Progression Of Mammary Carcinogenesis Research Articles

Lack of effect of the procarcinogenic 17β-estradiol on nutrient uptake by the MCF-7 breast cancer cell line

Breast cancer is one of the most frequent cancers in the population, especially in older women. Estrogen is known to be a key hormone in the development and progression of mammary carcinogenesis. In this study, we investigated if the procarcinogenic effect of 17β-estradiol (E2) in breast cancer MCF-7 cells is dependent on changes in glucose or folic acid cellular uptake.The effect of E2 on uptake of 3H-deoxy-d-glucose, 3H-folic acid, cell proliferation (3-thymidine incorporation assay), culture growth (sulforhodamine B assay), viability (lactate dehydrogenase activity assay), lactate production and migration capacity (injury assay) was evaluated.E2 (48h; 100nM) increased culture growth (16%), proliferation rate (24%), cellular viability (36%) and lactate production (38%). In contrast, E2 did not significantly affect the migration capacity of MCF-7 cells. The pro-proliferative, but not the cytoprotective effect of E2 was found to be ERβ-dependent. The polyphenols rutin and caffeic acid were not able to counteract the effect of E2 upon cell proliferation and viability. Uptake of 3H-deoxy-d-glucose was not affected by E2, either in the absence or presence of GLUT inhibitors (cytochalasin B plus phloridzin). Moreover, E2 did not change GLUT1 mRNA levels. Finally, 3H-folic acid uptake was also not affected by E2, both in the absence and presence of the RFC1 inhibitor, methotrexate.The pro-proliferative and cytoprotective effects of E2 are not dependent neither of stimulation of glucose cellular uptake (both GLUT and non-GLUT-mediated) nor of stimulation of folic acid uptake (both RFC1-and non-RFC1-mediated).

Abstract IA14: Breaking bad: Cancer-associated fibroblasts are reprogrammed from growth inhibitory to pro-inflammatory and tumor promoting in breast cancer

Abstract Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. We previously demonstrated that Cancer-Associated Fibroblasts (CAFs) orchestrate tumor-enhancing inflammation in multiple mouse and human malignancies, including breast cancer. Although breast cancer is one of the major tumor types where CAFs were shown to be tumor promoting, there is no detailed analysis of the dynamic changes in CAFs characteristics and function in correlation with tumor progression. We therefore set out to characterize the dynamic changes in CAFs during progression of mammary carcinogenesis, in a transgenic mouse model of human breast cancer. We profiled the transcriptome of fibroblasts isolated from normal mammary glands, or from mammary glands of transgenic mice at defined tumorigenic stages. Data analysis revealed unique CAF gene signatures that correspond to distinct tumor stages, with only partial overlap between the stages, suggesting co-evolution of the microenvironment with tumorigenic progression. Interestingly, the gene signature of fibroblasts isolated from hyperplastic lesions is inverse to that in CAFs from neoplastic stages, and has a growth inhibitory phenotype, while CAFs from mammary tumors express pro-inflammatory and tumor promoting gene signatures. Moreover, we functionally demonstrate that CAFs isolated from late carcinoma express a gene signature that links tissue damage with tumor-promoting inflammation. To identify tumor-secreted factors capable of reprogramming normal mammary fibroblasts to pro-inflammatory CAFs, we performed a proteomic screen of breast cancer cells secretome, and identified Osteopontin (OPN). Knockdown of OPN in tumor cells in vivo resulted in attenuated stromal activation, reduced immune cell recruitment and inhibition of tumor growth. Taken together, our findings reveal reciprocal crosstalk between fibroblasts and cancer cells which drives CAF reprogramming, to facilitate tumor progression in breast cancer. Citation Format: Yoray Sharon, Nour Ershaid, Yael Raz, Noam Cohen, Metsada Pasmanik-Chor, Tamar Geiger, Neta Erez. Breaking bad: Cancer-associated fibroblasts are reprogrammed from growth inhibitory to pro-inflammatory and tumor promoting in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA14.

Abstract B103: Proinflammatory signaling by cancer-associated fibroblasts co-evolves during mammary carcinogenesis

Abstract Cancer-associated fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. We demonstrated that CAFs also mediate tumor-enhancing inflammation: CAFs isolated from pre-neoplastic and tumor tissue expressed a pro-inflammatory gene signature and functioned to recruit macrophages and enhance tumor angiogenesis in a mouse model of squamous carcinoma. Moreover, we demonstrated that pro-inflammatory signaling by CAFs is operative also in mouse and human breast carcinogenesis. Although breast cancer is one of the major tumor types where CAFs were shown to be tumor promoting, there is no detailed analysis of CAF characteristics and function in correlation with tumor progression. We therefore set out to characterize the dynamic changes in CAFs during progression of mammary carcinogenesis, in a transgenic mouse model of human breast cancer, the MMTV-PyMT mice. We show that normal mammary fibroblasts can be “educated” by mammary carcinoma cells to become activated, pro-inflammatory CAFs. Furthermore, transcriptome profiling of fibroblasts isolated from normal mammary glands, or from mammary glands of transgenic mice at defined tumorigenic stages (hyperplasia, early carcinoma, invasive adenocarcinoma) revealed distinct CAF gene expression signatures that correspond to different tumor stages, with only partial overlap between the stages, suggesting co-evolution of the microenvironment with tumorigenic progression. Interestingly, the gene signature of fibroblasts isolated from hyperplastic lesions is inverse to that in CAFs from neoplastic stages, and has a putative growth inhibitory phenotype, while CAFs from early and late carcinoma express pro-inflammatory and tumor promoting gene signatures. Elucidating the molecular mechanisms by which CAFs mediate cancer-related inflammation in breast cancer will greatly increase our understanding of stromal pathways that contribute to tumorigenesis, in an effort to identify novel stromal molecular targets for combinatorial therapeutic approaches to combat breast cancer progression. Citation Format: Yoray Sharon, Yael Raz1,2, Lina Alon, Neta Erez. Proinflammatory signaling by cancer-associated fibroblasts co-evolves during mammary carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B103.

Inflammation and breast cancer. Inflammatory component of mammary carcinogenesis in ErbB2 transgenic mice

This review addresses genes differentially expressed in the mammary gland transcriptome during the progression of mammary carcinogenesis in BALB/c mice that are transgenic for the rat neu (ERBB2, or HER-2/neu) oncogene (BALB-neuT664V-E mice). The Ingenuity knowledge database was used to characterize four functional association networks whose hub genes are directly linked to inflammation (specifically, the genes encoding IL-1β, tumour necrosis factor, interferon-γ, and monocyte chemoattractant protein-1/CC chemokine ligand-2) and are increasingly expressed during such progression. In silico meta-analysis in a human breast cancer dataset suggests that proinflammatory activation in the mammary glands of these mice reflects a general pattern of human breast cancer.

Exercise-induced stress enhances mammary tumor growth in rats: Beneficial effect of the hormone melatonin

We hypothesized that intense exercise training (forced swimming for 30 min, 5 days/week) may enhance the progression of mammary carcinogenesis through the involvement of stress hormones, such as catecholamines and prolactin, which can promote breast cancer. After the appearance of the DMBA-induced tumors in Sprague-Dawley rats, the effect was evaluated of exercise-induced stress (with or without administration of the hormone melatonin) on the survival time, tumor multiplicity, and tumor growth until the death of the animals. In a second set of experiments, after one month of exercise, the NK cells count in blood, and the plasma concentrations of catecholamines and prolactin were determined. Although no significant change was found in either the survival time of the rats or the tumor multiplicity, exercise significantly increased the tumor growth rate. Stress was confirmed by the enhanced adrenaline and prolactin concentrations in the blood of the exercised rats. Exercise-induced stress did not change the percentage of NK cells in the tumor-bearing rats. Melatonin counteracted the increased tumor growth, returning the prolactin and adrenaline concentrations to their optimal physiological levels in the exercised tumor-bearing rats, thus confirming an "anti-stress" role of this hormone. In conclusion, intense exercise-induced stress enhances mammary carcinogenesis through the involvement of adrenaline and prolactin. The results also confirmed a role of melatonin as a therapeutic aid against breast cancer in general, and in particular during situations of stress.

TGF-alpha exerts biphasic effects on estrogen--and phytoestrogen-mediated gene expression in breast cancer cells.

Transforming growth factor-alpha (TGF-alpha) contributes to the progression of mammary carcinogenesis in part through synergistic augmentation of estradiol (E2) action. To investigate this further, we sought to determine (1) whether the duration of TGF-alpha treatment might influence the nature of the TGF-alpha/E2 interaction, and (2) whether TGF-alpha would behave in a similar manner when combined with phytoestrogens. To this end, we transfected T47-D breast cancer cells with an estrogen-responsive reporter and then treated the cells (for 4-48 h) with varying concentrations of TGF-alpha, E2, the antiestrogen 4-hydroxy-tamoxifen (HOT), and/or one of three phytoestrogens. Our findings revealed that TGF-alpha has short-term synergistic and long-term inhibitory effects on E2- and phytoestrogen-regulated gene expression. Furthermore, this secondary inhibition of E2 action by TGF-alpha was similar in magnitude to that imposed by HOT. These findings demonstrate a novel role for TGF-alpha and invite reevaluation of current models regarding TGF-alphas interactions with E2 in breast cancer cells. Our results also raise the possibility that phytoestrogens, which interact with TGF-alpha in a manner conceptually identical to that of E2, may subserve a regulatory function in breast cancer cells.

Animal models for breast cancer

Rodent mammary tumors induced by chemical carcinogens have proven to be very useful in the genetic analysis of initiation, promotion and progression of mammary carcinogenesis. We are studying rat mammary carcinomas induced by the chemical carcinogen, N-nitroso- N-methylurea. The earliest genetic event observed in the mammary gland is the activation of Ha- ras oncogenes, which is followed by promotion of the initiated cells by hormones involved in puberty. Preferential amplification of the mutated Ha- ras allele, of PRAD-1 and IGF2, loss of expression of the mitogenic growth factor gene, MK, and mutation in the tumor suppressor gene, p53, are seen in the mammary tumors during tumor progression.

Human Chorionic Gonadotropin and Rat Mammary Cancer Prevention

The observation that mammary cancer induced by 7,12-dimethylbenz[a]anthracene (DMBA) in young, virgin, Sprague-Dawley rats is abolished by pregnancy led us to test the possibility of protecting the mammary gland from chemically induced carcinogenesis by using the placental hormone human chorionic gonadotropin (hCG). Fifty-day-old, outbred, virgin, Sprague-Dawley rats were utilized in two different experimental protocols. In protocol 1, four groups of virgin rats received either no hCG (group I) or a daily intraperitoneal injection of hCG at 1 IU (group II), 10 IU (group III), or 100 IU (group IV) for 21 days; group I and groups II-IV, at 21 days after the last injection, were given a single intragastric dose of 8 mg of DMBA per 100 g of body weight. In protocol 2, 50-day-old rats were treated with a single intragastric dose of 8 mg of DMBA per 100 g of body weight; 21 days later, they were separated into groups V and VI. Group V rats remained undisturbed, except for palpation twice a week for detection of tumor development. Group VI rats received a daily intraperitoneal injection of 100 IU of hCG for 60 days. Tumorigenesis was evaluated 24 and 30 weeks after carcinogen administration in animals in protocols 1 and 2, respectively. In protocol 1, in which animals (with the exception of the control group) were treated with hCG prior to carcinogen administration, the incidence of adenocarcinomas decreased in a dose-dependent manner, from 43.8% in the controls (group I) to 34.4%, 18.2%, and 6.15% in groups II-IV treated with 1, 10, or 100 IU of hCG, respectively. Among animals in protocol 2, hCG treatment significantly reduced the incidence of adenocarcinomas, from 100% in DMBA-treated rats (group V) to 45.5% in rats treated with DMBA plus hCG (group VI). These data indicate that hCG can prevent both initiation and progression of mammary carcinogenesis.