Aromatase expression increases the survival and malignancy of estrogen receptor positive breast cancer cells.

In postmenopausal women, local estrogen produced by adipose stromal cells in the breast is believed to support estrogen receptor alpha (ERα) positive breast cancer cell survival and growth. This raises the question of how the ERα positive metastatic breast cancer cells survive after they enter blood and lymph circulation, where estrogen level is very low in postmenopausal women. In this study, we show that the aromatase expression increased when ERα positive breast cancer cells were cultured in suspension. Furthermore, treatment with the aromatase substrate, testosterone, inhibited suspension culture-induced apoptosis whereas an aromatase inhibitor attenuated the effect of testosterone suggesting that suspended circulating ERα positive breast cancer cells may up-regulate intracrine estrogen activity for survival. Consistent with this notion, a moderate level of ectopic aromatase expression rendered a non-tumorigenic ERα positive breast cancer cell line not only tumorigenic but also metastatic in female nude mice without exogenous estrogen supplementation. The increased malignant phenotype was confirmed to be due to aromatase expression as the growth of orthotopic tumors regressed with systemic administration of an aromatase inhibitor. Thus, our study provides experimental evidence that aromatase plays an important role in the survival of metastatic ERα breast cancer cells by suppressing anoikis

Increased expression of aromatase in suspension culture.

<p>Cells were plated at 1x10⁶ per well in regular plates for adhesion culture for 24 hrs (24h/adh) or in ultra-low adhesion plates for suspension culture for 24 hrs (24h/sus) and 48 hrs (48h/sus). Cells were harvested for RNA isolation, which was used in real time RT-PCR for quantification of aromatase and actin mRNA. Aromatase mRNA levels presented are normalized by actin mRNA levels. The data represents mean±SEM from three replicate measurements. Fig. A shows the increased aromatase expression in suspension culture of MCF-7, Clone 10 and CAMA-1 cells by real time PCR while Fig. B shows the same by Western blot analysis in CAMA-1 cells.</p

Moderate aromatase expression rendered ZR-75-1 cells tumorigenic without estrogen supplementation.

<p><b>A.</b> Luc-GFP expressing ZR-75-1/Aro Cl.10 and TT1 cells (2x10⁶) were inoculated into the inguinal mammary fat pads of 5-wk-old female nude mice. The tumor sizes were measured with a caliper in two dimensions. Tumor volumes were calculated with the equation V = (LxW²)/2, where L is length and W is width of a tumor. Values are mean±SEM of 10 tumors in 5 mice. B. The representative images (fluorescence on the left and bioluminescence on the right) C. The mice with growing tumors were divided into two groups and treated with vehicle or letrozole at 10 μg/mouse/day.</p

Increased expression of ERα in suspension culture.

<p>Cells were plated in either regular culture plates for adhesion culture or in ultra-low adhesion plates for suspension culture. Their RNA and protein were extracted and used for real-time RT-PCR (Panel A) or immunoblotting (Panel B). The ERα mRNA levels presented are normalized by actin mRNA. The data in Panel A represents mean±SEM from three replicate measurements. Two-tailed student t-tests were performed to determine the significant difference between control and experimental data. GAPDH protein was blotted to indicate equal loading in Panel B. The density of the ERα band from 24h adherent culture was set as one unit after being normalized with the corresponding GAPDH band for each cell line.</p

Aromatase expression rendered non-tumorigenic ZR-75-1 cells bone metastatic.

<p>The cells were inoculated through the left cardiac ventricle of female nude mice at 0.1x10⁶ cells/mouse. Bone metastases of ZR-75-1/Aro Cl.10 cells in the mandible and tibiae/femora were detected with fluorescence (panel A) and bioluminescence (panel B) imaging. Representative images of two mice inoculated with the Cl.10 cells are presented, which were taken 5 week post-inoculation.</p