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WO2018195273A1 - Protéine sam-1, composition et méthodes d'utilisation - Google Patents

Protéine sam-1, composition et méthodes d'utilisation Download PDF

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Publication number
WO2018195273A1
WO2018195273A1 PCT/US2018/028295 US2018028295W WO2018195273A1 WO 2018195273 A1 WO2018195273 A1 WO 2018195273A1 US 2018028295 W US2018028295 W US 2018028295W WO 2018195273 A1 WO2018195273 A1 WO 2018195273A1
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WIPO (PCT)
Prior art keywords
sam
protein
nucleic acid
biological sample
seq
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PCT/US2018/028295
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English (en)
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Himangshu S. Bose
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The Corporation Of Mercer University
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Publication of WO2018195273A1 publication Critical patent/WO2018195273A1/fr

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    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the present invention relates to a protein marker associated with healthy breast tissue.
  • the invention provides a protein for monitoring, diagnosing, screening, and prevention of breast cancer and hormonal disorders. More precisely, the invention relates a protein marker specifically down-regulated in breast cancer tissue.
  • estradiol i.e., estradiol
  • Estrogens are instrumental in breast development, fat distribution in the hips, legs, and breasts and the development of reproductive organs. Estrogens bind to estrogen receptor-a, generating a powerful stimulus for breast gland cell proliferation.
  • progesterone is essential for normal breast development during puberty and in preparation for lactation and breastfeeding.
  • the actions of progesterone are primarily mediated by its high-affinity receptors, which include the classical progesterone receptor (PR)-A and -B isoforms, located in various tissues, including the brain, where progesterone controls reproductive behavior, as well as the breast and reproductive organs.
  • PR progesterone receptor
  • Estradiol (abbreviated as E 2 ), or 17 -estradiol, is the primary steroid sex hormone in females. Estradiol, like other steroids, is derived from cholesterol. It regulates the estrous and menstrual female reproductive cycles and develops and maintains female reproductive tissues. Estradiol is biosynthesized by aromatase from ⁇ 4- androstenedione generating estrone, which upon further action of a dehydrogenase yields 17 -estradiol. (Miller, 2011) After side chain cleavage and using the ⁇ 5 or the ⁇ 4- pathway, A4-androstenedione is the key intermediary.
  • estradiol in women is produced by the granulosa cells of the ovaries by the aromatization of A4-androstenedione (produced in the theca folliculi cells) to estrone, followed by conversion of estrone to estradiol by 17 ⁇ - hydroxysteroid dehydrogenase. Part of the circulating estradiol is also produced by fat cells and this continues after menopause. During pregnancy, estriol becomes the predominant circulating estrogen and it is the only time when estriol is present in the body, whereas after menopause, estrone is the predominant form of estrogen.
  • Estradiol acts through estrogen receptors.
  • estradiol enters the cytoplasm, causes dissociation of heat-shock protein (HSP) and binds to HSP forming homodimers. The homodimers bind to the estrogen response element domains on the nucleus, allowing for gene transcription to take place.
  • HSP heat-shock protein
  • E 2 17 -estradiol
  • E2 17 -estradiol
  • E2 plasma levels decrease by 90%.
  • the concentrations of E2 in breast cancer tissues do not differ between these two groups of women, an indication that its uptake from the circulation might not contribute significantly to the total content of this hormone in breast tumors, but rather that possibly de novo biosynthesis, i.e., peripheral aromatization of ovarian and adrenal androgens or directly in the breast tissues possibly act to supplement as necessary for the E2 synthesis.
  • the present invention provides SAM-1 protein markers for diagnosing, screening, preventing and/or treating breast cancer and hormonal disorders, and compositions for monitoring changes to these protein markers.
  • the invention provides methods for screening drug candidates using these protein markers.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample comprising the steps of (i) contacting the biological sample with one or more probes, and (ii) detecting binding between the probes and the biological sample to determine the presence of SAM-1 protein.
  • the presence of SAM-1 protein in a human subject as determined directly by protein detection or indirectly by coding nucleic acid detection, can be quantified and compared to levels of SAM-1 protein in healthy tissue, to identify a disorder such as breast cancer, Polycystic Ovarian Syndrome, disorders associated with increased levels of estrogen, or other disorders of increased aromatase activity, in order to therapeutically treat the subject.
  • Therapeutically treating the subject can comprise administration of a composition to increase the level of SAM-1 in targeted tissue of the subject, or treatment through of a wide variety of other disease specific treatments now known (e.g., surgery, chemotherapy, radiation) or developed in the future.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample, wherein the presence of SAM-1 protein is determined by detecting the presence of a nucleic acid of SEQ ID NO: 1 encoding for SAM- 1 protein in a biological sample.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample, wherein the presence of a nucleic acid encoding for SAM- 1 protein in a biological sample comprising the steps of (i) contacting the biological sample with one or more labeled oligonucleotides that specifically bind to the nucleic acid encoding for SAM-1 protein, and (ii) detecting binding between the labeled oligunucleotides and the nucleic acid encoding for SAM-1 protein to determine the presence of the nucleic acid encoding for SAM-1 protein.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample comprising the steps of (i) contacting the biological sample with one or more labeled oligonucleotides that specifically bind to the nucleic acid encoding for SAM-1 protein, (ii) amplifying the nucleic acid between the bound labeled oligonucleotides, and (iii) detecting binding between the labeled oligunucleotides and the nucleic acid encoding for SAM-1 protein to determine the presence of the nucleic acid encoding for SAM- 1 protein.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample, wherein the presence of SAM-1 protein is determined by detecting the presence of RNA encoding for SAM-1 protein in a biological sample.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample comprising the steps of (i) contacting the sample with one or more labeled SAM- 1 antibodies that specifically bind to SAM-1 protein, and (ii) detecting binding between the labeled SAM-1 antibodies and SAM-1 protein to determine the presence SAM-1 protein.
  • the invention provides a method for determining the presence of SAM-1 protein in a human biological sample, wherein the detection of SAM-1 protein is used for early detection of breast cancer.
  • the invention provides a method for determining whether SAM-1 protein is present in a human biological sample at a level lower than in a healthy biological sample, and if the level is lower treating the human for breast cancer.
  • the invention provides a composition for detecting a nucleotide sequence encoding for SAM-1 protein comprising a pair of nucleic acid primers selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, and 14.
  • the invention provides a composition for detecting a nucleotide sequence encoding for SAM-1 protein wherein the primers are labeled with a detectable moiety.
  • the invention provides an isolated SAM-1 protein having an amino acid sequence as shown in SEQ ID NO: 2.
  • the invention provides a nucleic acid expression vector comprising a nucleic acid sequence encoding SAM-1 protein and a nucleic acid sequence encoding a promoter.
  • the invention provides a pharmaceutical composition comprising a SAM-1 protein or a functional fragment thereof and a pharmaceutically acceptable excipient.
  • the invention provides a method of preventing or treating breast cancer comprising administering to a human subject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • the invention provides a method of preventing or treating Polycystic Ovarian Syndrome comprising administering to a human subject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating or preventing hormonal disorders associated with increased levels of estrogens comprising administering to a human subject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating or preventing hormonal disorders associated with increased aromatase activity comprising administering to a humansubject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • FIGS. 1A-1I Aromatase expression in breast cell lysates.
  • FIG. 1A depicts a schematic presentation of the steroid synthesis pathway showing the specific catalytic activity of aromatase.
  • ER endoplasmic reticulum
  • aromatase catalyzes the conversion of androstenedione to estrone and testosterone to estradiol.
  • Androstenedione, testosterone, estrone and estradiol are inter-catalyzed by 17 -hydroxysteroid dehydrogenase (17 -HSD1).
  • FIG. IB depicts the metabolic activity with the microsomal membrane.
  • FIG. 1C depicts a quantitative analysis of the amount of estradiol synthesized.
  • FIGS. 1D-1F depict PCR amplification of the 3' (FIG. ID), 5 ' (FIG.
  • FIG. 1G depicts the open reading frame and the untranslated 3' region of the cDNA sequence.
  • FIG. 1H identifies the presence of SAM-1 (by staining) in patients with ER + /PR + /HER ⁇ ,ER /PR/ ⁇ HER + , ER + /PR7HER " , and ER7PR7HER " patient tissues and its comparison with the unaffected human breast tissue.
  • FIG. II depicts the determination of the presence of SAM- 1 in non-tumorigenic MCF12A and tumorigenic T47D cells by staining with SAM-1 antibody.
  • FIG. 2 Depicts the analysis of the SAM-1 protein sequence (SEQ ID NO:22), with the Blast search matching only with the first 42 N-terminal amino acids with the StAR protein.
  • FIGS. 3A-3M Depicts the localization of the SAM-1 protein in breast tissues.
  • FIGS. 3 A, 3B, and 3C depict different sections of the breast tissue stained with SAM-1 antibody in 1.0 mM scale, whereas a higher magnification is presented in FIG 3C.
  • Similar probing with aromatase antibody in FIGS. 3D, 3E, and 3F depicts the localization of the aromatase antibodies.
  • FIGS. 3G, 3H, and 31 depict different sections of the breast tissue stained with SAM-1 and aromatase antibodies showing co-localization of the aromatase and SAM-1 antibodies.
  • FIG. 3 J depicts the role of various protease inhibitors with the indicated protease inhibitors, including RVKR (SEQ ID NO:23) being incubated with tumorigenic T47D cells for 6 hours to determine the restoration of SAM-1 expression stained with antibody.
  • FIG. 3K depicts the localization of SAM-1 in different organelle fractions of the MCF12A cell (bottom panel) and unaffected breast tissue (top), where RER, MAM and mitochondria were isolated independently and then stained with SAM-1 antibody.
  • FIG. 3L depicts the localization of peroxisome in unaffected breast tissue stained with SAM-1 antibody, a higher magnification is presented in FIG. 3M.
  • FIGS. 4A-4F depict the localization of SAM-1 and aromatase in affected (tumorigenic tissues) and unaffected tissues.
  • FIGS. 4A and 4B depict the difference in organelle structure between the unaffected (FIG. 4 A) and affected breast tissue (FIG. 4B). In the unaffected breast tissue the mitochondria is well structured (FIG. 4A right) having OMM, IMM and christae but not the affected tissue mitochondria (FIG. 4B right).
  • FIG. 4C depict the localization of aromatase in affected tissue where it is localized in the ER, but not in the mitochondria (FIGS. 4C(2) and 4C(3)), where FIG. 4C(2) and 4C(3) are enlarged from FIG. 4C(1), and FIG.
  • FIG. 4C(4) depicts aromatase in the Golgi apparatus.
  • FIG. 4D depicts the localization of SAM-1 in affected tissue.
  • Fig. 4D(1) depicts SAM-1 localized in the ER, FIG. 4D(2) in the mitochondria and FIG. 4D(3) near the MAM section.
  • FIG. 4E depicts organelle fractionation of the affected breast tissue (top) and T47D cells stained with SAM-1 antibody.
  • FIG. 4F depicts colocalization of SAM-1 (55 nm) and aromatase (15nm) in tumorigenic tissue. An amplified version is on the right, which is mostly peroxisome.
  • FIGS. 5A-5E depict the effect of SAM-1 in the estradiol synthesis.
  • FIG. 5 A bottom depicts the effect of 30 and 60 pmol of SAM-1 siRNA in MCF12A cells determined by western staining with SAM-1 antibody.
  • FIG. 5 A top depicts the quantitative analysis of the estradiol synthesis from the bottom panel.
  • FIG. 5B depicts the metabolic activity measurement from testosterone to estradiol following incubation of the indicated siRNA.
  • FIG. 5C depicts the quantitative analysis of the amount of estradiol synthesized.
  • FIG. 5D depicts the quantitative measurement of aromatase expression in MCF12A and T47D cells.
  • FIG. 5E depicts the quantitative measurement of intensity of bands.
  • FIGS. 6A-6C depict the interaction between SAM-1 and aromatase through co-immunoprecipitation (CoIP) and independently staining with SAM-1 antibodies and aromatase antibodies.
  • Isolated Protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein” (1) is not associated with at least some of the proteins found in nature, (2) is free of other proteins from the same source, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • Conservative Substitutions are changes between amino acids of broadly similar molecular properties. Generally, conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine.
  • the hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • the hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.
  • Other families of amino acids include (i) serine and threonine, which are the aliphatic -hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
  • conservative substitution groups are aspartate-glutamate; asparagine - glutamine; valine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine; and lysine - arginine.
  • Naturally-Occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
  • Amino Acids As used herein "amino acids” refers to the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g.
  • D-amino acids of the twenty conventional amino acids, non-natural amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • unconventional amino acids include: 4 hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ - trimethyllysine, ⁇ - ⁇ -acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, a-N-methylarginine, and other similar amino acids and imino acids (e.g. , 4-hydroxyproline).
  • the left- hand direction is the amino terminal direction and the right-hand direction is the carboxy- terminal direction, in accordance with standard usage and convention.
  • the left-hand end of single-stranded polynucleotide sequences is the 5' end the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5' direction.
  • RNA transcripts The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences", sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • Polypeptide, Peptide, and Protein are used interchangeably herein to refer to polymers of amino acids.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • Substantial Identity As applied to polypeptides, peptides, and proteins the term "substantial identity” means that two peptide sequences, when optimally aligned share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.
  • variations in the amino acid sequences of polypeptides, peptides, and proteins are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. Certain percentages in between are included, such as 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. In particular, conservative amino acid replacements are contemplated.
  • amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs.
  • Polypeptide Fragment refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long.
  • SAM-1 analog refers to polypeptides which are comprised of a segment of at least 5 amino acids that has substantial identity to a portion of the deduced amino acid sequence of SAM-1 or a biologically active derivative thereof under suitable conditions.
  • polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence.
  • Analogs typically are at least 5 amino acids long, preferably at least 10 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.
  • Drug analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or “peptidomimetics”. Peptidomimetics are compounds based on, or derived from, peptides and proteins. Peptidomimetics can be obtained by for example structural modification of known peptide sequences using unnatural amino acids, conformational restraints, and isosteric replacement. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992)); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • Antibody refers to proteins that specifically bind to epitopes, including polyclonal antibody, monoclonal antibody, and recombinant antibody. Antibodies typically contain a conserved region and available region with heavy and light chains. The invention contemplates antibody fragments which functionally bind to the epitope of interest.
  • Antigen- antibody complex refers to the protein complex resulting from binding of specific antibody to SAM- 1 protein.
  • biological sample refers to cells, tissues, or bodily fluids that include but are not limited to breast tissue, urine, blood, plasma, and serum from a patient.
  • nucleic Acid or Polynucleotide The terms “nucleic acid” or “polynucleotide” or “oligonucleotide” as used herein refer to purine- and pyrimidine- containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, such as, for example, DNA-DNA, DNA-RNA and RNA-RNA hybrids. This also includes nucleic acids containing modified bases.
  • Complement A "complement" of a nucleic acid sequence as used herein refers to the antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
  • Primer is an isolated oligonucleotide between about 10 and about 50 nucleotides in length, preferably between about 12 and about 25 nucleotides in length and most preferably between about 12 and about 18 nucleotides in length, that forms a duplex with a single-stranded nucleic acid sequence of interest and allows polymerization of a complementary strand using, e.g., reverse transcriptase or DNA polymerase.
  • An "isolated" nucleic acid as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring or a reaction mixture if it is synthetic).
  • An isolated nucleic acid typically contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the components with which it was originally associated.
  • a nucleic acid sequence that is "derived from” a designated sequence refers to a sequence that corresponds to a region of the designated sequence. This encompasses sequences that are homologous or complementary to the sequence.
  • composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • therapeutically effective amount refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject's disease or condition, will have a desired therapeutic effect, e.g. , an amount which will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease or condition.
  • therapeutically effective amount or “effective amount” refers to an amount of a therapeutic agent that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the disease or condition or condition, or the progression of the disease or condition.
  • a therapeutically effective dose further refers to that amount of the therapeutic agent sufficient to result in amelioration of symptoms, e.g. , treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • Treating, Treatment, or Alleviation refers to therapeutic treatment wherein the object is to slow down (lessen) if not cure the targeted pathologic condition or disorder or prevent recurrence of the condition.
  • Preventative Treatment The term “preventative treatment” as used herein is meant to indicate a postponement of development of a disease, a symptom of a disease, or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurrence of a disease or symptom.
  • Subject or Patient refers to an animal, a non-human mammal or a human.
  • mammals include a pet, a farm animal, an economic animal, a sport animal and an experimental animal, such as a cat, a dog, a horse, a cow, an ox, a pig, a donkey, a sheep, a lamb, a goat, a mouse, a rabbit, a chicken, a duck, a goose, a primate, including a monkey and a chimpanzee.
  • Peptides may comprise a segment of the novel protein Savannah Anderson Mercer- 1 (SAM-1).
  • SAM-1 A nucleotide sequence encoding SAM-1 is set forth in SEQ ID NO: l and the SAM-1 protein encoded thereby is set forth as SEQ ID NO:2.
  • SAM-1 is a 207 amino acid sequence.
  • the invention provides functional fragements of SAM-1 protein, which may be polypeptides comprising about 5 to about 200 amino acids of the SAM-1 protein.
  • a SAM-1 peptide may comprise from about 10 to about 100, or from about 20 or 50 amino acids or from about 20 to about 30 or 35 amino acids of the SAM-1 protein.
  • a peptide may comprise an amino acid sequence identical to the SAM-1 protein sequence from about amino acid 1 to about amino acids 35, 30, 25, 20, 15, or 10 of the SAM-1 protein, such as a protein having SEQ ID NOs:3 or 4.
  • the amino acid sequences of these exemplary SAM-1 human peptides are LEVVVDQPMERL (SEQ ID NO:3) and LYEELVER (SEQ ID NO:4), respectively.
  • the peptide comprises about 5 to about 207 amino acids of the SAM-1 protein.
  • peptides may comprise an amino acid sequence starting with any of the first 203 amino acids of the SAM-1 protein such as starting at amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, or 203 or any other of the SAM-1 protein.
  • Other examples may be peptides comprising an amino acid sequence ending with any of the last 203 amino acids of the SAM-1 protein such as ending at amino acid 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, or 207.
  • Illustrative peptides may comprise amino acids; 2 to 27 of the SAM-1 protein; 3 to 207 of the SAM-1 protein; 203 to 207 of the SAM-1 protein; or 1 to 5 of the SAM-1 protein.
  • the peptides may comprise an amino acid sequence which terminates with 5 or more of the amino acids of the N-terminus amino acid sequence of the SAM- 1 protein.
  • the invention contemplates recombinant chimeric S AM- 1 proteins and peptides containing a portion of a SAM-1 protein and all or a portion of another protein.
  • peptides may comprise an amino acid sequence beginning with 5 or more of the amino acids of the C-terminus amino acid sequence of the SAM-1 protein.
  • Peptides may also comprise, consist of, or consist essentially of any of the amino acid sequences described herein.
  • peptides comprise, consist of, or consist essentially of an amino acid sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity or homology with the SAM-1 protein.
  • peptides that differ from the sequence in the naturally occurring SAM-1 protein in about 1, 2, 3, 4, 5 or more amino acids are also contemplated. The differences may be substitutions, e.g., conservative substitutions, deletions or additions.
  • the peptides comprise modified amino acids.
  • Exemplary peptides are peptides containing one or more amino acids modified by glycosylation, pegylation, phosphorylation or any similar process that retains at least one biological function of the peptide from which it was derived.
  • Peptides may also comprise one or more non-naturally occurring amino acids.
  • non-naturally occurring amino acids can be introduced as a substitution or addition into peptides.
  • Non-naturally occurring amino acids include, but are not limited to, the D-isomers of the twenty conventional amino acids, ⁇ -, ⁇ - disubstituted amino acids, N-alkyl amino acids, lactic acid 4 hydroxyproline, ⁇ - carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, ⁇ - ⁇ -acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, a-N- methylarginine, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2- amino butyric acid, 6-amino hexanoic acid, 2-amino is
  • Peptides may be modified during or after synthesis by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, or any similar process that retains at least one biological function of the peptide from which it was derived.
  • Peptide analogs such as chemically modified peptides and peptidomimetics are also contemplated.
  • peptides that are fused to a heterologous peptide or other signal sequences, such as a peptide that can be used for detecting; purifying; stabilizing; or solubilizing the peptide.
  • Peptides may be used as a substantially pure preparation, e.g., wherein at least about 90% of the peptides in the preparation are the desired peptide. Compositions comprising at least about 50%, 60%, 70%, or 80% of the desired peptide may also be used.
  • Peptides may be denatured or non-denatured and may be aggregated or non- aggregated as a result thereof. Peptides can be denatured according to methods known in the art.
  • the presence and expression levels of SAM-1 protein for early detection of breast cancer in the present invention can be detected by using a method which detects the presence and expression levels SAM-1 protein or of nucleic acid sequences that encode the SAM-1 protein relative to expression levels of a healthy subject or healthy tissue sample of the patient.
  • the detection method of the presence and expression levels of DNA and RNA typically use procedures which detect the presence and expression levels and/or patterns of mRNAs transcribed from these genes relative to expression levels of a healthy subject or healthy tissue sample of the patient.
  • Analytical methods that detect presence and expression levels and/or patterns of mRNAs include but are not limited to RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, northern blot, and DNA chip.
  • the levels of mRNAs in a biological sample from an unaffected person and a biological sample from patients at risk of breast cancer can be compared, and the expression levels and/or patterns of mRNAs that encode for SAM-1 protein for early stage breast cancer can be determined to diagnose breast cancer.
  • the invention provides methods for the detection of a nucleic acid encoding SAM-1 protein comprising.
  • a primer selected from the group consisting of: 5'-CTG GAG GTC GTG GTG GAC CAG CCC ATG GAG AGG CTC-3' (SEQ ID NO:6), 5'-AGCT AGATCT ACC CTG GAG GTC GTG GTG GAC CAG CCC ATG GAG AGG CTC-3' (SEQ ID NO:7), 5'-AGCTA GAATTC TCA ACA CCT GGC TTC AGA GGC AGG-3' (SEQ ID NO:8), 5'-CTC TAT GAA GAG CTC GTG GAG CGC-3' (SEQ ID NO:9), 5'-AGCTA AGATCT ACC ATG CTG CTA GCG ACA TTC-3' (SEQ ID NO: 10), 5'-GAG CCT CTC CAT GGG CTG GTC CAC CAC GAC CTC CAG-3' (SEQ ID NO: 11), 5'-GCG CTC CAC GAG CTC TTC ATA GAG-3' (SEQ ID NO: 6
  • the present invention relates to the nucleic acids encoding for SAM- 1 and also include vectors, such as expression vectors for producing a peptide as described herein. Also encompassed are cells comprising a nucleic acid sequence encoding for peptides described herein and methods for producing the peptides. The formation of such cells and methods can be performed using protocols that are known by those skilled in the art.
  • polynucleotide sequences encoding for SAM- 1 protein.
  • the polynucleotide sequence may also encode for a leader sequence.
  • the nucleic acid can be engineered such that the natural leader sequence is deleted and a heterologous leader sequence inserted in its place.
  • the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in expression and secretion of the polypeptide from the host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of the polypeptide from the cell.
  • polypeptides may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions to improve stability and persistence in the host cell. Additionally, peptide moieties may be added to the polypeptide to facilitate purification.
  • Another aspect of the present invention relates detecting the presence and expression level of SAM-1 protein.
  • the specific detection of presence and expression level of the SAM-1 protein for early detection of breast cancer in the present invention entails a process of confirming the presence and the expression level of SAM-1 protein within a biological sample.
  • specific antibodies that bind to SAM-1 protein can be used to detect the presence and expression level of SAM-1 protein.
  • Methods of detecting SAM-1 protein expression levels using antibodies include but are not limited to western blot, ELISA (enzyme linked immunosorbent assay), Radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion analysis, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, Complement Fixation Assay, FACS (fluorescent activated cell sorter), and protein chip.
  • the amount of the antigen-antibody complex can be measured by the levels and patterns of signals from detection labels of secondary antibodies.
  • detection labels include but are not limited to enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes.
  • the levels of antigen-antibody complexes in a biological sample from an unaffected person and a biological sample from patients at risk of breast cancer can be compared, and the expression levels of SAM-1 protein for early stage breast cancer could be determined, ultimately making it possible to diagnose breast cancer for patients at risk at early stage.
  • compositions may be provided in pharmaceutical compositions and administered to a patient to treat or prevent the development of breast cancer in the subject.
  • the invention provides pharmaceutical compositions which increase the levels or expression of SAM-1 in a patient.
  • Appropriate pharmaceutical agents can be administered to a subject in an amount that is therapeutically effective to prevent, inhibit, or decrease the development or growth of breast cancer.
  • the pharmaceutical agents may be administered alone or, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practices.
  • Pharmaceutical agents may be administered directly into breast tissue, orally or parenterally, including but not limited to intravenously, intramuscularly, intraperitoneally, subcutaneously, rectally and topically.
  • compositions containing a pharmaceutical agent may be in a form suitable for oral use, such as but not limited to tablets, troches, and suspensions.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more pharmaceutically acceptable soluble excipients.
  • the pharmaceutically acceptable excipients are known in the art, and the soluble excipients may include viscosity modifiers, surface active agents, diluents, and other non-active ingredients of the formulation intended to facilitate handling, stability, dispersibility, and/or release kinetics of the drug.
  • the pharmaceutical composition may contain other active agents may also be included in formulations, such as but not limited to anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents and other antibiotics.
  • the present invention provides pharmaceutical compositions and methods for preventing and/or treating breast cancer in a patient, which method comprises administering, to a patient in need an effective amount of a pharmaceutical agent for preventing breast cancer, treating breast cancer, diagnosing breast cancer, prognosing breast cancer and/or monitoring prevention or treatment of breast cancer.
  • the present invention provides pharmaceutical compositions and methods for preventing and/or treating Polycystic Ovarian Syndrome comprising administering to a human subject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • the present invention provides pharmaceutical compositions and methods for treating or preventing hormonal disorders associated with increased levels of estrogens comprising administering to a human subject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • the present invention provides pharmaceutical compositions and methods for treating or preventing hormonal disorders associated with increased aromatase activity comprising administering to a human subject in need thereof an effective amount of a pharmaceutical composition comprising a SAM-1 protein or functional fragment thereof and a pharmaceutically acceptable excipient.
  • endoplasmic reticulum fractions from MCF12A and T47D cells were, as previously described (Prasad, 2015), isolated. Isolated microsomes were collected by ultracentrifugation at 109,000 xg (Beckman TL- 100.2; Brea, CA) at 4°C for 30 min. The pellets (containing 10 ⁇ g of protein) were treated with freshly prepared 100 mM sodium carbonate (pH, 11.4) on ice for 15 min. The samples were ultracentrifuged at 80,000 x g to separate the soluble fraction from the membranous fraction. Washing the pellet with the buffer served as the control.
  • Protein (12.5 ⁇ g) was separated by 15% SDS-PAGE and transferred to a polyvinylidinedifluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 3% nonfat dry milk for 45 min, probed overnight with the primary antibodies, and then incubated with the peroxide-conjugated goat anti-rabbit IgG or anti mouse IgG (Pierce). Signals were developed with a chemiluminescent reagent (Pierce).
  • PVDF polyvinylidinedifluoride
  • siRNA 1 and siRNA 2 were independently applied to knockdown the expression of SAM-1. Expression levels were determined by western blotting.
  • SAM-1 protein The role of SAM-1 protein was elucidated by transfecting MC12A or T47D cells with siRNA 1 and siRNA 2 using oligofectamine in the absence of any serum. Twelve hours following transfection the incubation media was changed with a media containing serum and antibiotics and the cells were incubated for an additional 36 hours. After incubation the cells were washed with PBS two times and lysate was prepared for metabolic conversion. The reaction was initiated with 2 ⁇ g of cytochrome P450 and 2mM NADPH. For complete metabolic conversion, the reaction was chased with 10-fold cold testosterone and androstenedione independently.
  • reaction mixture was gently vortexed, and the reaction tubes, which were covered with parafilm to avoid any evaporation loss during incubation, were incubated in a shaking water bath (40 rpm) at 37°C for 4 h.
  • 4 niL of ether: acetone (9: 1) was added to each tube and gently vortexed to extract newly synthesized steroids.
  • the tubes were allowed to sit at room temperature for about 10 min to separate the aqueous and organic layers.
  • the upper, organic layer was gently collected without mixing the two layers using a Pasteur pipette and transferred to a new glass tube.
  • the remaining reaction mixture was again subjected to organic solvent extraction.
  • the extracted organic solvent layers were then mixed with 4 mL of basic water (0.01M NaOH), vortexed gently and allowed to remain for 15 min at room temperature to separate the layers.
  • the upper organic solvent layer was collected in a fresh 5 mL glass tube (VWR international, 12x75mm) and air-dried.
  • a cold testosterone: estradiol mixture was added to the completely dried reaction tubes at a final concentration of O.lmM resuspended in ethanol.
  • the tubes were gently rolled to dissolve all the dried steroids, and 2 iL was counted in 2.5 mL of scintillation cocktail (Beckmann Coulter, Beckman, CA) in triplicate.
  • silica-coated glass plate (20x20cm, 60W F254S, Millipore, Billerica, MA).
  • the silica plate was run in chroloform:ethylacetate (3: 1) for 1 h and dried in a 45 °C air incubator before exposing it to a 3 H screen.
  • the protein A-Sepharose pellets were washed with lx CoIP buffer and lOmM HEPES (pH 7.4), resuspended, and vortexed with lOOmM glycine (pH 3.0) for 10 seconds.
  • a pre-titrated volume of 1.0M Tris (pH 9.5) was added, and the beads separated from the soluble material by centrifugation at 2000xg for 2 min.
  • the supernatants (immune complexes) were analyzed by western blotting.
  • MCF12A or T47D cells (6xl0 6 ) were washed with PBS, gently scraped in the presence of PBS and transferred to 50 mL plastic disposable Corning tubes. Following centrifugation at 3500 rpm (Beckman Allegra 22R and rotor F630) for 10 min, the cells were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4, dehydrated with a graded ethanol series through 95%, and embedded in LR white resin.
  • Thin sections of the patient's breast or unaffected breast tissue of 75 nm thickness were cut with a diamond knife on a Leica EM UC6 ultramicrotome (Leica Microsystems, Bannockburn, IL, USA) and collected on 200 mesh nickel grids.
  • the cut sections were blocked in 0.1% BSA in PBS for 4 h at room temperature (RT) in a humidified atmosphere and incubated with Tim50 (1:100), Aromatase (1:1000), SAM-1 (1: 100), and GRP78 (1:1000) antibodies in 0.1% BSA overnight at 4°C.
  • the grids were washed with deionized 3 ⁇ 40 and air-dried.
  • the large gold particles were an average of 55 nm in diameter with 90% of the gold particles being between 45-65 nm in diameter.
  • the small gold particles were an average size of 15 nm with 90% of the gold particles being ⁇ 25 nm in diameter. All sections were observed in a JEM 1230 transmission electron microscope (JEOL USA, Peabody, MA, USA) at 110 kV and imaged with an UltraScan 4000 CCD camera and First Light Digital Camera Controller (Gatan, Pleasanton, CA, USA).
  • FIG. 1A The conversion of testosterone to estradiol is catalyzed by endoplasmic reticulum (ER) resident enzyme cytochrome P450 aromatase is shown in FIG. 1A.
  • ER endoplasmic reticulum
  • FIG. 1A To understand the factors or mechanism regulating higher estradiol production in affected patients, unaffected and affected breast tissues from the same patients as well as tumorigenic cell line, T47D, and non-tumorigenic cell line, MCF12A were selected, and the metabolic activity was measured by determining testosterone to estradiol conversion. The affected tissues and cells from both cell lines were collected and homogenized in HEPES and the cell debris was removed.
  • FIGS. IB and 1C The result of the metabolic conversion is shown in FIGS. IB and 1C which indicated a very high level of estradiol production in the affected breast tissue, but not in the unaffected tissue of the same person. Identical results were observed with the tumorigenic and non-tumorigenic cells, confirming the accuracy of the determination of metabolic activity in the unaffected and affected breast tissues.
  • P450 aromatase is an endoplasmic reticulum protein so the endoplasmic reticulum from all these patients were isolated and differential expression using mass spectrometry of the isolated endoplasmic reticulum was performed and analyzed through LC-MS/MS on a nanoAcquity UPLC coupled with a Q-TOF- Premier Mass Spectrometer. Two specific peptides, LEVVVDQPMERL (SEQ ID NO:3) and LYEELVER (SEQ ID NO:4) were found, which matched the sequence of StAR protein. However, this protein sequence was not observed with the tissues from any patients.
  • the second strand was amplified using primer (CTG GAG GTC GTG GTG GAC CAG CCC ATG GAG AGG CTC (SEQ ID NO:6)) and AP (FIG. ID), which generated a 600 bp fragment, and cloned into SP6 vector at the Bgl II and EcoRl sites (AGCT AGATCT ACC CTG GAG GTC GTG GTG GAC CAG CCC ATG GAG AGG CTC (SEQ ID NO:7); AGCTA GAATTC TCA ACA CCT GGC TTC AGA GGC AGG (SEQ ID NO:8)).
  • the sequencing result showed a completely new cDNA sequence with a new early stop codon at 361 bp.
  • the 3 'RACE from unaffected breast proceeded to the 5 '-RACE of the unaffected breast RNA and cDNA.
  • the cDNA was tail labelled with dCTPs and was amplified using 5 '-Abridged anchor primer (GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG (SEQ ID NO:20)) and (GAG CCT CTC CAT GGG CTG GTC CAC CAC GAC CTC CAG (SEQ ID NO: 11)) generating 450bp fragment (FIG. IE).
  • the combined 5' and 3' RACE amplified sequence was cloned (FIG. IF), which showed a 207 amino acid sequence not present in the open reading frame (FIG. 1G). This protein was named SAM-1 for Savannah (location of the lab), Anderson (Philanthropic support) and Mercer (University support).
  • the SAM-1 was present in a very short form 22.2 kDa protein in the affected breast tissues and also with the tumorigenic breast cells. Thirteen of the tissues were analyzed, including seven that were ER7PR7Her 2+ , three that were ER + /PR + /Her 2 ⁇ , and the three triple-negative tumors as well as two control breast tissue samples, which were obtained from the mirror image quadrant of the unaffected breast. Western blot analysis of the endoplasmic reticulum fraction showed a high level of SAM-1 expression in the control samples. However, SAM-1 was absent in the endoplasmic reticulum samples obtained from ER + /PR + /Her 2" and ER /PR7Her 2+ tumors even after loading up to 20 ⁇ g of total protein.
  • SAM-1 antibody a StAR protein antibody targeting this region was applied as a SAM- 1 antibody (hereinafter merely referred to as "SAM-1 antibody") (FIG. 2).
  • SAM-1 antibody A commercially available StAR antibody raised against StAR protein from amino acids 130 to 180 (Abeam, Cambridge, MA; Product code ab203193), and thus has no overlapping region with SAM-1, was used to confirm SAM-1 recognition and to rule out the possibility of any artifact of SAM-1 antibody. Staining with the commercial StAR antibody did not recognize SAM-1 protein, whereas the applied SAM-1 antibody did recognize SAM-1.
  • the non-tumorigenic MCF12A cells were stimulated with cAMP with two different concentrations of tenfold difference in concentration 0.1 and 10 mM. The incubation was continued from 4 to 12 hours to have optimum expression and determined the expression by western staining with SAM- 1 antibody. However, the result showed an unchanged expression of SAM-1 protein, suggesting that the expression was not acutely regulated (FIG. II).
  • the calculated molecular weight was 22.2 kDa but its migration was similar to the 17-18 kDa level.
  • FIG. 3F As expected staining with ER resident cytochrome P450 aromatase showed its presence mostly in the ER region shown in FIGS. 3D and 3E, with an enlarged view is shown in FIG. 3F. This was confirmed by immuno-staining breast tissue sections with GRP78 independently (data not shown). The colocalization of the SAM-1 and aromatase in the ER was also observed in the non- tumorigenic MCF12A breast cells (FIG. 3G-3I).
  • FIG. 4A Analysis of unaffected breast tissue showed that all the organelles are structured (FIG. 4A) with the mitochondria having outer and inner membrane (FIG. 4A).
  • FIG. 4B The analysis of tumorigenic tissue (FIG. 4B) showed both the organelles of the mitochondria (FIG. 4B) and ER are enlarged/swollen (FIG. 4B right hand panel).
  • FIG. 4C NANOGOLD labeled aromatase antibody
  • FIG. 4C NANOGOLD labeled aromatase antibody
  • SAM-1 antibody showed localization primarily in the MAM region and minimally at the ER.
  • Mitochondria-associated endoplasmic reticulum membrane regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction. /. Biol. Chem. , 290: 2604-16. Rajapaksha, M. J. (2016). An outer mitochondrial translocase, TOM22, is crucial for inner mitochondrial steroidogenic regulation in adrenal and gonadal tissues. Mol. Cell Biol, 36: 1032-47.

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Abstract

La présente invention concerne une nouvelle protéine, SAM-1, des fragments fonctionnels de celle-ci, des acides nucléiques codant pour ceux-ci, des compositions diagnostiques et pharmaceutiques, et leurs méthodes d'utilisation. La présente invention concerne la détection, la prévention et/ou le traitement du cancer du sein, du syndrome des ovaires polykystiques et des troubles hormonaux associés à des niveaux accrus d'œstrogènes et d'activité d'aromatase.
PCT/US2018/028295 2017-04-19 2018-04-19 Protéine sam-1, composition et méthodes d'utilisation WO2018195273A1 (fr)

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Citations (7)

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US20020132237A1 (en) * 2000-05-26 2002-09-19 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US20030105049A1 (en) * 1999-05-03 2003-06-05 Kinneret Savitzky StAR homologues
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US20060110730A1 (en) * 2002-05-20 2006-05-25 Bose Himangshu S Methods and compositions for regulation and manipulation of steroidogenesis
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US6783961B1 (en) * 1999-02-26 2004-08-31 Genset S.A. Expressed sequence tags and encoded human proteins
US20030105049A1 (en) * 1999-05-03 2003-06-05 Kinneret Savitzky StAR homologues
WO2001031342A2 (fr) * 1999-10-26 2001-05-03 Tularik Inc. DIAGNOSTIC ET TRAITEMENT DU CANCER UTILISANT DES POLYPEPTIDES ET DES POLYNUCLEOTIDES StAR
US20020132237A1 (en) * 2000-05-26 2002-09-19 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US20060110730A1 (en) * 2002-05-20 2006-05-25 Bose Himangshu S Methods and compositions for regulation and manipulation of steroidogenesis
US20100150871A1 (en) * 2004-10-27 2010-06-17 Centre National De La Recherche Scientifiqe Genetically-modified strain of yeast with an increased production and output of s-adenosylmethionine (sam)
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