Androgens act synergistically to enhance estrogen-induced upregulation of human tissue kallikreins 10, 11, and 14 in breast cancer cells via a membrane bound androgen receptor

Abstract

The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well as in the pathogenesis of endocrine-related cancers, especially breast cancer. However, clinical data suggest that combined testosterone and estrogen treatments on post-menopausal women increase the risk of breast cancer. Experiments have shown that many, if not all kallikreins are under steroid hormone regulation in breast cancer cell lines. Their implication as prognostic and diagnostic markers has also been well-documented. Thus, we investigated the effect of combined hormone stimulation with androgens and 17beta-estradiol on the ductal caricinoma cell line BT474. This cell line has been shown to be sensitive to both, androgens (secreting PSA) and estrogens (secreting a number of kallikreins including KLK10, 11, and KLK14). We found that PSA expression was downregulated upon combined hormone stimulation, confirming reports that estrogen can antagonize and block the activity of the androgen receptor. Upon analysis of estrogen-sensitive kallikreins 10, 11, and 14, all showed to be synergistically enhanced in their expression three- to fourfold, upon joint hormone treatment versus individual hormone stimulation. The enhancement is dependent upon the action of androgens as treatment with the androgen receptor antagonist cyproterone actetate normalized the expression of KLK10, 11, and KLK14 to estrogen-stimulation levels. The synergistic effects between estrogens and androgens on estrogen-sensitive genes may have implications on the role of the kallikreins in associated risk of breast cancer and progression.

Figure 1

Enhanced kallikrein expression upon combined DHT and estradiol stimulation. Cells were treated with either 100nM or 10nM DHT and 100nM or 10nM estradiol and combinations of the two hormone concentrations and quantified by ELISA of condition media supernatants. (A) Kallikrein protein expression profiles. PSA shows specific dose‐dependent DHT dependent expression, and is reduced significantly in the presence of estradiol. KLK10, 11, and KLK14 show specific estrogen‐dependent expression and are enhanced by the addition of DHT. (B) RT‐PCR analysis of KLK10, 11, and KLK14 genes also show enhanced transcriptional activation with joint DHT and estradiol stimulation. Actin expression was used as a control for all RT‐PCR analysis.

Figure 2

Aromatase inhibitor treatment. PSA, KLK10, 11, and KLK14 protein expression profiles were analyzed for synergistic enhancement upon stimulation with the aromatizable androgen, testosterone, and estradiol. BT474 cells were also subsequently treated with the aromatase inhibitor xanthenone in the presence of both steroid hormones.

Figure 3

Western blot analysis of hormone receptors. Cell lysates from BT474 were analyzed for changes in androgen (AR), estrogen (ER) and glucocorticoid (GR) and progesterone (PGR) receptor levels. β‐ACTIN is used as the loading control for this analysis.

Figure 4

RT‐PCR analysis of other estrogen upregulated genes. PS2, PGR, c‐fos, c‐myc, IGFBP4, and ADAMTS‐1 were analyzed by RT‐PCR for changes in expression upon joint DHT and estradiol hormone stimulations.

Figure 5

Hormone receptor antagonist treatment of combined hormone stimulated cells. Jointly hormone stimulated BT474 cells were treated with a single dose of 1μM final concentration of either ICI 182,780 (ICI) or cyproterone acetate (CPA). After 6 days, the cell supernatants were collected and ELISAs were carried out on, KLK10, KLK11, and KLK14.

Figure 6

BSA‐conjugated testosterone treatment also enhances estrogen‐dependent KLK expression. (A) BT474 cells were treated with BSA:Test and also together with estradiol and PSA, KLK10, 11 and KLK14 protein expression profiles were analyzed by ELISA. (B) RT‐PCR profiling of KLK10, 11, 14, and pS2 upon BSA:Test–estradiol co‐stimulations. (C) Western blot analysis of AR, ERα, GR and PGR of cell lysates from BT474 cells co‐treated with BSA:Test and estradiol.

Figure 7

AKT activation by androgens. Activity of ERK1/2 and AKT were analyzed by phospho‐specific Western blot analysis, over a time course, of androgen and estradiol stimulated BT474 cells. As a control of AKT and ERK1/2 activation BT474 cells were treated with 10ng/mL of EGF for 10min with and without the specific pathway inhibitors Wortmannin (500nM) and U0126 (10μM).

Figure 8

Schematic of enhanced estrogen‐dependent KLK expression by androgens via AKT pathway.