PhD Thesis proposal form
Discipline
Biology
Doctoral School
"Signaling and Integrated Networks in Biology"
Thesis subject title: Preclinical evaluation of selective progesterone receptor modulators
(SPRMs) and antiprogestins in breast cancer : mechanisms of antiproliferative action
 Laboratory name and web site:
INSERM U693
Récepteurs Stéroïdiens
Physiopathologie Endocrinienne et Métabolique
Faculté de Médecine Paris 11-sud
63 rue Gabriel Péri
94276-Le Kremlin-Bicêtre cedex
FRANCE
tel 33 (0)1 49 59 67 02
fax 33 (0)1 49 59 67 32
http://www.unites.inserm.fr/site/u693
PhD supervisor (contact person):
Name: Hugues lOOSFELT
Position: Research director
email: hugues.loosfelt@u-psud.fr
Phone number:
33 (0)1 49 59 67 10
 Thesis proposal (max 1500 words):
See text below
 Publications of the laboratory in the field (max 5):
Chabbert-Buffet, N., Meduri, G, Bouchard, P, Spitz, I. (2005 ) Selective Progesterone Receptor Modulators
and Progesterone Antagonists : mechanism of action and clinical applications. Human Reprod Updates
11, 293-307.
Georgiakaki, M., Chabbert-Buffet, N., Dasen, B., Meduri, G., Wenk, S., Rajhi, L., Amazit, L., Chauchereau,
A., Burger, C.W., Blok, L.J., Milgrom, E., Lombes, M., Guiochon-Mantel, A. and Loosfelt, H. (2006)
Ligand-controlled interaction of histone acetyltransferase binding to ORC-1 (HBO1) with the Nterminal transactivating domain of progesterone receptor induces steroid receptor coactivator 1dependent coactivation of transcription. Mol Endocrinol 20, 2122-40.
Chabbert-Buffet, N., Pintiaux-Kairis, A. and Bouchard, P. (2007) Effects of the progesterone receptor
modulator VA2914 in a continuous low dose on the hypothalamic-pituitary-ovarian axis and
endometrium in normal women: a prospective, randomized, placebo-controlled trial. J Clin Endocrinol
Metab 92, 3582-9.
Khan, J.A., Amazit, L., Bellance, C., Guiochon-Mantel, A., Lombes, M. and Loosfelt, H. (2011) p38 and
p42/44 MAPKs differentially regulate progesterone receptor A and B isoform stabilization. Mol
Endocrinol 25, 1710-24.
Amazit, L., Roseau, A., Khan, J.A., Chauchereau, A., Tyagi, R.K., Loosfelt, H., Leclerc, P., Lombes, M. and
Guiochon-Mantel, A. (2011) Ligand-dependent degradation of SRC-1 is pivotal for progesterone
receptor transcriptional activity. Mol Endocrinol 25, 394-408.
 Specific requirements to apply, if any:
English spoken
Biologist, veterinary, pharmacian or medical doctor student
Preclinical evaluation of selective progesterone receptor modulators (SPRMs) and
antiprogestins in breast cancer : mechanisms of antiproliferative action
Introduction
Breast cancer, a hormone dependent disease, is the first cause of cancer related death in women.
Breast cancer treatment includes hormone therapy (tamoxifene or aromatase inhibitors) to prevent
relapses. These may be inactive (due to acquired tumoral resistance or to BRCA1/2 mutations), or
contraindicated (in patients with high vascular risk). It is now established that progesterone receptor
(PR) isoforms (PRA and PRB) as well as progestins play a critical role in hormone-dependent cancer
development and metastasis evolution, suggesting the potential efficacy of PR antagonists to
selectively inhibit PR-dependent pathways affected in cancers.
Selective PR modulators (SPRMs) are steroid compounds with potential mixed agonist/antagonist
activity depending on the tissue or cell type, the dose and the hormonal context [1]. They are
currently marketed for termination of pregnancy, emergency contraception, and complicated uterine
myomas. SPRMs and antiprogestins are also evaluated for breast cancer treatment and could have
specific applications in breast cancer prevention in women bearing a mutation of the BRCA1/2
genes. Additional studies are currently ongoing. However, these molecules are not PR selective and
exhibit partial agonist activity on transcription with deleterious side effects impeding their use for
long term therapy.
Our group, in collaboration with the department of drug development in University Paris Sud
Pharmacology Unit, has developed new antiprogestins with a specific and original mechanism of
action (passive antagonism). We have also established a new cell line model conditionally expressing
PRA , PRB or both isoforms to characterize PR targets. We also use a preclinical murine model of
human breast cancer tumor xenograft to characterize the antitumoral effects of PR antagonists.
We will evaluate the antitumoral effects of a marketed SPRM (CDB/VA 2914 or Ulipristal
Acetate, UPA) as well as those of a new passive antagonist of PR (APR 19) developed in our group.
Finally the effect of UPA in normal breast will be evaluated in high breast cancer risk women. More
specifically the roles of PRA and PRB isoforms as well as interactions with their coregulators will be
evaluated. Antiproliferative and proapoptotic effects, impact on cell cycle progression and migration
capacity of tumor cells will be evaluated as well as the signaling transduction pathways involved.
Our group (Inserm U693) has acquired a large expertise on steroid hormone action notably both
on PR mechanisms of action [2-15](Dr H Losfelt PharmD, PhD ) as well as SPRMs evaluation [1619] and sex steroid effects in hormone dependent cancers [20-23](Dr N Chabbert-Buffet MD, PhD).
In this project the doctoral student will be under the supervision of Dr Loosfelt and Dr ChabbertBuffet.
Background
The mechanisms of action of PR ligands are still incompletely elucidated [24-26] and includes
genomic effects through nuclear progesterone receptors, interacting with a coregulating system of
proteins which modulates target genes transcription [27]. The progesterone receptor exists as two
major isoforms (PRA and PRB) with specific bioactivity and target genes [28-30]. PR isoforms have
differential effects in the control of breast development and differentiation [31,32]. Transcriptional
modulation and gene selectivity of the ligand-bound PR are highly dependent on PRA/PRB ratio
[33,34], [35-37] as well as ligand-induced PR transconformation, specifically modeling an interface
for co-regulator interactions [24,25]. PR isoform specificity for coregulators has been also
established [4]. Functional consequences on cell cycle, apoptosis, proliferation and invasiveness have
been largely evaluated, notably in particular in breast cancer cell lines [38-41]. Animal models have
shown the antiproliferative activity of non selective SPRMs in breast tumors, with additional
synergic effects with tamoxifen [42,43]. Finally a preventive effect of the SPRM mifepristone
(RU486) has been shown in a BRCA1 knock out model [44]. Breast cancer treatment with RU486
and onapristone (ZK 98299) has shown benefit [45,46], and new SPRMs such as ZK 211231 are
currently evaluated.
In the normal human breast, RU486 decreases cell proliferation [47]. Data from animal models
have opened new perspectives suggesting that BRCA proteins are involved in PR recycling [48] and
PRA/PRB ratio regulation. PRA is overexpressed in the normal tissue surrounding breast cancer of
women with BRCA1/2 mutations and in poor prognosis breast cancers [49,50].
PRELIMINARY RESULTS
1. A new mammary cancer cell line conditionally expressing PR isoforms
The new cell line MDA-iPRAB is a bi-inducible cell line expressing PRA and/or PRB generated
from the MDA-MB-231 mammary cancer cell line. The latter originated from a metastatic pleural
effusion sample and expresses neither the estrogen nor the progesterone receptor. It has a high
metastatic potential in murine models. The major challenge of this cell line relies on the possibility to
control PRA/PRB expression ratio in a dose-dependent manner using non-steroid inducer compounds
in vitro as well as in vivo. Using this model, we have already identified the genes differentially
regulated by unliganged and liganged PRA and PRB expression, as well as progesterone, by
microarray studies. We have also shown that PRB, but not PRA, is able to highly enhance cell
motility via non-genomic mechanisms involving focal adhesion kinase. Therefore this model is
actually a unique tool for studying the differential impacts of PR isoforms on tumor progression and
invasiveness as well as metastatic dissemination (Kahn et al, 2012, submitted). It might be also
critical to select PR antagonists exhibiting isoform specificity.
2. A new class of PR-selective antagonists
Based on comparative crystallographic studies of PR and other steroid receptors, we have
developed a new class of PR antagonists (APRn) exhibiting original properties as compared to
RU486-derived molecules. APRn are highly selective for PR binding, and do not induce the
conformations required for PR interactions with coregulators (Kahn et al PNAS 2012 in revision).
The homosteroid APR19 inhibits the anti-oestrogenic effect of progesterone on endometrial
proliferation in mice and has been extensively tested in vitro since it is devoid of any partial agonistic
activity, fully represses endogenous PR-responsive transcription and does not recognize other steroid
receptors .
3. Characterization of antiproliferative effects of APRn and VA 2914/UPA in a mouse
xenograft model of human breast tumors.
We are currently evaluating the HBCx-34 mouse model, which consists in xenografts of human
breast carcinoma in immunodeficient nude mice, developped by Xentech (Evry Genopole, France).
This tumor has been isolated from an invasive ductal carcinoma expressing PR (PRA and PRB
isoforms) and ER, and is devoid of p53 and BRCA1/2 gene mutations. Mice are treated by either the
APRn (30 mg/Kg) to be tested, the highly tumorigenic progestin medroxyprogesterone acetate
(MPA, 3 mg/kg), VA2914/UPA (at different doses) or VA2914/UPA plus progesterone for 42 days.
Results from these experiments are expected in April 2012.
D. PROJECT
a. Studies to be conducted in the MDA-iPRAB cell line
Biological activity of VA2914 and several APRn will be evaluated in our original cell line MDAiPRAB and after induction of co-expression of both PRA and PRB isoforms in variable ratios
following administration of exogenous inductors. We will evaluate signaling biomarkers (p42/44,
p38, JNK MAPKs and PI3k/Akt, STAT5, cyclinD1, Wnts, GSK3, uPA) as well as metastatic
process markers (uPA, FAK, E-Cadherin, integrins…) expression using a High Throughput
Microscopy, Scanning Image Cytometry [51].
The wound healing assay developped by Matsuda [52] will be used to measure the effect of the
studied molecules on progesterone dependent cell migration. Chemotaxis and haptotaxis studies will
be conducted as well using Boyden chambers. Evaluation of cell proliferation will use the
colorimetric MTS tetrazolium method [53]. Regulation of apoptosis will be evaluated by caspase
assays.
We will also evaluate the expression of various tumorigenesis and angiogenesis markers (TGFb,
EGFR, VEGF, Bcl2, Ki-67, PCNA, MCM2, IL-2, MMPs…) as well as the expression of different PR
target genes (Wnts, cyclin D1, amphiregulin, integrins, caspases…), in different conditions. These
will include progestin treatment, progestin + VA 2914/UPA and progestin + APRn. Real Time
quantitative RT-PCR, Western blot using the Odyssey equipment and immunohistochemistry will be
performed. Protein protein interactions of PR with co-regulators in the different conditions will be
evaluated using fluorescence resonance energy transfer technologies (FRET).
b. Studies to be conducted in the preclinical human tumor graft murine model
Tumors obtained from the HBCx 34 model after treatment as previously described will be studied.
Histological and immunohistochemichal evaluation of tumor growth, as well as expression of
different cancer biomakers (Ki-67, PCNA, MCM2…) or PR target genes (cyclin D1, RANKL,
VEGF, amphiregulin, TGF, IL-2, Wnts, DKK1) will be conducted.
c.Clinical sudy in women with genetic high breast cancer risk.
This study is currently being set up in collaboration with the HRA Pharma laboratory. This
prospective study, after validation by the local ethic committee, will enroll patients with BRCA1 or
BRCA2 mutations scheduled for prophylactic mastectomy. Patients will receive VA 2914/UPA 30
mg/day for one month preoperatively. Proliferation and apoptosis markers will be evaluated by RTPCR and immunohistochemistry. PR isoforms expression will be evaluated by western blot.
E. PERSPECTIVES
This project should enhance our knowledge on PR mediated regulation of breast proliferation and
its molecular mechanism. It will contribute to the design of new therapeutic and preventive strategies
for breast cancer.
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