Prolonged exposure to cadmium enhances the interactions of ERα with c-jun and c-fos.

<p>(<b>A</b>) MCF7, Cd7 and Cd12 cell lysates were immunoprecipitated with either α-ERα or normal rabbit IgG. Proteins interacting with ERα were analyzed with Western blot analysis. (<b>B</b>) Reverse co-IP was performed with α-c-jun, α-c-fos, or normal rabbit IgG. (<b>C</b>) MCF7, Cd7 and Cd12 were plated in 6-well plates and transfected with siRNA targeting either c-jun (Ji), c-fos (Fi) or a scramble siRNA control (Ci) and collected 48 hours later for protein analysis.</p

ERα, c-fos and c-jun are recruited to SDF-1 promoter.

<p>(<b>A</b>) 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>B</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). (<b>C</b>) 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). (<b>D</b>) 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).</p

Chronic Cadmium Exposure Stimulates SDF-1 Expression in an ERα Dependent Manner

<div><p>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 <i>chronic</i> 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.</p></div

SDF-1 is induced by acute cadmium and estrogen exposure.

<p>MCF7 cells were hormone deprived and treated with either 10⁻⁷ M E₂ (white bars) or 10⁻⁶ M CdCl₂ (black bars). Cells were harvested for gene expression analysis using (<b>A</b>) quantitative and (<b>B</b>) semi-quantitative RT-PCR. Quantitative PCR data is presented as relative SDF-1 fold increases.</p

Chronic cadmium exposure induces more aggressive cancer phenotypes: cell growth, migration and invasion.

<p>(<b>A</b>) MCF7-Cd cells (black bars) and parental MCF7 cells (white bars) were plated in 6 well plates under hormone-deprived conditions and cell growth was monitored 2, 4, and 6 days after plating by counting in triplicate. Experimental results are averages of three independent experiments (P<0.01). (<b>B</b>) A scratch wound assay was used to assess cell migration ability. MCF7 and MCF7-Cd cells were plated in 6-well plates and allowed to reach 70–80% confluence. A scratch was created using a p-200 pipette tip and a digital image was captured on Day 0. Cells were then allowed to grow and migrate for 3 days and images of the wound were captured 3 days later. Images are representative of three independent experiments done in triplicates. Arrows are indicating the same region on the scratch on day 0 and day 3. (<b>C</b>) Modified Boyden chamber assays were performed to measure the migration abilities of the cells. MCF7 (white bars) and MCF7-Cd (black bars) cells were seeded into upper chambers and cells were allowed to migrate to lower chamber for 16 hours. Data is representative of 3 independent experiments performed in triplicate (P<0.01). (<b>D</b>) An invasion assay was performed by seeding either MCF7 (white bars) or MCF7-Cd (black bars) cells in the upper chambers. Cells were allowed to invade through matrigel-coated membranes for 18 hours. Data is an average of 3 independent experiments done in triplicate (P<0.01).</p

Dendrogram of hierarchical clustering showing a clear distinction between control MCF-7 and cells chronically exposed to cadmium.

<p>Dendrogram of hierarchical clustering showing a clear distinction between control MCF-7 and cells chronically exposed to cadmium.</p

Heat map showing expression of 97 breast cancer associated genes that are differentially expressed between control and cadmium-adapted cells.

<p>Each row represents a gene and each column represents a sample. </p

Gene Ontology (GO) categories denoting Biological Process (BP) information for differentially expressed genes.

<p>Information is derived using Onto-Express (<a href="http://vortex.cs.wayne.edu/projects.htm" target="_blank">http://vortex.cs.wayne.edu/projects.htm</a>). Diagram shows the number of under- and over-expressed genes for GO BP. The BP marked with stars denotes a statistically significant group (<i>p</i><0.05). Only the groups with a gene number ≥ 5 are shown.</p

Gene Ontology (GO) categories denoting Molecular Function (MF) information for differentially expressed genes.

<p>Information is derived using Onto-Express (<a href="http://vortex.cs.wayne.edu/projects.htm" target="_blank">http://vortex.cs.wayne.edu/projects.htm</a>). Diagram shows the number of under- and over-expressed genes for each GO MF. The MF marked with stars denotes a statistically significant group (<i>p</i><0.05). Only the groups with a gene number ≥ 5 are shown.</p