WO2014009245A2

WO2014009245A2 - Novel drug target indentification methods

Info

Publication number: WO2014009245A2
Application number: PCT/EP2013/064132
Authority: WO
Inventors: Edgar Dahl; Michael Rose; Vera KLOTEN
Original assignee: Qithera Gmbh
Priority date: 2012-07-09
Filing date: 2013-07-04
Publication date: 2014-01-16
Also published as: WO2014009245A3

Abstract

The present disclosure relates to novel methods of identifying prophylactic or therapeutic agents, in particular for the treatment of cancer like breast cancer. In a further aspect, the present disclosure pertains to diagnostic methods as well as methods for stratification of cancer-therapy.

Description

NOVEL DRUG TARGET IDENTIFICATION METHODS

FIELD OF THE DISCLOSURE

BACKGROUND

Cancer is one of the most prominent causes of human death. For example, breast cancer is one of the most common causes of cancer in women. The likelihood of developing invasive breast cancer during a woman's lifetime is approximately 1 in 7. In this aspect it is expected that the global market for breast cancer in 2010 has been about 11 to 12 Billion US Dollar. There is an intense search for new compounds useful for the treatment and diagnosis of cancer, e.g. solid tumours, in particular breast cancer.

Anti-cancer drug discovery is now driven by the numerous molecular alterations that have been identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information in both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context matching those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context.

The recent remarkable progress in identifying molecular alterations in human tumors has unfortunately not been paralleled in the field of anti-cancer drug discovery. The shortage of effective anti-cancer drugs is due in part to the fundamental difficulties associated with the development of any safe and effective drug. For example, it remains a formidable task to design small molecules that alter the function of macromolecules towards desired properties, e.g. if the macromolecule represents an enzyme with a large active site. It is even more difficult to inhibit protein-protein interactions mediated over a larger surface, or to restore function to a defective protein (such as those encoded by inactive tumor suppressor genes). Even when successful, massive efforts are required - often measured in years to decades - from dozens of chemists, biochemists and toxicologists.

Considerable commercial and academic resources are directed to identification of candidate therapeutic agents for the treatment of various types of cancer. For example in case of breast cancer, herceptin was developed representing a humanized antibody approved for the treatment of H ER2- positive metastatic breast cancer. Other newly designed therapeutic agents include humanized anti- CD20-antibodies, like Rituximab. A further example, Tykerb is a dual kinase inhibitor which inhibits both ErbB-2 and EGFR kinases and may be more effective than e.g. the compound herceptin.

The most common molecular targets, which have proven useful in the identification of small molecule drugs, are enzymes, receptor-ligand pairs, and occasionally specific protein-protein interactions. Selective inhibitors of these types of molecular processes can readily be found that block the biochemical reactions carried out by these molecules (Gibbs, JB and Oliff A, Cell (1994) 79: 193-8). Unfortunately, the vast majority of novel molecules identified by molecular approaches do not present pharmaceutically tractable targets for the creation of small molecule therapeutics because of two reasons: First, potential target molecules such as important transcription factors contain interacting sites (e.g. protein to DNA) far too large to be blocked by small molecule drugs. Second, many of the important genetic and epigenetic alterations found in tumors represent loss of function aberrations that eliminate or severely reduce the biochemical function of the proteins encoded by the affected genes. Since these molecules have already lost their normal biochemical activities, blockade of their physiological functions by drug inhibitors would offer no therapeutic benefit. Especially in the case of lost tumor suppressor genes, it would instead be desirable to reactivate their function. Since the re-activation of mutated tumor suppressor genes e.g. by gene therapy has been proven difficult in the last decades, novel strategies to rescue the function of a tumor suppressor gene have been developed (e.g. Boeckler FM et al, Proc Natl Acad Sci U S A. (2008) 105: 10360-5). In one of these approaches, called "mimicry" screen or "mimetic rescue screen" (Sajjad A et al, Proc Natl Acad Sci U S A. (1999) 96:12156-61; Gaither A et al, Cancer Res (2007) 67:11493-11498), compound libraries are screened for those small molecules that are best able to simulate a cellular status, as if the putative tumor suppressor under investigation would be active. This can be best achieved by following the expression level of those genes normally involved in the signalling pathway regu lated by the putative tumor suppressor gene. Using this approach screening hits can be selected that are interfering with the important cellular pathways regulated by the putative tumor suppressor gene. In the case of the putative tumor suppressor gene ITI H5 we have strong evidence from our published data (Veeck J et al, Oncogene (2008) 27: 865-876) and novel unpublished data presented in Figure 1 to 4 that ITI H5 can suppress cancer metastasis, likely by stabilizing the extracellular matrix and inhibiting epithelial-to-mesenchymal transition (EMT).

Typically, the various types of cancers, for example breast cancer, are multifactorial and complex diseases with no standardized medication available for patients. In spite of some advances in early detection of cancer, optimized treatment with sufficient response rates remains a major clinical problem in cancer therapy.

Thus, the object of the present disclosure is to provide screening methods to identify novel candidate therapeutic agent for use in prophylaxis or treatment of diseases like cancers, in particular breast cancer.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to novel methods for identifying candidate therapeutic agents for use in prophylaxis or treatment of a disease like cancer comprising the steps of a) contacting a test agent with a target cell lacking endogenous ITIH5 expression and b) analyzing the effect of the test agent from step (a) on the level of expression of one or more genes, or products thereof, selected from the group of genes listed in Table 1, Table 2, Table 3 and Table 4. In another aspect, embodiments of the disclosure relate to methods for diagnosing cancer, in particular breast cancer, in a subject comprising the steps of a) analyzing the level of expression of two or more genes, or products thereof, selected from the group of genes listed in Table 5 and b) comparing the level of expression with a reference sample from a subject not afflicted with said cancer whereby an alteration of at least twofold of the expression level in the test sample compared to the reference sample is indicative for cancer.

In a further aspect, embodiments of this disclosure relate to methods for determining whether a subject is predisposed to cancer, in particular breast cancer, or suffering from cancer, in particu lar breast cancer, said method including the step of a) analyzing the level of expression of at least two or more genes, or products thereof, selected from the group of genes listed in Table 5 in a sample and b) determine whether said subject is predisposed to cancer or suffering from cancer, in particular breast cancer.

BRIEF DESCRIPTION OF THE DRAWING Figure 1A shows pictures of cell culture dishes with grown colonies (black spots) for the control group (MDA-MB-231 breast tumor cells without ITIH5 expression; #3 and #1, upper row) and the ITIH5 test group (MDA-MB-231 cells with manipulated (forced) ITIH5 re-expression, #11, #8, #4, #7, lower rows).

Figure IB is a diagram showing the densitometrical evaluation of colony growth. The Box plot graph presents averages of colony growth of triplicate experiments for each clone. Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, peak and minimum, ***p<0.001.

Figure 2A is a diagram showing the comparison of the migration capacity of a control cell set (MDA- MB-231 breast tumor cells without ITIH5 expression; black area) with that of the ITIH5 test set (MDA- MB-231 cells with manipulated ITIH5 re-expression, grey area) over 4 days. ITIH5 expressing cells close the wound area much slower. Vertical lines: standard deviation of triplicates. A_day:L: differences of cell-free areas on day 1.

Figure 2B shows pictures for the documentation of the migration process by scanning electron microscopy and illustrates that ITIH5 expressing tumor cells close the wound area much slower. Rectangle regions are separately enlarged. Scale bar = ΙΟΟμιη. Figure 3 shows pictures of the ITIH5 re-expression caused mesenchymal-to-epithelial shift in cell morphology.

Figure 4A is a 3D image of the entire lung after contrast-agent application and 3D volume rendering is shown. Macro-metastases foci (boxed area) were clearly verified in mice intravenously injected with control MDA-MB-231 breast tumor cells in the pleural space. Figure 4B is a diagram showing the comparison of the overall number of induced metastases between the ITIH5 set (n=ll) and the control set (n=ll), determined by non-invasive in vivo μ€Τ and histopathological evaluation. Horizontal lines: Grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, peak and minimum, **p<0.01. It is evident that ITIH5 re-expression strongly suppresses lung metastasis of MDA-MB-231 breast cancer cells in a nude mice model.

DETAILED DESCRIPTION OF THIS DISCLOSURE

The present application discloses a novel screening system for identifying candidate therapeutic agents for use in prophylaxis or treatment of diseases, in particular cancer diseases like breast cancer, using target cells lacking endogenous ITIH5 expression. Methods of the present disclosure involve determining whether a candidate agent can alter the level of expression of one or more genes, or products thereof, selected from the group of genes listed in Table 1, Table 2, Table 3 and Table 4 towards a pattern that is similar or identical to that present in healthy cells expressing ITIH5.

It is known that proteins of the group of "inter-alpha-trypsin inhibitor heavy chains" (ITIHs) are extracellular matrix (ECM) molecules responsible for maintaining the structural integrity of tissues (Zhuo L et al, J. Biol. Chem. (2004) 279: 38079-38082). Remodeling of the ECM network that consists of a variety of different structural components such as collagens or hyaluronans plays a crucial role in the progression of tumors by promoting metastasis (Lopez Jl et al, Cancer Res. (2005) 65: 6755- 6763). ITIH-molecules are able to covalently bind to hyaluronan and, thus, to form cable like structures that are thought to stabilize the ECM network (Salier JP et al, Biochem J. (1996) 315 (Pt 1): 1-9). As a consequence, it is thought that ITIH molecules serve as potential barrier for malignant progression and could serve as tumor suppressor genes (Hamm A et al, BMC Cancer (2008) 8: 25).

Previously, the fifth heavy chain member of the ITI family, namely ITIH5, was cloned and characterized and shown to be down regulated on the expression level in human breast cancer (Himmelfarb M et al, Cancer Lett. (2004) 204: 69-77). ITIH5 down regulation is due to hypermethylation of its gene promoter and is associated with clinical parameters of malignant progression and metastasis predicting reduced recurrence-free as well as overall patient survival (Veeck J et al, Oncogene (2008) 27: 865-876). Since loss of ITIH5 expression occurs during breast cancer development when tumor cells become invasive, the current hypothesis is that ITIH5 represents a novel tumor suppressor gene that can inhibit malignant progression (Veeck J et al, Oncogene (2008) 27: 865-876). This hypothesis is further supported by unpublished in vivo data presented in Figure 4 of this disclosure. With the methods according to the present disclosure the influence of candidate therapeutic agents for use in prophylaxis or treatment of a disease like cancer on ITIH5 mediated signalling pathways can be determined. The present inventors found new genes and/or gene products regulated by ITIH5 mediated cellular signalling (Table 1 to 4), and set of genes and/or gene products particular useful as surrogate markers for a ITIH5 mimetic rescue screen as shown in Tables 6 and 7. These genes can be used as surrogate markers to define candidate therapeutic agents that can change a target cells biochemical properties towards those found in ITIH5 expressing non-malignant healthy cells.

According to the present disclosure, the candidate therapeutic agents may be an isolated nucleic acid, a protein including polypeptides or oligopeptides, or in an advantageous embodiment a small molecule. For example, the protein may be a ligand of a transmembrane-receptor protein or, alternatively, the protein is an antibody, in particular, a monoclonal antibody. Depending on the subject, the antibody is genetically adapted to the organism to be applied to. That is, for the human being, the monoclonal antibody, typically derived from mice, is humanised according to methods known in the art. Preferably, the monoclonal antibody is a fully humanized antibody for application in human subjects.

Alternatively, the candidate agent, like a prophylactic or therapeutic agent, is an isolated nucleic acid, like DNA or RNA or modified DNA or RNA molecules with modifications known in the art. For example, the nucleic acid molecule is a DNA oligonucleotide or a modified version (e.g. morpholinos), silencer RNA, an interfering RNA, an antisense RNA, an artifical micro RNA, ribozyme, etc.

In this connection, the term "isolated nucleic acid" or "isolated nucleic acid molecule" refers to a nucleic acid molecule DNA or RNA that has been removed from its native environment. For example, recombinant nucleic acid molecules contained in a vector are considered isolated for the purpose of the present disclosure. Further, the term "gene" is used herein to describe a discrete nucleic acid or locus, unit or region within a gene known that may comprise one or more introns, exons, splice sites, open reading frames and 5' and/or 3' non-coding regulatory sequences such as a promoter and/or polyadenylation sequence. The skilled person is well aware of suitable nucleic acid molecules allowing modulation of the transcription or translation of genes or gene products. In an advantageous embodiment, the test agent, like the prophylactic or therapeutic agent, is a small molecule. In this context, the term "small molecule" particularly refers to small organic molecules. Typically, said small molecules are part of screening libraries comprising chemical, typically organical, synthetic compounds.

In one embodiment, the therapeutic agents may be identified by way of screening libraries of molecules such as synthetic chemical libraries, including combinatorial libraries, based on the use of the screening tools according to the present disclosure by methods known in the art.

It is also contemplated that libraries of naturally-occurring molecules may be screened by methodology such as reviewed in Kolb, Prog. Drug. Res. (1998) 51 185. More rational approaches to designing anti-cancer agents may employ X-ray crystallography, N MR spectroscopy, computer assisted screening of structural databases, computer-assisted modeling, or more traditional biophysical techniques which detect molecular binding interactions, as are well known in the art. According to the present disclosure, the test agent is contacted in a first step with a target cell lacking endogenous ITIH5 expression. The target cell may be a target cell completely lacking ITIH5 or showing reduced endogenous ITIH5 expression. In one embodiment, the target cells are stable ITIH5-transfectants and control transfectants (vector only, not expressing ITIH5) of the breast cancer cell lines MDA-MB231 and BT20. Both cell lines are model systems for basal-type breast cancer, which is of special importance, since it has the worst prognosis (Banerjee S et al, J Clin Pathol. (2003) 59:729-35) among all groups of breast defined by molecular profiles (Perou CM et al, Nature (2000) 406:747-52).

In some embodiments, a single test agent or a group of the same test agents are contacted with the target cells. In another embodiment a plurality of different test agents are contacted with the target cells simultaneously or in series. The target cells may be presented in a well, a cell culture plate, a flask or any suitable container for cultivating cells. In an example, the cells are presented on a biochip like a microfluidic system on a chip.

The effect of the test agent on the level of expression of one or more genes, or products thereof, selected from the group of genes listed in Table 1, Table 2, Table 3 and Table 4 can be analyzed by many known technologies known in the prior art. It will be readily appreciated by a person of skill in the art that a number of methods may be utilized to measure the expression levels of the protein of interest, in particular of transmembrane receptors, in a test sample. By way of example only, fluorescence activated cell sorting (FACS) analysis using labeled antibodies is readily amenable to quantitative measurement of cell surface expression of proteins. For example, immunofluorescence and other fluorescence microscopy methods can also be used to stain tissue to detect levels of the protein as well as other conventional immunohistochemistry techniques.

Alternatively, relative protein expression levels may be determined by other protein-based methods which include immunoassays, for example ELISA and immunoblotting to detect relative expression levels of one as more of said proteins.

The invention further contemplates use of microarray technology to determine the expression pattern profile of cells in order to analyze whether nucleic acid or protein expression is up-regulated, e.g. in patients with cancer. The screening tools according to the present invention are particularly useful for identifying the effect of the candidate agents on ITIH5 mediated cellular signaling and activity and, thus, on the possibility to have adverse (here: curing) effects on an organism when administered e.g. in form of a pharmaceutical. The principal in short is that the pharmaceutical compound reactivates biochemical pathways regulated by ITIH5 in healthy (non-malignant) cells, thus it mimics ITIH5 wildtype function.

Proteomic pattern analysis provides an alternative diagnostic method which is particularly useful for global expression pattern analysis of proteins. Methods of cancer diagnosis using proteomic patterns are provided in Conrads et al, Expert. Rev. Mol. Diagn. (2003) 3:411-20 and is incorporated herein by reference. In particular embodiments, a plurality of said proteins may be used in a protein library displayed in a number of ways, e.g., in phage display or cell display systems or by two-dimensional gel electrophoresis, or more specifically, differential two-dimensional gel electrophoresis (2D-DIGE). These particular embodiments may generally be referred to as "proteomic" or "protein profiling" methods, such as described in Chapters 3.9.1 and 22 of CU RRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, John Wiley & Sons NY USA (1996-2002).

In embodiments relating to protein arrays, preferably a cancer-associated protein of the invention is located at an identifiable address on the array. Preferably, the protein array comprises a substrate to which is immobilized, impregnated, bound or otherwise coupled cancer-associated protein, or a fragment thereof.

The substrate may be a chemically-derivatised aluminium chip, a synthetic membrane such as PVDF or nitrocellulose, a glass slide or microtiter plates. Detection of substrate-bound proteins may be performed using mass spectrometry, ELISA, immunohistochemistry, fluorescence microscopy or by colorimetric detection.

A person of skill in the art will contemplate that the diagnostic methods of the invention may involve measuring expression levels of a nucleic acid encoding the protein as defined herein for individual. It is also contemplated that relative levels of nucleic acids may be measured and/or compared in the diagnostic methods of the present invention. By way of example, mRNA levels may be measured. Measurement of relative levels of a nucleic acid level compared to an expressed level of a reference nucleic acid may be conveniently performed using a nucleic acid array. Nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECU LAR BIOLOGY Eds. Ausubel et al, John Wiley & Sons NY USA (1995-2001). An array con be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g. as described in U.S. Patent No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145). Reference is also made to Affymetrix nucleic acid array systems such as described in United States Patent 5,858,659 and United States Patent 6,300,063 which provide specific teaching in relation to nucleic acid array-based detection of disease-related polymorphisms.

In another particular form of this embodiment, quantitative or semi-quantitative PCR using primers corresponding to nucleic acids as defined herein may be used to quantify relative expression levels of said nucleic acid, the screening tools. Thus, it is possible to determine the influence of the candidate agent on ITIH5 mediated cellular signaling, the performance of ITIH5 signaling being a measure of the healthy status. Thus, for example, it is possible to determine whether an individual is predisposed to or suffering from cancer.

PCR amplification is not linear and, hence, end point analysis does not always allow for the accurate determination of nucleic acid expression levels. Real-time PCR analysis provides a high throughput means of measuring gene expression levels. It uses specific primers, and fluorescence detection to measure the amount of product after each cycle. Hydridization probes utilise either quencher dyes or fluorescence directly to generate a signal. This method may be used to validate and quantify nucleic acid expression differences in cells or tissues obtained from cancer sufferers compared to cells or tissues obtained from non-sufferers.

In a particular preferred embodiment, the method for diagnosis or cancer or determining whether a subject is predisposed to cancer or suffers from cancer is based on quantitative or semi-quantitative PCR, e.g. real-time PCR analysis.

An elevated level of the protein or nucleic acid molecules in test samples or other types of probes, e.g. tissue, compared to reference samples obtained from a healthy control subject will be indicative for cancer or predisposition of cancer. Based on the first model using MDA-MB-231 breast cancer cells with forced ITIH5 overexpression, genes have been identified which expression is decreased or increased at least twofold, preferably at least threefold, like at least fourfold compared to the highly malignant (empty vector) control MDA- MB-231 cell line wherein ITIH5 expression was not manipulated, Table 1 and Table 2.

In addition, based on the second model using BT20 breast cancer cells with forced ITIH5 overexpression, genes have been identified which expression is decreased or increased at least twofold, preferably at least threefold, like at least fourfold compared to the highly malignant (empty vector) control BT20 cell line wherein ITIH5 expression was not manipulated, Table 3 and Table 4.

As mentioned above, in a first aspect the present disclosure pertains to a method for identifying a candidate therapeutic agent for use in prophylaxis or treatment of cancer comprising the steps of: a) Contacting a test agent with a target cell lacking endogenous ITIH5 expression;

b) Analyzing the effect of the test agent from step (a) on the level of expression of one or more genes, or products thereof, selected from the group of genes listed in Table 1, Table 2, Table 3 and Table 4.

Table 1 shows the target genes suppressed by ITIH5 and Table 2 shows the target genes activated by ITIH5 in the MDA-MB-231 breast cancer cell model.

Table 1

14 Anterior gradient 2 homolog (Xenopus laevis) AGR2 S

15 Sema domain, immunoglobulin domain (Ig), short SEMA3A S

basic domain, secreted, (semaphorin) 3A

16 Galactosidase, beta 1-like 2 GLB1L2 E

17 Chemokine (C-X-C motif) ligand 11 CXCL11 S

18 Growth differentiation factor 15 GDF15 N

19 SH3 and multiple ankyrin repeat domains 2 SHAN K2 N

20 Zymogen granule protein 16 homolog B (rat) ZG16B N

21 Leupaxin LPXN N

22 Protocadherin beta 14 PCDH B14 T

23 Gamma-aminobutyric acid (GABA) B receptor, 1 GABBR1 T

24 Chromosome 10 open reading frame 116 C10orfll6 N

25 Chromosome 19 open reading frame 21 C19orf21 N

26 Matrix-remodelling associated 8 MXRA8 T

27 Solute carrier family 4, sodium bicarbonate SLC4A4 T

cotransporter, member 4

28 S100 calcium binding protein A4 S100A4 s

29 Wingless-type MMTV integration site family, WNT7B s

member 7B

Legend: HUGO name: Official gene abbreviation according to the international gene nomenclatu committee (http://www.genenames.org); Protein classes: S = secreted, T = transmembrane, E enzyme, N = other.

Table 2

18 T-box 3 TBX3

19 Scavenger receptor class A, member 3 SCARA3

20 Immunoglobulin superfamily, member 3 IGSF3

21 Coiled-coil domain containing 80 CCDC80

22 Nuclear RNA export factor 2 NXF2

23 Nephroblastoma overexpressed gene NOV

24 Myelin protein zero-like 2 MPZL2

25 Membrane protein, palmitoylated 2 (MAGU K p55 subfamily MPP2

member 2)

26 Syntrophin, beta 1 (dystrophin-associated protein Al, SNTB1

59kDa, basic component 1)

27 Clusterin CLU

28 Calpain 5 CAPN5

29 SLAIN motif family, member 1 SLAIN 1

30 Secreted protein, acidic, cysteine-rich (osteonectin) SPARC

Table 3 shows the target genes suppressed by ITIH5 and Table 4 shows the target genes activated by ITIH5 in the BT20 breast cancer cell model.

Table 3

Table 4

In an advantageous embodiment, the expression level are analyzed for genes, or products thereof, listed in Table 5 showing target genes activated (+) and suppressed (-) after forced ITIH5 re- expression. These target genes were additionally validated by real-time PCR analysis. Therefore, in an embodiment of the method according to the present disclosure, the test agent is analyzed for an effect on the level of expression of one or more genes, or products thereof, selected from the group of genes listed in Table 5.

Table 5

In further embodiments, the test agent is analyzed for an effect on the level of expression of two or more genes, or products thereof, selected from the group of genes listed in Table 1, Table 2, Table 3, Table 4 and Table 5.

The methods according to the present disclosure may be used for identifying a candidate therapeutic agent for use in prophylaxis, treatment and/or diagnosis of a disease or disorder based inter alia on an altered ITIH5 expression. In an advantageous embodiment the disease is a cancerous disease like colon cancer, lung cancer, prostate cancer, breast cancer, stomach cancer, bladder cancer, renal cell cancer, ovarian cancer, liver cancer and pancreatic cancer. In a further advantageous embodiment, the identified compounds may be useful for the treatment and/or diagnosis of breast cancer.

In a further embodiment, the target cell is contacted with a plurality of test agents and the test agents having an effect on the on the level of expression of the listed genes are selected as a candidate therapeutic agent. The different test agents may be contacted with the target cells simultaneously or in series.

As mentioned above, the target cell may be a normal or a cancer cell. In an advantageous embodiment the target cell is a breast cancer cell, preferably a MDA-MB-231 breast cancer cell and/or a BT20 breast cancer cell.

In further embodiments, the analyzed test agent from step (a) of the methods according to the present disclosure suppress the expression of at least one or more genes, or products thereof, selected from the group of genes listed in Table 1 and/or activate the expression of one or more genes, or products thereof, selected from the group of genes listed in Table 2 if the target cell is a MDA-MB-231 breast cancer cell.

In particular, the test agents suppress the expression of the matrix-remodeling associated 8 gene (MXRA8), or products thereof and activate the expression of NDRG family member 2 (NDRG2), or products thereof in a MDA-MB-231 breast cancer cell..

In another embodiment, the test agents activate the expression of one or more genes, or products thereof, selected from the group consisting of endoglin (ENG), UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14), gamma-aminobutyric acid (GABA) B receptor 1 gene (GABBR1) or any gene from Table 1 or Table 2 in a MDA-MB-231 breast cancer cell. In a further embodiment, the test agents suppress the expression of the matrix-remodeling associated 8 gene (MXRA8), or products thereof and activate the expression of NDRG family member 2 (NDRG2) and endoglin (ENG), or products thereof in a MDA-MB-231 breast cancer cell.

In a further embodiment, the test agents suppress the expression of the matrix-remodeling associated 8 gene (MXRA8), or products thereof and activate the expression of NDRG family member 2 (NDRG2), endoglin (ENG) and UDP-N-acetyl-alpha-D-galactosamine: polypeptide N- acetylgalactosaminyltransferase 14 (GALNT14), or products thereof in a M DA-M B-231 breast cancer cell. In a further embodiment, the test agents suppress the expression of the matrix-remodeling associated 8 (MXRA8) and the gamma-aminobutyric acid (GABA) B receptor 1 gene (GABBR1), or products thereof and activate the expression of NDRG family member 2 (NDRG2), endoglin (ENG) and UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) , or products thereof in a MDA-MB-231 breast cancer cell.

In another advantageous embodiment, the test agents suppress the expression of one or more genes, or products thereof, selected from the group of genes listed in Table 3 and/or activate the expression of one or more genes, or products thereof, selected from the group of genes listed in Table 4 if the target cell is a BT20 breast cancer cell. In particular, the test agents suppress the expression of one or more genes, or products thereof, selected from the group consisting, heparanase (HPSE), matrix-remodeling associated 8 (MXRA8) and hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD) in a BT20 breast cancer cell.

In a further embodiment, the test agents suppress the expression of the matrix-remodeling associated 8 (MXRA8) and hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD) genes, or products thereof in a BT20 breast cancer cell.

In a further embodiment, the test agents suppress the expression of the matrix-remodeling associated 8 (MXRA8), hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD) and heparanase gene (H PSE), or products thereof in a BT20 breast cancer cell.

In a further advantageous embodiment, the test agent suppresses the expression of the gene matrix- remodeling associated 8 (MXRA8).

In a second aspect, the present disclosure pertains to a method for diagnosing a disease like cancer, in particular breast cancer, in a subject comprising the steps of: a) Analyzing the level of expression of two or more genes, or products thereof, selected from the group of genes listed in Table 5; b) Comparing the level of expression with a reference sample from a subject not afflicted with said cancer whereby an alteration of at least twofold of the expression level in the test sample compared to the reference sample is indicative for the disease, in particular for cancer.

In a third aspect, the present disclosure pertains to a method for determining whether a subject is predisposed to cancer, in particular breast cancer, or suffering from cancer, in particular breast cancer, said method including the step of: a) analyzing the level of expression of at least two or more genes, or products thereof, selected from the group of genes listed in Table 5 in a sample b) determine whether said subject is predisposed to cancer or suffering from cancer, in particular breast cancer, preferably, wherein the level is an elevated level compared to a reference sample of a healthy control subject.

In an embodiment, the level of expression is analyzed on nucleic acid level, preferably by nucleic acid amplification methods, in particular RT-PCR. In a further embodiment, the level of expression of a gene is analyzed by using an amount of a mRNA in a sample and/or by using an antibody against the gene product.

Advantageous sets of analysed genes or products thereof are listed in Table 6 showing core target gene sets activated (+) and suppressed (-) after forced ITIH5 re-expression in M DA- M B- 231 target cells. The genes listed in the specific sets can be used also in test kits for the diagnosis of ITIH5 related disorders and/or diseases.

Table 6

Advantageous sets of analysed genes or products thereof are also listed in Table 7 showing core target gene sets activated (+) and suppressed (-) after forced ITIH5 re-expression in BT20 target cells. The genes listed in the specific sets can be used also in test kits for the diagnosis of ITIH5 related disorders and/or diseases.

Table 7

Figure 1 shows the ITIH5 re-expression suppresses tumor cell growth. A) Representative cell culture dishes with grown colonies (black spots) are shown for the control group (MDA-MB-231 breast tumor cells without ITIH5 expression; #3 and #1, upper row) and the ITIH5 test group (MDA-MB-231 cells with manipulated (forced) ITIH5 re-expression, #11, #8, #4, #7, lower rows). B) Densitometrical evaluation of colony growth clearly demonstrated an inhibitory effect of ITIH5 expression on tumor cell growth in vitro. Box plot graph presents averages of colony growth of triplicate experiments for each clone. Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, peak and minimum, ***p<0.001. Figure 2: ITIH5 re-expression inhibits tumor cell migration. Cell migration was analyzed by using a wound healing assay. It means that a cell layer (control or ITIH5 expressing tumor cells, respectively) was linearly scratched with a pipette tip and the dynamics of the wound closure was determined. A) Comparison of the migration capacity of a control cell set (MDA-MB-231 breast tumor cells without ITIH5 expression; black area) with that of the ITIH5 test set (MDA-MB-231 cells with manipulated ITIH5 re-expression, grey area) over 4 days. ITIH5 expressing cells close the wound area much slower. Vertical lines: standard deviation of triplicates. A_day:L: differences of cell-free areas on day 1. B) Documentation of the migration process by scanning electron microscopy also illustrates that ITIH5 expressing tumor cells close the wound area much slower. Rectangle regions are separately enlarged. Scale bar = ΙΟΟμιη. Figure 3: ITIH5 re-expression causes a mesenchymal-to-epithelial shift in cell morphology. Epithel ia I- to-mesenchymal transition (EMT) is a key process in tumor progression and metastasis (Micalizzi, DS et al, Mammary Gland Biol. Neoplasia (2010) 15 :117-34). Note that ITIH5 re-expression in M DA-MB- 231 breast tumor cells (upper row) induces the opposite process, a mesenchymal-to-epitehlial transition (MET), whose restored epithelial characteristics are thought to suppress the initiation of the metastasis process (Dusek RL and Attardi LD, Nat. Rev. Cancer (2011) 115:317-23). Scale bar = 20μηη.

Figure 4: ITIH5 leads to an effective suppression of metastasis in a xenograft mouse model. An in vivo metastasis assay was performed by using BALB/c nu/nu mice that were intravenously injected with human mock-transfected (tumor cells without ITIH5 expression = control set) and ITIH5-transfected MDA-MB-231 cells (tumor cells with manipulated ITIH5 re-expression = ITIH5 set), respectively. By whole-body in vivo μCT screening, differences in metastasis growth between the control and the ITIH5 test set was evident. Left picture: Representative 3D image of the entire lung after contrast- agent application and 3D volume rendering is shown. Macro-metastases foci (boxed area) were clearly verified in mice intravenously injected with control MDA-MB-231 breast tumor cells in the pleural space. Right graph: Comparison of the overall number of induced metastases between the ITIH5 set (n=ll) and the control set (n=ll), determined by non-invasive in vivo μCT and histopathological evaluation. Horizontal lines: Grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, peak and minimum, **p<0.01. It is evident that ITIH5 re-expression strongly suppresses lung metastasis of MDA-MB-231 breast cancer cells in a nude mice model.

Methods and Examples

In the following examples, materials and methods of the present disclosure are provided. It should be understood that these examples are for illustrative purpose only and are not to be construed as limiting this disclosure in any manner. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples: The impact of ITIH5 loss in human breast cancer cell lines was analyzed in two different highly invasive breast cancer models of basal-type breast cancer, i.e. M DA-M B231 breast cancer cells and BT20 breast cancer cells. These human breast cancer cell lines that do not endogenously express ITIH5, were stably transfected with an ITIH5 containing vector and served as the ITIH5-plus-model. Transfection experiments were done with Fugene 6 transfection reagents (Roche, Switzerland) by using the pBK CMV vector (Stratagene, Germany) alone or with the integrated ITIH5 coding region. This approach allowed elevation of ITIH5-mRNA levels in these cancer cell line clones to expression levels equivalent or larger to that of benign breast cells. In the following, some experimental results from the characterization of the MDA-MB-231 transfectants re-expressing ITIH5 are shown as examples: First, the effect of ITIH5 re-expression was analyzed in vitro by a colony formation assay (Figure 1). Interestingly, we found a very profound reduction in the tumor cells capability to grow and form colonies (compare upper row (minus ITIH5) and the two lower rows (plus ITIH5) in Figure 1). This growth inhibition was highly significant. Next, the migration capabilities of ITIH5-re- expressing tumor cells were analyzed by a "wound healing" assay (Figure 2) which measures the time to "heal" a scratch in a "lawn" of tumor cells. ITIH5-expressing tumor cells exhibit a dramatic loss of migration potential, a key feature of tumor cells. Closer analysis of the tumor cell morphology revealed an unexpected but very conclusive answer to the impaired growth and migration properties of ITIH5 expressing M DA-M B-231 tumor cells (Figure 3). The tumor cells change their morphology and perform a so-called mesenchymal-to-epithelial shift: Easy speaking, they acquire the morphology of more benign tumor cells, which are usually not being able to form metastatic tumors (Hanahan, D and Weinberg RA, Cell (2011) 144:646-74). Based on these data the influence of ITIH5 re-expression on the ability of tumor cells to form metastatic tumors was analyzed in a mouse model for human cancer metastasis (Figure 4). Very excitingly, a strong reduction of the number and size of lung metastases was observed in this in vivo model, arguing that ITIH5 re-expression and thus ITIH5 mediated cellular signaling could inhibit or revert metastasis in human tissues, especially breast cancer tissues as well.

Table 8

6 5,39 Transmembrane protein 45B TM EM45B T

7 5,32 Leucine rich repeat transmembrane neuronal 4 LRRTM4 T

8 5,15 TSPY-like 5 TSPYL5 N

9 4,72 Paraoxonase 3 PON3 N

10 4,67 Colony stimulating factor 2 receptor, alpha, low- CSF2RA T affinity (granulocyte-macrophage)

11 4,5 Amphiregulin AREG N

12 4,46 Ret finger protein-like 4A RFPL4A N

13 4,33 Transmembrane protein 163 TM EM163 T

14 4,32 Anterior gradient 2 homolog (Xenopus laevis) AGR2 s

15 4,28 Sema domain, immunoglobulin domain (Ig), short SEMA3A s basic domain, secreted, (semaphorin) 3A

16 3,88 Galactosidase, beta 1-like 2 GLB1L2 E

17 3,72 Chemokine (C-X-C motif) ligand 11 CXCL11 S

18 3,11 Growth differentiation factor 15 GDF15 N

19 3,08 SH3 and multiple ankyrin repeat domains 2 SHAN K2 N

20 3,07 Zymogen granule protein 16 homolog B (rat) ZG16B N

21 2,89 Leupaxin LPXN N

22 2,84 Protocadherin beta 14 PCDH B14 T

23 2,61 Gamma-aminobutyric acid (GABA) B receptor, 1 GABBR1 T

24 2,61 Chromosome 10 open reading frame 116 C10orfll6 N

25 2,29 Chromosome 19 open reading frame 21 C19orf21 N

26 2,2 Matrix-remodelling associated 8 MXRA8 T

27 2,14 Solute carrier family 4, sodium bicarbonate SLC4A4 T cotransporter, member 4

28 2,06 S100 calcium binding protein A4 S100A4 s

29 2 Wingless-type MMTV integration site family, WNT7B s member 7B

Table 8 shows the fold change of the target genes suppressed by ITIH5 in the MDA-MB-231 cell model.

Table 9

acetylgalactosaminyltransferase 14

13 4,31 N DRG family member 2 N DRG2

14 4,06 EH-domain containing 3 EH D3

15 3,82 Procollagen C-endopeptidase enhancer 2 PCOLCE2

16 3,74 Zinc finger protein 730 ZNF730

17 3,47 Transmembrane phosphatase with tensin homology TPTE

18 2,98 T-box 3 TBX3

19 2,87 Scavenger receptor class A, member 3 SCARA3

20 2,83 Immunoglobulin superfamily, member 3 IGSF3

21 2,69 Coiled-coil domain containing 80 CCDC80

22 2,59 Nuclear RNA export factor 2 NXF2

23 2,59 Nephroblastoma overexpressed gene NOV

24 2,5 Myelin protein zero-like 2 MPZL2

25 2,33 Membrane protein, palmitoylated 2 (MAGU K p55 subfamily MPP2

member 2)

26 2,31 Syntrophin, beta 1 (dystrophin-associated protein Al, SNTB1

59kDa, basic component 1)

27 2,22 Clusterin CLU

28 2,18 Calpain 5 CAPN5

29 2,15 SLAIN motif family, member 1 SLAIN 1

30 2,15 Secreted protein, acidic, cysteine-rich (osteonectin) SPARC

Table 9 shows the fold change of the target genes activated by ITIH5 in the M DA-MB-231 cell model.

Table 10

19 1,84 Microtubule-associated protein tau MAPT N

20 1,45 Heparanase H PSE E

21 1,50 Matrix-remodelling associated 8 MXRA8 T

Table 10 shows the fold change of the target genes suppressed by ITIH5 in the BT20 cell model.

Table 11

Table 11 shows the fold change of the target genes activated by ITIH5 in the BT20 cell model After a validation step, in which the modified expression of ITIH5 was confirmed by real-time PCR analysis and western blot analysis with an ITIH5 -specific antibody, the resulting changes in global RNA expression in both models were analyzed using Affymetrix expression arrays (U 133 PLUS 2.0 array). Results of Affymetrix expression analysis of ITI H5-plus- and ITIH5-minus-transfectants were clustered with DChip_2009 and grouped according to their pathway affiliation (KEGG Pathway http://www.genome.jp/kegg and www.genecards.org). Genes that are activated after loss of ITIH5 expression were collected if the fold change up regulation in the absence of ITIH5 was at least twofold In the tables above, the HGNC name (humane genome nomenclature committee; now www.gennames.org), a description of the gene, of the protein class as well as the fold change compared to the non-manipulated cell line are provided.

Table 12 shows the fold change of the target genes activated (+) and suppressed (-) after forced ITIH5 re-expression: Validation by real-time PCR analysis

Table 12

16 -1,4 Microtubule-associated protein tau MAPT BT20

17 -2 Heparanase H PSE BT20

18 -3 Matrix-remodelling associated 8 MXRA8 BT20

19 -190 Hydroxyprostaglandin dehydrogenase H PGD BT20

15-(NAD)

Table 13 shows the fold change of the core target gene sets activated (+) and suppressed (-) after forced ITIH5 re-expression in MDA-MB-231 target cells.

Table 13

Table 14 shows the fold change of the core target gene sets activated (+) and suppressed (-) after forced ITIH5 re-expression in BT20 target cells

Table 14

BT20 model Gene description HUGO name fold change gene set Heparanase H PSE -2

Matrix-remodelling associated 8 MXRA8 -3

Hydroxyprostaglandin dehydrogenase 15-(NAD) H PGD -190

gene set Matrix-remodelling associated 8 MXRA8 -3

Hydroxyprostaglandin dehydrogenase 15-(NAD) H PGD -190

Claims

1. A method for identifying a candidate therapeutic agent for use in prophylaxis or treatment of cancer comprising:

a) Contacting a test agent with a target cell lacking endogenous ITIH5 expression;

2. The method according to claim 1, wherein the test agent is analyzed for an effect on the level of expression of one or more genes, or products thereof, selected from the group of genes listed in Table 5.

3. The method according to any of claims 1 to 2, wherein the test agent is analyzed for an effect on the level of expression of two or more genes, or products thereof, selected from the group of genes listed in Table 1, Table 2, Table 3, Table 4 and Table 5.

4. The method according to any of claims 1 to 3, wherein the cancer is a cancer selected from the group of colon cancer, lung cancer, prostate cancer, breast cancer, stomach cancer, bladder cancer, renal cell cancer, ovarian cancer, liver cancer and pancreatic cancer.

5. The method according to any of claims 1 to 3, wherein the cancer is breast cancer.

6. The method according to any of claims 1 to 5, wherein the target cell is contacted with a plurality of test agents and the test agents having an effect on the on the level of expression of the listed genes are selected as a candidate therapeutic agent.

7. The method according to any of claims 1 to 6, wherein the target cell is a normal or cancer cell.

8. The method according to any of claims 1 to 6, wherein the target cell is a breast cancer cell.

9. The method according to any one of claims 1 to 6, wherein the target cell is i) a M DA-M B-231 breast cancer cell; or

ii) a BT20 breast cancer cell.

10. The method according to claim 9, wherein the test agent from step (a) suppress the expression of at least one or more genes, or products thereof, selected from the group of genes listed in Table 1 and/or activate the expression of one or more genes, or products thereof, selected from the group of genes listed in Table 2 if the target cell is a MDA-MB-231 breast cancer cell.

11. The method according to claim 10, wherein the test agents from step (a) suppress the expression of the gamma-aminobutyric acid (GABA) B receptor 1 and/or the matrix- remodeling associated 8 gene, or products thereof.

12. The method according to claim 10, wherein the test agents from step (a) activate the expression of one or more genes, or products thereof, selected from the group consisting of N DRG family member 2, endoglin, UDP-N-acetyl-alpha-D-galactosamine: polypeptide N- acetylgalactosaminyltransferase 14, nephroblastoma overexpressed gene, syntrophin beta 1 (dystrophin-associated protein Al, 59kDa, basic component 1), EH-domain containing 3, myelin protein zero-like 2, procollagen C-endopeptidase enhancer 2, SLAIN motif family, member 1, calpain 5, coiled-coil domain containing 80, membrane protein, palmitoylated 2 (MAGUK p55 subfamily member 2).

13. The method according to claim 10, wherein the test agents from step (a) suppress the expression of the matrix-remodeling associated 8 gene, or products thereof and activate the expression of N DRG family member 2 and endoglin, or products thereof.

14. The method according to claim 10, wherein the test agents from step (a) suppress the expression of the matrix-remodeling associated 8 gene, or products thereof and activate the expression of N DRG family member 2, endoglin and U DP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 14, or products thereof.

15. The method according to claim 10, wherein the test agents from step (a) suppress the expression of the matrix-remodeling associated 8 and the gamma-aminobutyric acid (GABA) B receptor 1 genes, or products thereof and activate the expression of NDRG family member 2, endoglin and U DP-N-acetyl-alpha-D-galactosamine: polypeptide N- acetylgalactosaminyltransferase 14, or products thereof.

16. The method according to claim 9, wherein the test agents from step (a) suppress the expression of one or more genes, or products thereof, selected from the group of genes listed in Table 3 and/or activate the expression of one or more genes, or products thereof, selected from the group of genes listed in Table 4 if the target cell is a BT20 breast cancer cell.

17. The method according to claim 16, wherein the test agents from step (a) suppress the expression of one or more genes, or products thereof, selected from the group consisting of microtubule-associated protein tau, heparanase, matrix-remodeling associated 8 and hydroxyprostaglandin dehydrogenase 15-(NAD).

18. The method according to claim 16, wherein the test agents from step (a) suppress the expression of the matrix-remodeling associated 8 and hydroxyprostaglandin dehydrogenase 15-(NAD) genes, or products thereof.

19. The method according to claim 16, wherein the test agents from step (a) suppress the expression of the matrix-remodeling associated 8, hydroxyprostaglandin dehydrogenase 15- (NAD) and heparanase genes, or products thereof.

20. The method according to any one of claims 1 to 9, wherein the test agent from step (a) suppress the expression of the transmembran protein matrix-remodeling associated 8.

21. A method for diagnosing cancer, in particular breast cancer, in a subject comprising the steps of: a) Analyzing the level of expression of two or more genes, or products thereof, selected from the group of genes listed in Table 5;

b) Comparing the level of expression with a reference sample from a subject not afflicted with said cancer whereby an alteration of at least twofold of the expression level in the test sample compared to the reference sample is indicative for cancer.

22. A method for determining whether a subject is predisposed to cancer, in particular breast cancer, or suffering from cancer, in particular breast cancer, said method including the step of: a) analyzing the level of expression of at least two or more genes, or products thereof, selected from the group of genes listed in Table 5 in a sample

b) determine whether said subject is predisposed to cancer or suffering from cancer, in particular breast cancer, preferably, wherein the level is an elevated level compared to a reference sample of a healthy control subject.

23. The methods according to any one of the proceeding claims, wherein the level of expression is analyzed on nucleic acid level, preferably by nucleic acid amplification methods, in particular RT-PCR.

24. The methods according to any one of the proceeding claims, wherein the level of expression of a gene is analyzed by using an amount of a mRNA in a sample.

25. The methods according to any one of the proceeding claims, wherein the level of expression of a gene product is analyzed by using an antibody against the gene product.