Estrogen Receptor Expression and Function in Long-Term Estrogen-Deprived Human Breast Cancer Cells

Abstract

Hormone-dependent breast cancer responds to primary therapies that block estrogen production or action, but tumor regrowth often occurs 12-18 months later. Additional hormonal treatments that further reduce estrogen synthesis or more effectively block its action cause additional remissions, but the mechanisms responsible for these secondary responses are not well understood. As a working hypothesis, we postulated that primary hormonal therapy induces adaptive changes, resulting in enhanced estrogen receptor (ER) expression and target gene activation and, further, that secondary treatment modalities interfere with these receptor-mediated transcriptional pathways. To test this hypothesis, we used an MCF-7 breast cancer model system involving deprivation of estradiol in culture for a prolonged period. These long-term estradiol-deprived (LTED) cells adapt by acquiring the ability to regrow in the absence of added estradiol. The experimental paradigm involved the comparison of wild-type cells with LTED cells. As endpoints, we directly assessed ER expression at the messenger RNA-, protein-, and ligand-binding levels and ER functionality by quantitating reporter gene activation and expression of endogenous estrogen target gene messenger RNA, as well as ER coactivator levels. Our data demonstrated an adaptive increase in ER expression and in basal ER functionality, as assessed by read-out of three different transfected reporters in LTED, as opposed to wild-type MCF-7 cells. Increased reporter gene read-out was dramatically inhibited by the pure antiestrogen ICI 182,780. As verification that endogenous (as well as transfected) estrogen target genes had enhanced transcription, we found that the basal levels of c-myb and c-myc message were substantially increased in LTED cells and could be inhibited by antiestrogen. Interestingly, the levels of c-myb and c-myc message in the LTED cells seemed to be increased out of proportion to the degree of ER reporter gene activation and were similar to those in wild-type cells maximally stimulated with estradiol. In addition, not all estrogen-responsive genes were activated, because transforming growth factor-alpha message level was not increased in LTED cells. Up-regulation of the steroid receptor coactivator SRC-1 did not seem to mediate the process of enhanced ER-induced transcription. Considering these observations together, we suggest that long-term estradiol deprivation causes adaptive processes that not only involve up-regulation of the ER but also influence the specificity and magnitude of activation of estrogen-responsive genes.

Biological reprogramming in acquired resistance to endocrine therapy of breast cancer

“…Consistent with previous studies (Jeng et al, 1998;Shim et al, 2000;Stephen and Darbre, 2000;Martin et al, 2003;Shaw et al, 2006;Santen et al, 2008;Sadler et al, 2009), progesterone receptor was found to be strongly underexpressed, whereas ERa was strongly overexpressed in MCF7-LTED extracts ( Figure 1c). Next, evaluation of activated forms of ERa revealed increased pS167 and pS305 but not pS118 (Figure 1c).…”

“…Activation of AKT and overexpression of EGF and ERBB receptors are consistent with previous observations in MCF7-LTED cells and related breast cancer conditions (Masamura et al, 1995;Jeng et al, 1998;Shim et al, 2000;McClelland et al, 2001;Knowlden et al, 2003;Osborne et al, 2005;Yue et al, 2005;Song et al, 2006;Beeram et al, 2007;LewisWambi et al, 2008;Santen et al, 2008;Ghayad et al, 2009). Notably, ERBB2 amplification was associated with resistance to endocrine therapies (Ellis et al, 2006), and treatment with the mTOR inhibitor RAD001 increased letrozole efficacy in a neoadjuvant setting of ERa-positive breast cancer (Baselga et al, 2009).…”

“…Molecular changes during MCF7-LTED adaptation are consistent with growth signaling pathway cross-talk To generate a breast cancer cell model of acquired resistance to AIs, MCF7 cells were cultured in a medium depleted of 17bE2, as described previously (Katzenellenbogen et al, 1987;Welshons and Jordan, 1987;Masamura et al, 1995;Jeng et al, 1998;Stephen et al, 2001;Chan et al, 2002). After initial quiescence for approximately 60 days, cell cultures adapted at 90 days, defined by a diminished proliferation-dependent response from 17bE2 ( Figure 1a).…”

The androgen metabolite 5α-androstane-3β,17β-diol (3βAdiol) induces breast cancer growth via estrogen receptor: implications for aromatase inhibitor resistance

“…These relatively low concentrations of circulating 3βAdiol may be sufficient to drive tumor growth in the absence of estrogen, particularly in women undergoing treatment with AIs. In addition, prior reports have demonstrated that breast cancer cells can become hypersensitive to extremely low concentrations of estrogens after long-term estrogen deprivation [31,32]. These data suggest that tumors may be sensitive to very low concentrations of a weak ERα agonist such as 3βAdiol.…”

C‐myc gene expression alone is sufficient to confer resistance to antiestrogen in human breast cancer cells

“…These changes are thought to allow estrogendeprived cells the ability to regrow in the absence of steroids. 14 Hormonal therapy using estrogen antagonists is an important therapeutic option in the treatment of hormone-responsive breast cancer and tamoxifen, a nonsteroidal estrogen antagonist/agonist, is the current choice. 15,16 The pure antiestrogen ICI 182,780 (ICI), however, has been reported to be effective even in patients with hormone-independent breast cancer.…”