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Local treatment using drug loaded implants allows decreasing seric concentrations of the active ingredient with the purpose of limiting side effects and reaching perfect observance. Nowadays, some diseases are already treated with implants, but generally, by subcutaneous or intra vaginal implantation. In this work, a new implant device dedicated to the intra-peritoneal cavity was developed. For this purpose, a core-membrane polymer implant was selected. We propose an original method to determine the most appropriate membrane to control the release based on the use of Franz cells. The ability of the implant to release a constant quantity of an active ingredient will be assessed by testing implants in vitro. Finally, intra peritoneal cavity and subcutaneous in vivo implantation has been achieved in order to confirm the controlled and local release of the active ingredient.

Accumulating data now suggest that ZO-1, once delocalized from tight junctions, could be implicated in the regulation of tumor-promoting genes. Because of their major implication in different steps of tumor progression, we investigated here the influence of ZO-1 on chemokines expression in breast cancer cells. Using GeneArray analysis to compare chemokine mRNA expression in breast tumor cells transfected with a siRNA against ZO-1, we identified CXCL-8IL-8 as a major potential target of ZO-1 signaling, being strongly downregulated following ZO-1 siRNA transfection. Examining further the relationship between ZO-1 and interleukin-8 (CXCL8/IL-8), we first showed that CXCL8/IL-8 expression correlates with a relocalization of ZO-1 in several breast cancer cell lines. Moreover, CXCL8/IL-8 is downregulated in invasive BT549 cells transfected with three different ZO-1 siRNA and overexpressed in noninvasive BT20 and SKBR3 cells transfected with vectors expressing ZO-1. We also provide evidence for an activation of the CXCL8/IL-8 promoter by ZO-1. Finally, we show that the regulation of CXCL8/IL-8 by ZO-1 is independent of the β-catenin pathway. Our results thus clearly show an implication of ZO-1 in CXCL8/IL-8 regulation. Because of the major implications of CXCL8/IL-8 in tumor invasion, such a regulation could play an important role in breast cancer progression. Mol Cancer Res; 10(1); 121–32. ©2011 AACR.

Increased risk of breast cancer is a critical side effect associated with the use of a menopausal hormone therapy (MHT). Estetrol (E4) is a natural estrogen produced by the human fetal liver and is a promising compound for clinical use in MHT. However, its impact on breast cancer is controversial and poorly defined. In this preclinical study, we show that E4 acts as a weak estrogen by stimulating the growth of hormone-dependent breast cancer only at concentrations exceeding menopausal therapeutic needs. E4 presents also an antitumor activity by decreasing the strong proliferative effect of estradiol (E2). While estrogen receptor alpha (ERα) is the predominant receptor mediating its effects, the dual weak-estrogenic/anti-estrogenic feature of E4 results from differential signaling pathways activation. Both nuclear and rapid extra-nuclear signaling pathway are necessary for a complete estrogenic effect of E4. However, the antitumor action of E4 is not due to a capacity to antagonize E2-induced nuclear activity. Altogether, our results highlight that E4 has a limited impact on breast cancer and may offer a safe therapeutic window for the treatment of menopausal symptoms. This work was supported by grants from the Fonds de la Recherche Scientifique - FNRS (F.R.S.-FNRS, Belgium) : FRSM 3.4557.12, FRSM 3.4567.11, Télévie 7.4524.11, Télévie 7.4604.13 ; the Fonds spéciaux de la Recherche (University of Liège) : FSRC- 12/64, FSRC-12/92, FSRC-14/89, FSRC-14/65, FSRC-14/109, FSRC-14/62 ; the Centre Anticancéreux près l’ Université de Liège, the Fonds Léon Fredericq (University of Liège), the Direction Générale Opérationnelle de l’Economie, de l’Emploi et de la Recherche from the Service Public de Wallonie (DGO6, SPW, Belgium) ; the Interuniversity Attraction Poles Programme - Belgian Science Policy (Brussels, Belgium) : IAP Phase VII - P7/03 ; the Plan National Cancer (Service Public Fédéral) and the Actions de Recherche Concertées (University of Liege, Belgium): A.R.C. 11/16-02.

The expression of Smad interacting protein-1 (SIP1; ZEB2) and the de novo expression of vimentin are frequently involved in epithelial-to-mesenchymal transitions (EMTs) under both normal and pathological conditions. In the present study, we investigated the potential role of SIP1 in the regulation of vimentin during the EMT associated with breast tumor cell migration and invasion. Examining several breast tumor cell lines displaying various degrees of invasiveness, we found SIP1 and vimentin expression only in invasive cell lines. Also, using a model of cell migration with human mammary MCF10A cells, we showed that SIP1 is induced specifically in vimentin-positive migratory cells. Furthermore, transfection of SIP1 cDNA in MCF10A cells increased their vimentin expression both at the mRNA and protein levels and enhanced their migratory abilities in Boyden Chamber assays. Inversely, inhibition of SIP1 expression by RNAi strategies in BT-549 cells and MCF10A cells decreased vimentin expression. We also showed that SIP1 transfection did not activate the TOP-FLASH reporter system, suggesting that the beta-catenin/TCF pathway is not implicated in the regulation of vimentin by SIP1. Our results therefore implicate SIP1 in the regulation of vimentin observed in the EMT associated with breast tumor cell migration, a pathway that may contribute to the metastatic progression of breast cancer.

Estetrol (E4) is a natural estrogen produced exclusively by the human fetal liver during pregnancy. Its physiological activity remains unknown. In contrast to ethinyl estradiol and estradiol (E2), E4has a minimal impact on liver cell activity and could provide a better safety profile in contraception or hormone therapy. The aim of this study was to delineate if E4exhibits an activity profile distinct from that of E2on mammary gland. Compared with E2, E4acted as a low-affinity estrogen in both humanin vitroand murinein vivomodels. E4was 100 times less potent than E2to stimulate the proliferation of human breast epithelial (HBE) cells and murine mammary glandin vitroandin vivorespectively. This effect was prevented by fulvestrant and tamoxifen, supporting the notion that ERα (ESR1) is the main mediator of the estrogenic effect of E4on the breast. Interestingly, when E4was administered along with E2, it significantly antagonized the strong stimulatory effect of E2on HBE cell proliferation and on the growth of mammary ducts. This study characterizes for the first time the impact of E4on mammary gland. Our results highlight that E4is less potent than E2and exhibits antagonistic properties toward the proliferative effect of E2on breast epithelial cells. These data support E4as a potential new estrogen for clinical use with a reduced impact on breast proliferation.

International audience; The loss of E-cadherin expression and the translocation of beta-catenin to the nucleus are frequently associated with the metastatic conversion of epithelial cells. In the nucleus, beta-catenin binds to the TCF/LEF-1 (T-cell factor/ lymphoid enhancer factor) transcription factor family resulting in the activation of several genes, some of them having important implications in tumour progression. In our study, we investigated the potential regulation of monocyte chemotactic protein-1 (MCP-1/CCL2) expression by the beta-catenin/TCF pathway. This CC-chemokine has been implicated in tumour progression events such as angiogenesis or tumour associated macrophage (TAM) infiltration. We thus demonstrated that MCP-1 expression correlates with the reorganization of the E-cadherin/beta-catenin complexes. Indeed, MCP-1 was expressed by invasive breast cancer cells (MDA-MB-231, BT549 and Hs578T), which do not express E-cadherin but was not produced by noninvasive breast cancer cell lines (MCF7 and T47D) expressing high level of E-cadherin. In addition, the MCP-1 promoter was activated in BT549 breast cancer cells transfected with beta-catenin and TCF-4 cDNAs. The MCP-1 mRNA level was similarly upregulated. Moreover, we showed that MCP-1 mRNA was downregulated after transfection with a siRNA against beta-catenin in both BT549 and Hs578T cells. Our results therefore identify MCP-1 as a target of the beta-catenin/TCF/LEF pathway in breast tumour cells, a regulation which could play a key role in breast tumour progression.

International audience; Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17β-estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications that should now be considered further.

The cytoplasmic/nuclear relocalization of β-catenin and ZO-1 from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumor invasion. Data are now accumulating to demonstrate that these molecules, which shuttle between the plasma membrane and the nucleus or the cytosol, are involved in signaling pathways, and contribute to the regulation of genes such as vimentin or matrix metalloproteinase-14 which are turned on during EMT.

FREE FULL TEXT http://cancerres.aacrjournals.org/cgi/reprint/63/10/2658; International audience; The cytoplasmic and nuclear redistribution of beta-catenin and the de novo expression of vimentin are frequently involved in the epithelial-to-mesenchymal transition associated with increased invasive/migratory properties of epithelial cells. Because beta-catenin can act as a coactivator of transcription through its binding to the T-cell factor (TCF)/lymphoid enhancer factor 1 transcription factor family, we have explored the possibility that beta-catenin/TCF could directly transactivate vimentin. We first compared vimentin expression in relation with the localization of beta-catenin in eight breast cancer cell lines displaying various degrees of invasiveness and in a model of cell migration using human mammary MCF10A cells. We could thus show a cytoplasmic and/or nuclear distribution of beta-catenin in invasive/migratory cells expressing vimentin, but not in noninvasive/stationary vimentin-negative cell lines. In addition, the human vimentin promoter was found to be up-regulated by beta-catenin and TCF-4 cotransfection. Varying with the cellular background, a diminution of this up-regulation was observed when the putative beta-catenin/TCF binding site of the vimentin promoter was mutated. Our results therefore demonstrate that the vimentin promoter is a target of the beta-catenin/TCF pathway and strongly suggest an implication of this regulation in epithelial cell migration/invasion.

To determinethe prevalence of spontaneous endometriosis andthe incidence of induced endometriosis after endocervical canal resection in baboons.Induction and follow-up of endometriosis in baboons, which is one of the primate species that develop spontaneous endometriosis. Forty-one baboons were checked for the presence of spontaneous endometriosis. We then attempted to induce endometriosis in 30 of them by endocervical canal resection.Institute of Primate Research, Nairobi, Kenya, and Catholic University of Louvain, Brussels, Belgium.Forty-one baboons were checked for spontaneous endometriosis and 30 of them were used to develop a model of induced endometriosis.A total of 41 baboons underwent diagnostic laparoscopy for 10 months. In a first step, 30 of this number subsequently underwent endocervical canal resection. In a second step, 20 of the 30 underwent uterine horn resection.Follow-up by laparoscopy.Two of the 41 baboons were diagnosed with spontaneous endometriosis (4.8%). Twelve months after the surgical procedure to induce endometriosis, 8 of 29 animals presented with endometriotic lesions diagnosed by using laparoscopy and confirmed by histologic examination. The incidence of induced endometriosis in our model was thus 27.6%. In 2 baboons, endometriosis disappeared over time, resulting in a final rate of 20.7% (6/29).The rate of spontaneous endometriosis is very low (4.8%). Endometriosis can be induced (with a rate of just 27.6%) by endocervical canal resection to stimulate retrograde menstruation.

Plasminogen activator inhibitor-1 is known to play a paradoxical positive role in tumor angiogenesis, but its contribution to metastatic spread remains unclear. We studied the impact of plasminogen activator inhibitor (PAI)-1 deficiency in a transgenic mouse model of ocular tumors originating from retinal epithelial cells and leading to brain metastasis (TRP-1/SV40 Tag mice). PAI-1 deficiency did not affect primary tumor growth or vascularization, but was associated with a smaller number of brain metastases. Brain metastases were found to be differentially distributed between the two genotypes. PAI-1-deficient mice displayed mostly secondary foci expanding from local optic nerve infiltration, whereas wild-type animals displayed more disseminated nodules in the scissura and meningeal spaces. SuperArray GEarray analyses aimed at detecting molecules potentially compensating for PAI-1 deficiency demonstrated an increase in fibroblast growth factor-1 (FGF-1) gene expression in primary tumors, which was confirmed by reverse transcription-polymerase chain reaction and western blotting. Our data provide the first evidence of a key role for PAI-1 in a spontaneous model of metastasis and suggest that angiogenic factors, such as FGF-1, may be important for primary tumor growth and may compensate for the absence of PAI-1. They identify PAI-1 and FGF-1 as important targets for combined antitumor strategies.

Estetrol (E4) is a natural estrogen produced by the human fetal liver only during pregnancy. Its distinctive biological profile offers potential benefits in women's health, such as contraception and hormone replacement therapy. The crystal structures of the estrogen receptor α (ERα) ligand binding domain with 17beta-estradiol (E2) and E4 were very similar, whereas these two estrogens showed distinct positioning within phospholipid bilayers. Using in vivo approaches, we demonstrated that high doses of E4 stimulate genomic, ERα-dependent effects in the uterus leading to epithelial proliferation and prevent atheroma in hypercholesterolemic mice to a similar extent as E2. In contrast to E2, however, E4 failed to promote membrane-initiated ERα signaling such as acceleration of endothelial healing. Moreover, E4 antagonized this endothelial effect of E2. We conclude that E4 is a weak estrogen able to modulate the nuclear/transcriptional activity of ERα, and is not only devoid of membrane-initiated steroid signal, but also able to antagonize the membrane effects of E2, thereby delineating a distinctive profile of ERα activation.