and SDG- or FSO- enriched products on the gut microbiota, to study gut microbial responses to FS and its components in different mouse strains and mammalian hosts to elucidate individualized effects, and to understand the importance of the gut microbiota for FS benefits.

Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components.

Gut microbial processing of dietary flaxseed (FS) contributes to its health benefits, but the relative effects of its bioactive components (lignans, omega-3 fatty acids, fiber) on the microbiota are unclear. We investigated the gut microbial compositional and functional responses to whole FS and its isolated components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG) (precursor to microbial-derived enterolignans) to help understand their contribution to whole FS benefits. Cecum content and fecal samples were collected from C57BL/6 female mice fed a basal diet (AIN93G) or isocaloric diets containing 10% FS or 10% FS-equivalent amounts of FSO or SDG for 21 days. Cecal and fecal microbiota composition and predicted genomic functions, and their relationship with serum enterolignans were evaluated. Only FS modified the community structure. Shared- and diet-specific enriched taxa and functions were identified. Carbohydrate and protein processing functions were enriched in FS mice, and there was a positive correlation between select enriched taxa, encompassing fiber degraders and SDG metabolizers, and serum enterolignans. This was not observed in mice receiving isolated FSO and SDG, suggesting that FS fiber supports SDG microbial metabolism. In conclusion, the cooperative activities of a diverse microbiota are necessary to process FS components and, when administered at the amount present in FS, these components may act together to affect SDG-derived enterolignans production. This has implications for the use of FS, FSO and SDG in clinical practice.

Enterolignan Production in a Flaxseed Intervention Study in Postmenopausal US Women of African Ancestry and European Ancestry.

Lignans are phytochemicals studied extensively as dietary factors in chronic disease etiology. Our goal was to examine associations between the gut microbiota and lignan metabolism and whether these associations differ by ethnicity. We conducted a flaxseed (FS) dietary intervention in 252 healthy, postmenopausal women of African ancestry (AA) and European ancestry (EA). Participants consumed ~10 g/d ground flaxseed for 6 weeks and provided overnight urine collections and fecal samples before and after intervention. The gut microbiota was characterized using 16S rRNA gene sequencing and differences in microbial community composition compared by ethnicity and intervention status. We observed a significant difference in the composition of the microbiota measured as beta diversity (p < 0.05) between AA and EA at baseline that was attenuated with FS consumption. Genera that were significantly associated with ENL production (e.g., Klebsiella, Lactobacillus, Slackia, Senegalimassilia) were unique to each group. Bacteria (e.g., Fusobacteria, Pyramidobacter and Odoribacter) previously associated with colorectal cancer and cardiovascular disease, both diet-related chronic diseases, were unique to either AA or EA and were significantly reduced in the FS intervention. This study suggests that ethnic variation in ENL metabolism may be linked to gut microbiota composition, and its impact on disease risk deserves future investigation.

Effects of Flaxseed and Its Components on Mammary Gland MiRNome: Identification of Potential Biomarkers to Prevent Breast Cancer Development.

Breast cancer is the most common cancer among women worldwide. We previously showed that early-life exposure to flaxseed (FS) or its components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG), affects the mammary gland (MG) and is associated with the reduction of breast cancer risk during adulthood. However, the underlying mechanisms are not understood. This study aimed to investigate the effect of FS, FSO, and SDG on the MG miRNA signature at a late stage of development. Female C57BL/6 mice, 4-5 weeks of age, were randomized into four groups to receive: (i) basal AIN-93G, (ii) 10% FS, (iii) 3.67% FSO, or (iv) 0.15% SDG. After 21 days, the mice were sacrificed and MG miRNAs were profiled. Diet-specific MG miRNA signatures were identified. Deregulated miRNAs were associated with breast cancer and targeted genes involved in MG development, growth, and cancer. The study allowed for the identification of potential biomarkers or novel therapeutic targets to prevent and/or reduce the risk of breast cancer.

Genetic Variation in Steroid and Xenobiotic Metabolizing Pathways and Enterolactone Excretion Before and After Flaxseed Intervention in African American and European American Women.

BACKGROUND: Metabolism and excretion of the phytoestrogen enterolactone (ENL), which has been associated with breast cancer risk, may be affected by variation in steroid hormone and xenobiotic-metabolizing genes. METHODS: We conducted a randomized, crossover flaxseed intervention study in 252 healthy, postmenopausal women [137 European ancestry (EA) and 115 African ancestry (AA)] from western New York. Participants were randomly assigned to maintain usual diet or consume 10 g/day ground flaxseed for 6 weeks. After a 2-month washout period, participants crossed over to the other diet condition for an additional 6 weeks. Urinary ENL excretion was measured by gas chromatography-mass spectrometry and 70 polymorphisms in 29 genes related to steroid hormone and xenobiotic metabolism were genotyped. Mixed additive genetic models were constructed to examine association of genetic variation with urinary ENL excretion at baseline and after the flaxseed intervention. RESULTS: SNPs in several genes were nominally (P < 0.05) associated with ENL excretion at baseline and/or after intervention: ESR1, CYP1B1, COMT, CYP3A5, ARPC1A, BCL2L11, SHBG, SLCO1B1, and ZKSCAN5. A greater number of SNPs were associated among AA women than among EA women, and no SNPs were associated in both races. No SNP-ENL associations were statistically significant after correction for multiple comparisons. CONCLUSIONS: Variation in several genes related to steroid hormone metabolism was associated with lignan excretion at baseline and/or after flaxseed intervention among postmenopausal women. IMPACT: These findings may contribute to our understanding of the differences observed in urinary ENL excretion among AA and EA women and thus hormone-related breast cancer risk.

Effect of Dietary Flaxseed Intake on Circulating Sex Hormone Levels among Postmenopausal Women: A Randomized Controlled Intervention Trial.

Lignan intake, and its richest food source, flaxseed, have been associated with reduced breast cancer risk. Endogenous sex hormones, such as estrogens, play a role in breast cancer development, and lignans may alter these sex hormone levels. To assess the effect of flaxseed on circulating sex hormones, a randomized controlled trial was conducted among 99 postmenopausal women in Toronto, Canada. The intervention arm consumed 2 tablespoons (15 g) of ground flaxseed daily for 7 weeks; the control arm maintained usual diet. Baseline and week 7 concentrations of 14 serum sex hormones were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay, and serum enterolignans (lignan biomarker) using LC-MS/MS. Intervention effects on sex hormone levels were assessed using analysis of covariance. Serum enterolignans increased among the flaxseed arm (+516%). Women consuming flaxseed (vs. controls) had increased serum 2-hydroxyestrone [treatment effect ratio (TER) = 1.54; 95% CI: 1.18-2.00] and 2:16α-hydroxyestrone ratio (TER =1.54; 95% CI: 1.15-2.06); effects on other hormones were not statistically significant. Within the flaxseed arm, change in enterolignan level was positively correlated with changes in 2-hydroxyestrone and 2:16α-hydroxyestrone ratio, and negatively with prolactin. Findings suggest flaxseed affects certain circulating sex hormone levels with possible implications for future breast cancer prevention research.

Omega-3 Polyunsaturated Fatty Acids Time-Dependently Reduce Cell Viability and Oncogenic MicroRNA-21 Expression in Estrogen Receptor-Positive Breast Cancer Cells (MCF-7).

The omega-3 polyunsaturated fatty acid (n-3 PUFA), α-linolenic acid (ALA), and its metabolites, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), independently reduce the growth of breast cancer cells in vitro, but the mechanisms, which may involve microRNA (miRNA), are still unclear. The expression of the oncomiR, miR-21, is reduced by DHA treatment, but the effects of ALA on miR-21, alone or combined with EPA and DHA under physiologically relevant concentrations, have not been investigated. The effects of ALA alone and +/-EPA and DHA at the blood molar ratios seen in either humans (1.0:1.0:2.5, ALA:EPA:DHA) or mice (1.0:0.4:3.1, ALA:EPA:DHA) post flaxseed oil consumption (containing ALA) were assessed in vitro in MCF-7 breast cancer cells. Cell viability and the expression of miR-21 and its molecular target, phosphatase and tension homolog (PTEN, gene and protein), at different time points, were examined. At 1, 3, 48 and 96 h ALA alone and 24 h animal ratio treatments significantly reduced MCF-7 cell viability, while 1 and 3 h ALA alone and human and animal ratio treatments all significantly reduced miR-21 expression, and 24 h animal ratio treatment reduced miR-21 expression; these effects were not associated with changes in PTEN gene or protein expressions. We showed for the first time that ALA alone or combined with EPA and DHA at levels seen in human and animal blood post-ALA consumption can significantly reduce cell viability and modulate miR-21 expression in a time- and concentration-dependent manner, with the animal ratio containing higher DHA having a greater effect. The time dependency of miR-21 effects suggests the significance of considering time as a variable in miRNA studies, particularly of miR-21.

α-linolenic acid and docosahexaenoic acid, alone and combined with trastuzumab, reduce HER2-overexpressing breast cancer cell growth but differentially regulate HER2 signaling pathways.

BACKGROUND: Diets rich in the n-3 fatty acid alpha-linolenic acid (ALA) have been shown to reduce breast tumor growth, enhance the effectiveness of the HER2-targeted drug trastuzumab (TRAS) and reduce HER2 signaling in mouse models. It is unclear whether this is due to direct effects of ALA or due to its long-chain n-3 fatty acids metabolites including docosahexaenoic acid (DHA). METHODS: The ability of HER2-overexpressing BT-474 human breast cancer cells to convert ALA to long-chain n-3 fatty acids was determined by measurement of phospholipid fatty acids by gas chromatography following treatment with 100 µM ALA. The effects of 96 h treatment with ALA or DHA, at serum levels seen in mice (50-100 µM), alone and combined with TRAS (10 µg/ml), on BT-474 cell growth measured by trypan blue exclusion, apoptosis measured by flow cytometric analysis of Annexin-V/7-AAD stained cells (ALA and TRAS treatment only) and protein biomarkers HER2 signaling measured by western blot were determined. RESULTS: ALA-treated BT-474 cells had higher phospholipid ALA but no increase in downstream n-3 metabolites including DHA. Both ALA and DHA reduced cell growth with and without TRAS. ALA had no effect on apoptosis. ALA and DHA showed opposite effects on Akt and MAPK phosphorylation; ALA increased and DHA decreased phosphorylation. CONCLUSIONS: Together these data suggest that, while both ALA and its DHA metabolite can reduce HER2-overexpressing breast cancer growth with and without TRAS, they demonstrate for the first time that DHA is responsible for the effects of ALA-rich diets on HER2 signaling pathways.

α-Linolenic Acid Reduces Growth of Both Triple Negative and Luminal Breast Cancer Cells in High and Low Estrogen Environments.

Flaxseed, rich in α-linolenic acid (ALA), is a complementary breast cancer (BC) therapy; however ALA effectiveness and mechanism are unclear. Variation in cellular expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and estrogen (E2) levels may alter ALA effectiveness. This research determined the effect of ALA on growth, apoptosis, and phospholipid fatty acids of 4 BC cell lines with varying receptor expression ± E2. MCF-7 (ER+/PR+/HER2-), BT-474 (ER+/PR+/HER2+), MDA-MB-231 (ER-/PR-/HER2-) and MDA-MB-468 (ER-/PR-/HER2-) cells were incubated with ALA (50-200 µM) ± 1 nM E2 for 48-72 h. ALA dose-dependently reduced growth, measured by trypan blue exclusion, of all cells (55-80% with 75 µM), and this effect was not altered by E2. ALA (75 µM)+E2 induced apoptosis, measured by flow cytometry (up to 111.2%). Decreased growth and increased apoptosis is related to increased cell phospholipid % ALA (up to 25.1%), measured by gas chromatography. ALA is shown for the first time to reduce cell growth and induce apoptosis regardless of receptor expression and E2 environment, by incorporating into BC phospholipids, supporting the use of ALA and ALA-rich foods as a safe, inexpensive complementary therapy for a wide range of BC.

Growth and gene expression differ over time in alpha-linolenic acid treated breast cancer cells.

SCOPE: Heterogeneity of breast cancer (BC) subtypes makes BC treatment difficult. α-linolenic acid (ALA), rich in flaxseed oil, has been shown to reduce growth and increase apoptosis in several BC cell lines, but the mechanism of action needs further understanding. METHODS AND RESULTS: Four BC cell lines (MCF-7, BT-474, MDA-MB-231 and MDA-MB-468) were incubated with 75 µM ALA+1 nM 17-ß estradiol (E2) or 1 nM E2 only (control) for 24 h. MDA-MB-231 cells were additionally incubated at 6 and 12 h. Viable cell number was measured, and expression of genes related to BC (signaling pathways, cell cycle, apoptosis) was quantified by real-time PCR array. There was a reduction in growth of all ALA treated cell lines after 24 h, and in MDA-MB-231 cells this was time-dependent. Many genes were altered after 24 h, and these differed between cell lines. In MDA-MB-231 cells, several gene expression changes were time-dependent. CONCLUSIONS: ALA reduces growth of BC cell lines, by modifying signaling pathways, which differ between BC molecular subtypes. The ALA effect on gene expression is dynamic and changes over time, indicating the significance of incubation period in detecting gene changes.

Flaxseed oil enhances the effectiveness of trastuzumab in reducing the growth of HER2-overexpressing human breast tumors (BT-474).

Flaxseed oil (FSO) reduces breast tumorigenesis and HER2 expression in animal models of luminal breast cancer. The primary treatment for HER2-overexpressing tumors is trastuzumab (TRAS). We aimed to determine the effect of 4% FSO alone and combined with TRAS on HER2-overexpressing tumor (BT-474) growth and to explore potential mechanisms with a specific focus on HER2, mitogen-activated protein kinase (MAPK) and Akt signaling and fatty acid profile. Athymic mice with established tumors were fed the basal diet (control) or 4% FSO diet, with or without TRAS (1 or 2.5 mg/kg) treatment for 4 weeks. Tumor growth, HER2 signaling biomarkers (mRNA and protein) and fatty acid profile were measured. Tumors treated with FSO alone showed no difference in tumor growth compared to control; however, compared to TRAS2.5 and other groups, FSO+TRAS2.5 caused significantly lower tumor growth and cell proliferation and higher apoptosis and the greatest lowering of signaling biomarker expressions (MAPK2, HER2 mRNA; pHER2 protein). Both TRAS and FSO had main effects of reducing the phosphorylated/total expression of Akt and MAPK protein expression. Dietary FSO altered the tumor fatty acid profile. In conclusion, 4% dietary FSO alone does not affect BT-474 tumor growth but enhances the tumor-reducing effect of TRAS (2.5 mg/kg). FSO×TRAS interactive effect may be modulated by their combined reductions of HER2 signaling through the Akt and MAPK pathways leading to reduced cell proliferation and increased apoptosis. FSO alters tumor fatty acid profile that likely contributes to effects on signaling pathways. This supports FSO as a complementary treatment for HER2+ breast cancer treated with TRAS.

17ß-estradiol increases liver and serum docosahexaenoic acid in mice fed varying levels of α-linolenic acid.

Docosahexaenoic acid (DHA) is considered to be important for cardiac and brain function, and 17ß-estradiol (E2) appears to increase the conversion of α-linolenic acid (ALA) into DHA. However, the effect of varying ALA intake on the positive effect of E2 on DHA synthesis is not known. Therefore, the objective of this study was to investigate the effects of E2 supplementation on tissue and serum fatty acids in mice fed a low-ALA corn oil-based diet (CO, providing 0.6 % fatty acids as ALA) or a high ALA flaxseed meal-based diet (FS, providing 11.2 % ALA). Ovariectomized mice were implanted with a slow-release E2 pellet at 3 weeks of age and half the mice had the pellet removed at 7 weeks of age. Mice were then randomized onto either the CO or FS diet. After 4 weeks, the DHA concentration was measured in serum, liver and brain. A significant main effect of E2 was found for liver and serum DHA, corresponding to 25 and 15 % higher DHA in livers of CO and FS rats, respectively, and 19 and 13 % in serum of CO and FS rats, respectively, compared to unsupplemented mice. There was no effect of E2 on brain DHA. E2 results in higher DHA in serum and liver, at both levels of dietary ALA investigated presently, suggesting that higher ALA intake may result in higher DHA in individuals with higher E2 status.

Flaxseed and its lignan and oil components: can they play a role in reducing the risk of and improving the treatment of breast cancer?

Flaxseed (FS), rich in the phytoestrogen lignans and α-linolenic acid-rich oil, has been suggested to have an anticancer effect. Questions remain whether FS and its lignan and oil components are effective in reducing breast cancer risk and tumour growth, and can interact beneficially with breast cancer drugs. To find answers, in vitro, animal, observational, and clinical studies on FS and its lignan and oil components were reviewed. The majority of studies in various rodent models show that 2.5%-10% FS diet or the equivalent amount of lignan or oil reduces tumour growth. Ten percent FS and equivalent lignans do not interfere with but rather increase the effectiveness of tamoxifen (80 mg/day) while the 4% FS oil increases trastuzumab/Herceptin (2.5 mg/kg) effectiveness. Observational studies show that FS and lignan intake, urinary excretion, or serum levels are associated with reduced risk, particularly in postmenopausal women. Lignans reduce breast cancer and all-cause mortality by 33%-70% and 40%-53%, respectively, without reducing tamoxifen effectiveness. Clinical trials show that FS (25 g/day with 50 mg lignans; 32 days) reduces tumour growth in breast cancer patients and lignans (50 mg/day; 1 year) reduces risk in premenopausal women. Mechanisms include decreased cell proliferation and angiogenesis and increased apoptosis through modulation of estrogen metabolism and estrogen receptor and growth factor receptor signalling pathways. More clinical trials are needed but current overall evidence indicates that FS and its components are effective in the risk reduction and treatment of breast cancer and safe for consumption by breast cancer patients.

Flaxseed enhances the beneficial effect of low-dose estrogen therapy at reducing bone turnover and preserving bone microarchitecture in ovariectomized rats.

Our previous research showed greatest protection to vertebral bone mineral density and strength in ovariectomized (OVX) rats when lignan- and α-linolenic acid-rich flaxseed (FS) is combined with low-dose estrogen therapy (LD) compared with either treatment alone. This study determined the effects of combined FS+LD on serum and tissue markers of bone turnover and microarchitecture to explain our previous findings. Three-month-old OVX rats were randomized to negative control (NEG), FS, LD or FS+LD for 2 or 12 weeks, meaningful time points for determining effects on markers of bone metabolism and bone structure, respectively. Ground FS was added to the AIN-93M diet (100 g/kg diet) and LD (0.42 µg 17ß-estradiol/(kg body weight·day)) was delivered by subcutaneous implant. Sham rats were included as positive control. Bone formation (e.g., osteocalcin), bone resorption (e.g., tartrate-resistant acid phosphatase-5ß (TRAP-5ß)), as well as osteoprotegerin (OPG) and receptor activator of nuclear factor κ-B ligand (RANKL) were analyzed from the 2-week study by commercial assays (serum) and (or) histology (vertebra). Vertebral bone microarchitecture was measured from the 12-week study using microcomputed tomography. In serum, FS+LD and LD induced lower TRAP-5ß and osteocalcin, and higher OPG and OPG/RANKL ratio versus NEG and FS (p < 0.05). In vertebrae, FS+LD induced higher OPG and lower osteocalcin versus NEG (p < 0.01) and did not differ from LD and FS. FS+LD improved bone microarchitecture versus NEG, FS, and LD (p < 0.05). In conclusion, FS+LD protects bone tissue because of a reduction in bone turnover. However, elucidating the distinctive action of FS+LD on bone turnover compared with LD requires further investigation.

A pilot study comparing the effect of flaxseed, aromatase inhibitor, and the combination on breast tumor biomarkers.

Use of complementary approaches is common among breast cancer survivors. Potential interactions between aromatase inhibitors (AI) and high phytoestrogen foods, such as flaxseed (FS), are not often described. We conducted a pilot 2 × 2 factorial, randomized intervention study between tumor biopsy and resection, in 24 postmenopausal women with estrogen receptor positive (ER+) breast cancer, to assess the effects of FS and anastrozole, and possible interactions between them, on serum steroid hormone and tumor-related characteristics associated with long-term survival (Roswell Park Cancer Institute, 2007-2010). The effect of each treatment vs. placebo on outcomes was determined by linear regression adjusting for pretreatment measure, stage, and grade. Although not statistically significant, mean ERß expression was approximately 40% lower from pre- to postintervention in the FS + AI group only. We observed a statistically significant negative association (ß ± SE -0.3 ± 0.1; P = 0.03) for androstenedione in the FS + AI group vs. placebo and for DHEA with AI treatment (ß ± SE -1.6 ± 0.6; P = 0.009). Enterolactone excretion was much lower in the FS + AI group compared to the FS group. Our results do not support strong effects of FS on AI activity for selected breast tumor characteristics or serum steroid hormone levels but suggest AI therapy might reduce the production of circulating mammalian lignans from FS.

Dietary flaxseed-trastuzumab interactive effects on the growth of HER2-overexpressing human breast tumors (BT-474).

Flaxseed (FS) reduces breast tumorigenesis and human epidermal growth factor receptor 2 (HER2) expression in postmenopausal patients and animal models. The primary treatment for HER2-overexpressing tumors is trastuzumab (TRAS). FS oil enhances TRAS effectiveness in athymic mice but the FS effect is unknown and was therefore determined. Athymic mice with established BT-474 tumors were fed the basal diet (control), or 10% FS diet, with or without TRAS (2.5mg/kg) treatment for 5 wk. After 2 wk, TRAS and FS reduced tumor size with a trend for an FS × TRAS interaction; however, after 5 wk, only TRAS reduced tumor size and increased tumor apoptosis. FS did not further improve TRAS effect but increased overall survival. TRAS reduced signaling biomarkers [phosphorylated HER2 and mitogen-activated protein kinase (MAPK) proteins; Akt1, Akt2, MAPK, and estrogen receptor-α mRNA], FS reduced phosphorylated-Akt1 protein, and FS × TRAS interactions were seen for HER2 mRNA and phosphorylated-Akt1 protein. FS, with and without TRAS, increased tumor n-3 PUFA levels and serum lignans indicating potential roles in the observed effect. In conclusion, TRAS reduces tumor growth by influencing HER2 signaling. Dietary FS has minimal tumor-reducing effect, does not interfere with TRAS action, but improves overall survival in athymic mice.

Use of isoflavone supplements is associated with reduced postmenopausal breast cancer risk.

Botanical supplements are widely used and contain diverse ingredients, including isoflavones. Food-based isoflavones have been associated with reduced breast cancer risk. However, no study has comprehensively evaluated supplements identified by isoflavone content and breast cancer risk. Associations between ever use of 28 isoflavone supplements and breast cancer risk in Ontario, Canada were evaluated using cases (n = 3,101) identified in 2002-2003 from the Ontario Cancer Registry and controls (n = 3,471) identified through random digit dialing methods. Multivariate logistic regression was used to estimate age-adjusted odds ratio (AOR) and 95% confidence intervals (CI). Several individual supplements were associated with reduced breast cancer risk (e.g., Natural HRT; AOR = 0.39; 95% CI: 0.22, 0.69; n(users) = 58). Use of any isoflavone supplements was associated with reduced risk when ≥ 3 were ever used (AOR = 0.68; 95% CI: 0.54, 0.86; n(users) = 332; p(trend) = 0.008) or any was taken >5 years (AOR = 0.75; 95% CI: 0.60, 0.94; n(users) = 325; p(trend) = 0.01); high content supplements were consistently associated with reduced risk. Risk reduction was confined to postmenopausal breast cancer for both individual and combined supplements, and was strongest in the latter among high content users who ever took ≥ 3 supplements (AOR = 0.55; 95% CI: 0.38, 0.81; n(users) = 118; p(trend) = 0.04) or any >5 years (AOR = 0.47; 95% CI: 0.27, 0.81; n(users) = 60; p(trend) = 0.03). Associations did not differ by estrogen-progesterone tumor receptor status. In conclusion, isoflavone supplements were associated with decreased postmenopausal breast cancer risk. Further research to examine these novel findings is warranted, given the low supplement use and potential limitations of our results.