Todd C. Skaar

Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis.

SummaryPrepubertal exposure to a pharmacological dose (500 mg kg–1) of the phyto-oestrogen genistein can reduce the incidence and multiplicity of carcinogen-induced mammary tumours in rats. However, such an exposure also disrupts the function of the hypothalamic–pituitary–gonadal axis, making it unsuitable for breast cancer prevention. We studied whether prepubertal exposure to genistein at a total body dose broadly comparable to the level typical of Oriental countries, approximately 1 mg kg–1 body weight, affects mammary tumorigenesis. We also studied whether prepubertal exposure to zearalenone, a major source for phyto-oestrogens in the USA, influences breast cancer risk. Prepubertal rats were treated between postnatal days 7 and 20, with 20 μg (~ 1 mg kg–1 body weight) of either genistein or zearalenone. Zearalenone exposure significantly reduced both the incidence and multiplicity of mammary tumours induced by 7,12-dimethylbenz(a)anthracene (DMBA). Genistein exposure significantly reduced tumour multiplicity, but not tumour incidence, when compared with vehicle-treated animals. Furthermore, 60% of the tumours in the genistein group were not malignant, while all the tumours analysed for histopathology in the vehicle and zearalenone groups were adenocarcinomas. A higher number of differentiated alveolar buds, and lower number of terminal ducts, were present in the DMBA-treated mammary glands of the phyto-oestrogen exposed rats. The concentration of oestrogen receptor (ER) binding sites after the DMBA treatment was low in the mammary glands of all groups but a significantly higher proportion of the glands in the zearalenone exposed rats were ER-positive (i.e. ER levels ≥ 5 fmol mg–1 protein) than the glands of the vehicle controls. Our data suggest that a prepubertal exposure to a low dose of either zearalenone or genistein may protect the mammary gland from carcinogen-induced malignant transformation, possibly by increasing differentiation of the mammary epithelial tree.

Molecular and pharmacological aspects of antiestrogen resistance.

Endocrine therapy is effective in approximately one-third of all breast cancers and up to 80% of tumors that express both estrogen and progesterone receptors. Despite the low toxicity, good overall response rates, and additional benefits associated with its partial agonist activity, most Tamoxifen-responsive breast cancers acquire resistance. The development of new antiestrogens, both steroidal and non-steroidal, provides the opportunity for the development of non-cross-resistant therapies and the identification of additional mechanisms of action and resistance. Drug-specific pharmacologic mechanisms may confer a resistance phenotype, reflecting the complexities of both tumor biology/pharmacology and the molecular endocrinology of steroid hormone action. However, since all antiestrogens will be effective only in cells that express estrogen receptors (ER), many mechanisms will likely be directly related to ER expression and signaling. For example, loss of ER expression/function is likely to confer a cross-resistance phenotype across all structural classes of antiestrogens. Altered expression of ERalpha and ERbeta, and/or signaling from transcription complexes driven by these receptors, may produce drug-specific resistance phenotypes. We have begun to study the possible changes in gene expression that may occur as cells acquire resistance to steroidal and non-steroidal antiestrogens. Our preliminary studies implicate the altered expression of several estrogen-regulated genes. However, resistance to antiestrogens is likely to be a multigene phenomenon, involving a network of interrelated signaling pathways. The way in which this network is adapted by cells may vary among tumors, consistent with the existence of a highly plastic and adaptable genotype within breast cancer cells.

Two-dimensional gel electrophoresis analyses identify nucleophosmin as an estrogen regulated protein associated with acquired estrogen-independence in human breast cancer cells.

We have used two-dimensional gel electrophoresis to identify proteins associated with estrogen-induced proliferation in MCF-7 breast cancer cells and their progression to estrogen-independent proliferation. We compared the total cellular proteins from MCF-7 cells and an estrogen independent derivative of the MCF-7 cells MCF-7/LCC1 (Brünner et al. Cancer Research 1993, 53, 283-290), each grown with and without estradiol. These comparisons reveal seven estrogen-regulated proteins. Three of these proteins (HI-1: 36 kDa/pI 4.5, HI-10: 40 kDa/pI 5.5 and HI-19: 62 kDa/pI 5.0) exhibit a progression-like pattern, being induced by estradiol in MCF-7 cells and constitutively present/upregulated in the MCF-7/LCC1 growing without estradiol. HI-11 (65 kDa/pI 5.5) is strongly induced by estradiol in MCF-7 cells but constitutively downregulated and unresponsive to estradiol in the MCF-7/LCC1 cells. Two proteins exhibit a suppressor pattern and are downregulated by estradiol in the estrogen-dependent MCF-7 cells (HI-3: 44 kDa/pI 4.4 and HI-4: 56 kDa/ pI 5.2) and present in MCF-7/LCC1 cells growing without estradiol at levels comparable to that seen in estrogen-treated MCF-7 cells. One protein (HI-9: 68 kDa/pI 5.5) exhibits a marked estrogen regulated pI shift, rather than changes in abundance. We purified and sequenced the HI-10 protein, which we identified as the nucleolar protein, nucleophosmin (NPM). One- and two-dimensional Western blot analyses of MCF-7/LCC1 cell lysates confirmed that HI-10 is immunoreactive with an antinucleophosmin antibody. Western blotting also confirmed the estrogenic regulation of NPM seen in the initial two-dimensional gel electrophoresis studies. Thus, NPM is induced by estradiol in the MCF-7 cells and upregulated in the MCF-7/LCC1 cells growing without estrogen, clearly associating its expression with an acquired estrogen-independent phenotype. NPM has several potentially important roles in regulating cell function and signaling. It is a substrate for phosphorylation by p34cdc2 kinase, protein kinase C and nuclear kinase II, and a repressor of the transcriptional regulating activities of both the IRF-1 tumor suppressor protein and the YY1 transcription factor. Studies are currently underway to determine which of these NPM functions may be involved in the hormonal progression of breast cancer.

Hormonal carcinogenesis in breast cancer: cellular and molecular studies of malignant progression

SummaryWe have established and characterized a series of variant cell lines in which to identify the critical factors associated with E2-induced malignant progression, and the acquisition to tamoxifen resistance in human breast cancer. Sublines of the hormone-dependent MCF-7 cell line (MCF7/MIII and MCF7/LCC1) form stable, invasive, estrogen independent tumors in the mammary fat pads of ovariectomized athymic nude mice. These cells retain expression of both estrogen (ER) and progesterone receptors (PGR), but retain sensitivity to each of the major structural classes of antiestrogens. The tamoxifen-resistant MCF7/LCC2 cells retain sensitivity to the inhibitory effects of the steroidal antiestrogen ICI 182780. By comparing the parental hormone-dependent and variant hormone-independent cells, we have demonstrated an altered expression of some estrogen regulated genes (PGR, pS2, cathepsin D) in the hormone-independent variants. Other genes remain normally estrogen regulated (ER, laminin receptor, EGF-receptor). These data strongly implicate the altered regulation of a specific subset or network of estrogen regulated genes in the malignant progression of human breast cancer. Some of the primary response genes in this network may exhibit dose-response and induction kinetics similar to pS2, which is constitutively upregulated in the MCF7/MIII, MCF7/LCC1 and MCF7/LCC2 cells.

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo

Many human breast tumors are driven by high intratumor concentrations of 17β-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of[ 3H]estrone sulfate to [3H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein·h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmo...

Acquisition of an Antiestrogen-Resistant Phenotype in Breast Cancer: Role of Cellular and Molecular Mechanisms

It is estimated that almost 11% of all women living to age 80 will develop breast cancer [1]. The annual worldwide incidence of breast cancer is estimated to be one million by the year 2000 [1]. The factors responsible for the genesis of breast cancer remain unclear, but estrogens have been strongly implicated [2]. Estrogens may function as carcinogens and/or cocarcinogens [2,3], but it is their role as promoters of the growth of estrogen-dependent and estrogen-responsive tumors through activation of their nuclear receptors [2] that has provided a rationale for the design of therapeutic strategies, that is, antiestrogens.

Use of ERE and reporter gene constructs to assess putative estrogenic activity

Estrogens primarily function through the activation of their receptors, which subsequently function as nuclear transcription factors. There are two estrogen receptor (ER) genes, now designated ERa (the classic ER gene) and ER/3. The key consequence of the activation of either gene product is the regulation of gene transcription. The extent and nature of transcription appear to be regulated by a series of coregulator proteins. One of the most sensitive assays for detection of potential estrogenic activity is measurement of the ability of a test compound to influence the transcription of reporter genes. In this regard, many investigators use promoter-reporter constructs. To assess putative estrogenic activity, an estrogen-responsive promoter is generally placed upstream of a reporter gene and transiently transfected into a target cell. When exposed to an estrogenic compound, expression of the reporter gene would normally be induced. We briefly discuss several issues pertinent to the use of these assays and the interpretation of resulting data, including estrogen-responsive, promoter-reporter constructs, reporter genes and measurements of activity, choice of target cell or cell line, transient introduction of promoter-reporter constructs into cells, basic statistical approaches to data analysis, and definitions of agonist, partial agonist, and antagonist.