WO2002046466A2

WO2002046466A2 - Complexes of brca and stat polypeptides and methods of use in the detection and treatment of cancer

Info

Publication number: WO2002046466A2
Application number: PCT/IB2001/002797
Authority: WO
Inventors: Sigridur Valgeirsdottir
Original assignee: Iceland Genomics Corporation
Priority date: 2000-12-04
Filing date: 2001-12-04
Publication date: 2002-06-13
Also published as: AU2002233572A1; WO2002046466A3

Abstract

The invention provides a method of inhibiting cellular proliferation mediated by a BRCA/STAT complex. The method consists of contacting a BRCA/STAT- containing cell with an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in the cell. The invention also provides a method of reducing the severity of cancer. The method consists of administering to an individual an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in cancer cells in the individual. Also provided are methods of detecting the risk of breast cancer in an individual. In one embodiment, the method consists of detecting an amount or activity of a BRCA/STAT complex in a sample obtained from an individual suspected of having breast cancer and comparing the amount or activity of the BRCA/STAT complex from the sample to an amount or activity form a reference sample, a difference in the amount or activity between the sample and the reference sample indicating the presence or susceptibility of breast cancer. The invention also provides methods of prognosing the severity or progression of breast cancer.

Description

COMPLEXES OF BRCA AND STAT POLYPEPTIDES AND METHODS OF USE IN THE DETECTION AND TREATMENT OF CANCER

BACKGROUND OF THE INVENTION

Cancer is currently the second leading cause of mortality in the United States. However, it is estimated that by the year 2000 cancer will surpass heart disease and become the leading cause of death in the United States. Breast cancer in particular is one of the most common cancers and a significant cause of female cancer mortality throughout the world. Cancerous tumors result when a cell escapes from its normal growth regulatory mechanisms and proliferates in an uncontrolled fashion. Tumor cells can metastasize to secondary sites if treatment of the primary tumor is either not complete or not initiated before substantial progression of the disease. Early diagnosis and effective treatment of tumors is therefore essential for survival.

Continuous developments over the past quarter century have resulted in substantial improvements in the ability of a physician to diagnose a cancer in a patient. Unfortunately, methods for treating cancer have not kept pace with those for diagnosing the disease. Thus, while the death rate from various cancers has decreased due to the ability of a physician to detect the disease at an earlier stage, the ability to treat patients presenting more advanced disease has progressed only minimally. A major hurdle to advances in treating cancer is the relative lack of agents that can selectively target the cancer, while sparing normal tissue. For example, radiation therapy and surgery, which generally are localized treatments, can cause substantial damage to normal tissue in the treatment field, resulting in scarring and, in severe cases, loss of function of the normal tissue. Chemotherapy, which generally is administered systemically, can cause substantial damage to organs such as bone marrow, mucosae, skin and the small intestine, which undergo rapid cell turnover and continuous cell division. As a result, undesirable side effects, for example, nausea, hair loss and reduced blood cell counts, occur as a result of systemically treating a cancer patient with chemotherapeutic agents. Such undesirable side effects often limit the amount of a treatment that can be administered.

A related hurdle prohibiting significant advances in cancer treatment is the relative lack of cancer specific targets. For example, treatments have been attempted where cytotoxic agents have been directed to tumor cell surface markers. However, such approaches similarly result in pleiotropic side effects and damage to normal tissue because of the lack of availability or specificity of tumor cell markers. Due to such shortcomings in treatment, cancer remains a leading cause of patient morbidity and death.

Thus, there exists a need for improved methods of treating cancer and other proliferative pathological conditions. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION

The invention provides a method of inhibiting cellular proliferation mediated by a BRCA/STAT complex.

The method consists of contacting a BRCA/STAT- containing cell with an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in the cell.

The invention also provides a method of reducing the severity of cancer. The method consists of administering to an individual an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in cancer cells in the individual.

Also provided are methods of detecting the risk of breast cancer in an individual. In one embodiment, the method consists of detecting an amount or activity of a BRCA/STAT complex in a sample obtained from an individual suspected of having breast cancer and comparing the amount or activity of the BRCA/STAT complex from the sample to an amount or activity from a reference sample, a difference in the amount or activity between the sample and the reference sample indicating the presence or susceptibility of breast cancer. In another embodiment, the method consists of exposing cells obtained from an individual suspected of having breast cancer to conditions that induce BRCA/STAT complex formation and detecting an amount or activity of uncomplexed BRCA in the exposed cells, wherein a decreased BRCA amount or activity compared to a reference sample indicates an increased susceptibility for breast cancer. The invention also provides a method of prognosing the severity or progression of breast cancer. The method consists of detecting an amount or activity of a BRCA/STAT complex in a sample obtained from an individual at a site suspected of containing cancer cells and comparing the amount or activity of the BRCA/STAT complex with a reference sample, a difference in the amount or activity of the BRCA/STAT complex in the sample indicating the level of severity or progression of cancer.

The invention further provides a method of screening for compounds that modulate the activity of a BRCA/STAT complex. The method consists of contacting a sample containing BRCA and STAT with a test compound under conditions that induce BRCA/STAT complex formation and detecting an amount of BRCA/STAT complex formation or an activity of a BRCA/STAT complex, a change in the amount of formation or activity of the BRCA/STAT complex in the sample compared to a reference sample indicating that the test compound has BRCA/STAT complex modulating activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that STAT5 coprecipitates with BRCAl in transiently transfected COS-7 cells.

Figure 2 shows that STAT5 coprecipitates with BRCA2 in transiently transfected COS-7 cells.

Figure 3 shows that STAT5 coprecipitates with BRCAl in prolactin-stimulated MCF-7 cells. Figure 4 shows that STAT5 coprecipitates with BRCA2 in prolactin-stimulated MCF-7 cells.

Figure 5A shows decreased transcriptional activity of STAT5 in C0S7 cells overexpressing BRCA2.

Figure 5B shows decreased transcriptional activity of STAT5 in PDGF-stimulated T47D cells overexpressing BRCAl or BRCA2.

Figure 5C shows decreased transcriptional activity of STAT5 in PDGF-stimulated T47D cells overexpressing BRCAl or BRCA2.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to BRCA/STAT complexes involved in the modulation of cell proliferation. A BRCA/STAT complex is useful as a diagnostic marker for neoplastic conditions and as a therapeutic target for proliferative diseases because BRCA/STAT complexes function in the control and maintenance of cellular proliferation. Therefore, pathological conditions involving unregulated proliferation can be diagnosed through the detection of an amount or activity of an abnormal BRCA/STAT complex and controlled by modulating the activity of BRCA/STAT complexes in the cell. The methods of the invention are applicable to the diagnosis and modulation of cellular proliferation for both normal and pathologically aberrant cells and include treatment of uncontrolled proliferative conditions such as neoplastic conditions, autoimmune diseases and cell or tissue degeneration. In one embodiment, the invention is directed to modulating the transcriptional activity of a STAT by inducing the formation of a complex between STAT and BRCA. The transcriptional activity of a STAT can mediate multiple cellular activities, such as growth, differentiation and apoptosis, in a variety of cell types. For example, the transcriptional activity of STAT5 can mediate growth factor responses in breast epithelial cell lines. Specifically, the activation of cell proliferation pathways in breast cancer cell lines by prolactin, PDGF and EGF results in the activation of STAT5 transcriptional activity. Transcriptional activity of a STAT can be affected by STAT participation in protein complexes. For example, the formation of a complex between STAT5 and either BRCAl or BRCA2 alters STAT5 transcriptional activity. The formation of a BRCA/STAT5 complex in response to growth factor stimulation, for example, decreases STAT5 transcriptional activity. A BRCA/STAT complex can thus modulate cell growth by altering a STAT-mediated growth signal such that a growth signaling pathways is either stimulated or inhibited. Both the formation of an aberrant BRCA/STAT complex that mediates a hyperproliferative response or the absence of a BRCA/STAT complex that inhibits a STAT-mediated growth response, can therefore alter the growth characteristics of a cell. Such alterations in cell growth can result in harm to involved tissues. Similarly, a BRCA/STAT complex can regulate cellular signaling pathways mediated by STAT that control differentiation and apoptosis. Thus, modulation of BRCA/STAT interactions can be used to alter the growth and differentiation of normal cells as well as those involved in neoplastic and cancerous conditions of cells. In particular, modulation of BRCA/STAT5 complex is useful for altering the growth characteristics of normal breast epithelial and breast tumor cells.

As used herein, the term "cellular proliferation mediated by a BRCA/STAT complex" is intended to mean a change in cellular growth characteristics mediated by interactions between BRCA and STAT polypeptides. Therefore, the growth characteristics of a cell, including cell viability, will be different before and after interaction between BRCA and STAT polypeptides. The interaction between BRCA and STAT can modulate the growth characteristics of a cell by, for example, promoting or inhibiting a growth, differentiation or apoptosis signal. Changes in growth characteristics can be affected, for example, either directly or indirectly by a BRCA/STAT polypeptide interaction. Direct changes include, for example, an increase or decrease in cell growth that is mediated by a BRCA/STAT polypeptide interaction that alters the expression level of a gene involved in cell growth control. For example, a BRCA/STAT complex can alter the expression of a polypeptide that regulates progression through the cell cycle or induction of apoptosis. Indirect changes include, for example, an increase or decrease in cell growth that is mediated by a BRCA/STAT polypeptide interaction that alters the expression level of a modulator of a growth regulatory gene or polypeptide. For example, a BRCA/STAT complex can alter the expression level of an activator or inhibitor of a growth stimulatory signaling protein.

As used herein, the term _^inhibiting" when used in reference to cellular proliferation mediated by a BRCA/STAT complex is intended to mean effecting a decrease in the extent, amount or rate of cell growth. Effecting a decrease in the extent, amount or rate of unregulated cell growth in BRCA/STAT containing cells is a specific example of inhibiting cellular proliferation.

As used herein, the term "BRCA/STAT complex" is intended to mean an association of BRCA and STAT polypeptides to form a heterocomplex sufficient to effect function of the associated polypeptides. The heterocomplex can be stable and long-lived, or it can be transient such as through brief interaction between the BRCA and STAT polypeptides. The time of association can therefore vary so long as BRCA/STAT complex function is induced by the interaction or association. For example, a BRCA/STAT complex can be an association of BRCA and STAT polypeptides having transcriptional activity that can remain associated before or after affecting transcriptional activity. A BRCA/STAT complex also can be, for example, a transient association of BRCA and STAT polypeptides that exists for a time sufficient to affect transcriptional activity or to induce transcriptional modulation through one or more effector molecules. The term is similarly intended to include functional fragments of BRCA or STAT that maintain the ability to associate into a complex and exhibit BRCA/STAT complex activity.

As used herein, the term "BRCA/STAT complex modulating compound" is intended to mean a compound that alters the amount or activity of a BRCA/STAT complex in a cell. A BRCA/STAT complex modulating compound can act directly or indirectly on a BRCA/STAT complex. For example, a BRCA/STAT complex modulating compound can act directly on a BRCA/STAT complex by altering the stability or activity of the complex. Alterations of BRCA/STAT complex can include, for example, induction of BRCA and STAT polypeptide association to form a complex or displacement of a BRCA or STAT polypeptide from a complex. Alterations of BRCA/STAT complex activity include, for example, modification of the complex or of a BRCA or STAT polypeptide combined in the complex.

Alterations of activity similarly include pre-complex modifications of BRCA or STAT polypeptides that affect or modulate activity once combined into a complex. Examples of polypeptide alterations include modifications such as phosphorylation or dephosphorylation, non-covalent or covalent binding of factors or regulatory molecules, a conformational change that alters affinity or activity, and polypeptide proteolysis or degradation.

Specific examples of BRCA/STAT complex modulating compounds that directly alter the amount or activity of a BRCA/STAT complex include small organic molecules, nucleic acids, and polypeptides such as those derived from combinatorial and random libraries, kinases, phosphatases, signal transduction factors and second messengers that bind to the BRCA/STAT complex and antibodies, including monoclonal antibodies. Functional fragments of such compounds that maintain complex modulating activity are similarly included. For example, functional fragments of an antibody includes single chain antibodies (scFv) , variable region fragments (Fv or Fd) , Fab and F(ab)₂. A BRCA/STAT complex modulating compound can act indirectly, for example, by altering the regulation of a BRCA/STAT complex amount or activity. A BRCA/STAT complex modulating compound can therefore include a ligand of a cell surface receptor that regulates BRCA/STAT complex amount or activity. For example, a growth factor, hormone or cytokine that binds to and activates a cell surface receptor can be a BRCA/STAT complex modulating compound. A BRCA/STAT complex modulating compound can also act indirectly by altering the activity of an intracellular polypeptide or factor that regulates the amount or activity of a BRCA/STAT complex in a cell. Such an intracellular polypeptide can be, for example, a kinase, phosphatase, transcription factor signal transduction factor or second messenger. Specific examples of modulators that can indirectly alter the amount or activity of a BRCA/STAT complex include prolactin, interleukins, SH2 and SH3 domain containing proteins and antibodies, including monoclonal antibodies. Similar functional fragments of such compounds can also be BRCA/STAT complex modulating compounds .

As used herein, the term "BRCA/STAT- containing cell" is intended to refer to a cell having an amount of BRCA and STAT polypeptides that is sufficient to form an effective amount of a BRCA/STAT complex exhibiting BRCA/STAT complex proliferation modulating activity or transcription modulating activity. A BRCA/STAT-containing cell can contain a BRCA/STAT complex or the formation of a BRCA/STAT complex can be induced. A BRCA/STAT-containing cell can also contain a naturally occurring mutant form of BRCA. As used herein, the term "modulating" is intended to mean causing an alteration in the amount or activity of a BRCA/STAT complex compared to a reference level of a BRCA/STAT complex. Such alterations include an increase or decrease in BRCA/STAT complex amount or activity. The amount or activity of a BRCA/STAT complex in a cell can be modulated by promoting or inhibiting the cellular processes that regulate BRCA/STAT complex formation. The formation of a BRCA/STAT complex can be modulated in a cell by activating a signal transduction pathway, such stimulating a cell with a growth or differentiation factor that induces a BRCA/STAT complex-mediated signaling pathway. For example, an extracellular ligand can be used to modulate the formation of a BRCA/STAT complex in a cell. Polypeptides that regulate the expression of BRCA and STAT polypeptides, or the affinity between BRCA and STAT polypeptides that results in BRCA/STAT complex formation similarly modulate the formation of a BRCA/STAT complex. Therefore, a BRCA/STAT complex amount or activity can be altered using a variety of methods including direct and indirect alteration of BRCA or STAT polypeptides or a BRCA/STAT complex.

The amount or activity of a BRCA/STAT complex can be modulated in a direct manner, for example, by modification or conformational change of a BRCA or STAT polypeptide that increases or decreases complex formation. An example of a modification of a BRCA or STAT polypeptide is phosphorylation or dephosphorylation that alters the formation or stability of a BRCA/STAT complex. A BRCA/STAT complex can be modulated indirectly, for example, by stimulating or inhibiting regulators of BRCA and STAT polypeptide expression or modification or by stimulating a signaling pathway that induces BRCA/STAT complex formation. Therefore, the term „modulating^λ refers to altering the amount or activity of a BRCA/STAT complex by both direct and indirect methods.

As used herein, the term "reducing the severity" is intended to mean an arrest or decrease in clinical symptoms, physiological indicators or biochemical markers of proliferative disease. Clinical symptoms include perceptible, outward or visible signs of disease. Physiological indicators include detection of the presence or absence of physical and chemical factors associated with a process or function of the body. Biochemical markers include those signs of disease that are observable at the molecular level, such as the presence of a disease marker, such as a tumor marker. A tumor marker is a substance in the body that usually indicates the presence of cancer. Tumor markers are usually specific to certain types of cancer and are usually found in the blood or other tissue samples. One skilled in the art will be able to recognize specific clinical symptoms, physiological indicators and biochemical markers associated with a particular proliferative disease.

As used herein, the term „effective amount" when used in reference to reducing the severity of a proliferative disease, such as cancer, is intended to mean an amount of BRCA/STAT complex modulating compound administered to an individual required to effect a decrease in the extent, amount or rate of spread of a neoplastic condition or pathology. The amount of a BRCA/STAT complex modulating compound required to be effective will depend, for example, on the type or types of BRCA/STAT modulating compounds administered, the pathological condition to be treated and the level of abundance of BRCA/STAT complex, as well as the weight and physiological condition of the individual, and previous or concurrent therapies . An amount considered as an effective amount for a particular application of BRCA/STAT complex modulating compound will be known or can be determined by those skilled in the art, using the teachings and guidance provided herein. One skilled in the art will recognize that the condition of the patient can be monitored throughout the course of therapy and that the amount of the modulating compound that is administered can be adjusted according to the individual's response to therapy.

As used herein, the term "cancer" is intended to mean a class of diseases characterized by the uncontrolled growth of aberrant cells, including all known cancers, and neoplastic conditions, whether characterized as malignant, benign, soft tissue or solid tumor. Specific cancers include breast and ovarian cancers. By exemplification, a list of known cancers is provided below in Table 1.

TABLE I Myelofibrosis with

HEMATOPORETIC NEOPLASMS Myeloid Metaplasia

Lymphoid Neoplasms Hemangioma Myeloid Neoplasms Lymphangioma Histiocytoses Glomangioma Precursor B lymphoblastic Kaposi Sarcoma leukemia/lymphoma (ALL) Hemanioendothelioma Precursor T lymphoblastic Angiosarcoma leukemia/lymphoma (ALL) Hemangiopericytoma Chronic lymphocytic leukemia/small HEAD & NECK lymphocytic Basal Cell Carcinoma lymphoma (SLL) Squamous Cell Carcinoma Lymphoplasmacytic Ceruminoma lymphoma Osteoma

Mantle cell lymphoma Nonchromaffin Follicular lymphoma Paraganglioma Marginal zone lymphoma Acoustic Neurinoma Hairy cell leukemia Adenoid Cystic Carcinoma Plasmacytoma/plasma cell Mucoepidermoid Carcinoma myeloma Malignant Mixed Tumors

Diffuse large B-cell Adenocarcinoma lymphoma Lymphoma

Burkitt lymphoma Fibrosarcoma T-cell chronic Osteosarcoma lymphocytic leukemia Chondrosarcoma Large granular Melanoma lymphocytic leukemia Olfactory Neuroblastoma Mycosis fungoids and Isolated Plasmocytoma sezary syndrome Inverted Papillomas Peripheral T-cell Undifferentiated lymphoma, unspecified Carcinoma Angioimmunoblastic T-cell Mucoepidermoid Carcinoma lymphoma Acinic Cell Carcinoma

Angiocentric lymphoma Malignant Mixed Tumor (NK/T-cell lymphoma) Other Carcinomas Intestinal T-cell Amenoblastoma lymphoma Odontoma Adult T-cell leukemia/lymphoma THYMUS Anaplastic large cell Malignant Thymoma lymphoma Type I (Invasive thymoma)

Hodgkin Diseases (HD) Type II (Thymic Acute myclogenous carcinoma) leukemia (AML) Squamous cell carcinoma Myclodysplastic syndromes Lymph epithelioma Chronic

Myclofroliferative THE LUNG Disorders Squamous Cell Carcinoma

Chronic Myclogenous Adenocarcinoma Leukemia (CML) Bronchial derived Polycythemia Vera Acinar; papillary; solid Essential Thrombocytosis Bronchioalveolar Small Cell Carcinoma THE KIDNEY Oat Cell Renal Cell Carcinoma

Intermediate Cell Nephroblastoma (Wilm/ s Large Cell Carcinoma Tumor) Undifferentiated; giant cell; clear cell THE LOWER URINARY TRACT Malignant Mesothelioma Urothelial Tumors Sarcomotoid Type Squamous Cell Carcinoma Epithelial Type Mixed Carcinoma Adenocarcinoma

THE GASTROINTESTINAL Small Cell Carcinoma TRACT Sarcoma

Squamous Cell Carcinoma

Adenocarcinoma THE MALE GENITAL TRACT

Carcinoid Squamous Cell

Malignant Melanoma CarcinomaSarcinoma

Adenocarcinoma Speretocytic Sarcinoma

Gastric Carcinoma Embyonal Carcinoma

Gastric Lymphoma Choriocarcinoma

Gastric Stromal Cell Teratoma

Tumors Leydig Cell Tumor

Lymphoma Sertoli Cell Tumor

Kaposi's Sarcoma Lymphoma

Intestinal Stromal Cell Adenocarcinoma

Tumors Undifferentiated

Carcinids Prostatic Carcinoma

Malignant Mesethelioma Ductal Transitional

Non-mucin producing Carcinoma adenocarcinoma

THE FEMALE GENITAL TRACT

THE LIVER AND THE BILIARY Squamous Cell Carcinoma TRACT Basal Cell Carcinoma

Hepatocellular Carcinoma Melanoma

Cholangiocarcinoma Fibrosarcoma

Hepatoblastoma Intaepithelial Carcinoma

Angiosarcoma Adenocarcinoma Embryonal

Fibrolameller Carcinoma Rhabdomysarcoma

Carcinoma of the Large Cell Carcinoma

Gallbladder Neuroendocrine or Oat

Adenocarcinoma Cell Carcinoma

Squamous Cell Carcinoma Adenocarcinoma

Papillary, poorly Adenosquamous Carcinoma differentiated Undifferentiated

Carcinoma

THE PANCREAS Carcinoma

Adenocarcinoma Adenoacanthoma

Cystadenocarcinoma Sarcoma

Insulinoma Carcinosarcoma

Gastrinoma Leiomyosarcoma

Glucagonamoa Endometrial Stromal

Sarcoma

Serous Cystadenocarcinoma Mucinous BONES, JOINTS, AND SOFT

Cystadenocarcinoma TISSUE TUMORS

Endometrioid Tumors Multiple Myeloma

Adenosarcoma Malignant Lymphoma

Celioblastoma (Brenner Chondrosacrcoma

Tumor) Mesenchymal

Clear Cell Carcinoma Chondrosarcoma

Unclassified Carcinoma Osteosarcoma

Granulosa-Theca Cell Ewing Tumor (Ewing

Tumor Sarcoma)

Sertoli-Leydig Cell Tumor Malignant Giant Cell

Disgerminoma Tumor

Teratoma Adamantinoma

Malignant Fibrous

THE BREAST Histiocytoma

Phyllodes Tumor Desmoplastc Fibroma

Sarcoma Fibrosarcoma

Paget's Disease Chordoma

Carcinoma Hemangioendothelioma

Insitu Carcinoma Memangispericytoma

Invasive Carcinoma Liposarcoma

Malignant Fibrous

THE ENDOCRINE SYSTEM Histiocytoma

Adenoma Rhabdomysarcorns

Carcinoma Leiomyosarcoma

Meningnoma Angiosarcoma

Cramiopharlingioma

Papillary Carcinoma NERVOUS SYSTEM

Follicular Carcinoma Schwannoma

Medullary Carcinoma Neurofibroma

Anoplastic Carcinoma Malignant Periferal Nerve

Adenoma Sheath Tumor

Carcinoma Astrocytoma

Pheochromocytoma Fibrillary Astrocytoma

Neuroblastome Glioblastoma Multiforme

Paraganglioma Brain Stem Glioma

Pineal Pilocytic Astrocytoma

Pineoblasto a Pleomorphic

Pineocytoma Xanthorstrocytoma

Oligodendroglioma

THE SKIN Ependymoma

Melanoma Gangliocytoma

Squamous cell carcinoma Cerebral Neuroblastoma

Basal cell carcinoma Central Neurocytoma

Merkel cell carcinoma Dysembryoplastic

Extramamary Paget's Neuroepithelial Tumor

Disease Medulloblastoma

Paget's Disease of the Malignant Meningioma nipple Primary Brain Lymphoma

Kaposi's Sarcoma Primary Brain Germ Cell

Cutaneous T-cell lymphoma Tumor THE EYE

Carcinoma

Squamous Cell Carcinoma

Mucoepidermoid Carcinoma

Melanoma

Retinoblastoma

Glioma

Meningioma

THE HEART

Myxoma

Fibroma

Lipoma

Papillary Fibroelastoma

Rhasdoyoma

Angiosarcoma

Other Sarcoma

HISTIOCYTOSES

Langerhans Cell Histiocytosis

As used herein, the term "sample" is intended to mean any cell, tissue, organ or portion thereof, that contains or is suspected of containing BRCA and STAT polypeptides or their encoding nucleic acids. The term includes samples present in an individual, such as when in vivo or in si tu detection methods are employed, as well as samples obtained or derived from the individual, such as when ex vivo detection methods are employed. For example, a sample can be a histologic section of a specimen obtained by biopsy, cells obtained from body fluids, or cells that are placed in or adapted to tissue culture. A sample further can be a subcellular fraction or extract prepared from such cells, such as a cytoplasmic lysate, a membrane preparation, a nuclear extract, or a crude or substantially pure protein preparation. A sample can be prepared by methods known in the art suitable for the particular format of the detection method. As used herein, the term "binding agent" when used in reference to BRCA, STAT or a BRCA/STAT complex, is intended to mean a small organic molecule or macromolecule, including, for example, a polypeptide, DNA, RNA or carbohydrate that selectively binds a BRCA, STAT or BRCA/STAT complex or a functional fragment thereof. Therefore, a binding agent is a compound or molecule that has selective binding affinity for BRCA, STAT or a BRCA/STAT complex. For example, a binding agent can be a polypeptide that selectively binds to a BRCA, STAT or BRCA/STAT complex polypeptide without substantial cross-reactivity to unrelated polypeptides. The affinity of a binding agent that selectively binds BRCA, STAT or a BRCA/STAT complex will generally be greater than about 10^"5 M and more preferably greater than about 10^"6 M. High affinity interactions can be preferred, and will generally be greater than about 10^"8 M to 10^"9 M. However, binding agents with low affinities or activities are also included within the meaning of the term where they can be made to selectively bind to the reference polypeptide or complex such as by inclusion of a competitive inhibitor or by modification and selection of more optimal variants .

Examples of selective binding agents for BRCA, STAT or BRCA/STAT complex polypeptides include a polyclonal or monoclonal antibody and functional fragments thereof, or a small organic compound, peptide, nucleic acid or analogs and derivatives thereof that have been identified or determined to bind BRCA, STAT or a BRCA/STAT complex without substantial cross reactivity to unrelated molecules. Such binding agents can be obtained from combinatorial and random libraries, for example. A binding agent that preferentially recognizes a particular conformational or post-translationally modified state of BRCA, STAT or a BRCA/STAT complex is also included within the meaning of the term as used herein so long as such agents exhibit selective binding to the referenced polypeptide or complex. The binding agent can be labeled with a detectable moiety, if desired, or rendered detectable by specific binding to a detectable secondary binding agent.

The invention is directed to heterocomplexes of STAT and BRCA polypeptides and to methods of controlling and diagnosing cellular proliferation mediated by BRCA/STAT heterocomplexes. STATs (signal transducers and activators of transcription) are a family of transcription factors that mediate transcriptional regulation in response to a diverse group of cytokines and growth factors . The family includes related subtypes termed STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, which function to effect a variety of cellular growth and differentiation processes through transcriptional activation and repression of gene products having cell regulatory activity. STATs are cytoplasmic transcription factors that, upon activation by tyrosine phosphorylation, form dimers and subsequently translocate to the nucleus to bind to specific regulatory elements that control gene expression. STATs play a role in transducing signals initiated by a wide variety of cytokines and growth factors that result in various changes in a cell including, for example, induction of proliferation, apoptosis, and differentiation.

STAT signal transduction and transcriptional activity can be modulated by forming complexes with BRCA polypeptides. The transcriptional activity of a STAT may be inhibited or enhanced by the formation of a BRCA/STAT complex. The cellular effect of BRCA/STAT complex formation will depend on the particular cell type containing the complex. For example, in a cell having a BRCA/STAT complex that participates in growth signaling, the rate or extent of proliferation can be altered by the formation of a BRCA/STAT complex. Similarly, in a cell having a BRCA/STAT complex that participates in cell differentiation, the process of differentiation can be inhibited or augmented by altering the formation of a BRCA/STAT complex. Using the methods and guidance provided herein, one skilled in the art can determine whether a BRCA/STAT complex is involved in a particular cellular process, such as cell growth.

BRCAl and BRCA2 are multifunctional proteins that have roles in homologous recombination and DNA repair, proliferation and transcriptional regulation. Mutations in BRCA genes predispose an individual to cancer, and in particular, to breast and ovarian cancers. Specific examples of BRCA/STAT complexes are BRCAl/STAT5 and BRCA2/STAT5 complexes that regulate terminal differentiation processes of epithelial cells during pregnancy and lactation.

The invention provides isolated BRCA/STAT complexes. The isolated complexes can consist of any of the STAT subtypes together with any of the BRCA subtypes. For example, any of STATl through STAT6 can be combined together with either of BRCAl or BRCA2 to be an isolated BRCA/STAT complex of the invention.

Isolated BRCA/STAT complexes include functional fragments of BRCA or STAT polypeptides so long as the BRCA or STAT fragment maintains the ability to associate with its cognate partner and the complex maintains BRCA/STAT complex modulatory activity of proliferation. A BRCA or STAT functional fragment will therefore include at least a cognate STAT or BRCA binding domain, respectively, and a regulatory domain that exhibits activity similar to the full length polypeptide when it is associated with its cognate BRCA or STAT heterocomplex partner. A regulatory domain can be the same or different than the cognate binding domain. For example, in the specific case where BRCA functions as a repressor of STAT transcriptional activity by binding and inhibiting STAT function, the binding domain and the regulatory domain can be the same because it is sufficient for BRCA to bind STAT to inhibit its transcriptional activation function. In contrast, where proliferation activity is mediated through the combined association of BRCA and STAT, such as in the specific case of transcriptional activation mediated by the complex, independent domains can be required to mediate complex association and complex function. The BRCA or STAT domain required to effect function of the complex is considered to be the regulatory domain.

Isolated BRCA/STAT complexes include BRCA and STAT polypeptides containing amino acid analogs, derivatives and mimetics. Such modifications are functional equivalents of amino acids are well known to those skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivitization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the ε-amino group of the side chain of the naturally occurring amino acid. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

An isolated BRCA/STAT complex can be a obtained from a cell that contains BRCA and STAT polypeptides that can form a BRCA/STAT complex. BRCA and STAT polypeptides that participate in a BRCA/STAT complex can be endogenously or exogenously expressed in a cell. The presence of BRCA and STAT polypeptides can be detected using any method of polypeptide detection known in the art, including those described below. Thus, a cell that contains endogenous BRCA and STAT polypeptides can be readily identified.

BRCA and STAT polypeptides can be exogenously expressed in a cell using essentially any method of recombinant polypeptide expression known in the art, including those methods described below. BRCA and STAT polypeptides can be co-expressed in one cell or can be expressed individually in two or more different cell populations . BRCA and STAT polypeptides that are expressed in different cell types can be combined, for example, by mixing cell lysates or preparations containing isolated BRCA and STAT polypeptides to obtain a BRCA/STAT complex.

A BRCA/STAT complex can pre-exist in a cell that endogenously or exogenously expresses BRCA and STAT polypeptides or can be induced by adding a compound that promotes the association of BRCA and STAT polypeptides to form a BRCA/STAT complex. Similarly, a BRCA/STAT complex can pre-exist in a cell lysate or to Ω CΛ C σ Ω TJ H- 3 Ό H- O D_ rt H- 3

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mediated through a BRCA/STAT complex, or a candidate cellular process identified or determined to be mediated through a BRCA/STAT complex, is therefore a target for modulation using the methods of the invention. For example, studies of the different STATs have revealed that they participate in diverse biological responses. Knockout studies have indicated that most stats have very specific essential function in vivo that are correlated with particular cytokine signaling pathways. Four of the STATS, (STAT1, STAT2, STAT4 and STATβ) have narrowactivation profiles and relatively restricted functions, centered around disease resistance. On the other hand, Stat3, STAT5a and STAT5b are activated by many different ligands and have broader functions.

STATl is activated by IFN-α, IFN-γ and many other cytokines and growth factors. STATl deficient mice are impaired in their ability to respond to interferons and become susceptible to infections from bacterial and viral pathogens because of a lack of the IFN response of the mice. STATl -/- animals kept in a pathogen-free environment appear normal and are capable of reproducing and they respond normally to several cytokines and growth factors such as EGF or GH.

STAT2 is activated by IFN-α. Homozygous deletion of STAT2 is embryonic lethal indicating a critical role for STAT2 in normal development.

STAT4 and STAT6 are key regulators of T cell differentiation involved in immune and inflammatory responses. STAT4 is activated in T cells in vitro in response to IL-12 a cytokine that stimulates the development of T helper 1 (Thl) cells. Inactivation of the STAT4 gene results in animals whose immunocytes are deficient in Thl cell function. The STAT6 gene can be activated by IL-4 which promotes the development of T helper 2 (Th2) cells. Therefore lymphocytes of STAT6 knockout mice lack Th2 cell function.

STAT3 is activated by IL-β, IL-10, leptin and many other cytokines and growth factors. Targeted disruption of STAT3, results in embryonic lethality, indicating that the protein is essential for early development. The STAT3 knockouts die before gastrulation, and have no obvious mesoderm. STAT3 is involved in many cellular processes, including proliferation, differentiation and apoptosis.

STAT5a and STAT5b share 96% sequence similarity, have a near identical expression pattern and are simultaneously activated by a number of cytokines and growth factors such as IL-2, prolactin, IL-3 and GH. In spite of the high sequence similarity, mice with STAT5a and STAT5b genes knocked out have quite different phenotypes. STAT5a -/- animals are normal except for the inability of females to develop normal breast tissue and to lactate. In STAT5a -/- animals, STATδb appears not to complement the absence of STAT5a. STAT5b -/- male mice grow slowly and have serum level of liver produced proteins similar to that of female mice and lack the characteristic sexually dimorphic responses in the liver. However STAT5b -/- females can lactate. These observations are consistent with an important role for STAT5b downstream of the growth hormone receptor.

Following the teachings and guidance provided herein, those skilled in the art will know, or can determine, which STAT associated cellular proliferation processes are mediated through a BRCA/STAT complex. Similarly, given the teachings and guidance provided herein, those skilled in the art will know or can determine which candidate proliferation processes are controlled through other STAT associated signal transduction processes. For example, a cell regulatory or proliferation process can be initiated by inducing the corresponding signal transduction event and the association or activity of newly formed BRCA/STAT complexes can be measured using any of a variety of methods described below. Those processes that exhibit BRCA/STAT complex formation are applicable for modulation using the methods of the invention. Therefore, the methods of the invention for modulating BRCA/STAT complexes in a cell can be used to either increase or decrease cell proliferation and related growth regulatory events. Similarly, depending on the cellular proliferation process which is to be targeted, increases or decreases can be effected by either augmenting or reducing the amount or activity of a BRCA/STAT complex.

The methods of the invention are applicable to the inhibition or promotion of cell proliferation mediated by a BRCA/STAT complex. Therefore, a cell useful in the methods of the invention contains a STAT polypeptide that can associate with a BRCA polypeptide to form a BRCA/STAT complex that mediates cell growth.

Cell growth mediated by a BRCA/STAT complex can be inhibited or promoted by a BRCA/STAT complex modulating compound of the methods of the invention. A BRCA/STAT complex modulating compound inhibits or promotes cell growth by increasing or decreasing the amount or activity of a BRCA/STAT complex, depending on the particular cell type. For example, an increase in the amount or activity of BRCA/STAT complex can induce an inhibition of cell proliferation in a particular cell type while a decrease in the amount or activity of a BRCA/STAT complex can induced an inhibition of cell proliferation in another cell type. Using the methods and guidance provided herein, the effect on cell proliferation of modulating BRCA/STAT complex amount or activity in a particular cell can be determined. A BRCA/STAT complex modulating compound is used in methods of the invention to inhibit or promote cellular proliferation mediated by a BRCA/STAT complex. Appropriate assays to determine whether a molecule of the invention alters cell proliferation are known in the art. Those of skill in the art will recognize that the molecular pathways involved in cell proliferation are generally well conserved among eukaryotic organisms. Therefore, a proliferative assay can be preformed in any eukaryotic cell type in which altered proliferation can be detected including, for example, primary mammalian cells, normal and transformed mammalian cell lines.

A BRCA/STAT complex modulating compound that inhibits proliferation can, for example, cause cell cycle arrest at a particular stage of mitosis or meiosis and induce apoptosis. Such qualitative changes in the cell cycle can be determined by methods known in the art. A molecule that inhibits cell proliferation can also, for example, slow the progression through the cell cycle, resulting in a decreased number of cells in a population after a given period of time. Those skilled in the art can choose an appropriate assay to determine whether and how a molecule of the invention inhibits cell proliferation.

The amount or activity of a BRCA/STAT complex in a cell can be modulated by a BRCA/STAT complex modulating compound. A BRCA/STAT modulating compound can be any molecule that selectively binds to a BRCA, STAT or BRCA/STAT complex or to a factor that regulates a BRCA, STAT or BRCA/STAT complex and that alters the amount or activity of a BRCA/STAT complex in a cell. Such a compound can be a naturally occurring macromolecule, such as an antibody, polypeptide, nucleic acid, carbohydrate, or lipid, or a modification ΓD fD C TJ

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are well known in the art and are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) . Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains.

A BRCA/STAT complex modulating compound can also be a polypeptide that selectively binds to BRCA, STAT, or a BRCA/STAT complex or a regulator of BRCA, STAT or BRCA/STAT complex. The amount or activity of a BRCA/STAT complex can be altered by a polypeptide through several mechanisms. The amount or activity of a BRCA/STAT complex can be altered directly by a BRCA/STAT complex modulating compound that selectively binds to BRCA, STAT, or a BRCA/STAT complex. A polypeptide can act indirectly, for example, by altering the amount or activity of a molecule that regulates the formation of a BRCA/STAT complex. Molecules that regulate BRCA/STAT complex formation can be cell surface, cytoplasmic, and nuclear molecules.

A polypeptide that regulates BRCA/STAT complex amount and activity can therefore include a ligand that activates a cell surface receptor which regulates the expression of BRCA and STAT polypeptides or formation and activation of a BRCA/STAT complex. Intracellular molecules that comprise a signal transduction pathway of a cell surface receptor can also be modulated by a polypeptide. Therefore, a potential BRCA/STAT modulating compound includes a compound that modulates the activity of an intracellular protein such as a kinase, phosphatase, or transcription factor that regulates BRCA/STAT complex amount or activity. A BRCA/STAT complex modulating compound can be a polypeptide that interacts directly with a BRCA/STAT complex. Polypeptides that interact directly with a BRCA/STAT complex can increase or decrease the amount or activity of the complex. For example, modification of a BRCA or STAT polypeptide, such as by a kinase, can affect the amount or activity of the complex. A specific example is an increase in STAT activity resulting from phosphorylation of a STAT polypeptide. The amount or activity of a BRCA/STAT complex in a cell can be altered by the introduction of STAT and BRCA polypeptide or functional fragment into a cell. The amount or activity of a BRCA/STAT complex can be increased by introducing, for example, a BRCA or STAT polypeptide into a cell. A BRCA polypeptide can act as a BRCA/STAT complex modulating compound, for example, by associating with and modulating the activity of a STAT. A structural variant of a BRCA or STAT polypeptide that can bind to a BRCA or STAT polypeptide in a cell to produce a BRCA/STAT complex that has decreased activity can be used to reduce the activity of a BRCA/STAT complex in a cell.

A functional fragment of a polypeptide can be a BRCA/STAT complex modulating compound. For example, a functional fragment of BRCA that can bind to a STAT polypeptide in a cell can be a BRCA/STAT modulating compound. A functional fragment of a BRCA can associate with and modulate the activity of a STAT polypeptide with at least about the same affinity as a native BRCA polypeptide. Functional fragments can be produced using several methods well known in the art, such as, for example, enzymatic or chemical cleavage of the full length polypeptide, chemical synthesis, and recombinant methods, as described below.

A polypeptide BRCA/STAT complex modulating compound useful in the methods of the invention can be prepared by essentially any method known in the art, including biochemical, recombinant and synthetic methods. For example, a polypeptide can be purified from any tissue or cell that expressing abundant amounts of the polypeptide, including cells containing recombinantly expressed polypeptide or functional fragment. Recombinant polypeptides or functional fragments can be expressed in essentially any compatible vector/host system, including for example, both prokaryotic and eukaryotic expression systems. BRCA and STAT polypeptides and functional fragments can be modified by the additions of epitope tags or other sequences that aid in polypeptide purification and which do not alter the activity of the BRCA/STAT complex .

Expression systems and corresponding host cell types are well known to those skilled in the art.

Similarly, promoters, enhancers and regulatory elements for augmenting or regulating expression and applicable cell types compatible with such expression elements are well known to those skilled in the art. Procaryotic expression systems are advantageous due to their ease in manipulation, rapid growth rate and relatively high yields. Eucaryotic systems are advantageous in that they are subject to eukaryotic post-translational modifications, including system, cell type or modification employed for producing user.

Expression systems include, for example, E coli, yeast, insect cell, and mammalian expression systems, including rodent and human vector/host systems.

Selection of a cell or tissue that is an appropriate starting material for purification of a BRCA/STAT complex modulating polypeptide can be determined by examining the level of polypeptide expression using a variety of methods known in the art, including the methods disclosed herein. Biochemical purification can include, for example, steps such as solubilization of the appropriate tissue, cell, or recombinant cell, isolation of the desired subcellular fractions, size or affinity chromatography, electrophoresis, and immunoaffinity procedures. The methods and conditions for biochemical purification of a polypeptide of the invention can be chosen by those skilled in the art, and purification monitored, for example, by an ELISA assay or a functional assay. Therefore, a BRCA/STAT complex modulating compound can be a polypeptide or functional fragment thereof that increases or decreases the amount or activity of a BRCA/STAT complex.

A BRCA/STAT complex modulating compound can be a nucleic acid, such as a DNA or RNA molecule, that selectively binds to BRCA, STAT, or a BRCA/STAT complex or a regulator of BRCA, STAT or BRCA/STAT complex. A nucleic acid that is useful in the methods of the invention can be a nucleic acid that effects transcription or translation of a molecule, such as an anti-sense nucleic acid. An anti-sense nucleic add can be used to modulate the expression level of a polypeptide in a cell. For example, a BRCA/STAT complex modulating compound can be an antisence nucleic acid that specifically binds to a BRCA or STAT nucleic acid to alter BRCA or STAT protein expression. The amount or activity of a BRCA/STAT complex can be decreased by an antisence BRCA/STAT complex modulating compound, for example, by decreasing the level of a BRCA or STAT polypeptide available in a cell to form a BRCA/STAT complex. An antisence nucleic acid can also be targeted to reduce the expression of a protein that regulates formation of a BRCA/STAT complex in a cell. Therefore, a BRCA/STAT complex modulating compound can be a nucleic acid that alters the amount or activity of a BRCA/STAT complex. A BRCA/STAT complex modulating compound can be a small organic compound that selectively binds to BRCA, STAT, or a BRCA/STAT complex or a regulator of BRCA, STAT or BRCA/STAT complex. A small organic compound that binds to a BRCA or STAT polypeptide can be used to increase or decrease the amount of BRCA or STAT polypeptide that forms a BRCA/STAT complex. Similarly, a small organic compound that binds to a BRCA, STAT or BRCA/STAT complex can increase or decrease the activity of a BRCA/STAT complex in a cell.

Such a compound can act directly or indirectly to modulate a BRCA/STAT complex. A small organic compound BRCA/STAT complex modulating compound can act directly, for example, by binding to a BRCA, STAT, or BRCA/STAT complex polypeptide and altering the formation or stability of the complex. Small organic compounds can alter the activity of a polypeptide, for example, by changing or blocking a site of polypeptide interaction with a substrate or polypeptide. A small organic molecule can also indirectly alter the amount or activity of a BRCA/STAT complex by altering the activity of regulators of BRCA/STAT complex formation.

A BRCA/STAT complex modulating compound can therefore be a small organic compound that increases or decreases the amount or activity of a BRCA/STAT complex.

A BRCA/STAT complex modulating compound can be contacted with a cell that contains BRCA and STAT polypeptides that form a BRCA/STAT complex that can mediate a cellular function. It is expected that different cell types express different STAT and BRCA polypeptides in varying amounts. A BRCA/STAT complex can include any BRCA family polypeptide or STAT family polypeptide that is expressed in a particular cell type. A BRCA polypeptide that can form a BRCA/STAT complex that can be modulated using the methods of the invention includes, for example, BRCAl and BRCA2. A STAT polypeptide that can form a BRCA/STAT complex that can be modulated by the methods of the invention includes, for example, STAT 5. Any BRCA/STAT complex that mediates a cellular function can be targeted by a BRCA/STAT complex modulating compound using the methods of the invention.

A variety of methods known in the art can be used to determine which BRCA and STAT polypeptides are expressed in a particular cell. Such methods include, for example, immunoblotting and immunoprecipitation as described in the Examples. An antibody that specifically recognize one member of the family of STAT polypeptides can be used, for example, to determine if that particular STAT is expressed in a cell or contained in a BRCA/STAT complex. Therefore, using methods described herein and those known in the art, the presence of BRCA and STAT polypeptides and a BRCA/STAT complex in a particular cell can be determined. A BRCA/STAT complex modulating compound can be used to modulate the amount or activity of a BRCA/STAT complex in such a cell.

A BRCA/STAT complex modulating compound can be added to a variety of cells containing BRCA and STAT polypeptides that form a BRCA/STAT complex. A BRCA/STAT-containing cell can be a cultured cell. Cultured cells applicable to the methods of the invention include culture of freshly prepared cells, a cell line, and a cell transduced with a recombinant nucleic acid. Cultured cells can be maintained in defined or undefined cell culture medium that can contain factors that alter BRCA/STAT complex formation. For example, a cell can be treated with a factor that induces BRCA/STAT complex formation. Such BRCA/STAT complex inducing factors can include growth factors, cytokines, hormones and other BRCA/STAT complex modulating compounds. Therefore, the methods of the invention can be applied to cultured BRCA/STAT- containing cells.

A BRCA/STAT-containing cell, including a hyperproliferative, neoplastic, or cancerous cell that can be obtained from any tissue or organ. For example, a cell can be dissociated from a tissue such as breast tissue, or an organ such as an ovary. Cells can be prepared from freshly harvested as well as cryopreserved tissues and organs. A variety of methods known in the art can be used for preparing cells from tissues and organs. A cell applicable to the methods of the invention can therefore be harvested from a tissue or organ and used directly or cryopreserved prior to use.

A BRCA/STAT complex modulating compound can be added to a cell that contains recombinantly expressed polypeptides that induce the formation of a BRCA/STAT complex that mediates a cellular function. For example, a cell can be transduced with nucleic acids encoding a polypeptide that controls BRCA/STAT complex formation or activity. Such a protein can be, for example, a kinase or phosphatase. Recombinant expression of a BRCA or STAT polypeptides can be used, for example, to increase the amount of a BRCA/STAT complex in a cell by increasing the amount of polypeptide available in a cell to form a BRCA/STAT complex. A cell can be transduced by methods well known in the art including transfection methods such as transfection mediated by calcium phosphate, lipofection, and electroporation. Therefore, BRCA/STAT-containing cells transduced with nucleic acids can also be used in the methods of the invention.

A BRCA/STAT complex in a cell can be modulated by adding an effective amount of a BRCA/STAT complex modulating compound that is sufficient to alter the amount or activity of a BRCA/STAT complex. An effective amount of a BRCA/STAT complex modulating compound is an amount required to affect an increase or decrease in the amount or activity of a BRCA/STAT complex. The amount of BRCA/STAT complex modulating compound required to affect a modulation of a BRCA/STAT complex can be determined by testing the effect of various concentrations of compound on complex formation, and determining an amount that results in an increased or decreased BRCA/STAT complex formation. BRCA/STAT complex formation in a cell can be detected using the methods described herein for determining BRCA, STAT and BRCA/STAT polypeptide amount, transcriptional activity, cell proliferation activity, and activity in animal models, or using any other method known in the art .

Time of incubation of BRCA/STAT complex modulating compound can similarly be assessed by incubating compound with cells for various time periods followed by determining the amount or activity of BRCA/STAT complex in a treated sample compared to untreated sample. For example, a cell can be incubated with BRCA/STAT complex modulating compound for 5-30 min, 30-90 min, 1-3 hours, or several hours. The time of incubation with a BRCA/STAT complex modulating compound will be determined by the physical characteristics of the particular compound, the delivery method used, and the characteristics of a particular cell type. For example, a compound that penetrates cell membranes and immediately binds to a BRCA, STAT, or BRCA/STAT complex polypeptide, will require a shorter incubation time than a nucleic acid molecule delivered using a standard lipid-mediated transfection method.

The invention further provides a method of reducing the severity of cancer, comprising administering to an individual an effective amount of a BRCA/STAT complex modulating compound sufficient to increase the amount or activity of a BRCA/STAT complex in said cancer cells.

A BRCA/STAT complex modulating compound that reduces the rate or extent of proliferation can be used to treat an individual having a proliferative disorder. Proliferative disorders include those diseases or abnormal conditions that result in unwanted or abnormal cell growth, viability or proliferation. Proliferative disorders include diseases such as cancer, in which the cells are neoplastically transformed, and diseases resulting from overgrowth of normal cells. For example, cell proliferative disorders include diseases associated with the overgrowth of connective tissues, such as various fibrotic diseases, including scleroderma, arthritis, alcoholic liver cirrhosis, keloid, and hypertropic scarring; vascular proliferative disorders, such as atherosclerosis; benign tumors, and the abnormal proliferation of cells mediating autoimmune disease. Those skilled in the art will be able to assess the severity of disease in an individual using an appropriate method for detecting and assessing the severity of a specific hyperproliferative disease.

By specific mention of the above categories of pathological conditions, those skilled in the art will understand that such terms include all classes and types of these pathological conditions. For example, the term cancer is intended to include all known cancers, as described previously. Specific types of cancer that can be treated using the methods of the invention are breast and ovarian cancers. The severity of breast or ovarian cancer can be indicated by an assessment of the stage of a particular tumor. For example, breast tumors can be classified according to 3' tr Ω ιQ CL Φ tr Ω a 3 Ω fD φ Φ hj P- X ^ O o o fD

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to cytoplasmic ratio, altered deposition of extracellular matrix, and a less differentiated phenotype. The unregulated proliferation of a cancer cell can be characterized by anchorage independent cell growth, proliferation in reduced-serum medium, loss of contact inhibition, and rapid proliferation compared to normal cells. Those skilled in the art will know how to determine if a BRCA/STAT complex modulating compound is effective in promoting a more normal phenotype in a cancer cell. Those skilled in the art will also be able to detect a cancer cell in a population of cells, tumor, or organ.

Animal models of hyperproliferative diseases similarly can be used to assess the activity of a BRCA/STAT complex modulating compound or an amount sufficient to induce the modulation of a BRCA/STAT complex. Animal models of such pathological conditions well known in the art which are reliable predictors of treatments in humans include, for example, animal models for tumor growth and metastasis and autoimmune disease.

There are animal tumor models known in the art which are predictive of the effects of therapeutic treatment. These models generally include the inoculation or implantation of a laboratory animal with heterologous tumor cells followed by simultaneous or subsequent administration of a therapeutic treatment. The efficacy of the treatment is determined by measuring the extent of tumor growth or metastasis. Measurement of clinical or physiological indicators can alternatively or additional be assessed as an indicator of treatment efficacy. Exemplary animal tumor models can be found described in, for example, Brugge et al. Origins of Human Cancer, Cold Spring Harbor Laboratory Press, Plain View, New York, (1991) . Similarly, animal models predictive for autoimmune disease and degenerative diseases are known in the art and can be used to assess the efficacy of treatment by measuring appropriate experimental endpoints or clinical or physiological indicators which will depend on the particular animal model selected. Those skilled in the art will know which other animal models can be used for determining the efficacy or effective amount of a BRCA/STAT complex modulating compound useful in the methods of the invention.

A BRCA/STAT complex modulating compound is applicable to the treatment of an individual having a type of breast cancer in which cancer cell proliferation is effected by the presence of a BRCA/STAT complex. The types of molecules that can modulate a BRCA/STAT complex are described above, and include, for example, small molecules, polypeptides, nucleic acids, and antibodies. A BRCA/STAT complex modulating compound can be tested in vitro or in vivo to determine potency and efficacy in inhibiting or reducing cell proliferation. A reduction or inhibition of cell proliferation can restore more normal characteristics to a cell having aberrant cell growth control, such as cancerous cell. To determine whether a compound restores more normal characteristics of proliferation of a cell, any method known in the art can be performed to compare a cell treated with a BRCA/STAT complex modulating compound with an untreated cell. For example, assays such as soft agar colony formation, overgrowth of a cell monolayer, proliferation, or tumor formation in an animal can be useful in assessing cell proliferation.

A BRCA/STAT complex modulating compound useful in the methods of the invention can be introduced or expressed in a cell in vitro by essentially any method appropriate for the physical characteristics of the particular compound. Those of skill in the art will be able to determine the appropriate method for delivery to a cell of small molecules, polypeptides and nucleic acids, as such methods are well known in the art.

The effect of a BRCA/STAT complex modifying compound on the amount or activity of a BRCA/STAT complex in a cancer cell can also be determined by observing the clinical symptoms, physiological indicators or biochemical markers associated with a hyperproliferative disease in an individual. For example, a reduction in severity of a hyperproliferative disease can include an arrest or a decrease in clinical symptoms, physiological indicators or biochemical markers associated with the particular disease. Those of skill in the art will know the appropriate symptoms, indicators and markers associated with specific types of hyperproliferative disease and will know how to determine if an individual is a candidate for treatment with a BRCA/STAT complex modulating compound.

The invention provides a method of treating, or reducing the severity of cancer by administering a BRCA/STAT complex modulating compound. The appropriate effective dose for a particular application of the methods can be determined by those skilled in the art, using the guidance provided herein. For example, the amount can be extrapolated from in vitro and in vivo assays as described previously. One skilled in the art will recognize that the condition of the patient can be monitored throughout the course of therapy and that the amount of a BRCA/STAT complex modulating compound that is administered can be adjusted accordingly.

A BRCA/STAT complex modulating compound can be delivered systemically, such as intravenously or intraarterially. A compound can also be administered locally at the site of cancerous cells. Appropriate sites for administration of a compound that modulate a BRCA/STAT complex can be determined by those skilled in the art depending on the clinical indications of the individual being treated.

A compound having BRCA/STAT complex modulating activity can be provided in various forms, including an isolated compound, substantially purified compound, and insoluble aggregate in a pharmaceutically acceptable formulation. Formulations of a BRCA/STAT complex modulating compound can be administered by standard routes, including for example, transdermal, intraperitoneal, oral, pulmonary or parenteral (e.g. intravenous, intraspinal, subcutaneous or intramuscular) routes. In addition, a compound can be incorporated into biodegradable polymers allowing for sustained release of the compound. Such polymers can be implanted in the vicinity of cancer cells, for example, at the site of a tumor, or implanted so that the compound is released systemically over time. An osmotic minipump can be used for controlled delivery of a high concentration of a compound to a site of interest. Biodegradable polymers and their uses are described in detail, for example, in Brem et al., J. Neurosurg. 74;441-446 (1991).

A BRCA/STAT complex modulating compound can be administered to an individual as a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. The choice of a pharmaceutically acceptable carrier depends on the route of administration of the compound and on its particular physical and chemical characteristics. Pharmaceutically acceptable carriers are well known in the art and include sterile aqueous solvents such as buffered saline and other solvents such as glycols, glycerol, oils and injectable organic esters. A pharmaceutically acceptable carrier can further contain physiologically acceptable compounds that stabilize the compound, increase its solubility, or increase its absorption. Such physiologically acceptable compounds include carbohydrates such as glucose, antioxidants such as ascorbic acid, surfactants such as polysorbate 80, chelating agents, and low molecular weight proteins .

A nucleic acid molecule that functions as a BRCA/STAT complex modulating compound can be delivered to a cell by gene therapy methods well known in the art. For example, a nucleic acid molecule encoding a polypeptide that inhibits cell proliferation can be inserted into a mammalian expression vector, such as a plasmid or viral vector, that contains all the necessary expression elements for the constitutive or inducible transcription and translation of the polypeptide, and administered to an individual having, or at risk of developing a breast cancer.

Useful mammalian expression vectors for gene therapy, and methods of introducing such vectors into cells are well known in the art. For example, a plasmid expression vector can be introduced into a cell by transfection, electroporation or any other method known in the art of introducing nucleic acids into a cell. A viral expression vector can be introduced into a cell in an expressible form by infection or transduction, for example, or by encapsulation in a liposome. An appropriate viral vector for gene therapy applications can be, for example, a retrovirus, an adenovirus, an adeno-associated virus or a herpes virus. Thus, a nucleic acid of the invention can be used to reduce or inhibit proliferation of cancer cells in an individual by modulating the amount or activity of a BRCA/STAT complex in a cell.

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The methods of predicting cancer risk and outcome provided by the invention include methods of assessing the amount or activity of a BRCA/STAT complex in a cell. A cell applicable to the methods of the invention will have a BRCA/STAT mediated cell proliferation pathway. In such a cell, the amount or activity of a BRCA/STAT complex will correlate with the proliferative state of the cell. For example, a hyperproliferative cell containing a BRCA/STAT complex that mediates cell proliferation can have an increased or decreased amount of a BRCA/STAT complex that can be detected by comparison with a normal cell.

The amount or activity of a BRCA/STAT complex can be indicative of an abnormal hyperproliferative state of a cell. For example, in a particular cell type in which STAT transcriptional activity can be activated in response to a stimulus, such as a growth signal, STAT can either positively or negatively regulate cell proliferation. In a normal cell in which STAT mediates a positive growth signal, the formation of a BRCA/STAT complex formation that alters the transcriptional activity of STAT can modulate cell proliferation, for example, by reducing STAT transcriptional activity. A reduction in STAT transcriptional activity caused by the formation of a BRCA/STAT complex can affect negative regulation of cell growth. In a hyperproliferative cell having STAT- mediated positive growth control, the absence of BRCA/STAT complex formation is therefore associated with unregulated growth because the negative regulatory function of a BRCA/STAT complex is absent. Alternatively, STAT-mediated negative growth control can be modulated by a BRCA/STAT complex such that the presence of a BRCA/STAT complex in a cell is associated with hyperproliferation. Using the methods and guidance provided herein and known in the art, those skilled in the art will be able to determine if a BRCA/STAT complex positively or negatively regulates a cellular process, such as cell growth, in a particular cell type.

As described above, the amount or activity of a BRCA/STAT complex can differ between normal and hyperproliferative cells. For example, as described above in reference to a cell having STA -mediated positive growth regulation, a cell that contains a BRCA/STAT complex amount or activity that is less than that of a normal cell indicates an increased potential for a higher level of STAT-mediated proliferation. Therefore, the amount or activity of a BRCA, STAT or BRCA/STAT polypeptide in a hyperproliferative cell, when compared to a normal cell, can correlate with severity of hyperproliferative disease.

The methods of detecting the risk of and prognosing breast cancer in an individual involve obtaining cells from that individual. A variety of different types of samples isolated or obtained from an individual having, or suspected of having breast cancer are applicable to the methods of the invention. For example, samples applicable for use in the methods of the invention include tissue and cell samples. A tissue or cell sample can be obtained, for example, by biopsy or surgery. As described below, and depending on the format of the method, the tissue can be used whole or subjected to various methods known in the art to disassociate the sample into smaller pieces, cell aggregrates or individual cells.

A tissue or cell sample can be obtained directly from an individual or, alternatively, can be obtained from other sources for testing. Similarly, the cell sample can be tested when it is freshly isolated or it can be tested following short or prolonged periods of cryopreservation without substantial loss in accuracy or sensitivity. If the sample is to be tested following an indeterminate period of time, it can be obtained and then cryopreserved, or stored at 4°C for short periods of time, for example. An advantage of the prognostic and risk assessment methods of the invention is that they do not require histological analysis of the sample. As such, the sample can be initially disaggregated, lysed, fractionated or purified and the active component stored for later diagnosis.

A sample that can be analyzed for increased or decreased BRCA/STAT polypeptide complex amount or activity includes a cell lysate, a fractionated portion of a cell lysate such as a nuclear fraction, or a purified component of a cell lysate. For example, transcriptional activity of a BRCA/STAT complex can be measured in a cell lysate or a further fractionated fraction of a cell lysate. A nuclear extract or fractionated nuclear extract can be prepared to enrich for BRCA/STAT complex and its corresponding activity. The level of BRCA/STAT complex polypeptides can be determined by, for example, using binding agents specific for BRCA, STAT or a BRCA/STAT complex. Other methods for measuring the level of BRCA/STAT complex in samples are known in the art and are similarly applicable.

The methods of detecting risk of and prognosing breast cancer involve comparing the amount or activity of a BRCA/STAT complex in a sample to that of a reference sample. A reference sample can be, for example, a normal tissue sample from the individual to be treated, or from another individual. A reference sample removed from the individual to be treated can be histologically similar to the cancerous sample, and can be obtained at the time of removal of the cancerous sample from the individual, or at another time. For example, a reference sample can be obtained prior to collection of the sample and can be preserved, for example, by cryopreservation. Similarly, a reference sample can be obtained after collection of the sample. A reference sample can also be obtained from a normal individual having no detectable pathology in a tissue or organ from which the reference sample is obtained. The normal individual can be a relative of, or unrelated to, the individual assessed for cancer risk or prognosis.

The amount or activity of BRCA/STAT complex in an individual compared to a reference sample can be determined as described above. An increase or decrease in the amount or activity of BRCA/STAT complex of about two-fold or more in a suspected breast cancer cell sample relative to samples obtained from normal tissue is indicative of increased risk of cancer.

As described above, BRCA/STAT complex amount and activity can be measured by a variety methods known in the art. For example, the amount of BRCA/STAT complex can be determined by measuring the amount of BRCA/STAT complex polypeptide in a sample from the individual. The amount of BRCA/STAT can be determined by measuring the amount of BRCA/STAT activity in the sample, the amount of activity being indicative of BRCA/STAT amount. A BRCA/STAT complex can be detected by contacting a sample with a binding agent selective for a BRCA/STAT complex, and detecting the amount of selective binding. The amount of selective binding correlates with the amount of BRCA/STAT complex in the sample.

A binding agent refers to a molecule that allows the detection of a BRCA/STAT complex by an analytical method. An appropriate binding agent depends on the particular detection format, and can be determined for a particular application of the method by those skilled in the art. For example, a binding agent specific for a BRCA/STAT complex can be an agent to Ω p. s P- hj P- o OJ rt H CL yQ P- to hi Qm Hi CQ Ω μ- o P- O J tr QJ tr Ω CO OJ OJ Ω tr rt

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BRCA/STAT regulated gene can additionally be a reporter gene that generates a detectable signal useful for measuring transcriptional activity of a STAT or BRCA/STAT complex, as described in Example III.

The invention provides another method of detecting the risk of breast cancer in an individual. The method consists of (a) exposing cells obtained from an individual suspected of having breast cancer to conditions that induce BRCA/STAT complex formation, and (b) detecting expression of BRCA in said exposed cells, wherein a decreased BRCA expression level compared to a reference sample indicates an increased susceptibility for breast cancer.

The expression level of a BRCA in normal cell and cells obtained from an individual suspected of having a neoplastic or cancerous breast cells can be measured using an agent specific for a BRCA nucleic acid. For example, an BRCA specific agent can be a complementary nucleic acid molecule, such as a hybridization probe or non-catalytic ribozyme that selectively hybridizes to BRCA nucleic acids. A hybridization probe or ribozyme can be labeled with a detectable moiety, such as a radioisotope, fluorochrome, chemiluminescent marker, biotin, or other detectable moiety known in the art that is detectable by an analytical method. A PCR or RT-PCR primer can also be used to detect a BRCA nucleic acid. For example, a PCR or RT-PCR primer can be used to selectively amplify all or a desired portion of the nucleic acid molecule, which can then be detected by methods known in the art. Further, a binding agent such as a peptide, nucleic acid analog, or small organic molecule can be used to detect a BRCA nucleic acid. Selective hybridization refers to the hybridization of a probe or binding agent to BRCA nucleic acids. The method of detecting the risk of breast cancer in an individual involves detecting expression of BRCA in cells exposed to BRCA/STAT complex inducing conditions. BRCA/STAT complex inducing conditions are described above. The expression of BRCA in the exposed cells that is decreased compared to a normal reference sample is indicative of increased susceptibility for breast cancer. BRCA gene expression that is lower than normal indicates an increased potential for STAT- mediated uncontrolled cell proliferation because the amount of BRCA polypeptide available in a cell to inhibit STAT-mediated proliferation by forming a BRCA/STAT complex is reduced.

A decrease in BRCA amount or activity can be further characterized by the presence of regulatory factors affecting BRCA/STAT complex activity. Regulatory factors that can affect BRCA/STAT complex activity include, for example, a kinase, phosphatase, or other regulator of BRCA/STAT complex activity as described above.

The invention further provides a method of prognosing the severity or progression of breast cancer. The method consists of (a) detecting an amount or activity of a BRCA/STAT complex in a sample obtained from an individual at a site suspected of containing cancer cells, and (b) comparing said amount or activity of said BRCA/STAT complex with a reference sample, a difference in said amount or activity of said BRCA/STAT complex in said sample indicating the level of severity or progression of said cancer.

A BRCA/STAT complex can be used quantitatively to determine the degree of susceptibility of a breast cancer. The relative amount or activity of BRCA/STAT complex can be measured in a series of samples collected over a period of time such 3 hi Ω I tr Qm rt fD Hi H Ω φ Φ P- hi o P- P- to O- Ω φ O o 3 OJ φ 3" 3 o Φ o X X 3 Φ Hi 3 3 50 Φ OJ

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breast cancer risk assessment or prognosis by the methods of the invention. For example, individuals suspected of having breast cancer can be identified by exhibiting presenting signs of breast cancer which include, for example, a hard irregular mass and skin dimpling or nipple retraction. Prognostic methods of this invention are applicable to individuals after diagnosis of breast cancer, for example, to monitor improvements or identify residual neoplastic breast cells using, for example, imaging methods known in the art which target a BRCA/STAT complex.

The determination of the amount of BRCA, STAT, or BRCA/STAT complex polypeptide in a sample can be performed by essentially all modes of affinity binding assays. Such methods are rapid, efficient and sensitive. Moreover, affinity binding methods are simple and can be adjusted to be perform under a variety of clinical settings and conditions to suit a variety of particular needs. Affinity binding assays which are known and can be used in the methods of the invention include both soluble and solid phase formats. A specific example of a soluble phase affinity binding assay is immunoprecipitation using a BRCA selective antibody or other binding agent. Solid phase formats are advantageous for the methods of the invention since they are rapid and can be performed more easily on multiple different samples simultaneously without losing sensitivity or accuracy. Moreover, solid phase affinity binding assays are further amenable to high throughput screening and automation.

Specific examples of solid phase affinity binding assays include immunoaffinity binding assays such as an ELISA and radioimmune assay (RIA) . Other solid phase affinity binding assays are known to those skilled in the art and are applicable to the methods of the invention. Although affinity binding assays are generally formatted for use with an antibody binding molecules that is selective for the analyte or ligand of interest, essentially any binding agent can be alternatively substituted for the selectively binding antibody. Such binding agents include, for example, macromolecules such as polypeptides, peptides, nucleic acids, lipids and sugars as well as small molecule compounds . Methods are known in the art for identifying such molecules which bind selectively to a particular analyte or ligand and include, for example, surface display libraries and combinatorial libraries.

Thus, for a molecule other than an antibody to be used in an affinity binding assay, all that is necessary is for the binding agent to exhibit selective binding activity for a polypeptide, such as, for example, a BRCA.

Immunoaffinity binding assays, include for example, solid phase ELISA and RIA as well as modifications thereof. Such modifications thereof include, for example, capture assays and sandwich assays as well as the use of either mode in combination with a competition assay format. The choice of which mode or format of immunoaffinity binding assay to use will depend on the intent of the user. Such methods can be found described in common laboratory manuals such as Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1999) .

Assay formats employing affinity binding can be used in conjunction with a variety of detection labels and systems known in the art to quantitate BRCA, STAT or BRCA/STAT complex polypeptide amounts in the analyzed sample. Detection systems include the detection of bound polypeptide by both direct and indirect means. Direct detection methods include labeling of a selective antibody or binding agent. Indirect detection systems include, for example, the use of labeled secondary antibodies and binding agents.

Secondary antibodies, labels and detection systems are well known in the art and can be obtained commercially or by techniques well known in the art. The detectable labels and systems employed with a selective binding agent should not impair binding of the agent to the polypeptide. Moreover, multiple antibody and label systems can be employed for detecting the bound selective antibody to enhance the sensitivity of the binding assay if desired.

Detectable labels can be essentially any label that can be quantitated or measured by analytical methods. Such labels include, for example, enzymes, radioisotopes, fluorochromes as well as chemi- and bioluminescent compounds. Specific examples of enzyme labels include horseradish peroxidase (HRP) , alkaline phosphatase (AP) , β-galactosidase, urease and luciferase.

A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB) , which yields a soluble product in the presence of hydrogen peroxide that is detectable by measuring absorbance at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable by measuring absorbance at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) , which yields a soluble product detectable by measuring absorbance at 410 nm, or a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, MO) . Luciferin is the substrate compound for luciferase which emits light following ATP-dependent oxidation.

Fluorochro e detection labels are rendered detectable through the emission of light of ultraviolet or visible wavelength after excitation by light or another energy source. DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine are specific examples of fluorochrome detection labels that can be utilized in the affinity binding formats of the invention. Particularly useful fluorochromes is fluorescein or rhodamine.

Signals from detectable labels can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a fluorometer to detect fluorescence in the presence of light of a certain wavelength; or a radiation counter to detect radiation, such as a gamma counter for detection of iodine-125. For detection of an enzyme-linked secondary antibody, for example, a quantitative analysis of the amount of bound agent can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices, Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

Given the teachings and guidance provided herein, the choice of measuring polypeptide amount or activity will be that of the user. Considerations such as the sample type, availability and amount will also influence selection of a particular assay format. For example, if the sample is a breast cell sample and there is only a small amount available, then formats which are more sensitive are suitable. Alternatively, μ- TJ to Ω TJ to 3 hj OJ Ω 3 OJ rt O OJ Ω Ω Hi d CQ Ω Hi a S Hi CO CL TJ OJ H 3 CQ μ-

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The method of screening for compounds that modulate the activity of a BRCA/STAT complex include the incubation of a sample containing BRCA and STAT with a test compound. A test compound can be any substance, molecule, compound, mixture of molecules or compounds, or any other composition which is suspected of being capable of inhibiting cellular regulator activity in vivo or in vi tro. The test compounds can be macromolecules, such as biological polymers, including proteins, polysaccharides and nucleic acids.

Sources of test compounds which can be screened for BRCA/STAT complex modulating activity include, for example, libraries of small molecules, peptides, polypeptides, RNA and DNA. Specific examples of polypeptides that can be test compounds include a cytokine, hormone, growth factor, kinase and phosphatase.

Additionally, test compounds can be preselected based on a variety of criteria. For example, suitable test compounds can be selected as having known BRCA/STAT complex modulating activity. Examples of such test compounds include, for example, a growth factor, cytokine, hormone, kinase and phosphatase. Alternatively, the test compounds can be selected randomly and tested by the screening methods of the present invention. Test compounds can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations to determine, for example, the optimal modulatory activity of the activity of a BRCA/STAT complex.

The method of screening for compounds that modulate the activity of a BRCA/STAT complex can involve groups or libraries of compounds. Methods for preparing large libraries of compounds, including simple or complex organic molecules, carbohydrates, peptides, peptidomimetics, polypeptides, nucleic acids, antibodies, and the like, are well known in the art. Libraries containing large numbers of natural and synthetic compounds can be obtained from commercial sources .

The number of different test compounds to examine using the methods of the invention will depend on the application of the method. It is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Large numbers of compounds can be processed in a high-throughput automated format.

The screening methods of the invention involve detecting the amount or activity of a BRCA/STAT complex in a sample exposed to a test compound and comparing the amount or activity of a BRCA/STAT complex to a reference sample. A sample that is useful in the screening methods of the invention includes a cell in which a BRCA/STAT complex mediates a cellular function, such as, for example, cell proliferation, apoptosis, or differentiation.

A sample can contain a wild-type BRCA polypeptide or an inactive mutant BRCA polypeptide characterized by one or more nucleotide changes compared to wild-type BRCA. Such altered BRCA polypeptides can be naturally-occurring mutant BRCA polypeptides that are encoded, for example, by BRCA genes having germline mutations that increase the risk for the development of early-onset breast cancer, ovarian cancer and other types of cancer. Several mutant BRCA polypeptides are known in the art, and can be prepared for use in the methods of the invention by recombinant methods known in the art and described herein. Cancer cells containing such BRCA mutants can lack the growth control provided by a wild-type BRCA in a normal cell. Growth control can be restored to a cancer cell containing a mutant BRCA by providing to the cell a compound that allows a mutant BRCA to bind to a STAT.

A compound that induces the formation of a mutant BRCA/STAT complex can increase the amount of functional BRCA/STAT complex in a cell, and thereby restore normal growth control to a cancer cell. Such a mutant BRCA/STAT complex modulating compound can increase the association of a mutant BRCA with a STAT, for example, by binding to a mutant BRCA and altering its conformation or supplying a missing STAT binding surface so that STAT binding can occur. The screening methods of the invention are applicable to obtaining a compound that modulates the formation of a mutant BRCA/STAT complex. A mutant BRCA/STAT complex modulating compound can be used therapeutically to restore or augment BRCA function in individuals having cancer cells that contain mutant BRCA polypeptides that have reduced, or lack STAT binding capability.

A sample to be used in screening for a BRCA/STAT complex modulating compound can be prepared using a variety of methods known in the art. For example, a sample prepared from a cell can include, for example, a cell lysate, nuclear extract, fractionated cell lysate, fractionated nuclear extract and an isolated mixture of BRCA and STAT polypeptides. A mixture of isolated BRCA and STAT polypeptides that can form a BRCA/STAT complex having a BRCA/STAT complex activity can also be used as a sample in the screening methods. Screening for compounds that alter the amount or activity of a BRCA/STAT complex can be performed under conditions that induce BRCA/STAT complex formation, as previously described. To determine the ability of a compound to modulate the amount of activity of a BRCA/STAT complex, a sample is compared to a reference sample. A reference sample is prepared in the same manner as test sample but is not exposed to a test compound. A reference sample may, however, be exposed to a carrier in which compounds are dissolved, such as, for example, a buffered saline solution containing DMSO.

A change in activity or amount of a BRCA/STAT complex treated with test compound compared to that of a BRCA/STAT complex in a reference sample can be an increase or decrease in measured signal. An increase or decrease can be expressed as, for example, percentage change. Samples having BRCA/STAT amount or activity increased or decreased compared to a reference sample by 10-30% 30-50%, 50-70%, 70-90%, and 90-100% can contain a BRCA/STAT complex modulating compound.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

Association of STAT5 with BRCAl and BRCA2

This Example shows that STAT5 associates with BRCAl and BRCA2 in transfected COS7 cells.

To determine if STAT5 interacts with BRCAl, cell lysates were prepared from cells expressing STAT5a, PDGF β-receptor and HA-tagged BRCAl, and protein complexes were immunoprecipitated using anti-HA antibodies. Proteins precipitated by anti-HA antibodies were separated by SDS-PAGE and immunoblotting was performed to detect STAT5a, BRCAl and PDGF β-receptor in anti-HA immunoprecipitates .

COS7 cells were transiently transfected with different combinations of STAT5a, PDGF β-receptor and HA-BRCA1. Transient transfections were performed using Fugene-6 (Boehringer Mannheim) with 0.7 μg of each cDNA. The total amount of cDNA used for transfections was made constant using empty vector. The BRCAl cDNA (pcDNA3 β/5'HABRCAl) was provided by Ralph Scully, Dana Farber^' Institute, Boston. The STAT5a cDNA (pXM-STAT5) was provided by Bernd Groner, The Institute for Biomedical Research, Georg-Spyer-Haus, Frankfurt (Wakao et al., 1994). The cDNA for PDGF β-receptor (pcDNA-3- PDGF β rec) was provided by Carl-Henrik Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden (Claesson-Welsh et al., 1988, Mol. Cell. Biol. 8, 3476- 3486). COS7 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

About 48 hours after transfection, the cells were stimulated with 100 ng/ml PDGF-BB for 10 min at 37°C. After washing with PBS, cells were solubilized in lysis buffer containing 1% Triton X-100, 1% Sodium Deoxycholate, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 150 mM EDTA, 1 mM PMSF, 1% Trasylol, 100 μM NaV0₃. The lysates were centrifuged and the supernatants subjected to immunoprecipitation with anti-HA antibodies for 2 h at 4°C, followed by incubation with protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech AB) for 30 minutes.

Immunoprecipitates were washed twice with lysis buffer, eluted from the Sepharose beads and subjected to SDS-gel electrophoresis in a gradient gel of 5-10% polyacrylamide. The samples were separated by SDS-PAGE and electrophoretically transferred to Immun- Blot PVDF Membrane (BIO-RAD) in a buffer consisting of 20% methanol, 0.2 M Glycine, and 25 mM Tris-HCl, at 400 mA for 3-4 h at 4°C. Blots were blocked by incubation in phosphate-buffered saline containing 5% dried milk and 0.05% Tween 20 (Merck) for 1 h at room temperature, before probing with anti-STAT5, anti-BRCAl, and anti- PDGF-βR antisera.

Rabbit antiserum against STAT5 (C-17 and N20) and rabbit antiserum against BRCAl (C-20) were purchased from Santa Cruz. The rabbit antiserum PDGFR- 33, has been described previously (Weima et al., 1990).

The blots were washed three times for 10 min in PBS containing 0.05% Tween-20 and then incubated with peroxidase-conjugated swine anti-rabbit or sheep anti- mouse immunoglobulins (1:5000 and 1:1000 dilutions, respectively) . Peroxidase-conjugated swine and anti- rabbit immunoglobulins and peroxidase-conjugated sheep anti-mouse immunoglobulin were from Amersham Pharmacia Biotech (Amersham Place, UK) . After washing, bound antibodies were visualized using an ECL Immunoblotting detection system.

Figure 1A is an anti-STAT5 immunoblot that shows STAT5a co-precipitation with HA-BRCAl in cells overexpressing these proteins. A STAT5 protein band at 97 kDa was seen in immunoprecipitations from cells expressing HA-BRCAl and Stat 5, with or without PDGF-β R expression, but not in cells expressing only STAT5 or control non-expressing cells. Figure IB is an anti- BRCAl immunoblot which demonstrates that HA-BRCAl was immunoprecipitated from all cells expressing HA-BRCAl.

Also shown in Figure IB is an increase in the expression level of the HA-BRCAl gene when HA-BRCAl was coexpressed with PDGF β-receptor. An increased amount of STAT5 and BRCAl complex was observed in lysates of cells that also expressed PDGF β-receptor. Cell lysates (10% of each sample) was immunoblotted with anti-STAT (Figure 1C) and PDGF-β-receptor (Figure ID) to equivalent levels of expressed proteins in samples.

These results indicated that an association of STAT5 with HA-BRCAl in transfected COS-7 cells is detectable in an anti-HA immunoprecipitation, and both STAT5 and HA-BRCAl are increased in an anti-HA immunoprecipitation from cells expressing the PDGF β- receptor.

To determine if Stat5 interacts with BRCA2, cell lysates were prepared from cells expressing STAT5a, PDGF β-receptor and flag-tagged BRCA2, and protein complexes were immunoprecipitated using anti- flag antibodies. Proteins precipitated by anti-flag antibodies were separated by SDS-PAGE and immunoblotting was performed to detect STAT5a, BRCA2 and PDGF β-receptor in anti-flag immunoprecipitates.

COS7 cells were transiently transfected with vector alone or different combinations of Flag-tagged BRCA2, STAT5a and PDGF-bR using the methods described above. The plasmid encoding flag-BRCA2 was provided by Alan Asworth and David Bertwistle, Institute of Cancer Research, Chester Beatty Laboratories, London.

Cell lysates, prepared as described above, were immunoprecipitated with anti-flag followed by SDS- PAGE and immunoblotting with anti-Stat5 (Figure 2A) or anti-BRCA2 (Figure 2B) . Cell lysates (10% of each sample) were immunobloted with anti-Stat5 (Figure 2C) or anti-PDGF-b R (Figure 2D) to confirm equivalent levels of proteins in samples.

As shown in Figure 2A, anti-flag immunoprecipitates were found to contain STAT5 and BRCA2. Figure 2B shows that flag-BRCA2 was efficiently immunoprecipitated using anti-flag antibodies. Figure 2C (anti-STAT5 immunoblot) and Figure 2D (anti-PDGF-b R immunoblot) show that STAT5 and PDGF-b R are expressed in transfected cells.

EXAMPLE II

Association of STAT5 with BRCAl and BRCA2 in breast cancer cells

To determine if STAT5 and BRCAl form a complex in breast epithelial cells, immunoprecipitations of BRCAl and BRCA2 complexes with STAT5 were performed in lysates from the breast cancer cell lines MCF-7 and T47D.

MCF-7 and T47D cell lines endogenously express STAT5, BRCAl, BRCA2, EGF receptor and prolactin receptor. T47D and MCF-7 cells were cultured in RPMI medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin and 2 μg/ml insulin.

MCF-7 cells were serum starved and then stimulated with 5 microgram/ml ovine prolactin for 15 and 30 minutes at 37 °C . Cell lysates were prepared as described above, and immunoprecipitation was performed using an anti-BRCAl monoclonal antibody (mAb, Oncogene Res. Prod.). The immunoprecipitated proteins were analyzed by SDS-PAGE followed by immunoblotting with anti-Stat5a (figure 3A) and anti-BRCAl (figure 3B) . In addition 10% of the cell lysates were run in parallel and immunoblotted with Stat5a, in order to confirm the expression of Stat5a in all lanes (Figure 3C) .

As shown in Figure 3A, endogenous STAT5a was co-immunoprecipitated with endogenous BRCAl in T47D cells stimulated with prolactin. Immunoblotting with Φ to tr P- CD 0J _^^ Hi rt pi^¬ Ω O s: CL CD d ι CL μ- Ω „-__^. O P- to

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This example shows that the transcriptional activity of STAT5 on the β-casein promotor is decreased in the presence of BRCAl and BRCA2.

Upon ligand stimulation, an activated STAT5 dimer can bind to a β-casein promoter, causing an increase in β-casein gene expression. To determine if the interaction of STAT5 with BRACl affects STAT5 function, the ability of BRCAl to modulate STAT5a transcriptional activity on the β-casein promoter was examined in a cotransfection assay.

C0S7 cells were transfected with the reporter gene β-casein-luc (pZZl) and β-galactosidase (CH-110) together with different combinations of cDNAs encoding STAT5a, PDGF β-receptor, or BRCA2. The pCHHO plasmid encoding the β-galactosidase gene under the control of the SV40 promoter was from Pharmacia Biotech. β-casein promoters (pZZl; -344 to -1 β-casein promoter 5' pLucDSS) (Gouilleux et al., 1994) were provided by Bernd Groner, The Institute for Biomedical Research, Georg-Spyer-Haus, Frankfurt. Transcriptional activity was then estimated by luciferase assay. β- galactosidase and luciferase activities were determined on triplicate samples using the β-galactosidase assay system (Promega) and luciferase assay system (Promega) as described by the manufacturer. Luciferase were corrected for the β-galactosidase values obtained in each experiment.

C0S7 cells were transfected with β-casein promoter luciferase construct (pZZl, all columns) , STAT5 (columns 1,3,5,6), PDGF β-R (columns 4-5), BRCA2 (columns 2,3,4,6). Transfected cells were stimulated with 100 ng/ml PDGF-BB for 8 h. All transfections included plasmids pCDllO and b-galactosidase assays were performed to determine transfection efficiency. As shown in Figure 5A, column 5, an induced transcriptional activity of exogenous STAT5a was observed upon stimulation with PDGF-BB, in agreement with previous studies (Valgeirsdottir, et. al., FEBS Letters, 450:1-7. 1999). Figure 5A, column 6 shows transcriptional activity of STAT5 was markedly decreased in cells overexpressing BRCA2. Transfection with STAT5a, BRCA2, or STAT5a and BRCA2 together in unstimulated cells did not induce expression of the β- casein luciferase reporter gene (Figure 5A, columns 1, 2, and 3) . Stimulation with PDGF of cells transfected with BRCA2 alone did not induce expression of the reporter gene (Figure 5A, column 4) .

To determine if both BRCAl and BRCA2 are capable of modulating transcriptional activity of STAT5, T47D cells were transiently transfected with the responsive reporter gene β-casein-luc (pZZl) and β- galactosidase (CH-110) together with different combinations of cDNAs encoding STAT5a, PDGF β-receptor, BRCAl or BRCA2. T47D cells were transfected with β- casein promoter luciferase construct (pZZl, all columns), STAT5 (all columns), PDGF β-receptor (columns 2-4), BRCAl (column 3) or BRCA2 (column 4). Transfected cells were stimulated with 100 ng/ml PDGF- BB for 8 h. at 37°C. Luciferase activity was determined in triplicate samples. All transfections included plasmid pCDllO and β-galactosidase assays were used to determine transfection efficiency.

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3' Φ rt 3 rt tr Ω CL H Hi O ^ Ω O O rt Φ 3 Hi 3- Φ hi to CO rt

Φ 3 tr Pi^¬ PI^¬ 3^" Φ Φ tr d rt ιQ rt hi co yQ π Ω PI Φ n P- rt φ CO co s: S' rt hi tr rt Φ rt μ- P- Φ 0J CQ Φ to hi X CO > 3 O

Hi P- tr P- Φ tr rt "< 3' Φ CQ <! PI^¬ CO 3 P- CQ rt pσ φ 0J CO [O 0J 3

O O 3 o CL h-¹ φ 3" rt Φ s rt. P- S' tr CO O li o 0J 3 P- • hi

3 OJ d Φ CL p. O P- CO 3^" 3 rt Φ o Hi 3 μ- 0J CO TJ o ? O

P" • P" rt μ- p. CO 3 Φ li P- O ^■ hi s: Φ 3 3 φ H 3 Φ Hi

O Φ CL J ϋ co 3 3 Ω φ co CL 3 Ω O CO CL φ -— . CL

■ P- Φ Ω <! P- o O TJ Hi d O PI^¬ • — rt Hi pi^¬ Ω o H " P¹ PI^¬

P- > t, tr P¹ OJ P¹ Φ 3 li hi d Φ OJ P¹ Hi S' • Φ s' H li P- H Φ P- ^■ S'

3 O P- Φ Φ α o 3 <J Φ TJ tr hi TJ OJ Φ CL to φ P- 3 _^ <! yQ Φ J iQ Ω rt μ- CQ rt Φ O H Φ TJ rt ω CQ to TJ to Φ d Qm

O 3' d μ-¹ φ P- 3 Hi hi P- 3 P" Φ m Φ Ω a TJ rt ta Ω < 1 H H Φ "CO

Ω li O 3 OJ ^•< CL o rt d OJ Ω Ω P- CL > Φ > H P- Ω Φ P- φ Ω 1

P¹ d CL li 3 P- rt OJ Φ Ω m CL H" O o h-^{1 y}Q O hi Ω

OJ P- rt Φ φ 0⁾ φ O Φ rt α fD tr Cπ OJ H rt 3 IO d Hi CJi Φ 0⁾

P- 3 li TJ 3 tr 3 ^■ Qm P- rt ^•<; rt CO o Φ 0J • CQ H O OJ CO

3 yQ CQ o TJ ¹ OJ o ■ P- P- OJ hi P- H φ Φ — ' CO Φ

CQ Φ rt 3 ϋ O CQ TJ CL tr 3 P- O to 3 OJ 3 rt CO Ω • φ P-

• ^•<; TJ O Φ CL Φ Φ •<: CO rt 3 - P- CQ to 0J HI 3" rt CL 3

OJ O ^■ Ω P- tr hi CO 3^* o li 3 to h-¹ Ω 3- 0J P> O li CL μ- 3 Φ rt Ω li P- P- Φ CL CO o Φ rt φ rt TJ P- li rt rt μ- OJ φ Φ 0⁾ hi φ 3 3 OJ CQ P- 3' > <J P- hi OJ P- 3 Φ tr P- rt rt 3 3 P- P- Hi hi TJ Ω O [O Φ <1 φ ■ 3 Ω TJ

Φ 3 3' P" Φ Pi^¬ 3 tr φ PI^¬ TJ P- OJ O OJ -t H P- Hi Φ CL TJ O ιQ OJ d co CL CO Φ li S' 0⁾ O 3 3 rt 3 φ rt o hi CΛ hi hi

P- rt CO rt -* Φ . φ Φ li d CL CO Φ JXJ O • hi Φ to Hi φ rt

3 Hi rt tr CO PI^¬ 3 P- Φ CO Φ tr φ Hi φ > CQ Φ

<! P> <5 hi J pi^¬ Ω S' Ω hi 3 to PI^¬ *< hi ≤ _^ Hi Φ li

Φ O OJ OJ PI- s' i Φ Φ PI^¬ to rt S' rt CΛ OJ -— . Cπ 3

3 3 hi rt o P- S' o O OJ Hi Φ Hi CO P" Ω yQ rt μ- P- PI^¬ CO tr CO P- Φ > rt (-^■ CL > 0J P- Φ Φ

P- PI^¬ o <l S' Φ Φ rt 3 CQ IO O Hi 3 3 3 3

O S' d Φ φ CL OJ φ • rt 3 π o Φ O Φ

3 Φ CO rt pi^¬ CO tr O • rt CO O Hi

O Φ s' p. Φ o

Hi P- CO CΛ

CO CO -J

Claims

What is claimed is:

1. A method of inhibiting cellular proliferation mediated by a BRCA/STAT complex, comprising contacting a BRCA/STAT-containing cell with an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in said cell.

2. The method of claim 1, further comprising contacting said BRCA/STAT-containing cells under conditions that induce BRCA/STAT complex formation.

3. The method of claim 1, wherein said BRCA/STAT complex modulating compound is selected from the group consisting of small molecule, polypeptide and nucleic acid.

4. The method of claim 3, wherein said polypeptide further comprises an antibody or functional fragment thereof.

5. The method of claim 1, wherein said BRCA/STAT complex modulating compound further comprises a STAT activating agent and a BRCA polypeptide, or functional fragment thereof.

6. The method of claim 1, wherein said cell further comprises a breast or ovarian cell.

7. The method of claim 1, wherein said effective amount of a BRCA/STAT complex modulating compound is sufficient to increase the amount or activity of a BRCA/STAT complex in said cell.

8. The method of claim 1, wherein said effective amount of a BRCA/STAT complex modulating compound is sufficient to decrease the amount or activity of a BRCA/STAT complex in said cell.

9. A method of reducing the severity of cancer, comprising administering to an individual an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in cancer cells in said individual.

10. The method of claim 9, wherein said BRCA/STAT complex modulating compound is selected from the group consisting of small molecule, polypeptide and nucleic acid.

11. The method of claim 9, wherein said polypeptide further comprises an antibody or functional fragment thereof.

12. The method of claim 11, wherein said BRCA/STAT complex modulating compound further comprises a STAT activating agent and a BRCA polypeptide, or functional fragment thereof.

13. The method of claim 9, wherein said effective amount of a BRCA/STAT complex modulating compound is sufficient to increase the amount or activity of a BRCA/STAT complex in said cancer cells.

14. The method of claim 9, wherein said effective amount of a BRCA/STAT complex modulating compound is sufficient to decrease the amount or activity of a BRCA/STAT complex in said cancer cells.

15. The method of claim 9, wherein said cancer further comprises breast or ovarian cancer.

16. A method of detecting the risk of breast cancer in an individual, comprising:

(a) detecting an amount or activity of a BRCA/STAT complex in a sample obtained from an individual suspected of having breast cancer, and

(b) comparing said amount or activity of said BRCA/STAT complex from said sample to an amount or activity from a reference sample, a difference in said amount or activity between said sample and said reference sample indicating the presence or susceptibility of breast cancer.

17. The method of claim 16, wherein said amount of said BRCA/STAT complex is detected by contacting said sample with a binding agent selective for said BRCA/STAT complex, and detecting the amount of selective binding, said amount of selective binding correlating with the amount of BRCA/STAT complex in said sample.

18. The method of claim 16, wherein said amount of said BRCA/STAT complex is detected by contacting said sample with a binding agent selective for BRCA or STAT, and detecting the amount of selective binding of said binding reagent, said amount of selective binding inversely correlating with the amount of said BRCA/STAT complex in said sample.

19. The method of claim 16, wherein said activity of said BRCA/STAT complex is detected by measuring the expression of a gene regulated by said BRCA/STAT complex.

20. The method of claim 19, wherein said gene regulated by said BRCA/STAT complex further comprises BRCAl or BRCA2.

21. The method of claim 12, wherein said activity of said BRCA/STAT complex is detected by measuring the expression of a STAT regulated gene.

22. The method of claim 16, wherein said BRCA/STAT complex further comprises a polypeptide selected from the group consisting of BRCAl, BRCA2 and STAT5.

23. The method of claim 16, wherein said difference in said amount or activity of said BRCA/STAT complex further comprises a decrease in said sample compared to said reference sample.

24. The method of claim 16, wherein said sample further comprises a cell lysate or a nuclear extract.

25. A method of detecting the risk of breast cancer in an individual, comprising:

(a) exposing cells obtained from an individual suspected of having breast cancer to conditions that induce BRCA/STAT complex formation, and

(b) detecting an amount or activity of uncomplexed BRCA in said exposed cells, wherein a decreased BRCA amount or activity compared to a reference sample indicates an increased susceptibility for breast cancer.

26. The method of claim 25, wherein said decreased BRCA amount or activity being further characterized by the presence of regulatory factors affecting BRCA/STAT complex activity.

27. The method of claim 25, wherein said conditions that induce BRCA/STAT complex formation further comprise a factor selected from the group consisting of cytokine, hormone, growth factor and kinase.

28. The method of claim 25, wherein said conditions that induce BRCA/STAT complex formation further comprise the step of expressing in said cells a BRCA or STAT encoding nucleic acid.

29. The method of claim 25, wherein said BRCA/STAT complex further comprises a polypeptide selected from the group consisting of BRCAl, BRCA2 and STAT5.

30. The method of claim 25, wherein said detecting the amount or activity of uncomplexed BRCA further comprises contacting said sample with a binding agent selective for BRCA, and detecting the amount of selective binding of said binding reagent.

31. The method of claim 25, wherein said detecting the amount or activity of BRCA further comprises contacting said sample with a nucleic acid probe selective for BRCA, and detecting the amount of selective hybridization of said nucleic acid probe.

32. A method of prognosing the severity or progression of breast cancer, comprising:

(a) detecting an amount or activity of a BRCA/STAT complex in a sample obtained from an individual at a site suspected of containing cancer cells, and

(b) comparing said amount or activity of said BRCA/STAT complex with a reference sample, a difference in said amount or activity of said BRCA/STAT complex in said sample indicating the level of severity or progression of said cancer.

33. The method of claim 32, wherein said BRCA/STAT complex further comprises a polypeptide selected from the group consisting of BRCAl, BRCA2 and STAT5.

34. The method of claim 32, wherein said reference sample is obtained from a normal individual.

35. The method of claim 32, wherein said reference sample is obtained from said individual at a temporally separate point compared to when said sample is obtained.

36. A method of screening for compounds that modulate the activity of a BRCA/STAT complex, comprising:

(a) contacting a sample containing BRCA and STAT with a test compound under conditions that induce BRCA/STAT complex formation, and

(b) detecting an amount of BRCA/STAT complex formation or an activity of a BRCA/STAT complex, a change in said amount of formation or activity of said BRCA/STAT complex in said sample compared to a reference sample indicating that said test compound has BRCA/STAT complex modulating activity.

37. The method of claim 36, wherein said BRCA/STAT complex modulating activity further comprises an increase in BRCA/STAT activity.

38. The method of claim 36, wherein said BRCA/STAT complex modulating activity further comprises a decrease in BRCA/STAT activity.

39. The method of claim 36, wherein said BRCA/STAT complex modulating activity further comprises BRCA/STAT complex formation promoting activity.

40. The method of claim 36, wherein said BRCA/STAT complex modulating activity further comprises BRCA/STAT complex formation inhibiting activity.

41. The method of claim 36, wherein said sample is selected from the group consisting of a cell, cell lysate, nuclear extract, fractionated cell lysate, fractionated nuclear extract and a mixture of isolated BRCA and STAT polypeptides.

42. The method of claim 36, wherein said sample further comprises an inactive BRCA polypeptide characterized by one or more nucleotide changes compared to wild-type BRCA.

43. The method of claim 36, wherein said test compound is selected from the group consisting of small molecule, polypeptide and nucleic acid.

44. The method of claim 36, wherein said test compound is selected from the group consisting of cytokine, hormone, growth factor, kinase and phosphatase.

45. The method of claim 36, wherein said conditions that induce BRCA/STAT complex formation further comprise increasing the amount of BRCA or STAT polypeptides in said sample.

46. The method of claim 36, wherein said BRCA/STAT complex further comprises a polypeptide selected from the group consisting of BRCAl, BRCA2 and STAT5.

47. The method of claim 36, wherein said amount of said BRCA/STAT complex formation is detected by contacting said sample with a binding agent selective for said BRCA/STAT complex, and detecting the amount of selective binding, said amount of selective binding correlating with the amount of BRCA/STAT complex in said sample.

48. The method of claim 36, wherein said activity of said BRCA/STAT complex is detected by measuring the expression of a gene regulated by said BRCA/STAT complex.

49. The method of claim 36, wherein said activity of said BRCA/STAT complex is detected by measuring the expression of a STAT regulated gene.