Marcel Garcia

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C19 steroids bearing a 17beta and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (greater than 0.4mg) of 5dihydrotestosterone provoked in vivo as in vitro the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation in vivo provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.

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 PKCα 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 PKCα observed in ER− cells. Int. J. Cancer 75:750–756, 1998.© 1998 Wiley‐Liss, Inc.

Increased cathepsin D level in the serum of patients with metastatic breast carcinoma detected with a specific pro‐cathepsin D immunoassay

An increased cathepsin D (cath‐D) level in breast carcinoma cytosol has been proposed as a prognostic parameter. However, no increase had been previously detected in serum when assaying total cath‐D concentration.

Androgens on the estrogen receptor. II--Correlation between nuclear translocation and uterine protein synthesis.

In the immature rat uterus, occupation of the androgen and estrogen receptor sties after injection of 5 alpha dihydrotestosterone (DHT = 17beta - hydroxy-5alpha-androstan-3-one) were compared to the biological responses induced by the androgen on protein synthesis. Injection of 100 mug DHT induced a maximal occupation of androgen receptor sites (RA) but was totally ineffective in translocating the estrogen receptor sites and in increasing general protein synthesis. Conversely, high doses of androgen (greater than or equal to 3 mg) translocated the estrogen receptor (RE) and stimulated general protein synthesis. In addition, these high doses induced a specific uterine protein undistinguishable from that induced by estradiol treatment (IP). These results strongly suggest that the uterotrophic response of the uterus to androgen is correlated with the nuclear transreceptor which is present in much smaller amounts.

Differential regulation of cathepsin D by sex steroids in mammary cancer and uterine cells

The precursor of cathepsin D, a lysosomal acidic protease, is secreted by human breast cancer cells, where its synthesis is specifically induced by estrogens and growth factors. In this study, we investigated the hormonal regulation of cathepsin D and its mRNA in uterine cells. In the Ishikawa endometrial cancer cell line, epidermal growth factor (EGF) increased the level of cathepsin D and its mRNA 2- to 3-fold. Although expression of the transiently transfected estrogen-responsive recombinant (Vit. tk. CAT) and the endogenous progesterone receptor was markedly increased by estradiol in Ishikawa cells, estradiol did not alter the level of cathepsin D or its mRNA. The progestin R5020 induced the expression of the LTR sp65 CAT, which contains the progesterone-responsive element of the MMTV but it too was without effect on cathepsin D. By contrast, the expression of cathepsin D gene, in normal rat uterus, was increased by R5020 but not by estradiol. We conclude that cathepsin D gene expression is regulated differently by sex steroid hormones in endometrial and breast cancer cell lines, whereas it is similarly induced by EGF in these cells.

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.

Synthesis and Biological Activity of Phosphonate Analogs of Mannose 6-Phosphate (M6P)

Two phosphonate analogs of mannose 6-phosphate (M6P) have been synthesized. The isosteric analog 1 was obtained by a Wittig–Horner reaction at position 6 of a sugar aldehyde. The non-isosteric analog 2 was obtained by a Michaelis–Arbuzov rearrangement of a 6-bromo derivative. In contrast to the non-isosteric analog 2, the isoster 1 was shown to bind to M6P receptors as effectively as does M6P itself, thus demonstrating the considerable potential of the system in drug design.

Role of estrogens and their receptors in adhesion and invasiveness of breast cancer cells.

Estrogen receptors (ERs) are overexpressed in human breast cancers (BCs) and associated with differentiated tumors and with a more favorable prognosis. Paradoxically, ERs mediate the mitogenic action of estrogens in human BC cells and the efficacy of antiestrogens in adjuvant therapy of primary tumors. The exact mechanism underlying the ER protection against cancer progression to metastasis remains to be investigated. Herein, we show that ERs decrease invasiveness of BC cells. Detailed studies revealed that the unliganded and the E2-activated ERs decrease cancer cell invasion in vitro through two distinct mechanisms. In the presence of ligand, ERalpha inhibits invasion through a mechanism requiring the functional ERalpha domains involved in the transcriptional activation of target genes. Moreover, using different approaches, we found that cell-cell contacts were markedly increased by 17beta-estradiol (E2) treatment and decreased by the pure antiestrogen, ICI182,780. This cell-cell adhesion was associated with an increase of the major intercellular junctions, desmosomes. Conversely, in the absence of ligand, ERalpha also inhibits invasion through a distinct mechanism involving protein-protein interaction with the region of the first zinc finger of ERalpha. The relationship of these data with clinical studies and their potential therapeutic consequences will be discussed.

Estradiol increases the production of α1-antichymotrypsin in MCF7 and T47D human breast cancer cell lines

alpha 1-Antichymotrypsin (Achy) is an antiprotease of the acute inflammation phase, which is also released by MCF7 human breast cancer cells in culture. Using a fluorimetric assay with the synthetic substrate L-Seryl-L-Tyrosyl-2-N-naphthylamide, we have shown that a medium conditioned by MCF7 cells treated by estradiol inhibits the activity of alpha-chymotrypsin. This inhibition increased when physiological concentrations of estradiol were added to the cells for 2 days. It was due to an increased production of Achy and not to a direct effect of estradiol on alpha-chymotrypsin activity as shown by double immunoprecipitation with an antiserum against human alpha 1-antichymotrypsin. An increased accumulation by estradiol of an antigen located in the cytoplasm of MCF7 cells, which was revealed by immunoperoxidase staining with antibodies to Achy, also indicated that estradiol increased the production of Achy in these cells. Similar immunostaining was observed in a breast cancer tissue. Most of the estrogen regulated 60-68 kDa protein secreted by T47D cells (Chalbos et al. (1982) J. Clin. Endocrinol. Metab. 55, 276-283) was also specifically immunoprecipitated by the antibodies to Achy. Thus, alpha 1-antichymotrypsin is the first protein to be identified which is induced by estradiol and secreted by breast cancer cells.

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.