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Characterization of MCF mammary epithelial cells overexpressing the Arylhydrocarbon receptor (AhR).

Wong PS, Li W, Vogel CF, Matsumura F - BMC Cancer (2009)

Bottom Line: The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics.The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2.This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental Toxicology and the Center for Environmental Health Sciences, University of California, One Shields Ave., Davis, CA 95616, USA. patwong@ucdavis.edu

ABSTRACT

Background: Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling.

Methods: We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics.

Results: MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2. Furthermore, we could demonstrate that suppression of AhR functions through anti-AhR siRNA or MNF causes the recovery of ERalpha functions.

Conclusion: One of the main causes for AhR overexpression in these MCF breast cancer cells appears to be the loss of ERalpha functions. This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR.

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ErbB2 expression in MCF10AT1 cells selected for various passages in the presence of 17-β-estradiol (E2), 4-OH-Tamoxifen (Tam) or β-hexachlorohexane (bHCH).
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Figure 3: ErbB2 expression in MCF10AT1 cells selected for various passages in the presence of 17-β-estradiol (E2), 4-OH-Tamoxifen (Tam) or β-hexachlorohexane (bHCH).

Mentions: To address the question on the possible cause(s) of AhR overexpression in mammary epithelial cells, we began this project by exposing MCF10AT1 cells to three estrogen receptor interacting chemicals, 1 nM E2, 10 nM 4-hydroxy-tamoxifen, or 1 μM β-hexachlorohexane (β-HCH), an estrogenic pesticide, throughout a number of passages under our standard culture conditions using "selection medium" which was phenol red-free and contained heat-inactivated serum (2.5%) [17]. After 20 passages, those selected cells have exhibited distinct signs of increased proliferation, when tested in "selection medium" (Figure 1). To gain insight into the main cause for such phenotypic changes in each of selected cell line, we have tested the effectiveness of two well known chemical inhibitors, AG879, a specific inhibitor for ErbB2 associated tyrosine kinase, and 3-methoxy-4-nitroflavone (MNF), a specific antagonist to the Ah receptor (AhR) on those cells (Table 1). It was found that MNF was particularly effective in suppressing the rate of proliferation of E2 selected lines, while AG879 was more effective in this regard on those selected by 4-hydroxy-tamoxifen (Tam) or by β-HCH (Table 1), suggesting perhaps that AhR plays an important role in inducing increased proliferation of the E2-selected line, and that contribution of ErbB2 may be more important in Tam- and β-HCH- selected cells than E2-selected ones in terms of promoting cell proliferation. This possibility was further checked by examining the changes in mRNA expressions of AhR (Figure 2) and ErbB2 (Figure 3) in these 3 sub-lines during their selection process. It is apparent from this study that the expression of AhR is indeed outstanding in E2-selected cells (Figure 2), and that the elevated expression of ErbB2 mRNA is most pronounced in β-HCH selected cells followed by those selected by Tam (Figure 3). In general, those selected MCF10AT1 cells passages showed the highest expression of AhR and ErbB2 mRNAs after 20 passages, except in the case of AhR expression in Tam-selected cells, and ErbB2 expression in β-HCH selected ones, both of which showed the sign of decline of their expression in 20 passage from that was found in 18 passage. In view of this observation, we have decided not to subject those cells for further selection by any of these selecting agents beyond 20 passages in the case of MCF10AT1.


Characterization of MCF mammary epithelial cells overexpressing the Arylhydrocarbon receptor (AhR).

Wong PS, Li W, Vogel CF, Matsumura F - BMC Cancer (2009)

ErbB2 expression in MCF10AT1 cells selected for various passages in the presence of 17-β-estradiol (E2), 4-OH-Tamoxifen (Tam) or β-hexachlorohexane (bHCH).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721847&req=5

Figure 3: ErbB2 expression in MCF10AT1 cells selected for various passages in the presence of 17-β-estradiol (E2), 4-OH-Tamoxifen (Tam) or β-hexachlorohexane (bHCH).
Mentions: To address the question on the possible cause(s) of AhR overexpression in mammary epithelial cells, we began this project by exposing MCF10AT1 cells to three estrogen receptor interacting chemicals, 1 nM E2, 10 nM 4-hydroxy-tamoxifen, or 1 μM β-hexachlorohexane (β-HCH), an estrogenic pesticide, throughout a number of passages under our standard culture conditions using "selection medium" which was phenol red-free and contained heat-inactivated serum (2.5%) [17]. After 20 passages, those selected cells have exhibited distinct signs of increased proliferation, when tested in "selection medium" (Figure 1). To gain insight into the main cause for such phenotypic changes in each of selected cell line, we have tested the effectiveness of two well known chemical inhibitors, AG879, a specific inhibitor for ErbB2 associated tyrosine kinase, and 3-methoxy-4-nitroflavone (MNF), a specific antagonist to the Ah receptor (AhR) on those cells (Table 1). It was found that MNF was particularly effective in suppressing the rate of proliferation of E2 selected lines, while AG879 was more effective in this regard on those selected by 4-hydroxy-tamoxifen (Tam) or by β-HCH (Table 1), suggesting perhaps that AhR plays an important role in inducing increased proliferation of the E2-selected line, and that contribution of ErbB2 may be more important in Tam- and β-HCH- selected cells than E2-selected ones in terms of promoting cell proliferation. This possibility was further checked by examining the changes in mRNA expressions of AhR (Figure 2) and ErbB2 (Figure 3) in these 3 sub-lines during their selection process. It is apparent from this study that the expression of AhR is indeed outstanding in E2-selected cells (Figure 2), and that the elevated expression of ErbB2 mRNA is most pronounced in β-HCH selected cells followed by those selected by Tam (Figure 3). In general, those selected MCF10AT1 cells passages showed the highest expression of AhR and ErbB2 mRNAs after 20 passages, except in the case of AhR expression in Tam-selected cells, and ErbB2 expression in β-HCH selected ones, both of which showed the sign of decline of their expression in 20 passage from that was found in 18 passage. In view of this observation, we have decided not to subject those cells for further selection by any of these selecting agents beyond 20 passages in the case of MCF10AT1.

Bottom Line: The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics.The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2.This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Environmental Toxicology and the Center for Environmental Health Sciences, University of California, One Shields Ave., Davis, CA 95616, USA. patwong@ucdavis.edu

ABSTRACT

Background: Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling.

Methods: We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics.

Results: MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2. Furthermore, we could demonstrate that suppression of AhR functions through anti-AhR siRNA or MNF causes the recovery of ERalpha functions.

Conclusion: One of the main causes for AhR overexpression in these MCF breast cancer cells appears to be the loss of ERalpha functions. This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR.

Show MeSH
Related in: MedlinePlus