Cadmium malignantly transforms normal human breast epithelial cells into a basal-like phenotype

Abstract

Background: Breast cancer has recently been linked to cadmium exposure. Although not uniformly supported, it is hypothesized that cadmium acts as a metalloestrogenic carcinogen via the estrogen receptor (ER). Thus, we studied the effects of chronic exposure to cadmium on the normal human breast epithelial cell line MCF-10A, which is ER-negative but can convert to ER-positive during malignant transformation.

Methods: Cells were continuously exposed to low-level cadmium (2.5 μM) and checked in vitro and by xenograft study for signs of malignant transformation. Transformant cells were molecularly characterized by protein and transcript analysis of key genes in breast cancer.

Results: Over 40 weeks of cadmium exposure, cells showed increasing secretion of matrix metalloproteinase-9, loss of contact inhibition, increased colony formation, and increasing invasion, all typical for cancer cells. Inoculation of cadmium-treated cells into mice produced invasive, metastatic anaplastic carcinoma with myoepithelial components. These cadmium-transformed breast epithelial (CTBE) cells displayed characteristics of basal-like breast carcinoma, including ER-alpha negativity and HER2 (human epidermal growth factor receptor 2) negativity, reduced expression of BRCA1 (breast cancer susceptibility gene 1), and increased CK5 (cytokeratin 5) and p63 expression. CK5 and p63, both breast stem cell markers, were prominently overexpressed in CTBE cell mounds, indicative of persistent proliferation. CTBE cells showed global DNA hypomethylation and c-myc and k-ras overexpression, typical in aggressive breast cancers. CTBE cell xenograft tumors were also ER-alpha negative.

Conclusions: Cadmium malignantly transforms normal human breast epithelial cells-through a mechanism not requiring ER-alpha-into a basal-like cancer phenotype. Direct cadmium induction of a malignant phenotype in human breast epithelial cells strongly fortifies a potential role in breast cancer.

Keywords: basal-type; breast cancer; cadmium; estrogen receptor; malignant transformation.

Figure 1

Chronic cadmium exposure induces a cancer phenotype in human breast epithelial cells exposed to 2.5 μM cadmium for up to 40 weeks compared with passage-matched, untreated control cells. (A) Active MMP-9 secretion during cadmium exposure. (B) Loss of contact inhibition at 40 weeks of cadmium exposure as indicated by formation of foci of cell mounding (arrows; bottom) that were rarely seen in control (top). Bars = 100 μm. (C) Increased colony formation in soft agar with chronic cadmium exposure. (D) Increased invasive ability with cadmium exposure. Numerical data are expressed as a percentage of control (set to 100%) ± SE. *Significantly different from control.

Figure 2

Tumor formation resulting from inoculation of CTBE cells into nude mice. (A) Tumor formation rate during 6 months after inoculation of CTBE or control cells under the renal capsules of 10 mice/group. (B) Representative section of an anaplastic carcinoma invading the normal kidney, which formed after CTBE inoculation; bar = 50 μm. The tumor is highly aggressive, with areas of epithelial, mesenchymal, and undifferentiated cells. (C) A representative metastasis to a peritoneal lymph node of a carcinoma produced by CTBE cell inoculation; bar = 500 μm. *Significantly different from control.

Figure 3

Expression of ER-α, ER-β, HER2, BRCA1, and MT in CTBE cells. (A) ER-α, ER-β, and HER2 protein levels showing clear negativity of control MCF-10A and CTBE cells. The positive controls were ER-α- and ER-β-positive MCF-7 cells and HER2-positive SKBR3 cells. Western blots are typical examples of triplicates using β-actin as the loading control and were not quantitated because of the very low protein levels in control and CTBE cells. (B) MT protein showing relatively low expression in control but increased expression in CTBE cells. (C) BRCA1 protein and transcript in CTBE and control cells. The data for MT and BRCA1 are expressed as a percentage of control (set to 100%; ± SE). *Significantly different from control.

Figure 4

Expression of genes in CTBE cells typical for basal-like human breast cancer phenotype and/or breast stem cells. (A) Protein expression for CK5 and p63 expressed as a percentage of control. Fluorescent microscopy was used to determine localization of stem cell marker protein expression in (B) control cells and (C) CTBE cells. Expression of CK5 (green) and p63 (red) were clearly co-localized to foci of cell mounding (yellow) in CTBE cells and, in comparison, barely detectable in similar structures from control cells. DAPI was used as a nuclear stain to show similar number of viable cells. Top images (gray) are relief contrast. Bars = 25 μm. *Significantly different from control.

Figure 5

Oncogene activation and global DNA hypomethylation during acquired malignant phenotype in CTBE cells. (A) K-ras expression. (B) c-myc expression. (C) DNA methylation. Protein or transcript data are expressed as percentage of control (set to 100% ± SE). Note broken scale in (C). *Significantly different from control.

Figure 6

Immunohistochemical analysis for ER-α in xenograft tumors produced by CTBE cell inoculation (A, C) and a positive control (B). (A) Example of a tumor formed by inoculation of CTBE injection, showing minimal ER-α protein in the cells of the tumor. (B) A commercially available, known ER-α-positive human breast carcinoma showing strong, nuclear staining (brown). (C) A lymph node metastasis from a CTBE-formed tumor showing minimal ER-α protein. Bars = 100 μm.