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Endocrine-Related Cancer (2003) 10 261–266

International Congress on Hormonal Steroids and and Cancer How to target receptor-negative ?

H Rochefort, M Glondu, M E Sahla, N Platet and M Garcia

Molecular and Cellular Endocrinology of Cancer, INSERM Unit 540 and Montpelier University, 60 rue de Navacelles, 34090 Montpelier, France (Requests for offprints should be addressed to H Rochefort; Email: [email protected]).

Abstract Estrogen receptor (ER)-positive breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy, which is the first example of a successful therapy targeted on a specific , the ER. Unfortunately ER-negative breast cancers are more aggressive and unresponsive to anti-. Other targeted therapies are thus urgently needed, based on breast cancer oncogene inhibition or suppressor activation as far as molecular studies have demonstrated the alteration of expression, or structure of these in human breast cancer. Using the MDA-MB.231 human breast cancer cell line as a model of ER-negative breast cancers, we are investigating two of these approaches in our laboratory. Our first approach was to transfect the ER or various ER-deleted variants into an ER-negative cell line in an attempt to recover anti-estrogen responsiveness. The unliganded receptor, and surprisingly estradiol, were both found to inhibit tumor growth and invasiveness in vitro and in vivo. The mechanisms of these inhibitions in ER-negative cancer cells are being studied, in an attempt to target the ER sequence responsible for such inhibition in these cancer cells. Another strategy is trying to inhibit the activity or expression of an oncogene specifically overexpressed in most breast cancers. This approach was recently shown by others to be efficient in breast cancer therapy with HER2-Neu oncogene amplification using Herceptin. Without excluding other molecular putative targets, we have focused our research on D as a potential target, since it is often overexpressed in aggressive human breast cancers, including ER-negative tumors, and rarely associated with HER2-Neu amplification. Our first results obtained in vitro on cell lines and in vivo in tumor xenografts in nude mice, illustrate that the mode of action of cathepsin D in breast cancer is useful to guide the development of these therapies. In the past 20 years we have learned that the action of cathepsin D is complex and involves both intracellular and extracellular activities due to its proteolytic activity and to interactions with membrane components without catalytic activity. Each of these mechanisms could be potentially inhibited in an attempt to prevent tumor growth. Breast cancer is a very heterogeneous and multigenic disease and different targeted therapies adapted to each category of breast cancer are therefore required. Validated assays in the primary tumor of molecular markers such as ER, HER2-Neu and cathepsin D should help to predict which targeted therapy should be applied to cure breast cancer patients. Endocrine-Related Cancer (2003) 10 261–266

Introduction hampered by toxicity or resistance problems. These targeted therapies should allow action with more efficacy and less The mechanism of breast cancer progression and toxicity, but only on the relevant breast cancer subset as is not fully understood. However, breast cancer frequency determined by assaying the corresponding predictive marker tends to increase. Moreover the ability of these cancers to in the tumor (Gibbs 2000). metastasize and their heterogeneity, clearly require new ther- There are at least three key requirements for developing apies, targeted on specific genes and actively a new molecular target in cancer treatments: (i) the avail- engaged in the physiopathology of breast cancer rather than ability of clinical studies relating gene overexpression in the the conventional cytotoxic chemotherapy, which is often tumor and prognostic value in clinical follow-up studies so

Endocrine-Related Cancer (2003) 10 261–266 Online version via http://www.endocrinology.org 1351-0088/03/010–261  2003 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 09:06:54AM via free access Rochefort et al.: Targeting ER-negative breast cancer as to determine which patients would benefit from this ther- breast cancer cell line in an attempt to recover normal estro- apy, and to tailor treatments adapted to each breast cancer gen and anti-estrogen responsiveness. The unliganded ERα subset in patients; (ii) a full understanding of the mode of was found to inhibit the invasiveness and growth of two action of the molecular target involved in breast cancer car- stably transfected MDA-MB 231 cell lines as compared with cinogenesis or progression; (iii) a means to specifically target cell lines transfected with the vector alone. This was consist- the drug in the tumor rather than in normal tissues, thus low- ent with the lower invasiveness of ER-positive breast cancer ering toxicity and increasing selectivity as compared with cells (Thompson et al. 1992). However, and surprisingly, traditional cytotoxic chemotherapy. estradiol inhibited cell growth and invasiveness to a greater Anti-estrogen therapy is the first successful cancer ther- extent, both in vitro and in vivo (Garcia et al. 1992), while apy targeting estrogen receptor (ER) expression (Rochefort tamoxifen had no effect and the pure anti-estrogen ICI- 1987, Jordan & Murphy 1990). However, its efficacy, as well 16438480 abolished the favourable effect of transfected ERα as that of aromatase inhibitors, is limited to ER-positive (Rochefort et al. 1998). In order to study the mechanism of breast cancers, which generally have a better prognosis these inhibitions and particularly to define the domain and (McGuire 1978, Osborne 1998). Unfortunately ER-negative sequence in ERα responsible for inhibition, Platet and Garcia breast cancers are more aggressive (Sheikh et al. 1994) in our laboratory developed a method for transient co- through a still unknown mechanism. Therefore other targeted transfection of ER or variants, with the luciferase expression therapies are urgently needed in these cancers. ER-negative vector, which enables monitoring of only the transfected cells invasive breast cancers are often considered to be the result without having to select stable transfectants (Platet & Garcia of tumor progression from ER-positive premalignant lesions 1999). The first zinc finger in the C domain of ER was found or ER-positive breast cancers by genetic alteration (gene to be responsible for this inhibition. The effect was specific instability, loss of heterozygosity LOH), exon deletion etc.) to ERα and ERβ, while transfected glucocorticoid receptors epigenetic alteration such as promoter methylation (Ferguson glucocorticoid receptors (GR), androgen receptors (AR), vit- et al. 1995) or ER protein degradation in proteasome after amin D receptors (VDR), retinoic acid receptors (RARα) hypoxia in non-vascularized tumor (Stoner et al. 2002). were inefficient (Platet et al. 2000). These studies suggest However, some invasive, human breast cancers may be that ERα is protective, which is in agreement with the better directly ER-negative via a -independent pathway. prognosis of ER-positive breast cancers. The protective effect This pathway is based on the results of gene knock out of ERβ might be even higher, since markedly decreased in studies in mice (Korach 1994, Hewitt et al. 2002) and on earlier steps of carcinogenesis (Roger et al. 2001) and immunohistochemical studies in humans, showing that some involved in the terminal differentiation of mammary glands proliferative ductal lesions and many high-grade ductal carci- (Forster et al. 2002). Estrogens and anti-estrogens unfortu- noma in situ (DCIS) are ERα-negative (Roger et al. 2000). nately had an opposite effect on growth in naturally ER- Targeted therapy of ER-negative hormone-independent positive breast cancer cells like MCF7 and in ER-negative breast cancers could be a priori based on two general breast cancer cells transfected with ERα like MDA-MB231 approaches: (i) to transform ER-negative into ER-positive cells. However, the transcriptional activity of reporter genes cancer cells by gene therapy or ER gene reexpression; (ii) to was stimulated by estrogen in both types of cancer and some target genes or proteins actively involved in the genesis or endogenous estrogen-regulated genes were also stimulated progression of the corresponding breast cancer subset. by estradiol after ER transfection (Touitou et al. 1990, Jeng et al. 1994). Understanding the difference in the two cell To transform ER-negative into types (ER-negative or ER-positive) responsible for the ER-positive breast cancer cells inverse effect of activated ER may provide a clue for developing new targeted therapy aimed at replacing ER in In our laboratory, we used the MDA-MB 231 breast cancer order to inhibit the factor involved in the stimulation of pro- cell line as a model of ER-negative breast cancers. This liferation and invasion in ER-negative breast cancers. More human cell line is particularly suitable for pre-clinical studies studies are required to pinpoint this factor, show its clinical since it is highly aggressive both in vitro and in vivo (Price significance in breast cancer patients and find a specific way et al. 1990), it does not overexpress HER2-Neu, but it does to target the inhibitory sequence of ER in breast cancer tissue overexpress and secrete cathepsin D (cath-D), a lysosomal in vivo. extensively studied both in our laboratory An interesting alternative is to reexpress the ER gene by (Rochefort et al. 1987, 1989) and in several independent demethylating its promoter or by using histone desacetylase clinical studies for its prognostic value (for reviews see inhibitors (Yang et al. 2001). Rochefort 1992, 1996, Rochefort et al. 2002). In both cases, however, an additional strategy will be Our first approach was very naively to transfect the ERα required to target the drug specifically in mammary cancer recombinant cDNA into the MDA-MB-231 ER-negative cells.

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To target a protease activelyengaged in metastasis was also increased. More recent studies showed that the pathogenicityof breast cancer cath-D antisense RNA, stably transfected in vitro into MDA- MB231 cancer cells, inhibited their proliferation, both in vitro There are a large number of possibilities to inhibit ER- (in a 3D matrigel system) and in vivo, when these cells are negative breast cancers since the nature of genes responsible injected s.c. or i.v. in nude mice. The size and number of exper- for the growth and spread of these tumors are not fully imental lung metastases were also increased. The in vitro defined and vary according to the tumor subset (Ingvarsson invasion conversely was not significantly decreased (Glondu 1999). For instance, overexpression and gene amplification et al. 2002). Therefore cath-D expressed in the human MDA- of HER2-Neu and epidermal receptor have led MB-231 breast cancer cell line is one of the rate-limiting fac- to the development of new therapies targeting these proteins tors required for the growth of micrometastases at distant sites. (Baselga et al. 1996, Pegram & Slamon 1999). These targets One can argue that similar results were obtained in similar were initially proposed on the basis of clinical studies indi- experiments with several growth factors and , indi- cating their bad prognostic significance in breast cancer. cating that a large number of proteins are needed to promote For 20 years our laboratory has been involved in study- tumor growth (Powis & Kozikowski 1991). However, since a ing another prognostic marker, cath-D, (for reviews see proteolytic cascade is necessary for a tumor to spread and grow Rochefort et al. 2000, 2002) and in developing an alternative at a remote site, the inhibition of only one actor of the cascade approach aimed at inhibiting cath-D expression or function may be sufficient to blockthe activation of other proteases. In in order to blockthe growth of breast cancer overexpressing this respect, cath-D has the potential to initiate a proteolytic this protease cascade in vitro (Rochefort et al. 2000). However, the key We will first present evidence that this lysosomal pro- argument is that very few potential breast cancer molecular tar- tease is a good potential therapeutic target and then review gets have been clinically studied as extensively as cath-D for the different approaches adopted in an attempt to inhibit its prognostic value when specifically overexpressed in breast breast cancer growth and progression based on the dual mode cancer (Ferrandina et al. 1997, Foekens et al. 1999). of action of cath-D in cancer.

Cath-D is a good potential target for Different approaches to inhibit cath-D ER-negative breast cancers production or action in breast cancer based on its mode of action Even though cath-D was initially shown to be induced by Cath-D can potentially act as a protease to degrade or acti- estrogen in ER-positive breast cancer cell lines, it is also vate substrates intracellularly and extracellularly. As an constitutively overexpressed in ER-negative tumors as shown extracellular protease, cath-D may liberate growth and angi- in both cell lines (Rochefort et al. 1989) and patients. Several ogenic factors entrapped in the extracellular matrix and con- clinical studies have shown an absence of correlation sequently stimulate angiogenesis as shown with fibroblast between ERα and cath-D level in invasive breast cancers growth factor-2 (Briozzo et al. 1991). This activity, however, (Rochefort 1992, Foekens et al. 1999) and DCIS (Roger et requires an acidic pH. It has also the potential, mostly after al. 2000). Cath-D overexpression was not correlated with secretion of the proenzyme to bind to membrane receptors at HER2-Neu amplification (Brouillet et al. 1990), another neutral pH through autocrine or paracrine mechanisms and target for ER-negative breast cancer. to act as a ligand to trigger mitogenic signals (for review When using a validated cytosolic assay, the specific see Rochefort & Liaudet-Coopman, 1999). A priori, one can overexpression of cath-D in ER-negative breast cancer therefore inhibit cath-D production or cath-D action as a pro- patients based on several independent clinical studies, is fre- tease or as a ligand. quently associated with shorter relapse-free survival and overall survival (for review see Rochefort et al. 2002). A relatively large fraction (about 60%) of ER-negative breast Cath-D can be inhibited via its cancers overexpressing cath-D might therefore benefit from gene regulation therapy targeted to cath-D. In ER-positive breast cancer the efficacy of anti-estrogens This protease is not only associated with breast cancer pro- and aromatase inhibitors may be partly, but not exclusively gression, but appears to be also a rate-limiting factor in stimul- due to this mechanism, since tamoxifen is a partial estrogen ating in vitro and in vivo tumor growth. Initial studies showed agonist for cath-D regulation and other mitogens are also that specific overexpression by stable transfection of human involved. In ER-negative breast cancer, the origin of cath-D pro-cath-D stimulated growth of a rat tumoral cell line derived over expression is unknown and might involve different from embryo fibroblasts transfected with an adenovirus, both mechanisms, including other enhancers stimulated by other in vitro and in vivo (Garcia et al. 1990). Experimental liver transcription factors (Giamarchi et al. 1999).

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Cath-D mRNA can be neutralized with antisense One characteristic common to all lysosomal proteases RNA expressed in cancer cells (cath-D, -B, -L) is that they are Antisense RNA, added in vitro to cancer cells, inhibits their secreted as proteolytically inactive pro-forms. They are then growth both in vitro and in vivo as described in Glondu et able to be re-endocytosed by tumoral cells (epithelial cancer al. (2002), but when added in vivo, it should be associated cells, stromal cells . . . etc.) by interacting with membrane with an additional means of targeting. In order to reduce tox- receptor(s). In fibroblasts the receptors to endocytose pro- icity, cath-D inhibitors should act more specifically in breast cath-D and other lysosomal are, as expected, the cancer cells than in normal tissue. Some normal tissues also Man6P receptors via the high mannose glycosylated chain(s) express cath-D such as liver, spleen etc. Liver toxicity was of the . However, in breast cancer cells, the lyso- not observed in the young gene knock out mice (cath-D−/−) somal routing and endocytosis of pro-cath-D are Man6P- which die, however, after weaning with intestinal necrosis independent (Capony et al. 1994, Laurent-Matha et al. 1998). and thymocytes apoptosis (Saftig et al. 1995). An additional We have successively excluded the Man6P/iinsulin-like targeting strategy is based on the fact that in solid tumors growth factor-II receptor and the low density lipoprotein such as breast cancer, pro-cath-D is secreted in an acidic (LDL) receptor related protein (Laurent-Matha et al. 2002) micro environment (pH down to 6.2) due to hypoxia but have not yet identified this interacting molecule. There (Gerweck& Seetharaman 1996) and that both macrophages are different potential means of interfering with this process, and ER-negative breast cancer cell lines such as MDA-MB- e.g. inhibiting secretion by neutralizing antibodies as 231 are able to acidify extracellular medium more strongly described for cath-L in (Guillaume-Rousselet et than an ER-positive breast cancer cell line such as MCF7 al. 2002), or inhibiting interaction with a Man6P-independent cells (Montcourrier et al. 1997). membrane receptor. Others have proposed that a receptor which binds part of Aspartyl protease inhibitors the cath-D pro-fragment is responsible and that neutralizing antibodies could inhibit its binding as well as tumor growth Other approaches are based on the dual mode of action of (Fusek& Vetvicka1994).This is to be confirmed by other this protease in cancer cells. It initially seemed obvious that laboratories. We propose that according to its site of cath-D, like most of proteases in cancer, acts intracellularly secretion, the cancer cell density, and the environmental or extracellularly through its proteolytic activity. This is extracellular pH of the tumor, cath-D could act in vivo both strongly suggested by the fact that the addition by directed as a ligand and as a protease. In this case the association of mutagenesis of a KDEL reticulum endothelial retention different types of inhibitors would be more efficient. signal at the C-terminal end of the protein, totally inhibited both the maturation of 52 kDa pro-cath-D into mature chains Conclusion (34 kDa + 14 kDa) and the stimulatory effect of cath-D over- expression on cell growth (Liaudet et al. 1994). Moreover, To conclude, the targeting of breast cancer on the ER has at high cell density, tumor cells secrete a growth inhibitor proven its efficacy and low toxicity with anti-estrogens and which is degraded or inactivated intracellularly in acidic aromatase inhibitors. Systemic treatment of ER-negative compartments by cath-D overexpression and maturation breast cancers requires other molecular targets, based on (Liaudet et al. 1995). Attempts have thus been made to use alteration or over expression of a breast cancer oncogene or aspartyl protease inhibitors such as pepstatin, but currently reexpression of a breast cancer suppressor gene. We have without great success. This failure might be due to solubility reviewed our attempts to transfect ER cDNA into ER- or pH problems or to the fact that cath-D may not be acting negative cell lines and to use cath-D as a target to inhibit the as a protease but rather as a ligand by interacting with a growth and progression of ER-negative breast cancer subset membrane receptor to trigger a mitogenic transduction path- overexpressing specifically this protease. In addition to be way for instance. potentially active on ER-negative breast cancers, cath-D inhibitors have also the potential to treat the anti-estrogen Inhibition of cath-D acting asa ligand resistant ER-positive breast cancers which overexpress cath-D. A better understanding of the mechanisms by which In order to determine whether cath-D proteolytic activity is cath-D overexpression stimulates tumor growth and pro- necessary to stimulate cancer cells growth, we mutated its gression and the development of means to inhibit these catalytic site by substituting the aspartic acid residue 231 of activities should in the near future, help in tailoring a treat- the 34 kDa chain with an asparagine residue. This ment adapted to each breast cancer subset totally abolished cath-D proteolytic activity, but affected neither its expression level, processing nor secretion, nor its Acknowledgements capacity to stimulate proliferation of cells embedded in Matrigel, colony formation in soft agar nor tumor growth in This review and the corresponding papers of our laboratory athymic nude mice (Glondu et al. 2001). have been supported by INSERM and research grants to H R

264 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 09:06:54AM via free access Endocrine-Related Cancer (2003) 10 261–266 from the Association pour la Recherche sur le Cancer (ARC hormone-dependent and -independent breast cancer cell lines. no. 5757) and Servier Laboratories. M Glondu and N P were Oncogene 18 533–541. also recipients of the Ligue Nationale contre le Cancer. Gibbs JB 2000 Mechanism-based target identification and drug discovery in cancer research. Science 287 1969–1973. Glondu M, Coopman P, Laurent-Matha V, Garcia M, Rochefort References H & Liaudet-Coopman E 2001 A mutated cathepsin D devoid of its catalytic activity stimulates the growth of cancer cells. Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Oncogene 20 6920–6929. 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