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Chronic cadmium exposure stimulates SDF-1 expression in an ERα dependent manner.

Ponce E, Aquino NB, Louie MC - PLoS ONE (2013)

Bottom Line: Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure.Data suggest that prolonged cadmium exposures result in the development of more aggressive cancer phenotypes - increased cell growth, migration and invasion.The results from this study show for the first time that chronic cadmium exposure stimulates the expression of SDF-1 by altering the molecular interactions between ERα, c-jun and c-fos.

View Article: PubMed Central - PubMed

Affiliation: Department of Natural Sciences and Mathematics, Dominican University of California, San Rafael, California, United States of America.

ABSTRACT
Cadmium is an omnipotent environmental contaminant associated with the development of breast cancer. Studies suggest that cadmium functions as an endocrine disruptor, mimicking the actions of estrogen in breast cancer cells and activating the receptor to promote cell growth. Although acute cadmium exposure is known to promote estrogen receptor-mediated gene expression associated with growth, the consequence of chronic cadmium exposure is unclear. Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure. This study aims to investigate the effects of chronic cadmium exposure on breast cancer progression. A MCF7 breast cancer cell line chronically exposed to 10(-7) M CdCl2 serves as our model system. Data suggest that prolonged cadmium exposures result in the development of more aggressive cancer phenotypes - increased cell growth, migration and invasion. The results from this study show for the first time that chronic cadmium exposure stimulates the expression of SDF-1 by altering the molecular interactions between ERα, c-jun and c-fos. This study provides a mechanistic link between chronic cadmium exposure and ERα and demonstrates that prolonged, low-level cadmium exposure contributes to breast cancer progression.

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ERα, c-fos and c-jun are recruited to SDF-1 promoter.(A) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. (B) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P<0.001). (C) In the ChIP re-ChIP assay, DNA/protein complexes from the first ChIP assay were extracted and re-ChIPed with a second antibody. The occupancy of ERα/c-jun or c-Jun/ERα complexes on the SDF-1 promoter was analyzed as in B (P<0.001). (D) To confirm the presence of c-fos in the ERα/c-jun complex, reChIPed DNA containing ERα/c-jun complexes were immunoprecipitated with a third antibody, α-c-fos, and the resulting DNA fragments were analyzed as in B (P<0.001).
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pone-0072639-g006: ERα, c-fos and c-jun are recruited to SDF-1 promoter.(A) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. (B) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P<0.001). (C) In the ChIP re-ChIP assay, DNA/protein complexes from the first ChIP assay were extracted and re-ChIPed with a second antibody. The occupancy of ERα/c-jun or c-Jun/ERα complexes on the SDF-1 promoter was analyzed as in B (P<0.001). (D) To confirm the presence of c-fos in the ERα/c-jun complex, reChIPed DNA containing ERα/c-jun complexes were immunoprecipitated with a third antibody, α-c-fos, and the resulting DNA fragments were analyzed as in B (P<0.001).

Mentions: Since the regulatory region of SDF-1 consists of only one full ERE site located 234 bp upstream of the promoter, five half ERE sites located within the proximal promoter, and multiple AP-1 and Sp-1 sites on the promoter [17], it is probable that ERα may interact with other transcription factors to regulate SDF-1 expression. Therefore, a chromatin immunoprecipitation (ChIP) assay was used to determine whether a greater fraction of c-jun and c-fos is recruited to the SDF-1 promoter in cells chronically exposed to cadmium. Cells were plated for 48 hours and harvested for ChIP analysis. The recruitment of ERα, c-jun and c-fos was analyzed by PCR. Results in figures 6A and B indicate that cells chronically exposed to cadmium (Cd7 and Cd12) had higher levels of ERα recruited to the SDF-1 promoter in comparison to parental MCF7 (M) cells (27% and 47% vs. 15%, respectively). Similarly, the occupancy of c-jun and c-fos on the SDF-1 promoter were significantly elevated in Cd7 and Cd12 cells when compared to parental MCF7 cells, with c-jun showing at least a 2-fold increase (Fig. 6A–B). The occupancy of all three transcription factors (ERα, c-jun, and c-fos) were also found elevated in the cyclin D1 and c-myc promoters of cells chronically exposed to cadmium (Fig. 6A–B). In contrast, Sp-1 was only found elevated on the c-myc promoter of cells chronically exposed to cadmium (Cd7 and 12); no significant difference in Sp-1 levels was observed on the SDF-1 and cyclin D1 promoters among the different cell lines (Fig. 6A).


Chronic cadmium exposure stimulates SDF-1 expression in an ERα dependent manner.

Ponce E, Aquino NB, Louie MC - PLoS ONE (2013)

ERα, c-fos and c-jun are recruited to SDF-1 promoter.(A) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. (B) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P<0.001). (C) In the ChIP re-ChIP assay, DNA/protein complexes from the first ChIP assay were extracted and re-ChIPed with a second antibody. The occupancy of ERα/c-jun or c-Jun/ERα complexes on the SDF-1 promoter was analyzed as in B (P<0.001). (D) To confirm the presence of c-fos in the ERα/c-jun complex, reChIPed DNA containing ERα/c-jun complexes were immunoprecipitated with a third antibody, α-c-fos, and the resulting DNA fragments were analyzed as in B (P<0.001).
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getmorefigures.php?uid=PMC3755996&req=5

pone-0072639-g006: ERα, c-fos and c-jun are recruited to SDF-1 promoter.(A) MCF-7, Cd7 and Cd12 cells were harvested for chromatin immunoprecititation (ChIP) analysis. ChIP analysis was done with α-ERα, α-c-fos, α-c-jun, and α-Sp1, and recruitment of proteins to the SDF-1, cyclin D1 and c-myc promoters was determined using promoter specific primers and semi-quantitative PCR. (B) Band intensities of PCR products for SDF-1, cycD, and c-myc were quantified and normalized to input using Quantity One (Bio-Rad) (P<0.001). (C) In the ChIP re-ChIP assay, DNA/protein complexes from the first ChIP assay were extracted and re-ChIPed with a second antibody. The occupancy of ERα/c-jun or c-Jun/ERα complexes on the SDF-1 promoter was analyzed as in B (P<0.001). (D) To confirm the presence of c-fos in the ERα/c-jun complex, reChIPed DNA containing ERα/c-jun complexes were immunoprecipitated with a third antibody, α-c-fos, and the resulting DNA fragments were analyzed as in B (P<0.001).
Mentions: Since the regulatory region of SDF-1 consists of only one full ERE site located 234 bp upstream of the promoter, five half ERE sites located within the proximal promoter, and multiple AP-1 and Sp-1 sites on the promoter [17], it is probable that ERα may interact with other transcription factors to regulate SDF-1 expression. Therefore, a chromatin immunoprecipitation (ChIP) assay was used to determine whether a greater fraction of c-jun and c-fos is recruited to the SDF-1 promoter in cells chronically exposed to cadmium. Cells were plated for 48 hours and harvested for ChIP analysis. The recruitment of ERα, c-jun and c-fos was analyzed by PCR. Results in figures 6A and B indicate that cells chronically exposed to cadmium (Cd7 and Cd12) had higher levels of ERα recruited to the SDF-1 promoter in comparison to parental MCF7 (M) cells (27% and 47% vs. 15%, respectively). Similarly, the occupancy of c-jun and c-fos on the SDF-1 promoter were significantly elevated in Cd7 and Cd12 cells when compared to parental MCF7 cells, with c-jun showing at least a 2-fold increase (Fig. 6A–B). The occupancy of all three transcription factors (ERα, c-jun, and c-fos) were also found elevated in the cyclin D1 and c-myc promoters of cells chronically exposed to cadmium (Fig. 6A–B). In contrast, Sp-1 was only found elevated on the c-myc promoter of cells chronically exposed to cadmium (Cd7 and 12); no significant difference in Sp-1 levels was observed on the SDF-1 and cyclin D1 promoters among the different cell lines (Fig. 6A).

Bottom Line: Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure.Data suggest that prolonged cadmium exposures result in the development of more aggressive cancer phenotypes - increased cell growth, migration and invasion.The results from this study show for the first time that chronic cadmium exposure stimulates the expression of SDF-1 by altering the molecular interactions between ERα, c-jun and c-fos.

View Article: PubMed Central - PubMed

Affiliation: Department of Natural Sciences and Mathematics, Dominican University of California, San Rafael, California, United States of America.

ABSTRACT
Cadmium is an omnipotent environmental contaminant associated with the development of breast cancer. Studies suggest that cadmium functions as an endocrine disruptor, mimicking the actions of estrogen in breast cancer cells and activating the receptor to promote cell growth. Although acute cadmium exposure is known to promote estrogen receptor-mediated gene expression associated with growth, the consequence of chronic cadmium exposure is unclear. Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure. This study aims to investigate the effects of chronic cadmium exposure on breast cancer progression. A MCF7 breast cancer cell line chronically exposed to 10(-7) M CdCl2 serves as our model system. Data suggest that prolonged cadmium exposures result in the development of more aggressive cancer phenotypes - increased cell growth, migration and invasion. The results from this study show for the first time that chronic cadmium exposure stimulates the expression of SDF-1 by altering the molecular interactions between ERα, c-jun and c-fos. This study provides a mechanistic link between chronic cadmium exposure and ERα and demonstrates that prolonged, low-level cadmium exposure contributes to breast cancer progression.

Show MeSH
Related in: MedlinePlus