Françoise Vignon

Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens.

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.

Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis.

Cathepsin-D is an independent marker of poor prognosis in human breast cancer. We previously showed that human wild-type cathepsin-D, as well as its mutated form devoid of proteolytic activity stably transfected in 3Y1-Ad12 cancer cells, stimulated tumor growth. To investigate the mechanisms by which human cathepsin-D and its catalytically-inactive counterpart promoted tumor growth in vivo, we quantified the expression of proliferating cell nuclear antigen, the number of blood vessels and of apoptotic cells in 3Y1-Ad12 tumor xenografts. We first verified that both human wild-type and mutated cathepsin-D were expressed at a high level in cathepsin-D xenografts, whereas no human cathepsin-D was detected in control xenografts. Our immunohistochemical studies then revealed that both wild-type cathepsin-D and catalytically-inactive cathepsin-D, increased proliferating cell nuclear antigen expression and tumor angiogenesis. Interestingly, wild-type cathepsin-D significantly inhibited tumor apoptosis, whereas catalytically-inactive cathepsin-D did not. We therefore propose that human cathepsin-D stimulates tumor growth by acting–directly or indirectly–as a mitogenic factor on both cancer and endothelial cells independently of its catalytic activity. Our overall results provide the first mechanistic evidences on the essential role of cathepsin-D at multiple tumor progression steps, affecting cell proliferation, angiogenesis and apoptosis.

IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells.

Estrogen-receptor (ER) status is an important parameter in breast cancer management as ER-positive breast cancers have a better prognosis than ER-negative tumors. This difference comes essentially from the lower aggressiveness and invasiveness of ER-positive tumors. Here, we demonstrate, that interleukin-8 (IL-8) was clearly overexpressed in most ER-negative breast, ovary cell lines and breast tumor samples tested, whereas no significant IL-8 level could be detected in ER-positive breast or ovarian cell lines. We have also cloned human IL-8 from ER-negative MDA-MB-231 cells, and we show that IL-8 produced by breast cancer cells is identical to monocyte-derived IL-8. Interestingly, the invasion potential of ER-negative breast cancer cells is associated at least in part with expression of IL-8, but not with IL-8 receptor levels. Moreover, IL-8 increases the invasiveness of ER-positive breast cancer cells by two fold, thus confirming the invasion-promoting role of IL-8. On the other hand, exogenous expression of estrogen receptors in ER-negative cells led to a decrease of IL-8 levels. In summary, our data show that IL-8 expression is negatively linked to ER status of breast and ovarian cancer cells. We also support the idea that IL-8 expression is associated with a higher invasiveness potential of cancer cells in vitro, which suggests that IL-8 could be a novel marker of tumor aggressiveness.

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

Mechanisms underlying differential expression of interleukin-8 in breast cancer cells

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-κB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-κB and AP-1 activities by adenovirus-mediated expression of an NF-κB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPα and C/EBPβ expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-κB, AP-1 and C/EBP transcription factors.

Involvement of Estrogen Receptor β in Ovarian Carcinogenesis

Knockout and expression studies suggest that estrogen receptor β (ERβ) plays a prominent role in ovarian function and pathology. Moreover, ovarian cancers are characterized by high morbidity and low responsiveness to anti-estrogens. Here we demonstrate, using quantitative PCR to measure ERα and ERβ levels in 58 ovarian cancer patients, that ERβ expression decreased in cysts and ovarian carcinomas as compared with normal ovaries and that this decrease is attributable only to a selective loss in ERβ expression during cancer progression. To address the question of a possible involvement of ERβ in ovarian cancers, we restored ERα and ERβ expression in two human ovarian cancer cell lines PEO14 (ERα-negative) and BG1 (ERα-positive) using adenoviral delivery. ERα, but not ERβ, could induce progesterone receptor and fibulin-1C. Moreover, ERα and ERβ had opposite actions on cyclin D1 gene regulation, because ERβ down-regulated cyclin D1 gene expression, whereas ERα increased cyclin D1 levels. Interestingly, ERβ expression strongly inhibited PEO14 and BG1 cell proliferation and cell motility in a ligand-independent manner, whereas ERα had no marked effect. Induction of apoptosis by ERβ also contributed to the decreased proliferation of ovarian cancer cells, as shown by Annexin V staining. This study shows that ERβ is an important regulator of proliferation and motility of ovarian cancer and provides the first evidence for a proapoptotic role of ERβ. The loss of ERβ expression may thus be an important event leading to the development of ovarian cancer.

The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene

Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) α expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERα cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17β-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER α and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.

The antiproliferative effect of tamoxifen in breast cancer cells: mediation by the estrogen receptor

The effects of tamoxifen (Tam) and its 4-hydroxylated metabolite (OH-Tam) on the growth of two human breast cancer cell lines ( MCF7 and BT20 ) were evaluated by fluorometric DNA assay. The effects of the antiestrogens were dependent upon their concentrations and the nature of the cells. At concentrations below 4 microM, the degree of inhibition was related to their relative affinities for the estrogen receptor and was totally reversed by estradiol in MCF7 cells. No inhibition was observed in the estrogen receptor negative cell line BT20 . This supports and extends the idea that the antiproliferative effect of Tam at these concentrations is mediated by the estrogen receptor even in the absence of measurable estradiol concentration. At concentrations greater than 4 microM, Tam was cytotoxic on MCF7 and BT20 mammary cell lines within 2 days of treatment. The cytotoxic effect was irreversible and was not prevented by occupation of the estrogen receptor with estradiol, suggesting that it was not mediated by the estrogen receptor. The cytotoxicity of the triphenylethylene drugs, however, has some specificity since it was not observed in a fibroblast rat cell line ( 49F ) or in the two mammary cell lines with similar high concentrations of estradiol and diethylstilbestrol.

MECHANISMS OF 4-HYDROXYTAMOXIFEN ANTI-GROWTH FACTOR ACTIVITY IN BREAST CANCER CELLS : ALTERATIONS OF GROWTH FACTOR RECEPTOR BINDING SITES AND TYROSINE KINASE ACTIVITY

We previously demonstrated that antiestrogen 4-hydroxytamoxifen (OH-Tam) blocks the mitogenic activity of growth factors in breast cancer. We now investigate this mechanism by evaluating how OH-Tam affects growth factor binding and receptor tyrosine kinase activity. We show here that OH-Tam has an opposite effect on epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) binding in estrogen receptor (ER) positive cells. A decrease in IGF-1 binding sites may explain the reduced IGF-I mitogenic effect, whereas an increase in high affinity EGF binding associated with a decrease in in vitro receptor autophosphorylation rather favors the possibility of an alteration in EGF receptor tyrosine kinase activity. We conclude that OH-Tam may prevent growth factor action in ER+ cells both by modulating the concentration of growth factor binding sites and by altering growth factor receptor functionality.

Difference between R5020 and the antiprogestin RU486 in antiproliferative effects on human breast cancer cells.

SummaryWe have compared the effects of the progestin R5020 and the antiprogestin RU486 on the growth of the MCF-7 and T47D breast cancer cell lines. Differences between the two compounds were demonstrated in several parameters.1.Estradiol was required for the efficient inhibition of cell growth of both lines by R5020 but not by RU486. Therefore in the total absence of estrogen (phenol-red free medium), the effects of the two drugs on cell growth were dissociated, RU486 remaining inhibitory while R5020 was inactive.2.The proteins secreted by cells were differently affected, since R5020 induced a 48K protein and decreased the production of the estrogen-regulated 52K protein, while RU486 had no effect on these two parameters.3.The morphology of cells treated by R5020 was more altered in the presence of estradiol than in its absence, while that of cells treated by RU486 was not affected whether or not estradiol was present.4.There was a greater reduction of estrogen receptor sites in MCF-7 cells produced by R5020 than by RU486. Even though the two drugs appear to act through the same progesterone receptor and to inhibit total protein secretion, it is likely that they exert their antiproliferative effects on cultured breast cancer cells by different mechanisms. R5020 antagonizes the stimulation produced by estradiol. RU486 by contrast exerts a more direct progesterone receptor mediated inhibitory effect requiring no synergism by estradiol and therefore does not act through a partial progestin activity.