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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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mRNA expression of selected proteins in long-term selected cells – MCF10AT1 and MCF-7 cell lines grown in the presence of 17-β-estradiol (E2, 1 nM), 4-OH-Tamoxifen (Tam, 10 nM) and β hexachlorohexane (β-HCH, 1 μM) for 20 and 34 passages respectively. PR = progesterone receptor. * p < 0.05 ** p < 0.01 ***p < 0.001 vs control
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Figure 4: mRNA expression of selected proteins in long-term selected cells – MCF10AT1 and MCF-7 cell lines grown in the presence of 17-β-estradiol (E2, 1 nM), 4-OH-Tamoxifen (Tam, 10 nM) and β hexachlorohexane (β-HCH, 1 μM) for 20 and 34 passages respectively. PR = progesterone receptor. * p < 0.05 ** p < 0.01 ***p < 0.001 vs control

Mentions: Since one of the main objectives of this study was to investigate the cause for AhR overexpression in breast cancer cells, we wanted to have at least another example of mammary epithelial cells developing AhR overexpression for the purpose of confirming the effect of E2. Fortunately we have already conducted a similar experiments on MCF-7 cells which had been selected by the same set of agents utilizing essentially identical procedures for 35 generations [18]. At that time, however, we had no knowledge on the expression of AhR or its consequences in those selected MCF-7 cell lines. The results of qRT-PCR assessments of selected marker mRNA expressions in these two E2-selected lines of MCF10AT1 and MCF-7 (Figure 4) show that for the given treatments, the basic mRNA expression profile of these two different cell lines are remarkably similar. For instance, in both cell lines, E2-selection induced the highest level of mRNA expression of AhR followed by those selected by 4-OH-tamoxifen (Tam) and β-HCH. A parallel assay on CYP1A1 expression, which is being used here as a marker for the functional activation of AhR, indicated that, while E2-selected ones (i.e. P35E and P20E for MCF-7 and MCF10AT1) still showed the highest expression among all sub-lines within each MCF cell group, β-HCH selected cells (as compared to "mock" selected control cells) exhibit also relatively higher expressions of the ratio of CYP1A1/AhR than Tam-selected ones. This set of data indicated that the level of induction of AhR is not exactly identical to that of CYP1A1, implying that the functional activation of AhR may be governed by a different set of cellular conditions or factors than its mRNA induction. Nevertheless, in both cases, E2-selected cell lines showed clearly the highest levels of AhR and CYP1A1 mRNA expressions among all selected lines. The observation that E2-selection in both MCF10AT1 and MCF-7 cell lines resulted in AhR overexpression and an increased proliferation indicates that estrogen signaling suppression might play a dominant role in these processes. In addition, these observations indicate some growth factor signaling such as that mediated by ErbB2 or their downstream signaling cascades are also likely to play important roles as well. As for the status of the estrogen receptor (ER), these two E2-selected cell lines show some different characteristics (Figure 4). E2-selected MCF-7 line (P35E) was found to show a drastically low mRNA level of ERα, but E2-selected MCF10AT1 line (i.e. P20E) did not show such a decrease in the mRNA expression of ERα as compared to the matched "mock"-selected control cell line (designated as P20C) (not shown). However, P20E MCF10AT1 cells showed no detectable level of expression of either progesterone receptor (PR) as in the case of P35E (shown in Figure 4) or PS2 (not shown). Such an observation suggests that in both cell lines ER functions are likely suppressed. The difference between these two cell lines appears to be that, in the case of P20E it is ER signaling that is impaired, but in the case of P35E it is the ERα mRNA titer that is suppressed.


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

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

mRNA expression of selected proteins in long-term selected cells – MCF10AT1 and MCF-7 cell lines grown in the presence of 17-β-estradiol (E2, 1 nM), 4-OH-Tamoxifen (Tam, 10 nM) and β hexachlorohexane (β-HCH, 1 μM) for 20 and 34 passages respectively. PR = progesterone receptor. * p < 0.05 ** p < 0.01 ***p < 0.001 vs control
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: mRNA expression of selected proteins in long-term selected cells – MCF10AT1 and MCF-7 cell lines grown in the presence of 17-β-estradiol (E2, 1 nM), 4-OH-Tamoxifen (Tam, 10 nM) and β hexachlorohexane (β-HCH, 1 μM) for 20 and 34 passages respectively. PR = progesterone receptor. * p < 0.05 ** p < 0.01 ***p < 0.001 vs control
Mentions: Since one of the main objectives of this study was to investigate the cause for AhR overexpression in breast cancer cells, we wanted to have at least another example of mammary epithelial cells developing AhR overexpression for the purpose of confirming the effect of E2. Fortunately we have already conducted a similar experiments on MCF-7 cells which had been selected by the same set of agents utilizing essentially identical procedures for 35 generations [18]. At that time, however, we had no knowledge on the expression of AhR or its consequences in those selected MCF-7 cell lines. The results of qRT-PCR assessments of selected marker mRNA expressions in these two E2-selected lines of MCF10AT1 and MCF-7 (Figure 4) show that for the given treatments, the basic mRNA expression profile of these two different cell lines are remarkably similar. For instance, in both cell lines, E2-selection induced the highest level of mRNA expression of AhR followed by those selected by 4-OH-tamoxifen (Tam) and β-HCH. A parallel assay on CYP1A1 expression, which is being used here as a marker for the functional activation of AhR, indicated that, while E2-selected ones (i.e. P35E and P20E for MCF-7 and MCF10AT1) still showed the highest expression among all sub-lines within each MCF cell group, β-HCH selected cells (as compared to "mock" selected control cells) exhibit also relatively higher expressions of the ratio of CYP1A1/AhR than Tam-selected ones. This set of data indicated that the level of induction of AhR is not exactly identical to that of CYP1A1, implying that the functional activation of AhR may be governed by a different set of cellular conditions or factors than its mRNA induction. Nevertheless, in both cases, E2-selected cell lines showed clearly the highest levels of AhR and CYP1A1 mRNA expressions among all selected lines. The observation that E2-selection in both MCF10AT1 and MCF-7 cell lines resulted in AhR overexpression and an increased proliferation indicates that estrogen signaling suppression might play a dominant role in these processes. In addition, these observations indicate some growth factor signaling such as that mediated by ErbB2 or their downstream signaling cascades are also likely to play important roles as well. As for the status of the estrogen receptor (ER), these two E2-selected cell lines show some different characteristics (Figure 4). E2-selected MCF-7 line (P35E) was found to show a drastically low mRNA level of ERα, but E2-selected MCF10AT1 line (i.e. P20E) did not show such a decrease in the mRNA expression of ERα as compared to the matched "mock"-selected control cell line (designated as P20C) (not shown). However, P20E MCF10AT1 cells showed no detectable level of expression of either progesterone receptor (PR) as in the case of P35E (shown in Figure 4) or PS2 (not shown). Such an observation suggests that in both cell lines ER functions are likely suppressed. The difference between these two cell lines appears to be that, in the case of P20E it is ER signaling that is impaired, but in the case of P35E it is the ERα mRNA titer that is suppressed.

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