FR2950076A1 - New human LIV21 complex sequences comprising specified nucleotide sequences, small interfering RNA sequence derived from RNA sequences and protein fraction, are protein kinase C epsilon inhibitors useful e.g. to diagnose cancer - Google Patents

Abstract

Human LIV21 complex sequences (I) comprising nucleotide sequence of SEQ ID NO: 171-175, small interfering (si)RNA sequence derived from RNA sequences of SEQ ID NO: 120 and SEQ ID NO 121, and SEQ ID NO: 171-175; and a protein fraction comprising at least SEQ ID NO: 1 and 181 or a sequence having 90%, preferably 70% identity with the SEQ ID NO: 1 and 181 and SEQ ID NO: 183 or a sequence having 70, 80 or 90% identity with the SEQ ID NO: 183, are new, where the sequences are not defined. An independent claim is included for a method for detecting human LIV21 complex comprising implementation of at least one specific probe of at least one LIV21 complex sequence. ACTIVITY : Cytostatic; Neuroprotective; Nootropic. MECHANISM OF ACTION : Protein kinase C epsilon inhibitor.

Description

Pharmaco diagnostic test targeting neuro oncology and Alzheimer's disease.

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 neuro oncology and for neurodegenerative diseases including Alzheimer's disease. More particularly, it relates to diagnostic and / or therapeutic tests in oncology and neurodegeneration. Molecular targeting with peptide vectors or siRNAs opens the door to a new understanding of the interdependence between diagnostic and therapeutic tools that now go hand in hand and open the way to pharmacodiagnostics. The understanding of equilibrium between overexpression and under expression of genes according to their localization in a cell compartment or another function of the proliferative state makes it possible to open perspectives of finer differential analysis and to adapt a targeted combination therapy. using siRNA and other biomolecules by molecular targeting. The inventor demonstrates the mechanisms of molecular interactions involving divalent cations and their roles in the formation of lesions due to Tau proteins and amyloid plaques. It also highlights the interest of a multi therapy supplemented with therapeutic adjuvants to slow the formation of these lesions in Alzheimer's disease.

DESCRIPTION OF THE STATE OF THE 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 disease, Huntington's, Parkinson's disease etc. On the other hand, the cancerization process corresponds to a blockage of apoptosis leading to an uncontrolled multiplication of cells. The link between these two 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.

In recent years, considerable progress has been made in understanding the means used 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. Among the genes expressed are: 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 set of markers that can therefore reveal cell proliferation would be very useful. The current therapeutic revolution makes it possible to find more targeted means of treatment with greater efficiency if one works with biomolecules having a smaller number of receptors, molecular targeting devices could allow a better efficiency as for example already we can see it with the RNA interference used to allow silencing of a molecule with a specific role.

DESCRIPTION OF THE STATE OF THE 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 Huntington diseases, Parkinson etc ... In contrast, the process of cancerization is 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 critical size. 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 is still 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. These cancers are of interest to be studied and diagnosed by biochip including the biomarkers of the major pathways of regulation to allow an adjustment of the combination therapy according to the results of under expression and overexpression of the Liv21 complex genes. Thus treatment with molecular targeting type siRNA or monoclonal antibodies combined or not with therapeutic therapeutic assets is of greater efficiency.

Neuroblasts are neuronal precursors derived from the division of neuroepithelial cells. These are immature embryonic neurons that can still divide unlike mature neurons that can not enter into mitosis. Neuroblastoma, also called sympathoblastoma, is a malignant tumor developed at the expense of neural crest cells, which give rise to the sympathetic nervous system. It is the most common solid tumor in children (8 to 10% of cancers under 15 years of age) and in particular the very young child (average age 4 years with 90% who have less than 5 years years). Its incidence is 1 to 3 cases per 100,000 children aged 0 to 14 years. There are no recognized causes or factors that promote the occurrence of neuroblastoma. Neuroblastoma is discovered from symptoms due to the primary tumor (tumor mass, in particular abdominal, or compression of a nearby organ, such as the spinal cord), to metastases or endocrine secretion (alteration of the condition). general, diarrhea, high blood pressure ...). Neuroblastoma can develop from any part of the sympathetic nervous system, most often at the abdominal level. At the time of diagnosis, the tumor can be localized at the level of a single organ, at the local or regional level, or be disseminated from the outset. The most common metastatic sites are bone, bone marrow, liver and skin. Thus, most localized tumors have an excellent prognosis. It is the same for those children under one year, regardless of the stage of the tumor. Some of these tumors even regress spontaneously. On the contrary, about 60% of children over one year of age have metastatic neuroblastoma, which has a poor prognosis. The variety of the clinical presentation is related to the expression of certain biological and molecular markers (DNA ploidy, amplification of the n-myc oncogene, expression of Trk receptors, loss of chromosome 1p, excess of 17q ...). Thus, diffuse tumors occurring in children over the age of one and overexpressing the myc oncogene are often chemoresistant and have a poor prognosis (Berthold et al., 1990, Schweigerer et al., 1990). Similarly, the expression of the TrkB and BDNF receptor allows an autocrine survival system of neuroblastic cells and induces neuritic growth. It is also associated with amplification of N-MYC and therefore poor prognosis (Nakagawara et al., 1994). In contrast, the expression of TrkA receptors, which induces neuroblastoma differentiation (Borrello et al., 1993, Eggert et al., 2000), and TrkC (Yamashiro et al., 1996) is rather associated with a good prognosis. Thus, it is common to say that the expression of the TrkA receptor is inversely correlated with the amplification of the n-myc oncogene. Glioblastomas During development, glial precursors give rise to astrocytes, oligodendrocytes, microglial cells, choroidal cells and ependymal cells in the CNS, and to Schwann cells in the peripheral nervous system. Thus, glial cells play a key role in differentiation (Lemke, 2001) and neuron survival (Bar, 2000). These cells ensure the nutrition of neurons, manage inter-neuronal connections, regulate neurotransmitters. Glioblastomas are the most undifferentiated astrocytic malignancies (grade IV according to the World Health Organization's classification) and are most often found in the cerebral hemispheres. In adults, they are the most common brain tumors (20% of all intracranial tumors) with an incidence of about 3 new cases per year and per 100,000 inhabitants, or about 2,400 new cases per year in France . They occur at any age but in 70% of cases between 45 and 70 years old.

Glioblastomas form soft masses, rich in blood vessels, 3 to 10 cm in diameter, vinous in color, heterogeneous with active compact zones and extensive areas of necrosis, interspersed with thrombosed vessels and infiltrating brain tissue. However, these tumors are almost never associated with the appearance of metastases. They surround themselves with edema that increases the suffering of the brain. Typically, they are expressed by signs of intracranial hypertension that is often associated with behavioral changes, seizures, focal neurological deficits. This tumor evolves rapidly, in 2-3 months, and even after surgery, 10 radiotherapy then chemotherapy, its prognosis remains dark unless the glioblastoma comes from the tumor can be localized at the level of a single organ, at the local or regional level, or be disseminated from the outset. The most common metastatic sites are bone, bone marrow, liver and skin. Thus, most localized tumors have an excellent prognosis. It is the same for those children under one year, regardless of the stage of the tumor. Some of these tumors even regress spontaneously. On the contrary, about 60% of children over one year of age have metastatic neuroblastoma, which has a poor prognosis. The variety of the clinical presentation is related to the expression of certain biological and molecular markers (DNA ploidy, amplification of the n-myc oncogene, expression of Trk receptors, loss of chromosome 1p, excess of 17q, etc.). . Thus, diffuse tumors occurring in children over 1 year of age and overexpressing the myc oncogene are often chemoresistant and have a poor prognosis (Berthold et al., 1990, Schweigerer et al., 1990). Likewise, expression of the TrkB and BDNF receptor permits an autocrine survival system of neuroblastic cells and induces neuritic growth. It is also associated with amplification of N-MYC and therefore poor prognosis (Nakagawara et al., 1994). In contrast, the expression of TrkA receptors, which induces neuroblastoma differentiation (Borrello et al., 1993, Eggert et al., 2000), and TrkC (Yamashiro et al., 1996) is rather associated with a good prognosis.

Thus, it is common to say that the expression of the TrkA receptor is inversely correlated with the amplification of the n-myc oncogene. TNF-R superfamily receptors have been studied in neuroblastic cells. Thus, the Fas receptor has been found in certain neuroblastic cells (Gross et al., 2001), cells that can be (Barthlen et al., 1999, Riffkin et al., 2001) or not (Bian et al. al., 2004) resistant to Fas-induced apoptosis. The sensitivity of neuroblastic cells does not depend solely on the expression of the Fas receptor but on the presence or absence of caspase-8 in the cells (Kisenge et al., 2003). TRAIL receptors are also expressed in neuroblastic cells. Neuroblastoma is one of the most common in children. The death rate is the highest of all cancers affecting children.

There are very few diagnostic markers that can detect neuroblastomas. There are also no markers that can detect or determine the invasiveness and aggressiveness of metastatic cancer cells or that allow therapeutic monitoring.

In recent years, considerable progress has been made in understanding the means used 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 (E2F1, E2F2, E2F3, E2F4, etc.) 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. Myc N and Aurora kinase A are also oncogenes involved in the proliferation of neuroblastoma. E2F3 modulates the expression of Aurora mRNA during the cell cycle. In addition, some data suggest that E2F transcription factors are critical for the total activation and repression of myc N in neuroblastomas. The combination of Myc N and RTPCR survivin assays is correlated with the clinical stage of neuroblastoma. Among the genes expressed are: - The over-expression of the transcription factor E2F4 and the oncogene c-myc which induce the apoptosis of post-mitotic cells by accumulation of oxygenated reagent (Tanaka, 2002) and the N Myc which is amplified in 35% of cases of neuroblastoma, ALK gene, DDX1 and CRABP II too. HMGA1 and survivin too. Suppression of HMGA1 by RNA interference reduces cellular proliferation of neuroblastoma. N myc induces the expression of FAK. N myc clown regulates mRNA expression of many genes involved in cell architecture. the p53 gene, belonging to the family of tumor suppressor genes, blocks the cell cycle in the event 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). A Zinc Finger transcription factor can interrupt the activity of cyclin D1 and block the cycle.

Evidence suggests that E2F transcription factors are critical for total activation and repression of MYCN in neuroblastomas. - chk1 and 2, crb2, p21 and other oncogenes and cytokines such as TNF alpha etc.

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. The work of Professor Jean Louis Mendel on glial markers and in particular on GFAP and NF70 have advanced the diagnosis, the expression of synemine in glial tumors is an important research path as is the study of mutations of the Ras / MAPK pathway. The deletion of chromosome 1 in the 1p36 region and the amplification of the MYC N gene signify the state of proliferation, a prognosis of neuroblastoma and an unfavorable histology. In 35% of cases MYC N is amplified, in 58% of cases it is chromosome 17 in q23 and in 35% of cases chromosome 1 in p36 is deleted. The LIV21 complex will be studied by RT PCR and by biochip and its cytoplasmic markers of interest and the membrane protein of the Liv 21 complex as well. 21 additional markers and new sequences of the LIV21 complex compared to the first and second patents filed will allow a significant improvement and improvement of the pharmaco diagnostic tests that we propose. It is thanks to this improvement that the therapeutic adjustment in HAART will allow a greater effectiveness of treatment and also it is thanks to this improvement that the diagnosis of glial tumors will be made more precisely with a more thorough knowledge on the prognosis, the grade and evolution of these tumors. Another improvement has made it possible to have a greater precision in the study of the expression levels of the various genes important for the diagnosis of glial tumor, it is the development of a standardization of the tissue samples as cerebrospinal fluid anointing. Thus the comparisons of expression profiles are more reliable and undergo fewer fluctuations due to observation bias. All of these new parameters that do not increase the number of variables but reduce background noise greatly improve the diagnosis and treatment of glial tumors.

SUMMARY OF THE INVENTION The present invention relates to novel polypeptide, ribonucleotide and nucleotide sequences to be incorporated into a new cell cycle reinduction screening test targeting oncology and the use of some of these same functionalized sequences as adjuvant therapy by molecular targeting. This is a major improvement without which the diagnosis and the pharmaco-diagnostic test could only be partial. The consequence would be a less effective treatment and less wide application depending on the heterogeneity of cancers. It is therefore a diagnostic test and a prognostic test for various brain cancers. More particularly, the invention relates to the use of genes or proteins of the LIV21 complex as well as their derivatives as therapeutic tools and as diagnostic and prognostic markers for cancers. The invention therefore relates to the detection of the LIV21 gene or protein with a kit comprising antibodies or probes specific for LIV21, but also the invention is to use all of the markers of proliferations and transcription factors which play a role. role in the process of cancerization and neurodegeneration for some. Thus the invention lies in the use of RT PCR and quantitative PCR (Q PCR) combined with the manufacture of diagnostic DNA, protein and antibody chips comprising the known antibodies of the different proteins of the associated protein complexes according to the phases. from the cell cycle to the LIV21 complex, that is to say without restriction to those: peptides and antibodies of RBP2, E2F4, E2F1, SUMO, HDAC1, crb2, Int2, cmd2, cycE / cdk2, cdk1, CREB1 and p300, Rb , p107, p130 of the protein pocket family. But also NFkB, cdc2A, mdm2, p21, p53, p65, BRCA1, TNFalpha, TGF beta. The new sequences are the polynucleotide and polypeptide sequences of Liv2IF, Liv 21H, etc. (additional list: SEQ Nos. 171 to 185) and the corresponding gene, protein and antibody sequences newly integrated into the pharmaco-diagnostic biochips are: Myc N, ALK, HMGA1, DDX1, GFAP, NF70, AD7cNTP, FAB3, Serin C2, Synemine, PDX1, HDAC6, TPX2, DKK1, DKK3, HUD, 1D2, SKP2, TP53INP1, VEGF, NLRR1, PAX3-FKHR, NDP kinase A, Bora ?, Aurora A, Survivine Protein chips will be used to study protein interactions and post-translational modifications, more particularly the phosphorylations and methylations of certain proteins, which signify a characteristic state of the diseased cell different from the protein interactions. of the metabolism of the healthy cell. The state of expression and silencing of certain genes being different. The level of expression of each biomolecule must be observed separately in each cell compartment in order to be able, in a second time, to be regulated (under expressed or expressed by an adjuvant treatment such as biomolecule or siRNA or vectorized peptides. to study the sub expression or overexpression of genes and the ratio between genes and between proteins of the Liv21 complex and its associated partners in nuclear and otherwise cytoplasmic or membrane cell extracts, the analysis of interactions within metabolic complexes is a key diagnosis and also by studying the functional areas.

A first objective of the present invention is to demonstrate a method for detecting and prognosing cancer and its metastatic potential that makes it possible to adjust a targeted combination therapy. Preferably, the cancer is selected from cerebral cancers, and more particularly glioblastoma, neuroblastoma, without being limited thereto.

One aspect of the present invention is the use of the novel LIV21 complex sequences as a cancer prognostic indicator and in its therapeutic monitoring. Indeed, when LIV21F is located in the cytoplasm, the cancer cells in the tissues are aggressive. Conversely, when the product of the expression of the LIV21F 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 LIV21 complex is defined by the extract of proteins and peptides studied in mass spectrometry type Maldi and ESI MSMS or Maldi Tof Tof. Which extract was obtained by the attachment of the Liv2l complex to one of these polyclonal antibodies LIV21 described in the patent (PCT / FR2006 / 000510). The Liv2l complex is also defined by its overall mass spectrometry profile (FIG. 5) and the number and the molecular weight of the protein extract bands obtained on the acrylamide gels of FIGS. 1A and 1B, a function of the temperature at which the submitted sample and described migration conditions. Indeed, when, for example, the LIV21F peptide is located in the cytoplasm and is revealed directly by in situ hybridization, for example, or by biochip analysis, its highest expression in the cytoplasm, the cancer cells in the tissues are aggressive. Conversely, when the product of the expression of the LIV21F gene or the expression of a peptide such as LIV21F 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. This observation associated with the study of expression, phosphorylation and localization of the other factors of the LIV21 complex and of these interaction partners. The efficacy of a cancer treatment can also be followed by the traceability of these new sequences. of these LIV21F and LIV21K proteins, its LIV21 complex, its derivatives and ratios with associated proteins. But also by diagmicroarray and sensorchip containing, among other things, this protein and its associated Liv21 complex (FIG. 20 and FIG. 21)

Moreover, the detection of the protein kinase C epsilon (PKCs) is also interesting since it has been determined that PKCs phosphorylates the LIV21 protein to maintain it in the cytoplasm. Thus, a significant increase in PKCs is indicative of the presence of cancer cells. In addition, the LIV21 / PKCs ratio increases in the cytoplasmic fraction of cancer cells. The same is true of the detection of HDAC1 which has been shown as LIV21 as involved in PML / SUMO / Rb / HDAC-1 complexes. More generally, the HDAC family plays a key role in regulating the expression of genes, when they are overexpressed they lead to 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 which contains LIV21.30 In addition the detection of E2F1, E2F2, E2F3 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 of cancer cells in a biological tissue sample (for example, breast, ovary, endometrium, bladder, melanoma, prostate, glioblastoma, etc.) of patients, this method comprising detecting LIV21 gene expression products in the nucleus compared to these same products in the cytoplasm and cell membranes in the biological tissue sample of said patient, which method comprises detecting the product of the expression of the LIV21F gene in the nucleus and / or the cytoplasm of the cells in the tissue biological sample of said patient, a location of said LIV21F gene expression product in the cytoplasm being indicative of the presence of cancer cells and a localization said product of LIV21F gene expression in the nucleus being indicative of the presence of non-cancerous cells. Preferably, a localization of said product of the expression of the LIV21F gene in the cytoplasm is indicative of the presence of invasive and / or metastatic cancer cells, the locations of the different expression products of the LIV21 complex genes and its associated partners. described in the examples of biochips indicate or not the presence of cancer cells.

Optionally, the method according to the present invention further comprises detecting the product of expression of at least one gene selected from the group consisting of the protein kinase C epsilon (PKCE) gene, the E2F1 gene and the gene. E2F4. The method may include the detection of the product of the expression of two of these genes or three genes. On the other hand, at least one of the LIV21 / PKCc, 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 separately preferably. Preferably, these ratios are determined in the nucleus. Preferably, these ratios are compared to those obtained in a healthy cell. The level of expression of each enzyme or polypeptide of the SUMO / Rb / HDAC complex or for certain cell types of the PML / SUMO / Rb / HDAC complex is an additional indicator of the proliferation state of the cell. These expression or silencing ratios can be detected via the level of expression or inhibition of the proteins themselves in microarrays (biochips) made according to the conventional methods described (Lubman David M, Qiao Tiecheng Alex, Mathew ABY J etc. ..) or new hybridization automation tools such as US2004152212 and Yu Xinglong US 2005019828 and novel polypeptides-hanging supports (US 2008 213130 and US 2005/0157445 or US 2006 17 09 25 or WO 2005 016515, Klages Claus Peter and example figure 20). Before redescribing the principle of these biochips which are well known to those skilled in the art, the following definitions will be given again: The biological sample can be in particular a sample of blood, serum, saliva, tissue, tumor, marrow bone, circulating cells of the patient. This biological sample is available by any type of sampling known to those skilled in the art. For the purposes of the present invention, the term "biological material" means any material making it possible to detect the expression of a target gene. The biological material may comprise, in particular, proteins, or nucleic acids such as in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). The nucleic acid may in particular be an RNA (ribonucleic acid). According to a preferred embodiment of the invention, the biological material comprises nucleic acids, preferentially RNAs, and even more preferably total RNAs. Total RNAs include transfer RNAs, messenger RNAs (mRNAs), such as mRNAs transcribed from the target gene, but also transcribed from any other gene and ribosomal RNAs. This biological material comprises material specific for a target gene, such as, in particular, the transcribed mRNAs of the target gene or the proteins derived from these mRNAs, but may also comprise non-specific material of a target gene, such as in particular the mRNAs transcribed from the target gene. a gene other than the target gene, the tRNAs, the R RNAs originating from other genes than the target gene and preferably the extracts to be studied will be analyzed when it comes to cell cultures directly on fresh cultures with or without prior treatment have undergone an extraction protocol separating the cellular compartments. This type of kit known to those skilled in the art allows by a subtle buffer set to extract only the membranes or only that which is contained in the cytoplasm or the nucleus or the cytoskeleton and that in a differential manner. The Proteo extract kit (ref 539790) from calbiochem can be used for example.

The other aspect of the present invention is the use of the above-mentioned genes and proteins as markers of the invasiveness and metastatic aggressiveness of cancer cancer cells in neurology. Indeed, successive pharmaco-diagnostic tests in the follow-up of a treatment will make it possible to observe, by comparison over time, the variations of the expression levels of the genes or of their silencing and thus to better evaluate the effectiveness of the treatments, to readjust these treatments in the context of HAART adapted so that the physiological balance of the different products of the genes involved in these metabolic complexes are maintained. . The plasticity of these equilibria justifies the use of diagnostic biochips and for therapeutic monitoring with the most relevant genes of the metabolic complexes involved in the physiology of anarchic proliferation in the case of breast cancer, which is very heterogeneous. So each individual or each phenotypic subgroup of individuals will show a profile of subexpressions and expressions of the genes involved in the metabolic complexes involved.

In one embodiment, the gene expression product is detected at the level of the mRNA. The mRNA can be detected by RT-PCR analysis (see examples attached). It can also be detected by Northern blot analysis or by SPR if the mRNAs and DNAs are functionalized on the surface of particles electrodeposited on a biochip support. (Techniques described inter alia in US 2008 213130 and US 2005/0157445 or US 2006 17 09 25 or WO 2005 016515). The MICAM technique that uses the piezoelectric effect in its biochips can also be used for the above-mentioned pharmacoecological biochip invention dedicated to neuroblastomas and other brain cancers.

In an alternative embodiment, the gene expression product is detected at the level of the protein or peptides characterizing the Liv21 complex and its interacting partners. Preferably, the protein and / or the associated LIV21 protein complex is detected with the aid of a specific antibody. For example, the protein can be detected by Western blot analysis and by SPR, by a biochip system using a cross-propagation wave (evanescent wave) of surface plasmon resonance (SPR). The interaction can be done with an electronic surface, the semiconductor surface creates an exiton by luminescence or fluorescence. In a preferred embodiment, it is detected by immunohistochemistry, immunocytochemistry, microfluidics, radiography or by peroxidase labeling or by any other optical, sonic or spectroscopic imaging means.

In a particular embodiment of the method comprising the detection of the expression product of the PKCs gene, a significant increase in PKCs is indicative of the presence of cancer cells. In addition, the method may also include determining the LIV21 / PKCs ratio in the nucleus and / or membranes and cytoplasm. This ratio can be compared to that observed in a healthy cell. An increase in the LIV21 / PKCs 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 LIV21 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. In a particular embodiment, the method comprises detecting a labeled RNA so as to target the specific sequence that characterizes it and thus signal the place of expression of the messenger RNA of the gene of interest. Thus, if the specific RNA can be used as a diagnostic marker as would be an antibody and allow to locate in a particular case as extemporaneous or any type of sample taken from a patient for example sample of cancerous tissue, fluorescence for any other labeling used on the si RNA and found in a cell compartment on the sample, a si RNA gene E2F1 and E24 and PKC epsilon would allow a complementary diagnosis. 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. the present invention relates to an isolated, purified or recombinant human LIV21 protein comprising a peptide sequence selected from SEQ ID Nos. 1 to 5 or, more generally, from the peptide sequences characterizing it obtained by MALDI (FIGS. 3, 4 and 5) and NanoLC. In a preferred embodiment, the polypeptide comprises the three peptide sequences SEQ ID Nos. 1 and 2 and 3. Preferably, the LIV21F protein and certain proteins of the LIV21 complex comprise a leucine zipper motif, a basic domain. characteristic of the DNA binding domains (Fig.), and a nucleation sequence.) In a particular embodiment, the present invention relates to polypeptides whose sequence consists of a peptide sequence characterized by The spectrograms of FIGS. 3,4,5 and 1,2 and 3 gel bands, selected from SEQ ID Nos. 1 and 2 and 3 and 4 and 5 and the score of additional unordered sequences (cf. sequences listed in the appendix), the other sequences of the protein being checked with respect to their homologies with contaminants and ordered during MSMS mass spectrometry on the unmatched fragments identified in the MALDI TOF analyzes (see FIG. 5), these unmatched fragments corresponding to unlabeled M (H +) masses partly characterize the LIV21 protein and some elements of its protein complex.

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 may specifically bind to a polypeptide comprising a peptide sequence selected from SEQ ID Nos 1-120 and preferably from SEQ ID No. 1 and 2 or 3 and / or 5 or 51 or a sequence exhibiting at least 80% identity with these. 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. 1-120, preferably from the sequences herein and 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 LIV21F. according to the present invention and an anti-LIV21 F serum according to the present invention, an oligonucleotide probe specific for LIV21F mRNA (hybridization probe) and a primer pair specific for LIV21F mRNA. In a particular embodiment of the invention, the kit further comprises means for detecting the product of the expression of a gene or an oligonucleotide probe specific for the factor mRNA selected from the group consisting of the protein gene. epsilon kinase C (PKCc), the E2F1 gene and the E2F4 gene. But also antibodies in the form of antibody microarray of RBP2, SUMO, HDAC, TNFalpha, crb2, cycE / cdk2, cdk1, CREB1 and p300, Rb, p107, p130, NFkB, cdc2A, mdm2, p21, p53, p65 and microarray with characteristic peptides known to those skilled in the art of the proteins mentioned above, their antigens being referenced. It is the combination of these different peptides corresponding to the specific interactions of the protein complexes which intervene in the dysregulation of the metabolism which generates the anarchic specific proliferation of the cancer or the neurodegeneration.

The invention relates to the use of an antibody specific for human LIV21 for the diagnosis of cancer and the antibodies of its protein complex, but also antibodies specific for RBP2, SUMO, HDAC, TNFalpha, crb2, cycE / cdk2, cdk1, CREB1 and p300, Rb, p107, p130, NFkB, cdc2A, mdm2, p21, p53, p65, p73. Similarly, the invention relates to the use of a primer or probe pair specific to LIV21 for the diagnosis of cancer. Preferably, the diagnosis is made ex vivo on samples of a patient. (puncture of the cerebrospinal fluid, blood test, biopsy, cell crushers, bronchial aspirations, DNA microarrays, Proteins, antibodies, hydrophobic, plasmionic (SPR) or metal-ionic support etc ...) 16. Method according to claim 15, characterized in that in addition it uses at least one of the probes specific for the sequences of said human Liv2I complex and its associated partners according to claims 3 to 7.

The method is characterized in that said biochip is: a protein biochip, or an antibody biochip, or a nucleic acid biochip, or an mRNA biochip, or siRNA biochip.

The method is characterized in that said detecting step uses any optical imaging means, or any sonic means or any means of spectroscopy.

The method is characterized in that said protein biochip, or said antibody biochip, consists of a microfluidic biochip and for which said detection step consists of SPR detection.

Method is characterized in that it comprises a step of amplification / retro-transcription by RT-PCR of at least one nucleotide sequence of said human Liv21 complex according to claim 1 or 2 or said Liv21 complex and its associated partners according to any of claims 3 to 7.

Method is characterized in that it comprises a pharmaco-diagnostic test for the diagnosis of cancer or the monitoring of the evolution of a cellular proliferation, said cancer being preferentially chosen from the group consisting of neuroblastomas, glioblastomas and others cancers affecting the tissues of the nervous system.

DESCRIPTION OF THE FIGURES FIG. 1A: one-dimensional gel (12% acrylamide gradient) revealing after three hours of migration of 11 bands with certain triplets and doubled with bands and a two-dimensional gel SDS Page of total cell extracts with spots between 15 and 20 KD and at 29 - 32kD and about 35 KD at basic pH and spots at 190 - 180 and 100kD at acid PH. Spots between 49 and 51 KD and 64 KD. Monodimensional: approximately 180 kD (TOFC), 100 KD, 64 kD, 51 to 49 kD, 49 kD, 35 kD, 29 kD, 25 to 17 kD bands. Figure 1B: Biomarker interactions pattern of the LIV21 complex and surrounding metabolic pathways. Figure 2: Schematic of nuclear protein interactions with the domain DNA binding and consequences on the study of therapy targeting the nucleus and the cell cytoplasm.

Figure 3A: list of monoisotopic peaks of band 1 at 50 kD and band 2 at 49-50 KD Figure 3B: is the protein profile of LIV21 by mass spectrometry (Maldi) M (H +) for the band of one-dimensional gel corresponding to band 2 migrating at 49 - 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 is taken from the two solutions, 1 microliter is mixed and deposited on the Maldi plate for analysis. FIG. 4 is a profile of the spectrogram of the band 6 called 6FC MS de novo analysis Maldi TOF TOF makes it possible to propose the sequences: RYLVTPVNA, RYVPSSNLP, RYVLSPVK, RYVPSSNPL, RYLPSANPD FIG. 4bis: MSMS analysis of the monoisotopic peak 1032.58: YRPGTVALR , RYVPSSNPL The monoisotopic peak 944.6 is a sequence: FAVAFPVGR The monoisotopic peak 1603.7: KPSHPKPSTK The peak 1328.69: AHNLFKT, TFKNLC The common monoisotopic peaks between the first spectro 2004 (230304 imagenium 20 03 Hl 1 a 001 and the peak 6FC of 2008 are: The following monoisotopic peaks: 1135,563, 1151,545, 1167,604, 1,206,589, 1324,634, 1336,658, 1507,735, 1604,710, 1,800,940, 2087,034, the protocol variation only takes three different steps, the first is the purification of the antibody used, the second is the separation of the cytoplasmic and nuclear fractions, the third is heating for two minutes at 100 ° of the sample before migration on acrylamide gel. Ure 5 is the third spectrogram corresponding to the 12% one-dimensional acrylamide gel band migrating at 51-52 KD and revealed by coumassie blue and LIV21 antibody.

Figure 5a is the list of the monoisotopic peaks of the third spectrogram corresponding to the 52 kD migrand one-dimensional acrylamide gel band and revealed by coumassie blue and LIV21 antibody. Monoisotopic peaks with an M H + value. 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. Figures 3-5 describe the MALDI analyzes 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 under 10 processors. Figure 6: analysis on the databases of the listing of monoisotopic peaks. Example of histatine 3. Mascot search parameters are: trypsin enzyme, variable modifications: carbamethylation and oxidation of methionines, no limit of molecular weight, without restriction of isoelectric point. Figure 7: Idem but second example: Type of mass: monoisotopic. Mass error (MS): depending on observer 50 ppm or 100ppm. Non-tryptic cut: 1 The masses entered are M (H +) / real masses. For spectrogram 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 chaperonin, deaminase, ferredoxin-NADP (+) reductase, pseudomonas polyribonucleotidyltransferase, chlathrin, dehydrolipoamide dehydrogenase. Figure 8: RNA pool Figure 9: PCR with housekeeping genes and Molecular weight analysis Figure 10: PCR with primers showing a 1400bp band Figure 11: Gel 2 with molecular weight analysis Figure 12: Gel 3 to 55 ° and molecular weight analysis Figure 13: Gel 4 at 45 ° and 55 ° and molecular weight analysis Figure 14: ligation screen band 400pb clones B1 to B10 Figure 15: Ligation band ligation 1400 bp clones C1 to c10 Figure 16: Gel 5: ligation screening on the five new clones FIG. 17: Gel 6 Screening of the recombinant clones S55T and S55M and molecular weight analyzes. Figure 18: Examples of comparison of nucleotide sequences between the sequenced clones. Figure 19: If RNA Design Figure 20 Protein Chip with Named Peptides of the Complexe 10 Proteins of Interest Studied in the Invention. Figure 21 Two microfluidic microarrays of four x2 wells with one control and three biomarkers. Overexpression of DDX1, MYCN and CRABPII is observed while the second sensorchip is observed to be under-expressed by HUD, TP53 INP1 and under major expression of p21. FIG. 22 Example of 20-spot biochip with 16 biomarkers of interest (and four controls) fixed on the sensorchip to see by SPR overexpression and under-expression of some genes of Liv2l complex and its interaction partners for example. Subexpression of DKK1, SKP2, DKK3, ID2, p21, SKP2, TP53INP1, ID2, P73 is observed while over expression of MYCN, ALK, NLRR1 (the leucine rich repeat neuronal is transactivated), CRABPII, is observed. DDX1, LIV21K, AURORA kinase A. FIGS. 23 RNA microarray for exploring the miniRNAs of the Liv2I complex and more particularly those derived from the LIV 12F and LIV21K polypeptide genes added to the Mir RNAs, microRNAs known to be involved in the pathology ((miR (= MIR) 34A MIR 9. MIR 125A .125B, MIR 128 MIR 184MIR 221)). Regulatory PTEN and TR AIL factors are also studied. FIG. 24 DNA microarray example from the abovementioned genes of interest targeting the pathology. 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 associated proteins in the Liv2I complex such as E2F4 and E2F1 was studied by co-immunoprecipitation of protein complexes. This analysis has highlighted new markers with a diagnostic and prognostic utility for cancer. From the one-dimensional and two-dimensional gel electrophoresis analysis (Figure 1 and 2), the protein samples corresponding to the putative protein and the elements of the complex were extracted from the gels and digested with trypsin (Proméga) in order to to be analyzed by MALDI mass spectrometry (Figure 3 to 5) and ESI MS / MS. The results confronted with proteomic data banks have made it possible to highlight several peptide sequences of interest, some of which are exemplary data (FIGS. 6 and 7), some of which are found in humans with very significant scores (splicing of the histatine etc ... Figure 8 and 9), these sequences served as primers (once retrotranscribed in cDNA) to screen a library made from MCF7 specific breast cancer cells (Figures 10 to 17). Cloning made it possible to reveal about 20 clones out of the 150 clones obtained, of which ten clones were sequenced and characterized the new LIV21 LIV21F gene and the LIV21K gene (FIG. 17 and FIG. From these sequences, siRNAs were determined to allow a silencing-type regulation within this metabolic complex of interest to develop therapeutic applications (Figure 18).

In postmitotic cells, apoptosis could be an aborted attempt at mitosis. It is in this context that the application of LIV21 has been developed. The inventor has identified sequences of the LIV21 gene. Using the LIV21 antibody on affinity columns he was able to extract peptides from the protein LIV21, he also used a second approach by coimmunoprecipitation kit (Pierce) to have larger quantities of proteins (Example 1) from of peptide sequences of the protein LIV21 obtained by mass spectrometry (Example 2), a primer design for amplifying a cDNA fragment was performed (Example 3). After culture and amplification of cells of MCF7 lines, extraction and purification of the RNAs, RT PCR and cloning in shuttle vector were carried out then a screening of the resistant colonies and sequencing made it possible to reveal sequences characterizing the LIV21F and LIV21K gene ( Examples 4 and 5). More than twenty characteristic clones on 150 clones were studied. . The cDNA of these clones was used to screen a library prepared from the total mRNA of MCF7 cells. The sequence of these new products are new transcription factors whose nuclear translocation for some changes their role and function, this being correlated for some of them to the establishment of quiescence cell such as LIV21F, E2F4 . On the other hand, it forms heterodimers with other transcription factors, and it binds to DNA.

The inventor then followed, using Northern blot, the expression of this new product during development, the embryonic stage. It has been observed that the amount of the LIV21 protein increases as the development progresses, that is, as the quiescent cell state is established. By the same strategy, he showed that the LIV21 / E2F4 complex was an inhibitor of E2F1 expression. This complex could correspond to a new checkpoint in the cell proliferation arrest.

The present invention relates to the Liv2l complex and its new nucleotide sequences SEQ ID No. 171 to SEQ ID No. also usable for a part of them in the form of siRNA for diagnostic and therapeutic applications and also the Liv21F and LIV21K polypeptides as well as derivatives and fragments and isoforms thereof (SEQ ID NO: SEQ ID No.). Human Liv2l complex, characterized in that it comprises: the nucleotide sequences SEQ ID No. 171 to 175, and a siRNA sequence derived from one of the RNA sequences of SEQ ID No. 120 and SEQ ID No. 121, and 171 to 175. - a protein fraction comprising at least the sequence SEQ ID No. 1 and 181 or a sequence having 90%, and preferably 80%, and more preferably 70% identity with said SEQ ID No. 1 and 181.

The human LIV21 complex, characterized in that it comprises: the nucleotide sequences SEQ ID No. 171, 172, and the protein fractions comprising at least the sequence SEQ ID No. 1 or 181 or a sequence exhibiting 70, 80 or 90 % identity with said SEQ ID No. 1 or 181and the sequence SEQ ID Na 183 or a sequence having 70, 80 or 90% identity with said SEQ ID No. 183.

The human LIV21 complex, characterized in that it also comprises: the nucleotide sequences SEQ ID No. 123, 124 and 127, and the protein fractions comprising at least the sequence SEQ ID No. 1 or a sequence having 70, 80 or 90% identity with said SEQ ID No. 1 and sequences 181 to 185 or a sequence having 70, 80 or 90% identity with said SEQ ID Na 181 to 185. The human complex Liv2I is characterized in that it furthermore comprises: any one of the nucleotide or ribonucleic sequences SEQ ID No. 119, 120, 121, 122, 123, 124, 125 126 or 127. or a sequence having 90%, and preferably 80%, and more preferably 70% identity with said sequences SEQ ID No. 119 to 127, or UUGGUAACGACCAUGCCAC, or UUCACUUAGAAUAAUGUCC, or UCUUUGUGAAUUUGACAAC, or UCAAGGUCCAGGCUACAAC, or any of the following siRNA sequences: GUGGCAUGGUCGUUACCAA dTdT dTdT CACCGUACCAGCAAUGGUU GGACAUUAUUCUAAGUGAA dTdT dTdT CCUGUAAU AAGAUUCACUU GUUGUCAAAUUCACAAAGA dTdT dTdT CAACAGUUUAAGUGUUUCU GUUGUAGCCUGGACCUUGA dTdT dTdT CAACAUCGGACCUGGAACU - or the following nucleotide sequence: CCGGCCGNNANNNNGGCCGCGGGAATTCGATTAAATATAAGCAATCCCAAC ACCTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATGGAGACC ATCCTGGCTAACACAGTGAAACCCTGTCTCTACTGAAAATACAAAAAAGTA GCCGGGCGTGGCGGCAGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGG CAGGAGAATGGCATGAACCCAGGAGGCAGAGCTTGCAGTGAGCCGAGATT GTGCCACTGCACTCCAGCCTGGGCAACAGAGCGAGACTCCATCTCAAAAAA AAAAAAAAATCACCCCAAAGCAATAAGGAGAACTAGAACAGGACATACAC TCCAACACTGGTGAAACTAGGAAAACATATGTAACCCCAAACCACAATATA TACACACAAAACTATACGAGATGTTGGGATTGCTTATATTTAAT. 3. Human Liv2l complex according to claim 1 or 2, characterized in that it further comprises at least: - any of the nucleotide sequences SEQ ID No. 123, SEQ ID No. 124 and SEQ ID No. 127 to 149, or - any of the amino acid sequences SEQ ID No. 1 to 118 and SEQ ID No. 150 to 170 and SEQ ID No. 180 to 185, or a sequence having 90%, and preferably 80%, and more preferably 70% identity with said sequences SEQ ID No. 180 to 185 and SEQ ID 1,2 and 5.

Human Liv2I complex characterized in that said protein fraction further comprises any of the following proteins: E2F1, E2F4, p130, p300, p107, Liv2l F, HDAC-1, PML, SUMO and pKCepsilon.

A human Liv2l complex characterized in that it interacts with at least one of its associated partners, said at least one of its associated partners being chosen from the group consisting of; any of the following proteins: RBP2, TNFalpha, crb2, cycE / cdk2, cdk1, CREB1, p300, p107, NFkB, cdc2A, mdm2, p21, p53, p65, p73 MYC, NMYC, TGFbeta, Chlatrin, Aurora, AKT, BRCA1, FOXO4 or cyclin A and D1, CHUK, HMGA2, IKBKB - an antibody of any one of the following proteins: RBP2, E2F4, E2F1, SUMO, HDAC-1, crb2, Int2, cmd2, cycE / cdk2, cdk1,, CREB1 and p300, Rb, p 107, p 130 of the pocket protein family NFkB, cdc2A, mdm2, p21, p53, p65, p73, cyclin A and D1, CHUK, MYC, NMYC, TGFbeta, Chlatrin ( 2), Aurora, AKT, BRCA1, FOXO4, HMGA2. in that it comprises an extract of proteins and peptides obtained by attachment to a polyclonal antibody of the Liv2l protein, and in that its acrylamide gel electrophoretic profile comprises at least the following three bands: 50 kD, the 51 kD band, and the 52 kD band.

Human Liv2I complex is also characterized in that after trypsin digestion, the MALDI mass spectrometry profile comprises at least the following mono isotopic peaks: 1135,563; 1151,545; 1167,604; 1206,589; 1324,634; 1336,658; 1507,735; 1604.710; 1800,940; 2087,034.

Method for detecting the human Liv2I complex, characterized in that it comprises the use of at least one probe specific for at least one sequence of said Liv2I complexes according to claim 1 or 2. Method for detecting the human Liv2l complex , characterized in that it further comprises the implementation of at least one probe specific for at least one sequence of said Liv2l complex and its associated partners according to any one of claims 3 to 7.

Detection method of LIV21 complex and its partners is in that it comprises at least the following steps: - a step of extracting the biological material from a biological sample taken from a patient, - a contacting step said biological material with at least said probe specific for any of the sequences of said human Liv2I complex or said Liv2I complex and its associated partners, and at least one control, and a step of detecting the expression of the products of expression of the genes of said human Liv2l complex or of said Liv2l complex and its associated partners, said expression products being constituted by messenger RNAs, or peptides, or proteins.

Detection method is also characterized in that it further aims to screen a candidate compound, said candidate compound being capable of modulating the activity of said human Liv2l complex, and further comprising: a step of contacting said biological material with said candidate compound, and a step of selecting said candidate compound.

The method is characterized in that said biological sample is taken from a cancer patient and a healthy patient, and said biological material comprises nuclear cell extracts, and cytoplasmic cell extracts, or membrane cell extracts, and in that it further comprises a step of determining the under expression and overexpression of the gene products of said human Liv2l complexes or said human Liv2l complex and its associated partners of said biological extracts.

The method is characterized in that it further comprises a step of determining the ratios of said under-expression and overexpression of said gene products: nuclear cell extracts, and cytoplasmic cell extracts, or membrane cell extracts, and in that it further comprises a step of paired analysis of said ratios of said biological material taken from a patient with cancer and from a healthy patient.

The method is characterized in that said specific probe comprises a rhodamine-labeled siRNA sequence.

A method according to any one of the above approaches is characterized in that it implements a biochip on which at least one of the sequence-specific probes of said human Liv2I complex according to claim 1 is deposited or 2.

The method is characterized in that in addition, it uses at least one of the sequence-specific probes of said human Liv2I complex and its associated partners according to claims 3 to 7. Method is characterized in that said biochip is: a protein biochip, or an antibody biochip, or a nucleic acid biochip, or an mRNA biochip, or siRNA biochip.

The method is characterized in that said detecting step uses any optical imaging means, or any sonic means or any means of spectroscopy. Figure 1 characterizing the protein complex, its PI and its PM is a function of temperature and migration conditions and its observation in total extracts or cell compartment extracts. Figure 1,2,3). It gives more than 54 peptides following digestion with the Proméga trypsin (Figure 7). The characteristics of LIV21F protein are also described in Figures 1, 2, 3, 4. Twenty particular peptides of LIV21 are described: LIV21a (SEQ ID No. 1) and LIV21b (SEQ ID No. 2) to the sequence 180. The gene of this protein is characterized by two main sequences (see sequences) and sequences representing alternative splicing. Liv2lK corresponds to a sequence of strong homology (greater than 60%) with AD7cNTP, the neural thread protein N terminal (position 193 to 299) of (060448_Human). SEQ N ° SVAQAGVQWRNLGSLQALPPGFMPFSCLSLLSSWDYRRLPPRPATFLYFPRQGF TVLARMVSISPRDPPASASQSVGIAYISNFFFFEMESRSVAQAGVQWHNLGSLQ ALPPGFMPF SCLSLLS S WDYRRLPPRPATFLYFPR With variants within the peptide in position (cf: figure ...): The structure of Liv2lK is like that of AD7C NTP, that is to say repeated patterns that we can also analyze in sub-patterns that correspond to functional areas. S VXQAGV Q WXNLGSLQXLPPGXXXFSCLSLXSS WDYXXLPPXPAXF or variant / S VXQAGV Q WXNLGS LQXLPPGFXXFS CXSLS S WDYRRXPPRXA 31 Between these two repeating units a peptide of 40 amino acids with a conserved motif out ISPXDXPASASQSXGIXXXSX THE LIV21I PEPTIDE: FLYFPRQGFTVLARMVSISPRDPPASASQSVGIAYISN 5 With variations in position: For example for LIV21I In position 31 an A instead of V and in position 34 a T instead of the amino acid A. So: the extremity PASASQSAGIT but also a variant where the A becomes T in position 25: ... DPPTSASQSVGI 10 Same for LIV21F or K: SEQ ID NO: FSCLSL L SSWDYRR L PPRPA T FLYF where the amino acid in position 7 here becomes a proline (P instead of L) and / or in position 15 too (with the possibility of an alanine variant, sometimes position 14 changes from R to H and / or position 21 to amino acid T becomes N. SEQ ID NO: FSCLSL P SSWDYRR P PPRPA N FLYF

. Moreover, two other LIV21 peptides are also described: the LIV21c peptide (SEQ ID No. 3) and the LIV21d peptide (SEQ ID No. 4). The peptide LIV21e (SEQ ID NO: 5) .KFFVFALILALMLSMCGADSHAKR whose terminal portion is homologous to a LIV21K sequence. Other particular peptides of LIV 21 are described in the ADDITIONAL LIST. A homology with a functional segment of the zinc finger protein 575 (ZN575-25 Human): score 13 to 48 and up to 91% overlap A homology of the sequence 6 and 56 and with the domain MF MR which is the domain bzip of a transcription factor containing a nuclear localization signal and a transregulatory activity in TAF 1 Within the meaning of the invention, a preferred protein comprises at least one sequence chosen from SEQ ID Nos: 1-180 or a sequence having 70%; 80% or, preferably, 90% homology therewith. The LIV21 complex has proteins comprising helical 3D structures that have a major functional role for its interactions between partners of this LIV21 complex,

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 and / or SEQ ID No. 3 and / or SEQ ID No. 4 and / or SEQ ID No. 5 Preferably, the LIV21F polypeptide comprises the sequences SEQ ID Nos. 1,2 and 5. In a particular embodiment, the LIV21 complex comprises a sequence selected from the peptide sequences characterizing it obtained by MALDI (FIG. ). The invention also relates to the three peptides LIV21a (SEQ ID No. 1) and LIV21b (SEQ ID No. 2) and LIV21e (SEQ ID No. 5). 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 LIV21F (see sequences 1- 180 attached). The present invention also relates to a polynucleotide encoding the human protein LIV21, LIV21a and / or LIV21b, more generally a polynucleotide encoding a polypeptide according to the present invention. The polynucleotide encoding LIV21F and that encoding LIV21K may be mRNA, cDNA or genomic DNA. The polynucleotides according to the present invention can be isolated from cells, more particularly from human cells, or from human cDNA libraries. They can also be obtained by a polymerase chain reaction (PCR) carried out on the total DNA of the cells or else by RT-PCR carried out on the total RNAs of the cells or by chemical synthesis. The probes and primers described in the present application can be used to isolate and / or prepare a polynucleotide encoding LIV21F. It further relates to a cloning vector or expression comprising such a polynucleotide. Such a vector may contain the elements necessary for the expression (expression vector) and optionally for the secretion of the protein in a host cell (secretory signal peptide). The vectors preferably include: a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. The vector may be a plasmid, a cosmid, a BAC, a phage, a virus or other. The invention also relates to a transgenic non-human host cell or animal comprising a vector or polynucleotide according to the present invention.

The invention further relates to derivatives of interest of LIV21F or LIV21K which are for example fusion proteins in which LIV21 is fused to marker proteins such as GFP. Furthermore, the LIV21F and LIV21K protein and all the petids described (cf.patent in listing) may be labeled by any means known to those skilled in the art. SiRNA also.

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. or the LIV21F or LIV21K polypeptides.

The method may include the detection of the product of the expression of two of these genes or three genes. Furthermore, at least one of the LIV21 / PKCs, 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.

In one embodiment, the gene expression product is detected at the level of the mRNA, the mRNA being detectable by any means well known to those skilled in the art.

Thus, the method according to the present invention also relates to the detection of a polynucleotide encoding the human LIV21 protein or a fragment thereof, for example LIV21a and / or LIV21b. The polynucleotide encoding LIV21 may be mRNA, cDNA or genomic DNA. The polynucleotides can be isolated from cells of the biological sample. They can also be obtained by a polymerase chain reaction (PCR) carried out on the total DNA of the cells or by RT-PCR carried out on the total RNAs of the cells or polyA RNAs. MRNA can be detected by RT-PCR analysis. For this purpose, the method uses a specific pair of primers of the expression product to be detected, in particular LIV21, PKCs, E2F1 or E2F4. By "specific primer pair" is meant that at least one of the primers is specific for the expression product to be detected. That is, this primer pair specifically amplifies a fragment of the desired mRNA. Preferably, the RT-PCR analysis is carried out on nuclear and / or cytoplasmic extracts of the cells contained in the sample of the patient.

Optionally, the RT-PCR assay can be a quatitative analysis. A pair of specific LIV21 primers may be prepared based on the teachings of the present application. For example, the primer pair may comprise the primers described in the sequences SEQ ID Nos. 3 and 4 and further sequences of the patented list are therefore numbered from 119 to 149 and from 171 to 180 with optionally all the additional nucleotide sequences of the additional list.

The specific primer pairs of PKCc, E2F1 and E2F4 are well known to those skilled in the art (Caroll JS 2000, Mundle SD 2003, Stevaux 0 2002, Cheng T 2002, Opalka B 2002) .30 The mRNA can also be detected by NoRthern blot analysis. For this, the method uses a specific probe of the expression product to be detected, in particular LIV21, PKCE, E2F1 or E2F4. A probe specific to LIV21 can be prepared based on the teachings of the present application. An example of a specific probe comprises the sequence SEQ ID No. 5. Preferably, the Northern blot analysis is performed on nuclear and / or cytoplasmic extracts of the cells contained in the sample of the patient. The nucleic probe is labeled. The technique for labeling oligonucleotides is well known to those skilled in the art. The labeling of the probes according to the invention can be carried out by radioactive elements or by non-radioactive molecules. Among the radioactive isotopes used, mention may be made of 32P, 33P, or 3H. Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, digoxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents. Specific PKCE, E2F1 and E2F4 probes are well known to those skilled in the art.

In an alternative 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 PKCE, E2F1 and E2F4 genes is to be detected, the method can use antibodies specific for PKCE, E2F1 and E2F4 proteins, respectively. Polyclonal and monoclonal antibodies against PKCE, E2F1 and E2F4 are commercially available. By way of example, mention may be made, for PKCE, 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 an immunoblot analysis (Western blot). 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 immunohitochemistry, 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 cell culture. 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-0.1% Triton X100 - 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 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.

In a particular embodiment of the method comprising the detection of the expression product of the PKCE gene, a significant increase in PKCE is indicative of the presence of cancer cells. Specifically, the amount of PKCE in healthy cells is compared to the amount of PKCE 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 / PKCE rate. This LIV21 / PKCs 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 the detection of the combination 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. 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 E2F 1 / LIV21 rate. This ratio E2F1 / LIV21 increases in the nuclear fraction of cancer cells compared to healthy cells. The method according to the present invention makes it possible, in particular, to detect metastasized cancer, therapeutic follow-up and / or recurrence. treatment and 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 derived 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 a cancer in a patient comprising, detection of 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 carrying out 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, an oligonucleotide probe specific for LIV21 mRNA and a primer pair specific for LIV21 mRNA. In a preferred embodiment, the kit comprises antibodies that specifically bind to human LIV21. In another preferred embodiment, the kit comprises an oligonucleotide probe specific for the LIV21 mRNA. It may also include a probe specific for a house-keeping gene. The kit according to the present invention may comprise reagents allowing the detection of an LIV21-antibody complex 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 (PKCs) gene, the E2F1 gene and the E2F4 gene. These detection means may be protein-specific antibodies, oligonucleotide probes specific for the mRNA concerned and / or a primer pair specific for the mRNA.

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, an oligonucleotide probe specific for LIV21 mRNA and a pair of specific primers of LIV21 mRNA. The LIV21F gene is located on the lp3I chromosome in the 30130K and 30132K region in the 30,081K and 30181K position between the TINAGL1 gene and LOC 284551 named for this exonic part NW 001838577.2 in 2009 (formerly referenced in March 2006 as a polypeptide derived from hsl 33153 , NT 032977.8) .Characterized by ring finger domains of Zn Finger type and a transduction signal.

Anti-Cancer Therapy In the context of anti-cancer therapy, one can consider increasing the amount of LIV21 present in the nucleus. For this, one could increase the expression of LIV21 in the nucleus of the cancer cells. LIV21 being capable of self-nuclearization, the production of an expression vector comprising a polynucleotide encoding human LIV21 could be envisaged with the aim of overexpressing this protein in the nucleus of the cells whose proliferation is to be regulated.

The human LIV21 coding expression vector can be administered in vivo to the patient by any means known to those skilled in the art. For example, the expression vector may be administered in the form of naked DNA (eg EP 465,529). Microinjection techniques, electroporation, calcium phosphate precipitation, nanocapsule or liposome formulations are other available techniques. The expression vector may also be in the form of a recombinant virus comprising, inserted into its genome, a polynucleotide encoding human LIV21. The viral vector may for example be chosen from an adenovirus, a retrovirus, in particular a lentivirus, as well as an adeno-associated virus (AAV), a herpes virus (HSV), a cytomegalovirus (CMV), a virus vaccinia, etc. Advantageously, the recombinant virus is a defective virus.

Preferably, the expression vector allows cell targeting. Thus, this vector could target cancer cells or the particular cell type that is affected by cancer. Targeting of a particular cell type can be achieved by placing the LIV21 coding polynucleotide under the control of a tissue-specific promoter. In another alternative, the expression vector may be targeted, for example by associating it with a specific molecule of a particular tissue or cancer cells, for example an antibody specific for an expressed molecule. specifically by the particular tissue or cancer cells. Moreover, the choice of the expression vector can also influence the targeting. Indeed, if the expression vector is a virus, the tropism of the natural or modified virus may also allow some targeting.

The present invention therefore relates to a pharmaceutical composition comprising a polynucleotide encoding LIV21, more particularly an expression vector coding for LIV21. It also relates to the use of a pharmaceutical composition comprising a polynucleotide encoding LIV21, in particular an expression vector coding for LIV21, as a drug. Preferably, the present invention relates to the use of a pharmaceutical composition comprising a polynucleotide encoding LIV21, in particular an expression vector encoding LIV21, for the preparation of a medicament for treating cancer. The present invention further relates to a method of treating cancer in a patient comprising administering to cancer cells a polynucleotide encoding LIV21, the expression of LIV21 to reduce or abolish the cancerous phenotype of the treated cells. Preferably, the cancer is selected from breast, bladder, ovarian, lung, skin, prostate, colon, and glioblastoma cancers, without being limited thereto.

Furthermore, one could also promote the nuclear localization of LIV21, for example by decreasing the activity of PKCc in cancer cells and using the HDAC inhibitors.

In another particular mode of anti-cancer therapy, it may be contemplated to decrease the activity of PKCE in cancer cells. This decrease in activity can be achieved by decreasing the activity of the PKCs protein or by decreasing its expression. A decrease in PKCs protein activity can be achieved by administering to the cancer cells inhibitors of the PKCc protein. Inhibitors of the PKCc protein are well known to those skilled in the art. A decrease in PKCE protein expression can be achieved by using antisense or siRNA specific for the PKC8 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 N 1998, Hughes 1987). The following RNAs can also be used: The present invention thus relates to a pharmaceutical composition comprising an inhibitor of PKCE protein. It also relates to the use of a pharmaceutical composition comprising an inhibitor of the PKCc 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 PKCE protein, the PKCE protein inhibitor for reducing or abolishing the cancerous phenotype of the treated cells. In a first embodiment, the PKCE protein inhibitor decreases the activity of the PKCE protein. In a second embodiment, the PKCE protein inhibitor decreases the expression of the PKCE 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. PKC epsilon inhibitors are published and used commercially for other applications.

In the context of therapy of a neurodegenerative disease, it is possible to reduce the amount of LIV21 present in the nucleus. For this, one could reduce or block the expression of LIV21 in the nucleus of the cells affected by the neurodegenerative disease. The cells affected by the neurodegenerative disease are generally neurons, motor neurons, etc. The inhibition or blocking of LIV21 expression can be achieved by any means known to those skilled in the art. In particular, by way of illustration, mention may be made of the antisense strategy, siRNA, and ribozymes. Thus, an antisense oligonucleotide or an expression vector encoding this antisense oligonucleotide could be prepared and used to block translation of the LIV21 coding mRNA in vivo. In addition, a ribozyme can be prepared to cut and destroy the LIV21 coding mRNA in vivo. It is also possible to envisage a triple helix strategy in which an oligonucleotide is designed to hybridize with the gene encoding LIV21 and thus block the transcription of this gene.

Furthermore, for this, one could also disadvantage the nuclear localization of LIV21, for example by increasing the activity of PKCE in the cells affected by the neurodegenerative disease.

In another particular mode of therapy against a neurodegenerative disease, it is possible to consider increasing PKCE activity in cells affected by neurodegenerative disease. This increase in activity can be achieved by increasing the activity of the PKCs protein or by increasing its expression. An increase in the activity of the PKCs protein can be obtained by administering to the cells affected by the neurodegenerative disease activators of the PKCc protein. Activators of PKCE protein are well known to those skilled in the art (Toma 0 (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 PKCs protein expression can be achieved using expression vectors encoding the PKCs protein and allowing overexpression in cells affected by neurodegenerative disease.

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

The invention relates to methods for the selection, identification, characterization or optimization of active compounds decreasing cell proliferation, based on the measurement of the nuclear or cytoplasmic localization of LIV21, or the binding of the LIV21 protein to 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 the 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 PKCc protein. A decrease in PKCs expression indicates that the candidate compound is active to decrease or abolish cell proliferation. An increase in the expression of PKCs 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 LIV21 / E2F4 complex. . 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 and LIV21 are brought into contact and, in in a second step, the complex formed between said candidate compound and LIV21 is detected by any appropriate means.

The present invention also relates to 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 Extraction of the LIV21 Complex Proteins The MCF-7 Line The MCF-7 line is a non-clonal human line of breast adenocarcinoma cells. 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 the induction of apoptosis, or TPA (12% -tetradecanoyl phorbol-13-SUMOate) for the induction of differentiation and thus for the study of cycle exit. cellular.

Extraction of LIV21 complex proteins

After culturing MCF7 cells (ATCC passage 15) and cell extraction, 5 mg of proteins are centrifuged after homogenization in 10 ml of RIPA buffer supplemented with antiprotease. These protein extracts were looped for 7 hours at the peristaltic pump on an affinity column (HITRAP NHS ACTIV HP 1x5 ml: article and catalog 17071701) where the antibody LIV 21 was fixed. Washings 3 times 10 ml in RIPA buffer then elution of fifteen fractions of 500 nl in PH2 / HCL glycine buffer, then control on SDS gel (deposition of 25 nl of the fraction) followed by an immunoblotting (western blot) verification. 51 kD gel band revelation framed by two other bands at 50 and 52 kD respectively and trace of a band at 80 and 100kD lower in one-dimensional gel fig 1A). Furthermore, 12 spots were revealed between 50 kD and 70 kD in two-dimensional gel (FIGS. 1B and 2). EXAMPLE 2 Mass Spectrometry Mass spectrometry (MALDI) was performed for the LIV21 protein and its complex. The LIV21 protein (LIV21F poly nucleotide) 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 was taken from both solutions, and 1 μl was mixed and plated on the MALDI plate for analysis. Mass spectrometry showed that the LIV21 protein and its complex digested with trypsin revealed a hundred peptides following the band of gel extracted between 49 and 54 KD and studied (see Figure 3 to 5). The LIV21 protein was characterized by a molecular weight of 501d), evidenced by Western Blot and two-dimensional SDS PAGE gel (Figure 2). But there is a product from 100kD to 130kD that could be a dimer of LIV21.

When it changes cell compartment and is sumoylated, it has a molecular weight of about 60kD. When phosphorylated in the cytoplasm, it has two forms differing by a few kilobases. We then observe a duplicate.

EXAMPLE 3 Sequence Analysis in the Proteomic Databases Several peptides listed in the patent application characterize it: PeptideL1V21b FVFALILALMLSMCG SEQ ID No 51 PeptideL1V21b KFFVFALILALMLSMCGADSHAKR SEQ ID No. 5 For example, the inventor obtained a very significant score of 81, Expected: 0.0012 for a variant of histatin 3-2 with 52% sequence recovery between the tested sample and histatin, this sequence SEQ ID No. 5 is common to LIV21 and HIS3-HUMAN ( Figure 6 and 7). Using a different library and a ppm which differs from 50 to 50 ppm, the identical sequence is obtained for their common part: SEQ ID No. 51 EXAMPLE 4: Reverse transcriptions: After thawing of MCF7 cells (ATCC passage 15) and cell culture up to 200 million cells were trypsinized and frozen at -80 ° C. The RNAs were extracted from two pools of 50 million cells with the Nucleospin RNA L Kit (Macherey Nagel) ref. 740,962.20 resulting in a pool 1 of 318 μg and a second pool of 182 μg. The poly A + RNAs were extracted from 313 μg of total pool 1 RNA using the oligo kit Tex RNA Midi kit (Qiagen) ref. 70042.

I Retrotranscription: RNAs were retro-transcribed with Revert Aid H minus M-MuLV Reverse Transcriptase Fermentas Ref. EPO451 lot 1124 with 3.64 μg of total RNA and 0.45 μg mRNA according to the supplier's conditions with an oligo dT primer. Reactions performed at 2 different temperatures at 45 ° C and 55 ° C so as to eliminate the structures of the RNA that may hinder retrotranscription. II PCR The PCRs were performed with the reverse transcripts as templates with primers Al + oligo dT in a first step. Nested PCRs were then carried out on these first PCRs with primers Al + Splicing, Al + GDBR1, or ATG + Splicing, ATG + GDBR1. Amplification by PCR with the specific primers of the genes to be detected from the cDNAs obtained after RT oligo dT. Enzyme: Taq DNA polymerase polymerase Fermentas. Thermocycler: Bio-Rad 25 iCycler. The quality of the cDNAs was tested by amplification of household genes GAPDH, Bactin and Histone H3.3 TABLE 1 Primers Reference Sequence 5'-3 'Amplified fragment size Histone N 5'-GTG GTA AAG CAC CCA GGA A-3' 347 bp Histone I (reverse) 5'-GCT AGC TGG ATG TCT TTT GC-3 'Reference Sequence 5'-3' Amplified fragment size Hum GAPDH sense 5'-TGA AGG TCG GAG TCA ACG G-3 '983 bp Hum GAPDH antisense 5'-CAT GTG GGC CAT GAG GTC-3 'Hum b-actin sense 5'-GGA CTT CGA GCA AGA GATGG-3' 234 bp Hum b-actin antisense 5'-AGC ACT GTG TTG GCG TAC AG-3 ' LIV21 (A1) 5'-TCCTATGCTTGAC-3 'LIV21 (A2) 5'-CCTGACATCCCTA-3' odT 5'-TTTTTTTTTTTTTTTTTTT-3 'ATG galgal 5'-ATGTATATTATA-3' Splicing sense 5'-TGTTGGGATTGCTTATTTA-3 'Splicing reverse 5'-AAATATAAGCAATTATATA-3 'G reverse 5'-CTTTATTATTTTTAATGA-3' In a particular mode of study by RT PCR of the cancerous tissues as of cell culture SH5Y, it is also possible to use the following primers Forward primer GCAGGTCGGCGTGACGGAAA Reverse primer

Forward primer 1 Reverse primer 1 Reverse primer Reverse primer Forward primer 1 Reverse primer 1

Forward primer Reverse primer Forward primer 1 Reverse primer 1 CGCCACGAACTCCAGCAGCA

GCAGGTCGGCGTGACGGAAA CGCCACGAACTCCAGCAGCA 20 CAGTGGAGAAGCGGCGGCAA TTTCCGTCACGCCGACCTGC CAGTGGAGAAGCGGCGGCAA 20 TTTCCGTCACGCCGACCTGC 20

CAGTGGAGAAGCGGCGGCAA CGCCACGAACTCCAGCAGCA CAGTGGAGAAGCGGCGGCAA CGCCACGAACTCCAGCAGCA 20 20 Forward primer Reverse primer GGCCTGCGCTCGCCTGTAAA CGCCACGAACTCCAGCAGCA Forward primer 1 GGCCTGCGCTCGCCTGTAAA 20 Reverse primer 1 CGCCACGAACTCCAGCAGCA 20 TABLE 2 Mix H2O PCR buffer dNTP ++ Primer forward Primer reverse Ta (pl) cDNA LOX ~ 25 mM Mg .mu.l) 10 mM microM (μl) 10 μM (μl) q (μl) (1, SmMfinal) 5U / μl 18.3 2.5 1.5 0.5 0.5 0.5 0.2 1 PCR Cycles 94 ° C 2 minutes 35 cycles Denaturation: 94 ° C 30 seconds Denaturation: 52-55 ° C 1 minute Annealing: Elongation: 72 ° C 1'30 min Ultimate elongation: 72 ° C 7 min Preservation: 4 ° C III Controls PCR products were checked on agarose gel and analyzed with Vilber Lourma's Biocapt 11,01 software. Gel 1 cf. Figure 7: RNA control on agarose gel Figure 8: PCR with household genes Figure 9: Molecular weight analysis Figure 10: PCR with primers showing a 1400 bp band Figure 11: Gel 2 with molecular weight analysis Figure 12: Gel 3 at 55 ° and molecular weight analysis Figure 13: Gel 4 at 45 ° and 55 ° and molecular weight analysis Figure 14: Ligation screen band 400pb clones B1 to B10 Figure 15: Ligation band ligation 1400 bp clones Figure 16: Screening ligation gels on the five new clones Figure 17: Gel 6 Screening of the S55T and S55M recombinant clones and molecular weight analyzes.

Gel 2: PCRs made from PCR matrices carried out with the Al + oligo dT primers on the RTs carried out at 45 ° C. on the total RNAs and poly A + (messengers). The primers used for these PCRs are Al + G or Al + Splicing reverse). . On the RTs carried out from the total RNAs, there is an amplification of a band of 1178-1253 bp with primers Al + G and Al + Splicing reverse. Poly A RNAs were used to perform RT and were poorly observed at 1400 bp size (FIG. 10) for amplification with Al + G primers and 415 bp with Al + Splicing reverse primers. In the PCR product Al + splicing there are other bands of 860 and 233 bp (FIG. 11).

Gel3: PCRs made from PCR matrices carried out with the Al + oligo dT primers on the RTs carried out at 45 ° C. on the total and poly A + (messenger) RNAs. The primers used for these PCRs are Al + GDBR1 or Al + Splicing reverse) .Figure 12 For the PCRs made from RT made at 55 ° C., we find generally the same pattern of bands as that obtained from RTs made at 45 ° C. ° C. Specific amplification was not observed when poly A RNAs were used to perform RT. On the RTs made from the total RNAs, there is a major band of 1554-1609 bp with primers Al + GDBR1 and Al + Splicing reverse. We expect a band at the theoretical size of 1455 bp for amplification with primers Al + GDBR1 and 415 bp with primers Al + Splicing reverse. In the 2 profiles we find very sharp bands of 1900-2100 bp and 1000-1300 bp but of lower intensity than that of the 1500-1600 bp band. In the PCR product Al + splicing reverse there is another major band of 263 bp. Gel 4: Nested PCRs made from the PCR matrices performed with primers Al + GDBR1 or Al + Splicing reverse) on the RTs carried out at 45 ° C. on the total RNAs and poly A + (messengers). The primers used for these PCRs are ATG + GDBR1 or ATG + Splicing reverse). FIG. 13 The nested PCRs carried out with the ATG + GDBR1 primers give bands at 1213 bp (RT 45 ° C.) and 1559 ± 1315 bp (RT at 55 ° C.), the theoretical size expected is 1455 bp. PCRs performed with the ATG + Splice reverse primers give more varied band profiles. These PCRs made from other PCRs made with primers Al + GDBR1 or Al + Splicing reverse.

The presence of a 400 bp band in the profiles obtained from messenger RNAs is noted (the band obtained from the reverse transcription carried out at 55 ° C. is of higher intensity). The profiles of the ATG + Splice reverse PCR carried out with total RNAs give a band of 424-437 bp of very strong intensity. There are also bands of 614 and 783 bp of very high intensity in the profile of the RT 45 and more numerous bands but of lower intensity in the profile of the RT 55 bands at 1118, 936 and 749 bp. The products of these different PCRs were cloned and sequenced. EXAMPLE 5: The PCR products of lanes 2, 4, 6, 7 and 8 were ligated with the plasmid pGEMT Easy Promega and screening for recombinant clones. FIG. 16 Track 2: ligations G45T Track 4: ligations S45T Track 6: ligations G55T Track 7: ligatures S55M Track 8: ligatures S55T

The recombinant clones obtained were screened (after extraction of the plasmid DNA) by restriction with the EcoRI enzyme whose sites flank the insertion site of the PCR products into the pGEMT Easy vector. Screening of the Recombinant Clones: The first experiments were made from the ten B clones and ten C clones Figure 14 and 15 and the results of the B2 and C8 clone sequences are given in the following example and have by sequence comparison between clones a great homology with the clones of the second series of experiments. Gel 5: Screening of the recombinant clones S45 Tet G45 T Molecular weight analysis The cloning of the clones by Eco RI shows that of the 9 clones S45T, 3 have inserts of 100 bp, 216 bp and 410 bp. On the G45T clones, of the 6 clones tested, 3 have inserts of 57, 71 and 148 bp. Figure 17: Screening of the recombinant clones S55T and S55M Molecular weight analysis The Eco RI screening shows that out of the 13 clones screened, 7 have inserts of sizes between 239 and 637 bp. Clones G45T5 (148bp), S45T9 (410bp), S45T3 (100bp), S55M1 (491bp), S55T6 (251bp) and S55T9 (637bp) were extracted for sequencing.

Primers used to find the Sequences of the different clones are described in the patent in listing: 25 ATG galgal 5'-atgtatattatatatctaa-3 'INV COMP ttagatatataatatacat Splicing reverse 5'- -3' and Splicing reverse 5'- -3 'inv comp GDBR1 reverse 5'- -3 'and GDBR1 reverse 5'-3' inv comp 55 Balance: The clone B2, the band which is about 400 bp is the same fragment as S55M1 which itself is the same fragment as S45T9 these fragments are between 400 and 450 bp approximately. This is not surprising because they were obtained after nested PCR performed with reverse primersplicing and Al primers (for B2 fragment) and ATG galgal primer for S45T9 and S55 M1 fragments. These fragments were obtained with RT made at different temperatures. On the other hand, the fragment S55T9 still has about 600 bp, one part of which (300 bp) has a rather strong identity with the other cloned fragments.

The biomarker test on extracts of neuroblastomas and SH5Y cell cultures reveals by RT PCR the following expression and expression set in this example of analysis: Example of 16 biomarkers of interest analyzed by RT PCR that crosses the results obtained by biochip on the sensorchip allowing to see by SPR the overexpression and the underexpression of certain genes of the complex Liv2l and its partners of interaction for example. Subexpression of DKK1, SKP2, DKK3, ID2, p21, SKP2, TP531NP1, ID2, P73 is observed while on expression of MYCN, ALK, NLRR1 (the leucine rich repeat neuron is transactivated), CRABPII, DDX1 is observed. EXAMPLE 6: From the cloning of the LIV21 gene described above, the new sequences are studied to draw the si RNA (see listing of si RNA and Figure 19) the most specific and effective for create the "silencing" of the gene that is to say inhibit its expression. Knowing that the effectiveness of the inhibition with each injection of siRNA remains short, that is to say most often less than one hour. The inventor has developed diagnostic products and therapeutic products from the same tool that is siRNA. EXAMPLE 6.1: The inventor uses rhodamine or fluorescein labeled siRNAs or any other marker that can be revealed and monitored by optical observation, by a microscope in order to locate the place in the cells, tissues or labeled sample where will be directed if labeled RNA in order to cling to the specific sequence that characterizes it and thus signal the place of expression of the messenger RNA of the gene of interest. Thus, if the specific RNA can be used as a diagnostic marker as would be an antibody and allow to locate in a particular case as extemporaneous or any type of sample taken from a patient for example sample of cancerous tissue, fluorescence for any other labeling used on the si RNA and found in a cell compartment on the sample. Thus, if a labeled RNA of LIV21 (FIG. 19) found in the cell nuclei under a microscope at the periphery of a surgical excision would signify a diagnosis of total excision of the cancerous tissue, on the other hand, this same marker found in the cytoplasm or Membranes of this same sample would require the surgeon to perform an immediate expanded resection if this test can be performed in the live operating room which would be the best case to remove all visible cancer cells only at this molecular and cellular level thanks to this siRNA marker. This could be a faster alternative of performance than the other application that the inventor developed with the LIV21 antibody or peptide that can be used in the same way. EXAMPLE 6.2: For therapeutic products that arise directly from observations made by biomarker expression results on biochips, the interest is well illustrated by the example of TGF beta and p27: The publication of Mr. Lecanda J and Gold Li from March 2009 is rich in teaching and illustrates very well the interest of pharmaco-diagnostic biochips as described above. TGFBeta inhibits cell proliferation by increasing the level of p27 via the activation of smad 2/3 transcription factors. TGFbeta increases the total level of p27 translocation in the nucleus and therefore accumulates p27 in the cell nucleus through the activation of smad 2 and smad 3. All these events are blocked by a TGFbeta inhibitor RI: serine kinase SD208 new targeted therapeutic axis for TGFBeta carriers decreases the level of skp2 components relative to the protein but not the level of mRNA. TGFbeta can mediate the degradation of p27 in the proteasome, therefore at the protein level because it maintains it in the nucleus, but on the other hand the inhibition of p27 transcription with a specific siRNA blocks TGF beta.

TGFBeta prevents the proteasomal degradation of the p27 inhibitory cycle kinase inhibitor. (Alvarez Rodriguez R Pon S J that sci 2009 March 1-122). The expression of the proneural gene encoding Mash 1 suppresses MYCN (mitotic activity). (2009 Liu W Wuz Guan M, Lu Y.) The inventor uses the if RNA of the complex LIV21 marked at first to objectify their expected presence in the cell compartment and to visualize them then if RNA LIV21 unmarked in a second time for their purely therapeutic action, in a particular case an injection of si RNA (Fig 19) into a neurodegenerative tissue or by a way that allows the if RNA to reach the tissue neurodegeneration (ex-ear, eye etc.). .) and to act by allowing the proliferation until the apoptosis and thus the death of the cell in neurodegeneration. An experiment on human cell cultures then on animal models with retinopathy giving rise to vitreoretinal degeneration (injection of direct siRNA into the eye) makes it possible to illustrate this method.

SiRNAs are also transfected into cells according to the Invitrogen protocol using lipofectamine 2000. The study of the specific role of PKC epsilon on the nuclear translocation of Liv 21F can use a peptide that inhibits PKC epsilon function and translocation as previously described but also according to the reference protocol of Si RNA PKC epsilon. The expression profile observed corresponds to a doublet of bands in the cytoplasm at approximately 51 KD (and at a 64 kD band according to the siRNAs used) whereas it corresponds to a single band at 50 kD in the nuclear fraction . The siRNA molecules derived from the nucleotide sequences of the Liv2I complex may be chosen from the group consisting of siRNA molecules comprising any of the sequences SEQ ID Nos. 119 to 126 and 171 to 175, and SEQ ID No. HAART based on the combination of several of these siRNAs for the treatment of cancer or neurodegenerative pathology. These siRNAs and the useful combination deduced from the results of over expression and under expression of biochips and RT PCR constitute an adjuvant treatment of the pathology and can be combined with small doses of chemical molecules that have already been proven in the treatment of cancer. pathology.

EXAMPLE 7 Pharmaco Diagnostic Test From the observations which follow in the appendix at the end of the description of the example on the properties of the LIV21 complex, a clinically applicable pharmaco-diagnostic test design was designed by the inventor with known technical means that can be declined differently according to the users and correspond for each mode to a new product. The invention lies in the manufacture of DNA, protein and antibody diagnostic chips comprising the known antibodies of the various proteins of the associated complex according to the phases of the cell cycle at LIV21, that is to say the antibodies, peptides or nucleotide sequences of the genes: RBP2, E2F4, E2F1, SUMO, 1NT2, CRB2, HDAC1, TGFbeta, integrin_alpha5 beta2, Myob, MyoD, cycE / cdk2, cdk1, chk1, chk2, TNFalpha, CREB1 and p300, Rb, p107, p130 of the families protein pockets. But also NFkB, cdc2A, mdm2, p21, p53, p65, p 73 RAS, Ki67, CAF1. The protein chips (Figure 20) will be able to study the overexpression or expression of gene products, protein interactions and post-translational modifications, especially the phosphorylations and methylations of certain proteins, which signify a characteristic state of the diseased cell. different from protein interactions and healthy cell metabolism. The state of expression and silencing of certain genes being different.

EXAMPLE 7.1 Pharmaco Diagnostic Chip (FIG. 20) Designed on the basis of nucleotide or peptide sequences fixed on conventional supports and according to the known Agilent or Affymetrix or Caliper type techniques without restriction to them and corresponding to the known sequences of the following and listed genes Furthermore, in the patent, preferably using the sequences which in 3D analysis have a 3D structure, preferably of the loop (100bp) or helix loop helix or basic loop (about 400bp) or zinc finger type, or else a 3D conformation similar to a helix. most often corresponding to functional sites, or nucleotide sequences corresponding to a methylation zone of a cytosine, methylation of the promoter region of the gene leading to a silencing potential (see general bibliography) as well as to new sequences of the LIV21F and LIV21K listed in the description and in appendix but also sequences co to some oncogenes and some viruses thought to be involved especially in breast cancer of Chinese populations: Mdm2 and HIV 1: GAVTSSNIAA DLDQSV; EGF and HPV16; EWWRLD; KNSLD MHIESLDS Who can also be tested in microfluidics fixed on the gold slide to study the protein interactions with the cell extracts of the patient. But which can also be studied by a MICAM technique applied to biochips that maintain on their surface (cavities) the biomarkers by electric piezo effect and not by physicochemical fixation.

Example 7.2: Microfluidic test, for example Biacore type, using the S.P.R. (Plasmonic surface resonance) known to those skilled in the art based on a support fixed with a gold film allowing once the light beam sent to the interface to obtain energy absorbed depending on the presence and size protein complexes (if protein protein or antibody protein interaction) or protein complexes DNA (if DNA protein interaction), or RNA or protein or peptide, and an evanescent wave perpendicular to the axis of the interface (use of a prism ). The inventor fixes the chosen peptide or DNA sequences on the gold particles and calculates for each interaction complex studied, the number of rU depending on the size of the molecules mentioned in this patent. In microfluidics, the liquid passing on these gold-plated chips can be both a cellular extract (see selective only cell nuclei or only cellular cytoplasm or only cytoskeletal proteins etc ... according to Extraction and fractionation separation type Calbiochem: subcellular proteom extraction or tissue or cell extracts without preparation other than anti proteases or even cerebrospinal fluid or serum from a sample of a patient to study the marker circulating (see list of nucleotide or peptide sequences described and / or known sequences of the genes involved in the proliferation cycle listed) Figure 21 illustrates the presence and quantification of certain biomarkers present in the samples of patients tested in interaction with the antibodies bound to biochips EXAMPLE 8 RNA biochip SEQ ID N sequences 119 to 127 and 180 to combined with the microRNA sequences of interest in the studied cancer: mir 21, mir 34a etc ... without the biochip being limited to those there. The technique of fixing the RNAs on the supports differs according to the type of support, revelation and biochip used. For example, the RNA sequences mentioned in biothynilated solution and then fixed on streptavidin-sensitive probes are used, if their interactions with cell or plasma extracts or cerebrospinal fluid passing through the (microfluidic) channels of an A100 or T100 are tested. Alternatively, as described in US Pat. No. 2008,045,418 A1, 3 'end-blocked RNAs can be used and a T4 RNA ligase and a labeled nucleic acid adaptor having a 5' phosphate residue can be added. EXAMPLE 9: Study of the Expression of LIV21 in Bed Biopsies To determine whether the observations obtained previously are applicable to human tissues, a large number of cancer biopsies obtained from patients were studied by reaction. immunohistochemistry with antibodies specific for the LIV21 complex. Immunohistochemical determination of LIV21 complex protein expression involved several patient biopsies. In addition, some paraffinic blades of cancer patients were also studied. The improvement of the invention is to standardize biopsies or punctures by a standardized and qualified sampling technique to then allow reliable comparisons between cell extracts of patients and controls or between cytoplasmic fractions and nuclear and membrane. This same standardization applied to samples allowing to compare the results of overexpression or under expressions of biomarkers by RTPCR is essential to obtain reliable and comparable results.

Immunocytochemistry analysis protocol: 1) Deparaffinize the slides, Rehydrate the tissues 2) Saturate the non-specific sites and permeabilize the membranes 3) Add the antibody in a humid chamber, Reveal the antibody Deparaffinize the slides under hood Two successive baths of toluene (rectapur Prolabo) 2x30 'or 2x20min; then dehydrate the tissues of the rectal alcohol 100% 15 min; then 95% ethanol rectapur for 10 min; then rectapur 70% 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 the 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 1m1 on each slide and avoid light and evaporation. Leave all night or at least three hours. Then rinse with normal PBS 1 X 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 the 250th so 10 μl in 2.5m1 of PBS with always 10% FCS and 0.1% Triton, Refit the slides on plate. Cover the cup with 2.5m1 to keep a humid chamber for one hour then wash with PBS 1X PH 7. Wash with Propidium Iodide at 0.5 microgram per microlitre to be diluted to 20 microgram per ml then again to 50th but this time, diluted in PBS alone 1X (50 microliter per 2.5 ml of PBS). Drain by removing them from the PBS and then 2.5 ml of propidium iodide distributed over the four slides for one minute then two rinsing with simple PBS. Slide the slides to the Moviol before reading. For an immuno labeling with peroxidase it is essential to mask the antigenic sites by a step in the water bath of 20 minutes in order to have interesting results when working on paraffinic slides. These results show that the cytoplasmic localization of LIV21 is a indicator of aggressiveness and the 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 or of well differentiated tissues.30 Annex: Study of the nuclear translocation of LIV21 in MCF-7 and SH5YH cells The study of sub-cellular distribution of LIV21 in different tumor lines of various origins showed an exclusively cytoplasmic localization of this protein. The presence of putative phosphorylation sites by Protein Kinase C (PKCs) in the LIV21 sequence reflects an implication of these proteins on its nuclear translocation. MCF-7 treated with TPA, modulates PKCs and is used here. 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 negatively controlling the retinoic acid receptor, both of which are expressed particularly prominently in these cells. TPA strongly and rapidly inhibits the expression and function of estrogen receptors (ERs) and induces the time-dependent and dose-dependent translocation of cytosolic protein kinase C (PKCs) to membranes.On the other hand, TPA increases the ability of MCF-7 cells to migrate in vitro and a short treatment of these cells by TPA induces cellular expansion and microtubule organization characteristic of their differentiation.

Expression of LIV21 in MCF-7 cells and in SH5YH cells In a first step, the inventor verified the expression of LIV21 in these cells, at the transcriptional level and at the protein level.

Expression of LIV21 mRNA was demonstrated by RT-PCR (sense primer: CCTGACATCCCTACA (SEQ ID No. 3) and antisense primer: TCCTATGCTTGA (SEQ ID No. 4)) in these cells compared to breast tissue cells. (Figure 2A). An mRNA of the same size as the mRNA detected in the mammary tissues and specific for LIV21 was detected. However, the level of LIV21 expression in MCF-7 cells is lower than in breast tissue. This first result shows that the MCF-7 line expresses LIV21 mRNA.

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. Anti-LIV21 antibodies were obtained by the described method. In this line, LIV21 is expressed in mammary tissues as well as in MCF-7 cells, in the form of a doublet migrating at an apparent molecular weight of 51 kDa.

Production of the purified anti-LIV21 serum The specific peptide sequences are sequences No. 1 and No. 2 and No. 180 These peptides were injected into rabbits (NZ W ESD 75 female 2.3 kg at day 0). standard immunization procedures, the effect of inhibitors on in neuroblastoma cells study of the influence of PKC on the nuclear translocation of LIV21 effect of TPA on the expression of PKCs Western blot study Since the protein sequence of LIV21 has putative PKC phosphorylation sites, two of which are specific for PKCs, the inventor has tested the variation in the expression of this PKC as a function of the duration of treatment with TPA. It has been observed that TPA acts very rapidly on the expression of PKCs, which decreases as early as 30 min. The expression PKCzeta (PKCE) 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 PKCE concomitant with the nuclear translocation of LIV21. It can be observed that PKCE 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 PKCs are weakly expressed at 12h, the time when we observe the least number of S-phase cells and the maximum of nuclear translocation of LIV21.

Study of the specific role of PKCs on the nuclear translocation of LIV21 using a peptide inhibiting the function and the translocation of PKCs. To determine the specific action of PKCs on the translocation of LIV21, the cultures were treated with a peptide, a selective antagonist of the function and translocation of this PKC (EAVSLKPT (SEQ ID No. 6), and the results were compared with those obtained by TPA treatment, this peptide is recognized by the enzyme and binds as a modified substrate at its catalytic site, which is non-phosphorylatable and acts as a specific inhibitor of PKCs activity.

The effect of selective inhibition of PKCs activity on LIV21 nuclear translocation was studied by immunocytochemistry. These experiments were carried out on cultures treated or not treated for 12 hours, with 25 nM TPA or with the peptide at two different concentrations, 1 and 2 μM (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 supported by cell fractionation experiments on cultures treated with 2 μM PKCE inhibitory peptide compared to TPA-treated cultures (the same expression pattern of LIV21 was observed in the form of a doublet in the cytoplasm and a single band in the nuclear fraction Specific inhibition of PKC * induces a nuclear translocation of LIV21, suggesting that LIV21 could be the target of PKCc, which would keep it in the cytoplasm EXAMPLE: Western Blot Analysis This example describes the conditions used for Western Blot analysis of brain 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, 1mM PMSF, (3-mercaptoethanol), and 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 hour under denaturing conditions (Migration buffer: 25mM Tris base, 192mM glycine, 1% SDS, pH 8.3). proteins are then transferred to nitrocellulose membrane (Schleicher & Schuell) for one hour in liquid transfer, in transfer buffer (25mM Tris, 192mM glycine, 20% methanol, pH 8.3) The membranes, saturated in PBS-Tween 0 , 1% -Triton X100 0.1% -5% skimmed milk for one hour are brought into contact with the primary antibody diluted in PBS-Tween 0.1% -Triton X100 0.1% -Milk 1% at temperature with mild agitation for one to two hours After washing, the secondary antibody coupled Eroxidase is incubated for 1 hour 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 made using synthetic peptides based on the sequence of LIV21F and PeptideLIV21a (SEQ ID No. 1 and the peptide LIV21b (SEQ ID No. 2 and / or peptide LIV21 e (SEQ ID NO: 51) The peptides were coupled to hemocyanin before being injected into rabbits for immunization The polyclonal antibody was obtained from two of these peptides by immunizing two rabbits and by blowing a rabbit to have a preimmune serum (in order 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 at 1 / 200.

The mouse anti-p21 monoclonal antibody (DAKO, M7202) diluted 1/150.

Mouse anti-p27 monoclonal antibody (Santa-Cruz technology sc-1641) diluted 1/100. It has previously been shown that the LIV21 protein is associated with the PML bodies and that, during sumoylation, LIV21 goes from a molecular weight of 50kd to 60kd. By immunoprecipitation, the inventor showed co-localization of LIV21 with SUMO in the PML-SUMO / LIV21 complex. (Figure 12)

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. It has been shown that PML associates with SUMO and HDAC-1 (histone deacetylase 1) and that its complex acts on the expression of E2F1 and PML thus acts on the stop of the proliferation by blocking E2F1. Thus the PML / HDAC-1 complex negatively regulates the expression of E2F1. PML associated with Rb (p130) binds to histone deacetylases and blocks E2F1 by binding to chromatin.

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.

It has been shown previously that LIV21 is phosphorylated by PKCs 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 PKCc-specific inhibitory peptide was used, it is the activity and not the expression of PKCc that was inhibited.

During this treatment, when E2F4, p130 and LIV21 (green fluorescence) were studied in the propidium iodide labeled nuclei (DNA) (red fluorescence), it was observed that, after 12h, intranuclear green fluorescence signals were observed. same pattern for E2F4, p130 and LIV21; After 48 hours when proliferation begins, E2F4 has a comparable location; but at 72 h, it disappears from the nucleus (in favor of E2F1).

By observing the co-localization by double labeling of PML and LIV21 at 24 h of PKC treatment (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 The hypothesis is that SUMO, which binds to LIV21, actually addresses LIV21 in PML bodies and that LIV21 is involved in PML / SUMO / complexes. Rb / HDAC-1.

LIV21 is physically associated with PML and SUMO in nuclear bodies, by immunoprecipitation and by co-localization in immunocytochemistry ((Rb, p130 and p107 are pocket proteins that have the same binding site). cell growth (Fabbro, Regazzi R Bioch BiophysResComm 1986 Feb 2, 135 (1): 65-73).

Physical Interaction of LIV21 with the E2F Family Proteins Co-immunoprecipitation experiments using anti-LIV21, anti-E2F1 and anti-E2F4 antibodies have shown that LIV21 associates with E2F4. It has been shown that BRCA1 also interacts with members of the E2F family and thus the LIV21 complex. Indeed E2F1 interacts with BRCA1 and Brcal interacts with MYC, ESR1, CHEK1 and MAP3K3.

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 has been shown in embryonic breast tissues (Espanel X, Gillet G 1998), 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 blot was made. These results suggest that the LIV21 / E2F4 complex appears to play an important role in establishing quiescence of cells. A co-immunoprecipitation study is also conducted with the PIERCE deep mammalian kit.

Functional interaction of LIV21 with the proteins of the E2F family. It has been demonstrated that the blocking of LIV21 protein expression correlates with a decrease in E2F4 expression and an increase in E2F1 expression.

In parallel, the functional aspect of the E2F1 increase was verified by studying the transcription of two of its target genes, DHFR and DNA polymerase a.

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 control point in cell proliferation arrest.

Functional interaction of LIV21 with the proteins of the E2F family. It has been demonstrated that the blocking of LIV21 protein expression correlates with a decrease in E2F4 expression and an increase in E2F1 expression. In parallel, the functional aspect of the E2F1 increase was verified by studying the transcription of two of its target genes, DHFR and DNA polymerase a.

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. The vectors for producing siRNAs: are described, E. g. , in Paul and others. (2002) Biotechnology 29 of nature: 505; Xia and others. (2002) Biotechnology of Nature: 1006; Zeng and others. (2002) Mole. Cell 9 1327; Thijn and others. (2002) Science 296: 550; BMC Biotechnol. Aug. 28 2002; 2 (1): 15; Lee and others. (2002) Biotechnology 19 of nature: 500; McManus and others. (2002) RNA 8: 842; Miyagishi and others. (2002) Biotechnology 19 by nature: 497; Sui and others. (2002) PNAS 99: 5515; Yu and others. (2002) PNAS 99 6047; Shi and others. (2003) Trends Genet. 19 (1) 9; Gaudilliere and others. (2002) Biol Chem. J. 277 (48): 46442; US2002 / 0182223; USA 2003/0027783; EO 01/36646 and EO 03/006477. Vectors are also commercially available. For example, pSilencer is available from Gene Therapy Systems, Inc. and the pSUPER RNAi system is available from Oligoengine. Also provided above are compositions comprising one or more siRNAs or nucleic acids encoding an siRNA RNA coding region. The compositions may be the pharmaceutical compositions and comprise a pharmaceutically acceptable carrier. The compositions may also be provided in a device for administering the composition to a cell or subject. For example, a composition may be present in a syringe or on a stent. In parallel, the functional aspect of the E2F1 increase was verified by studying the transcription of two of its target genes, DHFR and DNA polymerase a.

The present invention relates to methods and reagents useful in modulation gene expression associated with Alzheimer's disease in a variety of applications, including use in therapeutic, diagnostic, target, and therapeutic validation. genomic discovery applications. The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases that respond to the modulation of beta-secretase (BACE), amyloid expression and / or activity. of precursor protein (APP), pin-1, presenillin 1 (PS-1) and / or presenillin 2 (PS-2) in association with LIV21 gene and protein modulations. The present invention also relates to compounds, compositions, and methods relating to conditions and diseases that respond to modulation of the expression and / or activity of genes involved in the LIV21 complex and in association with genes. involved in beta-secretase (BACE), amyloid precursor protein (APP), pin-1, presenillin 1 (PS-1) and / or presenillin 2-way (PS-2). Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA molecules (shRNA) capable of interference mediated RNA (RNAi) against betasecretase (BACE), amyloid expression of precursor protein (APP) gene, pinch 1, presenillin 1 (PS-1) and / or presenillin 2 (PS-2) .25 (Z = CeSz Mayor N, Horman S, Mosselmans R, Galand P. Antisense inhibition of the 27 kDa heat shock protein producrion affects growth rate and cytoskeletal organism in MCF-7 cells (1996) Cell Biol Int, 20 (3): 205-12.Matunis MJ On the road to repair: PCNA encounters SUMO and ubiquitin 5 modifications (2002) Mol Cell, 10 (3): 441-2 Melchior F, Hengst L. SUMO-1 and p53 (2002) Cell Cycle, 1 (4): 245-9 Mundle SD, Saberwal G. Evolving intricacies and implications of E2F1 reg ulation (2003) FASEB J, 17 (6): 569-74. Opalka B, Dickopp A, Kirch HC. Apoptotic genes in cancer therapy. (2002) Cells 10 Tissues Organs, 172 (2): 126-32. Pardo FS, Su M, Borek C. Cyclin D1 induced apoptosis maintains the integrity of the G1 / S checkpoint following ionizing radiation irradiation. Somat Cell Mol Genet. 1996 Mar; 22 (2): 135-44. Pawson T, Gish GD, Nash P. SH2 domains, interaction modules and cellular 15 wiring. (2001) Trends Cell Biol, 11 (12): 504-11. Platet N, Prevostel C, Derocq D, Joubert D, Rochefort H, Garcia M. Breast cancer cell invasiveness: correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor positive and negative cis. (1998) Int J Cancer, 75 (5): 750-6. Ree AH, Bjornland K, Brunner N, Johansen HT, Pedersen KB, Aasen AO, Fodstad O. Regulation of tissue-degrading factors and in vitro invasiveness in progression of breast cancer cells. (1998) Clin Exp Metastasis, 16 (3): 205-15. Regazzi R, Fabbro D, Costa SD, Borner C, Eppenberger U. Effects of tumor promoters on growth and on cellular redistribution of phospholipid / Ca2 + -dependent protein kinase in human breast cancer. (1986) Int J Cancer, 37 (5): 731-737. Schneider SM, Offterdinger M, Huber H, Grunt TW. Involvement of nuclear steroid / thyroid / retinoid receptors and protein kinases in the regulation of growth and of c-erbB and retinoic acid receptor expression in MCF-7 breast cancer cells. (1999) Breast Cancer Res and Treat, 58: 171-181.

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Claims (11)

REVENDICATIONS1. The novel sequences of the human Liv2I complex, characterized in that it comprises: the nucleotide sequences SEQ ID No. 171 to 175, and an siRNA sequence derived from one of the RNA sequences of SEQ ID No. 120 and SEQ ID No. 121, and 171 to 175. a protein fraction comprising at least the sequence SEQ ID No. 1 and 181 or a sequence having 90%, and preferably 80%, and more preferably 70% identity with said SEQ. ID No. 1 and 181 and the sequence SEQ ID No. 183 or a sequence having 70, 80 or 90% identity with said SEQ ID No. 183. 2. Human Liv2I complex according to claim 1, characterized in that it further comprises: the nucleotide sequences SEQ ID No. 123, 124 and 127, and the protein fractions comprising at least the sequence SEQ ID No. 1 or a sequence having 70, 80 or 90% identity with said SEQ ID No. 1 and the sequences 181 to 185 or a sequence having 70, 80 or 90% identity with said SEQ ID No. 181 to 185. human Liv2I complex is characterized in that it further comprises: Any one of the nucleotide or ribonucleic sequences SEQ ID No. 119, 120, 121, 122, 123, 124, 125 126 or 127. or a sequence having 90% and preferably 80%, and more preferably 70% identity with said sequences SEQ ID NOS: 119-127, or UUGGUAACGACCAUGCCAC, or UUCACUUAGAAUAAUGUCC, or UCUUUGUGAAUUUGACAAC, or UCAAGGUCCAGGCUACAAC, or any of the following siRNA sequences: GUGGCAUGGUCGUUACCAA dTdT dTdT CACCGUACCAGCAAUGGUU 30 G GACAUUAUUCUAAGUGAA dTdTdTdT CCUGUAAUAAGAUUCACUU GUUGUCAAAUUCACAAAGA dTdT dTdT CAACAGUUUAAGUGUUUCU GUUGUAGCCUGGACCUUGA dTdT dTdT CAACAUCGGACCUGGAACU - or the following nucleotide sequence: CCGGCCGNNANNNNGGCCGCGGGAATTCGATTAAATATAAGCAATCCCAAC ACCTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATGGAGACC ATCCTGGCTAACACAGTGAAACCCTGTCTCTACTGAAAATACAAAAAAGTA GCCGGGCGTGGCGGCAGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGG CAGGAGAATGGCATGAACCCAGGAGGCAGAGCTTGCAGTGAGCCGAGATT GTGCCACTGCACTCCAGCCTGGGCAACAGAGCGAGACTCCATCTCAAAAAA AAAAAAAAATCACCCCAAAGCAATAAGGAGAACTAGAACAGGACATACAC TCCAACACTGGTGAAACTAGGAAAACATATGTAACCCCAAACCACAATATA TACACACAAAACTATACGAGATGTTGGGATTGCTTATATTTAAT. 3. Human Liv2I complex according to claim 1 or 2, characterized in that it further comprises at least: - any of the nucleotide sequences SEQ ID No. 123, SEQ ID No. 124 and SEQ ID 1 \ 1 127 to 149, or any of the amino acid sequences SEQ ID Nos. 1 to 118 and SEQ ID Nos. 150 to 170 and 180 to 185, or a sequence having 90%, and preferably 80%, and more preferably 70% identity with said sequences SEQ ID No. 1 to 185. 25 4. Human Liv2l complex according to any one of claims 1 to 3, characterized in that it interacts with at least one of its associated partners, said at least one of its associated partners being chosen from the group consisting of; any of the following proteins: RBP2, TNFalpha, crb2, cyce / cdk2, cdk1, CREB1, p300, p107, NFkB, cdc2A, mdm2, p21, p53, p65, p73 or an antibody of any one of of the following proteins: RBP2, E2F4, E2F1, SUMO, HDAC-1, crb2, Int2, cmd2, cycE / cdk2, cdk1,, CREB1 and p300, Rb, p 107, pi of the pocket protein family NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, CAF1, cyclin A and D1, CHUK. 5. Method for detecting the human Liv2l complex, characterized in that it comprises the use of at least one probe specific for at least one sequence of said Liv21 complexes according to claim 1 or 2. 10 6. Method for detecting the human Liv2I complex according to claim 5, characterized in that it further comprises the use of at least one probe specific for at least one sequence of said Liv2l complex and its associated partners according to the invention. any of claims 3 to 4. 7. Method of detection according to claim 5 or 6, characterized in that it comprises at least the following steps: - a step of extracting the biological material from a biological sample taken from a patient, - a step of setting contacting said biological material with at least said probe specific for any of the sequences of said human Liv2I complex or said Liv2I complex and its associated partners, and at least one control, and a product expression detection step expressing the genes of said human Liv2I complex or said Liv2I complex and its associated partners, said expression products being messenger RNAs, or peptides, or proteins. The detection method according to claim 7, characterized in that it is further intended to screen a candidate compound, said candidate compound being capable of modulating the activity of said human Liv21 complex, and in that it further comprises: a step of contacting said biological material with said candidate compound, and a step of selecting said candidate compound. 9. A method according to claim 7, characterized in that said biological sample is taken from a cancer patient and a healthy patient, and said biological material comprises nuclear cell extracts, and cytoplasmic cell extracts, or membrane cell extracts, and in that it further comprises a step of determining the under expression and overexpression of the gene products of said human Liv2I complexes or said human Liv2l complex and its associated partners of said biological extracts. 10. A method according to claim 9, characterized in that it further comprises a step of determining the ratios of said sub-expression and overexpression of said gene products: nuclear cell extracts, and cytoplasmic cell extracts, or membrane cell extracts and in that it further comprises a step of paired analysis of said ratios of said biological material taken from a cancer patient and from a healthy patient. 11. Method according to any one of claims 8 to 10, characterized in that it implements a biochip, on which is deposited at least one of the probes specific sequences of said human Liv2l complex according to claim 1 or 2 and 3 or 4. 305vik., Lce / t / w Cara v sfi ~ ce. 7A7C1 (, 1,9 ', G-IR-c c, 2I'S 62e-e .. "... 1, 4-A / Q ,, (1 ~ to-and-te. e .--: 4f 10 15 20 25 30