EP1869209A2 - Traceability of cellular cycle anomalies targeting oncology and neurodegeneration - Google Patents

Abstract

The invention concerns a novel diagnostic and/or prognostic test for cancer. More particularly, the invention concerns the human LIV21 protein, its use as cancer diagnostic and prognostic markers. The invention also concerns methods for treating cancers and neurodegenerative diseases as well as compositions therefor.

Description

 Traceability of cell cycle abnormalities targeting oncology and neurodegeneration.

FIELD OF THE INVENTION 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.

DESCRIPTION OF THE STATE OF ART

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.

Control of the balance between cell division (mitosis), differentiation and programmed cell death (apoptosis) is fundamental during normal physiological processes, such as embryonic development, tissue regeneration and aging. Impairment of this balance can lead to major pathological conditions such as tumor formation or certain neurodegenerative diseases.

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.

For a multitude of reasons, early diagnosis remains illusory for most forms of cancer. For some forms of cancer, disease-specific markers are either unavailable or only available at an advanced stage of the disease, making diagnosis difficult. For some other forms of cancer, markers are available but are not always specific to the disease or they may be associated with its benign form. In still other cases, the techniques exist but the prohibitive cost of applying them to the general population renders them inappropriate.

For example, 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. This is not just a disease but a whole range of interrelated conditions, which in many cases appear similar to visual inspection. A diagnosis based on such an inspection is therefore subjective. To better understand this subjectivity, consider an abnormal growth of the skin. This growth can be pigmented or not pigmented. If it is non-pigmented and malignant, then it is probably a basal cell carcinoma or squamous cell carcinoma. However, the clinical development of these 2 forms of cancer is very different. Basal cell carcinoma spreads laterally over the surface of the skin without penetrating the deeper layers of the skin. Thus, although disfiguring, basal cell carcinoma rarely develops metastases and is rarely fatal. However, squamous cell carcinoma causes metastases and is often fatal. It therefore becomes important to be able to distinguish these 2 types of skin cancer. 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.

Breast cancer is one of the most common in women with colon cancer. The death rate is the highest of all cancers affecting women.

There are very few diagnostic markers that can detect breast cancer and they only have a predictive value of 20%. There are also no markers that can detect or determine the invasiveness and aggressiveness of metastatic cancer cells. In recent years, considerable progress has been made in understanding the means implemented by oncogenes and tumor suppressor genes to regulate cell proliferation and apoptosis. One of the main targets of these regulators is the family of E2F type transcription factors in the E2Fs and RB protein signaling pathway. These proteins play a central role in the control of cell division by coupling the regulation of genes required for cell cycle progression to extracellular signals (mitogens, proliferation inhibitors). It behaves like an oncogene by stimulating the proliferation of tumor cells.

Among the genes expressed, we find:

- The over-expression of the transcription factor E2F4 and the c-myc oncogene that induce the apoptosis of post-mitotic cells by accumulation of oxygenated reagents (Tanaka, 2002) - the p53 gene, belonging to the family of tumor suppressor genes , blocks the cell cycle in case of DNA damage. It has now been shown that this gene is also involved in the course of apoptosis (Oren 1994, Yonish-Rouach 1996).

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).

It would be desirable to have new diagnostic methods that would detect the presence of cancer with more specificity and that would allow the distinction between aggressive cancer cells with a tendency to metastasize compared to more localized ones that have a low probability of metastasis. A marker that can therefore reveal cell proliferation would be very useful.

SUMMARY OF THE INVENTION

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. Preferably, 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.

One aspect of the present invention is the use of 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.

Moreover, the detection of the protein kinase C epsilon (PKCε) is also interesting since it has been determined that PKCε phosphorylates the LIV21 protein to maintain it in the cytoplasm. Thus, a significant increase in PKCε is indicative of the presence of cancer cells. In addition, the LIV21 / PKCε ratio increases in the cytoplasmic fraction of cancer cells.

In addition, the detection of E2F1 and / or E2F4 proteins is of interest. Indeed, 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. Thus, a decrease in the association of LIV21 with the E2F4 protein is indicative of the presence of cancer cells. Similarly, the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells.

Accordingly, 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. Preferably, a location of said product of LIV21 gene expression in the cytoplasm is indicative of the presence of invasive and / or metastatic cancer cells.

Optionally, 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 (PKCε) 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. Moreover, at least one of the LIV21 / PKCε, 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 same is true of the detection of HDACl which has been shown to be involved in PML / SUMO / Rb / HDAC-1 complexes. More generally, 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. Thus, in a particular embodiment, 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, ρ130 of the pocket protein family. Thus, 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). In addition, 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 (Amoulzy, Institut Curie) 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. Moreover, 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.

In one embodiment, the gene expression product is detected at the protein level. Preferably, the protein is detected using a specific antibody. For example, the protein can be detected by Western blot analysis. In a preferred embodiment, it is detected by immunohistochemistry, immunocytochemistry, radiography or peroxidase labeling.

In a particular embodiment of the method comprising the detection of the expression product of the PKCε gene, a significant increase in PKCε is indicative of the presence of cancer cells. In addition, the method may also comprise the determination of the LIV21 / PKCε 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 / PKCε ratio in the cytoplasmic fraction is indicative of cancer cells.

In another particular embodiment of the method comprising the detection of the expression product of the E2F4 gene, 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. In addition, 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.

In a further embodiment of the method comprising detecting the expression product of the E2F1 gene, the presence of the E2F1 protein in the nucleus is indicative of the presence of cancer cells. In addition, 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. 50-51 kD and / or a polypeptide characterized by one of the chromatograms of Figures 3-6 and / or 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 and / or one of the peptide sequences obtained by MALDI (Figure 7) and NanoLC-ESI-MS (Figure 9). In a preferred embodiment, the polypeptide comprises the two peptide sequences SEQ ID Nos. 1 and 2. In an even more preferred embodiment, the polypeptide comprises a third peptide sequence SEQ ID No. 3 and / or a fourth peptide sequence SEQ ID. No. 4 or a sequence having 70, 80 or 90% identity therewith. Optionally, LIV21 further comprises a sequence selected from SEQ ID Nos. 5-55 or a sequence having 70, 80 or 90% identity therewith. Preferably, 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. In a particular embodiment of the 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 (PKCε) gene, the E2F1 gene and the E2F4 gene. Preferably, the detection means is an antibody specific for the protein in question. In another preferred embodiment, 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. In a preferred embodiment, the kit comprises an antibody chip comprising an antibody specific for LIV21. In a preferred embodiment, the chip further comprises an antibody specific for a protein selected from PKCε, E2F1 and E2F4. In addition, it 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. Preferably, the diagnosis is made ex vivo on samples of a patient. DESCRIPTION OF THE FIGURES 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. The calibration with respect to autolysis and trypsin digestion peaks is not excellent and needs to be reviewed. 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. TITLE: Title. Elution from X to Y Period: x experiment: y = Elution of X to Y period: x experiment: y. Load not autodetermined = Load not determined automatically.

Legend illustrated by the first page describing the ion: molecular weight 523.25 Dalton. Title: Elution of 29.15 to 29.23 corresponds to the peptide eluted between 29.19 and 29.23 minutes. This molecule by ionization gives the dicharged ion (Charge = 2 +): MH2 ²⁺ of molecular mass m / z: 523.25 Dalton hence the mass of the molecule M: 1044.51. The peptide sequence of this ion is determined by MSMS. 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 (the son ion: 86.1373) 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. Common sequences with gallus gallus, histamine variant HIS3-HUMAN, HSP60 chaperonine, arginine deiminase, pseudomonas polyribonucleotide nucleotidyltransferase, dehydrogenase.

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.

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, dehydrolipoamide dehydrogenase.

Figure 11: Alignments between histatin-3-2 variant, PATF and Q7TCL4 (Turnip mosaic virus) AAN08045.2. By MALDI analysis TOF type Brucker.

12: Alignments between Gallus Gallus (gi 50732569) and PATF and common polypeptide sequence from LIV21 by MALDI TOF analysis. Figure 13: Example of interpretation diagram of the Maldi analysis for the histatin 3- 2.donnant the sequence n ° 5 by selecting the masses: 2383.2610 (delta with 0 .005); 2539.3280 (delta: -0.02); 2511.3740 (delta at 0.02) .score: 78 and expect: 0.0046.

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 PKCε and PKCζ proteins in total extracts. Pα-tubulin expression serves as a control of the amount of total protein deposited in the wells.

Figure 19 shows the comparative expression of PKCε 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-PKCε antibodies in green, and propidium iodide staining the DNA in red. LIV21 is translocated into the nucleus by specific inhibition of PKCε. PKCε is weakly expressed at 12h in the presence of TPA. Indeed, we observe the red nuclei and little green color in the cytoplasm. On the other hand, 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 PKCε inhibitory peptide on the expression pattern of LIV21 by immunocytochemistry on control cultures or treated with 1 μM peptide, 2 μM peptide, or 25 nM TPA. The treatments last 12 hours. The cells are labeled with anti-LIV21 (green) and propidium iodide (red). It is observed that 2 μM of peptide (image named 2 μM) have the same efficacy as "25 nM of TPA 12H": the nuclei (yellow) are predominantly labeled by both propidium iodide and the anti-LIV21 antibody ( in green) on these two images whereas the control and the cells treated with only 1 μM of PKC inhibiting peptide show a red staining of the nuclei showing the absence of nuclear translocation of LIV21 via its anti-LIV21 antibody (in green).

Figure 21 shows the effect of the PKCε 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 μM).

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. and PML (Merge: yellow nuclei), whereas 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 and 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.

Figure 28 is a sample

DETAILED DESCRIPTION OF THE INVENTION

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.

The discovery of this new molecule LTV21, could have diagnostic value for the following reasons. By performing a screening of the location of LIV21 in a dozen human tumors, 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.

The presence of LIV21 has thus been observed in mammals. The expression panel of LTV21 as a function of cell state (mitotic cycles, cell rest, differentiation) 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.

Thus, 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. The characterization of this factor suggests a new pathway of negative regulation of cell proliferation, through its association with one of the members of the EF (s) family: 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.

The location observed for 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.

The characterization of this molecule, the study of the chronology and the topology of its expression also indicate that 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.

In addition, it has been shown that the location of LIV21 in the cytoplasmic compartment is regulated by PKCε. Indeed, when LIV21 is phosphorylated by PKCε, LIV21 is located in the cytoplasmic compartment. Conversely, when the phosphorylation of LIV21 by PKCε is inhibited, LIV21 is located in the nuclear compartment.

LIV21

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). It gives more than 54 peptides following digestion with the Proméga trypsin (Figure 7). The characteristics of the LIV21 protein are also described in Figures 3-13. Several particular peptides of LIV21 have been characterized, and in particular the peptide LIV21a (SEQ ID No. 1), the peptide LIV21b (SEQ ID No. 2), the peptide LIV21c (SEQ ID No. 3), the peptide LIV21d (SEQ ID No. 4) and the peptide LIV21e (SEQ ID NO: 5). The longest sequence with the homology of The strongest sequence with PATF is LIV21e peptide KFFVFALILALMLSMTGADSHAKR (SEQ ID NO: 5).

Other particular peptides of LIV21 are described below:

RTLLLPAVSRQ (SEQ ID NO: 6)

LGFMEEWDVGEIMLR (SEQ ID NO: 7)

QIMAHFSDVAEAYIEK (SEQ ID NO: 8)

FYAWMIEQAPFSSLAQEGK (SEQ ID NO: 9) NLYTEIVYTPISTPDGTLVK (SEQ ID NO: 10)

GANNNLFGLDGNVGTTVENTER (SEQ ID NO: 11)

KFQFGQSTVTLETGRI (SEQ ID NO: 12)

KGFFPLSVHYQEKT (SEQ ID NO: 13)

RTVRPLNIEVGVLPKT (SEQ ID NO: 14) RRSVQAMLPGADVFPYTIRV (SEQ ID NO: 15)

KGITEEIMEIALGQALEARL (SEQ ID NO: 16)

RAICEETKASIDIEDDGSIKI (SEQ ID NO: 17)

KVTDILKEGQEVEVLVLDVDNRG (SEQ ID NO: 18)

KMLTGVNVLADAVKA (SEQ ID NO: 19) RAAVEEGWPGGGVALIRA (SEQ ID NO: 20)

KVIIVAVDWDLSKE (SEQ ID NO: 21)

KIFSPATVFFTSIEKH (SEQ ID NO: 22)

KNVWILTGFQQGQEFPKF (SEQ ID NO: 23)

RFNLFAGGSNKA (SEQ ID NO: 24) RAYSLLGTSERT (SEQ ID NO: 25)

AMAANDTGGFVK (SEQ ID NO: 26)

ASEEGIMWER (SEQ ID NO: 27)

FDWVIGAGPGGYVAAIK (SEQ ID NO: 28)

RPVTTDLLASDSGVTIDER (SEQ ID NO: 29) FYCGWDR (SEQ ID NO: 30)

DVAQEEGK (SEQ ID NO: 31)

SGIPSELR (SEQ ID NO: 32) EAHIQMK (SEQ ID NO: 33)

EGIWIPK (SEQ ID NO: 34)

YTFDSR (SEQ ID NO: 35)

LTHEIR (SEQ ID NO: 36) LYLDK (SEQ ID NO: 37)

YGLQR (SEQ ID NO: 38)

DSIIR (SEQ ID NO: 39)

LEAICAAMIESWGYDK (SEQ ID NO: 40)

GDLWFMSHQGHK (SEQ ID NO: 41) YAFDFYEMTSR (SEQ ID NO: 42)

EVNAGTSGTFSVPR (SEQ ID NO: 43)

NQDRPYMPR (SEQ ID NO: 44)

IVSILEWDR (SEQ ID NO: 45)

APYIAETALR (SEQ ID NO: 46) NMHNLLGVK (SEQ ID NO: 47)

NLTDMSLAR (SEQ ID NO: 48)

HTTEDVNR (SEQ ID NO: 49)

KFFVFAL (SEQ ID NO: 50)

FVFALILALMLSMCG (SEQ ID NO: 51) TLQIFNIEMKSK (SEQ ID NO: 52)

KDPELWAHVLEETNTSR (SEQ ID NO: 53)

KSWEVYQGVCQK (SEQ ID NO: 54)

HTSLVGCQVIHYR (SEQ ID NO: 55)

For the purposes of the invention, 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. Preferably, the polypeptide comprises the sequences SEQ ID Nos. 1 and 2. In one embodiment preferred, 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. In a particular embodiment, it comprises a sequence selected from one of the peptide sequences obtained by MALDI (FIG. 7) and NanoLC-ESI-MS (FIG. 9). 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. Moreover, the 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. In a particular embodiment, the antibody specifically binds to a LIV21a or LIV21b peptide. In a preferred embodiment, 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. In a particular embodiment, the animal has been immunized with the peptide LIV21a and / or LIV21b. In a preferred embodiment, 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. For example, the peptides may be coupled to a carrier protein such as hemocyanin, and then injected into an animal, for example a rabbit, for immunization. Polyclonal antibodies were obtained from these two peptides by immunizing two rabbits and blowing a rabbit to have a preimmune serum.

Methods for producing monoclonal antibodies from different animal species can be found for example in Harlow et al. (Harlow 1988) or in Kohler et al. (Kohler and Milstein 1975). These methods include immunization of an animal with an antigen, followed by recovery of the spleen cells which are then fused with immortalized cells, such as myeloma cells. The resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions to isolate the individual clones. The antibodies can also be produced by selection of combinatorial immunoglobulin libraries, such as those disclosed for example in Ward et al. (Ward, Gussow et al 1989).

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. In particular, 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. Generally for such assays, the antibodies used are labeled to be detectable. Alternatively, the antibodies can be labeled indirectly.

In a preferred embodiment, 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. In particular embodiments, the cytoplasmic and / or nuclear extracts are prepared, and these extracts are contacted with the human LIV21 protein specific antibody. Diagnostic

The present invention teaches the development of the diagnostic test also for monitoring the evolution of a cell proliferation. In particular, 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. Preferably, 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.

Four of these properties can be used: its passage from the cytoplasmic cell compartment to the nuclear compartment, the property of associating with the transcription factor E2F4 to form a complex inhibiting the expression of the factor E2F1 and the capacity of LIV21 to translocate in the nucleus by specific inhibition of PKCε, the sumoylation of LIV21 when this one is nuclear and integrated in the PML bodies and its interaction with HDAC.

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.

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 cancer cells, more particularly invasive, aggressive and / or metastatic cancer cells. These results also show that 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. Preferably, 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.

Optionally, 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 (PKCε) 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. Moreover, at least one of the LIV21 / PKCε reports,

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,

HDACl, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, p07, ρ130 of the pocket protein family. It 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. In a preferred embodiment, the gene expression product is detected at the level of the protein. Preferably, the protein is detected using a specific antibody. Thus, 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.

When the product of the expression of one of the PKCε, E2F1 and E2F4 genes is to be detected, the method can use antibodies specific for the PKCε, E2F1 and E2F4 proteins, respectively. The polyclonal and monoclonal antibodies directed against PKCε, E2F1 and E2F4 are commercially available. By way of example, mention may be made, for PKCε, 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). Preferably, the antibodies are labeled directly or via a secondary antibody. Antibody labeling techniques are well known to those skilled in the art.

In a particular embodiment, 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.

In another embodiment, 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.

By way of illustration, 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. In addition, 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.

In a preferred embodiment, the detection of the product of the expression of one or more genes or the interaction between several proteins is carried out using a protein chip. Thus, 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. Such 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 (PKCε), 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.

Moreover, the 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.

In a particular embodiment, an antibody specific for LIV21, a fragment or a derivative thereof retaining the binding specificity is immobilized on the solid support. Thus, this chip provides a convenient way to measure the LIV21 expression product. Preferably, the chip comprises at least one antibody specific for a polypeptide selected from the group consisting of PKCε, RBP2, E2F4, E2F1, SUMO, HDACl, CycE / cdk2, cdkl, CREBl, p300, Rb ₅ PML ₅ plO7, pl30 of the pocket protein family, preferably PKCε, 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, ρ65, 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. In a simplified manner, 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.

The term "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.

Applicable methods for protein chip synthesis are described for example in the following patents: WO2004 / 063719, WO2005 / 016515, US2005019828, WO03018773, US2002187464, US 5,143,854, US 5,242,974, US 5,252,743, US 5,324,633, US 5,384,261, US2006035387, US2005100947, US2005233473, WO0198458, WO0172458, WO0004382, WO0004389, WO9015070, WO9210092, WO9310161, WO9512808, WO9601836, the contents of these patents being incorporated by reference in the present application. For example, 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. By "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.

In a particular embodiment of the method comprising the detection of the expression product of the PKCε gene, a significant increase in PKCε is indicative of the presence of cancer cells. More specifically, the amount of PKCε in healthy cells is compared with the amount of PKCε 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 / PKCε rate. This LIV21 / PKCε ratio increases in the cytoplasmic fraction of cancer cells compared to healthy cells.

In another particular embodiment of the method comprising the detection of the expression product of the E2F4 gene, 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.

In a further embodiment of the method comprising detecting the expression product of the E2F1 gene, 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. Thus, 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.

Indeed, 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. Preferably, the biological sample is from the tissue affected by the treated cancer. Furthermore, 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. Preferably, 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. In a preferred embodiment, 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. Optionally, 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 (PKCε) gene, the E2F1 gene and the E2F4 gene. These detection means may be antibodies specific for the protein.

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 PKCε activity in cancer cells and using HDAC inhibitors.

In another particular mode of anti-cancer therapy, it is possible to reduce the activity of PKCε in cancer cells. This decrease in activity can be achieved by decreasing the activity of the PKCε protein or by decreasing its expression. A decrease in PKCε protein activity can be achieved by administering cancer inhibitors to the cancer cells. the PKCε protein. Inhibitors of the PKCε protein are well known to those skilled in the art. A decrease in the expression of the PKCε protein can be obtained by using antisense or siRNA specific for the PKCε gene. Kits are commercially available. Moreover, 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 PKCε protein. It also relates to the use of a pharmaceutical composition comprising an inhibitor of the PKCε 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 PKCε protein, the inhibitor of the PKCε protein making it possible to reduce or abolish the cancerous phenotype of the treated cells. In a first embodiment, the inhibitor of the PKCε protein decreases the activity of the PKCε protein. In a second embodiment, the inhibitor of the PKCε protein decreases the expression of the PKCε protein. Preferably, 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.

In the context of therapy of a neurodegenerative disease, it is possible to reduce the amount of LIV21 present in the nucleus. The cells affected by the neurodegenerative disease are usually neurons, motor neurons, etc. In a preferred embodiment, the neurodegenerative disease is selected from Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS). For that, one could also disadvantage the nuclear localization of LIV21, for example by increasing the activity of PKCε in the cells affected by the neurodegenerative disease. This increase in activity can be achieved by increasing the activity of the PKCε protein or by increasing its expression. An increase in the activity of the PKCε protein can be obtained by administering to the cells affected by the neurodegenerative disease activators of the PKCε protein. Activators of the PKCε 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 PKCε protein can be obtained by using expression vectors coding for the PKCε protein and allowing it to be overexpressed in the cells affected by the neurodegenerative disease.

Thus, the present invention relates to a pharmaceutical composition comprising an activator of the PKCε protein or an expression vector encoding the PKCε protein. It also relates to the use of an activator of the PKCε protein or of an expression vector encoding the PKCε protein for the preparation of a medicament for the treatment of a neurodegenerative disease.

Screening method

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.

In a first embodiment, 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.

In a second embodiment, 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 PKCε protein. A decrease in PKCε expression indicates that the candidate compound is active to decrease or abolish cell proliferation. An increase in PKCε expression indicates that the candidate compound is active to treat or prevent a neurodegenerative disease.

In a third embodiment, 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.

In a fourth embodiment, 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. In particular, the candidate compound could be an antibody, an antisense, a ribozyme or a siRNA.

Other advantages and features of the invention will appear in the examples and figures which follow, given in a non-limiting manner.

EXAMPLES 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 μl 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.

When it changes its cell compartment and is sumoylated, it has a molecular weight of about 6OkD. When it is phosphorylated in the cytoplasm, it presents two forms differing from a few kilobases. We then observe a duplicate.

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).

Only two peptides characterize in common PATF and LIV21:

The LIV21a peptide is located between an interaction site with the Rb / p107 / ρ130 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

The peptide LIV21 c SYMSMFLLLMAISCVLAK SEQ ID No 3

The peptide LIV21d PLMIIHHLDDCPHSQALK SEQ ID No. 4

Other peptides are provided in the sequences SEQ ID NOs: 5-55. EXAMPLE 2 Study of the Nuclear Translocation of LIV21 in MCF-7 Cells The study of the sub-cellular distribution of LIV21 in various tumor lines of various origins showed an exclusively cytoplasmic localization of this protein. It has been shown by which mechanism (s) LIV21 could be translocated into the nucleus to act on the cell cycle.

The presence of putative phosphorylation sites by Protein Kinase C (PKCs) for possible homology that PATF would have with the LIV21 sequence has directed the inventor's study towards a possible implication of these proteins on its nuclear translocation. The inventor has therefore chosen to study the MCF-7 line treated with TPA, which is known to modulate PKCs.

In parallel with this work, the inventor has also studied the expression and localization of cell cycle proteins involved in the signaling pathway in which LIV21 could act.

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.

In vivo, these cells are not very metastatic and this reduced invasiveness is due to a low constitutive activity of protein kinases C (PKCs) and a low level of expression of protein kinase C alpha.

This line is used in many studies on proliferation, differentiation and apoptosis. These studies use appropriate drugs such as

TNF for induction of apoptosis, or TPA (12-O-tetradecanoyl phorbol-13- SUMOate) for the induction of differentiation and thus for the study of the exit of the cell cycle.

The effect of TPA on the MCF-7 line TPA is a known activator of PKCs. It activates the growth of normal breast cells, does not alter the proliferation of benign tumor cells of the same tissue, but it drastically inhibits the proliferation of cells of human breast tumor lines such as the MCF-7 line. It reduces the cell growth of this line by positively controlling the c-erb-2 receptor and by negatively controlling the retinoic acid receptor, both of which are expressed particularly prominently in these cells. TPA strongly and rapidly inhibits estrogen receptor (ER) expression and function and induces the time-and dose-dependent translocation of cytosolic protein kinase C (PKCs) to membranes.

On the other hand, 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.

Expression of LIV21 in MCF-7 cells

In a first step, 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. In this line, 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.

Production of purified anti-LIV21 serum

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.

The effect of TPA in MCF-7 cells

It has been described in the literature that TPA induces proliferation arrest and differentiation of tumor cells of the MCF-7 line. To validate the culture conditions, the cell growth (by counting) 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.

At the same time, the inventor has been able to observe that TPA-treated cells rapidly acquire characteristics of differentiated mammary gland cells (FIG. 14): hypertrophy, membrane profusions and the tendency to dissociate from each other. However, in the time when the cells were studied, the secretory phenotype (appearances of granules and secretory canaliculi) was not observed.

FACS analysis of the effect of TPA

These preliminary studies of the effect of TPA showed in:

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).

A maximum of S-phase cells are thus observed in the early times of the kinetics (Oh at 6h), in the G2 / M phase at 12h and finally in the G0 / G1 phase at 24h. In the end, without new addition of TPA, the cells restart in phase S at 72h.

In conclusion, these results therefore show that TPA acts rapidly on the arrest of cell proliferation and its effect on the reduction of the number of cells in phase S is optimal at 12h. After 72 hours after the single addition of TPA, the cells re-initiate the cell cycle.

The effect of TPA on the nuclear localization of LIV21 The results obtained by flow cytometry led the inventor to study in parallel the expression of LIV21 in nuclear extracts carried out after 12h, 24h, 48h, and 72h of treatment with TPA (Figure 16A). During this kinetics, a maximum of anti-LIV21 immunoreactivity was observed from 12h, which is maintained until 48h and regains its initial intensity after 72h of treatment. These data are to be compared with those obtained by FACS. The immunoreactivity of LIV21 increases significantly at 12h, a time when the number of cells in phase S is minimal. It lasts until the recovery of the cell cycle observed at 72h.

In addition, it can be seen that this immunoreactivity is detected as a single band at an apparent molecular weight of 50 kDa, whereas it is predominantly expressed as a doublet in the total extracts. To find out which form of LIV21 corresponds to this single band, a total extract and a nuclear extract were compared on the same western blot at 12h of treatment (Figure 16B). The results obtained show that the nuclear form of LIV21 corresponds to the lower band of the doublet. These data suggest that the LIV21 protein may be in a different phosphorylation state depending on the cell compartment.

The results of the immunocytochemical study carried out using the anti-LIV21 antibody show that the nuclear translocation of LIV21 is maximal at 12h (FIG. 17) and the location of LIV21 is predominantly cytoplasmic at 72 hours, which is in agreement with observations in western blot. However, it is interesting to note that the expression of LIV21 already starts from Ih of treatment in some cells, because they are not synchronous.

All these observations show that the nuclear translocation of LIV21 is concomitant with the decrease in the number of cells in phase S. EXAMPLE 3 Study of the Influence of PKCs on the Nuclear Translocation of LIV21 Effect of TPA on the Expression of PKCε

Western blot study: Since the LIV21 protein sequence has putative PKC phosphorylation sites, two of which are specific for PKCε (, the inventor has tested the variation in the expression of this PKC as a function of the duration of the Treatment with TPA It has been observed that TPA acts very rapidly on the expression of PKCe ₅₁ which decreases as early as 30 min (Figure 18) .The expression of PKCzeta (PKCζ) is used as an internal control because it is not sensitive to TPA.

Immunocytochemistry: In parallel, immunofluorescence experiments on treated or non-treated cultures made it possible to demonstrate this decrease in PKCε concomitant with the nuclear translocation of LIV21 (FIG. 19). It can be observed that PKCε disappears from the cytoplasm when the cells are treated with TPA and that LIV21 is detected in the nucleus.

To conclude, it is interesting to note that PKCε 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 PKCε on the Nuclear Translocation of LIV21 Using a Peptide Inhibiting Function and the Translocation of PKCε In order to determine the specific action of PKCε on the translocation of LIV21, 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.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 PKCε activity.

The effect of selective inhibition of PKCε activity on LIV21 nuclear translocation was studied by immunocytochemistry. These experiments were carried out on cultures treated or not treated for 12h, with 25 nM TPA or with the two-fold peptide. different concentrations, 1 and 2 μM (Figure 20). The peptide used at a concentration of 2 μM has an identical effect to that of TPA on the nuclear translocation of LIV21.

These results were confirmed by cell fractionation experiments on cultures treated with the PKCε inhibitory peptide at 2 μM compared with cultures treated with TPA (FIG. 21). The same expression pattern of LIV21 was observed as a doublet in the cytoplasm and a single band in the nuclear moiety.

The specific inhibition of PKCε induces a nuclear translocation of LIV21, suggesting that LIV21 could be the target of PKCε, which would maintain it in the cytoplasm in a phosphorylated form.

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, β-mercaptoethanol). The migration is carried out in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). 10 to 20 μg 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: PeρtideLIV21a sequence (SEQ ID No. 1 and LIV21b peptide (SEQ ID

No 2). 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

1/200. The mouse anti-ρ21 monoclonal antibody (DAKO ₅ M7202) diluted 1/150.

Mouse anti-p27 monoclonal antibody (Santa-Cruz technology sc-1641) diluted

1/100.

The anti-PKCε rabbit polyclonal antibody (Santa-Cruz technology sc-214) diluted

1/200. The polyclonal rabbit anti-PKCδ antibody (Santa-Cruz technology sc-216) diluted

1/200.

Rat polyclonal anti-α tubulin antibody (Serotec, MCAP77) diluted 1/500.

The secondary antibodies used coupled to the peroxidase (Caltag) are:

Anti-rabbit IgG (H + L) goat F (ab ') 2 antibody diluted 1/2000. Anti-mouse IgG (H + L) goat F (ab ') 2 antibody diluted 1/2000.

EXAMPLE 6

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)

Antitumor role of 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. Thus the PML / HDAC-I complex negatively regulates the expression of E2F1. PML associated with Rb (pi 30) binds to histone deacetylases and blocks E2F1 by binding to chromatin (Figure 2).

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.

In the previous examples, it has been shown that LIV21 is phosphorylated by PKCε 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 PKCε-specific inhibitory peptide was used, it is the activity and not the expression of PKCε that was inhibited.

During this treatment with TPA (25 nM), when we studied E2F4, pl30 and LIV21

(green fluorescence) in labeled nuclei (DNA) with propidium iodide

(red fluorescence) (Figure 23 and 24), we observed:

After 12 hours, intranuclear green fluorescence signals of the same pattern

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).

By observing the co-localization by double labeling of PML and LIV21 at 24 h of treatment with TPA (see merge: yellow fluorescence), it was observed that they are colocalized in the nuclei. At 48h, co-localization is also observed between LIV21 and SUMO (see Figure 23. The hypothesis is that SUMO, which binds to LIV21, actually addresses LIV21 in PML bodies and LIV21 is involved in complex PML / SUMO / Rb / HDAC-1. Two different approaches have been made to demonstrate that LIV21 is physically associated with PML and SUMO in nuclear bodies, by immunoprecipitation (Figure 22) and by co-localization in immunocytochemistry (Figure 23 and 24) (Rb, pl30 and plO7 are proteins pockets (pocket proteins) that have the same attachment site). Rb proteins repress cell growth (Fabbro, Regazzi R Bioch BiophysResComm 1986 Feb 2; 135 (1): 65-73).

When you add only one time the TPA, its action is exhausted after 72 hours, the

: proliferation resumes and at that moment, the PML bodies dissociate, get excited and. become speckles thus leaving the proteins to ensure transcription, proliferation etc. LIV21 is no longer located in the nucleus because it is then rephosphorylated by PKCε and thus remains localized in the cytoplasm.

EXAMPLE 7 Study of the Expression of LIV21 in Breast Cancer and Skin Cancer Biopsies

To determine whether the previously obtained observations are applicable to human tissues, a large number of skin cancer biopsies obtained from patients were studied by immunohistochemistry reaction with antibodies specific for LIV21. The immunohistochemical determination of the expression of the LIV21 protein involved 60 biopsies of patients (9 patients having a biopsy of normal tissue versus a biopsy of cancerous tissue), the other biopsies corresponding to more or less advanced cancers and some metastatic ones. . (See Blade superbiochips Laboratories, Seoul, Korea). In addition some paraffinic blades of patients with bladder cancer, breast have also been studied.

Protocol of analysis by Immunocytochemistry:

Deparaffinize the blades

Rehydrate the tissues

Saturate nonspecific sites and permeabilize the membranes. Add the antibody in a humid chamber. Reveal the antibodies. Deparaffinize the slides under a hood

Two successive baths of toluene (Prolabo rectapur) 2x30 'or 2x20min; then dehydrate the tissues of the rectal alcohol 100% 15 min; then 95% ethanol rectapur for 10 min; then 70% rectapur for another 10 minutes. Thaw the antibody at the same time.

Rehydrate the tissues

Gently in PBS supplemented with 10% fetal calf serum and 0.1% Triton.

Saturate non-specific sites (eg with ovalbumin) and permeabilize membranes. Rehydrate for one hour.

Place one ml of this "PBS" per cut to cover the blade without drying at any time (when it comes to the blade with cells and not tissue, half an hour is enough).

Put the glass and the stainless steel lid and the water below to create a humid chamber.

Add the antibody in a humid chamber.

Dilute the rabbit serum in 200 ml in 4 ml of triton PBS to continue permeabilizing the membranes and FCS.

Put ImI on each slide and avoid light and evaporation. Leave all night or at least three hours.

Then rinse with normal PBS IX PH 7, wash twice for 5 to 10 minutes so that no trace remains of the first antibody.

While preparing the green Alexa 488 probe (cold at 4 ° protected from light) at

250 ^th so 10 μl in 2.5 ml of PBS with always 10% FCS and 0.1% Triton, Rest the slides on the plate. Cover the cup with 2.5ml to keep a humid chamber for one hour then wash with PBS IX PH 7.

Washing with propidium iodide 0.5 microgram per microliter diluted to 20 microgram per ml and then again at the ^50th but this time diluted in PBS alone IX (50 microliter per 2.5 ml PBS). Drain by removing them from the PBS and then 2.5 ml of propidium iodide distributed over the four slides for one minute and then two rinsing with simple PBS. Slide the slides to the Moviol before reading. The totality of the results is summarized in Table 1, below.

TABLE 1 Expression of the LIV21 protein determined by immunohistochemistry in 50 water cancer bio-sies and 9 healthy tissue bio-sies.

For example :

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.

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.

Members of the E2F family are transcription factors whose role has been widely described in the literature as key molecules in positive or negative control of the cell cycle (Slansky JE and Farnham PJ 1996, Helin K 1998 and Yamasaki L 1998), by their association with the pRb protein (WuCL, Zukerberg LR, Lees JA 1995) or pocket proteins. 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. In addition, strong expression of E2F1 in embryonic mammary tissues (Espanel X, Gillet G 1998) has been shown, whereas it is no longer expressed in postmitotic breast tissues in favor of a large increase expression of E2F4 (Kastner A Brown G 1998).

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 μ MACS PROTEIN to MICROBEADS (MILTENYIBIOTEC). 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. These results suggest that the LIV21 / E2F4 complex appears to play an important role in establishing quiescence of cells.

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 α-DNA polymerase.

In conclusion, these results suggest that the LIV21 / E2F4 complex acts as an inhibitory complex of E2F1 gene expression. This complex could correspond to a new checkpoint in the cell proliferation arrest.

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Claims

A method for detecting cancer cells in a biological sample of a patient comprising detecting the product of LIV21 gene expression in the nucleus and / or cytoplasm of cells in a sample of cells in the biological sample said patient, a location of said product of LIV21 gene expression in the cytoplasm being indicative of the presence of cancer cells and a location of said product of expression of LIV21 gene in the nucleus being indicative of the presence of non-cancerous cells .

2. Method according to claim 1, wherein a location of said product of the expression of LIV21 gene in the cytoplasm is indicative of the presence of invasive and / or metastatic cancer cells.

The method according to claim 1 or 2, further comprising detecting the product of expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKCε) gene, the E2F1 gene and the E2F4 gene.

4. The method according to claim 3, wherein at least one of the ratios LIV21 / PKCε, LIV21 / E2F4 and LTV21 / E2F1 is determined.

The method according to any one of the preceding claims, further comprising detecting the product of expression of at least one gene selected from the group consisting of RBP2, E2F4, E2F1, SUMO, HDAC1, cycE / cdk2, cdk1. , CREB1, ρ300, Rb, PML, p107, p130 of the pocket protein family. 6. Method according to any one of the preceding claims, further comprising detecting the product of the expression of at least one gene selected from NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, and CAF1.

7- Method according to any one of the preceding claims, wherein the gene expression product is detected at the level of the protein. The method of claim 7, wherein the protein is detected using a specific antibody.

The method according to claim 8, wherein the protein is detected by Western blot analysis, immunohistochemistry, immunocytochemistry, immunoradiography or peroxidase labeling.

10- Method according to any of the preceding claims, wherein the detection of gene expression products is made using a protein chip.

The method according to any one of claims 3-10, wherein a significant increase in PKCε is indicative of the presence of cancer cells.

12- Method according to any one of claims 3-10, wherein the combination of LIV21 with the E2F4 protein is detected, a decrease in this association being indicative of the presence of cancer cells.

13- Method according to any one of the preceding claims, for the detection of metastasized cancer, therapeutic monitoring and / or treatment recurrence.

14- Method according to any one of the preceding claims, in which the product of the expression of the LIV21 gene is a polypeptide having an apparent molecular weight of approximately 50-51 kD by Western Blot analysis and of approximately 60 kD when it is sumoylated and / or a polypeptide having an isoelectric point of 5.6 in its 50-51 kD form and / or a polypeptide characterized by one of the chromatograms of Figures 3-6 and / or a polypeptide comprising a selected peptide sequence from SEQ ID Nos 1-55 or a sequence having 70, 80 or 90% identity therewith. The method according to claim 8, wherein the antibody specifically binds to a polypeptide comprising a peptide sequence selected from SEQ ID Nos. 1-55, preferably from SEQ ID NOs: 1-5.

16. The method according to claim 15, wherein the antibody is an anti-LIV21 serum manufactured by immunizing an animal with a polypeptide comprising a peptide sequence selected from SEQ ID Nos. 1-55, preferably from SEQ ID NOs: 1-5. .

17- An isolated human polypeptide characterized in that the polypeptide has an apparent molecular weight of about 50-51 kD by Western Blot analysis and about 60 kD when it is sumoyled and / or has an isoelectric point of 5.6 in its 50-51 kD form and / or is characterized by one of the chromatograms of Figures 3-6 and / or comprises 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.

An antibody which specifically binds to a polypeptide according to claim 17.

An anti-LIV21 serum manufactured by immunizing an animal with the polypeptide of claim 17.

A kit for detecting cancer cells in a biological sample of a patient comprising one or more members selected from the group consisting of an antibody according to claim 18 and a serum according to claim 19.

The kit according to claim 20, further comprising means for detecting the product of the expression of a gene selected from the group consisting of the protein kinase C epsilon (PKCε) gene, the E2F1 gene and the E2F4 gene. .

The kit of claim 20 or 21, further comprising means for detecting the product of expression of a gene selected from the group consisting of RBP2, E2F4, E2F1, SUMO, HDAC1, cycE / cdk2, cdk1, CREB1, p300, Rb, PML, p107, p130 of the pocket protein family.

The kit of claim 20 to 22, further comprising means for detecting the product of expression of a gene selected from the group consisting of NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, and CAF1.

24- Method according to one of claims 1 to 16, wherein the cancer is selected from cancers of the breast, bladder, ovary, lung, skin, prostate, colon, liver , sarcoma, leukemia and glioblastoma.

Use of an antibody according to claim 18, or a serum according to claim 19 for the diagnosis of cancer.

26- A diagnostic chip comprising a means for detecting the product of the expression of the LIV21 gene.

27- The diagnostic chip according to claim 26, comprising means for 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, p107, pi of the pocket protein family.

The diagnostic chip of claim 26 or 27, comprising means for detecting the product of the expression of at least one gene selected from the group consisting of NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, and CAF1. .

29- A pharmaceutical composition comprising an inhibitor of the PKCε protein.

30- Use of a composition according to claim 29 for the preparation of a medicament for the treatment of cancer. 31- A pharmaceutical composition comprising an activator of the PKCε protein or an expression vector encoding the PKCε protein.

32- Use of a composition according to claim 31 for the preparation of a medicament for the treatment of a neurodegenerative disease.