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WO1999023252A1 - Gene du cancer - Google Patents

Gene du cancer Download PDF

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Publication number
WO1999023252A1
WO1999023252A1 PCT/GB1998/003375 GB9803375W WO9923252A1 WO 1999023252 A1 WO1999023252 A1 WO 1999023252A1 GB 9803375 W GB9803375 W GB 9803375W WO 9923252 A1 WO9923252 A1 WO 9923252A1
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hsc73
exon
dna
gene
protein
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PCT/GB1998/003375
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English (en)
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Christopher James Bakkenist
John Koreth
James O'donnell Mcgee
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Isis Innovation Limited
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Priority claimed from GBGB9723412.4A external-priority patent/GB9723412D0/en
Priority claimed from GBGB9818118.3A external-priority patent/GB9818118D0/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Priority to AU10451/99A priority Critical patent/AU1045199A/en
Publication of WO1999023252A1 publication Critical patent/WO1999023252A1/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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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

  • This invention relates to methods of diagnosing neoplasia or a susceptibility to neoplasia, in particular malignant neoplasia, which method is based on detecting abnormalities in or affecting the HSC73 (Heat Shock Cognate 73) gene (and/or gene products).
  • the invention further relates to reagents for use in such methods.
  • the invention also relates to genetically modified non-human mammals having a general weakness or a predisposition to cancer and/or other diseases, as a result of a genetic modification to or affecting a HSC73 gene.
  • the initiation of tumour development is considered an irreversible genetic change that occurs in cells either spontaneously or upon exposure to chemical and physical agents. A selective expansion of initiated cells then occurs with a concurrent increase in genetic lesions.
  • sufficient mutations in oncogenes and tumour suppresser genes accumulate to irreversibly remove the cells from growth control. Mutations which promote malignancy are expected to result in the dysfunction or loss of proteins which protect DNA from damage, proteins which repair DNA and proteins involved in cell cycle checkpoints which control the normal chromosome complement.
  • mice with a specific gene mutation e.g. TRP53
  • gene amplification e.g. Hras
  • expressing a viral gene e.g. HBV-X
  • a mouse is needed which is compromised in all pathways which can result in the malignant phenotype but which is not initiated or committed in any of them. What is needed is a hemizygous mouse which is compromised generally in homeostasis; compromised but not initiated to neoplasia by any specific pathway. Such a mouse model would detect all agents which can promote cancer, in a shorter period than an unmodified mouse, since less gene hits should be required.
  • D11S29 An anonymous locus at 11q22.3 (D11S29) was found to be deleted in a significant number of breast cancers. Subsequently the incidence of 11q allelic deletions in breast cancer was examined by Southern blot analysis in paired fresh normal and breast cancer tissue (1 ,2). Of 41 patients with sporadic breast cancer, 59% were shown to have lost one allele at the D11S29 locus. Four other probes (D11Z1 , INT2, DRD2, ETS1) proximal and distal to D11S29 did not show allelic deletion. This study was expanded by a microsatellite PCR study. Of 83 patients who were heterozygous at D11S29, 42 % demonstrated allelic imbalance (3).
  • deletion event(s) in breast cancer paraffin embedded archival tissue was examined by PCR with multiple microsatellite markers. It was found that the region 11q22-q23.1 (8cM) was deleted in 63% of sporadic breast cancers and a novel region of allelic deletion at chromosome 11q25 (2cM) was also identified in 51% of the cases studied (4). In all, deletions along 11q were present in 72% of sporadic breast cancers. When contrasted with a 10% "background" rate of allelic deletion in colorectal carcinomas, these data are strongly indicative of genetic events occurring at chromosome 11q22-q23.1 and 11q25 which may be of importance in the pathogenesis of breast cancer.
  • the deletion event at 11q22-23.1 has been examined in DCIS (a putative pre-invasive lesion) in patients with concomitant invasive cancer. Of 18 patients, 17 were concordant for deletion/or its absence at D11S29 (3). Further allelic deletion at this locus was present in lymph node metastasis (75%o) from carcinomas with the deletion. Allelic deletions in invasive breast cancer have been confirmed by others at 11q22-24 (5) or 11q22-23
  • allelic deletion at 11q22- 24 may be an early event in the neoplastic process.
  • Deletions of 11q have also been described in ovarian cancer (11q22-24) (10), gastric cancer (11) and lung cancer (12). Collating the allelic deletion data along 11q for all tumour types (see Figure 1), 3 minimal regions of allelic loss have been identified at 11q22-23.1 , 11q23.3-24 and 11q25-qter (13).
  • chromosome 11q may accumulate affecting up to 3 specific loci during the development of these carcinomas.
  • Several other lines of evidence implicate chromosome 11q in malignancy. Cytogenetic analysis of breast cancer has demonstrated anomalies at 11q22-25 (14). Numerical aberrations in chromosome 11 and 17, and chromosome 11 and 17 inequality, have been correlated with lymph node metastases or disseminated disease in breast cancer patients (15). Since the 1980's, micro-cell transfer has implicated chromosome 11 as a potent tumour suppresser source in cervical derived HeLa cervical cancer cells (16). Functional tumour suppression with chromosome 11q has also been shown in breast (6), lung (17) and melanoma (18) cell lines and metastasis suppression has been identified in the MDA breast cancer cell line (19).
  • chromosome 11q has been linked to cancer, no basis has been provided for a reliable test relating to this.
  • the regions of the chromosome involved have been until now too large and non-specific.
  • the region of the chromosome from 11q23.3 to 11q25 is greater than 10 megabases long and possibly contains 50 or more genes (most of which are as yet unidentified).
  • the first murine heat shock cognate sequence published was that of a clone obtained from a teratocarcinoma derived cell line (F9) from the 129/SvJ strain of mouse (27). This sequence was named HSC72. Subsequently a second heat shock cognate sequence was published and named HSC73 (28). These two sequences differ by a point mutation in exon 3 which results in a single amino change at position 129 from Asp to Asn.
  • HSC73 human Heat Shock Cognate 73
  • HSC73 has been fine-mapped to a YAC (yeast artificial chromosome) at 1 1 q23.3.
  • HSC73 expression is qualitatively aberrant by Northern blotting, RT- PCR (reverse transcriptase-PCR) and sequencing in 70% of RNA samples extracted from immortalised malignant breast cell lines and non-familial (i.e. sporadic) breast cancers, but not in paired constitutional samples (both breast and skin). Sequencing demonstrates that the aberrant transcripts are generally interstitial deletions of a large number of exons encoding the HSC73 protein (typically covering exon 2 or 3 to exon 9) with break points in exons.
  • HSC73 is therefore abberant at the RNA level in 70% of sporadic breast cancers.
  • tumour cells show either absent or reduced expression of HSC73 RNA in 70% of sporadic breast carcinomas and 50% of DCIS.
  • Breast tumours show increased expression of HSC73 protein in approximately 70% of cases (it is believed that this results from the accumulation of mutated protein).
  • HSC73 gene products has been found in cancers other than breast cancer, for example cervical and colorectal carcinoma and a malignant lymphocytic cell line (for which mutated transcripts have been detected).
  • the DNA mutations so far identified in HSC73 in clinical carcinomas of the breast are generally point mutations and small insertions/deletions of sequence.
  • the mutations are described in Example 1.
  • the mutations identified in HSC73 are in exon 3 (the second coding exon) of the gene and are not present in DNA purified from adjacent non- malignant breast tissue or skin from the same patient. These nucleotide changes result in amino acid substitutions.
  • the nucleotide changes and amino acid substitutions are listed in Example 1. This suggests that some change(s) in HSC73 protein function (either gain or loss) is acquired as a result of mutation in exon 3 which confers a growth advantage to the tumour cell.
  • Exon 3 is critical for the ATPase activity of the protein (O'Brien er a/ 1996, J. Biol. Chem. 271 ; 15874-8).
  • Allelic imbalance data implicates 11 q23.3 as the location of a multi-tumour suppressor gene.
  • the HSC73 gene maps to this location and is mutated in human clinical carcinomas of the breast.
  • HSC73 Disruption of HSC73 is expected to be involved in a wide variety of malignant and non-malignant diseases.
  • the protein encoded by HSC73 is an essential component of the centrosome (22), a genome caretaker (23), a participant in protein degradation pathways (24 - subsequently independently confirmed) and essential for maintaining proteins in a semi folded state in order to enable translocation through the mitrochondrial and endoplasmic reticulum (it is therefore essential for steroid hormone receptor activation) (25).
  • RNA encoding HSC73 which have been characterised in malignant disease also delete three small nucleolar RNAs 14 (SNURPs) which are essential for ribosome biosynthesis and 18S rRNA processing (26).
  • SNURPs small nucleolar RNAs 14
  • the three SNURPs are encoded by short sequences in introns 5, 6 and 8 of HSC73.
  • the invention provides in one aspect a method of diagnosis performed on a biological sample, which method comprises detecting the aberrant expression of a gene at the HSC73 locus, or detecting a mutation at the HSC73 locus, in the sample.
  • Such a method is useful in particular for diagnosing neoplasia or a susceptibility to neoplasia, especially malignant neoplasia.
  • the diagnosis may be a predictive diagnosis, for example it may predict whether or not in situ neoplasia such as DCIS will become invasive.
  • the invention provides novel reagents for use in the method according to the invention.
  • reagents include but are not limited to: i) Oligonucleotides and panels of oligonucleotides capable of indicating the presence of a mutation at the HSC73 locus by hybridising or by not hybridising to DNA or RNA.
  • Such oligonucleotides may be employed as primers or probes.
  • the invention is concerned in particular with oligonucleotides which hybridise specifically under stringent conditions, to exon 3 or a region of exon 3 of the HSC73 gene.
  • the oligonucleotides may be specifically complementary either to the wild type HSC73 sequence or to a mutant HSC73 sequence.
  • Monoclonal antibodies and panels of monoclonal antibodies capable of indicating the presence of a protein product of a mutant HSC73 are concerned in particular with antibodies which bind specifically to a region of the HSC73 protein encoded by exon 3, especially mutated exon 3. Such antibodies can be easily raised for example using recombinant HSC73 polypeptides or peptides generated using known techniques.
  • the invention further provides synthetic peptides capable of generating such antibodies and isolated or recombinant proteins or polypeptides capable of generating such antibodies.
  • the invention provides a genetically modified non-human mammal having genetic weakness to disease, in which the somatic cells and germ cells have at least one mutated or deleted copy of HSC73.
  • Such genetically modified animals will be susceptible to cancer by a variety of pathways, since their control of homeostasis is weak. At the neonatal stage, they will generally be healthy animals which are not initiated in any cancer pathway.
  • the invention further provides an embryonic stem cell transformed with a nucleic acid capable of replacing a copy of HSC73 in the cell.
  • the nucleic acid may be either a mutated copy of HSC73, or a marker such as a drug resistance marker.
  • Such a cell may be regenerated into a genetically modified animal according to the invention.
  • the invention provides in yet another aspect a cell line genetically engineered to have at least one mutated or deleted copy of HSC73. Genetically modified HSC73 cell lines will be useful as targets for therapeutic testing and development of anti-cancer agents.
  • the invention provides a method of testing a compound for biological activity, particularly toxicity, which method comprises exposing a cell line or a genetically modified non-human mammal according to the invention, to the compound.
  • kits for diagnostic and screening purposes in particular a kit for performing a method of diagnosis described herein which kit comprises an oligonucleotide which hybridises specifically to a DNA which encodes a mutated or unusual polymorphic form of an HSC73 mRNA or peptide, and not to a DNA which encodes wild type HSC73 mRNA or peptide.
  • the invention provides uses in gene therapy.
  • a method of treatment comprising introducing into a patient at least one copy of a nucleic acid which encodes a native HSC73 under the control of a suitable promoter; and a composition comprising a gene delivery vector which comprises a nucleic acid encoding an HSC73 protein under the control of a suitable promoter, together with a pharmaceutically acceptable carrier.
  • Suitable vectors for delivery of the nucleic acid are well known in the art of gene therapy, as also are approp ⁇ ate promoters for the expression of the HSC73 protein in vivo.
  • the introduction of a gene in gene therapy may be carried out on cells in vitro which cells are then introduced or reintroduced into the patient, or it may be carried out by delivery of the gene to cells in vivo.
  • the invention will be useful in the development of new anti-cancer therapies.
  • Pharmaceutical agents which can upregulate or replace a function of HSC73 or which are inhibitory to mutated HSC73 may be beneficial.
  • Mutated HSC73 may indicate tumour susceptibility to individualised therapeutic protocols.
  • Gene or protein replacement therapy for HSC73 in malignant tissue may also be beneficial, since HSC73 is ubiquitously expressed.
  • a suitable experimental model for the technical development of gene therapy would be a genetically modified mouse or other non-human mammal model, such as a hemizygous HSC73 "knockout" mouse model.
  • novel therapies directed to DNA gene therapy
  • therapies directed to RNA (antisense technology) and proteins binding, modifying agents and immunotherapy
  • the human HSC73 sequence is found at GenBank Accession Number Y00371 (see Figure 5).
  • the murine HSC73 sequence is found at GenBank Accession Number J 17982.
  • Figure 1 shows an ideogram of chromosome 11 , focusing on the 11q22-qter region. Marker loci are arrayed vertically (inter marker distance not to scale) and tumour types listed horizontally. Thick vertical lines indicate minimum regions of deletion reported (references in text), and barred boxes show consensus regions of deletion between different reports, for a given tumour type. Regions 1 and 2 show possible localisation of multi-tumour suppressor genes deleted in above tumour types. HSC73 maps to region 2.
  • HSC73 Immunohistochemistrv (on fresh and formalin fixed paraffin embedded tissue)
  • the gene which encodes HSC73 has nine exons, eight of which are coding.
  • the gene is mutated at the DNA and/or the RNA level in a high percentage of cases of several types of tumour (see below). This leads to aberrant expression of HSC73.
  • HSC73 in the tumours listed detected with the polyclonal antibody, is increased over normal tissue ⁇ in approximately 80% of cases.
  • Other malignant disease e.g. cervix, colon, stomach and skin
  • HSC73 can be mutated both at the DNA and/or RNA level
  • a panel of monoclonal antibodies raised against synthetic peptides with one antibody against each coding exon can be used to characterise the expression of HSC73 in malignant tissue.
  • the expression of HSC73 detected with each of these antibodies is correlated with diagnostic and prognostic data and with the expression detected by other antibodies.
  • Increased expression of the protein detected with an antibody against one exon may reflect an increased expression of the gene from one (or both) alleles or an accumulation of mutated HSC73 which cannot be degraded correctly.
  • a reduction in expression detected by an antibody may reflect a deletion in the gene either at the DNA and/or RNA level which removes an amino acid sequence from HSC73 expressed from one or both alleles; or it may reflect deletion of the entire gene from one (or possibly both) alleles.
  • the full sequences for both copies of the gene in malignant breast tissue and in paired normal tissue can be obtained by standard techniques. If a conserved point mutation(s) is detected which affects protein sequence(s), a synthetic peptide(s) corresponding to this sequence may be prepared and a monoclonal antibody raised against it which is specific to the mutant protein. The expression of HSC73 detected with one or more such antibodies is then correlated with diagnostic and prognostic data. It is anticipated that an antibody or a combination of such antibodies may be used for detection of aberrant HSC73 expression which will be of use in a routine histopathology laboratory for:
  • Diagnosis of neoplasia (invasive and in situ) Prognosis of malignancy (invasive disease).
  • - Prognosis of in situ neoplasias such as DCIS/LCIS and CIN3.
  • 2) RNA in situ hybridisation (on fresh and formalin fixed paraffin embedded tissue).
  • HSC73 mRNA in malignant disease of the breast, ovary, prostate and lung using a synthetic antisense oligonucleotide complementary to exon nine.
  • the expression of HSC73 mRNA in these tumours detected with this synthetic oligonucleotide is decreased in comparison to normal tissue in approximately 80% of cases.
  • the expression of HSC73 mRNA in other malignant disease e.g. cervix, colon, stomach and skin
  • an antisense oligonucleotide against each coding exon can be used to characterise the expression of HSC73 mRNA in malignant tissue from all tissues.
  • the expression of HSC73 mRNA detected with each of these synthetic oligonucleotides is correlated with diagnostic and prognostic data and with the other oligonucleotide.
  • Increased expression of the RNA detected with an oligonucleotide against one exon may reflect an increased expression of the gene from one (or both) alleles or it may reflect an accumulation of mutated HSC73 mRNA which cannot be degraded correctly.
  • a reduction in expression detected by a synthetic oligonucleotide may reflect mutation in the gene at the DNA and/or RNA level, which removes the target sequence from the HSC73 mRNA expressed from one or both alleles.
  • the mRNA detected may be decreased because it has a decreased half life as the result of a mutation or because it is not being expressed at its usual levels due to errors in the control of its transcription which may involve a positive feedback loop.
  • the gene may also be entirely deleted from one (or possibly both) alleles. If, in sequencing the HSC73 gene in malignant breast tissue and paired normal tissue, a conserved point mutation(s) is detected which affects mRNA sequence(s), a synthetic oligonucleotide(s) specific to it can be prepared.
  • HSC73 mRNA detected with each of these synthetic oligonucleotides is then correlated with diagnostic and prognostic data.
  • Synthetic oligonucleotides may be prepared which are antisense to each of the three U14 SNURPS, for use in examining the expression of the three SNURPS in malignant tissue from all tissues.
  • the expression of the three SNURPS detected using these synthetic oligonucleotides is then correlated with diagnostic and prognostic data.
  • a synthetic oligonucleotide staining procedure for HSC73 mRNA expression will be of use in a routine histopathology laboratory for: Diagnosis of neoplasia (invasive and in situ) Prognosis of malignancy.
  • HSC73 protein and HSC73 mRNA in malignant disease largely parallels that of p53 and TP53 mRNA (29). Staining techniques similar to those for p53 and TP53 mRNA can be devised for HSC73 and HSC73 mRNA.
  • a suitable method for detecting the numbers of copies of each HSC73 exon and each of the three SNURPS is as follows. At least two oligonucleotides, each labelled with a different detectable label, are provided. One oligonucleotide hybridises to a reference marker in the genome which is known not to be mutated in malignant disease. The second oligonucleotide hybridises to an exon of HSC73 or the three SNURPS. Comparison of the signals detected allows gene dosage measurements to be made for each exon of HSC73 and each of the SNURPS. Gene dosage measurements are correlated with diagnostic and prognostic data.
  • the detection systems that may be used include radiolabelled or fluorescence labelled oligonucleotide probes (including prompt [e.g. fluorescein] and time resolved fluorescence [lanthanide cheiates like Europium and Samarium]) with or without a prior gene amplification (e.g. by PCR) step.
  • radiolabelled or fluorescence labelled oligonucleotide probes including prompt [e.g. fluorescein] and time resolved fluorescence [lanthanide cheiates like Europium and Samarium]
  • prompt e.g. fluorescein
  • time resolved fluorescence lanthanide cheiates like Europium and Samarium
  • Detection of point mutations may also involve oligonucleotide hybridisation.
  • the oligonucleotides used for these experiments will normally be very short, and will be specific for the mutation. They will be designed such that they will not hybridise to the published normal sequence of the gene under stringent conditions. Point mutations are correlated with diagnostic and prognostic data. Detection systems may be as already described.
  • Suitable methods to detect mutation also include technologies such as PCR amplification, cycle sequencing and TaqmanTM PCR. Mutations will again be correlated with diagnostic and prognostic data.
  • Detection of mutations can be used for:
  • Diagnosis of neoplasia invasive and in situ
  • Prognosis of malignancy invasive disease
  • Mutations within the HSC73 coding region of the genome may be germline and therefore transmitted through the population predisposing individuals to cancer (and possibly other disease).
  • Detection systems may be as already described for the DNA analysis techniques. Point mutations may be detected using short oligonucleotide probes specific for the mutation. The probes are designed such that they will not hybridise to the published normal sequence of the gene under stringent conditions. Point mutations will be correlated with diagnostic and prognostic data. Detection systems used may be as already described. Suitable methods to detect mutations also include technologies such as PCR amplification, cycle sequencing and TaqmanTM PCR. Mutations will again be correlated with diagnostic and prognostic data.
  • Detection of mutations in the mRNA or SNURPS may be used for:
  • Diagnosis of neoplasia (invasive and in situ) Prognosis of malignancy (invasive disease). Prognosis of in situ neoplasias.
  • a mouse carrying a hemizygous deletion of HSC73 and U14 SNURPS is a good model for carcinogenesis testing since: General house keeping is compromised.
  • the HSC73 protein is expressed in every cell in the body
  • HSC73/U14 SNURPS are mutated both at the RNA and protein level in many different human cancers.
  • a mouse carrying a hemizygous deletion of HSC73 and U14 SNURPS is susceptible to cancer in many ways: - Increased mutation rate since there is reduced genome protection as a result of a reduced chaperone complement.
  • HSC73 is an essential component of the centrosome. Steroid hormone activation is compromised leading to aberrant inter and intra cell signalling and possible increased mitogenesis. Protein degradation in proteosomes is compromised since HSC73 is essential for the transport of a subset of proteins into the 28S proteosome.
  • HSC73 is essential for transport across membranes. Cells are therefore compromised in regard to self/self and self/antigen recognition.
  • Ribosome biosynthesis and RNA processing are compromised since U14 SNURPS are essential for pre-ribosomal RNA splicing. As a result translation is generally compromised.
  • a mouse carrying a hemizygous deletion of HSC73 and U14 SNURPS is a good model for carcinogenesis testing.
  • the pathway to neoplastic disease promoted by a chemical in this animal may be detectable.
  • As the hemizygous deletion of HSC73 and U14 SNURPS knock out mouse is characterised with respect to development the compromised cellular pathways and functions will be distinguished. It should be possible to demonstrate that a chemical is a carcinogen in the hemizygous deletion of HSC73 and U14 SNURPS mouse model of carcinogenesis testing if for example it damaged the remaining steroid hormone activation function to an extent which removed hepatocytes from normal growth control.
  • Manufacture of a mouse carrying a hemizygous deletion of HSC73 and U14 SNURPS The cloning of murine HSC73 has been described previously
  • Isolation of a 129/SvJ genomic BAC HSC73 clone requires PCR screening of the library with oligonucleotides which will not hybridise to processed pseudogenes elsewhere in the genome.
  • the best oligonucleotides therefore span the U14 SNURPS which are in the introns of HSC73 in mouse and man. This provides three autonomous reactions with which to screen the library.
  • Isogenic targeting constructs are then prepared from the BAC clone for homologous recombination.
  • the entire HSC73 gene and associated SNURPS may be replaced by neomycin or another drug marker or the "normal" HSC73 may be replaced by a mutated copy and the drug marker inserted into the BAC elsewhere.
  • the homologous recombination HSC73 clone is electroporated into RW-4 embryonic stem cells and plated onto MEF feeder cells. Transformed embryonic stem cells are selected for by antibiotic selection, and a minimum of 150 clones are screened by Southern blotting and PCR for appropriate homologous recombination between the engineered target and the "normal" HSC73 locus in the 129/SvJ mouse genome. Recombinant clones are expanded and karyotyped. Those in which satisfactory recombination has occurred are used for either blastocyst injection or morula aggregation in order to generate chimeric mice. These will be used to obtain founder mice. The mice strains will be maintained by conventional breeding. EXAMPLES EXAMPLE 1
  • RNA-ISH RNA in-situ hybridisation
  • IHC protein immunohistochemical
  • HSC73 RNA was expressed at a high level (RNA-ISH score 5/5) in all normal tissues examined (breast acini, lymphocytes, endothelium, skin). This was associated with moderate tissue levels of the HSC73 protein (IHC score 3/5). Assay of the 8 non malignant breast lesions revealed a similar uniform intense staining of HSC73 RNA in the lesional cells and adjacent histologically normal breast acini (RNA-ISH score 5/5). Increased protein staining (IHC score 4/5) was seen in 3 of 5 fibroadenomas (60%) and focally in the 3 fibrocystic samples (apocrine metaplastic cells only) ( Figure 1.1 A and B). As expected of a constitutive 'housekeeping' gene, the ⁇ 2.3kb HSC73 mRNA transcript is uniformly expressed in many normal tissues ( Figure 3 a).
  • RNA, protein and DNA analysis could not span the complete sequence, necessarily limiting mutation detection and data overlap from different analytic methods. Nonetheless, all cases with truncated bands on Northerns had RT-PCR mutations, and where no aberrant band was detected by Northern analysis and RT-PCR, no aberrant bands were detected by Western blotting.
  • HSC73 mRNA and protein expression occurred in 31 of 44 (70%) invasive breast cancers.
  • Aberrant HSC73 bands were also detected on Northern blots (44%) and RT-PCR (67%>). Sequencing revealed intra-exon breakpoints in most of the truncated tumour RNA, while Western blots and IHC showed aberrant protein expression patterns and mutation, with over-expression of wild-type (Wt) band and the presence of multiple truncated mutant (Mt) bands. Mutations in the DNA have been discovered as described above.
  • the table shows deduced HSC73 gene structure and expression data for the 18 fresh cases, with mutated cell lines listed below.
  • a contiguous chromosome 11q22-qterm YAC library plated in an ordered array was hybridised to oligonucleotides specific for HSC73, localised to an 11q23.3 YAC (y667F08) between D11S1336 and D11S1284 (11.7cR; ⁇ 500kb) and confirmed by PCR and southern analysis (data not shown).
  • RNA-ISH sections were dewaxed, rehydrated, proteinase K digested (1 mg/ml in 2mM CaCI 2 , 10mM Tris, pH 7.4) for 30min at 37°C and hybridised overnight at 37°C in 50% deionised formamide, 2X SSC, 20% dextran sulphate, 100mM antisense probe
  • RNA was resolved on 1.5% agarose-3% formaldehyde gels, integrity visualised by ethidium bromide, blotted to Hybond N+ (Amersham) and hybridised overnight to a ⁇ 32 P labelled cDNA probe from exon 5-3' polydenylation site (ATCC IB743), in 50% formamide, 0.75M NaCI, 36mM Na 2 HP0 4 , 4mM NaH 2 P0 4 , 0.15M Tris-HCI, pH 8.0, 10mM EDTA, 5X Denhardts, 200 ⁇ g/ml denatured salmon sperm DNA and 0.5% SDS (w/v) at 42°C.
  • Washes were for 15 min in 2X SSC, 0.1% SDS; 0.5X SSC, 0.1 % SDS and up to 4 times in 0.1X SSC, 0.1 % SDS at 65°C and autoradiographed on Xomat-MS film (Kodak) at -70°C for up to 7 days.
  • Multiple-tissue northerns were purchased (Clontech) and probed as above.
  • 1.5 ⁇ l cDNA was used in 35 cycles of hot-start PCR using l Opmoles of primers P1 (5' TTGTGGCTTCCTTCGTTA 3' [SEQ ID NO:3]) and P2 (5' CAGTGTATAAAGTGCAATG 3' [SEQ ID NO:4]) in 200 ⁇ M dNTPs, 1.5mM Mg(OAc) 2 , 1 X XL PCR buffer (Perkin-Elmer) and 0.5 units of rTth XL Taq (Perkin-Elmer) in a 25 ⁇ l reaction on a Perkin Elmer 9600 cycler at 94°C denaturation for 0.25 min, 54°C annealing for 0.25 min and 72°C extension for 1.25-2.75 min.
  • DNA fragments were excised, purified with a QIA quick kit (Qiagen), ⁇ 35 S cycle sequenced by the Amplicycle kit (Perkin Elmer) with each inner primer for 25-30 cycles, resolved on 6% denaturing urea-polyacrylamide gels and visualised on Xomat-AR film (Kodak).
  • PCR 50ng RNaseA treated DNA was used to amplify ⁇ 3.8kb of the HSC73 genomic sequence (intron 1-3' untranslated) by 42 cycles of hot- start PCR with 15pmoles of the primers P5 (5': TGGTTAAGTGTTCTGTTAAG 3' [SEQ ID NO: 7]) and P6 (5' CAATTGTATGGTGCCAATTT 3' [SEQ ID NO: 8]) in 200 ⁇ M dNTPs, 1.5mM MgCI 2 , 50mM KCI, 10mM Tris-HCI (pH 8.0), 0.1% Triton X-100 and 0.5units SuperTaq (HT Biotechnology, UK) in a 25 ⁇ l reaction at 94°C denaturation for 0.25 min, 56°C annealing for 0.25 min and 70°C extension for 2.5-4 min. PCR products were visualised on 0.7% agarose gels.
  • Immunohistochemistry Sections were dewaxed, rehydrated, pressure cooked for antigen retrieval (1.5 mins at maximum pressure; 10mM sodium citrate buffer, pH 6-0), incubated with a goat polyclonal antibody (Santa Cruz, USA) against HSC 73 amino acids 583-601 , at 1 :200 dilution in PBS/BSA for 60 minutes at 37°C. Secondary incubation used a biotinylated anti-goat antibody (DAKO) followed by a standard avidin-biotin complex (ABC, DAKO) for 30 minutes and developed with DAB.
  • DAKO biotinylated anti-goat antibody
  • ABS standard avidin-biotin complex
  • the membrane was incubated in horse radish peroxidase conjugated anti-goat antibody (1 :500) (DAKO), washed in 1X TBS, 0.1 % Tween 20 (v/v) and developed by ECL (Amersham). Coomassie Brilliant Blue gel staining and a monoclonal antibody against CD68 (KP1) controlled for protein integrity.
  • intron 2 of the gene may cause susceptibility to aberrant splicing of HSC73 in malignant cells; additional factors must be involved since aberrant transcripts have not been observed in normal breast. RNA truncations could occur if cryptic splice sites are produced or if mutated (and/or polymorphic) pre-mRNA primary structure affects tertiary structure bringing incorrect sequences into contact which may be spliced together.
  • the U14 SNURPs which are transcribed via an intron processing pathway concurrent with HSC73 mRNA splicing, and the repetitive nature of the intron/exon structure, may make HSC73 pre-mRNA splicing inherently susceptible to incorrect splicing in a malignant cell.
  • Valine 83 is changed to alanine gtc is changed to gcc
  • Glycine 99 is changed to Serine ggc is changed to age
  • Tyrosine 107 is changed to histidine tac is changed to cac
  • the polymorphism in intron 2 is: aaagtgttttcattgacaccttttacagAT which becomes aaagttttcattgacaccttttacagAT where AT is the start of exon 3 the sequence changes 23bp upstream of exon 3.
  • mda-mb435 a breast cancer cell line known to have aberrant hsc73 mRNA, as a model.
  • the HSC73 gene was transfected into the cell line.
  • Three stable subclones of this cell line expressing normal hsc73 from a genomic clone were produced and investigated for in vitro and in vivo tumour formation in mouse models.
  • Western blotting confirmed increased full length hsc73 protein in all three subcloned cell lines.
  • the clones were each introduced into three test animals. There were three control animals injected with untransfected cells of a breast cancer cell line.
  • Proliferation assays demonstrated that expression of wild type hsc73 in the mda-mb435 cell lines decreases the rate of cell growth both in vitro and in vivo in nude athymic mice.
  • In vitro proliferation rates for the three stable hsc73 expressing subclones and appropriate control cell lines were measured. The stable transfectants divide at approximately % of the rate of division of the wild type cell line (data not shown).
  • In vivo proliferation assays showed that 500,000 transfected cells produced no tumours in nude mice (except in a single case) while the wild type mda-mb435 produced tumours in 100% of cases ( Figure 7). It is likely that in the case where tumours did develop from the transfected cells, the HSC73 gene was for some reason no longer being expressed (see figure legend).
  • hsc73 prevents apoptosis in breast carcinoma cells.
  • Replacement of hsc73 function in the mda-md435 following stable transfection repairs the apoptotic function and gross proliferation of the cells is therefore reduced.
  • biochemical defects could include the degradation of c-fos in the 26S proteasome, a known early reaction in the cascade for the activation of caspase 3 by caspase 9 and cytochrome c.
  • Other explanations include the correct cellular localisation of apoptosis related proteins such as BAG and BUZ.
  • mda-mb435 expressing normal hsc73 could be stalled in a stage of their cell cycle progression.
  • the cell line could have impaired wild type hsc73 function as a means of overcoming such a cell cycle block.
  • Obvious classes of molecules to examine for their stability and localisation in the cell lines include cyclins and cdk inhibitors.
  • the fibroadenoma shows intense signal in epithelial cells on both analyses.
  • DCIS shows increased tumour cell HSC73 protein but low RNA compared to normal acini.
  • Invasive breast cancer cells show a similar phenomenon, with compressed normal ducts staining weakly for protein but intensely for RNA, compared to tumour cells with the opposite pattern.
  • Metastatic tumour in the lymph node stains highly for protein while RNA signal is virtually absent, contrasting sharply with the surrounding normal lymphocytes.
  • Scale bar 100 ⁇ m.
  • Figure 3- a) Multiple tissue northern blot (l-r: spleen, thymus, prostate, testis, ovary, small intestine, colon, leucocyte) probed with 1.1 kb cDNA probe (with ⁇ -actin control panel below) shows uniform expression of HSC73. b) Northern of paired normal (N) and tumour (T) samples (l-r: tumour cases 16, 20, 34, 26, 22) and cell lines MDA-MB435 (1 ), MDA- MB468 (3) and U937 (5) are shown with aberrant transcripts indicated (- ⁇ ).
  • HSC73 RT-PCR samples (l-r: tumour cases 20, 64, 54, 24, 22, 62, 58, 26, 34) with multiple aberrant bands in tumour lanes (N* indicates a 'normal' lane with aberrant transcript, subsequently shown to have tumour cells on cryosection).
  • HSC73 RT-PCR of the cell lines showing aberrant bands in MDA-MB435, MDA-MB468 and U937, but not in MDA- MB453 or T47D.
  • Figure 4- a) Genomic and mRNA structure of HSC73 showing the localisation of the probes (black bars) and PCR primers (arrows) used.
  • the upper genomic structure has 9 exons (coding exons: stippled boxes; non-coding regions of exons: stippled/black boxes).
  • Figure 7 Three BALB/c nude mice were injected subcutaneously with 500,000 cells from each cell line. After six weeks the mice were sacrificed and tumours evaluated.
  • Lane 1 mda435 parental line (tumour weights 8, 30 and 18mg)
  • Lane 2 mda435 HSC73 transfectant 1 (no tumours)
  • Lane 3 mda435 HSC73 transfectant 2 (one tumour of 68mg)
  • tumour which grew in one mouse injected with mda435 HSC73 transfectant 2 is thought to have arisen from a cell which had deleted the transfected clone. In the mice the cells are no longer under selection.
  • Post-translational protein translocation not all hsc70s are created equal.
  • TSG101 may be the prototype of a class of dominant negative ubiquitin regulators. Nat. Genet. 16, 330-31.

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Abstract

On décrit des procédés et des réactifs qui permettent de diagnostiquer une néoplasie ou la vulnérabilité à la néoplasie. Ces procédés consistent à détecter l'expression aberrante d'un gène au locus de l'analogue de choc thermique 73 (HSC73), ou à détecter une mutation au locus du HSC73. Ces procédés sont efficaces, par exemple, pour la détection et/ou la prévision de cancers du sein.
PCT/GB1998/003375 1997-11-05 1998-11-05 Gene du cancer WO1999023252A1 (fr)

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Cited By (2)

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WO2001036685A3 (fr) * 1999-11-17 2002-01-10 Nyxis Neurotherapies Inc Expression differentielle de genes dans le cancer
WO2001057270A3 (fr) * 2000-02-04 2003-02-13 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hbl 100

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WO1996010411A1 (fr) * 1994-09-30 1996-04-11 Mount Sinai School Of Medicine Of The City University Of New York Complexes immunotherapeutiques proteine de stress-peptide contre le cancer
WO1997006685A1 (fr) * 1995-08-18 1997-02-27 Sloan-Kettering Institute For Cancer Research Procede de traitement du cancer et de maladies infectieuses et compositions utiles pour ledit traitement

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036685A3 (fr) * 1999-11-17 2002-01-10 Nyxis Neurotherapies Inc Expression differentielle de genes dans le cancer
WO2001057270A3 (fr) * 2000-02-04 2003-02-13 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hbl 100
GB2385053B (en) * 2000-02-04 2004-12-22 Aeomica Inc Human genome-derived single exon nucleic acid probes useful for analysis of gene expression in human breast and HBL 100 cells

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