WO2001031342A2 - DIAGNOSTIC ET TRAITEMENT DU CANCER UTILISANT DES POLYPEPTIDES ET DES POLYNUCLEOTIDES StAR - Google Patents

DIAGNOSTIC ET TRAITEMENT DU CANCER UTILISANT DES POLYPEPTIDES ET DES POLYNUCLEOTIDES StAR Download PDF

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WO2001031342A2
WO2001031342A2 PCT/US2000/029556 US0029556W WO0131342A2 WO 2001031342 A2 WO2001031342 A2 WO 2001031342A2 US 0029556 W US0029556 W US 0029556W WO 0131342 A2 WO0131342 A2 WO 0131342A2
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star
cancer
level
biological sample
polypeptide
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PCT/US2000/029556
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WO2001031342A3 (fr
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Scott Powers
Allyson Servoss
Rong Wendy Zeng
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Tularik Inc.
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Publication of WO2001031342A3 publication Critical patent/WO2001031342A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to the field of cancer diagnosis and treatment.
  • Methods and diagnostic reagents are provided for diagnosing and treating cancers that involve abnormal or abnormally expressed StAR nucleic acids or polypeptides.
  • cancer remains the second leading cause of death in the United States.
  • epithelial cancers are among the most prevalent and deadly.
  • about 175,000 new invasive cases of breast cancer are expected to occur among women in the United States during 1999, and more than 43,000 women will die of this disease.
  • Cancer is a genetic disease of single cell origin caused by the accumulation of inherited and acquired mutations in specific cancer genes, which have normal cellular functions, but when mutated or present at abnormally high levels contribute to cancer.
  • One type of mutation that unlocks the cancer-causing potential of a cancer gene is gene amplification or overexpression, i.e., where a specific chromosomal region (including the cancer gene) has undergone a relative increase in DNA copy number, such that more copies of the cancer gene are present, or where the level of expression of a gene is increased, such that a correspondingly higher amount of mRNA and protein is produced, causing deleterious effects.
  • Gene amplification is one of the primary mutational mechanisms for causing primary genetic alterations in solid tumors, and most of the chromosomal regions that undergo amplification are not well characterized and do not harbor known oncogenes (Knuutila et al., (1998) Am. J. Pathol, 152:1107-23; Knuutila et al., (1998) Cancer Genet. Cytogenet., 100:25-30). Discovery of these amplified cancer genes will provide novel targets for diagnostic, and therapeutic applications.
  • RDA involves several steps, including isolating a "representative" population of nucleic acid from a cell or cell type or interest, e.g., a cancer cell. This "representative" population contains significantly less complexity than that found in the overall genome. Using a simultaneous hybridization and amplification step to enrich for those sequences not present in a control, or "tester" cell or cell population, RDA allows for the identification of sequences that are over- represented in the cell type of interest.
  • StAR The steroidogenic acute regulatory protein, or "StAR,” was identified in 1994 (Clark et al, (1994) J. Biol. Chem., 269:28314-22) based on its hormone-induced expression as well as its localization to the mitochondria. StAR is the rate-limiting protein in steroidogenesis, allowing entry of cholesterol into the mitochondria for the first enzymatic steps carried out by p450scc for hormone biosynthesis. The expression of StAR is normally strictly regulated, and usually only occurs in the presence of hormones, e.g., trophic hormones.
  • the present invention is based on the surprising discovery that StAR polynucleotide sequences are amplified and/or overexpressed in many types of cancer cells in mammals. As described herein, this discovery has provided novel and badly needed diagnostic, prognostic, and therapeutic tools for many types of cancers.
  • the present invention provides methods, reagents, and kits for diagnosing and treating cancer in a mammal, e.g., a human.
  • This invention is based upon the surprising discovery that the gene encoding the steroidogenic acute regulatory protein, or "StAR,” is overexpressed and/or amplified in a number of types of cancer cells. Accordingly, the present methods can be used to detect cancer or a propensity to develop cancer, to monitor the efficacy of a cancer treatment, and to treat cancer, e.g., by inhibiting the expression and/or activity of StAR in a cancer cell.
  • the present invention provides a method of detecting cancer in a biological sample from a mammal, the method comprising detecting the presence or absence of a StAR polypeptide or polynucleotide in the biological sample, wherein a diagnostic presence of the StAR polypeptide or polynucleotide indicates the presence of cancer in the biological sample.
  • the present invention provides a method of detecting cancer in a biological sample from a mammal, the method comprising detecting the level of a StAR polypeptide or polynucleotide in the biological sample, wherein an increased level of the StAR polypeptide or polynucleotide in the biological sample compared to the level expected in a control sample from a normal, cancer-free mammal indicates the presence of cancer in the biological sample.
  • the detection is performed under conditions that would not detect the StAR polypeptide or polynucleotide in a sample that is not cancerous, and wherein a diagnostic presence or an increased level comprises detecting any StAR polypeptide or polynucleotide in the biological sample.
  • the diagnostic presence or increased level comprises at least a 2-fold increase in the level of the StAR polypeptide or polynucleotide in the biological sample compared to a level expected in a sample from a control, cancer-free mammal. In another embodiment, the diagnostic presence or increased level comprises at least a 5-fold increase in the level of the StAR polypeptide or polynucleotide in the biological sample compared to a level expected in a sample from a control, cancer- free mammal. In another embodiment, the diagnostic presence or increased level comprises at least a 10-fold increase in the level of the StAR polypeptide or polynucleotide in the biological sample compared to a level expected in a sample from a control, cancer-free mammal.
  • the method comprises detecting a StAR polypeptide by immunoassay.
  • the immunoassay comprises i munohistochemistry.
  • the method comprises detecting the presence or absence of StAR gene amplification.
  • the method comprises detecting a StAR mRNA.
  • the cancer is an epithelial cancer.
  • the epithelial cancer is selected from the group consisting of breast cancer, lung cancer, colorectal cancer, prostate cancer, kidney cancer, stomach cancer, bladder cancer, ovarian cancer, and cancer of the gastrointestinal tract.
  • the mammal is a human.
  • the biological sample is selected from the group consisting of tissue biopsy, blood sample, and nipple discharge.
  • the present invention provides a method of monitoring the efficacy of a cancer treatment, the method comprising detecting the level of a StAR polypeptide or polynucleotide in a biological sample from a mammal undergoing treatment for cancer, wherein a reduced level of the StAR polypeptide or polynucleotide in the biological sample compared to a level in a sample taken from the mammal prior to, or earlier in, the treatment is indicative of efficacious treatment.
  • the cancer is an epithelial cancer.
  • the epithelial cancer is a breast cancer.
  • the mammal is a human.
  • the present invention provides methods for decreasing the proliferation of a cell with an elevated level of StAR, the method comprising reducing the level of StAR activity in the cell using an inhibitor of StAR.
  • the cell is a cancer cell.
  • the cancer cell is an epithelial cancer cell.
  • the epithelial cancer cell is a breast cancer cell.
  • the inhibitor produces a decrease in the production of steroid hormones by the cell.
  • Figure 1 shows a physical map of the 8pl l-pl2 amplified chromosomal region, indicated by the solid line marked with solid circles. Underneath are solid lines representing individual YACs, and a dashed line representing genomic sequence from GenBank (Accession numbers: AP000065 through AP000084). STSs on this map (solid circles) were derived from RDA probes or from the genomic sequence or public databases at the Whitehead Institute, Stanford University, and the Sanger Center. The STSs are ordered on the basis of genomic sequence; or by determination by PCR of their presence on these YACs and others not shown, and confirmed by radiation hybrid mapping using the GB3 panel (Stanford University RH server).
  • Figure 2 provides a schematic illustration of the role of StAR in steroid hormone biosynthesis.
  • the present invention provides methods of diagnosing and treating cancer in mammals and other animals. These methods are based upon the discovery that the StAR gene is amplified and/or overexpressed in cancer cells. Accordingly, cancer cells can be detected by virtue of amplification or overexpression of StAR.
  • the present invention provides methods for treating cancer, for example, by reducing the level of expression or activity of StAR in a cancer cell. The methods of the invention typically involve detecting the presence or absence of StAR in a biological sample taken from a mammal.
  • the level of StAR polypeptide or polynucleotide is detected in a sample and compared to a "control" level, where a "control" level is a level of StAR that is actually detected in a cancer-free sample or a level that is expected for a typical cancer-free sample.
  • a "control" level is a level of StAR that is actually detected in a cancer-free sample or a level that is expected for a typical cancer-free sample.
  • Such detection may involve quantitative or qualitative detection of the polypeptide or polynucleotide, and may involve an actual comparison with a control value or, alternatively, may be performed so that the detection itself inherently indicates an increased level, or "diagnostic presence" of the StAR.
  • a "diagnostic presence" indicates any level of StAR that is greater than that expected in a noncancerous sample.
  • assays for a StAR polypeptide or polynucleotide in a biological sample are conducted under conditions wherein a normal level of StAR polypeptide or polynucleotide, i.e., a. level typical of a noncancerous sample, i.e., cancer-free, would not be detected. In such assays, therefore, the detection of any StAR polypeptide or nucleic acid in the biological sample indicates a diagnostic presence, or increased level.
  • a diagnostic presence of StAR polypeptide or nucleic acid represents at least about a 1.5, 2, 5, 10, or greater fold increase in the StAR polypeptide or polynucleotide in the biological sample compared to a level expected in a noncancerous sample.
  • Detection of StAR can be performed in vitro, i.e., in cells within a biological sample taken from the mammal, or in vivo.
  • an increased level, or diagnostic presence, of StAR in a biologipal sample can be detected using any of a variety of forms of StAR, for example, StAR protein, StAR mRNA, or StAR genomic DNA, and can indicate an increase in, e.g., StAR gene copy number, StAR transcription, StAR translation, StAR RNA or protein stability, or StAR protein activity.
  • any of a large number of assays can be used to detect the StAR nucleic acids or polypeptides, such as StAR nucleic acid probes to detect StAR polynucleotides, or StAR-specific antibodies or other specific binding reagents to detect StAR polypeptides.
  • the ability to detect cancer cells by virtue of an increased level, or "diagnostic presence,” of StAR is useful for any of a large number of applications.
  • an increased level or diagnostic presence of StAR in cells of a mammal can be used, alone or in combination with other diagnostic methods, to diagnose cancer in the mammal or to determine the propensity of a mammal to develop cancer over time.
  • the detection of StAR can also be used to monitor the efficacy of a cancer treatment. For example, a level of a StAR polypeptide or polynucleotide after an anti-cancer treatment is compared to the level in the mammal before the treatment, wherein a decrease in the level of the StAR polypeptide or polynucleotide after the treatment indicates efficacious treatment.
  • An increased level or diagnostic presence of StAR can also be used to influence the choice of anti-cancer treatment in a mammal, where, for example, a strong increase in a level of StAR indicates the use of a more aggressive anti-cancer therapy, and a small increase or no increase indicates the use of a less aggressive anti-cancer therapy.
  • a level of StAR is used to guide the choice of an anti-cancer agent, in particular agents that affect the level or activity of a steroid hormone.
  • an increased level of StAR in breast or prostate tumor cells can indicate that the use of an agent that lowers the level or activity of estrogen or testosterone, respectively, would be effective in treating the tumor.
  • an increased or diagnostic level of StAR as detected by immunoassay in a breast tumor is used to indicate the use of tamoxifen in the treatment of the tumor.
  • the ability to detect cancer cells can be useful to monitor the number or location of cancer cells in a patient, in vivo or in vitro, for example, to monitor the progression of the cancer over time.
  • the level or presence or absence of StAR can be statistically correlated with the efficacy of particular anti-cancer therapies or with observed prognostic outcomes, thereby allowing the development of databases based on which a statistically-based prognosis, or a selection of the most efficacious treatment, can be made in view of a particular level or diagnostic presence of StAR.
  • the present invention also provides methods for treating cancer.
  • the growth, proliferation, or steroid production of a cell with an elevated level of StAR polynucleotides, polypeptides, or polypeptide activity is inhibited.
  • the growth, proliferation, or steroid production is decreased by, for example, contacting the cell with an inhibitor of StAR transcription or translation, or an inhibitor of the activity of a StAR polypeptide.
  • Such inhibitors include, but are not limited to, antisense polynucleotides, ribozymes, antibodies, dominant negative StAR polypeptides, and small molecule inhibitors of StAR activity.
  • the present methods can be used to diagnose, determine the prognosis for, or treat, any of a number of types of cancers.
  • the cancer is an epithelial cancer, e.g., breast, lung, colorectal, prostate, kidney, stomach, bladder, or ovarian cancer, or any cancer of the gastrointestinal tract.
  • the diagnostic methods of this invention can be used in animals including, for example, primates, canines, felines, murines, bovines, equines, ovines, porcines, lagomorphs, etc, as well as in humans. Suitable biological samples include, but are not limited to, tissue biopsy, blood sample, buccal scrape, saliva, nipple discharge, and urine. Kits are also provided for carrying out the herein-disclosed diagnostic and therapeutic methods.
  • detecting a cancer refers to the ascertainment of the presence or absence of cancer in an animal.
  • Detecting a cancer can also refer to obtaining indirect evidence regarding the likelihood of the presence of cancerous cells in the animal. Detecting a cancer can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the animal.
  • a “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may circulate in the blood stream as independent cells, such as leukemic cells.
  • Providing a biological sample means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo.
  • control sample refers to a sample of biological material representative of healthy, cancer-free animals.
  • the level of StAR in a control sample is desirably typical of the general population of normal, cancer-free animals. This sample can be removed from an animal expressly for use in the methods described in this invention, or can be any biological material representative of normal, cancer-free animals.
  • a control sample can also refer to an established level of StAR, representative of the cancer-free population, that has been previously established based on measurements from normal, cancer-free animals.
  • a detection method that only detects StAR when a level higher than that typical of a normal, cancer- free animal is present, i.e., an immunohistochemical assay giving a simple positive or negative result, this is considered to be assessing the StAR level in comparison to the control level, as the control level is inherent in the assay.
  • a level of StAR polypeptide or polynucleotide that is "expected" in a control sample refers to a level that represents a typical, cancer-free sample, and from which an elevated, or diagnostic, presence of StAR polypeptide or polynucleotide can be distinguished.
  • an "expected” level will be controlled for such factors as the age, sex, medical history, etc. of the mammal, as well as for the particular biological sample being tested.
  • an “increased,” or “elevated,” level of StAR refers to a level of StAR polynucleotide or polypeptide, that, in comparison with a control level of StAR, is detectably higher.
  • the method of comparison can be statistical, using quantified values for the level of StAR, or can be compared using nonstatistical means, such as by a visual, subjective assessment by a human.
  • a "StAR polynucleotide” or “StAR nucleic acid” is a DNA or RNA sequence of at least about 50 nucleotides that is at least about 70% identical, preferably at least about 80%, or more, identical over a region of at least about 50, 100, 200, 500, or more nucleotides to a StAR polynucleotide sequence, from a mammal including, but not limited to, human, rat, mouse, hamster, cow, pig, horse, sheep, or any mammal, as, e.g., provided in Clark et al., (1994) J. Biol.
  • a “StAR polynucleotide” encodes a "StAR polypeptide.”
  • a human StAR polynucleotide will comprise at least about 50%, 70%, 80%, 90%, or more nucleotide sequence identity to SEQ ID NO: 1.
  • the human StAR polynucleotide will comprise the endogenous StAR gene, present at genomic location 8pl 1.2, or will comprise an RNA transcribed from the endogenous StAR gene.
  • a "StAR polynucleotide” can comprise naturally occurring nucleotides, or any derivative or analog thereof, e.g., labeled or modified deoxyribo- or ribonucleotides.
  • StAR polynucleotide can refer to a mutated copy of any of the above sequences, or a fragment thereof, and can refer to any naturally occurring or synthetic StAR sequence.
  • a "StAR protein” or “StAR polypeptide” refers to a polypeptide of at least about 20 amino acids that is typically about 70% identical, preferably at least about 80%, more preferably at least about 90%, or more, identical over a region of at least about 20, 50, 100 or more amino acids, to one or more StAR polypeptide sequences from any mammal, as provided in, e.g., Clark et al, (1994) J. Biol.
  • a human StAR polypeptide will comprise at least about 50%, 70%, 80%, 90%, or more amino acid sequence identity to SEQ ID NO:2. Typically, the human StAR polypeptide will be expressed from the endogenous StAR gene located at genomic map position 8pl 1.2.
  • a “StAR protein” or “polypeptide” can comprise naturally occurring or synthetic amino acids, e.g., labeled or otherwise modified amino acids or amino acid analogs.
  • a “StAR protein” will typically contain one or more characteristic protein motifs, any of which can be used independently of other elements normally present in a full-length StAR protein, and will have one or more characteristic activities or properties, e.g., mitochondrial localization, phosphorylation patterns, or cholesterol translocating activity.
  • a “StAR protein” can refer to any naturally occurring or synthetic StAR polypeptide as described above.
  • the "level of StAR mRNA" in a biological sample refers to the amount of mRNA transcribed from an StAR gene that is present in a cell or a biological sample.
  • the mRNA generally encodes a functional StAR protein, although mutations or microdeletions may be present that alter or eliminate the function of the encoded protein.
  • a "level of StAR mRNA" need not be quantified, but can simply 'be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • the "level of S tAR protein or polypeptide” in a biological sample refers to the amount of polypeptide translated from a StAR mRNA that is present in a cell or biological sample.
  • the polypeptide may or may not have StAR protein activity.
  • a "level of StAR protein” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • a "full length" StAR protein or nucleic acid refers to a StAR polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type StAR polynucleotide or polypeptide sequences.
  • a "nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind.
  • a quantified level of StAR falls outside of a given confidence interval for a normal level of StAR
  • the difference between the two levels is said to be "statistically significant.” If a test value falls outside of a given confidence interval for a normal level of StAR, it is possible to calculate the probability that the test value is truly abnormal and does not simply represent a normal deviation from the average.
  • a difference between a test sample and a control can be termed "statistically significant" when the probability of the test sample being a normal deviation from the average can be any of a number of values, including 0.15, 0.1, 0.05, and 0.01.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%>, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol. Evol, 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989).
  • the program can align up to 300 sequences, each of a maximum length of 5 V 000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res., 12:387-395 (1984).
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873- 5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42°C or 5x SSC and 1% SDS incubated at 65°C, with a wash in 0.2x SSC and 0.1% SDS at 65°C. Washes can be performed, e.g., for 2, 5, 10, 15, 30, 60, or more minutes.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 ⁇ a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see, Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990))
  • any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature, 256:495-497 (1975); Kozbor et al. , Immunology Today, 4:72 (1983); Cole et al, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985)).
  • Techniques for the production of single chain antibodies can be adapted to produce antibodies to polypeptides of this invention.
  • transgenic mice, or other organisms such as other mammals may be used to express humanized antibodies.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature, 348:552-554 (1990); Marks et al, Biotechnology, 10:779-783 (1992)).
  • a “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, detect, and/or quantify the antigen.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to a StAR polypeptide can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with StAR and not with other proteins, except for polymorphic variants, orthologs, and alleles of StAR.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid- phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • the phrase "selectively associates with” refers to the ability of a nucleic acid to "selectively hybridize” with another as defined above, or the ability of an antibody to "selectively (or specifically) bind to a protein, as defined above.
  • the present invention is based on the discovery that a diagnosis and/or prognosis of cancer can be determined by detecting elevated levels of StAR polypeptide or polynucleotides in a biological sample from a mammal. As described below, any of a number of methods to detect the presence or level of StAR can be used.
  • a StAR polynucleotide level can be detected by detecting any StAR DNA or RNA, including S tAR genomic DNA, mRNA, and cDNA.
  • a StAR polypeptide can be detected by detecting an StAR polypeptide itself, or by detecting StAR protein activity, e.g., cholesterol translocation, steroid production, etc.
  • Detection can involve quantification of the level of StAR (e.g., gDNA, cDNA, mRNA, or protein level, or protein activity), or, alternatively, can be a qualitative assessment of the level, or of the presence or absence, of StAR, in particular in comparison with a control level.
  • StAR e.g., gDNA, cDNA, mRNA, or protein level, or protein activity
  • Any of a number of methods to detect any of the above can be used, as described infra. Such methods include, for example, hybridization, amplification, and other assays.
  • a level of StAR in a biological sample will be compared with a control sample taken from a cancer-free animal, or, preferably, with a value expected for a sample taken from a cancer-free animal.
  • an assay will be performed under conditions where only a higher than normal amount of StAR polynucleotide or polypeptide will be detectable in the assay.
  • an elevated level of StAR can be detected in a sample using a simple assay giving a simple, positive or negative result, with no need for quantification of StAR levels or direct comparison with a control level.
  • the level of StAR polynucleotide, polypeptide, or protein activity will be quantified.
  • the difference between an elevated level of StAR and a normal, control level will preferably be statistically significant.
  • an elevated level of StAR polynucleotide, polypeptide, and or protein activity will be at least about 2, 5, 10, or more fold greater than a control level, or a level expected of a control sample.
  • the ability to detect an increased level, or diagnostic presence, in a cell is used as a marker for cancer cells, i.e., to monitor the number or localization of cancer cells in a patient, as detected in vivo or in vitro.
  • the StAR polynucleotides or polypeptides detected herein will be at least about 70% identical, and preferably 80% or more identical, over a region of at least about 50, 100, 200, or more nucleotides, or 20, 50, 100, or more amino acids, to SEQ ID NO:l or 2, or to one or more StAR sequences available, e.g., from GenBank (see, e.g., GenBank Accession Nos: U66490, U53020, AB001349, L36062, L36061, S73839, AF031696, AF086814, Y17260, AB006007, AF038553, and NM_000349).
  • Such polynucleotides or polypeptides can represent functional or nonfunctional forms of StAR, or any variant, derivative, or fragment thereof.
  • a biological sample refers to a cell or population of cells or a quantity of tissue or fluid from an animal. Most often, the sample has been removed from an animal, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e., without removal from the animal. Typically, a “biological sample” will contain cells from the animal, but the term can also refer to noncellular biological material, such as noncellular fractions of blood, saliva, or urine, that can be used to measure StAR levels. Numerous types of biological samples can be used in the present invention, including, but not limited to, a tissue biopsy, blood sample, a buccal scrape, a saliva sample, or a nipple discharge.
  • tissue biopsy refers to an amount of tissue removed from an animal for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.
  • tissue biopsy can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.
  • a "buccal scrape” refers to a sample of cells removed from the inner lining of the mouth.
  • a “nipple discharge” refers to fluid originating from a nipple, which may contain cancerous cells or may contain elevated levels of StAR polypeptide indicating the presence of cancerous cells in the breast.
  • the presence of cancer is evaluated by determining the copy number of StAR genes, i.e., the number of DNA sequences in a cell encoding a StAR protein.
  • the copy number can be increased, however, by gene amplification or duplication, e.g., in cancer cells, or reduced by deletion.
  • Methods of evaluating the copy number of a particular gene are well known to those of skill in the art, and include, inter alia, hybridization and amplification based assays.
  • Hybridization-based Assays Any of a number of hybridization based assays can be used to detect the copy number of StAR genes in the cells of a biological sample.
  • One such method is by Southern blot.
  • genomic DNA is typically fragmented, separated electrophoretically, transfened to a membrane, and subsequently hybridized to a StAR specific probe.
  • Comparison of the intensity of the hybridization signal from the probe for the target region with a signal from a control probe for a region of normal genomic DNA (e.g., a nonamplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative StAR copy number.
  • Southern blot methodology is well known in the art and is described, e.g., in Ausubel et al, or Sambrook et al, supra.
  • An alternative means for determining the copy number of StAR genes in a sample is by in situ hybridization, e.g., fluorescence in situ hybridization, or FISH.
  • in situ hybridization assays are well known (e.g., Angerer (1987) Meth. Enzymol, 152:649).
  • in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments.
  • probes used in such applications are typically labeled, e.g., with radioisotopes or fluorescent reporters.
  • Preferred probes are sufficiently long, e.g., from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, so as to specifically hybridize with the target nucleic acid(s) under stringent conditions.
  • "comparative probe” methods such as comparative genomic hybridization (CGH), are used to detect StAR gene amplification.
  • CGH comparative genomic hybridization
  • a "test" collection of nucleic acids is labeled with a first label
  • a second collection e.g., from a healthy cell or tissue
  • The, ratio of hybridization of the nucleic acids is determined by the ratio of the first and second labels binding to each fiber in an array. Differences in the ratio of the signals from the two labels, e.g., due to gene amplification in the test collection, is detected and the ratio provides a measure of the StAR gene copy number.
  • Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J., 3:1227-1234; Pinkel (1988) Proc. Natl Acad. Sci. USA, 85:9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994), etc.
  • amplification-based assays are used to measure the copy number of StAR genes.
  • the StAR nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
  • an amplification reaction e.g., Polymerase Chain Reaction, or PCR.
  • the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the copy number of the StAR gene.
  • Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • a TaqMan based assay is used to quantify S tAR polynucleotides.
  • TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end.
  • the 5' nuclease activity of the polymerase e.g., AmpliTaq
  • the polymerase e.g., AmpliTaq
  • This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, literature provided by Perkin-Elmer, e.g., www2.perkin- elmer.com).
  • ligase chain reaction (LCR) (see, Wu and Wallace (1989) Genomics, 4:560, Landegren et al (1988) Science, 241 :1077, and Barringer et al. (1990) Gene, 89:117), transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA, 86: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA, 87:1874), dot PCR, and linker adapter PCR, etc.
  • LCR ligase chain reaction
  • StAR levels are characterized by detecting StAR gene expression by virtue of levels of StAR mRNA, StAR polypeptide and or StAR protein activity (e.g., cholesterol translocation activity) in a biological sample.
  • StAR cDNA involves a Southern Blot as described above. Briefly, StAR mRNA is isolated using standard methods and reverse transcribed to produce cDNA. The cDNA is then optionally digested, run on a gel, and transfened to a membrane. Hybridization is then carried out using nucleic acid probes specific for StAR cDNA and detected using standard techniques (see, e.g., Sambrook et al, supra).
  • a Northern blot may be used to detect an mRNA directly.
  • mRNA is isolated from a given biological sample, electrophoresed to separate the mRNA species, and transfened from the gel to a nitrocellulose membrane.
  • a StAR transcript e.g., StAR mRNA
  • amplification-based methods e.g., RT-PCR.
  • RT-PCR methods are well known to those of skill (see, e.g., Ausubel et al, supra).
  • quantitative RT-PCR is used, thereby allowing the comparison of the level of mRNA in a sample with a control sample or value.
  • StAR levels can also be detected and/or quantified by detecting or quantifying StAR polypeptide.
  • StAR polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like
  • immunological methods such as fluid or gel precipitin reactions,
  • an StAR polypeptide is detected using an immunoassay such as an ELISA assay (see, e.g., Crowther, John R. ELISA Theory and Practice. Humana Press. New Jersey, 1995).
  • an "immunoassay” is an assay that utilizes an antibody to specifically bind to the analyte (i.e., the StAR polypeptide). The immunoassay is thus characterized by detection of specific binding of a StAR polypeptide to an anti-StAR antibody.
  • StAR polypeptide in an immunoassay, can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241 ; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see, e.g., U.S. Patent Nos. 4,366,241 ; 4,376,110; 4,517,288; and 4,837,168.
  • Immunoassays typically rely on direct or indirect labeling methods to detect antibody-analyte binding.
  • an anti-StAR antibody can be directly labeled, thereby allowing detection.
  • the anti-StAR antibody may itself be unlabeled, but may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second or third antibodies can also be modified with a detectable moiety, e.g., as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • other antibody-binding molecules can be used, e.g., labeled protein A or G (see, generally Kronval, et al. (1973) J. Immunol, 111 :1401-1406, and Akerstrom (1985) J. Immunol., 135:2589-2542).
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
  • Immunoassays for detecting a StAR polypeptide can be competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured.
  • "sandwich" assays will be used, for example, wherein anti-StAR antibodies are bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the StAR protein present in a test sample. The StAR thus immobilized is then bound by a labeling agent, such as a second anti-StAR antibody bearing a label.
  • the amount of StAR protein present in a sample is measured indirectly, e.g., by measuring the amount of added (exogenous) StAR displaced (or competed away) from an anti-StAR antibody by StAR protein present in a sample.
  • a known amount of labeled StAR polypeptide is added to a sample and the sample is then contacted with an anti-StAR antibody.
  • the amount of labeled StAR polypeptide bound to the antibody is inversely proportional to the concentration of StAR polypeptide present in the sample.
  • any of a number of labels can be used in any of the immunoassays of this invention, including fluorescent labels, radioisotope labels, or enzyme-based labels, wherein a detectable product of enzyme activity is detected (e.g., peroxidase, alkaline phosphatase, ⁇ - galactosidase, etc.).
  • Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science, 246:1275-1281 (1989); Ward et al, Nature, 341 :544-546 (1989)).
  • StAR peptides or a full length protein may be used to produce antibodies specifically reactive with StAR protein.
  • recombinant StAR protein can be expressed in eukarvotic or prokaryotic cells and purified using standard methods.
  • Recombinant protein is the prefened immunogen for the production of monoclonal or polyclonal antibodies.
  • a synthetic peptide derived from any StAR amino acid sequence can be conjugated to a canier protein and used as an immunogen.
  • Naturally occurring protein may also be used either in pure or impure form.
  • the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
  • mice e.g., BALB/C mice
  • rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the StAR protein.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow & Lane, supra).
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol, 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Ban Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science, 246:1275-1281 (1989). '
  • Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • an immunoassay for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non-StAR proteins or even other related proteins from other organisms, using a competitive binding immunoassay.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a I of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • StAR polypeptide levels are determined by virtue of the StAR protein activity or behavior in a biological sample.
  • Such protein activity or behavior can be easily measured using standard techniques.
  • the StAR protein regulates the translocation of cholesterol from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis.
  • StAR protein activity can be indirectly detected in a biological sample by detecting the amount of cholesterol translocation, or steroid production, that is present in the sample.
  • assays are well known to those of skill in the art.
  • the present invention provides numerous methods for diagnosing any of a number of types of cancer, e.g., determining whether or not a mammal has cancer, whether or not a biological sample contains cancerous cells, estimating the likelihood of a mammal developing cancer, and monitoring the efficacy of anti-cancer treatment in a mammal with cancer.
  • Such methods are based on the discovery that cancer cells have an elevated level of StAR polynucleotide (i.e., gene copy number and or mRNA) and polypeptide level. Accordingly, by determining whether or not a cell contains elevated levels of StAR polynucleotide or polypeptide, it is possible to determine whether or not the cell is cancerous.
  • cancerous cells can be determined indirectly, i.e., in certain embodiments a biological sample that does not itself contain cancerous cells, but which has been taken from an animal with cancerous cells elsewhere in its body, may contain elevated levels of StAR reflecting the presence of the cancerous cells.
  • the level and/or presence or StAR polynucleotide or polypeptide will be detected in a biological sample, thereby detecting the presence or absence of cancerous cells in the biological sample, or, in certain embodiments, in the mammal from which the biological sample was removed.
  • the biological sample will comprise a tissue sample from a tissue suspected of containing cancerous cells. For example, in a woman suspected of having breast cancer, breast tissue is removed. Often, such methods will be used in conjunction with additional diagnostic methods, e.g., detection of other cancer markers, mammography, etc.
  • a tissue sample known to contain cancerous cells e.g., from a tumor, will be detected for StAR levels to determine information about the cancer, e.g., the efficacy of certain treatments, the survival expectancy of the animal, etc.
  • the amount of StAR polynucleotide or polypeptide that will indicate the presence of a cancer will depend on numerous factors, including the type of cancer, the age, sex, medical history, etc., of the patient, the cell type, the assay format, etc.
  • a level of StAR in a biological sample will not be quantified or directly compared with a control sample, but will rather be detected relative to a "diagnostic presence" of StAR, wherein a "diagnostic presence” refers to an amount of StAR polynucleotide or polypeptide that indicates the presence of cancer, or indicates a likelihood of cancer, in the mammal from which the sample was taken.
  • a "diagnostic presence” will be detectable in a simple assay giving a positive or negative result, where a positive “detection” of a "diagnostic presence” of StAR polynucleotide or polypeptide indicates the presence of cancer in the mammal.
  • the StAR level need not be quantified for a "diagnostic presence" to be detected, merely any method of determining whether StAR is present at levels higher than in a normal, cancer-free cell, sample, or mamml.
  • a "diagnostic presence” does not refer to any absolute quantity of StAR, but rather on an amount that, depending on the biological sample, cell type, assay conditions, medical condition of the mammal, etc., is sufficient to distinguish the level in a cancerous, or pre-cancerous sample, from a normal, cancer-free sample.
  • Such methods can be practiced regardless of whether any StAR polynucleotide or polypeptide is normally present, or "expected" to be present, in a particular control sample.
  • StAR may not be expressed in certain cell types, resulting in a complete absence of StAR in a control biological sample consisting of such cell types.
  • a "diagnostic presence” refers to any detectable amount of StAR, using any assay. In other tissues, however, there may be a detectable level of StAR present in normal, cancer-free cells, and a "diagnostic presence" represents a level that is higher than the normal level, preferably representing a "statistically significant" increase over the normal level.
  • a "diagnostic presence" of StAR polynucleotide, polypeptide, and or protein activity in a biological sample will be at least about 1.5, 2, 5, 10, or more fold greater than a level expected in a sample taken from a normal, cancer-free mammal.
  • the present methods can be used to assess the efficacy of a course of treatment.
  • the efficacy of an anti-cancer treatment can be assessed by monitoring, over time, StAR levels.
  • StAR levels For example, a reduction in StAR polynucleotide or polypeptide levels in a biological sample taken from a mammal following a treatment, compared to a level in a sample taken from the mammal before, or earlier in, the treatment, indicates efficacious treatment.
  • the level of StAR can be used to determine the prognosis of a mammal with cancer. For example, if cancer is detected using a technique other than by detecting StAR, e.g., tissue biopsy, then the presence or absence of StAR can be used to determine the prognosis for the mammal, i.e., an elevated level of StAR will indicate a reduced survival expectancy in the mammal compared to in a mammal with cancer but with a normal level of StAR.
  • survival expectancy refers to a prediction regarding the severity, duration, or progress of a disease, condition, or any symptom thereof.
  • an increased level, a diagnostic presence, or a quantified level, of StAR is statistically correlated with the observed progress of a disease, condition, or symptom in a large number of mammals, thereby providing a database wherefrom a statistically-based prognosis can be made in view of any detected level or presence of StAR.
  • a detection of a level of StAR that is, e.g., 2 fold higher than a control level may indicate, e.g., a 10% reduced survival expectancy in the human compared to in a similar human with a normal level of StAR, based on a previous study of the level of StAR in a large number of similar patients whose disease progression was observed and recorded.
  • the present methods can be used to determine the optimal course of treatment in a mammal with cancer.
  • the presence of an elevated level of StAR can indicate a reduced survival expectancy of a mammal with cancer, thereby indicating a more aggressive treatment for the mammal.
  • a conelation can be readily established between levels of StAR, or the presence or absence of a diagnostic presence of StAR, and the relative efficacy of one or another anti-cancer agent.
  • analyses can be performed, e.g., retrospectively, i.e., by detecting StAR levels in samples taken previously from mammals that have subsequently undergone one or more types of anti-cancer therapy, and conelating the StAR levels with the known efficacy of the treatment.
  • levels of S tAR polynucleotides or polypeptides in tumor cells of a mammal are used to guide the selection of an anti-cancer treatment based on the effects of the treatment on steroid hormone levels or activity.
  • StAR protein levels, as detected by anti- StAR antibodies, in breast tumors can be used to predict the response of a patient to tamoxifen or any other treatment that affects estrogen metabolism or activity, such as aromatase inhibitors or estrogen antagonists/agonists.
  • StAR protein levels, as detected by anti-StAR antibodies, in prostate tumors can be used to predict the response of a patient to a treatment that affects testosterone metabolism or activity.
  • a detection of an elevated or diagnostic level of StAR indicates the beneficial use of a treatment that inhibits the activity of a steroid hormone.
  • an increased ⁇ r diagnostic level of StAR indicates that the use of an agent that inhibits estrogen activity, such as tamoxifen or other estrogen antagonists, an agent that inhibits estrogen production, such as an aromatase inhibitor (e.g., letrozole or vorozole), or any other treatment that affects estrogen activity or levels, such as the surgical removal of the ovaries, would provide an efficacious treatment for the tumor.
  • an agent that inhibits estrogen activity such as tamoxifen or other estrogen antagonists
  • an agent that inhibits estrogen production such as an aromatase inhibitor (e.g., letrozole or vorozole)
  • any other treatment that affects estrogen activity or levels such as the surgical removal of the ovaries
  • an increased or diagnostic level of StAR indicates that the use of an antiandrogen such as flutamide, bicalutamide, or nilutamide, an agent that reduces testosterone levels such as a luteinizing hormone-releasing hormone (LHRH) analog, e.g., leuprolide or goserelin, other hormonal drugs such as megestrol acetate, medroxyprogesterone, or ketoconazole, or any other treatment that affects testosterone activity or levels, such as orchiectomy, would provide an effective treatment for the tumor.
  • an antiandrogen such as flutamide, bicalutamide, or nilutamide
  • an agent that reduces testosterone levels such as a luteinizing hormone-releasing hormone (LHRH) analog, e.g., leuprolide or goserelin
  • other hormonal drugs such as megestrol acetate, medroxyprogesterone, or ketoconazole, or any other treatment that affects testosterone activity or levels, such as orchi
  • the present invention provides numerous methods for treating a mammal with cancer.
  • methods are provided for treating a cancer by inhibiting the growth, proliferation, or steroid hormone production of cells within the mammal, e.g., cancer cells.
  • the methods are directed at reducing the level of StAR polypeptides, polynucleotides, or protein activity in a cancerous cell.
  • StAR polypeptides e.g., polypeptides, polynucleotides, or protein activity in a cancerous cell.
  • more than one of the methods described infra can be performed on a given animal, and may also be administered in conjunction with one or more traditional, well known anti-cancer therapies, e.g., chemotherapy, radiation therapy, surgery, hormone therapy, immunotherapy, etc.
  • a "method of treating cancer” refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in an animal, or to alleviate the symptoms of a cancer.
  • a method of treating cancer does not necessarily mean that the cancer cells will, in fact, be eliminated, that the number of cells will, in fact, be reduced, or that the symptoms of a cancer will, in fact, be alleviated.
  • a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an animal, is nevertheless deemed an overall beneficial course of action.
  • the present invention provides methods for treating cancer by detecting the level and/or a diagnostic presence of StAR polynucleotide or polypeptide in a biological sample, and, when a diagnostic presence or increased level is detected, applying one or more anti-cancer therapies, including, but not limited to, chemotherapy, radiation therapy, surgery, immunotherapy, hormone therapy, and gene therapy.
  • chemotherapeutic agents arrest the growth of or kill cells that are dividing or growing, such as cancer cells.
  • chemotherapeutic agents include, but are not limited to, genistein, taxol, busulfan, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), melphalan, carmustine, lomustine, 5-fluorouracil, methotrexate, gemcitabine, cytarabine (Ara-C), fludarabine, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), idarubicin, paclitaxel, docetaxel, etoposide, vinblastine, vincristine, vinorelbine, L-asparaginase, amsacrine, tretinoin, prednisone and dexamethasone.
  • chemotherapeutic agents include, but are not limited to, genistein, taxol, busulfan,
  • Radioactivity is administered to a mammal with cancer.
  • Radiation kills or inhibits the growth of dividing cells, such as cancer cells.
  • the administration of radiation may be from an external source (e.g., a gamma source, a proton source, a molecular beam source, etc.), or may be through an implantable radioactive material, or a radioactive molecule such as an antibody.
  • a tissue found to be cancerous using the present methods will be removed using surgery, i.e., the direct removal or ablation of cells, e.g., cancer cells, from a mammal.
  • the cancer cells will be in the form of a tumor (e.g., a mammary tumor), which is removed from the mammal.
  • the surgical methods may involve removal of healthy as well as cancerous tissue.
  • Hormone therapy can also be used to treat cancers, e.g., breast cancer.
  • compounds can be administered to a patient that counteract or inhibit hormones, such as estrogen or androgen, that have a mitogenic effect on cells and which often act to increase the cancerous properties of cancer cells in vivo.
  • Hormone therapy can also include methods of reducing or eliminating the production of hormones in an animal, e.g., the surgical removal of ovaries in an animal to prevent estrogen production.
  • immunotherapy will be used to treat a cancer following a diagnosis, based on detection of StAR, i.e., methods of enhancing the ability of an animal's immune system to destroy cancer cells within the animal.
  • StAR i.e., methods of enhancing the ability of an animal's immune system to destroy cancer cells within the animal.
  • This can involve the treatment with polyclonal or monoclonal antibodies (e.g., Herceptin) that bind to particular molecules located on, produced by, or indicative of, tumor cells.
  • polyclonal or monoclonal antibodies e.g., Herceptin
  • Immunotherapeutic methods are well know to those of skill in the art (see, e.g., Vasta et al. (1992) Ann. Rev. Biochem., 61 :331- 354, Brinkman and Pastan (1994) Biochimica Biphysica Ada, 1198:27-45, etc.).
  • gene therapy will be used to treat a cancer diagnosed based on a detection of StAR.
  • a nucleic acid is introduced into cells, e.g., cancer cells, to provide treatment for the cancer.
  • tumor suppressor genes that are often missing or mutated in a cancer cell, e.g., p53, RB, p21, pl6, and others, can be replaced or overexpressed by introducing a nucleic acid encoding a functional gene into the cells.
  • genes whose overexpression or increased activity contributes to cancer can be inhibited by any of a number of methods, including, but not limited to, antisense, ribozymes, and polynucleotides encoding dominant negative forms or other inhibiting polypeptides.
  • Such nucleic acids can be delivered using any of a variety of methods, e.g., liposomal formulations, viral vectors, naked DNA injection, etc., and can be performed in vivo or ex vivo.
  • this invention provides methods of treating a cancer by reducing StAR levels in a cell.
  • such methods are used to reduce an elevated level of StAR, e.g., an elevated level in a cancerous cell, and can be performed in any of a number of ways, e.g., lowering the copy number of StAR genes or decreasing the level of StAR mRNA, protein, or protein activity in a cell.
  • the level of StAR activity is lowered to a level typical of a normal, cancer-free cell, but the level may be reduced to any level that is sufficient to decrease the proliferation or steroid production of the cell, including to levels above or below those typical of normal cells.
  • inhibitors of StAR where an "inhibitor of StAR” is a molecule that acts to reduce StAR polynucleotide levels, StAR polypeptide levels and or StAR protein activity.
  • inhibitors include, but are not limited to, antisense polynucleotides, ribozymes, antibodies, dominant negative StAR forms, and small molecule inhibitors of StAR.
  • StAR levels will be reduced so as to reduce the growth or proliferation of a cancer cell with elevated StAR levels.
  • the proliferation of a cell refers to the rate at which the cell or population of cells divides, or to the extent to which the cell or population of cells divides or increases in number. Proliferation can reflect any of a number of factors, including the rate of cell growth and division and the rate of cell death.
  • StAR levels will be reduced so as to reduce the amount of steroid hormone production by the cell. StAR regulates the rate limiting translocation of cholesterol across the mitochondrial membranes, thereby controlling the rate of steroidogenesis in cells.
  • the amplification and/or overexpression of the StAR gene in cancer cells causes an increased level of steroid hormone, e.g., estrogen, production by the cells.
  • This increased steroid hormone appears to cause "autocrine" stimulation of cancer cell, e.g., breast cancer cell, growth and proliferation.
  • steroid hormone e.g., estrogen
  • any of the present compounds to affect StAR activity can be determined based on any of a number of factors, including, but not limited to, a level of StAR polynucleotide, e.g., mRNA or gDNA, the level of StAR polypeptide, the degree of binding of a compound to a StAR polynucleotide or polypeptide, StAR intracellular localization, or any functional properties of StAR protein, such as the ability of StAR activity to effect cholesterol translocation into the mitochondria and the resulting steroid hormone synthesis.
  • a level of StAR polynucleotide e.g., mRNA or gDNA
  • the level of StAR polypeptide e.g., the level of StAR polypeptide
  • StAR intracellular localization e.g., StAR intracellular localization
  • any functional properties of StAR protein such as the ability of StAR activity to effect cholesterol translocation into the mitochondria and the resulting steroid hormone synthesis.
  • StAR activity is downregulated, or entirely inhibited, by the use of antisense polynucleotide, i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g, StAR mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the StAR mRNA reduces the translation and or stability of the StAR mRNA.
  • antisense polynucleotide i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g, StAR mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the StAR mRNA reduces the translation and or stability of the StAR mRNA.
  • antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter- sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. All such analogs are comprehended by this invention so long as they function effectively to hybridize with StAR mRNA. '
  • antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
  • ribozymes can be used to target and inhibit transcription of StAR.
  • a ribozyme is an RNA molecule that catalytically cleaves other RNA molecules.
  • Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes (see, e.g., Castanotto et al. ( ⁇ 994) Adv. in Pharmacology, 25:289-317 for a general review of the properties of different ribozymes).
  • hairpin ribozymes are described, e.g., in Hampel et al. (1990) Nucl Acids Res., 18:299-304; Hampel et al (1990) European Patent Publication No. 0 360 257; U.S. Patent No. 5,254,678.
  • Methods of preparing are well known to those of skill in the art (see, e.g., Wong-Staal et al, WO 94/26877; Ojwang et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6340-6344; Yamada et al. (1994) Human Gene Therapy, 1 :39-45; Leavitt et al.
  • StAR activity can also be decreased by the addition of an inhibitor of the
  • StAR polypeptide This can be accomplished in any of a number of ways, including by providing a dominant negative StAR polypeptide, e.g., a form of StAR that itself has no activity and which, when present in the same cell as a functional StAR, reduces or eliminates the StAR activity of the functional StAR. Design of dominant negative forms is well known to those of skill and is described, e.g., in Herskowitz (1987) Nature, 329(6136):219-22. Also, inactive polypeptide variants (muteins) can be used, e.g., by screening for the ability to inhibit StAR activity. Methods of making muteins are well known to those of skill (see, e.g., U.S. Patent Nos.
  • any small molecule e.g., any peptide, amino acid, nucleotide, lipid, carbohydrate, or any other organic or inorganic molecule can be screened for the ability to bind to or inhibit StAR activity, as described below.
  • this invention provides methods of screening for agents that modulate and preferably dowrtregulate StAR protein activity.
  • Prefened "screening" methods of this invention involve (i) contacting an StAR-expressing cell (e.g., a cell capable of expressing StAR) with a test agent; and (ii) detecting the level of StAR activity (e.g., as described above), where a decreased level of StAR activity as compared to the level of StAR activity in a cell not contacted with the test agent indicates that the test agent inhibits or downregulates StAR.
  • any agent can be tested in such an assay.
  • agents include, but are not limited to, natural or synthetic nucleic acids, natural or synthetic polypeptides, natural or synthetic lipids, natural or synthetic small organic molecules, and the like.
  • test agents are provided as members of a combinatorial library.
  • a collection of small molecule inhibitors are tested for StAR inhibiting ability.
  • a "small molecule inhibitor" of StAR is any molecule, e.g., a carbohydrate, nucleotide, amino acid, oligonucleotide, oligopeptide, lipid, inorganic compound, etc. that inhibits StAR protein activity.
  • Such molecules can inhibit StAR protein activity by any of a number of mechanisms, e.g., by binding to an StAR protein and competitively inhibiting its interaction with mitochondria, with cholesterol, or with other proteins.
  • such "small molecule inhibitors" are smaller than about 10 kD. More preferably, such inhibitors are smaller than about 5, 2, or 1 kD or less.
  • test agents can be screened based on any of a number of factors, including, but not limited to, a level of StAR polynucleotide, e.g., mRNA or gDNA, the level of StAR polypeptide, the degree of binding of a compound to a StAR polynucleotide or polypeptide, StAR intracellular localization, or any functional properties of StAR protein, such as the ability of StAR activity to effect cholesterol translocation into the mitochondria and resulting steroid hormone synthesis.
  • a level of StAR polynucleotide e.g., mRNA or gDNA
  • the level of StAR polypeptide the degree of binding of a compound to a StAR polynucleotide or polypeptide
  • StAR intracellular localization or any functional properties of StAR protein, such as the ability of StAR activity to effect cholesterol translocation into the mitochondria and resulting steroid hormone synthesis.
  • Such direct and indirect measures of StAR activity in vivo can readily be used to detect
  • combinatorial libraries of potential StAR modulators will be screened for an ability to bind to a StAR polypeptide or to modulate StAR activity.
  • new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., StAR inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • a chemical compound called a "lead compound”
  • some desirable property or activity e.g., StAR inhibiting activity
  • HTS high throughput screening
  • high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al (1994) J. Med. Chem., 37(9):1233-1251). Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354:84-88), peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No.
  • a number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
  • any of the assays to identify compounds capable of modulating StAR levels described herein are amenable to high throughput screening. Prefened assays thus detect enhancement or inhibition of StAR gene transcription, inhibition or enhancement of StAR polypeptide expression, and inhibition or enhancement of StAR polypeptide activity.
  • an StAR inhibiting compounds i.e., a compound that reduces levels of StAR mRNA, polypeptide and/or protein activity
  • Such compounds can be administered by a variety of methods including, but not limited to, parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.
  • the StAR modulators e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc.
  • StAR modulators when administered orally, must be protected from digestion. This is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome. Means of protecting agents from digestion are well known in the art.
  • compositions for administration will commonly comprise a StAR modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous earner.
  • a pharmaceutically acceptable carrier preferably an aqueous earner.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980).
  • compositions containing modulators of StAR can be administered for therapeutic or prophylactic treatments.
  • compositions are administered to a patient suffering from a disease (e.g., a cancer) in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient.
  • An amount of a StAR modulator that is capable of preventing or slowing the development of cancer in a mammal is refened to as a "prophylactically effective dose.”
  • the particular dose required for a prophylactic treatment will depend upon the medical condition and history of the mammal, the particular cancer being prevented, as well as other factors such as age, weight, gender, etc.
  • Such prophylactic treatments may be used, e.g., in a mammal who has previously had cancer to prevent a recurrence of the cancer, or in a mammal who is suspected of having a significant likelihood of developing cancer.
  • any of the present StAR-inhibiting compounds can be administered alone or in combination with additional StAR-inhibiting compounds or with any other therapeutic agent, e.g., other anti-cancer agents or treatments.
  • one or more nucleic acids e.g., StAR polynucleotides, such as antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo.
  • the present invention provides methods, reagents, vectors, and cells useful for expression of StAR and other polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.
  • the particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific. Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger), F.M.
  • nucleic acids encoding StAR polypeptides, or inhibitors thereof will be inserted into vectors using standard molecular biological techniques.
  • Vectors may be used at multiple stages of the practice of the invention, e.g., for subcloning nucleic acids encoding StAR polypeptides or StAR inhibitors, e.g., StAR ribozymes or antisense sequences, or for subcloning additional elements used to control protein or mRNA expression, vector selectability, etc.
  • Vectors may also be used to maintain or amplify the nucleic acids, for example, by inserting the vector into prokaryotic or eukaryotic cells and growing the cells in culture.
  • vectors may be used to introduce and express StAR nucleic acids, or StAR-inhibiting nucleic acids, e.g., StAR ribozymes or antisense sequences, into cells for therapeutic or experimental purposes.
  • a variety of commercially or commonly available vectors and vector nucleic acids can be converted into a vector of the invention by cloning a polynucleotide of this invention into the commercially or commonly available vector.
  • a variety of common vectors suitable for this purpose are well known in the art.
  • common vectors include pBR322-derived vectors such as pBLUESCRIPTTM, and bacteriophage derived vectors.
  • vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2.
  • Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retro viruses).
  • lytic virus vectors e.g., vaccinia virus, adeno virus, and baculovirus
  • episomal virus vectors e.g., bovine papillomavirus
  • retroviral vectors e.g., murine retro viruses.
  • a nucleic acid subsequence encoding an StAR polypeptide is placed under the control of a promoter.
  • a nucleic acid is "operably linked" to a promoter when it is placed into a functional relationship with the promoter.
  • a promoter or enhancer is operably linked to a coding sequence if it increases or otherwise regulates the transcription of the coding sequence.
  • a "recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences.
  • Expression cassettes include promoters and, optionally, introns, polyadenylation signals, and transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.
  • an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.
  • promoters are well known, and can be used in the vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. For E. coli, example control sequences include the T7, tip, or lambda promoters, a ribosome binding site and preferably a transcription termination signal.
  • control sequences typically include a promoter which optionally includes an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, a retrovirus (e.g., an LTR based promoter) etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • epithelial cancers will be diagnosed and/or treated, e.g., breast cancer.
  • Other epithelial cancers include, e.g., ovarian, colorectal, kidney, stomach, bladder, and lung cancers.
  • a cancer at any stage of progression can be detected, such as primary, metastatic, and recurrent cancers.
  • Information regarding numerous types of cancer can be found, e.g., from the American Cancer Society (www3.cancer.org), or from, e.g., Wilson et al. (1991) Hanison's Principles of Internal Medicine, 12 th Edition, McGraw- Hill, Inc. VII. Kits for Use in Diagnostic and/or Prognostic Applications.
  • kits are also provided by the invention.
  • such kits may include any or all of the following: assay reagents, buffers, StAR specific nucleic acids or antibodies, hybridization probes and/or primers, antisense polynucleotides, ribozymes, dominant negative StAR polypeptides or polynucleotides, small molecules inhibitors of StAR, etc.
  • a therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
  • Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • Such media may include addresses to internet sites that provide such instructional materials.
  • Transgenic and chimeric non-human mammals and methods for generating them are described below.
  • the mammals are useful, inter alia, for testing the function of StAR in vivo, to generate models for the study of diseases and conditions associated with StAR (e.g., cancer), and for the development of potential treatments for diseases and conditions associated with StAR (e.g., cancer).
  • Transgenic and chimeric non-human mammals are generated that contain cells that lack at least one functional endogenous allele for StAR.
  • a "chimeric animal” includes some cells that lack the functional StAR gene of interest and other cells that do not have the inactivated gene.
  • a "transgenic animal,” in contrast, is made up of cells that have all incorporated the specific modification which renders the StAR gene inactive or otherwise altered. While a transgenic animal is typically always capable of transmitting the mutant StAR gene to its progeny, the ability of a chimeric animal to transmit the mutation depends upon whether the inactivated gene is present in the animal's germ cells.
  • the modifications that inactivate or otherwise alter the StAR gene can include, for example, insertions, deletions, or substitutions of one or more nucleotides. The modifications can interfere with transcription of the gene itself, with translation and/or stability of the resulting mRNA, or can cause the gene to encode an inactive or otherwise altered StAR polypeptide.
  • the methods of the present invention are useful for producing transgenic and chimeric animals of most vertebrate species.
  • species include, but are not limited to, nonhuman mammals, including rodents such as mice and rats, rabbits, ovines such as sheep and goats, porcines such as pigs, and bovines such as cattle and buffalo.
  • rodents such as mice and rats
  • rabbits ovines
  • porcines such as pigs
  • bovines such as cattle and buffalo.
  • Methods of obtaining transgenic animals are described in, for example, Puhler, A., Ed., Genetic Engineering of Animals, VCH Publ., 1993; Murphy and Carter, Eds., Transgenesis Techniques : Principles and Protocols (Methods in Molecular Biology, Vol. 18), 1993; and Pinkert, CA, Ed., Transgenic Animal Technology : A Laboratory Handbook, Academic Press, 1994.
  • transgenic mice will be produced as described in Thomas et al, (1999) Immunol, 163:978-84; Kanakaraj et al. (1998) J. Exp. Med., 187:2073-9; or Yeh et al., (1997) Immunity, 7:715-725.
  • a modified StAR gene is introduced, e.g., by homologous recombination, into embryonic stem cells (ES), which are obtained from preimplantation embryos and cultured in vitro.
  • ES embryonic stem cells
  • Hooper, ML Embryonal Stem Cells : Introducing Planned Changes into the Animal Germline (Modern Genetics, v, 1), IntT. Pub. Distrib., Inc., 1993; Bradley et al. (1984) Nature, 309, 255-258.
  • the transformed ES cell is combined with a blastocyst from a non-human animal, e.g., a mouse.
  • ES cells colonize the embryo and in some embryos form the germ line of the resulting chimeric animal. See, Jaenisch (1988) Science, 240:1468-1474.
  • ES cells or somatic cells that can reconstitute an organism can be used as a source of nuclei for transplantation into an enucleated fertilized oocyte giving rise to a transgenic mammal. See, e.g., Wilmut et al. (1997) N ⁇ twre 385:810-813.
  • Other methods for obtaining a transgenic or chimeric animal having a mutant StAR gene in its genome is to contact fertilized oocytes with a vector that includes a polynucleotide that encodes a modified, e.g., inactive, StAR polypeptide.
  • a vector that includes a polynucleotide that encodes a modified, e.g., inactive, StAR polypeptide.
  • fertilization is typically performed in vivo and fertilized ova are surgically removed.
  • other animals, particularly bovines it is preferably to remove ova from live or slaughterhouse animals and fertilize the ova in vitro. See, DeBoer et al, WO 91/08216. In vitro fertilization permits the modifications to be introduced into substantially synchronous cells.
  • Fertilized oocytes are typically cultured in vitro until a pre-implantation embryo is obtained containing about 16-150 cells.
  • the 16-32 cell stage of an embryo is described as a morula, whereas pre-implantation embryos containing more than 32 cells are termed blastocysts. These embryos show the development of a blastocoel cavity, typically at the 64 cell stage.
  • the presence of a desired StAR mutation in the cells of the embryo can be detected by methods known to those of skill in the art, e.g., Southern blotting, PCR, DNA sequencing, or other standard methods. Methods for culturing fertilized oocytes to the pre- implantation stage are described, e.g., by Gordon et al.
  • Pre-implantation embryos may also be stored frozen for a period pending implantation.
  • Pre-implantation embryos are transfened to an appropriate female resulting in the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is integrated.
  • Chimeric mammals can be bred to form true germline transgenic animals.
  • Chimeric mice and germline transgenic mice can also be ordered from commercial sources (e.g., Deltagen, San Carlos, CA).
  • recombinase systems which can be employed to delete all or a portion of a locus of interest.
  • recombinase systems include, the cre/lox system of bacteriophage PI (see, e.g., Gu et al. (1994) Science, 265:103-106; Terry et al. (1997) Transgenic Res., 6:349- 356) and the FLP/FRT site specific integration system (see, e.g., Dymecki (1996) Proc. Natl. Acad. Sci. USA, 93:6191-6196).
  • sites recognized by the particular recombinase are typically introduced into the genome at a position flanking the portion of the gene that is to be deleted.
  • Introduction of the recombinase into the cells then catalyzes recombination which deletes from the genome the polynucleotide sequence that is flanked by the recombination sites.
  • the presence of any mutation in a StAR gene in a cell or animal can be detected using any method described herein, e.g., Southern blot, PCR, or D ⁇ A sequencing. See, e.g., Ausubel et al, supra.
  • RDA representational difference analysis
  • the cell line BT483 is not amplified for FGFRI, but is amplified for the RDA probe.
  • one tumor (89-249) that was analyzed by quantitative RT-PCR does not overexpress the FGFRI gene, indicating that FGFRI is not the oncogene responsible for amplification of the region in this tumor.
  • the physical map of this region reveals that FGFRI is approximately 0.6 Mb away from the RDA probe, and there are sequences between the RDA probe and FGFRI that are not amplified or less amplified than the RDA probe and FGFRI ( Figure 1).
  • the region amplified near the RDA probe contains a separate oncogene.
  • ASH2 significant overexpression of ASH2, StAR, CaSM, andSODD in amplified tumors
  • the smallest area of common overlap of amplification was determined to encompass four genes: ASH2, the human homolog of a Drosophila trithorax gene (Ikegawa et al, (1999) Cytogenet.
  • StAR which encodes the steroidogenic acute regulatory protein
  • CaSM which encodes a protein with Sm-like motifs that are characteristic of small nuclear ribonucleoprotein particle proteins (Schweinfest et al, (1997) Cancer Res., 57:2961-5)
  • SODD which was recently discovered as a protein that binds to the TNF-receptor's death domain and that inhibits TNF-induced apoptosis (Jiang et al, 1999 Science, 283:543-6; 283:1852).
  • Relative expression level is the ratio of the value determined for the test gene relative to the value determined for the reference gene ⁇ -actin, after normalization to the ratio determined for normal human mammary gland (Clontech, Palo Alto, CA).
  • Relative expression level is the ratio of the value determined for the test gene relative to the value determined for the reference gene ⁇ -actin, after normalization to the ratio determined for normal human mammary gland (Clontech, Palo Alto, CA)
  • the overexpression of StAR in human tumors is strongly associated with estrogen receptor and progesterone receptor status. Tumors that overexpress StAR are almost exclusively ER or PR positive, whereas tumors that express normal levels of StAR are predominantly ER and PR negative (Table 3). Table 3.
  • the expression Of StAR in human breast tumors is associated with steroid receptor status
  • StAR is the cancer gene driving amplification of the 8pl 1 region, and that its overexpression upregulates estrogen biosynthesis causing "autocrine" stimulation of breast cancer cell growth.

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Abstract

La présente invention concerne des méthodes, des réactifs et des kits destinés au diagnostic et au traitement du cancer chez les mammifères, et notamment chez l'homme. L'invention repose sur l'étonnante découverte que le gène StAR est surexprimé et/ou amplifié dans plusieurs types de cellules cancéreuses. Ainsi, on peut utiliser les procédés de l'invention dans la détection du cancer ou d'une propension à développer le cancer, dans le suivi de l'efficacité d'un traitement anticancéreux et dans le traitement des cancers, notamment par l'inhibition de l'expression et/ou de l'activité du gène StAR dans une cellule cancéreuse.
PCT/US2000/029556 1999-10-26 2000-10-26 DIAGNOSTIC ET TRAITEMENT DU CANCER UTILISANT DES POLYPEPTIDES ET DES POLYNUCLEOTIDES StAR WO2001031342A2 (fr)

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AU14394/01A AU1439401A (en) 1999-10-26 2000-10-26 Diagnosis and treatment of cancer using star polypeptides and polynucleotides

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US16168799P 1999-10-26 1999-10-26
US60/161,687 1999-10-26
US16755699P 1999-11-23 1999-11-23
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US69673300A 2000-10-25 2000-10-25
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10251028A1 (de) * 2002-11-01 2004-05-19 Jenapharm Gmbh & Co. Kg Verwendung von Progesteron oder einem Progesteronrezeptor-Agonisten zur Hemmung der Steroidsynthese
WO2018195273A1 (fr) * 2017-04-19 2018-10-25 The Corporation Of Mercer University Protéine sam-1, composition et méthodes d'utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066728A1 (fr) * 1999-05-03 2000-11-09 Compugen Ltd. Homologues de star

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066728A1 (fr) * 1999-05-03 2000-11-09 Compugen Ltd. Homologues de star

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU J ET AL: "Expression of the steroidogenic acute regulatory protein mRNA in adrenal tumors and cultured adrenal cells." JOURNAL OF ENDOCRINOLOGY, vol. 150, no. 1, 1996, pages 43-50, XP001003088 ISSN: 0022-0795 *
MOOG-LUTZ CHRISTEL ET AL: "MLN64 exhibits homology with the steroidogenic acute regulatory protein (StAR) and is over-expressed in human breast carcinomas." INTERNATIONAL JOURNAL OF CANCER, vol. 71, no. 2, 1997, pages 183-191, XP001004431 ISSN: 0020-7136 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10251028A1 (de) * 2002-11-01 2004-05-19 Jenapharm Gmbh & Co. Kg Verwendung von Progesteron oder einem Progesteronrezeptor-Agonisten zur Hemmung der Steroidsynthese
WO2018195273A1 (fr) * 2017-04-19 2018-10-25 The Corporation Of Mercer University Protéine sam-1, composition et méthodes d'utilisation

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AU1439401A (en) 2001-05-08

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