Estrogen-induced Mammary Tumorigenesis Research Articles

EPA and EFSA approaches for benchmark dose modeling

Estrogen is reported to be involved in mammary tumorigenesis. To unveil metabolic signatures for estrogen-induced mammary tumorigenesis, we carried out serum metabolomic analysis in an estrogen-induced mammary tumor model, female August Copenhagen-Irish/Segaloff (ACI/Seg) rats, using liquid chromatography-mass spectrometry. In contrast to the control group, all rats with an implanted 17β-estradiol (E2) pellet developed mammary tumors during this experiment. E2 treatment significantly suppressed body weight gain. But no significant differences in food consumption were observed between the two groups, suggesting that metabolic alteration depended on E2 treatment. Serum metabolomic analysis detected 116 features that were statistically different (p < 0.01) between the groups. Quantitation analysis revealed that several phospholipids such as phosphatidylcholines and lysophosphatidylcholines (LPCs) were identified as significantly different metabolites. E2-treated rat serum stimulated the proliferation of human breast cancer MDA-MB-231 cells. In addition, the proliferation effect was diminished by pretreating cells with either autotaxin inhibitor or antagonist for lysophosphatidic acid receptor whose ligands are metabolites of LPCs via autotaxin-mediated hydrolysis. In summary, our results suggest that not only are phospholipids potential biomarkers for mammary tumors but importantly, LPCs themselves could be associated with E2-induced mammary tumorigenesis in female ACI/Seg rats.

Metabolomic analysis uncovered an association of serum phospholipid levels with estrogen-induced mammary tumors in female ACI/Seg rats

Estrogen is reported to be involved in mammary tumorigenesis. To unveil metabolic signatures for estrogen-induced mammary tumorigenesis, we carried out serum metabolomic analysis in an estrogen-induced mammary tumor model, female August Copenhagen-Irish/Segaloff (ACI/Seg) rats, using liquid chromatography-mass spectrometry. In contrast to the control group, all rats with an implanted 17β-estradiol (E2) pellet developed mammary tumors during this experiment. E2 treatment significantly suppressed body weight gain. But no significant differences in food consumption were observed between the two groups, suggesting that metabolic alteration depended on E2 treatment. Serum metabolomic analysis detected 116 features that were statistically different (p < 0.01) between the groups. Quantitation analysis revealed that several phospholipids such as phosphatidylcholines and lysophosphatidylcholines (LPCs) were identified as significantly different metabolites. E2-treated rat serum stimulated the proliferation of human breast cancer MDA-MB-231 cells. In addition, the proliferation effect was diminished by pretreating cells with either autotaxin inhibitor or antagonist for lysophosphatidic acid receptor whose ligands are metabolites of LPCs via autotaxin-mediated hydrolysis. In summary, our results suggest that not only are phospholipids potential biomarkers for mammary tumors but importantly, LPCs themselves could be associated with E2-induced mammary tumorigenesis in female ACI/Seg rats.

Abstract 5235: Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats

Abstract Tocopherols (T), members of the vitamin E family, occur in four forms designated as α, β, δ and γ. Epidemiological studies suggest that tocopherols could reduce the risk of cancer. α-T is known as the classical vitamin E, being the predominant form of tocopherol found in human blood and tissues. However, clinical studies with α-T to substantiate the role of tocopherols in cancer prevention have provided inconclusive results. Such outcomes indicate that the cancer preventive activity of tocopherols might be attributed to the other forms of tocopherols, but not α-T. γ-T, which is the most abundant tocopherol in vegetable oils, is our major dietary source of tocopherols. Recent studies have demonstrated that δ-T and γ-T have higher anti-cancer activity than α-T in several animal models of cancer. The present study investigated the cancer preventive activity of individual purified forms of tocopherols α-T, δ-T, γ-T as well as a naturally occurring γ-T rich mixture of tocopherols (γ-TmT) in an in vivo model of estrogen receptor (ER)-positive breast cancer, namely estrogen supplemented August-Copenhagen Irish (ACI) rats. ACI rats were subcutaneously implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and administered with diets containing 0.2% α-T, δ-T, γ-T or γ-TmT. Rats were euthanized at 30 weeks of mammary carcinogenesis when the E2 control group exhibited 100% tumor incidence. α-T had no effect on estrogen-induced mammary tumors. However, treatment with 0.2% δ-T, γ-T and γ-TmT reduced the tumor volume by 51% (p&amp;lt;0.05), 60% (p&amp;lt;0.01) and 59% (p&amp;lt;0.01), respectively. Administration of α-T, δ-T and γ-T resulted in a 2.1-, 80- and 62-fold increase of these tocopherols, respectively, in the serum of ACI rats. Supplementation with the individual tocopherols also increased the levels of their respective short chain metabolite, carboxyethyl hydroxychromans (CEHCs) in the serum. Administration of δ-T and γ-T was associated with 185- and 99-fold increase in their CEHC metabolites, respectively. The high serum levels of δ- and γ-CEHC metabolites could contribute to the enhanced chemopreventive activity of δ-T and γ-T. In conclusion, δ-T, γ-T and γ-TmT could be potential natural agents for the prevention of estrogen-induced mammary tumorigenesis. (This work was supported by the NIH grant R01 AT007036 and the New Jersey Commission on Cancer Research Postdoctoral Fellowship to Soumyasri Das Gupta). Citation Format: Soumyasri Das Gupta, Joseph Wahler, Min Ji Bak, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5235.

Dietary γ-Tocopherol-Rich Mixture Inhibits Estrogen-Induced Mammary Tumorigenesis by Modulating Estrogen Metabolism, Antioxidant Response, and PPARγ.

This study evaluated the anticancer activity and mechanism of action of a γ-tocopherol-rich tocopherol mixture, γ-TmT, in two different animal models of estrogen-induced breast cancer. The chemopreventive effect of γ-TmT at early (6 weeks), intermediate (18 weeks), and late (31 weeks) stages of mammary tumorigenesis was determined using the August-Copenhagen Irish rat model. Female rats receiving 17β-estradiol (E2) implants were administered with different doses (0%, 0.05%, 0.1%, 0.3%, and 0.5%) of γ-TmT diet. Treatment with 0.3% and 0.5% γ-TmT decreased tumor volume and multiplicity. At 31 weeks, serum concentrations of E2 were significantly decreased by γ-TmT. γ-TmT preferentially induced expression of the E2-metabolizing enzyme CYP1A1, over CYP1B1 in the rat mammary tissues. Nrf2-dependent antioxidant response was stimulated by γ-TmT, as evident from enhanced expression of its downstream targets, NQO1, GCLM, and HMOX1. Serum concentrations of the oxidative stress marker, 8-isoprostane, were also decreased in the γ-TmT-treated groups. Treatment with γ-TmT increased expression of PPARγ and its downstream genes, PTEN and p27, whereas the cell proliferation marker, PCNA, was significantly reduced in γ-TmT-treated mammary tumors. In an orthotopic model in which human MCF-7 breast cancer cells were injected into the mammary fat pad of immunodeficient mice, γ-TmT inhibited E2-dependent tumor growth at all the doses tested. In conclusion, γ-TmT reduced mammary tumor development, in part through decreased E2 availability and reduced oxidative stress in mammary tissues; γ-TmT could thus be an effective agent for the prevention and treatment of E2-induced breast cancer.

Abstract 4646: Cumin extract prevents estrogen-associated breast cancer in ACI rats

Abstract Spices are highly chemoprotective because of their antioxidant, antiproliferative, and anti-inflammatory activities. We have previously demonstrated significant chemoprotective effects of cumin seed powder (7.5%, w/w) against estrogen-induced mammary tumorigenesis in ACI rats by inhibiting cell proliferation as well as cellular and molecular markers. Here we report the inhibition of E2-mediated mammary tumorigenesis by cumin extract (1%, w/w) that is equivalent to the dietary dose in our previous study. Three extractions of cumin seeds with 80% aqueous ethanol followed by evaporation provided 13% of total dried extract (w/w). Groups of female ACI rats were given either AIN-93M diet or diet supplemented with cumin extract and then treated with subcutaneous 17ß-estradiol silastic implants (1.2 cm; 9 mg). Two additional groups were maintained on control or experimental diet. Rats received diet and water ad libitum. Five-six animals from each group were euthanized three weeks after the E2 treatment to determine the effect of interventions on early biomarkers. The remaining animals (n = 20-22) were euthanized when estrogen-treated group on control diet reached over 90% tumor incidence. Diet consumption and body weight gain was found to be largely unaffected by the cumin extract, suggesting that it was well tolerated. The first tumor incidence in control group was after 92 days, this was significantly delayed by 35 days in cumin extract treated group. Experimental diet significantly offset estrogen-related growth of pituitary prolactinomas as early as after three weeks (p = 0.019), the effect was more pronounced at tumor time point (p &amp;lt; 0.0001). At the end of the study (25 wks), the tumor incidence was found to be 96% in control group whereas only 45% animals had tumors in the intervention group. Significant reduction in average tumor volume (474 vs. 75 mm3) and tumor multiplicity (4.1 vs. 0.8 tumors/animal) was also observed by cumin extract compared to control. The mechanistic studies including effect on the plasma prolactin levels, and the cell proliferation marker, PCNA, estrogen-associated expression of the ERα and cyclin D1 in the mammary tissues are being evaluated. Together, our data show that cumin extract can significantly delay and prevent estrogen-mediated mammary tumorigenesis. This data further support our ultimate goal to develop these spice bioactives as clinically translatable chemopreventives and therapeutics against breast cancer. (Supported by CA-125152, Helmsley Fund, and Agnes Brown Duggan Endowment). Citation Format: Farrukh Aqil, Jeyaprakash Jeyabalan, Radha Munagala, Ramesh C. Gupta. Cumin extract prevents estrogen-associated breast cancer in ACI rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4646. doi:10.1158/1538-7445.AM2015-4646

Natural tocopherol mixtures as promising cancer preventive agents

Naturally occurring vitamin E, a family of fat-soluble antioxidants, exists in eight chemical forms of α-, β-, γ-, or δ-tocopherols and α-, β-, γ-, or δ-tocotrienols. Tocopherols are the major source of vitamin E in the U.S. diet. Tocopherols are widely occurring in corn, soybean, sesame and cottonseed oils and nuts. Vitamin E is also often consumed as a dietary supplement, with α-tocopherol as the most common form available. Numerous epidemiological studies suggest cancer preventive activity of vitamin E. However, recent large-scale randomized human trials with α-tocopherol did not demonstrate a cancer preventive effect. Importantly, each form of tocopherols has varying levels of biological activity. Our extensive studies in animal models of carcinogenesis have shown the cancer preventive activity of γ-tocopherol and δ-tocopherol as well as a naturally occurring mixture of tocopherols, but the lack of cancer preventive activity by α-tocopherol. Here, we present the anti-cancer activity of natural dietary γ-tocopherol rich tocopherol mixtures, γ-TmT, in two different animal models of estrogen-mediated breast cancer. γ-TmT inhibited estrogen-induced mammary tumorigenesis by lowering the levels of circulating estrogen, increasing estrogen metabolism via regulating metabolizing enzyme, CYP1A1, and facilitating the clearance of toxic metabolites and reactive oxygen species by stimulation of Nrf2-mediated antioxidant response. Activation of PPARγ signaling and inhibition of cell proliferation could also contribute to the chemopreventive effects of γ-TmT. Thus, dietary γ-tocopherol rich mixtures could be safe and effective natural agents for the prevention and treatment of estrogen-induced breast cancer.

Abstract 243: Tocopherols inhibit oxidative and nitrosative stress in estrogen-induced early mammary hyperplasia in ACI rats

Abstract Tocopherols (T), members of the vitamin E family, exist in four different forms designated as α, β, δ and γ. Due to their antioxidant properties, tocopherols are suggested to reduce the risk of cancer. α-T is known as the classic vitamin E as it is the major tocopherol found in plasma and tissues. However, some large scale clinical trials with α-T have provided inconclusive results. Recent studies from our laboratory and others indicate that δ-T and γ-T have stronger anti-cancer activities than α-T in vitro and in vivo. γ-T is the most common form of vitamin E in the U.S. diet, present in vegetable oils such as soybean, corn and cottonseed. The naturally occurring γ-T-enriched mixture of tocopherols (γ-TmT) has been reported to inhibit mammary tumorigenesis in estrogen receptor positive models of breast cancer. Using ACI rats, we investigated the chemopreventive activity of γ-TmT in the early stages of estrogen-induced mammary tumorigenesis. ACI rats provide an established model of rodent mammary carcinogenesis due to their high sensitivity to estrogen. Rats were subcutaneously implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and fed with 0.3% γ-TmT diet for 1, 3, 7 and 14 days. Administration of γ-TmT gradually increased the levels of tocopherols and their metabolites in the serum and mammary glands of the rats from day 1 to 14. Histological analysis showed that estrogen induced mammary hyperplasia starting at day 3. However, the induction of mammary hyperplasia was not altered by dietary γ-TmT. Interestingly, mRNA expression of PPARγ, a nuclear receptor which inhibits cell proliferation and survival, was significantly increased by γ-TmT administration at 14 days. The oxidative and nitrosative stress markers, 8-oxo-dG and nitrotyrosine, respectively, were induced in the mammary tissues of E2-treated rats as early as day 1. γ-TmT decreased the levels of these markers starting from day 7. Serum level of 8-isoprostane, a marker of oxidative stress, was also decreased by γ-TmT. Elevated mRNA levels of a transcription factor Nrf2 and its downstream antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, were observed in the γ-TmT fed groups as early as day 1, indicating the induction of antioxidant response by γ-TmT. In conclusion, inhibition of oxidative, nitrosative stress and stimulation of antioxidant response are the primary effects of γ-TmT administration in the early stages of E2-induced mammary tumorigenesis in ACI rats. Citation Format: Soumyasri Das Gupta, Jae Young So, Joseph Wahler, Mao-Jung Lee, Chung S. Yang, Nanjoo Suh. Tocopherols inhibit oxidative and nitrosative stress in estrogen-induced early mammary hyperplasia in ACI rats. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2014-243

Induction of quinone reductase by tamoxifen or DPN protects against mammary tumorigenesis

We have previously shown that estrogen receptor β (ERβ)-mediated up-regulation of quinone reductase (QR) is involved in the protection against estrogen-induced mammary tumorigenesis. Our present study provides evidence that the ERβ agonist, 2,3-bis-(4-hydroxy-phenyl)-propionitrile (DPN), and the selective estrogen receptor modulator tamoxifen (Tam), inhibit estrogen-induced DNA damage and mammary tumorigenesis in the aromatase transgenic (Arom) mouse model. We also show that either DPN or Tam treatment increases QR levels and results in a decrease in ductal hyperplasia, proliferation, oxidative DNA damage (ODD), and an increase in apoptosis. To corroborate the role of QR, we provide additional evidence in triple transgenic MMTV/QR/Arom mice, wherein the QR expression is induced in the mammary glands via doxycycline, causing a decrease in ductal hyperplasia and ODD. Overall, we provide evidence that up-regulation of QR through induction by Tam or DPN can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a novel mechanism of prevention against breast cancer. Thus, our data have important clinical implications in the management of breast cancer; our findings bring forth potentially new therapeutic strategies involving ERβ agonists.

Berries and Ellagic Acid Prevent Estrogen-Induced Mammary Tumorigenesis by Modulating Enzymes of Estrogen Metabolism

To determine whether dietary berries and ellagic acid prevent 17beta-estradiol (E(2))-induced mammary tumors by altering estrogen metabolism, we randomized August-Copenhagen Irish rats (n = 6 per group) into five groups: sham implant + control diet, E(2) implant + control diet (E(2)-CD), E(2) + 2.5% black raspberry (E(2)-BRB), E(2) + 2.5% blueberry (E(2)-BB), and E(2) + 400 ppm ellagic acid (E(2)-EA). Animals were euthanized at early (6 wk), intermediate (18 wk), and late (24 wk) phases of E(2) carcinogenesis, and the mammary tissue was analyzed for gene expression changes using quantitative real-time PCR. At 6 weeks, E(2) treatment caused a 48-fold increase in cytochrome P450 1A1 (CYP1A1; P < 0.0001), which was attenuated by both BRB and BB diets to 12- and 21-fold, respectively (P < 0.001). E(2) did not alter CYP1B1 levels, but both berry and EA diets significantly suppressed it by 11- and 3.5-fold, respectively, from baseline (P < 0.05). There was a 5-fold increase in 17beta-hydroxysteroid dehydrogenase 7 (17betaHSD7), and this was moderately abrogated to approximately 2-fold by all supplementation (P < 0.05). At 18 weeks, CYP1A1 was elevated by 15-fold in E(2)-CD and only E(2)-BB reduced this increase to 7-fold (P < 0.05). Catechol-O-methyltransferase expression was elevated 2-fold by E(2) treatment (P < 0.05), and all supplementation reversed this. At 24 weeks, CYP1A1 expression was less pronounced but still high (8-fold) in E(2)-treated rats. This increase was reduced to 3.2- and 4.6-fold by E(2)-BRB and E(2)-EA, respectively (P < 0.05), but not by E(2)-BB. Supplementation did not alter the effect of E(2) on steroid receptors. The diets also significantly suppressed mammary tumor incidence (10-30%), volume (41-67%), and multiplicity (38-51%; P < 0.05). Berries may prevent mammary tumors by suppressing the levels of E(2)-metabolizing enzymes during the early phase of E(2) carcinogenesis.

Abstract 5366: NAD(P)H-Quinone oxidoreductase (NQO1): Potential role in the prevention of estrogen-induced breast cancer

Abstract The importance of estrogens in the etiology of breast cancer is widely recognized. However, the exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. The objective of the current study was to characterize the role of NQO1 in the prevention of estrogen-induced mammary tumorigenesis by antioxidants vitamin C or butylated hydroxyanisole (BHA). NQO1 converts 17β-estradiol (E2)-quinones back to E2-catechols, thus making E2-quinones unavailable for oxidant damage. Female ACI rats were treated with E2 (3 mg); vitamin C (1% in drinking water); E2 + vitamin C; BHA (0.7% in diet); and E2 + BHA for up to 8 months. The female ACI rat is an established animal model to study the mechanisms of E2-induced breast cancer. The protein isolated from breast tissue and breast tumor tissue of rats treated with E2 and antioxidants was analyzed for NQO1 expression. NQO1 was suppressed in E2-exposed mammary tissue after treatment of rats with E2 for 8 months and in mammary tumors. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Co-treatment of rats with E2 and vitamin C resulted in a 4-fold induction of NQO1 compared to control mammary tissue. Similarly, co-treatment of rats with E2 and BHA induced NQO1 4.4-fold compared to control mammary tissue. There was no change in NQO1 expression during early time points of E2 exposure (7 and 15 days). The up-regulation of NQO1 by vitamin C or BHA was evident after 4 months of treatment. There was no effect of BHA or vitamin C treatment on NQO1 expression on other non-target organs like liver, lung and heart except kidney. These studies suggest a protective role of NQO1 in E2-induced breast carcinogenesis. NQO1 may thus have a great potential in the development of therapeutic strategies for the prevention of estrogen-induced neoplasia. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5366.

Mammary tumor induction in ACI rats exposed to low levels of 17β-estradiol

Animal models play a major role in understanding the etiology, molecular mechanisms, strategizing intervention and treatment of human diseases. ACI, an inbred line derived from August and Copenhagen strains, is unique for its susceptibility to estrogen-induced mammary tumors. Histologically and in many molecular aspects, the tumors formed in these rats are similar to human breast cancers. Previous studies have shown high mortality and significant weight loss in this model associated with pituitary gland abnormality. We hypothesized that this could be due to overwhelming the biological system with estrogen. Three groups of female ACI rats (7-8 weeks) received either 3-cm sham silastic implants, or the conventional 3-cm silastic implants containing 27 mg of 17beta-estradiol, or 1.2-cm silastic implants containing 9 mg 17beta-estradiol. The sham and 3-cm implant rats were euthanized at 180 days while the 1.2-cm implant rats were euthanized at 240 days. The 1.2-cm implants resulted in significantly reduced serum estrogen levels and pituitary gland size. Animals with 1.2-cm implants had 100% tumor incidence, while not all rats developed tumors with 3-cm implants. Both the tumor burden (from 1,011+/-402 to 2,324+/-454 mm(3); p=0.01) and tumor multiplicity (from 5.78+/-1.4 to 7.6+/-1.04) increased by lowering the estrogen dose, and the inter-animal variability in the tumor indices decreased. Finally, the weight of the pituitary gland was also significantly (p=0.0004) reduced (from 178+/-23.5 mg to 80+/-8.9 mg) and the mortality rate decreased from 42% to 0% (p=0.01). Our data indicate that the improvised model will provide valuable insights into the molecular alterations in the estrogen-induced mammary tumorigenesis and will be ideal for inhibition studies.

Protective roles of quinone reductase and tamoxifen against estrogen-induced mammary tumorigenesis

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.