FR2844597A1 - Diagnosis of embryonal rhabdomyosarcoma, by detecting expression of R-cadherin, also treatment with conjugates targeted to R-cadherin or muscle cells - Google Patents

Abstract

Method for diagnosing embryonal rhabdomyosarcoma by in vitro detection, in a patient sample, of the expression of R-cadherin (I). Independent claims are also included for the following: (1) kit for diagnosing embryonal rhabdomyosarcoma comprising a system for detecting expression of (I); (2) pharmaceutical composition for treating embryonal rhabdomyosarcoma comprising an anticancer agent, radioelement, fluorescent dye or toxin linked to an antibody (Ab1) directed against the extracellular domain of (I) to target (I)-expressing cells; and (3) pharmaceutical composition for treating embryonal rhabdomyosarcoma comprising a (I)-specific antisense RNA or small interfering RNA linked to an antibody (Ab2) directed against a membrane protein on the surface of skeletal muscle cells, to target such cells.

Description

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METHOD FOR DIAGNOSING EMBRYONIC RHABDOMYOSARCOMA AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT AND / OR PREVENTION OF EMBRYONIC RHABDOMYOSARCOMA
The present invention relates to a specific marker for cells derived from embryonic rhabdomyosarcoma (RMSe) as well as to methods and pharmaceutical compositions using this marker in the diagnosis, treatment and / or prevention of this pathology.

 Rhabdomyosarcoma is an uncommon striated muscle differentiation of the soft tissue (its incidence is estimated at 1/125000) and which mainly affects children since, in 70% of cases, rhabdomyosarcomas (RMS) occur during the first decade .

 These tumors are the result of a disruption in the growth or differentiation of cells of muscle origin during development. The primary location is variable (head and neck, urogenital tract, limbs, trunk). It is a pathology with a strong metastatic evolution (pulmonary, bone and medullary metastases) since 15 to 30% of patients already present metastases at the first consultation.

 Two major histologically different types of RMS are described. Embryonic rhabdomyosarcoma (RMSe) which represents 2/3 of these pathologies and concerns young subjects and alveolar rhabdomyosarcoma (RMSa) concerning older subjects. Different cytogenetic criteria are found in these two types of RMS. In particular, the RMSe show a loss of the heterozygosity of the 11p15 locus, region containing

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 many genes involved in oncogenesis. In the majority of RMSa, there is a reciprocal translocation 2-13 or 1-3 leading to the appearance of a chimeric protein Pax3-FKHR or Pax7-FKHR having an increased transcriptional activity. Other genetic alterations have been reported in rhabdomyosarcomas such as mutations of the p53 gene, H- and N-ras genes as well as amplifications of the N-myc and c-MET genes (Merlino and Helman, 1999, Oncogene, vol. 18, pages 5340-5348; et al., 1998, Bull Cancer, vol. 85, pages 1015-1042; and Pressey, 2000, Curr. Opin. Oncol., Vol. 12 pages 337-344).

 The state of the art knows methods for identifying RMSa described in US Pat. No. 5,605,278 based on the presence of a fusion transcript specific for a translocation, this transcript corresponding to a specific marker for RMSa. The positive diagnosis of an RMSe, meanwhile, is currently based on a heavy pathology and immunohistochemical study combining the use of different antibodies.

 The therapeutic strategy applied to RMSe depends on the location of the tumor and the existence or not of metastases. However, there is currently no specific therapeutic strategy for RMSe.

 The identification of a specific marker for RMSe therefore presents a major interest both in the field of diagnosis and treatment of this pathology.

By specific marker of an RMSe, is meant a substance or a compound which makes it possible to distinguish an RMSe from other pathologies and in particular from an RMSa. To date, no specific marker for RMSe has been described in the prior art.

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 R-cadherin belongs to a superfamily of transmembrane glycoproteins carrying out calcium-dependent homophilic cell-cell interactions, which are cadherins.

 The extracellular part of cadherins is composed of five homologous domains (EC) of approximately 110 amino acids. The cytoplasmic part, very conserved, interacts with different cytoplasmic proteins including catenins (a-, ss-, y- and pl20 catenins), themselves linked to actin filaments (see Figure 1) (Kemler, 1993, Trends Genet ., vol. 9, pages 317-321). Cadherins thus form, at the level of the adhesion sites, transmembrane bridges between the cytoskeleton of two neighboring cells. However, these structures are not frozen because they are constantly being remodeled. In addition, the establishment of intercellular contacts leads to the establishment of intracellular signaling allowing an adapted cellular response, in terms of growth and cell cycle regulation, migration, shape or differentiation (Fukata and Kaibuchi , 2001, Nat. Rev. Mol. Cell Biol., Vol. 2, pages 887-897).

 Skeletal muscle cells express N, R, M and 11 cadherins at different times in embryonic development. N-cadherin is expressed throughout skeletal muscle development and is required at various stages of this process (Charrasse et al., 2002, J. Cell Biol., Vol. 158, pages 953-965; George et al., 1997, Dev. Biol., Vol. 185, pages 14-24; Knudsen, 1990, Curr. Opin. Cell Biol., Vol. 2, pages 902-906; Knudsen et al., 1990, Exp. Cell Res., vol. 188, pages 175-184; Linask et al., 1998, Dev. Biol., vol. 202, pages 85-102). M-cadherin is expressed later and

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 participate in the fusion of myoblasts into myotubes, precursors of muscle fiber (Zeschnigk et al., 1995, J. Cell Sci., vol. 108, pages 2973-2981). R-cadherin and cadherin-11 are expressed during the early stages of muscle development (Inuzuka et al., 1991, Development, vol. 113, pages 959-967; Kimura et al., 1995, Dev. Biol., vol. 169, pages 347-358; et al., 1997, Dev. Biol., vol. 187, pages 55-70). These two cadherins are no longer expressed after the formation of myotubes.

 Indeed, the various published studies show an expression of R-cadherin during development only except for the brain where this protein has been found in adults (Inuzuka et al., 1991, Neuron, vol. 7, pages 69 -79; and Takeichi, 1995, Dev. Biol., Vol. 172, pages 466-478; et al., 1997, Histochem. Cell. Biol., Vol. 108, pages 35-43; Stoykova et al., 1997 , Development, vol. 124, pages 3765-3777; Ganzler and Redies, 1995, J. Neurosci., Vol. 15, pages 4157-4172; et al., 2001, Neuroscience, vol. 106, pages 505-533; and al., 2001, Anat.

Embryol., Vol. 204, pages 485-491; Gerhardt et al., 2000, Glia, vol. 31, pages 131-143; Inoue et al., 2001, Development, vol. 128, pages 561-569; et al., 2001, J. Neurobiol., vol. 15, pages 255-264). Indeed, the expression of R-cadherin in the striated muscle, the kidney and the pancreas was only described during development (Rosenberg et al., 1997, Dev. Biol., Vol.

187, pages 55-70; et al., 1998, J. Am. Soc. Nephrol., Vol. 9, pages 1234-1241; et al., 2002, Mol. Cell.

Biol., Vol. 22, pages 1474-1487; et al., 1995, Exp.

Cell Res., Vol. 221, pages 413-425).

 The formation and deformation of cadherin-dependent adherent junctions occurs at different

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 stages in the development of an organism (epithelium-mesenchyme transition) as well as during cell transformation mechanisms and more particularly during the loss of contact inhibition and the formation of metastases. One of the peculiarities of rhabdomyosarcoma lies in its strong aggressiveness. Indeed, 15 to 30% of patients present metastases during the first examination.

 The Applicant has therefore sought the expression profile of cadherins N, R, M and 11 in different rhabdomyosarcoma lines. These studies have enabled the Applicant to demonstrate, surprisingly, the expression of the protein R-cadherin specifically in the RMSe. The present invention therefore provides, for the first time, a specific RMSe marker that is Rcadherin.

 The present invention relates to a method for diagnosing embryonic rhabdomyosarcoma from a sample of an individual, consisting in demonstrating, in vitro, the expression of R-cadherin in said sample.

 In the context of the invention, the term “individual” means a man, a woman or an animal, adults, healthy or likely to be affected or suffering from embryonic rhabdomyosarcoma or alveolar rhabdomyosarcoma or any other pathology .

 Advantageously, the method for diagnosing embryonic rhabdomyosarcoma which is the subject of the present invention consists in bringing a sample of an individual into contact in vitro, after any treatment, with a means for detecting and / or measuring the expressed R-cadherin.

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 Since embryonic rhabdomyosarcoma affects the striated muscular differentiated soft tissue, the sample of an individual capable of being used in the context of the invention may be a cell, a set of cells or a tissue extracted from an individual .

More particularly, the extracted tissue can be a tissue suspected of carrying the pathology and, by way of example and in a non-exhaustive manner, a striated muscle. The cell (s) and the tissue can be cultured before determining the level of expression of Rcadherin in the latter.

 The possible treatments which the sample can undergo before the detection of the expression of Rcadherin are, for example, the treatments aimed at extracting from said sample the messenger RNAs and / or the proteins, so that the detection of the expression R-cadherin is carried out on these messenger RNAs or proteins extracted.

 The determination of the expression of Rcadherin can be carried out by various techniques known to those skilled in the art.

 In a first form of implementation of the invention, the expression of R-cadherin may be detected via the demonstration of the messenger RNA of Rcadherin. This messenger RNA can be demonstrated using molecular cytogenetics techniques (RT-PCR) using primers capable of specifically amplifying R-cadherin. Advantageously, the primers capable of specifically amplifying R-cadherin are the following: - primer meaning 5'-CCGACCAGCCCCCCATGGAG-3 '(SEQ ID No: 1 in the sequence list in the appendix) and

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 - antisense primer 5'-CCTGGCTTGGAGCCCTCGTCC-3 '(SEQ ID No: 2 in the sequence list in the appendix).

 Alternatively, the identification of the messenger RNA of R-cadherin can be carried out using a probe specific for this messenger RNA. More particularly, this probe specific for R-cadherin messenger RNA can be a RNA or a single stranded DNA capable of hybridizing under strong stringency conditions with the R-cadherin messenger RNA. Those skilled in the art know different techniques in order to demonstrate the double-strand structure resulting from this hybridization, such as, by way of example and in a non-exhaustive manner, using a probe specific for the labeled Rcadherin messenger RNA. .

 In a second form of implementation of the invention, the expression of R-cadherin can be detected via the demonstration of the protein corresponding to R-cadherin. This can be done using antibodies specific for this protein, for example, by the Western Blot technique.

 It is also possible to envisage highlighting the expression of R-cadherin on the cell or tissue sample from an individual by a simple immunohistochemical technique using an antibody directed against R-cadherin. This implementation requires no prior treatment of said sample such as those defined above.

 The present invention also contemplates the use of the specific marker of embryonic rhabdomyosarcoma (RMSe) to target in vivo the RMSe tumor foci (initial tumor and possible metastases). The

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 in vivo targeting method for tumor foci consists in administering to an individual at least one antibody directed against the extracellular domain of the radiolabelled (radio-immuno-targeting) or photomarked (immunophotomarking) R-cadherin. Such an antibody is capable of targeting the regions expressing R-cadherin.

 These different forms of implementation of the invention are given only by way of example and in a non-exhaustive manner. Those skilled in the art will be able to use, within the framework of the present invention, any technique which makes it possible to demonstrate the expression of a particular protein or the presence of particular messenger RNAs, and this, in a specific manner.

 The invention also relates to a diagnostic kit for embryonic rhabdomyosarcoma, characterized in that it comprises at least one means for detecting the expression of R-cadherin. Advantageously, this means is chosen from the group consisting of an antibody specific for R-cadherin, primers for RT-PCR capable of specifically amplifying R-cadherin, the following pair of primers: primer 5 ′ sense -CCGACCAGCCCCCCATGGAG-3 'and antisense primer 5'-CCTGGCTTGGAGCCCTCGTCC-3' (respectively SEQ ID No: 1 and No: 2 in the sequence list in the appendix) and a probe capable of hybridizing specifically to the messenger RNA of the R-cadherin.

 In addition, the identification of a specific marker for an embryonic rhabdomyosarcoma also makes it possible to envisage its use in a specific and targeted treatment method for embryonic rhabdomyosarcoma. Thus, the present invention contemplates a pharmaceutical composition for the treatment of

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 embryonic rhabdomyosarcoma comprising a molecule active in the treatment of embryonic rhabdomyosarcoma associated with a vector capable of addressing said active molecule to cells expressing R-cadherin. Such a pharmaceutical composition can, of course, also comprise one or more suitable excipients or adjuvants.

 The vector capable of addressing the active molecule to cells expressing R-cadherin is preferably an antibody directed against the extracellular domain of R-cadherin.

 The present invention implements antibodies directed against R-cadherin, in particular in the context of methods of diagnosis of embryonic rhabdomyosarcoma revealing the protein R-cadherin. Some of the antibodies used in the context of the present invention must, more specifically, be directed against the extracellular domain of R-cadherin, for example, in the context of pharmaceutical compositions useful for the treatment of embryonic rhabdomyosarcoma. Those skilled in the art can use any antibody directed against Rcadherin known in the state of the art in the context of the present invention. In addition, a person skilled in the art is also capable of generating such antibodies since he knows different standard techniques enabling him to produce monoclonal or polyclonal antibodies which specifically bind R-cadherin and, more particularly, the extracellular domain of Rcadherine.

 A molecule active in the treatment of embryonic rhabdomyosarcoma capable of being used in the therapeutic composition which is the subject of the present invention is chosen from anti-cancer agents, radioelements, fluorescent dyes and toxins.

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 The vector capable of addressing the active molecule to cells expressing R-cadherin and the active molecule can be associated with each other directly or indirectly by means of a chemical bond such as a hydrophobic or ionic bond. .

 As R-cadherin is also expressed in the adult brain, a person skilled in the art will be particularly vigilant that the molecule active in the treatment of embryonic rhabdomyosarcoma associated with a vector capable of addressing this active molecule to cells expressing R- cadherin should not be able to cross the blood-brain barrier.

 The present invention also contemplates a method of preventing embryonic rhabdomyosarcoma aimed at preventing expression of R-cadherin in skeletal muscle. Consequently, the present invention relates to a pharmaceutical composition for the prevention of embryonic rhabdomyosarcoma comprising a molecule preventing the expression of R-cadherin associated with a vector capable of addressing said molecule to skeletal muscle cells. Such a pharmaceutical composition can, of course, also comprise one or more suitable excipients or adjuvants.

 Any molecule capable of preventing the expression of R-cadherin can be used in the context of the present invention. By way of example and in a non-exhaustive manner, said molecule can be an antisense RNA or a siRNA (for small RNA interference) specific for R-cadherin.

 In addition, any vector capable of specifically addressing a molecule to skeletal muscle cells can be used in the context of the invention.

This vector can be an antibody specific for a protein

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 membrane present on the surface of skeletal muscle cells.

 Other advantages and characteristics of the invention will appear on reading the example which follows concerning the study of the expression of cadherins. This example is given by way of illustration and should not be interpreted as limiting the scope of the invention. This example refers to FIG. 2 which presents the study of the expression of R-cadherin in different cells by RT-PCR followed by the visualization of the PCR products in agarose gel (FIG. 2, part A) and quantification of the expression of the protein R-cadherin in different cells (Figure 2, part B).

 I. Materials and methods.

 1.1. Cells used.

 The cells used as control are: - myoblasts of C2C12 mice, - myoblasts of rat L6, and - human myoblasts derived from paravertebral striated muscle (Origin: Tissue bank for research, AFM-BTR).

The RMSe cells used are: - the RD line (ATCC origin, CCL-136), - the A-204 line (ATCC origin, HTB-82), - the A-673 line (ATCC origin, CRL-1598), - the CT-10 P8T line (Origine T. Triche, Childrens Hospital, Los Angeles, USA), and - the TTC-442 P8T line (Origine T. Triche, Childrens Hospital, Los Angeles, USA)
The RMSa cells used are:

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 - the Rh30 line (P. Houghton, St. Jude Childrens Hospital, Memphis, USA), - the Rh28 line (P. Houghton, St. Jude Childrens Hospital, Memphis, USA), - the JR / C line (J. Garvin , Medical University of South Carolina, Charleston, USA).

 The epithelial lines used are: - the MCF7 line (adenocarcinoma of the mammary gland), - the FT44 line (human tumor liver isolated in a hospital environment), - the Caco-2 line (colorectal tumor), - the line SW48 (colorectal adenocarcinoma , early adenoma), - the line SW480 (colorectal adenocarcinoma), and - the line SW620 (metastasis of the lymphoid nodule type originating from the tumor from which the line SW480 is derived).

 1.2. Quantitative RT-PCR.

 The RNAs are extracted by the method of Chomczynski and Sacchi (1987, Anal. Biochem., Vol. 162, pages 156-159) and their quality is checked in non-denaturing agarose gel.

 The cDNA is obtained by reverse transcription (Superscript II GIBCO) using oligo dT (Perkin Elmer). The PCR reaction contains 1 l of SYBR Green I (Light cycler Roche) Master mix (Taq DNA polymerase FastStart, buffer, deoxynucleotide trisphosphate mix, and SYBR Green I dye), 3 mM MgCl2, 0.5 M of each primer and 2 μl diluted cDNA. The primers used to amplify R-cadherin are:

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 - primer sense 5'-CCGACCAGCCCCCCATGGAG-3 '(SEQ ID No: 1 in the sequence list in the appendix) and - antisense primer 5'-CCTGGCTTGGAGCCCTCGTCC-3' (SEQ ID No: 2 in the list of sequences in the appendix). A standard concentration curve is established by series of dilutions of R-cadherin cDNA. Data are normalized using GAPDH as an index of cDNA content after reverse transcription. The samples are first denatured at 95 C for 8 min, then 45 cycles are carried out: denaturation at 95 C for 3 s, hybridization at 61-62 C for 8-10 s, and elongation at 72 C for 10-12 s. The temperature transition rate is 20 C / s. Fluorescence is measured at the end of each extension step. After amplification, the products are heated to 95 C, cooled and maintained at 70 C for 20 s, then gently (0.3 C / s) heated to 95 C to determine the specificity of the PCR products. This specificity is confirmed by visualization of the PCR products in agarose gel and by sequencing.

 1.3. Western blot.

 The cell extracts are lysed in a cold buffer containing 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM sodium PPi, 15 mM p-nitrophenyl phosphate, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium orthovanadate, 1% NP-40 and a protease inhibitor mixture (Sigma). The lysates are centrifuged at 14,000 rpm for 10 min at 4 C. The supernatant is collected and used. The protein concentration is determined with the "BCA protein assay" kit (Pierce).

 For the Western Blot, 20 g of proteins are loaded onto an 8% acrylamide gel (Laemmli gel) and transferred onto a PVDF membrane. R-cadherin is

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 revealed by a diluted monoclonal antibody 1: 200 (MRCD5 generously donated by Dr. Takaishi).

 II. Results.

 The inventors therefore sought the expression profile of cadherins N, R, M and 11 in 8 lines of rhabdomyosarcomas. Three normal lines were taken as control. Tumor lines of epithelial origin have also been tested. Two experimental approaches were used: quantitative RT-PCR and western-blot (for N and R-cadherin only).

 The results obtained show an overall decrease in the level of N, M and 11 cadherin mRNA in the rhabdomyoblasts (results not shown). The expression of R-cadherin was specifically detected in the RMSe (Figure 2 part A). The protein R-cadherin was also specifically detected in cells from RMSe (Figure 2 part B).

Claims (10)

 1. Method for diagnosing embryonic rhabdomyosarcoma from a sample of an individual, consisting in demonstrating, in vitro, the expression of R-cadherin in said sample.  2. Method of diagnosis according to claim 1, characterized in that it consists in bringing in vitro a sample of an individual, after a possible treatment, with a means for detecting and / or measuring the expressed Rcadherin.  3. Method according to any one of claims 1 or 2, characterized in that said sample is a cell, a set of cells or a tissue.  4. Method according to any one of claims 1 to 3, characterized in that the expression of R-cadherin is detected via the demonstration of the messenger RNA of R-cadherin.  5. Method according to claim 4, characterized in that said messenger RNA is demonstrated using molecular cytogenetics techniques (RT-PCR) using primers capable of specifically amplifying R-cadherin.  6. Method according to claim 4, characterized in that said messenger RNA is highlighted using a probe specific for this messenger RNA. <Desc / Clms Page number 16>  7. Method according to any one of claims 1 to 3, characterized in that the expression of R-cadherin is detected via the demonstration of the protein corresponding to R-cadherin by means of antibodies specific for this protein.  8. Kit for diagnosis of an embryonic rhabdomyosarcoma characterized in that it comprises at least one means for detecting the expression of R-cadherin chosen from the group consisting of antibodies specific for R-cadherin, primers for RT -PCR capable of specifically amplifying R-cadherin, the following pair of primers: primer sense 5'-CCGACCAGCCCCCCATGGAG-3 'and antisense primer 5'-CCTGGCTTGGAGCCCTCGTCC-3' (respectively SEQ ID No: 1 and No: 2 in the list of sequences in the appendix) and the probes capable of hybridizing specifically to the messenger RNA of R-cadherin.  9. Pharmaceutical composition for the treatment of embryonic rhabdomyosarcoma comprising a molecule active in the treatment of embryonic rhabdomyosarcoma chosen from anti-cancer agents, radioelements, fluorescent dyes and toxins and associated with a vector capable of addressing said active molecule to cells expressing R-cadherin which is an antibody directed against the extracellular domain of R-cadherin.  10. Pharmaceutical composition for the prevention of embryonic rhabdomyosarcoma comprising a molecule preventing the expression of R-cadherin chosen from an antisense RNA and a siRNA (for small interference RNA) specific for R-cadherin and associated with a vector capable of send said molecule to skeletal muscle cells which is an antibody specific for a <Desc / Clms Page number 17>  membrane protein present on the surface of skeletal muscle cells.