EP1023076A1 - Utilisation du gene suppresseur de tumeur p-ten dans le diagnostic et le traitement de cancer - Google Patents

Utilisation du gene suppresseur de tumeur p-ten dans le diagnostic et le traitement de cancer

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Publication number
EP1023076A1
EP1023076A1 EP98908523A EP98908523A EP1023076A1 EP 1023076 A1 EP1023076 A1 EP 1023076A1 EP 98908523 A EP98908523 A EP 98908523A EP 98908523 A EP98908523 A EP 98908523A EP 1023076 A1 EP1023076 A1 EP 1023076A1
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European Patent Office
Prior art keywords
nucleic acid
cancer
pten
altered
mutations
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German (de)
English (en)
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EP1023076A4 (fr
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Ramon E. Parsons
Michael H. Wigler
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Cold Spring Harbor Laboratory
Columbia University in the City of New York
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Cold Spring Harbor Laboratory
Columbia University in the City of New York
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Publication of EP1023076A1 publication Critical patent/EP1023076A1/fr
Publication of EP1023076A4 publication Critical patent/EP1023076A4/fr
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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • 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
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • This invention concerns the identification of a tumor suppressor gene designated PTEN and its use in the diagnosis and treatment of cancer.
  • Cancer cells have multiple genomic alterations not found in normal host DNA. These include point mutations, insertions, deletion, chromosomal rearrangements, amplifications (which increase gene copy number) , as well as a variety of karyotypic abnormalities (such as aneuploidy, supernumary chromosomes and losses of parental chromosomes) . Specific genomic changes are known to alter two main categories of cancer-related proteins: oncoproteins, which become activated or over- expressed; and tumor suppressor proteins, which become inactivated or diminished in expression. Loss of genetic information in cancer cells, a particular form of genomic alteration which is relevant here, is often characterized by loss of heterozygosity (LOH) , in which alleles from one parent are found missing, and homozygous deletion
  • LHO heterozygosity
  • HD tumor suppressor genes
  • the presence of a particular genetic lesion can be used to detect the presence of cancer cells, useful when the physician wants to monitor the patient's progress and response to therapy.
  • such hallmarks could be employed to screen for the presence of cancer cells in otherwise asymptomatic patients.
  • the example of the c-abl/bcr fusion resulting from the chromosome 9/21 rearrangement illustrates both of the first two uses. The presence of this alteration is used to diagnose chronic myelogenous leukemia, and to monitor recurrence .
  • oncoproteins which are found altered in cancers can be the target of therapeutic discovery and intervention.
  • Several clinical trials are now underway to test drugs that inhibit RAS function, ERB2 function, and CDK4 function.
  • Many drug discovery programs are targeting the tyrosine kinase type oncogene .
  • tumor suppressor proteins which are mutated in cancers can be the target of therapeutic discovery and intervention.
  • gene therapy experiments in animal models are underway to test the efficacy of restoring a tumor suppressor gene (p53) to cancer cells that have mutated p53.
  • the tumor suppressor protein is an extracellular protein, it may be replaceable therapeutically .
  • one may induce the expression of a tumor suppressor gene by chemical agents, or restore function to a mutated protein, or elevate activity of an attenuated protein, or activate another gene product of similar function.
  • altered oncoproteins or tumor suppressor proteins may be the target for immunotherapy and vaccination.
  • Detection of mutated genes is desirable, clinically, for the diagnosis of the mutation in patients, and, for research purposes, for the verification that a candidate oncogene or tumor suppressor gene is such.
  • a variety of techniques have been employed to detect mutation. These fall into two categories: the analysis of the gene itself and the analysis of the gene product .
  • the gene of a cancer cell may be sequenced and compared to the normal sequence. This can proceed by directly cloning, or producing by PCR, the subject gene from the cancer cell, and analyzing either the cDNA corresponding to the expressed mRNA or the genomic sequences in which the expressed sequences are embedded.
  • the presence of specific mutations within specific genes can be determined by oligonucleotide hybridization. Most recently, this has been accomplished by "chip" technology, wherein the subject oligonucleotide is placed on a solid surface, and then contacted with single- stranded DNA derived from the cancer cell. The thermal stability of the duplex formed is measured and used to detect the presence of mutation.
  • chip technology is that it allows for miniaturization and multiple queries in a high throughput format.
  • the protein When antibodies directed to the gene product are available, the protein can be examined directly from the cancer cell. Its level of expression may be assayed by immuno-cytochemistry, or, following its purification from the cancer cell, its altered electrophoretic mobility, or even its enzymatic function may be assessed. Antibodies to the subject gene-product can be obtained once the gene sequence is known by methods standard in the art.
  • This invention concerns the identification of a tumor suppressor gene, PTEN, which has homology to tyrosine phosphatase and tensin and which is mutated in multiple types of cancer, including breast, prostate, and brain cancer.
  • PTEN tumor suppressor gene
  • This invention further concerns use of this gene including altered forms thereof and its gene product
  • This invention provides a nucleic acid encoding P-TEN or an altered form thereof. This invention also provides a purified P-TEN or an altered form thereof.
  • This invention further provides the above-described P- TENs, wherein the P-TEN has an altered, truncated, or deleted phosphatase domain.
  • This invention additionally provides the above-described P-TENs, wherein the P-TEN is altered, truncated, or deleted at any of the amino acids between positions 90 and 141 inclusively, wherein the positions are set forth in figure 4A.
  • this invention provides a method of diagnosing whether a patient has cancer which comprises detecting the presence in a suitable sample from the patient of either an altered form of P-TEN or a nucleic acid encoding an altered form of P-TEN.
  • this invention provides the above-described method of diagnosis, wherein the altered form of P-TEN is detected based on a loss of heterozygosity or a homozygous deletion at the PTEN locus.
  • This invention provides a method of diagnosing whether a patient has Cowden disease which comprises detecting the presence in a suitable sample from the patient of either an altered form of P-TEN or a nucleic acid encoding an altered form of P-TEN.
  • This invention further provides a method of treating a cancer patient who has an altered form of P-TEN which comprises introducing into the patient wild-type P-TEN or a nucleic acid which expresses wild-type P-TEN in the subject .
  • This invention also provides the above-described methods, wherein the altered form of P-TEN has reduced or no phosphatase activity.
  • this invention provides a method of identifying a chemical compound which may be useful as a drug for the treatment of cancer which comprises testing compounds for effects on P-TEN activity and identifying compounds which exhibit such an effect.
  • Figure 2A The STS-based YAC map of the region surrounding CY17. Marker locations are taken from the Whitehead STS-based map. RH indicates the radiation hybrid interval for CY17. Yac Map indicates the YACs containing CY17 and their location on the map. Cen., centromere; Tel., telomere.
  • Map of homozygous deletions on 10q23 showing the STS markers spanning the deleted region, the four BACs overlapping the region, and the location of PTEN with respect to the STS markers.
  • STS markers Not-5', PTPD, and ET-1 contain exonic sequences of the PTEN gene. Absence of homozygous deletion is indicated with a "+" , and presence of homozygous deletion with a "-" . Numbers to the right indicate the fraction of tumor cell lines and xenografts with the deletion.
  • Breast cancer samples with deletion are xenografts Bxll and Bx38.
  • the glioblastoma line with a deletion encompassing markers JL25 through KP8 is A172, and that with the deletion affecting only ET-1 is DBTRG-05MG.
  • the glioblastoma samples with a deletion across the entire region are the cell line U105 and xenografts 2, 3, and 11, and the samples with deletion of only PTPD, which contains the phosphatase domain, are xenografts 22, 23, 24, 25, and 32.
  • the prostate cancer cell lines with homozygous deletion are NCI H660 and PC-3.
  • the 5' end of the PTEN cDNA was determined to be coincidental with the Not I site 20 Kb from the centromeric end of BAC D by sequencing this BAC with cDNA primers. These maps are not drawn to scale.
  • Figures 3A, 3B, and 3C Homozygous deletions in tumor cell lines and xenografts.
  • Amplification of AFMA086WG9 from breast cancer cell lines and xenografts A 6% polyacrylamide sequencing gel showing the products of PCR. Lane 1, MDA-MB-330, lane 2, MDA-MB-157, lane 3, MDA-MB-134-VI , lane 4, MDA-MB-435S, and breast xenografts , lane 5, Bxll, lane 6, Bxl5, lane 7, Bx38, and lane 8, Bx39.
  • FIG. 3C Homozygous deletions of exon ET-1 in glioblastoma cell lines. Genomic DNA samples were PCR amplified using intronic primers that amplify exon ET-1. The products were resolved on a 1.2% agarose gel and then stained with ethidium bromide. Lane 1 contains a DNA marker. The remaining lanes contain PCR products from control templates and seven glioblastoma cell lines: lane 2, lymphocyte DNA, lane 3, water , lane 4, U118MG, lane 5, A172, lane 6, DBTRG-05MG, lane 7, U373, lane 8, T-98G, lane 9, U-87MG, and lane 10, U138MG. Full length products are present for all templates except water, A172, and DBTRG-05MG.
  • Predicted amino acid sequence of P-TEN The putative phosphatase domain is underlined.
  • the nucleotide sequence has been deposited in the GenBank (accession number U93051) .
  • Abreviations for amino acids are A, Ala; C, Cys; D, Asp; E, Glu; F, Phe ; G, Gly; H, His; I, lie; K, Lys;, L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val ; W, Trp ; and Y, Tyr.
  • NCBI ID numbers are P53916 (Y50.2), M61194 (CDC14) , A56059 (PRL1) , 1246236 (PTP-IV1), 1125812 (CPTPH) , and P24656 (BVP) . Black boxes indicate amino acid identities and gray boxes indicate similarities.
  • FIG. 4C Homology of P-TEN to tensin and auxilin. Alignment was performed as in (B) over the region of highest homology.
  • NCBI ID numbers are A54970 (tensin) and 485269 (auxilin) .
  • glioblastoma cell line DBTRG-05MG Sequence of nucleotides 1039 to 1010 in the antisense orientation from prostate cancer cell lines DU145 (left) , LNCaP (middle) , and the glioblastoma cell line DBTRG-05MG (right) . Arrow indicates the in-frame deletion of nucleotides 822 to 1025 in DBTRG-05MG.
  • Meiotic recombinants define a minimal region for the Cowden disease locus.
  • Consensus critical interval containing Cowden disease locus Black bars represent the affected haplotype, while open bars indicate the unaffected haplotypes.
  • Cowden-critical interval 1 defined as between D10S215 and D10S564 , as of 1996 (Nelen et al . 1996).
  • Critical interval 2 as derived from panel a above.
  • Critical region 3 represents the shortest region of overlap between 1 and 2, placing the interval between D10S215 and
  • FIGS 7A, 7B, and 7C Mutations of PTEN in Cowden disease families. The start and stop codons are underlined.
  • nucleotides 463 to 476 (bottom to top) are shown in the sense orientation.
  • a single base change (G to T) at nucleotide 469 of the affected individual is seen in lane 4, indicated by the arrow.
  • An unaffected family member is in lane 5 and a unrelated control in lane "+" .
  • Case 25 and case 33 are presented. Dinucleotide repeat markers are indicated with the prefix "D10S" left off. C, control, shown from case 22 which is not informative at PTENCA. The potential homozygous deletion region was proposed based on the retention of heterozygosity at PTENCA. Case 25 and case 33 are not informative at D10S541 and D10S532, respectively. Dotted line, missing allele.
  • This invention provides a nucleic acid encoding P-TEN or an altered form thereof.
  • Nucleic acids encoding an altered form of P-TEN may have deletions, additions, or substitutions in the nucleic acid sequence, thereby, resulting an altered P-TEN product .
  • This invention further provides the above-described nucleic acid, wherein the nucleic acid is mRNA, cDNA, or genomic DNA.
  • this invention provides the above-described nucleic acids, wherein the nucleic acid has substantially the same sequence as the sequence shown in figures 1A and IB.
  • This invention further provides the above-described nucleic acids, wherein the nucleic acid encodes a protein having an altered, truncated, ' or deleted phosphatase domain.
  • An altered, truncated, or deleted phosphatase domain may have amino acid deletion, substitutions, or additions. In an embodiment these changes will reduce or eliminate the phosphatase activity.
  • This invention also provides an oligonucleotide probe having a sequence which renders it capable of specifically hybridizing with the above-described nucleic acids .
  • this invention provides a nucleic acid which upon transcription gives rise to one of the above- described nucleic acids .
  • This invention also provides a purified P-TEN or an altered form thereof.
  • altered forms of P- TEN may have amino acid substitutions, deletions, or additions .
  • This invention further provides the above-described P- TENs, wherein the P-TEN has an altered, truncated, or deleted phosphatase domain.
  • This invention additionally provides the above-described P-TENs, wherein the P-TEN is altered, truncated, or deleted at any of the amino acids between positions 90 and 141 inclusively, wherein the positions are set forth in figure 4A.
  • This invention also provides the above-described P-TENs, wherein the P-TEN is altered, truncated, or deleted at any of the amino acids between positions 82 and 131 inclusively, wherein the positions are set forth in figure 4B .
  • This invention further provides the above-described P- TENs, wherein the P-TEN is altered, truncated, or deleted at any of the amino acids between positions 15 and 64 inclusively, wherein the positions are set forth in f igure 4C .
  • this invention provides a peptide having a sequence which is present within the above-described proteins and absent from any other human protein.
  • This invention also provides an antibody or fragment thereof which specifically binds to the above-described proteins or peptides.
  • the term “antibody” includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies.
  • the term “antibody” includes polyclonal and monoclonal antibodies, and binding fragments thereof.
  • the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
  • this invention provides a method of diagnosing whether a patient has cancer which comprises detecting the presence in a suitable sample from the patient of either an altered form of P-TEN or a nucleic acid encoding an altered form of P-TEN.
  • a "suitable sample” may be obtained from blood, urine, or tissue samples.
  • this invention provides the above-described method of diagnosis, wherein the altered form of P-TEN is detected based on a loss of heterozygosity or a homozygous deletion at the PTEN locus.
  • This invention also provides the above-described methods, wherein the cancer results in a gliobastoma.
  • this invention provides the above-described methods, wherein the nucleic acid encoding an altered form of P-TEN has mutations or deletions in exons 4, 5, 6, 7, 8, or 9.
  • this invention provides the above-described methods, wherein the nucleic acid encoding an altered form of P-TEN has mutations or deletions in exons 5 or 8.
  • This invention also provides the above-described methods, wherein the cancer is breast, prostate, melanoma, or brain cancer.
  • This invention provides a method of diagnosing whether a patient has Cowden disease which comprises detecting the presence in a suitable sample from the patient of either an altered form of P-TEN or a nucleic acid encoding an altered form of P-TEN.
  • This invention further provides a method of treating a cancer patient who has an altered form of P-TEN which comprises introducing into the patient wild-type P-TEN or a nucleic acid which expresses wild-type P-TEN in the subject .
  • This invention also provides the above-described methods, wherein the altered form of P-TEN has reduced or no phosphatase activity.
  • this invention provides a method of identifying a chemical compound which may be useful as a drug for the treatment of cancer which comprises testing compounds for effects on P-TEN activity and identifying compounds which exhibit such an effect.
  • This invention further provides the compounds identified by the above-described methods This invention also provides the compounds identified by the above-described methods and a suitable carrier.
  • Suitable carriers are well known to those skilled in the art and include, but are not limited to, aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol , polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants , chelating agents, inert gases and the like.
  • this invention provides the compounds identified by the above-described methods and pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-O.lM and preferably 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • this invention provides the above-described methods, wherein the effect comprises inhibition, activation or enhancement of enzymatic activity.
  • this invention provides the above-described methods, wherein the enzymatic activity comprises phosphatase activity.
  • this invention provides the above-described methods, wherein the effect comprises inhibition, activation or enhancement of expression of the PTEN gene.
  • This invention also provides a previously unknown compound capable of mimicking the activity of P-TEN.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound capable of mimicking the activity of P-TEN and a pharmaceutically acceptable carrier.
  • PTEN tumor suppressor gene
  • LOH heterozygosity
  • RDA representational difference analysis
  • BAC bacterial artificial chromosome
  • the phosphatase domain of PTEN contained the critical (I/V) -H-C-X-A-G-X-X-R- (S/T) -G motif found in tyrosine and dual -specificity phosphatases ( 15) .
  • the phosphatase domain exon mapped within all four BACs and was deleted in all of the homozygously deleted samples with the exception of DBTRG-05MG, thus placing this exon within the region of homozygous deletion near JL25 and AFMA086WG9 (Fig 2B) .
  • the phosphatase domain of encoded by PTEN is most related in sequence to those of CDC14, Phosphatase of Regenerating Liver (PRL-1) , and Baculovirus PTP (BVP) (Fig. 4B) .
  • CDC14 and BVP are dual-specificity phosphatases that remove phosphate groups from tyrosine as well as serine and threonine ( 16) . These phosphatases can be distinguished from the more well characterized VHl-like enzymes by their sequence differences outside of the core conserved domain.
  • PRL-1 and CDC14 are involved in cell growth, and CDC14 appears to play a role in the initiation of DNA replication ( 17) . In contrast to PTEN, these phosphatases do not have extensive homology to tensin and auxilin.
  • PTEN is also homologous to the protein tyrosine phosphatase domains of three ORFs
  • PTEN is a tumor suppressor gene
  • the PTEN allele retained in tumor cells with LOH should contain inactivating mutations.
  • Two truncating mutations in PTEN were identified in the breast samples (Table 1) .
  • MDA-MB-468 cells had a deletion of 44 bp at codon 70, which resulted in a frameshift on the amino terminal side of the tyrosine phosphatase domain.
  • DBTRG-05MG cells had an in- frame deletion of 204 bp caused by the genomically deleted exon ET-1 (Fig. 5B)
  • U373MG had a 2-bp insertion at codon 242
  • U87MG had a frameshift at codon 54.
  • Both of the prostate tumor cell lines had PTEN mutations: LNCaP cells had a 2-bp deletion at codon 6, leading to a frameshift (Fig. 5C)
  • DBTRG-05MG*t Glioblastoma 274 - 342 delete 204 bp in-frame deletion
  • DBTRG-05MG has a genomic deletion of 180 bp within exon ET-1, which includes nearly the entire exon and the splice donor site. Because of the genomic deletion in this cell line, th transcript contains an in-frame deletion of codons 274-342. tTumor cell lines. tPrimary tumors. SMutations are indicated in the sense orientation. All mutations in primary tumors were not found in matched blood DNA.
  • P-TEN tumor suppressor as an enzyme that removes phosphate from tyrosines is intriguing, given that many oncoproteins function in the reverse process - to phophorylate tyrosines (22) .
  • P-TEN and tyrosine kinase oncoproteins may share substrates and the tight control of these substrates through phophorylation is likely to regulate a critical pathway that is altered late in tumor development .
  • the homology of P-TEN to tensin is also of interest. Tensin appears to bind actin filaments at focal adhesions - complexesy that contain intergrins, focal adhesion kinase (FAK) , Src, and growth factor receptors (23) .
  • FAK focal adhesion kinase
  • Integrins have been implicated in cell growth regulation (24) and in tumor cell invasion, angiogenesis, and metatasis (25) , so it is conceivable that PTEN regulates one or more of thes processes. Finally, the identification of P-TEN as a likely tumor suppressor raises the possibility that this protein and its substrates will be useful targets for the development of new therapeutics for cancer.
  • RDA was performed as described in Lisitsyn et al . , Proc Natl Acad Sci 92, 151 (1995) . Diploid and aneuploid nuclei from primary breast cancer cells were separated with a fluorescence activated cell sorter. DNA (100 ng) from each fraction was digested with Bgl II and used to prepare amplicons for 12 separate RDA reactions.
  • Probe CY17 was isolated from one of these reactions, was 236 bp long and was present in the diploid but not in the aneuploid amplicon from which it was derived. Hybridization of CY17 to normal genomic DNA samples digested with Bgl II revealed no evidence of restriction length polymorphism.
  • Amplification conditions were 95 °C for 30 s, 56 °C for 1 min, 70 °C for 1 min, for 35 cycles.
  • CEPH B library Research Genetics, Huntsville
  • the forward PCR primer was labeled with ( 32 P) ATP and used to amplify 40 ng of genomic DNA (11) .
  • the samples were then subjected to electrophoresis and autoradiography .
  • the samples included 25 human breast tumor cell lines available from American Type Culture Collection (ATCC) as well as 40 human primary breast tumors xenografted into nude mice.
  • ATCC American Type Culture Collection
  • the cell lines were HS578T, SK-BR-3, UACC812, UACC893, MDA-MB-453, MDA-MB- 175-VII , MDA-MB468, MDA-MB-361, MDA-MB-231, MDA-MB-436, MDA-MB-415, MDA-MB-330, MDA-MB-157, MDA-MB- 134 -VI , MDA-MB-435S,
  • BAC DNA was prepared using the Nucleobond kit (Nest Group, Southboro, MA) . BACs were digested with Not I and subjected to electrophoresis on a field inversion apparatus. BACs A, B, C, and D were 240, 200, 175, and 120 kb, respectively (see Fig. 2B) . A Not I site was present 20 kb from one end of BAC D. Twelve new STS sites were generated by sequencing both ends of BACs B, C, and D, and shotgun cloning Eco RI fragments. Plasmid DNA was prepared from the cloned Eco RI fragments. DNA was cycle sequenced with appropriate primers using a 33 P ddNTP cycle sequencing kit (Amersham, Cleveland) . STS primers were designed and the relative location of the STSs determined by testing for their presence in the BAC contig. Primer sequences are available upon request .
  • the glioblastoma lines included U105, U118MG, A172, DBTRG-05MG, U373MG, T-98G, U-87MG, and U138MG and 34 glioblastoma xenografts.
  • the prostate cancer cell lines tested were DU145, LNCaP, NCI H660, and PC-3. Microsatellite analysis was performed on the prostate lines and each was found to be unique. With the exception of U105, all lines were obtained from the ATCC.
  • DNA (10 (g) was digested with Eco RI , resolved on a 1% agarose gel and transferred to nylon. The JL25 3-kb probe and the 2-kb contol probe were randomly labelled and hybridized to the blot consecutively.
  • BACs C and D were digested with Bam HI, Bgl II, or both enzymes and ligated into the trapping vector pSPL3. Libraries were transfected into C0S1 cells with lipofectamine and poly(-A) + RNA was extracted after 2 days .
  • An exon trapping kit was purchased from GIBCO/BRL.
  • the map panel #2 monochromosome panel was purchased from the National Institute of General Medical Science (NIGMS) Human Mutant Genetic Cell Repository.
  • Randomly primed cDNA was prepared from each of the cell lines studied. Reverse transcription-PCR reactions were performed with 2 overlapping primer pairs to screen the entire ORF . Primer pairs were as follows :
  • RT-PCR products that produced truncated proteins were directly cycle sequenced (20 ng each) to identify potential mutations. All mutations were verified by repeating the RT-PCR and mutation analysis. cDNA primer sequences are available from upon request . 21. STSs Not-5', PTPD and ET-1 were amplified from primary glioblastoma and blood DNA and the exonic regions sequenced.
  • Cowden disease is an autosomal dominant cancer predisposition syndrome associated with an elevated risk for tumours of the breast, thyroid, and skin (1,2) .
  • Lhermitte-Duclos disease cosegregates with a subset of CD families and is associated with macrocephaly, ataxia, and dysplastic cerebellar gangliocytomatosis (3,4) .
  • the common feature of these diseases is a predisposition to hamartomas, benign tumours containing differentiated but disorganised cells indigenous to the tissue of origin. Linkage analysis has determined that a single locus within chromosome 10q23 is likely to be responsible for both of these diseases (5) .
  • a candidate tumour suppressor gene ( PTEN) within this region is mutated in sporadic brain, breast, and prostate cancer
  • Another group has independently isolated the same gene, which they call MMAC1 , and also found somatic mutations throughout the gene in advanced sporadic cancers (7) .
  • Cowden disease [MIM 158350]
  • MIM 158350 Cowden disease
  • breast cancers develop in approximately 30-50% of affected women and thyroid cancers are found in 10% of affected individuals (9,10) .
  • One-third of CD patients have macrocephaly; and brain tumours, especially meningiomas, are noted as well (9-11) .
  • LDD LDD-CD (4,11)
  • CD families and families with both CD and LDD were found to be linked to this region.
  • the PTEN open reading frame contains a putative protein tyrosine phosphatase domain as well as a region of homology to tensin, a protein that interacts with focal adhesions. Somatic frameshift and nonsense mutations were found throughout the gene in the tumours. In addition, two missense mutations were identified that are predicted to cause amino acid substitutions at codons 129 and 134 within the phosphatase domain. Based on its location on 10q23 and the identification of mutations in sporadic breast and brain tumours, we decided to examine the potential role of PTEN in CD.
  • exon 5 was amplified from an additional 28 unrelated individuals and subjected to restiction digestion analysis using the enzyme BsmFI .
  • This restriction site is lost (GGGAC to GGAAC) in the presence of the missense alteration at codon 129 found in families 2053 and BH. All of these control samples yielded two digestion products, while heterozygous loss of the restriction site was evident in samples from affected members of families 2053 and BH (data not shown) .
  • the mutations in families 2053, BH, C, and D fall within the putative tyrosine phosphatase domain of the gene and would be expected to disrupt its function (12) .
  • the codon G129E missense mutations observed in familes 2053 and BH occurred within the signature sequence found in all protein tyrosine phosphatases and dual-specificity phosphatases (13) . This motif is present within the catalytic core of these enzymes.
  • the glycine at codon 129 is typically conserved in protein tyrosine phosphatases but not in dual-specificity phosphatases, which have serine and threonine as their substrates.
  • PTEN Because germline mutations of PTEN predispose to a breast and thyroid cancer syndrome, and somatic mutations are found in sporadic breast cancer, PTEN becomes an obvious candidate for non-CD breast cancer predisposition. Molecular epidemiologic studies of PTEN in apparently sporadic breast and thyroid cancer cases will be useful to determine if PTEN plays a role as a common low penetrance susceptibility gene. The results of these studies may have important consequences in altering the management of patients with such common cancers as those of the breast and the thyroid.
  • CD cardinal phenotype of CD could hint at PTEN' s physiological function.
  • Multiple hamartomas are the sine qua non of CD; these are disorganised masses composed of cells and tissues normally found in the organ of origin. Therefore, PTEN may act in normal tissues to guide the development of cellular interactions such that cells, tissues, and organs are properly formed with respect to one another.
  • disruptive germline mutations in PTEN are associated with CD, one can postulate that heterozygous mutation, in combination with LOH of the wild type allele, could cause a proliferative and disorganisational tendency, leading to hamartoma formation. This cellular proliferation and disorganisation would be the background on which malignant transformation could occur due to somatic mutation of other tumour suppressor genes and oncogenes .
  • Families 0014, 2053, BH, C, and D are classic CD families that meet the consensus operational diagnostic criteria of the International Cowden Consortium (5,10). Families 0014, 2053, C, and D have been described previously (9,10,14) .
  • Family BH comprises two brothers, both of whom have multiple hamartomas of the skin, lung, kidney, follicular adenoma of the thyroid and hamarto atous polyps of the colon.
  • Genotyping Fourteen dinucleotide repeat markers located on lOq encompassing the Cowden critical interval were used for fine structure typing of Cowden family 0014: D10S537, D10S580 , D10S219 , D10S573 , D10S215, D10S541 , D10S2177, D10S2281 , D10S1739, D10S1615, D10S1571 , D10S1442 , D10S1753 , D10S564 .
  • Each forward primer was 5 'labelled with fluorescent dyes HEX or 6-FAM (Genosys Biotechnologies, Inc., The Woodlands, TX, USA). PCR and electrophoresis conditions as well as data analysis have been described previously (15) .
  • Intronic primers were designed for amplifying six exons of the PTEN gene (Table 3) .
  • Cycle sequencing was performed directly on PCR products using a nested sequencing primer and the 33 P- dideoxy-labelled terminators kit (Amersham, Cleveland, OH, USA) and the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit.
  • the products of cycle sequencing were electrophoresed on standard 6% acrylamide gels or analysed on an ABI 373A or 377 automated DNA sequencer (Perkin-Elmer Corp, Norwalk, CT, USA) . All mutations were independently confirmed ' using both sequencing methods .
  • Cowden disease gene marker studies and measurements of epidermal growth factor. Am. J. Hum . Genet . 38, 908-917 (1986) . 10. Starink, T.M. et al . The Cowden syndrome: a clinical and genetic study in 21 patients. Clnical Geneti cs . 29, 222-233 (1986) .
  • 34 consecutive glioblastomas were studied in detail. Sequencing each of the nine exons amplified from tumor DNA revealed 11 mutations. Analysis of polymorphic markers within and surrounding the PTEN gene identified an additional four homozygous deletion mutations. Loss of heterozygosity (LOH) was observed in 25 of 34 (74%) cases. All mutations occurred in the presence of LOH. PTEN was mutated in 44% (15/34) of all glioblastomas studied and 60% (15/25) of tumors with LOH on lOq. Thus, PTEN appears to be the major target of inactivation on chromosome lOq in glioblastoma multiforme.
  • Glioblastoma multiforme is the most aggressive form of glioma, and patients diagnosed with glioblastoma typically survive less than two years (1) .
  • Genetic analysis of glioblastoma tumorigenesis has identified alterations of pl5/pl6/Rb/CDK4 , p53/MDM2 and EGFR
  • the PTEN gene a tumor suppressor recently discovered on chromosome 10q23, contains a phosphatase domain, and its protein product has phosphatase activity (13-15) . Mutations of this gene have been detected in glioblastoma cell lines and tumors (13,14) . In addition, other types of cancer and the inherited predisposition to cancer, Cowden disease, are associated with PTEN mutations (13,14,16). To determine the frequency of PTEN alterations in glioblastomas, 34 normal-tumor pairs were investigated. The results showed that 74% (25/34) of glioblastomas demonstrated LOH at the PTEN locus, and 60%
  • BAC clone D that contains the 5' end of the gene was also included in the analysis (13) .
  • Each forward primer was labeled with ( ⁇ -32P) ATP by T4 polynucleotide kinase
  • Intronic primer pairs were designed to amplify and sequence each exon, including the splice junctions (16) .
  • Forward and reverse primer sequences for exon 2 are 5 ' - GTTTGATTGCTGCATATTTCAG- 3 ' and 5' -GGCTTAGAAATCTTTTCTAAATG-3' , respectively, for exon 3, 5 ' - AATGACATGATTACTACTCTA - 3 ' and 5' -TTAATCGGTTTAGGAATACAA-3 ' , respectively, and for exon 4 , 5 ' - CATTATAAAGATTCAGGCAATG - 3 ' and 5' -GACAGTAAGATACAGTCTATC-3' , respectively.
  • the sequencing primer for exon 2 is 5' -TCTAAATGAAAACACAACATGAA-3' (antisense), for exon 4, 5' -GATTCAGGCAATGTTTGTTAG-3' (sense).
  • the reverse PCR primer for exon 3 was used as a sequencing primer for the same exon. All primers used in this study were purchased from DNAgency (Malvern, PA) . 10-50 ng of genomic DNA were amplified at 94°C for 30 sec, 54 °C for 1 min and 72 °C for 1 min for 35 cycles.
  • PCR products Prior to sequencing reactions, PCR products were treated with 10 units of exonuclease I and 2 units of shrimp alkaline phosphatase for 25 min at 37 °C and incubated at 80 °C for 15 min. Cycle sequencing (Amersham Life Science) was performed for 25 cycles. Sequencing reactions were resolved on 5% polyacrylamide gels buffered with taurine . Each mutation has been verified independently at least 2 times.
  • Glioblastoma multiforme samples from 34 tumor cases were sequenced throughout all 9 exons of the PTEN gene, and mutations were found in 11 cases (Table 4) .
  • Frameshift mutations (Fig. 8A) that resulted in premature translational termininations were detected in three cases (2, 19 and 21) .
  • case 2 an A insertion at cysteine 136 resulted in a subsequent termination at codon 146; in case 19, an AT deletion at nucleotide positions 227-228 converted tyrosine 76 to a stop codon; in case 21, a 4-bp deletion from nucleotide positions 1011-1014 at codon 337 resulted in a subsequent termination at codon 343.
  • Nonsense mutations were detected at tyrosine 225, arginine 335 and tyrosine 336 in cases 10, 6, and 8, respectively (Fig. 8B) .
  • Five missense mutations were found (Fig. 8C) .
  • a G to T mutation in case 26 resulted in a glutamate 107 to tyrosine alteration, and a G to C change in case 30 resulted in alanine 121 to proline.
  • Glycine 129 and glycine 165 were mutated to arginine in case 20 and case 15, respectively.
  • leucine 345 was mutated to glutamine . No mutations were detected in the blood-derived DNA, indicating that all mutations were somatic.
  • Mutated nucleotides are in boldface type. Positions refer to the deduced PTEN protein sequence (13); fs, frameshift
  • D10S532, D10S1687, D10S541 and D10S583 were initially used to evaluate allelic loss in this locus (Fig. 9) .
  • the frequency of LOH was 83% (19/23) for D10S532, 81% (17/21) for D10S1687, 57% (12/21) for D10S541 and 64% (18/28) for D10S583.
  • Tumors with microsatellite instability were detected in two cases (7 and 18) . Further, there were 8 cases with no detectable chromosomal lesions.
  • a BAC spanning the 5' end was probed with a poly(CA)n oligonucleotide to identify novel CA repeats.
  • a highly polymorphic marker, PTENCA was obtained that is located within the PTEN gene. This marker was informative for 82% (28/34) of samples. LOH was detected in 68% (19/28) of the samples and all these cases were also hemizygously deleted in at least one of the flanking loci.
  • a point mutation polymorphism in intron eight of PTEN demonstrated LOH in 68% (15/22) of informative samples.
  • 25 cases (74%) showed loss of heterozygosity at two or more of these loci (Fig. 9) .
  • LOH frequency on chromosome lOq ranges from 83%- 95% with the epicenter of loss located at 10q24-25 (9-11) .
  • the epicenter of LOH on 10q24-25 may be a summation of deletions targeting PTEN and random partial chromosomal losses distal to the PTEN locus.
  • all examples of LOH indicate chromosomal break points that are centromeric to PTEN (Fig. 9) . These data indicate that LOH in this region typically includes the PTEN gene. Alternatively, another tumor suppressor may exist distal to PTEN.
  • glial tumor development clearly implicates chromosome lOq and therefore PTEN in the transition from anaplastic astrocytoma to glioblastoma multiforme (2) .
  • chromosome lOq PTEN in the transition from anaplastic astrocytoma to glioblastoma multiforme (2) .
  • p53 is altered in all grades of astrocytoma-derived tumors, suggesting that p53 inactivation is an early step in glial tumor formation (6) .
  • Progression to anaplastic astrocytoma is associated with mutations of pl5/pl6/Rb/CDK4 (4,5) .
  • the transition to glioblastoma correlates with loss of chromosome lOq and an increase in the freqency of alterations affecting the cyclin D pathway (4,5) .
  • glioblastomas are diagnosed in the absence of a prior lower grade glial tumor, however.
  • p53 mutations are not commonly observed, rather EGFR amplifications are seen at high frequency along with alterations of lOq and the cyclin D pathway.
  • p53 and EGFR alterations are not found in the same tumor (8) .
  • individual tumors accumulate alterations in the cyclin D regulatory pathway, chromosome lOq, for which PTEN is the likely target, and either p53 or EGFR.
  • CDK4 amplification occurs in the majority of glioblastomas. Cancer Res. 54:6321-6324, 1994.
  • Li, D., and Sun, H. TEP1 encoded by a candidate tumor suppressor locus is a novel protein tyrosine phosphatase regulated by transforming growth factor b. Cancer Res. 57: 2124-2129, 1997.

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Abstract

L'invention concerne un acide nucléique codant pour le gène P-TEN ou pour une forme modifiée de celui-ci. L'invention concerne également un gène P-TEN purifié ou une forme modifiée de celui-ci. L'invention concerne en outre les gènes P-TEN décrits, dans lesquels P-TEN possède un domaine phosphatase modifié, tronqué ou supprimé. De plus, l'invention concerne un procédé permettant de diagnostiquer un cancer chez un patient, qui comporte l'étape consistant à détecter la présence, dans un échantillon approprié provenant du patient, d'une forme modifiée de P-TEN ou d'un acide nucléique codant pour une forme modifiée de P-TEN. D'autre part, l'invention concerne un procédé permettant d'identifier un composé chimique pouvant être utile comme médicament pour le traitement de cancer, ce procédé comportant les étapes consistant à tester des composés pour rechercher des effets d'une activité de P-TEN, et à identifier des composés présentant un tel effet.
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US6777439B2 (en) * 2000-05-30 2004-08-17 Advanced Research & Technology Institute, Inc. Compositions and methods for identifying agents which modulate PTEN function and PI-3 kinase pathways
WO2004074459A2 (fr) * 2003-02-19 2004-09-02 Georgetown University Cellules deficientes en pten et utilisations de celles-ci
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Title
LI D-M ET AL: "TEP1, ENCODED BY A CANDIDATE TUMOR SUPPRESSOR LOCUS, IS A NOVEL PROTEIN TYROSINE PHOSPHATASE REGULATED BY TRANSFORMING GROWTH FACTOR BETA1-" CANCER RESEARCH, vol. 57, 1 June 1997 (1997-06-01), pages 2124-2129, XP002066157 ISSN: 0008-5472 *
LIAW DANNY ET AL: "Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome." NATURE GENETICS, vol. 16, no. 1, 16 May 1997 (1997-05-16), pages 64-67, XP001024445 ISSN: 1061-4036 *
LO SU HAO ET AL: "Molecular cloning of chick cardiac muscle tensin: Full-length cDNA sequence, expression, and characterization." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 35, 1994, pages 22310-22319, XP002178161 ISSN: 0021-9258 *
MYERS ET AL: "P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatse" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 94, 1 August 1997 (1997-08-01), pages 9052-9057, XP002088258 ISSN: 0027-8424 *
NELEN M R ET AL: "Localization of the gene for Cowden disease to chromosome 10q22-23." NATURE GENETICS, vol. 13, no. 1, 1996, pages 114-116, XP001024519 ISSN: 1061-4036 *
See also references of WO9834624A1 *

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