Amanda Coutts

Estrogen Receptor Variants and Mutations

There is a large and increasing body of experimental and clinical data supporting the existence or variant estrogen receptor (ER) proteins in both normal and neoplastic estrogen target tissues including human breast. Therefore, future examination of ER signal transduction and/or measurement of ER protein must take into account variant ER expression. The functions of variant ER proteins, either physiological or pathological, remain unclear, although a role(s) for some ER variants in breast tumorigenesis and breast cancer progression would be consistent with the accumulated data. Possible tissue specific expression leads to the speculation that ER variants may have a role in tissue specific estrogen action. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of estrogen receptor variants and mutations that have been identified to occur naturally in tissues and cell lines.

The Pathophysiological Role of Estrogen Receptor Variants in Human Breast Cancer

The accumulated evidence supports the expression of estrogen receptor variants at both the mRNA and protein levels. The relative level of expression of some estrogen receptor variant mRNAs and possibly progesterone receptor variant mRNAs is altered during breast tumorigenesis and breast cancer progression. The altered expression of estrogen receptor variants may effect estrogen signal transduction as well as the interpretation of assays where the estimation of estrogen receptor levels is used as a guide to treatment strategies and prognosis.

Estrogen regulation of nuclear matrix-intermediate filament proteins in human breast cancer cells

The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T‐47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix‐intermediate filament fraction (NM‐IF) of cells grown in estrogen‐replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen‐depleted conditions. Replacing estrogen in the medium of acute estrogen‐depleted cells restored expression of these proteins. T‐47D5 cells that are chronically depleted of estrogen (T5‐PRF) are estrogen‐nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5‐PRF cells with estrogen did not lead to further up‐regulation of these proteins. Treating T‐47D5 cells in estrogen‐replete conditions with the antiestrogens 4‐hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long‐term chronic estrogen‐depleted T‐47D5 cells. In conclusion, we have identified NM‐IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells.

Estrogen Regulates the Association of Intermediate Filament Proteins with Nuclear DNA in Human Breast Cancer Cells

In a previous study we showed that the levels of the intermediate filament proteins, cytokeratins 8, 18, and 19, in the nuclear matrix-intermediate filament (NM-IF) fraction from the hormone-dependent and estrogen receptor (ER)-positive human breast cancer cell line T-47D5 were regulated by estrogens. In contrast, estrogens did not regulate the cytokeratins in the NM-IF fraction of the hormone-independent and ER-positive cell line, T5-PRF. In this study, human breast cancer cells were treated withcis-diamminedichloroplatinum to cross-link protein to nuclear DNA in situ, and proteins bound to DNA were isolated. We show that cytokeratins 8, 18, and 19 of T-47D5 and T5-PRF were associated with nuclear DNA in situ. The levels of the cytokeratins 8, 18, and 19 bound to nuclear DNA or associated with the cytoskeleton of T-47D5 human breast cancer cells decreased when estrogens were depleted or the pure antiestrogen ICI 164,384 was added. In contrast, the cytokeratin levels associated with nuclear DNA or cytoskeleton were not significantly affected by estrogen withdrawal or antiestrogen administration in T5-PRF cells. These observations suggest that estrogen regulates the organization of nuclear DNA by rearrangement of the cytokeratin filament network in hormone-dependent, ER-positive human breast cancer cells and that this regulation is lost in hormone-independent, ER-positive breast cancer cells.

Oestrogen receptor variants and mutations in human breast cancer

Several oestrogen receptor variant and mutated mRNA species have been identified in human breast samples and cell lines. Over-expression and altered expression of some of these mRNAs have been correlated with breast tumourigenesis and progression. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of oestrogen receptor variants and mutations that have been identified as occurring naturally in human breast tissues and cell lines.

Mechanisms of growth inhibition by antiestrogens and progestins in human breast and endometrial cancer cells.

Marked changes in both growth factor and proto-oncogene expression occur due to treatment of hormonally-responsive human cancers with progestins and antiestrogens. In human endometrial cancer cell lines the antiproliferative effects of progestins and antiestrogens in a particular cell line appear to be associated with similar effects on growth factor and/or proto-oncogene expression. This suggests that although these compounds initially interact with different steroid hormone receptors, the molecular mechanisms of their growth inhibition may be essentially similar. In the case of human breast cancer cell lines, however, the effects of progestins and antiestrogens on gene regulation are often different, suggesting that the molecular mechanisms of progestin and antiestrogen growth inhibition may be essentially dissimilar.

Analysis of human breast cancer nuclear proteins binding to the promoter elements of the c-myc gene

The expression of the c‐myc gene is essential for the proliferation of both hormone‐dependent and ‐independent human breast cancer cells. The regulation of c‐myc gene expression in MCF‐7 (hormone‐dependent, estrogen‐receptor (ER)‐positive) and MDA MB 231 (hormone‐independent, ER‐negative) human breast cancer cells differs, with the c‐myc gene of MCF‐7 but not MDA MB 231 cells being regulated at the transcriptional level by estrogen. We have shown previously that the DNAase I hypersensitive (DH) sites in the c‐myc chromatin of hormone‐dependent and ‐independent human breast cancer cells were similar, with the exception of DH site II2, DH site II2, which maps near the P0 promoter, was less sensitive in hormone‐dependent than in hormone‐independent cells. As DH sites generally indicate the presence of sequence‐specific DNA‐binding proteins, we undertook a study to identify the nuclear proteins isolated from MCF‐7 and MDA MB 231 cells that bound to the P0 and P2 promoter regions of the c‐myc gene in vitro. The studies presented here provide evidence that Sp1 and/or Sp1‐like proteins bind to the P0 and P2 promoter regions of the c‐myc gene of MCF‐7 and MDA MB 231 cells. Furthermore, evidence is presented for the presence of several previously unidentified sequence‐specific DNA‐binding proteins binding to these promoters. The DNA‐binding activities of these latter proteins differed in the nuclear extracts of the MCF‐7 and MDA MB 231 human breast cancer cells.

Expression of insulin-like growth factor binding proteins by T-47D human breast cancer cells: regulation by progestins and antiestrogens

SummaryWe have used ligand blotting and Northern blotting techniques to examine the effects of progestins and antiestrogens on expression of insulin-like growth factor binding proteins (IGFBPs) by T-47D human breast cancer cells under conditions where these agents are growth inhibitory. Under basal conditions, conditioned medium from T-47D cells was found to contain IGFBPs of 39, 33, and 27 kDa. Northern blot and/or Western blot analysis have identified these as IGFBP 2, 5, and 4, respectively. Medroxyprogesterone acetate (MPA) treatment resulted in a time- and dose-dependent decrease in IGFBP 4 and 5 mRNA abundance and secretion of these proteins, while little if any effect was observed on IGFBP 2 expression. A decrease in the steady state mRNA levels for IGFBP 4 and 5 was observed with as little as 0.1 nM MPA. Using 10 nM MPA a maximum decrease in IGFBP 4 and 5 mRNA levels was observed between 12 and 24 hours. While RU 486 alone had little or no effect on IGFBP 4 expression, it inhibited the effect of MPA. However, in the same samples, IGFBP 5 expression was inhibited by RU 486, and RU 486 was unable to reverse the effects of progestins on the expression of IGFBP 5. Furthermore, another synthetic progestin, Org 2058, but not dexamethasone, inhibited IGFBP 4 and IGFBP 5 expression. The antiestrogen ICI 164384 also transiently decreased the steady state mRNA levels of both IGFBP 4 and IGFBP 5. Regulation of expression of the IGFBPs by these agents suggests a potential role for the IGFBPs in the growth response of T-47D cells to these agents.

Multiple Facets of Estrogen Receptor in Human Breast Cancer

Estrogen is a major regulator of mammary gland development and function, and affects the growth and progression of mammary cancers (1,2). In particular, the growth responsiveness of breast cancer (BC) cells to estrogen is the basic rationale for the efficacy of the so-called endocrine therapies, such as antiestrogens. Estrogens mediate their action via the estrogen receptor (ER), which belongs to the steroid/thyroid/retinoid receptor gene superfamily (3). The protein products of this family are intracellular, ligand-activated transcription factors regulating the expression of several gene products, which ultimately elicit a target tissue-specific response (4). Indeed, ER, together with progesterone receptor (PR), expression in human breast tumors, are important prognostic indicators, as well as markers of responsiveness to endocrine therapies (5, 6). However, although the majority of human BCs are thought to be initially hormone-responsive, it is well appreciated that alterations in responsiveness to estrogen occurs during breast tumorigenesis. During BC progression, some ER-positive BCs are de novo resistant to endocrine therapies, and of those that originally respond to antiestrogens, many develop resistance. This progression from hormonal dependence to independence is a significant clinical problem, because it limits the useful of the relatively nontoxic endocrine therapies, and is associated with a more aggressive disease phenotype (7). This occurs despite the continued expression of ER, and often PR (8,9). The ER is pivotal in estrogen and antiestrogen action in any target cell, but the nature of the ER is clearly multifaceted.