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WO2010115593A2 - Nouvelles cibles pour le diagnostic et le traitement de la dysplasie - Google Patents

Nouvelles cibles pour le diagnostic et le traitement de la dysplasie Download PDF

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WO2010115593A2
WO2010115593A2 PCT/EP2010/002130 EP2010002130W WO2010115593A2 WO 2010115593 A2 WO2010115593 A2 WO 2010115593A2 EP 2010002130 W EP2010002130 W EP 2010002130W WO 2010115593 A2 WO2010115593 A2 WO 2010115593A2
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gene
dysplasia
genes
lung
gene product
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WO2010115593A3 (fr
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Jϋrgen BORLAK
Astrid Rohrbeck
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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

Definitions

  • the invention relates to diagnostic targets and the use thereof in the diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung.
  • Areas of application are the life sciences: biology, biochemistry, biotechnology, medicine and medical technology.
  • Lung cancer is a multistage process with poor prognosis and high morbidity. Nonetheless, detection of early stages of disease significantly improves overall survival.
  • the genetic events associated with cellular lesion at the edge of malignant transformation are unknown. Such knowledge would, however be of great value for the development of novel disease diagnostic and therapeutic strategies.
  • the genetics of dysplasia, a facultative cancer are unknown but are of great concern and have not been investigated, so far.
  • non-small cell lung cancer Specifically, eighty percent of the lung cancers are classified as non-small cell lung cancer (NSCLC) whereas the remains 20% are small cell lung cancer (SCLC). Survival of patients diagnosed with non-small-cell lung cancer (NSCLC) is poor; over the last decades the 5-year survival rate remained less than 15%. Survival of lung cancer is, however, strongly associated with the stage of disease at the time of diagnosis. Indeed, 5- year survival rates range from 5% for patients with stage IV lesions to 70% at stage I [5]. Such encouraging outlooks have lead to renewed interest in the search and validation of biomarkers of disease to allow monitoring of individuals at risk for developing lung cancer [6]. Most frequently, diagnosis of lung cancer is at a late stage of disease with its classification being based on morphological appearance and immunohistological methods [7].
  • AAH atypical adenomatous hyperplasia
  • the aim of the present invention is therefore to make available an easy and efficient method for identifying diagnostic targets for the use in diagnosis or treatment monitoring of dysplasia, and the use of said targets and related substances for the diagnosis, treatment monitoring and treatment of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung.
  • the implementation of the actions and embodiments as described in the claims provides appropriate means to fulfill these demands in a satisfying manner.
  • the invention is based on the surprising finding, that a method comprising the steps of
  • RNA (1) and (2) determining, for each of the isolated RNA (1) and (2), a gene expression profile, preferably a genome wide gene expression profile, and
  • the laser microdissection is performed by using a focused laser beam for excising and extracting a precisely defined area from the tissue, whereby the desired cells are microdissected and collected.
  • the microdissection is performed by LMPC (Laser Microbeam Microdissection and Laser Pressure Catapulting).
  • the transgenic animal which is preferably a non-human mammal, such as a rodent may be, e.g. a mouse of the species mus musculus, comprises at least one foreign DNA sequence in its genome, wherein at least one DNA sequence codes for an oncogene and is operably linked to a regulatory sequence.
  • the oncogene is c-raf, such as in a SP-C/c-raf transgenic mouse.
  • the regulatory sequence is preferably a foreign DNA sequence, in particular a promoter, controlling gene expression in a tissue-dependent manner, such that the transgene will only be expressed in the tissue, preferably the lung, where the transgene product is desired, leaving the remaining tissues in the animal unmodified by the oncogene expression.
  • oncogene within the context of the invention relates to any suitable mutant of a proto-oncogene, whose expression induces an abnormal rate of cell division, particularly induces the formation of lung tumors.
  • the method according to the invention further comprises the generation of at least one ingenuity network by mapping the focus genes that are overexpressed in the gene expression profile of the isolated RNA (1).
  • the method comprises
  • oligonucleotide array in particular microarray, preferably covering over 34000 genes
  • PCA principal-component analysis
  • HCA hierarchical gene clustering
  • IPA - ingenuity pathways analysis
  • the implementation of the invention comprises a quantitative real-time PCR, for example for the corroboration of the RNA expression data, preferably by calculating CT values and expressing relative gene expression levels as the difference in CT values of the target gene and the control gene Actin beta.
  • microarray preferably at least two microarrays
  • the gene expression profile in particular to search genome wide for at least one, preferably two, gene regulatory network(s).
  • the term “dysplasia” also refers to interepithelial neoplasia, within the context of the invention.
  • the invention comprises an immunohistochemistry
  • the invention also concerns the use of at least one antibody or of antibodies specific for a gene product selected from the group of genes listed in Supplementary
  • Table 1 for the diagnosis and treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung.
  • the gene products encoded thereby in particular the genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoal, Arg2, Brunol4, Cldn2, Cldn4, Cldn ⁇ , CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfl , Rgs16, Stk39, Tnfsf9, preferably APOA1 , and/or the genes Btbd11 , C8orf13, Cyp1b1 , Fetub, Fut2, Klc3, Pcsk6, Pkhdi , Pla2gl1b, Psrd, Ptpm2, Rnf128, Ros1, Sdcbp2, Sult2b1 , preferably PKHD1 , are preferred, and the gene products of
  • the invention is further directed to a gene identified by the method described herein and to the gene product encoded thereby and to RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, for the diagnosis or treatment monitoring of dysplasia.
  • the invention is directed to genes identified by said method, more particular to the preferably in vitro use of least one of said genes, wherein the gene is selected from the group of the genes listed in Supplementary Table 1 , in particular selected from the coding regions thereof, or to the use of a gene product encoded thereby or of RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, and/or of an antibody directed against said gene product and/or of an antibody directed against said polypeptide, for the diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, and/or to screen for drugs against dysplasia, in particular against low grade or high grade dysplasia related to adenocarcinoma of the lung.
  • the invention provides the genes listed in Supplementary Table 1 and their gene products as biomarkers and/or provides antibodies directed against said gene products (for use) in the diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, preferably for the use in a blood serum analysis.
  • low grade dysplasia is particularly directed to a lesion having minimal aberration inside the cell.
  • high grade dysplasia as described herein also comprises mild or medium dysplasia.
  • the term "gene” according to the invention is directed to both the template strand, which refers to the sequence of the DNA that is copied during the synthesis of mRNA, and to the coding strand corresponding to the codons that are translated into a protein.
  • the genes according to the invention and gene products encoded thereby are described herein or can be easily derived from the common databases, as such are known to the person skilled in the art, wherein the UCSC Genome Database is particularly preferred: Karolchik D, Kuhn, RM, Baertsch R, Barber GP, Clawson H, Diekhans M, Giardine B, Harte RA, Hinrichs AS, Hsu F, Miller W, Pedersen JS, Pohl
  • genes described herein are murine genes or preferably the respective human genes.
  • biomarkers described herein such as the gene products encoded by said genes, concern gene products of mammalia, preferably gene products of the genome of mus musculus or homo sapiens, in particular the respective gene products of homo sapiens are preferred.
  • coding region is directed to the portion of DNA or RNA that is transcribed into the mRNA, which then is translated into a protein. This does not include gene regions such as a recognition site, initiator sequence, or termination sequence.
  • RNA sequences hybridizing to said gene or "RNA sequences, which hybridize to said gene", respectively, according to the invention relates to RNA molecules hybridizing with the template strand of said gene, in particular with the coding region.
  • DNA sequences hybridizing to said gene or “DNA sequences, which hybridize to said gene”, respectively, according to the invention preferably relates to DNA molecules hybridizing with the coding strand of said gene, in particular with the coding region thereof.
  • hybridizing refers to conventional hybridization conditions, preferably to hybridization conditions under which the Tm value is between 37°C to 70 0 C, preferably between 41 0 C to 66°C.
  • An example for low stringency conditions is e.g. hybridisation under the conditions 42°C, 2 ⁇ SSC, 0.1% SDS and an example for high stringency conditions is e.g. hybridization under the conditions: 65°C, 2 ⁇ SSC, 0.1% SDS, wherein in the case that washing is necessary for equilibrium, the hybridization solution is used for the washings.
  • hybridization refers to stringent hybridization conditions.
  • the gene according to the invention may be part of a recombinant DNA molecule for use in cloning a DNA sequence in bacteria, yeast(s) or animal cells.
  • the gene according to the invention may be part of a vector.
  • the invention is thus also directed to the use of a vector for the diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, and/or to screen for drugs against dysplasia, in particular against low grade or high grade dysplasia related to adenocarcinoma of the lung, wherein the vector comprises a gene selected from the group of genes listed in Supplementary Table 1 , or the vector comprises DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene.
  • function of said gene product is directed to biological activities of the polypeptide encoded by the selected gene, wherein the biological activities are described herein or may easily be derived from common gene or protein databases, such as the ncbi or the SWISS-Prot databases may be and the biological activities can be determined according to the literature provided therein.
  • the gene according to the invention is selected from the group of the focus genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoai , Arg2, BrunoW, Cldn2, Cldn4, Cldn8, CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfl , Rgs16, Stk39, Tnfsf9, preferably APOA1 or a gene product encoded by the selected gene or RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used, and/or an antibody directed against said gene product and/or an antibody directed against said polypeptide is used.
  • the focus genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoai , Arg2, BrunoW, Cldn2, Cldn4, Cldn8, CIu
  • the group of members of the EGF- pathway namely Areg and/or Ereg
  • the gene products encoded thereby and RNA or DNA sequences which hybridize to the gene(s) Areg and/or Ereg and which code for a polypeptide having the function of said gene product(s)
  • an antibody directed against said gene product and/or an antibody directed against said polypeptide is preferably used.
  • the group of claudins namely Cldn2, Cldn4, Cldn ⁇ , and the gene products encoded thereby and RNA or DNA sequences, which hybridize to the gene(s) Cldn2, Cldn4, and/or Cldn ⁇ and and which code for a polypeptide having the function of said gene product(s), is particularly preferred and/or an antibody directed against said gene product and/or an antibody directed against said polypeptide is preferably used.
  • the gene according to invention is selected from the group of the focus genes Btbd11 , C8orf13, Cyp1b1 , Fetub, Fut2, Klc3, Pcsk ⁇ , Pkhdi , Pla2gl1b, Psrd , Ptprn2, Rnf128, Ros1 , Sdcbp2, Sult2b1 , preferably PKHD1 , or a gene product encoded by the selected gene or RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used, and/or an antibody directed against said gene product and/or an antibody directed against said polypeptide is preferably used.
  • the gene is selected from the group of genes coding for a protein being involved in at least one metabolism reaction, preferably selected from
  • tight junction proteins in particular Claudins
  • gap junction proteins in particular GJA3 etc
  • EMT epithelial-mesenchymal transition
  • a diagnostically effective amount of the selected gene in particular the coding region of said gene, or of the gene product encoded thereby or of the RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, is used for the preparation of a diagnostic agent, in particular of a diagnostic standard for body fluid analysis or for tissue analysis, in particular for the production of a diagnostic agent for the diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, and/or an antibody directed against said gene product and/or an antibody directed against said polypeptide is used in this respect, in particular for the preparation of a diagnostic agent, preferably for the diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung.
  • body fluid according to the invention is directed to any body fluid of a subject, in particular
  • diagnostic agent as used herein relates to any solution, suspension or solid formulation, containing said composition in an acceptable amount for diagnostic purposes.
  • subject is directed to a mammal, in particular to a mouse or a human being having or being susceptible to dysplasia, more particular to a human dysplasia patient or a transgenic cancer mouse, such as a patient having low grade or high grade dysplasia related to adenocarcinoma of the lung or a c-raf- transgenic mouse may be.
  • the second aspect of the invention is used for the diagnosis and/or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, comprising determining the level of a gene selected from the group of the genes listed in Supplementary Table 1 (Table S1), in particular the coding region thereof, or of a gene product encoded thereby or of RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, and/or of an antibody directed against said gene product and/or of an antibody directed against said polypeptide, in a patient or in a sample, preferably in a tissue or body fluid sample, isolated from a patient who has or is susceptible to dysplasia, and comparing the level determined to a respective diagnostic standard or reference level, particularly (A) for the diagnosis of dysplasia, wherein a significantly elevated level of the gene, in particular the coding region thereof, or of the gene product encoded thereby or of the lung, comprising determining the
  • the gene is selected from the group of the focus genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoai , Arg2, BrunoW, Cldn2, Cldn4, Cldn ⁇ , CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfl , Rgs16, Stk39, Tnfsf9, preferably APOA1 , and/or an antibody directed against a gene product encoded by a gene selected from said genes is used.
  • the focus genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoai , Arg2, BrunoW, Cldn2, Cldn4, Cldn ⁇ , CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfl
  • the gene is selected from the group of the focus genes Btbd11 , C8orf13, Cyp1b1 , Fetub, Fut2, Klc3, Pcsk6, Pkhdi , Pla2gl1 b, Psrd , Ptpm2, Rnf128, Ros1 , Sdcbp2, Sult2b1 , preferably PKHD1 , and/or an antibody directed against a gene product encoded by a gene selected from said genes is used.
  • a dysplastic cell (over-)expressing the selected gene is contacted with a compound to be tested, such as Zileuton or Celecoxib may be, and the expression level of said gene is determined, preferably by using an antibody directed against the product encoded by said gene, and the compound that suppresses said expression level compared to a normal control level of said gene is identified as a drug inhibiting the expression of said gene.
  • a compound to be tested such as Zileuton or Celecoxib
  • a composition which is used for the preparation of a medicament, preferably a medicament against dysplasia, in particular against low grade or high grade dysplasia related to adenocarcinoma of the lung, wherein the composition comprises a therapeutically effective amount of
  • an antisense composition comprising a nucleotide sequence complementary to a coding sequence of a gene selected from the group of the genes listed in Supplementary Table 1 , and/or
  • siRNA composition reduces the expression of a gene selected from the group consisting of the genes listed in Supplementary Table 1 , and/or
  • the term "coding sequence” is directed to the portion of an mRNA which actually codes for a protein.
  • the term "nucleotide sequence complementary to a coding sequence” in particular is directed to an oligonucleotide compound, preferably RNA or DNA, more preferably DNA, which is complementary to a portion of an mRNA, and which hybridizes to and prevents translation of the mRNA.
  • the antisense DNA is complementary to the 5 1 regulatory sequence or the 5' portion of the coding sequence of said mRNA.
  • the antisense composition comprises a nucleotide sequence containing between 10-40 nucleotides , preferably 12 to 25 nucleotides, and having a base sequence effective to hybridize to a region of processed or preprocessed human mRNA.
  • the composition comprises a nucleotide sequence effective to form a base-paired heteroduplex structure composed of human RNA transcript and the oligonucleotide compound, whereby this structure is characterized by a Tm of dissociation of at least 45 0 C.
  • the present invention further employs siRNA oligonucleotides directed to said genes specifically hybridizing with nucleic acids encoding the gene products of said genes and interfering with gene expression of said genes.
  • the siRNA composition comprises siRNA (double stranded RNA) that corresponds to the nucleic acid ORF sequence of the gene product coded by one of said human genes or a subsequence thereof; wherein the subsequence is 19, 20, 21 , 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.
  • siRNA double stranded RNA
  • the subsequence is 19, 20, 21 , 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.
  • nucleotide sequences and siRNA according to the invention may be prepared by any standard method for producing a nucleotide sequence or siRNA, such as by recombinant methods, in particular synthetic nucleotide sequences and siRNA is preferred.
  • an antibody composition comprising a pharmaceutically effective amount of a labelled antibody directed against the gene product encoded by a gene selected from the group of the genes listed in Supplementary Table 1 , and is used for the preparation of a medicament or of a diagnostic agent, preferably of a medicament against dysplasia or of an agent for diagnosing dysplasia, in particular low grade or high grade dysplasia related to adenocarcinoma of the lung, wherein the antibody is preferably labeled with an isotope such as iodine-124 may be.
  • said antibody composition has been administered to the patient or to the dysplastic cell or to the transgenic animal for determining the level of the gene product, in particular wherein an imaging method, preferably PET or CT, is used for determining the level of the gene product, such as by administering a nanoparticle carrying 2-[18F] fluoro-2-deoxy-D-glucose, wherein said antibody is linked to said nanoparticle, for the use in FDG-PET imaging.
  • an imaging method preferably PET or CT
  • antibodies are understood to include monoclonal antibodies and polyclonal antibodies and antibody fragments (e.g., Fab, and F(ab') 2 ) specific for one of said polypeptides.
  • Polyclonal antibodies against selected antigens may be readily generated by one of ordinary skill in the art from a variety of warmblooded animals such as horses, cows, goats, rabbits, mice, rats, chicken or preferably of eggs derived from immunized chicken.
  • Monoclonal antibodies may be generated using conventional techniques (see Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are incorporated herein by reference).
  • the invention is furthermore directed to the use of primer sequences, preferably primer pairs, directed against the mRNA of a gene selected from the Supplementary Table 1 , for the diagnosis, prognosis and/or treatment monitoring of dysplasia, in particular against low grade or high grade dysplasia related to adenocarcinoma of the lung, wherein the production of adequate primer sequences and the use thereof, e.g. in a quantitative RT-PCR, is known to the person skilled in the art.
  • the invention provides a method of diagnosing, qualifying, and/or monitoring dysplasia in a subject, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, comprising determining in a body fluid sample of a subject being susceptible to cancer at least one biomarker selected from the group of the genes products encoded by the genes listed in Supplementary Table 1 , for the diagnosis or treatment monitoring of dysplasia, preferably by using an antibody directed against a gene product encoded by a gene selected from the group of genes listed in Supplementary Table 1 , in particular by performing a western blot, wherein the body fluid level of the at least one biomarker being significantly higher than the level of said biomarker(s) in the body fluid of subjects without dysplasia, in particular low grade or high grade dysplasia related to adenocarcinoma of the lung, is indicative of dysplasia in the subject.
  • this method is carried out for predicting the response of a dysplasia patient to a method of treating dysplasia, in particular by a chemoprevention therapy such as by administering Zileuton and/or Celecoxib to the patient, as by administering cancer comprising administering an EGFR kinase modulator, wherein the body fluid level of the at least one biomarker being significantly higher than the level of said biomarker(s) in the body fluid of subjects without dysplasia, in particular without low grade or high grade dysplasia related to adenocarcinoma of the lung, is indicative that the subject will respond therapeutically to the method of treating dysplasia.
  • this method is implemented for monitoring the therapeutically response of a dysplasia patient to a method of treating dysplasia comprising administering a chemopreventive drug, such as by administering Zileuton and/or Celecoxib, to the patient, wherein the body fluid level of the at least one biomarker before and after the treatment is determined, and a significant decrease of said body fluid level(s) of the at least one biomarker after the treatment is indicative that the dysplasia patient therapeutically responds to the administration of the chemopreventive drug.
  • a chemopreventive drug such as by administering Zileuton and/or Celecoxib
  • the method is implemented by performing an immunoassay, such as an enzyme immunoassay (EIA), a radio immunoassay (RIA) or a fluorescence immunoassay (FIA) may be, and/or by performing a western blot.
  • an immunoassay such as an enzyme immunoassay (EIA), a radio immunoassay (RIA) or a fluorescence immunoassay (FIA) may be, and/or by performing a western blot.
  • At least one antibody specific for the biomarker in particular selected from the group of the gene products of the genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoal , Arg2, BrunoW, Cldn2, Cldn4, Cldn ⁇ , CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfi , Rgs16, Stk39, Tnfsf9, preferably APOA1 , or selected from the group of the gene products of the focus genes Btbdi 1 , C8orf13, Cyp1 b1 , Fetub, Fut2, Klc3, Pcsk6, Pkhdi , Pla2gl1 b, Psrd , Ptprn2, Rnf128, Ros1 , Sdcbp2, Sult2b1 , preferably PKHD1 is used
  • this method is implemented by performing a peptide mass fingerprinting.
  • this method preferably comprises the steps of
  • the invention also relates to a procedure to screen for and to identify drugs against dysplasia, in particular against low grade or high grade dysplasia related to adenocarcinoma of the lung, comprising determining in a body fluid sample of a transgenic cancer mouse being treated with a compound to be tested, in particular of a mouse whose genome comprises a non natural c-raf sequence, at least one biomarker selected from the group of the genes products encoded by the genes listed in Supplementary Table 1 , preferably by using an antibody directed against a gene product encoded by a gene selected from the group of genes listed in Supplementary Table 1 , in particular by performing a western blot, wherein the body fluid level of the at least one biomarker being significantly higher than the level of said biomarker(s) in the body fluid of an untreated transgenic cancer mouse is indicative of the therapeutic effect of said compound as drug against dysplasia.
  • Lung cancer is a multistage process with poor prognosis and high morbidity. Importantly, the genetics of dysplasia, a facultative cancer, at the edge of malignant transformation is unknown.
  • the raf family (a-raf, b-raf and c-raf) of proteins code for serine/threonine kinases, originally isolated as a viral oncogene contributing to cellular transformation and are one of the best characterized Ras effectors to activate the mitogen-activated protein kinase (MAPK) signaling pathway [14].
  • RAF directly phosphorylates and activates MEK via two conserved serine residues in the kinase activation loop of MEK [15].
  • Activated MEK then directly phosphorylates a conserved tyrosine and threonine residue in the kinase activation loop of ERK [16].
  • the MAPK pathway is deregulated in many human malignancies through aberrant signaling upstream of the protein and by activating mutations of the protein itself, both of which induce a proliferative advantage. Indeed, mutations of the K-ras gene have been identified in up to 30% of lung adenocarcinomas and have been considered as a poor prognostic factor [17] underscoring the important role of this pathway in human lung cancer. As of today, the genetic events associated with dysplasia are basically unknown. Therefore, it was aimed for an identification of preneoplastic changes in a c-raf-1 lung cancer disease model.
  • mice predominately 5 month old mice were used to gain information at an early stage of tumor development where isolated foci of transformed cells in distinct areas of the lung are visible.
  • LMPC laser microdissection pressure catapulting
  • Dysplastic lesions were than compared with transgenic but otherwise normal cells or tumor cells.
  • Gene expression profiling was applied to determine differentially expressed genes as to identify the gene regulatory networks associated with dysplasia.
  • Figure 1 depicts lung tissue from transgenic mice where columnar epithelium, typical for bronchioles with vertically oriented nucleus is replaced by cells displaying horizontal orientation. Cytologically nuclei of different size and unevenly arranged chromatin are observed. Nuclei are hyperchromatic, often with prominent nucleoli. The shapes of the nuclei are irregular and the ratio of nucleus to cytosol area is grossly changed.
  • PCA Principal component analysis
  • HCA hierarchical gene cluster analysis
  • the expression levels were analyzed by the GCOS and ArrayTrack software.
  • the data in a 34,000 genes x 15 sample matrix were initially examined.
  • the PCA classified the data into 3 major groups, namely non-transgenic, transgenic and dysplasia ( Figure 3).
  • the Ingenuity Pathways Analysis software was employed and over 70% of regulated genes were mapped to different networks in the IPA database. These networks describe functional relationships among gene products based on findings presented in peer-reviewed biological pathways. Taken collectively, 12 and 16 networks could be defined for the comparison dysplasia vs transgenic and dysplasia vs non-transgenic cells. Based on pathway analysis the top 2 and top 3 networks reached a score of 25 or higher and contained 15 or more genes in the comparison dysplasia vs transgenic ( Figure 5) and dysplasia vs non-transgenic cells (Figure 6), respectively. This demonstrates the extensive relationship and interaction between the significantly regulated genes in dysplasia.
  • Cancer cells may produce their own growth factors to stimulate proliferation in neighbouring or parental cells (paracrine vs autocrine loops). Thus, it was searched for genes involved in cellular growth and proliferation and found in the case of dyspalsia 18 genes to be up-regulated ranging between 3.6 to 23.5-fold as compared to transgenic but morphologically unaltered lung tissue. Likewise 36 genes were up- regulated between 4.7 to 482.2-fold when compared to non-transgenic lung tissue.
  • the Areg (amphiregulin) and Ereg (epiregulin) ligands both members of the EGF-pathway, were highly significantly over expressed. These molecules play pivotal roles in an activation of the EGF receptor tyrosine kinase to foster proliferation and motility. Overexpressions of these EGF-ligands are observed in a wide variety of human cancers, including breast, prostate and lung cancer. Cell-to-cell interactions are also important for the regulation of cell proliferation and differentiation. Indeed, expression of cell adhesion molecules is programmed during development to provide positional and migratory information for cells. Disruption of these adhesion events leads to increased cell motility and potential invasiveness trough remodelling of the extra cellular matrix.
  • tight junction proteins Genes coding for tight junction proteins were found to be regulated. Identification of the molecular components of the tight junction evidenced that, in addition to their structural functions, these proteins play central roles in regulating cellular proliferation and differentiation. Under normal conditions, tight junctions act to segregate a growth factor within the apical membrane compartment away from its receptor in the basolateral membrane compartment, thereby precluding receptor activation [19]. The disruption of tight junctions in adjacent healthy cells permits the growth factor to bind and activate its receptor, thereby inducing cellular proliferation and migration.
  • gap junctional intercellular communication results in the inability of cells to receive apoptotic, growth suppressing or differentiation signals from their neighbours.
  • connexins a family of 20 trans-membrane proteins in humans, comprise the main subunits of gap junctions; - these specialised clusters of intercellular channels allow adjacent cells to directly share ions and hydrophilic molecules of up to ⁇ 1 KDa in size.
  • Gap junctional intercellular communication is thought to control tissue homeostasis and to coordinate cellular processes such as proliferation, migration and differentiation. Disruption of gap junctional intercellular communication or mutations in connexins is associated with several human diseases. Notably, gap junction expression is often up-regulated in hyperplastic tissues.
  • lipid metabolism is altered in cancer, including loss of body fat early in tumor growth, induction of hyperlipidemia and changes in a variety of serum lipid and lipoprotein fractions.
  • cancer patients were found to have higher rates of fat oxidation when compared with healthy individuals with equal weight loss [20, 21].
  • Genes involved in lipid metabolism were examined and found up to 13 genes in dysplasia to be up-regulated ranging from 3.6-28.1 -fold and 4.4-148.4-fold when unaltered transgenic and non-transgenic lung tissue was compared with dysplastic cells.
  • Apoai apolipoproteinA-1
  • HDL High Density Lipoprotein
  • Apoai is a cofactor for Lcat (lecithin-cholesterin-acetyltransferase), which is responsible for the formation of most cholesterol esters in plasma. Apoai also promotes efflux of cholesterol, phospholipids, sphingomyelin, sterol and phosphatidylcholin from cells and is involved in transport of cholesterol ester [38]. Recently, it has been observed that the HDL complex is capable of suppressing lymphocyte function, particularly in the host resistance to tumors [22].
  • B4galt6 UDP- GALBeta-GlcNAc Beta-1 ,4-Galactosyltransferase, polypeptide 6
  • Fut2 Fucosyltransferase 2
  • Gpc6 Glypican ⁇
  • 0rm1 orosomucoidi
  • glycosyltransferase such as B4galt6, which catalyzes the reaction UDP-galactose and N-acetylglucosamine for the production of galactose beta-1 ,4-N-acetylglucosamine. Strikingly, these carbohydrates are absent or have low activity in normal cells.
  • the secretor enzyme Fut2, an ⁇ r-1 ,2- fucosyltransferase is responsible for the transfer of fucose in an ⁇ -1 ,2 linkage to form the terminal H type 1 structure.
  • glycoproteins and glycolipids are associated with several pathological processes, such as tumor metastasis, inflammation and bacterial adhesion.
  • pathological processes such as tumor metastasis, inflammation and bacterial adhesion.
  • Orosomucoid 1 belongs to a group of highly glycosylated glycoproteins and appears to function in modulating the activity of the immune system.
  • glypican 6 was found to be induced (9.9-fold).
  • this protein belongs to a family of glycosylphosphatidylinositol-anchored heparin sulphate proteoglycans.
  • proteoglycans are high-molecular-weight glycoproteins and interact via their multiple binding domains with many other structural macromolecules. They are bound together with extracellular matrix components and act as cell adhesion factors by promoting organization of actin filaments in the cell cytoskeleton. Proteoglycans have been shown to undergo alterations during malignant transformation resulting in disrupted interaction between the extra cellular matrix and the transformed cells to simplify the invasion into the surrounding tissue. Further genes involved in developmental processes were investigated and 8 genes t were found to be 3.6-15.4-fold up-regulated in dysplasia as compared to transgenic lung tissue. Notably, 22 genes were 5.4-148.4-fold up-regulated in dysplasia as compared to non-transgenic lung tissue.
  • Hnf4 ⁇ hepatocyte nuclear factor 4-alpha
  • Foxa3 forkhead box gene A3
  • Foxp2 forkhead box gene P2
  • Hnf4 ⁇ plays a key role in a transcriptional hierarchy and controls the expression of other transcription factors such as Hnf1 (hepatocyte nuclear factor 1) [23]. Indeed, hundreds of genes are targeted by Hnf4 ⁇ .
  • Hnf4 ⁇ target genes are those involved in lipid transport (e.g., apolipoprotein genes) and glucose metabolism [24].
  • Foxa genes code the winged helix/forkhead transcription factor gene family that also includes Foxal , Foxa2 and Foxa3, as well [25].
  • Foxp2 is characterized as transcriptional repressor and is the first Fox gene that is expressed exclusively in the distal epithelium of the lung during pulmonary development [26].
  • Ros1 v-ros avian ur2 sarcoma virus oncogene homolog 1
  • Ros1 v-ros avian ur2 sarcoma virus oncogene homolog 1
  • This study aimed for a better understanding of the genetic events associated with dysplasia in a genetic model of lung cancer induced by overexpression of the c-Raf- 1 kinase.
  • qRT-PCR of selected genes was employed, and confirmed by immunohistochemistry regulated proteins in dysplastic foci.
  • RT-PCR data is suggestive for the microarray to underestimate changes in the gene expression even though both methods supported the general directions of changes.
  • An important finding of the study described herein was that about 10% of the differentially expressed genes regulated by >2 fold are already known to be associated with lung cancer.
  • EGF-ligands namely amphi- and epiregulin were highly significantly up-regulated. These ligands enable autocrine loops to foster undue EGF-signaling.
  • Areg is a 252-amino acid transmembrane glycoprotein and consist of two major soluble forms of 78 and 84 amino acids, respectively. Areg was originally isolated from conditioned medium of the human breast carcinoma cell line MCF-7 and was found to be a heparin-binding growth factor.
  • Areg promotes neoplastic growth in mammary epithelial cells, fibroblasts, and keratinocytes [29, 30] and was shown to function in an autocrine manner to drive the proliferation of malignantly transformed cells of colon, breast, cervix, prostate, and pancreas [31].
  • This ligand of the EGF tyrosine kinase is commonly over expressed in cancers of human colon, stomach, breast, and pancreas, in which the level of Areg correlates with tumor progression and poor patient survival [32, 33, 34, 35].
  • Ereg epiregulin
  • the gene codes for a transmembrane precursor before being proteolytically cleaved to release a 46-amino-acid activated protein [38].
  • Ereg is promiscuous, binds and activates the Egfr family member Erbb4 via heterodimeric interactions with Erbb2 [39].
  • Ereg expression is highly correlated with survival from bladder cancer [40]
  • others suggest epiregulin to be important for pancreatic and prostate cancer development [41].
  • transformed cells grow faster than unaltered cells is also sustained by the data described herein with regard to the number and strength of up-regulated genes affecting cellular growth.
  • the cell surface membrane proteins play an important role in the behaviour of cells to allow for communication with other cells, cell movement and migration, adherence to other cells or structures and recognition by the immune system. Alterations of the plasma membrane in malignant cells may thus be inferred from a variety of properties that characterize their growth and behaviour.
  • the study according to the invention is suggestive for changes in the glycosylation patterns and in cell surface glycolipids. Changes in glycosylation can include the presence of new carbohydrate structures that are not detected in the normal epithelial cells and/or short carbohydrate chains usually masked by larger epitopes.
  • St8sia6 was found to be up-regulated in dysplasia.
  • St8sia6 (alpha-2,8- sialyltransferase Vl) belongs to a family of sialyltransferases that synthesize sialylglycoconjugates.
  • the most frequently described aberrant glycosylation in cancer cells include the synthesis of highly branched and heavily sialyted glycans [43].
  • GIcNAc galactose beta-1 ,4-N-acetylglucosamine
  • the cytoskeleton which represents a complex of interconnected fibrillar elements, has been determined as an important factor in mediating adhesion-independent and dependent signalling.
  • morphogenesis they determine cell shape and polarity, and promote stable cell-cell and cell-matrix adhesions through their interactions with cadherins and integrins, respectively.
  • dysplastic cells regulation of genes involved in the cytoskeleton was not identified.
  • Gjb3 gap junction membrane channel protein beta 3
  • Gjb4 gap junction membrane channel protein beta 4
  • the regulation of connexins and gap junctions is a hallmark of carcinogenesis, while their induction in cancer cells leads to reversal of the cancer phenotype, induction of differentiation, and regulation of cell growth [46].
  • genes coding for surfactant lipids in respiratory epithelium were focused.
  • Surfactant is a complex mixture of lipids and proteins that reduces surface tension at the air-liquid interface and prevents alveolar collapse during respiration.
  • SP surfactant proteins
  • SP-A and SP- D are relatively hydrophilic proteins and contribute to innate defence of the lung and surfactant homeostasis.
  • SP-B and SP-C are hydrophobic proteins that enhance surface-active properties of surfactant phospholipid films [47].
  • dysplastic epithelium it was not possible to detect regulated genes coding for surfactant proteins. However, nearly all of the genes grouped under lipid metabolism that have been previously linked to transport and secretion of lipid were regulated.
  • Apoai apolipoprotein A-I
  • HDL high- density lipoprotein
  • Apoai apolipoprotein A-1
  • Adcyapi adenylate cyclase activating polypeptide 1
  • Ltb4dh leukotriene B4 12-hydroxydehydrogenase
  • Fst follistatin
  • lnhbb inhibin beta-B
  • CIu clusterin
  • Hnf4 ⁇ hepatic nuclear factor 4, alpha
  • Prokri prokineticin receptor 1
  • Pla2g1b phospholipase A2, group IB
  • Sult2b1 sulfotransferase family, cytosolic, 2B, member 1)
  • St ⁇ sia ⁇ ST8 alpha-N-acetyl-neuraminide alpha-2,8- sialyltransferase 6
  • Cyp1 b1 cytochrome P450, family 1 , subfamily b, polypeptide 1
  • Pthlh parathyroid hormone-like peptide
  • Adcyapi (adenylate cyclase activating polypeptide 1) is a member of the secretin/glucagons/vasoactive intestinal peptide (VIP) family of peptides. It has been localized by immunohistochemistry to the central nervous system, digestive tract and was shown to exhibit a variety of biological activities. It is involved in synthesis of sulfatides, glycolipid, sphingolipid and plays a role in the accumulation of estrogen and progesterone. It was also reported that Adcyapi can regulate the proliferation and differentiation [49] and was shown to be overexpressed in neuroblastoma [50] and breast cancer [51].
  • VIP vasoactive intestinal peptide
  • Ltb4dh leukotriene B4 12- hydroxydehydrogenase catalyzes the conversation of leukotriene B(4) into 12-oxo- leukotriene B(4) and is involved in reduction of 15-keto prostaglandin E1 and prostaglandin. It could be shown that Ltb4dh was overexpressed in Ta urothelial cell carcinoma [52]. Also Pthlh (parathyroid hormone-like peptide) was observed to be induced in dysplasia. This protein is responsible for most cases of humoral hypercalcemia of malignancy. Pthlh expression in tumor samples has also been correlated with poor prognosis in breast cancer [53], renal carcinoma [54] and colorectal tumors [55].
  • HNF3B hepatocyte nuclear factor 3 beta also termed Foxa2
  • Ttf1 transcription termination factor, RNA polymerase I
  • Foxfi forkhead box F1a
  • Gata ⁇ Gata binding protein 6
  • Gata ⁇ Gata ⁇ binding protein 5
  • Hnf4 ⁇ hepatocyte nuclear factor 4 alpha
  • a member of the steroid/thyroid hormone receptor superfamily is a transcriptional activator [62] whose ligand has been identified as tightly bound endogenous fatty acids [63].
  • Hnf4 ⁇ was found to be highly significantly induced (12.8-fold).
  • Hnf4 ⁇ regulates constitutive expression of a large number of target genes encoding enzymes, transporters and other nuclear receptors.
  • HNF4 ⁇ dysplasia i.e. Arg2 (arginase type 2), Apoai (apoliporotein A1), Cyp1b1 (cytochrome P450, family 1 , subfamily B, polypeptide 1), Cldn2 (claudin 2), Fetub (fetuin beta), Gpx2 (glutathione peroxidase 2), Gsta4 (glutathione S-transferase A4), Gpc6 (glypican 6), Hpn (hepsin), 0rm1 (orosomucoid 1) and Lad1 (ladinini) [64], therefore providing an important link between induction of this transcription factor and up-regulation of target genes.
  • Arg2 arginase type 2
  • Apoai apoliporotein A1
  • Cyp1b1 cytochrome P450, family 1 , subfamily B, polypeptide 1
  • Cldn2 claudin 2
  • Fetub fe
  • Foxa3 (forkhead box A3) was found to be induced 7.5-fold.
  • This protein belongs to a group of endoderm-related developmental factors that are members of the forkhead box (Fox) superfamily of transcription factors. They were first discovered by their ability to bind to promoters of liver specific genes encoding ⁇ 1- antitrypsin and transthyretin [65]. Foxa genes are regulated in early mouse embryo development [66] and are responsible, at least in part, for metabolic regulation. Mice that lack Foxa3 display an increase in the mRNA levels of various serum proteins and glycolytic enzymes and show a low serum glucagon level [67]. Also Foxp2 (forkhead box P2) was found to be up-regulated 4.5-fold.
  • This protein is a member of the new subfamily of winged-helix/forkhead DNA binding domain transcription factor. It could be shown that Foxp2 are expressed in the lung restricted to the distal epithelium and may regulate lung epithelial-specific gene transcription during embryonic development [68]. Foxp2 has been characterized as a transcriptional repressor. All the known Fox genes that were implicated in regulating lung expression were characterized as transcriptional activators [26]. This finding suggests that Foxp2 plays a role in the balance of transcriptional activation and repression that is involved in regulating epithelial cell identity and development of the lung. These processes are also crucial for the transdifferentiation of alveolar type I to type Il epithelial cells, which are responsible for gas exchange and surfactant protein expression essential for lung function.
  • dysplasia is a facultative cancer where additional events need to occur that enable malignant transformation.
  • the whole genome expression data provided important information in the multistage process of lung cancer.
  • the study according to the invention revealed interesting novel genes and pathways that dissected the programme of respiratory epithelium from transgenic into dysplasia and eventually lung cancer.
  • the additional genetic events are reported that take place from dysplasia to malignantly transformed cells and thus provide a molecular rational for the multistage process in lung cancer.
  • Lung samples were derived from 5 SP-C/c-raf mice (aged 5 - 10 months); dysplastic and unaltered lung tissue were always isolated from the same dysplasia- bearing transgenic mouse (aged 5 - 7 months). Endogenous normal lung tissue was studied of 5 non-transgenic mice (aged 7 - 10 months). The non-transgenic littermates (wild-type) served as control for transgenic effects.
  • mice were sacrificed and the lung tissues were immediately frozen on dry ice and stored at -80°C until further analysis.
  • the histopathological diagnosis was based on routinely processed hematoxylin- eosin stains.
  • the sections were fixed in methanol / acetic acid and stained in hematoxylin.
  • the desired cells were microdissected using the PALM MicroLaser systems (Zeiss, P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany) and collected in an adhesive cap (Zeiss, P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany).
  • Microdissected cells were resuspended in a guanidine isothiocyanate-containing buffer (RLT buffer from RNeasy MikroKit, Qiagen, Santa Clarita, CA, USA) with 10 ⁇ l/ml ⁇ -mercaptoethanol to ensure isolation of intact RNA.
  • Approximately an area of 6 x 10 6 ⁇ m 2 were pooled from a specific layer of interest in the same animal and used for RNA extraction.
  • RNA-extraction was performed with the RNeasy Micro Kit (RNeasy MicroKit Qiagen, Santa Clarita, CA, USA) according to the manufacturer's instruction.
  • a standard quality control of the total RNA was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).
  • RNA (median: 175ng; range: 150 - 200ng) was used to generate biotin- labeled cRNA (10 ⁇ g) by means of Message Amp aRNA Premium Amplification Kit (Ambion, Austin, TX). Quality control of cRNA was performed using a bioanalyzer (Agilent 2001 Biosizing, Agilent Technologies). Following fragmentation, labeled cRNA of each sample was hybridized to Affymetrix GeneChip ® Mouse Genome 430 2.0 Arrays covering over 34.000 genes and stained according to the manufacturer's instructions.
  • Array data was normalized using scaling or per-chip normalization to adjust the total or average intensity of each array to be approximately the same.
  • Microarray chips were analyzed by the GCOS (GeneChip Operating Software) from Affymetrix with the default settings except that the target signal was set to 500 and used to generate a microarray quality control and data report.
  • CEL files exported from GCOS were uploaded into ArrayTrack software (National Center for Toxicological Research, U.S. FDA, Jefferson, AR, USA (NCTR/FDA)) and normalized using Total Intensity Normalization after subtracting backgrounds for data management and analysis.
  • ArrayTrack software includes some tools common to other bioinformatics software (e.g., ANOVA, T-test and SAM).
  • PCA Principal component analysis
  • HCA hierarchical gene cluster analysis
  • PCA Principal-component analysis
  • HCA Hierarchical gene clustering
  • IPA Ingenuity Pathways Analysis
  • IPA Ingenuity Systems Inc., Redwood City, CA, USA
  • functional annotation and pathway analysis was performed.
  • IPA is a commercial, web-based interface that uses a variety of computational algorithms to identify and establish cellular networks that statistically fit the input gene list and expression values from experiments.
  • the analysis uses a database of gene interactions culled from literature and updated every quarter of the year.
  • Venn diagrams were used to examine the overlap of resulting lists of genes differentially expressed between the different sample sets.
  • RNA expression data was performed by realtime PCR using the ABI PRISM 7500 Sequence Detection System Instrument (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany).
  • Total RNA (200 ng) underwent reverse transcription using an Omniscript RT Kit (Qiagen, Santa Clarita, CA, USA) according to the manufacturer's instruction.
  • PCR reactions were performed according to the instructions of the manufacturer using commercially available assays-on-demand (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany).
  • CT values were calculated by the ABI PRISM software and relative gene expression levels were expressed as the difference in CT values of the target gene and the control gene Actin beta.
  • Each tumor section (8 ⁇ m in thickness) was deparaffinized in roti-histol for 2 times 8 minutes, these were dehydrogenated by means of a descending alcohol row.
  • the following incubation steps were accomplished: 2 times 3 minutes in 96% ethanol, 2 times 2 minutes in 70% Ethanol, and 2 minutes in Aqua dest.
  • the pre-treated slices were heated in a autoclave for 15 min in citrate buffer submitted of an antigen retrieval before the colouring first.
  • For blocking endogenous peroxidase activity the slices covered for 30 minutes with 3% hydrogen peroxide/Methanol peroxidase blocking solution.
  • the slices were incubated with the primary polyclonal anti-body against AREG, EREG, HNF4 ⁇ , FOXA3 and FOXP2 (Santa Cruz, Santa Cruz Biotechnologys Inc., CA, USA) for 45 minutes.
  • AREG primary polyclonal anti-body against AREG, EREG, HNF4 ⁇ , FOXA3 and FOXP2
  • a streptavidin horseradish peroxidase detection kit Envision DAKO, Hamburg, Germany
  • 3,3 ' -diaminobenzidine solution was used for immunohistochemical staining according to the manufacturer ' s instructions. Harris Hematoxylin was used as the counterstaining.
  • the specificity of the immunostaining was confirmed by negative control staining using mouse nonimmune immunoglobulin G instead of the primary antibody.
  • Serum samples 50 ⁇ g used for 2-DE were run on 12% gels by SDS-PAGE, blotted onto PVDF membranes and blocked with 10% RotiblockTM (Roth) in TBS for 1 hour at room temperature.
  • Primary antibodies (Santa Cruz) were diluted in TBS with 1% Rotiblock for 1 h each.
  • the membranes were incubated with goat anti-amphiregulin (1 :250), rabbit anti-ApoA1 (1 :200) and mouse anti- ⁇ -tubulin (1 :200).
  • HRP- conjugated IgGs (1 :10000, Chemicon) were used as secondary antibodies.
  • Rat liver total extracts and HeLa total extracts were used as positive controls, ⁇ -tubulin was used as a loading control.
  • Membranes were washed three times with TBS/0.1 % Tween between each antibody incubation and detected with enhanced chemiluminescence (Perkin Elmer) for 60 min with a CF440 imager (Kodak). In the western blot figure a part of the image with protein bands of interest cropped and marked by molecular weights is shown.
  • lung cancer is a multistage process with poor prognosis and high morbidity.
  • the genetics of dysplasia, a facultative cancer, at the edge of malignant transformation is unknown.
  • Laser microdissection was employed to harvest c-Raf1- induced dysplastic as opposed to transgenic but otherwise morphologically unaltered epithelium and compared findings to non-transgenic lung. Then, microarrays were employed to search genome wide for gene regulatory networks. A total of 120 and 287 genes were significantly regulated, respectively. Dysplasia was exclusive associated with up-regulation of genes coding for cell growth and proliferation, cell-to-cell signalling and interaction, lipid metabolism, development, and cancer. Likewise, when dysplasia was compared with non-transgenic cells up-regulation of cancer associated genes, tight junction proteins, xenobiotic defence and developmental regulators was observed.
  • dysplasia vs transgenic and dysplasia vs non-transgenic 114 genes were regulated in common. Additionally, regulation of some genes was confirmed by immunohistochemistry and therefore good concordance between gene regulation and coded protein is demonstrated.
  • the study according to the invention identified transcriptional networks at successive stages of tumor-development, i.e. from histological unaltered but transgenic lungs to nuclear atypia.
  • the SP-C/c-raf transgenic mouse model revealed interesting and novel genes and pathways that provide clues on the mechanism forcing respiratory epithelium into dysplasia and subsequently cancer, some of which are also useful in the molecular imaging and flagging of early stages of disease, thereby enabeling the invention described herein.
  • gene markers of dysplasia With the help of microarray studies several gene markers of dysplasia have been identified. Said gene markers in turn encode for proteins which in turn are involved in various metabolic reactions, such as in:
  • tight junction proteins more particular Claudins
  • gap junction proteins more particular GJA3 etc
  • transcription factors in the epithelial-mesenchymal transition preferably HNF4a etc , thereby allowing an easy identification of dysplasia by determining said metabolism reactions or proteins or protein functions.
  • biochemical reactions described herein are suitable for imaging procedures such as PET diagnostics ( “metabolic imaging”), optical imaging, which is applied for the bronchoscopy.
  • PET diagnostics “metabolic imaging”
  • optical imaging which is applied for the bronchoscopy.
  • reactions catalyzed by enzymes are appropriate.
  • the invention is easily implemented in the tumor-PET diagnostics, wherein preferably the glucose metabolism is investigated (FDG-PET).
  • the described reactions of dysplasia are particularly useful for implementing the invention by enzyme based assays (in vitro).
  • the described reactions of dysplasia are excellent targets for the therapy of facultative canceroses.
  • Figure 1 Histological analyses of lung tissue from 5-month-old transgenic mice.
  • Figure 2 Venn diagram for significantly regulated genes.
  • Figure 3 Principal component analysis for gene expression profiles of transformed cells.
  • Figure 4 Result of the hierarchical cluster analysis.
  • the normalized data were used for the Ward's Minimum Variance linkage clustering algorithm.
  • a total of 2909 differentially expressed genes (mean channel intensity > 100, FDR: 0.1 , Bad Flags: 5) were used in the cluster dendogram to obtain a clear segregation of the analyzed groups (dysplasia, transgenic and non-transgenic).
  • the similarity of gene expression profiles among experimental samples is summarized in a dendogram above the cluster, in which the pattern and the length of the branches reflect the relatedness of the samples.
  • Groups (dysplasia, transgenic and non- transgenic) are presented by columns, and genes in rows. Expression values were colour coded with a red green scale. Green, transcript levels below the median; black, equal to the median and red, greater than median.
  • Figure 5 Ingenuity networks: dysplasia versus transgenic mice
  • Ingenuity networks generated by mapping the focus genes that were differentially expressed between dysplasia and transgenic unaltered lung tissue.
  • Each network is graphically displayed with genes/gene products as nodes (different shapes represent the functional classes of the gene products) and the biological relationships between the nodes as edges (lines).
  • the length of an edge reflects the evidence in the literature supporting that node-to-node relationship.
  • the intensity of the node color indicates the degree of up- (red) or down-regulation (green) of the respective gene.
  • a solid line without arrow indicates protein-protein interaction. Arrows indicate the direction of action (either with or without binding) of one gene to another.
  • IPA networks were generated as follows: Upon uploading of genes and corresponding fold-change expression values (done separately for dysplasia vs transgenic and dysplasia vs non-transgenic differentially expressed genes), each gene identifier was mapped to its corresponding gene object in the IPA Knowledge Base (part of the IPA algorithm). Fold-change expression values were used to signed genes whose expression was differentially regulated; these "focus genes" were overlaid onto a global molecular network contained in the IPA Knowledge Base. Networks of these focus genes were then algorithmically generated based on their connectivity and scored according to the number of focus genes within the network as well as according to the strength of their associations.
  • Figure 7 Comparison quantitative RT-PCR and Oligonucleotid-Array.
  • Figure 8 lmmunohistochemical staining for dysplasia. lmmunohistochemical staining in dysplasia in the presence of primary antibody (a) 10x magnification, b) 4Ox magnification) and in the presence of primary antibody preincubated with blocking peptide (c).
  • 1 amphiregulin (AREG)
  • 2 epiregulin (EREG)
  • 3 hepatocyte nuclear factor 4 alpha (HNF4 ⁇ )
  • 4 forkhead box 3a (FOX3A/HNF3 ⁇ )
  • 5 forkhead box P2 (FOXP2)
  • Figure 9 Western immunoblotting of amphiregulin and apolipoprotein A-I in (blood-)serum of transgenic mice and healthy control mice, ⁇ -tubulin was used as a loading control (Fig. 9).
  • HNF4 alpha and the Ca-channel TRPC1 are novel disease candidate genes in diabetic nephropathy. Diabetes 57:1069-77.
  • Parathyroid hormone-related protein is an independent prognostic factor for renal cell carcinoma. Cancer 86:1028-34.
  • GATA-6 a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm. Dev Biol 177:309-22.
  • GATA-5 a transcriptional activator expressed in a novel temporally and spatially-restricted pattern during embryonic development. Dev Biol 183:21-36.
  • Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev 4:2353-2365.
  • Etv4 ets variant gene 4 (E1 A enhancer binding protein, E1AF) 35,16 1423232_at NM_008815 1810036H07R ⁇ k RIKEN cDNA 1810036H07 gene 31 ,07 1453132_a_at NM_025467
  • Gjb4 gap junction membrane channel protein beta 4 11 ,14 1422179_at NM_008127 CbInI cerebellin 1 precursor protein 11 ,13 1423288_s_at NM_019626 Fetub fetuin beta 10,81 1449555_a_at NM_021564
  • Gtl2 GTL2 imprinted maternally expressed untranslated mRNA 9,59 1428765_at NMJ44513
  • Gja3 gap junction protein alpha-3 9,32 1439793_at —
  • Gtl2 /// Lphni GTL2, imprinted maternally expressed untranslated mRNA /// latrophilin 1 8,27 1452905_at NM_144513
  • Gtl2 GTL2 imprinted maternally expressed untranslated mRNA 7,93 1426758_s_at NMJ44513
  • Myh6 /// LOC671894 myosin, heavy polypeptide 6, cardiac muscle, alpha 7,72 1448554_s_at NM_010856
  • Tnfsr ⁇ tumor necrosis factor hgand superfamily member 9 6,15 1422924_at —
  • AIbI albumin 1 4,74 1425260_at NM_009654
  • DIkI delta-like 1 homolog (Drosophila) 83,87 1449939_s_at NM_010052
  • Ndg1 /// LOC623189 Nur77 downstream gene 1 54,61 1455423_at NM_183322
  • Etv4 ets va ⁇ ant gene 4 (E1A enhancer binding protein, E1 AF) 52,64 1423232_at NM_008815 Ankrd22 ankyrin repeat domain 22 47,38 1453239_a_at NM_024204
  • Gtl2 /// Lphni GTL2, imprinted maternally expressed untranslated mRNA /// latrophilin 1 19,95 1452905_at NM_144513 Gtl2 GTL2, imprinted maternally expressed untranslated mRNA 19,57 1426758_S_at NMJ44513 Hnf4a hepatic nuclear factor 4, alpha 18,66 1427001_s_at NM_008261 Gtl2 gene trap locus 2 18,61 1436713_S_at — Gtl2 GTL2, imprinted maternally expressed untranslated mRNA 17,26 1439380_x_at NMJ44513 Mirg miRNA containing gene 17,26 1457030_at XM_488655 Atp6v0a4 ATPase, H+ transporting, lysosomal VO subunit A isoform 4 16,92 1422030 at NM 080467
  • Slc23a3 solute earner family 23 (nucleobase transporters), member 3 15,85 1460042_at NM_194333 Rgs16 regulator of G protein signaling 16 15,71 1455265_a_at — Tnfsf9 tumor necrosis factor hgand superfamily, member 9 15,68 1422924_at —
  • BC024561 cDNA sequence BC024561 11 ,87 1451610_at NM_153576
  • Myh6 /// LOC671894 myosin, heavy polypeptide 6, cardiac muscle, alpha 10,98 1448554_S_at NM_010856
  • Ttc9 tetrat ⁇ copeptide repeat domain 9 10,64 1436237_at XM_126933
  • Ptprn /// LOC669060 protein tyrosine phosphatase, receptor type, N 10,59 1416588_at NM_008985
  • Rab3c RAB3C member RAS oncogene family 7,99 1449494_at NM_023852
  • Rapigap Rap1 GTPase-activating protein 1 7,78 1428443_a_at XM_149500
  • Mapki 3 mitogen activated protein kinase 13 7,09 1448871_at NM_011950
  • SId 5a2 solute carrier family 15 H+/pept ⁇ de transporter
  • member 2 6,84 1447808_s_at NM_021301
  • B4galt6 /// LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 6,64 1423228_at NM_019737 Cldn3 claud ⁇ n 3 6,57 1426332_a_at NM_009902 Gm71 gene model 71 , (NCBI) 6,55 1455726_at XM_127052 Krt1-18 keratin complex 1 , acidic, gene 18 6,52 1448169_at NM_010664
  • Pcsk6 proprotein convertase subtilisin/kexin type 6 6,08 1426981_at XM_355911
  • Tnfrsf21 tumor necrosis factor receptor superfamily member 21 6,07 1450731_s_at NM_178589
  • Ly6g6c lymphocyte antigen 6 complex locus G6C 6,00 1422749_at NM_023463
  • Ly6g6e lymphocyte antigen 6 complex locus G6E 5,14 1429833_at NM_027366
  • Tacstdi tumor-associated calcium signal transducer 1 4,81 1416579_a_at NM_008532
  • Spbc24 spindle pole body component 24 homolog (S cerevisiae) 4,73 1431087_at NM_026282
  • Rab25 RAB25 member RAS oncogene family 4,55 1417738_at NM_016899
  • Ttc9 tetratricopeptide repeat domain 9 4,33 1455649_at XM_126933
  • Cox4 ⁇ 2 cytochrome c oxidase subunit IV isoform 2 0,15 1421373_at NM_053091
  • Chsti carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 0,15 1449147_at NM_023850
  • Emr4 EGF-like module containing, mucin-like, hormone receptor-like sequence 4 0,14 1451563_at NMJ39138
  • Slc7a10 solute earner family 7 (cationic amino acid transporter, y+ system), member 10 0,13 1421093_at NM_017394
  • Igfbp2 insulin-like growth factor binding protein 2 0,12 1454159_a_at NM_008342
  • Igfbp3 insulin-like growth factor binding protein 3 0,12 1458268_s_at NM_008343
  • AI841794 expressed sequence AI841794 0,12 1433744_at NM_172492
  • Lrat Lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) 0,10 1444487_at NM_023624
  • Igfbp3 insulin-like growth factor binding protein 3 0,08 1423062_at NM_008343
  • AI481121 expressed sequence AI481121 0,08 1456123_at
  • Gja3 gap junction protein alpha-3 16,27 1439793_at —
  • P2rx2 purinergic receptor P2X ligand-gated ion channel, 2 8,37 1435212_at NM_170682

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Abstract

L'identification de patients courant le risque de développer le cancer à des stades précoces de la maladie aurait un important impact sur la survie globale. Les perturbations moléculaires associées aux stades précoces du cancer du poumon sont, toutefois, inconnues, de même que les réseaux de régulation génique qui induisent la transformation des cellules dysplasiques en cellules malignes. Pour ce faire, la présente invention décrit un procédé simple et efficace pour identifier des cibles diagnostiques pouvant être utilisées dans le diagnostic, ou la surveillance des traitements de la dysplasie, et l'utilisation desdites cibles et substances apparentées pour le diagnostic, la surveillance des traitements et le traitement de la dysplasie, en particulier, la dysplasie de stade bas ou de stade élevé liée à l'adénocarcinome du poumon. En particulier, cette invention concerne un procédé permettant d'identifier des cibles pouvant être utilisées dans le diagnostic ou la surveillance des traitements de la dysplasie, en particulier, la dysplasie de stade bas ou de stade élevé liée à l'adénocarcinome du poumon, comprenant les étapes suivantes : microdissection au laser de cellules provenant du tissu d'un animal transgénique surexprimant un oncogène placé sous la commande d'une séquence de régulation provenant d'un gène spécifique du tissu, pour recueillir des cellules dysplasiques et des cellules morphologiquement non altérées, l'isolation de l'ARN (1) provenant desdites cellules dysplasiques et l'isolation de l'ARN (2) provenant desdites cellules morphologiquement inaltérées, la détermination, pour chacun des ARN (1) et (2) isolés, d'un profil d'expression génique, de préférence, génomique, et l'identification de la cible sous la forme d'un gène significativement surexprimé dans le profil d'expression génique de l'ARN isolé (1) par rapport au profil d'expression génique de l'ARN isolé (2). Plus particulièrement, l'invention concerne l'utilisation d'un gène identifié par ledit procédé, ledit gène étant, de préférence, choisi dans le groupe des Areg, Ereg, Adcyap1, Adora1, Afp, Apoa1, Arg2, Brunol4, Cldn2, Cldn4, Cldn8, CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1, Pcbd1, Pthlh, Rasgrf1, Rgs16, Stk39, Tnfsf9, de préférence, APOA1 ou dans le groupe des gènes Btbd11, C8orf13, Cyp1 b1, Fetub, Fut2, Klc3, Pcsk6, Pkhd1, Pla2gl1 b, Psrd, Ptprn2, Rnf 128, Ros1, Sdcbp2, Sult2b1, de préférence, PKHD1, ou d'un produit de gène codé par l'un de ceux-ci ou des séquences ARN ou ADN, qui s'hybrident audit gène et qui codent pour un polypeptide ayant la fonction dudit produit de gène, ou d'un anticorps dirigé contre ledit produit de gène ou d'un anticorps dirigé contre ledit polypeptide, pour le diagnostic ou la surveillance des traitements de la dysplasie, en particulier, la dysplasie de stade bas ou de stade élevé liée à l'adénocarcinome du poumon et/ou pour identifier par criblage des médicaments contre la dysplasie, en particulier, la dysplasie de stade bas ou de stade élevé liée à l'adénocarcinome du poumon.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011070150A1 (fr) * 2009-12-11 2011-06-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouvelles cibles pour le traitement de maladies prolifératives
JP5858391B2 (ja) * 2013-04-22 2016-02-10 株式会社栃木臨床病理研究所 抗腫瘍剤
CN107267518A (zh) * 2017-08-01 2017-10-20 浙江大学 特异性抑制CLDN8基因表达的siRNA及其重组载体和应用
WO2019035075A1 (fr) * 2017-08-17 2019-02-21 NantOmics, LLC. Changements dynamiques dans l'arn libre circulant de tumeurs neurales
CN113122626A (zh) * 2019-12-30 2021-07-16 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） Klc3基因作为标志物在肺癌、胃癌、结直肠癌、子宫内膜癌及卵巢癌中诊断及治疗的应用
CN119742082A (zh) * 2025-03-04 2025-04-01 天津市胸科医院 基于大数据的主动脉夹层术后对照模型构建方法及系统

Non-Patent Citations (74)

* Cited by examiner, † Cited by third party
Title
"Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
"Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses", 1980, PLENUM PRESS
ALESSI DR; SAITO Y; CAMPBELL DG; COHEN P; SITHANANDAM G ET AL.: "Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1", EMBO, vol. 13, 1994, pages 1610 - 9
ANG SL; WIERDA A; WONG D; STEVENS KA; CASCIO S ET AL.: "The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins", DEVELOPMENT, vol. 119, 1993, pages 1301 - 15
BABA I; SHIRASAWA S; IWAMOTO R; OKUMURA K; TSUNODA T ET AL.: "Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells", CANCER RES, vol. 60, 2000, pages 6886 - 6889
BALSARA BR; TESTA JR: "Chromosomal imbalances in human lung cancer", ONCOGENE, vol. 21, 2002, pages 6877 - 6883
BAVIK C; COLEMAN; DEAN JP; KNUDSEN B; PLYMATE S ET AL.: "The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms", CANCER RES, vol. 66, 2006, pages 794 - 802
BORLAK J; JENKE HS: "Cross-talk between aryl hydrocarbon receptor and mitogen-activated protein kinase signaling pathway in liver cancer through c-raf transcriptional regulation", MOL CANCER RES, vol. 6, 2008, pages 1326 - 36
BOYLE P; DRESLER C: "Editorial: Preventing the lung cancer epidemic", ANNALS OF OMCOLOGY, vol. 16, 2005, pages 1565 - 1566
CHATTERJI B; BORLAK J: "Serum proteomics of lung adenocarcinomas induced by targeted overexpression of c-raf in alveolar epithelium identifies candidate biomarkers", PROTEOMICS, vol. 7, 2007, pages 3980 - 91
CORDERO A; MUKHERJEE S; SUBRAMANIAN A; YOU H; ROIX JJ ET AL.: "An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis", NAT GENET, vol. 37, 2005, pages 48 - 55
COSTA RH; KALINICHENKO V; LIM L: "Transcription factors in mouse lung development and function", AM J PHYSIOL LUNG CELL MOL PHYSIOL, vol. 280, 2001, pages 823 - 838
DUDA K; CHI YI; SHOELSON SE: "Structural basis for HNF-4alpha activation by ligand and coactivator binding", J.BIOL.CHEM, vol. 279, 2004, pages 23311 - 23316
EBERT M; YOKOYAMA M; KOBRIN MS; FRIESS H; LOPEZ ME ET AL.: "Induction and expression of amphiregulin in human pancreatic cancer", CANCER RES, vol. 54, 1994, pages 3959 - 3962
EMMERT-BUCK MR; BONNER RF; SMITH PD: "Laser capture microdissection", SCIENCE, vol. 274, 1996, pages 998 - 1001
FONG KM; SEKIDO Y; GAZDAR AF; MINNA JD: "Lung cancer 9: Molecular biology of lung cancer: clinical implications", THORAX, vol. 58, 2003, pages 892 - 900
GARCIA-FERNANDEZ MO; BODEGA G; RUIZ-VILLAESPESA A; CORTES J; PRIETO JC ET AL.: "PACAP expression and distribution in human breast cancer and healthy tissue", CANCER LETTERS, vol. 205, 2004, pages 189 - 195
GRANVILLE CA; DENNIS PA: "An Overview of Lung Cancer Genomics and Proteomics", AM J RESPIR CELL MOL BIOL, vol. 32, 2005, pages 169 - 176
HAKOMORI S: "Glycosynapses: microdomains controlling carbohydrate- dependent cell adhesion and signalling", AN ACAD BRAS CIENC, vol. 76, 2004, pages 553 - 572
HURBIN A; DUBREZ L; COLL JL; FAVROT MC: "Inhibition of Apoptosis by Amphiregulin via an Insulin-like Growth Factor-1 Receptor-dependent Pathway in Non-small Cell Lung Cancer Cell Lines", JBC, vol. 277, 2002, pages 49127 - 49133
HYLTANDER A; DROTT C; KORNER U; SANDSTROM R; LUNDHOLM K: "Elevated energy expenditure in cancer patients with solid tumours", EUR J CANCER, vol. 27, 1991, pages 9 - 15
INOUE A; NUKIWA T: "Gene Mutations in Lung Cancer: Promising Predictive Factors for the Success of Molecular Therapy", PLOS MEDICINE, vol. 2, 2005, pages 5 - 13
ISHIKAWA N; DIAGO Y; TAKANO A; TANIWAKI M; KATO T ET AL.: "Increases of Amphiregulin and Transforming Growth Factor-a in Serum as Predictors of Poor Response to Gefitinib among Patients with Advanced Non-Small Cell Lung Cancers", CANCER RES, vol. 65, 2005, pages 9176 - 9184
ISOBE K; KANEKO M; KANEKO S; NISSATO S; NANMOKU T ET AL.: "Expression of mRNAs for PACAP and its receptor in human neuroblastomas and their relationship to catecholamine synthesis", J REGPEP, vol. 123, 2004, pages 29 - 32
IWAMURA M; WU W; MURAMOTO M; OHORI M; EGAWA S ET AL.: "Parathyroid hormone-related protein is an independent prognostic factor for renal cell carcinoma", CANCER, vol. 86, 1999, pages 1028 - 34
JACKSON DA; ROWADER KE; STEVENS K; JIANG C; MILOS P ET AL.: "Modulation of liver-specific transcription by interactions between hepatocyte nuclear factor 3 and nuclear factor 1 binding DNA in close apposition", MOL CELL BIOL, vol. 13, 1993, pages 2401 - 2410
JOHNSON GR; SAEKI T; AUERSPERG N; GORDON AW; SHOYAB M ET AL.: "Response to and expression of amphiregulin by ovarian carcinoma and normal ovarian surface epithelial cells: nuclear localization of endogenous amphiregulin", BIOCHEM BIOPHYS RES COMMUN, vol. 180, 1991, pages 481 - 488
KALINICHENKO W; LIM L; SHIN B; COSTA RH: "Differential expression of forkehead box transcription factors following butylated hydroxytoluene lung injury", AM J PHYSIOL LUNG CELL MOL PHYSIOL, vol. 280, 2001, pages L695 - 704
KAROLCHIK D; KUHN, RM; BAERTSCH R; BARBER GP; CLAWSON H; DIEKHANS M; GIARDINE B; HARTE RA; HINRICHS AS; HSU F: "The UCSC Genome Browser Database: 2008 update", NUCLEIC ACIDS RES., vol. 36, January 2008 (2008-01-01), pages D773
KERKHOFF E; FEDOROV LM; SIEFKEN R; WALTER AO; PAPADOPOULOS T; RAPP UR: "Lung-targeted expression of the c-Raf-1 kinase in transgenic mice exposes a novel oncogenic character of the wild-type protein", CELL GROWTH DIFFER, vol. 11, 2000, pages 185 - 90
KERR KM: "Pulmonary preinvasive neoplasia", J CLIN PATHOL, vol. 54, 2001, pages 257 - 271
KISSIN MW; HENDERSON MA; DANKS JA; HAYMAN JA; BENNETT RC ET AL.: "Parathyroid hormone related protein in breast cancers of widely varying prognosis", EUR J SURG ONCOL, vol. 19, 1993, pages 134 - 42
LI S; PLOWMAN GD; BUCKLEY SD; SHIPLEY GD: "Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells", J CELL PHYSIOL, vol. 153, 1992, pages 103 - 111
LIU ZZ; WADA J; KUMAR A; CARONE FA; TAKAHASHI M ET AL.: "Comparative role of phosphotyrosine kinase domains of c-ros and c-ret protooncogenes in metanephric development with respect to growth factors and matrix morphogens", DEV BIOL, vol. 178, 1996, pages 133 - 48
MAO ET AL., CANCER RES., vol. 54, no. 7, 1994, pages 1634 - 7
MASON RJ; GREENE K; VOELKER DR: "Surfactant protein A and surfactant protein D in health and disease", AM J PHYSIOL, vol. 275, 1998, pages L1 - 13
MENNECIER G; DERANGEON M; CORONAS V; HERVE JC; MESNIL M: "Aberrant expression and localization of connexin43 and connexin30 in a rat glioma cell line", MOL CARCINOG, vol. 47, 2008, pages 391 - 401
MINNA JD; ROTH JA; GAZDAR AF: "Focus on lung cancer", CANCER CELL, vol. 1, 2002, pages 49 - 52
MOLKENTIEN JD; LIN Q; DUNCAN SA; OLSON EN: "Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis", GENES DEV., vol. 11, 1997, pages 1061 - 72
MORRISEY EE; IP HS; LU MM; PARMACEK MS: "GATA-6: a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm", DEV BIOL, vol. 177, 1996, pages 309 - 22
MORRISEY EE; IP HS; TANG Z; LU MM; PARMACEK MS: "GATA-5: a transcriptional activator expressed in a novel temporally and spatially-restricted pattern during embryonic development", DEV BIOL, vol. 183, 1997, pages 21 - 36
MORRISON DK; CUTLER RE: "The complexity of Raf-1 regulation", CURR OPIN CELL BIOL, vol. 9, 1997, pages 174 - 9
NESBITT JC; PUTNAM JB; WALSH GL; ROTH JA; MOUNTAIN CF: "Survival in early- stage lung cancer", ANN THORAC SURG, vol. 60, 1995, pages 466 - 472
NIEHOF M; BORLAK J: "HNF4 alpha and the Ca-channel TRPC1 are novel disease candidate genes in diabetic nephropathy", DIABETES, vol. 57, 2008, pages 1069 - 77
NISHIHARA M; ITO M; TOMIOKA T; OHTSURU A; TAGUCHI T ET AL.: "Clinicopathological implications of parathyroid hormone-related protein in human colorectal tumours", J PATHOL, vol. 187, 1999, pages 217 - 22
ODOM DT; ZIZLSPERGER N; GORDON DB; BELL GW; RINALDI NJ ET AL.: "Control of Pancreas and Liver Gene Expression by HNF Transcription Factor", SCIENCE, vol. 303, 2004, pages 1378 - 1381
PANKIEWICZ ET AL., FOLIA HISTOCHEM CYTOBIOL., vol. 45, no. 2, 2007, pages 65 - 74
PAYNE DM; ROSSOMANDO AJ; MARTINO P; ERICKSON AK; HER JH ET AL.: "Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase)", EMBO, vol. 10, 1991, pages 885 - 92
POINTIS G; FIORINI C; GILLERON J; CARETTE D; SEGRETAIN D: "Connexins as precocious markers and molecular targets for chemical and pharmacological agents in carcinogenesis", CURR MED CHEM, vol. 14, 2007, pages 2288 - 303
REISHER SR; HUGHES TE; ORDOVAS JM; SCHAEFER EJ; FEINSTEIN SI: "Increased expressions of apolipoprotein genes accompanies differentiation in the intestinal cell line CACO-2", PNAS, vol. 90, 1993, pages 5757 - 61
ROTTERS H; ZURBIG P; HALTER R; BORLAK J: "Towards a lung adenocarcinoma proteome map: Studies with SP-C/c-raf transgenic mice", PROTEOMICS, vol. 6, 2006, pages 3127 - 3137
RUFIBACH LE; DUNCAN SA; BATTLE M; DEEB SS: "Transcriptional regulation of the human hepatic lipase (LIPC) gene promoter", J OF LIPID RESEARCH, vol. 47, 2001, pages 1463 - 1477
SCHULTZ IJ; WESTER K; STRAATMAN H; KIEMENEY LA; BABJUK M ET AL.: "Prediction of recurrence in Ta urothelial cell carcinoma by real-time quantitative PCR analysis: A microarray validation study", INT J CANCER, vol. 119, 2006, pages 1915 - 1919
SEHGAL; BAILEY J; HITZEMANN K; PITTELKOW MR; MAIHLE NJ: "Epidermal growth factor receptor-dependent stimulation of amphiregulin expression androgen- stimulated human prostate cancer cells", MOL BIOL CELL, vol. 5, 1994, pages 339 - 347
SHELLY M; PINKAS-KRAMARSKI R; GUARINO BC; WATERMAN H; WANG LM ET AL.: "Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes", J BIOL CHEM, vol. 273, 1998, pages 10496 - 10505
SHEN W; SCEARCE M; BRESTELLI JE; SUND NJ; KAESTNER KH: "Foxa3 (Hepatocyte Nuclear Factor 3gamma) Is required for the Regulation of Hepatic GLUT2 Expression and the Maintenance of Glucose Homeostasis during a Prolonged Fast", JBC, vol. 276, 2001, pages 42812 - 42817
SHIH DQ; NAVAS MA; KUWAJIMA S; DUNCAN SA; STOFFEL M: "Impaired glucose homeostasis and neonatal mortality in hepatocyte nuclear factor 3a-deficient mice", PNAS, vol. 96, 1999, pages 10152 - 10157
SHIRAKATA Y; KOMURASAKI H; TOYODA Y; HANAKAWA K; YAMASAKI S ET AL.: "Epiregulin, a novel memberof the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes", J BIOL CHEM, vol. 275, 2000, pages 5748 - 5753
SHU W; YANG H; ZHANG L; LU MM; MORRISEY E: "Characterization of a New Subfamily of Winged-helix/Forkhead (Fox) Genes That Are Expressed in the Lung and Act as Transcriptional Repressors", JBC, vol. 29, 2001, pages 27488 - 27497
SLADEK FM; ZHONG W; LAI E; DARNELL JE: "Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily", GENES DEV, vol. 4, 1990, pages 2353 - 2365
STAHLMAN MT; GRAY ME; WHITSETT JA: "Temporal-spatial distribution of hepatocyte nuclear factor-3beta in developing human lung and other foregut derivatives", J HISTOCHEM CYTOCHEM, vol. 46, 1998, pages 955 - 62
THOGERSEN VB; SORENSEN BS; POULSEN SS; ORNTOFT TF; WOLF H ET AL.: "A subclass of HER1 ligands are prognostic markers for survival in bladder cancer patients", CANCER RES, vol. 61, 2001, pages 6227 - 6233
THOMAS RK; WEIR B; MEYERSON M: "Genomic Approaches to Lung Cancer", CLIN CANCER RES, vol. 12, 2006, pages 4384S - 4391S
THOMAS SE; LINDA KC: "Plasma Lipoproteins with Bioregulatory Properties Including the Capacity to Regulate Lymphocyte Function and the Immune Response", CANCER RES, vol. 41, 1981, pages 3786 - 3788
TORRING N; JORGENSEN PE; SORENSEN BS; NEXO E: "Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines", ANTICANCER RES, vol. 20, 2000, pages 91 - 95
TUSHER VG; TIBSHIRANI R; CHU G: "Significance analysis of microarrays applied to the ionizing radiation response", PNAS, vol. 98, 2001, pages 5116 - 5121
VERMEER PD; EINWALTER LA; MONINGER TO; ROKHLINA T; KERN JA ET AL.: "Segregation of receptor and ligand regulates activation of epithelial growth factor receptor", NATURE, vol. 422, 2003, pages 267 - 8
WANG X; MASRI S; PHUNG S; CHEN S: "The role of amphiregulin in exemestane-resistant breast cancer cells: evidence of an autocrine loop", CANCER RES, vol. 68, 2008, pages 2259 - 65
WARREN L; BUCK CA; TUSZYNSKI GP: "Alterations of membrane glycoproteins in malignant cells", DEV BIOL STAND, vol. 42, 1979, pages 177 - 82
WEIR BA; WOO MS; GETZ G; PERNER S; DING L ET AL.: "Characterizing the cancer genome in lung adenocarcinoma", NATURE, vol. 450, 2007, pages 893 - 898
WOLMAN SR; HEPPNER GH; WOLMAN E: "New directions in breast cancer research", FED AM SOC EXP BIOL J, vol. 11, 1997, pages 535 - 543
WOODWORTH CD; MCMULLIN E; IGLESIAS M; PLOWMAN GD: "Interleukin 1 and tumor necrosis factor stimulate autocrine amphiregulin expression a proliferation of human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells", PROC NATL ACAD SCI USA, vol. 92, 1995, pages 2840 - 2844
YAM D; BEN-HUR H; FINK A; DGANI R; SHANI A ET AL.: "Insulin and glucose status, tissue and plasma lipids in patiens with tumours of the ovary or endometrium: possible dietary implications", BR J CANCER, vol. 70, 1994, pages 1186 - 7
ZARET K: "Developmental competence of the gut endoderm: Genetic potentiation by GATA and HNF3/fork head proteins", DEV BIOL, vol. 209, 1999, pages 1 - 10

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