Martin R. Schneider

Comparison of Proteomic and Genomic Analyses of the Human Breast Cancer Cell Line T47D and the Antiestrogen-resistant Derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex™, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.

Antitumor activity of the antiprogestins ZK 98.299 and RU 38.486 in hormone dependent rat and mouse mammary tumors: Mechanistic studies

SummaryIn the transplantable MXT mammary tumor model of the mouse and in the DMBA- and MNU-induced mammary tumor models of the rat, the progesterone antagonists ZK 98.299 and RU 38.468 were shown to have potent antitumor activity. The weight and/or morphology of the ovaries, uterus, and vagina, as well as the effects on serum hormone levels, indicate that the antitumor activity of both antiprogesterones in these models does not depend on a blockade of the ovarian and pituitary functions and does not depend on a non receptor-mediated cytotoxic effect.On the other hand, the morpholoy of the MXT and the DMBA-induced mammary tumors after treatment with the progesterone antagonists is completely different from that observed after ovariectomy. Treatment with the antiprogesterones seems to trigger differentiation of the mitotically active polygonal tumor cells towards glandular structures and acini with a massive sequestering of secretory products, as well as towards spindle-shaped necrobiotic subpopulations. By contrast, the induction of tumor cell degeneration and cytolysis is the predominant feature of the mammary tumors after ovariectomy.In conclusion, our results indicate that the main mechanism of the antitumor action of antiprogesterones in these models is a direct progesterone receptor-mediated antiproliferative effect at the level of the mammary tumor cells, most probably via the induction of terminal differentiation associated with terminal cell death. This antiproliferative effect seems to be dissociated from the antihormone (antiprogestational) activity of these progesterone antagonists.

The antitumor mechanism of progesterone antagonists is a receptor mediated antiproliferative effect by induction of terminal cell death

The antiprogesterones Onapristone, ZK 112.993 (Schering AG), and Mifepristone (Roussel-Uclaf) proved to possess progesterone receptor-mediated antiproliferative effects in experimental mammary carcinomas. In this study, the potency and mechanism of the antitumor action of Onapristone and ZK 112.993 is characterized by ovariectomized, progestagen and/or estradiol substituted mice bearing hormone-dependent MXT(+) mammary tumours. Medroxyprogesterone acetate (MPA, 0.8 mg/mouse, 3 times weekly, s.c.) could only induce a poor stimulation of tumour growth (% T/C = 40; intact control % T/C = 100), which was only marginally inhibited (% T/C = 21) by Onapristone (0.2 mg/mouse, 6 times weekly, s.c.) during a 6-week therapy. Therefore, the antitumor mechanism of antiprogesterones cannot preferably depend on a classical progesterone antagonism. In contrary, the pronounced stimulation of tumor growth (% T/C = 152) by estradiol benzoate (EB, 0.33 microgram/mouse, 3 times weekly, s.c.) was completely inhibited (% T/C = 7) by the antiprogesterones. An even more stimulated tumour growth was achieved by a combination of EB and MPA (% T/C = 365 using 0.17 mg; % T/C = 225 using 0.8 mg MPA). Onapristone dramatically blocked tumor growth (% T/C = 7) at the lower dose of MPA; no inhibition (% T/C = 203), however, was detected at the higher dose of MPA. These data and a morphological analysis indicate that the potent antitumor activity of the progesterone antagonists depends on the binding to a number of available progesterone receptors high enough to trigger an antiproliferative effect via the induction of terminal differentiation associated with terminal cell death.

Progesterone antagonists: Tumor-inhibiting potential and mechanism of action☆

A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical anti-hormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the G0G1 phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G1 arrest has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.

Estrogenic regulation of clusterin mRNA in normal and malignant endometrial tissue

Clusterin is a heterodimeric, 80kDa, glycoprotein that is synthesized in a wide variety of tissues in response to a number of diverse stimuli, including hormone ablation. We have investigated the regulation of clusterin expression by estradiol and anti‐estrogens in RUCA‐I rat endometrial adenocarcinoma cells in vitro and in vivo. We have also compared clusterin expression in endometrial tumors and in normal uterine tissue. Estradiol treatment significantly increases the steady state mRNA levels of clusterin in RUCA‐I cells cultured on a reconstituted basement membrane, with a maximal induction 24 hr after estradiol treatment. The inductive effects of estrogen on clusterin mRNA steady state levels in vitro are significantly more pronounced than the effects on fibronectin mRNA levels, an estrogen‐repressed gene in RUCA‐I. In vivo, induction of clusterin expression in primary and metastatic endometrial adenocarcinoma is also dependent on the presence of estradiol, in marked contrast to expression of clusterin in the normal endometrium of the same animals. These data suggest that clusterin mRNA expression in rat endometrial adenocarcinoma cells is tightly regulated by estrogens and anti‐estrogens in vitro and in vivo, and that there is a complex mechanism of regulation of clusterin expression in the normal and cancerous endometrium. Int. J. Cancer 76:684–688, 1998.© 1998 Wiley‐Liss, Inc.

Differentiation Therapy with Progesterone Antagonists

It is widely and readily accepted that a receptor antagonist may antagonize the functions of the specific receptor; therefore, it would be surprizing if a pure receptor antagonist possessed the ability to induce some of the physiological functions of this receptor: we will demonstrate that progesterone antagonists may indeed succeed in inducing the physiological function of the progesterone receptor. In females this function is accepted as being differentiation in steroid hormone-dependent tissue; in males the function has been neglected so far, although progesterone receptors have been localized in the male genital tract of different species.’-3

Entwicklung neuer Antiöstrogene vom Typ des 3,3′-Dihydroxy-α,β-diäthylstilbens und ihre Prüfung am DMBA-induzierten, hormonabhängigen Mammacarcinom der SD-Ratte

SummaryThe displacement of the phenolic OH-group of diethylstilbestrol into the 3,3′-position (trans-3,3′-dihydroxy-α,β-diethylstilbene compd. III) leads to a strong decrease of the estrogenic effect under conservation of the receptor affinity. In vitro, III inhibits the estradiol-receptor-interaction competitively and, in vivo, antagonises the uterotropic effect of estrone in the mouse. In tests with the DMBA-induced, hormone-dependent mammary carcinoma of the rat a dose-dependent strong decrease of tumor size and yield is achieved under the influence of III, due to the antiestrogenic, properties of III. The replacement of the α,β-bound ethyl groups in III by other alkyl chains leads to no further increase of the antiestrogenic and antitumor activity.ZusammenfassungDie Verlagerung der phenolischen OH-Gruppen des Diäthylstilböstrols in die 3,3′-position (trans-3,3′-Dihydroxy-α,β-diäthylstilben, Verb. Nr. III) führt unter Erhaltung der Rezeptoraffinität zu einer starken Abnhme der östrogenen Wirkung. III hemmt in vitro die östradiol-Rezeptor-Wechselbeziehung kompetitiv und antagonisiert in vivo bei der Maus die uterotrope Wirkung des Östrons. In Versuchen am DMBA-induzierten, hormonabhängigen Mammacarcinom der Ratte kommt es, unter III-Einwirkung dosisabhägig zu einer starken Abnahme von Tumorgröße und-zahl, die durch die antiöstrogenen Eigenschaften von III bedingt, ist. Der Austausch der α,β-ständigen Äthylreste in III durch andere Alkylketten führt zu keiner weiteren, Steigerung der antiöstrogenen und tumorhemmenden, Wirkung.

Studies on the development of resistance to the pure antiestrogen Faslodex in three human breast cancer cell lines.

In order to understand the mechanisms underlying the development of resistance to a pure antiestrogen we established three human breast carcinoma cell lines resistant to ZM 182780 (ZM) (Faslodex). Long-term cultivation of the ERalpha-positive, 17beta-estradiol (E(2))-responsive cell lines T47D, ZR-75-1, and MCF-7 with the pure antiestrogen ZM 182780 resulted in the T47D-r, ZR-75-1-r, and MCF-7-r cell lines, which proliferate continuously in the presence of 10(-6)M ZM 182780. The resulting antiestrogen-resistant cells grow equally well in medium with or without E(2) and in medium with or without ZM 182780 indicating that they are no longer estrogen-responsive. ERalpha expression was lost at the protein level in all three resistant cell lines. At the mRNA level, the ERalpha was only faintly detectable in T47D-r, whereas a weak signal was seen in ZR-75-1-r and MCF-7-r. By reverse transcription-polymerase chain reaction (RT-PCR) the ERbeta was detectable in the antiestrogen-sensitive and -resistant breast cancer cell lines, however, ZR75-1-r contained the smallest signal for ERbeta. In all three antiestrogen-resistant cells the PR was undetectable, whereas binding of epidermal growth factor (EGF) and protein expression of epidermal growth factor receptor (EGFR) were increased. To analyse alterations in the gene expression pattern in more detail Atlas arrays were hybridised with RNA isolated from T47D-r and T47D and the two Ca2+-binding proteins calgranulin A and B were found to be up-regulated in T47D-r compared to T47D. Calgranulin A and B were also both up-regulated in ZR-75-1-r and MCF-7-r compared to their antiestrogen-sensitive counterparts. Loss of ERalpha expression may be linked to the acquisition of antiestrogen resistance and enhanced expression of the EGFR and of proteins of the S100 family of Ca2+-binding proteins which may contribute to the outgrowth of resistant cells.

Antitumor activity of antiestrogenic phenylindoles on experimental prostate tumors

Two antiestrogenic phenylindoles (D 16726 and D 15413) were tested for their prostatic tumor-inhibiting activity. Both compounds exerted a strong inhibitory effect on prostate and seminal vesicle weight of intact rats and mice comparable to that of diethylstilbestrol. Their estrogenic properties, however, are much lower than those of DES. Therefore, there is no direct correlation between estrogenic potency and inhibition of accessory sex organ weights. The tumor-inhibiting activity of D 16726 and D 15413 on the androgen-dependent R 3327 Dunning prostatic carcinoma and the human prostatic tumor PC 82 implanted in nude mice equals that of castration or of diethylstilbestrol. Both 2-phenylindoles had good affinities for estrogen receptors from calf uterine and R 3327 tumor cytosol, but no affinities for androgen and progesterone receptors. As these 2-phenylindoles have much lower estrogenic properties than diethylstilbestrol, they may also have low side-effects, and can therefore be of interest for the therapy of the prostatic carcinoma.

Progesterone receptor repression by estrogens in rat uterine epithelial cells

Measurements performed using cell lines or animal tissues have shown that the progesterone receptor (PR) can be induced by estrogens. By use of immunohistochemistry we studied the effects of estrogens on the PR levels in the individual cell types of the target organs uterus and breast. In the uteri of rats, ovariectomy induced a decrease in PR immunoreactivity within the myometrium and outer stromal cell layers. In contrast, in the uterine luminal and glandular epithelium and surrounding stromal cell layers the PR immunoreactivity was significantly enhanced. The same picture emerged when intact rats were treated with the pure estrogen receptor antagonist, ZM 182780 (10 mg/kg/d). Treatment of ovariectomized rats with estradiol resulted in high PR levels in the myometrium and stroma cells but low PR immunoreactivity in the epithelial cells. The ER-mediated repression of the PR immunoreactivity was evidently restricted to the uterine epithelium, as we found that in the epithelial cells of the mammary gland and in cells of N-nitrosomethylurea-induced mammary carcinomas the PR expression was induced by estrogens and was blocked by the pure antiestrogen ZM 182780. These results clearly show that in the rat the activated ER induces diverging effects on PR expression in different cell types even within the same organ.