WO2000066767A1 - Estrogen signalling pathway regulators and uses thereof - Google Patents

Abstract

The present invention relates to estrogen receptor signalling and the regulation of estrogen receptor signalling for the treatment of cancer and proliferative disease. The invention further relates to the treatment of cancers associated with mutations in BRCA1 gene. Methods of identifying 'estrogen receptor signalling pathway regulators' are also part of the invention. Therapeutic compositions and methods of screening, diagnosis and therapy are also part of the invention. Methods of the invention can be used to inhibit cancer cell growth, especially in cancers of the breast, ovaries and prostate.

Description

ESTROGEN SIGNALLING PATHWAY REGULATORS AND USES

THEREOF

This application claims priority under 35 U.S.C. §119(e) to U.S. Application 5 No. 60/131,841, filed April 30, 1999, the contents of which are incoφorated herein by reference in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to estrogen receptor signalling and regulation of 10 estrogen receptor signalling for the treatment of cancer and proliferative disease. The invention relates to molecules and/or compounds that regulate the estrogen signalling pathway. More specifically, the invention relates to the treatment of cancers and diseases associated with alterations in hormone or hormone responsiveness. In a preferred embodiment, the invention relates to the treatment of breast cancer or cancers associated 15 with mutations in the BRCAl gene. The present invention relates, in part, to the discovery that BRCAl inhibits signalling by the ligand-activated estrogen receptor (ER-α) through the estrogen-responsive enhancer element (ERE) and blocks the transcriptional activation function of ER-α. The invention relates to compositions and methods for treating cancer and proliferative disease, such as breast cancer. The invention further relates to regulation 20 of the estrogen receptor signalling pathway with an "estrogen receptor signalling pathway regulator", or "ESP-regulator".

2. BACKGROUND OF THE INVENTION 2.1 BRCAl

25 BRCAl was the first breast cancer susceptibility gene to be identified and cloned. In individuals from high-risk families, mutations in BRCAl increase the lifetime risk of developing breast cancer eight to tenfold, compared to the general population. How the BRCAl protein product normally functions to suppress tumor formation and how defects in the gene can ultimately lead to breast cancer have been the focus of intense

30 scrutiny by the scientific and medical communities. BRCAl has intrinsic transactivation activity and is able to activate the p21 promoter. In addition, BRCAl is linked to a number of genes involved in transcriptional regulation, including CtlP. c-Myc, the RNA holoenzyme complex, and the histone deacetylase complex. Moreover, BRCAl is essential for cellular response to DNA damage repair. Inactivation of BRCAl in mouse embryonic

-^ stem and fibroblast cells results in increased cell sensitivity to DNA-damaging agents. Germ-line mutations of the BRCAl gene located on chromosome 17q21 (Miki et al., 1994, Science 266:66) account for 40-50% of hereditary breast cancers and confer increased risk for ovarian and prostatic cancers (Ford et al, 1994, Lancet 343:692; Gayther et al., 1995, Nat. Genet. 11 : 428; Streuwing et al., 1997. New Engl. J. Med. 336: 1401). The BRCAl gene encodes an 1863 amino acid (aa) protein with a highly conserved N-terminal RING finger domain and a C-terminal acidic transcriptional activation domain (Miki et al.,1994, Science 266:66; Monteiro et al., 1996. Proc. Natl. Acad. Sci. U.S.A. 93: 13595; Chapman and Verma, 1996, Nfltwre 382:678). The BRCAl gene product is a 220 kD nuclear phosphoprotein (Chen et al., 1986, Cancer Res. 56:3168) that has been implicated in regulation of cell proliferation (Hakem et al., 1996, Cell 85:1009; Holt et al.,

1996, Nat. Genet. 12:298), cell cycle progression (Chen et al.. 1996, Cancer Res. 56:3168; Somasundaram et al., 1997, Nature 389:187; Larson et al., 1997. Cancer Res. 57:3351), apoptosis induction (Shao et al., 1996, Oncogene 13:1 ; Fan et al., 1998, Oncogene 16:3069) 5 and DΝA repair and recombination (Fan et al., 1998, Oncogene 16:3069; Scully et al.,

1997, Cell 90:426; Husain et al., 1998, Cancer Res. 58: 1120; Gowen, 1998, Science 281 :1009). These functions of BRCAl are observed in various human epithelial cancer cell types and mouse fibroblasts and do not explain the association of BRCAl mutations with specific tumor types, such as breast, ovarian and prostate cancer. 0

2.2 ESTROGEN RECEPTOR

Nuclear hormone receptors are a family of hormone-activated transcription factors that can initiate or enhance the transcription of genes containing specific hormone response elements. The two closely related human estrogen receptors (ER-α and ER-β). 5 which belong to this family, were cloned and sequenced from MCF-7 human breast cancer cells (Green et al., 1986. Nature 320:134-139; and reviewed in Perlmann and Evans. 1997.

Cell 90:391). The ER-α protein, the major estrogen receptor of mammary epithelia, consists of 595 amino acids with a molecular weight of 66 kD (Green et al.. 1986) that has been separated into six different functional domains (Kumar et al.. 1987. Cell 51 :941-951 ; Kumar et al.. 1986, EMBO J. 5:9931-9936). Two of these functional domains are highly conserved in the primary sequence of members of the nuclear hormone receptor superfamily. One of the domains, the DNA binding domain, contains two zinc fingers that mediate receptor binding to hormone response elements in the promoters of hormone-responsive genes. In ,r the C-terminal region, the hormone binding domain contains two regions of sequence homology with other hormone receptors and bestows hormone specificity and selectivity (Carson- Jurica et al., 1990, Endocr. Rev. 11 :201-220; Kumar and Chambon, 1988, Cell 55:145-156; Orti et al., 1992, Endocr. Rev. 13:105-128). Estrogen responses are mediated by two closely related receptors, ER-α and ER-β (reviewed in Perlmann and Evans. 1997, Cell 90:391).

Estrogen diffuses through the plasma membrane of cells where it binds to the ER (Rao et al., 1981, Mol. Cell. Endocrinol. 21 :97-108). For many years, it generally was thought that estrogen bound to the ER in the cytoplasm and translocated into the nucleus, but it is known now that the ER is a nuclear transcription factor that initially interacts with estrogen in the nucleus (Welshons et al., 1984, Nature 307:747-749). Once estrogen binds to the ER, heat shock proteins dissociate and a change in conformation and homodimerization occurs.

Once the ER has bound estrogen and dimerized, it binds to estrogen response enhancers (EREs) present in the promoter region of genes. These EREs are 13 base pair palindromic sequences located upstream from the transcriptional start site. The EREs function by enhancing the transcriptional potential of a gene. EREs have been identified and defined using reporter systems to test the enhancer ability when exposed to different compounds (Gronemeyer, \99\, Annu. Rev. Genet. 25: 89-123). Also, deletional analysis has allowed the definition of the sequence of EREs. The exact sequence of EREs varies between species and genes (Klein-Hitpass et al., 1986, Cell 46:1053-1061).

ER-dependent transcription can be enhanced by coactivators. The first candidate for a transcriptional coactivator, SPT6, was isolated from Saccharomyces cerevisiae and was shown to be capable of modulating ER-mediated transcription in yeast and mammalian cells and to interact specifically with the carboxyl-terminal portion of the -. - ER (Baniahmad et al.. 1995, Mol. Endocrinol. 9:34-43). A member of the estrogen receptor coactivating complex is p300. This co-factor, acting in concert with ligand binding, is responsible for the conformational change required of the ER-α activation. The amino-and carboxy-terminal transactivation domains of the ER (AF-1 and AF-2) can now interact and cause further recruitment of coactivator proteins. These coactivators link ER-α to the basal 30 transcriptional machinery and convert chromatin to the active state via histone acetylation.

Another co-activator is the CREB-binding protein (CBP). This protein can interact specifically with members of the steroid hormone nuclear receptor family and is able to enhance transcriptional activity (Kamei et al., 1996. Cell 85:403-414). CBP has been shown to also interact with RNA polymerase II (Kee et al., 1996, J. Biol. Chem. 271 : 35 2373-2375), TFIIB (Kwok et al., 1994, Nature 370: 223-226), and with cAMP response element-binding protein (CREB) in its phosphorylated form (Chrivia et al., 1993. Nature 365:855-859). It has been postulated that the ability of CBP to stimulate transcription is through the targeted recruitment of RNA polymerase II to the promoters of genes.

3. SUMMARY OF THE INVENTION

The present invention relates to estrogen receptor signalling and the inhibition or regulation of estrogen receptor signalling for the treatment of cancer and proliferative disease. More specifically, the invention relates to the treatment of breast cancer or diseases associated with alterations in hormone or hormone responsiveness. The present invention relates, in part, to the suφrising discovery that BRCAl inhibits signalling by the ligand-activated estrogen receptor (ER-α) through the estrogen-responsive enhancer element (ERE) and blocks the transcriptional activation function of ER-α. The invention relates to compositions and methods for treating cancer and proliferative disease, such as breast cancer. The invention further relates to regulation of the estrogen signalling pathway as a method of treating and/or preventing breast cancer.

Mutations of the breast cancer susceptibility gene BRCAl are linked to development of breast, ovarian, and prostatic cancers; but it is not clear why BRCAl mutations lead to these particular tumor types. The present invention relates to the discovery that BRCAl modulates signalling by the ligand-activated estrogen receptor (ER- α). Inhibition of ER-α activity is demonstrated herein to require at least three structural domains of BRCAl including the N-terminal RING finger, a consensus retinoblastoma (RB) binding motif (³⁵⁸LXCXE), and a third domain at the C-terminus. The discoveries of the present invention suggest that the transcriptional adaptor p300. which is a component of the ER-α coactivator complex, may be a target for repression of ER-α by BRCAl . The present invention relates to the discovery that the loss of the ability to regulate ER-α contributes to the incidence of breast cancer in BRCAl mutation carriers. Thus, the methods and compositions of the present invention are useful for the treatment or amelioration of breast cancer or precancerous conditions by inhibition or regulation of the estrogen pathway in hosts in need of such treatment. In one embodiment, a host in need of treatment or amelioration of breast, ovarian or prostate cancer or precancerous condition carries a BRCAl mutation. In other embodiments, a host is screened for a BRCAl mutation prior to treatment with an estrogen signalling pathway regulator (ESP-regulator) of the present invention.

The present invention further provides for regulating the cellular proliferation of a cancer cell, dysplastic cell, tumorigenic cell, malignant cell, or precancerous cell. The present invention also encompasses the use of combinations of two or more estrogen signalling pathway regulators.

At the molecular genetic level the coding sequence for an ESP-regulator may be placed under the control of one or more of the following genetic elements: a naturally occurring strong, intermediate or weak constitutively expressed or regulated promoter from the targeted tissue, or an artificially contrived constitutively expressed or regulated promoter containing either a strong, intermediate or weak consensus sequence that delivers desired levels of the estrogen signalling pathway regulator expression. In a preferred embodiment of the invention, tissue-specific promoters can be used to direct expression of an ESP- regulator in a particular tissue. For example, the whey acidic protein promoter can be used to direct expression of an estrogen signalling pathway regulator in breast tissue.

This genetic information is delivered into the target cells by a either a biologic or abiologic delivery vehicle. In one embodiment of the present invention, a biologic viral vector is used to deliver an ESP-regulator. Alternatively, an abiologic delivery system (e.g., liposomes) can be used to package nucleic acid carrying the genetic elements necessary and sufficient for the proper expression of the ESP-regulator(s) into liposomes.

The present invention further encompasses the use of the ESP-regulators of the present invention for the treatment of disease, such as cancer or hypeφroliferative disorders. The present invention further relates to a method of treating a host having a proliferative disease of a specific tissue by inhibiting cell proliferation in the tissue, comprising administering to the host a ESP-regulator wherein the tissue-specific promoter binding sequence is specific for the diseased tissue, whereby the ESP-regulator encoded by the nucleic acid is expressed, estrogen receptor signalling is regulated in the tissue, cell proliferation is inhibited, and the proliferative disease treated is provided. The present invention encompasses the ESP-regulators of the present invention in pharmaceutical formulations.

The present invention further encompasses the use of the ESP-regulators of the present invention for research and screening puφoses. In one embodiment of the present invention, the ESP-regulators may be used to screen for gene products or molecules which effectively inhibit the proliferation of a cancer cell (such as a breast cancer cell with a

BRCAl mutation).

In yet another embodiment, the present invention relates to a vector encoding an ESP-regulator. 4. BRIEF DESCRIPTION OF THE DRAWINGS

Fig . 1. Inhibition of ER-α activity by BRCAl and mapping of involved domains, (a) BRCAl strongly inhibits ER-α signalling in prostate (DU-145) and breast cancer (T47D) cells. Cells were assayed for stimulation of ERE-TK-Luc reporter activity by 178-estradiol (E2). E2 induced a (lOO-lOOO)-fold increase in reporter activity when expression vector pSG5-ER-α (ER-α) was provided. wtBRCAl but not empty vector (pcDNA3), ablated E2- stimulated reporter activity. A control reporter missing the ERE (TK-Luc) gave no E2- stimulated activity. Note: Similar results were obtained whether assays were carried out in serum free-medium (as shown) or in DMEM supplemented with 2% charcoal-stripped serum (data not shown), (b) BRCAl weakly inhibits ER-α signalling in human cervical cancer cell lines. In three cervical cancer cell lines (C33A, CaSki. and SiHa), E2-stimulated reporter activity in the presence of wtBRCAl was reduced to 38-51% of that in the presence of empty vector. In contrast, two other prostate cancer cell lines (LNCaP and TsuPr-1) and two other breast cancer cell lines (MCF-7 and MDA-MB-231) showed virtual ablation of reporter activity by wtBRCAl . (c) Diagrams of proteins encoded by BRCAl genes.

Functional domains shown are: BRCT= BRCAl C-terminal homology region; RJNG= ring finger domain; LXCXE= consensus retinoblastoma binding motif; NLS1= nuclear localization signal; TAD= transactivation domain, (d) Expression of BRCAl genes in DU- 145 cells by semi-quantitative RT-PCR. All BRCAl cDNAs are expressed well. In addition, the BRCAl transgenes produce proteins of expected size when translated (data not shown), (e) Effect of BRCAl genes on ER-α activity in DU-145 and T47D. Activity is expressed as % of empty pcDNA3 vector positive control. Values are means ± SEMs of at least three experiments, (f) Dose-response for inhibition of ER-α activity by wtBRCAl vs

5677insA. The total transfected DNA content was kept constant by addition of the emptv pcDNA3 vector. Values are means ± SEMs of quadruplicate wells, (g) BRCAl inhibits the estrogen-stimulated expression of the endogenous pS2 and cathepsin D genes in MCF-7

(top panel) and T47D (bottom panel) breast cancer cells. Cells were transfected overnight with wtBRCAl or empty pcDNA3 vector, washed, and incubated in serum-free DMEM (5.0 ml per 100 mm dish) +/-E2 (1 μM). Conditioned medium was concentrated 10-fold and

1/10 of the concentrate (≥50 μl) was used for Western blotting.

Fig. 2. In vivo association of BRCAl and ER-α. (a) Association of endogenous BRCAl and ER-α in untransfected cells. Nuclear lysates from proliferating cells were subjected to immunoprecipitation (IP)-Western blotting using antibodies against BRCAl. ER-α. or control (mouse IgG). ER-α was detected in the BRCAl IP of MCF-7 cells and vice versa, but not in the BRCAl IP of DU-145 cells, which are ER negative, (b) Association of BRCAl and ER-α in transiently transfected DU-145 cells. Transfections were carried out under conditions parallel to those of the transcription assays. When expressed in DU-145 cells, ER-α does associate with BRCAl. (c) Restoration of ER-α signaling by 5 BRCAl ΔEcoRI. In inhibition of ER-α signaling by wtBRCAl could be decreased with the addition of increasing amounts of BRCAl ΔEcoRI.

Fig. 3. In vitro binding of BRCAl to ER-α by GST capture, (a) Schematic diagrams of ER-α proteins tested in GST binding assays (Wang et al., 1993, Science 260:1330-1335).

10 (b) GST:ER fusion proteins visualized by Western blotting, (c) Binding of in vitro translated (IVT) wtBRCAl to beads coated with GST-ER fusion proteins. The input lane shows 10% of the IVT wtBRCAl used in each assay, (d) IVT products of different BRCAl cDNAs (see Fig. lc), demonstrated by autoradiography. (e) Binding of IVT BRCAl proteins to beads coated with GST-ER 282-420. (f) Relative binding of different BRCAl

15 proteins to ER 282-420. Bars show the ratio of bound: input BRCAl derived from densitometric analysis of the data in panels d and e.

Fig. 4. wtBRCAl inhibits expression of p300 in prostate and breast cancer cells, (a) wtBRCAl down-regulates p300 levels in DU-145 cells. Subconfluent proliferating cells in

20 100 mm dishes were incubated overnight with 15 μg of each vector in 5.0 ml of DMEM containing Lipofectamine. Cells were washed, incubated in fresh DMEM for 24 hr and harvested for Western blotting. Of the various BRCAl genes tested, only wtBRCAl and 5677insA inhibited p300 expression, while none of these genes altered CBP expression (b) wtBRCAl and BRCAl-5677insA also inhibited p300 expression in T47D cells but was still

²⁵ incapable of affecting CBP levels, (c) Unlike DU-145 and T47D, three cervical cancer cell lines (C33A, CaSki and SiHa) did not decrease the levels of p300 (or CBP) in response to wtBRCAl . Note: SiHa cells had no detectable p300, but showed a band of slightly lower

M_r, consistent with p27O, a p300-like protein that cross-reacts with the p300 antibody

(Dallas et al, 1996, J Virol 71 :1726-1731). 30

Fig. 5. p300 and CBP overrides wtBRCAl inhibition of ER-α activity, (a) Diagrams of proteins encoded by p300 genes. Domains shown are: CHI, CH2, CH3 = cysteine/histidine rich regions-1, -2, -3; HAT = histone acetyl transferase domain; PCAF = p300-interacting cofactor; SRC-1 = steroid receptor coactivator- 1. (b) p300 overrides the inhibition of ER-α

35 by wtBRCAl . Co-transfection of wildtype (wt-p300) or mutant p300 genes missing the HAT or Bromo domains blocked the inhibition by wBRCAl . A p300 mutant missing the CH3 domain failed to block the inhibition; while a mutant consisting of only the CH3 region plus flanking sequences (p300 1514-1922) did override the inhibition, (c) Wild-type CBP (wt-CBP) also overrides wtBRCAl inhibition of ER-α activity, (d) Effect of co- transfection of both wtBRCAl and p300 on p300 levels in DU-145 cells. Co-transfection of wt-p300 blocked the down-regulation of p300 by wtBRCAl; but co-transfection of p300 1514-1922 did not block down-regulation of endogenous full-length p300 by wtBRCAl . Neither wt-p300 nor p300 1514-1922 inhibited BRCAl expression.

Fig. 6. The CR3 domain of CBP/p300 interacts with ER-α. (a) The carboxyl-terminal region of human CBP shows estrogen-independent binding to the AF-2/ligand binding domain region of mouse ER-α, as demonstrated by GST capture assay, (b) The binding site for ER-α maps to the CH3 region of human CBP. On the other hand, GRIPl binding maps to the pl60-interacting site located carboxyl-terminal to CH3. (c) Human ER-α binds to the CH3 region of CBP independently of estrogen. Estrogen (17β-estradiol) and the estrogen antagonists (tamoxifen, raloxifene, and ICI 182,780) were used at a concentration of 0.1 μM. (d) A p300 fragment containing the CH3 domain (p300 1514-1922) binds to ER 282-420. Competition assays in which GST-ER 282-420 was preincubated with IVT p300 1514-1922 or IVT wtBRCAl showed reduced binding of wtBRCAl and p300 1514- 1922, respectively. The amounts of competing protein represent μg of plasmid DNA. As a control, pre-incubation of GST-ER 282-420 with similar quantities of IVT empty vector (pcDNA3) reaction product failed to inhibit the binding of p300 1514-1922 to GST-ER 282- 420.

Fig. 7. Model for breast cancer suppression of BRCAl . Our findings suggest a model in which the amino-terminus of BRCAl interacts with ER-α, and the carboxy 1-terminus acts as an transcription inhibition domain. BRCAl and p300/CBP interact with the AF2/LBD region of ER-α. We propose that p300/CBP compete with BRCAl for a binding site on ER-α or otherwise neutralizes the ability of BRCAl to inhibit AF-2. The failure of mutant forms of BRCAl to inhibit ER-α activity can contribute to inappropriate cellular proliferation and breast cancer.

5. DETAILED DESCRIPTION OF THE INVENTION

Mutations of the breast cancer susceptibility gene BRCAl are linked to development of breast, ovarian, and prostatic cancers; but it is not clear why BRCAl mutations lead to these particular tumor types. The present invention relates to the discovery that BRCAl inhibits signalling by the ligand-activated estrogen receptor (ER-α) through the estrogen-responsive enhancer element (ERE) and blocks the transcriptional activation function of ER-α. Inhibition of ER-α activity requires at least three structural domains of BRCAl : the N-terminal RING finger, a consensus retinoblastoma (RB) binding motif (³⁵⁸LXCXE), and a third domain at the C-terminus. The studies describe herein suggest that transcriptional adaptor p300, which is a component of the ER-α coactivator complex, is a target for repression of ER-α by BRCAl . The loss of the ability to regulate ER-α may contribute to the incidence of breast cancer in BRCAl mutation carriers. Further, the present invention is based, in part, on the Applicants' discovery that the failure to repress ER-α activity may contribute to breast cancer.

The present invention relates to the estrogen signalling pathway and inhibition or regulation of estrogen receptor signalling as a means of treating the proliferative disease. More specifically, the invention relates to the treatment of breast cancer or cancers associated with alterations in hormone or hormone responsiveness. The present invention relates, in part, to the discovery that BRCAl inhibits signalling by the ligand-activated estrogen receptor (ER-α) through the estrogen-responsive enhancer element (ERE) and blocks the transcriptional activation function of ER-α. The invention relates to compositions and methods that regulate estrogen receptor signalling for treating cancer and proliferative disease, such as breast cancer. In particular, the invention relates to compositions and methods that regulate ER-α activity as a treatment for cancer or proliferative disease. The compositions of the invention include, but are not limited to, estrogen signalling pathway regulator (herein called an "ESP-regulator"). The invention further relates to regulation of the estrogen signalling pathway. In accordance with the invention, methods and compositions of the invention are used for the treatment of a host having one or more BRCAl mutations. The invention relates to the repression of ER-α activity as a treatment for cancer or proliferative disease.

An ESP-regulator of the invention may be any molecule (such as a compound, small molecule, nucleic acid, gene product, protein, hormone, recombinant protein, synthetic molecule, etc.) that is capable of modulating the estrogen signalling pathway. Such regulators include but are not limited to members of the estrogen signalling pathway (such as the ER-α, ER-β), ligands of an estrogen receptor (such as estrogen, recombinant estrogen, or modified forms of estrogen e.g., modified to bind the receptor with decreased efficacy), molecules which bind the estrogen receptor and prevent translocation of the receptor or receptor/ligand complex into the nucleus, molecules which bind to the DNA- binding domain of the receptor and inhibit signalling, molecules which prevent receptor dimerization, molecules which bind to the transactivation domain and inhibit transactivation, molecules which bind to the ligand binding domain and inhibit signalling. Such regulators also include dominant negative proteins (such as a dominant negative

5 estrogen receptor). Such regulators may also include molecules which act to compensate for a BRCAl mutation, or a molecule which interacts directly with a BRCAl protein to enhance BRCAl protein regulation of the estrogen receptor activation or signalling.

The present invention further relates to the use of a wide variety of vehicles to deliver the ESP-regulator of the invention to a target or target tissue, including virions

10 and viral vectors to package and deliver the DNA encoding an ESP-regulator non- viral expression vectors and abiologic vehicles, including liposomes and liposome-DNA and lipid-DNA complexes to deliver and target the DNA encoding an ESP-regulator to the host.

In accordance with the invention, the host to which the ESP-regulator is delivered may be cells in culture, tissues in culture, animal models, animals, mammals, or

15 preferably humans. In a preferred embodiment, the ESP-regulator is delivered to a female human host. In a more preferred embodiment, the ESP-regulator is delivered to a female human host, having breast cancer or at risk of acquiring breast cancer (such as one who has been identified as a carrier of a BRCAl mutation).

The present invention relates to pharmaceutical compositions comprising the

20 ESP-regulators of the present invention and their delivery vehicles. The ESP-regulators of the present invention may be engineered for the treatment of a wide variety of disorders and diseases related to expression of a particular gene or genes, cellular oveφroliferation, hereditary disorders, cancers, tumors, or proliferative disorders.

The present invention further relates to methods of screening hosts for

25 BRCAl mutations in order to identify those individuals that are more susceptible to developing cancer, in particular breast, ovarian or prostate cancer. The present invention also relates to methods of screening hosts to identify and diagnose individuals carrying BRCAl mutations that are in a precancerous stage or having cancer, in particular breast, ovarian or prostate cancer. The present invention further relates to methods of screening

30 hosts known to have cancer, in particular breast, ovarian or prostate cancer, to identify those individuals carrying BRCAl mutations that would benefit from methods of treatment and compositions described herein. Individuals to particularly benefit from treatment are those containing mutations in BRCAl domains essential for function (e.g., the N-terminal RING finger domain, the consensus retinoblastoma binding motif and a domain in the C-terminal

35 550 amino acids). In another embodiment the present invention also relates to in vitro screening or research puφoses, e.g., to identify gene products involved in cellular oveφroliferation such as a BRCAl mutation, or an estrogen signalling pathway gene mutation. For example. in vitro screening may be used to identify mutations that compensate for a BRCAl mutation and thus serve to protect from oveφroliferation due to the BRCAl mutation.

5.1 PRECANCEROUS/PREMALIGNANT CONDITIONS

The invention provides for the screening of individuals to ascertain their BRCAl nucleotide sequence and therefore the degree to which they are at risk of developing cancer, especially of the breast, ovary or prostate. Individuals found to be at risk can be treated prophylactically to decrease their chance of developing cancer. The invention also provides for the treatment of such precancerous or premalignant conditions. In one embodiment, precancerous or premalignant conditions is treated by induction of apoptosis. The precancerous or premalignant cells in which apoptosis is induced are generally ones which exhibit at least one functional p53 allele. "Functional" as used herein, refers to an ability of the p53 allele to contribute to differential apoptosis in cells. It is to be noted that in certain instances, administration of the tumor suppressor of the invention results in restoration of mutant p53 protein conformation and/or activity to normal. Thus, while precancerous or premalignant cells exhibiting at least one functional p53 allele are preferred targets of the methods of the invention, the methods described herein are not to be limited to such cells.

Precancerous or premalignant cells include, but are not limited to cells which present in conditions known or suspected to precede progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hypeφlasia. metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology. 2d Ed., W.B. Saunders Co.. Philadelphia, pp. 68-79).

Hypeφlasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hypeφlasia often precedes endometrial cancer.

Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. As but one example, the esophageal metaplasia of Barrett's esophagus often precedes esophageal cancer- Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomoφhism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the skin, cervix, respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growth characterized as hypeφlasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient, can indicate the desirability of therapeutic administration of the methods and compositions of the invention.

As mentioned above, such characteristics of a transformed phenotype include moφhology changes, loss of differentiation markers, reversion to a dedifferentiated phenotype, cell rounding, loss of adhesion, decreased spreading, loss of polarity, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, etc (see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 84-90 for characteristics associated with a transformed or malignant phenotype). In accordance with the invention, an ESP-regulator modulates estrogen receptor signalling activity or expression is used to treat or prevent hypeφroliferative or benign dysproliferative disorders. Specific embodiments are directed to treatment or prevention of benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hypeφlasia).

5.2 GENE THERAPY The present invention provides the therapeutic or prophylactic use of an ESP-regulator. In a specific embodiment, nucleic acids comprising a sequence encoding an ESP-regulator or functional derivative thereof, are administered to regulate or inhibit cell proliferation, by way of gene therapy. Gene therapy refers to therapy performed by the administration of a nucleic acid to a host. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect by to regulating or inhibiting cell proliferation of a cancerous, precancerous. of diseased cell.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991. Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11 :155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the Therapeutic comprises an ESP-regulator nucleic acid that is part of an expression vector that expresses a an ESP-regulator protein or fragment or chimeric protein thereof in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the ESP-regulator coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the ESP-regulator coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the ESP- regulator nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932- 8935; Zijlstra et al., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered in vivo. where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles. or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand host to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16,

5 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO92/20316 dated November 26, 1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incoφorated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989. Proc. Natl. Acad. Sci. USA

10 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, a viral vector that contains the ESP-regulator nucleic acid is used. For example, a retroviral vector can be used (see Miller et al., 1993. Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration

15 into host cell DNA. The ESP-regulator nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302; Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. 0 Opin. in Genetics and Devel. 3:110-114. Adenoviruses are other viral vectors that can be used in gene therapy. Targets for adenovirus-based delivery systems are epithelial cells, liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells, (see, Kozarsky and Wilson, 1993, Current Opinion Gen. Dev. 3:499-503). Adeno-associated virus (AAV) has also been 5 proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289- 300.

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection. calcium phosphate mediated transfection. or viral infection. Usually, the method of transfer includes the transfer of a 0 selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In a specific embodiment, the nucleic acid to be introduced for puφoses of gene therapy comprises an inducible promoter operably linked to the coding region, such 5 that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. In one embodiment, recombinant cells are injected, e.g., subcutaneously. In another embodiment, recombinant cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art. In accordance with the invention, delivery of such recombinant cells may be particularly desirable when used in combination with surgery or surgical treatment administered for the treatment of a tumor, a cancer, or a proliferative disease. For example, in a preferred embodiment of the invention, recombinant cells expressing an ESP-regulator are administered during or after a mastectomy.

Cells into which a nucleic acid can be introduced for puφoses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In a preferred embodiment, the cell used for gene therapy is autologous to the patient. In an embodiment in which recombinant cells are used in gene therapy, a ESP-regulator nucleic acid is introduced into the cells such that they are expressed by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.

5.3 ASSAYS OF ESP-REGULATORS The functional activity of ESP-regulators, derivatives and analogs can be assayed by various methods known to one skilled in the art.

For example, in one embodiment, where one is assaying for the ability of an ESP-regulator or an estrogen-receptor antibody to bind or compete for binding to an estrogen receptor, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. In another embodiment, where a estrogen receptor binding protein is identified, the binding can be assayed, e.g., by means well-known in the art. In another embodiment, physiological correlates of ESP- regulator binding to its substrates (e.g.. signal transduction molecules) can be assayed.

In another embodiment, an ESP-regulator or estrogen receptor is assayed for the ability to self association as a dimer, tetramer, or multimer. Assays for self-association include but are not limited to separation of complexes on a sucrose gradient or centrifugation gradient, analysis on a non-denaturing electrophoretic gel, or in vitro gel mobility shift assays. In another embodiment, detection of the association and disassociation of protein complexes is measured by Fluorescence Polarization (In Physical Biochemistry: Applications to Biochemistry and Molecular Biology 2^nd Ed., Freifelder, 1982, W.H. Freeman and Company, New York). Methods for detecting complexes may also be performed as described in Stenger et al., 1992, Mol. Carcinog. 5:102-106, including analysis by gradient gel electrophoresis and chemical cross-linking. In one embodiment, full cross-linking is combined with denaturing gel electrophoresis. In another embodiment, partial cross-linking is performed followed by dissociating tetramer complexes into monomers, dimers and trimers. In yet another embodiment, the subcellular localization of an ESP-regulator or estrogen receptor may be examined by any assay known in the art. For example, immunofluorescence staining with antibodies against an ESP-regulator protein can reveal the subcellular location of such protein. In a further embodiment, cells may be simultaneously stained for known markers of subcellular compartments, such that the location of the ESP-regulator protein may be co-localized by fluorescence. In another embodiment, physiological correlates of the subcellular location of an ESP-regulator protein (e.g., nuclear) can be assayed.

Another functional assay of a ESP-regulator is an assay for the ability of the ESP-regulator to bind DNA or nucleotide sequence, or the ability of an ESP-regulator to inhibit the binding of an estrogen receptor to DNA. Such assays include the electrophoretic mobility shift assay.

Other methods will be readily apparent to the skilled artisan and are within the scope of the invention.

5.4 BIOCHEMICAL ASSAYS USING AN ESP-REGULATOR The present invention provides for biochemical assays using the ESP- regulators of the invention. In one embodiment, ESP-regulators are useful for biochemical assays aimed at the identification and characterization of molecules which interact with a estrogen signalling pathway proteins, or are new estrogen signalling pathway proteins (e.g. proteins in the same signaling pathway as an estrogen receptor). For example, the cDNAs encoding the ESP-regulator proteins can be individually subcloned into any of a large variety of eukaryotic expression vectors permitting expression in insect and mammalian cells, described above. The resulting genetically engineered cell lines expressing ESP- regulator proteins can be assayed for production, activation, and interactions of ESP- regulator proteins, for example with antibodies ESP-regulator protein and Western blotting assays or ELISA assays. For assays of specific binding and functional activation of estrogen receptors or ESP-regulators, one can employ either crude extracts or lysates containing protein from genetically engineered cells, or partially purified extracts, or preferably highly purified ESP-regulator protein fractionated, for example, by chromatographic methods. Alternatively, an ESP-regulator protein can be synthesized using chemical methods (Nagata et al., \992, Peptides 13:653-62).

Specific binding of an ESP-regulator to an estrogen receptor (e.g., ER-α) can be assayed as follows, for example, following the procedures of Yamaguchi et al. (Yamaguchi et al., 1995. Biochemistry 34:4962-4968). Chinese hamster ovary cells, COS cells, or any other suitable cell line, can be transiently transfected or stably transformed with expression constructs that direct the production of an estrogen receptor. Direct binding of an ESP-regulator to such ER-α-expressing cells can be measured using a "labeled" purified ESP-regulator derivative, where the label is typically a chemical or protein moiety covalently attached to the ESP-regulator which permits the experimental monitoring and quantitation of the labeled ESP-regulator in a complex mixture.

Specifically, the label attached to the ESP-regulator can be a radioactive substituent such as an ¹²⁵I-moiety or ³²P-phosphate moiety, a fluorescent chemical moiety, or labels which allow for indirect methods of detection such as a biotin-moiety for binding by avidin or streptavidin, an epitope-tag such as a Myc-or FLAG-tag, or a protein fusion domain which allows for direct or indirect enzymatic detection such as an alkaline phosphatase-fusion or Fc-fusion domain. Such labeled ESP-regulator can be used to test for direct and specific binding to ER-α-expressing cells by incubating the labeled ESP-regulator with the ER-α-expressing cell lysates or subcellular fractions. Also, instead of a direct binding assay, a competition binding assay may be used.

This invention provides assays for detecting changes in the expression of estrogen signalling pathway genes and proteins. Such estrogen signalling pathway genes and proteins include but are not limited to genes and proteins within the estrogen pathway as well as estrogen responsive gene (e.g., genes that are transcriptionally activated by the ligand-activated estrogen receptor). Assays for changes in gene expression are well known in the art (see e.g., PCT Publication No. WO 96/34099, published October 31, 1996, which is incoφorated by reference herein in its entirety). Such assays may be performed in vitro using transformed cell lines, immortalized cell lines, or recombinant cell lines, or in vivo using animal models. In particular, the assays may detect the presence of increased or decreased expression of estrogen signalling pathway genes and proteins on the basis of increased or decreased mRNA expression (using, e.g., nucleic acid probes), increased or decreased levels of related protein products (using, e.g., the antibodies disclosed herein), or increased or decreased levels of expression of a marker gene (e.g., β-galactosidase or luciferase) operably linked to a 5' regulatory region in a recombinant construct.

In yet another series of embodiments, various expression analysis techniques may be used to identify genes which are differentially expressed between two conditions, such as a cell line or animal expressing a normal BRCAl gene compared to another cell line or animal expressing a mutant BRCAl gene. Such techniques comprise any expression analysis technique known to one skilled in the art, including but not limited to differential display, serial analysis of gene expression (SAGE), nucleic acid array technology, subtractive hybridization, proteosome analysis and mass-spectrometry of two-dimensional protein gels.

To elaborate further, the various methods of gene expression profiling mentioned above can be used to identify other genes (or proteins) that may have a functional relation to (e.g., may participate in a signaling pathway with) an estrogen signalling pathway gene. Gene identification of such other genes is made by detecting changes in their expression levels following mutation, /. e. , insertion, deletion or substitution in, or overexpression, underexpression, mis-expression or knock-out, of a BRCAl gene, as described herein. Expression profiling methods thus provide a powerful approach for analyzing the effects of mutation in a BRCAl gene or ESP-regulator gene.

Methods of gene expression profiling are well-known in the art, such as exemplified by the following references describing differential display (Liang and Pardee, 1992, Science 257:967-971), proteosome analysis (Humphery-Smith et al., 1997,

Electrophoresis 18:1217-1242; Dainese et al, 1997 ^', Electrophoresis 18:432-442). SAGE (Velculescu et al., 1995, Science 270:484-487), subtractive hybridization (Wang and Brown, 1991, Proc. Natl. Acad. Sci. U.S.A. 88:11505-11509), and hybridization-based methods of using nucleic acid arrays (Heller et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:2150-2155; Lashkari et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:13057-13062; Wodicka et al., 1997, Nαtwre Biotechnol. 15:1259-1267).

5.5 PROLIFERATION AND CELL CYCLE ASSAYS

An ESP-regulator has potential implications in regulation of cell proliferation and control of the cell cycle. Further, a number of signaling pathway molecules relating to ESP-regulator and cell cycle regulation are of interest in the instant invention. The present invention provides for cell cycle and cell proliferation analysis by a variety of techniques known in the art, including but not limited to the following techniques.

As one example, bromodeoxyuridine (BRDU) incoφoration may be used as an assay to identify proliferating cells. The BRDU assay identifies a cell population undergoing DNA synthesis by incoφoration of BRDU into newly-synthesized DNA. Newly-synthesized DNA may then be detected using an anti-BRDU antibody (see Hoshino et al.. 1986, Int. J. Cancer 38:369; Campana et al., 1988, J. Immunol. Meth. 107:79).

Cell proliferation may also be examined using [³H]-thymidine incoφoration (see e.g., Chen, 1996, Oncogene 13:1395-403; Jeoung. 1995. J. Biol. Chem.

270:18367-73). This assay allows for quantitative characterization of S-phase DNA synthesis. In this assay, cells synthesizing DNA will incoφorate[³H] -thymidine into newly synthesized DNA. Incoφoration can then me measured by standard techniques in the art such as by counting of radioisotope in a Scintillation counter (e.g. Beckman LS 3800 Liquid Scintillation Counter).

Detection of proliferating cell nuclear antigen (PCNA) may also be used to assay cell proliferation. PCNA is a 36 kilodalton protein whose expression is elevated in proliferating cells, particularly in early GI and S phases of the cell cycle and therefore may serve as a marker for proliferating cells. Positive cells are identified by immuno staining using an anti-PCNA antibody (see Li et al., 1996, Curr. Biol. 6:189-199; Vassilev et al., 1995, J. Cell Sci. 108:1205-15).

Cell proliferation may be measured by the counting samples of a cell population over time (e.g. daily cell counts). Cells may be counted using a hemacytometer and light microscopy (e.g. HyLite hemacytometer, Hausser Scientific). Cell number may be plotted against time in order to obtain a growth curve for the population of interest. In a preferred embodiment, cells counted by this method are first mixed with the dye Trypan- blue (Sigma), such that living cells exclude the dye, and are counted as viable members of the population. DNA content and/or mitotic index of the cells may be measured, for example, based on the DNA pioidy value of the cell. For example, cells in the GI phase of the cell cycle generally contain a 2N DNA pioidy value. Cells in which DNA has been replicated but have not progressed thru mitosis (e.g. cells in S-phase) will exhibit polidy value higher than 2N and up to 4N DNA content. Pioidy value and cell cycle kinetics may further be measured using propidium iodide assay (see e.g. Turner, et al., 1998, Prostate 34:175-81). Alternatively, the DNA pioidy may be determined by quantitation of DNA Feulgen staining (which binds to DNA in a stoichiometric manner) on a computerized microdensitometrystaining system (see e.g., Bacus, 1989, Am. J. Pathol.135:783-92). In an another embodiment, DNA content may be analyzed by preparation of a chromosomal spread (Zabalou, 1994, Hereditas.120:127-40; Pardue, 1994, Meth. Cell Biol. 44:333-351). Further, the expression of cell cycle proteins (e.g., CycA, CycB, CycE, CycD, CycJ, cdc2, Cdk4, Cdk6, E2f, E2f2, Rb, p21^c,pl, p27, etc.) also provide crucial information relating to the proliferative state of a cell or population of cells. For example, identification in an anti-proliferation signaling pathway may be indicated by the induction of p21^c,pl. Expression of p21 can be elevated by p53. In turn, increased levels of p21 expression in cells results in Cdk inhibition, resulting in delayed entry into GI of the cell cycle (Haφer et al., 1993, Cell 75:805-816; Li et al., 1996, Curr. Biol. 6: 189-199). p21 induction may be identified by immunostaining using a specific anti-p21 antibody available commercially (e.g. Santa Cruz). Similarly, cell cycle proteins may be examined by Western blot analysis using commercially available antibodies. In a specific embodiment, the role of an ESP-regulator in cell cycle regulation is examined by contacting a cell with said ESP- regulator and comparing the cell cycle characteristics (such as cell cycle length, duration of phases, expression of cell cycle proteins etc.) to a cell which has not been contacted by the ESP-regulator. In another embodiment, cell populations are synchronized prior to detection of a cell cycle protein. Cell cycle proteins may also be detected by FACS (fluorescence-activated cell sorter) analysis using antibodies against the protein of interest. Further assays include but are not limited to detection of changes in length of the cell cycle or speed of cell cycle. In one embodiment the length of the cell cycle is

5 determined by the doubling time of a population of cells. In another embodiment, FACS analysis is used to analyze the phase of cell cycle progression, or purify GI, S, and G2/M fractions (see e.g., Delia, et al., 1997, Oncogene 14:2137-47). In another embodiment assays of this type are be performed on synchronized cell populations. In a further embodiment, cells are synchronized by a nocodazole block (Knehr et al., 1995, Exp. Cell

10 Res. 217:546-553). In a further embodiment, length or speed of the cell cycle of a test population (such as BRCAl mutant population) is compared to wildtype populations.

Lapse of cell cycle checkpoint(s), and/or induction of cell cycle checkpoint(s), may be examined by the methods described herein, or by any method known in the art. Without limitation, a cell cycle checkpoint is a mechanism which ensures that a

15 certain cellular events occur in a particular order. Checkpoint genes are defined by mutations that allow late events to occur without prior completion of an early event (Weinert and Hartwell, 1993, Genetics 134:63-80). Induction or inhibition of cell cycle checkpoint genes may be assayed, for example, by Western blot analysis, or by immunostaining, etc. Lapse of cell cycle checkpoints may be further assessed by the

20 progression of a cell thru the checkpoint without prior occurrence of specific events (e.g. progression into mitosis without complete replication of the genomic DNA).

In addition to the effects of expression of a particular cell cycle protein, activity and post-translational modifications of proteins involved in the cell cycle can play an integral role in the regulation and proliferative state of a cell. The invention provides for

25 assays involved detected post-translational modifications (e.g. phosphorylation) by any method known in the art. For example, antibodies which detect phosphorylated tyrosine residues are commercially available, and may be used in western blot analysis to detect proteins with such modifications. In another example, modifications such as myristylation. may be detected on thin layer chromatography or reverse phase HPLC (see e.g., Glover.

30 1988. Biochem . J. 250:485-91 ; Paige. 1988. Biochem J. 250:485-91).

Activity of signaling and cell cycle proteins and/or protein complexes is often mediated by a kinase activity. In present invention provides for analysis of kinase activity by assays such as the histone HI assay (see e.g., Delia, et al., 1997, Oncogene 14:2137-47). In another embodiment, the kinase is a casein kinase. In one embodiment, the

35 kinase assay is performed in vitro. In a further embodiment, the in vitro kinase assay is performed with purified components. In a specific embodiment of the HI Kinase Assay is performed as follows: cell lysates are precleaned by incubation in protein G(or A)-agarose. Lysates are immunoprecipitated and washed three times with immunoprecipitation buffer and twice with IX kinase buffer (50 mM Tris-HCl 7.5, 10 mM MgC12, 5 mM EGTA, 2 mM DTT) without DDT. The kinase assay is then carried out on ice for 10 minutes in 35 μl of IX kinase buffer containing 15 μCi of γ-32P ATP, 1.6 μg of histone HI, and 1.5 μM ATP. The kinase activities are measured by quantifying the intensities of histone-Hl phosphorylation using a Phosphorlmager (Molecular Dynamics). In a preferred embodiment, the kinase assay experiments are repeated three times. Other methods will be apparent to one skilled in the art and are within the scope of the invention.

5.6 ASSAYS FOR APOPTOSIS

Without limitation to a particular mechanism, programed cell death, or apoptosis is a process by which a cell actively undergoes death by the induction of a series of biochemical and moφhological changes. Apoptosis plays an important role in development, homeostasis and disease (see e.g., Lowe et al., 1993, Cell 74:957-967).

A variety of techniques known in the art may be used in the present invention to examine apoptosis. One assay for apoptosis is the Terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (see e.g. Lazebnik et al., 1994, Nature 371 :346), by following the incoφoration of fluorescein-dUTP (Yonehara et al., 1989. J. Exp. Med. 169:1747). Alternately, apoptosis signaling may be detected by ApoAlert™ staining (Gavrieli et al.. 1992. J. Cell. Biol. 119:493). These assay kits are commercially available through suppliers such as Clontech and Boehringer

Mannheim. Apoptosis may further be assayed by acridine orange incoφoration (see e.g. Lucas et al., 1998, Blood 15:4730-41). Detection of a gene in the apoptotic pathway may be detected standard methods in the art such as by Western analysis using antibodies to the apoptotic gene of interest. Additional assays for detection of apoptosis include detection of DNA fragmentation by DNA laddering on an agarose gel (see e.g., Civielli et al., 1995, Int. J. Cancer 27:673-679; Yeung, 1998, J. Biol. Chem. 273:25198-202). In this technique, DNA fragmentation is detected on an agarose gel stained with ethidium bromide. Due to the specific sites at which DNA is fragmented during apoptosis, a characteristic pattern or "ladder" results when the cellular DNA is examined by electrophoresis. Other methods will be readily apparent to one skilled in the art and are within the scope of the invention.

5.7 CELL ADHESION AND LOSS OF INVASIVENESS A critical aspect of the formation of a metastatic cancer is the ability of a precancerous or cancerous cell to detach from primary site of disease, and establish a novel colony of growth at a secondary site. The ability of a cell to invade peripheral sites is reflective of a potential for a cancerous state. Invasion inhibition assays are within the scope of the invention. For example ESP-regulators may be assayed for their ability of inhibit invasion of a test cell. Loss of invasiveness may be measured by a variety of techniques known in the art including, for example, induction of E-cadherin-mediated cell-cell adhesion. Such E-cadherin-mediated adhesion can result in phenotypic reversion and loss of invasiveness (Hordijk et al., 1997. Science 278:1464-66).

Loss of invasiveness may further be examined by inhibition of cell migration. A variety of 2-dimensional and 3-dimensional cellular matrices are commercially available (Calbiochem-Novabiochem Coφ. San Diego, CA). Cell migration across or into a matrix may be examined by microscopy, time-lapsed photography or videography, or any method in the art allowing measurement of cellular migration. In a related embodiment, loss of invasiveness is examined by response to hepatocyte growth factor (HGF). HGF-induced cell scattering is correlated with invasiveness of cells such as Mardin-Darby canine kidney (MDCK) cells. This assay identifies a cell population which has lost cell scattering activity in response to HGF (Hordijk et al., 1997, Science 278:1464- 66).

Alternatively, invasion or loss of invasiveness may be measured by cell migration through a chemotaxis chamber (Neuroprobe/ Precision Biochemicals Inc. Vancouver , BC). In this assay, a chemo-attractant agent is incubated on one side of the chamber (e.g. the bottom chamber) and cells are plated on a filter separating the opposite side (e.g. the top chamber). In order for cells to pass from the top chamber to the bottom chamber, the cell must actively migrate through small pores in the filter. Checkerboard analysis of the number of cells which have migrated may then be correlated with invasivness (see e.g., Ohnishi, 1993, Biochem. Biophys. Res. Commun.193:518-25).

5.8 HOST CELLS

The present invention delivery of ESP-regulators to primary cells, or cell lines for in vitro screening assay and ex vivo gene therapy. In accordance with the present invention, a variety of primary or secondary cells or cell strains may be used including but not limited to cells isolated from breast, skin, bone marrow, liver, pancreas, kidney, adrenal and neurological tissue to name a few. Other cells types that may be used in accordance with the present invention are immune cells (such as T-cells, B-cells, natural killer cells. etc.), macrophages/monocytes, adipoctyes, pericytes, fibroblasts, neuronal cells, reticular cells etc. Breast cancer cell lines that may be used in accordance with the invention include but are not limited to UACC-893, UACC-812, ZR-75-1, ZR-75-30. MDA-MB-415. MDA-MB-415A, MDA-MB-435S, BT-483, and Hs 564(C).Mg. In a further embodiment, secondary cell lines may be used as engineered responsive cells and tissues in accordance with the present invention, including, but not limited to hepatic cell lines, such as C WSV. NR, Chang liver cells, or other cell lines such as CHO. VERO, BHK. Hela. COS, MDCK. 293, 373, CaSki and W138 cell lines.

5.9 THERAPEUTIC/PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS

As used herein, the novel compounds of the present invention, the compounds of the present compositions, and the compounds of the present methods are known collectively as "ESP-regulators". ESP-regulators include any compound that inhibits estrogen pathway signalling with the proviso that the ESP-regulator is not any of the chemotherapeutic or anti- cancer agents listed in Table 1. In a preferred embodiment the ESP-regulators inhibit the binding of p300 and/or CBP to the estrogen response enhancer with the proviso that the ESP-regulator is not any of the chemotherapeutic or anti-cancer agents listed in Table 1. In a most preferred embodiment the ESP-regulators inhibit transcription driven by estrogen response enhancers in cells having a mutation of BRCAl in an essential domain (e.g.. the N-terminal RING finger domain, the consensus retinoblastoma binding motif and a domain in the C-terminal 550 amino acids) with the proviso that the ESP-regulator is not any of the chemotherapeutic or anti-cancer agents listed in Table 1.

Due to the activity of the ESP-regulator, the ESP-regulator are advantageously useful in veterinary and human medicine. For example, the ESP-regulator are useful for the treatment or prevention of cancer or neoplastic disease or inhibiting the growth of a cancer cell or neoplastic cell.

When administered to a subject, e.g., an animal for veterinary use or to a human for clinical use, or when made to contact a cell or tissue, the ESP-regulator are preferably in isolated form. By "isolated" it is meant that prior to administration or contacting, a ESP-regulator is separated from other components of a synthetic organic chemical reaction mixture or natural product source, e.g., plant matter, tissue culture, bacterial broth, etc. Preferably, the ESP-regulator are isolated via conventional techniques, e.g. , extraction followed by chromatography, recrystalization, or another conventional technique. When in isolated form, the ESP-regulator are at least 90%, preferably at least 95%, of a single ESP-regulator by weight of that which is isolated.

The invention provides methods of treatment and prophylaxis by administration to a subject of an effective amount of a ESP-regulator. The subject is preferably an animal, including, but not limited, to an animal such a cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig, etc., and is more preferably a mammal, and most preferably a human.

The present compositions, which comprise one or more ESP-regulator. may be administered by any convenient route, for example by infusion or bolus injection, by absoφtion through epithelial or mucocutaneous linings (e.g.. oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a ESP-regulator of the invention. In certain embodiments, more than one ESP- regulator of the invention is administered to a subject. Methods of administration include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer).

In specific embodiments, it may be desirable to administer one or more ESP- regulator of the invention locally to the area in need of treatment. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue.

In certain embodiments, it may be desirable to introduce one or more ESP- regulator of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the ESP-regulator can be formulated as a suppository, with traditional binders and carriers such as triglycerides. In another embodiment, the ESP-regulator of the invention can be delivered in a vesicle, in particular a liposome (see Langer. 1990, Science 249:1527-1533; Treat et al.,1989, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365: Lopez-Berestein, ibid., pp. 317-327; see generally ibid. ) In yet another embodiment, the ESP-regulator can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al.,1989, N Engl J. Med. 321 :574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release. 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983, J. Macromol Sci. Rev. Macromol Chem. 23:61 ; see also Levy et al., 1985, Science 228:190; During et al, 1989, «n. Neurol 25:351; Howard et al., 1989, J. Neurosurg. 71 :105). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the ESP-regulator, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled-release systems discussed in the review by Langer (1990, Science 249:1527-1533) may be used.

The present compositions will contain a therapeutically effective amount of a ESP-regulator, preferably in purified form, together with a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient. or vehicle with which a ESP-regulator is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil. soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a subject, the ESP-regulator and pharmaceutically acceptable carriers are preferably sterile. Water is a preferred carrier when the ESP- regulator is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained- release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

In a preferred embodiment, the ESP-regulator are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, ESP-regulator for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the ESP-regulator is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the ESP-regulator is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions may contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions may be coated to delay disintegration and absoφtion in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered ESP-regulator. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade.

The amount of the ESP-regulator that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.

The ESP-regulator of the invention are preferably assayed in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of a specific ESP- regulator or combination of ESP-regulator is preferred.

In one embodiment, a patient tissue sample is grown in culture, and contacted or otherwise administered with a ESP-regulator, and the effect of such ESP- regulator upon the tissue sample is observed and compared to a non-contacted tissue. In other embodiments, a cell culture model is used in which the cells of the cell culture are contacted or otherwise administered with a ESP-regulator, and the effect of such ESP- regulator upon the tissue sample is observed and compared to a non-contacted cell culture. Generally, a lower level of proliferation or survival of the contacted cells compared to the non-contracted cells indicates that the ESP-regulator is effective to treat a the patient. Such ESP-regulator may also be demonstrated effective and safe using animal model systems. Other methods will be known to the skilled artisan and are within the scope of the invention.

5.10 INHIBITION OF CANCER AND NEOPLASTIC CELLS AND DISEASE

The ESP-regulator may be demonstrated to inhibit tumor cell proliferation, cell transformation and tumorigenesis in vitro and in vivo using a variety of assays known in the art, or described herein. Such assays may use cells of a cancer cell line, or cells from a patient. Many assays well-known in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring (³H)-thymidine incoφoration, by direct cell count, by detecting changes in transcription, translation or activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, DI, D2, D3, E, etc). The levels of such protein and mRNA and activity can be determined by any method well known in the art. For example, protein can be quantitated by known immunodiagnostic methods such as Western blotting or immunoprecipitation using commercially available antibodies (for example, many cell cycle marker antibodies are from Santa Cruz Inc.). mRNA can be quantitated by methods that are well known and routine in the art, for example by northern analysis, RNase protection, the polymerase chain reaction in connection with the reverse transcription, etc. Cell viability can be assessed by using trypan-blue staining or other cell death or viability markers known in the art. Differentiation can be assessed visually based on changes in moφhology, etc.

The present invention provides for cell cycle and cell proliferation analysis by a variety of techniques known in the art, including but not limited to the following:

As one example, bromodeoxyuridine (BRDU) incoφoration may be used as an assay to identify proliferating cells. The BRDU assay identifies a cell population undergoing DNA synthesis by incoφoration of BRDU into newly synthesized DNA. Newh' synthesized DNA may then be detected using an anti-BRDU antibody (see Hoshino et al.. 1986. Int. J. Cancer 38:369; Campana et al., 1988, J. Immunol Meth. 107:79).

Cell proliferation may also be examined using (Η)-thymidine incoφoration (see e.g., Chen, 1996. Oncogene 13:1395-403; Jeoung, 1995. J. Biol. Chem.

270: 18367-73). This assay allows for quantitative characterization of S-phase DNA synthesis. In this assay, cells synthesizing DNA will incoφorate (³H)-thymidine into newly synthesized DNA. Incoφoration may then be measured by standard techniques in the art such as by counting of radioisotope in a Scintillation counter (e.g. Beckman LS 3800 Liquid Scintillation Counter). Detection of proliferating cell nuclear antigen (PCNA) may also be used to measure cell proliferation. PCNA is a 36 kilodalton protein whose expression is elevated in proliferating cells, particularly in early GI and S phases of the cell cycle and therefore may serve as a marker for proliferating cells. Positive cells are identified by immunostaining using an anti-PCNA antibody (see Li et al., 1996, Curr. Biol. 6:189-199; Vassilev et al., 1995. J. Cell Sci. 108:1205-15).

Cell proliferation may be measured by counting samples of a cell population over time (e.g. daily cell counts). Cells may be counted using a hemacytometer and light microscopy (e.g. HyLite hemacytometer, Hausser Scientific). Cell number may be plotted against time in order to obtain a growth curve for the population of interest. In a preferred embodiment, cells counted by this method are first mixed with the dye Trypan-blue (Sigma), such that living cells exclude the dye, and are counted as viable members of the population.

DNA content and/or mitotic index of the cells may be measured, for example, based on the DNA pioidy value of the cell. For example, cells in the GI phase of the cell cycle generally contain a 2N DNA pioidy value. Cells in which DNA has been replicated but have not progressed through mitosis (e.g. cells in S-phase) will exhibit a pioidy value higher than 2N and up to 4N DNA content. Pioidy value and cell-cycle kinetics may be further measured using propidium iodide assay (see e.g. Turner, et al., 1998, Prostate 34:175-81). Alternatively, the DNA pioidy may be determined by quantitation of DNA Feulgen staining (which binds to DNA in a stoichiometric manner) on a computerized microdensitometrystaining system (see e.g., Bacus, 1989, Am. J. Pathol.135:783-92). In an another embodiment. DNA content may be analyzed by preparation of a chromosomal spread (Zabalou, 1994, Hereditas.120:127-40; Pardue, 1994, Meth. Cell Biol. 44:333-351 ). The expression of cell-cycle proteins (e.g., CycA. CycB. CycE, CycD, cdc2.

Cdk4/6, Rb, p21, p27, etc.) provide crucial information relating to the proliferative state of a cell or population of cells. For example, identification in an anti-proliferation signaling pathway may be indicated by the induction of p21^{C l}. Increased levels of p21 expression in cells results in delayed entry into GI of the cell cycle (Haφer et al.. 1993, Cell 75:805-816; Li et al., 1996. Curr. Biol 6:189-199). p21 induction may be identified by immunostaining using a specific anti-p21 antibody available commercially (e.g. Santa Cruz). Similarly, cell- cycle proteins may be examined by Western blot analysis using commercially available antibodies. In another embodiment, cell populations are synchronized prior to detection of a cell cycle protein. Cell cycle proteins may also be detected by FACS (fluorescence-activated cell sorter) analysis using antibodies against the protein of interest. Detection of changes in length of the cell cycle or speed of cell cycle may also be used to measure inhibition of cell proliferation by the ESP-regulator of the invention. In one embodiment the length of the cell cycle is determined by the doubling time of a population of cells (e.g., using cells contacted or not contacted with one or more ESP-regulator of the invention). In another embodiment, FACS analysis is used to analyze the phase of cell cycle progression, or purify GI, S, and G2/M fractions (see e.g., Delia et al., 1997, Oncogene 14:2137-47).

Lapse of cell cycle checkpoint(s), and/or induction of cell cycle checkpoint(s). may be examined by the methods described herein, or by any method known in the art. Without limitation, a cell cycle checkpoint is a mechanism which ensures that a certain cellular events occur in a particular order. Checkpoint genes are defined by mutations that allow late events to occur without prior completion of an early event (Weinert and Hartwell, 1993, Genetics 134:63-80). Induction or inhibition of cell cycle checkpoint genes may be assayed, for example, by Western blot analysis, or by immunostaining, etc. Lapse of cell cycle checkpoints may be further assessed by the progression of a cell through the checkpoint without prior occurrence of specific events (e.g. progression into mitosis without complete replication of the genomic DNA).

In addition to the effects of expression of a particular cell cycle protein, activity and post-translational modifications of proteins involved in the cell cycle can play an integral role in the regulation and proliferative state of a cell. The invention provides for assays involved detected post-translational modifications (e.g. phosphorylation) by any method known in the art. For example, antibodies that detect phosphorylated tyrosine residues are commercially available, and may be used in Western blot analysis to detect proteins with such modifications. In another example, modifications such as myristylation, may be detected on thin layer chromatography or reverse phase HPLC (see e.g.. Glover. 1988. Biochem. J. 250:485-91 ; Paige. 1988. Biochem J. 250:485-91 ).

Activity of signaling and cell cycle proteins and/or protein complexes is often mediated by a kinase activity. The present invention provides for analysis of kinase activity by assays such as the histone HI assay (see e.g., Delia et al.. 1997. Oncogene 14:2137-47).

The ESP-regulator can also be demonstrated to alter cell proliferation in cultured cells in vitro using methods which are well known in the art. Specific examples of cell culture models include, but are not limited to, for lung cancer, primary rat lung tumor cells (Swafford et al.. 1997, Mol Cell Biol. 17: 1366-1374) and large-cell undifferentiated cancer cell lines (Mabry et al.. 1991. Cancer Cells 3:53-58); colorectal cell lines for colon cancer (Park and Gazdar, 1996, J Cell Biochem. Suppl 24:131-141); multiple established cell lines for breast cancer (Hambly et al., 1997, Breast Cancer Res. Treat. 43:247-258; Gierthy et al, 1997, Chemosphere 34:1495-1505; Prasad and Church, 1997, Biochem. Biophys. Res. Commun. 232:14-19); a number of well-characterized cell models for prostate

5 cancer (Webber et al., 1996, Prostate, Part 1 29:386-394; Part 2 30:58-64; and Part 3 30: 136-142; Boulikas, 1997 ^', Anticancer Res. 17:1471-1505); for genitourinary cancers, continuous human bladder cancer cell lines (Ribeiro et al., 1997, Int. J. Radiat. Biol. 72:1 1-20); organ cultures of transitional cell carcinomas (Booth et al., 1997. Lab Invest. 76:843-857) and rat progression models (Vet et al., 1997, Biochim. Biophys Acta

10 1360:39-44); and established cell lines for leukemias and lymphomas (Drexler, 1994, Leuk Res. 18:919-927, Tohyama, 1997, Int. J. Hematol 65:309-317).

The ESP-regulator can also be demonstrated to inhibit cell transformation (or progression to malignant phenotype) in vitro. In this embodiment, cells with a transformed cell phenotype are contacted with one or more ESP-regulator, and examined for change in

1 characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo), for example, but not limited to, colony formation in soft agar, a more rounded cell moφhology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, or 0 expression of fetal antigens, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York, pp. 436-446).

Loss of invasiveness or decreased adhesion may also be used to demonstrate the anti-cancer effects of the ESP-regulator. For example, a critical aspect of the formation of a metastatic cancer is the ability of a precancerous or cancerous cell to detach from 5 primary site of disease and establish a novel colony of growth at a secondary site. The ability of a cell to invade peripheral sites is reflective of a potential for a cancerous state. Loss of invasiveness may be measured by a variety of techniques known in the art including, for example, induction of E-cadherin-mediated cell-cell adhesion. Such E-cadherin-mediated adhesion can result in phenotypic reversion and loss of invasiveness 0 (Hordijk et al. 1997. Science 278:1464-66).

Loss of invasiveness may further be examined by inhibition of cell migration. A variety of 2-dimensional and 3-dimensional cellular matrices are commercially available (Calbiochem-Novabiochem Coφ. San Diego. CA). Cell migration across or into a matrix may be examined by microscopy, time-lapsed photography or 5 videography. or by any method in the art allowing measurement of cellular migration. In a related embodiment, loss of invasiveness is examined by response to hepatocyte growth factor (HGF). HGF-induced cell scattering is correlated with invasiveness of cells such as Mardin-Darby canine kidney (MDCK) cells. This assay identifies a cell population that has lost cell scattering activity in response to HGF (Hordijk et al., 1997, Science 278:1464-66). Alternatively, loss of invasiveness may be measured by cell migration through a chemotaxis chamber (Neuroprobe/ Precision Biochemicals Inc. Vancouver, BC). In such assay, a chemo-attractant agent is incubated on one side of the chamber (e.g., the bottom chamber) and cells are plated on a filter separating the opposite side (e.g., the top chamber). In order for cells to pass from the top chamber to the bottom chamber, the cells must actively migrate through small pores in the filter. Checkerboard analysis of the number of cells that have migrated may then be correlated with invasiveness (see e.g.. Ohnishi, 1993, Biochem. Biophys. Res. Commun. 193:518-25).

The ESP-regulator can also be demonstrated to inhibit tumor formation in vivo. A vast number of animal models of hypeφroliferative disorders, including tumorigenesis and metastatic spread, are known in the art (see Table 317-1, Chapter 317, "Principals of Neoplasia," in Harrison's Principals of Internal Medicine, 13 th Edition, Isselbacher et al., eds., McGraw-Hill, New York, p. 1814; and Lovejoy et al., 1997, J. Pathol. 181 :130-135). Specific examples include for lung cancer, transplantation of tumor nodules into rats (Wang et al., 1997, Ann. Thorac. Surg. 64:216-219) or establishment of lung cancer metastases in SCID mice depleted of NK cells (Yono and Sone, 1997, Gan To Kagaku Ryoho 24:489-494); for colon cancer, colon cancer transplantation of human colon cancer cells into nude mice (Gutman and Fidler, 1995, World J. Surg. 19:226-234), the cotton top tamarin model of human ulcerative colitis (Warren, 1996. Aliment. Pharmacol. Ther. 10 Suppl 2:45-47) and mouse models with mutations of the adenomatous polyposis tumor suppressor (Polakis, 1997, Biochim. Biophys. Acta 1332:F127-F147); for breast cancer, transgenic models of breast cancer (Dankort and Muller, 1996, Cancer Treat. Res. 83:71-88; Amundadittir et al., 1996, Breast Cancer Res. Treat. 39:1 19-135) and chemical induction of tumors in rats (Russo and Russo, 1996, Breast Cancer Res. Treat. 39:7-20); for prostate cancer, chemically-induced and transgenic rodent models, and human xenograft models (Royai et al., 1996, Semin. Oncol. 23:35-40); for genitourinary cancers, induced bladder neoplasm in rats and mice (Oyasu, 1995. Food Chem. Toxicol 33:747-755) and xenografts of human transitional cell carcinomas into nude rats (Jarrett et al., 1995, J. Endourol 9: 1-7); and for hematopoietic cancers, transplanted allogeneic marrow in animals (Appelbaum, 1997 ^', Leukemia 11 Suppl. 4:S15-S17). Further, general animal models applicable to many types of cancer have been described, including, but not restricted to, the p53-deficient mouse model (Donehower, 1996, Semin. Cancer Biol. 7:269-278), the Min mouse (Shoemaker et al., 1997, Biochem. Biophys. Acta 1332:F25-F48), and immune responses to tumors in rat (Frey, 1997, Methods 12:173-188).

For example, a ESP-regulator can be administered to a test animal, preferably a test animal predisposed to develop a type of tumor, and the test animal subsequently examined for an decreased incidence of tumor formation in comparison with controls not administered the ESP-regulator. Alternatively, a ESP-regulator can be administered to test animals having tumors (e.g., animals in which tumors have been induced by introduction of malignant, neoplastic. or transformed cells, or by administration of a carcinogen) and subsequently examining the tumors in the test animals for tumor regression in comparison to controls not administered the ESP-regulator.

5.11 TREATMENT OR PREVENTION OF CANCER OR

A NEOPLASTIC DISEASE IN COMBINATION WITH CHEMOTHERAPY OR RADIOTHERAPY

Cancer or a neoplastic disease, including, but not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of a composition comprising a pharmaceutically acceptable carrier and a ESP-regulator compound. The compositions can comprise one or more ESP-regulator compounds, or a pharmaceutically acceptable salt thereof.

In certain embodiments, one or more ESP-regulator of the invention are used to treat or prevent cancer or neoplastic disease in combination with one or more anti-cancer, chemotherapeutic agents including, but not limited to, methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides. campathecins. bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone. asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel. and docetaxel. In a preferred embodiment, one or more ESP-regulator of the invention is used to treat or prevent cancer or neoplastic disease in combination with one or more chemotherapeutic or other anti- cancer agents including, but not limited to those presented in Table 1.

TABLE 1 CHEMOTHERAPEUTICS AND OTHER ANTI-CANCER AGENTS

Radiation: γ-radiation Alkylating agents Nitrogen mustards: cyclophosphamide

Ifosfamide trofosfamide

Chlorambucil

Nitrosoureas: carmustine (BCNU)

Lomustine (CCNU)

Alkylsulphonates busulfan

Treosulfan Triazenes: Dacarbazine

Platinum containing compounds: Cisplatin carboplatin

Plant Alkaloids Vinca alkaloids: vincristine

Vinblastine

Vindesine

Vinorelbine

Taxoids: paclitaxel

Docetaxol

DNA Topoisomerase Inhibitors Epipodophyllins: etoposide

Teniposide

Topotecan

9-aminocamptothecin campto irinotecan crisnatol mvtomvcins: mytomycin C Mytomycin C Anti-metabolites Anti-folates: DHFR inhibitors: methotrexate Trimetrexate IMP dehydrogenase Inhibitors: mycophenolic acid

- Tiazofurin

Ribavirin

EICAR

Ribonuclotide reductase Inhibitors: hydroxyurea deferoxamine

Pyrimidine analogs: Uracil analogs 5-Fluorouracil

Floxuridine

Doxifluridine

Ratitrexed

Cytosine analogs cytarabine (ara C)

Cytosine arabinoside fludarabine

Purine analogs: mercaptopurine Thioguanine

Hormonal therapies: Receptor antagonists: Anti-estrogens Tamoxifen

Raloxifene megestrol

LHRH agonists: goscrclin

Leuprolide acetate

Anti-androgens: flutamide bicalutamide

Retinoids/Deltoids Vitamin D3 analogs: EB 1089 CB 1093 KH 1060

Photodyamic therapies: vertoporfin (BPD-MA) Phthalocvanine photosensitizer Pc4 Demethoxy-hypocrellin A (2BA-2-DMHA)

Cytokines: Interferon-α Interferon-γ Tumor necrosis factor

Others: Isoprenylation inhibitors: Lovastatin Dopaminergic neurotoxins: 1 -methyl-4-phenylpyridinium ion Cell cycle inhibitors: staurosporine

Actinomycins: Actinomycin D Dactinomycin

Bleomycins: bleomycin A2 Bleomycin B2 Peplomycin

Anthracyclines: daunorubicin

Doxorubicin (adriamycin)

Idarubicin

Epirubicin

Pirarubicin

Zorubicin

Mitoxantrone

MDR inhibitors: verapamil

Ca²⁺ATPase inhibitors: thapsigargin

In other embodiments, a composition comprising one or more ESP-regulator is administered along with radiation therapy and/or with one or a combination of chemotherapeutic agents, preferably with one or more chemotherapeutic agents with which treatment of the cancer has not been found to be refractory. The ESP-regulator can be administered to a patient that has also undergone surgery as treatment for the cancer.

In another specific embodiment, the invention provides a method to treat or prevent cancer that has shown to be refractory to treatment with a chemotherapy and/or radiation therapy.

In a specific embodiment, a composition comprising one or more ESP- regulator is administered concurrently with chemotherapy or radiation therapy. In another specific embodiment, chemotherapy or radiation therapy is administered prior or subsequent to administration of a present composition, preferably at least an hour, five hours, 12 hours, a day. a week, a month, more preferably several months (e.g. , up to three months), subsequent to administration of a therapeutic of the invention.

The chemotherapy or radiation therapy administered concurrently with, or prior or subsequent to. the administration of a present composition can be accomplished by any method known in the art. The chemotherapeutic agents are preferably administered in a series of sessions, any one or a combination of the chemotherapeutic agents listed above can be administered. With respect to radiation therapy, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, may also be administered to expose tissues to radiation.

Additionally, the invention provides methods of treatment of cancer or neoplastic disease with a present composition as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or may prove too toxic. e.g., results in unacceptable or unbearable side effects, for the subject being treated. The subject being treated with the present compositions may, optionally, be treated with other cancer treatments such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.

5.12 CANCER AND NEOPLASTIC DISEASE TREATABLE OR PREVENTABLE

Cancers or neoplastic diseases and related disorders that can be treated or prevented by administration of the present compositions include but are not limited to those listed in Table 2 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed. J.B. Lippincott Co., Philadelphia):

TABLE 2CANCERS AND NEOPLASTIC DISORDERS

Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia , myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia 15 Polycythemia vera

Lymphoma

Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrom's macroglobulinemia Heavy chain disease 2 Solid tumors sarcomas and carcinomas fϊbrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma 25 angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor T leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma 5 basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine cancer testicular tumor lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma meningioma melanoma neuroblastoma retinoblastoma

In specific embodiments, cancer, malignancy or dysproliferative changes (such as metaplasias and dysplasias). or hypeφroliferative disorders, are treated or prevented in the ovary, breast, colon, lung, skin, pancreas, prostate, bladder, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented.

In a preferred embodiment the present compositions are used to treat or prevent cancers in which the cancer cells express the estrogen receptor.

In a highly preferred embodiment, the present compositions are used to treat or prevent cancers including those of the breast or prostate.

In specific embodiments of the invention, the present compositions are used to inhibit the growth of a cell, said cell being derived from a cancer or neoplasm in Table 2 or herein. 5.13 KITS

The invention further provides kits that facilitate the use of ESP-regulators described herein. The kits described herein may be conveniently used, e.g., in clinical settings to treat a patient suffering from a disorder involving inappropriate cell proliferation. The kits comprise one or more containers filled with one or more ESP- regulators of the invention. In certain preferred embodiments, e.g., when administered to a patient to treat cancer, the kit may also contain one or more other chemotherapeutic agents useful for inhibiting cancer to be administered in combination with the ESP-regulators of the invention. In various other embodiments, the kit can also comprise, e.g., a buffering agent, a preservative, or a stabilizing agent. The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Optionally associated with such container(s) can be instructions for use of the kit as a treatment along with a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

6. EXAMPLES 6.1 BRCAl GENE TO MODULATE THE TRANSCRIPTIONAL ACTIVITY OF ER-α To assess the effect of BRCAl on estrogen response, we measured the ability of the BRCAl gene to modulate the transcriptional activity of ER-α in transient transfection assays. Wild-type BRCAl expression plasmid (wtBRCAl) was created by cloning BRCAl cDNA into the pcDNA3 vector (Invitrogen) using artificially engineered 5' Hind III and 3' Not I sites. Cells were assayed for stimulation of ERE-TK-Luc reporter activity by 178- estradiol (E2). ERE-TK-Luc is composed of the vitellogenin A2 estrogen-responsive enhancer (ERE) controlling a minimal thymidine kinase promoter (TK81 ) that is operably linked to the luciferase gene, in plasmid pGL2 (Henttu et al.. 1997, Mol. Cell. Biol. 17:1832). Proliferating cells at 50-70% of confluency in 24-well dishes were incubated overnight with 0.5 μg of each vector (unless otherwise indicated) in serum-free DMEM containing Lipofectin (Life Technologies). The total transfected DNA was kept constant by addition of control vector. Cells were washed, incubated in serum-free phenolphthalein-free DMEM (0.2 ml per well) ± 17β-estradiol (E2, 1 μM) for 24 hr. and harvested for luciferase assays.

Human prostate cancer cell line DU-145 lacks ER-α; but when supplied with exogenous ER-α, these cells became competent for estrogenic signalling, as demonstrated by estradiol (E2) stimulated activation of estrogen-responsive reporter ERE-TK-Luc (Fig. la). Equally strong inhibition of ER-α signalling by wtBRCAl was observed in breast cancer cell line T47D, in two other human breast cancer cell lines (MCF-7 and MDA-MB- 231), and in two other human prostate cancer cell lines (LNCaP and TsuPr-1) (Figs, la and

5 lb). However, three cervical cancer cell lines showed relatively weak inhibition of ER-α signalling by wtBRCAl : E2-stimulated reporter activity in the presence of wtBRCAl was about 40-50% of that in the presence of empty vector (Fig. lb).

Mutations of BRCAl cause an increased risk for breast, ovarian, and prostatic cancers (Mild et al., 1994, Science 266:66-71 ; Streuwing, 1997, New Engl J Med

10 336:1401-1408; Gayther et al., 1995, Nat Genet 11 :428433). We tested a panel of mutant BRCAl genes, including tumor-associated and artificially engineered mutants (Fig. lc). to assess the impact of mutations on the ability of BRCAl to inhibit ER-α activity.

Mutant BRCAl vectors were created by modification of the BRCAl cDΝA in the pcDΝA3 vector. BRCAl mutants 5382insC and C53655G were a gift from Dr.

15 Barbara Weber. Expression of the BRCAl genes were confirmed by semi-quantitative RT- PCR, using three sets of primers mapping to different sites in BRCAl (Fig. Id). Semi- quantitative RT-PCR assays were performed as described earlier (Fan et al, 1998,. Oncogene 16:3069-3083). The BRCAl primer sequences (5' - 3') and the locations within the cDNA sequence (GenBank accession number U15595) were as follows: primer #1, ⁰ sense TTG CGG GAG GAA AAT GGG TAG TTA, antisense GAA GTA GTA AGT GGG AAC CGT GT (bp 5239 to 5525, 287-bp product); primer #2, sense GAG TAA CAA GCC AAA TGA ACA G, antisense TGG ATA CTT AAA GCC TTC TGT G (bp 2159 to 2616, 457-bp product); and primer #3, sense AAG GAA CCT GTC TCC ACA AAG T, antisense GTC AAG CTG AAA AGC ACA AAT G (bp 213 to 408. 196-bp product). The primers for the control gene β-actin were: sense TTG TTA CCA ACT GGG GAC GAT A. antisense TCG TCC TTC TAG TTC TAG (bp 265 to 1028. 764-bp) All three primers gave amplified products of the expected sizes. Primer #1 yielded increased levels of the 287-bp product in cells transfected with wtBRCAl, 5677insA. RXRXH. T300G. C5365G, and 5382insC.but mutations that truncate the BRCAl mRNA 5' to the primer (ΔBamHI, ΔKpnl, ΔEcoRI) failed to yield an increase in the 287-bp product, relative to untransfected or empty vector- transfected controls. Primer #2 yielded an increase in the 457-bp product in cells transfected with wtBRCAl, 5677insA, ΔBamHI. RXRXH, T300G, 5382insC, and C5365G, all of which yield mRNAs that include the amplified cDNA segment. On the other hand. ΔKpnl and ΔEcoRI. which encode mRNAs truncated 5' to the primer, failed to yield an increase in the 457-bp product. Primer #3. which maps to the amino-terminus of BRCAl . gave increased 196-bp product in all but control transfections. These findings confirm the expression of all BRCAl mRNAs, except 185delAG, which is not detected because it is truncated 5' to the primers. Because of the tiny size of the predicted product (38 aa), 185deLAG is likely to act as a null mutation.

5 We tested the ability of the BRCAl cDNAs to inhibit estradiol (E2) induced,

ER-α mediated ERE-TK-Luc reporter activation in DU-145 and T47D cells. BRCAl - 5677insA (Y1853term), which corresponds to a tumor-associated mutation that deletes 11 aa and ablates activity of the carboxyl-terminal transactivation domain (TAD) (Monteiro et al.. 1996, Proc Natl Acad Sci USA 93:13595-13599), abolished reporter activity (Fig. le).

10 However, two other carboxyl-terminal tumor mutations, 5382insC (Q1756term) and

C5365G (P1749R), failed to inhibit ER-α activity. These mutations also abolish the activity of the TAD.

Since the tumor-associated BRCAl mutant 5677insA strongly suppressed ER-α activity at the single plasmid dose tested, we compared the relative ability of

15 5677insA vs wtBRCAl to inhibit ER-α at a series of different plasmid doses. When tested side-by-side, the wtBRCAl gene was about 5-fold more effective (μg/μg) than the 5677insA gene at inhibiting ER-α transcriptional activity in DU-145 and T47D cells (Fig. If), suggesting that the 5677insA mutation significantly weakens the ability of BRCAl to inhibit ER-α activity. Taken together, these results suggest that a site within the minimal 0 TAD of BRCAl (last 100-120 aa) is required for the inhibition of ER-α activity.

A tumor-associated mutation of the amino-terminal RING domain (T300G) and a mutation of the retinoblastoma binding motif LXCXE (RXRXH) abolished ER-α inhibitory activity in both cell lines (Fig le). A second tumor-associated point mutation of the RING domain (T31OG) also failed to suppress ER-α activity (data not shown). 5 Examination of deletion mutants revealed that 185deAG. a tumor-associated mutation that truncates BRCAl in the RING domain, and BRCAl ΔEcoRI. which truncates BRCAl just before LXCXE, failed to inhibit ER-α activity (Fig. le). Mutants that truncate BRCAl after LXCXE but before the TAD (ΔKpnI. ΔBamHI) yielded 50% inhibition in DU-145 and no inhibition in T47D. Since E2 typically induces reporter activity by (lOO-lOOO)-fold and 0 wtBRCAl reduces the activity to near baseline (one-fold stimulation), a 50% reduction of activity is probably not meaningful. Differences in the degree of inhibition of ER-α activity by different BRCAl transgenes could not be attributed to differences in transfection efficiency, since similar results were obtained when a control plasmid (pRSV-β-gal) was cotransfected and reporter activity was normalized by β-galactosidase (data not shown). These 5 findings suggest that inhibition of ER-α requires several functional domains, including the RING and LXCXE motifs, as well as another domain located carboxyl-terminal to the BamHI site (aa 1313).

These experiments document the ability of BRCAl genes to inhibit an artificial E2-dependent reporter gene. We studied two endogenous E2-responsive breast cancer cell lines (MCF-7 and T47D) to determine if the wtBRCAl gene could inhibit the physiologic expression of two endogenous E2-responsive genes, pS2 and cathepsin D (Jeltsch et al., 1987. Nucleic Acids Res 15:1401-1414; Faust et al., Proc Natl Acad Sci USA 82: 4910-4914). Exposure to E2 (1 μM) for 24 hr induced significant increases in the production of pS2 and cathepsin D proteins as evidenced by Western blot analysis (anti- pS2= V3030, mouse monoclonal, Biomeda Coφ., 1 :100 and anti-cathepsin D= 06-467. rabbit polyclonal. Upstate Biotechnol.. 1 :200). This increase was abrogated or reduced in cells transfected with wtBRCAl. as compared with the pcDNA3 vector (Fig. lg). The E2- induced stimulation of pS2 expression and its inhibition by the wtBRCAl gene was also observed at the mRNA level, by Northern blotting (not shown). In these experiments, the wtBRCAl gene did not usually reduce pS2 or cathepsin D expression to below the unstimulated control levels.

These results demonstrate that BRCAl is a specific repressor of transcription. Thus, the discovery of the invention indicates that the regulation of the estrogen receptor activity plays an important role in the development of cancers associated with BRCAl mutation. The present invention discloses a novel use for the regulation of the estrogen receptor activity and signalling in the treatment or prevention of cancers or disease associated with BRCAl mutation.

6.2 BRCAl AND ESTROGEN RECEPTOR INTERACTING DOMAINS

To determine if a physical association of BRCAl and ER-α night contribute to suppression of ER-α activity, we tested an ER positive breast cancer cell line (MCF-7) b> immunoprecipitation (IP)-Western blotting. Subconfluent oroliferating cells in 150 cm² dishes were harvested, and nuclear extracts were prepared (Dignam et al.. 1983. Nucleic Acids Res 5:1475-1489). Each IP was carried out using 6 μg of antibody or antibody combination and 1000 μg of nuclear extract. The IP antibodies were as follows: BRCAl (Ab-1 + Ab-2 + Ab-3, Oncogene Research Products), ER-α (mouse monoclonal, catalog #17529, Upstate Biotechnology), and control (normal mouse IgG). Precipitated proteins were collected, washed, and boiled in Laemmeli sample buffer. These samples were then subjected to Western blotting- Equal aliquots of total protein (100 μg per lane) were electrophoresed on a 4-13% SDS-polyacrylamide gradient gel. transferred to nitrocellulose membranes (Millipore), and blotted using as primary antibodies: BRCAl (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1 :200 dilution), ER-α (11-184, rabbit polyclonal, Santa Cruz, 1 : 1000). Proteins were visualized using the enhanced chemiluminescence system, with colored markers (BioRad) as size standards.

ER-α was detected in the BRCAl IP of MCF-7 cell nuclei and vice versa (Fig. 2a). Control IPs using non-immune antibodies failed to yield BRCAl or ER-α protein bands. While BRCAl was precipitated from DU-145 cells, there was little or no ER-α detected in the ER-α or BRCAl IPs (Fig. 2a), consistent with the ER negative status of this cell line (Webber et al, 1997, The Prostate 30:58-64). On the other hand, DU-145 cells transfected with wtBRCAl (to enhance expression) and ER-α expression vectors showed association of BRCAl and ER-α, in the presence or absence of E2 (Fig. 2b). These findings indicate that BRCAl and ER-α associate within the same protein complex, independently of estrogen. To determine if the association of BRCAl and ER-α is due to a direct physical interaction, we performed GST capture assays using in vitro translated (IVT) BRCAl and beads coated with a series of GST-ER fusion proteins. GST bead assays were performed essentially as described before (Wang et al., 1993, Science 260:1330-1335). ³⁵S-methionine-labeled proteins were prepared by in vitro transcription (using the T7 promoter of pcDNA3) and translation. The GST fusion proteins were generated from cDNAs cloned into the pGEX vector, expressed in E. coli, and purified by affinity chromatography (Webb P et al., 1998, Mol Endocrinol 12:1605-1618). The GST-BRCA1 304-1863 vector was provided by Dr. Barbara Weber. Labeled proteins were incubated with GST (control) or GST fusion proteins for 4 hr at 4°C, recovered using GST agarose beads. eluted in boiling sample buffer, and analyzed by SDS-PAGE autoradiograph)'. GST fusion proteins were visualized by Western blotting, using an anti-GST mouse monoclonal (B-14. Santa Cruz. 1 :10,000).

Fig. 3a shows schematic diagrams of the series of ER-α constructs tested: Fig. 3b shows the various GST-ER fusion proteins visualized by Western blotting, using an anti-GST antibody. Among six GST-ER proteins tested, three carboxyl-terminal ER-α peptides (ER 282-420, ER 282-595, ER 393-595) captured IVT-wtBRCAl ; while three more amino-terminal ER-α peptides (ER 1-185, ER 1-282. ER 282-337) failed to capture wtBRCAl (Fig. 3c).

To delineate the binding site(s) for ER-α on BRCAl, we tested various IVT BRCAl proteins (see Fig. lc) for binding to GST-ER 282-420. An autoradiograph of the IVT BRCAl translation products is shown in Fig. 3d, which illustrates the relative translational efficiency for the different products. All BRCAl proteins tested bound to GST-ER 282-420, with the most binding for BRCAl ΔEcoRI (Fig. 3e). The ratio of bound:input BRCAl was higher for carboxyl-terminal peptides than for full-length or near full-length BRCAl proteins (Fig. 3f), suggesting interference to binding by a carboxyl- terminal portion of BRCAl, but this remains to be proven. Finally, a fusion protein of GST and N-terminally deleted BRCAl (GST-BRCA1 304-1863) failed to pull down IVT ER-α (data not shown). These findings suggest an E2-independent interaction of BRCAl and ER- α involving aa 1-302 of BRCAl and the AF-2/LBD region of ER-α. The failure of the BRCAl ΔEcoRI gene to inhibit ER-α activity suggests that binding to ER-α is insufficient for inhibition and that a more carboxyl-terminal domain of BRCAl is also required. However, co-transfection of BRCAl ΔEcoRI resulted in a dose- dependent rescue of the wtBRCAl -mediated inhibition of ER-α activity (Fig. 2c), suggesting that an amino-terminal fragment of BRCAl (aa 1-302) can function as a dominant inhibitor of the full-length wtBRC A 1.

6.3 EFFECT OF BRCAl ON PROTEINS IN THE

ESTROGEN RECEPTOR COACTIVATING COMPLEX

DU-145 cells stably expressing wtBRCAl or 5677insA showed decreased levels of the transcriptional adaptor p300 (Fan et al, 1998, Oncogene 16:3069-3083). p300 and its homolog CBP (CREB binding protein) are components of the ER co-activator complex (Hanstein et al., 1996, Proc Natl Acad Sci USA 93:11540-11545; Smith et al., 1996. Proc Natl Acad Sci USA 93:8884-8885; Kamei et al. . 1996, Cell 85:403-414). Therefore, we examined the ability of different BRCAl genes to regulate their expression. Cells were lysed and extracts were Western blotted as in Section 6.2 (anti-p300 = 05-267. mouse monoclonal, Upstate Biotechnology, 4 μg/ml; anti-CBP =C-20, rabbit polyclonal, Santa Cruz, 1 :400). wtBRCAl and 5677insA caused down-regulation of p300 at 24 hr post transfection in both DU-145 and T47D cells: while mutant BRCAl genes that gave partial or no inhibition of ER-α activity (T300G. RXRXH. ΔKpnI. ΔEcoRI. 185delAG, 5382insC. C536SG) had no effect on p300 protein levels (Fig. 4a & b). The down-regulation of p300 by wtBRCAl and 5677insA was also observed at the mRNA level and was not reversed by proteasomal inhibitors (data not shown). None of the BRCAl genes caused a reduction of CBP in either cell type (Fig. 4a & b). In contrast. wtBRCAl had no effect on p300 (or CBP) levels in three cervix cancer cell lines (Fig. 4c). consistent with the relatively weak inhibition of ER-α activity by BRCAl in this cell type (Fan et al., 1999, Science 284: 1354- 1356).

We reported that DU-145 cells transfected with wt BRCAl or BRCAl - 5677insA showed decreased expression of the transcriptional adaptor p300. compared to parental and vector transfected cells (Fan et al., 1998, Oncogene 16:3069). p300 and its functional homolog CBP (CREB binding protein) are components of the ER-α coactivator complex required for full transcriptional activation of ER-α (Kamei et al.. 1996. Cell 85:403, Hanstein et al, 1996., Proc. Natl. Acad. Sci. U.S.A. 93:11540; Smith et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8884). p300 interacts with ER-α and steroid receptor coactivator- 1 (SCR-1) via - and C-terminal domains, respectively, and may link ER-α to the basal transcriptional machinery via its E1A/TFIIB binding domain, which also binds p300/CBP-associated factor PCAF and transcription factors c-Jun and c-Fos ( Kamei et al.. 1996, Cell 85:403; Hanstein et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:11540; Smith et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8884; Wang et al, 1997, Mol. Cell. Biol. 17:519). Thus, the p300, PCAF, and CBP, or derivatives or mutants thereof, may act as ESP- regulators of the invention.

6.4 REGIONS OF THE ESTROGEN RECEPTOR COACTIVATING

COMPLEX IMPORTANT FOR INTERACTION WITH BRCAl AND ESTROGEN RECEPTOR

We tested the effects of wild-type (wt-p300) and mutant p300 expression vectors (Fig. 5a) on wtBRCAl inhibition of ER-α activity. In the absence of wtBRCAl, wt- p300 and several mutant p300 genes caused a moderate (up to a 2 or 3-fold) increase in E2-stimulated ER-α/ERE-TK-Luc reporter activity. Co-transfection of wt-p300 rescued the wtBRCAl inhibition of reporter activity in both DU-145 and T47D cells (Fig. 5b). p300 mutants missing the histone acetyl transferase (HAT) or Bromo domain also rescued the inhibition; but a mutant missing the El A/TFIIB binding site, corresponding to cysteine/histidine rich domain CH3. failed to rescue the inhibition.

Interestingly, a p300 mutant containing only CH3 and surrounding sequences p300 1514-1922) fully rescued the inhibition, suggesting that the El A/TFIIB site is essential for the rescue. A wt-CBP expression vector also rescued the wtBRCAl inhibition of ER-α in both cell types (Fig. 5c). indicating that p300 and CBP are similar with regard to the rescue function. In contrast top300 and CBP. expression vectors for two other nuclear receptor co-activators. PCAF (p300/CBP-associated co-factor) (Wang et al.. 1997, Mol Cell Biol 1 ':519-527) and GRIP 1 (glucocorticoid receptor-interacting protein) (Ding et al., 1998. Mol Endocrinol 12:302-313) failed to rescue the wtBRCAl inhibition of ER-α activity (data not shown). Co-transfection of wt-p300 but not p300 1514-1922 blocked the wtBRCA 1 -mediated down-regulation of p300 levels (Fig. 5d). The finding that p300 1514- 1922 rescues the wtBRCAl inhibition of ER-α activity but does not restore the levels of endogenous p300 suggests that the p300 rescue is distinct from its co-activator function. We performed GST capture assays to delineate the interaction between p300/CBP and ER-α. These assays revealed an E2-independent interaction between the AF- 2/LBD region of ER-α and the carboxyl-terminal, but not the amino-terminal, region of CBP (aa 1680-2441) (Fig. 6a). As a positive control, GRIPl exhibited E2-dependent binding to ER-α AF-2/LBD. Fig. 6b shows the binding of ER-α and other proteins to a series of overlapping fragments of the carboxyl-terminal region of CBP. The ER-α binding site of CBP mapped to the CH3 domain, the region found to be essential for rescue of the wtBRCAl inhibition of ER-α activity. The CH3 domain also interacted with c-Fos and TFIIB (positive controls) but not PR-B (progesterone receptor-B) or GRIPl; and the binding of ER-α to the two overlapping CBP fragments containing the CH3 region was

E2-independent (Fig. 6c). Lastly, we showed that the p300 1514-1922 fragment, which contains the CH3 region, interacts with the same region of ER-α (aa 282-420) that binds wtBRCAl. And preincubation of GST-ER 282-420 with IVT p300 1514-1922 or wtBRCAl competed out the binding of IVT wtBRCAl or p300 1514-1922, respectively (Fig. 6d). The GST competition assays were performed as described in Section 6.2, except that GST-ER 282-420 was pre-incubated for 3 hr at 4°C with different quantities of IVT competing protein.

These results suggest that other mechanisms besides the down-regulation of p300/CBP may contribute to inhibition of ER-α activity by BRCAl . Thus, the invention provides inhibition of the estrogen signalling pathway (e.g., via ER-α.) by one or more molecules. BRCAl is linked to the RNA polymerase II holoenzyme via its C-terminal

TAD; and p300 and CBP are also present within the RNA polymerase II complex (Scully et al.. 1997, Proc. Natl. Acad. Sci. U.S.A. 94:5605; Neish et al.. 1998, Nucleic Acids Res. 26:847). An alternatively spliced form of BRCAl may interact with CBP in vivo and in vitro, via the CH3 domain of CBP (Cui et al.. 1998. Oncol. Rep. 5:591 ). Further. BRCAl

(through an N-terminal domain) interacts with p53 and modulates p53 transcriptional activity (Zhang et al.. 1998. Oncogene 16:1713; Ouchi et al., 1998. Proc. Natl. Acad. Sci.

U.S.A. 95:2302). The present invention indicates that BRCAl may function, in pan, as an adaptor protein, with negative or positive transcriptional regulatory activity and such regulation is useful in the treatment and prevention of disease associated with BRCAl mutation.

5 The present invention demonstrates that regardless of mechanism, failure to repress ER-α activity contributes to breast cancer in BRCAl mutation carriers. This discovery is consistent with the finding that 8/9 mutant BRCAl genes (including four tumor-associated mutations (5382insC, C5365G. T300G, 185delAG), a mutation of the LXCXE site, and three deletion mutants) showed decreased or no ER-α repressor activity.

10 Two of these mutations, 5382insC and 185delAG, are very common and occur in 0.3% and 1%, respectively, of Ashkenazi Jews (Streuwing et al., 1997. New Engl J. Med. 336:1401). One rare tumor-associated mutant BRCAl gene (5677insA) (Friedman et al., 1994, Nat Genet. 8:399) retained ER-α repressor activity. Retention of this activity may explain the rarity of this mutation in breast cancer families. Additionally, associated genetic factors may also contribute to the high breast cancer incidence in BRCAl kindreds (Streuwing et al., 1997, New Engl. J. Med. 336:1401). Furthermore, the frequency of ER-α negativity (Johannsson et al., 1997, Ewr. J. Cancer 33:362; Loman et al., 1998, Cancer 83:310) and of chromosomal deletions (Tirkkonen et al., 1997, Cancer Res. 57:1222) are higher in BRCAl

2fl mutant than sporadic breast cancers. Thus, the accumulation of other genetic alterations may allow estrogen-independent growth despite a BRCAl mutation that retains the ability to repress ΕR-α.

Both in vivo and in vitro studies have linked expression of BRCAl with differentiation of specific cell types such as mammary epithelium; but molecular targets of

2- BRCAl for mammary differentiation have not been identified (Marquis et al.. 1995, Nat. Genet 1 1 : 17; Rajan et al.. 1996. Proc. Natl Acad. Sci U.S.A. 93:3078; Rajan et al.. 1997. Devel. Biol 184:385). The discovery of the present invention indicates that BRCAl may function, in part, to suppress estrogen-dependent mammary epithelial proliferation b> inhibiting ΕR-α mediated transcriptional pathways related to cell proliferation. Therefore.

30 modulation of the estrogen signalling pathway or the estrogen receptor activation by the methods of the invention is useful in the treatment and prevention of proliferative disease (such as breast cancer). A model for this putative tumor suppressor function of BRCAl is shown in Fig. 7. Two noteworthy features of this model are that: 1) induction of BRCAl expression that occurs when resting cells re-enter the cycle (Gudas et al.. 1995, Cancer Res

35 55:4561 : Marks et al., 1997. Oncogene 14: 1 15) serves as a feedback mechanism to limit estrogen stimulation of proliferation; and 2) BRCAl is also postulated to promote mammary cell differentiation, by an, as yet, unidentified transcriptional pathway(s). The present invention serves to illustrate the transcriptional pathway (e.g., via the ER-α activation) which plays a role in proliferation and differentiation.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incoφorated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Claims

WHAT IS CLAIMED:

A method of identifying modulators of the estrogen signalling pathway comprising;

(a) contacting a cell expressing an estrogen receptor and BRCAl with an estrogen receptor ligand and a test compound;

(b) measuring the amount of proliferation exhibited by the cell;

(c) comparing the amount of measured proliferation of step (b) to that of a cell contacted solely with the estrogen receptor ligand wherein a difference between the amount of proliferation exhibited by the two cells indicates that the test compound is a modulator of the estrogen signalling pathway.

A method of identifying modulators of the estrogen signalling pathway comprising;

(a) contacting a cell expressing an estrogen receptor, BRCAl and a reported gene whose expression is controlled by an estrogen response enhancer with an estrogen receptor ligand and a test compound;

(b) measuring the amount of transcription or translation of the reporter gene exhibited by the cell;

(c) comparing the amount of measured transcription or translation of step (b) to that of a cell contacted solely with the estrogen receptor ligand wherein a difference between the amount of transcription or translation exhibited by the two cells indicates that the test compound is a modulator of the estrogen signalling pathway.

3. The method of Claim 2. wherein the reporter gene is a luciferase gene, a chloramphenicol acetyltransferase gene, a human growth hormone gene, a β-galactosidase gene or a green fluorescent protein gene.

4. A method of identifying individuals at risk of developing cancer b} examining the nucleotide sequence their BRCAl domains essential for function, wherein the domains are any one in the group comprising the N-terminal RING finger domain, the consensus retinoblastoma binding motif and a domain in the C-terminal 550 amino acids.

5. The method of Claim 4 wherein the cancer is breast, ovarian, or prostate cancer.

6. A method of preventing or treating a proliferative disease in individuals identified in accordance with Claim 4, comprising administering an effective amount of a compound capable of modulating or inhibiting an estrogen signalling pathway in a host such that cell proliferation is decreased or inhibited.

7. The method of Claim 6, wherein the proliferative disease is cancer.

8. The method of Claim 6, wherein the cancer affects breast ovarian or prostate tissue.

9. The method of Claim 6 wherein the compound inhibits transcriptional activation through estrogen response enhancers.

10. The method of Claim 9 where in the compound inhibits binding of at least one component of the estrogen coactivating complex to estrogen response enhancers.

11. The method of Claim 10 wherein the component of the estrogen coactivating complex is p300 or CBP

12. A method for decreasing or inhibiting tumor growth in a mammal comprising administering to said mammal an amount of a compound that inhibits signalling by the estrogen receptor through estrogen responsive enhancers effective to decrease or inhibit tumor growth in said mammal.

- 5: