Johana E. Vanegas

Molecular basis of pregnancy-induced breast cancer protection.

We have postulated that the lifetime protective effect of an early pregnancy against breast cancer is due to the complete differentiation of the mammary gland characterized by a specific genomic signature imprinted by the physiological process of pregnancy. In the present work, we show evidence that the breast tissue of postmenopausal parous women has had a shifting of stem cell 1 to stem cell 2 with a genomic signature different from similar structures derived from postmenopausal nulliparous women that have stem cell 1. Those genes that are significantly different are grouped in major categories on the basis of their putative functional significance. Among them are those gene transcripts related to immune surveillance, DNA repair, transcription, chromatin structure/activators/co-activators, growth factor and signal transduction pathway, transport and cell trafficking, cell proliferation, differentiation, cell adhesion, protein synthesis and cell metabolism. From these data, it was concluded that during pregnancy there are significant genomic changes that reflect profound alterations in the basic physiology of the mammary gland that explain the protective effect against carcinogenesis. The implication of this knowledge is that when the genomic signature of protection or refractoriness to carcinogenesis is acquired by the shifting of stem cell 1 to stem cell 2, the hormonal milieu induced by pregnancy or pregnancy-like conditions is no longer required. This is a novel concept that challenges the current knowledge that a chemopreventive agent needs to be given for a long period to suppress a metabolic pathway or abrogate the function of an organ.

Human chorionic gonadotropin (hCG) prevents the transformed phenotypes induced by 17 β-estradiol in human breast epithelial cells.

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life‐long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF‐10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 β‐estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF‐10F induced by E2. MCF‐10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D‐cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF‐10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland.

Progressive increase of glucose transporter-3 (GLUT-3) expression in estrogen-induced breast carcinogenesis

IntroductionIncreased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. A family of glucose transporters (GLUTs) facilitates glucose movement across the plasma membranes in a tumor-specific manner. Glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12, have been previously shown in breast cancer cells and are found to be associated with poor prognosis. In addition, it has been shown that estrogen plays critical roles in GLUT regulation, however, the stage-specific GLUT regulation of mammary carcinogenesis is unclear.MethodsGLUT expression patterns were investigated in an in vitro–in vivo progressive, estrogen-induced, mammary carcinogenesis model which consisted of four cell lines, with same genetic background. In this model, different stages of tumor initiation and progression are represented, MCF-10F being the normal stage, E2 cells the transformed stage by estrogen, C5 cells, the invasive stage, and T4 cells the tumorigenic stage. In addition, loss of ductulogenesis and solid mass formation in collagen matrix and invasiveness of the cells were counted.ResultsReal time PCR showed that GLUT1 expression was downregulated in MCF10F after treatment with 17β-estradiol (E2), and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasive capacity, whereas, GLUT-12 was downregulated in C5 and T4 cells.ConclusionEstrogen-induced malignant transformation is associated with remarkable and progressive GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation.

Fish Oil Alters Tamoxifen-Modulated Expression of mRNAs That Encode Genes Related to Differentiation, Proliferation, Metastasis, and Immune Response in Rat Mammary Tumors

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publishers online edition of Nutrition and Cancer to view the supplemental file.

Abstract 5578: Transcriptomic changes in tamoxifen-treated mammary tumors in Sprague-Dawley rats fed with omega-3 or omega-6 rich diets

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We have demonstrated that administration of an omega-3 rich diet to Tamoxifen (Tam)-treated rats reduced the incidence of mammary tumors when compared to rats fed an omega-6 rich diet in the presence and in the absence of Tam treatment (A. Manni, et al. Cancer Prev Res 3:322, 2010). To understand the mechanisms of antitumor action of omega-3 fatty acids, we evaluated the transcriptomic changes in 1-Methyl-1-nitrosourea (MNU)-induced mammary tumors in Tam-treated rats fed with omega-3 (fish oil) or omega-6 (corn oil) fatty acids. Twenty one-days old Sprague Dawley rats received a single 50 mg/kg of body weight (bw) i.p. injection of MNU and were distributed into 4 groups: 1) CO group (20% corn oil diet); 2) COtam group (20% CO diet plus Tam 100 μg/Kg bw s.c., 5 days/week); 3) FO group (17% fish oil diet); 4) FOtam group (17% FO diet plus Tam). Animals were euthanized 8 weeks after MNU injection. RNA from cribiform tumors was extracted and Agilent oligonucleotide microarrays were performed in three samples per group. Tam-treated groups were compared to their respective controls (FOtam vs. FO and COtam vs. CO). Additionally, the FO-treated group was compared to the CO-treated group. Genes were considered differentially expressed when their level of expression differed by at least 3-fold with a P value <0.01. Genes of interest were validated by real time PCR. We found 32 differentially expressed genes between COtam and CO, 58 differentially expressed genes between FOtam and FO, and 19 differentially expressed genes between FO and CO. Real time PCR confirmed the differential expression of SerpinB10, Wisp2 and Apod in the FO-treated groups, which may be related to greater tumor differentiation. The down-regulation of Thrsp and Wnt5b genes in FOtam group may be related to tumor growth impairment and lower capacity of metastasis. Moreover, the up-regulation of IRF7, RT1-CE3, RT1-CE16 and RT1-Aw2 points to an improvement of the immune response against tumors in FO-treated animals. However, the up-regulation of FCER1A, HDC, MS4A2, SLP1, MCPT1 and MCTP2 suggests a shift of the immune response towards a Th2 pattern (mast cell-based immune response), which could be a mechanism of escape from the immune response caused by the combination of FO and Tam treatment. Our data suggests that an omega-3 rich diet could lead to a more differentiated tumor phenotype and an improved immune response against tumors. However, the administration of tamoxifen to FO fed rats promoted a Th2 pattern of immune response, which could represent an escape mechanism of the tumor cells from the improved immune response caused by FO per se. (Supported by the Susan G. Komen for the Cure, grant KG081632). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2011-5578

Abstract 5722: Effects of dietary omega-3 and omega-6 in the prevention of rat mammary cancer with tamoxifen

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC There is evidence that omega 3-rich diet strengthened the inhibitory effect of tamoxifen on the development of estrogen dependent tumors, pointing to omega 3 as a potential adjuvant to standard therapy (Manni A. et al. Cancer Prev Res, in press). However, the mechanistic basis of this observation is unknown. The objective of the present work is to analyze the genomic changes in chemically induced mammary tumors that developed in tamoxifen treated rats fed omega 3 or omega 6 fatty acids. Sprague Dawley rats, 21 days-old, were administered an i.p. injection of 1-Methyl-1-nitrosourea (MNU) 50 mg/kg body weight. The animals were distributed into 4 groups (G), and maintained under standard conditions and were given the following treatments for 8 weeks: G1 received a diet of 20% of corn oil (CO); G2 received a diet with 17% of fish oil (FO) and 3% CO; G3 received the CO diet and tamoxifen; G4 received FO diet and tamoxifen. Tamoxifen was administered s.c 100 μg/Kg b.w., 5 days/week. The animals were euthanized after 8 weeks. Mammary tumors were frozen in liquid nitrogen and stored at −80C. RNA from cribiform tumors was extracted and one-color Agilent 44K whole rat genome oligonucleotide microarray was performed in three tumor samples per group. Tamoxifen-treated groups (G3 and G4) were compared with their respective controls (G1 and G2). The genes were considered differentially expressed when the p value was lower or equal to 0.01 and had at least 1.50 fold change. A total of 488 genes were considered statistically different comparing G3 to G1 (G3/G1), whereas 206 genes were considered statistically different comparing G4 to G2 (G4/G2). Gene ontology analysis showed that 125 genes of G3/G1 comparison and 58 of G4/G2 comparison were related to cancer, apoptosis, cellular movement, cellular proliferation, inflammatory response or angiogenesis. In the G3 /G1 comparison, the expression pattern of genes related to cell proliferation and apoptosis was found to be divergent, as well as the genes related to cell proliferation in the G4/G2 comparison. In contrast, G4/G2 did not showed relevant genes expressed related to apoptosis. CO treatment in presence of tamoxifen (G3/G1) induced an up-regulation of C5r1, Hck, Il6Ra, Myo1f_predicted and Tlr6, and down-regulation of Mif, indicating an increase of the inflammatory host response. These changes were accompanied by up-regulation of Ets1, Tlr6 and Wdr44 genes controlling angiogenesis and cell migration, which can lead to metastasis. These changes were not observed in the FO and tamoxifen treated group. We conclude that an omega 6 rich-diet could modify the genomic profile of tamoxifen treated tumors making them more prone to an aggressive behavior or result in tamoxifen resistance. (Supported by the Susan G. Komen for the Cure, grant KG081632). The first two authors contributed equally to this work. 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 5722.

Stage-Specific Expressions of GLUT-1, GLUT-3 and GLUT-12 in Estrogen-Induced Breast Cancer.

Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. The glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12 have been shown in breast cancer cells in which estrogen play a critical role on its regulation and also associated with poor prognosis. However. the stage-specific GLUT regulation during the process of mammary carcinogenesis is unclear. In the present study we are aiming to study the GLUT expression pattern in an in vitro-in vivo model of estrogen induced carcinogenesis in which the human breast epithelial cell line MCF-10F is considered the normal counterpart E2 the transformed, C5 the invasive and T4 the tumorigenic stage. The gene expression of GLUTs was correlated with the ductulogenic pattern in a collagen matrix and the invasive properties using the Boyden chamber The RT-PCR data showed that the GLUT1 expression was downregulated in MCF10F after the treatment with 17β-estradiol (E2) and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasion capacity. Whereas, GLUT-12 is downregulated in invasive and tumorigenic cells. We conclude that the estrogen-induced malignant transformation is associated with remarkably GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation for transformed MCF-10F cells. (This work was supported by grant R21 ES015894from the NIEHS) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5152.