How to target estrogen receptor-negative breast cancer?

Estrogen receptor (ER)-positive breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy, which is the first example of a successful therapy targeted on a specific protein, the ER. Unfortunately ER-negative breast cancers are more aggressive and unresponsive to anti-estrogens. Other targeted therapies are thus urgently needed, based on breast cancer oncogene inhibition or suppressor gene activation as far as molecular studies have demonstrated the alteration of expression, or structure of these genes in human breast cancer. Using the MDA-MB.231 human breast cancer cell line as a model of ER-negative breast cancers, we are investigating two of these approaches in our laboratory. Our first approach was to transfect the ER or various ER-deleted variants into an ER-negative cell line in an attempt to recover anti-estrogen responsiveness. The unliganded receptor, and surprisingly estradiol, were both found to inhibit tumor growth and invasiveness in vitro and in vivo. The mechanisms of these inhibitions in ER-negative cancer cells are being studied, in an attempt to target the ER sequence responsible for such inhibition in these cancer cells. Another strategy is trying to inhibit the activity or expression of an oncogene specifically overexpressed in most breast cancers. This approach was recently shown by others to be efficient in breast cancer therapy with HER2-Neu oncogene amplification using Herceptin. Without excluding other molecular putative targets, we have focused our research on cathepsin D as a potential target, since it is often overexpressed in aggressive human breast cancers, including ER-negative tumors, and rarely associated with HER2-Neu amplification. Our first results obtained in vitro on cell lines and in vivo in tumor xenografts in nude mice, illustrate that the mode of action of cathepsin D in breast cancer is useful to guide the development of these therapies. In the past 20 years we have learned that the action of cathepsin D is complex and involves both intracellular and extracellular activities due to its proteolytic activity and to interactions with membrane components without catalytic activity. Each of these mechanisms could be potentially inhibited in an attempt to prevent tumor growth. Breast cancer is a very heterogeneous and multigenic disease and different targeted therapies adapted to each category of breast cancer are therefore required. Validated assays in the primary tumor of molecular markers such as ER, HER2-Neu and cathepsin D should help to predict which targeted therapy should be applied to cure breast cancer patients.

Estrogen regulated proteases and antiproteases in ovarian and breast cancer cells.

Cathepsin D (cath-D), an estrogen-regulated protease appears mostly to increase the number of tumor cells rather than their invasion or motility through the extracellular matrix. Estradiol is mitogenic but in vitro it also inhibits invasion and motility. In this review, we discuss the mechanism of this inhibition and the hormonal regulation of other proteases and protease inhibitors possibly involved in the control of tumor cell invasion by estrogens.

Unliganded and liganded estrogen receptors protect against cancer invasion via different mechanisms.

While estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor a (ERalpha) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ERalpha action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ERalpha expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ERalpha indicated that amino acids 179-215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ERalpha and ERbeta reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206-211 of ERalpha, reversed hormone-independent ERalpha inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ERalpha interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ERalpha-positive cell lines, where ERalpha content was inversely correlated with cell migration. We conclude that ERalpha protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.

Estrogen receptor mediated inhibition of cancer cell invasion and motility: an overview.

In this overview of results from our laboratory, we address the question of the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines was studied using a modified Boyden chamber assay. We observed, in all cases, estradiol induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER alpha was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however, it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies.

Breast cancer cell invasiveness: correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells.

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER+) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER- cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER+ cells, constitutive PKC activity and PKCalpha expression were very low as compared to ER- cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER+ and ER- cells could be due to the abnormal TPA regulation of PKCalpha observed in ER- cells.

A new bioassay using transient transfection for invasion-related gene analysis.

To understand the mechanisms of tumor invasion and metastasis, model systems are required that isolate the individual steps of these complicated, multifaceted processes. We propose a new procedure to identify genes involved in cell invasion and/or motility that features the combined advantages of transient gene transfection and Matrigel invasion assays. Cancer cells were transiently cotransfected with two vectors expressing the gene of interest and luciferase, as a marker of transfected cells, and then assayed for Matrigel invasion. Luciferase cotransfection appeared to be a sensitive semiquantitative assay for transfected cells and was maximal throughout the invasion assay. The proposed transfection procedure, using calcium phosphate precipitation, did not affect cell invasiveness and allowed cellular coexpression of both genes. When applying this method, we found that transient expression of the unliganded and liganded human estrogen receptor alpha prevented invasiveness of MDA-MB-231 breast cancer cells. In conclusion, we propose rapid and versatile in vitro procedure for studying the effects of individual cloned genes on cellular processes, such as invasion and motility.

Both estradiol and tamoxifen decrease proliferation and invasiveness of cancer cells transfected with a mutated estrogen receptor.

Previous studies have shown that, after wild-type estrogen receptor (ER) transfection in ER-negative breast cancer cells, estradiol but not tamoxifen prevents growth, invasiveness and metastasis of these cells in mice. Because an ER mutation at position 400 converts the triphenylethylene antiestrogen, OH-tamoxifen into a full estrogen agonist, we transfected this mutated form of human ER in an ER-negative rat cancer cell line. This was aimed at inducing an inhibitory, estrogen-like response of tamoxifen in these cells. In two stable ER-positive transfectants, OH-tamoxifen inhibited cell growth and invasiveness in vitro as efficiently as estradiol. The pure antiestrogen, ICI 164,384, was not agonistic alone and antagonized estrogen action. In contrast, the three compounds were ineffective in control mock-transfected cells. When injected into ovariectomized nude mice, ER-negative mock-transfected cells formed tumours which were significantly stimulated by estradiol and inhibited by tamoxifen treatment. This indicates that estradiol and tamoxifen altered the growth of ER-negative tumours via a general effect on the host response. Surprisingly, the hormone responsiveness of ER-positive tumours developed from ER-transfected cells did not significantly differ from that of ER-negative (mock-transfected) tumours. We conclude that transfection of a mutated human estrogen receptor inhibited, through an estrogenic activity of tamoxifen, the growth and invasiveness of these cancer cells in vitro. However, the low expression of ER did not allowed us to obtain the same effect of tamoxifen in vivo.

Biological and clinical significance of cathepsin D in breast cancer metastasis.

Cathepsin D (cath-D) is an aspartyl lysosomal protease expressed in all tissues. Most metastatic breast cancer cell lines, unlike normal cells, secrete high levels of pro-cath-D. This abnormal secretion is due to both overexpression of the cath-D gene and to an altered processing of the precursor protein. Cath-D gene transcription is increased by estrogen and growth factors in estrogen-receptor-positive breast cancer cells and by an unknown mechanism in estrogen-receptor-negative cells. A large number of independent clinical studies associated high cath-D concentrations in the cytosol of primary breast cancers with increased risk of subsequent metastasis. The amino acid sequence of cath-D analyzed in two breast cancer cell lines is normal, but glycosylation appears to be different with more acidic isoforms. To assess the potential role of this protease in cancer metastasis, we transfected a human cDNA cath-D expression vector in 3Y1-Ad12 embryonic rat tumorigenic cells which did not secrete the proenzyme. A moderate overexpression of human cath-D was sufficient to increase the metastatic potential of these cells in nude mice. The mechanism of cath-D-induced metastasis seems to require maturation of the proenzyme, in endosomes and in large acidic compartments identified as phagosomes. Rather than increase cancer cell escape from the primary tumor through basement membrane degradation as proposed for neutral proteinases, cath-D appears to facilitate cell growth at distant sites. The mechanism of this indirect mitogenic effect is discussed from results obtained in different models. Different cath-D substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.