WO2013121146A1 - Method for determining the activity of the lysophosphatidic acid (lpa) receptor lpa1 - Google Patents

Description

 METHOD OF DETERMINING LPAI RECEPTOR ACTIVITY OF LYSOPHOSPHATIDE ACID (LPA)

 The present invention relates to the field of biology in general, and in particular to lysophosphatidic acid receptors.

 More specifically, the invention relates in particular to a method for determining in vitro the specific activity of the LPAi receptor by measuring the expression of one or more specific markers, in particular for determining in vitro in vitro modulating activity. or in vivo of a compound vis-à-vis specifically the LPAi receptor, particularly in the context of monitoring the effectiveness of a therapeutic compound specifically targeting the LPAi receptor.

 Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits growth factor-like activity on a very wide variety of cells, regulating its proliferation, migration, and cellular invasion in vitro (Fukushima et al., 1998; Goetzl). ef a /., 1999). Platelets are the major but non-exclusive source of LPA in the body during platelet aggregation (Gerrard and Robinson 1989). The concentration of LPA passes from the order of Ο, ΙμΜ in the plasma to 5-25μ in the serum. Fibroblasts, preadipocytes and some tumor cells can themselves produce LPA,

LPA is involved in the control of many physiopathological functions (pulmonary fibrosis, renal fibrosis, obesity, rheumatoid arthritis, reproduction, urinary diseases, cancer). The cellular action of LPA passes through the activation of a series of receptors on the surface of many cells including cancer cells (LPA, LPA2, LPA _3, LPA _4, LPA _5, LPA LPA ₆ and _7). These receptors are very homologous to each other. They have a structure with seven transmembrane passages coupled to binding proteins GTP (G proteins), however There are two subclasses of receptors based on their phylogenetic origin and their genomic organization (LPA LPA1-3 and ₄ -7). It should be known that the messenger RNAs encoding these receptors are widely distributed at tissue (Contos et al., 2002) and that most cells express several types of receptors at the same time.

 Many laboratories and companies are currently developing therapeutically targeted molecules targeting LPA receptors, including the LPA1 or LPAi type 1 receptor.

 However, there is currently no test to validate the specific activation or inhibition of the LPAi receptor in biological samples taken in vivo, or in vitro in complex cellular systems expressing multiple receptors. LPA, which is largely in the majority.

 To date, it is therefore not possible to know if the LPAi receptor specifically is actually activated or inactivated. In particular in the body (human, mouse, ..), no test can validate that this LPAi receptor is effectively activated or inactivated under the action of these different therapeutic molecules, or to monitor the effectiveness of targeted therapies put in place.

 Currently, the validation of the activation and inhibition of the LPAi receptor is carried out only in vitro, and this under very limited conditions from cells expressing naturally no LPA receptor and which after genetic manipulations express this receptor. The tests consist in measuring either the intensity of the intracellular calcium flux by spectrophotometry in the presence of LPA, or the binding of gammaGTP * at the level of GTP-fixing proteins, present at the inner surface of the plasma membrane, by measuring the radioactivity associated with membranes following stimulation with LPA.

 It is in this context that the present invention proposes to be able to detect in vitro the specific activation or inhibition of the LPAi receptor by virtue of the use of a particular biological signature.

The present invention therefore fills the current impossibility of confirming the activation or specific inhibition state of the LPAi receptor in vivo in the body, but also in vitro in complex cellular systems that express different LPA receptors. Thus, according to a first aspect, the present invention relates to a method for determining in vitro the specific activity of the LPAi receptor comprising the steps of:

 measuring the level of expression of the HBEGF (Heparin-Binding EGF-like growth factor) and CXCL3 (Chemokine C-X-C Ligand 3 motif) markers in a sample,

 - conclude respectively the activation or inhibition of the LPAi receptor specifically, when a significant increase or decrease in the level of expression of these two markers is demonstrated with respect to a reference value.

 The inventors have in fact surprisingly found that the combined study of the expression of the gene encoding HBEGF (Heparin-Binding EGF-like growth factor) and of the gene encoding CXCL3 (Chemokine CXC motif Ligand 3) constitutes a signature of activation or inhibition of the LPAi receptor specifically. Thus, an increase in the expression of HBEGF and an increase in the expression of CXCL3 is associated with the activation of LPAi specifically, whereas a decrease in the expression of HBEGF and a decrease in the expression of CXCL3 is associated with the inhibition of LPAi specifically. Therefore, if the respective expression of the two markers HBEGF and CXCL3 is modified in the opposite direction, that is to say that the expression of one of these two markers is increased while the expression of the other of these two markers is decreased, it will not be possible to conclude to the activation or inhibition of the LPAi receptor specifically. Thus, it can not be concluded that the modification of the expression of these two markers is specific to the LPAi receptor signaling pathway of LPA.

According to a first alternative embodiment, the method of the invention allows the in vitro determination of the modulatory activity of a compound vis-à-vis the LPAi receptor, by measuring the expression of the two HBEGF markers. and CXCL3 as indicated above, and using as a reference value the level of expression of said markers before adding said compound whose modulator activity is at determine.

 According to a second variant of implementation, the method of the invention also makes it possible to monitor the effectiveness of a therapeutic compound for specifically targeting the LPAi receptor, by measuring the expression of the two markers HBEGF and CXCL3 as indicated. above, and using as a reference value the level of expression of said markers in an earlier sample.

 The type 1 receptor of LPA or LPAi is also sometimes called LPAR1 for "lysophosphatidic acid receptor 1", or else EDG2 "Endothelial Differentiation Gene 2", VZG1 "ventricular zone gene 1", GPR26, "G protein-coupled receptor 26" , Mred.3 (named after Macrae et al., 1996), Kdt2 (based on the Genome Informatîx website) http://www.informatics.iax.org.name: Lparl, ID: MGI: 108429 ).

In the context of the invention, the LPAi receptor preferably corresponds to the receptor as described by An et al in 1997. Furthermore, according to the invention, the word "specific" or "specifically" used in connection with the LPAi receptor. or its activity corresponds to an activity of the LPAi receptor, and not to the activity of other types of LPA receptors, and in particular LPA ₂ to LPA ₇ .

 In the context of the invention, the HBEGF marker (Heparin-Binding EGF-like growth factof), also called HEGFL, DTR (Diphtheria Toxin Receptor), preferably corresponds to that described by Besner et al.

 In particular, for the HBEGF marker according to the invention, the reference nucleic sequence is preferably the following: NM_001945.2

 Similarly, the reference protein sequence for the HBEGF marker according to the invention is preferably the following: EAW62069.1

In the context of the invention, the marker CXCL3 (Chemokine CXC motif Ligand S), also named GROG, GRO-gamma (Growth Regulated Oncogene Gamma), SCYB3 (Small Inducible Cytokine Subfamily B Member 3), MIP2B, MIP2beta (Macrophage Infiammatory Protein-2-beta), preferably corresponds to that described by Tekamp-Olson et al., In 1990. In particular, for the CXCL3 marker according to the invention, the preferred reference nucleic sequence is the following: NM_002090.2.

 Similarly, the reference protein sequence for the CXCL3 marker according to the invention is preferably the following: AAH16308.1.

 The method according to the invention makes it possible in particular to determine in vitro the specific activity of the LPAi receptor in vivo or in vitro, irrespective of the variant of the process according to the invention.

 According to the invention, its reference value is chosen according to the variant of implementation of the method.

 According to a second aspect, the subject of the present invention is also a method for determining, in vitro, the modulatory activity of a compound with respect to the LPAi receptor, characterized in that it comprises the steps of the determination method. in vitro the specific activity of the LPAi receptor as described above, and that the reference value corresponds to the level of expression of said markers before the addition of said compound whose modulator activity is to be determined.

 In the context of this method, the compound whose modulating activity is to be determined may correspond to a compound whose activity is to be determined as an LPAi receptor agonist or antagonist.

 In particular, the compound whose modulating activity is to be determined is preferably a therapeutic molecule.

 This method thus makes it possible to validate the efficacy of agonists or antagonists specifically directed against the LPAi receptor in complex cellular systems expressing multiple LPA receptors, and in particular in a therapeutic context.

According to a third aspect, the present invention also relates to a method for monitoring the efficacy of a therapeutic compound for specifically targeting the LPAi receptor, characterized in that it comprises the steps of the in vitro determination method of the specific activity of the LPAi receptor as described above, and that the reference value corresponds to the level of expression of said markers in an earlier sample. In this particularly preferred embodiment, the present invention thus makes it possible to validate the efficacy of therapeutic compounds specifically targeting the LPAi receptor and to monitor their effectiveness over time during the implementation of the treatment. Preferably, the previous sample corresponds to an earlier stage during treatment with the therapeutic compound whose efficacy is to be validated, and not at an earlier stage before the introduction of said treatment.

 This method of validation and monitoring of the effectiveness of therapeutic compounds specifically targeting the LPAi receptor finds application in numerous pathologies, and in particular in the treatment of cancers, rheumatoid arthritis, renal fibrosis, and pulmonary fibrosis. In oncology, for example, LPA receptors have recently been shown to be involved in carcinogenesis and metastatic dissemination of breast cancer (Boucharaba et al., 2004 and 2006, Liu et al., 2009). More particularly, the LPAi receptor is a target in the case of the treatment of bone metastases of breast cancer (Boucharaba et al., 2006).

 In addition to the expression of HBEGF and CXCL3, the method of the invention may further comprise a step of measuring the level of expression of one or more other marker (s), in particular chosen (s). ) among CSF2 / GMCSF (Colony Stimulating Factor 2), CXCL1 / Groa, (Chemokine CXC Motif Ligand 1), IL-6 (Interleukin 6) and IL-8 (Interleukin 8) as described by Boucharaba et al. in 2006. This particular embodiment can be applied irrespective of the variant of implementation of the method, namely to determine in vitro the specific activity of the LPAi receptor, but also to determine in vitro the modulating activity of a compound vis-à-vis specifically the LPAi receptor, or for monitoring the effectiveness of a therapeutic compound to specifically target the LPAi receptor.

For the purposes of the present invention, the terms "expression measurement" or "expression measurement" mean an in vitro or ex-vivo measurement of expression. Moreover, in these terms, the word "expression" means designate both the measurement of expression at the nucleic and protein levels. For this purpose, any measurement or quantification method well known to those skilled in the art can be used for the implementation of the invention.

 In particular, when the measurement of the expression is carried out at the nucleic level, and in particular from RNA or cDNA, this can in particular be carried out by PCR analysis, in particular quantitative PCR or qPCR, by micro hybridization. -arrays (biochips) or Northern-blot.

 When the measurement of the expression is carried out at the protein level, standard techniques such as Western blot, ELISA, RIA, IRMA, FIA, CLIA, ECL, flow cytometry or immunocytology can be used.

 According to a preferred embodiment of the invention, the measurement of the expression of the markers is carried out at the nucleic level, and in particular by a PCR technique.

 For the purposes of the present invention, any act defined in relation to the term "in vitro" also covers the same act in relation to the terms "ex vivo" or "in cellulo" or with any equivalent term as long as it excludes application to the human or animal body.

 "Significantly increased or decreased level of expression" means a statistically significant increase or decrease in the level of expression, preferably associated or correlated with a confidence index of at least 95% (p <0.05). )

The sample on which the process of the invention is implemented may be of biological origin, in particular animal or human, and preferably human. It may in particular correspond to a sample of biological fluid, for example serum, to a tissue sample or to isolated cells, in particular from such biological fluids or tissues. These samples may in particular be derived from a healthy or sick individual, in particular suffering from a disease that can be treated or improved by targeted therapy of LPAi receptor activity. In particular, the sample on which the method of the invention is implemented is derived from an individual suffering from prostate or breast cancer, preferably breast cancer. In the context of the present application, the terms "cancer" and "tumor" are used interchangeably to designate a cancer, that is to say that here the term "tumor" means to designate a malignant tumor that is to say cancerous.

 According to another aspect, the present invention also relates to the use of the in vitro measurement of the level of expression of the markers HBEGF and CXCL3 as a signature of the LPAi specific receptor activity of LPA, that is to say to say an activation or an inhibition of this receiver.

Indeed, in the context of this use, the demonstration of an increase in the level of expression of HBEGF and of CXCL3 is a signature of the activation of the specific receptor LPA _I whereas a decrease in the level of expression of HBEGF and CXCL3 is a signature of specific LPAi receptor inhibition.

 The use according to the invention thus makes it possible to determine, in vitro, the specific activity of the LPAi receptor, but also to determine in vitro the modulatory activity of a compound vis-à-vis the LPAi receptor, or the follow-up of the effectiveness of a therapeutic compound to specifically target the LPAi receptor.

 According to an advantageous embodiment of the invention, the measurement of the level of expression of HBEGF and of CXCL3 is used as a signature of the LPAi specific receptor activity of LPA for monitoring the in vivo efficacy of a therapeutic compound to specifically target the LPAi receptor.

 In the context of this aspect, the invention preferably relates to the use of measuring the level of expression of HBEGF and CXCL3 in a biological sample from a patient treated with said therapeutic compound.

All of the preferred embodiments and combinations thereof mentioned above relating to the method are also preferred embodiments with respect to use. In particular, in addition to measuring the level of expression of both HBEGF and CXCL3, the use according to the invention may also include level measurement. expressing one or more other marker (s), preferably selected from CSF2 / GMCSF, CXCL1 / Groa, IL-6 and IL-8.

 According to yet another aspect, the present invention also relates to the use of a kit comprising one or more specific reagents for measuring the level of expression of HBEGF and one or more specific reagents for measuring the level of expression. of CXCL3, for the implementation of the process of the invention, according to any of its variants described above, namely to determine in vitro the specific activity of the LPAi receptor, for the in vitro determination of the modulatory activity of a compound vis-à-vis specifically the LPAi receptor, or for monitoring the effectiveness of a therapeutic compound to specifically target the LPAi receptor.

 Preferably, the kit used according to the invention contains specific oligonucleotides as specific reagents for measuring the level of expression of the marker (s) chosen (s), in particular by a PCR technique. For example, such specific oligonucleotides may be:

 HBEGF-F: 5'-GG ACCCATGTCTTCGG AAAT-3 '(SEQ ID NO: 1); and or

HBEGF-R: 5'-CCCATGACACCTCTCTCCAT-3 '(SEQ ID NO: 2); and / or CXCL3-F; 5 '- ATCCCCC ATGGTTCAGAAA-3' (SEQ ID NO: 3); and or

CXCL3-R: 5 '- ACCCTGCAGG AAGTGTCAAT-3' (SEQ ID NO: 4).

 The kit used according to the invention may also contain one or more specific reagents for measuring the expression level of LPAI, and in particular the following oligonucleotides:

 LPA1-F: 5'-TGGCATTAAAAATTTTAC AAAAAC A-3 '(SEQ ID NO: 5); and or

LPA1-R: 5'-AATAGTTAACAACATGGGAATGG-3 '(SEQ ID NO: 6).

 Preferably, the kit used according to the invention further comprises one or more specific reagents making it possible to measure the level of expression of one or more other marker (s), preferably chosen from CSF2 / GMCSF, CXCL1 / Groa, IL-6 and IL-8.

According to a particularly preferred embodiment, the kit as defined above and in all its variants is used according to the invention for monitoring the effectiveness of a therapeutic compound to specifically target the LPAi receptor.

 In a particularly preferred manner, the use of the kit as defined above and in all its variants makes it possible to determine the LPAi specific receptor activity of LPA, and in particular the in vivo activity of this receptor.

 All of the preferred embodiments and combinations thereof mentioned above relating to the method and use are also preferred embodiments with respect to the kit used according to the invention.

 In particular, the invention preferably covers the use of a kit comprising one or more specific reagents making it possible to measure the level of expression of HBEGF and one or more specific reagents making it possible to measure the level of expression of CXCL3, and optionally that of one or more other marker (s), preferably chosen from CSF2 / GMCSF, CXCL1 / Groa, IL-6 and IL-8, for the implementation of the method according to invention, and particularly preferably the use of such a kit for monitoring the effectiveness of a therapeutic compound to specifically target the LPAi receptor or to determine the in vivo activity of LPAi specific receptor of LPA.

 Various other characteristics appear from the description given below with reference to the appended figures which show, by way of nonlimiting examples, embodiments of the present invention, and in which:

 FIG. 1 represents the effect of the antagonist VPC12249 on the expression of HBEGF and CXCL3 in PC3 cells,

 FIG. 2 represents the expression of LPAi, HBEGF and CXCL3 in MDA-B02, SK-BR-3 and MDA-B02 / LPAi, SK-BR-3 / LPAi cells,

FIG. 3 represents the expression of LPAi, HBEGF and CXCL3 in a RNA collection originating from primary breast tumors, FIG. 4 represents the expression correlation of LPAi as a function of the combined expression levels of CXCL3 and of HBEGF in an RNA collection originating from primary breast tumors,

 FIG. 5 represents the effect of the antagonist K16425 on the expression of HBEGF and CXCL3 in PC3 tumors induced in mice.

 The invention is not limited to the examples described and shown because various modifications can be made without departing from its scope. EXAMPLES

Methods

 Gene groups whose expression is stimulated early by specific activation of LPAi receptors in response to LPA stimulation have been identified in the following manner.

 Cross-analysis of transcriptomic data of cell lines stimulated or not by LPA for a short time was performed. In order to eliminate from the analysis all the genes that are specific to a single cell line, a series of three natural non-genetically modified tumor cell lines that are different at the level of their genus (male for PC3 and female for MDA- MB-231 and MCF-7) as their cancerous origin (hormone-independent prostate cancer for PC3, hormone-independent breast cancer for MDA-MB-231 and hormone-dependent breast cancer for MCF-7 ) were used.

The cells were cultured in the absence of serum for 24 hours and then placed with or without LPA (1 μl) for 45 minutes. Total RNAs from six independent replicates of each cell line were extracted using the Nucleospin RNAII kit (Macherey-Nagel) and the RNAs were treated with RNAse-free DNAse to remove any trace of contamination from the cell line. Genomic DNA. Two replicates for each condition were analyzed on Affixetrix U133 Plus2.0 biochips (54677 probes). After hybridization, according to the conditions recommended by the supplier (Affimetrix UK Ltd, United Kingdom), the average intensity of the signal emitted by the probes corresponding to each duplicate was calculated for each of the conditions. Duplicates with average expression intensity less than the median expression value (va (eur = 63)) of all 54677 probes in the biochip were removed from the analysis because of a low and therefore unreliable expression level. The probes designated as "Absent" in the presence of LPA were removed from the analysis by the Affimetrix biochip analysis software.

 In the absence of LPA stimulation, the analysis shows that each cell line used expresses a different panel of LPA receptors, as shown in Table 1 below.

Table 1

 Edg2 Edg4 Edg7 GPR23 GPR92 P2Y5 GPRS7

LPA LPA LPA LPA ₂ LPAj _S LPA LPA _{6 7}

 P2Y9

 PC3 ++++ ++ +++ 0 0 +++ ++

MCF-7 0 +++ 0 0 0 0 +++

MDA-MB-231 ++ ++ 0 0 0 0 0

In summary, the major LPA receptors in PC3 are

LPAi, 2, ₃ , 6.7, in MCF7 LPA ₂ , y and in MDA-MB231 LPA _ij2 .

 A microarray analysis was then carried out on the basis of the following hypothesis: the genes regulated in a similar way

(activation / inhibition) only in PC3 cells and MDA-MB-231 identify the specific activation of LPAi.

 For this, the three cell types were cultured in a medium containing no serum. Then they were put in the presence of LPA 1 μΜ for 45 minutes. The cells were then harvested and their contents

Total RNA was prepared. Each experimental condition was reproduced 6 times (6 replicates) independently. The microarray analysis is based on comparing the expression means of two replicates for each condition.

The analysis was carried out excluding the set of probes of the biochip corresponding to ESTs and genes coding for sequences identified as "hypothetical proteins". Thus, a set of 56 genes, not activated by LPA in MCF7 fold change = <1.0) and stimulated early (fold change> = 1.3) by LPA, was identified in PC3 and MDA cells. MB-231. According to the hypothesis formulated, this list of genes corresponds to the activation signature of the LPAi receptor.

 The expression of the two genes HBEGF and CXCL3 was analyzed in particular to study the potential link with the activity of the LPAi receptor.

Example 1: RT-OPCR Analysis of the Expression of HBEGF and CXCL3 from In Vitro Cell Lines

a) PC-3 independent hormonal prostate carcinoma cells (ATCC CRL-135 ™) were cultured at 37 ° C in a humid atmosphere at 5% CO ₂ in F-12K medium (Invitrogen) in the presence of fetal vera serum (10%) and the total RNAs were extracted using the Nucleospin RNAII kit (Macherey-Nagel) after 45 minutes or 16 hours after treatment of cells with a LPAi receptor (VPC12249, 5 or ΙΟμΜ, Avanti Polar Lipids, Alabaster, Alabama, USA). The RNAs were treated with RNAse-free DNAse to remove any trace of contamination by genomic DNA.

 Since there is no specific antagonist for the commercially available LPAi receptor, a non-specific LPA1 / LPA3 receptor antagonist (VPC 12249) was used for this study.

Expression of HBEGF and CXCL3 as specific markers of LPAi receptor activation was investigated by quantitative RT-PCR (QPCR) from total RNAs of 3 replicates. The cDNAs were synthesized from lpg RNA using the iScript cDNA Synthesis kit kit (Biorad). The mRNAs were amplified using SYBR Green kit (Finnzymes) using specific primers (HBEGF-F: 5'-GGACCCATGTCTTCGGAAAT-3 '(SEQ ID NO: 1); HBEGF-R: 5'-CCCATGACACCTCTCTCCAT -3 '(SEQ ID NO: 2); CXCL3-F: 5'-ATCCCCC ATG GTTC AG AAA-3' (SEQ ID NO: 3); CXCL3-R: 5 '-ACCCTGC AG GAAGTGTC AAT-3' (SEQ ID NO: 2); ID NO: 4) thanks to the Reaiplex Eppendorf Mastercycler (Invitrogen) The complementary DNAs were amplified by a 40 cycles PCR and the PCR products analyzed by electrophoresis in a 2% agarose gel labeled with ethidium bromide. The CT (Threshold Cycle or Cycle Threshold) values obtained were normalized by the expression of L32 ribon ribosomal RNA by the Ct method (L32-F: 5 '-C AAGG AGCTGG AAGTGCTGC-3' (SEQ ID NO: 7)). L32-R: 5'-CAGCTCTTTCCACGATGGC-3 '(SEQ ID NO: 8).

 The results are grouped in FIG. 1 and show that the expression of the LPAi HBEGF and CXCL3 target genes is inhibited by an LPA1 / LPA3 receptor antagonist.

b) From the MDA-B02 human breast cancer cell lines, a subclone of MDA-MB-231 cells (Peyruchaud et al., 2001) which express all the LPA receptors described to date (LPAi _-5 ) and SK-BR-3 (ATCC HTB-30 ™) that do not express LPA receptors, MDA-B02 / LPAi cell lines that overexpress the LPAi receptor and SK-BR-3 / LPAi that de novo LPAi were constructed by transfection as described by Boucharaba et al., 2004 and Boucharaba et al., 2006.

 The expression of LPAi, HBEGF and CXCL3 was studied by quantitative RT-PCR (QPCR) from the total RNAs extracted from these different MDA-B02, SK-BR-3, MDA-B02 / LPAi and SK-BR cell lines. 3 / LPAi cultured at 37 ° C. in DMEM medium (PAA) and McCoy's (Invitrogen) in the presence of fetal vesicle serum (10%). For the measurement of the expression of LPAi, the mRNAs were amplified at room temperature. using the following specific primers: LPA1-F: 5'-TGGOTTAAA TTTTACAAAAACA-3 '(SEQ ID NO: 5); LPA1-R: 5'-AATAGTTAACAACATGGGAATGG-3 '(SEQ ID NO: 6).

 The results are collated in FIG. 2, and show that the expression levels of HBEGF and of CXCL3 are directly correlated with that of LPAi in the human breast cancer MDA-B02 / LPAi and SK-BR-3 / cell lines. LPAi.

c) The level of expression of LPAi, HBEGF and CXCL3 was also measured in a series of cell lines from different cancers and grown in the presence of 10% FCS (fetal calf serum), such as PC3, LnCap (prostate cancer); MDA-MB231, T47D, MCF7 (breast cancer); MG63, KHOS, Mnng / HOS (osteosarcoma); and MDA-MB435S (melanoma).

 The results show that cell lines with the highest level of LPAi express the most strongly HBEGF and CXCL3. Conversely, cell lines with the lowest level of LPAi express the weakest HBEGF and CXCL3. These results indicate that the measurement of the expression of HBEGF and CXCL3 constitutes a signature of LPAi receptor activity, whether it is an activation or an inhibition of this receptor.

Example 2 Analysis by RT-OPCR of the Expression of HBEGF and CXCL3 from a RNA Series of Primary Breast Cancer Tumors

 A series of 260 RNA samples from primary tumors of breast cancer patients were analyzed by QPCR for expression levels of the gene encoding LPA1, CXCL3 and HBEGF.

 a) The analysis of the two markers HBEGF and CXCL3 taken individually was first performed. The results are grouped in Figure 3.

The population was stratified into four sub-groups of 65 individuals each (quartiles) corresponding to (i) ^1A of the population which has the level of LPA lowest (very low LPA +), (ii) ¼ of population which has the level attaining the median LPAi expression level (LPAi low ++), (iii) in ^" ¼ of the population with a higher than average median LPAi expression level (mean LPAi +++) , and (iv) au of the population with the highest LPAi level (LPAi high ++++) (Figure 3).

The analysis of different tumor populations shows a direct correlation between the level of expression of LPAi and HBEGF. This is observed by the unpaired serial t-test for the two populations with a lower than median LPAi expression level (very low LPAi and low LPAi) compared to the population with the highest expression level of LPAi. LPAi (LPAi high), p = 0.0215 and p = 0.021, respectively (Tables 2 and 3 below). The direct link between LPAi and HBEGF was also shown when the 4 groups are compared with each other by the Kruskal-Wallis test, p-0.0027 (Tables 4 and 5 below).

Table 2: Unmatched Serial T-Tests for HBEGF:

Table 3: Unmatched Serial T-Band Numbers for HBEGF:

 Table 4: Kruskal-Wallis for HBEGF:

Table 5: Kruskal-Wallis workforce for HBEGF

 number Sum of ranks Avg. Ranks

 LPAi very low 65 7360,500 113,238

 LPAi low 65 7760,500 119,392

 Average LPAi 65 8478,000 130,431

LPAi high 65 10331,000 158,938 Surprisingly, no significant correlation was found for the levels of expression of LPAi and CXCL3 taken individually (Tables 6 to 9 below). Table 6: Unmatched Serial T-Tests for CXCL3:

Table 7: Unmatched Serial T-Test Numbers for CXCL3:

Table 8: Kruskal-Wallis for CXCL3:

 DDL 3

 # Groups 4

 # ex-aequo 34

 H 3.568

 Value of p 0.3121

 H corrected for ex-aequo 3,568

p corrected for ex-aequo 0.3121 Table 9: Kruskal-Wallis workforce for CXCL3:

 b) A second analysis was then performed by combining the two markers HBEGF and CXCL3. The results are grouped in Figure 4.

 The median expression value of each marker has been defined as the threshold value. The population was thus stratified into four subgroups corresponding to (i) the population with CXCL3 below the median and HBEGF below the median (3BHB, 79 patients), (ii) the population with CXCL3 less than the median and HBEGF greater than the median (3BHH, 51 patients), (iii) the population with CXCL3 greater than the median and HBEGF lower than the median (3HHB, 51 patients) and (iv) the population with CXCL3 greater than the median and HBEGF greater than the median (3HHH, 79 patients). For each group, the expression level of LPAi was calculated (Figure 4).

Analysis by the unpaired serial t-test shows that the combination of the expression level of CXCL3 with that of HBEGF has a much higher correlation statistical power than HBEGF alone with the expression of LPAi: ρ = 0.0070 (3HHB-3HHH) versus p = 0.0215 (very low LPAi-LPAi high) (Tables 10 and 11 below and Tables 2 and 3 above). This analysis was confirmed by the Kruskal-Wallis test by comparing all four populations (p = 0.0144) (Tables 12 and 13 below). Table 10: Non-matched serial t-tests for LPAi:

 Table 11: Unpaired Serial T-Test Numbers for LPAi:

Tableau 12 : Kmskal-Wallis pour LPAi :

Table 12: Kmskal-Wallis for LPAi:

Table 13: Kmskal-Wallis workforce for LPAi:

 number Sum of ranks Avg. Ranks

3BHB 79 9200,200 116,456

3BHH 51 7164.500 140.480

3HHB 51 5831,500 114,343

3HHH 79 11734,000 148,532 Example 3; Effect of an LPA1 / LPA3 antagonist on expression of HBEGF and CXCL3 in tumors induced in mice

The expression of HBEGF and CXCL3 was analyzed by RT-QPCR from xenografts of PC3 cells generated by subcutaneous injection in Balb / c nude mice. To do this, human hormone-independent PC-3 prostate carcinoma cells (ATCC CRL-135 ™) were cultured at 37 ° C in a humid atmosphere at 5% CO ₂ in F-12K medium (Invitrogen) in the presence of fetal vision serum (10%).

An injection with 10 ⁶ PC3 cells was performed subcutaneously in the mice (n = 16). When tumors reached 1 mm ³ , some of the animals (n = 9) received treatment with the non-specific LPAi and LPA ₃ receptor antagonist (KI16425, Cayman) at a daily injection (20 mg / kg / day). ) by subcutaneous injection for 5 days. Tumors were harvested at the time of sacrifice. Total RNAs were prepared and analyzed for expression levels of HBEGF and CXCL3, as described above.

 The results are grouped together in FIG. 5 and show that the expression of HBEGF and of CXCL3 is significantly decreased (p <0.05) after treatment with the antagonist Ki 16425. Therefore, the in vitro study of the level of Expression of HBEGF and / or CXCL3 demonstrates the in vivo inhibition of LPAi in a therapeutic condition.

All of these results therefore indicate that the in vitro analysis of the level of HBEGF expression and the level of expression of CXCL3 makes it possible to show the activation or the inhibition in vivo of LPAi, in particular in a context therapeutic. Bibliographical references

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Claims

 A method for in vitro determination of the specific activity of the LPAi receptor comprising the steps of:  measure the level of expression of the HBEGF markers (Heparin-Binding EGF-Iike growth factor and CXCL3 (Chemokine C-X-C Ligand motif) 3) in a sample, and  - respectively conclude the activation or inhibition of the LPAi receptor specifically when a significant increase or decrease in the level of expression of these two markers is demonstrated with respect to a reference value.  2. Method for determining, in vitro, the modulatory activity of a compound vis-à-vis the LPAi receptor, characterized in that it comprises the steps of the method according to claim 1 and that the reference value corresponds to the level expressing said markers before adding said compound whose modulator activity is to be determined.  3. Method for monitoring the efficacy of a therapeutic compound for specifically targeting the LPAi receptor, characterized in that it comprises the steps of the method according to claim 1 or 2 and that the reference value corresponds to the level of expression. said markers in an earlier sample.  4. Method according to any one of the preceding claims, characterized in that it further comprises a step of measuring the level of expression of one or more other markers) chosen from CSF2 / GMCSF, CXCL1 / Groa, IL-6 and IL-8.  5. Method according to any one of the preceding claims, characterized in that the measurement of the expression level of the markers is performed at the nucleic level, and preferably by a PCR technique. 6. Method according to any one of the preceding claims, characterized in that the sample is of biological origin, and preferably human. 7. Use of in vitro measurement of the level of expression of HBEGF and CXCL3 markers as a signature of LPAI specific receptor activity of LPA, i.e., activation or inhibition of this receiver. 8. Use according to claim 7, characterized in that an increase in the level of expression of HBEGF and CXCL3 is a signature of the activation of the specific LPAi receptor, whereas a decrease in the level of expression of HBEGF and of CXCL3 is a signature of LPAi-specific receptor inhibition, 9. Use according to claim 7 or claim 8 for determining in vitro the specific activity of the LPAi receptor, for determining in vitro the modulatory activity of a compound vis-à-vis specifically the LPAi receptor, or for monitoring the in vivo efficacy of a therapeutic compound to specifically target the LPAi receptor.  10. Use according to any one of claims 7 to 9 also comprising measuring the level of expression of one or more other marker (s), preferably chosen from CSF2 / GMCSF, CXCLl / Groa, IL-6 and IL-8. 11. Use of a kit comprising one or more specific reagents for measuring the level of expression of HBEGF and one or more specific reagents for measuring the level of expression of CXCL3, and optionally one or more specific reagents for measuring the level of expression of one or more specific reagents for measuring the level of expression of one or more other marker (s), preferably chosen from CSF2 / G CSF, CXCLl / Groa, IL-6 and IL-8, for the implementation of the method as defined in any one of claims 1 to 6. 12. Use of a kit according to claim 11, for monitoring the effectiveness of a therapeutic compound to specifically target the LPAi receptor. 13. Use of the kit according to claim 11 or 12, characterized in that said kit makes it possible to determine the specific LPAi receptor activity of LPA, in particular the in vivo activity of this receptor.