FR3013847A1 - NEW BIOMARKER FOR PROSTATE CANCER. - Google Patents

Abstract

The invention relates to a method for the in vitro detection of prostate cancer and / or prostatic metastasis in a subject comprising the steps of: (i) measuring the concentration of α2δ2, or a fragment of α2δ2, in a biological sample obtained from said subject, (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 a fragment of α2δ2, in the biological sample greater than said predetermined value indicates the presence of a prostate cancer in said subject, and a concentration of α2δ2, or a fragment of α2δ2, lower at said predetermined value indicates the absence of prostate cancer in said subject.

Description

The invention relates to the use of a282 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).

Currently, the most common technique for diagnosing prostate cancer is based on anatomo-pathological criteria usually obtained from prostate biopsies indicated following elevated PSA (Prostate Specific 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.

There is therefore still a need for new, preferably non-invasive, methods of detecting prostate cancer and determining the stage 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 α-subunit, forming the pore, associated with a28, f3 and γ subunits. The α215 subunit is formed of two units, a2 and δ, encoded by the same annoyance. These two parts are interconnected via two disulfide bridges. The α215 subunit is essentially extracellular and is anchored to the plasma membrane by the δ portion of the protein using a glycosylphosphatidylinositol (GPI) group. Four subparts of a215 (a2151 to a284), each encoded by a gene, have thus been discovered. 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.

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). 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. Moreover, 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 CACNA 1E gene of a voltage-dependent calcium channel, coding for the am 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. Likewise, 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. The CACNA2D2 gene has also been studied in several studies. This gene encodes both the α2 and α2 subunits. The CACNA2D2 gene was notably located on the 3p21.3 locus, identified by many searches as a tumor suppressor gene locus (W00204511, 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 cancers, suggesting that one or more genes in this region would act as tumor suppressors. It has been discovered by these researchers that the expression of the α28 subunit is inhibited in the majority of lung cancer cells, indicating that the alteration of calcium homeostasis in cancer cells leads to 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 α282 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. It has been discovered that the a282 protein or a fragment thereof can serve as a biomarker for prostate cancer. DEFINITIONS The term "a282" as used herein refers to the a282 protein of the α215 family of voltages-dependent calcium channels and encompasses all synonyms referring to this subunit. The term "fragment" as used herein refers, unless otherwise indicated, to a fragment of the α282 protein and may for example relate to sub-part a2 or sub-part 82, preferably sub-part a2. The term "CANA2D2" as used herein refers to the gene encoding the? 282 subunit, and encompasses all synonyms referring to this gene.

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. 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.

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. 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"). To avoid confusion, the cell cycle phases usually known and defined by "GO", "G1", "G2" are identified here by "Go", "Gi", "G2", unless otherwise indicated. The terms "anti-α282 antibodies" refer to an antibody or fragment thereof that specifically recognizes a282 or a fragment of a282, for example an epitope of (12 or 82. The terms "predetermined value" refer herein to the amount of a2.52 or fragment of a282 in biological samples obtained from the general population or from a selected population of subjects.

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. In another example, the predetermined value may be the amount of a282 or fragment in subjects with stage prostate cancer.

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. The terms "subject" or "patient", as understood here, refer to a monkey, a chimpanzee or a human. Preferably, it is a human. The terms "normal" or "healthy" may be used herein to refer to a subject, patient, sample, tissue, organ, or other non-cancer presenting subject. The term "biological sample" refers to any biological sample derived from the patient, for example, but not limited to, 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. The term "biomarker" generally refers to a molecule, ie a gene (or a nucleic acid encoding that gene), a protein, the expression of which can be determined in a biological sample of a subject by methods known to the subject. state of the art (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. 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. Thus, "si-a282" is understood to mean interfering RNA against the a282 coding sequence. "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 a282 protein over normal LNCaP lines are referred to as "LNCaP-OE2152". The "PC3" cell line, a prostate cancer cell line, is obtained from a bone metastasis of a patient with androgen-resistant cancer. The cell line "DU145" is obtained from a cerebral metastasis in a patient with prostate cancer.

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) measuring the concentration of a2.52, or a fragment of a2.52, in a biological sample obtained from said subject, (ii) comparing the concentration of a2.52, or a fragment of a2.52, measured in step (i) to a predetermined value of the concentration at a282, or a fragment of a282, wherein a concentration of a2.52, or a fragment of a2.52, in the biological sample greater than said predetermined value indicates the presence of prostate cancer in said subject, and a concentration of a2.52, or a fragment of a282, lower than said predetermined value indicates the absence of a prostate cancer in said subject.

Advantageously, the predetermined value refers to the amount of a282 or a fragment of a2.52 in biological samples obtained from apparently healthy subjects, for example, subjects who have never previously had any signs or symptoms indicating presence of prostate cancer.

A method for detecting in vitro prostate cancer and / or prostatic metastasis in a subject may thus consist in performing the steps of: (i) measuring the concentration of a2.52, or a fragment thereof in a biological sample obtained from said subject, (ii) comparing the concentration of a2.52, or a fragment of a2.52, measured in step (i) to a predetermined value derived from the a282 concentration, or of a fragment of a282, in a biological sample of a healthy subject not suffering from prostate cancer, a high level of a2.52, or a fragment of a2.52, in the biological sample compared with at said predetermined value indicating that the subject is suffering from prostate cancer.

The inventors have shown that the a282 protein is expressed more strongly in the advanced stages of cancer (see FIG. 5, G2 and G3) than in the normal and early stages of cancer (see FIG. 5, 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) measuring the concentration of a2152, or a fragment of a2152, in a biological sample obtained said subject, (ii) comparing the concentration of a2152, or a fragment of a2152, measured in step (i) to a predetermined value of the concentration of a282, or a fragment of a282, in which, when the concentration of a282 , or a fragment of a282, in the biological sample is greater than said predetermined value of the concentration of a2152, or a fragment of a2152, the difference between said concentration of a2152, or a fragment of a282, in the biological sample obtained from said subject and said predetermined value indicates the stage of prostate cancer in said subject.

According to one embodiment, the predetermined value relates here to the amount of a282 or a fragment of a282 in biological samples obtained from apparently healthy subjects, for example, subjects who have never previously had signs or symptoms indicating the presence of prostate cancer.

In another embodiment, the predetermined value here is the amount of a282 or fragment of a282 in subjects with stage-established prostate cancer. The concentration of the a2152 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. 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. Once the biological sample is prepared, the protein concentration is measured by 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 a282 protein present in the biological sample. The binding molecule may be specific for the mature form of the a282 protein. The linker molecule can also recognize the two a2 and 82 parts of the? 282 subunit. Also advantageously, the binding molecule may be capable of binding specifically with a fragment of the a282 protein, for example with the a2 portion or with the 82 portion of the? 282 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 for recognizing a2.52 which can be used in the present invention are, for example but not limited to 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.

Thus, according to a particular embodiment of the invention, the concentration of the a282 protein, or a fragment of a282, is measured using an antibody or a fragment thereof, specific for said a2152 protein, or 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 assays. Such tests include, but are not limited to, agglutination tests, enzyme-labeled and enzyme-labeled immunoassays such as ELISA, biotin / avidin tests, radio immunoassays, immunoelectrophoresis, immunoprecipitation.35 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 on the affinity of the compound for the solid phase used). Once separated, the a282 protein can be identified by means of known protein separation profiles, 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 a2 or 82. Alternatively, the separated compounds may be detected and measured for example by mass spectrometry. . According to one embodiment of the invention, a control value or normalization may 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. Indeed, creatinine being a good indicator of renal function, it is for example used as a standard for narcotics or doping products to evaluate whether the urine sample is diluted, or as standard for heavy metal poisoning. Its value can therefore be used in the detection methods of the present invention. Once the urine is collected (for example over a period of 24 hours) and precipitated, the concentration of a2.52 protein, or a fragment, is measured. The value of increasing the ratio of a282 / creatinine (or a282 / creatinine fragment) to the ratio of a282 / creatinine (or a28282 / creatinine fragment) 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 without limitation, the analysis of the PSA assay. The present invention also relates to 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 as previously described. A method of treating prostate cancer in a subject can thus comprise the steps of: detecting the presence of a cancer of said subject by a detection method according to the present invention, administering to said subject a treatment adapted to treat 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. 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 estrogens. The present invention also relates to a method for monitoring a treatment of a subject with prostate cancer and / or prostatic metastasis comprising determining the concentration of a282, or a fragment, in a biological sample. obtained from said subject, and optionally comparing said α2152 concentration, or fragment, to a predetermined value representing a predetermined stage of prostate cancer, the concentration of a2.52, or fragment, relative 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 a2.52 protein, or fragment thereof, as a biomarker of prostate cancer of a subject. Preferably, said fragment is a2.

FIGURES FIG. 1: A: Analysis of the Expression of a2151, a282 and a283 in the LNCaP and DU145 Cell Lines by RT-PCR Screening. Pactin and a positive control sample ("brain") are used as a control.

B: Analysis of a282 Expression in LNCaP, PC3 and DU145 Cell Lines by RT-PCR Screening. Pactin is used as a control. Figure 2: Analysis of the expression of a282 in 10 cancerous tissues (numbered from C1 to C10) and 10 non-cancerous tissues (numbered N1 to N10) of the prostate by RT-PCR. The pactin is used as a control. Figure 3: Western blot analysis showing that si-a282 (50 nM, 4 days) well inhibited the expression of a282 in LNCaP cells. Calnexin is used as a control.

Figure 4: Analysis of expression of a282 in cancer ("non cancer", n = 18) and non-cancer ("cancer", n = 20) human prostate tissues by screening. Figure 5: Analysis of expression of a282 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: 20 hyperplasia, N: normal prostatic tissue. Immunohistochemical staining against a282 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) histogram showing mean optical density (ODDAB) for different stages of cancer. The number of cells analyzed is represented on each bar. Figure 6: Analysis of the effect of a282 protein on cell proliferation. A: a) Effect of a 4 day treatment using si-a282 (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) Effect of a 4 day treatment using si-a282 (50 nM) on the number of cells (manual counting of extruded cells by Tryptan Blue); B: Effect of si-1/282 on cell proliferation of two other prostate cancer cell lines (PC3 and DU145). C: Kinetic experiments on time inhibition by si-a282 of LNCaP cell growth relative to a control (si-ctl).

D: FACS analysis of the effect of si-1/282 on the number of LNCaP cells in the Go / Gi phase (represented by "GO / G1 in the figure), G2 / M and S with respect to a control, si-ctl . B: Effect of gabapentin (100 μM) on cell proliferation by MTS method. F: a) Kinetics of cell proliferation in two different clones of LNCaP-1/2152 (C9 and C11). b) FACS analysis of the effect of LNCaP-1/2152 (clone C11) on the number of cells in the Go / G1, G2 / M and S phase relative to a LNCaP cell line not overexpressing 1/2152 Figure 7: In vivo analysis of protein growth induced by 1/282 protein. A: Weight in grams of LNCaP (control) and LNCap-1/282 (clone C11) tumors at sacrifice; B: Kinetics of tumor growth LNCaP (control) and LNCap-1/2152 (clone C11); C: Mean doubling time of LNCaP (control) and LNCap-1/282 (clone C11) tumors. Figure 8: Histological analysis of LNCaP and LNCaP-1/2152 A tumors: Various magnified views of: a: the control LNCaP tumor; c: tumor LNCaP-1/282. The images b (LNCaP control) and tumor LNCaP-1/282) show by means of arrows the foci of necrosis and inflammation. 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 9: Analysis of the effect of 1/282 on blood vessel formation in LNCaP xenografted tumors A: CD31 immunohistochemical staining (peroxidase / DAB) (a: LNCaP tumor, b: LNCaP-1/282 tumor); B: Percentage of CD31-positive tissues in LNCaP and LNCaP-1 / 282.35 tumors Figure 10: Analysis of the effect of a282 on angiogenesis by VEGF secretion. A: a. Western blots of CD31 and VEGF in 8 different tumors from 4 different mice (M1 to M4) (LNCaP tumors: "T-LNCaP", LNCaP-a282 tumors: "T-a2152"). Pactin is used as a control. b. Relative density of CD31 bands observed by Western blot (Fig. 10A.a.) standardized with respect to actin (10 samples for each tumor). vs. Relative density of VEGF bands observed by Western blot (Fig. 10A.a.) normalized to actin (10 samples for each tumor). B: VEGF secretion measured by ELISA in LNCaP and LNCaP-a2152 cells (clones C9 and C11). Figure 11: Analysis of the secretion of a2 in the LNCaP-OE2152 A: Western blot cells of α2 in the culture medium of LNCaP cells, of two clones overexpressing a282 (clone C11 and C16) and in the culture medium alone (" RPMI "), without DTT (6 days of incubation) or with DTT, ie under reducing conditions (15 min of incubation). B: Western blot for two other culture media containing either 2% FCS (fetal calf serum) or 10% FCS (common culture conditions). EXAMPLES Materials and Methods Cell cultures The LNCaP, DU145 and P3 cell lines originate from the "American Type Culture Collection" and were grown, according to the supplier's recommendations, in RPMI 1640 (GIBCO, Life Technologies, France) supplemented with 10% fetal bovine serum (FBS, Sigma, L'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. Stable LNCaP cell lines expressing the a282 protein (hereinafter referred to as "LNCaP-a2152") were designed as previously reported in the publication of Florian Gackière et al (10).

Production of si-RNAs and cell preparation The small interfering RNAs come from Eurogentech (France). Si-a282 were validated in LNCaP cells. For this, it has been shown by Western-blot that si-a282 decreases the expression of the a282 protein in LNCaP cells (FIG. 3).

The siRNAs used herein include a non-specific control siRNA against luciferase (hereinafter referred to as "si-ctl" or "siCTL"). The siRNAs are shown in Table 1. siRNA Position siCTL Luciferase 153-171 si-112152 1868-1850 Table 1 Cells were transfected with 20 or 50 nM siRNA using HiPerFect Transfection Reagent (Qiagen) as previously reported in the publication of Florian Gackière et al (10).

Analysis of α2152 Subunit Gene Expression by RT-PCR or Quantitative RT-PCR The extraction of RNA 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).

For each PCR reaction, 1.1 g of total mRNA was treated with DNAse II, incubated at 70 ° C and reverse transcribed with MULV RNA polymerase (Applied Biosystems, 50U / μL) in the presence of 0. 1.1.M of dNTP and a ribonuclease inhibitor (Applied B io sy stems). The cDNA obtained was used for amplification by PCR using a Taq Gold kit (Applied Biosystems). The operating conditions of the PCR are shown in Table 2. 1 cycle 34 to 40 cycles 1 cycle 95 ° C 95 ° C 58 ° C 72 ° C 72 ° C 5 min 30 sec 30 sec 1 min / 1000bp 7 min Table 2 The sense and antisense primers (Invitrogen) used during the amplification are shown in Table 3.

Protein Gene Size (bp) 1/281 CACNA2D1 400 1/2152 CACNA2D2 505 500 112153 CACNA2D3 439 Pactin 220 Table 3 The PCR products were analyzed on an agarose gel (1.5% or 2%) including bromide of ethidium (0.5 μg / ml) then visualized under UV. The images were then captured using Gelcapture software (Bio-imaging System). Western blotting After washing in phosphate buffered saline, the cells were scraped into lysis buffer (1% Triton X-100, Na deoxycholate, 1%, 150 mM NaCl, 10 mM PO4NaK, 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).

The antibodies, used for the western blotting as well as for the immunohistochemical and immunofluorescence identifications, are presented in Table 4. Antibody Name Company Dilution Dilution Antibody MW in WB in IF / IH secondary (kDa) 112152 H210 Santa Cruz 1 / 200e 1 / 100th or Rabbit 130 1 / 50th 1/2152 A01 Abnova / 1 / 50th Mouse 130 PCNA Sc-56 Santa Cruz 1 / 200e / Mouse 36 f3-actin A-5441 Sigma 1 / 4000e / Mouse 43 Calnexin mab 3126 Millipore 1 / 2000e / Mouse 95 Ki-67 Sc-101861 Santa Cruz / 1 / 100th Rabbit CD31 Ab28364 Abcam 1 / 200e 1 / 50th Rabbit 130 VEGF Ab46154 Abcam 1 / 200e 1 / 50th Rabbit 43 Table 4 Cell Proliferation and Viability Tests The viability of cells was colorimetrically tested (CellTiter 96 Aqueous 5 Non-Radioactive Cell Proliferation Assay, Promega, USA) as recommended by the supplier. Cell proliferation was also tested by manual counting of Tryptan Blue extruded cells. The staining of Ki-67 or PCNA (Proliferating Cell Nuclear Antigen) antibodies was also used to discriminate quiescent cells (Go phase) and cyclic cells. Cell cycle analysis The cells were grown 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 emission measurement at 520 nm using a flow cytometer (Beckman Coulter Epics XL4-MCL with Expo32 acquisition). Data interpretation was done with Multicycle for Windows (Phoenix Flow system). Immunostaining and Confocal Analysis 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 and paraffin-embedded 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. The provider (Biomax) provides the clinical information (age of patients, grades of cancers and grades of Gleason of the 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 O 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. For immunofluorescence analyzes, the cells were incubated with Alexa fluor-labeled anti-rabbit IgG-488 secondary antibody (A-21206, Molecular tests, 1: 2000 dilution) 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 the tissue slides. Confocal and immunostaining observations were carried out as previously described by means of a Zeiss LSM 510 confocal microscope (Carl Zeiss, Le Pecq, France) connected to an inverted microscope for Zeiss Axiovert 200 M transmissive and epifluorescence microscopy with a microscope 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. Anti-α282 polyclonal antibodies were used to study the expression of α282 in prostate tissues and cells. VEGF secretion assay VEGF secretion 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 a2S2 In order to determine if a282 is secreted into 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). 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 a282 was analyzed after SDS-page electrophoresis (6 or 8% acrylamide) and a western blot. Expression of a282 in the cell culture medium was compared to culture medium alone (without cells).

In Vivo Trials Six week old male Swiss nude mice (Charles River Laboratories, France) were injected onto each flank of LNCaP cells or LNCaP cells overexpressing a282. 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.

Statistical Analyzes Charts 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: ***. Results Expression of a2152 in prostate cancer tissues and cell lines In previous studies it has been shown that Cav3.2 voltage-gated calcium channels are expressed in prostate cancer cell lines and in cancer tumors. the human prostate (5). It has been investigated here whether the supposed regulatory subunits can also be expressed in these cells. Attention has been focused on the a215 subunits since two of them (a282 and a283) are suspected of acting as tumor suppressors in different organs. As shown in FIG. 1, it was demonstrated by RT-PCR that the prostate cancer cell lines studied (LNCaP cells, DU145, PC3) express a product of the transcription of a282. The other α215 subunits sought (a2151 and a283) are not expressed in the prostate cell lines studied. Similarly, and as shown in Figure 2, RT-PCR assays demonstrated that a282 is present in normal and cancerous prostate tissues. Immunodetection assays were also performed to demonstrate the expression of α282 proteins in prostate cancer cell lines and in prostate tissue sections. Immunofluorescence assays showed that the LNCaP, DU145 and PC3 prostate cancer cell lines were strongly stained by anti-a282 antibodies. It is further observed that a282 staining is evident on the cell but is particularly pronounced on the periphery, i.e., near the plasma membrane, the anchor site of the a282 proteins (not shown).

In addition, by western blot, the expression of two bands around 160 kD was observed using anti-α282 antibodies (see FIG. 3). The expression of these bands is greatly reduced when the LNCaP cells are placed in the presence of si-a282. These 160 kD bands observed by western blot correspond to the expected size of a282, demonstrating that the antibodies used here correctly detect the a2b2-35 protein. Immunofluorescence (IF) and immunohistochemical (IH) staining of cancer Prostate obtained subjects were performed on sections of frozen prostate tissue (IF) or paraffin sections (reaction peroxidase IH). It has been shown that there is a strong staining of the glandular epithelial structures. Acini are frequently stained with anti-α282 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 a282 staining on the periphery of the cells. Α2S2 Expression Increases with Development of Prostate Cancer In order to evaluate possible differences in expression of α282 between normal and tumor tissues, a comparison of α282 expression using the RT technique -PCR was performed in 18 prostate cancer tissues and in 20 non-cancerous prostate tissues. As shown in FIGS. 2 and 4, a282 is more frequently expressed in cancerous tissues (95% of cancerous tissues express a282) than in non-cancerous tissues (40% of non-cancerous tissues express a282) (significant difference, p < 0.001, Chi2 test). Similarly, the expression of a282 was compared on macroarrays of 24 different tissues including 16 cancerous tissues and 8 non-cancerous tissues. As shown in Figure 5, epithelial cells overlying acini are stained by anti-α282 antibodies in normal, hyperplastic and cancerous samples. In the advanced stages of cancer, a number of stained cells leave the acini (see Figure 5B, 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. As shown in FIG. 5, the localization in the cell is different between the normal, BPH and early (G1) stages of cancer and the advanced stages of cancer (G2, G3). In BPH and early stages of cancer (G1), the 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 nucleus of the cells.

Staining density was also analyzed by means of color deconvolution to extract counterstain DAB (Azure Blue) staining, and DAB density was shown to be higher in advanced stages of cancer, thus making evidence that the expression of 112152 is increased in advanced prostate cancers (Figure 5C).

Inhibition of a2152 Reduces Cellular Proliferation While Overexpression Induces Cellular Proliferation Since 11282 is expressed in prostate cancer epithelial cells, it has been investigated whether the protein can participate in cell growth.

Different approaches have been used to evaluate cell proliferation. The use of ligands or si-α252 reduces tumorigenesis Firstly, 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-11282 compared with si-ctl (see Figure 6A a). In the same way, cell proliferation is decreased in the presence of si-11282 in the DU145 and PC3 lines (see Figure 6B). Similar results were obtained with hand counting of cells, which shows that a 4-day incubation in si-11282 decreases the number of LNCaP cells by 30% (see Figure 6A b). Kinetic experiments (FIG. 6C) illustrate that si-11282 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% reduction in cell proliferation). FACS cell cycle analyzes show that this inhibitory action of si-11282 on cell proliferation is correlated with an increase in the number of cells in G1 phase, as well as a decrease in the number of cells in G2 / M phase and in phase S (FIG. 6D). Gabapentin has been tested for cell proliferation, which is known to inhibit different voltage-gated calcium channels by its action on the regulatory subunit. Indeed, gabapentin has been shown to be a ligand of both subunits. units 112151 and 112152 (12). In the present tests, gabapentin, which was added to the 100 μM cell culture medium, reduced cell proliferation by 35% (Fig. 6E). 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. LNCaP-α252 Lines Induce Cell Proliferation Stonily overexpressing a282 LNCaP clones were similarly used in cell proliferation assays. It has been shown that two different clones overexpressing a282 (clone 9 "C9", and clone 11 "C 11") produce faster proliferation rates than LNCaP control cells, with a multiplication time of 2 of 47.5h for LNCaP against 40.5h for clone 9 and 36h for clone 11 (see Figure 6F a).

Ki-67 dye immunofluorescence assays on LNCaP and LNCaP cell lines overexpressing a282 (clone 11) were performed (see Figure 6F b). Ki-67 is a marker of proliferating cells. It is only expressed during G1, S, G2 and M phases. Go-phase cells do not express Ki-67 significantly. There may be either a punctual presence in the nucleus during G1 and S phases, or a more homogeneous localization in the nucleus during G2 and M phases. It has been shown here that a282 is associated with a decrease in the number of cells during the Go phase of the cell cycle. In these assays, 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 total LNCaP-α2152 cell population. This result is correlated with an increase in the number of cells during the Gi / S and G2 / M phases of the LNCaP-OE282 cell cycle. In view of all these results, it is therefore demonstrated that a282 increases the proliferation of LNCaP cells.

Overexpression of a252 induces tumor development in vivo In vivo assays were performed on immunodeficient nude mice. The mice were injected on each flank of LNCaP cells or LNCaP cells overexpressing a282 (Figure 4). The tumors obtained are hereinafter referred to as "LNCaP tumor" and "LNCaP-a282 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 7a) for LNCaP tumors, reaching an average size of 940 ± 146 mm3 ( 1.26 ± 0.22 g) (see Figures 7b and c). LNCaP-a2152 tumors grew with a doubling time of 6.6 +/- 0.8 days (see Figure 7a) to reach an average size of 1760 ± 154 mm3 (2.3 ± 0.21 g). ) (see Figures 7b 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.

Therefore, LNCaP-OE2152 tumors are found in vivo to increase faster and are larger than LNCaP tumors. The overexpression of a2S2 induces the formation of blood vessels and the secretion of VEGF.

The two LNCaP and LNCaP-a2152 tumors 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 8A).

No significant necrotic surface difference was detected between LNCaP tumors and LNCaP-a282 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-a2152 tumors than in tumors. LNCaP (Figure 8B). In addition, histological slides show that there are more blood vessels in LNCaP-a2152 tumors than in LNCaP tumors (Figures 8 A and B). The blood vessels were therefore stained with CD31 (Figure 9A) which is a commonly used marker for staining endothelial cells and for detecting angiogenesis in tumors. It has been shown that the stained area in LNCaP-a2152 tumors is twice as large compared with that of LNCaP tumors (FIG. 9B). This was confirmed by western blot analyzes which showed a 50% increase in CD31 expression (Figure 10A). Since a282 is associated with the formation of blood vessels, expression and secretion of VEGF in tumors and prostate cells were measured. As shown in Figure 10A, Western blot analyzes show that VEGF expression is slightly, but significantly, increased in tumors overexpressing a282. VEGF ELISA analyzes were therefore performed to determine if in vitro secretion is impaired in LNCaP cells overexpressing a282. As shown in Figure 10B, ELISA tests show that overexpression of a282 significantly increases VEGF secretion in two different LNCaP-a282 clones. This is not due to non-specific general stimulation of secretion by LNCaP cells since secretion of prostatic acid phosphatase (PAP) is not affected by overexpression of a282, although PAP is stimulated by an increase in intracellular calcium. a2S2 can be cleaved in LNCaP cells overexpressing a2S2. Under non-reducing conditions, a282 corresponds to a single 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 a2 and 82, derived from the same CACNA2D2 gene, are linked by disulfide bridges. a2 is a broad extracellular subunit (150kDa or greater, dependent on glycosylation) related to 82, a smaller transmembrane protein (approximately 20kDa). In order to evaluate whether a282 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 a282, or a2 in case of cleavage, was sought in the culture medium by western blot. Figure 11 shows that a2 is present in the culture medium in which the LNCaP-a282 cells were incubated for 6 days, while this subunit is barely detectable in the culture medium of LNCaP control cells.

Discussion The a282 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 a282 is capable of influencing itself. on cell proliferation in vitro and on tumorigenesis in vivo. This a282 protein, or a fragment thereof, represents a new biomarker for cancer, including prostate cancer. This is based on the following observations: (1) The a282 calcium channel subunit, encoded by the CACNA2D2 gene, is expressed in prostate cells; (2) This protein, or its gene, is more frequently expressed in cancerous tissues than in non-cancerous tissues of the prostate; (3) Targeting this protein with ligands or si-RNA can reduce tumorigenesis in vitro; (4) The expression of a282 is associated with secretion of VEGF and formation of blood vessels; (5) Its overexpression induces tumor development in nude mice; (6) a282 is overexpressed in other forms of cancer; (7) The a282 protein can be cleaved and the A2 sub-part of the a282 protein released into the extracellular medium of LNCaP-a282 cells. It is detectable with antibodies available on the market.

Thus, the a282 protein was detected in both cancer and non-cancer prostate cells by PCR and immunodetection, particularly on the apical membranes of the epithelium (observation (1)). PCR and immunohistochemical analyzes then showed that a282 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 targeting the protein with si-a282 or ligands like gabapentin reduces cell proliferation (observation (3)), it has been shown in vitro and in vivo that this overexpression of a282 induces tumor development in nude mice (observation (5)).

Histological, immunodetection, western blot and ELISA assays have also shown that this overexpression of a282 induces VEGF secretion and angiogenesis (observation (4)). Finally, it has been demonstrated that the a282 protein can be cleaved, thus releasing the a2 sub-part of the a282 protein in the extracellular medium of LNCaP-a282 cells. The a282 protein or fragments thereof, and more particularly the a2 subunit, are detectable with commercially available antibodies (observation (7)). It is also reasonable to say that the a282 protein, or fragments thereof, can similarly be detected in the urine. It is thus known that in vivo the GPI-anchored proteins at the membrane can be cleaved by different enzymes, some phospholipases C or D, hydrolyzing these GPI groups and thereby releasing the proteins in the extracellular medium.

More particularly, the in vivo cleavage of the a2 sub-part of the protein, releasing the a2 sub-part 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 disulfide isomerase or phosphoglycerate kinase (14), thus releasing a2 in the extracellular medium of 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).

Thus, similarly to the present case, the CD90 protein (Cluster of Differentiation 90) is an N-glycosylated surface protein, anchored to the membrane by a glycosylphosphatidylinositol (GPI) group, such as the a282 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 be used as a biomarker for non-invasive tests (22). It is known that the release of proteins by the prostatic cells into the seminal fluid takes place, 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 urine or seminal fluid, contain a large number of proteins, for example membrane proteins, such as GLUT5 (Glucose Transporter 5), VAT-1 (synaptic vesicle membrane protein homologue) 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 a282 protein is released into seminal fluid or blood plasma by these prostasomes and can be assayed in other body fluids than urine. 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 a282 in the blood, thus allowing find this protein in the plasma in greater quantity during tumor development. It has therefore been demonstrated here that the a2152 protein, or a fragment thereof, can be used as a biomarker of prostate cancer, as well as to detect the stage of advancement of cancer.

15 REFERENCES 1. "Guide - Long-term disease (malignant neoplasm, malignancy of the lymphatic or hematopoietic tissue, prostate cancer)", HAS and National Cancer Institute, January 2012. 20 2. Fiske JL, Fomin VP, Brown ML, Duncan RL, Sikes RA (2006) Voltage-sensitive ion channels and cancer. Cancer Metastasis Rev 25: 493-500. 3. Kunzelmann K (2005) Ion channels and cancer. J Membr Biol 205: 159-173. 4. Bennett ES, Smith BA, Harper JM (2004) Voltage-gated Na + channels confer invasive properties on human prostate cancer cells. Pflugers Arch 447: 908-914. 5. Marine P, Vanoverberghe K, Lalevee N, Rossier MF, Prevarskaya N (2002) Overexpression of an alpha 1H (Cav3.2) T-type calcium channel during neuroendocrine differentiation of human prostate cancer cells. J Biol Chem 277: 10824-10833. 6. Spitzner M, Ousingsawat J, Scheidt K, Kunzelmann K, Schreiber R (2007) Voltage-gated K + support channels proliferation of colonic carcinoma cells. Faseb J 21: 35-44. 7. Carboni GL, Gao B, Nishizaki M, Xu K, Minna JD, et al. (2003) CACNA2D2- mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity.

Oncogene 22: 615-626. 8. Lerman MI, Minna JD (2000) The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Chromosome Cancer 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 60: 6116-6133. 9. Chang JM, Lee HJ, SE Lee, Byun S-S, Choe Y G, Kim SH, Seong CK, Kim SH. Unusual tumors involving the prostate: radiological-pathological findings. The British Journal of Radiology, 81 (2008), 907-915. 10. Gackiere F, Bidaux G, Delcourt P, Van Coppenolle F, Katsogiannou M, et al. (2008) CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells. J Biol Chem 283: 10162-10173. 11. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156159. 12. Dooley DJ, Taylor CP, Donevan S, Feltner D (2007) Ca2 + channel alpha2delta ligands: novel modulators of neurotransmission. Trends Pharmacol Sci 28: 75-82. 13. Davies A, Hendrich J, Van Minh AT, Wratten J, Douglas L, et al. (2007) Functional biology of the alpha (2) delta subunits of voltage-gated calcium channels. Trends Pharmacol Sci 28: 220-228. 14. Hogg PJ (2003) Disulfide bonds as switches for protein function. Trends Biochem Sci 28: 210-214. 15. Lexander H, Palmberg C, Auer G, Hellstrom M, Franzen B, et al. (2005) Proteomic analysis of protein expression in prostate cancer. Anal Quant Cytol Histol 27: 263-272. 16. Wang J, Ying G, Jung Y, J Lu, Zhu J, et al. (2010) Characterization of phosphoglycerate kinase-1 expression of stromal cells derived from tumor microenvironment in prostate cancer progression. Cancer Res 70: 471-480. 17. Ades EW, Zwerner RK, Acton RT, Balch CM. Isolation and partial characterization of the human homolog of Thy-1. J Exp Med 1980; 151: 400-6. 18. Morris R. Thy-1 in developing nervous tissue. Dev Neurosci 1985; 7: 133-60. 19. Craig W, R Kay, Cutler RL, Lansdorp PM. Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med 1993; 177: 1331-42. 20. Koumas L, AE King, Critchley HO, Kelly RW, Phipps RP. Fibroblast heterogeneity: Existence of functionally distinct Thy 1 (+) and Thy 1 (-) human female reproductive tract fibroblasts. Am J Pathol 2001; 159: 925-35. 21. 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. 22. LD trick, Zhang H, Ye M, Huang CY, et al. CD90 / THY1 is overexpressed in prostate cancer associated fibroblasts and could serve as a cancer biomarker. Mod Pathol 2010; 23: 1346-56. 23. Tavoosidana, G., Ronquist, G., Darmanis, S., Yan, J., Carlsson, L., Wu, D., Conze, T., Ek, P., Semjonow, A., Eltze, E . et al. (2011). Multiple recognition assays reveals prostasomes as promising plasma biomarkers for prostate cancer. Proc Natl Acad Sci U S A 108, 8809-14.

Claims (7)

REVENDICATIONS1. A method for in vitro detection of prostate cancer and / or prostatic metastasis in a subject comprising the steps of: (i) measuring the concentration of a2152, or a fragment of a2152, in a biological sample obtained from said subject, (ii) comparing the concentration of a2152, or a fragment of a2152, measured in step (i) to a predetermined value of the concentration of a282, or a fragment of a282, in which a concentration of a282, or of a fragment of a282 in the biological sample greater than said predetermined value indicates the presence of prostate cancer in said subject, and a concentration of a2152, or a fragment of a2152, lower than said predetermined value indicates the absence of prostate cancer in said subject. A method for determining in vitro the stage of prostate cancer in a subject comprising the steps of: (i) measuring the concentration of a2152, or a fragment of a2152, in a biological sample obtained from said subject, (ii) ) comparing the concentration of a2152, or a fragment of a2152, measured in step (i) to a predetermined value of the concentration of a282, or a fragment of a282, in which, when the concentration of a282, or a fragment of a282, in the biological sample is greater than said predetermined value of the a2152 concentration, or a fragment of a2152, the difference between said a2152 concentration, or a fragment of a282, in the biological sample obtained said subject and said predetermined value indicates the stage of prostate cancer in said subject. The method of any one of claims 1 or 2, wherein said fragment of (12152 is (12. 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. The method according to any one of claims 1 to 4, wherein the concentration of a282 protein, or a fragment of a2152, is measured using an antibody or a fragment thereof, specific for said a282 protein. , or a fragment of a2b2- 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. Use of the a2152 protein, or fragment, as a biomarker of a subject's prostate cancer.