Patent Application
20110064739
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1. Field of the Invention
Embodiments of the invention relate to a medicament, compositions, and substances directed to novel c-myc modulated factors, and the use of said compositions and substances for labelling, identifying, and treating adenocarcinoma of the lung, in particular papillary adenocarcinoma of the lung. Areas of application are the life sciences: biology, biochemistry, biotechnology, medicine and medical technology.
2. Background of the Related Art
The most common cause of cancer-related death in industrial countries is lung cancer. Lung carcinoma is a leading cause of cancer morbidity with tumor entities being classified as small-cell (SCLC) or non-small-cell lung carcinomas (NSCLC). It is generally accepted that several genetic changes appear to be necessary for malignant transformation and these include, amongst others, overexpression of protooncogenes and loss of tumor-suppressor functions. However, despite intense research the molecular causes of lung adenocarcinoma are poorly understood. Nonetheless, there is evidence for the c-Myc protooncogene to play a central role in various types of human tumors.
However, the molecular and cellular mechanisms of carcinogenesis induced by c-myc in vivo are poorly understood, so far. In this regard, the identification of downstream target genes, which distinguish between physiologic and tumorigenic functions of c-Myc in vivo would allow novel approaches for the treatment, diagnosis, and study of cancer.
It would be desirable to make available a medicament and medication, compositions, and substances directed to novel c-myc modulated factors, and the use of said compositions and substances for labelling, identifying, and treating adenocarcinoma of the lung, in particular papillary adenocarcinoma of the lung.
Embodiments of the invention are based on the surprising finding that in mammalian lungs c-myc acts as a molecular switch, specifically inducing an expression pattern in vivo, which results in prototypical mammalian adenocarcinoma of the lung and liver metastasis. A set of factors essential for the processes of tumorigenesis and tumor progression, i.e. cell cycle and apoptosis, cell growth, extracellular signaling, angiogenesis and invasion, is identified, whose expression is significantly changed. In particular, the expression pattern found uncovers the network of molecules leading to mammalian papillary adenocarcinomas of the lung.
We make available a medicament, compositions, and substances directed to novel c-myc modulated factors, and the use of said compositions and substances for labelling, identifying, and treating adenocarcinoma of the lung, in particular papillary adenocarcinoma of the lung.
Embodiments concern a medicament for the treatment of lung carcinoma comprising a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Kra1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn and/or increases the expression or activity of a c-myc modulated gene selected from the group consisting of Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Caipn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, and Ablim1.
1—known c-Myc-targets and their relatives
2—known c-Myc-targets and their relatives containing potential c-Myc binding sites
3—known c-Myc-responsive genes and their relatives containing potential c-Myc binding sites
4—known c-Myc-responsive genes and relatives
5—new c-Myc-responsive genes containing potential c-Myc binding sites
6—new c-Myc-responsive genes
1—known c-Myc-targets and their relatives containing potential c-Myc binding sites
2—known c-Myc-targets and their relatives
3—known c-Myc-responsive genes and their relatives containing potential c-Myc binding sites
4—known c-Myc-responsive genes and relatives
5—new c-Myc-responsive genes containing potential c-Myc binding sites
6—new c-Myc-responsive genes
Normal subpleural parenchyma of non transgenic animals (a). Dispersed and immediately subpleural densely packed BAC: hyperchromatic low columnar lining cells of the alveoli (b). Initial invasive papillary lung adenocarcinoma (PLAC) besides foci of dispersed BAC (c)
BAC F—BAC, females
BAC M—BAC, males
PLAC S—small-sized PLACs
PLAC M—middlel-sized PLACs
PLAC L—large-sized PLACs
In one aspect, embodiments of the invention are directed to the use of at least one biomarker selected from the group consisting of Satb1, Hspa9a, Hey1, Gas1, Bnip2, Capn2, Anp32a, Ddit3, Ccnb2, Cdkn2d (p19), Prc1, Uck2, Srm, Shmt1, Slc19a1, Npm1, Npm3, Nol5, Lamr1/Rpsa, Arhu(Rhou), Traf4, Adam19, Bmp6, Rbp1, Reck, Ect2 (Group A) in the diagnosis of cancer and/or the prognosis of cancer and/or the treatment monitoring of cancer, in particular of lung cancer such as lung adenocarcinoma(s) may be.
For different purposes, it is preferred if the at least one biomarker is a combination of
(a) at least one biomarker selected from the group consisting of Group A, and
(b) the epithelial cell adhesion molecule (EpCAM).
Preferentially, a combination of Satb1 and at least one further biomarker selected from the group of Hspa9a, Hey1, Gas1, Bnip2, Capn2, Anp32a, Ddit3, Ccnb2, Cdkn2d (p19), Prc1, Uck2, Srm, Shmt1, Slc19a1, Npm1, Npm3, Nol5, Lamr1/Rpsa, Arhu(Rhou), Traf4, Adam19, Bmp6, Rbp1, Reck, Ect2, EpCAM is used as the at least one biomarker. Preferably, an appropriate amount of the at least one biomarker is used, in particular an amount for manufacturing a reference, more particular for manufacturing a reference comprising a reference level of said at least one biomarker, such as the level of said at least one biomarker in a sample of a normal healthy individual or the level of a said at least one biomarker in a sample of a patient suffering from cancer may be.
In particular, the at least one biomarker selected from the group consisting of Group A, or the combination of (a) at least one biomarker selected from the group consisting of Group A and (b) the epithelial cell adhesion molecule (EpCAM), is used for monitoring the therapeutic treatment of a patient, wherein the patient is preferably a human being or a transgenic c-myc mouse, suffering from lung cancer, in particular for monitoring the treatment of said patient with irinotecan, paclitaxel and/or 5-fluorouracil and/or the treatment with a drug binding to the epithelial cell adhesion molecule (EpCAM), such as an anti EpCAM antibody may be.
Preferably, at least one biomarker is selected from the group consisting of Ccnb2, Slc19a1, Uck2, Srm1, Nol5a, Arhu, Adam19, Ect2, Shmt1 (Group B) or from the group consisting of Gas1, Bmp6, Bnip2,_Capn2, Ddit3, Hey1 (Group C), and wherein the at least one biomarker preferably further comprises EpCAM, is used, in particular for the diagnosis, prognosis and/or treatment monitoring of PLAC. More preferably, at least one biomarker is selected from the group consisting of Group B and at least one biomarker selected from the group consisting of Group C are used, and wherein said at least two biomarkers preferably further comprise EpCAM.
Another aspect of the invention is directed to a method for diagnosing cancer and/or prognosing cancer and/or staging cancer and/or monitoring the treatment of cancer, in particular lung cancer, such as lung adenocarcinoma(s) may be, comprising the steps of:
(a) measuring the level of at least one biomarker selected from the group consisting of Group A, and wherein said at least one biomarker preferably further comprises EpCAM, in a patient or in a sample of a patient, wherein the patient is preferably a human being or a transgenic c-myc mouse, suffering from or being susceptible to cancer, and
(b) comparing the level of said at least one biomarker in said patient or in said sample to a reference level of said at least one biomarker.
Preferably, the method for diagnosing, prognosing and/or staging cancer and/or monitoring the treatment of cancer, is implemented for monitoring the therapeutic treatment of a patient suffering from lung cancer, in particular the treatment with irinotecan, paclitaxel and/or 5-fluorouracil and/or the treatment with a drug binding to the epithelial cell adhesion molecule (EpCAM), such as an anti EpCAM antibody may be.
In a particularly preferred embodiment, at least one biomarker is selected from the group consisting of Group B or from the group consisting of Group C, and wherein said at least one biomarker preferably further comprises EpCAM, in particular for the diagnosis, prognosis, staging and/or treatment monitoring of PLAC.
In particular, the method is preferably implemented to distinguish between different subtypes of lung cancer, such as (but not limited to) lung adenocarcinomas as defined by BAC or PLAC, wherein a significantly increased level of Group B in comparison with the level of a normal individual or a significantly decreased level of Group C in comparison with the level of a normal individual is indicative of PLAC.
More particularly, at least one biomarker selected from the group consisting of Group B and at least one biomarker selected from the group consisting of Group C are measured to distinguish between different subtypes of lung cancer, such as (but not limited to) lung adenocarcinomas as defined by BAC or PLAC.
A significantly increased level of Group B in comparison with the level of a normal individual and a significantly decreased level of Group C in comparison with the level of a normal individual is then indicative of PLAC.
According to embodiments of the invention, the medicament or medication for the treatment of lung carcinoma, in particular of papillary adenocarcinomas of the lung, comprises a pharmaceutically effective amount of a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn (Group D) and/or increases the expression or activity of a c-myc modulated gene selected from the group consisting of Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, and Ablim1 (Group E).
Preferably, the medicament or medication further comprises a pharmaceutically acceptable carrier and/or recipient and/or diluent. In particular, the medicament or medication for the treatment of lung carcinoma comprises a composition that increases and/or decreases the expression or activity a c-myc modulated gene in a mammal, preferably in a human being or a mouse, more preferably in a human being.
The genes according to embodiments of the invention concern genes of mammalia, preferably genes of the genome of mus musculus or homo sapiens, in particular the respective genes of homo sapiens are preferred. Within the context of the invention, it is in particular preferred if the c-myc modulated gene is selected from the group of genes being grayscaled in the Tables 2, 4 and 6 or preferably Table 6. Preferably, the medicament or medication according to the invention comprises a pharmaceutically effective amount of a composition as detailed below.
Within the context of the present invention an antisense composition is provided, wherein the antisense composition comprises a nucleotide sequence complementary to a coding sequence of a c-myc modulated gene selected from the group consisting of Group D.
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 5′ 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 mammalian, preferably human or murine, more preferably human mRNA.
In particular, the composition comprises a nucleotide sequence effective to form a base-paired heteroduplex structure composed of mammalian, preferably human or murine, more preferably human RNA transcript and the oligonucleotide compound, whereby this structure is characterized by a Tm of dissociation of at least 45° C.
Further, a siRNA composition is provided, wherein the siRNA composition comprises an siRNA reducing or preferably inhibiting the expression of a c-myc modulated gene selected from Group D. Embodiments may employ siRNA for use in modulating the level of protein presence in the cell. SiRNA oligonucleotides directed to said genes specifically hybridize nucleic acids encoding the gene products of said genes and interfere 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 mammalian, preferably human or murine, more preferably 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.
Nucleotide sequences and siRNA 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.
Furthermore, an antibody composition is provided, wherein the antibody composition comprises a pharmaceutically effective amount of an antibody or fragment thereof that binds to a polypeptide encoded by a gene selected from the group consisting of Group D.
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 warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Preferably, monoclonal antibodies are used in the antibody compositions of the invention which may be readily 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).
In particular, antibodies or fragments thereof specific for the gene products involved in extracellular signaling, angiogenesis and invasion, in particular specific for the gene products of Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Hpn, more particular for said gene products highlighted in Table 3, are preferred.
Further, a polypeptide composition (also termed as polypeptide composition (1)) is provided, wherein the polypeptide composition comprises a polypeptide coded by the sequence of a c-myc modulated gene selected from the group consisting of Group E.
Preferably, the polypeptide composition comprises a peptide coded by the entire ORF/coding sequence of one of said genes. Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, in particular 100% identity, to the entire amino acid sequence of the gene product of one of said genes.
In particular, a polypeptide composition comprising a polypeptide involved in extracellular signaling, angiogenesis and invasion is preferred. Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
Furthermore, a vaccine composition is provided comprising a polypeptide coded by the sequence of a c-myc modulated gene selected from the group consisting of Group D. The vaccine composition for immunogenic and vaccine purposes in eliciting lung adenocarcinoma-specific immune responses preferably provides polypeptides comprising antigenic determinants from said gene products. An antigenic determinant of the gene product of one of said genes can be provided either by the full sequence of the gene product concerned, or by such subsequences as may be desired, e.g. a sequence fragment comprising at least 25%, e.g. at least 50% or 75% of the full sequence of the gene product concerned, e.g. a N-terminal or C-terminal sequence fragment.
In particular, a vaccine composition is preferred that comprises an antigenic determinant of a polypeptide involved in extracellular signaling, angiogenesis and invasion. A further aspect of the invention concerns the use of a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Group D and/or increases the expression or activity of a c-myc modulated gene selected from the group consisting of Group E for the preparation of a medicament or medication, preferably for the preparation of a medicament or medication for preventing, treating, or ameliorating lung carcinoma, more preferably for preventing, treating, or ameliorating adenocarcinoma of the lung, in particular papillary adenocarcinomas of the lung.
In particular, an antisense composition, an siRNA composition, an antibody composition or the polypeptide composition (1) as detailed herein, or a combination thereof, is used for the preparation of the medicament or medication. In one embodiment, a nucleotide composition (also termed as nucleotide composition (1)) is used for the preparation of said medicament or medication, wherein the nucleotide composition comprises a nucleotide sequence of a c-myc modulated gene selected from the group consisting of Group E.
The nucleotide composition particularly comprises a nucleic acid being from about 20 base pairs to about 100,000 base pairs in length, wherein the nucleotide sequence is included. Preferably the nucleic acid is from about 50 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid is from about 50 base pairs to about 10,000 base pairs in length. Most preferred is a nucleic acid from about 50 pairs to about 4,000 base pairs in length. The nucleotide sequence can be a gene or gene fragment that encodes a protein, an oligopeptide or a peptide. Preferably, the nucleotide sequence of the present invention may comprise a DNA construct capable of generating a gene product coded by one of said genes and may further include an active constitutive or inducible promoter sequence.
In particular the nucleotide composition comprises a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of the gene product of one of said genes. In this regard, nucleotide sequences coding for polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred. In particular, it is preferred if the nucleotide sequence encodes a polypeptide with 100% identity to the entire amino acid sequence of the gene product of one of said genes.
In particular, the nucleotide composition comprises a DNA sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ORF (or coding sequence, respectively) of one of said genes over the entire coding region. In this regard, nucleotide sequences which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. In particular, it is preferred if the nucleotide sequence encodes a DNA sequence that has 100% identity to the entire ORF of one of said genes over the entire coding region.
The nucleotide composition may further comprise an enhancer element and/or a promoter located 5′ to and controlling the expression of said therapeutic nucleotide sequence or gene. The promoter is a DNA segment that contains a DNA sequence that controls the expression of a gene located 3′ or downstream of the promoter. The promoter is the DNA sequence to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene, typically located 3′ of the promoter.
Preferably, a pharmaceutically effective amount of one of the compositions specified above or a pharmaceutically effective amount of a combination thereof is used, in particular furthermore a pharmaceutically acceptable carrier and/or recipient and/or diluent is used for the preparation of the medicament or medication. In a further aspect of the invention, an immunogenically effective amount of the vaccine composition as detailed herein is used for the preparation of a vaccine, wherein preferably furthermore an adjuvant is used. In particular, it is preferred if the vaccine composition and the adjuvant are admixed with a pharmaceutically effective vehicle (excipient).
Yet another aspect of the invention concerns the use of novel c-myc modulated genes and/or gene products of thereof to screen for and to identify drugs against lung carcinoma, preferably against adenocarcinoma of the lung, more preferably against papillary adenocarcinoma of the lung, wherein at least one or more genes and/or one or more gene products of thereof selected from the group of Group D and/or Group E is used.
In one embodiment, a nucleotide composition (termed as nucleotide composition (2), respectively) is used as the one or more genes, wherein the nucleotide composition comprises a nucleotide sequence of a c-myc modulated gene selected from the group consisting of Group D.
Said nucleotide composition particularly comprises a nucleic acid being from about 20 base pairs to about 100,000 base pairs in length, wherein the nucleotide sequence is included. Preferably the nucleic acid is from about 50 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid is from about 50 base pairs to about 10,000 base pairs in length. Most preferred is a nucleic acid from about 50 pairs to about 4,000 base pairs in length. The nucleotide sequence can be a gene or gene fragment that encodes a protein, an oligopeptide or a peptide. Preferably, the nucleotide sequence of the present invention may comprise a DNA construct capable of generating a gene product coded by one of said genes and may further include an active constitutive or inducible promoter sequence.
Preferably, this nucleotide composition comprises a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of the gene product of one of said genes. In this regard, nucleotide sequences coding for polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred. In particular, it is preferred if the nucleotide sequence encodes a polypeptide with 100% identity to the entire amino acid sequence of the gene product of one of said genes.
In particular, the nucleotide composition (2) comprises a DNA sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ORF (or coding sequence, respectively) of one of said genes over the entire coding region. In this regard, nucleotide sequences which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. In particular, it is preferred if the nucleotide sequence encodes a DNA sequence that has 100% identity to the entire ORF of one of said genes over the entire coding region.
In another embodiment, a polypeptide composition (also termed as polypeptide composition (2)) is used as the one or more gene products, wherein the polypeptide composition comprises a polypeptide coded by the sequence of a c-myc modulated gene selected from the group consisting of Group D.
Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the entire amino acid sequence of the gene product of one of said genes. Preferably, the polypeptide composition comprises a peptide coded by the entire ORF/coding sequence of one of said genes. In particular, a polypeptide composition comprising a polypeptide involved in extracellular signaling, angiogenesis and invasion, as coded by Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Kra1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn is preferred.
Preferably, one or more of said genes and/or their gene products, in particular the nucleotide composition (2) and/or the polypeptide composition (2), is incubated with a compound to be tested and changes in the expression of said genes and/or derived sequences and/or the function of said gene products are determined. In particular, the antisense composition, the siRNA composition, the antibody composition, the nucleotide composition (1) or the polypeptide composition (1), as detailed herein, or a combination thereof, is used as test compound.
More preferably, said use for screening and identifying drugs comprises the steps of: (1) contacting a test cell expressing, preferably overexpressing, at least one of said genes with a test compound; (2) detecting the expression level of said gene; and (3) determining the compound that suppresses said expression level compared to a normal control level of said gene as an inhibitor of said gene.
In particular, a compound that enhances the expression or activity of one of said genes is identified by the inventive use comprising the steps of: (1) contacting a test cell expressing, in particular overexpressing, said non-small cell lung cancer-associated gene with a test compound; (2) detecting the expression level of said non-small cell lung cancer-associated gene; and (3) determining the compound that increases said expression level compared to a normal control level of said gene as an enhancer of said non-small cell lung cancer-associated gene. Within this context, it is particularly preferred that the use comprises the steps of: (1) contacting a test compound with a polypeptide encoded by the selected gene; (2) detecting the binding activity between the polypeptide and the test compound; and (3) selecting a compound that binds to the polypeptide.
More specifically, said use comprises the steps of (a) contacting a test compound with a polypeptide encoded by the selected gene; (b) detecting the biological activity of the polypeptide of step (a); and (c) selecting a compound that suppresses the biological activity of the polypeptide encoded by the gene selected from the group consisting of Group D in comparison with the biological activity detected in the absence of the test compound, and/or enhances the biological activity of the polypeptide encoded by the polynucleotide selected from the group consisting of Group E in comparison with the biological activity detected in the absence of the test compound. Advantageously, cell proliferation is detected as biological activity, or any other biological activity of the cell related to carcinogenesis or tumorigenesis, in particular the presence of tumor markers known in the art, is detected.
In a further preferred embodiment the use comprises the steps of: (1) contacting a test compound with a cell into which a vector comprising the transcriptional regulatory region of one or more of the selected genes and a reporter gene that is expressed under the control of the transcriptional regulatory region has been introduced; (2) measuring the activity of said reporter gene; and (3) selecting a compound that reduces the expression level of said reporter gene when the selected gene is an up-regulated gene selected from the group consisting of Group D, and/or that enhances the expression level of said reporter gene when the selected gene is a down-regulated gene selected from the group consisting of Group E, as compared to a control.
More particular, the use comprises drugs, in particular a test compound and/or a medicament or medication as specified above, wherein the drugs regulate the expression of one or more of said genes and/or the function of one or more of said gene products and/or their derived molecules, and said drugs are used for the production of means for treating, identifying or labeling adenocarcinoma, in particular papillary adenocarcinoma, of the lung.
In one embodiment of the use for screening and identifying drugs DNA and/or related molecules encoding one or more of said gene products and/or derived structures are used, and/or one or more polypeptides, peptides and/or derived molecules having the function of one or more of said gene products, are used.
Yet another aspect of the invention concerns a procedure for identifying, labelling and/or treating lung carcinoma, in particular adenocarcinoma of the lung, more particular papillary adenocarcinoma of the lung, wherein a biological and/or biotechnological system is contacted with a soluble substance, such as an oligonucleotide sequence or antibody may be, having affinity with at least one of the genes selected from the group of Group D and/or mRNA encoded thereby and/or their gene products and/or parts thereof and wherein the soluble substance is linked with a marker.
Preferably, the biological or biotechnological system is an organism, a tissue, a cell, a part of a cell, a DNA, a RNA, a cDNA, a mRNA, a cRNA, a protein and/or a peptide and/or a derived structure and/or contains the same. In particular, the biological system is a cell derived from a tumor or of a tumor metastasis, preferably a cell derived from a lung tumor or of a liver metastasis thereof or the biotechnological system is an oligonucleotide library derived thereof.
Particularly, the biological or biotechnological system employed for the procedure comprises cells of a tumor malignancy of the lung and/or an oligonucleotide library and/or a protein library and/or a peptide library derived thereof, such as a transgenic animal, in particular a c-myc mouse as described herein, or biological material received thereof or biological material received from a human lung cancer patient may be.
As the, at least partially, soluble substance in particular oligonucleotides, proteins, peptides or structures derived of thereof are suitable. These have the advantage that they can recognize specifically two or three-dimensional target structures on the molecular level. Beyond that, they provide the favourable characteristic that the recognition usually takes place in aqueous physiologically buffered solutions and leads to a specific association/binding with the targeted structure.
In one embodiment of the procedure, a nucleotide sequence according to the antisense composition, an siRNA according to the siRNA composition, an antibody according to the antibody composition, a nucleotide sequence according to the nucleotide composition (1) or a polypeptide according to the polypeptide composition (1), as described herein, or a combination thereof, is used as the soluble substance. In particular, complementary oligonucleotides, such as a primer or primer pairs described herein, is used as the substance having specific affinity.
Thereby, monoclonal and/or polyclonal antibodies and/or antibody fragments are particularly suitable, since they are formed as stable highly specific structures, which are, in principle, producible against all possible molecular target structures. In a favourable embodiment of the procedure human and/or bispecific antibodies or human and/or bispecific antibody fragments are used. In particular, monoclonal antibodies and/or antibody fragments are thereby suitable.
For implementing the invention it is preferred, if the marker according to invention is selected as an element, an isotope, a molecule and/or an ion or is composed of thereof, such as a dye, contrast means, chemotherapeutic agent, radionuclide, toxin, lipid, carbohydrate, biotin, peptide, protein, microparticle, vesicle, polymer, hydrogel, cellular organelle, virus and/or whole cell, in particular if the marker is formed as dye labeled and/or enzyme-labeled secondary antibodies and/or as protein A and/or as protein G or structures derived of thereof.
The linkage between the marker and the substance is favourably chemically, electrostatically and/or over via hydrophobic interactions, such as there is sufficient connection stability for the use of the marked substance for identifying, labelling and treating of metastatic cells, preferably if the linkage is covalent. For the increase of the sensitivity of the procedure according to invention also the simultaneous use of several substances is in particular suitable, in particular if they comprise two or more substances having each affinity with one of the targets or their mRNA sequences or their gene products.
In particular, for a specific recognition/identification substances are preferred, which bind with an affinity above the association constant Ka=1000 M-1 to the target structure. Preferably, the procedure according to the invention comprises contacting the biological or biotechnological system with a plurality of said soluble substances.
For implementing the procedure according to the invention it is favourable to use one of the following methods—PCR, in vitro translation, RT-PCR, gel electrophoresis, Western Blot, Northern Blot, Southern Blot, ELISA, FACS measurement, chromatographic isolation, UV microscopy, immunohistochemistry, screening of solid phase bound molecules or tissues and/or biosensory investigation—whereby by amplification, isolation, immobilization and/or detection and/or by combinations of thereof a particularly simple conversion of the procedure according to invention is made possible for the examined sample, in particular if furthermore a statistic analysis is accomplished.
The procedure according to invention is preferably implemented by using molecules, cells and/or tissue, in particular being immobilized or synthesized on a planar surface, e.g. spatially addressed. on a nitrocellulose or PVDF membrane, or is linked to the cavity of a microtiter/ELISA plate or on the bottom of a cell culture container, in particular on a glass or plastic chip (biochip).
Favourably, as solid phase bound molecules, substances are used, having affinity for at least one, in particular two, preferably three, more preferably four, most preferably five of the selected target genes and/or their variants and/or parts thereof and/or their mRNA and/or their gene products and/or cleavage products derived thereof, polypeptides or peptides.
The identification of the target structures, e.g. of factors in an immobilized section of tissue or of cells of a cell culture, can take place thereby with arbitrarily marked molecules, which are brought on the surface in solution, e.g. (fluorescence marked/labeled) antibodies. If a RT-PCR is accomplished, then favourably appropriate oligonucleotide probes and/or primers are used. For implementing the procedure according to invention immobilized molecule libraries, e.g. DNA or antibody libraries are used, which associate with the target structure(s) in solution, e.g. with a cDNA library, a PCR product library or with cells of a secondary tumor.
Substances bound to the molecule libraries are particularly identified by the use of appropriately marked probes, e.g. dye labeled oligonucleotides. If unabeled primary antibodies are used, preferably labeled secondary antibodies are used for detection. Furthermore, the use of other proteins, e.g. enzymes, or streptavidin or parts of thereof is suitable. For the diagnosis of a tumor, in particular by in vitro and/or in vivo diagnostics, with the help of the procedure according to invention preferably optically (or radiographically) sensitive equipment, is used, e.g. an UV microscope, scanner or ELISA reader, photometer, or szintigrafic equipment, e.g. X-ray gadget are appropriate.
A further aspect of the invention concerns the procedure according to the invention for diagnosing lung cancer, in particular adenocarcinoma of the lung, more particular papillary adenocarcinoma of the lung, or a predisposition to developing lung cancer in a subject, comprising determining an expression level of a c-myc modulated gene selected from the group consisting of Group D, in a biological sample derived from the subject, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing lung cancer, in particular if said increase is at least 200% greater than said normal control level, and whereby preferably said level is determined for a plurality of said genes.
Preferably, this procedure further comprises determining an expression level of a c-myc modulated gene selected from the group consisting of Group E in a biological sample derived from the subject, and wherein a decrease in said level compared to a normal control level indicates said subject suffers from or is at risk of developing non-small cell lung cancer, in particular if said decrease is at least 200% lower than said normal control level, and whereby preferably said level is determined for a plurality of said genes.
In practice, the procedure according to the invention preferably comprises any one of the methods: detecting the mRNA of the selected genes, detecting the protein encoded by the selected gene or detecting the biological activity of the protein encoded by the selected gene.
In one embodiment of the procedure it is preferred if the hybridization of a selected gene probe to a gene transcript of a biological sample is determined, in particular if a primer or primer pair, as described herein, is used as gene probe and/or if the hybridization step is carried out on a DNA array.
Another aspect of the invention concerns a method appropriate for the use and/or the procedure according to the invention, in particular for identifying adenocarcinoma of the lung, comprising the steps of isolating a biological sample from biological material received from an organism, in particular from a human patient, suffering from lung cancer or from an organism to be tested for its susceptibility to lung cancer, and determining the level of Group D and/or Group E or of fragments of thereof or of a selection of thereof in the biological sample by screening the presence of said proteins or of fragments of thereof or of mRNA coding for the same.
In one embodiment it is preferred, if the method comprises the steps of isolating a biological sample from biological material received from a healthy organism, in particular from a human being and pairwisely comparing the gene expression profiles determined for the isolated biological samples by correlating the levels measured; and/or a standard is used for determining the changes of expression, in particular the level of gene expression of at least one of the genes to be screened derived from a gene expression profile for a healthy tissue of the organ, wherein the tumor is formed, is used.
In particular, it is preferred, if
Moreover, the gene expression profiling preferably comprises the steps of
Particularly, the use of labelled ribonucleotides, such as biotin labelled ribonucleotides may be, is preferred for the in vitro transcription or the method wherein antibodies specific for one or more of said targets are used. In particular, it is preferred according to the invention, if microarrays are used for performing the hybridization assays.
Yet another preferable aspect of the invention concerns a testkit for identifying and/or determining mammalian, in particular human and/or murine, preferably human, tumor malignancies, in particular adenocarcinoma of the lung, more particular papillary adenocarcinoma of the lung, comprising a soluble substance as specified above, in particular comprising two or more detection reagents which bind to one or more genes selected from the group consisting of Group D, and/or Group E, and/or mRNA of thereof and/or polypeptides encoded thereby, for a fast and easy implementation of the invention.
Histopathology of Lung Tumors.
Lung specimens of non-transgenic and transgenic mice were examined at the age of 9-13 months. Mostly a medium sized or large solid tumor nodule was found in transgenic lung and such nodule may filled an entire lobe, while the remaining parenchyma appeared to be macroscopically unaffected. Histologically a ubiquitous proliferation of the peripheral pulmonary epithelium was evident, and the solid tumors were only part of it (
Genome Wide Transcript Profiling
To better understand the transforming capacity of over expressed c-Myc, gene expression profiles in c-Myc-induced PLACS of transgenic mice with histologically confirmed normal lung of non transgenic animals were compared. To identify key transcriptional events associated with malignant growth RNA was prepared from tumor sets of small, middle and large size (see Methods) and analyzed by oligonucleotide microarrays.
Specifically, c-Myc transcript level was more than 50 fold induced in tumor tissues of transgenic mice when compared with lung tissues of normal non-transgenic mice Induction of c-Myc protein expression was confirmed by Western blot analysis (
Statistical analysis of replicates with T-test and more stringent comparative ranking was performed to identify significantly induced and repressed genes in tumor sets in comparison with non transgenic lungs (see Methods). Array analyses yielded 162 genes (not functionally characterized ESTs were not taken into consideration) with significantly increased expression levels (mean FC >3, p-value in T-test <0,05 and 100% of “Increase” calls in comparative ranking analyses in one or more sets of tumors—see Methods for more details) and about twice as many genes (301) were identified with repressed expression (FC <−3, p-value in T-test <0,05 and 100% “Decrease” accordingly) in c-Myc-induced lung tumors (Table 1). Then, genes differently expressed in lung tumors with published data using the Myc Target Gene database were compared. This database provides valuable information on Myc-responsive genes for various cell types and species (Zeller et al., 2003; Zeller et al., 2003; Zeller et al., 2003). Known c-Myc-responsive genes were identified, i.e. genes whose expression was changed in response to c-Myc activation as well as known c-Myc targets, e.g. genes known to bind c-Myc directly. The results are included in Table 1 and Table 2. From 162 up regulated genes in tumors 29% proved to be previously reported as c-Myc-targets (designated as “T” in the Table 2) or their relatives (“rT”), i.e. these belong to gene families, which include known c-Myc-target (
To identify genes which may be directly regulated by c-Myc a computational tool was applied to search for potential c-Myc-binding sites in promoter regions of differently regulated genes. A specific set of mononunucleotide weight matrices with E-box as a core sequence available in TRANSFAC® professional database (see Methods) was applied. The results of this search are included in Table 2a,b and summarized in Table 1 and
The functional analysis of regulated genes in c-Myc induced lung adenocarcinomas indicated that they code for proteins involved in a broad spectrum of cell metabolic and signalling pathways. In the first part of the study genes were described which are associated with cell cycle regulation and intracellular control for cell division as well as those coding for transcription factors. The genes are compiled in Table 2a, b and known c-Myc responsive genes and c-Myc targets are indicated (see above for definition). In addition the number of identified c-Myc-binding sites in promoter regions is also included. Additionally, RT-PCR was performed for 9 genes involved in cell cycle regulation and apoptosis to verify their regulation in tumors by an alternative method The changes in gene expression found in lung tumors by both methods were in good agreement (
No qualitative differences in gene expression profiles in relation to tumor size were observed. This agrees with the histological examination where all tumors were classified as papillary adenocarcinomas of various sizes. However, some quantitative differences (up to 2-3 fold) in expression level of several genes between small and large tumors were observed. Among those genes whose expression vel was substantially higher in small size tumors were members involved in cell cycle regulation and included the proto-oncogene ect2. Evidently, this underlines their important role in the onset of tumor growth. Furthermore, the expression of some genes was not detected in tumor samples while their expression was abundant in non-transgenic healthy control lungs (highlighted by grey row accordingly in Table 2b). These included, among others, pro-apoptotic Ddit3 and transcription factor Nr2f1 which exerts anti-AP-1 activity and is a mediator of anti-cancer effect of retinoic acid (Lin et al., 2002).
Thus, in this first study on the oncogenomics of c-Myc-induced lung tumours activation of Myc-target genes was identified greatly contributing to the promotion of cell cycle as well as activation of genes involved in inhibition of apoptosis, therefore, providing a selective advantage for tumor growth, as discussed below.
Genome-wide expression of transcripts in solid tumors induced by targeted c-Myc-expression in alveolar epithelial cells was studied. Note, these tumors were classified as papillary lung adenocarcinomas. To follow the dynamic changes in the gene expression during carcinogenesis, sets of small-, middle- and large-sized tumors were examined separately. By use of the mouse whole genome microarrays it was possible to investigate expression of more than 12480 genes in c-Myc-induced lung tumors and compared findings with normal non-transgenic lungs. Stringent criteria were applied to determine significantly regulated genes in tumors (see “Material and Methods”). As shown in Table 4, many of these genes coded for cell metabolism, DNA and RNA synthesis, ribosomal biogenesis, protein synthesis and transport. Additionally, regulated genes were subjected to bioinformatic analysis and for comparison with findings deposited in a publicly available database termed the Myc Target Gene. The results are included in the Table 4 and summarized in
Importantly, no qualitative differences could be determined in the gene expression profiles of papillary adenocarcinomas of various sizes. However, quantitative differences in expression of Tk1, Top2a, Hmgb2, Rrm2, Kpna2, H1fx were observed which were found to be most strongly induced in small-sized adenocarcinomas. This suggests a specific role of these genes at the onset of tumor growth. Other genes, i.e. arginase1, hexokinase2 and anion exchanger Slc4a4 were expressed at higher level in large tumors (more than 2 fold) when compared with small sized tumors pointing to their essential function in malignant growth (Table 4).
It is also of considerable importance that induction of genes in tumors was identified whose expression was absent in non transgenic control lungs (marked by a gray background for the gene title in Table 4); some were consistently increased in all 10 tumors studied (marked by grey rows accordingly in Table 4). Likely, these uniquely expressed genes such as arginase1 and serine hydroxymethyl transferase, are good candidates for novel tumor therapy.
Also, reverse transcription-polymerase chain reaction (RT-PCR) for 12 genes was performed to confirm their regulation in c-Myc induced lung tumors by an alternative method. Notably, the results from both methods agreed well (
The solid tumors isolated from the lungs of c-Myc-transgenic mice were identified as papillary lung adenocarcinomas arising from lung epithelial cells, as discussed in part 1 of the study. To follow gene expression changes during lung tumor progression RNA was prepared from tumor sets of small, middle and large size (see Methods) and analyzed using the oligonucleotide microarrays MG u74Av2 to study simultaneously nearly 12500 genes. Statistical analysis of replicates with T-test and stringent comparative ranking was performed to identify regulated genes in tumors as compared with non transgenic healthy lungs (see Methods). The analysis was confined to up regulated genes which were expressed in all tumor samples of at least one set of tumors and down regulated genes which were expressed in all non-transgenic control lungs. The thresholds for significance testing was defined as mean FC>3, p-value in T-test <0,05 and 100% “Increase” call in comparative ranking analysis at least in one set of tumors for up regulated genes and FC ≦−3, p-value in T-test ≦0,05 and 100% “Decrease” for down regulated genes (see Methods). 162 genes with significantly increased expression and 301 genes with significantly decreased expression in c-Myc-expressing lung adenocarcinomas were further analyzed in regard to their biological functions and known c-Myc responsiveness based on publicly available databases and the presence of in silico determined c-Myc binding sites in their promoters. Here the focus was directed on regulated genes involved in extracellular signaling, adhesion, angiogenesis and invasion. In Table 6a, b regulated genes are divided into functional groups. Also, the number of c-Myc-binding sites and known c-Myc responsiveness for each gene analysed is reported. Remarkably, in tumors regulation of many genes was observed implicated in the main extracellular signaling pathways thereby contributing to cell proliferation. Thus, some genes coding for proteins involved in the Ras/Raf/MEK/ERK signaling cascade like the autocrine growth factor amphiregulin, Map2k1 (MEK) were specifically up regulated, and several genes that negatively influence Erk signaling like phosphatases Dusp1, Ptpre and Rassf5, a proapoptic Ras-effector, were found to be repressed. Elevated gene expression of Arhu and Frat2 as well as repressed expression of Tcf7 and Sox, involved in Wnt/beta-catenin signaling was detected. Importantly, in lung adenocarcinomas multiple gene expression changes were found which can lead to excess of=survival signals and inhibition of antigrowth signaling and, in such a way, contribute to the emerging of a malignant phenotype. These changes included up regulation of Tnf receptor associated factor 4 (Traf 4) and down regulation of the death receptor protein Tnfrsf6 to result in reduction of signaling through cell death receptor. Remarkably, the numerous gene transcript repressed in c-Myc-over expressing lung tumors included many coding for growth-inhibiting functions. Obviously disabling growth inhibitor function contributed to malignant transformation in the lung. Importantly, among repressed genes several genes coding for proteins interfering with survival signaling pathway were detected. These included Igfbp 4, Igfbp5, Igfbp 6 and inhibitor of NFk-beta Nfkbia with reduced expression leads to excessive survival signaling through IGFR and in such a way to suppression of the apoptic capacity of c-Myc.
Furthermore, in lung adenocarcinomas of transgenic mice a considerable decline in expression of genes involved in anti-growth TGFbeta signaling like Bmp6 and Tgfβ1 conveying antigrowth signals, Acvrl1, a member of Tgfβ receptor family, Gadd45b and others was found. Genes whose expression was decreased in lung adenocarcinomas also included inhibitors of proliferation like Gas3, Ndr2, Lox, Meox2, Ptprb and Ptprg as well as transformation repressors like Lats2, scaffolding proteins Akap 12 and caveolin-1.
In c-Myc-over expressing lung tumors repression of genes coding for cell-cell and cell-ECM junctions like laminin alpha 3, integrin alpha 8, cadherin 5, protocadherin alpha 4 and 6, and others was detected. Up regulation of Ect2 and down regulation of Vsnl1 and calpain2 in lung tumors demonstrates a role for c-Myc in regulation of cytoskeleton dynamics of transformed cells.
Increased expression of several proteases like HGF activator, Adam 19, lipocalin, hepsin, thimet oligopeptidase 1 and down regulation of Reck, visinine-like1 and semaphorins may well contribute to invasion and angiogenesis in c-Myc-induced lung tumors.
Some of the differently regulated genes were uniquely expressed in tumors_(expressed in all tumor samples, not detected in healthy non-transgenic control lungs) and coded for extracellular proteins, for example, HGF activator, thimet oligopeptidase and kininogen, which makes them good candidates as tumor markers. Inversely, expression of some other genes detected in all normal lungs was lost in all tumor samples. These included, among others, genes coding for cell adhesion proteins protocadherin alpha 4 and 6, Lama3, procollagen XIIIalpha1, dermatopontin, claudin5 as well as antiproliferative proteins like Bmp6, Ndr2 and Igfbp4, Sema3f, fibulin1, the loss of which in lung epithelial tissues may be essential for the development towards malignancy. These genes are highlighted in the Table 6 a,b.
Quantitative differences in gene expression from small to large size tumors included 2-3 fold changes in expression level of several genes. Among those genes whose expression level was substantially higher in small size tumors were adenosine A2b receptor and kininogen presumably involved in angiogenic signaling. In large size tumors expression levels of proto-oncogene Ros1, protease Hgfac, procollagen type XV and claudin2, were significantly increased, while gene expression of Tgfb1, Gadd45b, Igfbp6, Akap12, Ptpns1 coding for proteins involved in inhibition of growth were significantly decreased when compared with small size tumors that infers an important role for tumor progression.
Among differently regulated genes in lung adenocarcinomas some known c-Myc-responsive genes as Tnfrsf6 and Hoxa5, involved in Tnf-signaling, scaffolding proteins Akap12 and Cav, phosphatase Dusp1, ECM enzyme Lox as well as established c-Myc targets which included both up regulated (for example, mucin1, Icam1) and down regulated genes (for example, Tcf7, Notch4) were found. The remaining regulated genes represent newly identified c-Myc responsive genes and are of great interest for their role in cancer development.
Search of the putative c-Myc-binding sites in the promoter region yielded new putative c-Myc-targets which may play a role in tumor development like Arhu involved in Wnt-signaling, anti-apoptic Traf4, membrane-anchored peptidase Adam19, anti-growth Bmp6 and Rbp1, anti-invasive Reck and previously unsuspected classes of c-Myc-responsive proteins-connexins (Gjb3, Gja1) and semaphorines such as Sema3c.
To confirm the microarray data RT-PCR analysis for 5 up regulated and 6 repressed genes in 11 individual lung tumors and 4 control lungs (
Hispopatology
By use of the surfactant protein C promoter it was possible to selectively target alveolar epithelium and achieved >50-fold c-Myc expression. This led to an atypical proliferation with malignant cell transformation resulting ultimately in invasive PLACs. The WHO-Classification of human lung tumors (Travis et al., 1999) was used because it fitted well with the observed alterations in c-Myc-induced lung cancer. The resulting hyperchromatic monolayers of bronchiolar alveolar cells were typical for a non-mucinous BAC. The growth of the invasive PLAC was not hindered by the surrounding non-invasive BAC-formations. All invasive tumors of the c-Myc-transgenic model were invariably pure PLAC from the very onset of invasion. Likely, the non-mucinous BAC and pure PLAC observed in the study are derived from the same ancestral tumor progenitor, as suggested by others (Shimosato et al., 1982).
Based on histopathology, the disease model as described herein is highly relevant for the study of human lung malignancies. This is of considerable importance as primary lung adenocarcinoma (LAC) is on the rise. Furthermore, the c-Myc-transgenic disease model mimics subtypes of LAC and, therefore, enables novel insight into the molecular pathology of lung cancer.
c-Myc Activates Cell Cycle
In c-Myc overexpressing lung epithelial tumor cells an altered expression of some key cell cycle regulators was found that can be responsible for the cell cycle reentry as discussed below.
G1/S Promotion
The progression of cell cycle is catalyzed by cyclin-dependent kinases which are activated by cyclins. In c-Myc-induced lung tumors Cdk4 was up regulated, and gene expression of its inhibitor, Cdkn2d (p19), was repressed. Additionally, the expression of cyclin D1 was also increased in tumors. Further, expression of the heterodimer partner of E2Fs, transcription factor Dp1 (Tfdp1), was significantly elevated. These changes are highly suggestive for c-Myc-over expression in the lung tumors to abrogate Rb protein/E2F regulated pathway which controls G1 progression and S-phase initiation and therefore allow inappropriate reinititiation of DNA synthesis.
G2/M Promotion
The final M-phase of cell cycle is triggered by Cdk1 (Cdc2a) associated with mitotic cyclin B. In constitutively c-Myc expressing lung tumor cells, significant up regulation of genes coding for both cyclin B1, cyclin B2 and associated kinase cdc2a (cdk1), the complex of which promotes the transition G2/M, was observed. Besides, over expression of several other genes essential for progression of mitosis like serine/threonine kinases Nek6 and Stk6 (Aurora-A kinase), Cks1 (cdc28 protein kinase), Cks2 (cdc28 protein kinase regulator subunit 2), cdc20, regulators of cytokinesis Prk1 (protein 1), and three kinesin family members coding for molecular motors involved in various kinds of spindle dynamics was detected in lung tumors. Most of these mitotic genes were shown the first time to be activated in response to c-Myc.
Loss of Intracellular Control of Cell Division
Inactivation of p53-mediated apoptosis: C-Myc was found to be a powerful inducer of apoptosis. Moderate elevation of Trp53 gene transcription level in lung tumors of transgenic mice was observed that was expected because the gene is a known direct target for c-Myc. Its expression may be induced by c-Myc directly or indirectly, as a response to signaling imbalance provoked by the oncogene. Importantly, in lung adenocarcinomas deregulation of several genes that interfere with the p53 response was identified. Among these a dramatic decrease of gene expression of epithelially enriched transcription factor Klf-4, an essential mediator of Trp53 in controlling cell cycle progression following DNA damage was detected Repression of intrinsic apoptotic machinery:
In addition to genes linked to the silencing of Trp53 activity, changes in expression of several genes interfering with the cell apoptotic machinery were observed in tumor tissues. One of these was up regulation of Birc5 (survivine), which is a member of the inhibitor of apoptosis protein (IAP) family and interferes directly with the function of caspases (Chiou et al., 2003). Several down regulated genes in tumors coded for proteins interfering with the anti-apoptotic Bcl-2 protein. This is of particular importance because Bcl-2 inhibits almost all types of cell death. One of these repressed genes was Bnip-2, coding for a protein which interacts directly with death-inhibiting Bcl-2 and induced apoptosis via a caspase-dependent mechanism (Zhang et al., 2002). The other repressor calpain-2 is an intracellular protease that was demonstrated to promote decrease of Bcl-2 proteins by cleavage and thereby triggers the intrinsic apoptotic pathway (Gil-Parrado et al., 2002). Proapoptotic effect of Ddit3 (=Gadd153) was linked to down regulation of Bcl-2 and enhanced oxidant injury (McCullough et al., 2001). Furthermore, the tumor suppressor Lats2 was down regulated which was reported to induce apoptosis in lung cancer cells through decrease of the protein level of anti-apoptotic proteins BCL-2 and BCL-x(L) and activation of caspase 9-cascade (Ke et al., 2004). At last, expression of the tumor suppressor Anp32a (=PHAP) was reduced which promotes caspase-9 activation after apoptosome formation (Jiang et al., 2003). Markedly, some of these proteins with regulatory role in apoptosis, have other functions that can also contribute to cell proliferation and motility. For example, Birc5 is implicated not only in anti-apoptosis but also in cytokinesis by stimulating Aurora-B kinase activity (Chen et al., 2003a), Lats2 inhibits cell cycle by controlling different check points (s. above). Anp-32 is involved in repression of transcription as part of the INHAT (inhibitor of histone acetyltransferases) complex (Seo et al., 2002) and calpain-2 plays a role in cell migration through regulation of membrane activity and morphology (Franco et al., 2004).
In summary, the gene expression changes discussed above indicate essential molecular events that allowed c-Myc-overexpressing tumor cells to suppress intracellular control for cell division.
The Presence of c-Myc-Binding Sites in Promoters of Deregulated Gene
C-Myc binding sites were not always identified in genes whose expression was altered in response to c-Myc over expression. Likely, some genes changed their expression as a result of regulation by other transcriptions factors targeted by c-Myc (see Table 2a,b). It was of no surprise that most of the down regulated genes did not contain c-Myc-binding sites in their promoters. Indeed repression of gene transcription by c-Myc is thought to occur independently of binding to the E-box, but is driven by interaction of c-Myc with other transcription factors, notably Miz-1, which recognizes other regulatory sequences (Wanzel et al., 2003). Besides, the repression of several transcription factors by c-Myc, some of them, E-boxes containing, may be one of the reasons for the remarkable large number of down regulated genes in c-Myc-induced lung tumors.
Unexpectedly, a small number of genes known to bind c-Myc directly, e.g. cyclin B1, Cdc2a, Cks2, did not contain c-Myc recognition sites in the bioinformatic analyses. This requires further consideration. First, the selected promoter region was restricted to 2000 bp upstream and 100 bp downstream of the putative transcription start site. It is possible that some c-Myc binding sites are positioned outside this region. Second, the matrices similarity cut-offs were set stringently so that false positive matches would be less than 1 per 10,000 bp. On the other side, such cut-off enables recognition of only 10-30% of putative sites. Finally, for human E-box containing genes a distribution into two groups with low and high affinity binding for c-Myc has been reported (Fernandez et al., 2003). Remarkably, overexpression of c-Myc enhanced its association with low affinity E-boxes and at very high level to non targeted chromatin. Possible, c-Myc overexpression of >50-fold, as achieved in the study, resulted in perturbation of transcriptional control and extended activity of c-Myc beyond E-box containing promoters. The consequence may be up regulation of such genes as Birc5, Ect2 and others found to be uniquely expressed in lung tumors.
Orthologs Regulated in Human Malignancies.
A number of genes regulated in c-Myc-induced papillary lung adenocarcinomas that contribute to cell cycle progression, growth and block of apoptosis, i.e. Ccnd1, Stk6, Klf-4, Gas1, Hey1 and Stat-1 were similarly regulated in various human malignancies (He et al., 2004; Anand et al., 2003; Chen et al., 2003a). Apart from remarkable agreement in histological phenotypes the disease model mimic closely genetic events in human lung cancer. Therefore, its significance in the study of lung tumor development is emphasized. Specifically, altered expression of some genes in lung tumors of the mouse model, were identified in human NSCLC as well. This included overexpression of CDK4, CDC2A, TFDP1, CCNB1, CDC20 (Singhal et al., 2003), and contributed to promotion of cell cycle. In addition, overexpression of anti-apoptotic BIRC5 (Escuin and Rosell, 1999) and heat shock protein HSP70 (Volm et al., 1995) as well as repression of the tumor suppressor LATS2 (Li et al., 2003) were also found in human lung cancer. Up regulation of Cdk4 and down regulation of pro-apoptotic Bnip2 were likewise detected in chemically induced murine lung tumors (Bonner et al., 2004). These changes in gene expression point to similarities in the development of lung adenocarcinoma in mouse and human malignancies and demonstrate the relevance of the c-Myc-induced lung tumor model for investigating human NSCLCs.
To the best of our knowledge, some genes identified in the study were not previously associated with human lung carcinogenesis like the newly identified c-Myc responsive genes Hey-1, Anp32a, Bnip-2, calpain-2, amongst others. Their altered expression may lead to inhibition of apoptosis and these genes represent interesting candidates for further validation as therapeutic targets.
Central Role of c-Myc in Activation of Cell Metabolism
Previously, changes in gene expression induced by targeted c-Myc-overexpression in lung epithelium were reported to result in an activation of genes coding for cell cycle promotion and inhibition of cell cycle arrest and death. Here the oncogenomics associated with different metabolic pathways are presented to enable rapid growth and proliferation of tumor cells. Notably, induction of genes coding for DNA and RNA metabolism, protein synthesis, ribosomal biogenesis, glycolysis and transport in c-Myc induced lung tumors was observed (see Table 4). The ability of c-Myc to control transcription of genes involved in both cell cycle progression and growth is in agreement with other studies (Lutz et al., 2002). A significant number (53,7%) of identified genes are known to be c-Myc responsive in other cell types (
Metabolic Genes Activated by c-Myc Contributes to Regulation of Cell Proliferation
Recent data provided evidence that several c-Myc target genes which were found to be upregulated in lung adenocarcinoma code for metabolic enzymes and may contribute otherwise to cell proliferation. For example, thymidilate synthase which maintains the dTMP pool critical for DNA replication functions also as an RNA binding protein. It forms a complex with a number of cellular mRNAs, including the RNA corresponding p53 tumor suppressor gene, to cause translational repression (Liu et al., 2002), and, in such a way, may regulate cell cycle and apoptosis. Furthermore, fatty acid synthase (Fasn) is a key metabolic enzyme catalyzing the synthesis of long-chain saturated fatty acids and plays a central role in the production of surfactant in lung. On the other hand overexpression of Fasn has been linked to dysregulation of membrane composition and modulation of lipid rafts functioning in tumor cells. Lipid rafts are membrane microdomains involved in signal transduction, intracellular trafficing and cell migration (Swinnen et al., 2003). The Fasn is expressed at high level in numerous human malignancies (Evert et al., 2005), and it was found that the ability of flavonoids to induce apoptosis in cancer cells is strongly associated with their properties to inhibit Fasn (Brusselmans et al., 2005). In addition, nucleophosmin (Npm1) is a nucleolar phosphoprotein which functions as a molecular chaperone and plays an important role in ribosomal protein assembly and transport through prevention of proteins from aggregating in the nucleolus. Besides, it inhibits apoptosis by suppression of the protein kinase PKR. This kinase effects apoptosis in normal cells in response to various extra and intracellular cues (Pang et al., 2003). Up regulation by c-Myc of spermidine synthase (Srm) in lung tumor cells could lead to elevated intracellular level of polyamines which are substantial for cell proliferation. Polyamines are not only of critical importance for protein biosynthesis, membrane stability, chromatin structure or modulation of ion channels but they have been shown in breast cancer cells to regulate the interaction of transcription factors such as NFkB with their responsive elements, thereby leading to an up regulation of genes involved in proliferation (Shah et al., 2001). In the case of Fas, Npm1 and Srm multiple (2,3 and 5, respectively) c-Myc-binding sites were found in the promoter region (Table 5). This suggests direct up regulation of these genes by c-Myc in lung adenocarcinomas. Unlike normal cells many tumors rely entirely on glycolysis for ATP production even in a not hypoxic environment, known as the Warburg effect (Garber, 2004). Though being less efficient, glycolysis provides ATP quickly which is needed for the many metabolic processes associated with rapid tumor growth. Note, several glycolytic enzymes were over expressed in lung adenocarcinoma and contained c-Myc-recognition sites in their promoters or were previously shown to bind c-Myc directly (see Table 1). This clearly demonstrates a direct role of c-Myc in the activation of glycolysis (Table 4). It is highly interesting that glycolytic enzymes, similarly to other metabolic enzymes discussed above, have been shown to have additional functions in cell proliferation. For example, activity of hexokinase 2 associated with the outer membrane of mitochondria, has been implicated in protection from apoptosis (Garber, 2004; Majewski et al., 2004). Gpi1/AMP (glucose phosphate isomerase 1/autocrine motility factor), a ubiquitous cytosolic enzyme that plays a key role in glycolysis, is a multifunctional protein that acts in the extracellular milieu as a potent mitogen/cytokine. Indeed, its over expression contributes to motility, invasion and metastasis (Garber, 2004; Tsutsumi et al., 2004) and was correlated with aggressive tumor growth and poor prognosis in human pulmonary adenocarcinomas (Garber, 2004; Takanami et al., 1998). These examples demonstrate how expression of metabolic genes induced by c-Myc contribute to promotion of cell proliferation in lung tumorigenesis.
Remarkably, 9 genes coding for enzymes involved in purine and pyrimidine nucleotide synthesis pathways (Tk1, Tyms, Shtm1, Srm, Impdh2, Gart, Uck2, Rrm2, Rrm1) were identified as well as folate transporter Slc19a1 which were found to be over expressed in lung adenocarcinomas and are essential for rapid cell proliferation. Indeed, inhibition of thymidilate syntase by 5-fluorouracil is an established therapy against certain tumors. Notably, Shmt1, both mRNA (Table 4) and protein (
Altered expression of several genes coding for proteins important in chromatin remodeling.
The increased expression of linker histone H1fx and repression of core histone H2b1 could change the structure of nucleosome and access to nucleosomal DNA for gene expression, DNA replication and repair. Besides, increased expression of genes coding for helicase and Smarcc1 was found. Both have c-Myc-binding sites in their promoters and are involved in all processes including DNA strand separation like gene transcription, DNA replication, recombination and repair. Satb1 and Anp32a, which are found to be repressed, are involved in repression of gene transcription through interfering with histone acetylation (Seo et al., 2002). Altered expression of such genes could lead to remodeling of large chromatin domains and initiate undue-expression of numerous genes in c-Myc-overexpressing cells. In addition, elevated level of the SNRP transcript, the gene coding for a protein involved in RNA editing (the evolutionary conserved mechanism for regulation of gene expression at the posttranscriptional level that plays an essential role in development) may prove to be of great importance for lung carcinogenesis. These genes were identified as new c-Myc responsive genes and first demonstrate their regulation in cancer (except for helicase which was linked to cancer previously).
Orthologues Regulated in Human Malignancies.
Several genes regulated in c-Myc-induced lung adenocarcinomas contribute to cell growth e.g. Hk2, Fasn, Uck2, Impdh2, Tk1 and were shown previously to be similarly regulated in human malignancies (Shen et al., 1998; Swinnen et al., 2003; Goel et al., 2003; Natsumeda et al., 1990). Strikingly, altered expression of some of these orthologues were specifically identified in human NSCLC. This included induction of RRM2 and TOP2A transcript expression with defined roles in DNA synthesis and transcription (Wikman et al., 2002), as well as overexpression of ARG1 which fosters polyamine synthesis (Suer et al., 1999). High activity of TYMS activity and GPI1 expression was associated with poor prognosis of patients with NSCLC (Nakagawa et al., 2004; Takanami et al., 1998) as was exaggerated activity of LDH with tumor stage (Rotenberg et al., 1988). Clearly, this demonstrates the relevance of the findings obtained in transgenic mice for human NSCLCs, as described herein. To the best of our knowledge, certain genes identified in the study were not previously associated with human lung carcinogenesis and these included the c-Myc targets Srm, Shmt1, Npm1, Slc19a1 as well as the newly identified c-Myc responsive genes Satb1 amongst others. Their specific regulation represents new and important therapeutic targets worthy for further exploration to combat this highly malignant disease.
In the effort to better understand the transforming capacity of c-Myc in lung adenocarcinomas firstly gene networks directly involved in cell cycle progression and cell growth as well as regulation of cell cycle arrest and programmed cell death were examined. As intrinsic programs to regulate cell proliferation, growth and cell death are also governed by extracellular signaling, regulation of genes coding for proteins involved in the main pro- and anti-growth extracellular signaling pathways was studied.
In the following, the consequences of c-myc overexpression on extracellular signaling, angiogenesis and invasion are discussed.
Incapacitating of Extrinsic Pathways of Growth-Inhibitory Signaling
Decrease of Cell Death Receptor Signaling
In lung tumors gene expression changes were identified that could have a negative impact on the death receptor signaling, for example, decrease in expression of the death receptor protein Tnfrsf6 (FAS) and up regulation of uniquely expressed gene transcripts in tumors, such as Traf 4 which codes for a putative signal-transducing protein and was shown to inhibit Fas-induced apoptosis in transformed embryonic kidney cells (Fleckenstein et al., 2003). Both Tnfrsf6 and Traf 4 possess c-Myc-binding site in their promoters, and presumably are directly regulated by c-Myc. Besides, in lung tumors decreased expression of proapoptic transcription factor Hoxa5 was found which was shown to sensitize cells more than 100-fold to TNF-alpha-induced death mediated by caspase 2 and 8 (Chen et al., 2004). Expression of this transcription factor led to apoptotic death in breast tumor cells and was lost in the majority of breast tumors (Raman et al., 2000). The expression of these genes could make lung epithelial cells resistant to apoptosis induced by cytokines like FasL and TNFalpha.
Evading the Tgf-Beta Growth-Suppression Signaling
Gene expression of several components of the Tgf-beta (TGFb) signaling pathway was significantly repressed in c-Myc-induced lung adenocarcinomas. Members of the TGFb superfamily, including TGFb and BMPs are important extracellular signaling proteins which inhibit proliferation of several cell types, either by blocking cell cycle progression through G1 or by stimulating apoptosis. Down regulated genes in lung tumors of transgenic mice included genes coding for type I receptor for TGFb superfamily of ligands Acvrl1 and a Tgfb coreceptor endoglin as well as the ligand molecules Tgfb1 and Bmp6. Notable, gene expression of Bmp6, a new putative c-Myc target, was lost in all lung tumor tissues of transgenic mice. Additionally, expression of dermatopontin was completely abrogated in lung tumors. This gene codes for a component of extracellular matrix that increases the cellular response to TGF-beta (Okamoto et al., 1999). Besides, significant decrease in expression of transcription factor Zfhx1a that is crucial regulator of TGFb/BMP signaling through synergizing with SMAD to induce growth arrest (Postigo et al., 2003) was detected. Additionally, an immediate early response gene for TGFb inhibitory growth factors Gadd45b, which functions as a positive effector of Tgfb-induced apoptosis (Yoo et al., 2003), was found to be repressed in tumors of c-Myc transgenic mice. Gadd45b is a likely c-Myc target gene based on the c-Myc recognition site which was identified in its promoter. A strong repression of Gadd45 was also observed in RAT-1 overexpressing c-Myc cells (Amundson et al., 1998) and therefore agrees well with the finding as described herein. The inhibited expression of discussed genes could likely disrupt growth-inhibitory signaling by Tgfb family proteins in lung tumor tissues.
Promotion of Cell Survival Through IGFR-Signaling Pathway
Apoptosis can be antagonized by exogenous survival factors like insulin-like growth factors (IGF) I/II which bind to cell-surface receptors and activate PI3 kinase-Akt/PKB signaling pathway that keeps the cell death program suppressed. Gene expression changes in c-Myc induced lung adenocarcinomas indicated significant activation of the cell survival signaling pathway. The prominent feature in lung adenocarcinomas was the significant decrease of gene expression levels of three Igf-binding proteins—Igfbp 5, 6, 4 which were expressed at high level in control lungs. These IGFBPS bind to IGFs and regulate its biological availability and function. Besides, they exert also IGF-independent growth-inhibitory effects (Clemmons, 1998). Thus, decline of the level of the IGFBPs could provide for a local excess of available IGFs in tumor tissue and thereby contribute to antiapoptotic signaling through IGFR. It is of considerable importance that amongst several growth factors tested only IGFs proved to be survival factors for c-Myc-over expressing fibroblasts (Harrington et al., 1994). Furthermore, repression of additional genes involved in negative regulation of IGF1R signaling pathway was found. These were Socs2 coding for a protein which interacts with IGF1R and suppresses IGF1-induced growth (Dey et al., 1998) and Nfkbia, which inhibited NFkB (a transcription factor activated by PI3K-Akt signaling) by trapping it in the cytoplasm. Activation of IGFR-mediated survival signaling through inhibition of expression of several negative regulators of the pathway could antagonize c-Myc-mediated apoptosis and allow proliferation and growth of c-Myc-overexpressing lung epithelial cells without concomitant apoptosis.
Activation of Growth Factor Signaling Through Tyrosine Kinase Receptors and Related Genes
Many of the regulated genes identified in c-Myc-induced lung adenocarcinomas coded for molecules involved in the main cell signaling pathways with a positive effect on cell division and growth. A key role in extracellular stimulation of cell growth and proliferation belongs to the signaling through EGFR (epidermal growth factor receptor). One of the up regulated genes in lung adenocarcinomas of c-Myc transgenic mice coded for amphiregulin (AR), an autocrine growth factor of the EGF family. AR was shown to stimulate proliferation of human lung epithelial cells through binding to EGFR (Lemjabbar et al., 2003) as well as survival of the human NSCLC cells through activation of IGF1R-dependent pathway (Hurbin et al., 2002). Besides, AR was demonstrated to be an essential mediator of G-protein-coupled receptor (GPCR)-induced transactivation of EGFR signal and down stream cell response in squamous cell carcinoma. In turn, the secretion of AR could be induced through an activation of the EGFR-signaling pathway in bronchiolar epithelial cells (Blanchet et al., 2004) and IGF1R in NSCLC (Hurbin et al., 2002). These findings suggest that AR growth factor may play an important role in lung cancer development and, like in human cells, increased AR expression in c-Myc-induced lung tumors may resulted in stimulation of autocrine growth and suppression of apoptosis.
One further component of the EGFR/Ras/Raf/Mek/Erk-signaling cascade found to be up regulated in lung tumors was Map2k1 (Mek) while Rassf5, the pro-apoptic Ras effector, was down regulated, therefore evidencing the suppression of Ras-mediated apoptotic signaling. Notable, Rassf5 was not expressed in 80% of human lung adenocarcinomas and was specifically repressed in lung epithelial tumor cells (Vos et al., 2003). Additionally, among down regulated genes in lung adenocarcinomas several phosphatases like Dusp1, Ptpre, Ptpns1, Ptprb and Ptprg were identified. Likely, specific dephosphorylation of signaling proteins such as MAPKs negatively regulate receptor tyrosine kinase-coupled signaling processes. Notably, the phosphatases Ptprb and Ptprg excert growth inhibitory effect (Gaits et al., 1995); (Liu et al., 2004), and expression of Ptprb was dramatically decreased expression of Ptprb in human lung adenocarcinomas (Gaits et al., 1994).
Among uniquely expressed genes in c-Myc-induced tumors the proto-oncogene Ros1 coding for a transmembrane tyrosine kinase which may function as a growth factor receptor (Keilhack et al., 2001 was identified).
Another gene uniquely expressed in tumors coded for the HGF activator, a secreted protease that converts precursor of HGF in its active form which functions as a potent mitogenic factor for epithelial cells and was found to induce angiogenesis (Sengupta et al., 2003). Up regulation of HGFac was also observed in human lung adenocarcinomas (McDoniels-Silvers et al., 2002). The unique expression of these genes which in tumorous lungs may be essential for promotion of lung carcinogenesis. Besides, up regulation of Adam19, a member of a disintegrin and metalloprotease family that is involved in signal transduction through processing of transmembrane growth factor precursors (Fischer et al., 2003) may foster activation of EGFR-signaling in lung tumors of transgenic mice. Presence of c-Myc-binding sites in the promoters of Adam19, Dusp 1 and Map2k1 likely indicates their direct regulation by c-Myc.
Activation of Wnt/Beta-Catenin Signaling
The study of differently expressed genes in lung tumors of transgenic mice provide evidence for Wnt signaling to be affected in c-Myc-induced lung adenocarcinomas. In tumor tissues enhanced expression of Arhu, a member of the Rho family that mediates in part the effects of Wnt1 signaling in the regulation of cell morphology, cytoskeleton organization and cell proliferation (Tao et al., 2001) was found. Also, unique expression of Frat2, a positive regulator of Wnt signaling and stabilizer of beta-catenin (Saitoh and Katoh, 2001) was observed. In contrast, repressed expression of the transcription factors Tcf7 and Sox7, which are negative regulators of Wnt/beta-catenin-induced gene transcription, was observed. Down regulation of these genes was shown to be associated with malignant transformations (Roose et al., 1999). Modulation of Wnt signaling in c-Myc transgenic mice contributed to promotion of cell proliferation, cytoskeleton dynamic and motility of tumor cells. It is of considerable importance that c-Myc-binding sites were found in promoters of Arhu and Tcf7, the latter was shown to be a c-Myc target gene, therefore linking c-Myc directly to the activation of Wnt-signaling pathway.
Regulation of Cytoskeleton Contributing to Transformation, Increased Motility and Invasiveness
The ability of malignant cells to migrate and invade surrounding tissues is associated with changed motility based on modifications of the cytoskeleton. A central role in rearrangement of the actin cytoskeleton in response to diverse external signals is ascribed to members of the Rho protein family. Many genes involved in regulation of cytoskeleton and transformation were found to be differentially expressed. These included the strongly up regulated proto-oncogene Ect2, a growth-regulatory molecule, which, in addition to its ability to regulate cell cycle, activates as a guanine nucleotide exchange factor the Rho family of Ras-related GTPases to bring about remodelling of actin cytoskeleton and to contribute to cell transformation (Saito et al., 2004); (Westwick et al., 1998).
In lung tumors the loss of expression of invasion suppressor Vsnl1, which was shown to decrease RhoA activity and MMP-9 level through cAMP-mediated signaling (Mahloogi et al., 2003), may enhance the malignant phenotype. Down regulation of calpain-2, an intracellular protease required for proteolysis of the cytoskeleton and focal adhesion proteins was shown to restrict membrane protrusions and lamellipodia dynamics (Franco et al., 2004), to further strengthen promigratoric capacity of lung tumor cells.
Changes in Cell Junctions and Adhesion: Increased Motility and Invasiveness
Cell-cell and cell-extracellular matrix (ECM) junctions do not only bind cells mechanically through adhesion but also transmit signals to the cell interior thereby playing an important role in regulation of cell survival, proliferation and migration.
For example, up regulation of the c-Myc target gene mucin1, a transmembrane glycoprotein defined as a tumor antigen (VanLith et al., 2002) that has been implicated in activation of Wnt/beta-catenin-signaling (Huang et al., 2003) was observed in lung tumors of transgenic mice. Over expression of mucin1 was shown to be involved in invasive tumorigenesis in the breast (Schroeder et al., 2003) and colon (Baldus et al., 2004). The other c-Myc target gene Icam1 (intercellular adhesion molecule1) which is the only known direct ligand of the mucin extracellular domain was also over expressed in c-Myc-induced lung tumors. It was shown that mucin1, on the contact with Icam1 produced calcium-based signals that might affect the cytoskeleton and increased cell motility (Rahn et al., 2004). In contrast, strong decrease in expression of genes coding for cell-cell junction proteins like cadherin-5, protocadherins alpha 4 and 6 may contribute to cell proliferation and migration in lung tumor. Additionally, in lung adenocarcinomas of transgenic mice repression of several genes coding for proteins implicated in cell-ECM interactions was observed. These included fibulin-1, an extracellular matrix (ECM) protein which affect the Erk-mediated signaling transduction cascades to inhibit motility and invasiveness of cancer cells (Twal et al., 2001), and Lama3 (laminin 5), a ECM glycoprotein shown to be repressed in various human cancers (Sathyanarayana et al., 2003). The decreased expression of matrix receptor alpha8integrin which negatively affects cell migration and proliferation (Bieritz et al., 2003) could therefore contribute to advanced malignant phenotype of lung tumors.
Tumor specific regulation of genes involved in cell adhesion may impact control of cell growth through changes in cell-cell and cell-matrix attachment to foster increased cell proliferation and motility in lung tumor tissue.
Degradation of ECM
Based on the histopathology of PLACs an invasive nature of the lung tumor was evidenced. Note, proteolysis of matrix proteins is essential for cell migration. Matrix components are degraded by extracellular proteolytic enzymes, most of which are matrix metalloproteinases while others are serine proteases. In lung tumors up regulation of gelatinase-associated protein lipocalin 2, a regulator of activity of matrix metalloproteinase (MMP) was found, which was suggested to be involved in invasion of tumor cells (Liu et al., 2003; Huang et al., 2003) Additionally, repressed expression of the metastasis suppressor Reck was observed (Liu et al., 2003) which suppresses invasiveness of carcinoma cells through inhibition of the release of MMP. Likewise, upregulation of the metalloproteinase Adam 19 impacts EGFR signaling as discussed above. Furthermore, the known c-Myc responsive gene Thop1 was significantly overexpressed in tumor tissues of transgenic mice. This gene codes for a member of the metalloendopeptidase family but was not as yet linked to cancer. Among serine proteases over expressed in c-Myc-induced lung tumors the cell surface protease hepsin was found over expressed. This protein was demonstrated to cause disorganization of basement membrane and promote progression of metastasis in a mouse prostate cancer model (Klezovitch et al., 2004). The other serine protease, i.e. HGF activator, a serine protease specific for activation of HGF (Tjin et al., 2004) with key roles in invasion and metastasis of cancer (Hurle et al., 2005), was upregulated as well. At last, gene transcription of extracellular enzyme lysyl oxidase (Lox) indicated an inhibition of cross-linking of collagen and elastin in ECM that makes the matrix fragile and prone to tear. Mutations or reduction of the LOX transcript level in human cancer suggests this protein may excert tumor suppressive activity (Rost et al., 2003).
Gap Junctions
Over expression of connexins 43 and 31 in lung tumors revealed an unsuspected role for c-Myc in cell-cell communication.
Note, there connexins contain c-Myc binding sites in their promoters as evidenced by in silico annotations. These proteins form intercellular channels, which allow the exchange of small molecules (such as inorganic ions, sugars, nucleotides, intracellular mediators like cyclic AMP and IP3) among coupled cells. Connexin43 is essential for survival, and its expression was altered in malignant tissues and in the presence of carcinogenic factors. Besides, it was reported that connexins can be induced through Ras-signaling (Carystinos et al., 2003). The consequences of perturbed connexin 31 expression on cell proliferation (Kibschull et al., 2004) and cell death (Di et al., 2002) were discussed elsewhere.
Tight Junctions
Tight junctions seal epithelial cells together and function as selective permeability barriers. They are formed exclusively by integral membrane proteins of the claudins family. Their role in cancer is not well understood. High level of claudin-5 gene expression was observed in control lung which was progressively suppressed and eventually absent in large tumors. As a consequence loosening of intercellular adhesion and increased motility can be expected. Inversely, gene expression of claudin 2, absent in normal lungs, was strongly induced in tumors, probably, due to the activation of Wnt signaling (Mankertz et al., 2004). Claudin 2 and some other claudin family members were shown to be involved in activation of proMMP through recruitment of enzymes on the cell surface (Miyamori et al., 2001) and so may contribute to invasion.
Proangiogenic Signaling
Some of the differently expressed genes in lung adenocarcinomas of transgenic mice can be linked to angiogenesis. Indeed, a remarkable up regulation of kininogen, a haemostatic factor that exerts its angiogenic effect (Colman et al., 2003) through signaling of its cleaved product form bradykinin through the B (2) receptor, was observed (Yang et al., 2003). Increased transcription level of G-protein-coupled receptor Adora2b detected in tumor tissues might additionally contribute to a stimulation of angiogenesis because it was shown to mediate secretion of angiogenic factors like VEGF and IL-8 (Feoktistov et al., 2003). Furthermore, in all lung tumors the loss of expression of Sema3f, a secreted signaling molecule from the immunoglobulin family with antagonistic action towards Vegf due to competitive binding to neuropilin1/2 receptors (Nasarre et al., 2003), was observed. Sema3f was demonstrated to inhibit angiogenesis (Kessler et al., 2004), metastasis (Bielenberg et al., 2004) and motility of primary tumor cells (Nasarre et al., 2003). Decreased expression of Sema3f correlated with advanced stage and more aggressive type of human NSCLC (Roche and Drabkin, 2001) that indicates its important role in lung carcinogenesis. At last, up regulation of Hgfac in c-Myc-induced tumors could result in activation of HGF, whose multiple functions include also a potent angiogenic capacity.
From the study the inferred link of c-Myc to angiogenesis is in agreement with an observation that—c-Myc had a potent angiogenic capacity. This was shown in skin (Pelengaris et al., 1999), pancreatic β-cells (Pelengaris et al., 2002) and lymphoma (Brandvold et al., 2000). The reversible activation of conditional alleles of c-Myc in these studies induced hyperproliferation, dedifferentiation and angiogenesis in a reversible manner, suggesting that these complex events may be directly affected by c-Myc.
Deregulation of Signaling Pathways Through Scaffold Proteins
Gene expression changes discussed above link c-Myc to the regulation of the main intra- and extracellular signaling pathways. To this question, in lung adenocarcinomas of c-Myc-transgenic mice significantly decreased expression of several scaffolding proteins involved in spatiotemporal regulation of signaling pathways was found. They included two known c-Myc responsive genes caveolin1 and Akap12 (gravin), both proteins being frequently absent in a number of carcinomas (Williams et al., 2003), including lung cancer (Wikman et al., 2002). Caveolin-1 is the principal structural component of caveolae—the sites of membrane responsible for concentrating an array of signaling molecules including growth factor receptors and G-proteins as well as calcium channels and pumps, and thought to be a major regulator of signaling complexes in caveolae. The loss of caveolin-1 was demonstrated to confer a significant growth advantage to mammary epithelial cells in vitro and in vivo (Williams et al., 2004). Decreased expression of the c-Myc responsive genes caveolin-1 together with overexpression of FAS, points to an important role of derangement of the membrane rafts in the malignant transformation induced by c-Myc. The transformation suppressor potential of Akap 12 seems to be based on its ability to reorganize actin-based cytoskeleton architecture and control G1 phase signaling proteins (Gelman, 2002). Scaffold proteins guide the interactions between successive components of signaling complexes to relay the signal with precision, speed and efficiency and to avoid an unwanted cross-talk between signaling pathways therefore providing for a specificity of cell response to many different signals. The loss of such spatiotemporal regulators of signaling processes in response to c-Myc could likely resulted in an amplification and spreading of proliferative signals in lung epithelial cells thereby accelerating malignant transformation.
2. Some genes identified in this study were known to be similarly changed in human malignancies and specifically in lung tumors which makes the SPC/c-Myc-transgenic mouse model useful for investigating human lung cancer.
3. Many other genes differently expressed in lung tumors are novel targets for mechanism based therapies and serve as useful tumor markers.
4. New c-Myc-responsive genes and putative c-Myc-targets identified in this study contributed to the collective examination of distinct c-Myc transcriptomes that are partly cell type- and species-specific and therefore represent one more important result of this study.
c-Myc is a transcription factor and frequently overexpressed in diverse malignancies. It's targeted overexpression (>50-fold) in mouse alveolar epithelium induced lung cancer. To identify new myc target genes and to explore the molecular basis of c-Myc transforming capacity were investigated genome wide regulation of genes in histologically well defined tumors. Essentially, expression of 463 genes differed significantly when compared with healthy non-transgenic lung. Then promoters of regulated genes for c-Myc recognition sites to link c-Myc binding to gene expression were annotated and interrogated. Notably, many of the genes could be traced back to regulators of cell cycle and included c-Myc targets, e.g. Cdk4, Ccnb1, Cks2 in addition to c-Myc responsive genes, e.g. Tfdp1, Cdc2a, Stk6, Nek 6, Prc1, Ect2. Furthermore, Several genes whose regulation in tumors interfere with the p53- and Bcl2-mediated cell cycle arrest and cell death program were identified, notably Birc5, KLf4, Stat1, Ddit3, Lats2 therefore providing a selective advantage for tumor growth. There was considerable agreement among the regulation of c-Myc target genes in mouse and human lung malignancies. Notably, targeting new lung cancer genes i.e. Prc1, Klt4, Ect2, Hey-1, Gas-1, Bnip-2, calpain-2 and Anp32a may enable mechanism based therapies.
The c-Myc transcription factor regulates a wide set of genes in response to mitogenic stimuli. Its targeted overexpression in alveolar epithelial cells led to malignant transformation and development of papillary lung adenocarcinomas (PLAC) in mice. In the first part of the study cell cycle and apoptosis networks were investigated in papillary adenocarcinomas. Now regulation of genes coding for DNA and RNA metabolism and processing, ribosomal biogenesis and protein biosynthesis, nuclear and cellular transport, glycolysis, lipid metabolism and chromatin remodeling is reported. The c-Myc target genes Tyms, Tk, Srm, Fasn and the c-Myc-responsive genes Hells, Rpo1-3, Gpi1, Impdh were found to be significantly induced. Their regulation in PLAC could contribute to an accelerated cell proliferation and growth. Notably, increased expression of some orthologues in human lung carcinomas was observed as well. In addition, bioinformatics to search for c-Myc-binding sites in promoters of regulated genes were applied thereby identifying new c-Myc targets; these included, amongst others, Uck2, Smarcc1, Npm3, Nol5, Lamr1, next to the well established c-Myc targets Srm, Shmt1, Npm1, Slc19a1. These newly identified lung cancer genes are interesting targets for the development of novel antineoplastic therapies.
To better understand the molecular basis of c-Myc transforming capacity genome-wide expression analysis of lung adenocarcinomas in a transgenic mouse model was performed. In part 1 and 2 of the study regulatory gene networks associated with cell cycle and apoptosis as well as tumor growth and metabolism was reported.
Here it was focussed on regulated genes involved in extracellular signaling pathways, e.g. activation of EGFR-mediated mitogenic, IGFR-mediated survival and Wnt-signaling as well as disruption of Fas-mediated death and anti-mitogenic Tgf-β-signaling. Additionally, altered gene transcription associated with cell adhesion, cell-cell junctions, regulation of cytoskeleton, proangiogenic signaling and invasion provided a molecular rationale for c-Myc invasive and angiogenic capacities. An identification of novel c-Myc target genes associated with lung tumors (e.g., Rbp1, Reck) along with known c-Myc-responsive or c-Myc-target genes (e.g., Akap12, Cav1, Hoxa5, Lox, Muc1) and genes linked to lung cancer (e.g., Rassf5, Hgfac, Sema3f, Ptprb), provide highly interesting targets for mechanism based therapies.
Materials and Methods
Maintenance of the Transgenic Mouse Line
SPC/myc transgenic mice (Ehrhardt et al., 2001) were maintained as hemizygotes in the CD2F1-(DBA/2xBalb/C) background and identified by PCR of DNA extracted from tail biopsies (Hogan et al., 1994) (
Sample Collection and Preparation
Mice were anasthesized by an overdose of CO2 at the age of 9-13 months. The tumors were inspected macroscopically, separated from the surrounding lung tissue of SPC/myc transgenic mice and frozen immediately in liquid nitrogen. The tumors were divided into groups according to size 1 mm, 5 mm and >10 mm. Small tumors were pooled in three groups of 3, 3 and 4 separate tumors because of the low yield of RNA. Normal lung from four non transgenic mice of about the same age were used as a control.
Histology
For histopathology tissue specimen of 5 transgenic animals and 2 non-transgenic controls at the age of 9 to 13 months were investigated. The specimens were rinsed in PBS and fixed in 4% PBS-buffered formaldehyde, and processed for paraffin embedding as usual. 5 μm thick serial sections were stained with hematoxylin and eosin (H and E), hematoxylin only (H) and PAS for light microscopic evaluation.
Isolation of RNA, Production of c-RNA, Array Hybridization and Scanning.
The cRNA-samples were prepared following the Affymetrix Gene Chip® Expression Analysis Technical Manual (Santa Clara, Calif.). 10 μg of biotinylated fragmented cRNA was hybridized onto the Murine Genome U74Av2 Array (MG-U74Av2)—a GeneChip® expression oligonucleotide probe array from Affymetrix which contains 12 488 probe sets, that represents annotated sequences in the Mouse UniGene database as well as EST clones. The procedures for isolation of RNA, production of c-RNA, array hybridization and scanning were done according to Affymetrix's manual and as described (Borlak et al., 2005). Each hybridization image was scaled to all probes set intensity of 250 for comparison between chips.
Gene Expression Data Analysis
The data analysis was performed basically as described earlier (Borlak et al., 2005). For single arrays a statistical expression algorithm within the Affymetrix® Microarray Suite software (version 5) yielded gene expression values as numeric values (signal intensities) and detection calls (“Present” or “Absent”) produced with different algorithms. A comparison between two arrays (tumor and control lung) resulted in the signal logarithm ratio (log 2 ratio) and a change call (“Increase” or “Decrease”) for expression level of each gene. Data from replicate samples were evaluated and compared using statistical analyses with the Affymetrix® Data Mining Tool 2 (DMT-2). To summarize the expression level (the signal values) for each transcript across the replicates were used the average and standard deviation statistics. Mean fold change values were calculated as the ratio of the average expression levels for each gene between two tissue sets. To determine significance of change in mean gene expression level between comparison groups the unpaired two-sided T-Test was used, with the p-value cutoff determined as 0.05. Additionally to parametric T-test, which uses for analysis numeric expression values (signal intensities), comparison ranking analysis was done to study the concordance between “Increase” or “Decrease” calls for tumors in one set. The results are shown as % of concordance between all comparisons, e.g. 16 analyses (4 tumors versus 4 controls) for tumors of median size and 12 (3 tumors versus 4 controls) for tumors of large size. Three pools of small tumors (1 mm) were compared with the 4 individual controls, resulting in 12 comparisons. The group of differently regulated genes in tumors was restricted to those genes, which were detected (“Present” call) in all samples of a tumor sets for the up regulated genes and in all control lungs for the down regulated genes. Further criteria were FC≧3, p-value in T-test ≦0,05 and 100% of “Increase” call in comparative ranking analysis for one or more sets of tumors versus control lungs for up regulated genes and FC ≦−3, p-value in T-test ≦0,05 and 100% of “Decrease” call accordingly for down regulated genes.
Gene Expression Studies by RT-PCR
The primer 3 software (Rozen S and Skaletsky H J, 2000) was used to design the primers. The amplification fragments spanning an intron were chosen to distinguish between amplification from contaminating DNA.
Total RNA was isolated with the Qiagen RNA purification kit according to the manufacturers instructions. Reverse transcription was carried out using Omniscript (Qiagen), Oligo-dT primers (InVitrogen) and RNasin (Promega) followed by PCR amplification (see above) with the following primer pairs: Ccnb1, fp: CAGTTGTGTGCCCAAGAAGA (SEQ ID NO 3); rp: TCCATTCACCGTTGTCAAGA (SEQ ID NO 4) (57°, 32 cycles). Cdc2a,fp: CTCGGCTCGTTACTCCACTCGAGCATCAAGAAAGAGGTCAAAGG (SEQ ID NO 5);rp: CCATTTTGCCAGAGATTCGT (SEQ ID NO 6) (56° C., 34 cycles). Stk6:fp:GCCCACTAGGAAAAGGGAAG (SEQ ID NO 7). rp: CGTTTGCCAACTCAGTGATG (SEQ ID NO 8) (56° C., 34 cycles). Cdk4, fp: AACTGATCGGGACATCAAGG (SEQ ID NO 9), rp:CACGGGTGTTGCGTATGTA (SEQ ID NO 10) (58° C., 30 cycles). Nek6, fp:TTGAGATGATGGATGCCAAA (SEQ ID NO 11), rp: AGCTGTGATGAACACGTTGG (SEQ ID NO 12) (56° C., 32 cycles). Prc1, fp: CATGATGCCGAGATTGTACG (SEQ ID NO 13), rp: CAGCCGATGTAATTCCCACT (SEQ ID NO 14) (57° C., 32 cycles). Birc5, fp: GAATCCTGCGTTTGAGTCGT (SEQ ID NO 15), rp:CAGGGGAGTGCTTTCTATGC (SEQ ID NO 16) (56° C., 38 cycles). Ddit3, fp:CTGCCTTTCACCTTGGAGAC (SEQ ID NO 17), rp: GGGCACTGACCACTCTGTTT (SEQ ID NO 18) (58° C., 34 cycles). Satb1 fp: GTGATGGCTCAGTTGCTGAA (SEQ ID NO 19), rp:CATAGCCCGAAGGTTTACCA (SEQ ID NO 20) (57° C., 32 cycles). Hey1 fp:GAGACCATCGAGGTGGAAAA (SEQ ID NO 21), rp: ACCCCAAACTCCGATAGTCC (SEQ ID NO 22) (58° C., 32 cycles); β-actin fp:GGCATTGTTACCAACTGGGACG (SEQ ID NO 23), rp: CTCTTTGATGTCACGCACGATTTC (SEQ ID NO 24) (65° C., 23 cycles). β-actin was used as a housekeeping gene because of the equal expression in all lung samples. PCR reactions were done using Taq Platinum PCR Super-Mix Kit (Invitrogen), and amplification products were separated on 1% agarose gels. A semiquantitative measurement of band intensities was done using Kodak 1D Image Analysis Software. The gene expression values in each samples were normalized to the beta-actin expression and then used to calculate mean expression values for the sets of tumors of various sizes and control lungs. The mean fold changes was computed as a ratio between mean gene expression values for tumor sets and control non transgenic lung as a baseline.
Western Blotting
Frozen lung tumors of about 0.5-1 cm from three different SPC/c-Myc transgenic mice and non transgenic lung from three control mice were thawed on ice. Protein extracts were prepared by sonications of the samples in 500 μl 2D-loading buffer with the following incubation with benzonase according to (Molloy et al., 1999) and stored at −80° C. 75 μg (c-Myc) or 100 μg (Hspa9a, Nek6) of total protein extracts were separated on 10% (Hspa9a), or 12.0% SDS-polyacrylamide gel (c-Myc, Nek6) and blotted onto PVDF membranes in 25 mM Tris and 190 mM Glycin at 4° C. for 2 h (10%, 12% gel) at 350 mA. Specific antibodies to detect the selected proteins were anti-c-Myc rabbit polyclonal (1:500), anti-GRP75 (Hspa9a) goat polyclonal (1:200) purchased from Santa Cruz and anti-NEK6 rabbit polyclonal (1:100) purchased from Abgent. Antigen-antibody complexes were visualized using the ECL detection system as recommended by the manufacturer NEN Life Science Products (PerkinElmer Life Science, Rodgau-Juegesheim, Germany) and recorded with Kodak IS 440 CF (Kodak, Biostep GmbH, Jahnsdorf, Germany).
Bioinformatic search for potential c-Myc-binding sites in 5′-UTR region of the differently expressed genes in tumors.
In order to identify c-Myc targets among genes differently regulated in lung adenocarcinoma were applied positional weight matrices (PWMs) that is the most widely used method for recognition of transcription factor binding sites (Quandt et al., 1995) in a bioinformatic analysis of putative promoter sequences of deregulated genes. For that purpose TRANSFAC® Professional rel. 8.3 database was used which contains the largest collection of weight matrices for eukaryotic transcription factors (Wingender et al., 2001). The UCSC Mouse Genome Browser was used to extract the putative promoter regions of the corresponding genes. The beginning of the first exon was considered to be a tentative TSS (transcription start site), and 2000 bp upstream and 100 bp downstream of TSS were extracted. For the analysis of the extracted sequences a search profile which included five PWMs for c-Myc transcription factor with accession numbers M01034, M00322, M00118, M00123, M00615 was applied. The MATCH™ algorithm calculating scores for the matches by using the so-called information vector (Kel et al., 2003) was employed. The matrix similarity cut-offs for the matrices were set to different values (1.0, 0.96, 0.93, 0.98, 0.995 respectively), so that the rate of false positive matches for each matrix was less than 1 per 10,000 bp.
Maintenance of the transgenic mouse line, sample collection and preparation, isolation of RNA, production of c-RNA, array hybridization and scanning, gene expression data analysis, bioinformatic search for c-Myc-binding sites in 5′-UTR region of differently expressed genes in tumors, gene expression studies by RT-PCR and Western blotting were performed as described in the first part of the study.
RT-PCRs were Performed with the Following Primer Pairs (fp;rp):
Shmt1, fp.GCAACTCTGAACCAGTGCAA (SEQ ID NO 25), rp:TGAGGATCCAGATGGTAGGC (SEQ ID NO 26) (56° C., 36 cycles). Satb1, fp:GTGATGGCTCAGTTGCTGAA (SEQ ID NO 27), rp:CATAGCCCGAAGGTTTACCA (SEQ ID NO 28) (57° C., 32 cycles); Arg1, fp: AAGCTGGTCTGCTGGAAAAA (SEQ ID NO 29), rp: CTGGTTGTCAGGGGAGTGTT (SEQ ID NO 30), (55° C., 36 cycles); Fasn, fp: TCTGCAGAGAAGCGAGCATA (SEQ ID NO 31), rp: GTCATTGGCCTCCTCAAAAA (SEQ ID NO 32) (55° C., 31 cycles); Srm, fp: GTCCAGTGCGAGATTGATGA (SEQ ID NO 33), rp: GCAGAAGTGCCTCATCTCCT (SEQ ID NO 34) (57° C., 32 cycles); Hk2, fp: GGTGGAGATGGAGAACCAGA (SEQ ID NO 35), rp: TCATTCACCACAGCCACAAT (SEQ ID NO 36) (55° C., 32 cycles); Tk1, fp: CCAGATCGCCCAGTACAAGT (SEQ ID NO 37), rp: GAAGCACTCCATGCACACAG (SEQ ID NO 38) (59° C., 34 cycles); Impdh2, fp: GGAAGTGGTTCCATCTGCAT (SEQ ID NO 39), rp: TGGCTGCTGAGATGTTTGTC (SEQ ID NO 40) (57° C., 32 cycles); Uck2, fp: CAGATCCCCGTGTACGACTT (SEQ ID NO 41), rp: GTCGGCACCTCTAGGAATGA (SEQ ID NO 42) (59° C., 32 cycles); Smarcc1, fp: TGATCCAAGTCGCTCAGTTG (SEQ ID NO 43), rp: TCACAGCACACACATCTCCA (SEQ ID NO 44) (57° C., 32 cycles); Npm3, fp: GGCTGCTTTAGCGTTCTTGA (SEQ ID NO 45), rp: AAGGTGACAGGTGGTTGGAG (SEQ ID NO 46) (57° C., 33 cycles); Slc19a1, fp: ATGTGCATGTCCTGTGGAGA (SEQ ID NO 47), rp: AGGGAAGACGCAATCTGAAA (SEQ ID NO 48) (55° C., 37 cycles); β-actin was used as a housekeeping gene, because its expression was found to be unchanged in lung tumors of SPC/c-Myc mice (microarray analyses) compared with non-transgenic lungs: fp:GGCATTGTTACCAACTGGGACG (SEQ ID NO 49), rp: CTCTTTGATGTCACGCACGATTTC (SEQ ID NO 50) (65° C., 23 cycles).
Western blotting was done using specific antibodies for selected proteins: anti-Arg1 (1:100), anti-cShtm (1:100), anti-HxkII goat polyclonal (1:100) and anti-Fasn rabbit polyclonal (1:100) antibodies purchased from Santa Cruz Biotechnology, Inc.
Maintenance of the transgenic mouse line, sample collection and preparation, isolation of RNA, production of c-RNA, array hybridization and scanning, gene expression data analysis, bioinformatics search for potential c-Myc-binding sites in 5′-UTR region of the differently expressed genes in tumors, gene expression studies by RT-PCR and Western blot were performed as described.
RT-PCRs were Performed with the Following Primer Pairs:
Ros1, fp: CAGTGGCACACGGTACAATC (SEQ ID NO 51); rp: CTCCGTGAAAGTCCAGCTTC (SEQ ID NO 52) (59°, 36 cycles). Ect2, fp: AGGACCTTCCATTCGAACCT (SEQ ID NO 53). rp: GACTCGGGTGTGTTGGAGAT (SEQ ID NO 54) (58° C., 34 cycles). Traf4: fp: CGGCTTCGACTACAAGTTCC (SEQ ID NO 55). rp: TAGGGCAGGGGACTACATTG (SEQ ID NO 56) (59° C., 34 cycles). Hgfac, fp: GCTTCCTGGGAAATGGTACA (SEQ ID NO 57), rp: CCTCTTGCCACAGGTAGGAC (SEQ ID NO 58) (58° C., 36 cycles). Adam19, fp: TTTACCGCTCCCTGAACATC (SEQ ID NO 59), rp: AGCAGTGTGCAGAATCATGG (SEQ ID NO 60) (57° C., 32 cycles). Bmp6, fp: TTCTTCAAGGTGAGCGAGGT (SEQ ID NO 61), rp: CTCGGGATTCATAAGGTGGA (SEQ ID NO 62) (57° C., 32 cycles). Acvrl1, fp: CCACAACGTGTCTCTGATGC (SEQ ID NO 63), rp: ACACTCTACCAGCGCAACCT (SEQ ID NO 64) (59° C., 32 cycles). Igfbp5, fp: CTACTCCCCCAAGGTCTTCC (SEQ ID NO 65), rp: TTGTCCACACACCAGCAGAT (SEQ ID NO 66) (57° C., 29 cycles). Sema3f, fp: GCTTCCAGCCACACCTAGAG (SEQ ID NO 67), rp: TACAGGTGTGTTCGGTTCCA (SEQ ID NO 68) (57° C., 34 cycles). Cav fp: GGGAACAGGGCAACATCTAC (SEQ ID NO 69), rp: GTGCAGGAAGGAGAGAATGG (SEQ ID NO 70) (59° C., 32 cycles). Cdh5 fp: CAATGACAACTTCCCCGTCT (SEQ ID NO 71), rp: TGTTTTTGCCTGAAGTGCTG (SEQ ID NO 72) (57° C., 29 cycles). β-actin was used as a housekeeping gene, because its expression was found to be unchanged in lung tumours of SPC/c-Myc mice (microarray analyses) compared with controls (non-transgenic lungs. The following primer pair was used: fp:GGCATTGTTACCAACTGGGACG (SEQ ID NO 23), rp: CTCTTTGATGTCACGCACGATTTC (SEQ ID NO 24) (65° C., 23 cycles).
Western blotting was done using specific antibodies for selected proteins: anti-Traf4 (1:100), anti-hepsin (1:100), anti-Alk1 (=Acvrl1) (1:100) goat polyclonal antibodies and anti-Igfbp6 (1:100) rabbit polyclonal antibody purchased from Santa Cruz Biotechnology, Inc., anti-claudin2 (1:500) rabbit polyclonal antibody purchased from Zymed Laboratories Inc., and Adam19 (1:1000) rabbit polyclonal antibody purchased from Cedarlane Laboratories.
The characteristics of the invention being disclosed in the preceding description, the subsequent drawings and claims can be of importance both singularly and in arbitrary combination for the implementation of the invention in its different embodiments.
The foregoing description of preferred embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the teaching above. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the claims.
Targeted overexpression of c-Myc to alveolar epithelium of mice induced multifocal non-invasive bronchioloalveolar carcinoma (BAC) and solitary invasive papillary lung adenocarcinoma (PLAC). Initially, we reported regulation of genes in papillary adenocarcinomas (Meier et al., part1-3). Here we report genes specifically regulated in BAC. Strikingly, only a few genes were commonly regulated in both subtypes of c-Myc-induced adenocarcinomas and included genes involved in cell cycle progression, e.g. Ccnb1, Cdc2a, Ect2, Shtm1 (induced), and Igf binding protein 2 with growth-modulating activity (repressed). The common regulation of these genes in BAC and PLACs is suggestive for their primary role in c-Myc-induced lung carcinogenesis. Notable, the majority of regulated genes differed for BAC and PLAC. Thus, activation of genes involved in angiogenesis like Angpt1, Calcrl, Edg3, and negative regulators of cell proliferation, e.g. Rasa1, Ptpns1, Mapk14, was found specifically in BAC. Repression of genes with growth-inhibitory functions like Tgfbr3, Lats2, Akap12 and induction of genes enhancing cell growth like Arg1, Hk2, Fasn, the proteases Adam19, Hgfac and hepsin were characteristic for PLAC. In conclusion, an identification of genes attributable to BAC and PLAC enables molecular finger printing of the two lung adenocarcinoma subtypes.
Lung cancer is a leading cause of death and a major public health problem throughout the world. Histologically adenocarcinomas differ and are subdivided into distinct subtypes on the basis of predominant cell morphology and growth pattern, i.e.—acinar, papillary, bronchioalveolar and mucinous adenocarcinomas (Notably, lung tumors induced by c-Myc were heterogenous and could be classified as either multicentric non-invasive bronchioloalveolar carcinoma (BAC) or solitary invasive papillary lung adenocarcinoma (PLAC) (Part1). Strikingly, regulation of genes differed mostly amongst BAC and PLAC when compared with control non-transgenic lung. Therefore, oncogenomics enabled an identification of genes specifically associated with either BAC or PLAC adenocarcinomas.
Results
Histology of Lung Tumors.
We specifically studied mice at the age of 8 months as they didn't contain macroscopically visible solid tumors (PLACs). This was confirmed by histopathology (
Oncogenomics of BAC
To study genetic events underlying the development of BAC we investigated genome wide transcript expression in the lungs of c-Myc-transgenic males and females at the age of 8 months. BAC growth was confirmed by histopathology. By use of the murine genome array 430 2.0 we identified 246 genes and ESTs differently expressed in transgenic lungs as compared with healthy non-transgenic lung (see Methods for the significant expression change criteria). Note 243 genes were up regulated but 3 genes were repressed. Gene expression changes were more prominent with females resulting in 241 significantly regulated genes. In males the same genes were regulated (
Discussion
1. Genes Activated in Either c-Myc-Induced Subtype of Lung Adenocarcinoma Code for Essential Regulators of Cell Proliferation
Comparison of gene expression profiles of c-Myc-induced BAC and PLAC revealed that only a limited number of genes were induced in both subtypes of lung adenocarcinoma when compared with healthy lung of non-transgenic mice (Table 8.1a, b). Notably, some of these genes were expressed at higher level in PLAC than in BAC (Table 8.1a) like the c-Myc transgene itself which was induced 3-5-fold in lungs with BAC and 23- to 58-fold in solid PLACLikely, expression of the oncogenic transgene may initially be kept under control, for instance, by recruitment of transcriptional repressors. In advanced stages of tumor growth the ability to suppress activation of the transgene may become less efficient because of impaired control mechanisms or/and autocrine stimulation by oncogene, e.g. c-Myc, of its own transcription through activation of appropriate signaling circuits giving rise to high level of transgene expression. Most genes whose expression was coherent with c-Myc expression coded for proteins essential for cell cycle progression. Thus, genes significantly induced in BAC and still stronger upregulated in PLAC were cyclin b1 (Ccnb1) and its kinase Cdc2a forming the complex which initiates the M phase of cell cycle. We additionally identified genes moderately increased in BAC and significantly induced in PLAC (Table 8, 1a) which are associated with cell cycle progression and proliferation. Identification of the above named genes point to molecular events induced by c-Myc to enhance cell proliferation in PLAC as compared with BAC. Besides, the PDZ-binding kinase (Pbk), which is a novel mitotic kinase up regulated in a variety of highly proliferative malignant cells, fetal tissues and hematologic malignancies (Simons-Evelyn et al., 2001), (Nandi et al., 2004), and dCMP deaminase (Dctd) involved in pyrimidine biosynthesis were significantly induced in BAC (Table 8, 1c) and may well represent new targets of c-Myc to stimulate cell division. Among those genes whose expression was significantly increased in BAC and similarly changed in PLAC (Table 8, 1b) we identified two receptors for growth factors, i.e. the c-Met proto-oncogene (Met) and the fibroblast growth factor receptor 2 (Fgfr2), the hyaluronic acid receptor Cd44 and syndecan1 (Sdc1) with roles in cell proliferation, cell migration and cell-matrix interactions in addition to the lipase-related Pnilprp1. This suggests that overexpression of members of a gene family of lipases may be of great importance for inappropriate cell proliferation through as yet unestablished mechanism. Increased expression of some genes with growth-, metastasis- and angiogenesis supressor functions like Ppp2r5c, Gja1, Rrm1 and Nedd 4 may be part of an anti-tumor response in lung cancer.
Thus, the comparison of genes differently expressed in BAC and PLAC proved to be instrumental in identifying particular genes with similar regulation in different subtypes of c-Myc-induced lung adenocarcinomas which suggest their primary role in c-Myc-trigged lung carcinogenesis. Tracing individual genes specific for tumor pathologies enables an identification of disease candidate genes.
Genes Up Regulated in BAC Contributing to Cell Growth
Several new genes which may prove to be essential in lung cancer development were identified in BAC by use of the MG 430 2.0 array (Table 8.1c). We identified regulation of the thymoma viral proto-oncogene 3 (Akt3), the platelet-derived growth factor, D (Pdgfd), the sphingomyelin synthase 1 (Tmem23=Sms1), the Adducin 3 (Add3), the S-adenosylmethionine decarboxylase 1 (Amd1), and the ELL associated factor 1 (Eaf1
2. Genes with Different Regulation in BAC and PLAC (See Also Table 8).
2.1 Genes Significantly Enhanced in BAC and Repressed in PLAC Included Those Coding for Angiogenesis, Growth Inhibitory Response and Adhesion
Among genes whose expression was strongly induced in BAC but repressed in PLAC (Table 8.2.1) we identified endothelial-specific growth factors angiopoietin 1 (Angpt1), (Zadeh et al., 2004), and the G-protein-coupled receptors, Edg3 (endothelial differentiation, sphingolipid G-protein-coupled receptor, 3) and Calcrl (calcitonin receptor-like receptor) which likely contribute to angiogenesis through increase of endothelial cell proliferation and survival (Fieber et al., 2006). Besides, genes whose expression was significantly increased in BAC and only slightly in PLAC (Table 8.1b) included hypoxia-inducible factor1 Hif1a. Hif1 plays an essential role in cellular and systemic homeostatic response to hypoxia, directly activating transcription of genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation and matrix production, and can also be induced by hypoxia-independent factors like growth factor receptor (e.g. Egfr) signaling (Yudoh et al., 2005). Thus, Hif1a is a transcription factor that can directly transactivate genes important for the enhanced growth and metabolism. Activation of proangiogenic genes in BAC argues for the angiogenic capacity of c-Myc.
Furthermore, among genes activated in BAC but not in PLAC (Table 8.2.1) we observed regulation of transcripts coding for anti-growth capacities, e.g. the receptor for TGFβ family members Activation of the growth-inhibitory genes, may represent a part of a feed-back regulatory response to an inappropriate activation of proliferative signaling induced by constitutive c-Myc over expression. This feedback mechanism was absent in PLAC to foster aggressive tumor growth. Notably, genes oppositely regulated in BAC and PLAC, i.e., induced in BAC and repressed in PLAC, included cytoskeleton coding genes Utrn, Vcl and adhesion molecules Itga8, Cd38. Opposite regulation of these genes may be related to different growth pattern and invasive behaviour of BAC and PLAC.
Further, genes specifically regulated in BAC, but not in PLAC included the docking protein Gab1 utilized for Egfr- and c-Met signaling (Meng et al., 2005), the proto-oncogene Yes1 involved in integrin signaling (Klinghoffer et al., 1999), and the protease Adam 17 shown to mediate activation of Egfr in vivo in tumor development by nude mice (Borrell-Pages et al., 2003). Likewise, in BAC the apoptosis inhibitors Birc 4 (Tenev et al., 2005) and Api 5 (Kim et al., 2000), the proliferation-associated transferrin receptor 1 (Tfrc1) (Wang et al., 2005b) and the G-proteins Gna12 and Gna13 which interact with cadherins to release transcriptional activator β-catenin (Meigs et al., 2001) were significantly upregulated. Activation of the genes discussed herein represents unique features of BAC oncogenomics.
2.2 Genes Induced or Repressed in PLAC but not Regulated in BAC May Contribute to a More Aggressive and/or Invasive Phenotype of PLAC
We identified genes whose expression was significantly regulated in PLAC but unchanged in BAC (
In conclusion, we investigated the oncogenomics of distinct subtypes of lung adenocarcinoma in a c-Myc transgenic mouse model. The lung tumors arise from the same progenitor cells, e.g. alveolar epithelium. Few solitary PLACs were observed in mice at much later time points than multi-centric BAC which we found dispersed throughout the whole lung. Furthermore, genome wide expression profiling revealed more gene expression changes in PLAC than in BAC with a number of genes linked to cell cycle progression being stronger induced in PLAC than in BAC therefore indicating enhanced cell proliferation in PLAC. Besides, genes activated or repressed in PLAC could be linked to a more aggressive phenotype to enable invasive and metastatic growth. Collectively, these observations allowed us to hypothesize that BAC is an earlier stage and PLAC an advanced stage of the lung adenocarcinoma induced by c-Myc. Importantly, by genome wide expression profiling of different pathologies of c-Myc-induced lung tumors, e.g. non-invasive BAC and invasive PLAC, genes could be identified which represent, most likely, the molecular basis for malignant growth induced by c-Myc. Genes specifically regulated in BAC or PLAC can therefore be held responsible for the more aggressive phenotype observed with PLACs.
Methods
Maintenance of the Transgenic Mouse Line and Sample Collection
SPC/Myc transgenic mice (Ehrhardt et al., 2001) were maintained in the C57B1/6 background and identified as described previously (Meier et al., 2006, Part1). To study the transcriptome of BAC total RNA was isolated from the lungs of the transgenic mice at the age of 8 months. Normal lungs of non-transgenic animals served as a control. Mice were anesthetized by an overdose of CO2 and the lung were isolated, one part of each lung being shock frozen in dry ice and stored at −80° for further gene expression studies and the other fixed in 4% formaldehyde and processed for histological investigation as described previously (Meier et al., 2006, Part1). 16 c-Myc transgenic males and 16 females were studied separately and compared with equivalent group of non-transgenic animals. For microarray analyses, equal amount of total RNA isolated from 4 individual mice were pooled, i.e. 4 pools of RNA, each pool combining RNAs from 4 mice, were analysed with microarrays per each animal group.
Isolation and microarray analysis of PLACs was described previously (Meier et al., 2006).
Transcriptome Analyses
Total RNA was isolated using the RNeasy total RNA isolation Mini Kit (Qiagen), pooled as described above, and a second clean-up of pooled RNA was done with the same RNeasy Kit. 8 mkg of total RNA were used to produce cRNA using One-Cycle Target Labeling and Control Reagents (Affymetrix) according to the Affymetrix Gene Chip® Expression Analysis Technical Manual (Santa Clara, Calif., 2005). Purified cRNA was quantified and checked for quality using the NanoDrop ND-1000 and the Agilent 2100 Bioanalyzer, then cleaved into fragments of 35-200 bases by metal-induced hydrolysis.
10 μg of biotinylated fragmented cRNA were hybridized onto the GeneChip® Mouse Genome 430 2.0 array from Affymetrix, washed and stained according to the manufacturer's recommendation. The arrays were scanned using the GeneChip® Scanner 3000. Scanned images were analyzed with the GeneChip® Operating Software (GCOS), each image being scaled to the same all probe set intensity for proper comparison between chips. Default parameters provided in the Affymetrix data analysis software package were applied for analysis.
Data Analysis
Identification of Significantly Regulated Genes
Gene expression data analysis for a single microarrays and a comparison between two arrays were performed using GCOS software. It applies two different algorithms to produce for each gene on the array numeric expression value and detection call (Present, Absent) and, by comparison of two samples (=arrays), additionally a signal logarithm ratio and a change call (“Increase”, “Decrease” or “No Change”).Statistical evaluation of replicate data was done with the Affymetrix® Data Mining Tool 3.1 (DMT). Fold change values were calculated as the ratio of the average expression levels for each gene between two tissue sets, i.e. BAC and normal lung, for males and females separately. To determine significance of gene expression changes in BAC compared with normal lung the unpaired two-sided T-Test with the p-value=0.05 as a threshold of significance was done using numeric gene expression values. Additionally, comparison ranking analysis was done to determine the concordance between change calls (“Increase” or “Decrease”) for regulated genes in BAC derived from different mice. The results are shown in Table 8 as % of change calls in 16 analyses (each of 4 tumor pools versus each of 4 normal lung pools). The thresholds for significantly up/down regulated genes in BAC were defined as a mean FC ≧=2,5/<=−2,5, a p-value in T-test <=0,05 and number of change calls >=87,5% (i.e. more than 14 out of 16) in comparative ranking in the group of transgenic females or males. The criteria for genes significantly up/down regulated in PLAC were defined more stringent and represented FC≧3 or <−3, p-value in T-test ≦0,05 and 100% of “Increase”/“Decrease” calls in comparative ranking in at least one group of tumors of different size, i.e. small- (n=3), middle- (n=4) and large-sized tumors (n=3), in comparison to normal lung (n=4) (Meier et al., 2006. Part1). Besides; both in BAC and PLAC analyses, we restricted our consideration for those up or down regulated genes, which were detected (“Present” call) correspondingly in all tumor or in all control lung replicates. In Table 8 the statistical values for all 10 PLACs are reported, whereas by hierarchical clustering the data for PLACs of different sizes were analysed separately (see below).
Hierarchical Gene Cluster Analysis,
To get an overall view of similarity and differences in regulation of gene expression in c-Myc-induced BAC and PLAC hierarchical gene cluster analysis implemented in ArrayTrack software (Tong et al., 2004) was done. Ward fusion algorithm with Euclidean distance was applied to the log transformed (with the base 2) ratios of gene expression values. The list of analysed genes combined all 597 genes significantly deregulated in BAC, males or females, and in PLAC, in small-middle- and large-sized tumors.
10.3
21.6
−3.8
−6.2
−19.4
−31.6
−16.4
1% Increase/Decrease - concordance of change calls in the pairwise comparisons (each tumor to each normal lung sample), by which genes are up or down regulated respectively
U51805
Arg1
arginase 1, liver
6.1
10.8
14.8
94
100
100
0.01
0.09
0.27
T, R
2
rR
T, R
5
AA913994
Shmt1
serine hydroxymethyl transferase 1
T, R
4
7.5
7.3
9.4
100
100
100
0.01
0.04
0.04
(soluble)
T, R
T, R
R
4.8
2
1
R
1
1
phosphatidylinositol glycan, class N
T, R
thymidine kinase 1
T, R
R
1
R
2
1
meiotic recombination 11 homolog A
topoisomerase (DNA) II alpha
R
T, R
2
ribonucleotide reductase M1
R
T
helicase, lymphoid specific
R
1
1
rR
R
2
R
3
T, R
4
T, R
3
U64450
Npm3
nucleoplasmin 3
2
6.0
5.5
7.4
100
94
100
0.01
0.05
0.04
T, R
2
R
1
1
rT
rT
rT
T
2
1
1
2
glutamate-rich WD repeat containing 1
T, R
T, R
1
T, R
T, R
1
rT
2
T, R
1
T, R
1
rT
1
1
2
1
rT
translocase of inner mitochondrial
rR
1
membrane 10 homolog (yeast)
rR
2
translocase of outer mitochondrial
membrane 40 homolog (yeast)
R
1
solute carrier family 19 (sodium/
T
1
hydrogen exchanger), member 1
4.3
rR
RAN guanine nucleotide release factor
1
rT
1
1
rT
1
1
−3.8
rT
Areg
Map2k1
rT
1
Myc
51.0
58.6
Ros1
Ros1 proto-oncogene
21.9
Arhu
1
Frat2
frequently rearranged in
advanced T-cell lymphomas 2
AV109962
Traf4
Tnf receptor associated factor 4
1
100
100
0.01
0.00
0.03
Cldn2
claudin 2
20.7
45.3
Gjb3
1
Gja1
2
Krt1-18
Col15a1
Icam1
intercellular
adhesion
molecule*
T
AW228162
Dsc2
desmocollin 2
rR
100
0.00
0.00
0.00
Dsg2
1
Ect2
10.3
Muc1
mucin
1,
transmembrane*
T
Lcn2
AI786089
Kng
kininogen
22.4
37.1
12.6
100
100
0.02
0.03
0.09
AI042655
Hgfac
hepatocyte growth factor
9.6
100
100
0.01
0.01
0.01
Adora2b
Spint1
Adam19
1
Hpn
AW047185
Thop1
thimet
oligopeptidase
1
R
2.8
100
100
0.00
0.01
0.02
Tnfrsf6
tumor
necrosis
factor
receptor
R
1
−4.2
superfamily,
member
6
Hoxa5
homeo
box
A5
R
−6.1
−7.7
AI850533
Bmp6
bone morphogenetic protein 6
1
−15.5
−34.8
−22.8
100
100
100
0.00
0.00
0.00
Tgfb1
−2.9
−7.2
AA717826
Dpt
dermatopontin
−5.0
−6.7
−16.6
100
100
100
0.02
0.01
0.01
Acvrl1
−5.9
−7.6
Eng
−6.7
−7.6
Zfhx1a
−6.8
−8.5
Gadd45b
rR
1
−3.5
Igfbp5
−6.5
−4.9
Igfbp6
−8.8
−18.9
X76066
Igfbp4
insulin-like growth factor
−2.8
−3.3
−5.3
100
100
100
0.03
0.03
0.01
binding protein 4
Socs2
−3.7
Nfkbia
−3.1
Tcf7
transcription
factor
7,
T-cell
T
1
−4.9
−8.5
specific*
Sox7
−4.4
−6.3
Dusp1
dual
specificity
phosphatase
1
R
1
Ptpre
−4.5
Ptpns1
−4.7
Rassf5
−3.7
Akap12
A
kinase
(PRKA)
anchor
protein
R
1
−5.8
−11.9
(gravin)
12
AF033275
Akap2
A kinase (PRKA) anchor
rR
−3.3
−3.3
−4.8
100
93
91
0.01
0.01
0.01
protein 2
Cav
caveolin,
caveolae
protein
R
1
−4.0
−6.5
Dnajb9
rR
1
−3.0
Fkbp7
1
−3.6
−4.1
AF080090
Sema3f
sema domain, immunoglobulin
−4.9
−5.4
−5.6
100
100
100
0.01
0.00
0.00
domain(Ig), short basic
domain, secreted,
(semaphorin) 3 F
X70854
Fbln1
fibulin 1 !!!
rR
−3.8
−3.1
−10.1
100
100
100
0.00
0.00
0.00
D21165
Vsnl1
visinin-like 1
−7.1
−7.1
−7.1
100
87
100
0.00
0.00
0.00
Reck
1
−4.1
−5.2
Capn2
−3.0
−3.8
Cdh5
−7.2
−10.0
Spock2
−6.6
−22.3
Thbd
1
−16.0
−55.8
Tie1
−8.4
−12.2
X65493
Icam2
intercellular adhesion molecule 2
−12.0
−30.6
−48.1
100
100
100
0.00
0.00
0.00
Osf2-
osteoblast
specific
factor
2
R
−3.7
−5.4
pending
(fasciclin
I-like)
Tek
−12.6
−26.3
Nes
−3.1
−4.9
Cd97
CD97
antigen*
T
−8.4
−9.7
Vwf
−8.4
−16.9
Xlkd1
−12.2
−28.2
Sparcl1
−5.4
Marcks
−5.8
Tenc1
−5.6
−6.8
Mfap2
rT
−5.2
−5.8
U30292
Col13a1
procollagen, type XIII, alpha 1
rR
−6.2
−7.3
−16.7
100
100
100
0.01
0.01
0.01
D86917
Pcdha6
protocadherin alpha 6
−7.6
−8.6
−9.0
100
100
100
0.00
0.00
0.00
Lama4
−3.0
X84014
Lama3
laminin, alpha 3
−3.5
−4.8
−5.2
100
100
100
0.01
0.00
0.01
Itga8
rT
−7.5
−8.8
D86916
Pcdha4
protocadherin alpha 4
−9.9
−9.9
−9.9
100
100
100
0.00
0.00
0.00
Fbln2
R
−3.7
M77123
Vtn
vitronectin
−6.6
−6.6
−6.6
100
93
100
0.00
0.00
0.00
Vcam1
1
−4.7
X79199
Tna
tetranectin (plasminogen
−11.1
−11.1
−11.1
100
100
100
0.00
0.00
0.00
binding protein)
Stab1
−3.0
−3.4
Cldn5
−9.1
−45.9
Pmp22
peripheral
myelin
protein
−6.2
−16.3
Ptprb
−14.3
−40.9
Ptprg
−5.7
−4.0
Slfn2
−3.3
?
−3.7
AV349686
Ndr2
N-myc downstream regulated 2
−5.8
−4.4
−4.8
100
100
100
0.00
0.00
0.00
Ets1
−4.2
−5.3
Lox
lysyl
oxidase
R
1
−3.8
−4.9
Sipa1
−3.0
−3.9
Ndn
−7.6
−10.4
Notch4
Notch
gene
homolog
4
T
−5.7
−5.8
(
Drosophila
)*
?
X74134
Nr2f1
nuclear
receptor
subfamily
2,
R
−3.1
−3.1
−3.1
100
87
100
0.00
0.00
0.00
group
F,
member
1
Meox2
−8.6
−12.6
Rbp1
2
−3.3
Sema7a
−3.9
Sema3c
1
−5.1
Sema3e
−3.5
Tagln
−3.9
−4.9
Ablim1
−4.4
−6.8
| Number | Date | Country | Kind |
|---|---|---|---|
| 08075243.9 | Mar 2008 | DE | national |
This application is a continuation of and claims priority to PCT international patent application no. PCT/EP2009/002441, filed on Mar. 30, 2009, claiming priority to European application no. 08075243.9, filed on Mar. 28, 2008. Those applications are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2009/002441 | Mar 2009 | US |
| Child | 12891025 | US |
