NOVEL BIOMARKERS OF LIVER CANCER

The invention is directed to biomarkers of dysplasia and cancer and the use thereof, in particular in the diagnosis, prognosis and/or monitoring of treatment of liver cell dysplasia or hepatocellular carcinoma (HCC). Areas of application are the life sciences: biology, biochemistry, biotechnology, medicine and medical technology.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. It causes approximately one million deaths each year. Discovery of novel HCC markers for early detection of the disease has been the aim of much research.

Disease proteomics is an evolving science and has been applied especially in the fields of cancer research. However, in order to identify regulated proteins a reference map needs to be established. In the case of the liver the inventors and others reported a proteome map and in the efforts of the inventors >150 novel proteins were identified [1]. Studying quantitative differences in protein expression is a delicate but an important part of proteomics and such research requires efficient methods to allow for reproducible and robust data generation. In this regard two-dimensional electrophoresis (2-DE) and MALDI mass spectrometry (MALDI-MS-TOF) are techniques widely applied to proteomic research to probe for disease regulated proteins in cancer such as hepatocellular carcinoma (HCC). So far, most research focused on viral HBV/HCV-associated HCC, but little is know about non-viral HCC. Liver malignancies are common cancers worldwide and are responsible for approximately one million deaths each year, i.e. most HCC patients died quickly because of rapid tumor progression [2]. Early diagnosis of disease will thus improve overall survival. Recent research from the lab of the inventors identified exaggerated EGF signaling as a possible route for hepatocarcinogenesis, but the EGF receptorkinase plays a much wider role in the immortalisation of different cell types [3]. Notably, the epidermal growth factor (EGF) is highly expressed in a number of solid tumors, and its expression correlates well with tumor progression, resistance to chemotherapy, and poor prognosis; consequently, it is an obvious target for the rational design of novel anticancer agents. In the past, the serum proteome of EGF-tumor-bearing mice has been studied to gain more information about disease-regulated proteins in HCC and to search for novel biomarkers at different stages of disease [4]. Next to alpha-fetoprotein (AFP) only a few serological markers are available (e.g. Lens culinaris agglutinin-reactive AFP (AFP-L3) and protein induced by vitamin K absence or antagonist-II (PIVKA-II)), but its diagnostic accuracy is unsatisfactory, because of its low sensitivity and the non-specific correlation between the clinical behavior of HCC and, for instance, AFP blood levels. For this reason, new biomarkers for the diagnosis of HCC are in strong demand by clinicians [5, 6], and more selective tests, such as soluble interleukin-2 receptor levels, are currently being investigated [7].

The aim of the present invention is therefore to provide novel biomarkers and molecules binding to said biomarkers, compositions and a kit, as well as methods for the diagnosis, prognosis and/or monitoring of treatment of dysplasia and cancer patients, in particular of liver cell dysplasia and hepatocellular carcinoma (HCC) patients.

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

The invention is based on the surprising finding that proteins regulated by excessive EGFR signalling in the liver may be used as biomarkers, in particular as immunohistochemical markers, in the diagnosis, prognosis and/or monitoring of treatment, preferably of/in the early stage, of diseases, including liver cell dysplasia or hepatocellular carcinoma (HCC), in particular of non-viral HCC, wherein the protein is selected from a first group consisting of

In a first aspect, the invention is thus directed to a protein regulated by excessive EGFR signalling in the liver for use as biomarker in the diagnosis, prognosis and/or monitoring of treatment, preferably in the early stage, of diseases, including liver cell dysplasia or hepatocellular carcinoma (HCC), in particular of non-viral HCC, wherein the protein is selected from a first group consisting of

The term hepatocellular carcinoma (HCC) according to the invention is in particular directed to all primary malignancies of the liver, including malignancies induced by cirrhosis or by viral hepatide, in particular hepaptitis B or C, infections, more particular the term hepatocellular carcinoma (HCC) is directed to non-viral HCC, preferably HCC caused by excessive EGFR signalling in the liver.

The proteins according to the invention concern gene products of mammalia, preferably gene products of the genome of mus musculus or homo sapiens, in particular the respective gene products of homo sapiens are preferred, or, respectively, sequence fragments of said gene products as described herein.

Within the context of the invention the term EGFR is directed to mammalian epidermal growth factor receptor (EGFR, also named ErbB-1), preferably murine or human EGFR, in particular human EGFR, and also to oligomers composed of proteins of the epidermal growth factor receptor family (EGFR, HER2, HER3, and HERF2) wherein the oligomers comprise EGFR.

According to the invention the term “regulated by excessive EGFR signalling in the liver” as common technical feature of the 41 biomarker proteins mentioned herein, said term is particularly directed to the regulation of said proteins by exaggerated EGFR tyrosine kinase activity in the liver, in particular induced by overexpression of EGFR in the liver and/or induced by the activation of EGFR by a growth factor selected from the group consisting of EGF, amphiregulin, epiregulin, TGF-α, HB-EGF, betacellulin, epigen, more particular selected from the group consisting of EGF, amphiregulin, epiregulin.

The term “dysplasia” according to the invention is directed to low grade and/or high grade dysplasia, wherein “low grade dysplasia” is particularly directed to a lesion having minimal aberration inside the cell, and “high grade dysplasia” also comprises mild or medium dysplasia. The term “liver cell dysplasia” according to the invention is in particular directed to premalignant lesions of HCC, as described for example by Kojiro M. J Hepatobiliary Pancreat Sum. 2000; 7(6):535-41.

In one aspect, the invention is directed to a protein for use as biomarker in the diagnosis, prognosis and/or treatment monitoring of dysplasia or cancer, in particular bladder, breast, cervical, colorectal, endometrial, gastric, head and neck, ovarian and oesophageal dysplasia or cancer, wherein the protein is selected from a first group consisting of

4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Alpha glucosidase 2, Inosine triphosphatase, Interleukin 25, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, RIKEN cDNA 2410004H02, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, 170 kDa glucose regulated protein GRP170 precursor,

or from a second group consisting of

2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein.

In another aspect, the invention is directed to a protein regulated by excessive EGFR signalling in the liver for use as serum marker in the diagnosis, prognosis and/or treatment monitoring of liver cell dysplasia or hepatocellular carcinoma (HCC) wherein the protein is selected from a first group consisting of

Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component

or from a second group consisting of

Major urinary protein 1.

The term “serum marker” as used herein is in particular understood as a specific indicator found in a blood test, including tests on plasma or serum, that identifies a disease.

The term “serum” according to the invention is in particular directed to blood, plasma, and serum, more particular to blood plasma and serum, wherein blood serum is particularly preferred within the context of the invention.

The term “tissue” according to the invention is directed to the cellular organizational level intermediate between cells and the complete organism, in particular to an ensemble of cells from the same origin that together carry out a specific biological function, and wherein the tissue may be either part of an animal or human body or wherein preferably the tissue has been removed from an animal or human body.

Accordingly, the proteins according to the invention are preferably used as immunohistochemical markers, such as for a immunohistochemical staining, or as blood plasma markers or particularly as blood serum markers.

The invention is further directed to molecules specifically binding to the protein biomarkers mentioned herein or to mRNA coding for said proteins, and wherein said molecules are selected from the group consisting of antibodies and siRNA. The terms “specifically binding” or “specific for” as mentioned herein is particularly related to an association of the biomarker or mRNA with the binding molecule being established via an association constant Ka>1000 M⁻¹.

Thus, the invention is also directed to an antibody specific for a protein regulated by excessive EGFR signalling in the liver for use in the diagnosis, prognosis and/or treatment monitoring of liver cell dysplasia or HCC.protein, wherein the protein is selected from a first group consisting of Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, or from a second group consisting of

2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein,

and wherein the use of an antibody directed against a protein selected from said first group is particularly preferred.

Further, the invention is thus directed to an antibody specific for a protein for use in the diagnosis, prognosis and/or treatment monitoring of dysplasia or cancer, in particular bladder, breast, cervical, colorectal, endometrial, gastric, head and neck, ovarian and oesophageal dysplasia or cancer, wherein the protein is selected from a first group consisting of

or from a second group consisting of

2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein,

and wherein the use of an antibody directed against a protein selected from said first group is particularly preferred.

Moreover, the invention is thus directed to an antibody for serum profiling in the diagnosis, prognosis and/or treatment monitoring of liver cell dysplasia or HCC, wherein the antibody is specific for a protein selected from a first group consisting of Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component

or selected from a second group consisting of Major urinary protein 1,

and wherein the use of an antibody directed against a protein selected from said first group is particularly preferred.

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 proteins. 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, goats, rabbits, mice, rats, chicken or preferably of eggs derived from immunized chicken. Monoclonal antibodies may be generated using conventional techniques (see Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are incorporated herein by reference).

The term “serum profiling” according to the invention is in particular directed to the analysis of blood plasma or blood serum for the presence or concentration of the selected protein in said plasma or serum, such as by using a biosensor, performing an ELISA, a Western Blot, a magnetic bead separation/purification, a ZipTip approach, and wherein said procedures, if applicable, may be combined with a mass spectrometry analysis.

The invention is also directed to siRNA, which reduces or preferably inhibits the expression of a protein regulated by excessive EGFR signalling in the liver, for use in the treatment of liver cell dysplasia or HCC, wherein the protein is selected from the group consisiting of Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component.

Within this context, the present invention employs siRNA for use in modulating the level of protein presence in the cell, wherein siRNA oligonucleotides specifically hybridize nucleic acids encoding the selected protein and interfere with the expression of the gene coding for said protein.

Preferably, the siRNA comprises double stranded RNA including a sense RNA strand and an antisense RNA strand, wherein the sense RNA strand comprises a subsequence being 15-30, preferably 19, 20, 21, 22, 23, 24, or 25 contiguous RNA nucleotides in length, preferably corresponding to the ORF sequence encoding the selected protein, and wherein said subsequence contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding for the selected protein.

In another aspect, the invention is directed to a nucleotide sequence coding for a protein regulated by excessive EGFR signalling in the liver for use in the treatment of liver cell dysplasia or HCC, wherein the protein is selected from the group consisting of 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, Major urinary protein 1.

The nucleotide sequence particularly comprises a nucleic acid being from about 20 base pairs to about 100,000 base pairs in length. 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 the protein, an oligopeptide or a peptide. Preferably, the nucleotide sequence of the present invention may comprise a DNA construct capable of generating the selected protein 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 selected protein. 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 selected protein.

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 the selected protein over the entire coding region. In this regard, nucleoetide 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 the selected protein over the entire coding region.

In another aspect the nucleotide sequence 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.

Further, within the context of the present invention an antisense composition is provided for use in the treatment of liver cell dysplasia or HCC, wherein the antisense composition comprises a nucleotide sequence complementary to a coding sequence of a protein regulated by excessive EGFR signalling in the liver, wherein the protein is selected from the group consisting of Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component.

Said nucleotide sequences and siRNA according to the invention may be prepared by any standard method for producing a nucleotide sequence or siRNA, such as by recombinant methods, in particular synthetic nucleotide sequences and siRNA is preferred.

Said nucleotide sequences and siRNA are preferably for the use in the treatment of liver cell dysplasia or HCC by administering an amount of said nucleotide sequences and/or siRNA to a subject suffering from or being susceptible to liver cell dysplasia or HCC for decreasing or increasing the expression or biological activity of the targeted protein to a normal level.

The invention thus also relates to a composition comprising a substance that

- decreases or inhibits the expression or biological activity of a protein selected from the group consisting of Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, and/or
- increases the expression or biological activity of a protein selected from the group consisting of 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, Major urinary protein 1,
  
  for the treatment of liver cell dysplasia and HCC, wherein the substance is preferably selected from the group consisting of said nucleotide sequences and siRNA according to the invention.

Preferably, the compositions according to the invention further comprises a pharmaceutically acceptable carrier and/or recipient and/or diluent.

The term “biological activity” within the context of the invention is particularly directed to the interaction of the selected protein with other biomolecules, in particular with proteins, carbohydrates and lipids or with a combination thereof.

The term “subject”, as used herein, is directed to a mammal, in particular to a mouse or a human being having or being susceptible to dysplasia or cancer, preferably liver cell dysplaisa or HCC, more particular to a human dysplasia or cancer patient or a transgenic cancer mouse, including a HCC bearing mouse, such as a patient having liver cell dysplasia or HCC or a transgenic mouse overexpressing Egf, in particular a mouse whose genome comprises a non natural sequence coding for IgEGF, may be.

The invention is also directed to a method of detecting liver cell dysplasia or HCC, in particular non-viral HCC, or of predicting the susceptibility or resistance to liver cell dysplasia or HCC, in particular to non-viral HCC, comprising testing a sample isolated from the liver of a subject for the presence or concentration of a protein selected from a first group consisting of Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor,

or from a second group consisting of

2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein,

or comprising testing a serum sample of a subject for the presence or concentration of a protein from a first group consisting of Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, or from a second group consisting of Major urinary protein 1,

and wherein, in particular, the sample is tested for the increase of a protein selected from said first group(s) and/or the decrease of a protein selected from said second group(s).

The invention is further directed to a method of detecting the response of a subject to a compound in the treatment of liver cell dysplasia or HCC, or of predicting the responsiveness of subject to a compound in the treatment of liver cell dysplasia or HCC, comprising determining in a sample isolated from the liver of said subject the presence or concentration of a protein regulated by excessive EGFR signalling in the liver, wherein the protein is selected from a first group consisting of Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor,

or from a second group consisting of

2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein,

or comprising determining the presence or concentration of a protein in a serum sample of said subject, wherein the protein is selected from a first group consisting of Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, or from a second group consisting of Major urinary protein 1,

and wherein the compound is selected from the group of EGF receptor tyrosine kinase activity modulator, siRNA, in particular above-mentioned siRNA, nucleotide sequence, in particular above-mentioned nucleotide sequence, and if applicable said compound is combined with a chemotherapeutic drug,

and wherein the sample is preferably tested for the decrease of the presence or concentration of a protein selected from said first group(s) and/or the increase of the presence or concentration of a protein selected from said second group(s).

The term “EGF receptor tyrosine kinase activity modulator” or “compound modulating EGF receptor tyrosine kinase activity” or “compound to be tested” according to the invention is in particular directed to a compound selected from the group consisting of Sorafenib, Sunitinib, Gefitinib, Erlotinib, anti-EGF antibody, anti-HER1 antibody, anti-HER2 antibody, anti-HER3 antibody, anti-HER4 antibody, Trastuzumab (Herceptin), Cetuximab, Panitumumab, Matuzumab, Nimotuzumab, MDX-447, Pertuzumab.

According to the invention, the term “chemotherapeutic drug” is in particular directed to any antineoplastic chemotherapy drug usable for treating HCC and any chemopreventive drug usable for treating liver cell dysplasia, and wherein the antineoplastic chemotherapy drug is preferably selected from the group consisiting of Taxol, 5-fluorouracil, doxorubicin and vinblastine, or wherein the chemopreventive drug is preferably selected from the group consisiting of Zileuton and Celecoxib.

The invention further concerns a method to screen for and to identify a compound for modulating EGF receptor tyrosine kinase activity in the liver of a subject suffering from or being susceptible to liver cell dysplasia or HCC, comprising the use of a protein biomarker selected from a first group consisting of

Thus, the invention is also directed to the use of at least one of said proteins and/or of at least one of said antibodies to screen for and to identify a compound for modulating EGF receptor tyrosine kinase activity in the liver of a subject suffering from or being susceptible to liver cell dysplasia or HCC, in particular by a serum analysis of the subject to which a compound, in particular a (at least putative) EGF receptor tyrosine kinase activity modulator, to be tested has been administered.

In another aspect, the invention is directed to a method of qualifying the EGFR kinase activity in a subject comprising determining in a sample of the liver of a subject suffering from or being susceptible to liver cell dysplasia or HCC at least one protein selected from a first group consisting of

Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor

and/or at least one protein selected from a second group consisting of

2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondria!), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein,

and/or comprising determining in a serum sample of a subject suffering from or being susceptible to liver cell dysplasia or HCC at least one protein selected from a first group consisting of

Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component

and/or at least one protein selected from a second group consisting of Major urinary protein 1,

wherein the level of the at least one protein of said first group(s) being significantly higher and/or the level of the at least one protein of said second group(s) being significantly lower than the level of said protein(s) in the liver of subjects without cancer associated with increased activity of EGFR is indicative of induced EGFR kinase activity in the subject,

and optionally further comprising the comprising the above-mentioned screening method to identify a compound for modulating the increased EGF kinase activity in the liver of the subject.

The invention also concerns a method, in particular the aforementioned method, for predicting the response of a liver cell dysplasia or HCC patient to the administration of a EGF receptor tyrosine kinase activity modulator, wherein the level of at least one protein selected from said first group(s) of proteins being significantly higher and/or the level of at least one protein selected from said second group(s) of proteins being significantly lower in the liver tissue or serum (or sample sample, respectively) of said patient than the level of said protein(s) in the liver tissue or serum (or sample sample, respectively) of subjects without liver cell dysplasia or HCC associated with increased activity of EGF receptor tyrosine kinase is indicative that the patient will respond therapeutically to a method of treating cancer comprising administering a EGF receptor tyrosine kinase activity modulator.

In a preferred embodiment, the methods of the invention are implemented by performing an immunoassay, such as an enzyme immunoassay (EIA), a radio immunoassay (RIA) or a fluorescence immunoassay (FIA) may be, in particular by using the kit according to the invention and/or by performing an immunohistochemical analysis or a western blot.

Preferably, at least one antibody specific for a protein selected from the group consisting of

Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, and Major urinary protein 1, is used for the immunoassay and/or reagents effective to detect said biomarker(s) in a serum sample, such as a blocking buffer for reducing unspecific antibody binding or an enzyme substrate for imaging enzyme labelled antibodies may be, is used for the immunoassay.

In particular it is preferred if an immunohistochemical analysis and/or a western blot is performed for determining the presence or concentration of at least one of said proteins, and wherein preferably at least one of said antibodies is used, and/or wherein dysplastic or malignantly transformed cells isolated from liver tissue by laser microdissection are used.

In another preferred embodiment, the method is implemented by performing a peptide mass fingerprinting, in particular by using the kit described herein.

In one embodiment, the methods according to the invention comprise the steps of

- adding lysis buffer to a sample, preferably a serum sample or a liver tissue sample, isolated from a subject suffering from or being susceptible to liver cell dysplasia or HCC;
- separating the proteins of the lysed serum sample by 2D gel electrophoresis;
- excising from the gel at least one 2-D spot containing a differentially regulated protein;
- adding digesting buffer, preferably a buffer containing trypsin, to the at least one excised sample;
- determining the identity of the protein by analyzing the digested 2-D spot by mass spectrometry.

According to the invention, the identity, or the presence and/or the concentration, respectively, of the proteins Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, and Major urinary protein 1, may be determined by determining the presence or concentration of fragments, in particular being 7-24 amino acid residues in length, of said proteins, preferably in a tissue or body fluid sample, which may have been further processed, such as by 2-DE, and wherein a protease, preferably trypsin, has been added to said preferably further processed sample.

In particular, a method according to the invention is preferred, wherein peptide mass fingerprinting is performed, preferably based on mass spectrometry with 2D tryptic digested spots selected by recognition and identified by MALDI-TOF, ESI-TOF or ITMS, for determining the presence or concentration of the selected protein, preferably in the serum or tissue.

In another embodiment, a method according to the invention is preferred, wherein the expression of the gene coding for the selected protein is determined by means of a PCR, preferably a RT-PCR and/or a quantitative real time PCR, for determining the presence or concentration of said protein, preferably in the sample of the tissue isolated from of the liver.

The invention is further directed to a kit for the use in qualifying the EGF receptor tyrosine kinase activity in a subject suffering from or being susceptible to cancer or dysplasia, in particular liver cell dysplasia or HCC, preferably for use in a method according to the invention, in particular for predicting or monitoring the response of a liver cell dysplasia or HCC patient to a method of treating cancer comprising administering a EGF receptor tyrosine kinase activity modulator, wherein the kit comprises at least one standard indicative of the level of a protein selected from the group consisting of

Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, and Major urinary protein 1 in the liver or in a serum sample, of normal individuals or in the liver or serum of individuals having liver cell dysplasia or HCC associated with increased EGF receptor tyrosine kinase activity, and/or at least one preferably synthetic fragment, being 7-24 amino acids in length, and/or at least one antibody specific for said protein(s), and/or at least one primer pair for determining the mRNA coding for the protein, and preferably instructions for the use of the kit.

The invention also concerns the use of at least one biomarker selected from the group consisting of the proteins

Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, and Major urinary protein 1 and/or of at least one antibody directed against said at least one biomarker, in the diagnosis, prognosis and/or treatment monitoring of cancer or dysplasia, in particular of HCC or liver cell dysplasia.

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 subject or the level of a said at least one biomarker in a sample of a patient suffering from HCC or having liver cell dysplasia may be.

In particular, at least one of said biomarkers and/or at least one antibody directed against said at least biomarker, is used for monitoring the therapeutic treatment of a patient suffering from HCC or having liver cell dysplasia, in particular the treatment with a chemotherapeutic drug, preferably with an antineoplastic chemotherapy drug, or with a chemopreventive drug.

Further, a composition for diagnosing or treatment monitoring of dysplasia or cancer, in particular of liver cell dysplasia or HCC, associated with increased EGF receptor tyrosine kinase activity in a patient, preferably by an in vitro body fluid analysis, is provided according to the invention, comprising an effective amount of at least one biomarker selected from the group consisting of the proteins Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, and Major urinary protein 1, or of a preferably synthetic fragment, being 7-24 amino acids in length, of at least one of said proteins, or comprising at least one antibody directed against said at least one biomarker, in particular for use in diagnosing or treatment monitoring of dysplasia or cancer, preferably of liver cell dysplasia or HCC, associated with increased EGF receptor tyrosine kinase activity in a patient.

The term “body fluid” according to the invention is directed to any body fluid of a subject, in particular to intracellular fluid (or cytosol, respectively), blood, plasma, serum or urine, whereas blood serum or plasma is the preferred body fluid within the context of the invention.

Said composition is preferably used for the production of a diagnostic agent, in particular of a diagnostic standard for body fluid analysis, or, more particular, for the production of a diagnostic agent for qualifying the EGF receptor tyrosine kinase activity in a patient suffering or being susceptible to cancer or for classifying a patient suffering from or being susceptible to HCC.

Within this context, said composition is particularly used for the production of a diagnostic agent for predicting or monitoring the response of a cancer patient to a method of treating cancer comprising administering a EGF receptor tyrosine kinase activity modulator, e.g. Sorafenib.

In yet another preferred embodiment, said composition further comprises an effective amount of a protease, in particular of trypsin, thus enabling a further enhancement of the system sensitivity.

Said composition, in particular the protease digest thereof, may be preferably used for producing a vaccine for the immunization of an animal in order to produce polyclonal antibodies specific for the at least one biomarker.

Within the context of the invention, also a method of qualifying the EGF receptor tyrosine kinase activity in a patient suffering or being susceptible to cancer or for classifying a patient suffering from or being susceptible to HCC is provided, comprising determining in a body fluid sample of a subject suffering from or being susceptible to cancer at least one biomarker selected from a first group consisting of the proteins Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, and/or at least one biomarker selected from the second group consisting of the proteins 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondrial), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, Major urinary protein 1, wherein the body fluid level of the at least one biomarker of said first group being significantly higher and/or the body fluid level of the at least one biomarker of said second group being significantly lower than the level of said biomarker(s) in the body fluid of subjects without cancer associated with increased activity of EGF receptor tyrosine kinase is indicative of induced EGF receptor tyrosine kinase activity in the subject.

In one embodiment, said method is preferably used for predicting the response of a cancer patient to a method of treating cancer comprising administering a EGF receptor tyrosine kinase activity modulator, wherein the body fluid level of the at least one biomarker of said first group being significantly higher and/or the body fluid level of the at least one biomarker of said second group being significantly lower than the level of said biomarker(s) in the body fluid of subjects without cancer associated with increased activity of EGF receptor tyrosine kinase is indicative that the subject will respond therapeutically to a method of treating cancer comprising administering a EGF receptor tyrosine kinase activity modulator.

In another embodiment, said method is used for monitoring the therapeutically response of a cancer patient to a method of treating cancer comprising administering an EGF receptor tyrosine kinase activity modulator, wherein the body fluid level of the at least one biomarker of said first group before and after the treatment and/or the body fluid level of the at least one biomarker of said second group before and after the treatment is determined, and a significant decrease of said body fluid level(s) of the at least one biomarker of said first group and/or a significant increase of said body fluid level(s) of the at least one biomarker of said second group after the treatment is indicative that the subject therapeutically responds to the administration of the EGF receptor tyrosine kinase activity modulator.

Moreover, a procedure to screen for and to identify drugs against cancer associated with an increased EGF receptor tyrosine kinase activity is provided, comprising determining in a body fluid sample of a transgenic cancer mouse, including a HCC bearing mouse, being treated with a compound to be tested, in particular of a mouse whose genome comprises a non natural sequence coding for IgEGF, at least one biomarker selected from the first group consisting of the proteins Arginase type II, 4931406C07Rik (Ester hydrolase C11orf54 homolog), Akr1c12 protein, Alanyl-tRNA synthetase, Aldo-keto reductase family 1 member C14, Aldo-keto reductase family 1 member C6, Aldolase 3, Alpha glucosidase 2, Beta 5-tubulin, Cai protein (Pdia4), cDNA sequence BC021917 (dihydroxyacetone kinase 2 homolog), Farnesyl diphosphate synthetase, Fatty acid binding protein 5 epidermal, Inosine triphosphatase, Interleukin 25, Kininogen 1, LIM and SH3 protein 1, Major vault protein, Nucb1 protein, Poly(rC) binding protein 2; heterogeneous nuclear ribonucleoprotein X, Psmd11 protein, RIKEN cDNA 2410004H02, Rps12 protein, Sars1 protein, Sorcin, T43799 proteasome protein p45/SUG [imported], Uap1l1 protein, v-crk sarcoma virus CT10 oncogene homolog, 170 kDa glucose regulated protein GRP170 precursor, Apolipoprotein A1, Apolipoprotein E, Carboxylesterase precursor, Fibrinogen-alpha polypeptide, Fibrinogen-beta polypeptide, Fibrinogen-gamma polypeptide, Pzp (A2mg protein), Serum amyloid P-component, and/or at least one biomarker selected from the second group consisting of the proteins 2-hydroxyphytanoyl-CoA lyase, Branched chain ketoacid dehydrogenase E1 alpha polypeptide, Butyryl Coenzyme A synthetase 1, Dhdh protein, Diacetyl/L-xylulose reductase, Dmgdh protein (Dimethylglycine dehydrogenase, mitochondria!), Enoyl coenzyme A hydratase 1 peroxisomal, Hypothetical protein LOC68347, Lysophospholipase 1, Mitochondrial acyl-CoA thioesterase 1, PREDICTED: agmatine ureohydrolase (agmatinase), RIKEN cDNA 1810013B01 (abhydrolase domain containing 14b), Serpinb1a protein, Major urinary protein 1,

wherein the body fluid level of the at least one biomarker of said first group being significantly lower and/or the body fluid level of the at least one biomarker of said second group being significantly higher than the level of said biomarker(s) in the body fluid of an untreated transgenic cancer mouse is indicative of the therapeutic effect of said compound as a EGF receptor tyrosine kinase activity modulator.

For implementing the methods or uses according to the invention, in particular for determining the presence, concentration or expression of a protein, it may be 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, mass spectrometra, and/or biosensory investigation—whereby by amplification, isolation, immobilization and/or detection and/or by combinations of thereof a particularly simple conversion of the methods or according to invention is made possible for the examined sample, in particular if furthermore a statistic analysis is accomplished.

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

Since the liver produces many blood proteins as pre-proteins which are then delivered into the blood and activated by proteolytic enzymes, serum proteins may be used for disease diagnosis and prognosis. In the present study, two-dimensional electrophoresis (2-DE) and MALDI-MS were employed to identify disease regulated proteins involved in a HCC-transgenic mouse model. A total of 98 proteins showed significant differences in expression levels between non-transgenic healthy controls and HCC-bearing mice. These included 3-phosphoglycerate dehydrogenase, the capping protein alpha 1 subunit, the fibrinogen alpha polypeptide, and the interleukin 1 receptor antagonist protein, which were found exclusively in HCC-bearing mice. On the contrary, arginosuccinate synthetase 1, dimethylglycine dehydrogenase, and glycine N-methyltransferase are examples of proteins identified only in non-transgenic healthy control samples. A total of 42 new proteins differentially expressed have been related for the first time to HCC, among them two aldo-keto reductase family 1 proteins, members C14 and C6, interleukin 25 and the v-crk sarcoma virus CT10 oncogene homolog. Several works on proteomic analysis of HCC-employing cell lines [8-12] and/or animal models [13-15] already reported novel disease-associated biomarkers, but further validation of the results in human clinical samples will be necessary [16]. Finally, serum and liver proteomes of HCC bearing mice were compared and 10 proteins were found to have the same regulation both in sera and liver tissue, thus, providing a direct link between regulated proteins of the tumor and serum proteomes. Obviously, these are highly interesting biomarker candidates for HCC.

Materials and Methods

Materials:

A UP 200S Sonicator (Dr. Hielscher GmbH, Stuttgart, Germany) was used to homogenize the samples. For the first dimension, immobilized pH-gradient (IPG) strips (17 cm, pH 5-8, 7-10) were purchased from Bio-Rad (Hercules, Calif., USA). The pre-fractionation was carried out with a Rotofor Cell (Bio-Rad). The focusing chamber was a Protean Isoelectric Focusing (IEF) Cell (Bio-Rad). For the second dimension, a Protean plus Dodeca Cell (Bio-Rad) was used. Reagents (tris, urea, thiourea, CHAPS, dithiothreitol, bromophenol blue, glycerin, sodium dodecyl sulfate, glycine, temed, ammoniumperoxodisulfate, ammonium sulfate, ammonium

bicarbonate, colloidal Coomassie Blue, and acrylamide) were purchased from Roth (Karlsruhe, Germany). Iodacetamide was from SERVA (Heidelberg, Germany). Benzonase was purchased from Novagen (Darmstadt, Germany). Ampholytes (Biolyte 3-10) were purchased from Bio-Rad. DeStreak was purchased from Amersham Bioscience (Freiburg, Germany).

Animal Care:

A total of n=12 C57/BI6 male mice (aged 6-8 months), weighing 25-33 g, were housed in Makrolon Type III cages. Drinking water and food (V1124-000, SSNIFF, The Netherlands® were given ad libitum. The temperature and relative humidity were 22±2° C. and 40-70%, respectively. Furthermore, a 12-h day and night cycle was used. For liver explantation, mice were anesthetized with ketamine 10% 100 μL/100 g and xylazine 2% 50 μL/100 g, and after surgical removal the liver was washed until free of blood.

Mouse Liver Sample Preparation:

Approximately 0.1 g of the liver sample was ground in a mortar under liquid nitrogen flow. Then, the samples were processed with 0.5 mL of a buffer containing 40 mM tris base, 7 M urea, 4% CHAPS, 100 mM DTT, and 0.5% (v/v) biolyte 3-10 first (LB2). The suspensions were homogenized by sonication (3×20 s) and after addition of 3 μL of benzonase (endonuclease that degrades DNA and RNA) were incubated at room temperature for 20 min. The samples were then centrifuged at 12,000 g for 20 min. The pellets were washed and sonicated for 5 min with a further 0.5 mL of LB2 and centrifuged at 12,000 g for another 20 min, and the resulting two fractions of supernatant were collected (extract A). Finally, the pellets were redissolved with 0.5 mL of buffer containing 40 mM tris base, 5 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, and 0.5% (v/v) biolyte 3-10 (LB3), sonicated, and centrifuged at 12,000 g for 20 min. The pellet was collected, and the supernatant was marked as extract B. From the same animals, a further 0.1-g portion was ground in a mortar, but was now treated with 0.5 mL of LB3. The suspensions were sonicated, incubated with benzonase, and centrifuged. The pellets were then washed with another 0.5 mL of LB3, sonicated, and centrifuged, and the supernatants were collected (extract C). Proteome mapping was done under a variety of conditions, e.g. extraction with lysis buffers 2 and 3. In addition, proteins were separated at two different pH ranges (5-8 and 7-10). A total of 4 experiments were carried out, and duplicate measurements were run for each experiment. The protein concentration of all extracts was determined using the Bradford assay.

Liquid-Phase IEF Pre-Fractionation:

Liquid-phase IEF pre-fractionation was performed in the Rotofor Cell system (Bio-Rad) following the supplier's instructions. Ion exchange membranes were equilibrated overnight in the appropriate electrolyte (anion exchange membranes in NaOH 0.1 M and cation exchange membranes in H3PO4 0.1 M). After four runs ion exchange membranes were always discarded and new membranes were replaced for the other samples. For each run, the electrode chambers were filled with appropriate fresh electrolytes (30 mL). Initially, the cell was filled with pure water and run for 5 min at 5 watts constant power to remove residual ionic contaminants from the membrane core and ion exchange membranes. Approximately 32 mL of LB2 were used to fill the cell. A total of 60 mg of total proteins in approximately 2 mL of LB2 were added to the cell to reach the maximum loadable volume (40 mL). Focusing started at 12 watts constant power. After approximately 4 hours the voltage increased to 3000 V and the wattage decreased to 3 W. The focused proteins were harvested in 20˜1.5 mL fractions, and pH values were checked. Fractions having pH values between 3 and 7.0 were collected and denoted “A-a” (acid). Fractions having pH values >7.0 were collected and denoted “A-b” (basic). Again, the protein concentration was determined for both fractions (A-a and A-b) by the Bradford method. Approximately 30 mg of protein were recovered at the end of the liquid-phase IEF pre-fractionation from an initial 60-mg load. The losses are accounted for by the multi-step pre-fractionation procedure, but are not the result of a precipitate that could not be dissolved in the lysis buffer described herein. After each run the membrane core was cleaned with NaOH 0.1 M overnight and sonicated for 5 min in water before the new focusing.

Two-Dimensional Gel Electrophoresis Isoelectric Focusing (IEF)—First Dimension:

IEF was performed using precast linear IPG strips. The 17-cm IPG strips 7-10 and 5-8 were loaded with 1.5 mg of proteins by active rehydration (12 h, 50 V). Samples destined to be separated by IPG strips 7-10 received an excess of hydroxyethyldisulphide (HED) (DeStreak™) prior to the focusing run. Focusing began at 250 V for 20 min in rapid mode, 10,000 V for 5 h in linear mode, and 10,000 V for 50,000 Vh in rapid mode (for the IPG strips 5-8). IEF for the strips 7-10 was carried out at 250 V for 60 min in rapid mode, 10,000 V for 3 h in linear mode, and 10,000 V for 50,000 Vh in rapid mode. Each sample was analyzed in duplicate. Control and HCC samples were run always at the same time (6 control and 6 HCC samples).

2-DE—Second Dimension:

After IEF, the IPG strips were either stored at −80° C. or transferred to 10 mL of equilibration buffer (6 M urea, 30% w/v glycerin, 2% w/v SDS, 50 mM Tris-HCl pH 8.8) with 2% w/v DTT and 0.5% v/v bromophenol blue solution (0.25% w/v bromophenol blue, 1.5 M Tris-HCl pH 8.8, 0.4% w/v SDS) and incubated for 20 min at room temperature. Strips were removed and incubated in equilibration buffer with 4% w/v iodoacetamide and 0.5% v/v bromophenol blue solution for further 20 min at room temperature. Finally, the strips and 10 μL SDS-PAGE molecular weight standard on filter paper were placed on top of the 20 cm×20.5 cm 12% second-dimension gel (12% v/v acrylamide/bis solution, 375 mM Tris, pH 8.8, 0.1% v/v SDS, 1/2000 TEMED, 0.05% v/v APS). Both were fixed in place with a 0.5% w/v agarose overlay. Gels were run in PROTEAN Plus Dodeca cell from Bio-Rad at 70 V for approximately 14 h, followed by 200 V until the bromophenol blue dye reached the bottom of the gel. The running buffer (25 mM Tris, 0.2 M glycin, 0.1% SDS) was cooled externally to 16° C. Gels/proteins were fixed overnight in 30% ethanol, 2% phosphoric acid, and washed 3×20 min with 2% phosphoric acid. The gels were equilibrated with 15% ammoniumsulfate, 18% ethanol, 2% phosphoric acid for 15 min and finally stained with colloidal Coomassie Blue for 48 h.

Gel Scanning And Image Analysis:

After staining, gels were washed 10 min with pure water and scanned on a Molecular FX Scanner Bio-Rad at 100 μm resolution. Protein spots were imaged first automatically and then manually and analyzed using the PDQues™ software Bio-Rad The normalization was carried out in total density in gel mode according to the manufacturer's recommendation.

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS):

Gels were excised using the spot cutter of Bio-Rad and placed into 96-well microtiter plates. Excised gel spots were washed manually with 20 μL of water for 10 min and destained twice, first with 15 μL ammonium bicarbonate 50 mM for 5 min and then with 15 μL 50% ammonium bicarbonate 50 mM—50% acetonitrile for 5 min. Finally, the gel particles were covered by acetonitrile until gel pieces shrunk and left to dry for 10 min. All gels/proteins were digested manually in situ with 4 μL of ammonium bicarbonate 50 mM containing 20 ng rypsin (Sequencing Grade Modified Trypsin Promega). After 15 min each gel piece was re-swelled with 10 μL of ammonium bicarbonate 50 mM and incubated for 4 h at 37° C. After 4 h the reaction was stopped by adding 10 μL of trifluoroacetic acid 1% containing 1.5% (w/v) n-octyl-beta-D-glucopyranoside (OGP) (AppliChem). For the application of the samples, 4 μL of peptide solution were loaded onto an MTP Anchor Chip Target 600/384 (Bruker Daltonics) previously prepared with a saturated solution of matrix, alpha-cyano-4-hydroxy-cinnamic acid (alpha-HCCA) (Bruker Daltonics). An external calibration was performed by spotting on the 96 calibration positions of the Anchor Chip Target 1 μL of the peptide calibration standards (Bruker Daltonics) containing the following peptides: angiotensin 11 (1046.5420 Da), angiotensin I (1296.6853 Da), substance P (1347.7361 Da), bombesin (1619.8230 Da), ACTH clip 1-17 (2093.0868 Da), ACTH clip 18-39 (2465.1990 Da), somatostatin 28 (3147.4714 Da), and OGP 1.5% (w/v). Samples were analyzed in a MALDI-TOF-TOF spectrometer (Ultraflex, Bruker Daltonics) using an accelerating voltage of 25 kV for the Peptide Mass Fingerprint (PMF) mode. When necessary, MALDI-Post Source Decay (PSD) analysis was carried out using the LIFT special technique delivered by Bruker (the basic idea of LIFT is to lift the potential to fragment the selected peptides of interest). Peptide matching and protein searches were performed automatically with the MASCOT software. For the PMF search the parameters were the following: C-carbaimidomethyl (fixed modification), M-oxidation (variable modification), monoisotopic (mass value), 100 ppm (peptide mass tolerance), 1 (max missed cleavege), mammalia (taxonomy). Five matching peptides and at least 10% peptide coverage of the theoretical sequences was the minimal requirement for an identity assignment. For the MS/MS search (PSD) the parameters were the same except the peptide mass tolerance, which was 200 ppm. The identified Proteins™ individually, and only mouse proteins or highly homologous sequences from other mammalian species, like Homo sapiens or Rattus Norvegicus, having pl and Mw values close to the theoretical, were considered.

Immunohistochemistry:

Livers, dissected from EGF-overexpressing mice aged between 7-9 month, were fixed in 4% buffered paraformaldehyde and embedded in paraffin. 5 μm thick sections were deparaffinized and rehydrated through a descending alcohol series followed by a 4 min washing step in destilled H2O. Then, antigen retrieval was performed in citrate buffer (pH 6) by autoclaving the sections 15 min at 121° C. The Envision kit (Dako; Ham burg; Germany) was used for immunohistochemistry. The slides were rinsed with destilled H2O and after a 5 min incubation step in tris-buffered saline (washing buffer), endogenous peroxidase activity was blocked with DAKO Peroxidase blocking Reagent for 5 min followed by a second washing step. Thereafter, the sections were blocked for 10 min with protein-block serum free (Dako) and incubated with primary antibodies for 45 min. Details of antibody dilutions with washing buffer are given in table 1. In the case of goat primary antibodies a rabbit-anti-goat bridging antibody (Dako) was employed. Specifically, the bound primary antibodies or bridging antibodies were detected by use of labelled polymer HRP Anti-Rabbit secondary antibody (Envision Kit; Dako) and the immunoreactivity was visualized by DAKO Liquid DAB Substrate Chromogen System in an 5 min incubation. Finally, the sections were couterstained with Harris Haematoxylin for 2 min, dehydrated in an ascending alcohol series, coverslipped and examined under a light microscope. (Leica; Jülich; Germany)

Antibody
Cat. Nr.
Dilution
Company

Antibody to GDI 2
10116-1-AP
1:100
Proteintech Europe

Ltd.; Manchester, UK

Arginase II (H-64)
sc-20151
1:100
Santa Cruz;

Heidelberg; Germany

Antibody to CAPZA1
11806-1-AP
1:400
Proteintech Europe

Ltd.; Manchester; UK

hnRNP L antibody
ab65049
1:1000
Abcam;

Cambridge; UK

Rabbit Anti-beta
ab52623
1:100
Abcam;

Tubulin Monoclonal

Cambridge; UK

Antibody

Amphiregulin (M-18)
sc-5797
1:150
Santa Cruz;

Heidelberg; Germany

Epiregulin (T-19)
sc-25232
1:150
Abcam;

Cambridge; UK

HNF-4α
sc-6556
1:250
Santa Cruz;

Heidelberg; Germany

Results

Image Analysis of Differentially Expressed Proteins:

Gels were scanned on a Molecular FX Scanner Bio-Rad at 100 μm resolution. Then, protein spots were imaged first automatically and then manually, and analyzed using the PDQuest™ software Bio-Rad (version 8.1). A total of 122 spots/proteins showed differences in expression levels between non-transgenic controls and HCC mice, and detailed information on these spots is listed in Table 1. Notable, FIG. 1 depicts some examples. Among them, 98 spots/proteins were statistically significantly regulated (p≦0.05); 62 of these spots/proteins were significantly upregulated (ratioHCC/control≧2), such as fibrinogen β, vimentin, Cu/Zn superoxide dismutase, and apolipoprotein E (FIG. 2-A, B, C, D), whereas 36 spots/proteins were significantly down-regulated (ratioHCC/control≦0.6), among them arginase 1, Dhdh protein, glutathione peroxidase 1, and predicted: agmatine ureohydrolase (FIG. 3-A, B, C, D) (see Table 1).

Identification of Proteins by MS Analysis:

Previously, a reference 2-DE map of mouse liver proteins has been created, consisting of more than 500 proteins [4]. Moreover, a reference 2-DE map of mouse serum proteins was reported in the same transgenic disease model and 25 serum proteins were detected to be differentially expressed, which are involved in a variety of cellular and metabolic pathways. Among them alpha-fetoprotein, clusterin, fibrinogen-α, fibrinogen-γ, serum amyloid component P, and some apolipoproteins were significally overexpressed [4]. In this work, a total of 122 differentially expressed spots/proteins were identified (Table 1), of which 98 were statistically significant. Interestingly, differentially expressed protein spots were found to be products of the same gene. They were: albumin (5 upregulated spots), alpha enolase (4 downregulated spots), apoliproptein A-I (2 upregulated spots), ATP synthase, H+ transporting, mitochondrial (2 downregulated spots), fibrinogen beta (2 upregulated spots), glycine N-methyltransferase (3 spots, in controls only), hsp60 (2 downregulated spot spots), nit protein 2 (2 downregulated spots), peroxiredoxin 6 (1 upregulated spot and 1 downregulated spot), and 4931406C07Rik (2 upregulated spots) (see Table 1). A total of 47 novel proteins were found to be HCC-associated. They are marked with an asterisk in Table 1. When disease regulated serum and liver proteins were compared, a total of 10 proteins were found to be regulated in common, therefore providing a direct link between regulated proteins of the tumor and serum proteome (Table 2). Among them, serum Afp was upregulated. Regulation of Afp and other proteins was confirmed by Western blot analysis. (FIG. 4-A, B, C, D, E).

An isoform of apolipoprotein 1 (gi|26345182), carboxylesterase precursor, fibrinogen alpha and fibrinogen gamma, were expressed exclusively in tumor samples. Fibrinogen beta was expressed exclusively in HCC serum, but displayed upregulation in HCC liver samples as well (ratio HCC/control=3.5). Apolipoprotein E was upregulated both in serum and liver samples, the ratio HCC/control being 2.2 and 3.9, respectively. Two spots of alpha-2-macroglobulin were upregulated in serum of HCC-bearing mice (spot 1: ratioHCC/control=1.8; spot 2: ratioHCC/control=3.2). In liver tissues, alpha-2-macroglobulin was exclusively expressed in tumor samples. Finally, serum amyloid component P was upregulated in serum up to 10-fold, and was expressed in HCC liver tissue only (Table 2).

Immunohistochemistry of Disease-Regulated Proteins:

To further confirm disease regulation in HCC a total of 8 proteins were selected for immunohistochemistry. Five of them were picked from the list of new but differently expressed proteins (see table 1). Additionally amphiregulin and epiregulin were chosen because of their importance as ligands in the EGF-signaling pathway. Also, HNF4α was selected for immunohistochemistry. This protein was shown to be significantly downregulated in HCC (74). As shown in FIGS. 5 and 6 immunohistochemistry confirms the regulation of the selected proteins. Indeed, arginase II, Capza1 GDI2 and amphiregulin were detected predominantly in the cytoplasmic compartment, whereas for hnRNPL and HNF4α nuclear expression was predominant. In tumors HNF4α was repressed and expression of tubulin β was observed particularly beneath the liver capsule.

Discussion

This study aimed for an identification of novel disease regulated proteins in HCC. The proteome of healthy and liver tissues from HCC tumor-bearing mice was analyzed using the 2-DE technique coupled with MALDI-TOF MS/MS and 122 mouse liver proteins were identified to be differentially expressed. Here, 42 novel proteins are reported to be HCC disease-associated with prominent examples being discussed below.

Extra-Cellular Space or Secreted Proteins were Upregulated in HCC Livers

In this study, 62 upregulated proteins were identified. Among these, 18 (29%) were extra-cellular or secreted proteins, including albumin (main function transport), three apolipoproteins, apoE, apoA4, and apoA-I (cholesterol and lipid metabolic processes), α-, β-, and γ-fibrinogen, plasminogen, kininogen (main function in fibrinolysis and coagulation), interleukin 25 and interleukin 1 receptor antagonist protein (IL-1ra) (main functions immune and inflammatory responses and proliferation). Notably, an isoform of apoA1 has been proposed to be a serum marker of HCC [17]. Whereas the interleukin 1 receptor antagonist protein was immunohistochemically detected in tumor cells in approximately 70% of hepatocellular adenomas and carcinomas, eventhough early preneoplastic hepatocytic foci as well as normal hepatocytes surrounding the lesions were negative; consequently, this protein might be used to stage liver tumors. Additionally, RT-PCR analysis confirmed that mouse hepatic tumors contain both secreted and intracellular forms of IL-1ra [18]. Indeed, changes at the proteome level in serum have been used to monitor the effect of treatments applied to HCC patients [19].

Mitochondrial Proteins Involved in Biosynthetic Processes were Down-Regulated

Notably, in the results 14 (39%) mitochondrial proteins were down-regulated. This is in agreement with the results of Chignard and Wei Sun, which suggested that mitochondria were altered significantly during carcinogenesis, mitochondrial proteins being the second largest proportion of dysregulated proteins identified in HCC [20, 21]. These include NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, prohibitin (DNA replication), glutathione peroxidase 1 (induction of apoptosis by oxidative stress, response to oxidative stress), and argininosuccinate synthetase 1 (ASS) (urea cycle, amino acid biosynthetic process). This protein is the first of two enzymes to convert citrulline to arginine. This pathway allows cells to synthesize arginine from citrulline, making this amino acid non-essential for the growth of most mammalian cells. Previous studies demonstrated that several human tumor cell lines were auxotrophic for arginine due to an inability to express ASS [22-27]. In the study, as described herein, ASS was found in 15 out of 24 control gels, but none in tumor gels (see Table 1). Another protein that was found exclusively in control gels (23 out of 24) is the glycine N-methyltransferase (GNMT). This protein is strongly downregulated in HCC eventhough its expression is abundant in liver [28]. This multifunctional protein is involved in maintenance of genetic stability and frequently downregulated in HCC [29] (see Table 1).

Newly Identified Disease-Related Proteins

We identified 122 mouse liver proteins to be differentially expressed. Some of these proteins have already been described in previous proteomic studies or are already known to be involved in hepatocarcinogenesis.

We therefore confirm earlier findings in an EGF-disease model of liver cancer. For these proteins references are reported in Table 1. The present study, however, compiles 42 newly identified and differentially expressed proteins which so far have not been described by previous proteomic studies to be involved in HCC. These proteins are marked with an asterisk in Table 1. Their functions cover a broad range spanning from metabolism to translation and signalling. For instance an elevated expression of proteins belonging to the carbohydrate metabolism was detected, like pyruvate kinase 3, aldolase 3 or alpha glucosidase 2 as well as of those involved in translation, like alanyl-tRNA synthetase (↑), eukaryotic translation elongation factor 2 (↑), or sars1 (↑). Proteins responsible for synthesis and degradation of lipids, steroids, fatty acids, and cholesterol, like the aldo-keto reductase family 1 (↑), butyryl coenzyme A synthetase 1 (↓), 2-hydroxyphytanoyl-CoA-lyase, enoyl coenzyme A hydratase 1, dihydrodiol dehydrogenase, lysophosphopholipase, mitochondrial acyl-CoA thioesterase 1 or farnesyl diphosphate synthetase (↑), which was also found to be upregulated in colonic cancers [71] are in contrast partially elevated in expression and partially downregulated. It was also found that RNA binding proteins like hnRNPL and Poly (rC) binding protein 2 were uniformly upregulated in HCC. Notably, hnRNPL was also upregulated in serum of patients diagnosed with HCC [72]. In contrast the ribosom-compononent rps12 and components of aminoacid metabolism like branched chain ketoacid dehydrogenase E1 or dimethyl glycine dehydrogease were repressed but proteasom components like psmd11 that was also found to be regulated in breast cancer [85] and p45/SUG was upregulated in tumor tissues. The results also show an enhanced expression of cytoskeletal proteins such as tubulin β 5 and Capza1 but immunohistochemical staining evidences differences in the localisation of these proteins beeing primarily associated with cells proximal to the capsule of the liver whereas Capza1 was strongly associated with the tumor foci. Also GDI2, a protein that functions in the cycling of Rab GTPases and arginase II, an non liver isoform of the urea cycle protein, were upregulated in HCC bearing mice (see FIG. 5). Notably the regulation of arginase II in lung cancer was already reported [73]. Moreover the actin-binding protein LASP1 was only found in tumorous tissues.

It is known to be upregulated in breast cancer [86] and could be responsible for migration of cancer cells. [87]. Other proteins with elevated expression are Kininogen, a part of the blood coagulation system and the precurser of kinin, and Pdia4 a disulfide bond isomerase, whereas Serpinb1a a serinproteinsase inhibitor was found to be downregulated in HCC mice. Also the calcium binding protein Sorcin and Nucleobindin 1, a protein that may play a role in calcium mediated signaling, could be a proteins of further interest. Sorcin was found to be correlated with multidrug resistance in human leukemia cells [88] were as nucleobindin1 is a potential biomarker for coloncancer [89]. Transthyretin, also upregulated in HCC, is a transport protein of thyroid hormons and synthesized by the liver. Interestingly its regulation was shown in thyroid cancer [90]. Noteworthy, among the newly identified 42 HCC-related proteins are the following: v-crk sarcoma virus CT10, an oncoprotein involved in the intracellular signaling cascade and in the activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway [30], and the 170 kDa glucose-regulated protein GRP170, an endoplasmic reticulum lumenal glycoprotein that may play a role in immunoglobulin folding. In fact, GRP170 was found to be precipitated with immunoglobulin in four different B cell hybridomas [31]. A summary of the biological functions and their previous reported tumor association is given in table 3. In a previous study to map the serum proteome of hepatocellular carcinoma induced by targeted overexpression of Egf to liver cells of transgenic mice, many immunoglobulins were found to be repressed or absent in serum samples of tumor-bearin mice, like the Ig K and L classes [4]. Whether GRP170 is responsible for the repression of immunoglobulins in the sera of Egf tumor-bearing mice needs further studies. (See table 3)

To identify disease proteins regulated in EGF-HCC-bearing transgenic mice, a proteomic approach has been used, as described herein, that consisted of two-dimensional electrophoresis (2-DE) and MALDI-MS/MS. A total of 98 proteins showed significant differences in expression levels between non-transgenic controls and HCC-bearing mice; 62 proteins were upregulated, whereas 36 proteins were downregulated. Although some of these proteins have already been described in previous proteomic studies or are already known to be involved in hepatocarcinogenesis, this work reports 42 new proteins differentially expressed in HCC-bearing mice. Additionally, regulation of the proteins was confirmed by immunohistochemistry and western blotting. Moreover, the results were compared and correlate with those of the previous work of the inventors, where sera of EGF induced HCC-bearing mice and of non-transgenic controls were studied. Thus, a total of ten disease-regulated liver proteins were identified as commonly regulated in sera and tumor tissue of HCC-bearing mice.

In conclusion, the study within the context of the invention identified a total of 42 proteins which so far have been unknown to be regulated in hepatocellular carcinoma and may enable an improved understanding of HCC disease. Many previously reported HCC-related proteins were also identified in the study. Serum and liver proteomes of tumor-bearing mice were studied and compared. Ten proteins were found to have the same regulation both in sera and liver tissue of the same EGF-HCC mouse model, providing a direct link between regulated proteins of the tumor and serum proteomes. Obviously, these are highly interesting biomarkers for HCC.

FIGURE CAPTIONS

FIG. 1. Upregulation and downregulation of some of the 122 deregulated mouse liver proteins. Spot 1: glycine N-methyltransferase, identified in control samples only; Spot 2: peroxiredoxin 6, upregulated in tumor samples (ratioHCC/control=2.3); Spot 3: peroxiredoxin 6, downregulated in tumor samples (ratioHCC/control=0.3); Spot 4: lysophospholipase 1, downregulated in tumor samples (ratioHCC/control=0.2); Spot 5: hypothetical protein LOC68347, downregulated in tumor samples (ratioHCC/control=0.4); Spot 6: glutathione peroxidase 1, downregulated in tumor samples (ratioHCC/control=0.3).

FIG. 2. Some of the 62 upregulated mouse liver proteins: fibrinogen 8 (FIG. 2-A), vimentin (FIG. 2-B), Cu/Zn superoxide dismutase (FIG. 2-C), and apolipoprotein E (FIG. 2-D).

FIG. 3. Some of the 36 downregulated mouse liver proteins: arginase 1 (FIG. 3-A), Dhdh protein (FIG. 3-B), glutathione peroxidase 1 (FIG. 3-C), and predicted: agmatine ureohydrolase (FIG. 3-D).

FIG. 4. Western blots of alpha-fetoprotein (A), fibrinogen gamma (B), serum amyloid component P (C), epidermal growth factor (D), and apolipoprootein M, which was identified in serum samples only (E); C=control, T=tumor.

FIG. 5 Immunohistochemical stainings of Arginase II, Capza1, GDI 2 and Tubulin β Conrols were treated with washing buffer instead of primary antibody.

FIG. 6
a-b Immunohistochemical stainings of hnRNPL, Amphiregulin HNF4a and Epiregulin Conrols were treated with washing buffer instead of primary antibody. For amphiregulin the control staining was done with blocking peptide.

Table 1. List of the 122 differentially regulated proteins. The proteins are sorted in alphabetical order, and the NCB, annotation is given in the accession number column. Molecular weight, pl, and MASCOT scores are also reported in the table. The column “Gels” indicates in how many different gels of the total 48 cut gels each protein was identified; the columns “C” (C=control) and “T” (T=tumor) indicate in how many control and tumor gels/samples each protein was identified; the columns “LB2” and “LB3” (LB=lysis buffer) indicate how many times each protein was identified in LB2 and/or LB3. Data like p-value, ratio T/C, and cellular location are also reported in the table. The column “references” reports citations for those proteins which have already been described to be HCC-associated proteins. * Proteins which so far have been unknown to be regulated in hepatocellular carcinoma Abbreviations: C, cytosol; Ck, cytoskeleton; M, mitochondria; N, nucleus; P, peroxisome; ES, extracellular space; ER, endoplasmic reticulum; G, golgi; L, lysosome; MEM, membrane; MM, mitochondrial matrix; S, secreted.

Table 2 The ten differentially expressed mouse proteins common to serum and liver samples. For each sample the ratioHCC/control is given.

Table 3 Biological function and tumor association of new and differentially regulated proteins.

Table 4: Overview of the 122 proteins in comparison with the state of the art, wherein “x” denotes “References” ([ . . . ]) with regard to “HCC” or “Other cancer” or the protein is “unknown”, so far, in the context of cancer.

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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 following claims.

TABLE 1

Accession

Mascot

Ratio
Cellular

No.
Protein
number
Mr
pl
score
Gels
C
T
LB2
LB3
p-value
T/C
location
References

1*
170 kDa glucose regulated
gi|7643979
111
5
214
10
0
10
8
2

T
ER, ES
[31, 32]

protein GRP170 precursor

2*
2-hydroxyphytanoyl-CoA
gi|18204150
63.6
5.9
261
32
19
13
22
10
0.027
0.38
P

lyase

3
3-phosphoglycerate
gi|52353955
56.6
6
204
14
0
14
11
3

T

[33]

dehydrogenase

4*
4931406C07Rik (Ester
gi|71059921
35
5.8
184
35
17
18
24
11
0.033
1.5
N

hydrolase C11orf54

homolog) (spot 4342)

5*
4931406C07Rik (Ester
gi|71059921
35
5.8
195
33
15
18
22
11
0.244
1.9
N

hydrolase C11orf54

homolog) (spot 4349)

6
Acylpeptide hydrolase; N-
gi|22122789
79.9
5.2
182
5
0
5
0
5

T

[34]

acylaminoacyl peptide

hydrolase

7*
Akr1c12 protein
gi|15215042
37
6.1
135
24
14
10
15
9
0.301
1.9

8
Akr1c18 protein (aldo-keto
gi|19527284
37.2
5.9
189
11
0
11
5
6

T
C
[21]

reductase family 1,

member C18)

9*
Alanyl-tRNA synthetase
gi|34610207
106.9
5.4
178
4
0
4
0
4

T
C

10
Albumin 1 (spot 3707)
gi|33859506
68.7
5.7
444
43
21
22
31
12
0.068
3.88
C, ES
[21, 35]

11
Albumin 1 (spot 3712)
gi|33859506
68.7
5.7
416
40
20
20
30
10
0.25
2.04
C, ES
[21, 35]

12
Albumin 1 (spot 4506)
gi|33859506
68.7
5.7
423
40
20
20
31
9

T
C, ES
[21, 35]

13
Albumin 1 (spot 4702)
gi|33859506
68.7
5.7
355
42
20
22
31
11
0.069
1.85
C, ES
[21, 35]

14
Albumin 1 (spot 5509)
gi|33859506
68.7
5.7
473
38
18
20
30
8
0.392
3.59
C, ES
[21, 35]

15*
Aldo-keto reductase family
gi|19527294
37.2
5.9
189
11
0
11
5
6

T

1, member C14

16*
Aldo-keto reductase family
gi|13487925
37
8.5
120
10
0
10
7
3

T
C

1, member C6

17
Aldolase 1, A isoform
gi|53733633
39.3
9.2
213
12
1
11
11
1

T

[36]

18*
Aldolase 3
gi|60687506
39.4
6.5
153
5
0
5
4
1

T
M

19
Alpha enolase (spot 4501)
gi|58476212
47.1
6.4
341
45
22
23
33
12
0.24
0.22
C
[33, 37-39]

20
Alpha enolase (spot 4516)
gi|58476212
47.1
6.4
338
40
21
19
30
10
0.161
0.47
C
[33, 37-39]

21
Alpha enolase (spot 4524)
gi|58476212
47.1
6.4
338
42
22
20
31
11
0.021
0.53
C
[33, 37-39]

22
Alpha enolase (spot 5510)
gi|58476212
47.1
6.4
330
45
22
23
34
11
0.234
0.76
C
[33, 37-39]

23*
Alpha glucosidase 2
gi|26326711
106.9
5.6
269
5
0
5
0
5

T
ER, G

24
Annexin A6
gi|31981302
75.9
5.3
267
5
0
5
0
5

T
C
[34, 35]

25
Apoa4 protein
gi|14789706
45
5.3
224
21
6
15
18
3
0.011
2.77
ES
[40]

26
Apoe
gi|71060041
35.9
5.5
220
31
14
17
21
10
0.01
3.97
ES
[34, 41]

27
Apolipoprotein A-I (spot
gi|26345182
30.6
5.4
173
38
21
17
28
10
0.001
5.4
S
[33, 40, 42]

2215)

28
Apolipoprotein A-I (spot
gi|26345182
30.6
5.4
170
30
18
12

T
S
[40, 42]

3204)

29
Arginase 1, liver
gi|7106255
34.8
6.6
306
38
23
15
28
10
0.013
0.32
C
[37, 43-45]

30*
Arginase type II
gi|6753110
38.9
6.1
161
6
0
6
6
0

T
M

31
Argininosuccinate
gi|6996911
46.6
8.5
175
15
15
0
10
5

C
M
[22-27, 46]

synthetase 1

32
ATP synthase, H+
gi|51980458
18.7
5.3
148
37
20
17
25
12
0.002
0.33
M
[47]

transporting, mitochondrial

F0 complex, subunit d (spot

2120)

33
ATP synthase, H+
gi|51980458
18.7
5.3
145
35
19
16
22
13

C
M
[47]

transporting, mitochondrial

F0 complex, subunit d (spot

3203)

34*
Beta 5-tubulin
gi|18088719
49.7
4.7
267
11
1
10
7
4

T
Ck

35*
Branched chain ketoacid
gi|31982494
50.4
8.4
237
26
18
8
20
6
0.018
0.5
MM

dehydrogenase E1, alpha

polypeptide

36*
Butyryl Coenzyme A
gi|16905127
64.8
6.6
119
4
4
0
0
4

C
MM

synthetase 1

37*
Cai protein (Pdia4)
gi|45219865
65
5.9
267
5
0
5
1
4

T
ER

38
Capping protein alpha 1
gi|595917
32.8
5.2
114
11
0
11
7
4

T
Ck
[33]

subunit (Capza1)

39
Carbamoyl-phosphate
gi|8393186
164.6
6.3
350
31
22
9
22
9
0.023
0.2
M, C
[21, 34, 46, 48,

synthetase 1, mitochondrial

49]

40
Carboxylesterase MH1
gi|14331135
61.8
6.2
168
4
0
4
0
4

T
ER
[36, 50]

41*
cDNA sequence BC021917
gi|21703976
59.7
6.3
170
4
0
4
1
3

T

(dihydroxyacetone kinase 2

homolog)

42
Creatine kinase, brain
gi|10946574
42.7
5.3
181
4
0
4
0
4

T
M, C
[33]

43
Cryz protein
gi|13277837
35.3
8.2
84
5
5
0
5
0

C
C
[51]

44
Cu/Zn superoxide
gi|226471
15.8
5.9
186
39
21
18
27
12
0.007
2.92

[33, 42]

dismutase 1513495A

45
DEMSMC malate
gi|319837
36.5
5.9
138
31
17
14
19
12
0.00004
0.27
C
[52]

dehydrogenase, cytosolic

46*
Dhdh protein
gi|21618806
36.6
5.7
209
26
17
9
16
10
0.00012
0.17

47*
Diacetyl/L-xylulose
gi|50400594
25.7
7.8
228
5
5
0
0
5

C
MEM

reductase

48*
Dmgdh protein
gi|12836171
97.3
7.6
184
8
8
0
5
3

C
M

(Dimethylglycine

dehydrogenase,

mitochondrial)

49*
Enoyl coenzyme A
gi|7949037
36.1
7.4
162
26
11
15
21
5
0.026
0.49
M, P

hydratase 1, peroxisomal

50
Eukaryotic translation
gi|33859482
95.3
6.3
208
6
0
6
3
3

T
C
[91]

elongation factor 2

51
Eukaryotic translation
gi|124231
16.8
5.1
115
10
5
5
7
3
0.106
3.4
C, N
[34]

initiation factor 5A (eIF-5A)

52*
Farnesyl diphosphate
gi|19882207
40.6
5.4
158
5
0
5
0
5

T
C

synthetase

53*
Fatty acid binding protein 5,
gi|6754450
15.1
6.2
149
15
2
13
13
2
0.09
27.9
C

epidermal

54
Fibrinogen, alpha
gi|33563252
61.3
7
144
15
0
15
10
5

T

[46, 48]

polypeptide

55
Fibrinogen, B beta
gi|33859809
54.8
6.5
238
35
15
20
23
12
0.0011
2.41
ES
[48]

polypeptide (spot 5602)

56
Fibrinogen, B beta
gi|33859809
54.8
6.5
244
33
15
18
22
11
0.187
3.54
ES
[48]

polypeptide (spot 5612)

7
Fibrinogen, gamma
gi|18044708
49.4
5.5
196
12
1
11
5
7

T
ES
[43, 46]

polypeptide

58
FK506 binding protein 4
gi|6753882
51.6
5.5
154
4
0
4
1
3

T
C, N
[33, 34]

59
GDP dissociation inhibitor 2
gi|38197560
50.5
5.8
220
5
0
5
0
5

T
G, C,
[34]

(GDI 2)

MEM

60
Glutathione peroxidase 1
gi|6680075
22.3
6.2
215
25
18
7
18
7
0.00002
0.28
C, M
[53-55]

61
Glutathione S-transferase,
gi|6680121
25.7
7.6
222
11
1
10
7
4

T
C
[21, 34]

mu 2

62
Glycine N-
gi|34013296
32.7
6.9
214
23
23
0
17
6

C
C
[21, 29, 43, 56]

methyltransferase (spot

4256)

63
Glycine N-
gi|34013296
32.7
6.9
226
22
22
0
15
7

C
C
[21, 29, 43, 56]

methyltransferase (spot

5269)

64
Glycine N-
gi|34013296
32.7
6.9
218
20
20
0
15
5

C
C
[21, 29, 43, 56]

methyltransferase (spot

9105)

65
Glycyl-tRNA synthetase
gi|21264024
81.9
6.2
180
5
0
5
0
5

T
C
[57]

66
Haao protein (3-
gi|15277547
32.8
6
420
35
22
13
27
8
0.006
0.24
C
[34]

hydroxyanthranilate 3,4-

dioxygenase)

67
Hal protein
gi|35505393
72.2
5.9
301
6
6
0
0
6

C
C
[58]

68
Hemopexin
gi|23956086
51.3
9
231
16
2
14
11
5
0.323
7.27
ES
[59]

69
Heterogeneous nuclear
gi|33667042
60.1
6.6
156
8
0
8
8
0

T
N
[33]

ribonucleoprotein L

(hnRNPL)

70
HSP60 (spot 2604)
gi|1334284
57.9
5.3
298
15
7
8
6
9
0.008
0.4
MM
[33, 34, 50]

71
HSP60 (spot 2610)
gi|1334284
57.9
5.3
298
14
7
7
6
8
0.012
0.5
MM
[33, 34, 50]

72*
Hypothetical protein
gi|58037115
22.7
5.8
125
29
15
14
21
8
0.014
0.39

LOC68347

73*
Inosine triphosphatase
gi|31982664
21.9
5.4
139
5
0
5
5
0

T
C

74
Interleukin 1 receptor
gi|238585
17.7
5.5
121
7
0
7
6
1

T
S, C
[18, 50-62]

antagonist protein

75*
Interleukin 25
gi|18250288
18
5.9
89
13
6
7
9
4
0.23
2.1
ES

76*
Kininogen 1
gi|12963497
47.9
5.7
217
21
8
13
19
2
0.091
2.8
ES

77
Lamin-A/C
gi|15929761
74.2
6.6
199
12
0
12
10
2

T
N
[21, 33]

78*
LIM and SH3 protein 1
gi|6754508
30
6.7
156
7
0
7
6
1

T
Ck

79
Liver fructose-1,6-
gi|6688689
36.9
6.2
281
29
18
11
17
12
0.013
0.35

[34, 43, 48]

bisphosphatase

80*
Lysophospholipase 1
gi|6678760
24.7
5.9
134
25
13
12
20
5
0.25
0.25
M

81
Major urinary protein
gi|1839508
18.7
4.7
138
6
6
0
6
0

C
S
[18, 63]

82*
Major vault protein
gi|12003287
96
5.4
238
4
0
4
0
4

T
C

83
MAWD binding protein
gi|31560132
32
5.1
318
25
19
6
19
6
0.065
0.38

[34, 37, 43]

homolog 1

84*
Mitochondrial acyl-CoA
gi|40538846
49.6
7
123
6
6
0
1
5

C
M

thioesterase 1

85
NADH dehydrogenase
gi|21312012
20
8.8
145
4
4
0
4
0

C
M
[48, 64]

(ubiquinone) 1 alpha

subcomplex, 8

86
NADH dehydrogenase
gi|26331822
79.7
5.5
240
5
5
0
0
5

C
M
[34]

(ubiquinone) Fe—S protein 1

(Ndufs1)

87
NADH dehydrogenase
gi|19526814
51
8.5
151
4
0
4
3
1

T
M, ES
[64]

(ubiquinone) flavoprotein 1

88
Nit protein 2 (spot 5315)
gi|12963555
30.5
6.2
347
34
18
16
24
10

C

[33, 37]

89
Nit protein 2 (spot 6201)
gi|12963555
30.5
6.2
345
30
16
14
22
8
0.295
0.8

[33, 37]

90*
Nucb1 protein
gi|49117484
53.4
5
184
8
0
8
8
0

T
G, C,

MEM

91
Peroxiredoxin 6 (spot 4207)
gi|6671549
24.8
5.7
282
40
21
19
30
10
0.132
2.3
C, L
[37]

92
Peroxiredoxin 6 (spot 5216)
gi|6671549
24.8
5.7
280
38
20
18
28
10
0.018
0.3
C, L
[37]

93
Phosphatidylethanolamine
gi|53236978
20.8
5.2
135
12
2
10
11
1
0.008
2.2
C
[42, 43, 64]

binding protein

94
Plasminogen
gi|200403
90.8
6
243
9
0
9
6
3

T
S
[49, 65-67]

95*
Poly(rC) binding protein 2;
gi|6754994
37.5
6.4
189
8
0
8
8
0

T
N

heterogeneous nuclear

ribonucleoprotein X

96*
PREDICTED: agmatine
gi|20848362
38.3
8
108
24
16
8
19
5
0.003
0.1
ES

ureohydrolase

(agmatinase)

97
Prohibitin
gi|54035592
29.8
5.4
335
39
21
18
28
11
0.018
0.6
M
[33]

98*
Psmd11 protein
gi|33585718
46.9
6.1
258
4
0
4
0
4

T
C

99
Pyridoxine 5′-phosphate
gi|19527238
30.1
8.4
120
24
14
10
16
8
0.026
0.3

[21, 43]

oxidase

100
Pyruvate kinase 3
gi|31981562
57.8
7.9
274
7
0
7
4
3

T
M
[80]

101
Pzp protein (a2-
gi|34785996
165.9
6.2
151
14
2
12
9
5

T
ES
[68]

macroglobulin)

102
Retinol binding protein 4,
gi|33859612
23.2
5.6
144
19
2
17
14
5
0.129
12.7
ES
[37]

plasma

103*
RIKEN cDNA 1810013B01
gi|27753960
22.5
5.6
172
31
16
15
23
8
0.042
0.6
N, C

(abhydrolase domain

containing 14b)

104*
RIKEN cDNA 2410004H02
gi|26080429
84.8
5.8
114
4
0
4
0
4

T

105*
Rps12 protein
gi|34849622
14.5
7.3
90
8
1
7
6
2
0.008
2
C

106*
Sars1 protein
gi|14250361
58.4
5.9
199
4
0
4
0
4

T
C

107
Selenium binding protein 1
gi|22164798
52.5
5.9
336
31
19
12
21
10
0.061
0.5
C,
[34]

MEM, N

108
Serine (or cysteine)
gi|6678097
42.6
5.4
137
5
0
5
0
5

T
C
[33]

proteinase inhibitor, clade

B, member 6a

109*
Serpinb1a protein
gi|12834891
42.6
5.7
255
8
8
0
4
4

C
C

110
Serum amyloid P-
gi|38174334
26.2
6
141
4
0
4
0
4

T
ES
[21, 34, 35]

component

111*
Sorcin
gi|13385076
20.3
4.9
129
7
0
7
6
1

T
C, MEM

112*
T43799 proteasome protein
gi|11265288
45.6
7.6
222
6
0
6
6
0

T
C, N

p45/SUG [imported]

113
T-complex protein 1, theta
gi|12846632
49.9
5.5
189
5
0
5
0
5

T
C
[21]

subunit (TCP-1-theta)

(CCT-theta)

114
Transglutaminase 2, C
gi|6678329
77.1
5
240
6
0
6
3
3

T
C, MEM
[34]

polypeptide

115
Transthyretin
gi|7305599
15.8
5.5
127
18
6
12
12
6
0.169
3
ES
[35, 40]

116
Tumor metastatic process-
gi|51980604
17.2
6.4
134
9
0
9
7
2

T

[33, 34]

associated protein NM23

117*
Uap1I1 protein
gi|28175154
56.6
5.2
168
4
0
4
0
4

T

118
UDP-glucose
gi|6678499
54.8
7.4
307
14
0
14
13
1

T

[57]

dehydrogenase

119
Unnamed protein product
gi|52987
27.5
8.8
136
6
0
6
5
1

T
N, C
[33]

(Galectin-3)

120*
v-crk sarcoma virus CT10
gi|56205173
33.8
5.3
187
8
0
8
8
0

T
C, MEM

oncogene homolog

121
Vimentin
gi|31982755
53.7
5
260
26
11
15
21
5
0.022
4
Ck
[33, 50]

122
Vitamin D-binding protein
gi|193446
53.1
5.2
229
32
15
17
22
10
0.175
1.6
ES
[69, 70]

TABLE 2

Accession
Ratio_HCC/control
Ratio_HCC/control

N°
Protein
number
(serum)
(liver)

1
Alpha-fetoprotein
gi|42542817
2
up

(by Western

blot)

2
Apolipoprotein A1
gi|26345182
tumor
Spot 1: 5.4

Spot 2: tumor

3
Apolipoprotein E
gi|6753102
2.2
3.9

4
Carboxylesterase precursor
gi|2921308
tumor
tumor

5
Fibrinogen, alpha polypeptide
gi|33563252
tumor
tumor

6
Fibrinogen, beta polypeptide
gi|33859809
tumor
Spot 1: 2.4

Spot 2: 3.5

7
Fibrinogen, gamma
gi|19527078
tumor
tumor

polypeptide

8
Major urinary protein 1
gi|8569601
0.1
control

9
Pzp (A2mg protein)
gi|34785996
Spot 1: 1.8
tumor

Spot 2: 3.2

10
Serum amyloid P-component
gi|38174334
10
tumor

TABLE 3

Accession
Ratio

No.
Protein name
Proteinfunction
number
T/C
Tumor association

18*
Aldolase 3
Carbohydrate metabolism
gi|60687506
T
occurrance in brain

cancer [92]

23*
Alpha glucosidase 2
Carbohydrate metabolism
gi|26326711
T

9*
Alanyl-tRNA synthetase
Translation
gi|34610207
T

106*
Sars1 protein
Translation
gi|14250361
T

7*
Akr1c12 protein
Fat metabolism
gi|15215042
1.9

15*
Aldo-keto reductase family 1,
Fat metabolism
gi|19527294
T
Aldo-keto-reductase 1

member C14

B10 in human HCC [81]

16*
Aldo-keto reductase family 1,
Fat metabolism
gi|13487925
T
Aldo-keto-reductase 1

member C6

B10 in human HCC [81]

36*
Butyryl Coenzyme A synthetase 1
Fat metabolism
gi|16905127
C

49*
Enoyl coenzyme A hydratase 1,
Fat metabolism
gi|7949037
0.49
occurrance in gastric

peroxisomal

carcinoma [93]

52*
Farnesyl diphosphate
Fat metabolism
gi|19882207
T
upregulated in colon

synthetase

rectal cancer [71]

53*
Fatty acid binding protein 5,
Fat metabolism
gi|6754450
27.9
occurrance in brain

epidermal

cancer [92]

80*
Lysophospholipase 1
Fat metabolism
gi|6678760
0.25

84*
Mitochondrial acyl-CoA
Fat metabolism
gi|40538846
C

thioesterase 1

34*
Beta 5-tubulin
Cytoskeletal proteins
gi|18088719
T
Elevated levels of β4b

tubulin in rat HCC [82]

Lung cancer [94]

78*
LIM and SH3 protein 1
This protein functions as an
gi|6754508
T
occurrance in breast

actin-binding protein and

cancer [95]

possibly in

cytoskeletal organization

[75).

105*
Rps12 protein
Ribosom component
gi|34849622
2
occurrance in breast

cancer [95]

98*
Psmd11 protein
Proteasom component
gi|33585718
T
occurrance in breast

cancer [85]

112*
T43799 proteasome protein
Proteasom component
gi|11265288
T

p45/SUG [imported]

4*
4931406C07Rik [Ester
Hydrolase
gi|71059921
1.5

hydrolase C11orf54 homolog)

[spot 4342)

5*
4931406C07Rik [Ester
Hydrolase
gi|71059921
1.9

hydrolase C11orf54 homolog)

[spot 4349)

73*
Inosine triphosphatase
Hydrolase
gi|31982664
T

96*
PREDICTED: agmatine
Hydrolase
gi|20848362
0.1
Downregulation in _“Clear

ureohydrolase [agmatinase)

cell type” in renal cell

carcinoma[83]

103*
RIKEN cDNA 1810013B01
Hydrolase
gi|27753960
0.6

[abhydrolase domain containing

14b)

95*
Poly[rC) binding protein 2;
RNA binding Protein
gi|6754994
T

heterogeneous nuclear

ribonucleoprotein X

35*
Branched chain ketoacid
Aminoacid metabolism
gi|31982494
0.5

dehydrogenase E1, alpha

polypeptide

48*
Dmgdh protein [Dimethylglycine
Aminoacid metabolism
gi|12836171
C

dehydrogenase, mitochondrial)

1*
170 kDa glucose regulated

gi|7643979
T

protein GRP170 Precurser

2*
2 hydroxyphytanoyl-CoA lyase
A peroxisomal enzyme
gi|18204150
0.38

involved in the catabolism

of phytanoic acid [76)

30*
Arginase type II
Arginase converts L-
gi|6753110
T
Upregulated in human

arginine into L-ornithine

pulmonary cancer [73]

and urea.[77)

Arginase II is usually not

expressed in liver tissue.

37*
Cai protein [Pdia4)
Disulfide bond isomerase
gi|45219865
T

46*
Dhdh protein
oxidizes trans-dihydrodiols
gi|21618806
0.17
occurrance in gastric

of aromatic hydrocarbons to

Cancer [96]

the corresponding catechols

(99)

47*
Diacetyl/L-xylulose reductase
Aldo-keto reductase
gi|50400594
C
occurrance in prostate

andenocarcinoma [93]

72*
Hypothetical protein

gi|58037115
0.39

LOC68347

75*
Interleukin 25
Cytokine
gi|18250288
2.1

76*
Kininogen 1
Precurser to kinin
gi|12963497
2.8
occurrance in lung

adenocarcinoma [97]

82*
Major vault protein
Resistance related protein
gi|12003287
T
occurrance in lung

cancer [98]

90*
Nucb 1 protein
CALNUC [nucleobindin) is
gi|49117484
T
occurrance in colon

an EF-hand, Ca²⁺-binding

carcinoma [89]

protein[79)

104*
RIKEN cDNA 2410004H02

gi|26080429
T

109*
Serpinb1a protein
Serinproteaseinhibitor
gi|12834891
C

111*
Sorcin
Ca-binding protein
gi|13385076
T

associated with cardiac

ryanodine receptors and L-

type Ca²⁺ channels (100)

117*
Uap1l1

gi|28175154
T

120*
v-crk sarcoma virus CT10
intracellular signaling
gi|56205173
T
Delays apoptoses in P12

oncogene homolog
cascade and in the

renal tumor-zellen [84]

activation of the

phosphoinositide 3-kinase

PI3K/AKT pathway [30]

TABLE 4

No.
Protein
References
HCC
Other cancer
unknown

1*
170 kDa glucose regulated
[31, 32]

x

protein GRP170 precursor

2*
2-hydroxyphytanoyl-CoA

x

lyase

3
3-phosphoglycerate
[33]
x
Breast cancer

dehydrogenase

[A19]

4*
4931406C07Rik (Ester

x

hydrolase C11orf54 homolog)

(spot 4342)

5*
4931406C07Rik (Ester

x

hydrolase C11orf54 homolog)

(spot 4349)

6
Acylpeptide hydrolase; N-
[34]
x

acylaminoacyl peptide

hydrolase

7*
Akr1c12 protein

x

8
Akr1c18 protein (aldo-keto
[21]
x

reductase family I, member

C18)

9*
Alanyl-tRNA synthetase

x

10
Albumin 1 (spot 3707)
[21, 35]
x

11
Albumin 1 (spot 3712)
[21, 35]
x

12
Albumin 1 (spot 4506)
[21, 35]
x

13
Albumin 1 (spot 4702)
[21, 35]
x

14
Albumin 1 (spot 5509)
[21, 35]
x

15*
Aldo-keto reductase family I,

x

member C14

16*
Aldo-keto reductase family I,

x

member C6

17
Aldolase I, A isoform
[36]
x
Pancreatic ductal

adenocarcinoma

[A1]

18*
Aldolase 3

Brain cancer [A2]
x

19
Alpha enolase (spot 4501)
[33, 37-39]
x

20
Alpha enolase (spot 4516)
[33, 37-39]
x

21
Alpha enolase (spot 4524)
[33, 37-39]
x

22
Alpha enolase (spot 5510)
[33, 37-39]
x

23*
Alpha glucosidase 2

x

24
Annexin A6
[34, 35]
x
Inhibits

rassignalling in

breast cancer

[A20]

25
Apon4 protein
[40]
x

26
Apoe
[34, 41]
x
Gastric cancer

[A3]

Lung

adenocarcinoma

[A19]

27
Apolipoprotein A-1
[33, 40, 42]
x
Brain cancer [A2]

(spot 2215)

Lung

adenocarcinoma

[A19]

Thyroid cancer

[A29]

28
Apolipoprotein A-1
[40, 42]
x
Brain cancer [A2]

(spot 3204)

Lung

adenocarcinoma

[A19]

Thyroid cancer

[A29]

29
Arginase 1, liver
[37, 43-45]
x
Gastric

cancer[,A30, A31]

colorectal

cancer[A32, A33]

Breast

cancer[A34, A35]

skin cancer lung

cancer[A36, A37]

Prostate

cancer[A38]

30*
Arginase type II

Lung cancer
x

31
Argininosuccinate sythetase 1
[22-27, 46]
x
Ovarian cancer

[A21]

32
ATP synthase, H+
[47]
x
Brain cancer [A2]

transporting, mitochondrial FD

complex, subunit d (spot

2120)

33
ATP synthase, H+
[47]
x
Brain cancer [A2]

transporting, mitochondrial F0

complex, subunit d (spot

3203)

34*
Beta 5-tubulin

Lung cancer [A5]
x

↓

35*
Branched chain ketoacid

x

dehydrogenase B1, alpha

polypeptide

36*
Butyryl Coenzyme A

x

synthetase I

37*
Cai protein (Pdia4)

x

38
Capping protein alpha 1
[33]
x

subunit

39
Carbamoyl-phosphate
[21, 34, 46, 48,
x

synthetase 1, mitochondrial
49]

40
Carboxylesterase MH1
[36, 50]
x

41*
cDNA sequence BOD21917

x

(dihydroxyacetone kinase 2

homolog)

42
Creatine kinase, brain
[33]
x
Bran cancer [A2]

43
Cryz protein
[51]
x

44
Cu/Zn superoxide diamutase
[33, 42]
x

1513495A

45
DEMSMC malate
[52]
x

dehydrogenase, cytosolic

46*
Dhdh protein

Gastric Cancer
x

[A7]

47*
Diacetyl/L-xyulose reductase

Prostate
x

adenocarcinoma

[A22]

48*
Dmgdh protein

x

(Dimethylglycine

dehydrogenase

mitochondrial)

49*
Enoyl coenzyme A hydralase

Gastric

1, peroxisomal

carcinoma [A10]

50
Eukaryotic translation
[91]
x [A11]

elongation factor 2

51
Eukaryotic translation
[34]
x
Ovarian Cancer

initiation factor 5A (eIF-5A)

[A23]

52*
Farnesyl diphosphate

Colon rectal
x

synthetase

carcinoma

53*
Fatty acid binding protein 5,

Brain cancer [A2]
x

epidermal

54
Fibrinogen, alpha polypeptide
[46, 48]
x

55
Fibrinogen, B beta
[48]
x
Pancreatic ductal

polypeptide (spot 5602)

adenocarcinoma

[A1]

Thyroid cancer

[A29]

56
Fibrinogen, B beta
[48]
x
Pacreatic ductal

polypeptide (spot 5612)

adenocarcinoma

[A1]

Thyroid cancer

[A29]

57
Fibrinogen, gamma
[43, 46]
x

polypeptide

58
FK506 binding protein 4
[33, 34]
x
Glioma tumors

[A24]

59
GDP dissociation inhibitor 2
[34]
x
Pancreatic

carcinoma [A9]

60
Glutathione peroxidase l
[53-55]
x

61
Glutathione S-transferase, mu 2
[21, 34]
x

62
Glycine N-methyltransferase
[21, 29, 43, 56]
x
Cholangiocarcarcinoma

(spot 4256)

[A8]

63
Glycine N-methyltransferase
[21, 29, 43, 56]
x
Cholangiocarcarcinoma

(spot 5269)

[A8]

64
Glycine N-methyltransferase
[21, 29, 43, 56]
x
Cholangiocarcarcinoma

(spot 9105)

[A8]

65
Glycyl-tRNA sythetase
[57]
x

66
Hano protein (3-
[34]
x

hydroxyanthranilate 3,4-

dioxygenase)

67
Hal protein
[58]
x

68
Hemopexin
[59]
x

69
Heterogeneous nuclear
[33]
x
Pancreatic ductal

ribonucleoprotein L

adenocarcinoma

[A1]

70
HSP60 (spot 2604)
[33, 34, 50]
x
Brain cancer [A2]

71
HSP60 (spot 2610)
[33, 34, 50]
x
Brain cancer [A2]

72*
Hypothetical protein

x

LOC68347

73*
Inosine triphosphatase

x

74
Interleukin 1 receptor
[18, 50-62]
x

antagonist protein

75*
Interleukin 25

x

76*
Kininogen 1

Lung
x

adenocarcinoma

[A19]

77
Lamin-A/C
[21, 33]
x
Colorectal Cancer

[A12]

78*
LIM and SH3 protein 1

Breast cancer
x

[A13]

79
Liver fructose-1,6-
[34, 43, 48]
x

bisphosphatase

80*
Lysophospholipase 1

x

81
Major urinary protein
[18, 63]
x

82*
Major vault protein

Lung cancer [98]
x

83
MAWD binding protein
[34, 37, 43]
x
Breast Cancer

homolog 1

[A14]

84*
Mitochondrial acyl-CoA

x

thioesterase 1

85
NADH dehydrogenase
[48, 64]
x

(ubiquinone) 1 alpha

subcomplex, 8

86
NADH dehydrogenase
[34]
x
Gastric

(ubiquinone) Fe—S protein 1

carcinoma [A25]

(Ndufs1)

87
NADH dehydrogenase
[64]
x

(ubiquinone) flavoprotein 1

88
Nit protein 2 (spot 5315)
[33, 37]
x

89
Nit protein 2 (spot 6201)
[33, 37]
x

90*
Nucb1 protein

Colon cancer
x

[A41]

91
Peroxiredoxin 6 (spot 4207)
[37]
x
Breast cancer

[A15]

92
Peroxiredoxin 6 (spot 5216)
[37]
x
Breast cancer

[A15]

93
Phosphatidylethanolamine
[42, 43, 64]
x

binding protein

94
Plasminogen
[49, 65-67]
x
Lung

adenocarcinoma

[A19]

95*
Poly(rC) binding protein 2;

x

heterogeneous nuclear

ribonucleoprotein X

96*
PREDICTED: text missing or illegible when filed

x

ureohydrolase ( text missing or illegible when filed

)

97
Prohibitin
[33]
x
Gastric cancer

[A27]

98*
Psmd11 protein

Breast cancer
x

[A16]

99
Pyridoxine 5′-phosphate
[21, 43]
x

oxidase

100
Pyruvate kinase 3
[80]
Serum HCC

101
P2p protein (a2-
[68]
x

macroglobulin)

102
Retinol binding protein 4,
[37]
x

plasma

103*
RIKEN cDNA 1810013B01

x

(abhydrolase domain

containing 14b)

104*
RIKEN cDNA 2410004H02

x

105*
Rps12 protein

Breast cancer
x

[A28]

106*
Sars1 protein

x

107
Selenium binding protein 1
[34]
x
Gastric

carcinoma [A25]

108
Serine (or cysteine) proteinase
[33]
x

inhibitor, clade B, member 6a

109*
Serpinb1a protein

x

110
Serum amyloid P-component
[21, 34, 35]
x
Neuroblastoma[A29]

111*
Sorcin

Malignant glioma
x

[A17]

112*
T43799 proteasome protein

x

p45/SUG [imported]

113
T-complex protein 1, theta
[21]
x

subunit (TCP-1-theta) (CCT-

theta)

114
Transglutaminase 2, C
[34]
x

polypeptide

115
Transthyretin
[35, 40]
x
Thyroid cancer

[A28]

116
Tumor metastatic process-
[33, 34]

x

associated protein NM23

117*
Unp1l1 protein

x

118
UDP-glucose dehydrogenase
[57]
x

119
Unnamed protein product
[33]
x
Brain cancer [A2]

(Galectin-3)

120*
v-crk sarcoma virus CT10

Delays apoptosis
x

oncogene homolog

in renal tumor

121
Vimentin
[33, 50]
x
Prostate cancer

[A18]

122
Vitamin D-binding protein
[69, 70]
x
Lung

adenocarcinoma

[A19]

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kann. Hab die info aus dem englishen Abstract)

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text missing or illegible when filed

indicates data missing or illegible when filed

NOVEL BIOMARKERS OF LIVER CANCER

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