HK1110246A - Diagnostic marker for cancer - Google Patents

Description

Diagnostic markers for cancer

The invention relates to the use of a plurality of different proteins as diagnostic markers for cancer, the use of active substances for the treatment of cancer, and related pharmaceutical preparations and kits.

Cancer diseases are generally characterized by the development of one or more tumors. A tumor means a growth of tissue or a local increase in tissue volume. Broadly speaking, each localized swelling belongs to this category, such as edema, acute or chronic inflammation, dilatation due to aneurysms, inflammatory growth of organs (e.g. so-called splenic tumors). More narrowly, a tumor means the formation of new tissues (excrescence, blastoma, neoplasia) by spontaneous, uncontrolled, uninhibited to varying degrees, autonomous and irreversible growth of body tissues, together with the loss of specific functions of the cells and tissues.

For better classification, tumors were classified according to the following criteria:

I. its biological properties

1. Benign tumors with slowly growing differentiated cells and replacement of normal tissue

2. Nuclei exhibiting polymorphism, atypical cells, degenerative changes, invasive and destructive growth and metastatic malignancies

3. Semi-malignant tumors that show histological features of malignancy and local invasive growth but are often devoid of metastasis.

II. Criteria for tissue occurrence：

In this regard, tumors are classified according to the embryonic tissue of their developmental origin. Is provided with

1. Epithelial tumors originating from ectoderm and endoderm:

a) benign tumours, like adenomas, papillomas or polyps

b) Malignant tumors, e.g. carcinoma

2. Mesenchymal tumors originating in the mesoderm

a) Benign tumors, analogous to lipomas, fibroids, osteomas, myomas, leiomyomas, rhabdomyomas, chondromas

b) Malignant tumors, e.g. sarcomas

3. Embryonic tumors that develop from undifferentiated tissues. Nephroblastoma, neuroblastoma, medulloblastoma, retinoblastoma, embryonal rhabdomyosarcoma, teratoma are part of this group.

III. Classification according to clinical and pathological results. Among these are TNM-taxonomy, hierarchy, Lauren-taxonomy, Dukes-taxonomy, Kieler taxonomy, Rappaport-taxonomy, and the like.

A short overview of this tumor classification shows exactly how different types of tumors are varied, overlapping and even contradictory. Not only do there be differences between benign and malignant tumors; but also the lethality of certain tumors and the possibility that benign tumors may become malignant must be considered.

Some tumors, e.g., breast carcinoma (breast cancer), the most common malignancy in women, especially occurring in large numbers between the ages of 45 and 70. Early symptoms are suspicious results during examinations in cancer prevention regimes or periodic self-examinations of the breast. Depending on the stage of tumor development and differentiation, the prognosis can range from very positive to truly severe. Rapid diagnosis as early as possible is essential to initiate therapeutic measures due to early lymphogenic and hematopoietic metastasis of breast cancer.

Prostate cancer (carcinoma of the prostate) is the most common tumor in men, most of which occurs between the ages of 50 and 70. The main cases are adenocarcinoma. This type of malignancy first spreads by invasive growth in the prostate, later infiltrating cells in the transition zone and the connective tissue of the renal pelvis, and cells that rarely infiltrate the intestine, bladder, or urethra. Metastasis occurs via the lymphogenic and/or hematopoietic pathways. The therapeutic measures depend on the histological grade of differentiation and clinical stage and usually imply radical surgery, with complete removal of the prostate and lymphatic junction zones; androgen withdrawal during the developmental stage is a measure. Prognosis also depends on the stage of the tumor. Radical surgery cures approximately 90% of prostate cancer in the early stages, while the prognosis is often poor in the developing stages.

Prostate cancer must be differentiated from prostatic hyperplasia by diagnostic means. Prostatic hyperplasia is a benign tumor of the prostate; the gland becomes enlarged by the increase in the number of stromal cells and glands. Prostatic hyperplasia is the most common cause of dysuria in elderly men. Clinical symptoms usually occur between the ages of 40 and 50, and the disease progresses slowly and gradually. Symptoms often appear after a gradual reduction in the ejection of urine and a delay in urination for several years. The application of phytotherapy may be therapeutic use and alleviate clinical symptoms.

In general, early diagnosis of tumors is necessary for rapid initiation of therapeutic measures. If the tumor is detected early; the prognosis is improved; many so-called tumor markers are therefore used in clinical practice. Tumor markers are molecular or cellular alterations that can be identified or quantified to obtain information about the presence, progression and prognosis of a (malignant) disorder. Tumor markers are classified as:

1. cellular tumor markers：

This group comprises, inter alia, tumor antigens of the cell membrane, receptors (e.g. hormone receptors, receptors for growth-stimulating substances in leukemia), and cell markers representing an increased oncogene expression and growth of monoclonal cells, as well as genetic alterations, in particular chromosomal aberrations.

2. Humoral tumor markers：

Under physiological conditions, increased concentrations of these substances (which are mostly part of the normal physiological component) can be detected in biological samples, in particular in serum, urine and other body fluids. They are synthesized and/or secreted in tumor tissues, either by destruction of tumor cells or released in the tissue's response to the tumor. There is little understanding of the physiological relevance of tumor markers. In humans, it generally has no immunogenic properties. Their clinical (diagnostic) significance depends on specificity and sensitivity. Humoral tumor markers are divided into two categories. One group includes markers produced by tumors, such as tumor-associated antigens, specific hormones (e.g., gastrin, cortisol, etc.), enzymes (e.g., neuron-specific enolase, NSE), or proteins (e.g., Bence-Jones-protein). Tumor markers produced by tumor induction rather than by tumor cells belong to the second group. Important members of the second group are Alkaline Phosphatase (AP), LDH (lactate dehydrogenase), neopterin, etc.

Recent publications disclose two representative cells of a brain tumor, a myeloblastoma, which may be the most common in childrenList of proteins detected in lines and possibly used as tumor markers (a. peyrol et al, 2003, Proteomics, 3, 1781-. US6,645,465 discloses that it belongs to Ca²⁺Annexins a1 and a2, which bind proteins, are useful as tumor markers for lung, breast and esophageal cancers and can be identified by detecting auto-antibodies directed against them. In animal experiments it could be shown that the use of radiolabeled antibodies against annexin a1 results in loss of tumor mass, which may promote cell necrosis of tumor cells (p.oh, y.li, j.yu, e.durr, k.m.krasinska, l.a.carver, j.e.testa, j.e.schnitzer, 2004, Nature, 429, 629-35).

Differential abundance analysis has recently been performed in pancreatic epithelial cells where annexin a3 is indicated as malignant and non-malignant (benign) in identifying proteins (a.r. shekouh et al, 2003, Proteomics, 3, 1988-. The abundance of proteins in malignant and non-malignant prostate tissue has also been studied. Considering the proteins identified in this regard, essentially no further details are given regarding the possible over-or under-expression of the proteins listed in cancer tissues compared to healthy tissues (a.a. alaiya et al, 2001, cell.mol.life sci., 58, 307-.

Reports to date have demonstrated the importance of selective and sensitive methods for tumor detection. Furthermore, there is a great need for new targets for tumor and cancer therapy, respectively.

The present invention therefore relates to the problem of developing new markers for cancer diagnosis as well as new targets and drugs for cancer therapy.

This problem is solved by the solution of the independent claims. Preferred embodiments are given in the dependent claims. By reference, the wording of all claims is an integral part of the description.

By focused comparative analysis between malignant degenerated (cancerous) and normal tissues, unique proteomes can be identified that exhibit markedly different abundances and concentrations in different types of tissues. The characteristic abundance of certain proteins compared to controls represents an important indicator of degenerated cell growth, i.e., cancerous tissue. According to the present invention, these specific proteins are used as diagnostic markers for cancer.

To identify these proteins, samples from tumor tissue (prostate cancer) and healthy tissue were prepared, both samples labeled with two different radioisotopes. The samples were pooled and the mixtures were separated by electrophoresis on a two-dimensional polyacrylamide gel. The signal of each isotope was detected separately and the corresponding protein spots were further analyzed. This approach identifies and quantifies several unique proteins that apparently have different abundances in cancer or healthy tissue. Some of these proteins are significantly more abundant in cancer tissues, they are up-regulated, while others are present in significantly lower abundance, they are down-regulated.

The present invention encompasses the use of the protein annexin, in particular annexin a3, as a diagnostic marker for cancer. The inventors were able to demonstrate that this protein is upregulated on average 2.4-fold, and in some cases more than 5-fold, in tumor tissue from a defined set of patients. In a particularly preferred embodiment, annexin a3 can therefore be used as a diagnostic marker for a specific subtype (patient group) of prostate cancer. Therefore, the up-regulation of this protein relative to the control is preferably investigated as a characteristic index of cancer tissue. Annexins are members of a family of structurally related proteins that bind phospholipids and form calcium pores in the presence of calcium. The precise function of annexin is not fully understood to date.

There is evidence that annexin is involved in intracellular and extracellular processes. However, it is not known how annexin is secreted, e.g. membrane trafficking, cell motility Ca²⁺Influx and signal transduction. For example, they do not have the classical leader sequence for transfer into the lumen of the endoplasmic reticulum. However, annexin is found in small secretory vesicles, so-called exosomes, and therefore it is presumed that annexin reaches the outside of cells by lysis of exosomes. The cleavage of the vesicles may result inPresentation of modified antigens in tumors. In general, extranuclear is involved in the presentation of antigens in the immune system, which involves the MHC class I/T-cell system.

Interestingly, annexins are involved in bone mineralization (Wang W.xu J., Kirsch T2003 J.biol.chem.2003, 278: 3762-9). Annexin-mediated Ca²⁺Influx regulates growth surface chondrocyte maturation and apoptosis. This is particularly significant because metastasis of prostate cancer typically produces a high frequency of osteoblastic bone lesions compared to other cancer types. Most cancer metastases are characterized by their osteolytic activity, which means degeneration of bone. In contrast, prostate cancer metastasis shows destructive bone (destructive) and osteogenic (proliferative) activity. In this case, normal bone crystals are deconstructed and then re-established as irregular bone deposits. Even with little understanding of this mechanism, the physiological processes of mineralization play an important role. Mineralization is caused by vesicles secreted by the plasma membrane of mineralized osteoblasts, so-called stroma vesicles. In the early stages, crystals of calcium phosphate appear inside the matrix vesicles. These vesicles are covered with a film; channel proteins are therefore necessary to transport minerals into the vesicles. Important components of the vesicles are the proteins annexin a2, a5 and a6 and the collagens type II and X on the outer surface of the vesicles that bind annexin a5 to adhere to the outer surface of the vesicles. Annexin channels through the membrane of matrix vesicles, which allow Ca²⁺Into the interior of the vesicle. Collagen amplification channel activity binding to annexin A5 and mediating Ca together with other annexins²⁺And the formation of an initial crystalline phase within the vesicle. This leads to the initiation of mineralization. When the intracellular crystals have reached a critical size, they break the membrane and lyse the vesicles. The crystals grow further (growth phase of mineralization) and promote bone establishment. According to the results of the present inventors, this function of annexin in irregular mineralization of bone caused by prostate cancer metastasis is presumably associated with upregulation of annexin a3 in cancer tissues. In this context, inorganic pyrophosphatase has to be considered, which according to the inventors' results releases phosphate and is up-regulated in cancer, in particular prostate cancer. Annexin A3 from cancer cellsUp-regulation of annexin a3 could lead to the conclusion that annexin a3 has a biological function in the exosomes of prostate cancer cells. This may be due to ion channel relationships. The preferred use of the protein annexin a3 relates to the activity of the protein in the exosomes. Preferably, this results in a change in the immunological control of the tumor cells. Since the extracellular concentration of annexin A3 is higher in the vicinity of tumor cells, affinity agents-in particular antibodies with high affinity for annexin A3-are suitable for targeting active substances similar to toxins or radioactive compounds of adjacent tumors. The drug does not pass through the cell membrane, so that only healthy cells expressing intracellular annexin a3 are not affected. Interestingly, matrix vesicles have also been observed to be associated with osteoarthritic cartilage and atherosclerotic lesions.

The release of cytoplasmic proteins into extracellular mediators, which occurs after ectosomal lysis, can induce an inflammatory response similar to cellular necrosis. Inflammation is known to reduce the adaptive T-cell-induced immune response known to characterize many cancer cells. Furthermore, the presence of annexin in the extracellular space can also affect this pattern (a. bonanza et al, 2004 j. exp. med. 2001157-65). Vaccination against cancer can therefore be determined by understanding and influencing this system.

A particularly preferred embodiment of the use of annexin A3 relates to the up-regulation of this protein and the simultaneous down-regulation of annexin a1, annexin a2 and/or annexin a 5. Preferably this is done in comparison to a control. It has recently been demonstrated that annexin a1, annexin a2 and annexin a5 are down-regulated in cancer tissues, particularly prostate cancer. It is therefore particularly advantageous to assay for down-regulation of one or more other annexins in combination with up-regulation of annexin a 3. Based on these results, annexin a3 can replace other annexins during prostate carcinogenesis and is therefore a surrogate marker or target for prostate cancer therapy.

The invention also encompasses the use of protein ubiquitin isopeptidase T and/or Protein Disulfide Isomerase (PDI) as a diagnostic marker for cancer. Advantageously, down-regulation of ubiquitin isopeptidase T and/or up-regulation of Protein Disulfide Isomerase (PDI) compared to controls should be used as a characteristic marker for cancer tissue. The present inventors demonstrated that ubiquitin isopeptidase T was in average 5-6 fold lower in abundance in cancer tissues compared to healthy tissues, while PDI was approximately 2 fold higher in abundance. This demonstrates the inverse correlation between PDI and ubiquitin isopeptidase T.

Ubiquitin isopeptidases are enzymes involved, among other enzymes, in ubiquitin-dependent proteolytic cleavage of proteins. After the polyubiquitin chain is added to the target protein, the ubiquinated protein is degraded by a protein complex consisting of multiple subunits, the 26S proteasome. Subsequent removal of polyubiquitin chains is mediated by the zinc-binding ubiquitin enzyme isopeptidase T. Thus down-regulation of ubiquitin isopeptidase T can affect the rate of proteolysis by ubiquitin, or the rate of degradation of specific proteins, in prostate cancer. Furthermore, ubiquitin isopeptidase T PDI is involved in the controlled hydrolysis of proteins, i.e., the process of apoptosis. Inside the endoplasmic reticulum, PDI interacts with ubiquitin-like and ubiquitin-associating domains under certain conditions. This interaction is functionally related to obtaining tolerance to ischemic stress and apoptosis (Ko h.s., et al, 2002, j.biol.chem.277: 35386-92).

The association of the two enzymes with apoptosis makes the observation of up-or down-regulation suitable as a marker characteristic of cancer tissues. On the other hand, it may be advantageous to analyze the abundance of only one protein, in particular ubiquitin isopeptidase T as a diagnostic marker. The great advantage is that ubiquitin isopeptidase in cancer tissues is significantly down-regulated and shows only about 1/5 to about 1/6 abundance in healthy controls. The observed decrease in the abundance of ubiquitin-isopeptidase T in cancer tissues is more pronounced than in mammalian fatty acid-binding proteins (M-FABPs) identified as anti-carcinogens.

The possible link between annexin antigen presentation by MHC (major histocompatibility complex) by ubiquitin-isopeptidase T and influencing T-cell activity by altered activity of the systemic immune system due to the absence of immunoglobulin domain containing SAP in prostate tissue may be of paramount importance for the survival of tumor cells in the presence of the immune system.

The invention also encompasses the use of mitochondrial enoyl-coa-hydratase as a diagnostic marker for cancer and/or as a therapeutic target molecule. This protein may also be used in combination with fatty acid-binding protein 3(FABP-3) and/or epidermal fatty acid-binding protein (E-FABP) and/or annexin a 3. Particular preference is given to up-regulation of mitochondrial enoyl-coa-hydratase and/or epidermal fatty acid-binding protein (E-FABP) and/or down-regulation of fatty acid-binding protein 3(FABP-3) and/or annexin a3 as compared to a control, since this up/down regulation of these proteins according to the invention is revealed as a characteristic feature of cancer tissue.

The present inventors have been able to show that mitochondrial enoyl-coa-hydratase has an average increased abundance in cancer tissues of about 2.8 to 4 fold. This enzyme has been described to be synergistic with the beta-oxidation of fatty acids, and this occurs mainly in the mitochondria. Enoyl-coa-hydratases participate in non-oxidative metabolism. It has long been known that cancer cells have increased, non-oxidative metabolism even when there is an excess of oxygen, and that both oxidation and de novo synthesis of fatty acids is increased in cancer patients. The cancers listed herein are associated with many changes in fatty acid metabolism. Fatty acid synthases responsible for fatty acid biosynthesis have recently been suggested as therapeutic pharmacological targets. The results presented show that enoyl-coa-hydratase is a similarly suitable target. This link in fatty acid metabolism represents a functional link between enoyl-coa-hydratase and FABP-3 and E-FABP. The abundance of these fatty acid-binding proteins in cancer tissue is also characteristically altered. FABP-3 was approximately 2.5 fold down-regulated, while E-FABP was approximately 2.3 fold up-regulated. In addition to the association with fatty acids, the role of FABP-3 in a relationship with cell cycle control has been described (Seidita G. et al 2000, Carcinogenesis 21: 2203-10). The association of E-FABP with different types of cancer has been described and has been detected in the urine of cancer patients (Brouard M.C. et al 2002, Melanoma-Research 12: 627-31). The increase in abundance of this protein observed by the present inventors makes it particularly suitable as a diagnostic marker for cancer when combined with the other markers mitochondrial enoyl-coa-hydratase and/or FABP-3 and/or annexin a3, as there is a functional link between these different proteins. Thus, one or particularly advantageously two or three of these proteins can be observed in abundance compared to a control, so that by characteristic up/down-regulation of these proteins, conclusions can be drawn about the presence of cancerous tissue. As will be indicated hereinafter, the link to fatty acid metabolism is also applied to the further proteins described herein.

The invention also encompasses the use of the protein serum-amyloid P-component (SAP) as a diagnostic marker or therapeutic agent for cancer. The inventors were able to show that this protein in cancer tissues revealed on average a reduction in its incidence of about 2.7 to 5.1 fold. SAP is mainly present in the interstitial cells of benign prostate tissue, so that its relatively low incidence in cancer tissue may be interpreted as a relatively small number of interstitial cells in cancer tissue. Studies of SAP down-regulation compared to controls are therefore particularly suitable as a characteristic feature of cancerous tissue. SAP is an acute phase protein (results from mice) similar to the lectin of the pentraxin family and is associated with several clinical phenomena of amyloid. Amyloid deposits are sometimes observed in the male urinary system, but are poorly understood biologically. Native SAP, which folds correctly beyond the amyloid fibrils, also binds polysaccharides, including bacterial polysaccharides and matrix components, by means of acid carbohydrate determinants, phosphoethanolamine and phosphocholine. SAP is a constituent of simple membranes and may cause its interaction with laminin and phospholipids. It is involved in target recognition by phagocytes of the evolved or systemic immune system, such as polymorphonuclear leukocytes, binding to phospholipids on apoptotic cells, leading to their phagocytosis by macrophages. It has long been known that SAP levels in the serum of malignant humans are increased, and IL-6 appears to be responsible for this, at least in the serum of some cancer patients. In summary, SAP can be speculated to be involved in the regulation of non-cancer cell interactions with its environment and possibly in immune monitoring, a function which may be disturbed in many cancer cells. Pentraxins can be induced by cytokines, the concentrations of which in the blood are significantly elevated during infection and trauma, and therefore they play a role in immune defense. The observations provided suggest that the association between annexin a3, ubiquitin isopeptidase T and serum-amyloid P-component in prostate immune monitoring results in an altered immune monitoring regulation by exosomes.

The invention also encompasses the use of protein 14-3-3 protein tau (tau) as a diagnostic marker for cancer. This protein is known to be involved in the apoptotic process. This process has been described in relation To cancer, but an anti-carcinogenic state has been established (He H.1997, Gan-To-Kagaku-Ryoho 24: 1448-53). However, the present inventors have surprisingly found increased levels of the 14-3-3 protein tau (1.8 fold) in cancer tissues. Immunohistochemical staining reactions have revealed that the 14-3-3 protein tau is present predominantly in healthy epithelial cells and in cancer cells of prostate tissue. However, in the interstitium, the protein 14-3-3. tau is present only in lymphocytes (only lymphocytes are stained). According to the invention, the upregulation of this protein is therefore investigated as a characteristic feature of cancer tissue in comparison with controls.

The invention also encompasses the use of the protein nuclear chloride channel protein (CLIC-1) as a diagnostic marker for cancer, in particular prostate cancer. The present inventors confirmed that the abundance of this protein increased by about 1.5-fold in cancer tissues compared to controls. It is therefore preferred to study the upregulation of this protein as a characteristic feature of cancerous tissue in comparison to controls. This intracellular anion channel has been described to be associated with cell division and apoptosis (Ashley r.h., 2003, mol.membr.biol.20: 1-11).

Furthermore, the present invention encompasses the use of the protein HES1 as a diagnostic marker for cancer. The inventors could demonstrate that the abundance of this protein in cancer tissue is about 4-fold higher compared to controls. The p-value of the probability of corresponding up-regulation in the T-test was < 0.0001. Preferably, the upregulation of this protein compared to a control is considered to be a characteristic marker of a cancer disease. This protein is a specific splice variant with unknown function (HES 1/Kpn-la). It comprises the DJ 1-Pfdl-domain; presumably, it is located in the mitochondria, which may indicate a possible link to enoyl-coa-hydratase function. This protein is expressed in many human tissues. Its relationship to cancer has been demonstrated for the first time by the inventors.

Furthermore, the invention encompasses the use of proteasome α 2-subunit as a diagnostic marker for cancer. The relationship of this protein to cancer diseases has been demonstrated for the first time by the inventors. The abundance doubled in cancer tissues compared to controls. T-test for cancer-related changes of this protein was significant, p < 0.009. Preferably, the up-regulation of this protein is tested in comparison with a control. Proteasomes are known to function in processing peptides for antigen presentation in the MHC class 1 system, which promotes the activity of killing t cells.

Furthermore, the invention encompasses the use of the protein adenine-phosphoribosyltransferase as a diagnostic marker for cancer, in particular prostate cancer. The relationship of this protein to cancer has been recently discussed. For example, the down-regulation of this protein in lymphocytes from breast cancer patients has been described. Furthermore, overexpression of this protein in colorectal cancer has been observed. The inventors could demonstrate that the abundance of this protein in prostate cancer tissue is about 2-fold higher compared to controls. These results were significant for differential expression in the t-test, p < 0.007. According to the invention, the upregulation of this protein compared to a control is considered as a characteristic marker of cancer tissue.

Furthermore, the invention encompasses the use of a proteinaceous inorganic pyrophosphatase as a diagnostic marker for cancer, in particular prostate cancer. This protein has recently been shown to be up-regulated in lung and colorectal cancers. The present inventors could demonstrate that the abundance of this protein is 1.6 times higher in prostate cancer tissues compared to normal tissues. These results were significant for differential expression in the t-test, p < 0.005. Inorganic pyrophosphatase 1 catalyzes a reaction of releasing inorganic phosphoric acid. This relates to the involvement of annexins, in particular annexin A3, in Ca²⁺A process of calcification of the flow. There is thus a functional link, in particular, between the upregulation of annexin A3 and the upregulation of inorganic pyrophosphatase。

The various proteins referenced herein, as well as the proteins further mentioned below, may be used for diagnostic purposes alone or in combination with other proteins.

Furthermore, the invention encompasses the use of at least one of the following proteins as a diagnostic marker for cancer: ubiquitin-isopeptidase T, serum amyloid P component (SAP), fatty acid-binding protein 3(FABP-3), galectin-1, heat shock protein 27(HSP27), 14-3-3 protein beta, 14-3-3 protein zeta (zeta), nuclear chloride channel protein 1(CLIC-1), 14-3-3 protein tau, heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), epidermal fatty acid-binding protein (E-FABP), mitochondrial enoyl-coa-hydratase, nucleophosmin, annexin, in particular annexin a3, tranexamin, triose phosphate isomerase, aldolase a HES1, the α 2-subunit of proteasome, adenine-phosphoribosyl-transferase. Preferably, downregulation of at least one of the protein isopeptidase T, serum amyloid P component (SAP), fatty acid-binding protein 3(FABP-3), galectin-1, microgrinin beta, heat shock protein 27(HSP27), or a transgelin, as compared to a control, is considered a marker characteristic of a cancer disease. Furthermore, it is preferred that upregulation of at least one of the proteins 14-3-3 protein β, 14-3-3 protein ζ, nuclear chloride channel protein 1(CLIC-1), 14-3-3 protein τ, heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), epidermal fatty acid-binding protein (E-FABP), mitochondrial enoyl-CoA-hydratase, nucleophosmin, annexin particular annexin 3, triose phosphate isomerase, aldolase A, HES1, alpha 2-subunit of proteasome, adenine-phosphoribosyl-transferase and inorganic pyrophosphatase 1 in addition or alternatively compared to controls is considered a characteristic marker of cancer disease. It is particularly preferred to study other annexin downregulations in addition to one or more of these proteins. It is particularly preferred to study at least two proteins.

Particular benefits are provided by the use of two of the following proteins as diagnostic markers: ubiquitin-isopeptidase T, heat shock protein 27(HSP27), heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), mitochondrial enoyl-coenzyme A-hydratase and/or nucleophosmin.

It has been demonstrated according to the present invention that the expression of a unique set of proteins is characteristically down-or up-regulated, respectively. Details are shown in table 1 below summarizing the results for identifying and quantifying proteins differentially expressed between benign and malignant tissues. The selection of proteins is based on statistically significant differential expression analysis of proteins in benign (benign parts) or malignant tissues (cancerous parts). Accession numbers refer to the respective numbers in the NCBI database. Theoretical Molecular Weights (MW) were calculated from sequences in the database. "integration" refers to the number of samples determined using MASCOT techniques. The details given with respect to PMF-points refer to the Mouse-points used by MASCOT-servers; typically a PMF-integral in excess of 65 represents significant identity. The last two columns summarize the intensity quantification of protein spots found in benign and malignant tissue samples.

TABLE 1

Numbering	Login number	Description of the invention	Theoretical molecular weight	PMF integration	Benign segment	Cancer part
							1	gi|1732411	Isopeptidase T [ human]	94104	115	83.6	16.4
2	gi|576259	Chain A; serum amyloid P component (Sap)	23598	106	73.1	26.9
							3	gi|494781	Fatty acid-binding protein (intact form, human muscle) (M-Fabp)	14775	87	71.6	28.4
4	gi|4504981	Beta-galactosidase binding to a lectin precursor; a lectin; galactose-binding; is soluble; 1; galectins [ human beings ]]	15769	177	66.2	33.8
							5	gi|225159	Microseminal protein beta	12238	92	63.9	36.1
6		n.i.			60.6	39.4
							7	gi|662841	Heat shock protein 27 (human)]	22667	182	60.2	39.8
8	gi|4507949	Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein, beta polypeptide; 14-3-3pr	27946	160	41.2	58.8
							9	gi|4507953	Tyrosine 3/tryptophan 5-monooxygenase activating protein, zeta polypeptide; protein kinase C inhibitors	27810	160	41.1	58.9
10	gi|2073569	Nuclear chloride channel protein (human)]	27249	139	40.1	59.9
							11		n.i.			39.5	60.5
12		n.i (annexin A3)	36524	160	37.4	62.6
							13	gi|5803227	Tyrosine 3/tryptophan 5-monooxygenase	28032	130	35.6	64.4

		An activator protein, a theta polypeptide; 14-3-3 protein tau
							14	gi|13129150	Heat shock 90kDa protein 1, alpha, heat shock 90kD protein 1, alpha [ human]	85006	147	32.6	67.4
gi|20149594	Heat shock 90kDa protein 1, beta, heat shock 90kD protein-1, beta	83554	164
						15		gi|20070125	Prolyl 4-hydroxylase, beta subunit, v-erb-a avian erythroblastic leukemia virus oncogene homolog 2	57480	235	31.2	68.8
16	gi|4557581	(NM 001444) fatty acid binding protein 5 (psoriasis-associated); E-FABP (human)]	15497	94	27.9		72.1
						17		gi|12707570	Mitochondrial short chain enoyl-coenzyme A-hydratase I precursors [ human]	31807	101	26.2	73.8
18	gi|16307090	Similar to nucleophosmin (nucleolar phosphoprotein B23, nuclear matrix protein) [ human]	29617	77	21.9		78.1
						19		gi|7768772	HES1 protein, homologous to E.coli and zebrafish ES1 protein, anti-sigma cross-reactive protein homolog I alpha precursor, KNP-Ia, GT335, analogous to E.coli SCRP27A and zebrafish ES1[ human]	29215	95	＜20	＞80
20	gi|4506181	Proteasome α 2 subunit; proteasome subunit HC 3; proteasome synthesisC3; macropain subunit C3; multicatalytic endopeptidase complex subunit C3[ human]	26236	105	32.6		67.4
						21		gi|4502171	Adenine-phosphoribosyltransferase; AMP pyrophosphorylase; AMP diphosphorylase; transphosphoribosylase	20127	134	33	67
22	gi|11056044	Inorganic pyrophosphatase [ human)]			38.6		61.4

We refer herein to fig. 5 and 10, in which mean significantly differentially expressed protein point results for 21 patients and 31 patients accompanied by statistical data are shown in tabular form.

An overview of the different proteins in english synonyms is listed below. The numbers listed above correspond to the numbers in table 1.

1, gi | 1732411: ubiquitin-isopeptidase T; isopeptidase t (siot); ubiquitin-specific protease 5; ubiquitin carboxy-terminal hydrolase 5; a ubiquitin thioesterase 5; ubiquitin-specific processing protease 5; deubiquitinase 5; a deubiquitinase.

Gi | 576259: serum-amyloid P-component; chain A; serum amyloid P component (SAP).

3, gi | 494781: fatty acid-binding protein 3 (FABP-3); mammary gland-derived growth inhibitor (MDGI); fatty acid binding protein 3 (FABP-3); heart fatty acid binding protein (H-FABP); muscle fatty acid binding protein (M-FABP).

Gi | 4504981: a galectin; galectin-1; kDa β -galactoside-binding lectin; beta galactoside soluble lectin; beta-galactoside binding lectin 1-14-1; a galactin; a soluble galactoside binding lectin; S-Lac lectin 1.

Gi | 225159: microsamin protein beta; beta-microservoloprotein; a micro-seminal protein beta; immunoglobulin binding factor (IGBF); PN 44; seminal albumin secreted by the prostate; a prostate secretory protein of 94 amino acids (PSP-94); refined beta-inhibin; refined albumin.

6. And is not identified.

7, gi/662841: heat shock protein 27(HSP 27); heat shock protein 27; 27kDa heat shock protein 1 (HSP-27); stress-responsive protein 27(SRP 237); an estrogen-regulated 24kDa protein; a 28kDa heat shock protein.

Gi | 4507949: 14-3-3 protein beta; 14-3-3 protein beta (14-3-3 beta); 14-3-3 protein alpha (14-3-3 alpha); protein kinase C inhibitor protein-1; PKC inhibitor protein-1 (KCIP-1: also 14-3-3 ζ); RNH-1.

Gi | 4507953: 14-3-3 protein ζ; 14-3-3 ζ; 14-3-3 sigma; KCIP-1 (also 14-3-3. beta.); YWHAZ; mitochondrial import stimulation factor S1(MSF S1); factor-activated exoenzyme S; tyrosine monooxygenase activating protein ζ; tryptophan monooxygenase activating protein ζ.

Gi | 2073569: nuclear chloride channel proteins; chloride intracellular channel 1 (CLIC-1); nuclear chloride channel protein (p64 CLCP); a nuclear chlorine channel; a chloride channel ABP; nuclear chloride channel 27(NCC 27); an RNCC protein; nuclear chloride channel 27(NCC 27).

11. And is not identified.

(annexin a3, see 23).

Gi | 5803227: 14-3-3 protein τ; 14-3-3 θ; s15076 protein kinase modulator 14-3-3; HS 1; tyrosine 5-monooxygenase activating proteins; tryptophan 3-monooxygenase activating proteins.

Gi | 13129150: heat shock protein 90(HSP 90); heat shock protein 90(HSP 0-90); heat shock protein HSP 90-alpha; heat shock protein 90-alpha; a 90kDa heat shock protein; heat shock protein 86(HSP 86); hspca; heat shock 90kDa protein 1; heat shock protein 1; tumor specific transplantation 86kDa antigen (TSTA).

Gi | 20070125: protein Disulfide Isomerase (PDI); protein Disulfide Isomerase (PDI); prolyl-4-hydroxylase β; a protein disulfide oxidoreductase; thyroid hormone binding protein p 55; glutathione (glutamhlone) insulin transhydrogenase.

Gi | 4557581: epidermal fatty acid-binding protein (E-FABP); fatty acid binding protein 5 (FABP-5); epidermal fatty acid-binding protein (E-FABP); psoriasis-associated fatty acid-binding protein (PA-FABP); skin fatty acid-binding protein (C-FABP); keratinocyte acid-binding protein (KLBP); DA 11.

Gi | 2707570: mitochondrial enoyl-coenzyme-a-hydratase; mitochondrial enoyl-coa hydratase; mitochondrial enoyl-CoA-hydratase; short-alkenoyl-CoA hydratase, mitochondrial; short-alkenoyl-coa hydratase (SCEH).

Gi | 6307090: nucleophosmin; nucleophosmin; nucleolar phosphoprotein B23; nucleolin NO 38; nuclear matrix protein; NPM (1).

Gi | 7768772: HES1 protein, homologs of e.coli and zebrafish ES1 protein; anti-sigma cross-reactive protein homolog I α precursor, KNP-la/Kpn-1 α, GT335 (analogous to E.coli SCRP27A and zebrafish ES1[ human ].

Gi | 4506181: proteasome α 2-subunit; proteasome subunit HC3, proteasome component C3; macropain subunit C3; multicatalytic endopeptidase complex subunit C3[ human ].

Gi | 4502171: adenine-phosphoribosyltransferase; AMP pyrophosphorylase; AMP diphosphorylase; a transphosphoribosylase.

22, gi | 11056044: inorganic pyrophosphatase; a cylosolic inorganic pyrophosphatase; inorganic pyrophosphatase 1; pyrophosphatase phospho-hydrolase [ human ].

In addition, more than four proteins that are up-or down-regulated in cancer tissue compared to controls in certain patient sets were identified (clustering analysis). These are the proteins annexin a3, the transgelins, the triosephosphate isomerase and the aldolase a. Annexin a3 was up-regulated by approximately 5-fold and the transcoagulant protein was down-regulated by approximately 5-fold in cancer tissues. Triose phosphate isomerase and aldolase a were up-regulated in cancer tissues by approximately 20% and 10%, respectively.

In this respect we refer to fig. 3 which shows a graphical representation of the results of the clustering analysis. The figure illustrates the up-or down-regulation of different proteins in cancer tissue compared to healthy tissue in certain patient groups (or clusters, respectively), each represented by a circle.

The english synonyms for annexin and transcoagulant are as follows:

gi | 4826643: annexin a 3; annexin III; lipocortin III; anticoagulant protein III; placental anticoagulant protein iii (pap iii); 35 alpha calmette-guerin.

24, gi | 4507359: a protein is converted into a coagulin; SM 22-alpha smooth muscle protein, 22Da actin-binding protein, smooth muscle 22 protein, actin-associated protein p27, 25kDa F-actin-binding protein.

In addition, more proteins that show different abundance levels (down or up) in cancer tissues of certain patient populations compared to controls are identified. These proteins are ATP synthase, cholecycline reductase B, glucose-regulated proteins, prolyl-4-hydrolase beta and dnak-like molecular chaperones. ATP synthase is down-regulated and other proteins are up-regulated.

Interestingly, many of the proteins we identified are involved in lipid metabolism. Direct binding of annexin a3 and SAP to lipids has been reported. Both proteins are involved in phagocytosis. FABP-3 and E-FABP are fatty acid binding proteins. Mitochondrial enoyl-coa hydratase involves beta-oxidation of fatty acids. Phospholipases interacting with phospholipids induce protein kinase C which stimulates HSP27 activity. HSP90 is also involved in phospholipid metabolism, as inhibition of HSP90 leads to changes in phospholipid metabolism (Chun Y. et al, 2003, J.Natl.cancer Inst.95: 1624-33). Furthermore, PDI is also presumed to be involved in lipid metabolism because it functions as a multifunctional protein and is involved in triglyceride transfer in addition thereto (Horiuchi R. andYamauchi K.1994, Nippon-Rinsho 52: 890-5). Furthermore, the 14-3-3 protein inhibits protein kinase C and contains a conserved sequence that appears similar to the pseudo-substrate domain and C-terminus of protein kinase C. This suggests a functional relationship between those different proteins.

The diagnostic markers according to the invention can be used to identify different types of tumors and cancer diseases. In a preferred embodiment of the invention, the cancer disease to be diagnosed is prostate cancer, in particular prostate carcinoma. As mentioned earlier, prostate cancer is the most common malignancy in men. Only if detected at an early stage, prostate cancer can be successfully treated by prophylactic surgical removal of the prostate. If the disease progresses and is no longer confined to one organ, preventive surgical removal of the prostate is insufficient. For prostate tumors that cannot be removed by surgery, androgen suppression may be considered. This inhibition, preferably accompanied by surgery or drug detasseling, can sometimes inhibit tumor proliferation and metastasis and allow control of the tumor for a particular time. But most prostate tumors soon became resistant to this endocrinological treatment. Other therapeutic modalities, such as the use of cytotoxic agents, gene therapy or immunotherapy, are still in clinical research and have not been successful. Tumors of the prostate must therefore be detected early enough to be able to be successfully removed by surgery. The marker proteins described according to the present invention provide great benefits for the early detection of prostate cancer.

In a preferred embodiment of the method of the invention, certain subtypes of cancer, in particular of prostate cancer, can be diagnosed by quantifying preferably several of the above-mentioned proteins. The inventors could demonstrate that by so-called clustering analysis, unique protein patterns reflect characteristic up-or down-regulation of different proteins corresponding to a unique set of patients. Patients belonging to a particular cohort all showed the same unique subtype of cancer, in particular prostate cancer. According to the present invention, it is contemplated that patients will be characterized according to the relationship of a particular subtype of cancer to the protein pattern determined by application of the methods of the present invention to selectively treat this subtype of cancer. Preferably, the defined combination of proteins should be analyzed. In this regard, we refer to fig. 3 which shows a graphical representation of characteristic protein patterns corresponding to different patient pools. The table in figure 4 illustrates a summary of the protein patterns representing different patient pools and prostate cancer subtypes, respectively.

For diagnosing different subtypes of prostate cancer, it should be preferred to determine the abundance of different combinations of proteins. Thus at least one of the following should be determined as a common cancer marker: up-regulation of nuclear phosphoprotein, protein disulfide isomerase, heat shock protein 90, mitochondrial coenzyme a hydratase; down-regulation of heat shock protein 27 and/or ubiquitin isopeptidase T. These can be used together with at least one of the following proteins for the analysis of three subtypes of prostate cancer.

Subtype a: up-regulation of the tranagulin; substantial down-regulation of galectins and microgrinins beta; down-regulation of fatty acid binding protein 3; there was no or little change in epidermal fatty acid-binding protein, no or little change in nuclear chloride channel protein, 14-3-3 protein beta, zeta and tau, aldolase A, serum amyloid P component, triosephosphate isomerase and/or annexin A3.

Subtype b: substantial upregulation of protein disulfide isomerase, heat shock protein 90; substantial down-regulation of ubiquitin isopeptidase T; upregulation of 14-3-3 protein β, ζ and τ, aldolase A, triose phosphate isomerase, annexin A3; down-regulation of tranglutinin, galectin, microgrinin beta, serum amyloid P components; fatty acid binding protein 3 and/or nuclear chloride channel protein has no or less change.

Subtype c: substantial upregulation of nuclear chloride channel proteins; down-regulation of serum amyloid P component; fatty acid binding protein 3, 14-3-3 protein beta, zeta and tau, aldolase a, triose phosphate isomerase, annexin a3, epidermal fatty acid-binding protein; micro-seminal protein beta, galectin, transgelin were not or less altered.

According to the present invention, for the diagnosis of different subtypes of prostate cancer, it is possible to analyze at least one common cancer marker in combination with at least annexin a3 as a further protein.

In another particularly preferred embodiment, it is possible to diagnose a specific prostate cancer subtype present in a particular patient population by exclusively studying annexin a3 and/or mitochondrial enoyl-coa-hydratase.

For the purposes of the present invention, where the protein is described as a diagnostic marker, its use can consist of a variety of methods in order to analyze the incidence and abundance of the protein in cancerous tissue (or in the tissue under investigation) as compared to control tissue. It is particularly advantageous if the proteins in the sample under investigation and the control sample are separated by gel electrophoresis, for example on a conventional polyacrylamide gel. The abundances of a given protein in the sample and control are then compared. Bi-directional gels are particularly preferred due to the necessary disintegration. However, it is also possible to carry out a preliminary purification before the gel electrophoresis separation, so that sufficient separation and analyzability is obtained with, for example, one-way polyacrylamide gel electrophoresis. Other methods of protein separation may also be advantageously used, for example, conventional chromatographic methods, particularly column chromatography. It is particularly advantageous if the sample to be investigated and the control sample are labeled in different ways, for example with different isotopes or labels. This facilitates comparison of the abundance for a given protein in the sample to be investigated and the control. In another preferred embodiment, the protein to be analyzed is mass spectrometrically examined to provide accurate identification of the protein. Thus, for example, surface-enhanced laser desorption ionization (SELDI method) can be used for tissue or body fluid preparations. However, it may be advantageous to use in vivo imaging methods, in particular electron emission tomography (PET).

The protein to be investigated can also be characterized qualitatively and quantitatively by means of molecules which point in the opposite direction to the protein to be investigated and which are used as diagnostic markers. In a particularly advantageous manner, the molecule is an antibody, in particular a polyclonal and/or monoclonal antibody. However, the invention also covers all known affinity reagents of this aspect.

For qualitative and, in particular, quantitative identification, conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISA), can be used. It is also possible to use immunohistochemical methods and/or protein chips, such as the SELDI method. For identification purposes, body fluids or tumor tissue may be investigated, for example. Antibodies are particularly suitable for identifying annexin a3, 14-3-3 protein beta, tau and zeta and/or SAP. For example, the pan anti-14-3-3 β/ζ monoclonal antibody (Stressgen catalog number KAM-CC012C) stains epithelial and cancer cells, as well as certain lymphocytes in the stroma. Mesenchymal, but not epithelial or cancer cells were stained with a monoclonal antibody (Stressgen catalog number HYB 281-05, working dilution 1: 10) against the protein serum-amyloid P component (SAP).

In a further preferred embodiment, the qualitative and quantitative measurement of the diagnostic marker protein is carried out with the aid of oligonucleotides, for example during the usual Polymerase Chain Reaction (PCR). PCR belongs to the methodological skill of molecular genetics for the selective amplification of defined DNA-sequences. The method gives a qualitative or quantitative detection of the test protein at the DNA-or RNA-level, respectively. It is possible to use suitable oligonucleotide hybridization assays, such as, for example, common Northern-or Southern blots; they also yield qualitative or quantitative information about the protein at the DNA-or RNA-level. The method of detection with oligonucleotides can be easily automated, which is one of its advantages. On the other hand they deliver only information about the abundance of certain DNA-or RNA-sequences, but not about the true abundance of the corresponding proteins. In this case, it must be ensured that the characteristic up-and down-regulation of the diagnostic marker protein is obtained at the mRNA-level or whether this regulation takes place at the post-transcriptional level.

The characteristic changes in the abundance of the different marker proteins determined according to the invention also affect the activity of the respective protein, for example its enzymatic activity. It is therefore advantageous to determine the activity of the protein alternatively or in parallel with its abundance compared to a control. This is also understood by the term up-or down-regulation of various proteins. The individual assays can be performed by ordinary enzymatic testing of the respective enzymes, which is well known to the skilled person. In addition, a binding assay or comparative test can be performed with the fatty acid-binding protein to obtain information about its activity and its up-or down-regulation, respectively. The same assay is performed for other proteins, for example, the channel activity of the nuclear chloride channel protein (CLIC-1) can be measured. This can be used for the use of various proteins as diagnostic markers or for the diagnostic kit according to the invention described hereinafter. Furthermore, measuring the activity of the corresponding protein can be used to test the effect of the drug for cancer treatment according to the present invention, as described below:

in a further preferred embodiment of the use of the invention for testing at least one protein, exosomes, e.g. from patient material, are isolated and the target protein is analyzed. In particular, protein patterns corresponding to at least one protein inside the extranuclear body are tested to determine diagnostically relevant up-and/or down-regulation of one or more proteins. Suitable methods for preparing exosomes from patient material may be carried out using standard methods known to the skilled person.

Furthermore, the present invention encompasses a diagnostic kit comprising at least one compound for determining the activity and/or expression of at least one protein as a diagnostic marker according to the above-mentioned reports of the present description. This diagnostic kit is preferably used for determining the activity and/or expression of at least one of the following proteins: isopeptidase T, serum amyloid P component (SAP), nuclear chloride channel protein channel 1(CLIC-1), mitochondrial enoyl-CoA hydratase and/or annexin A3. Importantly, such diagnostic kits are useful for determining the respective abundance of at least one of these proteins that is characteristically up-or down-regulated compared to a control. The first and initial abundance reflects the expression of the protein. The kits developed according to the present invention are preferably suitable for detecting or screening for cancer diseases, in particular prostate cancer; it provides particular benefits for the early diagnosis of these diseases. For example, such kits allow for distinguishing benign or healthy tissue from malignant tissue, such as benign tissue in prostate hyperplasia or prostate cancer. Preferably such a kit comprises one or several antibodies or one or several oligonucleotides or oligonucleotide pairs, respectively, interacting with one or more of the described proteins or related nucleic acids. By means of these compounds, qualitative and in particular quantitative information can be obtained about the comparison of the protein with a control.

The sample to be tested and the control were taken from the same patient. For example, a tissue sample, or a body fluid sample, such as blood, lymph or urine, is obtained and prepared by methods well known to the skilled artisan. Preferably, the potentially malignant tissue, i.e. the sample to be tested, and the control tissue, i.e. the benign tissue, are taken from the same patient and directly compared. On the other hand it is possible to compare the abundance of individual proteins with other criteria that have been statistically determined from a large number of independent control samples. In the case of prostate cancer, it is advantageous to take benign and possibly malignant prostate tissue from a patient who has had the prostate removed by surgery. Benign tissue from prostate hyperplasia can be used as a control.

Furthermore, the invention encompasses methods of diagnosing cancer by analyzing the abundance of at least one of the described proteins. The results of the up-and down-regulation analysis in cancerous tissue according to the present invention convey information about the presence of cancerous tissue. For further features of the process of the invention, reference is made to the above description.

The invention also covers the use of at least one active substance that interacts with the protein annexin A3 and in particular affects, preferably inhibits, the activity and/or abundance of annexin, in particular annexin A3, for the manufacture of a medicament for the treatment of prostate cancer, preferably of a specific prostate cancer patient group. According to the invention, it is preferred that the active substance interacts directly with the protein annexin a3, thereby influencing, preferably inhibiting, its activity and/or abundance. In another embodiment of the invention it is advantageous that the active substance interacts indirectly with the protein annexin A3, wherein the active substance is for example an activator, inhibitor, modulator and/or bioprecursor directed directly to annexin A3.

In a particularly preferred embodiment of this application, the active substance is at least one derivative of the benzodiazepine * type (Hofmann et al, 1998, J.biol.chem.273 (5): 2885-94). Particularly preferred are BDA250(1, 3-dihydro-1-methyl-5-phenyl-2H-1, 4-benzodiazepine * -2-one), BDA452(3- (R, S) - (L-tryptophanyl) -1, 3-dihydro-1-methyl-5-phenyl-2H-1, 4-benzodiazepine * -2-one) and/or BDA753(3- (R, S) -all-L (NH-Trp-Gly-Tyr-Ala-H) -1, 3-dihydro-1-methyl-5-phenyl-2H-1, 4-benzodiazepine * -2-one). Furthermore, it is preferred to use/employ benzodiazepine * (Diazepam) (7-chloro-1, 3-dihydro-1-methyl-5-phenyl-2H-1, 4-benzodiazepine * -2-one). Other molecules derived from these substances may preferably be used according to the invention. In particular those molecules which block the activity of annexin a 3.

In a particularly preferred manner, annexin a 3-specific antibodies are suitable as active substances, particularly preferred are therapeutic antibodies. These are preferably blocking antibodies and/or radiolabeled and/or toxin-labeled antibodies. The radiolabeled antibody may, for example, carry¹³¹I. Such antibodies advantageously make possible radioimmunotherapy as known to the skilled person. However, any other agent known to the skilled person is also suitable as active substance.

In a particularly preferred manner, such active substances can be used to influence the activity and/or abundance of annexin a3 in nuclear exosomes.

Active substances which influence the activity and/or expression of annexin a3, in particular active substances which exhibit an inhibitory effect, can advantageously be used for the production of a medicament for the treatment of osteoarthritis degradation and/or atherosclerotic lesions.

Furthermore, the present invention encompasses the use of at least one active substance affecting the activity and/or expression of isopeptidase T and/or the activity and/or expression of Protein Disulfide Isomerase (PDI) for the development of a medicament for the treatment of cancer. As previously described, the abundance of these proteins in cancer tissue has been characteristically altered. In particular, the abundance of ubiquitin-isopeptidase T is significantly reduced, while the abundance of Protein Disulfide Isomerase (PDI) is increased. Alteration of the expression or activity of these proteins to normal levels shown in control tissues represents a possible way to cure cancer diseases. The use of active substances which increase the activity and/or abundance of ubiquitin-isopeptidase T is therefore claimed. Furthermore, the use of active substances inhibiting the PDI activity and/or abundance is claimed. By this active substance, the activity of the protein is regulated to a normal level so that the cancer disease can be effectively treated.

The invention also covers the use of at least one active substance that affects the activity and/or abundance of mitochondrial enoyl-coa-hydratase for the manufacture of a medicament for the treatment of cancer. Preferably this can be done in combination with affecting fatty acid-binding protein 3(FABP-3) and/or epidermal fatty acid-binding protein (E-FAPB). Preferably, the use consists of an inhibitory active substance of the activity and/or abundance of mitochondrial enoyl-coa-hydratase and/or E-FABP, respectively an increasing active substance of the activity and/or abundance of FABP-3.

The invention also covers the use of at least one active substance affecting and in particular increasing the active abundance and/or localization of serum amyloid P component (SAP) for the manufacture of a medicament for the treatment of cancer. It has been shown that the location of SAP in cancerous tissue can be altered. Thus inventively it is preferred to influence the positioning of the SAP by using at least one active substance. The active substance may for example be a fusion protein comprising the cancer cell binding domain of annexin a3 and the immune response-influencing domain of SAP. SAP is located, for example, on mesenchymal cells in prostate tissue, but not on healthy epithelial cells or transformed cancer cells. Annexin a3 is abundant in cancer tissues. Annexins are also known to occur on the cell surface. Since SAP is involved in the immune system as a protein component without cancer cells being eliminated from the immune system, the immune response-affecting domain of SAP on the surface of cancer cells produces an altered immune response relative to cancer cells.

The invention furthermore encompasses the use of at least one active substance which influences, preferably inhibits, the activity and/or expression of the 14-3-3 protein tau for the development of a medicament for the treatment of cancer.

The invention furthermore encompasses the use of at least one active substance which influences, preferably inhibits, the activity and/or expression of nuclear chloride channel protein 1(CLIC-1) for the development of a medicament for the treatment of cancer, in particular for the treatment of prostate cancer.

The invention furthermore encompasses the use of at least one active substance which influences-preferably inhibits-the activity and/or expression of HES1 for the development of a medicament for the treatment of cancer.

The invention furthermore encompasses the use of at least one active substance which influences the activity and/or expression of the-preferably inhibitory-proteasome α 2-subunit for the development of a medicament for the treatment of cancer, in particular prostate cancer.

The invention furthermore encompasses the use of at least one active substance influencing, preferably inhibiting, the activity and/or expression of adenine-phosphoribosyl-transferase for the development of a medicament for the treatment of prostate cancer.

The invention furthermore encompasses the use of at least one active substance which influences, preferably inhibits, the activity and/or expression of inorganic pyrophosphatase, in particular in exosomes, for the development of a medicament for the treatment of prostate cancer.

The invention furthermore encompasses the use of at least one active substance which influences-preferably stimulates-the activity and/or expression of at least one of the following proteins for the development of a medicament for the treatment of prostate cancer: ubiquitin-isopeptidase T, serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), galectin-1, Microseminal protein beta, Heat shock protein 27(HP27), and Transgelin. Furthermore, the invention encompasses the use of at least one active substance influencing-preferably inhibiting-the activity and/or expression of at least one of the following proteins for the development of a medicament for the treatment of cancer: 14-3-3 protein beta, 14-3-3 protein zeta, nuclear chloride channel protein 1(CLIC-1), 14-3-3 protein tau, heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), epidermal fatty acid-binding protein (E-FABP), coenzyme A hydratase, nucleophosphoprotein, annexin, in particular annexin A3, triose phosphate-isomerase, aldolase A, proteasome alpha-2-subunit, adenine-phosphoribosyl-transferase and inorganic pyrophosphatase. Particular preference is given to combinations of two or more active substances with respect to at least two different proteins. Furthermore, it is preferred that the active substance is used together with one or more of these active substances which increase the activity and/or abundance of annexin a1, a2, a4, a7 and/or a10, in particular in vitro.

For each of these proteins, according to the invention, it was demonstrated that they are characteristically up-or down-regulated in cancer tissue compared to controls. Thus, according to the invention it is claimed that these proteins are down-or up-regulated in contrast by the respective active substances in order to obtain an activity, in particular an enzymatic activity of healthy tissue, or to inhibit and/or kill cancer cells. This makes it possible to successfully treat various cancers. It is particularly preferred to produce a medicament for the treatment of prostate cancer, preferably a specific subtype of prostate cancer.

The active substances used according to the invention can be peptides, proteins, small molecule compounds or polynucleotides. Known active substances with known mechanisms affecting the activity and/or expression of different proteins are considered as new active substances. These active substances directly address the proteins described. On the other hand, it may be advantageous if these active substances address modulators, in particular activators or inhibitors, and/or bioprecursors of these different proteins. Depending on whether certain active substances act to inhibit or stimulate the activity and/or expression of the respective protein, they may be agonists or antagonists. Further examples of antagonists are defective or dominant negative mutants which can be constructed by genetic engineering. They show no enzymatic activity, but they compete for the respective substrate of the protein or enzyme to be inhibited, which results in a reduction of the activity of the protein. Another example of an antagonist is an antisense molecule which can reduce the expression of a particular protein in a known manner. An agonist may be a substance that promotes the expression of a particular gene or promotes the translation of mRNA into an active gene product. This may be a specific transcription factor, or a similar compound that modulates the expression level of the mentioned proteins. In particular, small molecule compounds can be advantageously used for this purpose.

In a particularly preferred embodiment of the invention, the active substance may be a therapeutic antibody which acts as an inhibitory antibody reducing or blocking the activity of a given protein, preferably annexin a 3. The therapeutic antibodies are also characterized, for example, by carrying a toxic or radioactive label that is brought to the cancer cells solely by interaction with, for example, annexin a 3. This can be used, for example, during radioimmunotherapy, with a radioactive label, for example¹³¹An antibody of I. According to the invention, the active substance may be a small molecule compound with a Molecular Weight (MW) < 1000, inhibiting the activity of ion channels in membranes, preferably exosomes and/or matrix vesicles.

To increase the activity of the described proteins, in particular isopeptidase T, FABP-3, galectin-1, microgrinin beta, HSP27 and the transdegenins, compounds with comparable or similar enzymatic activity can be used. Furthermore, the activity of the enzyme molecules present can be induced or increased by the respective compounds. On the other hand it is possible to use active substances which are suitable for inducing or increasing the expression which means the synthesis of the corresponding enzymatic molecule. The active substance may also address certain precursor molecules, modulators, activators or inhibitors of enzymes or other proteins.

Furthermore, hormones or substances with similar effects can be used as active substances if they influence the activity of the respective proteins in the desired manner. For example, molecules similar to progesterone can be used to inhibit enoyl-coa hydratase.

In a particularly preferred embodiment of the use according to the invention, the active substance is at least one protein per se: ubiquitin-isopeptidase T, serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), galectin-1, Microseminal protein beta, Heat shock protein 27(HSP27) and/or Transgelin. The inventors could prove that the abundance of these proteins is lower in cancer tissues and therefore aim according to the invention to use the proteins themselves as active substances for stimulating their activity and/or expression. For this purpose, a single protein or preferably a combination of several different proteins can be used. Furthermore, it is contemplated that portions of these proteins, such as peptides or molecules derived from the proteins, may be used as the active agents of the present invention.

In a particularly preferred embodiment of the use according to the invention, the one or more active substances can be delivered as nuclear bodies or the application of the active substances can be mediated by nuclear bodies. This may preferably affect the immune response of the patient, in particular by modulating the T-cell response. Exosomes are membrane-coated vesicles that are preferably secreted by hematopoietic cells. It is known that nuclear exosomes produced by dendritic cells stimulate potent anti-tumor responses, for example, in mice.

By treating cancer according to the invention by applying the active substance, a reduction or inhibition of tumor development or growth can advantageously be obtained, and/or metastasis of the tumor can be partially reduced or completely avoided.

Furthermore, the present invention encompasses pharmaceutical compositions comprising at least one active substance as described above and at least one pharmaceutically acceptable carrier. For details concerning the pharmaceutical composition or the active substance, reference is made to the above description. Suitable pharmaceutically acceptable carriers will be apparent to the skilled person.

Furthermore, the invention encompasses methods of treating cancer diseases, such as prostate cancer, by applying at least one of the described active substances. For further details of this cancer treatment we refer to the above description.

For the active substance to be administered, different modes of administration can be used, for example, oral, intravenous, topical or by inhalation. The respective formulations are known to the skilled worker. The mode of administration depends on the disease to be treated and, of course, on the physical condition of the patient. The details are well known to the skilled person.

Finally, the invention encompasses methods for finding active substances for cancer therapy, in particular prostate cancer therapy. Using this method, at least one protein will be used which may be selected from: ubiquitin-isopeptidase T, serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), galectin, microgrinin beta, heat shock protein 27(HSP27), 14-3-3 protein beta, 14-3-3 protein zeta, nuclear chloride channel protein 1(CLIC-1), 14-3-3 protein tau, heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), epidermal fatty acid-binding protein (E-FABP), mitochondrial enoyl-coenzyme A-hydratase, nucleophosmin, annexin, in particular annexin A3, a transgelin, a triose phosphate isomerase, an aldolase A, HES1, the alpha 2-subunit of a proteasome, an adenine-phosphoribosyl-transferase and an inorganic pyrophosphatase 1. Preference is given to isopeptidase T, serum-amyloid P-component (SAP), nuclear chloride channel protein 1(CLIC-1), 14-3-3 protein tau, mitochondrial enoyl-coenzyme A-hydratase and/or annexin A3. Furthermore, derivatives of these proteins, in particular homologous sequences or mutated forms of the proteins which have been produced by molecular biological methods, can be used. Furthermore, portions or subregions of each of these proteins or combinations of multiple proteins and/or derivatives thereof may be used. The method is well known to the skilled person and for example the protein or derivative thereof may be expressed using a suitable expression system. By means of this system, interactions with potential ligands, in particular inhibitors or activators, can be investigated. For example, two-hybrid systems are suitable for studying these interactions.

The described features and further characteristics of the invention will be apparent from the following description of preferred embodiments, taken in conjunction with the following claims and the accompanying drawings. The individual features can be identified individually or in combination with one another.

Examples

In order to identify the proteins of interest according to the invention, tissue samples of two patient groups (group A: 23 patients, group B: 33 patients) were examined. Cancer tissues and control tissues were prepared in each case and subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Isoelectric focusing was performed at pH 4-7 and pH 6-11. The results of gel electrophoresis from two patients from group a were not suitable for further analysis. For the other two patients, the results at pH 6-11 were not satisfactory. Thus, it is possible to assess the results for 21 patients in the pH range 4-7, and 19 patients in the pH range 6-11. Results from both patients in group B at pH 4-7 were not suitable for further analysis. Thus, the overall results of 31 patients in the pH range of 4-7 can be assessed.

Two different samples of each patient were labeled with a mixture of different isotopes in each case and electrophoretically separated on a single two-dimensional polyacrylamide gel. The signals of the individual isotopes are then detected independently of one another, so that protein samples of the two tissue samples can be compared directly (ProteTope-technique).

The final identification of the amount of protein analyzed (< 1. mu.g) for the radiolabeled samples was separated in a preparative gel along with the amount of unlabeled protein prepared (> 200. mu.g) for the same samples. Relevant protein spots of the silver-stained preparative gels were excised, enzymatically digested with trypsin, and identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) on Bruker Biflex or Ultra-Flex. Electrospray ionization ion trap mass spectrometry (ESI-MS) (Bruker Esquire) was performed in part.

By this method, a plurality of proteins are identified which specifically show a corresponding up-or down-regulation of abundance in cancer tissue compared to control tissue.

For these analyses, specific patient populations were developed in part in which different protein abundances were studied. In this so-called clustering analysis (Clustan Graphics 6.4), 3 patient groups from group a and 2 patient groups from group B were determined, which in each case revealed a characteristic protein expression/abundance pattern. With this method, the proteins annexin a3, the tranexamins, the triose phosphate isomerases and the aldolases a showing characteristic abundance for specific patients are identified.

Tissue sample

After prostectomy, healthy and malignant prostate tissue was obtained from the patient. Patients were screened for PSA (prostate specific antigen) and tumors were confirmed by ultrasonography. The first patient was obtained before surgery.

Immediately after resection, the prostate bodies were transferred to a sterile cassette and cooled. Tissue sections of 0.5-1cm thickness were prepared and divided into left and right halves. It was embedded in a freezing matrix and shock frozen. The remaining prostate was fixed in formaldehyde solution and further treated according to standard methods of routine. To prepare tissue samples, thin sections of the prostate on both sides were taken and stained with hematoxylin eosin. These sections were stored at-80 ℃ until use. Control tissue samples were taken from the tumor-free area and treated identically.

Sample preparation

The protein was lysed with 100. mu.l of boiling solution containing 2% SDS, 0.1M Tris pH8.8 and the concentration of the protein was determined by the bicinchoninic acid method.

Are used separately¹²⁵I or¹³¹I iodination, two-dimensional polyacrylamide gel electrophoresis and data analysis were performed according to the general protocol (Cahill et al, 2003 Rapidcommunication in Mass Spectrometry 17: 1283-1290). Radioactive iodine was purchased from Amersham Bioscience (Freiburg, germany). In equal concentration¹²⁵I or¹³¹I, respectively carrying out iodination.

Polyacrylamide gel electrophoresis

For loading onto polyacrylamide gels, the same amount of labeled sample (cancer tissue and control tissue) protein was mixed. For isoelectric focusing (IEF) in the range of pH 4-7 and 6-11, samples were diluted in common sample buffer and loaded onto 18cm pH-gradient (IPG) strips (Amersham Bioscience). IEF first-pass separation of proteins was performed by 2D-PAGE on a Multiphor device (Amersham Bioscience). The second pass (SDS-PAGE) was carried out on an ISO-DALT apparatus (H ö fer). The gel was dried on 80 μ M plastic film, laminated, and the final measurement of the two radioisotope signals was performed.

Using selection methods that analyze different radioisotopes, quantitative multi-color differential displays of proteins from different samples can be obtained. Thus, a direct comparison of the accumulated intensity of protein spots separated on one gel can be used for further analysis. Analysis on a single gel offers the advantage of making systematic errors of variation among two or more gels irrelevant. The largest possible source of error is the different chemical equivalents when labeled with either isotope. This can be excluded by performing a counter-labelling of the gel, i.e. the control sample and the cancer sample are each labelled with either isotope and compared in reverse. The protein expression patterns of the opposite labeling steps were matched, thus completing the quantitative criteria. The signals of the different isotopes are visually observed in different colors (blue or orange) by computer-assisted methods, so that consistent differences in abundance among the samples are displayed in one or two colors corresponding to the isotopes used for labeling. Details on this methodology are set forth in Cahill et al, 2003 Rapid Communications in Mass Spectrometry 17: 1283 and 1290.

Imaging analysis

Differential analysis of protein expression was based on the described reliable quantification of differences in protein spots in polyacrylamide-gels. For quantitative imaging analysis, the software Phoretix 2D Advanced (Nonlinear Dynamics) specially modified by the present inventors was used.

Identification of proteins by mass spectrometry

In principle two different methods of mass spectrometry are used. On the other hand, rapid and reliable identification of high-abundance proteins is aided by peptide mass spectrometry fingerprinting with MALDI-TOF MS. The identification of very low abundance proteins was performed using the ESI-Ion-Trap-MS/MS or MALDI-TOF-TOF method, which takes more time. In summary, gel pieces containing the selected protein spots were cut and the proteins in the gel pieces were digested with trypsin. The resulting solution was first fingerprinted by peptide mass spectrometry with MALDI-TOF MS. For protein spots that could not be so unambiguously identified, slower fragmentation analysis based on MALDI-TOF-TOF or LC-ESI-Ion-Trap-MS/MS was performed. A detailed description of these methods can be found in Vogt et al, 2003, molecular cellular protein 2: 795.

Identification of proteins

For the identification of proteins, their peptide mass spectra, which have been found by mass spectrometry, are analyzed using the NCBI-database. This is done with the program MASCOT version 1.9 (Matrix Science, London, UK).

Quantitative imaging analysis

Quantitative analysis is performed using digital data that has been recorded by a radiographic photomultiplier tube for each pixel of the image matrix. The limits of the protein spots are defined by means of the software Phoretix 2 dadchanged (nonlinear dynamics), and the pixel results in the spot area are accumulated after subtraction of a suitable background signal. Based on the complete data generated, a detailed quantification of the protein spots detected was performed. Table 1 summarizes these results.

FIGS. 1 and 2 show the positions of selected spots after isoelectric focusing at pH 4-7 (FIG. 1) and in the second case at pH 6-11 (FIG. 2), respectively.

Illustration of the drawings:

FIG. 1: image of a two-dimensional polyacrylamide gel with isolated proteins. Isoelectric focusing was performed at pH 4-7. The protein spots marked by numbers show proteins whose abundance differs from those of cancerous and control tissues, respectively. The numbers refer to the numbers in table 1.

FIG. 2: presentation of a two-dimensional polyacrylamide gel with isolated proteins. Isoelectric focusing was performed at pH 6-11. The protein spots marked by numbers show proteins whose abundance differs from those of cancerous and control tissues, respectively. The numbers point to the numbers in table 1.

FIG. 3: a graphical representation of the protein expression patterns characterizing a unique patient pool, i.e., a unique subtype of prostate cancer. Statistically significant p < 0.01 results are depicted in black, and results with t-test p-values of 0.01 < p < 0.1 are depicted in gray. Proteins with different expression within different clusters are shown in box.

FIG. 4: the different levels of protein expression in the patient groups 1 to 3 are tabulated compared to benign (healthy). The data refer to the percentage of protein spots in cancer tissue that are associated with standard deviation in size relative to the total volume of protein spots (benign + malignant). the t-test probability represents the likelihood that the point score distributions of the two given sets are significantly different. T-test results with a probability of more than 99% are printed in bold. Results with a probability below 95% are printed in light gray.

FIG. 5: the table lists protein spots with significantly different expression in all patients based on a two-color ProteoTope analysis comparing benign (healthy) and malignant tissues. "No. obs. represents the number of patients in which spots were observable. The point-scores with standard error for benign tissue (benign part) and malignant tissue (cancer part) refer to the percentage of protein spots of the total volume (benign + malignant). the t-test probability represents the likelihood that the point score distribution in benign tissue differs significantly from the distribution in cancerous tissue when all patients are considered. The points are selected under the condition that the t-test probability reaches at least 99%.

FIG. 6: presentation of a two-way polyacrylamide gel with isolated proteins from 14 patients from group B. For both control and cancer tissue samples¹³¹I and¹²⁵i labeled and compared in reverse. The different isotopic signals become visible in each case in the other colour (blue/orange), so that differences in e.g. protein abundance between samples of the respective colours end up as a function of the chosen isotopic label.

FIG. 7: graph showing the accuracy and statistical significance of the Proteo-Tope measurements using the B set of examples:

a. for reproduction¹²⁵I and¹³¹(iii) Bland and Altman plots of the ratio between the difference in the differential abundance ratio M of the I-labeled gel and its arithmetic mean,

b. for reproduction¹²⁵I and¹³¹a plot of the ratio between the difference in the differential abundance ratio M of I-labeled gels and the arithmetic mean of the intensities A,

c. for reproduction¹²⁵I and¹³¹MA plot of the ratio between the differential abundance ratio M and the intensity a for I-labelled gels, where M ═ log2 · (I2/I1) and a ═ 0.5 · log2 · (I1 · I2) (I ═ measured intensity).

FIG. 8: volcano plots showing the difference between the mean intensity of the opposite detection marker proteins from cancer and healthy tissue.

FIG. 9: a graph of Pavlidis template matching analysis, which provides two subgroups of protein abundance ratio patterns for cancer patients from group B. One group consisted of 22 patients, while another group, significantly different from it, consisted of 9 patients. The numbering of the proteins corresponds to the numbering in the table of figure 10. In a subgroup of 22 patients, there was a significant difference in the relative abundance of annexin a3 (protein 14). With patients 14, 11, 10, 21, 3, 1, 6, 22, 23, 7, 4, 19 and 27, the protein is more abundant in malignant prostate tissue than in patients 29, 28, 32, 15, 31, 24, 25, 30 and 33.

FIG. 10: tables of protein spots from differential analysis of all 31 patients (group B), groups with 22 and 9 patients (obtained by Pavlidis template matching analysis) are displayed. The accession number corresponds to the number from the NCBI database. The integration is obtained using MASCOT techniques. The metrics corresponding to PMF points are related to the mouse points used by the MASCOT server and, in general, PMF points in excess of 65 represent a clear equivalence. The identity of the asterisked protein was determined by LC/MS/MS. The mean point score of cancerous tissue with standard error is given as a percentage of the total point mass (healthy + malignant). The P value of this model is also given. The bars in the table give the average percent abundance of each protein in benign (dark blue) and cancerous (light orange) samples in the indicated patient groups.

Claims (53)

1. Use of the protein annexin a3 as a diagnostic marker for prostate cancer.

2. Use according to claim 1, characterized in that it relates to a specific subtype of prostate cancer.

3. Use according to claim 1 or 2, characterized in that the upregulation of annexin a3 is investigated in comparison to a control.

4. Use according to one of the preceding claims, characterized in that the upregulation of annexin A3 in combination with downregulation of annexin a1, annexin a2 and/or annexin a5 is investigated.

5. Use of at least one active substance that interacts with the protein annexin A3 and in particular influences, preferably inhibits, the activity and/or abundance of the protein annexin A3 for the manufacture of a medicament for the treatment of prostate cancer, preferably of a specific prostate cancer patient group.

6. Use according to claim 5, characterized in that the active substance is an agonist, an antagonist, a defective mutant, a dominant negative mutant and/or an antisense molecule.

7. Use according to claim 5 or 6, characterized in that the active substance is an antibody, preferably a therapeutic antibody.

8. Use according to one of claims 5 to 7, characterized in that the active substance is at least one benzodiazepine * derivative, preferably BDA250 and/or BDA 452.

9. Use according to one of claims 5 to 8, characterized in that the activity and/or abundance of the protein annexin A3 in exosomes is affected.

10. Use according to one of claims 5 to 9, characterized in that the active substance is a "small molecule compound" with a Molecular Weight (MW) < 1000 for inhibiting the activity of ion channels in membranes, preferably exosomes and/or matrix vesicles.

11. Use of the protein mitochondrial enoyl-coa-hydratase as a diagnostic marker for cancer.

12. Use according to claim 11, characterized in that the upregulation of mitochondrial enoyl-coa-hydratase is investigated in comparison to a control.

13. Use of the protein ubiquitin-isopeptidase T and/or protein-disulfide-isomerase (PDI) as a diagnostic marker for cancer.

14. Use according to claim 13, characterized in that the downregulation of ubiquitin-isopeptidase T and/or the upregulation of protein-disulfide-isomerase (PDI) is investigated in comparison to a control.

15. Use of the protein serum-amyloid P-component (SAP) as a diagnostic marker for cancer.

16. Use according to claim 15, characterized in that the downregulation of serum-amyloid P-component (SAP) is investigated in comparison to a control.

17. Use of a protein-nuclear chloride channel protein as a diagnostic marker for prostate cancer.

18. Use according to claim 17, characterized in that the upregulation of nuclear chloride channel proteins is investigated when compared to a control.

19. Use of the protein HES1 as a diagnostic marker for cancer.

20. Use according to claim 19, characterized in that the up-regulation of HES1 compared to a control is investigated.

21. Use of proteasome α 2-subunit as a diagnostic marker for cancer.

22. Use according to claim 21, characterized in that the up-regulation of the proteasome α 2-subunit is studied in comparison with a control.

23. Use of the protein adenine-phosphoribosyl-transferase as a diagnostic marker for prostate cancer.

24. Use according to claim 23, characterized in that the upregulation of adenine-phosphoribosyl-transferase is investigated in comparison with a control.

25. Use of a proteinaceous inorganic pyrophosphatase as a diagnostic marker for prostate cancer.

26. Use according to claim 25, characterized in that the up-regulation of inorganic pyrophosphatase is investigated in comparison with a control.

27. Use of the protein ubiquitin-isopeptidase T and the serum-amyloid P-component (SAP) as diagnostic markers for cancer, wherein the down-regulation of said protein compared to a control is preferably investigated.

28. Use of at least two proteins selected from ubiquitin-isopeptidase T, heat shock protein 27(HSP27), heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), mitochondrial enoyl-coa-hydratase and nucleophosmin as diagnostic markers for cancer, wherein the down-regulation of ubiquitin-isopeptidase T and/or heat shock protein 27(HSP27), and/or the up-regulation of heat shock protein 90(HSP90), protein-disulfide-isomerase (PDI), mitochondrial enoyl-coa-hydratase and/or nucleophosmin, compared to a control, is investigated.

29. Use according to one of the preceding claims, characterized in that the cancer is prostate cancer.

30. Use according to one of the preceding claims, characterized in that diagnosis is made by studying one or more protein subtypes of cancer, in particular prostate cancer.

31. Use according to claim 30, characterized in that the study of the at least one protein according to claim 28 in combination with at least one protein selected from the group consisting of serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), galectin, microgrinin β, 14-3-3 protein ζ, nuclear chloride channel protein, 14-3-3 protein τ, epidermal fatty acid-binding protein (E-FABP), annexin a3, transcoactin, triosephosphate isomerase and aldolase a is carried out as a result of no or minor changes in SAP, down-regulation of FABP-3, strong down-regulation of galectin, strong down-regulation of microgrinin β, no or minor changes in 14-3-3 protein β, no or minor change in the 14-3-3 protein zeta, no or minor change in the nuclear chloride channel protein, no or minor change in the 14-3-3 protein tau, no or minor change in the E-FABP, no or minor change in the annexin A3, upregulation of the tranagulin, no or minor change in the triose phosphate isomerase, and/or no or minor change in the aldolase A.

32. Use according to claim 30, characterized in that the investigation of the at least one protein according to claim 28 in combination with at least one protein selected from the group consisting of serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), galectin, microgrinin β, 14-3-3 protein ζ, nuclear chloride channel protein, 14-3-3 protein τ, epidermal fatty acid-binding protein (E-FABP), annexin a3, transgelin, triose phosphate isomerase and aldolase a occurs as a strong up-regulation of PDI, a strong up-regulation of HSP90, a strong down-regulation of ubiquitin-isopeptidase T, down-regulation of SAP, no or minor changes of FABP-3, down-regulation of galectin compared to controls, Down-regulation of microgrinin beta, up-regulation of 14-3-3 protein zeta, up-regulation of 14-3-3 protein tau, no or minor change in nuclear chloride channel protein, up-regulation of annexin A3, down-regulation of transcoagulant protein, up-regulation of triosephosphate isomerase and/or up-regulation of aldolase A.

33. Use according to claim 30, characterized in that the study of the at least one protein according to claim 28 in combination with at least one protein selected from the group consisting of serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), galectin, microgrinin β, 14-3-3 protein ζ, nuclear chloride channel protein, 14-3-3 protein τ, epidermal fatty acid-binding protein (E-FABP), annexin a3, transcoactin, triosephosphate isomerase and aldolase a is carried out as a down-regulation of SAP, no or small change in FABP-3, no or small change in galectin, no or small change in microgrinin β, no or small change in 14-3-3 protein β, as compared to a control, No or minor change in 14-3-3 protein zeta, strong upregulation of nuclear chloride channel protein, no or minor change in 14-3-3 protein tau, no or minor change in E-FABP, no or minor change in annexin A3, no or minor change in a tranagulin, no or minor change in triose phosphate isomerase, and/or no or minor change in aldolase A.

34. Use according to one of the preceding claims, characterized in that at least one protein is detected by means of polyacrylamide gel electrophoresis, in particular two-dimensional gel electrophoresis, mass spectrometry, positron-emission tomography (PRT), antibodies, ELISA, immunohistochemistry, protein chips and/or oligonucleotides, in particular Polymerase Chain Reaction (PCR).

35. Use according to one of the preceding claims, characterized in that exosomes are isolated and/or at least one protein is investigated analytically.

36. Diagnostic kit comprising at least one substance for detecting the activity and/or abundance of at least one protein selected from ubiquitin-isopeptidase T, serum-amyloid P-component (SAP), nuclear chloride channel protein, mitochondrial enoyl-coa-hydratase and annexin a3, for the identification of cancer diseases, in particular prostate cancer.

37. Use of at least one active substance affecting the activity and/or abundance of ubiquitin-isopeptidase T and protein-disulfide-isomerase (PDI), wherein preferably the active substance increases the activity and/or abundance of ubiquitin-isopeptidase T and/or the active substance inhibits the activity and/or abundance of protein-disulfide-isomerase (PDI), for the manufacture of a medicament for the treatment of cancer.

38. Use of at least one active substance that influences the activity and/or abundance of the protein mitochondrial enoyl-coa-hydratase for the manufacture of a medicament for the treatment of cancer.

39. Use according to claim 38, characterized in that the active substance inhibits the activity and/or abundance of mitochondrial enoyl-coa-hydratase.

40. Use of at least one active substance influencing and in particular increasing the activity, abundance and/or localization of the protein serum-amyloid P-component (SAP) for the manufacture of a medicament for the treatment of cancer.

41. Use of at least one active substance influencing, in particular inhibiting, the activity and/or abundance of the protein nuclear chloride channel protein for the manufacture of a medicament for the treatment of prostate cancer.

42. Use of at least one active substance influencing, in particular inhibiting, the activity and/or abundance of the protein HES1 for the manufacture of a medicament for the treatment of cancer.

43. Use of at least one active substance influencing, in particular inhibiting, the activity and/or abundance of the proteasome α 2-subunit for the manufacture of a medicament for the treatment of cancer.

44. Use of at least one active substance influencing, in particular inhibiting, the activity and/or abundance of adenine-phosphoribosyl-transferase for the production of a medicament for the treatment of prostate cancer.

45. Use of at least one active substance influencing, in particular inhibiting, the activity and/or abundance of the protein inorganic pyrophosphatase for the manufacture of a medicament for the treatment of prostate cancer.

46. Use according to one of claims 37 to 45, characterized in that the cancer is prostate cancer, preferably a specific prostate cancer subtype.

47. Use according to one of claims 37 to 46, characterized in that the active substance is an agonist, an antagonist, a defective mutant, a dominant negative mutant and/or an antisense molecule.

48. Use according to one of claims 37 to 47, characterized in that the active substance is an antibody, preferably a therapeutic antibody.

49. Use according to one of claims 37 to 48, characterized in that the active substance is a "small molecule compound" with a Molecular Weight (MW) < 1000 for inhibiting the activity of ion channels in membranes, preferably exosomes and/or matrix vesicles.

50. Use according to one of the preceding claims, characterized in that the active substance is at least one protein selected from the group consisting of ubiquitin-isopeptidase T, serum-amyloid P-component (SAP), fatty acid-binding protein 3(FABP-3), annexin a3, galectin, microgrinin β, heat shock protein 27(HSP27) and a transgelin.

51. Use according to one of the preceding claims, characterized in that the active substance is provided in the form of an exosome.

52. Pharmaceutical composition comprising at least one active substance according to one of the preceding claims and at least one pharmaceutically acceptable carrier.

53. Method for the search for active substances for the treatment of cancer, characterized in that at least one protein and/or at least one derivative thereof selected from ubiquitin-isopeptidase T, serum-amyloid P-component (SAP), nuclear chloride channel protein, 14-3-3 protein tau, mitochondrial enoyl-coa-hydratase, annexin a3, HES1, proteasome alpha 2-subunit, adenine-phosphoribosyl-transferase and inorganic pyrophosphatase is used.