Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • G01N2333/91205—Phosphotransferases in general

Definitions

• the present invention relates to the field of medicine and biology. It concerns a new screening test and therapeutic monitoring in oncology. More particularly, it relates to diagnostic and / or therapeutic tests in oncology and neurodegenerative diseases.
• Age-related neurodegenerative diseases and cancers both involve a change in the physiological process of programmed cell death or apoptosis. Neuronal death is abnormally accelerated during neurodegenerative diseases such as Alzheimer 's, Huntington' s, Parkinson 's etc. In contrast, the process of cancerization corresponds to a blockage of apoptosis leading to an uncontrolled multiplication of cells. The link between these two processes has now become a major field of research in aging research.
• Cancer is one of the leading causes of death worldwide. While over the last generation the percentage of deaths related to heart disease, cardiovascular disease and many other diseases have been declining, the number of deaths related to various forms of cancer is on the rise. Despite the rapid advancement of our understanding of different forms of cancer, low survival rates are generally attributable to inadequate diagnosis and treatment. Most tumors can only be detected when they reach a size of about 1 cm. Since there is a relatively short period of time for the continuous development of a tumor to a stage that becomes incompatible with survival, this leaves little time for therapeutic intervention. So, early diagnosis becomes the key to success for cancer treatment.
• skin cancer is the most common cancer in Canada. In 1992 alone, 50,300 new cases of skin cancer were reported, compared to 19,300 cases of lung cancer, 16,200 cases of colorectal cancer and 15,700 cases of breast cancer. In other words, skin cancer is as common as the top 3 types of cancers combined. Its incidence continues to grow with its 64,200 new cases in 1997, an increase of 14,000 cases annually in 5 years. In particular, the incidence of malignant melanoma is growing at a rate of 2% per year. Early diagnosis remains the key to effective treatment. A malignant tumor is easily accessible and can be removed with minor surgery. In fact, the cure is 100% if skin cancer is detected early enough. However, early diagnosis of skin cancer remains difficult.
• a definitive diagnosis of skin cancer requires biopsy and histological analysis. However, the decision to send a biopsy for analysis (or even if a patient needs to be referred to a dermatologist) becomes very subjective. There are several biopsies that are not taken when they should have been.
• Colon cancer is the third leading cause of cancer-related mortality among men and women in North America (16,200 cases per year). Early detection, leading to early intervention, has shown that treatment success and survival can be improved. For example, the 5-year survival rate is 92% for a patient whose disease has been detected at an early stage, while the rate drops to about 60% in patients with localized cancer, and to about 6% in those with metastases. However, only one-third of colon cancers are detected at an early stage. One of the reasons for this delay in diagnosis is the lack of a sensitive screening test, inexpensive and non-invasive.
• cyclin D1 one of the proteins constituting the regulatory subunits of the kinases of the cell cycle, essential for the progression of the cell cycle. This protein is also expressed during apoptosis in various cell types (Han et al, 1996, Pardo et al, 1996).
• the present invention relates to a new cell cycle reinduction screening test targeting oncology. This is a diagnostic test and a prognostic test for different cancers (cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, glioblastoma, sarcoma, leukemia, etc.). More particularly, the invention relates to the use of the protein LIV21 as well as their derivatives as markers diagnostics and prognosis of cancers. The invention thus relates to the detection of the LFV21 protein with a kit comprising antibodies specific for LIV21.
• a first objective of the present invention is to demonstrate a method for detecting and prognosing cancer and its metastatic potential.
• the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, sarcoma, leukemia and glioblastoma, without being limited to these.
• LIV21 as a prognostic indicator of cancer. Indeed, when LIV21 is located in the cytoplasm, the cancer cells in the tissues are aggressive. Conversely, when the product of the expression of the LIV21 gene is preferentially localized in the cell nucleus, this is a prognostic indicator that the cells of the tissue are differentiated and quiescent and therefore non-invasive. The effectiveness of a cancer treatment can also be followed by the traceability of this protein, its derivatives and ratios with the associated proteins.
• PKC ⁇ protein kinase C epsilon
• the detection of E2F1 and / or E2F4 proteins is of interest.
• the LIV21 protein forms a complex with E2F4 which is capable of inhibiting the expression of the E2F1 gene in the nucleus, the expression of the E2F1 gene being a sign of cell proliferation.
• a decrease in the association of LIV21 with the E2F4 protein is indicative of the presence of cancer cells.
• the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.
• the present invention relates to a method for the detection (in vitro or ex vivo) of cancer cells in a biological tissue sample (for example, breast, ovary, endometrium, bladder, melanoma, prostate, glioblastoma, etc.) of patients, which method comprises detecting the product of the expression of the LIV21 gene in the nucleus and / or the cytoplasm of the cells in the tissue biological sample of said patient, a location of said product of the expression of the LIV21 gene in the cytoplasm being indicative of the presence of cancer cells and a location of said LIV21 gene expression product in the nucleus being indicative of the presence of non-cancerous cells.
• a location of said product of LIV21 gene expression in the cytoplasm is indicative of the presence of invasive and / or metastatic cancer cells.
• the method according to the present invention further comprises the detection of the product of the expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene. .
• the method may include the detection of the product of the expression of two of these genes or three genes.
• at least one of the LIV21 / PKC ⁇ , LIV21 / E2F4 and LIV21 / E2F1 ratios can be determined in the present method. This ratio can be determined in the cytoplasm and / or in the nucleus. Preferably, these ratios are determined in the nucleus. Preferably, these ratios are compared to those obtained in a healthy cell.
• the HDAC family plays a key role in the regulation of gene expression. When they are overexpressed, they result in the silencing of tumor suppressor genes hence the interest of using HDAC inhibitors in therapy associated with other inhibitors regulating the metabolic cascade involving the protein complex that contains LIV21.
• the level of expression of each enzyme or polypeptide of the complex SUMO / Rb / HDAC or for certain cell types of the PML / SUMO / Rb / HDAC complex is an additional indicator of the proliferation state of the cell.
• the method according to the present invention further comprises detecting the product of the expression of at least one gene selected from the group consisting of SUMO, Rb, HDAC and PML.
• the methods according to the present invention also consists in using the detection of the LIV21 protein in combination with all the markers of proliferations and transcription factors which play a role in the cancerization and neurodegeneration process.
• the method therefore further comprises detecting the product of the expression of at least one gene selected from the group consisting of RBP2, E2F4, E2F1, SUMO, HDAC1, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, plO7, ⁇ 130 of the pocket protein family.
• the invention lies in the manufacture and use of antibody diagnostic chips comprising an antibody specific for LIV21 and antibodies of the various proteins of the complex associated with LIV21 according to the phases of the cell cycle, that is to say without restriction to those there, antibodies specific for RBP2, E2F4, E2F1, SUMO, HDAC1, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, pi07, p130 of the pocket protein family (FIG. 1).
• the diagnostic chips according to the present invention may comprise antibodies specific for NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, and CAF1.
• Ki67 and CAFl are nuclear markers that sign the proliferation state of many cancers.
• the protein chips will be used to study the expression of proteins, protein interactions and post-translational modifications, more particularly the phosphorylations and methylations of certain proteins, which signify a characteristic state of the diseased cell.
• the state of expression and silencing of certain genes is different in diseased cells and in healthy cells.
• the protein interactions and the metabolism of the diseased cell are different from those of the healthy cell.
• the other aspect of the present invention is the use of the proteins mentioned above as markers of the invasiveness and metastatic aggressiveness of the cells. cancerous prostate, colon, bladder, melanoma, ovary, endometrium, cervix and cancers in neurology etc.
• the gene expression product is detected at the protein level.
• the protein is detected using a specific antibody.
• the protein can be detected by Western blot analysis. In a preferred embodiment, it is detected by immunohistochemistry, immunocytochemistry, radiography or peroxidase labeling.
• a significant increase in PKC ⁇ is indicative of the presence of cancer cells.
• the method may also comprise the determination of the LIV21 / PKC ⁇ ratio in the nucleus and / or the cytoplasm. This ratio can be compared to that observed in a healthy cell. An increase in the LIV21 / PKC ⁇ ratio in the cytoplasmic fraction is indicative of cancer cells.
• the method comprises the detection of the combination of LFV21 with the E2F4 protein, and a decrease of this association in the cell nucleus being indicative. of the presence of cancer cells.
• the method may also include determining the LIV21 / E2F4 ratio in the nucleus and / or the cytoplasm. This ratio can be compared to that observed in a healthy cell.
• the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.
• the method may also include determining the LIV21 / E2F1 ratio in the nucleus and / or the cytoplasm. This ratio can be compared to that observed in a healthy cell.
• the method according to the present invention allows in particular the detection of metastasized cancer, therapeutic monitoring and / or treatment recurrences.
• a second aspect of the invention relates to the human LIV21 protein as well as fragments thereof. More particularly, the present invention relates to isolated, purified or recombinant human LIV21 protein. It relates in particular to an isolated polypeptide having an apparent molecular weight of about 50-51 kD by Western Blot analysis and about 60 kD when it is sumoyled and / or a polypeptide having an isoelectric point of 5.6 in its form.
• polypeptide comprises the two peptide sequences SEQ ID Nos. 1 and 2.
• polypeptide comprises a third peptide sequence SEQ ID No. 3 and / or a fourth peptide sequence SEQ ID. No.
• LIV21 further comprises a sequence selected from SEQ ID Nos. 5-55 or a sequence having 70, 80 or 90% identity therewith.
• the LIV21 protein comprises a leucine zipper motif, a basic domain characteristic of the DNA binding domains, and a nucleation sequence. Trypsin digestion of the LIV21 protein yields more than 54 peptides corresponding to the monoisotopic peaks among all the peptides as specified in Figures 3-6; Figure 7 or 9 (Table); Figure 8 (SDS PAGE gel).
• a third aspect of the invention relates to an antibody that specifically binds to a polypeptide according to the present invention. More particularly, the antibody can specifically bind to a polypeptide comprising a peptide sequence selected from SEQ ID Nos. 1-55, preferably from SEQ ID NOs: 1-5 or a sequence having 70, 80 or 90% identity with those -this.
• the present invention relates in particular to an anti-LIV21 serum manufactured by immunizing an animal or a human with a polypeptide according to the present invention, in particular a polypeptide. comprising a peptide sequence selected from SEQ ID Nos. 1-55, preferably from SEQ ID NOs: 1-5 or a sequence having 70, 80 or 90% identity therewith.
• a fourth aspect of the invention relates to a kit for detecting cancer cells in a biological sample of a patient, the kit comprising one or more members selected from the group consisting of an antibody that specifically binds to human LFV21. according to the present invention and an anti-LIV21 serum according to the present invention.
• the kit further comprises means for detecting the product of the expression of a gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene.
• the detection means is an antibody specific for the protein in question.
• the kit also comprises a means for detecting the product of the expression of a gene selected from the group consisting of RBP2, SUMO, HDAC, PML, cycE / cdk2, cdk1, CREB1, p300, Rb 5 PL07, pl30, NFkB, cdc2A, mdm2, p21, p53, p65, Ki67 and CAFL.
• the kit comprises an antibody chip comprising an antibody specific for LIV21.
• the chip further comprises an antibody specific for a protein selected from PKC ⁇ , E2F1 and E2F4.
• the chip may comprise an antibody specific for a protein selected from RBP2, SUMO, HDAC1, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, pI07, p130 of the pocket protein family.
• the chip may also comprise an antibody specific for a protein selected from NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, and CAF1.
• the invention relates to the use of an antibody specific for human LIV21 for the diagnosis of cancer and / or of one or more antibodies specific for a protein complex containing LIV21, for example antibodies specific for RBP2, E2F4, E2F1, SUMO, HDACl, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, p107, p130 of the pocket protein family.
• the diagnosis is made ex vivo on samples of a patient.
• FIG. 1 Antibody chip.
• Figure 2 diagram of nuclear protein interactions and consequences on the study of therapeutics.
• Figure 3 Profile of the LJV21 protein by mass spectrometry (Maldi) M (H +) for the one-dimensional gel band corresponding to the protein doublet migrating at 50kD.
• the peptides resulting from the digestion are solubilized in a solvent: acetonitrile / water (1/1) containing 0.1% of TFA (trifluoroacetic acid).
• a saturated solution of the alpha cyano-4-hydroxycinnamic acid matrix is made in the same solvent.
• the same volume of the two solutions is taken, mixed and 1 microliter is deposited on the Maldi plate for analysis.
• the spectrum was made on a Voyager with a software type Waters.
• Figure 4 is a zoomed profile of the chromatogram of the 49 KD band without smoothing.
• Figure 5 is the second chromatogram corresponding to the 12% one-dimensional acrylamide gel band migrating at 49 KD and revealed by coumassie blue and LIV21 antibody. The spectrum was made on a Brucker.
• Figure 6 is the third chromatogram corresponding to the one-dimensional acrylamide gel band migrating at 53 kD and revealed by coumassie blue and LIV21 antibody. The spectrum was also performed on a Brucker.
• Figure 7 a table of monoisotopic peaks with a value M H +. The masses are given with three digits behind the comma by the proteomic platforms because they consider that it is the limit of the acquisition accuracy of MALDI TOF machines.
• Figure 8 shows a 2D SDS PAGE gel separating the twelve polypeptides hooked by the LI V21 antibody.
• Figure 9 depicts Nano LC-ESI MS characterized peptides including LIV21a and LIV21b.LIV21c, LIV21d, LIV21e.
• the table describes an experiment of NanoLC-ESI MS.
• NanoLC allows the separation of peptides derived from the tryptic hydrolysis of the protein. The eluted peptide fragments are ionized by electrospray, the formed ions detected by mass spectrometry (Q-TOF analyzer). These ions characterize for each a peptide specific for the protein.
• Caption BEGIN IONS: Starting Ions.
• PEPMASS Peptide mass.
• the corresponding MSMS spectrum is defined by the column of numbers between Begin ions and End ions which corresponds to an amino acid sequence (each Amino Acid having a specific mass except Leucine and Isoleucine which have the same molecular weight).
• the first column with a 4 decimal number corresponds to the mass of the "son” ions.
• the second column (1:45) corresponds to the intensity of these "son” ions.
• Figure 9 describes the analyzes in MSMS giving a set of polypeptides assignable to the protein LIV21 and its complex or contaminants according to the different observers of the different platforms of proteomics sub-contractors.
• Figure 10 describes the MALDI TOF assays giving a set of polypeptide sequences assignable to LIV21. and its complex and sometimes different contaminants according to the observers of the different platforms of proteomics subcontractors.
• the Mascot research parameters are: trypsin enzyme, variable modifications: carbamethylation and oxidation of methionines, no limit of molecular weight, without restriction of isoelectric point. Mass type: monoisotopic. Mass error (MS): depending on the observer 50 ppm or 100 ppm.
• Non-tryptic cut 1 The masses entered are M (H +) / real masses. For chromatogram 1 the cysteines are blocked by iodoacetamide. The possibility of digestion of Proméga bovine trypsin may be incomplete with missed cut.
• Gallus Gallus gi 50732569
• mouse Syntaxin Histatin-3-2 variant (P15516-00-01-00)
• ZN575-Human G6 P translocase
• HSP60 chaperonine arginine deiminase
• ferredoxin-NADP (+) reductase pseudomonas polyribonucleotidyl transferase
• dehydrolipoamide dehydrogenase common sequences with Gallus Gallus (gi 50732569), mouse Syntaxin, Histatin-3-2 variant (P15516-00-01-00), ZN575-Human, G6 P translocase, HSP60 chaperonine
• arginine deiminase ferredoxin-NADP (+) reductase
• pseudomonas polyribonucleotidyl transferase pseudomonas polyribonucleotidyl transferase
• Figure 11 Alignments between histatin-3-2 variant, PATF and Q7TCL4 (Turnip mosaic virus) AAN08045.2.
• Figure 14 is the morphology of MCF7 cells treated or not treated with 25 nM TPA.
• Figure 15 is a FACS analysis; representation for each phase of the cell cycle of the percentage of cells as a function of processing time: Figure 15 A. Phase S, Figure 15B. Phase G2 / M, Figure 15C. phase G0 / G1. The scale of the abscissa is not proportional to the duration of treatment.
• Figure 16 is a Western Blot comparing total extracts (ET) and nuclear extracts (EN) and showing TPA inhibition of expression of LIV21 phosphorylation.
• Figure 16 A as a function of treatment time with 25 nM TPA;
• Figure 16B compared to LIV21 in protein extracts, at 12h treatment with 25 nM TPA.
• Figure 17 is a study of the nuclear translocation of LIV21 by immunocytochemistry with an anti-LIV21 primary antibody (in green) in cultures treated or not treated with 25 nM TPA.
• the nuclei are stained red with propidium iodide.
• the nuclei are mostly stained yellow at 12H until 24H because the primary anti-LIV21 antibody (in green) is nuclear while it is predominantly cytoplasmic at 72H (red nuclei and green cytoplasm).
• Figure 18 shows the expression as a function of the 25nM TPA processing time of the PKC ⁇ and PKC ⁇ proteins in total extracts.
• P ⁇ -tubulin expression serves as a control of the amount of total protein deposited in the wells.
• Figure 19 shows the comparative expression of PKC ⁇ and LIV21 in immunocytochemistry on cultures of MCF-7 cells treated and untreated by TPA at 25 nM for 12h, performed with anti-LIV21 and anti-PKC ⁇ antibodies in green, and propidium iodide staining the DNA in red.
• LIV21 is translocated into the nucleus by specific inhibition of PKC ⁇ .
• PKC ⁇ is weakly expressed at 12h in the presence of TPA. Indeed, we observe the red nuclei and little green color in the cytoplasm.
• the expression of LIV21 is strong in the nuclei which are stained yellow (merge) by both the anti-LIV21 antibody (green) and the propidium iodide specific for the nuclei.
• Figure 20 shows the effect of the PKC ⁇ inhibitory peptide on the expression pattern of LIV21 by immunocytochemistry on control cultures or treated with 1 ⁇ M peptide, 2 ⁇ M peptide, or 25 nM TPA. The treatments last 12 hours. The cells are labeled with anti-LIV21 (green) and propidium iodide (red).
• Figure 21 shows the effect of the PKC ⁇ inhibitory peptide on the expression pattern of LIV21 in cytoplasmic (C) and nuclear (N) cell fractions, after a 12h treatment with TPA (25 nM) or by the peptide (2 ⁇ M).
• FIG. 22 shows by immunoprecipitation (IP) the coexistence between PML / SUMO and LIV21: a yellow fluorescence (merge) corresponding to the co-localization of PML / SUMO and LIV21 in the cell nuclei is observed.
• FIGS. 23 and 24 show by immunocytochemistry the coexistence between PML / SUMO and LIV21.
• the co-labeling (by fluorescent immunostaining) of LIV21 (green) and SUMO-I (red) in the nuclei of cells treated or not treated with TPA for 24h and 72h show that 24h LIV21 is translocated in the nucleus where SUMO coexists.
• PML Merge: yellow nuclei
• at 72H, LIV21 (green) is cytoplasmic.
• the nuclear bodies containing the SUMO1 protein are called PML bodies.
• Figure 25 shows that from a chip of 60 biopsies including 50 of skin cancer and 9 of normal tissue and a control T; the nuclear expression of LIV21 in healthy tissue biopsies and the expression of cytoplasmic LIV21 in biopsies of metastatic cancers.
• Figure 43 T2N0M0, poorly differentiated skin cancer
• image 58 healthy tissue from the same subject with mildly differentiated skin cancer (cell nuclei stained yellow)
• image 40 metastatic carcinoma 10 cm
• image 17 metastatic carcinoma of 3.5cm.
• the cell nuclei are stained red.
• Figure 26 is, as in Figure 25, a second example of nuclear localization of LIV21 in the control and healthy tissue (# 52) (the cells nuclei are stained yellow), the sick subject # 7 of a carcinoma of squamous pharynx cells (moderately differentiated T4N0M0) In images # 7 of cancerous tissue the cell nuclei are stained red.
• Figure 27 is a sample of advanced bladder cancer on cystectomy (urothelial grade III carcinoma infiltrating the chorion and muscularis) versus healthy bladder tissue of the same patient with internal control (PI): preimmune PI serum before the rabbit is immunized against LIV21, red marking of nuclei with propidium iodide.
• PI internal control
• the invention relates to the identification of antigens in cell lysates by immunoprecipitation.
• the analysis of the physical interaction of different proteins associated with E2F4 and E2F1 was studied by co-immunoprecipitation of protein complexes. This analysis has highlighted a new marker with a diagnostic and prognostic utility for cancer.
• a marker of PATF proliferation associated with the E2F family had been demonstrated (Crisanti) by the characterization of exons without the gene being cloned in humans, or a corresponding protein found in humans.
• this new molecule LTV21 could have diagnostic value for the following reasons.
• the inventor was able to observe that, in all the proliferating tumor cells, this protein is cytoplasmic instead of nuclear. It is therefore not in the good cell compartment to be active on stopping the multiplication of cells.
• LIV21 has thus been observed in mammals.
• the expression panel of LTV21 as a function of cell state was studied on tissues from different mammals. Protein analyzes on the different tissue samples confirmed that expression of this transcription factor appears to be associated with progression to a quiescent cell state (mitosis arrest and differentiation initiation).
• LIV21 is present in actively proliferating tumor cell lines and its expression is essentially cytoplasmic. The same results are obtained on human mammary adenocarcinoma.
• the present invention relates to a new screening test for abnormalities of reinduction of the cell cycle.
• This diagnostic test is based on the study of the mechanism of action of the new gene, coding for a new potential transcription factor called LIV21, which negatively regulates proliferation.
• LIV21 is involved in stopping cell proliferation.
• LFV21 is cytoplasmic when cells proliferate as it becomes nuclear when cells become shedding.
• E2F4 The latter is known to negatively regulate the cell cycle in association with the P 130 protein or pocket protein of the RB family.
• LIV21 in tumor cells (cytoplasmic localization) and in physiological cells (nuclear localization) suggests in any case that its functioning is disrupted when cellular development becomes anarchic.
• this ubiquitous transcription factor has an expression and a location regulated according to the cellular state: stronger expression and nuclear localization for the cells leaving the mitotic cycles, weak expression and cytoplasmic localization for actively proliferating cells such as human tumor cells.
• LIV21 appears as a key molecule to stabilize another transcription factor (E2F4) in the cell nucleus and thus induce cell proliferation arrest.
• LIV21 in the cytoplasmic compartment is regulated by PKC ⁇ . Indeed, when LIV21 is phosphorylated by PKC ⁇ , LIV21 is located in the cytoplasmic compartment. Conversely, when the phosphorylation of LIV21 by PKC ⁇ is inhibited, LIV21 is located in the nuclear compartment.
• the present invention thus relates to the LIV21 protein as well as derivatives and fragments thereof.
• the LIV21 protein is a human protein of about 300 amino acids. However, depending on the alternative splicing it undergoes, it occurs in at least three different size forms ( Figure 8). Moreover, it can be phosphorylated or sumoylated. It has an apparent molecular weight between 50 kD and 51 kD in Western Blot analyzes. This apparent molecular weight is 60 kD when LIV21 is sumoylated. In its 51 kD form, phosphorylated or otherwise, its isoelectric point is 5.6 and its intensity is 13632. This protein has been characterized by mass spectrometry (Maldi) (Example 1, Figures 3-13 3).
• RTLLLPAVSRQ (SEQ ID NO: 6)
• LGFMEEWDVGEIMLR (SEQ ID NO: 7)
• FYAWMIEQAPFSSLAQEGK (SEQ ID NO: 9)
• NLYTEIVYTPISTPDGTLVK (SEQ ID NO: 10)
• RTVRPLNIEVGVLPKT (SEQ ID NO: 14)
• RRSVQAMLPGADVFPYTIRV (SEQ ID NO: 15)
• GDLWFMSHQGHK (SEQ ID NO: 41)
• YAFDFYEMTSR (SEQ ID NO: 42)
• FVFALILALMLSMCG (SEQ ID NO: 51)
• TLQIFNIEMKSK (SEQ ID NO: 52)
• a preferred LIV21 protein comprises at least one sequence chosen from SEQ ID Nos: 1-55 or a sequence exhibiting 70%; 80% or, preferably, 90% homology therewith.
• the LIV21 protein comprises a leucine zipper motif, a basic domain characteristic of DNA binding domains, and a nucleation sequence.
• the present invention relates to an isolated, purified or recombinant human polypeptide having a sequence comprising the sequence SEQ ID No. 1 and / or SEQ ID No. 2.
• the polypeptide comprises the sequences SEQ ID Nos. 1 and 2.
• the polypeptide comprises (in addition) a sequence selected from SEQ ID Nos. 3-55, preferably from SEQ ID NOs 3-5, or a sequence having 70, 80 or 90% identity therewith.
• it comprises a sequence selected from one of the peptide sequences obtained by MALDI (FIG.
• the invention also relates to the two peptides LIV21a (SEQ ID No. 1) ) and LIV21b (SEQ ID No. 2).
• the invention also relates to a peptide having a sequence selected from SEQ ID Nos. 3-55, preferably from SEQ ID NOs 3-5, or a sequence having 70, 80 or 90% identity therewith. It also relates to peptides comprising at least 10 consecutive amino acids of human LIV21, preferably at least 20, 30 or 50 consecutive amino acids of LIV21.
• the invention further relates to derivatives of interest of LIV21 which are for example fusion proteins in which LIV21 is fused to marker proteins such as GFP.
• LTV21 protein can be labeled by any means known to those skilled in the art.
• the present invention also relates to an antibody that specifically binds to a polypeptide according to the present invention, preferably human LIV21, a fragment or a derivative thereof.
• the antibody specifically binds to a LIV21a or LIV21b peptide.
• the antibody specifically binds to a polypeptide comprising a sequence selected from SEQ ID Nos. 1-55, preferably from SEQ ID NOs 1-5, or a sequence having 70, 80 or 90% of identity with these.
• the antibodies may be polyclonal or monoclonal. It may also be fragments and derivatives of antibodies having substantially the same antigenic specificity, in particular antibody fragments (eg Fab, Fab'2, CDRs), humanized, polyfunctional, single-stranded antibodies ( ScFv), etc.
• Antibodies the invention may be produced using conventional methods, including immunizing an animal and recovering its serum (polyclonal) or spleen cells (so as to produce hybridomas by fusion with appropriate cell lines) .
• Said antibodies can be obtained directly from human serum or from serum of animals immunized with the proteins or peptides according to the present invention.
• Methods for producing polyclonal antibodies from a variety of animal species including rodents (mice, rats, etc.), primates, horses, pigs, sheep, rabbits, poultry, etc. are described for example in Vaitukaitis et al. (Vaitukaitis, Robbins et al., 1971).
• the antigen is combined with an adjuvant (eg, Freud's adjuvant) and administered to an animal, typically by subcutaneous injection. Repeated injections can be performed. Blood samples (immune serum) are collected and immunoglobulins are separated.
• the present invention relates to an anti-LIV21 serum manufactured by immunizing an animal with the polypeptide according to the present invention.
• the animal has been immunized with the peptide LIV21a and / or LIV21b.
• the animal is immunized with these two peptides.
• the present invention also relates to an anti-LIV21 serum manufactured by immunizing an animal or a human with a polypeptide according to the present invention, in particular a polypeptide comprising a peptide sequence selected from SEQ ID Nos 1-55, preferably from SEQ ID NOs 1- 5, or a sequence having 70, 80 or 90% identity therewith.
• the peptides may be coupled to a carrier protein such as hemocyanin, and then injected into an animal, for example a rabbit, for immunization.
• a carrier protein such as hemocyanin
• Polyclonal antibodies were obtained from these two peptides by immunizing two rabbits and blowing a rabbit to have a preimmune serum.
• the subject of the invention is also the use of the antibodies according to the invention for the detection and / or purification of the human LIV21 protein.
• antibodies specific for LIV21 can be used for the detection of these proteins in a biological sample. They thus constitute a means of immunocytochemical or immunohistochemical analysis of LIV21 expression on tissue sections.
• the antibodies used are labeled to be detectable.
• the antibodies can be labeled indirectly.
• the antibodies are labeled.
• Markers include radiolabels, enzymes, fluorescent, luminescent, chemical, magnetic particle markers, gold tagging, biotin / avidin labeling, peroxidase, etc.
• the subject of the invention is also a method for detecting the LIV21 protein in a biological sample, comprising a step of suitable treatment of the cells by any appropriate means making it possible to make the intracellular medium accessible, a step of bringing the said intracellular medium into contact as well as obtained with an antibody specific for the human LIV21 protein and a step of detection by any appropriate means of the LIV21 complex-formed antibody.
• the cytoplasmic and / or nuclear extracts are prepared, and these extracts are contacted with the human LIV21 protein specific antibody.
• the present invention teaches the development of the diagnostic test also for monitoring the evolution of a cell proliferation.
• the present invention makes it possible to monitor the evolution of cell proliferation on fresh cells or tissues, on frozen cells or tissues and on tissues treated, inter alia, with paraffin.
• the applications can be the diagnosis of cancer as well as the follow-up of the evolution of a cellular proliferation.
• the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, sarcoma, leukemia and glioblastoma, without being limited to these.
• the predominantly cytoplasmic state of this protein in cancer cases compared to its nuclear situation in healthy cells is a geographical and structural difference which makes it possible, without the need for a fluorescent marker, to differentiate spectral profiles of the functional pattern of the tissue. cancerous versus healthy tissue and thus to make the diagnosis.
• the cytoplasmic localization of LIV21 is an indicator of the aggressiveness and metastatic potential of cancer.
• the detection of LIV21 expression indicates the presence of cancer cells, more particularly invasive, aggressive and / or metastatic cancer cells.
• the nuclear localization of LIV21 is an indicator of healthy quiescent cells or well differentiated tissues.
• the invention further provides cancer diagnostic or prognostic methods for detecting the cytoplasmic localization of a nuclear localized transcription factor in healthy cells.
• the present invention relates to a method for the detection of cancer cells in a biological sample of a patient comprising detecting the product of the expression of the LIV21 gene in the nucleus and / or the cytoplasm of the cells in the biological sample of said patient, a location of said LIV21 gene expression product in the cytoplasm being indicative of the presence of cancer cells and a location of said product of LIV21 gene expression in the nucleus being indicative of the presence of non-cancerous cells.
• a location of said product of LIV21 gene expression in the cytoplasm is indicative of the presence of invasive and / or metastatic cancer cells.
• the method preferably comprises a prior step of suitably treating the cells contained in the sample by any suitable means for making the intracellular medium accessible.
• the method optionally includes a step of comparing with a biological sample that does not contain cancer cells.
• the method according to the present invention further comprises the detection of the product of the expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene.
• the method may include the detection of the product of the expression of two of these genes or three genes.
• LIV21 / E2F4 and LIV21 / E2F1 can be determined in the present method. This ratio can be determined in the cytoplasm and / or in the nucleus. Preferably, these ratios are determined in the nucleus. Preferably, these ratios are compared to those obtained in a healthy cell.
• the method may also include detecting the product of expression of at least one gene selected from the group consisting of RBP2, E2F4, E2F1, SUMO,
• the method may further comprise the detection of the product of expression at least one gene selected from the group consisting of NFkB, cdc2A, mdm2, p21, p53, p65, ⁇ 161, and CAF1.
• the method may include detecting an interaction between some of these proteins and / or detecting a post-translational modification of one of these proteins.
• the gene expression product is detected at the level of the protein.
• the protein is detected using a specific antibody.
• the method comprises a step of contacting the cells of the biological sample with an anti-human LIV21 antibody.
• the antibodies can be monoclonal or polyclonal.
• the anti-LIV21 antibody may for example be an anti-LIV21 serum.
• the method can use antibodies specific for the PKC ⁇ , E2F1 and E2F4 proteins, respectively.
• the polyclonal and monoclonal antibodies directed against PKC ⁇ , E2F1 and E2F4 are commercially available.
• PKC ⁇ of a polyclonal rabbit antibody (Santa Cruz Technology, sc-214), for E2F1, a polyclonal rabbit antibody (Santa Cruz Technology, sc-860), and for E2F4, a polyclonal rabbit antibody (Santa Cruz Technology, sc-866).
• the antibodies are labeled directly or via a secondary antibody. Antibody labeling techniques are well known to those skilled in the art.
• the protein can be detected by Western blot analysis.
• Western blot analysis can be performed on nuclear and / or cytoplasmic extracts of the cells contained in the patient's sample. Briefly, the proteins are migrated in a gel and then transferred to a membrane. Then, this membrane is incubated in the presence of antibodies and antibody binding is eventually revealed using labeled secondary antibodies.
• the protein is detected by immunohistochemistry, immunocytochemistry or immunoradiography. These techniques are well known to those skilled in the art. Immunocytochemistry analysis may be performed on whole cells from the sample or derived therefrom, for example by culture cellular. It can also be performed on isolated nuclei. Immunohistochemistry analysis can be done on sections of breast tissue.
• an immunocytochemical analysis may comprise the following steps. However, it is understood that other preparatory modes can be implemented. Cells from the biological sample are cultured, preferably on slides (Lab Tek, Nunc, Germany), then washed with buffer and fixed with paraformaldehyde (eg 4%). A saturation step is preferably performed by incubating the cells with S buffer (PBS-Triton X100 0.1% - 10% FCS). The cells are then incubated with the primary antibody and then washed and incubated with a fluorescent secondary antibody, if necessary. The nuclei can be labeled with propidium iodide (Sigma). The slides are moviol mounted for observation by fluorescence microscopy.
• isolated nuclei removed during nuclear extraction can be fixed with paraformaldehyde (eg 4%).
• the suspensions of nuclei are deposited between lamella and lamella and the observation is made by fluorescence microscopy and confocal microscopy.
• the primary antibodies are, for example, rabbit antibodies and the secondary antibodies are labeled antibodies directed against rabbit IgG.
• the present invention also relates to the use of a protein chip for detecting the expression of one or more of these proteins, and / or an interaction between two or more of these proteins, and / or post-translational modification. one or more of these proteins.
• a polypeptide according to the present invention in particular LIV21 or a fragment thereof, or an antibody specific thereto, a fragment or a derivative thereof retaining the binding specificity, may advantageously be immobilized on a support, preferably a protein chip.
• a protein chip is an object of the invention.
• This chip may also contain at least one polypeptide selected from the group consisting of protein kinase C epsilon (PKC ⁇ ), RBP2, E2F4, E2F1, SUMO, HDAC1, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, p107, p130 of the pocket protein family or at least one antibody specific for one of these polypeptides, a fragment or a derivative thereof that retains the binding specificity.
• the chip may further comprise other polypeptides well known to those skilled in the art as being of interest for the detection and / or prognosis of cancer, or antibodies specific thereto. These polypeptides may for example be selected from the following list: NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, and CAF1.
• the protein chips according to the present invention can be prepared according to the techniques well known to those skilled in the art. In practice, it is possible to synthesize the polypeptides attached directly to the protein chip, or to carry out an ex situ synthesis followed by a step of fixing the polypeptide synthesized on said chip.
• polypeptides or antibodies to be fixed can be purified from a cell.
• Supports include smooth supports (eg, metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials.
• Such carriers also include, but are not limited to, gels, rubbers, polymers, and other soft materials. The supports do not need to be flat.
• the proteins or antibodies of the chip may be attached directly to the support or attached via a spacer or link.
• an antibody specific for LIV21, a fragment or a derivative thereof retaining the binding specificity is immobilized on the solid support.
• this chip provides a convenient way to measure the LIV21 expression product.
• the chip comprises at least one antibody specific for a polypeptide selected from the group consisting of PKC ⁇ , RBP2, E2F4, E2F1, SUMO, HDACl, CycE / cdk2, cdkl, CREBl, p300, Rb 5 PML 5 plO7, pl30 of the pocket protein family, preferably PKC ⁇ , E2F1, and E2F4.
• the chip further comprises at least one antibody specific for a polypeptide known to those skilled in the art as being of interest for the detection and / or prognosis of a cancer, for example NFkB, cdc2A, mdm2, p21, p53, ⁇ 65, Ki67, and CAF1.
• the chip may comprise an antibody or a fragment or derivative thereof having the same specificity.
• the protein chips according to the invention are also extremely useful for proteomic experiments, which studies the interactions between different proteins.
• peptides representing the various proteins on a support are fixed. Then, said support is brought into contact with labeled proteins, and after an optional rinsing step, interactions between said labeled proteins and the peptides attached to the protein chip are detected.
• protein chip is intended to mean a support on which are fixed polypeptides or antibodies, each of which can be identified by its geographical location. These chips differ mainly in size, support material, and possibly the number of polypeptides attached thereto.
• Protein chips may also be useful for screening test compounds.
• the present invention also relates to a method for the detection of cancer cells in a biological sample of a patient comprising detecting the product of the expression of the LIV21 gene in the nucleus and / or the cytoplasm of the cells in a sample of cells in the biological sample of said patient, which method is characterized in that it comprises at least: (a) bringing said biological sample into contact with a protein chip as defined above, and (b) revealing by any suitable means of the antigen-antibody complexes formed in (a), for example by EIA, ELISA, RIA, or immunofluorescence.
• Other detection methods are described in detail in the following documents: US2004152212.
• the protein chips can be manufactured using conventional methods described (Lubman David M 5 QIAO Tiecheng Alex Mathew J ABY etc ..) or new hybridization automation tools like reading US2004152212 and Yu Xinglong US 2005019828 and novel carriers that cleave Klages Claus Peter polypeptides and (example, Figure 2).
• the biological samples come from a patient potentially suffering from cancer or cancer in a proven way.
• biological sample is meant in particular a sample of the biological fluid type, living tissue, tissue fragment, slime, organ or organ fragment, or any culture supernatant obtained using a sample.
• the method according to the present invention may comprise a step of taking a biological sample from the patient.
• the detection step can be performed directly on a section of tissue of the sample, on a culture of cells from the sample, on total cell extracts, nuclear and / or cytoplasmic extracts.
• the patient's specimen may come from a puncture, a biopsy, a crushed cell, a bronchial aspiration, a blood sample or a urine sample.
• a significant increase in PKC ⁇ is indicative of the presence of cancer cells. More specifically, the amount of PKC ⁇ in healthy cells is compared with the amount of PKC ⁇ in the cells of the sample and the significant increase is determined by this comparison.
• the method according to the present invention may optionally comprise the measurement of the LIV21 / PKC ⁇ rate. This LIV21 / PKC ⁇ ratio increases in the cytoplasmic fraction of cancer cells compared to healthy cells.
• the method comprises detecting the association of LIV21 with the E2F4 protein, and a decrease in this association being indicative. of the presence of cancer cells. Detection of the association of LIV21 with the E2F4 protein can be achieved by concomitant detection of LIV21 and E2F4 and / or concomitant HDAC1 measurement.
• the method according to the present invention may optionally comprise the measurement of the E2F4 / LIV21 level. This ratio E2F4 / LIV21 decreases in the nucleus of cancer cells compared to healthy cells.
• the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.
• the method according to the present invention may optionally comprise the measurement of the E2F1 / LIV21 level. This ratio E2F1 / LIV21 increases in the nuclear fraction of cancer cells compared to healthy cells.
• the method according to the present invention allows in particular the detection of metastasized cancer, therapeutic monitoring and / or treatment recurrences and to determine the degree of invasiveness of a cancer.
• the specificity of the detection can be linked to the crossings of the information obtained by the existence and the topography of LIV21 by all the means of imaging and spectroscopy and obtained by the association with other known oncological indicators via protein chips or proteins. microarrays.
• detection based on LIV21 may be associated with the detection of other markers of cancer, in particular breast cancer, known to those skilled in the art.
• the present invention relates to a method of therapeutic follow-up of an anti-cancer treatment in a patient suffering from a cancer comprising administering the anti-cancer treatment to said patient and detecting cancer cells in a biological sample. of the patient according to the method of the present invention. A decrease in cancer cells will be indicative of the effectiveness of the treatment.
• the detection of cancer cells in a biological sample of the patient according to the method of the present invention may be carried out once or several times during the anti-cancer treatment or after the anticancer treatment.
• the biological sample is from the tissue affected by the treated cancer.
• the present invention also relates to a method for detecting recurrence following anti-cancer treatment of cancer in a patient comprising detecting cancer cells in a biological sample of the patient according to the method of the present invention.
• the detection of cancer cells in a biological sample of the patient according to the method of the present invention can be carried out once or several times after the anti-cancer treatment.
• the detection of cancer cells is indicative of recurrence.
• the biological sample is from the tissue affected by the treated cancer.
• the present invention also describes a kit for implementing a method according to the invention. More particularly, the invention relates to a kit for the detection of cancer cells in a biological sample of a patient comprising one or more members selected from the group consisting of an antibody that specifically binds to human LIV21 according to the present invention. and an anti-LIV21 serum according to the present invention.
• the kit comprises antibodies that specifically bind to human LIV21.
• the kit according to the present invention may comprise reagents allowing the detection of a LIV21 complex-an antibody produced during an immunological reaction.
• the kit according to the present invention further comprises means for detecting the product of the expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKC ⁇ ) gene, the E2F1 gene and the E2F4 gene.
• PLC ⁇ protein kinase C epsilon
• the present invention also relates to a diagnostic composition comprising one or more elements selected from the group consisting of an antibody according to the present invention and a serum according to the present invention.
• Anti-cancer Therapy In the context of anti-cancer therapy, one can consider increasing the amount of LIV21 present in the nucleus. For that, we could favor the nuclear localization of LIV21, for example by decreasing PKC ⁇ activity in cancer cells and using HDAC inhibitors.
• PKC ⁇ in cancer cells it is possible to reduce the activity of PKC ⁇ in cancer cells.
• This decrease in activity can be achieved by decreasing the activity of the PKC ⁇ protein or by decreasing its expression.
• a decrease in PKC ⁇ protein activity can be achieved by administering cancer inhibitors to the cancer cells.
• Inhibitors of the PKC ⁇ protein are well known to those skilled in the art.
• a decrease in the expression of the PKC ⁇ protein can be obtained by using antisense or siRNA specific for the PKC ⁇ gene. Kits are commercially available.
• the techniques concerning inhibitions by antisense or siRNA are well known to those skilled in the art. (Arya R 2004, Lee W 2004, Sen A 2004, Platinum 1998, Hughes 1987)
• the present invention therefore relates to a pharmaceutical composition comprising an inhibitor of the PKC ⁇ protein. It also relates to the use of a pharmaceutical composition comprising an inhibitor of the PKC ⁇ protein as a medicament, in particular for the preparation of a medicament for treating a cancer. Finally, it relates to a method of treating cancer in a patient comprising administering to the cancer cells an inhibitor of the PKC ⁇ protein, the inhibitor of the PKC ⁇ protein making it possible to reduce or abolish the cancerous phenotype of the treated cells.
• the inhibitor of the PKC ⁇ protein decreases the activity of the PKC ⁇ protein.
• the inhibitor of the PKC ⁇ protein decreases the expression of the PKC ⁇ protein.
• the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver, sarcoma, leukemia and glioblastoma, without being limited to these.
• the cells affected by the neurodegenerative disease are usually neurons, motor neurons, etc.
• the neurodegenerative disease is selected from Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS).
• ALS amyotrophic lateral sclerosis
• An increase in the activity of the PKC ⁇ protein can be obtained by administering to the cells affected by the neurodegenerative disease activators of the PKC ⁇ protein.
• Activators of the PKC ⁇ protein are well known to those skilled in the art (Toma O (2004), Activation of PKC by DAG, PUFA: oleic acid, linoleic acid, arachidonic acid, etc. Activation and proteolysis of PKCs in gonadotropic cells: Communication 2004 by Macciano H, Junoy B, Mas JL Drouva SV UMR6544 Marseille).
• An increase in the expression of the PKC ⁇ protein can be obtained by using expression vectors coding for the PKC ⁇ protein and allowing it to be overexpressed in the cells affected by the neurodegenerative disease.
• the present invention relates to a pharmaceutical composition comprising an activator of the PKC ⁇ protein or an expression vector encoding the PKC ⁇ protein. It also relates to the use of an activator of the PKC ⁇ protein or of an expression vector encoding the PKC ⁇ protein for the preparation of a medicament for the treatment of a neurodegenerative disease.
• the invention relates to methods for the selection, identification, characterization or optimization of cell-depleting active compounds based on the measurement of the nuclear or cytoplasmic localization of LIV21, or the binding of LIV21 protein to the E2F4 protein.
• the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the nuclear or cytoplasmic localization of the cell.
• LIV21 expression product An increase in Nuclear localization of LIV21 indicates that the candidate compound is active to decrease or abolish cell proliferation. A decrease in the nuclear localization of LIV21 indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
• the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the level of expression of the gene. encoding the PKC ⁇ protein. A decrease in PKC ⁇ expression indicates that the candidate compound is active to decrease or abolish cell proliferation. An increase in PKC ⁇ expression indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
• the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the level of complex
• LIV21 / E2F4 An increase in the level of LIV21 / E2F4 complex indicates that the candidate compound is active to decrease or abolish cell proliferation. A decrease in the level of LIV21 / E2F4 complex indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
• the selection, identification, characterization or optimization of active compounds of therapeutic interest comprises contacting a candidate compound with a cell and determining the level of expression of the gene. encoding the E2F1 protein. A decrease in E2F1 expression indicates that the candidate compound is active to decrease or abolish cell proliferation. An increase in E2F1 expression indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.
• the subject of the invention is also a method for screening a compound capable of interacting in vitro, directly or indirectly, with LIV21, characterized in that: in a first step, the candidate compound is contacted with LIV21 and, in a second step, the complex formed between said candidate compound and LI V21 is detected by any suitable means.
• the subject of the present invention is also a method for screening a compound capable of modulating (activating or inhibiting) the activity of the LIV21 protein, characterized in that: in a first step, cells of a biological sample expressing the LIV21 protein with a candidate compound, in a second step, the effect of said candidate compound on the activity of said LIV21 protein is measured by any appropriate means, and in a third step candidate compounds capable of modulating said activity.
• the activity of LIV21 can for example be estimated through the evaluation of the ability of the cell to divide, by measuring the expression of the E2F1 gene, or by the cytoplasmic and / or nuclear localization of LIV21. .
• the candidate compound can be a protein, a peptide, a nucleic acid (DNA or RNA), a lipid, an organic or inorganic compound.
• the candidate compound could be an antibody, an antisense, a ribozyme or a siRNA.
• EXAMPLE 1 The inventor has carried out mass spectrometry (MALDI) for the LIV21 protein from a one-dimensional acrylamide gel.
• LIV21 protein was digested with trypsin.
• the peptides resulting from the digestion are solubilized in a solvent: acetonitrile / water (1/1) containing 0.1% TFA (trifluoroacetic acid).
• a saturated solution of the alpha cyano-4-hydroxycinnamic matrix was prepared in the same solvent. The same volume of the two solutions was taken, 1 ⁇ l was mixed and deposited on the MALDI plate for analysis.
• Mass spectrometry showed that the trypsin-digested LIV21 protein showed 54 peptides (see Figures 3-4).
• the LIV21 protein was characterized by a molecular weight of 50 kD, highlighted by Western Blot, the first MALDI results are not probands, the inventor has made a two-dimensional gel SDS PAGE ( Figure 8) more than ten proteins were revealed by staining with silver nitrate but the very small amount of material did not allow testing of samples from this MALDI gel or MSMS.
• the inventor carried out a third MALDI analysis which yielded interesting results, especially on the 49KD gel band on gallus gallus and a variant of histatin (figure, the inventor then looked at the sequence alignments which allowed confirm homologies between gallugallus, PATF, Q7TCL4 and the histatin and gallus gallus polypeptides (Figure 10-13).
• the LIV21a peptide is located between an interaction site with the Rb / p107 / ⁇ 130 protein (ITCCE) and an SUMO1 sumoylation site.
• the sequence of this peptide is the following: PeptideLIV21a RVYGPLTNPKPQ SEQ ID No. I
• the LIV21b peptide is located between a sumoylation site (PKPG) and a phospholipase C (YVKI) site followed by (KKKRK) NLS.
• the sequence of this peptide is the following: PeptideLIV21b CYRSILHTKV SEQ ID No 2
• PKCs Protein Kinase C
• the cell line MCF-7 The cell line MCF-7
• the MCF-7 line is a non-clonal human breast adenocarcinoma cell line. During their differentiation induced by exogenous factors, these cells develop hypertrophy, membrane protrusions and a tendency to dissociate from each other. They acquire a secretory phenotype characterized by the appearance of numerous granules and secretory canaliculi.
• PLCs protein kinases C
• TNF for induction of apoptosis
• TPA (12-O-tetradecanoyl phorbol-13- SUMOate) for the induction of differentiation and thus for the study of the exit of the cell cycle.
• TPA cytosolic protein kinase C
• TPA increases the capacity for migration of MCF-7 cells in vitro and a short treatment of these cells by TPA induces cell expansion and microtubule organization characteristic of their differentiation.
• the inventor verified the expression of LIV21 in these cells at the protein level.
• the inventor has discussed the study of the expression of the protein LIV21 by the technique of the western blot, with an anti-LIV21 antibody, in the MCF-7 cells compared to the mammary tissues.
• the anti-LIV21 antibodies were obtained by the method described below.
• LIV21 is expressed as well in mammary tissues as in MCF-7 cells, in the form of a doublet migrating at an apparent molecular weight of 50 kDa.
• the specific peptide sequences are sequences No. 1 and No. 2 These peptides were injected into rabbits (NZ W ESD 75 female 2.3kg at day 0) In accordance with standard immunization procedures, such as:
• the reactivity of the serum obtained was tested to bind peptide sequences # let 2. At J60 the serum showed good reactivity with each sequence.
• the serum produced is not related to any member of the E2F family.
• TPA induces proliferation arrest and differentiation of tumor cells of the MCF-7 line.
• the cell growth was previously followed over three days of culture in the presence or absence of TPA at a concentration of 25 nM (FIG. 13).
• Cell counts show variation in growth kinetics between untreated and TPA treated cultures. Indeed, from the second day of culture, the number of cells between the two treatment conditions is significantly different, the TPA already inducing cell proliferation arrest. After 3 days of treatment, the control cultures have twice as many cells as the treated cultures. TPA at a concentration of 25 nM therefore inhibits proliferation well in our culture conditions.
• TPA-treated cells rapidly acquire characteristics of differentiated mammary gland cells (FIG. 14): hypertrophy, membrane profusions and the tendency to dissociate from each other.
• FOG. 14 characteristics of differentiated mammary gland cells
• Phase S The FACS study shows that the number of cells in S phase decreases to reach a limit value between 12h and 24h of treatment with TPA (Figure 15A). But, without new addition of TPA, the number of cells in phase S increases again to return to the initial state at 72 hours of treatment.
• Phase G2 / M The number of cells in G2 / M phase increases from the beginning of treatment with a maximum observed at 12 hours (Figure 15B).
• Phase G0 / G1 The number of cells in G0 / G1 phase is minimal at 6 hours and maximum at 24 hours (Figure 15C).
• PKC ⁇ is weakly expressed at 12h, at which time we observe the least number of cells in S phase and the maximum of nuclear translocation of LIV21.
• EXAMPLE 4 Study of the Specific Role of PKC ⁇ on the Nuclear Translocation of LIV21 Using a Peptide Inhibiting Function and the Translocation of PKC ⁇
• cultures have were treated with a peptide, selective antagonist of the function and translocation of this PKC (EAVSLKPT (SEQ ID No. 6), and the results were compared with those obtained by treatment with TPA.
• EAVSLKPT SEQ ID No. 6
• This peptide is recognized by the enzyme and It binds as a modified substrate at its catalytic site and is non-phosphorylatable and acts as a specific inhibitor of PKC ⁇ activity.
• EXAMPLE 5 Western Blot Analysis This example describes the conditions used for Western Blot analysis of breast cancer cells.
• the protein extracts are heated for 5 minutes at 80 ° C. in Laemmli buffer (pH 7.4, 0.06M Tris, 3% SDS, 10% glycerol, 1M PMSF, ⁇ -mercaptoethanol).
• Laemmli buffer pH 7.4, 0.06M Tris, 3% SDS, 10% glycerol, 1M PMSF, ⁇ -mercaptoethanol.
• SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 10 to 20 ⁇ g of proteins migrate in a 12% polyacrylamide gel for 1 h under denaturing conditions (Migration buffer: 25 mM Tris base, 192 mM glycine, 1% SDS, pH 8.3).
• the proteins are then transferred to a nitrocellulose membrane (Schleicher & Schuell) for one hour in liquid transfer, in a transfer buffer (25mM Tris, 192mM glycine, 20% methanol, pH 8.3).
• the membranes saturated with PBS-Tween 0.1% -Triton X100 0.1% -5% skimmed milk for one hour, are contacted with the primary antibody diluted in PBS-0.1% Tween -Triton X100 0.1% -1% milk at room temperature with gentle stirring for one hour to two hours. After washing, the secondary antibody coupled to the peroxidase is incubated with the membranes.
• the revelation is by a chemiluminescence reaction using the ECL kit according to the supplier's protocol (Amersham).
• the primary antibodies used are:
• the anti-LIV21 serum which was manufactured using two synthetic peptides based on the LIV21: Pe ⁇ tideLIV21a sequence (SEQ ID No. 1 and LIV21b peptide (SEQ ID No.
• the peptides were coupled to hemocyanin before being injected into rabbits for immunization.
• the polyclonal antibody was obtained from these two peptides by immunizing two rabbits and blowing a rabbit to have a preimmune serum (to be sure that this antibody did not already exist in this rabbit).
• the anti-CDK2 rabbit polyclonal antibody (Santa-Cruz technology sc-163) diluted
• the mouse anti- ⁇ 21 monoclonal antibody (DAKO 5 M7202) diluted 1/150.
• the anti-PKC ⁇ rabbit polyclonal antibody (Santa-Cruz technology sc-214) diluted
• Rat polyclonal anti- ⁇ tubulin antibody (Serotec, MCAP77) diluted 1/500.
• the secondary antibodies used coupled to the peroxidase (Caltag) are:
• the LIV21 protein has been shown to be associated with PML bodies and, during sumoylation, LIV21 to change from a molecular weight of 50kd to 60kd. Immunoprecipitation showed co-localization of LIV21 with SUMO in the PML-SUMO / LIV21 complex. ( Figure 22)
• PML bodies In the proliferation stage, there are visualized changes in PML bodies because these PML bodies dissociate and degrade: (speekles), proteins are then available in the nucleus to ensure transcription, proliferation, immune responses and anything that requires gene transcription.
• PML has been shown to associate with SUMO and HDAC-I (histone deacetylase 1) and that its complex acts on the expression of E2F1 and PML thus acts on the stop proliferation by blocking E2F1.
• HDAC-I histone deacetylase 1
• PML thus acts on the stop proliferation by blocking E2F1.
• PML / HDAC-I complex negatively regulates the expression of E2F1.
• PML associated with Rb binds to histone deacetylases and blocks E2F1 by binding to chromatin ( Figure 2).
• PML In acute promyelocytic leukemias, PML is truncated and becomes a fusion protein with the retinoic acid receptor. This fusion protein (PMLRARalpha) is due to a chromosomal translocation 15/17. It has been reported in the literature a new treatment of this disease by combination of arsenic and retinoic acid to induce cancer cells in apoptosis. The PML protein regulates proliferation in cancers and lymphomas. The inventor has shown by immunoprecipitation the SUMO-PML association in which LIV21 is located.
• LIV21 is phosphorylated by PKC ⁇ and that TPA is a PKC inhibitor.
• MCF7 lines treated with TPA show inhibition of cancer proliferation and cell differentiation and LIV21 is translocated into the nucleus. If a PKC ⁇ -specific inhibitory peptide was used, it is the activity and not the expression of PKC ⁇ that was inhibited.
• E2F4, p130 and LIV21 After 48 hours when proliferation begins, E2F4 has a comparable location; but at 72 h, it disappears from the core (in favor of E2Fl).
• Figure 25 Results: image 43: poorly differentiated cancer; Image 58: healthy tissue from the same subject with cancer; Image 40: metastatic carcinoma 10 cm and image 17: metastatic carcinoma 3.5 cm.
• FIG. 26 is like FIG. 25 a second example of nuclear localization of LIV21 in the control and healthy tissue (No. 52) of the patient No. 7 of squamous cell carcinoma of pharynx (moderately differentiated T4N0M0).
• Figure 27 is a sample of advanced bladder cancer on cystectomy (urothelial grade III carcinoma infiltrating the chorion and muscularis) versus healthy bladder tissue of the same patient with internal control (PI): preimmune PI serum before the rabbit is immunized against LIV21 labeling nuclei with propidium iodide.
• PI internal control
• Figure 28 is a breast cancer sample for demonstrating the cyplasmic labeling of the LFV21 antibody in cancer cells. These results show that the cytoplasmic localization of LIV21 is an indicator of the aggressiveness and metastatic potential of cancer. The detection of LIV21 expression indicates the presence of invasive, aggressive and metastatic cancer cells. These results also show that the nuclear localization of LIV21 is an indicator of healthy quiescent cells of well differentiated tissues.
• EXAMPLE 8 Physical Interaction of LIV21 with the Proteins of the E2F Family Co-immunoprecipitation experiments carried out using anti-LIV21, anti-E2F1 and anti-E2F4 antibodies made it possible to demonstrate that LIV21 associates at E2F4.
• E2F1 positively controls the cell cycle by transactivating the promoter of the genes responsible for cell proliferation (DNA polymerase alpha, thymidine kinase, DHFR, etc.), whereas E2F4 is described as one of the members of the EF family that negatively controls the cell proliferation. cycle.
• E2F1 embryonic mammary tissues
• Antigen identification was made in cell lysates by immunoprecipitation.
• the analysis of the physical interaction of different proteins associated with E2F4 and E2F1 was demonstrated by co-immunoprecipitation of protein complexes.
• the study of the complex by ⁇ MACS PROTEIN to MICROBEADS (MILTENYIBIOTEC).
• MILTENYIBIOTEC MILTENYIBIOTEC
• S aureus lysates By addition of S aureus lysates, the A proteins interact with the Fc portion of the specific antibodies and the immune complexes become insoluble, thus recovering them by centrifugation. After breaking the bonds (heating) between AG AC and protein-rich membranes A, a western was made blot.
• EXAMPLE 9 Functional Interaction of LIV21 with the Proteins of the E2F Family It has been demonstrated that the blocking of the expression of the LF / 21 protein is correlated with a decrease in the expression of E2F4 and an increase in the expression of E2F1. In parallel, the functional aspect of the E2F1 increase was verified by studying the transcription of two of its target genes, DHFR and ⁇ -DNA polymerase.
• HSP27 27 kDa heat shock protein

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