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WO2015079185A1 - Novel biomarker of prostate cancer - Google Patents

Novel biomarker of prostate cancer Download PDF

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Publication number
WO2015079185A1
WO2015079185A1 PCT/FR2014/053079 FR2014053079W WO2015079185A1 WO 2015079185 A1 WO2015079185 A1 WO 2015079185A1 FR 2014053079 W FR2014053079 W FR 2014053079W WO 2015079185 A1 WO2015079185 A1 WO 2015079185A1
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Prior art keywords
fragment
prostate cancer
concentration
cells
lncap
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PCT/FR2014/053079
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French (fr)
Inventor
Pascal MARIOT
Morad ROUDBARAKI
Natalia Prevarskaya
Marine WARNIER
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Universite Des Sciences Et Technologies De Lille
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Publication of WO2015079185A1 publication Critical patent/WO2015079185A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57434Specifically defined cancers of prostate
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/112Disease subtyping, staging or classification
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/56Staging of a disease; Further complications associated with the disease

Definitions

  • the invention relates to the use of ⁇ 2 ⁇ 2 protein or a fragment thereof as a biomarker of prostate cancer of a subject.
  • Prostate cancer is one of the most common types of cancer in men, and is one of the leading causes of cancer morbidity and mortality worldwide. Prostate cancer is an increasingly important public health problem due in part to increased life expectancy. For example, in France, it ranks first among cancers with about 71,000 new cases in 2011 (1).
  • prostate cancer Currently, the most common technique for diagnosing prostate cancer is based on anatomo-pathological criteria usually obtained from prostate biopsies indicated following elevated blood levels of PSA (Prostate Specifies Antigen) and / or Rectal touch.
  • PSA Prostate Specifies Antigen
  • This PSA protein is secreted by the prostate and its rate rises when there is a benign or malignant inflammatory or tumoral prostatic pathology (cancer).
  • the measurement of PSA level in the blood thus indicates the probability of developing prostate cancer, but there may be errors, especially in the screening of PSA, or because of the fact that this test is not specific to prostate cancer.
  • the digital rectal exam leads to great divergences according to the practitioner, and is more used for the detection of advanced stages of prostate cancer.
  • Calcium channels are one of the main pathways of calcium entry into the nerve cell, actively participating in the cellular excitability and molecular processes of synaptic transmission.
  • Calcium channels are glycoprotein complexes whose central component is the ⁇ 1 subunit ; forming the pore, associated with auxiliary subunits ⁇ 2 ⁇ , ⁇ and ⁇ .
  • the subunit ⁇ 2 ⁇ is formed of two units, a 2 and ⁇ , coded by the same gene. These two parts are interconnected via two disulfide bridges.
  • the ⁇ 2 ⁇ subunit is essentially extracellular and is anchored to the plasma membrane by the part ⁇ of the protein by means of a group GlycosylPhosphatidylInositol (GPI).
  • GPI GlycosylPhosphatidylInositol
  • voltage-gated calcium channel subunits or their gene are often considered as serious factors in tumor growth.
  • Patent application EP 2 062 591 thus describes the possibility of using the CACNA1E gene of a voltage-dependent calcium channel, coding for the ⁇ 1 protein, as a marker for the diagnosis of cancers, in that it would be overexpressed in many cancers, such as lymphoma, breast, liver, lung, colon cancer, rectal cancer, ovarian, pancreatic, prostate, skin, uterine cancer.
  • CACNA1H gene encoding the am subunit, has been identified in US patent application 2008/0160009 A1 as a target for the treatment of prostate and breast cancer.
  • the CACNA2D2 gene has also been studied in several studies. This gene codes for the two subunits a 2 and ⁇ 2.
  • the CACNA2D2 gene has notably been located on the 3p21.3 locus, identified by numerous searches as a tumor suppressor gene locus (WO0204511, US 2003/0044911). Indeed, mutations of the 3p21 region of chromosome 3 are found in cancers of the lung, kidney, breast, pancreas and uterus. This locus has been shown to undergo many genetic modifications. The identification of mutations in the 3p21 region is thus the first genetic abnormality currently detected in lung cancer, suggesting that one or more genes in this region would act as tumor suppressors.
  • ⁇ 2 ⁇ subunit is inhibited in the majority of lung cancer cells, thus indicating that the alteration of calcium homeostasis in cancer cells leads to to a malignant tumor.
  • the subunit has been considered a tumor suppressor, inducing cell apoptosis (7, 8) and the origin of cancer, including lung and breast cancer.
  • the inventors have now discovered, very surprisingly and after much research, that the ⁇ 2 ⁇ 2 protein is closely related to tumorigenesis and angiogenesis in prostate cancer. This action on tumor growth goes against research demonstrating that the gene CACNA2D2, encoding this protein, is located in the region 3p21.3 which would be a cluster of tumor suppressor genes.
  • ⁇ 2 ⁇ 2 protein or a fragment thereof can serve as a biomarker for prostate cancer.
  • ⁇ 2 ⁇ 2 refers to the protein ⁇ 2 ⁇ 2 family subunit ⁇ 2 ⁇ voltages-dependent calcium channels and includes all synonyms referring to this subunit such as Cacna2d2, subunit 2 of voltages-dependent calcium channels alpha 2 / delta (or ⁇ 2 ⁇ ), alpha 2 / delta 2, a2d2.
  • This protein is encoded by the CACNA2D2 gene (Gene ID: 9254).
  • fragment refers, unless otherwise indicated, to a fragment of the ⁇ 2 ⁇ 2 protein and may for example relate to sub-part a 2 or sub-part ⁇ 2 , preferably sub-part -part a 2 .
  • the term "CANA2D2" as used herein refers to the gene encoding the ⁇ 2 ⁇ 2 subunit, and encompasses all synonyms referring to this gene (Gene ID: 9254).
  • prostate cancer refers to a prostate tumor, for example a malignant tumor, in a given subject, wherein the tumor is generally of epithelial origin.
  • tumor should be understood in its general meaning of the state of the art, and refers to the extent of a tumor of the prostate to another non-adjacent organ or element.
  • prostate cancer is understood to mean both adenocarcinoma (95% of malignant prostate tumors) and the rarer morphological variants, such as the precursor of prostate cancer, known as prostatic intraepithelial neoplasia, or neuroendocrine tumors (eg, small cell carcinoma, carcinoid tumors, adenocarcinoma with neuroendocrine differentiation).
  • prostatic intraepithelial neoplasia eg, small cell carcinoma, carcinoid tumors, adenocarcinoma with neuroendocrine differentiation.
  • the "aggression" of prostate cancer which can be used to determine the stage of cancer progression, can be quantified according to Gleason's classification based on the degree of differentiation, ie the existing gap. between a tumor tissue and a healthy prostate tissue, and the number of mitoses.
  • the classification of Gleason is noted with increasing aggression from grade 1 to grade 5, the higher the grade, the more aggressive the cancer, and the worse the prognosis.
  • the grade, indicated in the application before the parenthesis corresponds to the grade of Gleason of the cut studied.
  • the addition of the two most frequently observed grades on the different biopsies from a patient makes it possible to determine the Gleason score for each patient (between 0 and 10) and is indicated in parentheses (for example "2 + 3").
  • anti- ⁇ 2 ⁇ 2 antibodies refer to an antibody or fragment thereof that specifically recognizes ⁇ 2 ⁇ 2 or a fragment of ⁇ 2 ⁇ 2 , for example an a 2 or ⁇ 2 epitope.
  • predetermined value refers here to the amount of ⁇ 2 ⁇ 2 or a fragment of ⁇ 2 ⁇ 2 in biological samples obtained from the general population or from a selected population of subjects.
  • the selected population may be comprised of apparently healthy subjects, for example subjects who have never previously had any signs or symptoms indicating the presence of prostate cancer.
  • the predetermined value may be the amount of ⁇ 2 ⁇ 2 or a fragment in subjects with prostate cancer whose stage is established.
  • the predetermined value may be a threshold or a range.
  • the predetermined value can be established on the basis of comparative measurements between healthy subjects and subjects with established prostate cancer.
  • subject or “patient”, as understood here, refer to a monkey, a chimpanzee or a human. Preferably, it is a human.
  • normal or “healthy” may be used herein to refer to a subject, patient, sample, tissue, organ, or other non-cancer presenting subject.
  • biological sample denote any biological sample derived from the patient, for example but without limitation, biological fluids.
  • biological fluids such as blood, serum, plasma, urine or sperm. More preferably, it is a sample of urine or sperm.
  • biomarker generally refers to a molecule, ie a gene (or a nucleic acid encoding that gene), a protein whose expression can be determined in a biological sample of a subject by methods known in the state of the technique (as well as those described here) and which makes it possible to predict or deduce the state of the subject from which it has been obtained.
  • RNA is meant herein a small non-coding double-stranded interfering RNA that interferes with the translation of the messenger RNA into protein and destroys it, thereby inhibiting gene expression at the post-transcriptional stage.
  • si-a 2 ⁇ 2 is understood to mean interfering RNA against the coding sequence ⁇ 2 ⁇ 2 .
  • LNCaP refers to a human prostate cancer cell line derived from a supraclavicular ganglion of a patient with androgen-resistant prostate cancer. LNCaP lines overexpressing ⁇ 2 ⁇ 2 protein relative to normal LNCaP lines are referred to as “LNCaP-a 2 ⁇ 2 ".
  • the cell line "PC3" a prostate cancer cell line, is obtained from a bone metastasis of a patient with androgen-resistant cancer.
  • the cell line "DU 145" is obtained from a cerebral metastasis in a patient with prostate cancer.
  • the present invention relates to a method for in vitro detection of prostate cancer and / or prostatic metastasis in a subject comprising the steps of:
  • step (ii) comparing the concentration of ⁇ 2 ⁇ 2 , or a fragment of ⁇ 2 ⁇ 2 , measured in step (i) with a predetermined value of the concentration of ⁇ 2 ⁇ 2 , or a fragment of ⁇ 2 ⁇ 2 ,
  • a concentration of ⁇ 2 ⁇ 2 , or of a fragment of ⁇ 2 ⁇ 2 , in the biological sample greater than said predetermined value indicates the presence of a prostate cancer in said subject, and a concentration of ⁇ 2 ⁇ 2 , or a fragment of ⁇ 2 ⁇ 2 , less than said predetermined value indicates the absence of a prostate cancer in said subject.
  • the predetermined value relates to the amount of ⁇ 2 ⁇ 2 or a fragment of ⁇ 2 ⁇ 2 in biological samples obtained from apparently healthy subjects, for example subjects which have never previously presented any signs or symptoms indicating the presence of prostate cancer.
  • a method for detecting in vitro prostate cancer and / or prostatic metastasis in a subject may thus consist in carrying out the steps of:
  • step (ii) comparing the concentration of ⁇ 2 ⁇ 2 , or a fragment of ⁇ 2 ⁇ 2 , measured in step (i) with a predetermined value derived from the ⁇ 2 ⁇ 2 concentration, or a fragment of ⁇ 2 ⁇ 2 , in a biological sample of a healthy subject not suffering from prostate cancer, a high level of ⁇ 2 ⁇ 2 , or of a fragment of ⁇ 2 ⁇ 2 , in the biological sample by to said predetermined value indicating that the subject is suffering from prostate cancer.
  • the inventors have shown that the ⁇ 2 ⁇ 2 protein is expressed more strongly in the advanced stages of cancer (see FIG. 6, G2 and G3) than in the normal and early stages of cancer (see FIG. 6, normal stages, BPH). and metastasis).
  • the present invention therefore also relates to a method for determining in vitro the stage of prostate cancer in a subject comprising the steps of:
  • step (ii) comparing the concentration of ⁇ 2 ⁇ 2 , or a fragment of ⁇ 2 ⁇ 2 , measured in step (i) with a predetermined value of the concentration of ⁇ 2 ⁇ 2 , or a fragment of ⁇ 2 ⁇ 2 ,
  • the predetermined value relates here to the quantity of ⁇ 2 ⁇ 2 or of a fragment of ⁇ 2 ⁇ 2 in biological samples obtained from apparently healthy subjects, for example subjects which have never before presented signs or symptoms indicating the presence of prostate cancer.
  • the predetermined value here is the amount of ⁇ 2 ⁇ 2 or a fragment of ⁇ 2 ⁇ 2 in subjects with prostate cancer whose stage is established.
  • the biological sample is preferably a biological fluid, preferably a blood sample, a serum sample, a plasma sample, a urine sample or a sperm sample, more preferably a urine or sperm sample. According to a particularly advantageous embodiment, it is a urine sample possibly collected after massage of the prostate of the subject to be tested.
  • such a method comprises the step of contacting the sample with a binding molecule capable of binding specifically with the ⁇ 2 ⁇ 2 protein present in the biological sample.
  • the binding molecule may be specific for the mature form of the ⁇ 2 ⁇ 2 protein.
  • the binding molecule can also recognize the two parts a 2 and ⁇ 2 of the ⁇ 2 ⁇ 2 subunit.
  • the binding molecule may be capable of binding specifically with a fragment of the ⁇ 2 ⁇ 2 protein, for example with the a 2 part or with the ⁇ 2 part of the ⁇ 2 ⁇ 2 subunit.
  • the binding molecule may be a polyclonal or monoclonal antibody, preferably monoclonal.
  • the binding molecule may also be an aptamer.
  • Polyclonal antibodies of the invention, or fragments thereof, can be determined by methods known to those skilled in the art, for example by administering an appropriate antigen or epitope to an animal, for example a pig, a cow, a horse, a rabbit, a goat, a sheep, a mouse or other. Adjuvants known from the state of the art can be used to improve the production of antibodies.
  • Polyclonal antibodies which make it possible to recognize ⁇ 2 ⁇ 2 and which can be used in the present invention are, for example but without limitation, H210 (Santa Cruz) or A01 (Abnova). Although polyclonal antibodies are convenient for carrying out the invention, monoclonal antibodies are preferred.
  • Monoclonal antibodies of the invention, or fragments thereof, can be prepared and isolated using methods known to those skilled in the art.
  • the binding molecules of the invention such as antibodies or aptamers, can be labeled with a molecule or substance, for example a fluorescent molecule, an enzyme capable of producing a colored product, a radioactive molecule or other markers. known from the state of the art.
  • a molecule or substance for example a fluorescent molecule, an enzyme capable of producing a colored product, a radioactive molecule or other markers. known from the state of the art.
  • the concentration of the ⁇ 2 ⁇ 2 protein, or of a fragment of ⁇ 2 ⁇ 2 is measured using an antibody or a fragment thereof, specific for said ⁇ 2 ⁇ 2 protein, or a fragment of ⁇ 2 ⁇ 2 .
  • the protein concentration can be measured using standard techniques known to those skilled in the art, generally immunodiagnostic techniques. including immunoassays such as competition, direct reaction or sandwich type tests. Such tests include, but are not limited to, agglutination tests, enzyme-labeled and mediated immunoassays such as ELISA, biotin / avidin, radio immunoassays, immunoelectrophoresis, immunoprecipitation.
  • immunoassays such as competition, direct reaction or sandwich type tests.
  • Such tests include, but are not limited to, agglutination tests, enzyme-labeled and mediated immunoassays such as ELISA, biotin / avidin, radio immunoassays, immunoelectrophoresis, immunoprecipitation.
  • the measurement of the protein concentration may also include a step of separating the compounds such as by HPLC (based on hydrophobicity), size exclusion chromatography (size-based), and / or solid phase affinity (based on size). affinity of the compound for the solid phase used).
  • the ⁇ 2 ⁇ 2 protein can be identified by means of known separation profiles for the protein, for example the retention time, and measured according to standard techniques. According to another embodiment, it may be an identification and a measurement of a fragment, for example a 2 or ⁇ 2 .
  • the separated compounds can be detected and measured for example by mass spectrometry.
  • a control value or normalization can be used.
  • the amount of creatinine can be used to normalize the result obtained. Since creatinine is a good indicator of renal function, it is used, for example, as a standard for the determination of narcotic drugs or doping substances to evaluate whether the urine sample is diluted or as a standard for poisoning. heavy metals. Its value can therefore be used in the detection methods of the present invention.
  • the value of the increase in the ratio of ⁇ 2 ⁇ 2 / creatinine (or a fragment of ⁇ 2 ⁇ 2 / creatinine) to the ratio of ⁇ 2 ⁇ 2 / creatinine (or a fragment of ⁇ 2 ⁇ ) 2 / creatinine) of a healthy subject indicates the presence of prostate cancer.
  • a 50% increase may indicate that the subject is suffering from prostate cancer.
  • the detection methods according to the invention can also be used in addition to another detection method known to those skilled in the art, for example, but in a non-limiting manner, analysis of the PS A assay.
  • the present invention also relates to an anti-cancer agent for use in the treatment of prostate cancer in a subject characterized by detecting said prostate cancer using a method as previously described.
  • a method of treating prostate cancer in a subject can thus include the steps of:
  • Treatment prostate cancer can be understood as any treatment that can, for example, suppress tumor or metastasis, reduce the risk of recurrence, slow tumor development or metastasis, and / or treat symptoms of cancer. disease.
  • the treatment may, for example but not limited to, consist of hormone treatment, for example gonadotropin releasing hormone (LHRH) agonists (such as leuprolide, goserelin or buserelin), antiandrogen treatment (such as flutamide). or nilutamide), treatment with drugs that prevent the adrenal glands from producing androgens (such as ketoconazole or aminoglutethimide), or estrogen.
  • LHRH gonadotropin releasing hormone
  • antiandrogen treatment such as flutamide
  • nilutamide treatment with drugs that prevent the adrenal glands from producing androgens (such as ketoconazole or aminoglutethimide), or estrogen.
  • the present invention also relates to a method for monitoring a treatment of a subject suffering from prostate cancer and / or prostatic metastasis comprising determining the concentration of ⁇ 2 ⁇ 2 , or a fragment, in a biological sample obtained from said subject, and optionally comparing said concentration of ⁇ 2 ⁇ 2 , or a fragment, to a predetermined value representing a predetermined stage of prostate cancer, the concentration of ⁇ 2 ⁇ 2 , or of a fragment, with respect to said predetermined value indicating the evolution of prostate cancer, and thus the effectiveness of the treatment.
  • the present invention finally relates to the use of the ⁇ 2 ⁇ 2 protein, or a fragment, as a biomarker of prostate cancer of a subject.
  • said fragment is ⁇ 2 . It has indeed been demonstrated that the detection of ⁇ 2 ⁇ 2 protein or of a fragment makes it possible to conclude on the presence or absence of prostate cancer, as well as on the stage of cancer of the prostate. prostate.
  • FIGURES It has indeed been demonstrated that the detection of ⁇ 2 ⁇ 2 protein or of a fragment makes it possible to conclude on the presence or absence of prostate cancer, as well as on the stage of cancer of the prostate. prostate.
  • FIG. 1 Representation of the fluorescence of the secondary antibody as a function of the ⁇ 2 ⁇ 2 peptide concentration over the entire concentration range of the ⁇ 2 ⁇ 2 peptide (from 6.25 ⁇ g / ml to 3 ⁇ g / ml) (" Alexa488 ": fluorescein).
  • Figure 2 A: Analysis of the Expression of ⁇ 2 ⁇ 1; ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 in the LNCaP and DU145 cell lines by RT-PCR screening. Pactin and a positive control sample (“brain”) are used as a control.
  • PC3 and DU! 45 by RT-PCR screening are used as a control.
  • Pactin is used as a control.
  • Figure 3 Analysis of the expression of ⁇ 2 ⁇ 2 in 10 cancer tissues (numbered from C1 to C10) and 10 non-cancerous tissues (numbered NI to N10) of the prostate by RT-PCR. Pactin is used as a control.
  • Figure 4 Western blot analysis showing that si-a 2 ⁇ 2 (50 nM, 4 days) well inhibited the expression of ⁇ 2 ⁇ 2 in LNCaP cells. Calnexin is used as a control.
  • Figure 6 Analysis of the expression of ⁇ 2 ⁇ 2 at different stages of prostate cancer by immunohistochemical analysis.
  • a and B an overview of prostate tissues from normal, hyperplastic and cancerous tissues (A: objective * 5, B: objective * 40). The grades shown before the parenthesis correspond to the most common Gleason grade in the cut shown. Between parentheses are indicated Gleason scores of the corresponding patient. M: rectal metastasis, BPH: hyperplasia, N: normal prostatic tissue. Immunohistochemical staining against ⁇ 2 ⁇ 2 was revealed by DAB-peroxidase reaction.
  • C a) Overview of prostate tissue from normal ("normal"), metastatic ("metastatic"), hyperplastic (“BHP”) or cancerous (“cancer”) tissues. The stained images were deconvolved and the staining intensity of the cell was analyzed and converted to optical density. The optical density is increased in the acini surrounding the cells of the cancerous tissues;
  • Figure 7 Analysis of the effect of the ⁇ 2 ⁇ 2 protein on cell proliferation.
  • A a) Effect of a 4-day treatment using si-a 2 ⁇ 2 (50 nM) on cell growth (MTS method) compared to a control, si-ctl, which, at the same concentration, has no influence on cell proliferation;
  • Figure 8 In vivo analysis of cell growth induced by the ⁇ 2 ⁇ 2 protein.
  • A Weight in grams of LNCaP (control) and LNCap-a 2 ⁇ 2 (clone Ci1) tumors at sacrifice;
  • FIG. 9 Histological analysis of LNCaP and LNCaP-a 2 ⁇ 2 A tumors: Various magnified views of: a: the control LNCaP tumor; c: the tumor LNCaP-a 2 ô 2 .
  • the images b (LNCaP control) and tumor LNCaP-a 2 ô 2 ) show by means of arrows the foci of necrosis and inflammation.
  • PCNA nuclei of dividing cells
  • Figure 10 Analysis of the effect of ⁇ 2 ⁇ 2 on the formation of blood vessels in LNCaP xenografted tumors.
  • Figure 11 Analysis of the effect of ⁇ 2 ⁇ 2 on angiogenesis by the secretion of VEGF.
  • A a. Western blots of CD31 and VEGF in 8 different tumors from 4 different mice (Ml to M4) (LNCaP tumors: “T-LNCaP", LNCaP-a 2 ô 2 tumors: "T- ⁇ 2 ⁇ 2 "). Pactin is used as a control.
  • A Western blot of ⁇ 2 in the culture medium of LNCaP cells, two clones overexpressing ⁇ 2 ⁇ 2 (clone Ci and C16) and in the culture medium alone ("RPMI"), without DTT (6 days d incubation) or with DTT, ie under reducing conditions (15 min incubation).
  • A Representation of the fluorescence of the secondary antibody as a function of the ⁇ 2 ⁇ 2 peptide concentration in the concentration range close to the detection threshold, with representation of the concentration of peptide ⁇ 2 ⁇ 2 in total urine and in proteins obtained from urine (proteins precipitated with acetone and suspended in PBS buffer) of a man suffering from cancer of the prostate (see B).
  • the LNCaP, DU 145 and P3 cell lines originate from the American Type Culture Collection and were cultured, according to the supplier's recommendations, in RPMI 1640 (GIBCO, Life Technologies, France) supplemented with 10% fetal bovine serum ( FBS, Sigma, Isle d'Abeau, France) and 2 mM L-glutamine (Sigma, L'Isle d'Abeau, France).
  • the cells were systematically cultured in 75 cm 2 containers (Nunc, Poly-Labo, France) in a humidified atmosphere at 37 ° C. (95% air - 5% CO2). The culture medium was then changed every three days.
  • LNCaP- ⁇ 2 ⁇ 2 LNCaP stable cell lines expressing the ⁇ 2 ⁇ 2 protein
  • the small interfering RNAs come from Eurogentech (France).
  • si-a 2 ⁇ 2 were validated in the LNCaP cells. For this, it has been shown by Western-blot that si-a 2 ⁇ 2 decreases the expression of the ⁇ 2 ⁇ 2 protein in LNCaP cells (FIG. 3).
  • siRNAs used herein include a non-specific control siRNA against luciferase (hereinafter referred to as "si-ctl” or “siCTL”).
  • siRNAs are presented in Table 1. siRNA Position
  • the cells were transfected with 20 or 50 nM siRNA using HiPerFect Transfection Reagent (Qiagen) as previously reported in the publication by Florian Gackière et al (10).
  • RNA extraction was performed with Trizol® using a method originally described by Chomczynski and Sacchi (11).
  • the RT-PCR method is equivalent to that described in the publication by Florian Gackière et al (10).
  • the cDNA obtained was used for amplification by PCR using a Taq Gold kit (Applied Biosystems).
  • PCR products were analyzed on an agarose gel (1.5% or 2%) comprising ethidium bromide (0.5 ⁇ g / ml) then visualized under UV. The images were then captured using Gelcapture software (Bio-imaging System).
  • the cells were scraped into lysis buffer (1% Triton X-100, 1% Na deoxycholate, 150 mM NaCl, 10 mM PO 4 NaK, pH 7, 2) with a mixture of anti-proteases and then incubated in ice for 45 min.
  • the lysates were centrifuged at 12000G for 10 min at 4 ° C.
  • the protein concentration of the supernatant was determined by BCA test (Pierce Chemical Company).
  • the Western blot analyzes of the expression of the proteins were carried out as described in the publication by Florian Gackière et al (10).
  • Ki-67 or PCNA Proliferating Cell Nuclear Antigen
  • the cells were cultured in three 60mm wells per state, and the treatments or siRNAs were applied as previously described. After treatment, the cells were trypsinized, harvested and resuspended in 0.2 ml of sterile PBS. 1 ml of 70% cold ethanol was added to the cells in suspension during the vortexing step. The samples were centrifuged, washed in sterile PBS and then incubated with ribonuclease (2 ⁇ g / ml) for 15 min at room temperature. Propidium iodide (25 ⁇ g / ml final in PBS-0.1% triton X-100) was added and incubated for an additional 30 min at room temperature.
  • the DNA content was measured by excitation of propidium iodide at 488 nm and measured emission at 520 nm using a flow cytometer (Beckman Coulter Epies XL4-MCL with Expo32 acquisition). Data interpretation was performed with Multicycle for Windows (Phoenix Flow System).
  • PCA prostate cancer cells and tissues
  • Tissue microarrays and tumor tissues of mice were studied using immunohistochemical methods.
  • Multi-tumor tissue DNA microarray slides are from Biomax (USA). Tumors were analyzed, as well as the corresponding normal tissues. Supplier (Biomax) provides clinical information (age of patients, grades of cancers and grades of
  • the cells were incubated with Alexa fluor-labeled anti-rabbit IgG-488 secondary antibody (A-21206, Molecular tests, dilution 1: 2000) diluted in PBS for 1h at room temperature. After three rinses in PBS, the slides were mounted with Mowiol ® . The DAB-horseradish peroxidase detection technique was used in immunohistochemical analyzes. Azure B was used to counter-stain tissue slides.
  • Alexa fluor-labeled anti-rabbit IgG-488 secondary antibody A-21206, Molecular tests, dilution 1: 2000
  • the confocal and immunostaining observations were performed as previously described by means of a Zeiss LSM 510 confocal microscope (Cari Zeiss, Le Pecq, France) linked to an inverted microscope for Zeiss Axiovert 200 M episodopy and epifluorescence with a lens. oil immersion objective x63 (numerical aperture 1.4).
  • Anti-Ki67 antibody was used to evaluate the percentage of proliferating cells. At least 500 cells per slide and three slides per state were counted. Anti-PCNA antibody was used to evaluate the percentage of S-phase cells in the tumor samples. An immunofluorescence of anti-CD31 was used to evaluate the number of blood vessels in xenografted tumors. Polyclonal anti 2 O 2 were used to study the expression of ⁇ 2 ⁇ 2 in the tissues and cells of the prostate.
  • VEGF vascular endothelial growth factor
  • the cells were incubated for 6 days without changing the culture medium. At the end of this period, the culture medium was removed and centrifuged (cell culture medium).
  • the cells were then incubated for 15 min with 10mM DTT to cleave the disulfide bridges and the medium was again removed and centrifuged. Proteins from both supernatants (with and without DTT) were precipitated with acetone. The concentrate was resuspended in RIPA. The protein content was measured and ⁇ 2 ⁇ 2 was analyzed after SDS-page electrophoresis (6 or 8% acrylamide) and a western blot. The expression of ⁇ 2 ⁇ 2 in the cell culture medium was compared with the culture medium alone (without cells).
  • mice Six weeks old male Swiss nude mice (Charles River Laboratories, France) received an injection on each flank of LNCaP cells or LNCaP cells overexpressing ⁇ 2 ⁇ 2 . Six million cells were injected on both flanks of each mouse. The cells were prepared in a mixture of 50% PBS and 50% BD-Matrigel ® . The tumors were measured twice a week and the animals were sacrificed when the tumor size reached 10% of the weight of the animal. On the day of sacrifice, the tumors were weighed and divided for immunohistochemistry, western blot and additional RT-PCR. At least 10 animals per state were used.
  • the test was conducted according to Da Silva et al. (2000) with some modifications.
  • the wells of the plates (of 96 wells) were incubated with 200 ⁇ l PBS (phosphate-buffered saline solution) containing the ⁇ 2 ⁇ 2 peptide (AVIVA SYSTEM BIOLOGY reference AAP64646) diluted at different concentrations (from 6.25 ⁇ g / ml to 3 ⁇ g / ml). pg / ml) with the exception of control wells containing only 200 ⁇ l of PBS buffer.
  • the plates were subsequently incubated for 1h at 37 ° C and then at 4 ° C overnight.
  • the plates were then washed 3 times with PBS wash buffer. Then, the wells were incubated with 200 ⁇ l of aspecific site blocking buffer (PBS + 1% BSA) for 1 hour at room temperature. Then, the wells are emptied and incubated with the anti- ⁇ 2 ⁇ 2 antibody (reference AVIVA SYSTEM BIOLOGY antibody ARP64646_P050) (developed in the rabbit) diluted to 1/500 in blocking buffer (100 ⁇ ). The plates are incubated at 4 ° C overnight.
  • aspecific site blocking buffer PBS + 1% BSA
  • the wells are emptied and incubated with the anti- ⁇ 2 ⁇ 2 antibody (reference AVIVA SYSTEM BIOLOGY antibody ARP64646_P050) (developed in the rabbit) diluted to 1/500 in blocking buffer (100 ⁇ ).
  • the plates are incubated at 4 ° C overnight.
  • the wells are incubated 1 h at 4 ° C. in the presence of a secondary antibody (Goat Anti-Rabbit IgG, Jackson Immunoreasearch Lab) conjugated to fluorescein (Alexa Fluor 488 from Lifetechnologies) at a dilution of l / 2000e in a total volume of 100 ⁇ of the blocking buffer.
  • a secondary antibody Goat Anti-Rabbit IgG, Jackson Immunoreasearch Lab conjugated to fluorescein (Alexa Fluor 488 from Lifetechnologies) at a dilution of l / 2000e in a total volume of 100 ⁇ of the blocking buffer.
  • the wells are washed three times with wash buffer (PBS) and the fluorescence is measured at the wells using a fluorescence plate reader. After the fluorescence subtraction of the controls, the fluorescence variation is plotted as a function of the concentrations of the ⁇ 2 ⁇ 2 peptide. This made it possible to estimate the sensitivity of the assay method.
  • FIG. 1 shows the fluorescence of the secondary antibody as a function of the ⁇ 2 ⁇ 2 peptide concentration over the entire concentration range of the ⁇ 2 ⁇ 2 peptide.
  • FIG. 13A shows an enlargement of this curve in the concentration range close to the detection threshold. This threshold, determined by derivation of the experimental curve obtained, is about 10 ng / ml. Determination of the protein o ⁇ in the urine
  • Urine samples from patients obtained without prostate massage (20 ml) are concentrated 10 times using the acetone concentration method. After the determination of the urinary samples, 4 ⁇ g of each urinary sample is incubated in the wells (96-well plate) in a total volume of 100 ⁇ PBS ("urine proteins"). In order to check whether a direct dose of 2 2 is possible in the urine, at the same time
  • total urine 50 ⁇ of urine from each patient are deposited in wells in a total volume of 100 ⁇ in the presence of PBS ("total urine"). From this step, all the operations for all the samples are carried out as previously described for the realization of the ⁇ 2 ⁇ 2 peptide detection range.
  • ⁇ 2 ⁇ 2 in prostate cancer tissues and cell lines II has been shown in previous studies that Cav3.2 voltage-gated calcium channels are expressed in prostate cancer cell lines and in prostate cancer cells. tumors of the human prostate (5). It has been investigated here whether the supposed regulatory subunits can also be expressed in these cells. Attention has been given to ⁇ 2 ⁇ subunits since two of them ( ⁇ 2 ⁇ 2 and ⁇ 2 ⁇ 3 ) are suspected of acting as tumor suppressors in different organs.
  • the RT-PCR analyzes demonstrated that ⁇ 2 ⁇ 2 is present in the normal and cancerous tissues of the prostate. Immunodetection tests were also performed to demonstrate the expression of ⁇ 2 ⁇ 2 proteins in prostate cancer cell lines and in prostate tissue sections. Immunofluorescence assays showed that the LNCaP prostate cancer cell lines, DU! 45 and PC3 are strongly stained by anti- ⁇ 2 ⁇ 2 antibodies. It is further observed that the ⁇ 2 ⁇ 2 staining is evident on the cell, but it is particularly pronounced on the periphery, that is to say near the plasma membrane, where the ⁇ 2 ⁇ 2 proteins are anchored ( not shown).
  • Immunofluorescence (IF) and immunohistochemical (IH) staining of prostate cancer sections obtained from the subjects were performed on sections of frozen prostate tissue (IF) or paraffin sections (IH reaction peroxidase). It has been shown that there is a strong staining of the glandular epithelial structures. Acini are frequently stained with anti- ⁇ 2 ⁇ 2 antibodies (not shown). In addition, the apical membranes of the epithelium are more strongly stained than basolateral membranes (not shown).
  • the immunostaining of the prostate cancer epithelial cells in the primary culture medium thus revealed a strong ⁇ 2 ⁇ 2 staining on the periphery of the cells.
  • the expression of ⁇ 2 ⁇ 2 increases with the development of prostate cancer
  • ⁇ 2 ⁇ 2 is more frequently expressed in the cancerous tissues (95% of the cancerous tissues express ⁇ 2 ⁇ 2 ) than in the non-cancerous tissues (40% of the non-cancerous tissues express ⁇ 2 ⁇ 2 ) (significant difference, p ⁇ 0.001, Chi2 test).
  • ⁇ 2 ⁇ 2 was compared on macroarrays of 24 different tissues including 16 cancerous tissues and 8 non-cancerous tissues.
  • the epithelial cells covering the acini are stained with anti- ⁇ 2 ⁇ 2 antibodies in normal, hyperplastic and cancerous samples.
  • a number of colored cells leave the acini (see Figure 6B, G3, Metastasis).
  • Several grades of differentiation were analyzed. Colored area and staining intensity increase in advanced stages of prostate cancer compared to normal or hyperplastic prostate.
  • the localization in the cell is different between the normal, BPH and early stages (G1) of cancer and the advanced stages of cancer (G2, G3).
  • BPH and early stages of cancer Gl
  • staining is predominantly apical and intracellular
  • advanced stages of cancer G2 and G3
  • staining increases in all compartments of the cell, and more particularly in the cell nucleus.
  • Staining density was also analyzed using color deconvolution to extract counterstain DAB staining (azure blue) and DAB density was shown to be higher in advanced stages of cancer, thus making evidence that ⁇ 2 ⁇ 2 expression is increased in advanced prostate cancers (Figure 6C).
  • ⁇ 2 ⁇ 2 is expressed in epithelial cells of prostate cancer, it has been investigated whether the protein can participate in cell growth.
  • FIG. 7C Kinetic experiments illustrate that si-a 2 ⁇ 2 is a significant inhibitor of cell proliferation after only three days of incubation and that a maximal effect is reached after 5 days to 6 days (40% of reduction of cell proliferation).
  • FACS cell cycle analyzes show that this inhibitory action of si-a 2 ⁇ 2 on cell proliferation is correlated with an increase in the number of cells in G 1 phase ; as well as a decrease in the number of cells in G 2 / M phase and in S phase ( Figure 7D).
  • Gabapentin has been tested for cell proliferation, which is known to inhibit different voltage-gated calcium channels by its action on the ⁇ 2 ⁇ regulatory subunit. Indeed, gabapentin has been demonstrated to be a ligand of the two subunits 2 ⁇ i and ⁇ 2 ⁇ 2 (12). In the present tests, gabapentin, which has been added to cell culture medium at 100 ⁇ , reduces cell proliferation by 35%
  • LNCaP clones stably overexpressing ⁇ 2 ⁇ 2 were similarly used in cell proliferation assays. It has been shown that two different ⁇ 2 ⁇ 2 overexpressing clones (clone 9 "C9", and clone 11 "Ci l”) produce faster proliferation rates than LNCaP control cells, with a doubling time of 47. , 5h for LNCaP against 40.5h for clone 9 and 36h for clone 11 (see Figure 7F a).
  • Ki-67 dye immunofluorescence assays on LNCaP and LNCaP cell lines overexpressing ⁇ 2 ⁇ 2 (clone 11) were performed (see Figure 7F b).
  • Ki-67 is a marker of proliferating cells. It is only expressed during G 1 phases ; S, G 2 and M. Go-phase cells do not express Ki-67 significantly. There may be either a punctual presence in the nucleus during the G ⁇ and S phases, or a more homogeneous localization in the nucleus during the G 2 and M phases. It has been shown here that ⁇ 2 ⁇ 2 is associated with a decrease. of the number of cells during the Go phase of the cell cycle.
  • LNCaP tumor LNCaP- 2 ⁇ 2 tumor
  • Tumor size which was measured twice a week, began to increase usually after 4-6 weeks. Once the tumor was detectable, they grew with a doubling time of 12.2 ⁇ 1.9 days (see Figure 8a) for LNCaP tumors, reaching an average size of 940 ⁇ 146 mm (1.26 ⁇ 0.22 g) (see Figures 8b and c).
  • LNCaP-a 2 ⁇ 2 tumors grew with a doubling time of 6.6 ⁇ 0.8 days (see Figure 8a), reaching an average size of 1760 ⁇ 154 mm (2.3 ⁇ 0). 21 g) (see Figures 8b and c). For both types of tumors, the maximum tumor size was reached usually 4 weeks after the beginning of tumor growth for each mouse.
  • LNCaP-a 2 ⁇ 2 tumors are found in vivo to increase faster and are larger than LNCaP tumors.
  • the overexpression of ⁇ 2 ⁇ 2 induces the formation of blood vessels and the secretion of VEGF.
  • the two tumors LNCaP and LNCaP-a 2 ô 2 obtained in the in vivo tests described above are highly irrigated and are characterized by abundant blood vessels but also by signs of hemorrhages with leakage of red blood cells in neighboring tissues and many foci of inflammation and necrosis (see Figure 9A).
  • VEGF in two different clones of LNCaP-a 2 ⁇ 2 . This is not due to a general non-specific stimulation of secretion by LNCaP cells since the secretion of prostatic acid phosphatase (PAP) is not affected by the overexpression of ⁇ 2 ⁇ 2 , although the PAP is stimulated by an increase in intracellular calcium.
  • PAP prostatic acid phosphatase
  • ⁇ 2 ⁇ 2 can be cleaved in LNCaP cells overexpressing ⁇ 2 ⁇ 2 .
  • ⁇ 2 ⁇ 2 corresponds to a simple band of 175 kDa whereas under reducing conditions, two bands of 150 and 22 kDa are observed by western blots (13). It has thus been shown that a 2 and ⁇ 2 , derived from the same CACNA2D2 gene, are linked by disulfide bridges. a 2 is a broad extracellular subunit (150kDa or more dependent on glycosylation) related to ⁇ 2 , a smaller transmembrane protein (approximately 20kDa).
  • FIG. 12 shows that a 2 is present in the culture medium in which LNCaP-a 2 ⁇ 2 cells have been incubated for 6 days, whereas this subunit is hardly detectable in the culture medium of the control cells.
  • LNCaP. ⁇ 2 ⁇ 2 can be assayed in the urine of prostate cancer patients.
  • Figure 13B shows the concentrations of the protein encoded by the CACNA2D2 gene in total urine and in proteins obtained from urine of a man with prostate cancer.
  • This concentration is about 20 ng / ml, which is greater than the detection threshold as can be seen in FIG. 13A.
  • the ⁇ 2 ⁇ 2 protein was detected as regulating the activity of voltage-gated calcium channels. Although many studies have shown that the CACNA2D2 gene, encoding this protein, is located in the 3p21 locus, known as a cluster of tumor suppressor genes, it has been shown here that ⁇ 2 ⁇ 2 is capable of influencing itself on cell proliferation in vitro and on tumorigenesis in vivo. This ⁇ 2 ⁇ 2 protein, or a fragment thereof, represents a new biomarker for cancer, and in particular for prostate cancer. This is based on the following observations: (1) The ⁇ 2 ⁇ 2 calcium channel subunit, encoded by the CACNA2D2 gene, is expressed in prostate cells;
  • This protein, or its gene, is more frequently expressed in cancerous tissues than in non-cancerous tissues of the prostate;
  • ⁇ 2 ⁇ 2 is associated with secretion of VEGF and formation of blood vessels
  • the protein ⁇ 2 ⁇ 2 may be cleaved and the sub-portion 2 of the ⁇ 2 ⁇ protein 2 released into the extracellular medium of LNCaP cells 2-a O 2. It is detectable with antibodies available on the market.
  • the ⁇ 2 ⁇ 2 protein was detected in both cancerous and non-cancerous prostate cells by PCR and immunodetection, particularly on the apical membranes of the epithelium (observation (1)). PCR and immunohistochemical analyzes have shown that the ⁇ 2 ⁇ 2 protein is more expressed in prostate cancer tissue than in non-cancerous tissues (observation (2)).
  • the protein ⁇ 2 ⁇ 2 can be cleaved, releasing Subpart 2 of the protein ⁇ 2 ⁇ 2 in the extracellular medium of cells LNCaP-2 O 2.
  • the ⁇ 2 ⁇ 2 protein or the fragments of this protein, and more particularly the a 2 sub-part, are detectable with antibodies available on the market (observation (7)).
  • ⁇ 2 ⁇ 2 protein or fragments of this protein can similarly be detected in blood, serum, plasma, sperm and urine.
  • proteins anchored by GPI groups to the membrane can be cleaved by different enzymes, including certain phospholipases C or D, hydrolyzing these GPI groups and thus releasing the proteins in the extracellular medium. It has thus been demonstrated that the ⁇ 2 ⁇ 2 protein is present in the urine of prostate cancer patients.
  • the in vivo cleavage of the a 2 sub-part of the protein, releasing sub-part a 2 in the extracellular medium is strongly presumed. It is indeed known that, in the extracellular medium, the disulfide bridges can be cleaved by numerous enzymes such as disulphide isomerase or phosphoglycerate kinase (14), thus releasing 2 in the extracellular medium epithelial cells of the prostate. However, it has already been shown that these two enzymes are detected in the prostate and that their expression is increased during cancerous development (15, 16).
  • the CD90 (Cluster of Differentiation 90) protein is an N-glycosylated surface protein, anchored to the membrane by a GlycosylPhosphatidylInositol (GPI) group, such as the ⁇ 2 ⁇ 2 protein. It was originally found as a thymocyte antigen (17), and has been described as expressed in nerve tissue (18), hematopoietic progenitor cells (19), and in fibroblasts of many tissues such as the lungs or myometrium. lung (20). High levels of CD90 have been detected in malignant gliomas and hepatocarcinoma (21).
  • GPI GlycosylPhosphatidylInositol
  • the CD90 protein is multiplied by a factor of 5 in prostate cancer tissues compared to non-cancerous tissues (22).
  • a peptide derived from CD90 has thus been identified in the urine of prostate cancer patients by mass spectrometry, confirming that CD90 is secreted by prostate cancer tissue and can therefore serve as a biomarker for non-invasive tests (22). It is known that the release of proteins by the prostatic cells into the seminal fluid is effected on the one hand by the secretion of soluble proteins by exocytosis and, on the other hand, by the production of prostasomes, or exosomes, released by exocytosis from multivesicular bodies of epithelial cells.
  • exosomes found in the seminal fluid, contain a large number of proteins, for example membrane proteins, such as GLUT5 (Glucose Transporter 5), VAT-1 (synaptic vesicle membrane homologous protein) or SCA2 (synaptic carrier associated membrane protein).
  • membrane proteins such as GLUT5 (Glucose Transporter 5), VAT-1 (synaptic vesicle membrane homologous protein) or SCA2 (synaptic carrier associated membrane protein).
  • these prostasomes which are normally released in the seminal fluid, can be found in blood plasma during the development of prostatic cancers (23).
  • the ⁇ 2 ⁇ 2 protein is released into seminal fluid or blood plasma by these prostasomes and can be assayed in other body fluids than urine.
  • the ⁇ 2 ⁇ 2 protein, or a fragment thereof can be used as a biomarker of prostate cancer, as well as to detect the stage of cancer progression.
  • Voltage-gated K + channels support proliferation of colony carcinoma cells. Faseb J 21: 35-44.
  • He J, Liu Y, Zhu T, Zhu J, et al. CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays. Mol Cell Proteomics 2012; 11.

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Abstract

The invention relates to a method for in vitro detection of prostate cancer and/or prostatic metastasis in a patient, which includes the following steps: (i) measuring the concentration of α2δ2, or a fragment of α2δ2, in a biological sample obtained from said patient; (ii) comparing the concentration of α2δ2, or a fragment of α2δ2, measured in step (i) with a predetermined value of the concentration of α2δ2, or a fragment of α2δ2, in which a concentration of α2δ2, or of a fragment of α2δ2, in the biological sample that is higher than said predetermined value indicates the presence of prostate cancer in said patient, and a concentration of α2δ2, or of a fragment of α2δ2, that is lower than said predetermined value indicates the absence of prostate cancer in said patient.

Description

NOUVEAU BIOMARQUEUR DU CANCER DE LA PROSTATE  NEW BIOMARKER OF PROSTATE CANCER
L'invention concerne l'utilisation de la protéine α2δ2 ou un fragment de celle-ci comme biomarqueur du cancer de la prostate d'un sujet. The invention relates to the use of α 2 δ 2 protein or a fragment thereof as a biomarker of prostate cancer of a subject.
Le cancer de la prostate est un des types de cancer les plus fréquents chez les hommes, et est l'une des principales causes de morbidité et mortalité cancéreuse dans le monde. Le cancer de la prostate constitue un problème de santé publique de plus en plus important en partie du fait de l'augmentation de la durée de vie. Il se situe par exemple en France au premier rang des cancers avec environ 71 000 nouveaux cas en 2011 (1). Prostate cancer is one of the most common types of cancer in men, and is one of the leading causes of cancer morbidity and mortality worldwide. Prostate cancer is an increasingly important public health problem due in part to increased life expectancy. For example, in France, it ranks first among cancers with about 71,000 new cases in 2011 (1).
Actuellement, la technique la plus répandue pour diagnostiquer le cancer de la prostate est basée sur des critères anatomo-pathologiques habituellement obtenus à partir de biopsies de la prostate indiquées suite à une élévation du taux sanguin de PSA (Prostate Spécifie Antigen) et/ou un toucher rectal. Cette protéine PSA est sécrétée par la prostate et son taux s'élève lorsqu'il existe une pathologie prostatique inflammatoire ou tumorale bénigne ou maligne (cancer). La mesure du taux de PSA dans le sang indique ainsi la probabilité de développer un cancer de la prostate, mais il peut y avoir des erreurs, notamment dans le screening de la PSA, ou encore à cause du fait que ce test n'est pas spécifique du cancer de la prostate. Le toucher rectal, quant à lui, aboutit à de grandes divergences selon le praticien, et est plus utilisé pour la détection de stades avancés du cancer de la prostate. Currently, the most common technique for diagnosing prostate cancer is based on anatomo-pathological criteria usually obtained from prostate biopsies indicated following elevated blood levels of PSA (Prostate Specifies Antigen) and / or Rectal touch. This PSA protein is secreted by the prostate and its rate rises when there is a benign or malignant inflammatory or tumoral prostatic pathology (cancer). The measurement of PSA level in the blood thus indicates the probability of developing prostate cancer, but there may be errors, especially in the screening of PSA, or because of the fact that this test is not specific to prostate cancer. The digital rectal exam, on the other hand, leads to great divergences according to the practitioner, and is more used for the detection of advanced stages of prostate cancer.
Il existe donc toujours un besoin pour de nouvelles méthodes, de préférence non- invasives, de détection du cancer de la prostate et de détermination du stade du cancer de la prostate.  There is therefore still a need for new, preferably non-invasive, methods of detecting prostate cancer and determining the stage of prostate cancer.
Les canaux calciques représentent une des principales voies d'entrée du calcium dans la cellule nerveuse, participant activement à l'excitabilité cellulaire et aux processus moléculaires de la transmission synaptique. Les canaux calciques sont des complexes glycoprotéiques dont le composant central est la sous-unité a1; formant le pore, associée à des sous-unités auxiliaires α2δ, β et γ. La sous-unité α2δ est formée de deux unités, a2 et δ, codées par le même gêne. Ces deux parties sont reliées entre elles via deux ponts disulfures. La sous- unité α2δ est essentiellement extracellulaire et est ancrée à la membrane plasmique par la partie δ de la protéine au moyen d'un groupement GlycosylPhosphatidylInositol (GPI).Calcium channels are one of the main pathways of calcium entry into the nerve cell, actively participating in the cellular excitability and molecular processes of synaptic transmission. Calcium channels are glycoprotein complexes whose central component is the α 1 subunit ; forming the pore, associated with auxiliary subunits α 2 δ, β and γ. The subunit α 2 δ is formed of two units, a 2 and δ, coded by the same gene. These two parts are interconnected via two disulfide bridges. The α 2 δ subunit is essentially extracellular and is anchored to the plasma membrane by the part δ of the protein by means of a group GlycosylPhosphatidylInositol (GPI).
Quatre sous-parties de α2δ ( 2ôi à α2δ4), chacune codée par un gène, ont ainsi été découvertes. Four subparts of α 2 δ ( 2 δ 1 to α 2 δ 4 ), each encoded by a gene, have thus been discovered.
Alors que l'implication des canaux calciques dans les maladies cardiovasculaires, neurologiques ou métaboliques est connue depuis longtemps, leur rôle dans d'autres pathologies comme le cancer n'a été admis que récemment. Des études in vivo ou in vitro ont ainsi démontré l'implication des canaux calciques dans le développement cancéreux de différents tissus ou organes, comme la prostate, le sein, ou le cerveau (2, 3). Les canaux calciques participeraient en effet à l'invasion cellulaire, la migration, la différenciation et/ou à la prolifération (4, 5, 6). Il a ainsi été mis en évidence que différents canaux calciques, et notamment les canaux calciques voltage-dépendant, pouvaient être impliqués, soit en étant surexprimés, soit en étant sous-exprimés, dans la tumorigénèse. Dans la plupart des cas, la surexpression des canaux calciques accélère la prolifération cellulaire tandis que l'inhibition et la régulation à la baisse des canaux calciques diminuent la croissance cellulaire. While the involvement of calcium channels in cardiovascular, neurological or metabolic diseases has been known for a long time, their role in other pathologies such as cancer has only recently been acknowledged. In vivo or in vitro studies have demonstrated the involvement of calcium channels in the cancerous development of various tissues or organs, such as the prostate, breast, or brain (2, 3). Calcium channels would indeed participate in cell invasion, migration, differentiation and / or proliferation (4, 5, 6). It has thus been demonstrated that different calcium channels, and in particular the voltage-dependent calcium channels, could be involved, either by being overexpressed or by being under-expressed, in tumorigenesis. In most cases, calcium channel overexpression accelerates cell proliferation while inhibition and downregulation of calcium channels decreases cell growth.
II a, par exemple, été précédemment démontré par les inventeurs que les canaux calciques de type T voltage-dépendants Cav3.2 sont surexprimés dans les lignées cellulaires du cancer de la prostate, aboutissant vers un phénotype plus agressif (5).  For example, it has been previously demonstrated by the inventors that voltage-gated Cav3.2 T-type calcium channels are overexpressed in prostate cancer cell lines, resulting in a more aggressive phenotype (5).
Il a également précédemment été démontré par les inventeurs que ces canaux permettent la sécrétion de facteurs paracrines comme la sérotonine ou la phosphatase prostatique acide, pouvant jouer un rôle dans la progression cancéreuse.  It has also previously been demonstrated by the inventors that these channels allow the secretion of paracrine factors such as serotonin or acidic prostatic phosphatase, which may play a role in cancer progression.
Par ailleurs, les sous-unités des canaux calciques voltage-dépendants ou leur gène sont souvent considérés comme des facteurs sérieux dans la croissance tumorale. Moreover, voltage-gated calcium channel subunits or their gene are often considered as serious factors in tumor growth.
La demande de brevet EP 2 062 591 décrit ainsi la possibilité d'utiliser le gène CACNA1E d'un canal calcique voltage-dépendant, codant pour la protéine α^, en tant que marqueur pour le diagnostic de cancers, en ce qu'il serait surexprimé dans de nombreux cancers, comme le lymphome, le cancer du sein, du foie, du poumon, du colon, le cancer rectal, le cancer des ovaires, du pancréas, de la prostate, de la peau, de l'utérus.  Patent application EP 2 062 591 thus describes the possibility of using the CACNA1E gene of a voltage-dependent calcium channel, coding for the α 1 protein, as a marker for the diagnosis of cancers, in that it would be overexpressed in many cancers, such as lymphoma, breast, liver, lung, colon cancer, rectal cancer, ovarian, pancreatic, prostate, skin, uterine cancer.
De la même façon, le gène CACNA1H, codant pour la sous-unité am, a été identifié dans la demande de brevet US 2008/0160009 Al comme cible pour le traitement du cancer de la prostate et du sein.  Similarly, the CACNA1H gene, encoding the am subunit, has been identified in US patent application 2008/0160009 A1 as a target for the treatment of prostate and breast cancer.
Le gène CACNA2D2 a, lui aussi, fait l'objet de plusieurs études. Ce gène code pour les deux sous-unités a2 et δ2. Le gène CACNA2D2 a notamment été localisé sur le locus 3p21.3, identifié par de nombreuses recherches comme un locus de gènes suppresseurs de tumeurs (WO0204511, US 2003/0044911). En effet, des mutations de la région 3p21 du chromosome 3 sont retrouvées dans les cancers du poumon, du rein, du sein, du pancréas et de l'utérus. Il a été montré que ce locus subit de nombreuses modifications génétiques. L'identification de mutations dans la région 3p21 est ainsi la première anomalie génétique détectée actuellement dans les cancers du poumon, suggérant qu'un ou plusieurs gènes de cette région agiraient comme suppresseurs de tumeurs. Il a par ailleurs été découvert par ces chercheurs que l'expression de la sous-unité α2δ est inhibée dans la majorité des cellules du cancer du poumon, indiquant ainsi que l'altération de l'homéostasie du calcium dans les cellules cancéreuses conduit à une tumeur maligne. La sous-unité a ainsi été considérée comme suppresseur de tumeur, induisant l'apoptose des cellules (7, 8) et l'origine du cancer, notamment le cancer du poumon et du sein. Les inventeurs ont à présent découvert, de manière très surprenante et après de nombreuses recherches, que la protéine α2δ2 a un lien étroit avec la tumorigénèse et l'angiogénèse dans le cancer de la prostate. Cette action sur la croissance tumorale va à l'encontre des recherches démontrant que le gène CACNA2D2, codant pour cette protéine, est localisé dans la région 3p21.3 qui serait un cluster de gènes suppresseurs de tumeurs. The CACNA2D2 gene has also been studied in several studies. This gene codes for the two subunits a 2 and δ 2. The CACNA2D2 gene has notably been located on the 3p21.3 locus, identified by numerous searches as a tumor suppressor gene locus (WO0204511, US 2003/0044911). Indeed, mutations of the 3p21 region of chromosome 3 are found in cancers of the lung, kidney, breast, pancreas and uterus. This locus has been shown to undergo many genetic modifications. The identification of mutations in the 3p21 region is thus the first genetic abnormality currently detected in lung cancer, suggesting that one or more genes in this region would act as tumor suppressors. It has also been discovered by these researchers that the expression of the α 2 δ subunit is inhibited in the majority of lung cancer cells, thus indicating that the alteration of calcium homeostasis in cancer cells leads to to a malignant tumor. The subunit has been considered a tumor suppressor, inducing cell apoptosis (7, 8) and the origin of cancer, including lung and breast cancer. The inventors have now discovered, very surprisingly and after much research, that the α 2 δ 2 protein is closely related to tumorigenesis and angiogenesis in prostate cancer. This action on tumor growth goes against research demonstrating that the gene CACNA2D2, encoding this protein, is located in the region 3p21.3 which would be a cluster of tumor suppressor genes.
II a ainsi été découvert que la protéine α2δ2 ou un fragment de celle-ci pouvait servir de biomarqueur au cancer de la prostate. It has thus been discovered that the α 2 δ 2 protein or a fragment thereof can serve as a biomarker for prostate cancer.
DEFINITIONS Le terme «α2δ2 », tel qu'utilisé ici, se réfère à la protéine α2δ2 de la famille des sous- unités α2δ des canaux calciques voltages-dépendants et englobe tous les synonymes se référant à cette sous-unité comme Cacna2d2, la sous-unité 2 des canaux calciques voltages- dépendants alpha 2/delta (ou α2δ), alpha 2/delta 2, a2d2. Cette protéine est codée par le gène CACNA2D2 (Gene ID: 9254). DEFINITIONS The term "α 2 δ 2" as used herein refers to the protein α 2 δ 2 family subunit α 2 δ voltages-dependent calcium channels and includes all synonyms referring to this subunit such as Cacna2d2, subunit 2 of voltages-dependent calcium channels alpha 2 / delta (or α 2 δ), alpha 2 / delta 2, a2d2. This protein is encoded by the CACNA2D2 gene (Gene ID: 9254).
Le terme « fragment » tel qu'utilisé ici se réfère, sauf indication contraire, à un fragment de la protéine α2δ2 et peut par exemple concerner la sous-partie a2 ou la sous-partie δ2, de préférence la sous-partie a2. Le terme « CANA2D2 », tel qu'utilisé ici, se réfère au gène codant pour la sous-unité α2δ2, et englobe tous les synonymes se référant à ce gène (Gene ID: 9254). The term "fragment" as used herein refers, unless otherwise indicated, to a fragment of the α 2 δ 2 protein and may for example relate to sub-part a 2 or sub-part δ 2 , preferably sub-part -part a 2 . The term "CANA2D2" as used herein refers to the gene encoding the α 2 δ 2 subunit, and encompasses all synonyms referring to this gene (Gene ID: 9254).
Le terme « cancer de la prostate », tel qu'utilisé ici, se réfère à une tumeur de la prostate, par exemple une tumeur maligne, chez un sujet donné, dans laquelle la tumeur est généralement d'origine épithéliale. The term "prostate cancer" as used herein refers to a prostate tumor, for example a malignant tumor, in a given subject, wherein the tumor is generally of epithelial origin.
Le terme « métastase » doit être entendu dans sa signification générale de l'état de la technique, et se réfère à l'étendue d'une tumeur de la prostate à un autre organe ou élément non-adjacent.  The term "metastasis" should be understood in its general meaning of the state of the art, and refers to the extent of a tumor of the prostate to another non-adjacent organ or element.
On entend ainsi par « cancer de la prostate » aussi bien l'adénocarcinome (95% des tumeurs malignes de la prostate) que les variantes morphologiques plus rares, comme le précurseur du cancer de la prostate, connu comme la néoplasie intraépithéliale prostatique, ou encore les tumeurs neuroendocrines (par exemple, carcinome à petites cellules, tumeurs carcinoïdes, adénocarcinome avec différenciation neuroendocrine). On peut ainsi se référer à Chang et al. (9) pour les différentes formes de cancer.  The term "prostate cancer" is understood to mean both adenocarcinoma (95% of malignant prostate tumors) and the rarer morphological variants, such as the precursor of prostate cancer, known as prostatic intraepithelial neoplasia, or neuroendocrine tumors (eg, small cell carcinoma, carcinoid tumors, adenocarcinoma with neuroendocrine differentiation). We can thus refer to Chang et al. (9) for different forms of cancer.
L' « agressivité » du cancer de la prostate, qui peut permettre de déterminer le stade d'avancement du cancer, peut être quantifiée selon la classification de Gleason fondée sur le degré de différenciation, c'est-à-dire l'écart existant entre un tissu tumoral et un tissu prostatique sain, et le nombre de mitoses. La classification de Gleason est notée selon une agressivité croissante du grade 1 au grade 5, plus le grade étant élevé, plus le cancer étant agressif et plus le pronostic étant pessimiste. Le grade, indiqué dans la demande avant la parenthèse, correspond au grade de Gleason de la coupe étudiée. L'addition des deux grades les plus fréquemment observés sur les différentes biopsies provenant d'un patient permet de déterminer le Score de Gleason pour chaque patient (entre 0 et 10) et est indiqué entre parenthèses (par exemple « 2+3 »). The "aggression" of prostate cancer, which can be used to determine the stage of cancer progression, can be quantified according to Gleason's classification based on the degree of differentiation, ie the existing gap. between a tumor tissue and a healthy prostate tissue, and the number of mitoses. The classification of Gleason is noted with increasing aggression from grade 1 to grade 5, the higher the grade, the more aggressive the cancer, and the worse the prognosis. The grade, indicated in the application before the parenthesis, corresponds to the grade of Gleason of the cut studied. The addition of the two most frequently observed grades on the different biopsies from a patient makes it possible to determine the Gleason score for each patient (between 0 and 10) and is indicated in parentheses (for example "2 + 3").
Pour éviter toute confusion, les phases de cycles cellulaires habituellement connues et définies par « G0 », « Gl », « G2 » sont ici identifiées par « Go », « G », « G2 », sauf indication contraire. To avoid confusion, the cell cycle phases usually known and defined by "G0", "G1", "G2" are identified here by "Go", "G", "G 2 ", unless otherwise indicated.
Les termes « anticorps anti-a2ô2 » se rapportent à un anticorps ou un fragment de celui-ci qui reconnaît spécifiquement α2δ2 ou un fragment de α2δ2, par exemple un épitope de a2 ou δ2. Les termes « valeur prédéterminée » se rapportent ici à la quantité de α2δ2 ou d'un fragment de α2δ2 dans les échantillons biologiques obtenus de la population en général ou d'une population sélectionnée de sujets. The terms "anti-α 2 δ 2 antibodies" refer to an antibody or fragment thereof that specifically recognizes α 2 δ 2 or a fragment of α 2 δ 2 , for example an a 2 or δ 2 epitope. The term "predetermined value" refers here to the amount of α 2 δ 2 or a fragment of α 2 δ 2 in biological samples obtained from the general population or from a selected population of subjects.
Selon un exemple, la population sélectionnée peut être comprise de sujets apparemment sains, par exemple des sujets n'ayant jamais présenté auparavant de signes ou de symptômes indiquant la présence d'un cancer de la prostate.  In one example, the selected population may be comprised of apparently healthy subjects, for example subjects who have never previously had any signs or symptoms indicating the presence of prostate cancer.
Selon un autre exemple, la valeur prédéterminée peut être la quantité de α2δ2 ou d'un fragment chez des sujets ayant un cancer de la prostate dont le stade est établi. In another example, the predetermined value may be the amount of α 2 δ 2 or a fragment in subjects with prostate cancer whose stage is established.
La valeur prédéterminée peut être un seuil ou une fourchette.  The predetermined value may be a threshold or a range.
La valeur prédéterminée peut être établie sur la base de mesures comparatives entre des sujets sains et des sujets ayant un cancer de la prostate établi.  The predetermined value can be established on the basis of comparative measurements between healthy subjects and subjects with established prostate cancer.
Les termes « sujet » ou « patient », tels qu'entendus ici, désignent un singe, un chimpanzé ou un humain. De préférence, il s'agit d'un humain. The terms "subject" or "patient", as understood here, refer to a monkey, a chimpanzee or a human. Preferably, it is a human.
Les termes « normal » ou « sain » peuvent être utilisés ici pour désigner un sujet, patient, échantillon, tissu, organe ou autre ne présentant pas de cancer.  The terms "normal" or "healthy" may be used herein to refer to a subject, patient, sample, tissue, organ, or other non-cancer presenting subject.
Les termes « échantillon biologique » désignent tout échantillon biologique issu du patient, par exemple mais de façon non limitative, des fluides biologiques. De manière préférentielle, il s'agit de fluides biologiques comme le sang, le sérum, le plasma, l'urine ou le sperme. De préférence encore, il s'agit d'un échantillon d'urine ou de sperme. The terms "biological sample" denote any biological sample derived from the patient, for example but without limitation, biological fluids. Preferably, it is biological fluids such as blood, serum, plasma, urine or sperm. More preferably, it is a sample of urine or sperm.
Le terme « biomarqueur » désigne en général une molécule, i.e. un gène (ou un acide nucléique codant pour ce gène), protéine, dont l'expression peut être déterminée dans un échantillon biologique d'un sujet par des méthodes connues de l'état de la technique (aussi bien que celles décrites ici) et qui permet de prédire ou déduire l'état du sujet duquel elle a été obtenue. The term "biomarker" generally refers to a molecule, ie a gene (or a nucleic acid encoding that gene), a protein whose expression can be determined in a biological sample of a subject by methods known in the state of the technique (as well as those described here) and which makes it possible to predict or deduce the state of the subject from which it has been obtained.
On entend ici par « siRNA » un petit ARN interfèrent bicaténaire non codant qui interfère avec la traduction de l'ARN messager en protéine et le détruit, inhibant ainsi l'expression génique au stade post-transcriptionnel. By "siRNA" is meant herein a small non-coding double-stranded interfering RNA that interferes with the translation of the messenger RNA into protein and destroys it, thereby inhibiting gene expression at the post-transcriptional stage.
Ainsi, on entend par « si-a2ô2 » l'ARN interférant contre la séquence codant α2δ2. Thus, "si-a 2 δ 2 " is understood to mean interfering RNA against the coding sequence α 2 δ 2 .
« LNCaP » désigne une lignée cellulaire humaine de cancer de la prostate provenant d'un ganglion sus-claviculaire d'un patient atteint d'un cancer de prostate androgénorésistant. Les lignées LNCaP surexprimant la protéine α2δ2 par rapport aux lignées LNCaP normales sont désignées par « LNCaP-a2ô2 ». "LNCaP" refers to a human prostate cancer cell line derived from a supraclavicular ganglion of a patient with androgen-resistant prostate cancer. LNCaP lines overexpressing α 2 δ 2 protein relative to normal LNCaP lines are referred to as "LNCaP-a 2 δ 2 ".
La lignée cellulaire « PC3 », lignée cellulaire du cancer de la prostate, est obtenue à partir d'une métastase osseuse d'un patient atteint de cancer androgénorésistant.  The cell line "PC3", a prostate cancer cell line, is obtained from a bone metastasis of a patient with androgen-resistant cancer.
La lignée cellulaire « DU 145 » est obtenue à partir d'une métastase cérébrale chez un patient atteint d'un cancer de la prostate.  The cell line "DU 145" is obtained from a cerebral metastasis in a patient with prostate cancer.
DESCRIPTION DE L'INVENTION La présente invention concerne une méthode de détection in vitro d'un cancer de la prostate et/ou d'une métastase prostatique chez un sujet comprenant les étapes consistant à : DESCRIPTION OF THE INVENTION The present invention relates to a method for in vitro detection of prostate cancer and / or prostatic metastasis in a subject comprising the steps of:
(i) mesurer la concentration de α2δ2, ou un fragment de α2δ2, dans un échantillon biologique obtenu dudit sujet, (i) measuring the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in a biological sample obtained from said subject,
(ii) comparer la concentration de α2δ2, ou un fragment de α2δ2, mesurée à l'étape (i) à une valeur prédéterminée de la concentration en α2δ2, ou un fragment de α2δ2, (ii) comparing the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , measured in step (i) with a predetermined value of the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 ,
dans laquelle une concentration de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique supérieure à ladite valeur prédéterminée indique la présence d'un cancer de la prostate chez ledit sujet, et une concentration de α2δ2, ou d'un fragment de α2δ2, inférieure à ladite valeur prédéterminée indique l'absence d'un cancer de la prostate chez ledit sujet. in which a concentration of α 2 δ 2 , or of a fragment of α 2 δ 2 , in the biological sample greater than said predetermined value indicates the presence of a prostate cancer in said subject, and a concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , less than said predetermined value indicates the absence of a prostate cancer in said subject.
De manière avantageuse, la valeur prédéterminée se rapporte à la quantité de α2δ2 ou d'un fragment de α2δ2 dans les échantillons biologiques obtenus de sujets apparemment sains, par exemple des sujets n'ayant jamais présenté auparavant de signes ou de symptômes indiquant la présence d'un cancer de la prostate. Advantageously, the predetermined value relates to the amount of α 2 δ 2 or a fragment of α 2 δ 2 in biological samples obtained from apparently healthy subjects, for example subjects which have never previously presented any signs or symptoms indicating the presence of prostate cancer.
Une méthode de détection in vitro d'un cancer de la prostate et/ou d'une métastase prostatique chez un sujet peut ainsi consister à effectuer les étapes consistant à : A method for detecting in vitro prostate cancer and / or prostatic metastasis in a subject may thus consist in carrying out the steps of:
(i) mesurer la concentration de α2δ2, ou d'un fragment, dans un échantillon biologique obtenu dudit sujet, (i) measuring the concentration of α 2 δ 2 , or a fragment, in a biological sample obtained from said subject,
(ii) comparer la concentration de α2δ2, ou d'un fragment de α2δ2, mesurée à l'étape (i) à une valeur prédéterminée dérivée de la concentration en α2δ2, ou d'un fragment de α2δ2, dans un échantillon biologique d'un sujet sain ne souffrant pas de cancer de la prostate, un niveau élevé de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique par rapport à ladite valeur prédéterminée indiquant que le sujet souffre d'un cancer de la prostate. Les inventeurs ont montré que la protéine α2δ2 est exprimée de façon plus forte dans les stades avancés du cancer (voir figure 6, G2 et G3) que dans les stades normaux et précoces du cancer (voir figure 6, stades normaux, BPH et métastase). La présente invention concerne donc également une méthode pour déterminer in vitro le stade d'un cancer de la prostate chez un sujet comprenant les étapes consistant à : (ii) comparing the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , measured in step (i) with a predetermined value derived from the α 2 δ 2 concentration, or a fragment of α 2 δ 2 , in a biological sample of a healthy subject not suffering from prostate cancer, a high level of α 2 δ 2 , or of a fragment of α 2 δ 2 , in the biological sample by to said predetermined value indicating that the subject is suffering from prostate cancer. The inventors have shown that the α 2 δ 2 protein is expressed more strongly in the advanced stages of cancer (see FIG. 6, G2 and G3) than in the normal and early stages of cancer (see FIG. 6, normal stages, BPH). and metastasis). The present invention therefore also relates to a method for determining in vitro the stage of prostate cancer in a subject comprising the steps of:
(i) mesurer la concentration de α2δ2, ou un fragment de α2δ2, dans un échantillon biologique obtenu dudit sujet, (i) measuring the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in a biological sample obtained from said subject,
(ii) comparer la concentration de α2δ2, ou un fragment de α2δ2, mesurée à l'étape (i) à une valeur prédéterminée de la concentration en α2δ2, ou un fragment de α2δ2, (ii) comparing the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , measured in step (i) with a predetermined value of the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 ,
dans laquelle, lorsque la concentration de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique est supérieure à ladite valeur prédéterminée de la concentration en α2δ2, ou un fragment de α2δ2, l'écart entre ladite concentration de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique obtenu dudit sujet et ladite valeur prédéterminée indique le stade du cancer de la prostate chez ledit sujet. in which, when the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in the biological sample is greater than said predetermined value of the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , the difference between said concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in the biological sample obtained from said subject and said predetermined value indicates the stage of prostate cancer in said subject.
Selon un mode de réalisation, la valeur prédéterminée se rapporte ici à la quantité de α2δ2 ou d'un fragment de α2δ2 dans les échantillons biologiques obtenus de sujets apparemment sains, par exemple des sujets n'ayant jamais présenté auparavant de signes ou de symptômes indiquant la présence d'un cancer de la prostate. According to one embodiment, the predetermined value relates here to the quantity of α 2 δ 2 or of a fragment of α 2 δ 2 in biological samples obtained from apparently healthy subjects, for example subjects which have never before presented signs or symptoms indicating the presence of prostate cancer.
Selon un autre mode de réalisation, la valeur prédéterminée est ici la quantité de α2δ2 ou d'un fragment de α2δ2 chez des sujets ayant un cancer de la prostate dont le stade est établi. According to another embodiment, the predetermined value here is the amount of α 2 δ 2 or a fragment of α 2 δ 2 in subjects with prostate cancer whose stage is established.
La concentration de la protéine α2δ2, ou d'un fragment, dans un échantillon biologique obtenu du sujet, selon les méthodes de l'invention, permet ainsi de déterminer le niveau d'expression du gène CACNA2D2. The concentration of the α 2 δ 2 protein, or of a fragment, in a biological sample obtained from the subject, according to the methods of the invention, thus makes it possible to determine the level of expression of the CACNA2D2 gene.
L'échantillon biologique est avantageusement un fluide biologique, de préférence un échantillon de sang, un échantillon de sérum, un échantillon de plasma, un échantillon d'urine ou un échantillon de sperme, de préférence encore un échantillon d'urine ou de sperme. Selon un mode de réalisation particulièrement avantageux, il s'agit d'un échantillon d'urine éventuellement recueilli après massage de la prostate du sujet à tester. The biological sample is preferably a biological fluid, preferably a blood sample, a serum sample, a plasma sample, a urine sample or a sperm sample, more preferably a urine or sperm sample. According to a particularly advantageous embodiment, it is a urine sample possibly collected after massage of the prostate of the subject to be tested.
Une fois que l'échantillon biologique est préparé, la concentration en protéines est mesurée au moyen de toute technique connue de l'homme du métier. Dans un mode de réalisation particulier de l'invention, une telle méthode comprend l'étape de mise en contact de l'échantillon avec une molécule de liaison capable de se lier spécifiquement avec la protéine α2δ2 présente dans l'échantillon biologique. La molécule de liaison peut être spécifique de la forme mature de la protéine α2δ2. La molécule de liaison peut également reconnaître les deux parties a2 et δ2 de la sous-unité α2δ2. De manière également avantageuse, la molécule de liaison peut être capable de se lier spécifiquement avec un fragment de la protéine α2δ2, par exemple avec la partie a2 ou avec la partie δ2 de la sous-unité α2δ2. La molécule de liaison peut être un anticorps polyclonal ou monoclonal, de préférence monoclonal. La molécule de liaison peut également être un aptamère. Once the biological sample is prepared, the protein concentration is measured using any technique known to those skilled in the art. In a particular embodiment of the invention, such a method comprises the step of contacting the sample with a binding molecule capable of binding specifically with the α 2 δ 2 protein present in the biological sample. The binding molecule may be specific for the mature form of the α 2 δ 2 protein. The binding molecule can also recognize the two parts a 2 and δ 2 of the α 2 δ 2 subunit. Also advantageously, the binding molecule may be capable of binding specifically with a fragment of the α 2 δ 2 protein, for example with the a 2 part or with the δ 2 part of the α 2 δ 2 subunit. The binding molecule may be a polyclonal or monoclonal antibody, preferably monoclonal. The binding molecule may also be an aptamer.
Des anticorps polyclonaux de l'invention, ou des fragments de ceux-ci, peuvent être déterminés au moyen de méthodes connues de l'homme du métier, en administrant par exemple un antigène ou un épitope approprié à un animal, par exemple un cochon, une vache, un cheval, un lapin, une chèvre, un mouton, une souris ou autre. Des adjuvants connus de l'état de la technique peuvent être utilisés pour améliorer la production d'anticorps. Des anticorps polyclonaux, permettant de reconnaître α2δ2, et pouvant être utilisés dans la présente invention sont, par exemple mais de manière non limitative, H210 (Santa Cruz) ou A01 (Abnova). Bien que les anticorps polyclonaux soient pratiques à la réalisation de l'invention, les anticorps monoclonaux sont préférés. Polyclonal antibodies of the invention, or fragments thereof, can be determined by methods known to those skilled in the art, for example by administering an appropriate antigen or epitope to an animal, for example a pig, a cow, a horse, a rabbit, a goat, a sheep, a mouse or other. Adjuvants known from the state of the art can be used to improve the production of antibodies. Polyclonal antibodies which make it possible to recognize α 2 δ 2 and which can be used in the present invention are, for example but without limitation, H210 (Santa Cruz) or A01 (Abnova). Although polyclonal antibodies are convenient for carrying out the invention, monoclonal antibodies are preferred.
Des anticorps monoclonaux de l'invention, ou des fragments de ceux-ci, peuvent être préparés et isolés au moyen de méthodes connues de l'homme du métier. Les molécules de liaison de l'invention, comme les anticorps ou aptamères, peuvent être marquées au moyen d'une molécule ou substance, par exemple une molécule fluorescente, une enzyme capable de produire un produit coloré, une molécule radioactive ou d'autres marqueurs connus de l'état de l'art. Ainsi, selon un mode de réalisation particulier de l'invention, la concentration de la protéine α2δ2, ou d'un fragment de α2δ2, est mesurée en utilisant un anticorps ou un fragment de celui-ci, spécifique de ladite protéine α2δ2, ou d'un fragment de α2δ2. Monoclonal antibodies of the invention, or fragments thereof, can be prepared and isolated using methods known to those skilled in the art. The binding molecules of the invention, such as antibodies or aptamers, can be labeled with a molecule or substance, for example a fluorescent molecule, an enzyme capable of producing a colored product, a radioactive molecule or other markers. known from the state of the art. Thus, according to a particular embodiment of the invention, the concentration of the α 2 δ 2 protein, or of a fragment of α 2 δ 2 , is measured using an antibody or a fragment thereof, specific for said α 2 δ 2 protein, or a fragment of α 2 δ 2 .
La concentration en protéines peut être mesurée au moyen de techniques standards connues de l'homme du métier, généralement des techniques d'immunodiagnostic comprenant des immunoessais comme la compétition, la réaction directe ou les tests de type sandwich. De tels tests incluent, sans y être limité, les tests d'agglutination, les immunoessais médiés et marqués par enzyme comme les tests ELISA, les tests de type biotine/avidine, les immunoessais par radio, l'immunoélectrophorèse, l'immunoprécipitation. The protein concentration can be measured using standard techniques known to those skilled in the art, generally immunodiagnostic techniques. including immunoassays such as competition, direct reaction or sandwich type tests. Such tests include, but are not limited to, agglutination tests, enzyme-labeled and mediated immunoassays such as ELISA, biotin / avidin, radio immunoassays, immunoelectrophoresis, immunoprecipitation.
La mesure de la concentration en protéines peut aussi comprendre une étape de séparation des composés comme par HPLC (basé sur hydrophobie), par chromatographie avec exclusion par taille (basée sur la taille), et/ou par affinité de phase solide (basé sur l'affinité du composé pour la phase solide utilisée). The measurement of the protein concentration may also include a step of separating the compounds such as by HPLC (based on hydrophobicity), size exclusion chromatography (size-based), and / or solid phase affinity (based on size). affinity of the compound for the solid phase used).
Une fois séparée, la protéine α2δ2 peut être identifiée au moyen de profils de séparation connus pour la protéine, par exemple le temps de rétention, et mesurée selon des techniques standards. Selon un autre mode de réalisation, il peut s'agir d'une identification et d'une mesure d'un fragment, par exemple a2 ou δ2. Once separated, the α 2 δ 2 protein can be identified by means of known separation profiles for the protein, for example the retention time, and measured according to standard techniques. According to another embodiment, it may be an identification and a measurement of a fragment, for example a 2 or δ 2 .
De manière alternative, les composés séparés peuvent être détectés et mesurés par exemple par spectrométrie de masse. Alternatively, the separated compounds can be detected and measured for example by mass spectrometry.
Selon un mode de réalisation de l'invention, une valeur témoin ou une normalisation peut être utilisée. Par exemple, selon un mode de réalisation particulier de l'invention dans lequel l'échantillon biologique est un échantillon d'urine, la quantité de créatinine peut être utilisée pour normaliser le résultat obtenu. En effet, la créatinine étant un bon indicateur de la fonction rénale, elle est par exemple utilisée comme standard en matière de dosage de stupéfiant ou de produits dopants pour évaluer si l'échantillon d'urine est dilué, ou encore comme standard pour les intoxications aux métaux lourds. Sa valeur peut donc être utilisée dans les méthodes de détection de la présente invention. Une fois les urines récoltées (par exemple sur une durée de 24h) et précipitées, la concentration en protéine α2δ2, ou un fragment, est mesurée. La valeur de l'augmentation du ratio de a2ô2/créatinine (ou d'un fragment de a2ô2/créatinine) par rapport au ratio de a2ô2/créatinine (ou d'un fragment de a2ô2/créatinine) d'un sujet sain indique la présence d'un cancer de la prostate. Une augmentation de 50% peut par exemple indiquer que le sujet souffre d'un cancer de la prostate. Les méthodes de détection selon l'invention peuvent également être utilisées en complément d'une autre méthode de détection connue de l'homme du métier, par exemple, mais de façon non limitative, l'analyse du dosage de PS A. La présente invention se rapporte également à un agent anticancéreux pour son utilisation dans le traitement du cancer de la prostate chez un sujet, caractérisé en ce qu'on détecte ledit cancer de la prostate au moyen d'une méthode comme précédemment décrite. According to one embodiment of the invention, a control value or normalization can be used. For example, according to a particular embodiment of the invention in which the biological sample is a urine sample, the amount of creatinine can be used to normalize the result obtained. Since creatinine is a good indicator of renal function, it is used, for example, as a standard for the determination of narcotic drugs or doping substances to evaluate whether the urine sample is diluted or as a standard for poisoning. heavy metals. Its value can therefore be used in the detection methods of the present invention. Once the urine has been collected (for example over a period of 24 hours) and precipitated, the concentration of α 2 δ 2 protein, or a fragment, is measured. The value of the increase in the ratio of α 2 δ 2 / creatinine (or a fragment of α 2 δ 2 / creatinine) to the ratio of α 2 δ 2 / creatinine (or a fragment of α 2 δ) 2 / creatinine) of a healthy subject indicates the presence of prostate cancer. For example, a 50% increase may indicate that the subject is suffering from prostate cancer. The detection methods according to the invention can also be used in addition to another detection method known to those skilled in the art, for example, but in a non-limiting manner, analysis of the PS A assay. The present invention also relates to an anti-cancer agent for use in the treatment of prostate cancer in a subject characterized by detecting said prostate cancer using a method as previously described.
Une méthode de traitement du cancer de la prostate chez un sujet peut ainsi comprendre les étapes consistant à : A method of treating prostate cancer in a subject can thus include the steps of:
détecter la présence d'un cancer dudit sujet au moyen d'une méthode de détection selon la présente invention,  detecting the presence of a cancer of said subject by means of a detection method according to the present invention,
administrer audit sujet un traitement apte à traiter le cancer de la prostate. « Traiter le cancer de la prostate » peut être entendu comme tout traitement capable, par exemple, de supprimer la tumeur ou les métastases, réduire le risque de récidive, ralentir le développement de la tumeur ou des métastases, et/ou traiter les symptômes de la maladie.  administering to said subject a treatment for treating prostate cancer. "Treat prostate cancer" can be understood as any treatment that can, for example, suppress tumor or metastasis, reduce the risk of recurrence, slow tumor development or metastasis, and / or treat symptoms of cancer. disease.
Tout agent anticancéreux connu de l'homme du métier peut ainsi être utilisé. Le traitement peut, par exemple mais sans s'y limiter, consister en un traitement hormonal, par exemple des agonistes de l'hormone de libération des gonadotrophines (LHRH) (comme leuprolide, goserelin ou buserelin), un traitement par antiandrogènes (comme flutamide, ou nilutamide), un traitement par des médicaments empêchant les glandes surrénales de produire des androgènes (comme ketoconazole ou aminoglutethimide), ou encore par des œstrogènes. Any anti-cancer agent known to those skilled in the art can thus be used. The treatment may, for example but not limited to, consist of hormone treatment, for example gonadotropin releasing hormone (LHRH) agonists (such as leuprolide, goserelin or buserelin), antiandrogen treatment (such as flutamide). or nilutamide), treatment with drugs that prevent the adrenal glands from producing androgens (such as ketoconazole or aminoglutethimide), or estrogen.
La présente invention concerne également une méthode pour surveiller un traitement d'un sujet atteint d'un cancer de la prostate et/ou d'une métastase prostatique comprenant la détermination de la concentration de α2δ2, ou d'un fragment, dans un échantillon biologique obtenu dudit sujet, et éventuellement la comparaison de ladite concentration de α2δ2, ou d'un fragment, à une valeur prédéterminée représentant un stade prédéterminé du cancer de la prostate, la concentration de α2δ2, ou d'un fragment, par rapport à ladite valeur prédéterminée indiquant l'évolution du cancer de la prostate, et ainsi l'efficacité du traitement. The present invention also relates to a method for monitoring a treatment of a subject suffering from prostate cancer and / or prostatic metastasis comprising determining the concentration of α 2 δ 2 , or a fragment, in a biological sample obtained from said subject, and optionally comparing said concentration of α 2 δ 2 , or a fragment, to a predetermined value representing a predetermined stage of prostate cancer, the concentration of α 2 δ 2 , or of a fragment, with respect to said predetermined value indicating the evolution of prostate cancer, and thus the effectiveness of the treatment.
La présente invention concerne enfin l'utilisation de la protéine α2δ2, ou d'un fragment, comme biomarqueur du cancer de la prostate d'un sujet. De préférence, ledit fragment est α2. Il a en effet été mis en évidence que la détection de la protéine α2δ2 ou d'un fragment permet de conclure sur la présence ou l'absence d'un cancer de la prostate, ainsi que sur le stade du cancer de la prostate. FIGURES The present invention finally relates to the use of the α 2 δ 2 protein, or a fragment, as a biomarker of prostate cancer of a subject. Preferably, said fragment is α 2 . It has indeed been demonstrated that the detection of α 2 δ 2 protein or of a fragment makes it possible to conclude on the presence or absence of prostate cancer, as well as on the stage of cancer of the prostate. prostate. FIGURES
Figure 1 : Représentation de la fluorescence de l'anticorps secondaire en fonction de la concentration de peptide α2δ2 sur toute la gamme de concentration du peptide α2δ2 (de 6,25μg/ml à 3 pg/ml) (« Alexa488 » : fluorescéine). FIG. 1: Representation of the fluorescence of the secondary antibody as a function of the α 2 δ 2 peptide concentration over the entire concentration range of the α 2 δ 2 peptide (from 6.25 μg / ml to 3 μg / ml) (" Alexa488 ": fluorescein).
Figure 2 : A : Analyse de l'expression de α2δ1; α2δ2 et α2δ3 dans les lignées cellulaires LNCaP et DU145 par criblage par RT-PCR. La Pactine et un échantillon contrôle positif (« cerveau ») sont utilisés comme témoin. Figure 2: A: Analysis of the Expression of α 2 δ 1; α 2 δ 2 and α 2 δ 3 in the LNCaP and DU145 cell lines by RT-PCR screening. Pactin and a positive control sample ("brain") are used as a control.
B : Analyse de l'expression de α2δ2 dans les lignées cellulaires LNCaP,B: Analysis of the Expression of α 2 δ 2 in the LNCaP Cell Lines,
PC3 et DU! 45 par criblage par RT-PCR. La Pactine est utilisée comme témoin. PC3 and DU! 45 by RT-PCR screening. Pactin is used as a control.
Figure 3 : Analyse de l'expression de a2ô2 dans 10 tissus cancéreux (numérotés de Cl à C10) et 10 tissus non-cancéreux (numérotés de NI à N10) de la prostate par RT-PCR. La Pactine est utilisée comme témoin. Figure 3: Analysis of the expression of α 2 δ 2 in 10 cancer tissues (numbered from C1 to C10) and 10 non-cancerous tissues (numbered NI to N10) of the prostate by RT-PCR. Pactin is used as a control.
Figure 4 : Analyse par western-blot montrant que si-a2ô2 (50 nM, 4 jours) inhibe bien l'expression de a2ô2 dans les cellules LNCaP. La calnexine est utilisée comme témoin. Figure 5 : Analyse de l'expression de α2δ2 dans les tissus cancéreux (« non cancer », n=18) et non cancéreux (« cancer », n=20) de prostate humaine par criblage. Figure 4: Western blot analysis showing that si-a 2 δ 2 (50 nM, 4 days) well inhibited the expression of α 2 δ 2 in LNCaP cells. Calnexin is used as a control. Figure 5: Analysis of the expression of α 2 δ 2 in cancer tissues ("non cancer", n = 18) and non-cancerous ("cancer", n = 20) of human prostate by screening.
Figure 6 : Analyse de l'expression de α2δ2 à différentes stades du cancer de la prostate par analyse immunohistochimique. Figure 6: Analysis of the expression of α 2 δ 2 at different stages of prostate cancer by immunohistochemical analysis.
A et B : aperçu de tissus de la prostate provenant de tissus normaux, hyperplasiques et cancéreux (A : objectif *5, B : objectif *40). Les grades indiqués avant la parenthèse correspondent aux grades de Gleason le plus fréquent de la coupe montrée. Entre-parenthèses sont indiqués les scores de Gleason du patient correspondant. M : métastase rectale, BPH : hyperplasie, N : tissu prostatique normal. La coloration immunohistochimique contre α2δ2 a été révélée par réaction DAB-peroxydase. C : a) aperçu de tissus de la prostate provenant de tissus normaux (« normal »), de métastase (« métastase »), tissu hyperplasique (« BHP ») ou tissus cancéreux (« cancer »). Les images colorées ont été déconvolutionnées et l'intensité de coloration de la cellule a été analysée et convertie en densité optique. La densité optique est augmentée dans les acini entourant les cellules des tissus cancéreux ; A and B: an overview of prostate tissues from normal, hyperplastic and cancerous tissues (A: objective * 5, B: objective * 40). The grades shown before the parenthesis correspond to the most common Gleason grade in the cut shown. Between parentheses are indicated Gleason scores of the corresponding patient. M: rectal metastasis, BPH: hyperplasia, N: normal prostatic tissue. Immunohistochemical staining against α 2 δ 2 was revealed by DAB-peroxidase reaction. C: a) Overview of prostate tissue from normal ("normal"), metastatic ("metastatic"), hyperplastic ("BHP") or cancerous ("cancer") tissues. The stained images were deconvolved and the staining intensity of the cell was analyzed and converted to optical density. The optical density is increased in the acini surrounding the cells of the cancerous tissues;
b) histogramme montrant la densité optique moyenne (ODDAB) pour différents stades de cancer. Le nombre de cellules analysées est représenté sur chaque barre. b) Histogram showing mean optical density (OD DAB ) for different stages of cancer. The number of cells analyzed is represented on each bar.
Figure 7 : Analyse de l'effet de la protéine α2δ2 sur la prolifération cellulaire. Figure 7: Analysis of the effect of the α 2 δ 2 protein on cell proliferation.
A : a) Effet d'un traitement de 4 jours utilisant si-a2ô2 (50 nM) sur la croissance cellulaire (méthode MTS) par rapport à un témoin, si-ctl, qui, à la même concentration, n'a aucune influence sur la prolifération cellulaire ; A: a) Effect of a 4-day treatment using si-a 2 δ 2 (50 nM) on cell growth (MTS method) compared to a control, si-ctl, which, at the same concentration, has no influence on cell proliferation;
b) Effet d'un traitement de 4 jours utilisant si-a2ô2 (50 nM) sur le nombre de cellules (comptage manuel des cellules extradées par Tryptan Blue) ; b) Effect of a 4-day treatment using si-a 2 δ 2 (50 nM) on the number of cells (manual counting of cells extradited by Tryptan Blue);
B : Effet de si-a2ô2 sur la prolifération cellulaire de deux autres lignées cellulaires du cancer de la prostate (PC3 et DU! 45). B: Effect of si-a 2 δ 2 on the cell proliferation of two other prostate cancer cell lines (PC3 and DU! 45).
C : Expériences cinétiques sur l'inhibition en fonction du temps par si-a2ô2 de la croissance cellulaire de LNCaP par rapport à un témoin (si-ctl). C: Kinetic Experiments on the inhibition function of time by si-2 O 2 LNCaP cell growth relative to a control (so-ctl).
D : Analyse FACS de l'effet de si-a2ô2 sur le nombre de cellules LNCaP en phase Go/Gi (représenté « G0/G1 sur la figure), G2/M et S par rapport à un témoin, si-ctl D: FACS analysis of the effect of si-a 2 δ 2 on the number of LNCaP cells in Go / Gi phase (represented by "G0 / G1 in the figure), G 2 / M and S with respect to a control, if -ctl
E : Effet de la gabapentine (100 μΜ) sur la prolifération cellulaire par méthode E: Effect of gabapentin (100 μΜ) on cell proliferation by method
MTS. MTS.
F : a) Cinétique de la prolifération cellulaire chez deux clones différents de LNCaP-a2ô2 (C9 et Ci l). F: a) Kinetics of cell proliferation in two different clones of LNCaP-a 2 δ 2 (C9 and Ci I).
b) Analyse FACS de l'effet de LNCaP-a2ô2 (clone Ci l) sur le nombre de cellules en phase Go/Gi, G2/M et S par rapport à une lignée cellulaire LNCaP ne surexprimant pas α2δ2. b) FACS analysis of the effect of LNCaP-a 2 δ 2 (clone Ci I) on the number of cells in Go / Gi, G 2 / M and S phase relative to an LNCaP cell line not overexpressing α 2 δ 2 .
Figure 8 : Analyse in vivo de la croissance cellulaire induite par la protéine α2δ2. A : Poids en gramme des tumeurs LNCaP (témoin) et LNCap-a2ô2 (clone Ci l) au sacrifice ; Figure 8: In vivo analysis of cell growth induced by the α 2 δ 2 protein. A: Weight in grams of LNCaP (control) and LNCap-a 2 δ 2 (clone Ci1) tumors at sacrifice;
B : Cinétique de croissance des tumeurs LNCaP (témoin) et LNCap-a2ô2 (clone Ci l) ; C : Temps moyen de multiplication par 2 des tumeurs LNCaP (témoin) et LNCap-a2ô2 (clone Ci l). Figure 9 : Analyse histologique des tumeurs LNCaP et LNCaP-a2ô2 A : Différentes vues à grossissement *20 de : a : la tumeur LNCaP témoin ; c : la tumeur LNCaP-a2ô2. Les images b (LNCaP témoin) et d (tumeur LNCaP-a2ô2) montrent au moyen des flèches les foyers de nécroses et d'inflammation. B: Kinetics of growth of LNCaP (control) and LNCap-a 2 δ 2 tumors (clone Ci l); C: Average multiplication by 2 LNCaP tumors (control) and LNCap-2 O 2 (clone Ci l). Figure 9: Histological analysis of LNCaP and LNCaP-a 2 δ 2 A tumors: Various magnified views of: a: the control LNCaP tumor; c: the tumor LNCaP-a 2 ô 2 . The images b (LNCaP control) and tumor LNCaP-a 2 ô 2 ) show by means of arrows the foci of necrosis and inflammation.
B : Quantification du pourcentage des noyaux positifs à l'analyse par immunofluorescence par PCNA (noyaux des cellules en division), normalisé par rapport au nombre total de noyaux (DAPI).  B: Quantification of the percentage of nuclei positive for analysis by immunofluorescence by PCNA (nuclei of dividing cells), normalized to the total number of nuclei (DAPI).
Figure 10 : Analyse de l'effet de α2δ2 sur la formation de vaisseaux sanguins dans les tumeurs LNCaP xénogreffées. Figure 10: Analysis of the effect of α 2 δ 2 on the formation of blood vessels in LNCaP xenografted tumors.
A : Coloration immunohistochimique (peroxydase/DAB) de CD31 (a : tumeur LNCaP, b : tumeur LNCaP-a2ô2) ; A: Immunohistochemical staining (peroxidase / DAB) of CD31 (a: tumor LNCaP, b: tumor LNCaP-a 2 δ 2 );
B : Pourcentage de tissus positifs au CD31 dans les tumeurs LNCaP et LNCaP-a2ô2. B: Percentage of CD31 positive tissue in LNCaP and LNCaP-a 2 δ 2 tumors.
Figure 11 : Analyse de l'effet de α2δ2 sur l'angiogénèse par la sécrétion de VEGF. A : a. Western blots de CD31 et VEGF dans 8 tumeurs différentes provenant de 4 souris différentes (Ml à M4) (Tumeurs LNCaP : « T-LNCaP », Tumeurs LNCaP-a2ô2 : « T- α2δ2 »). La Pactine est utilisée comme témoin. Figure 11: Analysis of the effect of α 2 δ 2 on angiogenesis by the secretion of VEGF. A: a. Western blots of CD31 and VEGF in 8 different tumors from 4 different mice (Ml to M4) (LNCaP tumors: "T-LNCaP", LNCaP-a 2 ô 2 tumors: "T-α 2 δ 2 "). Pactin is used as a control.
b. Densité relative des bandes CD31 observées par Western blot (fig lOA.a.) normalisées par rapport à Pactine (10 échantillons pour chaque tumeur).  b. Relative density of CD31 bands observed by Western blot (FIG. 10A.a.) standardized with respect to actin (10 samples for each tumor).
c. Densité relative des bandes VEGF observées par Western blot (fig lOA.a.) normalisées par rapport à Pactine (10 échantillons pour chaque tumeur).  vs. Relative density of the VEGF bands observed by Western blot (FIG. 10A.a.) standardized with respect to actin (10 samples for each tumor).
B : Sécrétion de VEGF mesurée au moyen du test ELISA dans les cellules LNCaP et LNCaP-a2ô2 (clones C9 et Ci l). B: VEGF secretion measured by ELISA in LNCaP and LNCaP-a 2 δ 2 cells (clones C9 and Ci1).
Figure 12 : Analyse de la sécrétion de a2 dans les cellules LNCaP-a2ô2 Figure 12: Analysis of secretion of α 2 in LNCaP-a 2 δ 2 cells
A : Western blot de a2 dans le milieu de culture des cellules LNCaP, de deux clones surexprimant α2δ2 (clone Ci l et C16) et dans le milieu de culture seul (« RPMI »), sans DTT (6 jours d'incubation) ou avec DTT, i.e. en conditions réductrices (15 min d'incubation). A: Western blot of α 2 in the culture medium of LNCaP cells, two clones overexpressing α 2 δ 2 (clone Ci and C16) and in the culture medium alone ("RPMI"), without DTT (6 days d incubation) or with DTT, ie under reducing conditions (15 min incubation).
B : Western blot pour deux autres milieux de culture contenant soit 2% de SVF (sérum de veau fœtal), soit 10% de SVF (conditions courantes de culture).  B: Western blot for two other culture media containing either 2% FCS (fetal calf serum) or 10% FBS (current culture conditions).
Figure 13 : Figure 13:
A : Représentation de la fluorescence de l'anticorps secondaire en fonction de la concentration de peptide α2δ2 dans la gamme de concentration proche du seuil de détection, avec représentation de la concentration de peptide α2δ2 dans l'urine totale et dans les protéines obtenues à partir d'urine (protéines précipitées à l'acétone et suspendues dans tampon PBS) d'un homme atteint d'un cancer de la prostate (cf. B). A: Representation of the fluorescence of the secondary antibody as a function of the α 2 δ 2 peptide concentration in the concentration range close to the detection threshold, with representation of the concentration of peptide α 2 δ 2 in total urine and in proteins obtained from urine (proteins precipitated with acetone and suspended in PBS buffer) of a man suffering from cancer of the prostate (see B).
B : Concentrations de la protéine α2δ2 codée par le gène CACNA2D2 dans les urines totales et dans les protéines obtenues à partir d'urine d'un homme atteint d'un cancer de la prostate. B: Concentrations of the α 2 δ 2 protein encoded by the CACNA2D2 gene in total urine and in proteins obtained from urine of a man suffering from prostate cancer.
EXEMPLES Matériels et méthodes EXAMPLES Materials and methods
Cultures cellulaires Cell cultures
Les lignées cellulaires LNCaP, DU 145 et P3 proviennent de la « American Type Culture Collection » et ont été cultivées, selon les recommandations du fournisseur, dans du RPMI 1640 (GIBCO, Life Technologies, France) additionné de 10% de sérum fœtal bovin (FBS, Sigma, L'Isle d'Abeau, France) et de 2 mM de L-glutamine (Sigma, L'Isle d'Abeau, France). Les cellules ont été systématiquement cultivées dans des récipients de 75 cm2 (Nunc, Poly-Labo, France) en atmosphère humidifiée à 37°C (95% d'air - 5% de C02). Le milieu de culture a ensuite été changé tous les trois jours. The LNCaP, DU 145 and P3 cell lines originate from the American Type Culture Collection and were cultured, according to the supplier's recommendations, in RPMI 1640 (GIBCO, Life Technologies, France) supplemented with 10% fetal bovine serum ( FBS, Sigma, Isle d'Abeau, France) and 2 mM L-glutamine (Sigma, L'Isle d'Abeau, France). The cells were systematically cultured in 75 cm 2 containers (Nunc, Poly-Labo, France) in a humidified atmosphere at 37 ° C. (95% air - 5% CO2). The culture medium was then changed every three days.
Des lignées cellulaires LNCaP stables exprimant la protéine α2δ2 (ci-après désignées sous le terme « LNCaP-a2ô2 ») ont été conçues comme précédemment reporté dans la publication de Florian Gackière et al (10). LNCaP stable cell lines expressing the α 2 δ 2 protein (hereinafter referred to as "LNCaP-α 2 δ 2 ") were designed as previously reported in the publication by Florian Gackière et al (10).
Production de si-RNAs et préparation cellulaire Production of si-RNAs and cell preparation
Les petits ARN interférents proviennent de Eurogentech (France).  The small interfering RNAs come from Eurogentech (France).
Les si-a2ô2 ont été validés dans les cellules LNCaP. Pour cela, il a été montré par western-blot que si-a2ô2 diminue l'expression de la protéine α2δ2 dans les cellules LNCaP (figure 3). The si-a 2 δ 2 were validated in the LNCaP cells. For this, it has been shown by Western-blot that si-a 2 δ 2 decreases the expression of the α 2 δ 2 protein in LNCaP cells (FIG. 3).
Les siRNAs utilisés ici comprennent un siRNA témoin non- spécifique contre la luciférase (ci-après désigné sous le terme « si-ctl » ou « siCTL »).  The siRNAs used herein include a non-specific control siRNA against luciferase (hereinafter referred to as "si-ctl" or "siCTL").
Les siRNAs sont présentées dans le tableau 1. siRNA Position The siRNAs are presented in Table 1. siRNA Position
siCTL Luciferase 153-171  siCTL Luciferase 153-171
si-a2ô2 1868-1850 if-a 2 ô 2 1868-1850
Tableau 1 Table 1
Les cellules ont été transfectées avec 20 ou 50 nM de siRNA en utilisant HiPerFect Transfection Reagent (Qiagen) comme précédemment reporté dans la publication de Florian Gackière et al (10). The cells were transfected with 20 or 50 nM siRNA using HiPerFect Transfection Reagent (Qiagen) as previously reported in the publication by Florian Gackière et al (10).
Analyse de l'expression du gène de la sous-unité α2δ2 par RT-PCR ou RT-PCR quantitative Analysis of Gene Expression of the α 2 δ 2 Subunit by RT-PCR or Quantitative RT-PCR
L'extraction de l'ARN a été effectuée avec du Trizol® au moyen d'une méthode décrite à l'origine par Chomczynski et Sacchi (11). La méthode de RT-PCR est équivalente à celle décrite dans la publication de Florian Gackière et al (10).  RNA extraction was performed with Trizol® using a method originally described by Chomczynski and Sacchi (11). The RT-PCR method is equivalent to that described in the publication by Florian Gackière et al (10).
Pour chaque réaction de PCR, 1 μg d'ARNm total a été traité avec du DNAse II, incubé à 70°C et a subi une transcription inverse avec la polymérase MULV RNA (Applied Biosystems, 50υ/μί) en présence de 0,5 μΜ de dNTP et d'un inhibiteur de la ribonucléase (Applied Biosystems).  For each PCR reaction, 1 μg of total mRNA was treated with DNAse II, incubated at 70 ° C and reverse transcribed with MULV RNA polymerase (Applied Biosystems, 50 μ / μί) in the presence of 0.5 μΜ of dNTP and a ribonuclease inhibitor (Applied Biosystems).
L'ADNc obtenu a été utilisé pour l'amplification par PCR au moyen d'un kit Taq Gold (Applied Biosystems).  The cDNA obtained was used for amplification by PCR using a Taq Gold kit (Applied Biosystems).
Les conditions opératoires de la PCR sont présentées dans le tableau 2.  The operating conditions of the PCR are presented in Table 2.
Figure imgf000017_0001
Figure imgf000017_0001
Tableau 2  Table 2
Les amorces sens et antisens (Invitrogen) utilisées pendant l'amplification sont présentées dans le tableau 3. Protéine Gène Taille (bp) The sense and antisense primers (Invitrogen) used during the amplification are shown in Table 3. Protein Gene Size (bp)
α2δ1 CACNA2D1 400  α2δ1 CACNA2D1 400
α2δ2 CACNA2D2 505 α 2 δ 2 CACNA2D2 505
500  500
α2δ3 CACNA2D3 α 2 δ 3 CACNA2D3
439  439
Pactine 220  Pactine 220
Tableau 3 Table 3
Les produits de la PCR ont été analysés sur un gel d'agarose (1,5% ou 2%) comprenant du bromure d'éthidium (0,5 μg/ml) puis visualisés sous UV. Les images ont ensuite été capturées au moyen d'un logiciel Gelcapture (Bio-imaging System). The PCR products were analyzed on an agarose gel (1.5% or 2%) comprising ethidium bromide (0.5 μg / ml) then visualized under UV. The images were then captured using Gelcapture software (Bio-imaging System).
Western blotting Western blotting
Après un lavage dans une solution saline tamponnée au phosphate, les cellules ont été collectées par grattage dans du tampon de lyse (Triton X-100 1%, Na déoxycholate, 1%, NaCl 150 mM, P04NaK 10 mM, pH 7,2) avec un mélange d'anti-protéases, puis ont été incubées dans de la glace pendant 45 min. Les lysats ont été centrifugés à 12000G pendant 10 min à 4°C. La concentration en protéines du supernageant a été déterminée par test BCA (Pierce Chemical Compagny). Les analyses par Western-blot de l'expression des protéines a été effectuée comme décrit dans la publication de Florian Gackière et al (10). After washing in phosphate buffered saline, the cells were scraped into lysis buffer (1% Triton X-100, 1% Na deoxycholate, 150 mM NaCl, 10 mM PO 4 NaK, pH 7, 2) with a mixture of anti-proteases and then incubated in ice for 45 min. The lysates were centrifuged at 12000G for 10 min at 4 ° C. The protein concentration of the supernatant was determined by BCA test (Pierce Chemical Company). The Western blot analyzes of the expression of the proteins were carried out as described in the publication by Florian Gackière et al (10).
Les anticorps, utilisés pour le western blotting ainsi que pour les identifications immunohistochimiques et par immunofluorescence, sont présentés dans le tableau 4.  The antibodies used for western blotting as well as for immunohistochemical and immunofluorescence identifications are shown in Table 4.
Anticorps Nom Entreprise Dilution Dilution Anticorps MW dans WB dans IMH secondaire (kDa) α2δ2 H210 Santa Cruz 1/2006 1/1006 ou Rabbit 130 l/50e Antibody Name Company Dilution Dilution Antibody MW in WB in secondary IMH (kDa) α 2 δ 2 H210 Santa Cruz 1/200 6 1/100 6 or Rabbit 130 l / 50 th
α2δ2 A01 Abnova / l/50e Mouse 130α 2 δ 2 A01 Abnova / l / 50 th Mouse 130
PCNA Sc-56 Santa Cruz 1/2006 / Mouse 36 β-actin A-5441 Sigma 1/40006 / Mouse 43PCNA Sc-56 Santa Cruz 1/200 6 / Mouse 36 β-actin A-5441 Sigma 1/4000 6 / Mouse 43
Calnexine mab 3126 Millipore 1/20006 / Mouse 95Calnexine mab 3126 Millipore 1/2000 6 / Mouse 95
Ki-67 Sc-101861 Santa Cruz / 1/1006 Rabbit
Figure imgf000019_0001
Ki-67 Sc-101861 Santa Cruz / 1/100 6 Rabbit
Figure imgf000019_0001
Tableau 4  Table 4
Prolifération cellulaire et tests de viabilité  Cell proliferation and viability tests
La viabilité des cellules a été testée par méthode colorimétrique (CellTiter 96 Aqueous Non-Radioactive Cell Prolifération Assay, Promega, USA) selon les recommandations du fournisseur.  Viability of the cells was assayed by colorimetric method (CellTiter Aqueous Non-Radioactive Cell Proliferation Assay, Promega, USA) according to the supplier's recommendations.
La prolifération cellulaire a également été testée par comptage manuel des cellules extradées par Tryptan Blue.  Cell proliferation was also tested by hand counting extruded cells by Tryptan Blue.
La coloration des anticorps Ki-67 ou PCNA (« Proliferating Cell Nuclear Antigen ») a également été utilisée pour discriminer les cellules en quiescence (phase Go) et les cellules cycliques.  The staining of Ki-67 or PCNA ("Proliferating Cell Nuclear Antigen") antibodies was also used to discriminate cells in quiescence (Go phase) and cyclic cells.
Analyse du cycle cellulaire Cell cycle analysis
Les cellules ont été cultivées dans trois puits de 60mm par état, et les traitements ou siRNA ont été appliqués comme précédemment décrit. Après traitement, les cellules ont été trypsinisées, récoltées et remises en suspension dans 0,2 ml de PBS stérile. 1 ml d'éthanol froid 70% a été ajouté sur les cellules en suspension pendant l'étape dans le vortex. Les échantillons ont été centrifugés, lavés dans du PBS stérile puis incubés avec de la ribonucléase (2 μg/ml) pendant 15 min à température ambiante. Du iodure de propidium (25 μg/ml final dans du PBS-triton X-100 0,1%) a été ajouté et mis à incuber pour 30 min supplémentaires à température ambiante. Le contenu en ADN a été mesuré par excitation d'iodure de propidium à 488 nm et mesure de l'émission à 520 nm au moyen d'un cytomètre en flux (Beckman coulter Epies XL4-MCL avec acquisition Expo32). L'interprétation des données a été effectuée avec Multicycle pour Windows (Phoenix Flow System).  The cells were cultured in three 60mm wells per state, and the treatments or siRNAs were applied as previously described. After treatment, the cells were trypsinized, harvested and resuspended in 0.2 ml of sterile PBS. 1 ml of 70% cold ethanol was added to the cells in suspension during the vortexing step. The samples were centrifuged, washed in sterile PBS and then incubated with ribonuclease (2 μg / ml) for 15 min at room temperature. Propidium iodide (25 μg / ml final in PBS-0.1% triton X-100) was added and incubated for an additional 30 min at room temperature. The DNA content was measured by excitation of propidium iodide at 488 nm and measured emission at 520 nm using a flow cytometer (Beckman Coulter Epies XL4-MCL with Expo32 acquisition). Data interpretation was performed with Multicycle for Windows (Phoenix Flow System).
Immunocoloration et analyse confocale Immunostaining and confocal analysis
L'analyse de l'expression de protéine dans les cellules et les tissus du cancer de la prostate (PCA) a été effectuée au moyen d'analyse d'immunofluorescence indirecte sur des cellules fixées à l'acétone et des tissus fixés dans du formol et enrobés de paraffine.  The analysis of protein expression in prostate cancer cells and tissues (PCA) was performed by means of indirect immunofluorescence analysis on acetone-fixed cells and formalin-fixed tissues. and coated with paraffin.
Des puces à ADN (« microarrays ») de tissus et des tissus tumoraux de souris ont été étudiées au moyen de méthodes immunohistochimiques.  Tissue microarrays and tumor tissues of mice were studied using immunohistochemical methods.
Les lames de puces à ADN de tissus multi-tumoraux proviennent de Biomax (USA). 30 tumeurs ont été analysées, ainsi que les tissus normaux correspondants. Le fournisseur (Biomax) fournit les informations cliniques (âge des patients, grades des cancers et grades deMulti-tumor tissue DNA microarray slides are from Biomax (USA). Tumors were analyzed, as well as the corresponding normal tissues. Supplier (Biomax) provides clinical information (age of patients, grades of cancers and grades of
Gleason des coupes) correspondant aux différents tissus. Toutes les tumeurs ont été fixées dans du formol et enrobées de paraffine. L'activité peroxydase endogène a été inhibée au moyen d'un traitement avec 0,3% de H202. Les lames de tissus ont été passées au micro-onde pendant 20 min, et lavées trois fois dans 10 mM de tampon au citrate (pH 6,0). Afin d'inhiber les liaisons non-spécifiques des anticorps et de perméabiliser les cellules, les lames ont été incubées avec du PBS contenant 0,2% de BSA, 0,1% de Triton X-100 et 5% de sérum d'âne pendant 30 min à température d'ambiante. Ils ont ensuite été incubés toute la nuit à 4°C avec PBS/5 % de sérum non-immunisé contenant les anticorps pertinents. Les dilutions sont présentées dans le tableau 4. Les cellules ont ensuite été lavées avec du PBS. Gleason cuts) corresponding to the different tissues. All tumors were fixed in formalin and paraffin-embedded. Endogenous peroxidase activity was inhibited by treatment with 0.3% H 2 0 2 . The tissue slides were microwaved for 20 min, and washed three times in 10mM citrate buffer (pH 6.0). In order to inhibit the nonspecific binding of the antibodies and to permeabilize the cells, the slides were incubated with PBS containing 0.2% BSA, 0.1% Triton X-100 and 5% donkey serum. for 30 minutes at room temperature. They were then incubated overnight at 4 ° C with PBS / 5% non-immune serum containing the relevant antibodies. The dilutions are shown in Table 4. The cells were then washed with PBS.
Pour les analyses d'immunofluorescence, les cellules ont été incubées avec le IgG secondaire Alexa fluor labellisé-488 anti-lapin (A-21206 ; Tests moléculaires ; dilution 1 :2000) dilué dans du PBS pendant lh à température ambiante. Après trois rinçages dans du PBS, les lames ont été montées avec du Mowiol®. La technique de détection DAB- peroxydase de raifort a été utilisée dans les analyses immunohistochimiques. De l'azuré B a été utilisé pour contre-colorer les lames de tissus. For immunofluorescence analyzes, the cells were incubated with Alexa fluor-labeled anti-rabbit IgG-488 secondary antibody (A-21206, Molecular tests, dilution 1: 2000) diluted in PBS for 1h at room temperature. After three rinses in PBS, the slides were mounted with Mowiol ® . The DAB-horseradish peroxidase detection technique was used in immunohistochemical analyzes. Azure B was used to counter-stain tissue slides.
Les observations confocales et d'immunocoloration ont été réalisées comme précédemment décrit au moyen d'un microscope confocal Zeiss LSM 510 (Cari Zeiss, Le Pecq, France) relié à un microscope inversé pour diascopie et épifluorescence Zeiss Axiovert 200 M avec une lentille d'objectif à immersion en huile x63 (ouverture numérique 1.4).  The confocal and immunostaining observations were performed as previously described by means of a Zeiss LSM 510 confocal microscope (Cari Zeiss, Le Pecq, France) linked to an inverted microscope for Zeiss Axiovert 200 M episodopy and epifluorescence with a lens. oil immersion objective x63 (numerical aperture 1.4).
L'anticorps anti-Ki67 a été utilisé pour évaluer le pourcentage de cellules proliférantes. Au moins 500 cellules par lame et trois lames par état ont été comptées. L'anticorps anti-PCNA a été utilisé pour évaluer le pourcentage de cellules en phase S dans les échantillons de tumeurs. Une immunofluorescence de l'anti-CD31 a été utilisée pour évaluer le nombre de vaisseaux sanguins dans les tumeurs xénogreffées. Les anticorps polyclonaux anti-a2ô2 ont été utilisés pour étudier l'expression de α2δ2 dans les tissus et les cellules de la prostate. Anti-Ki67 antibody was used to evaluate the percentage of proliferating cells. At least 500 cells per slide and three slides per state were counted. Anti-PCNA antibody was used to evaluate the percentage of S-phase cells in the tumor samples. An immunofluorescence of anti-CD31 was used to evaluate the number of blood vessels in xenografted tumors. Polyclonal anti 2 O 2 were used to study the expression of α 2 δ 2 in the tissues and cells of the prostate.
Analyse de la sécrétion de VEGF Analysis of the secretion of VEGF
La sécrétion de VEGF a été mesurée in vitro au moyen d'une technique ELISA comme décrit par le fournisseur (Abcam). Après mise en culture, les cellules ont été cultivées dans plaques à 12 puits pendant 3 jours avant changement du milieu de culture. VEGF a ensuite été analysé dans le milieu de culture après 24 heures d'incubation (3 puits par état, et l'expérience a été répétée au moins trois fois). Analyse de la sécrétion de α2δ2 Secretion of VEGF was measured in vitro using an ELISA technique as described by the supplier (Abcam). After culturing, the cells were cultured in 12-well plates for 3 days before changing the culture medium. VEGF was then assayed in the culture medium after 24 hours of incubation (3 wells per state, and the experiment was repeated at least three times). Analysis of the secretion of α 2 δ 2
Afin de déterminer si α2δ2 est sécrétée dans le milieu de culture, les cellules ont été incubées pendant 6 jours sans changement du milieu de culture. A la fin de cette période, le milieu de culture a été retiré et centrifugé (milieu de culture cellulaire). In order to determine if α 2 δ 2 is secreted in the culture medium, the cells were incubated for 6 days without changing the culture medium. At the end of this period, the culture medium was removed and centrifuged (cell culture medium).
Les cellules ont ensuite été incubées pendant 15 min avec 10 mM de DTT afin de cliver les ponts disulfure et le milieu a encore une fois été retiré et centrifugé. Les protéines des deux supernageants (avec et sans DTT) ont été précipitées avec de l'acétone. Le concentré a été remis en suspension dans RIPA. Le contenu en protéines a été mesuré et α2δ2 a été analysée après une électrophorèse SDS-page (6 ou 8% d'acrylamide) et un western-blot. L'expression de α2δ2 dans le milieu de culture cellulaire a été comparé au milieu de culture seul (sans cellules). The cells were then incubated for 15 min with 10mM DTT to cleave the disulfide bridges and the medium was again removed and centrifuged. Proteins from both supernatants (with and without DTT) were precipitated with acetone. The concentrate was resuspended in RIPA. The protein content was measured and α 2 δ 2 was analyzed after SDS-page electrophoresis (6 or 8% acrylamide) and a western blot. The expression of α 2 δ 2 in the cell culture medium was compared with the culture medium alone (without cells).
Essais in vivo In vivo tests
Des souris Swiss nude mâles âgées de 6 semaines (Laboratoires Charles River, France) ont reçues une injection sur chaque flanc de cellules LNCaP ou de cellules LNCaP surexprimant α2δ2. Six millions de cellules ont été injectées sur les deux flancs de chaque souris. Les cellules ont été préparées dans un mélange composé de 50% de PBS et de 50% de BD-Matrigel®. Les tumeurs ont été mesurées deux fois par semaine et les animaux ont été sacrifiés quand la taille de la tumeur a atteint 10% du poids de l'animal. Le jour du sacrifice, les tumeurs ont été pesées et divisées pour des analyses immunohistochimiques, par western blot et par PCR-RT supplémentaires. Au moins 10 animaux par état ont été utilisés. Six weeks old male Swiss nude mice (Charles River Laboratories, France) received an injection on each flank of LNCaP cells or LNCaP cells overexpressing α 2 δ 2 . Six million cells were injected on both flanks of each mouse. The cells were prepared in a mixture of 50% PBS and 50% BD-Matrigel ® . The tumors were measured twice a week and the animals were sacrificed when the tumor size reached 10% of the weight of the animal. On the day of sacrifice, the tumors were weighed and divided for immunohistochemistry, western blot and additional RT-PCR. At least 10 animals per state were used.
Analyses statistiques Statistical analyzes
Des graphiques ont été reproduits au moyen de Origin 7.0 (Microcal Software, Inc., Northampton, MA). Les résultats ont été exprimés en moyenne ± écart-type moyen. Des analyses statistiques ont été effectuées au moyen de tests t non appariés (pour comparer deux groupes) ou des tests ANOVA suivis de Dunnett (pour contrôle multiple vs tests de comparaisons) ou de post-tests de Student-Newman-Keuls (pour les comparaisons multiples) ou de tests du Chi2 (pour comparer des proportions). Les différences ont été considérées comme significatives avec p<0,05 : *, p<0,01 : **, p<0,001 :  Graphics were reproduced using Origin 7.0 (Microcal Software, Inc., Northampton, MA). The results were expressed as mean ± standard deviation. Statistical analyzes were performed using unpaired t-tests (to compare two groups) or ANOVA tests followed by Dunnett (for multiple control vs. comparison tests) or Student-Newman-Keuls post-tests (for comparisons). multiples) or Chi2 tests (to compare proportions). The differences were considered significant with p <0.05: *, p <0.01: **, p <0.001:
Test d'ELISA (Enzyme Linked ImmunoSorbent Assay) Indirect pour la détection de α2δ2 dans les urines des patients atteints de cancers prostatiques Indirect Enzyme Linked ImmunoSorbent Assay (ELISA) Test for the Detection of α 2 δ 2 in the Urine of Prostate Cancer Patients
Gamme de détection du peptide o^ô? Le test a été conduit selon Da Silva et al. (2000) avec quelques modifications. Les puits des plaques (de 96 puits) ont été incubés avec 200μ1 PBS (phosphate-buffered saline solution) contenant le peptide α2δ2 (AVIVA SYSTEM BIOLOGY de référence AAP64646) dilué à différentes concentrations (de 6,25μg/ml à 3 pg/ml) à l'exception des puits du contrôle contenant uniquement 200 μΐ du tampon PBS. Les plaques ont par la suite été incubées pendant lh à 37° C puis à 4°C pendant une nuit. Range of detection of the peptide o? The test was conducted according to Da Silva et al. (2000) with some modifications. The wells of the plates (of 96 wells) were incubated with 200 μl PBS (phosphate-buffered saline solution) containing the α 2 δ 2 peptide (AVIVA SYSTEM BIOLOGY reference AAP64646) diluted at different concentrations (from 6.25 μg / ml to 3 μg / ml). pg / ml) with the exception of control wells containing only 200 μl of PBS buffer. The plates were subsequently incubated for 1h at 37 ° C and then at 4 ° C overnight.
Les plaques ont été ensuite lavées 3 fois avec du tampon de lavage PBS. Puis, les puits ont été incubés avec 200 μΐ du tampon de blocage des sites aspécifiques (PBS + 1% BSA) pendant 1 heure à la température ambiante. Puis, les puits sont vidés et incubés avec l'anticorps anti-a2ô2 (anticorps AVIVA SYSTEM BIOLOGY de référence ARP64646_P050) (développé chez le lapin) dilués à l/500e dans du tampon de blocage (100 μΐ). Les plaques sont incubées à 4°C pendant la nuit. Deux expériences contrôles sont effectuées ; un contrôle en absence des anticorps primaires et secondaires et un contrôle en absence de l'anticorps primaire avec la concentration la plus haute de la gamme (6,25μg/ml), ce dernier afin d'estimer la fixation aspécifique de l'anticorps secondaire. The plates were then washed 3 times with PBS wash buffer. Then, the wells were incubated with 200 μl of aspecific site blocking buffer (PBS + 1% BSA) for 1 hour at room temperature. Then, the wells are emptied and incubated with the anti-α 2 δ 2 antibody (reference AVIVA SYSTEM BIOLOGY antibody ARP64646_P050) (developed in the rabbit) diluted to 1/500 in blocking buffer (100 μΐ). The plates are incubated at 4 ° C overnight. Two control experiments are performed; a control in the absence of the primary and secondary antibodies and a control in the absence of the primary antibody with the highest concentration of the range (6.25 μg / ml), the latter in order to estimate the aspecific binding of the secondary antibody .
Après 3 lavages avec du tampon de lavage, les puits sont incubés lh à 4°C en présence d'un anticorps secondaire (Goat Anti-Rabbit IgG, Jackson Immunoreasearch Lab) conjugué à la fluorescéine (Alexa Fluor 488 de Lifetechnologies) à une dilution de l/2000e dans un volume total de 100 μΐ du tampon de blocage. Après cette incubation, les puits sont lavés trois fois avec du tampon de lavage (PBS) et la fluorescence est mesurée au niveau des puits à l'aide d'un lecteur de plaque fluorescence. Après la soustraction de la fluorescence des contrôles, la variation de la fluorescence est tracée en fonction des concentrations du peptide α2δ2. Ceci a permis d'estimer la sensibilité de la méthode de dosage. After washing with washing buffer for 3 times, the wells are incubated 1 h at 4 ° C. in the presence of a secondary antibody (Goat Anti-Rabbit IgG, Jackson Immunoreasearch Lab) conjugated to fluorescein (Alexa Fluor 488 from Lifetechnologies) at a dilution of l / 2000e in a total volume of 100 μΐ of the blocking buffer. After this incubation, the wells are washed three times with wash buffer (PBS) and the fluorescence is measured at the wells using a fluorescence plate reader. After the fluorescence subtraction of the controls, the fluorescence variation is plotted as a function of the concentrations of the α 2 δ 2 peptide. This made it possible to estimate the sensitivity of the assay method.
La figure 1 montre la fluorescence de l'anticorps secondaire en fonction de la concentration de peptide α2δ2 sur toute la gamme de concentration du peptide α2δ2. La figure 13 A montre un agrandissement de cette courbe dans la gamme de concentration proche du seuil de détection. Ce seuil, déterminé par dérivation de la courbe expérimentale obtenue, est d'environ 10 ng/ml. Dosage de la protéine o^dans les urines FIG. 1 shows the fluorescence of the secondary antibody as a function of the α 2 δ 2 peptide concentration over the entire concentration range of the α 2 δ 2 peptide. FIG. 13A shows an enlargement of this curve in the concentration range close to the detection threshold. This threshold, determined by derivation of the experimental curve obtained, is about 10 ng / ml. Determination of the protein o ^ in the urine
Les échantillons urinaires des patients obtenus sans massage de la prostate (20 ml) sont concentrés 10 fois en utilisant la méthode de la concentration à l'acétone. Après le dosage des protéines des échantillons urinaires, 4μg de chaque échantillon urinaire sont incubés dans les puits (plaque 96 puits) dans un volume total de 100 μΐ PBS (« protéines d'urine »). Afin de vérifier si un dosage direct d'a2ô2 est possible dans les urines, parallèlement,Urine samples from patients obtained without prostate massage (20 ml) are concentrated 10 times using the acetone concentration method. After the determination of the urinary samples, 4μg of each urinary sample is incubated in the wells (96-well plate) in a total volume of 100 μΐ PBS ("urine proteins"). In order to check whether a direct dose of 2 2 is possible in the urine, at the same time
50 μΐ d'urines de chaque patient sont déposés dans des puits dans un volume total de 100 μΐ en présence de PBS (« urine totale ») . A partir de cette étape, toutes les opérations pour tous les échantillons sont effectuées comme décrites précédemment pour la réalisation de la gamme de détection du peptide α2δ2. 50 μΐ of urine from each patient are deposited in wells in a total volume of 100 μΐ in the presence of PBS ("total urine"). From this step, all the operations for all the samples are carried out as previously described for the realization of the α 2 δ 2 peptide detection range.
Résultats Results
Expression de α2δ2 dans les tissus et les lignées cellulaires du cancer de la prostate II a été démontré dans des études précédentes que les canaux calciques voltage- dépendants Cav3.2 sont exprimés dans les lignées cellulaires du cancer de la prostate et dans les tumeurs de la prostate humaine (5). Il a ici été recherché si les sous-unités régulatrices supposées peuvent aussi être exprimées dans ces cellules. L'attention a été portée sur les sous- unités α2δ étant donné que deux d'entre elles (α2δ2 et α2δ3) sont suspectées d'agir comme suppresseurs de tumeurs dans différents organes. Expression of α 2 δ 2 in prostate cancer tissues and cell lines II has been shown in previous studies that Cav3.2 voltage-gated calcium channels are expressed in prostate cancer cell lines and in prostate cancer cells. tumors of the human prostate (5). It has been investigated here whether the supposed regulatory subunits can also be expressed in these cells. Attention has been given to α 2 δ subunits since two of them (α 2 δ 2 and α 2 δ 3 ) are suspected of acting as tumor suppressors in different organs.
Comme montré à la figure 2, il a été mis en évidence par RT-PCR que les lignées cellulaires du cancer de la prostate étudiées (cellules LNCaP, DU145, PC3) expriment un produit de la transcription de α2δ2. Les autres sous-unités α2δ recherchées ( 2ôi et α2δ3) ne sont pas exprimées dans les lignées cellulaires de la prostate étudiées. As shown in FIG. 2, it has been demonstrated by RT-PCR that the prostate cancer cell lines studied (LNCaP cells, DU145, PC3) express a α 2 δ 2 transcription product. Other subunits α 2 δ sought (2 Oi and α 2 δ 3) are not expressed in the cell lines examined prostate.
De la même façon, et comme montré à la figure 3, les analyses par RT-PCR ont démontré que α2δ2 est présente dans les tissus normaux et cancéreux de la prostate. Des tests d'immunodétection ont également été effectués pour mettre en évidence l'expression des protéines α2δ2 dans les lignées cellulaires du cancer de la prostate et dans les coupes de tissus de la prostate. Les analyses par immunofluorescence ont montré que les lignées cellulaires du cancer de la prostate LNCaP, DU! 45 et PC3 sont fortement colorées par les anticorps anti-a2ô2. Π est en outre observé que la coloration α2δ2 est évidente sur la cellule mais elle est particulièrement prononcée sur la périphérie, c'est-à-dire près de la membrane plasmique, lieu d'ancrage des protéines α2δ2 (non représenté). In the same way, and as shown in FIG. 3, the RT-PCR analyzes demonstrated that α 2 δ 2 is present in the normal and cancerous tissues of the prostate. Immunodetection tests were also performed to demonstrate the expression of α 2 δ 2 proteins in prostate cancer cell lines and in prostate tissue sections. Immunofluorescence assays showed that the LNCaP prostate cancer cell lines, DU! 45 and PC3 are strongly stained by anti-α 2 δ 2 antibodies. It is further observed that the α 2 δ 2 staining is evident on the cell, but it is particularly pronounced on the periphery, that is to say near the plasma membrane, where the α 2 δ 2 proteins are anchored ( not shown).
De plus, par western blot, il a été observé, au moyen d'anticorps anti-a2ô2, l'expression de deux bandes autour de 160 kD (cf. figure 4). L'expression de ces bandes est fortement réduite lorsque les cellules LNCaP sont mises en présence de si-a2ô2. Ces bandes de 160 kD observées par western blot correspondent à la taille attendue de α2δ2, ce qui démontre que les anticorps utilisés ici détectent bien la protéine α2δ2. In addition, by western blotting, the expression of two bands around 160 kD was observed by means of anti-α 2 δ 2 antibodies (see FIG. 4). The expression of these bands is greatly reduced when the LNCaP cells are placed in the presence of si-a 2 δ 2 . These bands of 160 kD observed by western blot correspond to the expected size of α 2 δ 2 , which demonstrates that the antibodies used here correctly detect the α 2 δ 2 protein.
Des colorations par immunofluorescence (IF) et immunohistochimiques (IH) de coupes du cancer de la prostate obtenus des sujets ont été effectuées sur des coupes de tissus de la prostate congelées (IF) ou des coupes en paraffine (réaction peroxydase IH). Il a été montré qu'il y a une forte coloration des structures glandulaires épithéliales. Les acini sont fréquemment colorés avec les anticorps anti-a2ô2 (non représenté). De plus, les membranes apicales de l'épithélium sont plus fortement colorées que des membranes basolatérales (non représenté). Immunofluorescence (IF) and immunohistochemical (IH) staining of prostate cancer sections obtained from the subjects were performed on sections of frozen prostate tissue (IF) or paraffin sections (IH reaction peroxidase). It has been shown that there is a strong staining of the glandular epithelial structures. Acini are frequently stained with anti-α 2 δ 2 antibodies (not shown). In addition, the apical membranes of the epithelium are more strongly stained than basolateral membranes (not shown).
L'immunocoloration des cellules épithéliales du cancer de la prostate dans le milieu de culture primaire a donc mis en évidence une forte coloration de α2δ2 sur la périphérie des cellules. L'expression de α2δ2 augmente avec le développement du cancer de la prostateThe immunostaining of the prostate cancer epithelial cells in the primary culture medium thus revealed a strong α 2 δ 2 staining on the periphery of the cells. The expression of α 2 δ 2 increases with the development of prostate cancer
Afin d'évaluer les éventuelles différences d'expression de α2δ2 entre les tissus normaux et les tissus tumoraux, une comparaison de l'expression de α2δ2 au moyen de la technique RT-PCR a été effectuée dans 18 tissus du cancer de la prostate et dans 20 tissus non-cancéreux de la prostate. Comme montré aux figures 3 et 5, α2δ2 est plus fréquemment exprimée dans les tissus cancéreux (95% des tissus cancéreux expriment α2δ2) que dans les tissus non-cancéreux (40% des tissus non-cancéreux expriment α2δ2) (différence significative, p<0,001, test du Chi2). In order to evaluate the possible differences in expression of α 2 δ 2 between normal tissues and tumor tissues, a comparison of the expression of α 2 δ 2 by means of the RT-PCR technique was carried out in 18 tissues of the prostate cancer and in 20 non-cancerous prostate tissues. As shown in FIGS. 3 and 5, α 2 δ 2 is more frequently expressed in the cancerous tissues (95% of the cancerous tissues express α 2 δ 2 ) than in the non-cancerous tissues (40% of the non-cancerous tissues express α 2 δ 2 ) (significant difference, p <0.001, Chi2 test).
De la même façon, l'expression de α2δ2 a été comparée sur des macroarrays de 24 tissus différents dont 16 tissus cancéreux et 8 tissus non cancéreux. Comme montré à la figure 6, les cellules épithéliales recouvrant les acini sont colorées par les anticorps anti-a2ô2 dans les échantillons normaux, hyperplasiques et cancéreux. Dans les stades avancés de cancer, un certain nombre de cellules colorées quittent les acini (cf. figure 6B, G3, Métastase). Plusieurs grades de différenciation ont été analysés. La surface colorée ainsi que l'intensité de coloration augmentent dans les stades avancés du cancer de la prostate en comparaison à la prostate normale ou hyperplasique. In the same way, the expression of α 2 δ 2 was compared on macroarrays of 24 different tissues including 16 cancerous tissues and 8 non-cancerous tissues. As shown in Figure 6, the epithelial cells covering the acini are stained with anti-α 2 δ 2 antibodies in normal, hyperplastic and cancerous samples. In the advanced stages of cancer, a number of colored cells leave the acini (see Figure 6B, G3, Metastasis). Several grades of differentiation were analyzed. Colored area and staining intensity increase in advanced stages of prostate cancer compared to normal or hyperplastic prostate.
Comme montré à la figure 6, la localisation dans la cellule est différente entre les stades normaux, BPH et précoces (Gl) du cancer et les stades avancés du cancer (G2, G3). Dans les stades BPH et précoces du cancer (Gl), la coloration est majoritairement apicale et intracellulaire, alors que dans les stades avancés du cancer (G2 et G3), la coloration augmente dans tous les compartiments de la cellule, et plus particulièrement dans le noyau des cellules. La densité de coloration a également été analysée au moyen de la déconvolution de couleur pour extraire la coloration DAB de contre-coloration (Bleu azuré) et il a été montré que la densité DAB est plus importante dans les stades avancés du cancer, mettant ainsi en évidence que l'expression de a2ô2 est augmentée dans les cancers avancés de la prostate (figure 6C). As shown in Figure 6, the localization in the cell is different between the normal, BPH and early stages (G1) of cancer and the advanced stages of cancer (G2, G3). In BPH and early stages of cancer (Gl), staining is predominantly apical and intracellular, whereas in the advanced stages of cancer (G2 and G3), staining increases in all compartments of the cell, and more particularly in the cell nucleus. Staining density was also analyzed using color deconvolution to extract counterstain DAB staining (azure blue) and DAB density was shown to be higher in advanced stages of cancer, thus making evidence that α 2 δ 2 expression is increased in advanced prostate cancers (Figure 6C).
L'inhibition de α2δ2 réduit la prolifération cellulaire alors que sa surexpression induit la prolifération cellulaire Inhibition of α 2 δ 2 reduces cell proliferation whereas its overexpression induces cellular proliferation
Etant donné que α2δ2 est exprimée dans les cellules épithéliales du cancer de la prostate, il a été recherché si la protéine peut participer à la croissance cellulaire. Since α 2 δ 2 is expressed in epithelial cells of prostate cancer, it has been investigated whether the protein can participate in cell growth.
Différentes approches ont été utilisées pour évaluer la prolifération cellulaire. L'utilisation de ligands ou de si-a,2Ô2 réduit la tumorigénèse  Different approaches have been used to evaluate cell proliferation. The use of ligands or si-α, ₂2 reduces tumorigenesis
Premièrement, il a été démontré par une méthode MTS que la prolifération cellulaire est réduite de 20% au bout de 4 jours d'incubation dans 50 nM de si-a2ô2 en comparaison avec si-ctl (cf. figure 7A a). De la même façon, la prolifération cellulaire est diminuée en présence de si-a2ô2 dans les lignées DU 145 et PC3 (cf. figure 7B). First, it has been demonstrated by an MTS method that cell proliferation is reduced by 20% after 4 days of incubation in 50 nM of si-a 2 δ 2 compared to si-ct1 (see Figure 7A a). . In the same way, cell proliferation is decreased in the presence of si-a 2 δ 2 in DU 145 and PC3 lines (see Figure 7B).
Des résultats similaires ont été obtenus avec un comptage manuel de cellules, ce qui montre qu'une incubation de 4 jours dans si-a2ô2 diminue de 30% le nombre de cellules LNCaP (cf. figure 7 A b). Des expériences cinétiques (figure 7C) illustrent le fait que si-a2ô2 est un inhibiteur significatif de la prolifération cellulaire après seulement trois jours d'incubation et qu'un effet maximal est atteint après 5 jours à 6 jours (40% de réduction de la prolifération cellulaire). Des analyses FACS du cycle cellulaire montrent que cette action inhibitrice de si-a2ô2 sur la prolifération cellulaire est corrélée à une augmentation du nombre de cellules en phase G1; ainsi qu'à une diminution du nombre de cellules en phase G2/M et en phase S (figure 7D). Similar results were obtained with hand counting of cells, which shows that a 4-day incubation in si-a 2 δ 2 decreases the number of LNCaP cells by 30% (see Figure 7 A b). Kinetic experiments (FIG. 7C) illustrate that si-a 2 δ 2 is a significant inhibitor of cell proliferation after only three days of incubation and that a maximal effect is reached after 5 days to 6 days (40% of reduction of cell proliferation). FACS cell cycle analyzes show that this inhibitory action of si-a 2 δ 2 on cell proliferation is correlated with an increase in the number of cells in G 1 phase ; as well as a decrease in the number of cells in G 2 / M phase and in S phase (Figure 7D).
La gabapentine a été testée sur la prolifération cellulaire, celle-ci étant connue pour inhiber différents canaux calciques voltage-dépendants par son action sur la sous-unité régulatrice α2δ. En effet, la gabapentine a été démontrée comme étant un ligand des deux sous-unités 2ôi et α2δ2 (12). Dans les présents essais, la gabapentine, qui a été ajoutée au milieu de culture cellulaire à 100 μΜ, réduit de 35% la prolifération cellulaire d'incubationGabapentin has been tested for cell proliferation, which is known to inhibit different voltage-gated calcium channels by its action on the α2δ regulatory subunit. Indeed, gabapentin has been demonstrated to be a ligand of the two subunits 2 δi and α 2 δ 2 (12). In the present tests, gabapentin, which has been added to cell culture medium at 100 μΜ, reduces cell proliferation by 35%
(figure 7E). L'inhibition induite par la gabapentine diminue ensuite légèrement jusqu'à 20% après 4 jours, ce qui peut être dû soit à une désensibilisation de la gabapentine, soit à une dégradation de la gabapentine. (Figure 7E). The inhibition induced by gabapentin then slightly decreases to 20% after 4 days, which may be due to either desensitization of gabapentin or degradation of gabapentin.
Les lignées LNCaP-a,2Ô2 induisent la prolifération cellulaire LNCaP-a, 2Ô2 Lines Induce Cell Proliferation
Des clones de LNCaP surexprimant de manière stable α2δ2 ont été utilisés de manière similaire dans les essais de prolifération cellulaire. Il a été montré que deux clones différents surexprimant α2δ2 (clone 9 « C9 », et clone 11 « Ci l ») produisent des taux de prolifération plus rapides que les cellules témoins LNCaP, avec un temps de multiplication par 2 de 47,5h pour LNCaP contre 40,5h pour le clone 9 et 36h pour le clone 11 (cf. figure 7F a). LNCaP clones stably overexpressing α 2 δ 2 were similarly used in cell proliferation assays. It has been shown that two different α 2 δ 2 overexpressing clones (clone 9 "C9", and clone 11 "Ci l") produce faster proliferation rates than LNCaP control cells, with a doubling time of 47. , 5h for LNCaP against 40.5h for clone 9 and 36h for clone 11 (see Figure 7F a).
Des essais d'immunofluorescence colorant Ki-67 sur des lignées cellulaires LNCaP et LNCaP surexprimant α2δ2 (clone 11) ont été effectués (cf. figure 7F b). Ki-67 est un marqueur de cellules proliférantes. Il est seulement exprimé pendant les phases G1; S, G2 et M. Les cellules en phase Go n'expriment pas Ki-67 de façon significative. Il peut y avoir soit une présence ponctuelle dans le noyau pendant les phases G\ et S, soit une localisation plus homogène dans le noyau pendant les phases G2 et M. Il a ici été démontré que α2δ2 est associée à une diminution du nombre de cellules pendant la phase Go du cycle cellulaire. Dans ces essais, les cellules non proliférantes représentent 35,6 ± 1,6% de la population cellulaire LNCaP totale, alors qu'elles ne représentent que 21,6% ± 3,5% de la population cellulaire LNCaP-a2ô2 totale. Ce résultat est corrélé avec une augmentation du nombre de cellules pendant les phases Gi/S et G2/M du cycle cellulaire des cellules LNCaP-a2ô2. Au vu de tous ces résultats, il est donc démontré que α2δ2 augmente la prolifération des cellules LNCaP. Ki-67 dye immunofluorescence assays on LNCaP and LNCaP cell lines overexpressing α 2 δ 2 (clone 11) were performed (see Figure 7F b). Ki-67 is a marker of proliferating cells. It is only expressed during G 1 phases ; S, G 2 and M. Go-phase cells do not express Ki-67 significantly. There may be either a punctual presence in the nucleus during the G \ and S phases, or a more homogeneous localization in the nucleus during the G 2 and M phases. It has been shown here that α 2 δ 2 is associated with a decrease. of the number of cells during the Go phase of the cell cycle. In these trials, nonproliferative cells accounted for 35.6 ± 1.6% of the total LNCaP cell population, whereas they accounted for only 21.6% ± 3.5% of the LNCaP-a 2 δ 2 cell population. total. This result is correlated with an increase in the number of cells during the Gi / S and G 2 / M phases of the cell cycle of LNCaP-a 2 δ 2 cells. Given all these results, it is therefore demonstrated that α 2 δ 2 increases the proliferation of LNCaP cells.
La surexpression de a,2Ô2 induit le développement tumoral in vivo Overexpression of α, ₂2 induces tumor development in vivo
Des essais in vivo ont été effectués sur des souris nude immunodéficientes. Les souris ont subi une injection sur chaque flanc de cellules LNCaP ou de cellules LNCaP surexprimant α2δ2 (figure 5). Les tumeurs obtenues sont respectivement nommées ci-après « tumeur de LNCaP » et « tumeur de LNCaP- 2δ2». La taille des tumeurs, qui a été mesurée deux fois par semaine, a commencé à augmenter habituellement après 4-6 semaines. Une fois que la tumeur était détectable, elles ont grandi avec un temps de multiplication par 2 de 12,2 ± 1,9 jours (cf. figure 8a) pour les tumeurs des LNCaP, pour atteindre une taille moyenne de 940 ± 146 mm (1,26 ± 0,22 g) (cf. figures 8b et c). Les tumeurs des LNCaP-a2ô2 ont grandi avec un temps de multiplication par 2 de 6,6 ± 0,8 jours (cf. figure 8a), pour atteindre une taille moyenne de 1760 ± 154 mm (2,3 ± 0,21 g) (cf. figures 8b et c). Pour les deux types de tumeurs, la taille maximale de la tumeur a été atteinte habituellement 4 semaines après le début de croissance de la tumeur pour chaque souris. In vivo assays were performed on immunodeficient nude mice. The mice were injected on each flank of LNCaP cells or LNCaP cells overexpressing α 2 δ 2 (FIG. 5). The tumors obtained are hereinafter referred to as "LNCaP tumor" and "LNCaP- 2 δ 2 tumor," respectively. Tumor size, which was measured twice a week, began to increase usually after 4-6 weeks. Once the tumor was detectable, they grew with a doubling time of 12.2 ± 1.9 days (see Figure 8a) for LNCaP tumors, reaching an average size of 940 ± 146 mm (1.26 ± 0.22 g) (see Figures 8b and c). LNCaP-a 2 δ 2 tumors grew with a doubling time of 6.6 ± 0.8 days (see Figure 8a), reaching an average size of 1760 ± 154 mm (2.3 ± 0). 21 g) (see Figures 8b and c). For both types of tumors, the maximum tumor size was reached usually 4 weeks after the beginning of tumor growth for each mouse.
Par conséquent, il est mis en évidence in vivo que les tumeurs de LNCaP-a2ô2 augmentent plus vite et sont plus grosses que les tumeurs de LNCaP. La surexpression de α2δ2 induit la formation de vaisseaux sanguins et la sécrétion de VEGF. Therefore, LNCaP-a 2 δ 2 tumors are found in vivo to increase faster and are larger than LNCaP tumors. The overexpression of α 2 δ 2 induces the formation of blood vessels and the secretion of VEGF.
Les deux tumeurs de LNCaP et LNCaP-a2ô2 obtenues lors des essais in vivo décrits précédemment sont fortement irriguées et sont caractérisées par d'abondants vaisseaux sanguins mais aussi par des signes d'hémorragies avec des fuites de globules rouges dans les tissus voisins et de nombreux foyers d'inflammations et de nécroses (cf. figure 9A). The two tumors LNCaP and LNCaP-a 2 ô 2 obtained in the in vivo tests described above are highly irrigated and are characterized by abundant blood vessels but also by signs of hemorrhages with leakage of red blood cells in neighboring tissues and many foci of inflammation and necrosis (see Figure 9A).
Aucune différence significative de surface nécrotique n'a été détectée entre les tumeurs de LNCaP et les tumeurs de LNCaP-a2ô2. Pourtant, les études d'immunofluorescence par PCNA, permettant l'identification des cellules en phase S du cycle cellulaire, montrent au contraire qu'm vivo, il y a plus de cellules proliférantes dans les tumeurs de LNCaP-a2ô2 que dans les tumeurs de LNCaP (figure 9B). De plus, les lames histologiques montrent qu'il y a plus de vaisseaux sanguins dans les tumeurs de LNCaP-a2ô2 que dans les tumeurs de LNCaP (figures 9 A et B). Les vaisseaux sanguins ont donc été colorés avec du CD31 (figure 10A) qui est un marqueur couramment utilisé pour colorer les cellules endothéliales et pour détecter l'angiogénèse dans les tumeurs. Il a été montré que la surface colorée dans les tumeurs de LNCaP-a2ô2 est deux fois plus importante en comparaison avec celle des tumeurs de LNCaP (figure 10B). Ceci a été confirmé par des analyses par western blot qui ont montré une augmentation de 50% dans l'expression de CD31 (figure 11 A). Puisque α2δ2 est associée à la formation de vaisseaux sanguins, l'expression et la sécrétion de VEGF dans les tumeurs et les cellules de la prostate ont été mesurées. Comme montré à la figure 11A, les analyses par western blot montrent que l'expression de VEGF est légèrement, mais significativement, augmentée dans les tumeurs surexprimant α2δ2. Des analyses ELISA de VEGF ont donc été effectuées pour déterminer si la sécrétion in vitro est altérée dans les cellules LNCaP surexprimant α2δ2. Comme montré à la figure 11B, les tests ELISA montrent que la surexpression de α2δ2 augmente significativement la sécrétion deNo significant necrotic surface difference was detected between LNCaP tumors and LNCaP-a 2 δ 2 tumors. However, PCNA immunofluorescence studies, allowing the identification of cells in the S phase of the cell cycle, show that, in vivo, there are more proliferating cells in LNCaP-a 2 δ 2 tumors than in LNCaP tumors (Figure 9B). In addition, histological slides show that there are more blood vessels in LNCaP-a 2 δ 2 tumors than in LNCaP tumors (Figures 9 A and B). The blood vessels were therefore stained with CD31 (Figure 10A) which is a marker commonly used to stain endothelial cells and to detect angiogenesis in tumors. It has been shown that the stained surface in LNCaP-a 2 δ 2 tumors is twice as large compared to that of LNCaP tumors (FIG. 10B). This was confirmed by western blot analyzes which showed a 50% increase in CD31 expression (Fig. 11A). Since α 2 δ 2 is associated with the formation of blood vessels, the expression and secretion of VEGF in tumors and prostate cells were measured. As shown in Figure 11A, western blot analyzes show that VEGF expression is slightly, but significantly, increased in overexpressing α 2 δ 2 tumors. VEGF ELISA analyzes were therefore performed to determine if in vitro secretion is impaired in LNCaP cells overexpressing α 2 δ 2 . As shown in Figure 11B, the tests ELISA show that the overexpression of α 2 δ 2 significantly increases the secretion of
VEGF chez deux différents clones de LNCaP-a2ô2. Ceci n'est pas dû à une stimulation générale non- spécifique de la sécrétion par les cellules LNCaP puisque la sécrétion de la phosphatase d'acide prostatique (PAP) n'est pas affectée par la surexpression de α2δ2, bien que la PAP soit stimulée par une augmentation en calcium intracellulaire. α2δ2 peut être clivée dans les cellules LNCaP surexprimant α2δ2. VEGF in two different clones of LNCaP-a 2 δ 2 . This is not due to a general non-specific stimulation of secretion by LNCaP cells since the secretion of prostatic acid phosphatase (PAP) is not affected by the overexpression of α 2 δ 2 , although the PAP is stimulated by an increase in intracellular calcium. α 2 δ 2 can be cleaved in LNCaP cells overexpressing α 2 δ 2 .
En conditions non-réductrices, α2δ2 correspond à une bande simple de 175 kDa alors qu'en conditions réductrices, deux bandes de 150 et 22 kDa sont observées par western blots (13). Il a ainsi été montré que a2 et δ2, dérivés du même gêne CACNA2D2, sont liés par des ponts disulfure. a2 est une sous-unité large extracellulaire (150kDa ou plus, dépendant de la glycosylation) reliée à δ2, une plus petite protéine transmembranaire (environ 20kDa). Under non-reducing conditions, α 2 δ 2 corresponds to a simple band of 175 kDa whereas under reducing conditions, two bands of 150 and 22 kDa are observed by western blots (13). It has thus been shown that a 2 and δ 2 , derived from the same CACNA2D2 gene, are linked by disulfide bridges. a 2 is a broad extracellular subunit (150kDa or more dependent on glycosylation) related to δ 2 , a smaller transmembrane protein (approximately 20kDa).
Afin d'évaluer si a2ô2 peut être utilisée comme marqueur tumoral, il a été recherché si ces ponts disulfure peuvent être clivées dans les cellules LNCaP pour permettre à la protéine d'être libérée dans le milieu de culture. Pour cela, la présence de α2δ2, soit a2 en cas de clivage, a été recherchée dans le milieu de culture par western blot. La figure 12 montre que a2 est bien présente dans le milieu de culture dans lequel les cellules LNCaP-a2ô2 ont été incubées pendant 6 jours, tandis que cette sous-unité est à peine détectable dans le milieu de culture des cellules témoins LNCaP. α2δ2 peut être dosée dans les urines de patients atteints de cancer de la prostate.In order to evaluate whether a 2 δ 2 can be used as a tumor marker, it has been investigated whether these disulfide bridges can be cleaved in LNCaP cells to allow the protein to be released into the culture medium. For this, the presence of α 2 δ 2 , or a 2 in case of cleavage, was sought in the culture medium by western blot. FIG. 12 shows that a 2 is present in the culture medium in which LNCaP-a 2 δ 2 cells have been incubated for 6 days, whereas this subunit is hardly detectable in the culture medium of the control cells. LNCaP. α 2 δ 2 can be assayed in the urine of prostate cancer patients.
La figure 13 B montre les concentrations de la protéine codée par le gène CACNA2D2 dans les urines totales et dans les protéines obtenues à partir d'urine d'un homme atteint d'un cancer de la prostate. Figure 13B shows the concentrations of the protein encoded by the CACNA2D2 gene in total urine and in proteins obtained from urine of a man with prostate cancer.
Cette concentration est d'environ 20 ng/ml, valeur supérieure au seuil de détection comme visible à la figure 13 A.  This concentration is about 20 ng / ml, which is greater than the detection threshold as can be seen in FIG. 13A.
Discussion Discussion
La protéine α2δ2 avait été détectée comme régulant l'activité des canaux calciques voltage-dépendants. Bien que de nombreuses recherches aient montré que le gène CACNA2D2, codant pour cette protéine, est localisé dans le locus 3p21, connu comme étant un cluster de gènes suppresseurs de tumeurs, il a ici été démontré que α2δ2 est capable d'influer elle-même sur la prolifération cellulaire in vitro et sur la tumorigénèse in vivo. Cette protéine α2δ2, ou un fragment de celle-ci, représente un nouveau biomarqueur du cancer, et notamment du cancer de la prostate. Ceci est basé sur les observations suivantes : (1) La sous-unité de canaux calciques α2δ2, codée par le gène CACNA2D2, est exprimée dans les cellules de la prostate ; The α 2 δ 2 protein was detected as regulating the activity of voltage-gated calcium channels. Although many studies have shown that the CACNA2D2 gene, encoding this protein, is located in the 3p21 locus, known as a cluster of tumor suppressor genes, it has been shown here that α 2 δ 2 is capable of influencing itself on cell proliferation in vitro and on tumorigenesis in vivo. This α 2 δ 2 protein, or a fragment thereof, represents a new biomarker for cancer, and in particular for prostate cancer. This is based on the following observations: (1) The α 2 δ 2 calcium channel subunit, encoded by the CACNA2D2 gene, is expressed in prostate cells;
(2) Cette protéine, ou son gène, est plus fréquemment exprimée dans les tissus cancéreux que dans les tissus non-cancéreux de la prostate ; (2) This protein, or its gene, is more frequently expressed in cancerous tissues than in non-cancerous tissues of the prostate;
(3) Cibler cette protéine avec des ligands ou si-RNA peut réduire la tumorigénèse in vitro ; (3) Targeting this protein with ligands or si-RNA can reduce tumorigenesis in vitro;
(4) L'expression de α2δ2 est associée à une sécrétion de VEGF et une formation de vaisseaux sanguins ; (4) The expression of α 2 δ 2 is associated with secretion of VEGF and formation of blood vessels;
(5) Sa surexpression induit le développement tumoral chez la souris nude ; (5) Its overexpression induces tumor development in nude mice;
(6) α2δ2 est surexprimée dans d'autres formes de cancers ; (6) α 2 δ 2 is overexpressed in other forms of cancer;
(7) La protéine α2δ2 peut être clivée et la sous-partie a2 de la protéine α2δ2 libérée dans le milieu extracellulaire de cellules LNCaP-a2ô2. Elle est détectable avec des anticorps disponibles sur le marché. Ainsi, la protéine α2δ2 a été détectée dans les cellules de la prostate à la fois cancéreuses et non-cancéreuses par PCR et immunodétection, en particulier sur les membranes apicales de l'épithélium (observation (1)). Des analyses PCR et immunohistochimiques ont ensuite montré que la protéine α2δ2 est plus exprimée dans les tissus cancéreux de la prostate que dans les tissus non-cancéreux (observation (2)). En partant du constat, établi par des techniques de MTS, comptage manuel ou des expériences cinétiques, que le ciblage de la protéine avec si-a2ô2 ou des ligands comme la gabapentine réduit la prolifération cellulaire (observation (3)), il a été montré in vitro et in vivo que cette surexpression de α2δ2 induit le développement tumoral chez la souris nude (observation (5)). Des analyses histologiques, d' immunodétection, par western blot et des tests ELISA ont également montré que cette surexpression de α2δ2 provoque la sécrétion de VEGF et l'angiogénèse (observation (4)). Il a enfin été mis en évidence que la protéine α2δ2 peut être clivée, libérant ainsi la sous-partie a2 de la protéine α2δ2 dans le milieu extracellulaire de cellules LNCaP-a2ô2. La protéine α2δ2 ou les fragments de cette protéine, et plus particulièrement la sous-partie a2, sont détectables avec des anticorps disponibles sur le marché (observation (7)). (7) The protein α 2 δ 2 may be cleaved and the sub-portion 2 of the α 2 δ protein 2 released into the extracellular medium of LNCaP cells 2-a O 2. It is detectable with antibodies available on the market. Thus, the α 2 δ 2 protein was detected in both cancerous and non-cancerous prostate cells by PCR and immunodetection, particularly on the apical membranes of the epithelium (observation (1)). PCR and immunohistochemical analyzes have shown that the α 2 δ 2 protein is more expressed in prostate cancer tissue than in non-cancerous tissues (observation (2)). Starting from the finding, established by MTS techniques, manual counting or kinetic experiments, that the targeting of the protein with si-a 2 δ 2 or ligands such as gabapentin reduces cell proliferation (observation (3)). has been shown in vitro and in vivo that this overexpression of α 2 δ 2 induces tumor development in nude mice (observation (5)). Histological, immunodetection, western blot and ELISA assays have also shown that this overexpression of α 2 δ 2 induces the secretion of VEGF and angiogenesis (observation (4)). Finally, it was shown that the protein α 2 δ 2 can be cleaved, releasing Subpart 2 of the protein α 2 δ 2 in the extracellular medium of cells LNCaP-2 O 2. The α 2 δ 2 protein or the fragments of this protein, and more particularly the a 2 sub-part, are detectable with antibodies available on the market (observation (7)).
Il est par ailleurs raisonnable d'affirmer que la protéine α2δ2> ou les fragments de cette protéine, puisse, de la même façon, être détectée dans le sang, le sérum, le plasma, le sperme et les urines. It is also reasonable to say that the α 2 δ 2 protein or fragments of this protein can similarly be detected in blood, serum, plasma, sperm and urine.
Il est ainsi connu qu'm vivo, les protéines ancrées par des groupements GPI à la membrane peuvent être clivées par différentes enzymes, dont certaines phospholipases C ou D, hydrolysant ces groupements GPI et libérant ainsi les protéines dans le milieu extracellulaire. Il a ainsi été mis en évidence que la protéine α2δ2 était bien présente dans les urines de patients atteints de cancer de la prostate. It is thus known that, in vivo, proteins anchored by GPI groups to the membrane can be cleaved by different enzymes, including certain phospholipases C or D, hydrolyzing these GPI groups and thus releasing the proteins in the extracellular medium. It has thus been demonstrated that the α 2 δ 2 protein is present in the urine of prostate cancer patients.
Plus particulièrement le clivage in vivo de la sous-partie a2 de la protéine, libérant la sous-partie a2 dans le milieu extracellulaire, est fortement présumé. II est en effet connu que, dans le milieu extracellulaire, les ponts disulfures peuvent être clivés par de nombreuses enzymes comme la disulfure isomérase ou la phosphoglycérate kinase (14), libérant ainsi a2 dans le milieu extracellulaire des cellules épithéliales de la prostate. Or, il a déjà été montré que ces deux enzymes sont détectées dans la prostate et que leur expression est accrue lors du développement cancéreux (15, 16). More particularly, the in vivo cleavage of the a 2 sub-part of the protein, releasing sub-part a 2 in the extracellular medium, is strongly presumed. It is indeed known that, in the extracellular medium, the disulfide bridges can be cleaved by numerous enzymes such as disulphide isomerase or phosphoglycerate kinase (14), thus releasing 2 in the extracellular medium epithelial cells of the prostate. However, it has already been shown that these two enzymes are detected in the prostate and that their expression is increased during cancerous development (15, 16).
Ainsi, de façon similaire au présent cas, la protéine CD90 (Cluster of Differentiation 90) est une protéine de surface N-glycosylatée, ancrée à la membrane par un groupement GlycosylPhosphatidylInositol (GPI), comme la protéine α2δ2. Elle a été découverte initialement comme antigène de thymocytes (17), et a été décrite comme étant exprimée dans le tissu nerveux (18), les cellules progénitrices hématopoïétiques (19), et dans les fibroblastes de nombreux tissus tels que les poumons ou le myomètre lung (20). Des niveaux élevés de CD90 ont été détectés dans des gliomes malins et l'hépatocarcinome (21). La protéine CD90 est multipliée par un facteur 5 dans les tissus de cancers de la prostate par rapport aux tissus non cancéreux (22). Les mêmes auteurs ont étudié la présence de CD90 dans les urines afin de déterminer si cette protéine était sécrétée. Un peptide issu de CD90 a ainsi été identifié dans les urines de patients atteints de cancer de la prostate par spectrométrie de masse, confirmant que CD90 est sécrétée par le tissu cancéreux prostatique et peut donc servir de biomarqueur pour des tests non invasifs (22). II est connu que la libération de protéines par les cellules prostatiques dans le liquide séminal s'effectue d'une part par la sécrétion de protéines solubles par exocytose et d'autre part par la production de prostasomes, ou exosomes, libérés par exocytose à partir des corps multivésiculaires des cellules épithéliales. Ces exosomes, retrouvés dans le liquide séminal, contiennent un grand nombre de protéines, par exemple des protéines membranaires, comme GLUT5 (Glucose Transporter 5), VAT-1 (synaptic vesicle membrane protein homologue) ou SCA2 (synaptic carrier associated membrane protein). D'autre part, ces prostasomes, qui sont normalement libérés dans le liquide séminal peuvent se retrouver dans le plasma sanguin au cours du développement de cancers prostatiques (23). Ainsi, il est très probable que la protéine α2δ2 soit libérée dans le liquide séminal ou le plasma sanguin par ces prostasomes et puisse être dosée dans d'autres liquides organiques que l'urine. Thus, in a similar manner to the present case, the CD90 (Cluster of Differentiation 90) protein is an N-glycosylated surface protein, anchored to the membrane by a GlycosylPhosphatidylInositol (GPI) group, such as the α 2 δ 2 protein. It was originally found as a thymocyte antigen (17), and has been described as expressed in nerve tissue (18), hematopoietic progenitor cells (19), and in fibroblasts of many tissues such as the lungs or myometrium. lung (20). High levels of CD90 have been detected in malignant gliomas and hepatocarcinoma (21). The CD90 protein is multiplied by a factor of 5 in prostate cancer tissues compared to non-cancerous tissues (22). The same authors studied the presence of CD90 in urine to determine if this protein was secreted. A peptide derived from CD90 has thus been identified in the urine of prostate cancer patients by mass spectrometry, confirming that CD90 is secreted by prostate cancer tissue and can therefore serve as a biomarker for non-invasive tests (22). It is known that the release of proteins by the prostatic cells into the seminal fluid is effected on the one hand by the secretion of soluble proteins by exocytosis and, on the other hand, by the production of prostasomes, or exosomes, released by exocytosis from multivesicular bodies of epithelial cells. These exosomes, found in the seminal fluid, contain a large number of proteins, for example membrane proteins, such as GLUT5 (Glucose Transporter 5), VAT-1 (synaptic vesicle membrane homologous protein) or SCA2 (synaptic carrier associated membrane protein). On the other hand, these prostasomes, which are normally released in the seminal fluid, can be found in blood plasma during the development of prostatic cancers (23). Thus, it is very likely that the α 2 δ 2 protein is released into seminal fluid or blood plasma by these prostasomes and can be assayed in other body fluids than urine.
Enfin, il peut être appuyé le fait que, de la même façon que la PSA, l'architecture des tissus, et notamment la plus grande fragilité des vaisseaux sanguins, entraîne une fuite de PSA et d' 2δ2 dans le sang, permettant ainsi de retrouver cette protéine dans le plasma sanguin en plus grand quantité lors de développement tumoral. Finally, it can be argued that, in the same way as PSA, the architecture of the tissues, and in particular the greater fragility of the blood vessels, leads to a leak of PSA and 2 δ 2 in the blood, allowing thus to find this protein in the blood plasma in greater quantity during tumor development.
Il a donc été ici mis en évidence que la protéine α2δ2, ou un fragment de celle-ci, peut être utilisée comme biomarqueur du cancer de la prostate, ainsi que pour détecter le stade d'avancement du cancer. It has therefore been demonstrated here that the α 2 δ 2 protein, or a fragment thereof, can be used as a biomarker of prostate cancer, as well as to detect the stage of cancer progression.
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Claims

REVENDICATIONS
1. Méthode de détection in vitro d'un cancer de la prostate et/ou d'une métastase prostatique chez un sujet comprenant les étapes consistant à : A method for in vitro detection of prostate cancer and / or prostatic metastasis in a subject comprising the steps of:
(i) mesurer la concentration de α2δ2, ou un fragment de α2δ2, dans un échantillon biologique obtenu dudit sujet, (i) measuring the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in a biological sample obtained from said subject,
(ii) comparer la concentration de α2δ2, ou un fragment de α2δ2, mesurée à l'étape (i) à une valeur prédéterminée de la concentration en α2δ2, ou un fragment de α2δ2, (ii) comparing the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , measured in step (i) with a predetermined value of the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 ,
dans laquelle une concentration de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique supérieure à ladite valeur prédéterminée indique la présence d'un cancer de la prostate chez ledit sujet, et une concentration de α2δ2, ou d'un fragment de α2δ2, inférieure à ladite valeur prédéterminée indique l'absence d'un cancer de la prostate chez ledit sujet. in which a concentration of α 2 δ 2 , or of a fragment of α 2 δ 2 , in the biological sample greater than said predetermined value indicates the presence of a prostate cancer in said subject, and a concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , less than said predetermined value indicates the absence of a prostate cancer in said subject.
2. Méthode pour déterminer in vitro le stade d'un cancer de la prostate chez un sujet comprenant les étapes consistant à : 2. A method for determining in vitro the stage of prostate cancer in a subject comprising the steps of:
(i) mesurer la concentration de α2δ2, ou un fragment de α2δ2, dans un échantillon biologique obtenu dudit sujet, (i) measuring the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in a biological sample obtained from said subject,
(ii) comparer la concentration de α2δ2, ou un fragment de α2δ2, mesurée à l'étape (i) à une valeur prédéterminée de la concentration en α2δ2, ou un fragment de α2δ2, (ii) comparing the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , measured in step (i) with a predetermined value of the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 ,
dans laquelle, lorsque la concentration de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique est supérieure à ladite valeur prédéterminée de la concentration en α2δ2, ou un fragment de α2δ2, l'écart entre ladite concentration de α2δ2, ou d'un fragment de α2δ2, dans l'échantillon biologique obtenu dudit sujet et ladite valeur prédéterminée indique le stade du cancer de la prostate chez ledit sujet. in which, when the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in the biological sample is greater than said predetermined value of the concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , the difference between said concentration of α 2 δ 2 , or a fragment of α 2 δ 2 , in the biological sample obtained from said subject and said predetermined value indicates the stage of prostate cancer in said subject.
3. Méthode selon l'une quelconque des revendications 1 ou 2, dans laquelle ledit fragment de α2δ2 est a2. 3. Method according to any one of claims 1 or 2, wherein said fragment of α 2 δ 2 is a 2 .
4. Méthode selon l'une quelconque des revendications 1 à 3, dans laquelle l'échantillon biologique est un fluide biologique, de préférence un échantillon de sang, un échantillon de sérum, un échantillon de plasma, un échantillon d'urine ou un échantillon de sperme, de préférence encore un échantillon d'urine ou de sperme. The method according to any one of claims 1 to 3, wherein the biological sample is a biological fluid, preferably a blood sample, a serum sample, a plasma sample, a urine sample, or a sample. sperm, preferably a sample of urine or sperm.
5. Méthode selon l'une quelconque des revendications 1 à 4, dans laquelle la concentration de la protéine α2δ2, ou d'un fragment de α2δ2, est mesurée en utilisant un anticorps ou un fragment de celui-ci, spécifique de ladite protéine α2δ2, ou d'un fragment de α2δ2. 5. Method according to any one of claims 1 to 4, wherein the concentration of the α 2 δ 2 protein, or of a fragment of α 2 δ 2 , is measured using an antibody or a fragment thereof. , specific for said α 2 δ 2 protein, or a fragment of α 2 δ 2 .
6. Agent anticancéreux pour son utilisation dans le traitement du cancer de la prostate chez un sujet, caractérisé en ce qu'on détecte ledit cancer de la prostate au moyen d'une méthode selon l'une quelconque des revendications 1 à 5. An anti-cancer agent for use in the treatment of prostate cancer in a subject, characterized in that said prostate cancer is detected by a method according to any one of claims 1 to 5.
7. Utilisation de la protéine α2δ2, ou d'un fragment, comme biomarqueur du cancer de la prostate d'un sujet. 7. Use of the α 2 δ 2 protein, or fragment, as a biomarker of prostate cancer of a subject.
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