Novel Targets For The Diagnosis And Treatment Of Dysplasia

Novel targets for the diagnosis and treatment of dysplasia

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. As today 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. Importantly, the genetics of dysplasia, a facultative cancer, are unknown but are of great concern and have not been investigated, so far.

The lung cancer epidemic was the subject of a recent editorial [1]. Indeed, in Europe alone more than 340 000 death per annum are attributable to this cancer, but this disease is by large preventable. There is concluding evidence for tobacco smoke to be the primary cause of lung cancer and more than 4,800 compounds have been identified in the particulate and gas phases of cigarette smoke. Major lung carcinogens in smoke include some of the polycyclic aromatic hydrocarbons, such as benzo/a/pyrene, as well as tobacco-specific Λ/-nitrosamines. Although a lot of evidence supports the relationship between cigarette smoking and lung cancer, the molecular events associated with early stages of disease remain somewhat elusive. A diverse range of genetic abnormalities are seen in different stages of lung cancer, some of which may be employed as markers of disease progression; others may have a direct role in lung cancer etiology in the context of gene-environment interactions. Characterisation of the cancer genome in lung adenocarcinoma was the subject of a recent study and several reviews [2, 3, 4].

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]. An identification of patients at risk for developing cancer at early stages of disease would have a big impact on overall survival. Notably, there has been significant progress in an understanding of the molecular pathogenesis of lung cancer, which includes an identification of genetic and epigenetic events in cancer subgroups [8]. The molecular perturbations associated with early stages of lung cancer are, however, unknown as are the gene regulatory networks forcing dysplastic cells into malignant transformation.

Previous studies had defined atypical adenomatous hyperplasia (AAH) as a preinvasive lesion that progresses from low to high grade dysplasia to invasive adenocarcinoma [9]. Strikingly, the genetic events associated with dysplasia and its progression into invasive carcinomas remains uncertain. Specifically, with low grade dysplasia the architectural and cytological changes of the cell are minimal, while with high grade dysplasia gross cytological irregularities become obvious with larger, columnar cells, cytoplasmic pleomorphism, large hyperchromatic nuclei, uneven chromatin structure and higher mitotic rates. Although these morphological changes are well recognized, the underlying molecular alterations are at best poorly understood.

Hence, the identification of targets allowing the diagnosis, treatment monitoring or treatment of dysplasia, is of great importance.

Different immunohistochemical markers of cancerogenesis in the lung have been described (Pankiewicz et al. Folia Histochem Cytobiol. 2007 45(2):65-74). Further, the detection of p53 gene mutations in sputum may precede the diagnosis of lung cancer (Mao et al. Cancer Res. 1994 54(7): 1634-7). However, systematic methods for a goal-directed identification of targets in dysplastic cells and means directed against said targets are urgently needed but are still a great challenge.

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. To this end, the implementation of the actions and embodiments as described in the claims provides appropriate means to fulfill these demands in a satisfying manner.

Thus, the invention in its different aspects and embodiments is implemented according to the claims.

The invention is based on the surprising finding, that a method comprising the steps of

- laser microdissection of cells from the tissue of a transgenic animal overexpressing an oncogene placed under the control of a regulatory sequence from a tissue specific gene, wherein dysplastic cells and morphologically unaltered cells are harvested,

- isolating RNA (1) from the dysplatic cells and isolating RNA (2) from the morphologically unaltered cells,

- determining, for each of the isolated RNA (1) and (2), a gene expression profile, preferably a genome wide gene expression profile, and

- identifying the target as a gene being significantly overexpressed or being significantly underexpressed in the gene expression profile of the isolated RNA (1) in comparison with the gene expression profile of the isolated RNA (2) enables an easy and efficient identification of targets for the use in diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung. According to this first aspect of the invention 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. Preferably, 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. In particular it is preferred if 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.

The term "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.

In particular, it is preferred, if 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).

Still further, it is preferred, if the method comprises

- a total RNA-extraction,

- a standard quality control of the total RNA,

- using the total RNA to generate biotin-labeled cRNA,

- quality control of the cRNA,

- fragmentation of the cRNA, - hybridizing the labeled c-RNA to a oligonucleotide array, in particular microarray, preferably covering over 34000 genes,

- staining of the hybridized oligonucleotide array,

- quantification of the array data,

- statistical analyses of the array data,

- normalizing the array data, preferably by using scaling or per-chip normalization to adjust the total or average intensity of each array to be approximately the same,

- comparing the normalized data from dysplasia, normal lung tissue from transgenic mouse, tumor cells and non-transgenic of different mice, preferably by using Significance Analysis of Microarrays (SAM) algorithm (ArrayTrack), in particular by chosing as cut-off for significance an estimated FDR of 0.001 and using a cut-off for fold-change of differential expression of 2,

- principal-component analysis (PCA),

- hierarchical gene clustering (HCA),

- ingenuity pathways analysis (IPA), and/or

- using a Venn diagram to examine the overlap of resulting lists of genes differentially expressed between the different sample sets.

In particular, it is preferred, if 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.

In particular it is preferred if at least one microarray, preferably at least two microarrays, is/are employed for 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.

In another preferred embodiment, the invention comprises an immunohistochemistry

(IHC) analysis, in particular by using antibodies directed against the gene products of the genes according to Supplementary Table 1 (Table S1) in a direct or an indirect IHC.

Thus, 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.

It is understood according to the invention that within the context of the group of genes listed in Supplementary Table 1 or the group the gene products encoded thereby, in particular the genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoal, Arg2, Brunol4, Cldn2, Cldn4, Cldnδ, 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 said genes are preferred, respectively. Accordingly, within the context of an antibody directed against said gene product or of an antibody directed against said polypeptide, it is preferred if an antibody directed against a gene product encoded by a gene selected from the group consisting of Areg, Ereg, Adcyapi , Adoral , Afp, Apoal , Arg2, Bruno!4, Cldn2, Cldn4, Cldn8, CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi, Pthlh, Rasgrfl , Rgs16, Stk39, Tnfsf9, preferably APOA1 , and/or if an antibody directed against a gene product encoded by a gene selected from the group consisting of Btbdi 1 , C8orf13, Cyp1 b1 , Fetub, Fut2, Klc3, Pcskθ, Pkhdi , Pla2gl1 b, Psrd , Ptprn2, Rnf128, Ros1 , Sdcbp2, Sult2b1 , preferably PKHD1 , is used.

According to a second aspect 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.

In particular, 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.

Thus, 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.

The term "low grade dysplasia" according to the invention is particularly directed to a lesion having minimal aberration inside the cell. The term "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

A1 Raney BJ, Rhead B, Rosenbloom KR, Smith KE, Stanke M, Thakkapallayil A,

Trumbower H, Wang T, Zweig AS, Haussler D, Kent WJ. The UCSC Genome

Browser Database: 2008 update. Nucleic Acids Res. 2008 Jan;36:D773, which is incorporated herein by reference.

In particular, it is understood that the genes described herein are murine genes or preferably the respective human genes.

Within the context of the invention it also understood that the 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.

The term "coding region" according to the invention 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.

The term "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.

The term "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.

The term "hybridizing" as used herein refers to conventional hybridization conditions, preferably to hybridization conditions under which the Tm value is between 37°C to 700C, preferably between 410C to 66°C. An example for low stringency conditions is e.g. hybridisation under the conditions 42°C, 2χSSC, 0.1% SDS and an example for high stringency conditions is e.g. hybridization under the conditions: 65°C, 2χSSC, 0.1% SDS, wherein in the case that washing is necessary for equilibrium, the hybridization solution is used for the washings. Most preferably, the term hybridization refers to stringent hybridization conditions.

Within the context of the invention it is further understood that the gene according to the invention, or the DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene, may be part of a recombinant DNA molecule for use in cloning a DNA sequence in bacteria, yeast(s) or animal cells.

Within the context of the invention it is moreover understood that the gene according to the invention, or the DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene, 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.

The term "function of said gene product" according to the invention 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.

Preferably, 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.

Within the context of the invention, in particular the group of members of the EGF- pathway, namely Areg and/or Ereg, and 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), is preferred, and/or an antibody directed against said gene product and/or an antibody directed against said polypeptide is preferably used.

Further, within this context the group of claudins, namely Cldn2, Cldn4, Cldnδ, and the gene products encoded thereby and RNA or DNA sequences, which hybridize to the gene(s) Cldn2, Cldn4, and/or Cldnδand 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.

In another preferred embodiment, the gene according to invention is selected from the group of the focus genes Btbd11 , C8orf13, Cyp1b1 , Fetub, Fut2, Klc3, Pcskβ, 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.

For putting the method or use described into practice, it is particularly preferred, if the gene is selected from the group of genes coding for a protein being involved in at least one metabolism reaction, preferably selected from

- lipid metabolism

- glycosylation - fucosylation

- receptor tyrosine kinase activity

- regulation of Amphiregulin and/or Epiregulin,

- cell interactions, preferably the tight junction proteins (in particular Claudins) or gap junction proteins (in particular GJA3 etc)

- transcription factors in the epithelial-mesenchymal transition (EMT), preferably HNF4a etc

, and/or wherein said metabolism reaction or protein function is determined.

Further, the use or methods described herein is/are preferably used for or in

- bronchoscopy, in particular for monitoring enzyme catalytic reactions,

- in vitro developing of enzyme based assays,

- the therapy of facultative canceroses, and/or

- immunohistochemistry, in particular a immunohistochemical staining.

Within the context of the second aspect of the invention it is preferred, if 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. The term "body fluid" according to the invention is directed to any body fluid of a subject, in particular to blood, plasma, serum or urine, whereas blood serum is the preferred body fluid within the context of the invention.

The term "diagnostic agent" as used herein relates to any solution, suspension or solid formulation, containing said composition in an acceptable amount for diagnostic purposes.

The term "subject", as used hereinafter, 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.

Further, it is preferred if 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 RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, in comparison with the respective diagnostic standard or reference level is indicative that the patient has dysplasia, in particular low grade or high grade dysplasia related to adenocarcinoma of the lung or (B) for monitoring the treatment of a patient having dysplasia to a method of treating dysplasia, in particular by a chemoprevention therapy such as by administering Zileuton (= 1-(1-Benzothiophen-2-ylethyl)-1-hydroxy-urea) and/or Celecoxib (= 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1 -yl]benzenesulfon- amide) to the patient, wherein the level of the gene, in particular the coding region thereof, 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 determined in the sample before and after the treatment, and wherein a significant decrease of said level is indicative that the patient therapeutically responds to the method of treating dysplasia.

In a further embodiment according to the second aspect of the invention it is preferred to screen for drugs targeting cellular growth and proliferation and/or hair and skin development and function, wherein the gene is selected from the group of the focus genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoai , Arg2, BrunoW, Cldn2, Cldn4, Cldnδ, 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.

In another embodiment according to the second aspect of the invention it is preferred to screen for drugs targeting cell death and/or cancer and/or gastrointestinal diseases, wherein 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..

In yet a further embodiment according to the second aspect of the invention, it is preferred if 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.

In a particular embodiment according to the second aspect of the invention, it is preferred 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 the level of the gene, in particular the coding region thereof, 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, in a sample, preferably a tissue sample or a plasma sample, isolated from a transgenic animal to which a compound to be tested has been administered and wherein said level being significantly lower than the respective level in a corresponding sample of an equivalent transgenic animal to which said compound has not been administered is indicative of the therapeutic effect of said compound as a drug directed against dysplasia.

According to another aspect of the invention a composition is provided, 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

- an siRNA composition, wherein the siRNA composition reduces the expression of a gene selected from the group consisting of the genes listed in Supplementary Table 1 , and/or

- an antibody directed against a gene product encoded by a gene selected from the group of genes listed in Supplementary Table 1 , wherein the antibody is preferably linked to a drug carrier, such as a nanoparticle may be. In this regard, 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. Preferably, the antisense DNA is complementary to the 51 regulatory sequence or the 5' portion of the coding sequence of said mRNA.

It is preferred that 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.

In particular, 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 450C.

In this regard, 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.

Preferably, 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.

The 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. According to a further aspect of the invention an antibody composition is provided, 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.

Within the context of the second aspect of the invention, as aforementioned, it is preferred, if 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.

Within the inventive context, 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.

In a further aspect, 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.

In particular, it is preferred, if the use according to the claims, is used for this method.

Preferably, 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.

In one embodiment, 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.

In a preferred embodiment, 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. Preferably, 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, Cldnδ, 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 for the immunoassay and/or reagents effective to detect said biomarker(s) in a blood serum sample, such as a blocking buffer for reducing unspecific antibody binding or an enzyme substrate for imaging enzyme labelled antibodies may be, is used for the immunoassay.

In another preferred embodiment, this method is implemented by performing a peptide mass fingerprinting.

Within the context of peptide mass fingerprinting, this method preferably comprises the steps of

- isolating a serum sample from a blood sample of a subject suffering from or being susceptible to cancer,

- adding lysis buffer to the serum sample;

- separating the proteins of the lysed serum sample by 2-D gel electrophoresis;

- excising from the gel at least one sample containing a protein of interest;

- adding digesting buffer to the at least one excised sample, and - determining the amount of the at least one protein of interest by analyzing the at least one digest mixture by mass spectrometry.

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.

In particular, it is preferred wherein the use or methods according to the claims are performed for putting the procedure into practice.

In particular it is preferred, and surprisingly sufficient, to implement the uses, methods and procedures according to the invention by performing a western blot, thus further simplifying the accomplishement of the invention in its different embodiments.

Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Detailed description

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.

Through the use of refined experimental models new insight into the molecular events associated with dysplasia and its progression into cancers can be obtained, as described herein.

Here findings with a transgenic mouse model are reported, where targeted overexpression of c-RAF to respiratory epithelium resulted in lung cancer development [10]. A serum and lung adenocarcinoma proteome map of the SP-C/c- raf transgenic line was recently published [11 , 12]. Also the molecular organisation of the c-raf promoter [13] has been published. Specifically, 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. In the study according to the invention, 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. By use of laser microdissection pressure catapulting (LMPC) dysplastic cells in well defined lesions [18] could be isolated. 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.

Overall, the study according to the invention revealed interesting and novel genes and pathways that contribute to the early stages of lung cancer development, many of which are worthy for their exploitation as biomarkers and for molecular imaging of early stages of disease.

Results Histological changes

Animals at the age of 5 month displayed morphological changes typical for dysplasia. Note, 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.

SAM (Significance Analysis of Microarrays)

120 genes were significantly regulated when transgenic but otherwise unaltered cells were compared with dysplastic cells (Supplementary Table S1). Strikingly, dysplasia is exclusively associated with induction of transcript expression. In contrast, when transcript expression in dysplasia was compared with non-transgenic lung tissue 234 up-regulated and 53 down-regulated genes could be determined (Supplementary Table S2). For this comparison, at least 2-fold differentially expressed genes at an estimated false discovery rate of ≤ 0.001 (Table 1) were requested. Notably, transgenicity alone was associated with 16 up-regulated and 2 down-regulated genes (Supplementary Table S3). It was searched for differentially expressed genes by comparing the data sets of dysplasia vs transgenic and dysplasia vs non-transgenic. In such a comparison 114 genes were regulated in common (Figure 2).

Notably, this stringent cut-off was used because a smaller number of genes with very low false positive rates was desired on which to focus attention. With an estimated false discovery rate of 0.1 in dysplasia 2352 significantly regulated genes (867 up-regulated and 1485 down-regulated) were obtained compared with transgenic but unaltered cells and 3311 genes (1279 up-regulated and 2032 down- regulated) in dysplasia versus non-transgenic cells. Comparison of these data sets resulted in 2207 genes regulated in common in dysplasia (data not shown).

Principal component analysis (PCA) and hierarchical gene cluster analysis (HCA)

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).

Also a hierarchical gene cluster analyses was applied after SAM analysis. The closest pair of expression values of 2909 differentially expressed genes was grouped together. Consequently, the data are organized in a phylogenetic tree in which the branch lengths represent the degree of similarity between the expression values. A clear segregation of the analyzed groups (dysplasia, transgenic but unchanged lung tissue and non-transgenic lung tissue) was obtained (Figure 4). The PCA and HCA grouped data according to their biological state. Correspondingly, gene expression data of transgenic SP-C/c-raf lung were well separated from non- transgenic and dysplastic cells, suggesting a large difference between these groups.

Pathway analysis of differentially expressed genes

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. These networks were associated with the following pathways: cellular growth and proliferation, cell-to-cell signalling and interaction, cancer, lipid metabolism, development, cellular movement amongst others (Figure 10). In the following, prominent examples are described. 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. In particular, 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.

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. In dysplasia the claudins Cldn2, Cldn4 and Cldnδ were significantly up-regulated, their expression ranged from 4.6-13.31 -fold and 4.9-122.38-fold, when transgenic but healthy and non-transgenic lung tissue were compared, respectively. Overexpression of these tight junction proteins suggest remodelling of tight junctions.

Next to claudins, up-regulation of gap junction proteins was observed in dysplasia by 11 and 9-fold as well (Gja3, gap junction protein alpha-3 and Gjb4, gap junction protein beta-4). Alteration in gap junctional intercellular communication results in the inability of cells to receive apoptotic, growth suppressing or differentiation signals from their neighbours. Specifically, 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.

Furthermore, 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. Thus, 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. In particular Apoai (apolipoproteinA-1) the major apoprotein of High Density Lipoprotein (HDL) was significantly over expressed. 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].

In this regard also changes in the expression of cell surface glycolipids were found in dysplastic cells as compared to unaltered transgenic or to non-transgenic lung tissue. For example, Sult2b1 (sulfotransferase family 2b, member 1) was significantly over expressed. This enzyme catalyzes the sulphate conjugation of pregnenolone and cholesterol. Moreover, Stδsiaδ (Stδ alpha-n-acetyl-neuraminide alpha-2,8-sialyltransferase 6), an enzyme which synthesize sialylglycoconjugates of glycolipids was significantly up-regulated, as well. These changes may result in new surface antigens and glycolipids in addition to altered cell-cell and cell-extracellular matrix communication followed by decreased adhesiveness and invasiveness through normal tissue barriers.

Further evidence for changes in glycosylation patterns during transformation of normal cells into malignant ones stems form the up-regulation of B4galt6 (UDP- GALBeta-GlcNAc Beta-1 ,4-Galactosyltransferase, polypeptide 6), Fut2 (Fucosyltransferase 2), Gpc6 (Glypicanθ) and 0rm1 (orosomucoidi) as observed in dysplastic cells. The synthesis of new carbohydrate chains in dysplastic cells are due to activation of 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. Moreover, the secretor enzyme Fut2, an αr-1 ,2- fucosyltransferase, is responsible for the transfer of fucose in an σ-1 ,2 linkage to form the terminal H type 1 structure. The extra fucosylations that appear on membrane glycoproteins and glycolipids are associated with several pathological processes, such as tumor metastasis, inflammation and bacterial adhesion. As the expression of glycoproteins is increased in many cancers, it was of no surprise that 0rm1 was 11.1-fold overexpressed in dysplastic cells. Orosomucoid 1 belongs to a group of highly glycosylated glycoproteins and appears to function in modulating the activity of the immune system. Furthermore, glypican 6 was found to be induced (9.9-fold). Notably, this protein belongs to a family of glycosylphosphatidylinositol-anchored heparin sulphate proteoglycans. It is known that 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.

In particular Hnf4α (hepatocyte nuclear factor 4-alpha), Foxa3 (forkhead box gene A3) and Foxp2 (forkhead box gene P2) were significantly over expressed in dysplasia (4.5-12.8-fold). These genes codes for proteins that belong to a family of winged-helix/forkhead DNA binding domain transcription factors and are expressed in defined neural, intestinal, and cardiovascular cell types during embryogenesis and differentiation of epithelium. Hnf4α 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 Hnf4α. Since many of those genes contain more than one Hnf4α binding site, these genes can be grouped into several different categories, according to function, such as nutrient transport and metabolism, blood maintenance, immune function, cellular differentiation and growth factors. The best characterized Hnf4α target genes are those involved in lipid transport (e.g., apolipoprotein genes) and glucose metabolism [24]. In this regard Foxa genes code the winged helix/forkhead transcription factor gene family that also includes Foxal , Foxa2 and Foxa3, as well [25]. Notably, 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].

Finally, it was searched for expression of oncogenes and the proto-oncogene Ros1 (v-ros avian ur2 sarcoma virus oncogene homolog 1), which encodes a transmembrane protein with a sequence typical of tyrosine kinases was found to be 5.8-fold up-regulated in dysplasia. Ectopic expression of this receptor tyrosine kinase Ros1 has been reported in many tumors of the central nervous system and recently in lung and stomach cancers [27] as well. It has been suggested that Ros1 plays a role in the mesenchymal epithelial transition during development of kidney, lung and small intestine [28]. Quantitative real-time PCR

To confirm the microarray data, expression of 8 genes in dysplasia, transgenic and non-transgenic samples was examined by quantitative real-time PCR using TaqMan Technology. qRT-PCR confirmed that amphiregulin (Areg), epiregulin (Ereg), fetuin beta (Fetub), apolipoprotein A1 (Apoal), claudin 2 (Cldn2), hepatic nuclear factor 4, alpha (Hnf4α), glutathione S-transferase, alpha 4 (Gsta4) and forkhead box A3 (Foxa3) were up-regulated in dysplasia (Figures 11 and Figure 12). The expression data generated by the oligonucleotid array and RT-PCR agreed well, therefore supporting the reliability of the array analysis (Figure 7).

lmmunohistochemistry

In order to validate array data as well as to add subcellular localization, immunohistochemical staining was performed for some differentially expressed genes, lmmunohistochemistry using antibodies towards amphiregulin, epiregulin, hepatocyte nuclear factor 4 alpha, forkhead box A3 and forkhead box P2 showed a consistent difference in immunoreactivity between dysplastic and transgenic but otherwise unaltered cells (Figure 8). For all five up-regulated proteins, patterns of immunoreactivity confirmed the array data and quantitative real-time PCR results.

DISCUSSION

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.

It was aimed to determine the regulatory gene networks associated with dysplasia. This surprisingly enabled identification of genes specifically regulated at the edge of malignant transformation. Based on hierarchical gene cluster analysis and principal- component analysis it was possible to distinguish the dysplastic cells from transgenic and non-transgenic lung tissue.

To further validate the microarray data, qRT-PCR of selected genes was employed, and confirmed by immunohistochemistry regulated proteins in dysplastic foci. Notably, 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.

Specifically, in dysplasia genes coding for nucleic acid metabolism or cell cycle regulation were unchanged but two EGF-ligands, namely amphi- and epiregulin were highly significantly up-regulated. These ligands enable autocrine loops to foster undue EGF-signaling. Notably, Areg (amphiregulin) 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]. Notably, it was shown in patients with advanced non-small cell lung cancer that the survival time of amphiregulin-negative patients treated with gefitinib was significantly longer than that of amphiregulin-positive patients. Therefore, an increase in serum amphiregulin can be viewed as an predictor of resistance to gefitinib [36]. Moreover, Areg was also reported as an inhibitor of apoptosis in non-small cell lung cancer cell lines [37]. The data shows that Areg secreted from dysplastic cells are in an autocrine loop to foster malignant transformation.

A further growth factor strongly induced in dysplasia is 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]. Although Ereg expression is highly correlated with survival from bladder cancer [40], others suggest epiregulin to be important for pancreatic and prostate cancer development [41]. The strongest evidence for a role of epiregulin during tumorigenesis was obtained in Ki-ras-mediated signaling of colon cancer cells [42]. The fact that 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. Specifically, 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. For example, 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]. Because of the negative electric charge of sialic acid at the terminal non- reducing end of glycoprotein oligosaccharides, it plays a key role in mediating biological recognition events, including those responsible for metastasis. Next, an overexpression of B4galt6 (UDP-GALBeta-GlcNAc Beta-1 ,4-Galactosyltransferase, polypeptide 6) was found. This enzyme is involved in the production of GIcNAc (galactose beta-1 ,4-N-acetylglucosamine). GIcNAc is the first sugar residue to be linked to serine or threonine and serves as acceptor for the elongation with further GIcNAc molecules. The resulting branched structure may be elongated by the sequential addition of galactose, fucose and sialic acid [44].

Changes of the glycosylation pattern in dysplasia enables further variability in molecular assembly of membranes and offers a wide range of flexibility in response to cellular environment. Their detection on the cancer cell surface may provide useful diagnostic or prognostic information but until now is incompletely understood and therefore requires further elucidation.

Changes in the expression pattern of cell-adhesion molecules and components of intercellular junctions are related to loss of epithelial organization, proper cell layer and tissue polarity. With regard to adhesion molecules, up-regulation of ChM (cell adhesion molecule with homology to L1 CAM), a member of the L1 gene family of neural cell adhesion molecules, was found and of several claudins as exemplified by the up-regulation of Cldn2 (claudin2), Cldn4 (claudin4), and Cldnδ (claudinδ), which are important components of the tight junctions. Adhesion properties greatly impacts cell-to-cell interaction and growth of cancer cells.

In this regard, the cytoskeleton, which represents a complex of interconnected fibrillar elements, has been determined as an important factor in mediating adhesion-independent and dependent signalling. During morphogenesis, they determine cell shape and polarity, and promote stable cell-cell and cell-matrix adhesions through their interactions with cadherins and integrins, respectively. Surprisingly, in dysplastic cells regulation of genes involved in the cytoskeleton was not identified.

Instead, changes in cell to cell communication were identified. In the study according to the invention Gjb3 (gap junction membrane channel protein beta 3) and Gjb4 (gap junction membrane channel protein beta 4) were significantly up- regulated. It was shown that the gap junctional intercellular communication is a form of cell-to-cell signalling thereby mediating the exchange of small molecules between neighbouring cells [45]. 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]. Also, 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. Four surfactant proteins (SP) with unique properties have been identified. 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]. In 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. For example, Apoai (apolipoprotein A-I) is known to be the major protein moiety of high- density lipoprotein (HDL) and is mainly produced in the liver and the intestine [36] but is also produced in vitro by some differentiated cell lines established from human colorectal tumors [48].

Apoai (apolipoprotein A-1), Adcyapi (adenylate cyclase activating polypeptide 1), Ltb4dh (leukotriene B4 12-hydroxydehydrogenase), Fst (follistatin), lnhbb (inhibin beta-B), CIu (clusterin), Hnf4α (hepatic nuclear factor 4, alpha), Prokri (prokineticin receptor 1), Pla2g1b (phospholipase A2, group IB), Sult2b1 (sulfotransferase family, cytosolic, 2B, member 1), Stδsiaδ (ST8 alpha-N-acetyl-neuraminide alpha-2,8- sialyltransferase 6), Cyp1 b1 (cytochrome P450, family 1 , subfamily b, polypeptide 1) and the Pthlh (parathyroid hormone-like peptide) were found to be regulated with inferred roles in lipid metabolism.

For example, 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]. Likewise, 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].

Because cellular growth and differentiation is depended on highly co-ordinated transcriptional networks it was also searched for master regulatory proteins of respiratory epithelium. Notably, morphogenesis and branching of respiratory epithelium depends on the timely expression and activity of transcription factors. Dynamic changes in transcriptional activation of lung-specific genes are required to get an appropriate positioning of respiratory epithelial cells with the mesenchyme- derived endothelial cells. If this critical process is disturbed, this can lead to malignancy [56]. Transcription factors involved in differentiation of pulmonary epithelium includes HNF3B (hepatocyte nuclear factor 3 beta also termed Foxa2) [57], Ttf1 (transcription termination factor, RNA polymerase I) [58], Foxfi (forkhead box F1a) [59], Gataβ (GATA binding protein 6) [60] and Gataδ (GATA binding protein 5) [61].

In the study according to the invention some of these transcription factors were highly significantly regulated in dysplasia i.e. Hnf4α, Foxa3 and Foxp2. Notably, Hnf4α (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]. Hnf4α was found to be highly significantly induced (12.8-fold). Hnf4α regulates constitutive expression of a large number of target genes encoding enzymes, transporters and other nuclear receptors.

Among the genes regulated by HNF4α at least 11 target genes were found up- regulated in 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.

Likewise, 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 α1- 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.

Additionally, the up-regulation of Areg, Ereg, Hnf4α, Foxa3 and Foxp2 was confirmed by immunohistochemical staining. The immunostaining showed a distinct pattern of immunoreactivity in dysplastic cells with no staining in unaltered transgenic cells. For example, immunohistochemical staining of Areg showed a cytoplasmic immunoreactivity in dysplastic cells but not each cell expressed this molecule equally strong.

In dysplasia, however, it was not possible to evidence altered transcriptional networks of the MAPK pathway and regulated genes coding for cell cycle or nucleotide metabolism were not observed. Both processes are highly active in tumor cells but do not play a big role in dysplastic cells prior to malignant transformation. In this context two EGF ligands, Areg and Ereg were found to be specifically up- regulated in dysplasia. While amphiregulin binds exclusively to EGFR epiregulin binds to HER4 and mediates aberrant activation of these receptor. Indeed, exaggerated EGF tyrosine kinase activity is a probable cause for lung cancer. Now, de novo expression of two ligands of EGFR in dysplasia that are frequently over expressed in cancers are evidenced. However, increased transcript expression of the cancer stem cell markers CD44, CD133 and the epithelial cell adhesion molecule EpCAM were not observed. This supports the notion that dysplasia is a facultative cancer where additional events need to occur that enable malignant transformation. Taken collectively, 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. In the second part of the study 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.

Materials and Methods SP-C/c-raf model

Animals were kept according to the Public Health Service Policy on Humane Care and Use of Laboratory Animals and SP-C/c-raf transgenic mice were obtained from the laboratory of Prof. UIf Rapp (University of Wϋrzburg, Germany), who bred the mice in the C57BL/6/DBA/2 hybrid background. The SP-C/c-raf transgenic mice were kept in the C57BL/6 background for at least five generations.

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.

Microdissection (LMPC - Laser Microbeam Microdissection and Laser Pressure Catapulting)

From each frozen lung tissue 10-μm thick sections were prepared and transferred on polyethylene napthalate foil-covered slides (Zeiss, P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany).

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 μl/ml β-mercaptoethanol to ensure isolation of intact RNA. Approximately an area of 6 x 106 μm2 were pooled from a specific layer of interest in the same animal and used for RNA extraction.

Following microdissection, total 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).

cRNA labeling and hybridization to microarrays

Total RNA (median: 175ng; range: 150 - 200ng) was used to generate biotin- labeled cRNA (10 μg) 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.

Quantification, normalization and statistical analysis

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). SAM

To compare the normalized data from dysplasia, normal lung tissue from transgenic mouse, tumor cells and non-transgenic of different mice, the Significance Analysis of Microarrays (SAM) algorithm (ArrayTrack) was used, which contains a sliding scale for false discovery rate (FDR) of significantly up- and down-regulated genes [69]. All data were permuted 100 cycles by using the two classes, unpaired data mode of the algorithm. As cut-off for significance an estimated FDR of 0.001 was chosen. Moreover, a cut-off for fold-change of differential expression of 2 was used. The full description of the extraction protocol, labeling and hybridization protocol and data processing is obtainable in the GEO DATA base under http://www.ncbi.nlm.nih.gov/geo/ [accession number GSE13963].

Principal component analysis (PCA) and hierarchical gene cluster analysis (HCA)

Two clustering approaches were used to determine components of variation in the data in this study as follows.

A) Principal-component analysis (PCA) that was used to obtain a simplified visualization of entire datasets. PCA is a useful linear approach to obtain a simplified visualization of entire datasets, without losing experimental information (variance). PCA allowed the dimension of complex data to be reduced and highlights the most relevant features of a given dataset to be highlighted.

B) Hierarchical gene clustering (HCA) where the data points were organized in a phylogenetic tree in which the branch lengths represent the degree of similarity between the values. HCA were conducted using the ward's minimum variance linkage clustering algorithm within ArrayTrack. After normalisation and SAM analysis a total of 2909 significant genes were used for hierarchical clustering.

Functional analysis of the significant genes with IPA

Lists of significantly differentially expressed genes were uploaded to Ingenuity Pathways Analysis (IPA, Ingenuity Systems Inc., Redwood City, CA, USA) (www.lngenuity.com) and 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.

Additionally, Venn diagrams were used to examine the overlap of resulting lists of genes differentially expressed between the different sample sets.

Quantitative real-time PCR

Corroboration of 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.

lmmunohistochemistry

Each tumor section (8μm 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. After a wash step, the slices were incubated with the primary polyclonal anti-body against AREG, EREG, HNF4α, FOXA3 and FOXP2 (Santa Cruz, Santa Cruz Biotechnologys Inc., CA, USA) for 45 minutes. After washing, a streptavidin horseradish peroxidase detection kit (Envision DAKO, Hamburg, Germany) containing 3,3'-diaminobenzidine solution as substrate 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.

Western blotting

Expression of selected proteins was further validated by Western blotting. Serum samples (50 μg) used for 2-DE were run on 12% gels by SDS-PAGE, blotted onto PVDF membranes and blocked with 10% Rotiblock™ (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-α-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, α-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.

Western immunoblotting of amphiregulin and apolipoprotein A-I in (blood-)serum of transgenic mice and healthy control mice showed that amphiregulin and apolipoprotein A1 were exclusively expressed in transgenic animals, α-tubulin was used as a loading control (Fig. 9).

In summary, 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.

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. Further, in a comparison of the data sets of 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.

In conclusion, 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.

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:

- lipid metabolism

- glycosylation

- fucosylation

- receptor tyrosine kinase activity

- regulation of Amphiregulin and/or Epiregulin,

- cell cell interactions: in particular the tight junction proteins (more particular Claudins) or gap junction proteins (more particular GJA3 etc)

- transcription regulation: transcription factors in the epithelial-mesenchymal transition (EMT), preferably HNF4a etc , thereby allowing an easy identification of dysplasia by determining said metabolism reactions or proteins or protein functions.

In particular, the biochemical reactions described herein are suitable for imaging procedures such as PET diagnostics ( "metabolic imaging"), optical imaging, which is applied for the bronchoscopy. Within this context reactions catalyzed by enzymes are appropriate. Further, 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 Legends

Figure 1 : Histological analyses of lung tissue from 5-month-old transgenic mice.

Histological analyses of lung tissue from mice transgenic for lung-targeted expression of the cRaf-1 protein. Lung tissue from a 5-month-old SP-C-c-Raf mouse were sectioned at 10μm, fixed in methanol /acetig acid and stained with H&E. Single dysplastic foci were detected, whereas in 10-month-old SP-C-c-Raf mice were multiple foci. A: Overview presentation (magnification: x 50), B: dysplastic foci (magnification: x 200)

Figure 2: Venn diagram for significantly regulated genes.

Venn diagram for significantly up-regulated genes. Comparison of dysplasia and transgenic unaltered lung tissue with non-transgenic samples. 114 genes were found in dysplasia, respectively, which were at least 2-fold differentially expressed (FDR=0.001). Figure 3: Principal component analysis for gene expression profiles of transformed cells.

Principal component analysis of transformed cells from transgenic SP-C/c-raf mouse model in comparison to unaltered lung tissue of transgenic and non-transgenic mice were conducted using the autoscaled method within ArrayTrack. orange; dysplasia; blue, transgenic non-tumor sample; green, non-transgenic samples

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 6: Ingenuity networks: dysplasia versus non-transgenic mice

Ingenuity networks generated by mapping the focus genes that were differentially expressed between dysplasia and non-transgenic unaltered lung tissue (descriptions see Fig.6).

Figure 7: Comparison quantitative RT-PCR and Oligonucleotid-Array.

Summary of differential expression of the eight genes verified by quantitative RT- PCR in comparison with Oligonucleotid-Array analysis. The data were analyzed statistically using Student's t-test (*p < 0.05; **p < 0.01). Error bars indicate standard deviation of five samples and two independent assays for each gene.

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 (HNF4α), 4 = forkhead box 3a (FOX3A/HNF3γ), 5 = forkhead box P2 (FOXP2)

Strong, mainly cytoplasmic immunoreactivity was found in dysplastic cells, whereas unaltered cells shown only weak positivity using the amphiregulin antibody (1). Epiregulin immunostaining showed a cytoplasmatic pattern of immunoreactivity in dysplastic cells (2). The FOX3A antibody (3) showed strong nuclear positivity in dysplastic cells, while unaltered transgenic cells were negative. The HNF4α antibody showed nuclear immunoreactivity (4). Nuclear positivity was also restricted to dysplastic cells with no staining in unaltered transgenic cells using the FOXP2 antibody (5).

Figure 9: Western immunoblotting of amphiregulin and apolipoprotein A-I in (blood-)serum of transgenic mice and healthy control mice, α-tubulin was used as a loading control (Fig. 9).

Figure 10

Ingenuity - Canonical Pathways. This figure shows the canonical pathways which were overrepresented in the group of significantly regulated genes in dysplasia versus transgenic mice.

Figure 11 and 12

Title: Quantitative real-time PCR. Real-time PCR curves of eight genes assessed by Taqman technology as well as of the reference gene ACTB of a representative experiment are shown. The differences of the Ct values of target and ACTB (ΔCT) are indicated. The smaller the ΔCT, the higher the relative expression level of the target mRNA.

Tables

Table 1 : Overview of significant differentially expressed Genes (FDR=0.001 , FC

<2>).

Supplementary Table 1 (Table S1)

List of genes with changed expressions that are significantly overexpressed in dysplasia versus non-altered transgenic mice: 120 significantly regulated genes. This table shows the RefSeq transcript IDs, Unigene IDs, gene titles, gene symbols, and fold changes of the significantly regulated genes.

Table S2

List of genes with changed expressions that are significantly over- or under- expressed in dysplasia versus non-transgenic mice: 287 significantly regulated genes. This table shows the RefSeq transcript IDs, Unigene IDs, gene titles, gene symbols, and fold changes of the significantly regulated genes.

Table S3

List of genes with changed expressions that are significantly over- or under- expressed in unaltered transgenic versus non-transgenic mice: 18 significantly regulated genes. This table shows the RefSeq transcript IDs, Unigene IDs, gene titles, gene symbols, and fold changes of the significantly regulated genes.

References

1 Boyle P, Dresler C (2005) Editorial: Preventing the lung cancer epidemic. Annals of Omcology 16: 1565-1566.

2 Weir BA, Woo MS, Getz G, Perner S, Ding L et al. (2007) Characterizing the cancer genome in lung adenocarcinoma. Nature 450:893-898.

3 Thomas RK, Weir B, Meyerson M (2006) Genomic Approaches to Lung Cancer. Clin Cancer Res 12:4384s-4391s.

4 Granville CA, Dennis PA (2005) An Overview of Lung Cancer Genomics and Proteomics. Am J Respir Cell MoI Biol 32:169-176.

5 Nesbitt JC, Putnam JB, Walsh GL, Roth JA, Mountain CF (1995) Survival in early- stage lung cancer. Ann Thorac Surg 60:466-472.

6 Minna JD, Roth JA, Gazdar AF (2002) Focus on lung cancer. Cancer Cell 1 :49- 52.

7 Fong KM, Sekido Y, Gazdar AF, Minna JD (2003) Lung cancer 9: Molecular biology of lung cancer: clinical implications. Thorax 58: 892-900. 8 Balsara BR, Testa JR (2002) Chromosomal imbalances in human lung cancer. Oncogene 21 :6877-6883.

9 Kerr KM (2001) Pulmonary preinvasive neoplasia. J Clin Pathol 54:257-271.

10 Kerkhoff E, Fedorov LM, Siefken R, Walter AO, Papadopoulos T, Rapp UR (2000) 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 11 :185-90.

11 Rϋtters H, Zϋrbig P, Halter R, Borlak J (2006) Towards a lung adenocarcinoma proteome map: Studies with SP-C/c-raf transgenic mice. Proteomics 6:3127-3137.

12 Chatterji B and Borlak J (2007) Serum proteomics of lung adenocarcinomas induced by targeted overexpression of c-raf in alveolar epithelium identifies candidate biomarkers. Proteomics 7:3980-91.

13 Borlak J and Jenke HS (2008) Cross-talk between aryl hydrocarbon receptor and mitogen-activated protein kinase signaling pathway in liver cancer through c-raf transcriptional regulation. MoI Cancer Res 6:1326-36.

14 Morrison DK, Cutler RE (1997) The complexity of RaM regulation. Curr Opin Cell Biol 9:174-9.

15 Alessi DR, Saito Y, Campbell DG, Cohen P, Sithanandam G et al. (1994) Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO 13:1610-9.

16 Payne DM, Rossomando AJ, Martino P, Erickson AK, Her JH et al. (1991) Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO 10:885-92.

17 lnoue A, Nukiwa T (2005) Gene Mutations in Lung Cancer: Promising Predictive Factors for the Success of Molecular Therapy. PLoS Medicine 2:5-13.

18 Emmert-Buck MR, Bonner RF, Smith PD (1996) Laser capture microdissection. Science 274: 998-1001.

19 Vermeer PD, Einwalter LA, Moninger TO, Rokhlina T, Kern JA et al. (2003) Segregation of receptor and ligand regulates activation of epithelial growth factor receptor. Nature 422:267-8.

20 Hyltander A, Drott C, Korner U, Sandstrόm R, Lundholm K (1991) Elevated energy expenditure in cancer patients with solid tumours. Eur J Cancer 27:9-15.

21 Yam D, Ben-Hur H, Fink A1 Dgani R, Shani A et al. (1994) Insulin and glucose status, tissue and plasma lipids in patiens with tumours of the ovary or endometrium: possible dietary implications. Br J Cancer 70:1186-7. 22 Thomas SE, Linda KC (1981) Plasma Lipoproteins with Bioregulatory Properties Including the Capacity to Regulate Lymphocyte Function and the Immune Response 1. Cancer Res 41 : 3786-3788.

23 Rufibach LE, Duncan SA, Battle M, Deeb SS (2001) Transcriptional regulation of the human hepatic lipase (LIPC) gene promoter. J of Lipid Research 47:1463-1477.

24 Niehof M and Borlak J (2008) HNF4 alpha and the Ca-channel TRPC1 are novel disease candidate genes in diabetic nephropathy. Diabetes 57:1069-77.

25 Kalinichenko W, Lim L, Shin B, Costa RH (2001) Differential expression of forkehead box transcription factors following butylated hydroxytoluene lung injury. Am J Physiol Lung Cell MoI Physiol 280:L695-704.

26 Shu W, Yang H, Zhang L, Lu MM, Morrisey E (2001) Characterization of a New Subfamily of Winged-helix/Forkhead (Fox) Genes That Are Expressed in the Lung and Act as Transcriptional Repressors. JBC 29:27488-27497.

27 Cordero A, Mukherjee S, Subramanian A, You H, Roix JJ et al. (2005) An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis. Nat Genet 37:48-55.

28 Liu ZZ, Wada J, Kumar A, Carone FA, Takahashi M et al. (1996) 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 178:133-48.

29 Johnson GR, Saeki T, Auersperg N, Gordon AW, Shoyab M et al. (1991) Response to and expression of amphiregulin by ovarian carcinoma and normal ovarian surface epithelial cells: nuclear localization of endogenous amphiregulin. Biochem Biophys Res Commun 180:481-488.

30 Li S, Plowman GD, Buckley SD, Shipley GD (1992) Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells. J Cell Physiol 153:103-111.

31 Wang X, Masri S, Phung S, Chen S (2008) The role of amphiregulin in exemestane-resistant breast cancer cells: evidence of an autocrine loop. Cancer Res 68:2259-65.

32 Bavik C, Coleman I, Dean JP, Knudsen B, Plymate S et al. (2006) The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms. Cancer Res 66:794-802.

33 Sehgal I, Bailey J, Hitzemann K, Pittelkow MR, Maihle NJ (1994) Epidermal growth factor receptor-dependent stimulation of amphiregulin expression androgen- stimulated human prostate cancer cells. MoI Biol Cell 5:339-347. 34 Ebert M, Yokoyama M, Kobrin MS, Friess H, Lopez ME et al. (1994) Induction and expression of amphiregulin in human pancreatic cancer. Cancer Res 54:3959- 3962.

35 Woodworth CD, McMullin E, lglesias M1 Plowman GD (1995) lnterleukin 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 92:2840-2844.

36 Ishikawa N, Diago Y, Takano A, Taniwaki M, Kato T et al. (2005) Increases of Amphiregulin and Transforming Growth Factor-α in Serum as Predictors of Poor Response to Gefitinib among Patients with Advanced Non-Small Cell Lung Cancers. Cancer Res 65:9176-9184.

37 Hurbin A, Dubrez L, Coll JL, Favrot MC (2002) Inhibition of Apoptosis by Amphiregulin via an Insulin-like Growth Factor-1 Receptor-dependent Pathway in Non-small Cell Lung Cancer Cell Lines. JBC 277:49127-49133.

38 Shirakata Y, Komurasaki H, Toyoda Y, Hanakawa K, Yamasaki S et al. (2000) Epiregulin, a novel memberof the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes. J Biol Chem 275:5748-5753.

39 Shelly M, Pinkas-Kramarski R, Guarino BC, Waterman H, Wang LM et al. (1998) Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes. J Biol Chem 273:10496-10505.

40 Thogersen VB, Sorensen BS, Poulsen SS, Orntoft TF, Wolf H et al. (2001) A subclass of HER1 ligands are prognostic markers for survival in bladder cancer patients. Cancer Res 61 :6227-6233.

41 Torring N, Jorgensen PE, Sorensen BS, and Nexo E (2000) Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines. Anticancer Res 20:91-95.

42 Baba I, Shirasawa S, Iwamoto R, Okumura K, Tsunoda T et al. (2000) Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells. Cancer Res 60:6886-6889.

43 Warren L, Buck CA, Tuszynski GP (1979) Alterations of membrane glycoproteins in malignant cells. Dev Biol Stand 42:177-82.

44 Hakomori S (2004) Glycosynapses: microdomains controlling carbohydrate- dependent cell adhesion and signalling. An Acad Bras Cienc 76: 553-572. 45 Mennecier G, Derangeon M, Coronas V, Herve JC, Mesnil M (2008) Aberrant expression and localization of connexin43 and connexin30 in a rat glioma cell line. MoI Carcinog 47:391-401.

46 Pointis G, Fiorini C, Gilleron J, Carette D, Segretain D (2007) Connexins as precocious markers and molecular targets for chemical and pharmacological agents in carcinogenesis. Curr Med Chem 14:2288-303.

47 Mason RJ, Greene K, Voelker DR (1998) Surfactant protein A and surfactant protein D in health and disease. Am J Physiol 275:L1-13. Review.

48 Reisher SR, Hughes TE, Ordovas JM, Schaefer EJ, Feinstein SI (1993) Increased expressions of apolipoprotein genes accompanies differentiation in the intestinal cell line CACO-2. PNAS 90: 5757-61.

49 Wolman SR, Heppner GH, Wolman E (1997) New directions in breast cancer research. Fed Am Soc Exp Biol J 11 :535-543.

50 lsobe K, Kaneko M, Kaneko S, Nissato S, Nanmoku T et al. (2004) Expression of mRNAs for PACAP and its receptor in human neuroblastomas and their relationship to catecholamine synthesis. J RegPep 123:29-32.

51 Garcia-Fernandez MO, Bodega G, Ruiz-Villaespesa A, Cortes J, Prieto JC et al. (2004) PACAP expression and distribution in human breast cancer and healthy tissue. Cancer Letters 205:189-195.

52 Schultz IJ, Wester K, Straatman H, Kiemeney LA, Babjuk M et al. (2006) Prediction of recurrence in Ta urothelial cell carcinoma by real-time quantitative PCR analysis: A microarray validation study, lnt J Cancer 119: 1915-1919.

53 Kissin MW, Henderson MA, Danks JA, Hayman JA, Bennett RC et al. (1993) Parathyroid hormone related protein in breast cancers of widely varying prognosis. Eur J Surg Oncol 19:134-42.

54 Iwamura M, Wu W, Muramoto M, Ohori M, Egawa S et al. (1999) Parathyroid hormone-related protein is an independent prognostic factor for renal cell carcinoma. Cancer 86:1028-34.

55 Nishihara M, lto M, Tomioka T, Ohtsuru A, Taguchi T et al. (1999) Clinicopathological implications of parathyroid hormone-related protein in human colorectal tumours. J Pathol 187:217-22..

56 Costa RH, Kalinichenko V, Lim L (2001) Transcription factors in mouse lung development and function. Am J Physiol Lung Cell MoI Physiol 280:823-838.

57 Ang SL, Wierda A, Wong D, Stevens KA, Cascio S et al. (1993) The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development. 119:1301-15. 58 Stahlman MT, Gray ME, Whitsett JA (1998) Temporal-spatial distribution of hepatocyte nuclear factor-3beta in developing human lung and other foregut derivatives. J Histochem Cytochem 46:955-62.

59 Molkentien JD, Lin Q, Duncan SA, Olson EN (1997) Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev.11 :1061-72.

60 Morrisey EE, Ip HS, Lu MM, Parmacek MS (1996) GATA-6: a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm. Dev Biol 177:309-22.

61 Morrisey EE, Ip HS, Tang Z, Lu MM, Parmacek MS (1997) GATA-5: a transcriptional activator expressed in a novel temporally and spatially-restricted pattern during embryonic development. Dev Biol 183:21-36.

62 Sladek FM, Zhong W, Lai E, Darnell JE (1990) Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev 4:2353-2365.

63 Duda K, Chi Yl, Shoelson SE (2004) Structural basis for HNF-4alpha activation by ligand and coactivator binding. J.Biol.Chem 279: 23311-23316.

64 Odom DT, Zizlsperger N, Gordon DB, Bell GW, Rinaldi NJ et al. (2004) Control of Pancreas and Liver Gene Expression by HNF Transcription Factor. Science 303: 1378-1381.

65 Jackson DA, Rowader KE, Stevens K, Jiang C, Milos P et al. (1993) Modulation of liver-specific transcription by interactions between hepatocyte nuclear factor 3 and nuclear factor 1 binding DNA in close apposition. MoI Cell Biol 13: 2401-2410.

66 Zaret K (1999) Developmental competence of the gut endoderm: Genetic potentiation by GATA and HNF3/fork head proteins. Dev Biol 209:1-10.

67 Shih DQ, Navas MA, Kuwajima S, Duncan SA, Stoffel M (1999) Impaired glucose homeostasis and neonatal mortality in hepatocyte nuclear factor 3α-deficient mice. PNAS 96: 10152-10157.

68 Shen W, Scearce M, Brestelli JE, Sund NJ, Kaestner KH (2001) 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 276: 42812-42817.

69 Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. PNAS 98: 5116 - 5121. The features of the invention being disclosed in the preceding description, the subsequent tables, drawings, and claims can be of importance both singularly and in arbitrary combination for the implementation of the invention in its different embodiments.

Supplementary

Table 1 (Table S1):

Gene Symbols Gene Title Fold Change Gen_id_mfr RefSeq Transcript ID

Gzme granzyme E 47,45 1421227_at NM_010373 CbInI cerebellin 1 precursor protein 35,86 1423287_at NM_019626

Etv4 ets variant gene 4 (E1 A enhancer binding protein, E1AF) 35,16 1423232_at NM_008815 1810036H07Rιk RIKEN cDNA 1810036H07 gene 31 ,07 1453132_a_at NM_025467

Gzme granzyme E 30,82 1450171_x_at NM_010373

0710001A04Rιk RIKEN cDNA 0710001 A04 gene 28,84 1454126_at — Apoai apolipoprotein A-I 28,06 1455201_x_at NM_009692 CbInI cerebellin 1 precursor protein 27,33 1423286_at NM_019626 Pkhdi polycystic kidney and hepatic disease 1 23,73 1419820_at NMJ53179 Apoai apolipoprotein A-I 23,52 1419233_x_at NM_009692 Ndg1 /// LOC623189 Nur77 downstream gene 1 21 ,01 1455423_at NM_183322

Itιh2 inter-alpha trypsin inhibitor, heavy chain 2 17,23 1417618_at NM_010582

Mcpt2 mast cell protease 2 16,59 1449989_at NM_008571

Rgs16 regulator of G protein signaling 16 16,46 1426037_a_at —

BC048546 cDNA sequence BC048546 16,21 1436503_at XM_132895

Areg amphiregulin 15,38 1421134_at NM_009704 Cldn2 claudin 2 13,32 1417231_at NM_016675 Hnf4a hepatic nuclear factor 4, alpha 12,81 1427001_S_at NM_008261

Pthlh parathyroid hormone-like peptide 12,48 1422324_a_at NM_008970

Ereg epiregulin 12,32 1419431_at NM_007950

ChH cell adhesion molecule with homology to L1CAM 12,31 1435190_at NM_007697

Ccdcβ3 RIKEN cDNA 4932423M01 gene 12,13 1453425_at NM_029256

0rm1 orosomucoid 1 11 ,77 1451054_at NM_008768

St8sιa6 ST8 alpha-N-acetyl-neuramidine alpha-2,8-sιalyltransferase 11 ,71 1438566_at —

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 Ankrd22 ankyrin repeat domain 22 10,73 1453239_a_at NM_024204 Pbp2 RIKEN cDNA 1700023A18 gene 10,72 1424793_a_at NM_029595

Gtl2 GTL2, impπnted maternally expressed untranslated mRNA 10,61 1452183 a at NM 144513

Rgs16 regulator of G protein signaling 16 10,49 1455265_a_at — Gpc6 glypican 6 9,91 1428774_at NM_011821

9130213B05Rιk RIKEN cDNA 9130213B05 gene 9,75 1424214_at NM_145562

Apoal apolipoprotein A-I 9,75 1438840_x_at NM_009692

Gtl2 GTL2, imprinted maternally expressed untranslated mRNA 9,59 1428765_at NMJ44513

Gja3 gap junction protein, alpha-3 9,32 1439793_at —

Gtl2 gene trap locus 2 9,27 1436713_s_at —

WdM 6 RIKEN cDNA 1700019F09 gene 9,22 1429552_at NM_027963

Rhbdl2 rhomboιd-lιke2 9,18 1442819_at —

St8sιa6 ST8 alpha-N-acetyl-neuramidine alpha-2,8-sιalyltransferase 9,13 1456440_s_at —

Rasgrfi RAS protein-specific guanine nucleotide-releasing factor 1 9,09 1435614_s_at NM_011245

Adcyapi adenylate cyclase activating polypeptide 1 8,79 1441778_at NM_009625

Slc35f1 solute carrier family 35, member F1 8,77 1436719_at NM_178675

Rian RNA imprinted and accumulated in nucleus 8,77 1452899_at —

Mirg miRNA containing gene 8,58 1457030_at XM_488655

1700027A23Rιk RIKEN cDNA 1700027 A23 gene 8,47 1453320_at NM_029604

Cldn4 claudin 4 8,44 1418283_at NM_009903

Gtl2 /// Lphni GTL2, imprinted maternally expressed untranslated mRNA /// latrophilin 1 8,27 1452905_at NM_144513

Ul K> 9130213B05Rιk RIKEN cDNA 9130213B05 gene 8,05 1428891_at NM_145562

Gtl2 GTL2, imprinted maternally expressed untranslated mRNA 7,93 1426758_s_at NMJ44513

Stk39 serine/threonine kinase 39, STE20/SPS1 homolog (yeast) 7,89 1419551_s_at NM_016866

Stδsiaβ ST8 alpha-N-acetyl-neuraminide alpha-2,8-sιalyltransferase 6 7,81 1456147_at NM_145838

Myh6 /// LOC671894 myosin, heavy polypeptide 6, cardiac muscle, alpha 7,72 1448554_s_at NM_010856

S100a14 S100 calcium binding protein A14 7,67 1449166_at NM_025393

1110006E14Rιk RIKEN cDNA 1110006E14 gene 7,65 1431094_at —

Atp13a4 ATPase type 13A4 7,58 1438707_at NMJ 72613

Hecwi HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 7,56 1456527_at XM_484217

Foxa3 forkhead box A3 7,45 1431900_a_at NM_008260

Ptprn /// LOC669060 protein tyrosine phosphatase, receptor type, N 7,44 1416588_at NMJ308985

Ckmti creatine kinase, mitochondrial 1 , ubiquitous 7,33 1417089_a_at NM_009897

Lad1 ladinin 7,23 1418449_at NM_133664

Tmem54 RIKEN cDNA 1810017F10 gene 7,21 1417895_a_at NM_025452

Brunol4 bruno-like 4, RNA binding protein (Drosophila) 7,19 1452240_at NMJ33195

Cdsn Similar to corneodesmosin precursor, S protein, differentiated keratinocyte S protein precursor 7,17 1444607_at NM_001008424

D630002J15Rιk RIKEN cDNA D630002J15 gene 7,14 1453480_at XM_485742 Brunol4 bruno-like 4, RNA binding protein (Drosophila) 7,12 1426930_at NMJ 33195 Rasgrfi RAS protein-specific guanine nucleotide-releasing factor 1 7,12 1422600_at NM_011245

Afp alpha fetoprotein 6,88 1416646_at NM_007423

Prss22 protease, seπne, 22 6,87 1420352_at NM_133731 LOC671894 /// LOC674761 6,83 1448553_at —

Gtl2 GTL2, imprinted maternally expressed untranslated mRNA 6,73 1439380_x_at NMJ44513 Cd 177 RIKEN cDNA 1190003K14 gene 6,68 1424509_at NM_026862 Ptpm2 protein-tyrosine phosphatase, receptor-type, N, polypeptide 2 6,60 1435968_at — Ptprn2 protem-tyrosine phosphatase, receptor-type, N, polypeptide 3 6,54 1441971_at — Slc23a3 solute carrier family 23 (nucleobase transporters), member 3 6,51 1460042_at NM_194333 Cyp1b1 cytochrome P450, family 1 , subfamily b, polypeptide 1 6,50 1416612_at NM_009994 Pcsk6 proprotein convertase subtilisin/kexin type 6 6,42 1426981_at XM_355911 Sdcbp2 syndecan binding protein (syntenin) 2 6,40 1424090_at NM_145535 Oacti O-acyltransferase (membrane bound) domain containing 1 6,33 1435323_a_at NM_153546 Sult2b1 sulfotransferase family, cytosolic, 2B, member 1 6,30 1417335_at NM_017465

Afp alpha fetoprotein 6,27 1416645_a_at NM_007423

Arg2 arginase type Il 6,25 1418847_at NM_009705

Gjb3 gap junction membrane channel protein beta 3 6,17 1416715_at NM_008126

Tnfsrø tumor necrosis factor hgand superfamily, member 9 6,15 1422924_at —

Akr1c19 similar to 3(20)alpha-hydroxysteroιd/dιhydrodιol/ιndanol dehydrogenase 6,13 1455454_at NM_001013785

Ul Adoral adenosine A1 receptor 5,98 1435495_at NM_001008533

Psrd RIKEN cDNA 5430413102 gene 5,95 1417323_at NM_019976

Fut2 fucosyltransferase 2 5,88 1434862_at NM_018876

Ros1 Ros1 proto-oncogene 5,86 1425970_a_at NM_011282

Prokri G protein-coupled receptor 73 5,82 1456543_at NM_021381

Ltb4dh leukotπene B4 12-hydroxydehydrogenase 5,71 1417777_at NM_025968

BC065085 hypothetical protein A030013D21 5,68 1455872_at NM_177628

LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 5,55 1435758_at NM_019737

Gsta4 glutathione S-transferase, alpha 4 5,49 1416368_at NM_010357

Rnf128 ring finger protein 128 5,49 1449036_at NM_023270

Sertad4 SERTA domain containing 4 5,44 1454877_at NMJ 98247

Psrd RIKEN cDNA 5430413102 gene 5,40 1425416_s_at NM_019976 4930579J09Rιk RIKEN cDNA 4930579J09 gene 5,38 1418870_at NM_133689

Fst Follistatin 5,21 1434458_at NM_008046

Arg2 arginase type Il 5,16 1438841_s_at NM_009705 Ly6g6c lymphocyte antigen 6 complex, locus G6C 5,16 1422749_at NM_023463 AdssH adenylosuccinate synthetase like 1 5,14 1449383_at NM_007421

Hpn hepsin 5,08 1420712_a_at NM_008281 B4galt6 /// LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 4,85 1423228 at NM 019737

Tspani tetraspan 1 4,79 1417957_a_at NM_133681

AIbI albumin 1 4,74 1425260_at NM_009654

Gpx2 glutathione peroxidase 2 4,74 1449279_at NM_030677

Akr1b8 aldo-keto reductase family 1 , member B8 4,67 1448894_at NM_008012

Klc3 kinesin light chain 3 4,66 1425558_at NM_146182

Cldnβ claudin 8 4,60 1449091_at NM_018778

Foxp2 forkhead box P2 4,60 1438232_at NM_053242

LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 4,50 1460329_at NM_019737

Krt1-18 keratin complex 1 , acidic, gene 18 4,49 1448169_at NM_010664

CIu clustenn 4,46 1418626_a_at NM_013492

Kcnk2 potassium channel, subfamily K, member 2 4,34 1449158_at NM_010607

Pla2g1b phospholipase A2, group IB, pancreas 4,08 1416626_at NM_011107

Pcbdi pterin 4 alpha carbinolamine dehydratase/dimeπzation cofactor of hepatocyte nuclear factor 1 alpha 4,06 1418713_at NM_025273

Btbdi 1 BTB (POZ) domain containing 11 3,97 1428377_at NM_001017525

Golph2 golgi phosphoprotein 2 3,95 1415698_at NM_027307 lnhbb inhibin beta-B 3,62 1426858 at XM 148966

Table 2 (Table S2):

Gene Symbols Gene Title Fold Change Gen_id_mfr RefSeqTranscript ID

Gzme granzyme E 1160,72 1421227_at NM_010373

Ereg epiregulin 482,20 1419431_at NM_007950

1810036H07Rιk RIKEN cDNA 1810036H07 gene 280,79 1453132_a_at NM_025467

CbInI cerebellin 1 precursor protein 239,44 1423287_at NM_019626

Gzme granzyme E 227,58 1450171_x_at NM_010373

CbInI cerebellin 1 precursor protein 178,86 1423286_at NM_019626

Gja3 gap junction protein, alpha-3 151 ,62 1439793_at

Apoai apolipoprotein A-I 148,43 1455201_x_at NM_009692

Cldn2 claudin 2 122,38 1417231_at NM_016675

Mcpt2 mast cell protease 2 109,92 1449989_at NM_008571

Apoai apolipoprotein A-I 102,84 1419233_x_at NM_009692

0rm1 orosomucoid 1 90,18 1451054_at NM_008768

DIkI delta-like 1 homolog (Drosophila) 83,87 1449939_s_at NM_010052

Apoai apolipoprotein A-I 74,39 1438840_x_at NM 009692

Rhbdl2 rhomboid-like 2 65,30 1442819 at

PkhcM polycystic kidney and hepatic disease 1 65,30 1419820_at NMJ53179

0710001 A04Rιk RIKEN cDNA 0710001 A04 gene 62,95 1454126_at —

Stδsiaβ ST8 alpha-N-acetyl-neuraminide alpha-2,8-sιalyltransferase 6 56,45 1456440_s_at —

Fetub fetuin beta 56,15 1449555_a_at NM_021564

Ndg1 /// LOC623189 Nur77 downstream gene 1 54,61 1455423_at NM_183322

Etv4 ets vaπant 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

Itιh2 inter-alpha trypsin inhibitor, heavy chain 2 45,75 1417618_at NM_010582

Stδsiaβ ST8 alpha-N-acetyl-neuraminide alpha-2,8-sιalyltransferase 6 44,18 1438566_at —

Rgs16 regulator of G protein signaling 16 42,74 1426037_a_at —

St8sιa6 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sιalyltransferase 6 41 ,71 1456147_at NM_145838

Adcyapi adenylate cyclase activating polypeptide 1 41 ,19 1441778_at NM_009625

Pbp2 RIKEN cDNA 1700023A18 gene 37,72 1424793_a_at NM_029595

Cd 177 RIKEN cDNA 1190003K14 gene 37,21 1424509_at NM_026862

Areg amphiregulin 35,63 1421134_at NM_009704

Gjb4 gap junction membrane channel protein beta 4 35,37 1422179_at NM_008127

ChM cell adhesion molecule with homology to L1CAM 30,54 1435190_at NM_007697

Gtl2 GTL2, impπnted maternally expressed untranslated mRNA 30,49 1452183_a_at NMJ 44513

BC048546 cDNA sequence BC048546 28,46 1436503_at XM_132895

Ul Kng1 kininogen 1 28,25 1416676_at NM_023125 Ul

1110014F24Rιk RIKEN cDNA 1110014F24 gene 28,13 1428781_at NM_028618

Rian RNA impπnted and accumulated in nucleus 27,14 1427580_a_at —

Rian RNA imprinted and accumulated in nucleus 27,03 1452899_at —

GM2 GTL2, impπnted maternally expressed untranslated mRNA 26,00 1428765_at NM_144513

Rasgrfi RAS protein-specific guanine nucleotide-releasing factor 1 25,78 1422600_at NM_011245

Atp13a4 ATPase type 13A4 25,35 1438707_at NMJ72613

Gjb3 gap junction membrane channel protein beta 3 24,77 1416715_at NM_008126

9130416B15 22,98 1445233_at —

D630002J15Rιk RIKEN cDNA D630002J15 gene 22,61 1453480_at XM_485742

Stk39 senne/threonine kinase 39, STE20/SPS1 homolog (yeast) 22,57 1419551_s_at NM_016866

Rasgrfi RAS protein-specific guanine nucleotide-releasing factor 1 22,04 1435614_s_at NM_011245

FgM fibnnogen-like protein 1 21 ,77 1424599_at NM_145594

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

Tmem54 RIKEN cDNA 1810017F10 gene 16,56 1417895_a_at NM_025452

Ptprn2 protein-tyrosme phosphatase, receptor type N, polypeptide 2 16,35 1435968_at —

Cldn4 claudin 4 16,23 1418283_at NM_009903

Lad1 ladinin 16,05 1418449_at NM_133664

Afp alpha fetoprotein 15,88 1416645_a_at NM_007423

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 —

Afp alpha fetoprotein 15,07 1416646_at NM_007423 Prss22 protease, serine, 22 14,71 1420352_at NM_133731

Oιt1 oncoprotein induced transcript 1 14,67 1424502_at NM_146050 1110006E14Rιk RIKEN cDNA 1110006E14 gene 14,51 1431094_at —

Rnf128 ring finger protein 128 14,36 1449036_at NM_023270 Smurfi /// LOC640390 SMAD specific E3 ubiquitin protein ligase 1 14,16 1428396_at NM_029438

Pthlh parathyroid hormone-like peptide 14,07 1422324_a_at NM_008970

Sult2b1 sulfotransferase family, cytosohc, 2B, member 1 13,58 1417335_at NM_017465

S100a14 S100 calcium binding protein A14 13,42 1449166_at NM_025393

Ccdc83 RIKEN cDNA 4932423M01 gene 13,34 1453425_at NM_029256

Brunotø bruno-like 4, RNA binding protein (Drosophila) 13,22 1426930_at NMJ33195

Brunotø bruno-like 4, RNA binding protein (Drosophila) 12,98 1452240_at NM_133195

Stk39 seπne/threonine kinase 39, STE20/SPS1 homolog (yeast) 12,77 1419550_a_at NM_016866

Ckmti creatine kinase, mitochondrial 1 , ubiquitous 12,72 1417089_a_at NM_009897

CbInI cerebellin 1 precursor protein 12,33 1423288_s_at NM_019626

Foxa3 forkhead box A3 12,30 1431900_a_at NM_008260

BC024561 cDNA sequence BC024561 11 ,87 1451610_at NM_153576

Rnf128 ring finger protein 128 11 ,62 1418318_at NM_023270

Gjb1 gap junction membrane channel protein beta 1 11 ,47 1448766_at NM_008124

Pglyrpi peptidoglycan recognition protein 1 11 ,31 1449184_at NM_009402

Gpc6 glypican 6 11 ,23 1428774_at NM_011821

Myh6 /// LOC671894 myosin, heavy polypeptide 6, cardiac muscle, alpha 10,98 1448554_S_at NM_010856

Prokri G protein-coupled receptor 73 10,82 1456543_at NM_021381

Slc26a4 solute carrier family 26, member 4 10,80 1419725_at NM_011867

LOC671894 /// LOC674761 10,73 1448553_at —

Ttc9 tetratπcopeptide repeat domain 9 10,64 1436237_at XM_126933 Ptprn /// LOC669060 protein tyrosine phosphatase, receptor type, N 10,59 1416588_at NM_008985

Ltb4dh leukotriene B4 12-hydroxydehydrogenase 10,49 1417777_at NM_025968

9130213B05Rιk RIKEN cDNA 9130213B05 gene 10,45 1424214_at NM_145562

3110049J23Rιk RIKEN cDNA 3110049J23 gene 10,33 1449462_at NM_026085

Gjb1 gap junction membrane channel protein beta 1 10,18 1448767_s_at NM_008124

Shmti seπne hydroxymethyl transferase 1 (soluble) 10,09 1425179_at NM_009171 LOC433393 10,06 1439888_at XM_488930

Arg2 arginase type Il 10,04 1418847_at NM_009705

Cdsn Similar to corneodesmosin precursor, S protein, differentiated keratinocyte S protein precursor 9,93 1444607_at NM_001008424

Cyp1 b1 cytochrome P450, family 1 , subfamily b, polypeptide 1 9,71 1416612_at NM_009994

Hecwi HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 9,68 1456527_at XM_484217

LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 9,37 1435758_at NM_019737

Fut2 fucosyltransferase 2 9,36 1434862_at NM_018876

1700027A23Rιk RIKEN cDNA 1700027 A23 gene 9,28 1453320_at NM_029604

Psrd RIKEN cDNA 5430413102 gene 9,18 1417323_at NM_019976

IvI RIKEN cDNA 1110019C06 gene 9,14 1439878_at —

5730559C18Rιk RIKEN cDNA 5730559C18 gene 8,96 1436345_at NM_028872

Psrd RIKEN cDNA 5430413102 gene 8,91 1425416_s_at NM_019976

— Mus musculus transcribed sequences 8,84 1441971_at —

Hpn hepsin 8,70 1420712_a_at NM_008281

Adcyapi adenylate cyclase activating polypeptide 1 8,61 1423427_at NM_009625

Sdcbp2 syndecan binding protein (syntenin) 2 8,53 1424090_at NM_145535

D17H6S56E-5 DNA segment, Chr 17, human D6S56E 5 8,34 1417821_at NM_033075

Arg2 arginase type Il 8,23 1438841_s_at NM_009705

Ul SId 3a2 solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 2 8,07 1418857_at NM_022411

C430004E15Rιk RIKEN cDNA C430004E15 gene 8,02 1426809_at NMJ 75286

Rab3c RAB3C, member RAS oncogene family 7,99 1449494_at NM_023852

E030010A14Rιk hypothetical protein E030010A14 7,95 1437595_at NM_183160

9130213B05Rιk RIKEN cDNA 9130213B05 gene 7,92 1428891_at NM_145562

Ros1 Ros1 proto-oncogene 7,92 1425970_a_at NM_011282

Syt12 synaptotagmin 12 7,84 1422878_at NMJ34164

Rapigap Rap1 , GTPase-activating protein 1 7,78 1428443_a_at XM_149500

Plek2 pleckstπn 2 7,66 1449424_at NM_013738

Hgfac hepatocyte growth factor activator 7,65 1418405_at NM_019447

4931408D14Rιk RIKEN cDNA 4931408D14 gene 7,46 1431806_at —

Adoral adenosine A1 receptor 7,39 1435495_at NM_001008533

Phlda2 pleckstrin homology-like domain, family A, member 2 7,35 1417837_at NM_009434

SId 5a2 solute earner family 15 (H+/peptιde transporter), member 2 7,25 1424730_a_at NM_021301

Gsta4 glutathione S-transferase, alpha 4 7,20 1416368_at NM_010357

Foxp2 forkhead box P2 7,10 1438231_at NM_053242

Mapki 3 mitogen activated protein kinase 13 7,09 1448871_at NM_011950

MaI myelin and lymphocyte protein, T-cell differentiation protein 6,99 1417275_at NM_010762

Foxqi Forkhead box Q1 6,98 1438558_x_at NM_008239

Eva1 epithelial V-like antigen 1 6,95 1416236_a_at NM_007962

Dfnaδh deafness, autosomal dominant 5 homolog (human) 6,95 1417903_at NM_018769

HdC histidine decarboxylase 6,94 1451796_s_at NM_008230

Pcbdi pterin 4 alpha carbinolamine dehydratase/dimeπzation cofactor of hepatocyte nuclear factor 1 alpha 6,93 1418713_at NM_025273

Eva1 epithelial V-hke antigen 1 6,92 1448265_x_at NM_007962

Dyrk3 dual-specificity tyrosιne-(Y)-phosphorylatιon regulated kinase 3 6,92 1424229_at NM_145508

AdssM adenylosuccinate synthetase like 1 6,86 1449383_at NM_007421

Kcnfi potassium voltage-gated channel, subfamily F, member 1 6,86 1454768_at NM_201531

SId 5a2 solute carrier family 15 (H+/peptιde transporter), member 2 6,84 1447808_s_at NM_021301

Foxp2 forkhead box P2 6,80 1438232_at NM_053242

Golph2 golgi phosphoprotein 2 6,79 1415698_at NM_027307

4930579J09Rιk RIKEN cDNA 4930579J09 gene 6,77 1418870_at NM_133689

CIu clusteπn 6,76 1418626_a_at NM_013492

Akr1b8 aldo-keto reductase family 1 , member B8 6,76 1448894_at NM_008012

WdM 6 RIKEN cDNA 1700019F09 gene 6,75 1429552_at NM_027963

MaI myelin and lymphocyte protein, T-cell differentiation protein 6,73 1432558_a_at NM_010762

Wnk4 protein kinase, lysine deficient 4 6,64 1427196_at NM_175638

B4galt6 /// LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 6,64 1423228_at NM_019737 Cldn3 claudιn 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

OO Acsl4 acyl-CoA synthetase long-chain family member 4 6,44 1451828_a_at NM_019477 Chia RIKEN cDNA 2200003E03 gene 6,38 1416456_a_at NM_023186 Cd14 CD14 antigen 6,34 1417268_at NM_009841

LOC675709 UDP-GaI betaGlcNAc beta 1 ,4-galactosyltransferase, polypeptide 6 6,27 1460329_at NM_019737 Ace2 angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 6,27 1425102_a_at NM_027286 Cideb cell death-inducing DNA fragmentation factor, alpha subunit-like effector B 6,25 1418976_s_at NM_009894

D17H6S56E-5 DNA segment, Chr 17, human D6S56E 5 6,24 1417822_at NM_033075

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

Klc3 kinesin light chain 3 6,04 1425558_at NM_146182

BC065085 hypothetical protein A030013D21 6,04 1455872_at NMJ77628

Scara3 scavenger receptor class A, member 3 6,03 1427020_at NM_172604

Elf5 E74-lιke factor 5 6,02 1419555_at NM_010125

Oacti O-acyltransferase (membrane bound) domain containing 1 6,01 1435323_a_at NM_153546

Ly6g6c lymphocyte antigen 6 complex, locus G6C 6,00 1422749_at NM_023463

Fgg fibrinogen, gamma polypeptide 5,97 1416025_at NM_133862

Akr1c19 similar to 3(20)alpha-hydroxysteroιd/dιhydrodιol/ιndanol dehydrogenase 5,95 1455454_at NM_001013785 Aqp3 aquaporιn 3 5,92 1450460_at NM_016689 Kcnki potassium channel, subfamily K, member 1 5,89 1448690_at NM_008430

Bnipl BCL2/adenovιrus E1B 19kD interacting protein like 5,89 1420683_at NM_134253

Chc6 chloride intracellular channel 6 5,89 1454866_s_at NM_172469

6330530A05Rιk RIKEN cDNA 6330530A05 gene 5,88 1434094_at NM_172383

Sp5 trans-acting transcπption factor 5 5,82 1422914_at NM_022435

Mtac2d1 membrane targeting (tandem) C2 domain containing 1 5,81 1439045_x_at — Dnase2a deoxyπbonuclease Il alpha 5,75 1430135_at NM_010062

CrIfI cytokine receptor-like factor 1 5,70 1418476_at NM_018827

Tspan11 tetraspanin 11 5,69 1430310_at NM_026743

Tspani tetraspan 1 5,69 1417957_a_at NM_133681

Tspani tetraspan 1 5,68 1417958_at NM_133681

Gjb2 gap junction membrane channel protein beta 2 5,65 1423271_at NM_008125

Mt2 metallothionein 2 5,62 1428942_at NM_008630

Hs3st3b1 heparan sulfate (glucosamine) 3-0-sulfotransferase 3B1 5,62 1433977_at NM_018805

Kcnk2 potassium channel, subfamily K, member 2 5,55 1449158_at NM_010607

Celsri cadherin EGF LAG seven-pass G-type receptor 1 5,53 1418925_at NM_009886

Gcnti glucosaminyl (N-acetyl) transferase 1 , core 2 5,52 1460431_at NM_010265

Matia methionine adenosyltransferase I, alpha 5,42 1423147_at NMJ 33653

Spinti serine protease inhibitor, Kunitz type 1 5,40 1416627_at NM_016907

Kctd14 potassium channel tetrameπsation domain containing 14 5,39 1426633_s_at NM_001010826

Mt1 metallothionein 1 5,32 1422557_s_at NM_013602

Psati phosphoseπne aminotransferase 1 5,32 1451064_a_at NM_177420

Cldn3 claudin 3 5,29 1460569_x_at NM_009902

Foxp2 forkhead box P2 5,24 1440108_at NM_053242

Dos downstream of StM 1 5,17 1433494_at XM_125771

Shmti serine hydroxymethyl transferase 1 (soluble) 5,17 1425178_s_at NM_009171

Modi /// LOC624892 malic enzyme, supernatant 5,16 1430307_a_at NM_008615

Ly6g6e lymphocyte antigen 6 complex, locus G6E 5,14 1429833_at NM_027366

2310057J16Rιk RIKEN cDNA 2310057J16 gene 5,11 1432464_a_at XM_133997

Ddιt4l DNA-damage-inducible transcπpt 4-lιke 5,11 1439332_at NM_030143

Tmc4 transmembrane channel-like gene family 4 5,09 1427178_at NM_181820

Hdc histidine decarboxylase 5,07 1454713_s_at NM_008230

Sertad4 SERTA domain containing 4 5,06 1454877_at NM_198247

Dos downstream of Stk11 4,99 1438370_x_at XM_125771

3300001A09Rιk RIKEN cDNA 3300001 A09 gene 4,98 1453326_at XM_134869

Glrx glutaredoxin 1 (thioltransferase) 4,94 1416592_at NM_053108

Cldn3 claudin 3 4,94 1451701_x_at NM_009902

ViII villin-like 4,93 1426022_a_at NM_011700

12 days embryo spinal cord cDNA, RIKEN full-length enriched library, clone C530045D16 product unknown EST, full insert sequence 4,92 1445501_at

Nek6 /// LOC674247 NIMA (never in mitosis gene a)-related expressed kinase 6 4,87 1423596 at NM 021606

Cep55 RIKEN cDNA 1200008012 gene 4,84 1452242_at NM_028760

Azιn1 ornithine decarboxylase antizyme inhibitor 4,82 1422702_at NM_018745

Shmti serine hydroxymethyl transferase 1 (soluble) 4,82 1425177_at NM_009171

Tacstdi tumor-associated calcium signal transducer 1 4,81 1416579_a_at NM_008532

Ube2c ubiquitin-conjugating enzyme E2C 4,78 1452954_at NM_026785

Spbc24 spindle pole body component 24 homolog (S cerevisiae) 4,73 1431087_at NM_026282

Tspan11 tetraspanin 11 4,69 1441776_at NM_026743

Cks 1b CDC28 protein kinase 1b 4,61 1448441_at NM_016904

Fst Follistatin 4,58 1434458_at NM_008046

Rab25 RAB25, member RAS oncogene family 4,55 1417738_at NM_016899

Mlph melanophilin 4,49 1449896_at NM_053015

Pla2g1b phospholipase A2, group IB, pancreas 4,45 1416626_at NM_011107

Cdkn3 cyclm-dependent kinase inhibitor 3 4,34 1430574_at XM_484366

Ttc9 tetratricopeptide repeat domain 9 4,33 1455649_at XM_126933

St14 suppression of tumoπgenicity 14 (colon carcinoma) 4,28 1418076_at NM_011176

Atp6v0a1 ATPase, H+ transporting, lysosomal VO subunit a isoform 1 4,24 1425227_a_at NM_016920 Gch1 GTP cyclohydrolase 1 4,23 1420499_at NM_008102 CbIc Casitas B-lιneage lymphoma c 4,21 1422666_at NM_023224

Tmem139 RIKEN cDNA A930027H06 gene 4,18 1436556_at NM_175408

Olfml2b olfactomedin-like 2B 4,16 1423915_at NM_177068 o

Med12l mediator of RNA polymerase Il transcription, subunit 12 homolog (yeast)-lιke 4,09 1452864_at

Krt2-8 keratin complex 2, basic, gene 8 3,99 1423691_x_at NM_031170

Azιn1 ornithine decarboxylase antizyme inhibitor 3,92 1450714_at NM_018745

Rbm35a RIKEN cDNA 2210008M09 gene 3,84 1454681_at NM_194055

Crιspld2 cysteine-rich secretory protein LCCL domain containing 2 0,20 1460458_at NM_030209

Centd3 centauπn, delta 3 0,19 1419833_s_at NM_139206

BC030477 cDNA sequence BC030477 0,19 1455845_at NM_177618

Edιl3 EGF-like repeats and discoidin l-like domains 3 0,19 1433474_at NM_010103

AcvrM Activin A receptor, type ll-like 1 0,18 1435825_at NM_009612

Ankrd38 cDNA BC060737 0,18 1436425_at NM_172872

Aard alanine and arginine rich domain containing protein 0,17 1434528_at NM_175503

Centd3 centauπn, delta 3 0,17 1451282_at NM_139206

2210023G05Rιk RIKEN cDNA 2210023G05 gene 0,16 1424968_at NM_197999

Stard9 /// LOC668856 RIKEN cDNA 4831403C07 gene 0,16 1436324_at

1700110N18Rιk RIKEN CDNA 1700110N18 gene 0,16 1430596_s_at XM_283372

CfIr cystic fibrosis transmembrane conductance regulator homolog 0,16 1420579_s_at NM_021050

Vsnl1 visinin-like 1 0,16 1420955_at NM_012038

TmemlOO RIKEN cDNA 1810057C19 gene 0,16 1449533_at NM 026433

LOC623121 0,16 1437636 at

2310016C08Rιk RIKEN cDNA 2310016C08 gene 0,16 1421031_a_at NM_023516

Cox4ι2 cytochrome c oxidase subunit IV isoform 2 0,15 1421373_at NM_053091

Scn3a sodium channel, voltage-gated, type III, alpha 0,15 1439204_at XM_141275

Mcc Transcribed locus 0,15 1438081_at —

Chsti carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 0,15 1449147_at NM_023850

Upkib uroplakin ib 0,15 1435831_at —

Prkg2 Protein kinase, cGMP-dependent, type Il 0,14 1435162_at NM_008926

TremW tπggeπng receptor expressed on myeloid cells-like 4 0,14 1460014_at NM_172623

Bmp6 bone morphogenetic protein 6 0,14 1450759_at NM_007556

Lipg lipase, endothelial 0,14 1450188_s_at NM_010720

Cacnaid calcium channel, voltage-dependent, L type, alpha 1D subunit 0,14 1427974_s_at NM_028981

Emr4 EGF-like module containing, mucin-like, hormone receptor-like sequence 4 0,14 1451563_at NMJ39138

Cttnbp2 cortactin binding protein 2 0,14 1435435_at XM_289703

Tcf21 transcπption factor 21 0,13 1417447_at NM_011545

G0s2 G0/G1 switch gene 0,13 1448700_at —

Wfdcθa gene model 122, (NCBI) 0,13 1457766_at XM_130716

Wnt2 wingless-related MMTV integration site 2 0,13 1449425_at NM_023653

Adcyδ adenylate cyclase 8 0,13 1418754_at NM_009623

Slc7a10 solute earner family 7 (cationic amino acid transporter, y+ system), member 10 0,13 1421093_at NM_017394

BB114106 expressed sequence BB114106 0,13 1439527_at —

Clec14a C-type lectin domain family 14, member a 0,13 1419468_at NM_025809

Gm1337 Gene model 1337, (NCBI) 0,12 1443287_at XM_357250

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

Ptgfr 15 days embryo head cDNA, RIKEN full-length enriched library, clone D930037F10 0,12 1446331_at —

Gpm6a glycoprotein m6a 0,12 1456741_s_at NM_153581

AI841794 expressed sequence AI841794 0,12 1433744_at NM_172492

Scube2 signal peptide, CUB domain, EGF-like 2 0,11 1453486_a_at NM_020052

Fgfr4 fibroblast growth factor receptor 4 0,11 1418596_at —

Adult male olfactory brain cDNA, RIKEN full-length enriched library, clone 6430530M09 product

— unclassifiable, full insert sequence 0,10 1460061_at

Lrat Lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) 0,10 1444487_at NM_023624

Serpιna3c seπne (or cysteine) proteinase inhibitor, clade A, member 3C 0,10 1421564_at NM_008458

ChrdH chordιn-lιke1 0,10 1434201_at —

Itgaβ integrin alpha 8 0,10 1427489_at NM_001001309

1500016O10Rιk RIKEN cDNA 1500016010 gene 0,09 1438641_x_at XM_133706

Hpcal4 hippocalcin-like 4 0,09 1433987_at NM_174998

Igfbp3 insulin-like growth factor binding protein 3 0,08 1423062_at NM_008343

AI481121 expressed sequence AI481121 0,08 1456123_at

Table 3 (Table S3):

Gene Symbol Gene Title Fold Change Gen_id_mfr RβfSβqTranscript ID

Ereg epiregulin 39,13 1419431_at NM_007950

Cyp4a12 cytochrome P450, family 4, subfamily a, polypeptide 12 27,16 1424352_at NM_177406

DIM delta-like 1 homolog (Drosophila) 18,61 1449939_s_at NM_010052

Gja3 gap junction protein, alpha-3 16,27 1439793_at —

1810036H07Rιk RIKEN cDNA 1810036H07 gene 9,04 1453132_a_at NM_025467

P2rx2 purinergic receptor P2X, ligand-gated ion channel, 2 8,37 1435212_at NM_170682

1110014F24Rιk RIKEN cDNA 1110014F24 gene 7,57 1428781_at NM_028618

Rhbdl2 rhomboid-like 2 7,11 1442819_at —

Stδsiaβ ST8 alpha-N-acetyl-neuraminide alpha-2,8-sιalyltransferase 6 6,18 1456440_S_at —

Cd177 RIKEN cDNA 1190003K14 gene 5,57 1424509_at NM_026862

Stβsiaθ ST8 alpha-N-acetyl-neuraminide alpha-2,8-sιalyltransferase 6 5,34 1456147_at —

Fetub fetuin beta 5,19 1449555_a_at NM_021564

Mfsd2 major facilitator superfamily domain containing 2 3,88 1428223_at NM.032793

Lrg1 hypothetical protein 3,23 1417290_at NM_029796

BC024561 cDNA sequence BC024561 2,91 1451610_at NM_153576

C430004E15Rιk RIKEN cDNA C430004E15 gene 2,28 1426809_at NM_175286

K> 9330184L24Rιk 9330184L24Rιk RIKEN cDNA 9330184L24 gene 0,12 1441550_at —

5033428C03Rιk 5033428C03Rιk RIKEN cDNA 5033428C03 gene 0,08 1454381 at

What is claimed is:

1. A method for identifying targets for the use in diagnosis or treatment monitoring of dysplasia, in particular of low grade or high grade dysplasia related to adenocarcinoma of the lung, comprising the steps of:

- laser microdissection of cells from the tissue of a transgenic animal overexpressing an oncogene placed under the control of a regulatory sequence from a tissue specific gene, wherein dysplastic cells and morphologically unaltered cells are harvested,

- isolating RNA (1) from the dysplatic cells and isolating RNA (2) from the morphologically unaltered cells,

- determining, for each of the isolated RNA (1) and (2), a preferably genome wide gene expression profile, and

- identifying the therapeutical target as a gene being significantly overexpressed in the gene expression profile of the isolated RNA (1) in comparison with the gene expression profile of the isolated RNA (2).

2. Method according to claim 1 further comprising 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).

3. Method according to one of the claims 1-2, wherein at least one microarray is employed for the gene expression profile, in particular to search genome wide for at least one gene regulatory network.

4. Use of a gene identified by the method according to one of the claims 1-3 or 6, wherein the gene is selected from the group of the genes listed in Supplementary Table 1 , 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, or of an antibody directed against said gene product 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.

5. Use according to claim 4, wherein the gene is selected from the group of the focus genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoal , Arg2, Brunol4, Cldn2, Cldn4, Cldnδ, CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfl , Rgs16, Stk39, Tnfsfθ, preferably APOA1 , or wherein the gene is selected from the group of the focus genes Btbdi 1 , C8orf13, Cyp1b1 , Fetub, Fut2, Klc3, Pcskβ, Pkhdi , Pla2gl1b, Psrd , Ptprn2, Rnf128, Ros1 , Sdcbp2, Sult2b1 , preferably PKHD1.

6. Method according to one of the claims 1-3 or use according to one of the claims 4-6, wherein the gene is selected from the group of genes coding for a protein being involved in at least one metabolism reaction, preferably selected from

- lipid metabolism

- glycosylation

- fucosylation

- receptor tyrosine kinase activity

- regulation of Amphiregulin and/or Epiregulin,

- cell interactions, preferably the tight junction proteins (in particular Claudins) or gap junction proteins (in particular GJA3 etc)

- transcription factors in the epithelial-mesenchymal transition (EMT), preferably HNF4a etc

, and/or wherein said metabolism reaction or protein function is determined.

7. Use according to one of the claims 4-6, wherein a diagnostically effective amount of the 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, or of the antibody directed against said gene product or of the antibody directed against said polypeptide, is used for the preparation of a diagnostic agent, in particular of a diagnostic standard for body fluid analysis or for tissue analysis.

8. Use according to claim 7 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.

9. Use according to one of the claims 4-6, preferably of the antibody directed against said gene product or of the antibody directed against said polypeptide, 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 , 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, 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.

10. Use according to claim 9 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 RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, in comparison with the respective diagnostic standard or reference level is indicative that the patient has dysplasia, in particular low grade or high grade dysplasia related to adenocarcinoma of the lung.

11. Use according to claim 9 for monitoring the treatment of a patient having dysplasia to a method of treating dysplasia, in particular by a chemoprevention therapy such as by administering Zileuton or Celecoxib to the patient, wherein the level of the gene, in particular the coding region thereof, 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 determined in the sample before and after the treatment, and wherein a significant decrease of said level is indicative that the patient therapeutically responds to the method of treating dysplasia.

12. Use according to claim 4, preferably of the antibody directed against said gene product or of the antibody directed against said polypeptide, to screen for drugs targeting cellular growth and proliferation and/or hair and skin development and function, wherein the gene is selected from the group of the genes Areg, Ereg, Adcyapi , Adoral , Afp, Apoal , Arg2, BrunoW, Cldn2, Cldn4, Cldn8, CIu, Fst, Gpx2, Gsta4, Hnf4a, Inhbb, Lad1 , Pcbdi , Pthlh, Rasgrfl , Rgs16, Stk39, Tnfsf9, preferably APOA1 or to screen for drugs targeting cell death and/or cancer and/or gastrointestinal diseases, wherein the gene is selected from the group of the genes Btbdi 1 , C8orf13, Cyp1b1 , Fetub, Fut2, Klc3, Pcsk6, Pkhdi , Pla2gl1b, Psrd , Ptprn2, Rnf128, Ros1 , Sdcbp2, Sult2b1 , preferably PKHD1.

13. Use according to one of the claims 4 or 12, wherein an dysplastic cell (over-)expressing said gene is contacted with a compound to be tested and the expression level of said gene is determined 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.

14. Use according to one of the claims 4, 12, or 13 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 the level of the gene, in particular the coding region thereof, 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, in a sample, preferably a tissue sample or a plasma sample, isolated from a transgenic animal to which a compound to be tested has been administered and wherein said level being significantly lower than the respective level in a corresponding sample of an equivalent transgenic animal to which said compound has not been administered is indicative of the therapeutic effect of said compound as a drug directed against dysplasia.

15. Use of a composition 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

- an siRNA composition, wherein the siRNA composition reduces the expression of a gene selected from the group consisting of the genes listed in Supplementary Table 1 , and/or

- an antibody directed against a gene product encoded by a gene selected from the group of genes listed in Supplementary Table 1.

16. Use of an antibody composition comprising a pharmaceutically effective amount of a labeled antibody, preferably labeled with an isotope such as iodine-124 may be, directed against the gene product encoded by a gene selected from the group of the genes listed in Supplementary Table 1 , 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.

17. Use according to one the claims 9-11 or 13-14, wherein an antibody composition according to claim 16 has been administered to the patient or to the dysplastic cell or to the transgenic animal for determining the level of the gene product.

18. Use according to claim 17, wherein an imaging method, preferably PET, such as by examining the glucose metabolism in tumor-PET, wherein FDG-PET is particularly preferred, or CT is used for determining the level of the gene product.

19. Use according to one of the claims 4-18 for or in

- bronchoscopy, in particular for monitoring enzyme catalytic reactions,

- in vitro developing of enzyme based assays, and/or

- therapy of facultative canceroses

- immunohistochemistry, in particular a immunohistochemical staining.

20. A method of qualifying 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 iisted in Supplementary Table 1 , for the diagnosis or treatment monitoring of dysplasia, 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.

21. 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 gene products encoded by the genes listed in Supplementary Table 1 , 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.

22. Use, method or procedure according to one of the preceding claims, wherein at least one western blot is performed for detecting the gene product or the polypeptide.

23. Use according to any one of the preceding claims, wherein the compositions, drugs or compounds are related from the group consisting of the tyrosine kinase inhibitors, preferably consisting of Dasatinib, Erlotinib, Imatinib, Lapatinib, Nilotinib, Sorafinib and Sunitinib, in particular to inhibit tyrosine kinase activity in dysplasia or other precancerous iesions.

24. Use according to one of the claims 4 - 14, wherein the diagnosis or treatment monitoring, in particular in a patient, is performed by an imaging method, preferably selected from the group of optical or nuclear traces, in particular positron emission tomography (PET) or single photon emission computed tomography (SPECT) or other optical dyes.

Download Citation


Sign in to the Lens

Feedback