Shiuan Chen

Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010

Received 12/18/97 Accepted 7/15/98

4. CONSEQUENCE OF AROMATASE EXPRESSION IN BREAST TUMORS

Kitawaki et al. (14) reported that by converting androgen to estrogen (at physiological concentrations), the endogenous aromatase enzyme in the MCF-7 breast cancer cells (ER positive cells) stimulated DNA synthesis, and the stimulation was abolished by the administration of aromatase inhibitors. In 1993, using an aromatase expressing MCF-7 cell line [expressing aromatase at a level 10 times that of the untransfected MCF-7 cell line (15)], androgen-dependent cell growth was observed (16). In addition, tumors were grown in nude mice inoculated with the aromatase transfected MCF-7 cells together with Matrigel (17). The tumor growth was accelerated by injections of androstenedione. These cell culture and nude mouse experiments using aromatase transfected MCF-7 cells demonstrate, in a direct fashion, that aromatase expressed in breast cancer cells can play a role in stimulating the growth of tumors, in an autocrine manner. Similar cell culture and nude mouse studies were also performed by Dowsett and his colleagues (18-20). In addition, results obtained from studies using a transgenic mouse model in which aromatase is over-expressed in mammary tissues indicate that in situ produced estrogen plays a more important role than circulating estradiol in breast tumor promotion (21). The above described investigations have generated critical results supporting the theory that in situ aromatase can play a role in promoting breast tumor growth. However, the studies could not address important issues as to interactions between aromatase-expressing cell and non-aromatase-expressing cell subpopulations that may affect growth rates and drug (e.g., aromatase inhibitor) sensitivity. In addition, it has not yet been shown whether the tumor cell subpopulations which express aromatase manifest a growth advantage within the tumors. We have carried out co-culture experiments with two aromatase expressing breast cancer cell lines (MCF-7aro and T-47Daro) together with an untransfected MCF-7 cell line, using the spheroid culture method (22).

Stable aromatase expressing MCF-7 and T-47D cell lines (i.e., MCF-7aro and T-47Daro) have been prepared by aromatase cDNA transfection and G418 (neomycin) selection. MCF-7aro was subjected to clonal purification. Testosterone (1 nM) increased cell growth to a similar degree for MCF-7/MCF-7aro co-culture (0.75 million cells each type) as with MCF-7aro only (2 to 3 fold). In addition, the enzyme specific activities remained unchanged for MCF-7/MCF-7aro co-culture samples with and without androgen treatment, indicating that estrogen produced by transfected cells can also stimulate the growth of untransfected cells. The androgen response could be inhibited by addition of an aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) (0.01 - 0.1 mM). For MCF-7/T-47Daro co-culture experiments, a clear induction of cell growth by androgen was observed and the level of the increase was similar to that on T-47Daro only. However, for both the T-47D only or the MCF-7/T-47Daro co-culture, the aromatase activity was found to increase significantly after testosterone treatment. Since T-47Daro cells were not subjected to clonal purification, it is thought that the androgen treatment may selectively stimulate the growth of high aromatase-expressing T-47Daro cells. These results indicate that estrogen synthesized by the tumor aromatase can stimulate breast tumor growth in both an autocrine and a paracrine manner.

It is thought that in normal breast tissue aromatase is mainly expressed in adipose stromal cells and fibroblasts. The estrogens stimulate epithelial cell growth through a paracrine fashion. However, in breast tumor tissue, aromatase is found to be expressed in both stromal and cancer cells. Therefore, aromatase stimulates breast tumor growth in both an autocrine and a paracrine manner.