HK1172351A - Estrogen receptors and methods of use - Google Patents

Description

Estrogen receptors and methods of use

The application is a divisional application of Chinese patent application 'estrogen receptor and use method' with application number 200580007467.8(PCT/US2005/007857) and application date 3, 10, 2005.

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This application claims the benefit of U.S. provisional application serial No. 60/552,067 filed on 3/10/2004 and 60/643,469 filed on 13/1/2005, each of which is incorporated herein by reference.

Government funding

The invention described herein was made with the support of the Department of health and Human Services (Department of health and Human Services) grant number CA 84328. The united states government has certain rights in the invention.

Background

Estrogens are a generic term for steroid compounds formed in the ovary, testis and possibly adrenal cortex. Examples of estrogens and compounds having estrogenic activity include diethylstilbestrol, diethylstilbestrol diphosphate, diethylstilbestrol, estragole, polyestradiol, bromophenestrol, cletholenetetrol, hexadienestrol, diethylstilbestrol, proestrol, hydroxyestrone diacetate, 6, 9-dehydroequilenin, equilenin, estradiol, estriol, estrone, ethinyl estradiol, mestranol, mexestrol, estriol cyclopentyl ether, and ethinyl estradiol. Estrogens regulate a variety of physiological processes in reproductive tissues and breast, cardiovascular, bone, liver and brain tissues. Estrogens are also used in oral contraceptives. Other uses of estrogens include alleviating menopausal complaints, inhibiting lactation, and treating osteoporosis, threatened abortion, and various functional ovarian disorders. Antiestrogens are used to treat metastatic breast cancer and advanced prostate cancer.

The action of estrogens is mediated by estrogen receptors. The first Estrogen Receptor (ER) was cloned in 1986 (Green et al, Nature, 320: 134 (1986)) and Greene et al, Science, 231: 1150 (1986)). Prior to 1995, it was thought that there was only one estrogen receptor responsible for all the physiological and pharmacological effects of natural and synthetic estrogens and antiestrogens. However, in 1995, a second estrogen receptor was cloned (Kuiper et al, PNAS, 93: 5925 (1996)). The first estrogen receptor found is now called estrogen receptor- α (ER- α), while the second estrogen receptor is called estrogen receptor- β (ER- β).

ER- α and ER- β share a common structural system (Zhang et al, FEBS Letters, 546: 17(2003) and Kong et al, biochem. Soc. trans., 31: 56 (2003)). Both consist of 3 independent but interacting functional domains: an N-terminal A/B domain, a C or DNA-binding domain and a D/E/F or ligand-binding domain (FIG. 1). The N-terminal domain of ER- α encodes a ligand-independent activation function (AF-1), a region that participates in the interaction with co-activators and transcriptional activation of target genes. The DNA-binding domain or C-domain contains 2 zinc fingers, which play an important role in receptor dimerization and binding to specific DNA sequences. The C-terminal D/E/F domain is a ligand-binding domain that mediates ligand binding, receptor dimerization, nuclear translocation, and ligand-dependent transactivation function (AF-2). The relative contribution of AF-1 and AF-2 to transcriptional control varies in a cell-specific and DNA promoter-specific manner (Berry et al, EMBO J., 9: 2811(1990) and Tzukerman et al, mol. endogrin., 8: 21 (1994)).

It has been shown that the 46-kDa ER-alpha isoform (A/B or AF-1 domain) lacking the first 173 amino acids of the full-length gene product of the ER-alpha gene results from alternative splicing of the ER-alpha gene skipping exon 1 (Flouriot et al, EMBO J., 19: 4688 (2000)). This alternative splicing event results in an mRNA with AUG in frame with the remainder of the original open reading frame within the Kozak sequence that facilitates translation initiation. Thus, this new isoform of ER- α is referred to as ER- α 46, while the original isoform is referred to as ER- α 66(Flouriot et al, EMBOJ., 19: 4688 (2000)). ER- α 46 forms a homodimer and binds to the Estrogen Response Element (ERE), and it can also form a heterodimer with ER- α 66(Flouriot et al, EMBO J., 19: 4688 (2000)). ER- α 46 homodimers showed higher affinity for ERE than ER- α 66 homodimers. Moreover, ER- α 46/66 heterodimers form preferentially over ER- α 66 homodimers, and ER- α 46 acts competitively to inhibit transactivation mediated by the AF-1 domain of ligand-bound-ER- α 66, but does not affect AF-2-dependent transactivation (Floutiot et al, EMBO J., 19: 4688 (2000)). Thus, ER- α 46 is believed to be a naturally occurring isoform of ER- α that regulates estrogen signaling mediated by the AF-1 domain of ER- α 66.

ER- α can be expressed in approximately 15-30% of luminal (luminal) epithelial cells, but not in any other cell type in normal human breast at all. Double-labeled immunofluorescence techniques revealed that ER- α -expressing cells can be separated from cells labeled with proliferation markers in normal human and rodent mammary glands (Clarke et al, Cancer Res., 57: 4987 (1997)). ER-alpha expression increases in the earliest stages of ductal hyperplasia, even more with increasing allotypes, so that most cells in atypical ductal hyperplasia and ductal carcinoma in situ at the low and intermediate nuclear levels contain ER-alpha (Khan et al, Cancer Res., 54: 993(1994) and Lawson et al, Lancet, 351: 1787 (1994)). With increased ER- α expression, the inverse correlation between receptor expression and cell proliferation becomes dysregulated (Shoker et al, am. Jour. Path., 155: 1811 (1999)). Approximately 70% of invasive breast cancers express ER- α, and the majority of these tumors contain ER- α -positive proliferating cells (Clarke et al, Cancer Res., 57: 4987 (1997)).

Estrogen receptors are members of the ligand-activated nuclear receptor superfamily of transcription factors that control many physiological processes. This control often occurs by modulating gene transcription (Katzenllenbogen and Katzenllenbogen, Breast Cancer Res., 2: 335 (2000); Hull et al, J.biol.chem., 276: 36869 (2001); McDonnell and Norris, Science, 296: 1642 (2002)). The estrogen receptor uses a variety of mechanisms to activate or inhibit transcription of its target genes. These mechanisms include: (a) direct interaction of ligand-occupied receptors with DNA at the estrogen response element, followed by recruitment of transcription co-regulators (coreregulator) or mediator complexes, (B) interaction of ligand-occupied ERs with other transcription factors such as AP-1(Kushner et al, j.stemid biochem. mol. biol., 74: 311(2000)), Sp1(Safe, vitam. horm., 62: 231: 2001) or NF- κ B (McKay and cidowski, endocr. rev., 20: 435(1999)), or (c) indirect regulation of gene transcription by isolation of general/normal transcription components (Harnish et al, Endocrinology, 141: 3403(2000) and Speir et al, circ.1006: res., 87: res. (2000)). In addition, the ability of estrogen receptors to regulate transcription by these different mechanisms appears cell type specific, possibly due to differences in the complement of transcriptional co-regulators available in each cell type (Cerillo et al, J.Steroid biochem. mol.biol., 67: 79 (1998); Evans et al, circ.Res., 89: 823 (2001); Maret al, Endocrinology, 140: 2876 (1999)). Again, transcriptional regulation depends on the nature of the ligand, with various natural and synthetic selective estrogen receptor modulators acting as estrogen receptor agonists or antagonists through each of these different mechanisms (Shang and Brown, Science, 295: 2465 (2002); Katzenllenbogen and Katzenllenbogen, Science, 295: 2380 (2002); Marget et al, J.mol.biol., 326: 77 (2003); Dang et al, J.biol.chem., 278: 962 (2003)).

There is another signaling pathway mediated by estrogen, also known as the "non-classical", "non-genomic" or "membrane signaling" pathway, which comprises cytoplasmic proteins, growth factors and other membrane-initiated signaling pathways (Segars et al, Trends endocrin. met., 13: 349 (2002)). Several intracellular signaling pathways have been shown to communicate with a rapid estrogen-initiated action: the adenylate cyclase pathway (Aronica et al, PNAS, 91: 8517(1994)), the phospholipase C pathway (Le Mellay et al, J.cell.biochem., 75: 138(1999)), the G-protein-coupled receptor-activated pathway (Razandi et al, mol.Endocrin., 13: 307(1999)), and the mitogen-activated protein kinase (MAPK) pathway (Watters et al, Endocrinology, 138: 4030 (1997)). However, all membrane forms described so far are associated with ER- α but not with ER- β (Segars et al, Trends Endocrin. Met., 13: 349 (2002)).

Pathologically, estrogen signaling has been associated with a high risk of breast and endometrial cancer (Summer and Fuqua, semin. cancer biol., 11: 339 (2001); Turner et al, endocr. rev., 15: 275 (1994); farcat et al, faeb j., 10: 615 (1996); Beato et al, Cell, 83: 851 (1995); Dobrzycka et al, endo.rel. cancer, 10: 517 (2003)). As a result, estrogen receptors have been found to be essential for the initiation and progression of most such cancers. Current endocrine therapy for estrogen receptor-positive breast cancer is primarily designed for estrogen levels, estrogen receptor levels, or the activity of estrogen and estrogen receptors. The use of part of the antiestrogen tamoxifen in the management of early breast cancer has clearly demonstrated disease-free and increased overall survival. In addition, recent studies have demonstrated that tamoxifen can be used as a chemopreventive agent for hormone-dependent breast cancer. The major problem with long-term therapy with tamoxifen is its uterotrophic effect, which leads to a high risk of endometrial cancer, and acquired clinical resistance to tamoxifen. This has led to an urgent search for better Selective Estrogen Receptor Modulators (SERMs) that exhibit optimal agonistic or antagonistic activity in various estrogen responsive target tissues.

Thus, there is a need for additional methods and materials that can be used to screen for modulation of estrogen signaling, and methods and materials that can be used to modulate estrogen signaling.

Summary of The Invention

The present invention provides isolated antibodies that specifically bind to SEQ ID NO: 1, or an immunogenic fragment thereof, preferably, the amino acid sequence of SEQ ID NO: 1, amino acid sequence as set forth in amino acids 13-27 of seq id no. The antibody may be a monoclonal antibody or a polyclonal antibody. Optionally, the antibody is a humanized antibody. The antibody may be covalently attached to a compound, e.g., a chemotherapeutic agent or a detectable label, e.g., a fluorescent label. The antibody may be present in a composition, and the composition may comprise a pharmaceutically acceptable carrier. Kits comprising the antibodies of the invention are also provided.

The invention also provides methods of making the antibodies. The antibody may be polyclonal or monoclonal. The method comprises administering to the animal a peptide having the sequence of SEQ ID NO: 1, or an immunogenic fragment thereof, preferably, the amino acid sequence of SEQ ID NO: 1, amino acid sequence as set forth in amino acids 13-27 of seq id no. The method further comprises isolating the antibody from the animal, wherein the isolated antibody specifically binds to the amino acid sequence. The polypeptide or immunogenic subunit thereof may be covalently attached to a carrier polypeptide. Isolation may include obtaining cells producing the antibody from the animal and using the cells to prepare hybridomas producing monoclonal antibodies. The invention also includes polyclonal antibodies produced by the method and monoclonal antibodies produced by the method.

The present invention also relates to a cell comprising a foreign coding region, wherein the coding region encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20. The coding region may encode a polypeptide having an amino acid sequence substantially identical to SEQ ID NO: 20, wherein the polypeptide has ER- α 36 activity. The coding region may be operably linked to a constitutive promoter. The cell may be a eukaryotic cell or a prokaryotic cell. The invention also provides cells expressing such polypeptides.

The invention also provides methods of identifying agents that bind to polypeptides. The method comprises, in combination, combining a polypeptide comprising SEQ ID NO: 1, detecting complex formation between the agent and the polypeptide, detecting a change in activity of the polypeptide, or a combination thereof. Binding of a reagent to a polypeptide can be detected by directly detecting binding of the reagent to the polypeptide, detecting binding of the reagent to the polypeptide using a competitive binding assay, or a combination thereof. Optionally, the method also includes determining whether the agent binds to a polypeptide comprising SEQ ID NO: 18.

The invention also provides methods for detecting polypeptides. In one aspect, the method comprises providing a cell, analyzing the cell for a polypeptide having ER- α 36 activity and a 36kDa molecular weight as determined by electrophoresis on a Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel, and determining whether the cell expresses the polypeptide. The cells may be ex vivo (ex vivo) or in vivo. The cell may be, for example, a tumor cell, such as a breast tumor cell. The assay may comprise contacting the cell with a nucleic acid molecule capable of specifically binding to SEQ ID NO: 1, or an immunogenic fragment thereof. The analysis can include amplifying the mRNA polynucleotide to form an amplified polynucleotide. Amplification comprises contacting a polynucleotide obtained from a cell with a primer pair capable of amplifying an mRNA polynucleotide comprising the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 25, or a combination thereof, wherein the presence of the amplified polynucleotide indicates that the cell expresses the polypeptide. One primer of the primer pair may be selected from SEQ ID NO: 22, and SEQ ID NO: 25, or a combination thereof.

The invention also provides methods of inhibiting ER-alpha 36 activity in a cell. The method comprises, allowing expression of a polypeptide having the sequence of SEQ ID NO: 1 with a compound capable of inhibiting ER- α 36 activity. Such a compound may be a polypeptide that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1 of amino acids 13-27. The cells may be in vivo or ex vivo, and optionally may be ER- α 66 negative, ER- α 46 negative, or a combination thereof. In some aspects, the compound is not an antiestrogen.

The invention also provides an isolated polypeptide comprising SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1, more preferably, SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof. In another aspect, the isolated polypeptide is substantially identical to SEQ ID NO: 20, wherein the polypeptide has ER- α 36 activity, is at least 70% identical. The invention also includes SEQ ID NO: 1.

The term "comprising" and its variants do not have a limiting meaning when these terms appear in the description and claims. Unless otherwise defined, "a," "an," "the," and "at least one" are used interchangeably and refer to one or more than one.

Brief Description of Drawings

FIG. 1 illustrates a domain structure representation of the human estrogen receptor- α (ER- α) isoform. The domains (labeled with A-F), amino acid sequence numbering, AF-1 and AF-2, DNA binding domain, ligand-binding domain and dimerization domain are shown. The phosphorylation sites and the function of each domain are also indicated.

FIG. 2 is a schematic demonstrating possible communication between the membrane and genomic signaling pathways of ER- α. Cav-1 represents caveolin-1, ER-alpha, estrogen receptor-alpha; RTKs, receptor tyrosine kinases; ras, Ras oncogene; mek, MAP/ERK kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide-triphosphate kinase; AKT, protein kinase 13; PDK1, phosphoinositide-dependent protein kinase; RSK, p90 ribosomal S6 kinase.

FIG. 3 is a photograph showing pRET-infected MCF10A cells growing into large colonies in soft agar in the presence of estradiol (E2). The ST1 clone showed rapid growth in soft agar containing E2. MCF7 and MCF10A cells were included as positive and negative controls, respectively.

FIG. 4 is a Western blot showing down-regulation of caveolin-1 (Cav-1) expression in pRET-infected MCF10A cells. Equal amounts of whole cell extracts from different cell lines were analyzed by western blotting using a rabbit anti-Cav-1 antibody (N20). The position of Cav-1 is indicated by an arrow, and the cell extract analyzed in each lane is indicated above each lane.

FIG. 5 is a Western blot showing down-regulation of ER- α expression in pRET-infected MCF10A cells. Equal amounts of whole cell extracts from different cell lines were analyzed by western blotting using antibodies against ER- α (H222) and ER- β. The positions of ER-alpha and ER-beta are indicated by arrows, and the cell extracts analyzed in each lane are indicated above each lane.

FIG. 6 is a Western blot showing activation of ERK1/2 phosphorylation in pRET-infected MCF10A cells. Equal amounts of whole cell extracts from cell lines were analyzed by western blotting using antibodies against ERK1/2 and phosphorylated ERK 1/2.

FIG. 7 is a Western blot showing the presence of 3 ER- α proteins in Cav-1 haploid deficient (haploinsufficient) cells ST1 and ST3 and MCF7 breast cancer cells. Equal amounts of whole cell extracts from cell lines were analyzed by western blotting using H222 antibody against ER- α. The positions of ER- α 66, ER- α 46 and ER- α 36 are indicated by arrows, and the cell extracts analyzed in each lane are indicated above each lane.

FIG. 8 depicts the genomic organization of the human ER-alpha gene. The positions of the various promoters are indicated by arrows. Translation start and stop positions are indicated by AUG and UGA. Exons are shown in numbered boxes. Intron 1 is also shown with exon 1' in the box. The lower panel shows the mRNA structure of the ER-alpha isoform. The polyadenylation site is indicated by AAA.

FIG. 9 is a photograph of an agarose gel showing the isolation of cDNA encoding the open reading frame of ER-. alpha.36 by PCR. The position of the cDNA in the gel is indicated by arrows.

FIG. 10 shows the predicted amino acid sequence of the ER-. alpha.36 open reading frame. The amino acid positions are indicated by the numbering on the left side of the amino acid sequence (SEQ ID NO: 20). The last 27 amino acids unique to ER- α 36 are underlined (SEQ ID NO: 1).

FIG. 11 shows Western blot analysis of ER- α 66, ER- α 46 and ER- α 36. Lanes labeled ER- α 66, ER- α 46, and ER- α 36 represent isolated cultures of HEK293 cells transfected with expression plasmids encoding the estrogen receptor isoforms and lysed 2 days after transfection. Lysates from each transfectant were immunodetected with anti-ER-alpha antibody (H222). Cell extracts from MCF7 cells were used as positive controls. The positions of ER- α 66, ER- α 46 and ER- α 36 are indicated by arrows.

Fig. 12 shows: (a) a DNA sequence (SEQ ID NO: 22) encoding ER- α 36 and comprising the 5 'flanking sequence of the gene of the ER- α 36 promoter, and (b) a DNA sequence (SEQ ID NO: 25) encoding ER- α 36 and comprising the 3' flanking sequence of the gene of the nucleotides encoded by exon 9. In the 5' flanking sequence, the putative transcription binding site is underlined and also indicates the protein capable of binding the nucleic acid sequence. The starting site of the cDNA is also indicated by an arrow.

FIG. 13 shows northern blot analysis of ER- α 36 in different breast cancer cells MCF10A, T47D, MCF7, and MDA-MB-231. The positions of ER-alpha 36 and actin are indicated by arrows.

FIG. 14 shows the inhibition of the activity of the AF-1 and AF-2 domains of ER- α 66 and ER- β mediated transactivation of transcription by ER- α 36. (+ E2), E2 treated cells; (-E2), cells not treated with E2.

Figure 15 shows that ER- α 36 mediates the membrane-initiated MAPK kinase pathway stimulated by E2. (a) Western blot shows that treatment of ER36-293 cells with estradiol-17 beta (E2 beta) induces rapid phosphorylation of Mek1/2 and ERK 1/2. P-Mek1/2 and P-ERK1/2, the phosphorylated forms of Mek1/2 and ERK1/2, respectively. (b) Serum (but not E2. beta.) induced phosphorylation of ERK1/2 in control vector-293 cells. P-ERK1/2 is a phosphorylated form of ERK 1/2. (c) Different estrogens and antiestrogens induce rapid phosphorylation of ERK1/2 in ER36-293 cells. P-ERK1/2 is a phosphorylated form of ERK 1/2. (d) Tamoxifen treatment stimulated ERK1/2 phosphorylation constitutively in ER36-293 cells. P-ERK1/2 is a phosphorylated form of ERK 1/2.

FIG. 16 shows that ER- α 36 mediates E2 β -induced MAPK kinase nuclear signaling and stimulates cell growth. (a) Effect of E2 β on MAPK kinase nuclear signaling. ER36-293 and control vector-293 cells were transiently transfected with 5XGAl4-LUC, a luciferase reporter plasmid containing 5 Gal4DNA binding sites, and a Gal-ELK expression vector containing an ELK transcriptional activation domain fused to a Gal4DNA binding domain (upper panel). After transfection, cell cultures were maintained in estrogen-free medium for 36 hours, followed by the addition of E2 β (1nM or 10nM) for 12 hours. Luciferase activity with standard deviation represents more than 3 experiments performed in duplicate. (b) E2 beta and antiestrogens stimulate the growth of ER36-293 cells. The absorbance data at 490nm is shown. The results of more than 5 independent experiments were averaged; mean values and SEM are shown. The statistical significance of these results was also assessed by paired t-tests. The P-value of ER36-293 and vector-293 cells was less than 0.001.

FIG. 17 shows that ER- α 36 is primarily a membrane-based estrogen receptor. (a) Western blot analysis ER- α 36 expression in different established breast cancer cell lines. The same blot was stripped and probed with anti-actin antibody to ensure equal loading. (b) Subcellular localization of ER- α 36 in ER- α 36 transfected 293 cells. ER- α 36 was immunoblotted in different subcellular fractions with an antibody specific for ER- α 36. W, whole cell lysate; PM, plasma membrane; c, cytosol; n, nuclear. The purity of the subcellular fraction was checked by immunoblotting the plasma membrane, cytosol, nucleus and various protein markers of golgi apparatus. 5 'NT, 5' nucleotidase; D4-GDI, inhibitor of GDP dissociation; mSin3A, a component of histone reconstitution complex; COPB, β coat protein.

FIG. 18 shows that E2 β promotes the growth of ER- α 66 negative breast cancer cells MDA-MB-231 in soft agar. MDA-MB-231 cells were grown on soft agar for 3 weeks in the absence of E2 β (0), in the presence of 10nM E2 β (E2), in the presence of 10nM E2 β and 10nM tamoxifen (E2+ TAM) and in the presence of 10nM Tamoxifen Alone (TAM).

FIG. 19 shows that E2 β induces membrane-initiated estrogen signaling in ER- α 66 negative breast cancer cells MDA-MB-231. Treatment of MDA-MB-231 cells with estradiol-17 beta (E2 beta) induced rapid phosphorylation of ERK 1/2. Cells were treated with E2 β (10nM) at different time points, lysed, and analyzed by western blot using phosphorylation-dependent and independent antibodies.

Detailed description of the preferred embodiments of the invention

It has been found that down-regulation of the caveolin-1 (Cav-1) system constitutively activates the mitogen-activated protein kinase (MAPK) pathway, activates expression of estrogen receptor- α (ER- α), and triggers positive estrogen signaling. This finding for the first time provides a clear link between activated MAPK signaling and breast tumorigenesis, especially breast cancer progression stimulated by estrogen. This finding strongly suggests that Cav-1 plays an important role in maintaining normal growth of breast epithelial cells by coordinating communication between MAPK and estrogen signaling pathways, and that its down-regulation may contribute to dysregulation of these 2 important pathways, which ultimately leads to breast tumorigenesis. Figure 2 shows a schematic representation of the estrogen signaling pathway and the MAPK signaling pathway.

Estrogen receptor-alpha isoforms have also been identified and cloned. This 36-kDa isoform of estrogen receptor-alpha (ER-alpha 36) was generated from a promoter located in the first intron of the original 66-kDa ER-alpha (ER-alpha 66) gene. ER- α 36 differs from ER- α 66 in that it lacks the 2 transcriptional activation domains (AF-1 and AF-2), but retains the DNA-binding, dimerization and most of the ligand-binding domains. The structure of ER- α 36 suggests that ER- α 36 is a modulator of estrogen signaling. ER- α 36 may also mediate the membrane effects of estrogen signaling because it is expressed primarily in the plasma membrane, and also in the cytosol and nucleus.

ER- β has been considered to be a constitutive regulator of ER- α 66-mediated estrogen signaling. It was found that ER- α 46 lacking the AF-1 domain can dimerize to ER- α 66 and inhibit transactivation activity mediated by the AF-1 domain of ER- α 66, suggesting that ER- α 46 plays a regulatory role in functional activity mediated by the AF-1 domain of ER- α 66. ER-alpha 36 lacks the AF-1 and AF-2 domains. Thus, ER- α 36 is believed to inhibit biological functions mediated by AF-1 and AF-2 of ER- α 66, as well as functions mediated by AF-2 of ER- α 46. Utilizing ER- α 36 and ER- α 46 mediated regulation, both of which may be expressed at different levels in different tissues, ER- α 66 may function differently in different target tissues. Such mechanisms are thought to explain the pleiotropic effects of estrogen signaling in different biological processes.

Polypeptides and peptidomimetics (peptidomimetics) of the invention

The present invention provides polypeptides. As used herein, the term "polypeptide" broadly refers to a polymer of 2 or more amino acids linked together by peptide bonds. The terms peptide, oligopeptide and protein are included in the definition of polypeptide and these terms are used interchangeably. It will be understood that these terms do not imply a particular length of amino acid polymer, nor are they intended to imply or distinguish whether a polypeptide is produced by recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. Various examples of polypeptides within the scope of the invention are disclosed and described herein. In the case of naturally occurring polypeptides or polynucleotides, preferably such polypeptides or polynucleotides are isolated and optionally purified. An "isolated" polypeptide or polynucleotide is a polypeptide or polynucleotide that is separated and isolated from its natural environment. A "purified" polypeptide or polynucleotide is one that is at least 60% free, preferably 75% free, and most preferably 90% free of other components with which it is naturally associated. Polypeptides and nucleotides that are produced outside of the organism in which they naturally occur (e.g., by chemical or recombinant methods) are considered to be defined isolated and purified because they never exist in the natural environment. "exogenous polypeptide" refers to a foreign polypeptide, i.e., a polypeptide that is not normally present in a cell, or a polypeptide that is normally present in a cell but has been introduced into a cell experimentally (e.g., by introducing a polynucleotide encoding the polypeptide).

The polypeptides of the invention may be biologically active. Such biological activity is referred to herein as "ER- α 36 activity". Examples of bioassays that can be used to determine whether a polypeptide of the invention is biologically active include contacting a cell expressing the polypeptide with an estrogen or an anti-estrogen and determining whether the activity of the MAPK pathway is increased or decreased in the presence of estrogen or anti-estrogen compared to the MAPK activity in a control cell not expressing the polypeptide of the invention. Preferably, the MAPK activity is phosphorylation of ERK1/2 and Mek1/2, and preferably, the polypeptide of the invention induces no reduction in phosphorylation of ERK1/2 in the presence of an anti-estrogenic agent. Preferably, ER- α 36 activity is membrane-initiated. ER- α 36 activity can be measured by exposing cells expressing polypeptides having ER- α 36 activity against different ligands. Examples of ligands that may be used include, but are not limited to, estrone (E1), 17 α -estradiol (E2 α), 17 β -estradiol (E2 β), estriol (E3), estetrol (E4), or estrogen attached to a membrane impermeable molecule, such as Bovine Serum Albumin (BSA). Typically, estrogens attached to membrane impermeable molecules are used when the ER- α 36 activity to be measured is limited to membrane initiated ER- α 36 activity. The amount of estrogen used may vary and is preferably in the range of 1nM to 10 nM. The estrogen-exposed cells are preferably quiescent cells. This exposure is allowed to occur for 5-90 minutes, after which the cells are lysed and the polypeptides present in the cells are separated by SDS-polyacrylamide gel electrophoresis. After transfer of the isolated polypeptide to a membrane, activation of the MAPK pathway was assessed using antibodies to the non-phosphorylated and phosphorylated forms of ERK1/2 and Mek 1/2. Optionally, an anti-estrogen such as tamoxifen, 4 OH-tamoxifen or ICI-182, 780 may be included to determine whether phosphorylation of ERK1/2 is insensitive to anti-estrogen.

The invention provides a polypeptide having the sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof. The polypeptides, and related polypeptides described herein, are also referred to herein as ER- α 36, ER- α 36 isoforms, and ER receptor α 36-subunits. As shown in FIG. 1, the ER- α 36 isoform lacks the amino-terminal amino acid residues 1-183, the carboxy-terminal amino acid residue 430-595 and has the addition of 27 amino acid residues at its C-terminus when compared to the ER- α 66 isoform (see Table 1). The alpha isomers of estrogen receptors include ER-alpha 36, ER-alpha 46, ER-alpha 66. The estrogen receptor beta isomer includes ER-beta. The invention also provides estrogen receptors, including the ER- α 36 isoform. Without intending to be limiting, it is believed that the ER- α 36 isoform modulates cellular responses to estrogen by modulating estrogen receptor function through the formation of dimers with ER- α 66, ER- α 46, or ER- β. Furthermore, ER- α 36 is believed to lack both activating factor 1(AF-1) and activating factor 2(AF-2) activity, and thus lack intrinsic transcriptional activity. However, ER- α 36 is believed to retain the intact dimerization domain, which allows ER- α 36 to dimerize with ER- α 46, ER- α 66, or ER- β. This interaction is thought to allow ER- α 36 to modulate the activity of ER- α 46, ER- α 66, and ER- β, which contain estrogen receptors.

TABLE 1

Amino acid and nucleotide sequences

The polypeptide of the invention comprises a polypeptide corresponding to SEQ ID NO: 20 a polypeptide having an amino acid sequence with at least 70% identity. Such polypeptides include polypeptides that are identical to SEQ ID NO: 20, such as a polypeptide having an amino acid sequence with at least a single percentage of identity greater than 70% to SEQ id no: 20 have 71%, 72%, 73% identity, and so on, up to 100% identity. Preferably, the polypeptide comprises a polypeptide having an amino acid sequence which, on an increasing order of preference, differs from the amino acid sequence of SEQ ID NO: 20 have at least about 80% identity, at least about 90% identity, or at least about 95% identity. Preferably, the polypeptide is biologically active. Preferably, the polypeptide has a molecular weight of 36kDa, as determined by electrophoresis on a Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel. In general, the residues involved in phosphorylation of ER- α 66, e.g., S236, K302, and K303 are conserved, as are those residues involved in the function of the DNA binding domain, ligand binding domain, and dimerization domain of ER- α 66. Residues that function in DNA binding, ligand binding and/or dimerization are known in the art.

Typically, the percent identity between 2 polypeptide sequences is determined by aligning the residues of the 2 amino acid sequences to optimize the number of identical amino acids along their sequence length; gaps in the alignment are allowed in either or both of the 2 sequences to optimize the number of identical amino acids, although the amino acids in each sequence must maintain their correct order. Preferably, 2 amino acid sequences are compared using the BLASTP program of the BLAST2 search algorithm, version 2.0.9, as described by Tatusova et al (FEMS Microbiol. Lett., 174, 247-charge 250(1999)) and available from the world Wide Web at http:// www.ncbi.nlm.nih.gov/BLAST/bl2seq/bl2. html. Preferably, default values for all BLAST2 search parameters are used, including matrix (matrix) ═ BLOSUM 62; open gap penalty of 11, extended gap penalty of 1, gap x _ dropoff of 50, expect of 10, word length of 3, and optionally, the filter is on. When 2 amino acid sequences are compared using the BLAST search algorithm, structural similarity is referred to as "identity".

The invention also provides polypeptides that are fragments of the ER-alpha 36 estrogen receptor isoform. Preferably, the fragment is immunogenic. In some aspects, the fragment has ER- α 6 activity. An example of an immunogenic fragment is the fragment of SEQ ID NO: 1, more preferably, the amino acid sequence set forth in amino acids 13-27 of SEQ ID NO: 1-27 of 1. Such fragments can be used to prepare antibodies that specifically bind to the ER- α 36 estrogen receptor isoform. Examples of fragments include estrogen receptor isoforms that have been truncated by 1 or more amino acids at the N-terminus or C-terminus or both, so long as the fragment contains at least 5 contiguous amino acids, more preferably at least 7 contiguous amino acids, even more preferably at least 10 contiguous amino acids, and most preferably at least 12 contiguous amino acids.

The invention provides fusion polypeptides having a carrier polypeptide coupled to a polypeptide of the invention. The carrier polypeptide can be used to increase or decrease the solubility of the fusion polypeptide. The carrier polypeptide may also be used to increase the immunogenicity of the fusion polypeptide to increase the production of antibodies capable of binding to the polypeptide of the invention. For example, a carrier polypeptide can be fused to a polypeptide fragment of the invention to facilitate production of antibodies that specifically bind to ER- α 36. Examples of such fragments are those having seq id NO: 1, amino acids 13-27. The present invention is not limited by the type of carrier polypeptide used to produce the fusion polypeptides of the present invention. Examples of carrier polypeptides include keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serum albumin, and the like. The carrier polypeptide may also be used to isolate or detect the fusion polypeptide. Examples of such carrier proteins include glutathione-S-transferase, maltose-binding protein, chitin-binding protein and polypeptides having the following amino acid sequences: QFGLM (SEQ ID NO: 2), EQKLISEEDL (SEQ ID NO: 3), KAEDESS (SEQ ID NO: 4), YPYDVPDYA (SEQ ID NO: 5), DYKDDDDK (SEQ ID NO: 6), YTDIEMNRLGK (SEQ ID NO: 7), MASMTGGQQMG (SEQ ID NO: 8), DTYRYI (SEQ ID NO: 9), TDDYLK (SEQ ID NO: 10), HHHHHHHHHHHH (SEQ ID NO: 11), HPOL (SEQ ID NO: 12), QYPALT (SEQ ID NO: 13), QRQYGDVFKGD (SEQ ID NO: 14), EYMPME (SEQ ID NO: 15), EFMPME (SEQ ID NO: 16) and RYIRS (SEQ ID NO: 17). Thus, the fusion polypeptide can be detected or isolated by interaction with other components of the carrier polypeptide portion to which the fusion polypeptide binds. For example, a fusion polypeptide having avidin as a carrier polypeptide can be detected or isolated with biotin by using a known method. The carrier polypeptide can also be used to cause inclusion bodies to be formed upon expression of the fusion polypeptide in a cell. The carrier polypeptide can also be an export signal that causes the fusion polypeptide to be exported out of the cell, or directs the fusion polypeptide to a compartment in the cell, such as the periplasm.

The invention also provides 2 or more polypeptides of the invention linked consecutively into a single amino acid chain. Such polypeptides are referred to herein as polypeptides. The polypeptides may be linked by a linker (see Stahl et al, U.S. Pat. No. 6,558,924). Such polyproteins can be isolated and then cleaved to produce the polypeptides or conjugated polypeptides of the invention. The polyprotein can be cleaved using a number of methods, such as chemical or protease cleavage. Thus, the linker may be designed to be cleaved by a particular protease or chemical agent. Examples of compounds that can be used to cleave the polyprotein of the invention include chemical agents and enzymes. Examples of chemical agents include cyanogen bromide, formic acid and heat, hydroxylamine and heat, iodoxybenzoic acid-2- (2-nitrophenyl) -3-methyl-3-bromoindolenine dissolved in acetic acid, and the like. Examples of enzymes include Ala-64 subtilisin, clostripain, collagenase, enterokinase, coagulation factor Xa, renin, alpha-thrombin, trypsin, chymotrypsin, tobacco etch virus protease, and the like. The polyprotein can be used to increase the efficiency of production of the polypeptides of the invention. Methods for producing polyproteins are known in the art (see Coolidge et al, U.S. Pat. No. 6,127,150).

The polypeptides of the invention include analogs that have been modified by the addition, substitution, or deletion of one or more contiguous or non-contiguous amino acids, or analogs that have been chemically or enzymatically modified, for example, by attachment of a reporter group, by modification or derivatization of the N-terminus, C-terminus, or other functional group, or by cyclization, so long as the analog retains biological activity or stimulates production of antibodies that bind ER- α 36. Analogs may thus include additional amino acids at one or both termini of the polypeptide. Preferably, the analog is immunogenic, more preferably, the analog is immunogenic and has ER- α 36 activity. In some aspects, the invention provides non-analog polypeptides.

Amino acid substitutions in the polypeptides of the invention are preferably conservative substitutions selected from other members of the class to which the amino acid belongs. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a group of amino acids having a particular size or characteristic (e.g., charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without substantially changing the structure of the polypeptide. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of preferred conservative substitutions include the substitution of Lys for Arg, and vice versa, to maintain a positive charge; glu for Asp and vice versa to maintain a negative charge; ser for Thr to maintain free-OH; and Gln substituted Asn to maintain free NH₂. The relevant amino acids (e.g.3-hydroxyproline, 4-hydroxyproline, homocysteine, 2-aminoadipic acid, 2-aminopimelic acid, gamma-carboxyglutamic acid, beta-carboxyaspartic acid), amino acid amides (ornithine, homoarginine, N-methyllysine, dimethyllysine, trimethyllysine, 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, homoarginine, sarcosine and hydroxylysine) and substituted phenylalanines, norleucine, norvaline, 2-aminocaprylic acid, 2-aminoheptanoic acid, statine, beta-valine, naphthylalanine, tetrahydroisoquinoline-3-carboxylic acid and halogenated tyrosines can be exchanged for similar amino acids.

The invention provides peptidomimetics of the polypeptides of the inventionA compound (I) is provided. Peptidomimetics refer to polypeptides in which at least one peptide bond has been replaced with a non-peptide bond, such as those often used as non-peptide drugs in the pharmaceutical industry, which have similar properties to the template polypeptide (Fauchere, J., adv. drug Res., 15: 29(1986), Evans et al, J.Med. chem., 30: 1229(1987), and Janda et al, U.S. Pat. No. 6,664,372). The peptidomimetic is similar in structure to a polypeptide having a peptide bond, but has one or more peptide bonds optionally replaced by the following bonds by methods known in the art: for example, - -CH₂NH--、--CH₂S--、--CH₂--CH₂-, - -CH- - - - (cis and trans) - -, - -COCH₂--、--CH(OH)CH₂- - -and- -CH₂SO- -. Advantages of peptidomimetics over native polypeptide embodiments include more economical production, greater chemical stability, altered specificity and enhanced pharmacological properties such as half-life, absorption, potency and efficacy.

Substitution of one or more amino acids in a polypeptide or peptidomimetic with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can result in a polypeptide or peptidomimetic that is, for example, more stable and more resistant to endogenous proteases.

The polypeptides and peptidomimetics of the invention may be modified for in vivo use by the addition of blocking agents at the amino-terminus and/or the carboxy-terminus that reduce in vivo degradation. This can be used in situations where the polypeptide termini are prone to degradation in vivo by proteases. Such blocking agents include, but are not limited to, other related or unrelated peptide sequences that may be attached to the amino and/or carboxy terminal residues of a polypeptide or peptidomimetic of the invention. This can be done during chemical synthesis or by recombinant DNA techniques by methods familiar to the person skilled in the art. Alternatively, blocking agents such as pyroglutamic acid, or other molecules known in the art, may be attached to the amino and/or carboxy terminal residues, or the amino terminal amino or carboxy terminal carboxy group may be replaced with a different moiety. Thus, the invention provides blocked polypeptides and peptidomimetics, and amino-terminal, carboxy-terminal, or combinations thereof.

The polypeptides of the invention can be produced on a small or large scale by using a number of expression systems, including, but not limited to, cells or microorganisms transformed with a recombinant vector into which a polynucleotide of the invention has been inserted. Such recombinant vectors and methods of use thereof are described below. These vectors can be used to transform a number of organisms. Examples of such organisms include bacteria (e.g., escherichia coli (e.coli) or bacillus subtilis)); yeasts (e.g., Saccharomyces (Saccharomyces) and Pichia (Pichia)); insects (e.g., baculovirus); a plant; or mammalian cells (e.g., COS, CHO, BHK, 293, VERO, HeLa, MDCK, W138, and NIH 3T3 cells). Primary or secondary cells obtained directly from a mammal transfected with the vector may also be used as host cells.

Synthetic methods may also be used to produce the polypeptides and peptidomimetics of the invention. Such methods are known and conventional in the art. For example, solid phase peptide synthesis methods are well-established and widely used methods. The polypeptide can be easily purified by fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or an anion exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, ligand affinity chromatography, or the like. The polypeptide can also be readily purified by binding the fusion polypeptide to a separation medium and then cleaving the fusion polypeptide to release the purified polypeptide. For example, a fusion polypeptide can be generated that includes a factor Xa cleavage site between the polypeptide and a carrier polypeptide. The fusion polypeptide may also be bound to an affinity column to which the carrier polypeptide portion of the fusion polypeptide is bound. The fusion polypeptide can then be cleaved with factor Xa to release the polypeptide. Such systems have been used in combination with factor Xa removal kits for purifying polypeptides of the invention.

Polynucleotide

The present invention provides polynucleotides encoding the polypeptides of the present invention. The term "polynucleotide" broadly refers to a polymer of 2 or more nucleotides covalently linked in a 5 'to 3' direction. Polynucleotides may include nucleotide sequences having different functions, including, for example, coding sequences, and non-coding sequences, such as regulatory sequences. Coding, non-coding and regulatory sequences are defined below. The terms nucleic acid, nucleic acid molecule, and oligonucleotide are included within the definition of polynucleotide, and these terms are used interchangeably. It will be understood that these terms do not imply a particular length of nucleotide polymer, nor are they intended to imply or distinguish whether a polynucleotide is produced by recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

The polynucleotide may be single-stranded or double-stranded, and the sequence of the second complementary strand is determined by the sequence of the first strand. Thus, the term "polynucleotide" should be broadly construed to include single-stranded nucleic acid polymers, the complement thereof, and duplexes formed thereby. "complementarity" of a polynucleotide refers to the ability of 2 single-stranded polynucleotides to base pair with each other, wherein adenine on one polynucleotide base pairs with thymidine (or uracil, in the case of RNA) on the other, and cytidine on one polynucleotide base pairs with guanine on the other. When the nucleotide sequence on one polynucleotide is base paired with the nucleotide sequence on a second polynucleotide, the 2 polynucleotides are complementary to each other. For example, 5 '-ATGC and 5' -GCAT are perfectly complementary, as are 5 '-GCTA and 5' -TAGC.

An example of a polynucleotide of the invention is SEQ ID NO: 21 (see table 1, nucleotide 234-. Preferred polynucleotides of the invention also include polynucleotides having a nucleotide sequence "substantially complementary" to a nucleotide sequence capable of encoding a polypeptide according to the invention, or the complement of such a nucleotide sequence. "substantially complementary" polynucleotides may include at least one base pair mismatch, but 2 polynucleotides still have the ability to hybridize. For example, the intermediate nucleotides of each of the 2 DNA molecules 5 '-AGCAAATAT and 5' -ATATATGCT are not capable of base pairing, but the 2 polynucleotides are still substantially complementary as defined herein. If they hybridize under the hybridization conditions exemplified by 2 XSSC (SSC: 150mM NaCl, 15mM trisodium citrate, pH7.6) at 55 ℃, the 2 polynucleotides are substantially complementary. Substantially complementary polynucleotides for the purposes of the present invention preferably share at least one region of at least 20 nucleotides in length, which shared region has at least 60% nucleotide identity, preferably at least 80% nucleotide identity, more preferably at least 90% nucleotide identity and most preferably at least 95% nucleotide identity. Particularly preferred substantially complementary polynucleotides share many such regions. Preferably, the polynucleotide is identical to SEQ ID NO: 21 having at least 70% identity. More preferably, the polynucleotide has a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 21 has at least a single percentage identity of more than 70%, e.g. to SEQ ID NO: 21 have 71%, 72%, 73% identity, and so on, up to 100% identity. Even more preferably, the polynucleotide has a sequence identical to seq id NO: 21, a nucleotide sequence that is at least 80% identical, at least 90% identical, or at least 95% identical. Most preferably, the polynucleotide has a sequence identical to SEQ ID NO: 21 having 100% identity. And SEQ ID NO: 21 has ER- α 36 activity, and a polynucleotide having at least 70% identity thereto.

Typically, the percent identity between 2 polynucleotide sequences is determined by aligning the bases of the 2 polynucleotide sequences to optimize the number of identical bases along their sequence length; gaps in any one or 2 sequences are allowed when aligning to optimize the number of identical bases, although the bases in each sequence must maintain their correct order. Preferably, 2 polynucleotide sequences are aligned using the Blastn program of the BLAST2 search algorithm, version 2.0.11, also described by Tatusova et al (FEMS Microbiol. Lett, 174, 247-K250 (1999)) and available from the world Wide Web at http:// www.ncbi.nlm.nih.gov/BLAST/bl2seq/bl2. html. Preferably, default values for all BLAST2 search parameters are used, including a match reward (reward for match) of 1, a mismatch penalty (penalty for mismatch) of-2, an open gap penalty of 5, an extended gap penalty of 2, gap x _ drop of 50, expect of 10, word length of 11, and optionally, the filter is on. CLUSTALW multiple sequence alignment software (J.Thompson et al, Nucl. acids Res., 22: 4673-4-4680 (1994)) was used, which was available at www.ebi.ac.uk/CLUSTALW/from the world Wide Web and also readily determined the position and level of nucleotide sequence identity between 2 polynucleotide sequences.

It is understood that a polynucleotide capable of encoding a polypeptide of the present invention is not limited to a polynucleotide containing all or part of a naturally occurring genomic or cDNA nucleotide sequence, but also includes the class of polynucleotides that encode such polypeptides due to the degeneracy of the genetic code. For example, the naturally occurring polynucleotide sequence of SEQ ID NO: 21 can only encode a polypeptide having the amino acid SEQ ID NO: 20, or a member of the nucleotide sequence class of polypeptides. The class of nucleotide sequences that can encode a selected polypeptide sequence is large but limited, and the nucleotide sequence of each member of the class can be readily determined by one skilled in the art with reference to the standard genetic code, where different nucleotide triplets (codons) are known to encode the same amino acid.

A polynucleotide capable of "encoding" a polypeptide of the present invention optionally comprises coding and non-coding regions, and thus it is understood that unless otherwise stated to the contrary, a polynucleotide capable of "encoding" a polypeptide is not structurally limited to a nucleotide sequence capable of encoding a polypeptide, but also includes other nucleotide sequences outside (i.e., 5 'or 3') the coding region. A "coding region" or "coding sequence" is a nucleotide sequence capable of encoding a polypeptide and, when placed under the control of appropriate regulatory sequences, is capable of expressing the encoded polypeptide. The boundaries of the coding region are generally determined by a translation start codon at its 5 'end and a translation stop codon at its 3' end. "foreign coding region" refers to a foreign coding region, i.e., a coding region that is not normally found in a cell, or a coding region that is normally present in a cell but has been introduced into a cell experimentally, operably linked to a regulatory region to which it is not normally operably linked, or a combination thereof.

The polynucleotides of the invention may be linear or circular in topology. The polynucleotide may be, for example, part of a vector. The vector may provide for further cloning (amplification of the polynucleotide), i.e., cloning the vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector. Vectors may include, but are not limited to, plasmids, phagemids, factors F, viruses, cosmids, or phages. The vector may be in the form of a linear or circular double-or single-stranded vector. Vectors may also be used to transform prokaryotic or eukaryotic hosts by integration into the cellular genome or by extrachromosomal presence (e.g., as autonomously replicating plasmids with origins of replication). The polynucleotide in the vector may be under the control of, and operably linked to, an appropriate promoter or other regulatory sequence for in vitro transcription or transcription in a host cell, e.g., a eukaryotic cell, or a microorganism, e.g., a bacterium. Examples of preferred eukaryotic cells include the MDA-MB-231, Hela, CHO and MCF10A cell lines. A regulatory sequence, or regulatory region, refers to a nucleotide sequence located upstream, within, or downstream of a coding sequence, and is operably linked to the coding sequence. Examples of regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include both natural and synthetic sequences, as well as sequences that may be combined with synthetic and natural sequences. The regulatory sequence is not limited to a promoter. However, some suitable regulatory sequences useful in the present invention include, but are not limited to, constitutive promoters, tissue-specific promoters, development-specific promoters, inducible promoters, and viral promoters. The term "operably linked" refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is linked in a manner that achieves expression of the coding region under conditions compatible with the regulatory sequence.

The vector may be a shuttle vector capable of functioning in a variety of hosts. The vector may also be a cloning vector, which typically contains one or a few restriction endonuclease recognition sites, where foreign DNA sequences may be inserted in a determinable fashion. Such insertion can occur without loss of the basic biological function of the cloning vector. The cloning vector may also contain a marker gene suitable for use in identifying and selecting cells transformed with the cloning vector. Examples of marker genes are tetracycline resistance or ampicillin resistance. Many cloning vectors are commercially available (e.g., Stratagene, New England Biolabs, Clonetech). The vector may be an expression vector comprising regulatory sequences capable of directing the expression of a polynucleotide inserted into the expression vector. A number of vectors are commercially available and known in the art (Stratagene, LaJolla, Calif.; New England Biolabs, Beverly, Mass.). The expression vectors can be used in vitro transcription and translation assays.

Methods for introducing polynucleotides into vectors are well known in the art (Sambrook et al, Molecular Cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Briefly, a vector into which a polynucleotide is to be inserted is treated with one or more restriction enzymes (restriction endonucleases) to produce a linearized vector having blunt ends, "sticky" ends with 5 'or 3' overhangs, or any combination of the above. The vector may also be treated with a restriction enzyme followed by another modifying enzyme, such as a polymerase, exonuclease, phosphatase or kinase, to produce a linearized vector having features useful for ligating the polynucleotide into the vector. Treating the polynucleotide to be inserted into the vector with one or more restriction enzymes to generate a linearized fragment having blunt ends, "sticky" ends with 5 'or 3' overhangs, or any combination thereof. The polynucleotide may also be treated with a restriction enzyme followed by treatment with another DNA modifying enzyme. Such DNA modifying enzymes include, but are not limited to, polymerases, exonucleases, phosphatases, or kinases to generate polynucleotides having features useful for ligating polynucleotides into vectors.

The treated vector and polynucleotide are then ligated together to form a polynucleotide-containing construct according to methods known in the art (Sambrook et al, molecular cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Briefly, the treated nucleic acid fragments and the treated vector can be combined in the presence of a suitable buffer and ligase. The mixture is then incubated under appropriate conditions to allow the ligase to ligate the nucleic acid fragments into the vector.

The invention also provides methods for producing the polypeptides of the invention and for producing polynucleotides encoding them. Methods include biological, enzymatic, and chemical methods, as well as combinations thereof, and are well known in the art. For example, a polynucleotide can be expressed in a host cell using standard recombinant DNA techniques, it can be synthesized enzymatically in vitro using cell-free RNA-based systems, or it can be synthesized using chemical techniques, such as solid phase peptide synthesis. When recombinant DNA technology is used, the host cell may be, for example, a bacterial cell, an insect cell, a yeast cell, or a mammalian cell.

The present invention also provides polynucleotides having promoter activity. In one aspect, the promoter of the invention comprises SEQ ID NO: 22, or a portion thereof. In another aspect, the promoter of the present invention has a nucleotide sequence that is identical to the nucleotide sequence of SEQ ID NO: 22 has at least a single percentage identity of more than 70%, e.g., to SEQ ID NO: 22 have 71%, 72%, 73% identity, and so on, up to 100% identity. Methods of determining percent identity are described herein. Even more preferably, the promoter has a sequence identical to SEQ ID NO: 22, or at least 80% identity, at least 90% identity, or at least 95% identity. The promoters of the present invention have Estrogen Receptor (ER) regulated activity. As used herein, ER-regulated activity refers to enhanced expression of an operably linked coding region in the presence of ER- α 66, preferably in the presence of ER- α 66 that is capable of binding estrogen. The promoters of the invention are expressed in an estrogen-dependent and estrogen-independent manner. The promoters of the present invention may be operably linked to a coding region capable of encoding a polypeptide, including a marker polypeptide. Examples of marker polypeptides include detection markers (e.g., fluorescent proteins, enzymes, antigenic markers, etc.) and selection markers (polypeptides that cause drug resistance, drug susceptibility, or nutritional deficiencies or correct nutritional deficiencies, etc.).

Antibodies

The invention provides antibodies that specifically bind to the polypeptides and peptidomimetics of the invention. As used herein, an antibody that "specifically binds" to a polypeptide is an antibody that interacts only with an epitope of the antigen that induces synthesis of the antibody, or interacts with a structurally related epitope. An antibody that "specifically binds" to an epitope can interact with the epitope under appropriate conditions, even in the presence of multiple potential binding targets. In some aspects, antibodies of the invention are capable of specifically binding to ER- α 36 or a portion thereof, and do not specifically bind to ER- α 66 or ER- α 46.

Thus, the polypeptides and peptidomimetics and fragments thereof of the invention can be used as antigens to produce antibodies, including vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, altered antibodies, monovalent antibodies, monoclonal and polyclonal antibodies, Fab proteins, and single domain antibodies. For example, a polypeptide having the sequence of SEQ ID NO: 1 or a fragment thereof, such as SEQ ID NO: 1, can be used to generate antibodies that specifically bind ER- α 36. The polypeptides or peptidomimetics of the invention or fragments thereof may be modified by covalently linking them to an immunogenic carrier, such as Keyhole Limpet Hemocyanin (KLH), bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serum albumin, and the like.

If polyclonal antibodies are desired, the selected animal (e.g., mouse, rabbit, goat, horse or bird, e.g., chicken) may be immunized with the desired antigen. Serum from the immunized animal is collected and processed according to known and conventional methods. If the serum containing polyclonal antibodies to the polypeptide of the invention contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art (see, e.g., Harlow et al, Antibodies: A Laboratory Manual, Cold spring Harbor Pub. 1988)).

Monoclonal antibodies directed against the polypeptides or peptidomimetics or fragments thereof of the present invention can also be readily produced by those skilled in the art. The general method of making monoclonal Antibodies by hybridomas is well known (see, e.g., Harlow et al, Antibodies: A laboratory Manual, Cold Spring Harbor Pub. 1988). Immortalized antibody-producing cell lines (hybridomas) can be generated by cell fusion, and also by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with EB virus. The panel of monoclonal antibodies produced against the polypeptides and peptidomimetics of the invention can be screened for various properties, such as epitope affinity. Other well-known methods of making antibodies include the use of Phage display technology (see, e.g., Kay et al, phase display of peptides and proteins: A laboratory Manual. san Diego: Academic Press (1996)).

The antibodies of the invention may be derived from "humanized" monoclonal antibodies. Humanized monoclonal antibodies can be produced by transferring mouse complementarity determining regions from the heavy and light variable chains of a mouse immunoglobulin into a human variable domain, and then substituting human residues of the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies eliminates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described (Orlandi et al, Proc. Natl Acad. Sci. USA, 86: 3833(1989), and techniques for producing humanized monoclonal antibodies are also described (Jones et al, Nature, 321: 522 (1986); Riechmann et al, Nature, 332: 323 (1988)).

The antibody fragments of the invention may be prepared by conventional known methods, including proteolysis of antibodies, or by expression of polynucleotides encoding the fragments in E.coli. Antibody fragments can be obtained by digestion of intact antibodies by conventional methods (with, for example, pepsin or papain). For example, by enzymatic cleavage of the antibody with pepsin,providing a mixture called F (ab')₂The 5S fragment of (5), an antibody fragment can be produced. This fragment can be further cleaved using a thiol reducing agent and optionally a blocking agent for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce a 3.5 SFab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin would directly yield 2 monovalent Fab' fragments and 1 Fc fragment.

Other methods of cleaving antibodies, such as isolating the heavy chain to form monovalent light-heavy chain fragments, further cleaving fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to an antigen that is recognized by the intact antibody.

Antibodies can be screened to determine the identity of the epitope to which they bind. An epitope refers to a site on an antigen (e.g., a polypeptide of the invention) to which a paratope of an antibody binds. Epitopes are usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Methods that can be used to identify epitopes are known in the art (Harlow et al, Antibodies: A Laboratory Manual, page319(Cold Spring Harbor Pub. 1988).

Antibodies can be screened for the ability to specifically bind to a polypeptide or peptidomimetic of the invention. For example, Antibodies that specifically bind to the ER- α 36 isoform or a portion thereof, but do not bind to either the ER- α 46 or the α 66 isoform, can be selected by using methods routine in the art (see Kitajima et al, U.S. Pat. No. 6,534,281, and Harlow et al, Antibodies: antibody Manual, Cold Spring Harbor pub. 1988).

The antibodies of the invention can be conjugated to a variety of compounds. Examples of compounds include detection labels. Examples of such detection labels include fluorescent labels, enzymes, radioisotopes, etc., such as avidin or biotin, which allow for the detection of antibodies. Methods of coupling antibodies to detection labels, and useful detection labels, are known in the art. Such antibodies can be used in an automated system for the detection of ER-alpha 36. The antibody may be covalently attached to the chemotherapeutic agent. Chemotherapeutic agents useful in the treatment of cancers such as breast and prostate cancer are known in the art. Examples of chemotherapeutic agents include chromans, damascenone esters, droloxifene, idoxifene, tamoxifen, raloxifene, toremifene, fulvestrant (fulvestrant) and foslodex, bisphosphonates, calcitonin, tribolone, parathyroid hormone or strontium ranelate (strontium ranelate). Other examples include cytokines, or toxins such as diphtheria toxin a chain.

Composition comprising a metal oxide and a metal oxide

The invention also provides compositions comprising a polynucleotide, peptidomimetic or antibody of the invention. Such compositions typically comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Other active compounds may also be incorporated into the compositions.

The compositions of the present invention can be prepared in a number of forms including tablets, hard or soft capsules, aqueous solutions, suspensions and liposomes, and other sustained release formulations such as shaped polymeric gels. The oral dosage form may be formulated so that the polypeptide, peptidomimetic or antibody is released into the intestine after passing through the stomach. Such formulations are described in Hong et al, U.S. patent No. 6,306,434 and the literature contained therein.

Oral liquid compositions may be in the form of: for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous carriers (which may include edible oils), or preservatives.

The compositions may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers, or in multi-dose containers with an added preservative. The composition may take the form of: suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Compositions suitable for rectal administration may be prepared as unit dose suppositories. Suitable carriers that may be included in the compositions include those exemplified by saline solutions, and other materials commonly used in the art.

For inhalation administration, the composition may be conveniently delivered from an insufflator, nebulizer or pressurized pack or other convenient means of delivering an aerosol spray. The pressurized pack may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder composition, for example, a powder mix of the conditioning agent and a suitable powder base (e.g., lactose or starch). The powder compositions may be presented in unit dosage form, for example in capsules or cartridges, or for example, gelatin or blister packs from which the powder may be administered with the aid of an inhaler or insufflator. For intranasal administration, the composition may be administered by liquid spray, for example by a plastic bottle nebulizer.

The composition may be formulated for transdermal administration. The composition may also be formulated as an aqueous solution, suspension or dispersion, aqueous gel, water-in-oil emulsion or oil-in-water emulsion. Transdermal formulations may also be prepared by encapsulating the composition in a polymer. The dosage form may be applied directly to the skin peptide as a lotion, cream, ointment, or by use of a patch.

The compositions of the present invention may also contain other ingredients such as flavouring agents, colouring agents, antimicrobial agents and preservatives. In addition, the composition of the present invention may contain pharmaceutically active ingredients such as hormones, anti-necrosis agents, vasodilators, pharmaceutical agents and the like.

By standard pharmaceutical procedures in cell cultures or experimental animals, e.g. determining LD₅₀(dose lethal to 50% of the population) and ED₅₀(a therapeutically effective dose for 50% of the population), toxicity and therapeutic efficacy of such compositions can be determined. The dose ratio between toxic and therapeutic effects, which is the therapeutic index, and which can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used to formulate a range of human doses. The dosage of such compounds, preferably within the range of circulating concentrations, includes the ED with little or no toxicity₅₀. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For compounds used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Doses can be formulated in animal models to achieve inclusion of IC₅₀The circulating plasma concentration range (i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms) of (a), as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In one aspect, the dosage range for human use is an amount sufficient to produce a serum concentration of at least 10 micromolar (μ M, preferably at least 25 μ M, more preferably 50 μ M.

The composition may be administered from 1 or more times per day to 1 or more times per week, including 1 time every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and/or age of the subject, and other diseases present. Furthermore, treatment of a subject with an effective amount of the composition may include a single treatment, or preferably, a series of treatments.

Detection method

The invention provides methods for detecting the polypeptides of the invention. The method generally includes providing a cell, analyzing the cell for a polypeptide of the invention, and determining whether the cell expresses the polypeptide. The cells may be ex vivo or in vivo. As used herein, the term "ex vivo" refers to cells that have been removed (e.g., isolated) from the body of a subject. Ex vivo cells include, for example, primary cells (e.g., cells that have recently been removed from a subject and are capable of limited growth or maintenance in tissue culture) and cultured cells (e.g., cells that are capable of long-term growth or maintenance in tissue culture). As used herein, the term "in vivo" refers to a cell in a subject. The in vivo cell may be a cell present in an organ or a tumor. The cell is preferably a mammalian cell, e.g., mouse, rat, or primate (e.g., monkey, human), preferably human. Examples of preferred cells include breast cells, such as breast tumor cells, and prostate cells, such as prostate tumor cells. Cells can be obtained from a subject by, for example, biopsy of human breast or prostate tissue. Samples obtained from almost all types of tissue can be used. Control cells can be cultured in vitro according to methods known in the art. Cells that do not express the ER-alpha 36kDa estrogen receptor and thus can be used as negative controls include HEK293 cells. Positive control cells include cells that can grow at low cell density in the presence of serum and BRCA1 negative cells. Preferably, the cells are grown at low density in the presence of serum. Control cells can also be obtained from a tissue sample by, for example, biopsy.

In one aspect, the method comprises analyzing the cell by contacting the cell with an antibody of the invention. Whether a cell expresses a polypeptide of the invention can be determined using conventional and art-known assays. Examples of immunoassays include competitive and non-competitive assays, such as radioimmunoassays, immunoenzyme assays, immunofluorescence assays, or enzyme immunoassays. Chemiluminescent methods utilizing horseradish peroxidase, alkaline phosphatase, or other chemiluminescent detection agents may also be used. In the methods of the invention, western blotting and chromatographic assays may also be used. The antibody of the present invention for detecting the polypeptide of the present invention may be coupled to a detection label and thereby detected directly, or a second antibody may be used. When using detection methods that allow detection of polypeptides in different cellular regions, cells expressing ER- α 36 are generally considered to be ER- α 36 positive when the polypeptides are predominantly associated with the plasma membrane and cytoplasm and less than 20% of the signal is associated with the nucleus.

In another aspect, the method comprises analyzing the cell by amplifying a polynucleotide, preferably an RNA polynucleotide (e.g., mRNA), to form an amplified polynucleotide. Preferably, the polynucleotide is amplified by Polymerase Chain Reaction (PCR), preferably by Reverse Transcriptase (RT) PCR. Methods for synthesizing DNA polynucleotides from RNA polynucleotides are known and conventional in the art. Contacting a polynucleotide obtained from a cell with a primer pair capable of amplifying a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 25, or a combination thereof. The presence of the amplified polynucleotide produced by such a primer pair indicates that the cell is capable of expressing an estrogen receptor. As used herein, the term "primer pair" refers to 2 oligonucleotides designed to flank a region of a polynucleotide to be amplified. One primer is complementary to a nucleotide present on the sense strand at one end of the polynucleotide to be amplified, and the other primer is complementary to a nucleotide present on the antisense strand at the other end of the polynucleotide to be amplified. The polynucleotide to be amplified may be referred to as a template polynucleotide. The nucleotide of the polynucleotide to which the primer is complementary may be referred to as the target sequence. The primer may have at least about 15 nucleotides, preferably, at least about 20 nucleotides, and most preferably, at least about 25 nucleotides. The conditions for amplifying a polynucleotide by PCR vary depending on the nucleotide sequence of the primer used, and methods for determining such conditions are conventional in the art.

Following amplification, the presence of the amplified polynucleotide can be determined, for example, by gel electrophoresis. By dyeing (e.g. with brominating)Ethidium) or labeled with a suitable label known to those skilled in the art, including radioactive and non-radioactive labels, the amplified polynucleotide can be visualized. Typical radiolabels include³³And P. Non-radioactive labels include, for example, ligands such as biotin or digoxigenin, and enzymes such as phosphatase or peroxidase, or various chemiluminescent agents (chemiluminescers) such as luciferin, or fluorescent compounds such as fluorescein and its derivatives.

Optionally, the presence of an ER- α 66 polypeptide, an ER- α 46 polypeptide, an ER- β, or a combination thereof in a cell can also be determined. Methods of detecting the presence of ER- α 66, ER- α 46, ER- β, or a combination thereof include the use of immunoassays using antibody or polynucleotide based detection methods, such as amplification of polynucleotides. When using detection methods that allow detection of polypeptides in different cellular regions, cells expressing ER- α 66 or ER- α 46 are generally considered to be ER- α 66 or ER- α 46 positive when the polypeptide is predominantly associated with the nucleus, e.g., more than 90% of the signal is associated with the nucleus.

Approximately 70-80% of all breast cancers express ER- α 66 and are termed ER-positive breast cancers. These tumors usually grow more slowly, differentiate better, and are associated with a better overall prognosis (Clark, In: Harris JR, edition. diseases of the Breast, volume 2.Lippincott Williams & Wilkins, 38: 103-116 (2000)). The method of detecting the presence of the polypeptide of the invention can be used as a diagnostic marker for differentiating between estrogen-positive and estrogen-negative cancers, preferably breast cancer. The results disclosed in the examples herein strongly suggest that estrogen signaling mediated by ER- α 36 contributes to breast tumorigenesis and suggest that ER- α 36 may be involved in tumorigenesis of ER- α 66 negative breast cancers. The results disclosed in the examples herein also indicate that cells highly expressing the polypeptides of the invention are more resistant to low doses of anti-estrogen drugs, e.g., tamoxifen, than cells expressing high levels of ER- α 66, but lower levels of ER- α 36. Thus, the method of detecting the presence of the polypeptides of the invention allows the identification of new patient types, i.e., ER- α 66-negative and ER- α 36-positive. Methods of detecting the presence of the polypeptides of the invention may also be used to determine the sensitivity of cells to anti-estrogen drugs and provide information about, for example, the appropriate course of treatment of an individual. For example, a physician may decide that a subject with ER- α 36-positive breast tumor cells may require a higher dose of tamoxifen to overcome resistance to lower levels.

Detecting the presence of ER- α 66, ER- α 46, ER- α 36, ER- β or combinations thereof also allows for the comparison of estrogen receptor ratios. Determining the ratio of 2 or more estrogen receptors allows prediction of the sensitivity of cells to anti-estrogen drugs (e.g., tamoxifen). For example, in this aspect of the invention, the ratio of ER- α 36 to ER- α 46, the ratio of ER- α 36 to ER- α 66, the ratio of ER- α 36 to ER- β, or a combination thereof, in a cell is determined and compared to the corresponding ratio in a control cell. Such a ratio is referred to herein as the ER- α 36 ratio. In one example, the control cells can be cells that are refractory to treatment with an anti-estrogen. In another embodiment, the control cells are cells that are not refractory to treatment with an anti-estrogen. If the above-mentioned ER-. alpha.36 ratio determined in the test cells is the same as the ER-. alpha.36 ratio described in the control cells, the test cells are classified according to the state of the control cells. For example, if the ratio of ER- α 36 to ER- α 66 in the test cells is the same as the ratio of ER- α 36 to ER- α 66 in control cells known to be resistant to tamoxifen treatment, the test cells are classified as resistant to tamoxifen treatment. However, if the ratio of ER- α 36 to ER- α 66 in the test cells is the same as the ratio of ER- α 36 to ER- α 66 in control cells that are not resistant to tamoxifen treatment, the test cells are classified as being non-resistant to tamoxifen treatment. In another example, the ratio of ER- α 36 determined in experimental cells is compared to the corresponding ratio in control cells known to be resistant to tamoxifen treatment and to the ratio in control cells known to be not resistant to tamoxifen treatment. The test cells were then classified as either tamoxifen-resistant or tamoxifen-intolerant as described above. An example of a control cell known to be intolerant to tamoxifen treatment is MCF7(ATTC deposit accession number HTB-22). An example of a control cell known to be resistant to low dose tamoxifen treatment is MDA-MB-231. Breast cancer cells known to be resistant to tamoxifen treatment can also be used as control cells by comparing the ER- α 36 ratio in experimental cells.

Reagents for identifying polypeptides capable of binding to the invention

The invention also provides methods of identifying agents that bind to the polypeptides of the invention. Such methods are also known as screening assays. The method comprises combining a polypeptide of the invention with an agent, and determining whether the agent binds to the polypeptide. Typically, determining whether the agent binds to the polypeptide comprises detecting complex formation between the agent and the polypeptide. Methods for assaying complexes include, for example, directly detecting binding of an agent to a polypeptide, and detecting binding of an agent to a polypeptide using a competitive binding assay. The assay may be a cell-free assay. The determination may be made in the presence or absence of an estrogen or anti-estrogen. Optionally, the method also includes determining whether the agent binds to an ER- α 66 polypeptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof. Preferably, the agent is incapable of binding to the ER- α 66 polypeptide.

Reagents can be obtained using any of a number of combinatorial library methods known in the art, including biological libraries, spatially addressable parallel solid or liquid phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. Biological library methods include peptide libraries, while the other 4 methods are applicable to peptide, non-peptide oligomer or small molecule compound libraries (Lam, Anticancer Drug Des.12: 145 (1997)). Examples of methods for synthesizing libraries of molecules can be found in the art (see, e.g., DeWitt et al Proc. Natl. Acad. Sci. USA 90: 6909 (1993); Erb et al Proc. Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al J. Med. chem. 37: 2678 (1994); Cho et al Science 261: 1303 (1993); Carrell et al Angel. chem. int. Ed. Engl. 33: 2059 (1994); Carell et al Angel. chem. int. Ed. Cheng. 33: 2061 (1994); and Gal lop et al J.Med. chem. 37: 1233 (1994)). Sources of potential agents to be screened include, for example, fermentation media for bacteria and fungi, and cell extracts of plants and other proliferators.

The compound library may be present, for example, in solution (e.g., HoughtenBio/Techniques 13: 412-.

The ability of a reagent to bind to a polypeptide of the invention can be determined by, for example, labeling the coupling reagent with a radioisotope or an enzyme, thereby determining the binding of the reagent to the polypeptide of the invention by detecting the labeled compound in the complex. For example, can use¹²⁵I、³⁵S、¹⁴C or³H directly or indirectly labels the reagents and detects the radioisotopes either by direct counting of the radioimmunity or by scintillation counting. Alternatively, the reagents may be labeled enzymatically, for example with horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label detected by measuring the conversion of the appropriate substrate to product.

In a similar manner, one can determine the ability of an agent to alter (e.g., stimulate or inhibit) the binding of a polypeptide of the invention to a known ligand of the polypeptide (e.g., a molecule to which the polypeptide of the invention naturally binds or interacts). Examples of such ligands are estrogens, and anti-estrogens, such as tamoxifen. In a preferred aspect, the ability of an agent to alter the binding of a polypeptide of the invention to a ligand can be determined by monitoring the activity of the polypeptide of the invention.

In another aspect, the assay of the invention comprises contacting a polypeptide of the invention with a reagent, and assaying the ability of the reagent to bind to the polypeptide. As described above, binding of the agent to the polypeptide can be determined directly or indirectly. In a preferred aspect, the assay comprises contacting a polypeptide of the invention with a ligand known to bind to a polypeptide of the invention to form an assay mixture, and contacting the assay mixture with a reagent, and determining the ability of the reagent to preferentially bind to the polypeptide (as compared to the ligand).

In another aspect, the assay comprises contacting a polypeptide of the invention with an agent, and assaying the ability of the agent to alter (e.g., stimulate or inhibit) the activity of the polypeptide of the invention.

In another aspect, the assay comprises screening for agents that alter (e.g., stimulate or inhibit) the ability of a polypeptide of the invention to modulate transcriptional transactivation of an estrogen response element, including, for example, activity mediated by the AF-1 and/or AF-2 domains of ER- α 66. Preferably, a fusion polypeptide is used, which comprises a polypeptide of the invention and a polypeptide having a transcription activation domain or a transcription repression domain. One example of a polypeptide having a transcriptional activation domain is VP-16, and other useful polypeptides having a transcriptional activation domain or transcriptional repression domain are known in the art. Typically, such fusion polypeptides are used in combination with a polynucleotide having an estrogen responsive element upstream of a promoter, and an operably linked coding sequence. Many promoters may be used, including, for example, the thymidine kinase promoter. Preferably, the operably linked coding region is capable of encoding a detectable marker, such as luciferase, or a fluorescent polypeptide, such as green fluorescent protein. In one aspect, a fusion polypeptide having a transcription activation domain, a polynucleotide, and an agent are combined in the absence of the agent under conditions that promote expression of a coding region present on the polynucleotide, and the effect of the agent on altering transcription is determined. Optionally, an ER- α 66 polypeptide and/or an ER β polypeptide can also be present, and the assay used to identify agents that alter (e.g., stimulate or inhibit) the ability of a polypeptide of the invention to modulate the transcriptional activity of a ligand-dependent and ligand-independent ER- α 66 polypeptide or ER β. Preferably, the fusion polynucleotide is present in the cell, and the polynucleotide comprises an estrogen responsive element upstream of the promoter and an operably linked coding sequence. Without intending to be limiting, it is contemplated that agents that alter the ability of the polypeptides of the invention to modulate transcriptional transactivation may include agents that alter the conformation of the polypeptides of the invention.

In an assay, it may be desirable to immobilize a polypeptide of the invention, its ligand, or a reagent to facilitate separation of one or 2 molecules in complexed and uncomplexed form and to automate the assay. In one embodiment, fusion proteins can be provided that can add domains that allow the polypeptides of the invention to bind to a substrate. For example, fusion polypeptides with glutathione-S-transferase can be adsorbed onto glutathione sepharose beads (Sigma Chemical, st. louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with reagents and the mixture incubated under conditions conducive to complex formation (e.g., physiological conditions for salt and pH). After incubation, the bead or microtiter plate wells can be washed to remove any unbound components and to measure complex formation directly or indirectly, e.g., as described above. Alternatively, the complex can be dissociated from the matrix and the level of binding determined.

Other techniques for immobilizing polypeptides onto a substrate may also be used in the screening assays of the invention. For example, the polypeptide of the present invention may be immobilized using a conjugate of biotin and streptavidin. The polypeptides of the invention can be biotinylated using biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, il.), and immobilized, for example, in wells of a streptavidin-coated 96-well plate (Pierce Chemicals). Alternatively, an antibody reactive with a polypeptide of the present invention may be derivatized to the wells of the plate and unbound polypeptide of the present invention captured into the wells by antibody conjugation. In addition to those methods for GST-immobilized complexes described above, methods for detecting such complexes include immunodetection of the complexes using antibodies reactive with antibodies that specifically bind to the polypeptides of the invention.

The invention also includes novel agents identified by the above screening assays, and their use in the treatment described herein.

Method of treatment

The invention also relates to methods of treating certain diseases in a subject. The subject is a mammal, preferably a human. As used herein, the term "disease" refers to any deviation from or disruption of the normal structure or function of a part, organ or system, or a combination thereof, of a subject exhibiting a characteristic symptom or set of symptoms. Diseases include cancers that rely on signaling through steroid hormone receptors (e.g., estrogen receptors). Examples of such diseases are referred to as estrogen-related cancers and include breast and prostate cancer. Generally, by evaluating symptoms associated with a disease, it is determined whether the subject has the disease, and whether the subject is able to respond to the treatment. As used herein, the term "symptom" refers to objective evidence of a disease present in a subject. The symptoms associated with the diseases and the assessment of such symptoms, referred to herein, are conventional and known in the art. Examples of symptoms of cancer that depend on signaling through steroid hormone receptors include, for example, the presence and size of tumors, and the presence and amount of biomarkers. Biomarkers are compounds, typically polypeptides, present in a subject and are indicative of the progression of cancer. Examples of biomarkers include, for example, Her-2 expression, and cyclin D1 expression.

Treatment of the disease may be prophylactic or, alternatively, may be initiated after the disease has progressed. Prophylactic treatment, e.g., treatment of a subject who is at "risk" for disease development, is initiated before the subject exhibits symptoms of the disease. An example of a subject at risk for disease progression is a person with risk factors (e.g., genetic markers) associated with the disease. Examples of genetic markers that can indicate that a subject has a predisposition to develop certain cancers (e.g., breast or prostate cancer) include alterations in the BRAC1 and/or BRAC2 genes. Treatment may be administered before, during or after the onset of the diseases described herein. Treatment initiated after the disease has progressed may reduce the severity of symptoms of one of the conditions, or eliminate the symptoms altogether.

In some aspects, the methods generally comprise contacting a cell with a composition comprising an effective amount of an agent that inhibits the activity of a polypeptide of the invention, e.g., an agent identified using the methods described herein. Preferably, such an agent is capable of binding to a polypeptide of the invention. In some aspects, the agent is preferably not an anti-estrogen. As used herein, an "effective amount" is an amount effective to inhibit the activity of a polypeptide of the invention, reduce symptoms associated with a disease, or a combination thereof in a cell. In one aspect, the composition can include an effective amount of an antibody of the invention. Preferably, the antibody is covalently attached to a chemotherapeutic agent, e.g., tamoxifen. The composition may optionally comprise other chemotherapeutic agents. Using ex vivo and animal models, it can be assessed whether an agent or antibody (preferably, an antibody) can be expected to function in this aspect of the invention. Such models are known in the art and are generally recognized as representative of a disease or method of treating a human. A preferred example of such an animal model is a nude mouse. For example, breast cancer cells can be seeded into the mammary fat pad of ovariectomized female nude mice, lesions subsequently formed, and evaluated by, for example, palpation, vernier caliper measurement, and tumor weight. Transgenic animal models are also available. For example, models for studying prostate cancer such as TRAMP model (see, e.g., Greenberg et al, Proc. Natl. Acad. Sci. USA, 92: 2429-.

In another aspect, a cell is contacted with a composition comprising a polynucleotide that causes post-transcriptional silencing of a coding region that encodes a polypeptide of the invention. Such polynucleotides are referred to herein as silencing polynucleotides. The silencing polynucleotide may be introduced into the cell as an RNA polynucleotide, or as a vector comprising a DNA polynucleotide capable of encoding and expressing an RNA polynucleotide. More than one type of polynucleotide may be administered, for example, 2 or more polynucleotides designed to silence the same mRNA may be combined and used in the methods herein. Alternatively, 2 or more polynucleotides may be used together, wherein each polynucleotide is individually designed to silence a different mRNA.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length (ribonucleotides, deoxynucleotides, or combinations thereof), and includes single-stranded molecules and duplexes that are double-stranded. Polynucleotides may be obtained directly from natural sources, or may be prepared with the aid of recombinant, enzymatic, or chemical techniques. Preferably, the polynucleotide of the invention is isolated. As used herein, "target coding region" and "target coding sequence" refer to a coding region whose expression is inhibited by a silencing polynucleotide. As used herein, a "target mRNA" is an mRNA encoded by a target coding region. Examples of target coding regions are nucleotide sequences encoding ER- α 36 (SEQ ID NO: 21), 5 '-flanking nucleotide sequences (SEQ ID NO: 20) or 3' -flanking nucleotide sequences, including the nucleotide sequence encoded by exon 9 (SEQ ID NO: 25).

Silencing polynucleotides include double stranded rna (dsrna) polynucleotides. The sequence of the silencing polynucleotide includes one strand, referred to herein as the sense strand, having 16 to 30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The sense strand is substantially identical, preferably identical, to the target mRNA. As used herein, the term "identical" refers to a nucleotide sequence of the sense strand having a nucleotide sequence identical to a portion of the target mRNA. The term "substantially identical" as used herein means that the sequence of the sense strand differs from the sequence of the target mRNA by 1, 2 or 3 nucleotides, preferably 1 nucleotide, and the remaining nucleotides are identical to the sequence of the mRNA. These 1, 2, or 3 nucleotides of the sense strand are referred to as non-complementary nucleotides. When the silencing polynucleotide comprises a sense strand that is substantially identical to the target mRNA, 1, 2, or 3 non-complementary nucleotides are preferably located in the center of the sense strand. For example, if the sense strand is 21 nucleotides long, the non-complementary nucleotide is generally at nucleotide 9, 10, 11, or 12, preferably at nucleotide 10 or 11. The other strand of the dsRNA polynucleotide, referred to herein as the antisense strand, is complementary to the sense strand. The term "complementary" refers to the ability of 2 single-stranded polynucleotides to base pair with each other, wherein an adenine on one polynucleotide base pairs with a thymine or uracil on a second polynucleotide, and a cytosine on one polynucleotide base pairs with a guanine on the second polynucleotide. Silencing polynucleotides also include double-stranded DNA polynucleotides corresponding to dsRNA polynucleotides. Also included are single-stranded RNA polynucleotides and single-stranded DNA polynucleotides corresponding to the sense and antisense strands disclosed herein. It will be appreciated that a sequence disclosed herein as a DNA sequence may be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uracil nucleotide. Without intending to be limiting, the polynucleotides of the invention cause post-transcriptional silencing of the target coding region. Modifications of polynucleotides used in silencing are known in the art, and silencing polynucleotides may be so modified.

Sense and antisense strands of the dsRNA silencing polynucleotides of the invention can also be covalently attached, typically by spacers made of nucleotides. Such polynucleotides are often referred to in the art as short hairpin rnas (shrnas). Upon base pairing of the sense and antisense strands, the spacer region forms a loop. The number of nucleotides constituting the loop may vary, and loops of 3 to 23 nucleotides have been reported (Sui et al, Proc. Nat' l.Acad. Sci. USA, 99, 5515-.

Silencing a polynucleotide can cause post-transcriptional suppression of expression of the target coding region, also referred to as silencing. Without wishing to be bound by theory, upon introduction into a cell, the silencing polynucleotide hybridizes to the target mRNA and triggers an endonuclease of the cell to cleave the target mRNA. As a result, expression of the polypeptide encoded by the mRNA is inhibited. Whether expression of the target coding region is inhibited can be determined, for example, by measuring a decrease in the amount of the target mRNA in the cell, measuring a decrease in the amount of the polypeptide encoded by the mRNA, or by measuring a decrease in the activity of the polypeptide encoded by the mRNA. The silencing polynucleotide may be present in a vector. The silencing polynucleotide may also be present in the vector as 2 separate complementary polynucleotides, each of which may be expressed to produce the sense and antisense strands of the dsRNA, or as a single polynucleotide containing the sense, loop and antisense strands, which may be expressed to produce an RNA polynucleotide having the sense and antisense strands of the dsRNA.

Silencing polynucleotides can be designed using methods that are routine and known in the art. For example, silent polynucleotides can be identified by scanning the coding region AA dinucleotide sequence; the 16 to 30 contiguous nucleotides downstream (3') of each of AA and mRNA may be used as the sense strand of the candidate polynucleotide. Candidate polynucleotides are polynucleotides that are tested to determine whether they can reduce the expression of one of the polypeptides of the invention. The candidate polynucleotide may be identical to the nucleotide located in a region encoding the polypeptide, or in a 5 'or 3' untranslated region of the mRNA. Optionally and preferably, the candidate polynucleotide is modified to comprise 1, 2 or 3, preferably 1, non-complementary nucleotides. Other methods are known in the art and are routinely used to design and select candidate polynucleotides. The silencing polynucleotide may, but need not, begin with a dinucleotide AA at the 5' end of the sense strand. Candidate polynucleotides may also comprise an overhang of 1, 2 or 3 nucleotides, typically at the 3' end of the sense strand, antisense strand or both. Typically, candidate polynucleotides are screened for alignment of candidate polynucleotide sequences to coding sequences using publicly available algorithms (e.g., BLAST). Based on other considerations, those that may form duplexes with mrnas expressed from non-target coding regions are generally eliminated. The remaining candidate polynucleotides can then be tested to determine whether they are capable of inhibiting the expression of one of the polypeptides described herein.

Typically, candidate polynucleotides are individually tested by introducing them into cells capable of expressing a polypeptide of the invention. Candidate polynucleotides can be prepared in vitro and then introduced into cells. Methods for in vitro synthesis include, for example, chemical synthesis using conventional DNA/RNA synthesizers. Commercial suppliers of synthetic polynucleotides and reagents for such syntheses are well known. Methods of in vitro synthesis also include, for example, in vitro transcription using circular or linear vectors in cell-free systems.

Candidate polynucleotides can also be prepared by introducing into a cell a construct that encodes a candidate polynucleotide. Such constructs are known in the art and include, for example, vectors capable of encoding and expressing the sense and antisense strands of a candidate polynucleotide, and RNA expression cassettes comprising sequences capable of encoding the sense and antisense strands of a candidate polynucleotide flanked by operably linked regulatory sequences, such as an RNA polymerase III promoter and an RNA polymerase III terminator, that result in the production of an RNA polynucleotide. The cells may be ex vivo or in vivo. Candidate polynucleotides may also be tested in animal models.

When assessing whether a candidate polynucleotide is capable of acting to inhibit the expression of one of the polypeptides described herein, the amount of target mRNA in a cell containing the candidate polynucleotide can be measured and compared to a cell of the same type that does not contain the candidate polynucleotide. Methods for measuring mRNA levels in cells are known and conventional in the art. Such methods include quantitative RT-PCR. The primers and specific conditions used to amplify the mRNA will vary depending on the mRNA and can be readily determined by the skilled artisan. Other methods include, for example, northern blotting and array analysis.

Other methods for assessing whether a candidate polynucleotide can function to inhibit expression of one of the polypeptides described herein include monitoring the polypeptides. For example, an assay can be used to measure the amount of a polypeptide encoded by an mRNA, or to measure a decrease in the activity of a polypeptide encoded by an mRNA. Methods for measuring the decrease in the amount of a polypeptide include determining the polypeptide present in a cell containing a candidate polynucleotide and comparing it to a cell of the same type that does not contain the candidate polynucleotide. For example, the antibodies of the invention may be used, for example, in western immunoblotting, immunoprecipitation, or immunohistochemistry. Antibodies to each of the polypeptides described herein are commercially available. Methods that measure a decrease in activity of one of the polypeptides of the invention may also be used.

Reagent kit

The invention provides kits comprising reagents that can be used in the methods of the invention, including, for example, determining whether a cell expresses a polypeptide of the invention. Such kits may contain packaging materials and an antibody of the invention. Such kits may also be used by medical personnel to formulate compositions, e.g., pharmaceutical compositions, containing the antibodies of the invention.

The packaging material can provide a protective environment for the antibody. For example, the packaging material may protect the antibody from contamination. In addition, the packaging material may keep the antibody in solution to avoid drying out. Examples of suitable materials that may be used as packaging materials include glass, plastic, metal, and the like. Such materials may be silanized to avoid adhesion of the antibody to the packaging material.

In one example, the invention provides a kit comprising packaging material, a first antibody that specifically binds to a polypeptide of the invention, and a second antibody that specifically binds to an ER- α 66 polypeptide. The kit may optionally comprise other components, such as buffers, reaction vessels, secondary antibodies and syringes.

Examples

Example 1

Haploid insufficiency of caveolin-1 results in activation of ER-alpha expression and estrogen-stimulated transformation of normal mammary epithelial cells

A gene-capture library of cell clones from normal human mammary epithelial MCF10A cells was prepared using poly-A capture retroviral vector (RET) obtained from the laboratory of Dr. Philip Leder of Harvard Medical School (Ishida et al, nucleic acid Res., 27: 580 (1999)). Brief description of the drawingsMoreover, the vector uses an improved poly A trapping strategy to efficiently identify functional genes regardless of their expression state in target cells. The combination of a strong splice acceptor and an efficient polyadenylation signal ensures complete disruption of the function of the "trapped" gene. The inclusion of promoterless GFP cDNA in the RET vector allows the expression pattern of the captured genes to be easily monitored in living cells. MCF10A cells were infected with a retrovirus containing the RET vector. The cells were then screened for G418-resistance to establish a gene-trapped MCF10A cell library. After 3 weeks of selection by G418, GFP expression under the control of the endogenous promoter of the "trapped" gene was monitored, and G418 resistant and GFP expressing clones were selected. The library represents 3X10⁵Independent infected clones, in which the RET vector disrupts one allele of the functional gene.

It is thought that loss of expression of the gene with tumor suppressor activity confers a transformed phenotype on normal MCF10A cells. Soft agar cloning assays were performed and cells from the obtained gene-captured cell library that grew anchorage-independent (a feature of the transformed phenotype) were identified. More than 100 positive colonies (. gtoreq.30 cells) from the G418-resistant cell library could grow on soft agar, whereas the parental MCF10A cells could not. 20 cell clones were isolated, expanded and selected for 3 weeks on soft agar containing normal serum +10^-8M17 b estradiol (E2) and dextrin-coated activated charcoal-adsorbed serum, which lacks steroid hormones. 4 cell clones (ST1, ST3, ST4 and ST6) that were able to express accelerated growth in soft agar containing additional E2 were isolated and expanded (FIG. 3).

The use of 3 '-RACE, which allows the capture of unknown 3' mRNA sequences located between the exons and the polya tail of the candidate gene, clones potential genes whose disruption would result in MCF10A cell transformation. Transformation of MCF10A cells is thought to be due to positive estrogen signaling. The purified PCR fragments generated by the RACE method were cloned and sequenced. Using a BLASTN search, the DNA sequences from 2 clones (ST1 and ST3) matched identically to the sequence of exon-3 of caveolin-1 (Cav-1) located on chromosome 7 (GenBank accession No. XM 048940). This result indicates that the allele of the Cav-1 gene was disrupted in at least 2 clones. Using the same technique, 2 other genes were identified, except for Cav-1. The gene disrupted in clone ST4 is SPRR1B (GenBank accession No. NT-004441.5), a member of the keratin/small proline-rich protein family involved in the structural organization of keratinized cell membranes. Another gene from clone ST6 is a putative novel gene (GenBank accession number 6599139), whose function is unknown.

Cav-1 protein levels were analyzed in parental MCF10A cells to test whether the expression level of Cav-1 was reduced in cells captured by the Cav-1 gene. The 4 cell clones described above (ST1, 3,4 and 6) and MCF10A-Ha-ras (MCF 10A cells transformed with Ha-ras mutant) were analyzed. Cav-1 protein levels were about 1/2 of all transformed cells compared to levels in parental MCF10A cells, as confirmed by western blot analysis (fig. 4). This data is consistent with reduced Cav-1 expression when only one functional allele of the Cav-1 gene is functional in ST1 and ST3 cells. This indicates that the Cav-1 haploidy produced by "gene trapping" is insufficient, resulting in E2-stimulated transformation. This data also suggests that Cav-1 down-regulation is also involved in transformation caused by disruption of other genes in ST4 and ST6 cells.

To determine the mechanism by which Cav-1 haploinsufficiency produces estrogen-stimulated cell growth and transformation, the expression levels of ER- α and ER- β in the above transformed cells were examined. All 4 transformed cell clones were found to express ER- α at levels comparable to Ha-ras transformed cells, whereas parental MCF10A cells and HBL-100 cells (another normal mammary epithelial cell line) expressed undetectable levels of ER- α (FIG. 5). There was no change in ER- β expression in all cells tested (fig. 5). This data indicates that ER- α expression is activated and that estrogen signaling in these transformed cells is responsible for estrogen-stimulated cell growth on soft agar. It has been previously shown that ER- α expression and estrogen signaling are both activated in Ha-ras transformed cells (Shekhar et al, int.J.Oncol., 13: 907 (1998)) and Shekhar et al, am.J.of Pathl., 152: 1129 (1998)). This result indicates that the Ras/MAPK pathway is involved in regulating ER- α expression and positive estrogen signaling.

The activation of the MAPK pathway in these transformed cells was analyzed by examining the phosphorylation level of ERK1/2 using phospho-specific antibodies. ERK1/2 was found to be highly and constitutively phosphorylated in all transformed cells, but not in MCF10A cells (FIG. 6).

Taken together, these data indicate that the Cav-1/Ras/MAPK pathway is involved in the activation of ER- α expression during human breast cancer development and cooperates with the estrogen signaling pathway to stimulate transformed cells to proliferate.

Example 2

Identification, cloning, expression and characterization of an isoform of estrogen receptor alpha (ER-alpha 36)

In the course of the above work, 3 protein bands (66-kDa, 46-kDa and 36-kDa) were observed consecutively in Western blot analysis using rat anti-ER-alpha antibody from Research Diagnostic, INC. The H222 antibody recognizes the ligand-binding domain of ER- α. To exclude the possibility that the 46-kDa and 36-kDa protein bands are degradation products of ER- α 66, cells were lysed in culture plates using a buffer containing 8M urea and tested by Western blot analysis as suggested by previous reports (Abbondanza et al, Steroids, 58: 4 (1993)). In the Cav-1 haploid-deficient cells ST1 and ST3, and MCF7 breast cancer cells, 3 different bands were readily observed (fig. 7). These results indicate the presence of an ER- α isoform sharing a similar epitope recognized by antibody H222.

Through literature search, it has been found that a 46-kDa isoform of ER- α has been cloned that functions as a dominant negative inhibitor of transactivation mediated by the AF-1 domain of ER- α 66(Flouriot et al, EMBO J., 19: 4688 (2000)). Subsequent searches identified a clone from the normal human endometrial cDNA library (RZPD clone No.: DKFZp686N23123) that contained 5.4kb cDNA. This cDNA clone carries a 310 amino acid open reading frame which theoretically produces a protein with a predicted molecular weight of 35.7 kDa. The cDNA sequence of the open reading frame is 100% matched to the DNA sequence of exon 26 of the ER- α 66 gene. The 5 'untranslated region (5' UTR) of the cDNA showed 100% homology with the DNA sequence 734-907 of the first intron of the ER-. alpha.66 gene (the first base pair of the 34,233bp first intron of the ER-. alpha.66 gene is referred to as 1). Thus, it was confirmed that the transcript of ER- α 36 started from a previously unidentified promoter in the first intron of the ER- α 66 gene.

The small, non-coding new exon from the first intron 734-907 of the ER- α 66 gene is referred to as "exon 1'". Exon 1' is then spliced directly into exon 2 of the ER- α 66 gene and continues from exon 2 to exon 6 of the ER- α 66 gene. Exon 6 was then spliced into an exon located 64,141bp downstream of the ER- α 66 gene (GeneBank accession No. AY425004, see table 1). The cDNA sequence encoding the last 27 amino acids and the 4,293bp 3' untranslated region matched 100% to the contiguous sequence from the genomic sequence of clone RP1-1304 on chromosome 6q24.2-25.3(GeneBank accession number AL078582), indicating that the remaining cDNA sequence of the new ER- α isoform was transcribed from an 4,374bp exon located downstream of the previously reported ER- α 66 gene. Thus, this exon was called exon 9 to reflect an additional exon beyond the 8 exons previously reported (fig. 8). All of these splicing events are supported by the identification of perfect splice donors and acceptors at the splice junctions. The protein ER- α 36 can be produced from a perfect Kozak sequence located in the second exon, i.e.with the same start codon as for the production of ER- α 46 (Flouriot et al, EMBO J., 19: 4688 (2000)). ER- α 36 differs from ER- α 66 by the absence of the transcriptional activation domains AF-1 and AF-2, but by the retention of the dimerization, DNA-binding and local ligand-binding domains. It also has an additional, unique 27 amino acid domain to replace the last 138 amino acids encoded by exons 7 and 8 of ER- α 66 (fig. 1). This new isoform of ER- α is referred to herein as ER- α 36.

The open reading frame encoding ER-. alpha.36 was obtained by PCR using MarathonReady cDNA (Clonetech) prepared from human placental RNA according to the manufacturer's protocol. PCR primer pairs are designed according to the cDNA sequence of DKFZp686N 23123. The 5 'primer was 5'-CGGAATTCCGAAGGGAAGTATGGCTATGGAATCC-3'(SEQ ID NO: 23) with an EcoRI site at the end, and the 3' primer was 5'-CGGGATCCAGAGGCTTTAGACACGAGGAAAC-3' (SEQ ID NO: 24) with a BamHI site at the end. The PCR product was subjected to electrophoresis on a 1% agarose gel, and the expected 1.1kb DNA fragment was observed (FIG. 9). The DNA fragment was purified, digested with EcoRI and BamHI, cloned into the pBluescript vector (pBS-ER-. alpha.36), and completely sequenced. The sequence showed 100% identity with the cDNA clone DKFZp686N23123, indicating that ER- α 36 is a naturally occurring isoform of ER- α that can be cloned from another source. FIG. 10 shows the predicted amino acid sequence encoded by the open reading frame.

Transient transfection assays were performed in human embryonic kidney 293 cells using expression vectors containing ER- α 66, ER- α 46 and ER- α 36 to test whether the cloned cDNAs were capable of producing ER- α 36 protein. Whole cell extracts from these transfected cells and MCF7 cells were subjected to Western blot analysis in which monoclonal antibody H222 was directed against the ligand-binding domain of ER- α (Abbondanza et al, Steroids, 58: 4 (1993)). In cells transfected with ER- α 36 vector, a 36kDa protein recognized by the H222 antibody was produced (FIG. 11). The size of this protein, and its inability to react with antibody H226 directed to the B-domain of ER- α 66, as well as with antibody HC 20 recognizing the C-terminus of ER- α 66, indicates that the ER- α isoform lacks the N-terminus and C-terminus of ER- α 66, resulting in an ER- α lacking the AF-1 and AF-2 domains.

On the ER-alpha 36 protein, a series of computer searches were performed. The FindMod and SCANPROSITE algorithms predicted 3 myristoylation sites in ER- α 36, suggesting that it may be located in peripheral membranes. This is consistent with the k-adjacent (PSPORT II) algorithm, which predicts 21.7%, 34.8%, 17.4% and 26% of ER- α 36 to be located in nuclear, cytoplasmic, mitochondrial and membrane fractions, respectively. This is similar to the prediction for ER- α 46 (26.1%, 30.4%, 17.4% and 26.1%, respectively). In contrast, 73.9% 8.7%, 0.1% and 17.3% of ER- α 66 have comparable predictions. Thus, differential compartmentalization of ER- α 66, ER- α 46 and ER- α 36 suggests that the functional site and primary role of each receptor may be different.

A computer search was also performed on the putative 5' flanking region of the gene encoding ER- α 36 located in the first intron of the ER- α 66 gene. The TATA Binding Protein (TBP) recognition sequence was found upstream of the cDNA initiation site, and several Sp1, NF-. kappa.B, and Ap1 binding sites were located in the 5' flanking region (FIG. 12). In the 5' upstream region of ER- α 36, a perfect semiestrogen response element (ERE) was identified, indicating that ER- α 36 is capable of E2-mediated transcriptional regulation.

Example 3

ER-alpha 36 mediates membrane-initiated estrogen signaling and is expressed in ER-negative breast cancers

Method of producing a composite material

Cell culture, establishment of stable cell lines, and membrane labeling with E2-BSA-FITC. MCF10A cells were obtained from the Karmanos Cancer Institute at Detroit, MI, and human embryonic kidney 293 cells and all breast Cancer cells were obtained from the ATCC. In appropriate tissue culture medium, 5% CO at 37 deg.C₂All cells were maintained in the atmosphere. To establish stable cells expressing recombinant ER-. alpha.36, 1X10 was used⁵Cells/density of 60-mm dishes, HEK293 cells were plated and 24 hours later transfected with ER- α 36 expression vector driven by Cytomegalovirus (CMV) promoter using FuGene6 transfection reagent (Roche molecular biochemicals). An ER- α 36 expression vector was constructed by cloning a 1.1-kb EcoRI-BamHI cDNA fragment of ER- α 36 from pBS-ER- α 36 into EcoRI and BamHI sites of a mammalian expression vector pCB6 +. Empty vector was also transfected into cells to serve as a control. 48 hours after transfection, the cells were re-plated and selected with 500. mu.g/ml G418(Invitrogen) for 2 weeks. The resulting clonal G418-resistant cell population is expanded to generate cells for further analysis. To label the cell surface of stable cells expressing recombinant ER-. alpha.36, covalently attached to E2. beta. -hemisuccinate at 4 deg.Cmu.M Fluorescein Isothiocyanate (FITC) -labeled BSA (Sigma) on perp, labeled cells for 15 min, fixed in freshly prepared 4% paraformaldehyde, and mounted with mounting solution containing DAPI for microscopic evaluation.

Cell stimulation with estrogen and antiestrogens, and MTT assay. Prior to treatment, cells were cultured in phenol red-free medium containing 2.5% dextrin-coated activated charcoal-adsorbed fetal bovine serum for 48-72 hours, then washed with PBS, and placed in fresh phenol red-free, serum-free medium containing 0.1. mu.g/ml BSA and 5. mu.g/ml insulin for 12 hours. Dormant cells were stimulated at 37 ℃ for various periods of time in serum-free medium. Different estrogens and antiestrogens were purchased from Steraloids inc. BSA-E2 β was purchased from Sigma.

For 3- (4, 5-dimethylthiazol-2-yl) -2, 5-biphenyltetrazoleBromide (MTT) assay, suspension cells were added to each well of 96-well culture plates to 1X10³Final concentration of cells/well at 37 ℃ in CO₂The culture was carried out in an incubator for 24 hours. Media containing 10nME2 β, 10nM tamoxifen or 4 OH-tamoxifen or 7.2nM UO126(Calbiochem) was added to each well for 48 hours. MTT assays were performed using the CellTiter96Aqueous One Solution cell proliferation assay kit (Bio Rad) as recommended by the manufacturer. Absorbance at 490nm was measured using a microplate reader (Promega).

Cell fractionation assay. Cell fractionation was performed as described by Marquez et al (Oncogene, 20, 5420-5430 (2001)).

Western blot analysis, indirect immunofluorescence and antibodies. For western blot analysis, cells were disrupted with RIPA buffer, boiled in gel loading buffer, and separated on a 10% SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane (Millipore). The filters were probed with various antibodies and visualized with the appropriate HRP-conjugated secondary antibody (Santa cruz biotechnology) and ECL reagent Perkin Elmer Life Sciences.

Antibodies to ERK1/2(K-23) were purchased from Santa Cruz Biotechnology. Antibodies used to analyze activation of the MAP kinase pathway include the phosphorylated forms of Mekl and ERK1/2, and were purchased from Cell Signaling Technology. Rat anti-ER-alpha antibody (H222) was purchased from Research Diagnostic Inc. Antibodies to COPB (Y-20), mSin3A (AK-11) and 5' nucleotidase (H-300) were purchased from Santa Cruzbiotechnology Inc. D4-GDI (clone 97A1015) was purchased from upstateBiotechnology.

In rabbits, a polyclonal anti-ER- α 36 antibody was generated against a synthetic peptide antigen of the last 15 amino acids of the C-terminal region of ER- α 36, which is unique to ER- α 36 (Alpha Diagnostic Inc.). The antibodies were purified using an affinity column for the synthetic peptides used to produce the antibodies. The specificity of the antibodies has also been tested in HEK293 cells transfected with ER- α 36 expression vectors incapable of expressing endogenous ER- α 36. Immunofluorescence assays showed that the immunoreactive signal for anti-ER- α 36 antibody was detected only in transient transfectants with ER- α 36-expression vector, but not in transfectants expressing mutant ER- α 36 lacking the C-terminus, indicating that ER- α 36 antibody is highly specific.

DNA transfection and luciferase assay. For transient transfection assays, sub-seeded (subled) HEK293 cells in 6-well dishes were grown to 60-70% confluence in phenol red free medium containing 2.5% steroid free fetal bovine serum. Cells were washed and transiently transfected with a total of 5 μ g plasmid (2 μ g reporter plasmid 2 × ERE-tk-Luc, and 1.5 μ g expression vector pSG5, 1.5 μ g pSG herr α 66 or 1.5 μ g pSG herr β alone, or in combination with 1.5 μ g ER- α 36 expression vector) and FuGene6 reagent (Roche molecular biochemicals). A reporter plasmid (2XERE-tk-Luc, available from Katarine Pettersson, Ph.K., Karolinska Institute, Sweden) containing 2 EREs (the-331 to-289 sequences of the chicken vitellogenin A2 gene) placed upstream of the thymidine kinase promoter was used. Expression vectors containing ER- α 66 and β were also obtained from doctor Katarine Pettersson. Expression vectors for ER- α 46 were obtained from the Zafar Nawaz doctor (Creighton University medical center, Omaha, Nebraska). Cells were treated with or without E2(10nM) for 12 hours prior to determination of luciferase activity. Luciferase assays were performed using luciferase assay kit from Promega. Values correspond to mean ± standard deviation of more than 3 separate transfection experiments.

RNA extraction and northern blot analysis. Total cellular RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. Mu.g of total RNA were separated by electrophoresis on a 1.2% formamide/formaldehyde gel and blotted on a nylon membrane (Hybond-N, Amersham pharmacia Biotech). The blot was prehybridized for 1 hour and hybridized in Quick-Hybridization solution (Amersham Pharmacia Biotech) for 2 hours at 65 ℃. The probe contained a 410bpcDNA fragment from the untranslated region of ER- α 363', unique to ER- α 36, and a β -actin DNA probe from BD Clonetech. The DNA probe was labeled with 32P dCTP and Rediprime II DNA labeling kit (Amersham Biotech). Blots were autoradiographed using an intensifying screen overnight at-70 ℃. The same membrane was stripped and re-probed with a labeled β -actin DNA probe to confirm equivalent loading.

Breast cancer specimens and immunohistochemical assays. Paraffin-embedded human breast cancer specimens were obtained from the Pathology department of the Chinesian admirable Yi Fu Hospital, Hangzhou, China. Immunohistochemical staining was performed using an Ultrasensitive S-P kit (Maxin-Bio, China) according to the manufacturer' S instructions, using an ER- α 66-specific antibody (LabVision corporation, USA) and an ER- α 36-specific antibody as primary antibodies, respectively.

Results

To further confirm that ER- α 36 is a naturally occurring isoform of ER- α 66, northern blot analysis was performed on total RNA from the normal mammary epithelial cell line MCF10A, and ER-positive and-negative (i.e., ER- α 66-positive and-negative) breast cancer cells. DNA probes were synthesized using RT-PCR method using a primer pair (5 ' GCAAAGAAGAGAATCCTGAACTTGCATCCT (SEQ ID NO: 26) and 5 ' TTAGTCAGGTATTTAATAACTAGGAATTG (SEQ ID NO: 27)) designed based on the ER-. alpha.363 ' untranslated region unique to ER-. alpha.36. Northern blot analysis indicated that in the ER-positive (i.e., ER- α 66-positive) breast cancer cell, MCF7, a single mRNA of estimated 5.6kb in size was identified, but not in the MCF10A cell (fig. 13). Surprisingly, ER- α 36 was also expressed in MDA-MB-231 cells, which is a well-known ER-negative (i.e., ER- α 66-negative) breast cancer cell line (FIG. 13). This data indicates that transcripts of the predicted size of ER- α 36mRNA can be expressed in breast cancer cells, and even in breast cancer cells lacking ER- α 66.

ER- α 36 inhibits the transactivation activity of ligand-bound-and ligand-unbound-ER- α 66 and- β. We first tested whether ER- α 36, lacking both the AF-1 and AF-2 domains, retains any transcriptional activity. Transient transfection assays were performed in HEK293 cells using luciferase-expressing reporter constructs containing 2 Estrogen Response Elements (ERE) upstream of the thymidine kinase promoter (2 XERE-tk-Luc). The HEK293 cell line was chosen because it was previously reported that AF-1 and-2 of ER- α 66 functioned equally well in HEK293 cells (Denger et al, mol. Endocrinol., 15, 2064-2077 (2001)). As shown in FIG. 14, we found that ER- α 36 did not exhibit intrinsic transcriptional activity in the presence and absence of E2 β, consistent with the finding that ER- α 36 lacks a transcriptional activation domain. We then assessed the regulatory function of ER- α 36 in transcriptional transactivation activity mediated by the AF-1 and-2 domains of ER- α 66. Co-expression of ER- α 36 in the presence and absence of E2 β strongly inhibited the transactivation activity of ER- α 66 (FIG. 14), indicating that ER- α 36 is able to inhibit the transactivation activity mediated by the AF-1 and-2 domains of ER- α 66. Furthermore, ER- α 36 also inhibited ligand-dependent and ligand-independent transactivation activity of ER- β (fig. 14).

ER- α 36 mediates membrane-initiated estrogen signaling pathways. Previous reports have shown that E2 β stimulates rapid activation of the MAPK/ERK pathway (Razandi et al, mol. Endocrinol., 13, 307-. To determine whether ER- α 36 is involved in this signaling pathway, we established stable cells expressing exogenous ER- α 36 in HEK293 cells that are incapable of expressing endogenous ER- α. All ER- α 36 transfected HEK293 cells were incubated with membrane-impermeable Fluorescein Isothiocyanate (FITC) -conjugated E2 β -BSA (E2 β -BSA-FITC). The cell surface of ER-. alpha.36 transfected cells was strongly labeled with E2. beta. -BSA-FTIC, and control cells transfected with the empty vector were not labeled with E2. beta. -BSA-FITC. Cell lysates were prepared from resting cells treated with or without E2 β (10nM) for various lengths of time. ERK activation was measured by immunoblotting using phosphorylation state-dependent and phosphorylation state-independent antibodies. Within 5 minutes, a 10-fold increase in phosphorylation of ERK1/2 lasting about 45 minutes was observed in cells transfected with ER- α 36 expression vector, but not in control cells transfected with the empty vector (fig. 15a and 15 b). However, serum (20%, 10 min) activated ERK1/2 in these control cells (fig. 15b), indicating that the MAPK signaling pathway is not overall defective in these cells. Furthermore, in ER- α 36 transfected cells, Mek1, a kinase that phosphorylates and activates ERK1/2, was also activated in response to E2 β (FIG. 15 a). To further demonstrate that membrane-initiated estrogen signaling activates ERK1/2, ER- α 36 transfected cells were also treated with E2 β -BSA (membrane impermeable form of E2 β). A strong activation of ERK1/2 phosphorylation was also observed in E2 β -BSA treated cells (FIG. 15 a).

ER-36 mediates the activation of MAPK signaling pathways stimulated by different estrogens and antiestrogens. We also treated ER- α 36 transfected cells with estrone (E1), 17 β -estradiol (E2 β), 17 α -estradiol (E2 α), estriol (E3) or estetrol (E4) for 10 min and found that all these estrogens except E1 activated ERK1/2 phosphorylation at very similar levels, indicating that ER- α 36 could recognize these estrogens at similar levels (fig. 15 c). We then included antiestrogens in experiments, including tamoxifen, 4 OH-tamoxifen, ICI-182, 780, to test whether ER- α 36 mediated estrogen signaling is sensitive to antiestrogens. Tamoxifen, 4 OH-tamoxifen and the pure anti-estrogen ICI-182, 780 were unable to block ERK1/2 activation mediated by ER- α 36. In contrast, the effect was even stronger compared to the effect mediated by E2 β alone (fig. 15 c). When ER- α 36 transfected cells were treated with 1 μ M tamoxifen alone (which can inactivate the concentrations of ER- α 66 and β), a strong and persistent activation of ERK1/2 was observed for more than 8 hours (fig. 15 d). However, the same concentration of tamoxifen had no effect in control 293 cells transfected with the empty expression vector.

ER-alpha 36 mediates E2-stimulated cell proliferation. To further determine whether ER- α 36-mediated estrogen-activated MAPK pathway can lead to transcriptional signaling in the nucleus, we examined the ability of membrane-initiated estrogen signaling to activate the transcription factor Elk (a downstream effector of the MAPK/ERK signaling pathway). We used an ERK-responsive GAL-Elk chimeric transcription factor consisting of the DNA-binding domain of the yeast transcription factor GAL4 fused to the ERK-responsive transactivation domain of human Elk1, transiently transfected 293 cells expressing ER- α 36, and measured its in vivo activity on expression of GAL 4-binding reporter in the presence of E2 β. The reporter gene was 5X Gal4-LUC, a luciferase reporter plasmid containing 5 Gal4DNA binding sites. Bacterial beta-galactosidase expression vectors were used to control transfection efficiency. After transfection, the cell culture was maintained in estrogen-free medium for 36 hours, followed by the addition of E2 β (10nM) for 12 hours. Luciferase activity with standard deviation represents more than 3 experiments performed in duplicate. Estrogen treatment of ER- α 36 transfected cells induced an approximately 2-fold increase in Elk/Gal4 fusion-mediated transactivation of the reporter gene, whereas E2 β had no effect on transcriptional activity of the Elk/Gal4 fusion protein in control cells transfected with empty vector (fig. 16 a).

We next investigated whether ER- α 36 could mediate estrogen-stimulated cell proliferation. Proliferation of ER- α 36 transfected cells and control cells in the presence and absence of E2 β was assessed by MTT assay. E2 β treatment stimulated proliferation of ER-. alpha.36 transfected cells, whereas E2 β had no effect on growth of control cells transfected with the empty expression vector (FIG. 16 b). Containing antiestrogens, including tamoxifen and 4 OH-tamoxifen, failed to block E2 β -stimulated cell growth (fig. 16 b). Tamoxifen alone, or 4 OH-tamoxifen, strongly stimulates the growth of ER- α 36 transfected cells. However, UO126, a specific inhibitor of the MAPK pathway, strongly inhibited E2 β -stimulated cell growth. These data indicate that ER- α 36-mediated membrane estrogen signaling stimulates cell growth through activation of the MAPK/ERK signaling pathway. The data also indicate that antiestrogens can also stimulate cell growth via ER- α 36.

ER- α 36 is primarily a membrane-based estrogen receptor. To further characterize ER- α 36, we have successfully developed a polyclonal anti-ER- α 36 antibody directed against the 15 amino acids of the C-terminal region of ER- α 36, which is unique to ER- α 36. The antibodies were purified using an affinity column for the synthetic peptides used to produce the antibodies. Western blot analysis of proteins prepared from normal breast epithelial cells and established breast cancer cell lines using this antibody confirmed a single protein band of 37-kDa molecular weight in some breast cancer cells, but not in normal breast epithelial cells (fig. 17 a). ER- α 36 was expressed in MDA-MB-231, MDA-MB436 and HB3396 cells, 3 well-known ER- α 66-negative breast cancer cell lines, and also in ER- α 66-positive breast cancer cell MCF7, but not in T47D (FIG. 17a), consistent with our northern blot data for ER- α 36 expression in ER- α 66-negative breast cancer cells. To further evaluate the likelihood of expression of ER- α 36 in ER- α 66 negative breast cancer cells, indirect immunofluorescence assays and confocal microscopy using anti-ER- α 36 specific antibodies in permeabilized MDA-MB-231 cells indicated that ER- α 36 is expressed on the plasma membrane, cytoplasm, and nucleus of ER- α 66 negative breast cancer cells MDA-MB-231.

To further assess ER- α 36 compartmentalization in cells, subcellular fractionation assays were performed to isolate the nucleus, plasma membrane and cytosol from ER- α 36 transfected HEK293 cells. ER- α 36 was identified from the different fractions by immunoassay. A high percentage of ER- α 36 (about 50%) is located on the plasma membrane and a low percentage is located in the cytosol (about 40%) and nucleus (about 10%). To exclude cross-contamination of the different fractions, the purity of the fractions was checked by western blot analysis with different marker proteins including mSin3A (nuclear), GDP dissociation inhibitor (cytosol), 5' nucleotidase (plasma membrane) and β -COP (golgi apparatus). These results confirm that there is no contamination in the different fractions. This experiment confirmed that ER- α 36 is mainly membrane-based estrogen receptor (fig. 17 b).

ER- α 36 is expressed in breast cancer specimens that are ER- α 66 negative. To further determine the association of ER- α 36 with human breast cancer, we examined the expression pattern of ER- α 36 in human breast cancer specimens by immunohistochemical assay using specific anti-ER- α 36 antibodies. In situ analysis of human breast tissue indicates upregulation of ER- α 36 in breast cancer. Cells positive for ER protein were stained brown and nuclei were stained blue with hematoxylin. Of the 35 breast cancer specimen cases examined, 21 (60%) were positive for ER- α 36 staining and 21 (60%) were positive for ER- α 66 (table 2). Consistent with our RNA and Western blot analysis, 11 out of 14 (78%) breast cancer specimens that were negative for ER- α 66 stained positive for ER- α 36, indicating that a majority of ER-negative (i.e., ER- α 66-negative) breast cancers still express ER- α 36. In situ staining of ER- α 36 in normal tissue sections found in human ductal carcinoma indicated that ER- α 36 is expressed only in luminal epithelial cells and is predominantly localized to the cytoplasm and plasma membrane. Tumor sections were from human invasive ductal carcinoma and invasive ductal carcinoma. All 21 cases of samples positive for ER-. alpha.36 showed an immunostaining pattern of ER-. alpha.36 predominantly outside the cell nucleus, in contrast to the predominant nuclear staining of ER-. alpha.66. Similar to ER- α 66, some of the luminal epithelial cells in adjacent normal tissue also stained positive for ER- α 36. These results confirm that ER- α 36, similar to ER- α 66, is expressed in 2/3-examined human breast cancer, and suggest that ER- α 36 may be involved in the development of ER- α 66-negative breast cancer.

In this study, a new variant of ER- α, ER- α 36, has been identified, cloned and characterized. The ER- α isoform is a transcript from a previously unidentified promoter in the first intron of the ER- α 66 gene. The putative promoter region of ER- α 36 contains a TATA-binding protein (TBP) recognition sequence located upstream of the initiation site of the ER- α 36cDNA, and several Sp1, NF-kB, and Ap1 binding sites (FIG. 12). We have cloned the 5' flanking region of ER-alpha 36 and confirmed that it has strong promoter activity. Furthermore, in the 5' flanking region of ER- α α 36, a perfect half of the ERE site was identified, indicating that ER- α 36 is ER-mediated transcriptional regulation.

The ER- α 36 protein is identical to the ER- α 66 protein encoded by exons 2-6 of the ER- α 66 gene. This isoform does not have previously identified domains with transactivation activity, AF-1 and-2. Indeed, analysis of ER- α 36 transactivation activity confirmed the lack of intrinsic transcriptional activity of ER- α 36. However, ER- α 36 was effective in inhibiting transactivation activity mediated by the AF-1 and-2 domains of ligand-bound-and ligand-unbound-ER- α 66 and- β, indicating that ER- α 36 is a potent inhibitor of genomic estrogen signaling. This finding is similar to previous reports that ER- α 46, lacking the AF-1 domain, can function as a potent competitor to inhibit AF-1 activity of ER- α 66.

The presence of plasma membrane-based ERs, which trigger rapid estrogen signaling, has long been controversial because the molecular identity of the receptor has not been established. Previously, Razandi reported that ER- α 66 and- β both could initiate membrane estrogen signaling using transfection assays, although only a very small percentage of them were expressed on the cell surface (Razandi et al, mol. endocrinol., 13, 307-319(1999)), suggesting that these ERs may be involved in membrane-initiated estrogen signaling in addition to their traditional role in genomic estrogen signaling. Recently, the 46-kDa isoform of ER- α has been localized to the cell surface and was found to mediate estrogen-stimulated eNOS phosphorylation (Li et al, Pro.Natl.Acad.Sci.USA, 100, 4807-. Here, we demonstrate that another ER- α variant, ER- α 36, is predominantly localized to the plasma membrane and mediates the activation of the MAPK/ERK pathway induced by membrane-initiated estrogen signaling. Moreover, since ER- α 36 lacks entirely intrinsic transactivation activity and functions only as a modulator of genomic estrogen signaling, ER- α 36 may function primarily as a membrane-based estrogen receptor to mediate membrane-initiated estrogen signaling. Previously, it has been reported that some E2 β -mediated rapid effects occur in neurons in ER- α knockout (aERKO) mice and that these effects cannot be blocked by ICI 182, 780 (Gu et al, Endocrinology, 140, 660-666(1999)), suggesting the existence of more than one membrane-initiated estrogen signaling pathway. We demonstrate here that anti-estrogens cannot block ER- α 36-mediated MAPK/ERK activation, suggesting that ER- α 36 is involved in the previously described anti-estrogen insensitive signaling pathway. Since α ERKO mice were generated by disruption of the first coding exon (exon skipped when producing transcripts of ER- α 36) of the mouse ER- α gene by insertion, production of mouse counterparts of ER- α 36 may remain normal in these knockout mice. Thus, ER- α 36 may contribute to the residual estrogenic effects observed in these mice. More recently, Toran-Allerand et al (J.neuroscience 22, 8391-8401(2002)) reported the presence of a novel plasma membrane-associated estrogen receptor (ER-X) with an estimated molecular weight of 63-65 kDa. ER-X shows some similarities to ER- α 36, e.g., antibody reaction with the ligand-binding domain of ER- α 66, and responds equally well to E2 α and β. However, the molecular identity of these 2 receptors is expected for the cloning and sequencing of ER-X.

We have also shown that ER- α 36 promotes membrane-initiated activation of the MAPK/ERK pathway, which leads to estrogen-stimulated cell proliferation. Thus, ER- α 36, which lacks intrinsic transcriptional activity, is sufficient to promote estrogen-stimulated cell growth, supporting previous reports that transcriptionally inactive mutants of ER- α 66 induce DNA synthesis. Together, these data suggest that transcriptional activity of the ER may not be required to promote estrogen-stimulated cell growth. Surprisingly, we have also observed that during the experimental phase (48 hours), antiestrogens such as tamoxifen, strongly activate MAPK/ERK signalling and stimulate cell growth. This finding is well consistent with the notion that tamoxifen acts as an agonist and antagonist of estrogen signaling and suggests that ER- α 36 may also be involved in membrane-initiated anti-estrogen signaling.

It is well known that breast cancer cells with an ER- α 66 positive phenotype (ER-positive breast cancer) are more differentiated and have lower metastatic potential than ER- α -negative tumors (McGuire, W.L. Prognotic factors in primary breast cancer. cancer Surv.5, 527-536 (1986)). Interestingly, ER- α 36 was expressed not only in the ER- α 66-positive breast cancer subtype, but also in most of the examined ER- α 66-negative breast cancers. To confirm these results, it has been reported that estrogen signaling induces rapid activation of the PI3K/Akt pathway in MDA-MB-231 cells, which cannot be blocked by estrogen antagonists (Tsai et al, Cancer Res..61, 8390-8392(2001)), which can be explained as estrogen signaling through ER-independent pathways. It has also been shown that high concentrations of tamoxifen induce Apoptosis in MDA-MB-231 cells (Mandleker et al, Apoptosis 6, 469-477 (2001)). These data strongly suggest that ER- α 66-negative breast cancer may still retain the estrogenic or anti-estrogenic effects mediated by membrane-initiated signaling.

It can alter the ligand-binding specificity and affinity of ER- α 36 by replacing the last 5 helices (helices 8-12) of the 12 helices of ER- α 66 with a unique 27 amino acid domain, ER- α 36 also having a unique ligand-binding domain. In fact, we found that ER- α 36 is able to induce membrane-initiated signaling, which is as good as the response to E2 α and β, E3 and E4. Thus, ER- α 36 appears to have a broader ligand-binding spectrum than ER- α 66, which makes ER- α 36 a potentially more effective mediator of mitogenic signaling. Further analysis of ligand-binding specificity and affinity of ER- α 36 facilitates the design of anti-estrogen agents specific for ER- α 36, which can be used to treat ER- α negative (i.e., ER- α 66-negative) breast cancer.

Example 4

E2 beta promotes the growth of ER-alpha 66-negative MDA-MB-231 cells in soft agar

To determine anchorage-independent growth in soft agar in the presence and absence of combined or separate E2 β and tamoxifen, 500MDA-MB-231 cells were suspended in 3ml of 3.5% (wt/vol) agar containing phenol red-free DMEM/F12 medium supplemented with 10% E2-free fetal bovine serum. The cells were then plated onto 0.7% (wt/vol) agar in 5 replicate 60mm dishes containing phenol red free DMEM/F12 medium supplemented with 10% E2 free fetal bovine serum. Cell coverage on soft agar was supplemented with medium of 10% E2-free fetal bovine serum with or without 1nM E2 β, or in combination with 1nM tamoxifen. After 3 weeks, colonies were scored using an inverted microscope. As shown in fig. 18, we found that treatment with E2 strongly promoted anchorage-independent growth of ER- α 66-negative MDA-MB-231 cells in soft agar, whereas anti-estrogen tamoxifen inhibited the effect of E2 β, indicating that ER- α 66-negative MDA-MB-231 cells maintained responsiveness to estrogen signaling, presumably through ER- α 36.

Example 5

E2 beta can induce membrane-initiated estrogen signaling in ER-alpha 66 negative MDA-MB-231 cells

To determine whether E2 β could induce membrane-initiated estrogen signaling in ER- α 66 negative MDA-MB-231, serum-starved MDA-MB-231 cells were treated with 1nM E2 β for different time periods. For western blot analysis, cells were disrupted with RIPA buffer, boiled in gel loading buffer, and separated on a 10% SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane (Millipore). The filters were probed with an antibody directed against ERK1/2(K-23) (Santa Cruz Biotechnology), or against the antibody used for the phosphorylated form of ERK1/2 (Cell Signaling Technology). As shown in FIG. 19, treatment of MDA-MB-231 cells with estradiol-17 beta (E2 beta) induced rapid phosphorylation of ERK 1/2. These data strongly suggest that E2 β stimulates the activation of the MAPK pathway in ER- α 66-negative MDA-MB-231 cells.

All publications, patents and patent applications are herein incorporated by reference. While the foregoing description of the invention has been described with reference to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is capable of being practiced in other embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

Claims (55)

1. An isolated antibody that specifically binds to SEQ ID NO: 1, said immunogenic fragment comprising at least 5 contiguous amino acids.

2. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody of claim 1, wherein the antibody is a polyclonal antibody.

4. The antibody of claim 1, wherein the antibody is a humanized antibody.

5. The antibody of claim 1, wherein the antibody is an antibody fragment.

6. The antibody of claim 1, wherein the antibody is covalently attached to a compound.

7. The antibody of claim 6, wherein the compound is a chemotherapeutic agent.

8. The antibody of claim 6, wherein the compound is a detection label.

9. The antibody of claim 8, wherein the detection label is a fluorescent label.

10. A composition comprising the antibody of claim 1.

11. The composition of claim 10, further comprising a pharmaceutically acceptable carrier.

12. A method of making an antibody comprising:

administering to the animal a composition comprising SEQ ID NO: 1, and isolating antibodies from the animal,

wherein the immunogenic fragment comprises at least 5 contiguous amino acids, and the isolated antibody specifically binds to the immunogenic fragment.

13. The method of claim 12, wherein the polypeptide is covalently attached to a carrier polypeptide.

14. The method of claim 12, wherein said isolating comprises obtaining cells from the animal that produce the antibody, the method further comprising using the cells to make hybridomas that produce monoclonal antibodies.

15. A polyclonal antibody produced by the method of claim 12.

16. A monoclonal antibody produced by the method of claim 14.

17. A cell comprising a foreign coding region, wherein the coding region encodes a polypeptide comprising SEQ id no: 20, or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 20, wherein said second polypeptide has ER- α 36 activity.

18. The cell of claim 17, wherein said coding region is operably linked to a constitutive promoter.

19. The cell of claim 17, wherein the cell is a eukaryotic cell.

20. The cell of claim 17, wherein the cell is a prokaryotic cell.

21. A cell capable of expressing an exogenous polypeptide, wherein the polypeptide comprises SEQ ID NO: 20, or comprises a sequence identical to SEQ ID NO: 20 and having ER- α 36 activity, and at least 90% identity.

22. The cell of claim 21, wherein said coding region is operably linked to a constitutive promoter.

23. The cell of claim 21, wherein the cell is a eukaryotic cell.

24. The cell of claim 21, wherein the cell is a prokaryotic cell.

25. A method of identifying an agent that binds to a polypeptide, the method comprising:

a combination comprising SEQ ID NO: 1, and reagents, and

detecting complex formation between the reagent and the polypeptide.

26. The method of claim 25, wherein binding of the agent to the polypeptide is detected by a method selected from the group consisting of: directly detecting binding of the agent to the polypeptide, and detecting binding of the agent to the polypeptide using a competitive binding assay.

27. The method of claim 25, further comprising, determining whether the agent binds to a polypeptide comprising seq id NO: 18.

28. The method of claim 25, further comprising determining whether the agent inhibits ER- α 36 activity of the polypeptide.

29. A method of detecting a polypeptide, comprising:

providing a cell in vitro, wherein the cell is,

determining whether a polypeptide having a molecular weight of 36kDa and having the amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence corresponding to SEQ ID NO: 20, having at least 90% identity as determined by electrophoresis on a Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel.

30. A method of detecting a polypeptide, comprising:

providing a cell in vitro, wherein the cell is,

determining whether a polypeptide having a molecular weight of 36kDa and comprising SEQ ID NO: 1, as determined by electrophoresis on a Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel.

31. The method of claim 29 or 30, wherein the cell is a tumor cell.

32. The method of claim 31, wherein the tumor is a breast tumor.

33. The method of claim 29 or 30, wherein said determining comprises contacting the cell with a polypeptide capable of specifically binding to SEQ ID NO: 1, or an immunogenic fragment thereof.

34. The method of claim 33, wherein the antibody is covalently attached to a detection label.

35. The antibody of claim 34, wherein the detection label is a fluorescent label.

36. The method of claim 29 or 30, wherein said determining comprises amplifying the mRNA polynucleotide to form an amplified polynucleotide, wherein said amplifying comprises contacting a polynucleotide obtained from the cell with a primer pair capable of amplifying the mRNA polynucleotide comprising the sequence of SEQ id no: 22 or SEQ ID NO: 25, or a combination thereof, wherein the presence of the amplified polynucleotide indicates that the cell expresses the polypeptide.

37. The method of claim 36, wherein one primer of the primer pair is selected from the group consisting of SEQ ID NO: 22, and SEQ ID NO: 25, or a combination thereof, and wherein each primer has at least 15 nucleotides.

38. A method of inhibiting ER- α 36 activity in a cell, comprising: enabling expression of a polypeptide comprising seq id NO: 1, with a compound that inhibits ER- α 36 activity.

39. The method of claim 38, wherein the compound comprises an antibody of any one of claims 1-9.

40. The method of claim 39, wherein the compound comprises a peptide that specifically binds to a polypeptide comprising SEQ ID NO: 1 of amino acids 13-27.

41. The method of claim 38, wherein the cell is ex vivo.

42. The method of claim 38, wherein the cell is ER- α 66 negative.

43. The method of claim 38, wherein the cell is ER- α 46 negative.

44. The method of claim 38, wherein the compound is not an anti-estrogen.

45. A method of inhibiting ERK1/2 or MEK1/2 phosphorylation in a cell in vitro comprising: contacting a cell with the antibody of any one of claims 1-9;

wherein the cell expresses a polypeptide having a molecular weight of 36kDa, binds estrogen and comprises SEQ ID NO: 1, the molecular weight as determined by electrophoresis on a Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel, and the antibody is at a dose capable of inhibiting estrogen and the amino acid sequence set forth in amino acids 13-27 of SEQ ID NO: 20 in combination.

46. The method of claim 45, wherein the antibody specifically binds to a polypeptide comprising SEQ ID NO: 1, amino acid 13-27.

47. An isolated polypeptide consisting of SEQ ID NO: 1 or a modified sequence thereof which is a sequence of SEQ ID NO: 1, and the modified sequence is capable of stimulating production of a polypeptide that binds to amino acids 1-27 of SEQ ID NO: 1, amino acids 1-27.

48. An isolated polypeptide consisting of SEQ ID NO: 1 or a modified sequence thereof which is a sequence of SEQ ID NO: 1, and the modified sequence is capable of stimulating production of a polypeptide that binds to amino acids 13-27 of SEQ ID NO: 1, amino acids 13-27.

49. An isolated polypeptide comprising the polypeptide of claim 47 or 48 covalently attached to a carrier polypeptide.

50. An isolated polynucleotide encoding the polypeptide of any one of claims 47-49.

51, SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 1 of at least 5 consecutive amino acids.

52. A kit comprising the isolated antibody of any one of claims 1-9 and packaging material.

53. Use of the antibody of any one of claims 1-9 for the preparation of a diagnostic reagent for distinguishing between estrogen-positive and estrogen-negative cancers.

54. The use of claim 53, for distinguishing between estrogen-positive and estrogen-negative cancers by detecting whether a polypeptide having a molecular weight of 36kDa, as determined by electrophoresis on a Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel, and capable of binding estrogen or antiestrogen is expressed in the cells.

55. Use of an antibody according to any one of claims 1 to 9 for the manufacture of a medicament for treating an estrogen-related cancer in an individual, wherein the antibody is capable of inhibiting the binding of estrogen to a cellular polypeptide of SEQ ID NO: 20 in combination.