WO2010135475A2 - Phosphorylation de l'acide gras synthétase et cancer - Google Patents

Phosphorylation de l'acide gras synthétase et cancer Download PDF

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WO2010135475A2
WO2010135475A2 PCT/US2010/035483 US2010035483W WO2010135475A2 WO 2010135475 A2 WO2010135475 A2 WO 2010135475A2 US 2010035483 W US2010035483 W US 2010035483W WO 2010135475 A2 WO2010135475 A2 WO 2010135475A2
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fas
phosphorylated
seq
cancer
polypeptide
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PCT/US2010/035483
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WO2010135475A3 (fr
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Susan M. Medghalchi
Francis P. Kuhajda
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Fasgen Diagnostics Llc
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    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)

Definitions

  • FIELD OF THE DISCLOSURE This disclosure relates to the early and accurate diagnosis of cancer to enable more effective therapy and to enhance patient survival and quality of life.
  • This disclosure provides an assay for cancer based upon the identification of fatty acid synthase (FAS), particularly in a phosphorylated form, to improve detection methods for the presence, course, and treatment of cancer.
  • FAS fatty acid synthase
  • FAS fatty acid synthase
  • Table 1 illustrates the expression of FAS in a number of human cancers.
  • FAS is a complex, multifunctional enzyme that contains seven catalytic domains and a 4'-phosphopantetheine prosthetic group on a single polypeptide with a relative molecular weight of about 27OkDa (Smith, FASEBJ, 8: 1248-1259 (1994), Wakil, Biochem. 28: 4523-4530 (1989)).
  • the human FAS amino acid sequence is known and deposited with accession number AAA73576 (with a length of 2509 amino acid residues) and NP_004095 (with a length of 251 1 amino acid residues).
  • FAS is the sole mammalian enzyme that catalyzes the NADPH dependent condensation of malonyl-CoA and acetyl-CoA to produce the 16-carbon saturated free fatty acid palmitate (see Figure 1).
  • FAS performs the de novo synthesis of fatty acid from carbohydrates, the immediate proximal enzyme in the pathway, acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme of fatty acid synthesis (Ruderman et al., Am. J. Physiol., 276: E1-E18 (1999).
  • the disclosure relates to detecting or measuring expression of phosphorylated fatty acid synthase (FAS) as an indicator, or marker, of the presence of cancer in a subject.
  • FAS phosphorylated fatty acid synthase
  • the disclosure is based in part on the observation that FAS has been observed at high (above normal) levels in most common cancers, including colon, lung, prostate and breast cancers.
  • the disclosure is also based in part on detecting phosphorylated FAS produced by cancer cells in comparison to non- phosphorylated FAS produced by non-cancer, or normal, cells.
  • the disclosure provides methods and compositions for detecting or measuring expression of a phosphorylated FAS polypeptide (containing one or more phosphorylated amino acid residues) or a fragment of the FAS polypeptide containing one or more phosphorylated residues.
  • a fragment may be produced from a larger polypeptide by human manipulation.
  • the methods and compositions include a complex comprising a binding agent which specifically binds the phosphorylated FAS polypeptide, or phosphorylated fragment thereof, to form a detectable complex.
  • the specificity of the binding agent may be such that the agent detectably binds the phosphorylated FAS polypeptide, or phosphorylated fragment thereof, to the exclusion of the counterpart non- phosphorylated polypeptide or fragment.
  • the binding agent is an antibody, such as a monoclonal antibody.
  • the FAS polypeptide, or fragment thereof is phosphorylated at a serine residue.
  • Non-limiting examples include the serine residue at position 1237 in SEQ ID NO: 1 (or SEQ ID NO: 2), at position 1473 in SEQ ID NO: 1 , and/or at position 1475 in SEQ ID NO: 2 as described herein.
  • Other non-limiting examples include the serine residue at position 2279 in SEQ ID NO: 1, and/or at position 2281 in SEQ ID NO: 2 as described herein.
  • the FAS polypeptide, or fragment thereof is phosphorylated at a threonine residue.
  • Non-limiting examples include the threonine residue at position 1827 in SEQ ID NO: 1 or at position 1829 in SEQ ID NO: 2.
  • Other non-limiting examples include the threonine residue at position 126 in SEQ ID NO: 1 (or SEQ ID NO: 2).
  • the disclosure provides a method of identifying the presence of cancer in a subject based on the presence of a phosphorylated FAS polypeptide, or phosphorylated fragment thereof, in a biological sample from the subject.
  • the method may comprise detecting or measuring the presence of a phosphorylated FAS polypeptide, or a phosphorylated fragment of the polypeptide, in a biological sample obtained from a subject.
  • a fragment may be produced from a larger polypeptide by human manipulation.
  • the method may be used as a screening method or assay to identify individuals afflicted with cancer.
  • the method may be used to confirm a diagnosis of the presence of cancer, such as in combination with one or more other diagnostic methods or protocols.
  • the disclosure provides a method of selecting subjects with phosphorylated FAS for treatment.
  • the method may be used to select or identify a subject as having tumor cells expressing phosphorylated FAS and then administering one or more treatments against the tumor cells.
  • the method may comprise detecting the presence of a phosphorylated FAS polypeptide, or a phosphorylated fragment thereof, in a biological sample obtained from a subject and administering an anti-cancer or antitumor treatment to the subject.
  • the disclosure provides a method of detecting or measuring disease progression, or efficacy of treatment, based on the expression of a phosphorylated FAS polypeptide, or a phosphorylated fragment thereof.
  • the method may comprise measuring the level of a phosphorylated FAS polypeptide, or a phosphorylated fragment thereof, in a biological sample from said subject; and repeating the measuring over time.
  • a fragment may be produced from a larger polypeptide by human manipulation.
  • An increase or decrease in the level of expression over time indicates an increase or decrease, respectively, in tumor cells, or tumor cell activity.
  • the method may be used to monitor cancer or tumor cell burden.
  • the measurements over time are made before, during and/or following therapy to monitor the course of treatment and outcome for a subject.
  • the methods and compositions of the disclosure are practiced in relation to specific cancers, such as cancers of the colon, lung, pancreas, prostate, ovary and breast. In some cases, the cancer is not cervical cancer. In additional embodiments, the methods and compositions are practiced in relation to human subjects and patients.
  • the methods and compositions of the disclosure are based on the detection of a phosphorylated FAS polypeptide, or phosphorylated fragment thereof, in a blood, serum, or tumor cell containing sample.
  • a fragment may be produced from a larger polypeptide by human manipulation.
  • a number of phosphorylated serum proteins have been reported in humans, although phosphorylated FAS is not among them.
  • a portion of human fetuin ( ⁇ 2-Heremans-Schmid protein) is phosphorylated on serine (Haglund et al., Biochem. J., 357: 437-445 (2001 ) which may affect insulin signal transduction.
  • C3 is phosphorylated by casein kinase released from platelets which enhances its binding to complement receptor 1 (Nilsson-Ekdahl and Nilsson, Eur. J. Immunol., 31 : 1047-1054 (2001)).
  • Other reported circulating phosphoproteins include cardiac troponin I and T (Labugger et al., Circulation 102: 1221 -1226 (2000)), tumor type M2 pyruvate kinase (Luftner et al., Anticancer Res., 23: 991-997 (2003)), complement C3c (Goldknopf, et al., Biochem. Biophys. Res.
  • An additional aspect of the disclosure is a detectable complex comprising a phosphorylated FAS polypeptide, or a phosphorylated fragment of the polypeptide, and a binding agent.
  • a fragment may be produced from a larger polypeptide by human manipulation.
  • the binding agent is specific for the phosphorylated, as opposed to the unphosphorylated, polypeptide or fragment thereof.
  • the complex includes a phosphorylated FAS polypeptide with a relative molecular weight (MW) of about 270 kiloDaltons (kDa).
  • MW relative molecular weight
  • the complex includes a polypeptide with a length of 2509 or 251 1 amino acid residues.
  • the polypeptide may have the same relative MW but a length shorter than that of 2509 or 2511 , such as by truncation or loss of one or a few amino acid residues from one or both ends of the FAS polypeptide.
  • the 2509 or 2511 residue polypeptide has the sequence represented by SEQ ID NO: 1 or 2 as disclosed herein, respectively.
  • the complex includes a phosphorylated fragment of a phosphorylated FAS polypeptide.
  • the fragment may have a length of at least five, or about five, amino acid residues that present a sufficient epitope for recognition by the antibody.
  • the fragment may be longer than five residues, up to one residue less than the full-length of a FAS polypeptide, so long as the fragment contains at least one phosphorylated residue.
  • a fragment may be produced from a larger polypeptide by human manipulation.
  • Embodiments of the disclosure include those wherein the phosphorylated FAS polypeptide or a phosphorylated fragment thereof, contains at least one phosphothreonine residue and/or at least one phosphoserine residue.
  • animals for the application of the present disclosure are mammals, particularly those important to agricultural applications (such as, but not limited to, cattle, sheep, horses, and other "farm animals"), animal models of cancer, and animals for human companionship (such as, but not limited to, dogs and cats).
  • Figure 1 is a schematic of the fatty acid synthesis pathway
  • FIG. 1 illustrates FAS expression in prostate (A), colon (B), and breast cancer (C);
  • Figure 3 shows a representative FAS ELISA standard curve with human FAS
  • Figure 4 illustrates FAS levels in cancer and normal subjects
  • Figure 5 illustrates FAS expression in normal human and human cancer cell lines
  • Figures 6A and 6B illustrate FAS from human cancer cells is phosphorylated on Thr/Pro
  • Figure 7 shows a Western blot analysis of FAS phosphorylation in human cancer cell lines
  • Figure 8 shows phosphoprotein gel stain detection of FAS in human cancer cell lines
  • Figures 9A and 9B illustrate that phosphorylated FAS increases with okadaic acid treatment (9A) while total FAS protein is reduced (9B);
  • Figures 1OA and 1OB show the sequences of exemplary FAS proteins of the disclosure with a length of 2509 amino acid residues (SEQ ID NO: 1) and 251 1 amino acid residues (SEQ ID NO: 2).
  • FAS is the enzyme which catalyzes the de novo synthesis of fatty acids predominantly from dietary carbohydrates.
  • FAS circulates at high (above normal) levels in the blood of colon, breast, lung, ovarian, and prostate cancer patients as compared to normal (cancer-free) subjects.
  • increased FAS expression is associated with aggressive disease in breast, prostate, ovary, endometrium, urinary bladder, pediatric malignancies, and soft tissue sarcomas.
  • Figure 2 illustrates the high levels of FAS expression in prostate, colon, and breast carcinomas with immunohistochemistry.
  • FAS from normal murine mammary cells were not phosphorylated.
  • SKBR3 human breast cancer cells were also studied and reported to be phosphorylated on serine, threonine and tyrosine residues.
  • earlier reports of normal tissues demonstrated that FAS purified from rat liver and adipose tissue was not phosphorylated (Rous, FEBS Lett., 44: 55-58 (1974), Ramakrishna and Benjamin, Prep. Biochem., 13: 475-488 (1983)).
  • the disclosure includes methods and compositions for detecting or measuring the expression level of phosphorylated FAS polypeptide, or a fragment thereof, where the polypeptide or fragment contains one or more phosphorylated amino acid residues distinct from the 4'-phosphopantetheine prosthetic group found on FAS polypeptides.
  • Embodiments of the disclosure include a FAS polypeptide, or fragment thereof, containing one or more of a phosphothreonine or phosphoserine residue. More specifically, the disclosure includes detecting or measuring a phosphorylated FAS polypeptide, or fragment thereof, as it may be present in a subject, such as a human patient. Of course a fragment may be produced from a larger polypeptide by human manipulation. In many embodiments, the detection or measurement is used in cases of elevated FAS levels, such as that observed in obese subjects.
  • the FAS polypeptide, or fragment thereof is phosphorylated at a serine residue.
  • Non-limiting examples include the serine residue at position 1237 in SEQ ID NO: 1 (or SEQ ID NO: 2), at position 1473 in SEQ ID NO: 1 , and/or at position 1475 in SEQ ID NO: 2 as described herein.
  • Other non-limiting examples include the serine residue at position 2279 in SEQ ID NO: 1, and/or at position 2281 in SEQ ID NO: 2 as described herein.
  • the FAS polypeptide, or fragment thereof is phosphorylated at a threonine residue.
  • Non-limiting examples include the threonine residue at position 1827 in SEQ ID NO: 1 or at position 1829 in SEQ ID NO: 2.
  • Other non-limiting examples include the threonine residue at position 126 in SEQ ID NO: 1 (or SEQ ID NO: 2).
  • a method of the disclosure may be used to qualitatively detect the presence of a phosphorylated FAS polypeptide, or fragment thereof. In some cases, such a method may be used to determine whether the polypeptide or fragment is present or not. In other embodiments, a method may be used to qualitatively measure the amount of the polypeptide or fragment.
  • such a method may be used to determine the expression level of the polypeptide or fragment, and optionally provide a measurement in the form of amount per volume like nanogram of polypeptide or fragment per milliliter of volume. Alternatively, a measurement may be based on the number of molecules per volume, like one expressed in terms of molarity. Of course, a fragment may be produced from a larger polypeptide by human manipulation.
  • the methods of the disclosure are practiced with the use of a biological sample from a subject, such as a fluid or cell containing sample from a human patient.
  • the fluid sample may be a blood, plasma, or serum sample.
  • Non-limiting methods of the disclosure include determinations of phosphorylated FAS polypeptide, or a fragment thereof, in amounts based on ng/ml, such as above 4 ng/ml or above 10 ng/ml.
  • a method may be practiced with the use of a cell containing sample, or extract thereof, obtained from a subject.
  • the sample may contain tumor cells, and use of the sample in a method of the disclosure may be used to confirm a determination of the presence of tumor cells or cancer.
  • the sample may be from a subject suspected as having cancer, and so the sample is suspected to contain tumor cells.
  • Non- limiting embodiments of the disclosure include the use of such a sample to determine, or diagnose, the presence of tumor cells in the sample and so cancer in the subject.
  • the detection or measurement of a phosphorylated FAS polypeptide, or a fragment thereof, in a sample may be made directly or indirectly.
  • a non-limiting example of a direct method is with Pro-Q® Diamond gel stain (Molecular Probes) and analysis using a Typhoon 940 Laser Scanner.
  • an indirect method is used, such as via detection or measurement of a complex containing the polypeptide or fragment bound to a binding agent that specifically recognizes the polypeptide or fragment.
  • the binding agent binds to the phosphorylated, but not the non-phosphorylated, form of the polypeptide or fragment, and so may also be termed a detection agent.
  • the binding agent is an antibody or antigen binding fragment thereof.
  • Non-limiting examples include the use of and F v or F ab fragment of an antibody that specifically binds a phosphorylated FAS polypeptide, or fragment thereof, as described herein.
  • the antibody is a monoclonal antibody that binds in part, a phosphorylated residue in the FAS polypeptide or fragment thereof.
  • the antibody may be referred to as a detection antibody.
  • a polyclonal antibody, or a combination of monoclonal antibodies may be used.
  • the binding agent is detectably labeled to facilitate the detection of the phosphorylated FAS polypeptide or fragment thereof.
  • the detection antibody may itself be a "primary antibody” which is unlabeled and so detected by binding of one or more detectably labeled secondary antibodies that recognize the primary antibody.
  • the label is an enzyme that produces a detectable signal by catalyzing a reaction with a substrate.
  • Non-limiting examples include the use of horseradish peroxidase (HRP) or alkaline phosphatase (AP).
  • a complex of the disclosure comprises the detection agent and a phosphorylated FAS polypeptide or fragment thereof.
  • the complex further comprises an additional binding agent which immobilizes, or captures, either the detection agent or the phosphorylated FAS polypeptide, or fragment thereof.
  • the immobilization, or capture may be mediated by the immobilization of the additional binding agent on a solid phase substrate, such as the surface of a plastic or glass plate or a bead as non- limiting examples.
  • the additional binding agent is also an antibody, termed a capture antibody, which binds the FAS polypeptide, or fragment thereof, in a manner that does not interfere with the interaction(s) between the detection agent and the polypeptide or fragment thereof.
  • a capture antibody of the disclosure may be a monoclonal or polyclonal antibody. Alternatively, it may be a combination, or cocktail, of monoclonal antibodies. In many cases, a capture antibody recognizes FAS polypeptides based upon a conserved or consensus sequence present even in FAS polypeptides with sequence in other regions of the molecule.
  • a phosphorylated FAS polypeptide or fragment thereof
  • a method or process includes the formation and/or detection of the "sandwich” complex.
  • Alternatives to a "sandwich” format include a competitive format, which may also be used in the disclosed methods and processes.
  • Embodiments of the disclosed methods include a diagnostic assay, such as an ELISA (enzyme-linked immunosorbent assay).
  • the assay is used to diagnose human cancer based on FAS phosphorylation.
  • the assay includes the feature of differentiating the indication, provided by phosphorylated FAS in a biological sample from a subject, from normal FAS present from normal tissues, such as the liver.
  • the differentiation may be of phosphorylated FAS polypeptide, or fragment thereof, from non-phosphorylated FAS.
  • the ability to differentiate is applied in cases of elevated FAS levels, such as that observed in obese subjects.
  • ELISA may be used in either a "sandwich” or competitive format.
  • a competitive radioimmunosorbent assay RIA may also be used.
  • the methods and assays of the disclosure may be used to identifying the presence, or absence, of cancer (or tumor cells that express phosphorylated FAS) in a subject based on the presence, or absence, of a phosphorylated FAS polypeptide, or phosphorylated fragment thereof, in a biological sample from the subject.
  • the method or assay screens individuals, optionally asymptomatic, to identify those afflicted with, or free of, cancer or tumor cells that express phosphorylated FAS.
  • the method or assay may be used to confirm a diagnosis of the presence of cancer, such as in combination with one or more other diagnostic methods or protocols.
  • the method or assay is used to confirm a diagnosis of the absence of cancer, or tumor cells that express phosphorylated FAS.
  • the cancer may be early stage and/or characterized by a pre-neoplastic lesion.
  • Non-limiting examples of cancers, or the tumor cells thereof include cancer of the breast, prostate, colon, stomach, lung, mesothelium (mesothelioma), oral cavity, esophagus, head and neck (squamous cancer), ovary, pancreas, endometrium, thyroid, parathyroid, kidney, or urinary bladder; or a cancer selected from retinoblastoma, nephroblastoma (Wilm's tumor), or a soft tissue sarcoma.
  • a subject identified as having cancer, or tumor cells, as described above may be selected for treatment based upon the FAS expression phenotype.
  • a method may include selecting a subject identified as having cancer, or tumor cells, expressing phosphorylated FAS and then administering one or more treatments against the cancer or tumor cells.
  • treatments include surgery, radiation, and/or chemotherapy.
  • Additional embodiments of the disclosure include a method or assay to detect or measure disease progression, or efficacy of treatment, over time based on the expression of a phosphorylated fatty acid synthase (FAS) polypeptide, or a phosphorylated fragment thereof.
  • FAS phosphorylated fatty acid synthase
  • the method or assay includes more than one detection or measurement over time for comparative purposes such that an increase or decrease in the level of expression over time indicates an increase or decrease, respectively, in tumor cells, or tumor cell activity, or cancer activity.
  • the method or assay may be used to monitor treatment progress or effectiveness when a FAS targeted therapy or other anti-cancer or anti-tumor therapy (as disclosed herein) is applied to a subject.
  • the detections or measurements over time may be made before, during and/or following therapy to monitor the course of treatment and outcome for a subject.
  • the disclosure includes a method or process based on detecting or measuring FAS phosphorylation by immunohistochemistry (IHC).
  • the method may include the use of antibodies specific for phosphorylated FAS polypeptide, or a fragment thereof, to detect or measure expression of the phosphorylated form in a cell containing sample.
  • the disclosure includes the detecting or measuring of a full-length FAS protein, such as those with a relative MW of about 270 kDa, with one or more phosphorylated amino acid residues.
  • FAS polypeptides with variations in the sequence such as in the size of the full-length sequence as a non-limiting example, may be detected or measured in the practice of the disclosure based on the presence of the phosphorylated amino acid residues in the polypeptide.
  • polypeptides with other sequence variations such as those due to polymorphism as a non-limiting example, may also be detected or measured based on the phosphorylated amino acid residue.
  • the full-length FAS sequence may be 2509 or 251 1 amino acids long.
  • the lengths may be longer or shorter than 2509, or 251 1 , residues based on the size of the full length FAS polypeptide as present in a subject.
  • the disclosure may also be practiced with a phosphorylated fragment of FAS produced from a larger polypeptide by human manipulation.
  • the size will necessarily be less than full-length, and so non-limiting examples include fragments of less than 2509 or 251 1 residues as described herein.
  • the fragments will be fragments of SEQ ID NOS: 1 or 2, as described herein.
  • Various fragments of these described sequences are recognized by the skilled person based upon knowledge in the field.
  • the fragments will simply be truncations of one or a few amino acid residues from one or both ends of a FAS polypeptide.
  • the fragments will continue to have a relative molecular weight of about 27OkDa.
  • fragments of a FAS polypeptide are disclosed herein or readily known to the skilled person in the field.
  • sequences of SEQ ID NOS: 1 and 2 can be readily scanned for the presence of lysine or arginine residues to select tryptic digestion fragments of a FAS polypeptide.
  • the digestion may be partial, to produce larger fragments, or to completion, where all seven threonine-proline sites and the 4'-phosphopantetheine prosthetic group would be on separate and distinct peptides.
  • FAS fragments produced by V8 protease, chymotrypsin, subtilisin, clostripain, endoproteinase Lys-C, endoproteinase GIu-C, endoproteinase Asp-N, and thermolysin may be used to generate additional fragments that can be detected by the methods and assays disclosed herein.
  • these fragments would contain one or more phosphorylated residues of FAS based on the location of the cleavage sites and the residue positions.
  • the fragments may be termed FAS phosphopeptides of the disclosure, which are at least five amino acid residues in length (as found sequentially in a FAS polypeptide) to define an epitope recognized by a binding agent as described herein.
  • the epitope need not be produced by five or more sequential residues but may instead be the result of protein folding to form an epitope from non-sequential residues.
  • Additional embodiments include fragments of at least 10 or about 10, at least 15 or about 15, at least 20 or about 20, at least 25 or about 25, at least 30 or about 30, at least 35 or about 35, at least 40 or about 40, at least 45 or about 45, at least 50 or about 50, at least 75 or about 75, at least 100 or about 100, at least 200 or about 200, at least 300 or about 300, at least 400 or about 400, at least 500 or about 500, at least 750 or about 750, at least 1000 or about 1000, at least 1250 or about 1250, at least 1500 or about 1500, at least 1750 or about 1750, at least 2000 or about 2000, at least 2250 or about 2250, or at least 2500 or about 2500 sequential residues of a FAS polypeptide.
  • a fragment must contain one or more phosphorylated residue for use in the disclosed methods and assays.
  • a phosphorylated residue include a phosphothreonine residue or a phosphoserine residue.
  • Additional FAS phosphopeptides of the disclosure are those found in a biological sample of a subject, such as a human patient.
  • a phosphorylated FAS polypeptide or fragment thereof may be present in a complex, such as a "sandwich" complex as disclosed herein.
  • the complex is immobilized on a solid phase substrate as described herein.
  • the complex is present in combination with other blood, serum, or plasma components, or other cellular components, present in the biological sample containing the complexed FAS polypeptide or fragment thereof.
  • kits comprising agents for the detection of expression of the disclosed phosphorylated FAS polypeptides and fragments.
  • kits optionally comprise the agents with an identifying description or label or instructions relating to their use in the methods and assays of the present disclosure.
  • a kit may comprise containers, each with one or more of the various reagents (typically in concentrated form) utilized in the methods and assays, including, for example, antibodies, buffers, wash solutions, etc.
  • a set of instructions will also typically be included.
  • Example 1 FAS binding antibodies and a representative ELISA format
  • FAS purified from human ZR-75-1 human breast cancer cells were used as an immunogen to produce hybridomas for reactivity to purified human FAS.
  • FAS from other human or animal sources may also be used.
  • Figure 3 is a representative standard curve summarizing within-plate standard curves with purified FAS.
  • the assay is linear through FAS concentrations of 1.6 - 50 ng/ml with a CV of 3%.
  • the day-to-day variability over 2 weeks with 8 assays generated a CV of 5.9% + 1.9%.
  • Analytical sensitivity is 0.301 ng/ml within 95% confidence using EP evaluator software.
  • a FAS ELISA assay was used to confirm serological studies of FAS in cancer patients.
  • Serum FAS levels were measured in 79 patients with active disease representing most common human cancer types and were compared to 30 male and female control subjects (that were clinically free of cancer). The results are shown in Figure 4.
  • the relationship between FAS phosphorylation and cancer was investigated with a panel of the following human immortalized non-transformed cell lines: IMR-90 fetal lung, hPS human prostate, and human cancer cell lines: HCT-1 16 colon, PPC-I prostate, and SKBr3 breast.
  • FAS expression was quantified by immunoblot as shown in Figure 5.
  • FAS enzyme levels (adjusted to total cellular protein) were quantitated by immunoblot and normalized to IMR-90.
  • Both IMR-90 and hPS non-transformed cell lines had relatively low levels of FAS expression compared to the cancer cell lines, which ranged from 4.3 to 22 fold elevations compared to IMR-90 cells.
  • the high level of FAS expression in SKBr3 cells is consistent with the observation that 28% of its cytosolic protein is FAS (Thompson et al., Biochem. Biophys Acta, 662: 125-130 ( 1981 )).
  • Figure 6A illustrates the results from immunoprecipitating cellular contents, from the cell lines, with anti-FAS followed by immunoblotting of the immunoprecipitates with anti-FAS (upper panel) or anti-phosphothreonine-proline (lower panel) where the phosphothreonine requires an adjacent proline for antibody reactivity (Cell Signaling). All three tumor cell lines show evidence of FAS phosphorylation while the two non-transformed cell lines are negative. No immunoreactivity was detected with an anti-phosphotyrosine or an anti-phosphoserine antibody, although additional antibodies and FAS proteins were not tested.
  • Additional human cancer cell lines such as LnCAP, OVCAR-3, SKOV3 (ovary), RKO (colon), H460, LX7 (lung), CAPAN-I , and PANC-I (pancreas) can also be assessed for differential FAS phosphorylation as described above.
  • Figure 6B illustrates the results of SKBr3 cell material immunoblotted (labeled) with an anti-phosphosphoserine/threonine antibody preparation which confirms the phosphothreonine reactivity shown in Figure 6A.
  • Purified FAS from ZR-75-1 breast cancer cells, which were used as an immunogen for antibody production, are also immunolabeled by the antibody preparation.
  • Example 4 Phosphorylated FAS in other cancer cell lines
  • FAS phosphorylation was investigated in human cancer cell lines.
  • Specific phosphorylation site(s) on FAS in human cancer cell lines were then identified and sequenced using tandem mass spectroscopy. Phosphorylation sites both common and unique to the individual cell lines were identified.
  • FAS phosphorylation was detected by these three independent methods. Phosphorylated threonine and serine residues were identified and modeled to be on the surface of FAS protein.
  • Lysates were sonicated and pre-cleared with protein A/G beads. Lysates were incubated with an anti-FAS monoclonal antibody (FASgen, Inc.) overnight at 4 0 C. ImmunoPure Immobilized Protein A/G (Pierce) was added and incubated for 2 hours. Following washing with RIPA buffer, the beads were boiled in sample loading buffer and proteins were separated by electrophoresis through a 4-15% gradient SDS-PAGE gel.
  • Phosphoprotein Gel Staininp The immunoprecipitated proteins were separated by electrophoresis through a 4-15% gradient SDS-PAGE gel. The gel was processed and analyzed as recommended by the manufacturer of the Pro-Q Diamond Gel Stain. The bands were visualized using a Typhoon 940 Laser Scanner. The imaging was done at the AB Mass Spectrometry/Proteomics Facility at The Johns Hopkins School of Medicine. The results are shown in Figure 8.
  • Tandem mass spectroscopy was used to identify the location of phosphor-threonine and phosphor-serine residues described above.
  • the peptide from positions 1 188 to 1275 of SEQ ID NO: 1 (identical positions in SEQ ID NO: 2) was used to identify the phosphorylated serine at position 1237.
  • sequence of that peptide is as follows: llsaacrlql ngnlqlelaq vlaqerpklp edpllsglld spalkacldt avenmpSlkm kvvevlaghg hlysripgll sphpllqlsy tatdr (SEQ ID NO: 3).
  • SKBr3 (breast adenocarcinoma) cells.
  • the peptide contains residues 1226 to 1239 of SEQ ID NO: 1 (identical positions in SEQ ID NO: 2) and has the following sequence: acldtavenm pSlk (SEQ ID NO: 4).
  • SEQ ID NO: 2 was used to identify the phosphorylated serine at position 1473 in SEQ ID NO: 1 and at position 1475 in SEQ ID NO: 2 in SKBr3 and 0VCAR3 cells.
  • the sequence of that peptide is as follows: cvllSnlsst shvpevdpgs aelqk (SEQ ID NO: 5)
  • a peptide was used to identify phosphorylated serine at residue 2279 in SEQ ID NO: 1 (residue 2281 in SEQ ID NO: 2) in SKBr3 cells.
  • the peptide contains residues 2274 to 2292 of SEQ ID NO: 1 (residues positions 2276 to 2294 in SEQ ID NO: 2) and has the following sequence: aapldSihsl aayyidcir (SEQ ID NO: 6).
  • the peptide from positions 1826 to 1839 in SEQ ID NO: 1 was used to identify the phosphorylated threonine at position 1827 in SEQ ID NO: 1 and at position 1829 in SEQ ID NO: 2 in MCF7, SKBr3, OVCAR3, ZR75, HCTl 16, and RKO (colon carcinoma) cells.
  • a peptide was used to identify phosphorylated threonine at position 126 in SEQ ID NO: 1 (identical position in SEQ ID NO: 2) in MCF7 cells.
  • the peptide contains residues 123 to 137 of SEQ ID NO: 1 (identical positions in SEQ ID NO: 2) and has the following sequence: dpeTlvgysm vgcqr (SEQ ID NO: 8).
  • peptides or other peptides containing the same phosphorylated serine or threonine residue, may be used to detect or identify the presence of the phosphorylated residue as disclosed herein.
  • This distance is from the residue of one KS monomer to the active site of the other KS monomer.
  • Figure 9 A shows results demonstrating that a brief treatment with 100 nM okadaic acid, a concentration that is specific for protein phosphatase 2A inhibition (Yan and Mumby, J. Biol. Chem., 21 A: 31917-31924 (1999)), the ratio of phosphorylated FAS to total FAS increases substantially in both PPC-I and HCT-116 cell lines.
  • Figure 9B shows that increased phosphorylation of FAS leads to a reduction in total cellular FAS.

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Abstract

Cette invention concerne la détection de l'acide gras synthétase phosphorylée utilisée comme élément de diagnostic et comme composant dans l'identification et le traitement du cancer. Les méthodes décrites permettent le diagnostic précoce et précis du cancer, assurent un traitement plus efficace et améliorent la survie du patient et sa qualité de vie.
PCT/US2010/035483 2009-05-19 2010-05-19 Phosphorylation de l'acide gras synthétase et cancer WO2010135475A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2217929A2 (fr) * 2007-11-13 2010-08-18 Fasgen, Inc. Acide gras synthase phosphoryle et traitement du cancer
US8729239B2 (en) 2009-04-09 2014-05-20 Nuclea Biotechnologies, Inc. Antibodies against fatty acid synthase

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763164A (en) * 1993-04-16 1998-06-09 Northwestern University Immunogenic cancer proteins and peptides and methods of use
US20070134687A1 (en) * 2005-09-12 2007-06-14 Aurelium Biopharma Inc. Focused microarray and methods of diagnosing cancer
US20070148718A1 (en) * 2005-08-17 2007-06-28 Fasgen Diagnostics, Llc Fas binding antibodies
US20070212736A1 (en) * 2004-04-08 2007-09-13 Cornell Research Foundation, Inc. Functional Immunohistochemical Cell Cycle Analysis as a Prognostic Indicator for Cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763164A (en) * 1993-04-16 1998-06-09 Northwestern University Immunogenic cancer proteins and peptides and methods of use
US20070212736A1 (en) * 2004-04-08 2007-09-13 Cornell Research Foundation, Inc. Functional Immunohistochemical Cell Cycle Analysis as a Prognostic Indicator for Cancer
US20070148718A1 (en) * 2005-08-17 2007-06-28 Fasgen Diagnostics, Llc Fas binding antibodies
US20070134687A1 (en) * 2005-09-12 2007-06-14 Aurelium Biopharma Inc. Focused microarray and methods of diagnosing cancer

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2217929A2 (fr) * 2007-11-13 2010-08-18 Fasgen, Inc. Acide gras synthase phosphoryle et traitement du cancer
EP2217929A4 (fr) * 2007-11-13 2011-10-05 Fasgen Inc Acide gras synthase phosphoryle et traitement du cancer
US8729239B2 (en) 2009-04-09 2014-05-20 Nuclea Biotechnologies, Inc. Antibodies against fatty acid synthase
US9732158B2 (en) 2009-04-09 2017-08-15 Nmdx, Llc Antibodies against fatty acid synthase

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