WO2006061430A2

WO2006061430A2 - Bone metastasis markers as a target regulating bone metastasis and bone development

Info

Publication number: WO2006061430A2
Application number: PCT/EP2005/056636
Authority: WO
Inventors: Philippe Clement-Lacroix; Philippe Clezardin; Amanda Jackson; Philippe Pujuguet
Original assignee: Proskelia; Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2004-12-10
Filing date: 2005-12-09
Publication date: 2006-06-15
Also published as: WO2006061430A3

Abstract

The present invention concerns a method of, and products for, diagnosing bone disorders using bone metastasis specifie gene markers, including ITGBL1.

Description

ITGBL-1 AS A TARGET REGULATING BONE METASTASIS AND

BONE DEVELOPMENT

GENERAL DISCLOSURE OF THE INVENTION Metastases occur in 80% of breast cancer and represent the major cause of treatment failure. Breast cancer cells disseminate from the malignant breast through the blood and lymphatic circulation to invade specific host organs, mainly bone, liver and lung. There is a urgent clinical need to interfere with the specific cellular and molecular mechanisms used by the tumor cells to grow in their target organ. An animal model was used to address this question by gene profiling, and a pool of genes thought to be involved in this late step of the tumorigenic process has been identified. Human MDA-MB-231 breast cancer cells were injected into the heart of Nude mice allowing the cells to home to bone. The cells from this location were harvested, cultured and re-injected into the animal. This was repeated six times producing a cell line, B02, which homes to bone 100% of the time within three weeks after a tail vein injection (1-3). The whole genome expression profiles of the B02 cell line and its parental MDA-MB- 231 line were compared using the Affymetrix U133 chip set which represents more than 39,000 transcripts. A list of up-regulated and down- regulated genes was produced and compared to relevant literature data. A subset of overlapping genes required for bone homing were found when comparing the resulting data set to a previous work (4), which helps to validate the results described herein. In addition, these data were also validated by showing that a number of genes regulated in the aforementioned model are common to the recently described "metastatic signature" found in human breast metastasis (5). A further subset of genes regulated in this model is also common to genes recently implicated in the metastatic homing to human bone (6). This latter study only used a small number of human samples and DNA arrays with relatively few genes on them, making the gene profiling comparison limited. However, a large number of genes were unique to the model. Of interest, the gene ITGBL1 was found to be up-regulated three times in the B02 cell line compared to the MDA cell line. None of the other integrin family members were regulated in this system. Retrospectively, ITGBL1 has been searched for in archive data comprising genes modulated during bone cell differentiation. ITGBL1 was hereby consistently found to be up-regulated during calvaria maturation and during mesenchymal cell differentiation induced by Wnt.

According to the results described herein, ITGBL1 appears to be a target relevant to the homing of breast cancer cells to bone and during bone development.

BACKGROUND OF THE INVENTION

Metastases occur in 80% of breast cancer and represent the major cause of treatment failure. Breast cancer cells disseminate from the malignant breast through the blood and lymphatic circulation to invade specific host organs, mainly bone, liver and lung. In the case of breast cancer, most patients with advanced disease develop osteolytic (loss of bone) bone meatastasis, which are a common cause of morbidity (11 ). The classical view arguing that acquisition to metastasize to a target organ is a late mutational event is now challenged. Differential analysis of whole genome expression between distant metastasis and matched (from the same patient) primary tumors indicate that the ability to metastasize is already present in the parental tumor (5) while its ability to grow into a specific tissue, such as bone, is a late event (4). Some genes have been identified whose regulated expression is involved in the establishment of bone metastasis (5, 6). At least four key cellular functions are now causally involved in bone metastasis: homing, invasion, angiogenesis and osteolysis regulated respectively through CXCR4, MMP1 , CTGF/FGF5 and IL11/OPN (4). However, an incomplete understanding of the cellular and molecular mechanisms underlying bone metastasis hinders the development of effective therapies that would eliminate or ameliorate this condition (11 ).

Therefore, there is a urgent clinical need to interfere with the specific cellular and molecular mechanisms used by the tumor cells to grow in their target organ. An animal model has been used to address this question by gene profiling (see Materials and Methods), and a pool of genes thought to be involved in this bone-specific late step of the breast tumorigenic process has been identified. Some of these genes are shared in some developmental stages of bone formation.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention, there is provided a method of and products for diagnosing bone disorders by detecting the presence of RNA transcribed from the bone metastasis specific genes (also referred to herein as "markers") of the present invention, or DNA corresponding to such RNA in a sample derived from a host. In accordance with another aspect of the present invention, there is provided a method of and products for diagnosing bone disorders by detecting an altered level of a polypeptide corresponding to the bone metastasis specific markers of the present invention in a sample derived from a host.

In accordance with yet another aspect of the present invention, there are provided processes for using one or more of the polypeptides corresponding to the markers of the present invention to prevent and/or to treat bone disorders.

In accordance with another aspect of the present invention, there are provided processes for using the polypeptides corresponding to the markers of the present invention to screen for compounds which interact with the polypeptides, for example, compounds which modulate (inhibit or activate) said polypeptides. In accordance with yet another aspect of the present invention, there are provided compounds which modulate the biological activity of one or more of the polypeptides corresponding to the markers of the present invention, which may be used therapeutically, for example, for preventing and/or treating bone disorders.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Ratio measurements and red/green plot representations of ITGBL1 expression profiles observed by Affymetrix chip hybridization. Figure 2: ITGBL1 profiling in osteoblast. ITGBL1 is induced by Wnt3a in C3H10T1/2 and calvaria osteoblasts and consistently increased during calvaria maturation. Figure 3: Determination of ITGBL1 mRNA expression levels in MDA-MB- 231 and BO2 cells measured by quantitative real-time PCR.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

By the terms "bone metastasis", it is meant secondary cancers located at the bone level. Indeed, many cancers spread through the blood-stream, a process known in the art as "metastasis". Small collections of cells break off from the original tumour and are carried to other organs where they are deposited. These secondary deposits, called "metastases", also grow and may extend locally into the surrounding tissues. Certain types of cancer metastasize in characteristic ways. For example, cancers of the thyroid gland, lung, breast, kidney, and prostate frequently metastasize to the bones. Metastases often occur after the primary cancer has attained considerable size.

As used herein, a "bone metastasis (specific) marker" (also referred to as a "bone development (specific) marker") means a marker selected in the group consisting of the markers listed in appended Table 1 , i.e., a marker selected in the group consisting of: EDN1 , ENPP1 , FGF13, GJA1 , GMFG, ITGBL1 , LPHN2, MAGEA3, MAGEA6, NR2F1 , NSBP1 , PAEP, PGF, PHC1 , PLCB1 , ROBO1 , TITF1 , AKR1C3, AREG, ARHD, CPO, MUC1 , S100A2, TPD52L1. According to the present invention, either one single marker is used, preferably ITGBL1 , or two or more markers are used, including preferably ITGBL1.

By the term "marker", it is referred to a gene as identified in appended Table 1. The term "marker" may also be used for the polypeptide encoded by the gene (also "the polypeptide corresponding to the marker" or "the marker polypeptide"). In any case, the skilled artisan will clearly understand, depending on the context, whether a "marker" refers to a gene or the polypeptide corresponding to a gene.

By "modulate", it is meant either induce (equivalents of "induce" being herein "increase", "promote", "enhance", and "stimulate"), or inhibit (equivalents of "inhibit" being "reduce", "decrease", "suppress", and "block"). This may reflect, for instance, (i) an increase or decrease in expression or in activity of the polynucleotides or polypeptides corresponding to the markers of the present invention; or (ii) a change in the amount of said polynucleotides or polypeptides, in the cellular distribution thereof, in the level of expression thereof, in the type of activity thereof.

The terms "bone disorders" encompass any disorders of bone metabolism, including without any limitation bone metastases (which may be seen as excessive bone developments) and bone defects (regarded as insufficient bone developments).

In accordance with one aspect of the present invention there are provided diagnostic assays for detecting bone disorders in a host. The presence of active transcription of at least one, preferably several, more preferably all bone metastasis specific markers of the present invention in cells in the host is indicative of bone disorders. In other words, it is the detection of this enhanced transcription or enhanced protein expression in cells which is indicative of disorders of bone. An embodiment of the present invention thus relates to a method for diagnosing a bone disorder in a host comprising determining transcription of at least one gene in a sample of a host, said gene having a coding portion which includes DNA having at least 90% identity (preferably at least 95%, more preferably at least 98%, yet more preferably at least 99%, and even more preferably at least 99.5%) to the DNA of a bone metastasis marker as set forth in Table 1 , whereby said transcription indicates a bone disorder in the host.

In particular embodiments, transcription is determined by detecting the presence of:

- an altered level of RNA transcribed from said gene; or

- an altered level of DNA complementary to the RNA transcribed from said gene; or

- an altered level of an expression product of said gene.

The aforementioned embodiments are described with more details below. Preferably, more than one marker will be searched for when performing the diagnostic assays. In this case, at least two markers will be selected in appended Table 1. In particular embodiments, the diagnostic assays will advantageously be performed searching for all the markers disclosed in Table 1. In yet particular embodiments, not the full-length marker is used, but one or more fragment(s) thereof only, said fragment(s) being long enough to be so discriminatory that the same results are equally obtained when performing the diagnostic method of the invention using full-length marker sequences or fragments thereof.

In one example of such a diagnostic assay, an RNA sequence in a sample derived from a human bone metastasis is detected by hybridization to a probe. The sample contains a nucleic acid or a mixture of nucleic acids, at least one of which is suspected of containing at least one bone metastasis specific marker of the present invention, or fragment(s) thereof, which is transcribed and expressed in said human bone metastasis. Thus, for example, in a form of an assay for determining the presence of a specific RNA in cells, initially RNA is isolated from the cells. A sample may be obtained from cells derived from tissues including but not limited to blood, tissue biopsy and autopsy material.

The detection of enhanced transcription to mRNA from at least one bone metastasis marker of the present invention, or fragment(s) thereof, in a sample obtained from cells derived from a human bone metastasis is well within the scope of those skilled in the art from the teachings herein. A non-limiting example of suitable experimental procedure is briefly reported here. The isolation of mRNA comprises isolating total cellular RNA by disrupting a cell and performing differential centrifugation. Once the total RNA is isolated, mRNA is isolated by making use of the adenine nucleotide residues known to those skilled in the art as a poly(A) tail found on virtually every eukaryotic mRNA molecule at the 3' end thereof. Oligonucleotides composed of only deoxythymidine [oligo(dT)] are linked to cellulose and the oligo(dT)-cellulose packed into small columns. When a preparation of total cellular total RNA is passed through such a column, the mRNA molecules bind to the oligo (dT) by the poly (A) tails while the rest of the RNA flows through the column. The bound mRNAs are then eluted from the column and collected.

One example of detecting isolated mRNA trancribed from a specific marker of the present invention comprises screening the collected mRNAs with gene specific oligonucleotide probes. It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe. Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalysing the formation of a detectable product. An example of detecting a polynucleotide complementary to the mRNA sequence (cDNA) utilizes the polymerase chain reaction (PCR) in conjunction with reverse trancriptase. PCR is a conventional method for the specific amplification of DNA or RNA stretches. One application of this technology is in nucleic acid probe technology to bring up nucleic acid sequences present in low copy numbers to a detectable level. RT-PCR is a combination of PCR with the reverse transcriptase enzyme. Reverse transcriptase is an enzyme which produces cDNA molecules from corresponding mRNA molecules. This is important since PCR amplifies nucleic acid molecules, particularly DNA, and this DNA may be produced from the mRNA isolated from a sample derived from the host. Oligonucleotide primers and probes are prepared with high specificity to the DNA sequences of the markers of the present invention. The probes are at least 10 base pairs in length, preferably at least 30 base pairs in length and most preferably at least 50 base pairs in length or more. The reverse transcriptase reaction and PCR amplification are performed sequentially without interruption.

Another embodiment of the diagnostic assays of the invention relates to assays which detect the presence of an altered level of the expression products of the bone metastasis specific genes of the present invention. Thus, for example, such an assay involves detection of the polypeptides corresponding to the markers of the present invention, or fragments thereof.

In yet another embodiment of the diagnostic assays of the invention, there is provided a method of diagnosing a disorder in bone development, for example bone metastasis, by determining altered levels of the polypeptides corresponding to the specific markers of the present invention in a biological sample, derived from, e.g., a human bone metastasis. Elevated levels of the specific polypeptides indicates active transcription and expression of the corresponding bone metastasis specific gene product. Assays used to detect levels of a specific marker polypeptide in a sample derived from a host are well-known to those skilled in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, ELISA assays and "sandwich" assays. A biological sample may include, but is not limited to, tissue extracts, cell samples or biological fluids. A conventional ELISA assay initially comprises preparing an antibody specific to a specific marker polypeptide of the present invention, preferably a monoclonal antibody. In addition, a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme. A sample is removed from a host and incubated on a solid support, e.g., a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein, such as BSA. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to the specific marker polypeptide attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the specific marker polypeptide. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of the specific marker polypeptide present in a given volume of patient sample when compared against a standard curve. A competition assay may be employed where antibodies specific to a specific marker polypeptide are attached to a solid support. The specific marker polypeptide is then labeled. The labeled polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected, for example, by liquid scintillation chromatography, can be correlated to a quantity of the specific marker polypeptide in the sample. A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay, specific marker polypeptides are passed over a solid support and bind to antibody attached to the solid support. A second antibody is then bound to the specific marker polypeptide. A third antibody which is labeled and is specific to the second antibody, is then passed over the solid support and binds to the second antibody and an amount can then be quantified. In alternative methods, labelled antibodies to a bone metastasis specific marker polypeptide are used. In a one-step assay, the target molecule, if it is present, is immobilized and incubated with a labeled antibody. The labeled antibody binds to the immobilized target molecule. After washing to remove the unbound molecules, the sample is assayed for the presence of the label. In a two-step assay, immobilized target molecule is incubated with an unlabeled antibody. The target molecule-labeled antibody complex, if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label.

The choice of label used to label the antibodies will vary depending upon the application. However, this choice is readily determinable to one skilled in the art. The labeled antibodies may be used in immunoassays as well as in histological applications to detect the presence of the proteins. The labeled antibodies may be polyclonal or monoclonal.

Another aspect of the present invention concerns a diagnostic composition comprising at least one compound selected in the group of : bone metastasis marker genes and bone metastasis marker polypeptides as set forth in Table 1 , and antagonists and agonists thereof. Such antagonists and agonists will be further described hereunder.

In accordance with another aspect of the present invention, there are provided assays which may be used to screen for therapeutics to inhibit

("antagonist compounds") or, on the contrary, to promote ("agonist compounds") the action of the bone metastasis specific genes or protein products of the present invention. Antagonist and agonist compounds are herein encompassed by the terms "modulating compounds". A particular embodiment of such an assay is related to a method for selecting a modulating compound useful for preventing and/or treating a bone disorder in a mammal, preferably a human, in need of such treatment, wherein said method comprises: a) testing the ability of a candidate compound to modulate the biological activity of a bone metastasis marker in vitro and/or in vivo; and b) if said candidate compound modulates said biological activity, selecting said compound.

Advantageously, this method further comprises purifying the selected compound. One assay takes advantage of ligand/receptor interacting or binding functions of the markers.

In this respect, methods for detecting an inhibition or an enhancement of the biological activity of a marker such as ITGBL1 include both in vitro and in vivo procedures (e.g., protein-protein binding assays, biochemical screening assays, immunoassays, cell-based assays, animal model experiments, which are well-characterized in the art). For instance, the person skilled in the art may use only one in vitro and/or one in vivo selection technique. However, in order to strengthen the validity and reproducibility of the results, this person may prefer to use at least two in vitro and/or at least two in vivo selection methods. Examples of in vitro and in vivo procedures for showing an inhibitory or an enhancer activity on ITGBL1 are described below.

For instance, the present invention discloses methods for selecting a therapeutic which forms a complex with bone metastasis specific proteins with sufficient affinity to prevent their biological action. The methods include various assays, including competitive assays where the proteins are immobilized to a support, and are contacted with a natural substrate and a labeled therapeutic candidate, either simultaneously or in either consecutive order, and determining whether the therapeutic candidate effectively competes with the natural substrate in a manner sufficient to prevent binding of the protein to its substrate.

In another embodiment, the substrate is immobilized to a support, and is contacted with both a labelled specific marker polypeptide and a therapeutic candidate (or, conversely, unlabelled marker proteins and a labelled therapeutic candidate), and it is determined whether the amount of the specific marker polypeptide bound to the substrate is reduced in comparison to the assay without the therapeutic added. The specific marker polypeptide may be labelled with antibodies. Potential therapeutic compounds capable of inhibiting the biological activity of bone metastasis markers (also referred to as "antagonists") include but are not limited to antibodies and anti-idiotypic antibodies and, in some cases, oligonucleotides which bind to the marker polypeptides. Another example is an antisense construct prepared using well-known antisense technology, which is directed to a bone metastasis specific polynucleotide to prevent transcription. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature marker polypeptides, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription, thereby preventing transcription and the production of a bone metastasis specific polynucleotide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the corresponding specific marker polypeptide; The oligonucleotides can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the specific marker polypeptides. Another example is a small molecule which binds to and occupies the active site of the specific marker polypeptide, thereby making the active site inaccessible to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.

An example of an agonist compound capable of promoting the biological activity of bone metastasis markers is a small molecule which binds to and occupies the active site of the specific marker polypeptide, thereby transactivating the active site such that the resulting biological activity is enhanced compared to normal. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.

According to another aspect, the present invention relates to modulating compounds, i.e., antagonist and agonist compounds of bone metastasis markers selected in Table 1.

According to yet another aspect of the present invention, the modulating (antagonist and agonist) compounds of bone metastasis markers (genes and/or polypeptides) may be employed to prevent and/or treat bone disorders, since they interact with the normal metabolism of bone. Indeed, antagonists or agonists are capable of interacting with the function of specific markers in a manner sufficient to inhibit or promote, respectively, their natural function which is necessary for the normal metabolism of bone.

More specifically, the modulating compounds (antagonists or agonists) are useful for preventing and/or treating bone disorders (e.g., bone metastases or bone defects) by modulating (inhibiting or promoting) bone development. At least one modulating compound (selected among antagonists and agonists, depending on the expected effect and the bone disorder to prevent and/or treat) may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described. The compounds may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of at least one compound, and at least one pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit to mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described above. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitonel, intramuscular, subcutaneous, intranasal, intra-anal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for the treatment and/or prophylaxis of the specific indication.

The bone metastasis specific genes and compounds which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is conventionally referred to as "gene therapy". In addition, the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunigen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples of well-known techniques include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV- hybridoma-technique to produce human monoclonal antibodies. All these techniques belong to the general knowledge in the art.

Techniques known for producing single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptide products of the invention. Transgenic mice may also be used to generate antibodies. Besides being useful as therapeutics per se, the antibodies may also be employed to target bone metastasis cells, for example, in a method of homing interaction agents which, when contacting bone metastasis cells, destroy them. A linking of the interaction agent to the antibody would cause the interaction agent to be carried directly to the bone metastases. The antibodies may also be employed to label bone metastasis to support metastasis detection using medical imaging technologies. In order to fully illustrate the present invention and advantages thereof, the following specific examples are given, it being understood that the same are intended only as illustrative and in no way as limitative.

EXAMPLES

I- Materials and Methods

• Animal model of bone metastasis using the breast cancer MDA-MB-231 and B02 cell lines

Cells: The human breast carcinoma cell line MDA-MB-231 was obtained from the European Type Culture Collection, Salisbury, UK (ECACC 92020424). Cell line MDA-MB-231 /B02 (B02) has been selected from bone metastases caused by MDA-MB-231 cells (1 ). Briefly, anesthetized nude mice were inoculated with a tumor cell suspension of MDA-MB-231 cells into the left ventricle of the heart. Three weeks after the inoculation, metastatic MDA-MB-231 cells were harvested from metastatic sites in bones, maintained in culture until confluence, and then once again inoculated into the left ventricle of the heart of nude mice. The B02 cell line was established after six in vivo passages of MDA-MB-231 cells in bones (1 ). MDA-MB-231 and B02 cell lines were routinely culture in RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% penicillin and 1% streptomycin at 37⁰C in humidified atmosphere containing 5% CO2. Total RNAs were extracted in duplicates from sub-confluent and confluent cultures using Trizol™. Animals:

All procedures were performed with female BALB/c nu/nu mice of 4 weeks of age (Charles River). Studies involving animals, including housing and care, method of euthanasia, and experimental protocols, were conducted in accordance with a code of practice established by the Experimentation Board Review of Proskelia. Anesthetized mice were inoculated with tumor- cell suspension (5 X 10⁵ cells in 100 μl of PBS) into the tail vein. A the end of the experiments, animals were examined by radiography using a X-ray cabinet (Faxitron).

• Chip hybridizations data assembly and calculation of gene expression ratios.

For every resulting sample, labeled cRNA probes were generated by reverse transcription followed by in vitro transcription incorporating biotin labeling as part of the standard Affymetrix protocol. The probes were then hybridized in duplicate to the Affymetrix U133A and B human gene chips and the chips scanned by laser after hybridization and staining. The final data set consisted of a total of 16 scan files, each obtained using the Affymetrix GENECHIP software, which for each qualifier in the file assigns an intensity that is a measure of the corresponding transcript abundance.. The final step of the data assembly consisted of obtaining for each qualifier the expression ratios of B02/ MDA-MB-231 , which were obtained taking into account both intensity and noise.

• Quantitative RT-PCR

Total RNA samples were obtained from duplicates of non confluent and confluent MDA-MB-231 and MDA-MB-231 B02 cell lines using Trizol™ extraction protocol (Invitrogen). Two micrograms of total RNAs were then reverse transcribed using the "Advantage RT for PCR" Kit (BD Biosciences #PT1107-1 ). Quantitative real time PCR was performed with TaqMan PCR reagent kits by using the ABI PRISM 7700 sequence detection system (Applied Biosystems) as recommended by the supplier. The following primer/probe set was designed using Primer-Express Version 1.0 software from Applied Biosystems: forward, 5'-CGC TGT GTT TGT GAG AGA GGA T-3¹ (SEQ ID N°1 ); reverse , 5'-TTG TTC TTC CGT CAT GTT ACA CTT C-3¹ (SEQ ID N°2); probe, 5'-TTT GGA AAG CTC TGC CAA CAT CCG C-3¹ (SEQ ID N°3).

• DNA constructs, transfection and western blot analysis

Mouse ITGBL1 (N M_145467) full length cDNA was obtained from ATCC collection (cDNA clone, MGC-28057). ITGBL1 ORF was amplified using primers allowing directional cloning into the gateway acceptor mammalian expression plasmid pEF-Dest 51 (Invitrogen). The resulting pEF-Dest51-

ITGBL1 plasmid was transfected into Cos7 cells using FuGene 6 transfection reagent (Roche diagnostics). Two days later, cells were lysed and protein extracts were subjected to SDS-PAGE migration. V5-tagged

ITGBL1 was revealed after blotting using HRP conjugated anti-V5 antibodies (Invitrogen).

• Calvaria osteoblasts differentiation Murine calvaria cells were obtained from the calvariae of neonatal mice one day after birth by sequential collagenase digestion at 37⁰C. Calvariae were removed from the animals under aseptic conditions and incubated at 37⁰C in DMEM containing trypsin (0.5 mg/ml) and EDTA (1.5 mg/ml) under continuous agitation. The cells released between 20-40 minutes were collected and cultured in proliferation medium (DMEM supplemented with 20% FCS and 2 mM glutamine) at a density of 2.5 x10⁴ cells per cm² in Petri dishes (100 mm diameter). Calvaria cells were cultured until 80% confluence (time 0) and proliferation medium was replaced by differentiation medium (ocMEM containing 10% FCS, 2 mM glutamine, 50 μg/ml ascorbic acid and 10 mM b-glycerolphosphate). Total RNAs were extracted at days 0, 2, 7, 14, and 21. II- Results

• Set of genes up- or down-regulated in the B02 cell line compared to the MDA-MB-231 cell line, not reported in the profiles described in (4) The whole genome expression profiles of the B02 cell line and its parental MDA-MB-231 line were compared using the Affymetrix U133 chip set which represents more than 39,000 transcripts. A list of up-regulated and down-regulated genes was produced and compared to relevant literature data. A subset of overlapping genes required for bone homing were found when comparing this data set to a previous work (4), which helps to validate these results. In addition, these data were also indirectly validated by showing that a number of genes regulated in this model are common to the recently described "metastatic signature" found in human breast metastasis (5). A further subset of genes regulated in this model is also common to genes recently implicated in the metastatic homing to human bone (6). This latter study only used a small number of human samples and DNA arrays with relatively few genes on them, making the gene profiling comparison limited. Interestingly, no genes in the angiogenesis and chemokine families were found to be regulated in this model. However, a large number of genes were unique to this model compared to the profile described in (4).

• ITG BL1 is the only member of the integrin family regulated in the B02 cell line compared to the MDA-MB-231 cell line Of interest, the gene ITGBL1 was found to be up-regulated three times in the B02 cell line compared to the MDA cell line (confirmed by quantitative RT-PCR). None of the other integrin family members were regulated in the system. ITGBL1 is a protein recently identified via a family member cloning approach (7). This integrin-like protein encompasses part of the integrin structure around the extracellular domain of the receptor and neither encodes a trans-membrane domain nor a RGD docking site. There is only one literature entry for ITGBL1 (7). From a structural point of view, ITGBL1 belongs to the beta-1 integrin family and has homologies to beta- PAT from C. elegans indicating that it is a conserved integrin protein (8). lntegrins are a family of hetero-dimers composed of alpha and beta sub- units of about 24 members (9). They are involved in cell adhesion and transduction of a number of signals regulating apoptosis, proliferation and differentiation. Clinical trials are aimed at inhibiting alpha v beta 3 in particular to block the neo-angiogenic process induced by a number of tumor during growth (10).

• Sub-cloning of the mouse ITGBL1 in a mammalian expression vector ITGBL1 was proposed to be a target relevant to the homing of breast cancer cells to bone. The following approaches have been performed to start demonstrating a functional role for this protein in bone metastasis. Firstly, since there is no reagent available to test this protein, the commercially available mouse ITGBL1 gene has been cloned and tagged in a mammalian expression vector (Invitrogen). The human gene is being cloned at present. The mouse ITGBL1 protein has already been demonstrated to be expressed at the correct molecular weight after transient tranfections.

• ITG BL1 is up-regulated during development: osteoblasts from calvaria Retrospectively, a search for ITGBL1 was performed in archive data comprising genes modulated during bone cell differentiation. ITGBL1 was consistently found to be up-regulated during calvaria maturation and during mesenchymal cell differentiation induced by Wnt. Table 1

QUAL Ugtile Ugid x xJ_BBQQ22_jcGonf x_BQ2_su&corif 1834 1834 ATCC-1 ATCC-I ATGC-2 218995_S_AT endothelin 1 Hs.437313 3,11 4,32 153,7 A 197,9 A 152,7 ectonucleotide pyrophosphatase/phosphodi

205066_S_AT esterase 1 Hs.213840 5,52 1 ,50 644,6 P 626,6 P 858,2 205110_S_AT fibroblast growth factor 13 Hs.6540 17,85 12,39 18,8 A 19,7 A 24,2 gap junction protein, alpha

201667_AT 1 , 43kDa (connexin 43) Hs.74471 4,56 4,53 95,8 P 95 P 100,2 glia maturation factor, 204220_AT gamma Hs.5210 11 ,02 8,25 9,2 A 12,1 A 13,9 integrin, beta-like 1 (with

205422_S_AT EGF-like repeat domains) Hs.311054 2,36 3,38 34,2 A 9,3 A 18,5 206953_S_AT latrophilin 2 Hs.24212 4,82 3,85 58,5 A 103,9 A 132,8 melanoma antigen, family A, 37,1

209942_X_AT 3 Hs.417816 43,55 92,72 40,8 A 36 A

Melanoma antigen, family 214612_X_AT A, 6 NONE 44,41 81 ,20 2,6 A 13,4 A 19 nuclear receptor subfamily

209505_AT 2, group F, member 1 Hs.361748 7,84 9,94 21,4 A 51 ,9 A 74,3 nucleosomal binding protein 221606 S AT 1 Hs.282204 4,91 5,28 26,4 A 35,1 A 25,3 progestagen-associated endometrial protein

(placental protein 14, pregnancy-associated endometrial alphas- globulin, alpha uterine

206859 S AT protein) Hs.82269 4,78 2,19 30,3 A 9,5 7,4 placental growth factor, vascular endothelial growth

209652 S AT

factor-related protein Hs.252820 3,50 4,77 123,3 P 104,5 83,5

Table 1 (continued) polyhomθotic-likθ 1

218338_AT (Drosophila) Hs.305985 3,35 5,22 131,5 A 117 44,2

Phospholipase C, beta 1 5,51

(phosphoinositide- 213222_AT specific) NONE 7,68 68,6 A 2,3 37,2 roundabout, axon guidance 3,05 receptor, homolog 1

213194_AT (Drosophila) Hs.301198 3,94 71 P 175,7 P 103,5

211024_S_AT

hyroid transcription factor 1 Hs.94367 8,31 6,00 11,2 A 15,1 A 3,4

Table 1 (continued)

ATCO2 2279 2279 2260 28001 2287 1833 MDA_PC

A 161 ,7 A 173,4 A 568,3 M 778,7 P A

P 571 ,9 P 496,8 P 1093,7 P 1128,6 P P

A 71,3 P 30,9 A 90,4 A 39,1 P A

P 304,8 P 259,6 P 251 ,1 P 117,7 P P

A 131 P 18,7 A 62,2 A 58,6 A A

A 29,5 A 5,4 A 17,6 A 49,8 A A

P 56,3 A 28,8 A 173,5 P 188,2 P P

A 12,8 A 121 ,4 A 27,7 A 7,7 A A

A 9,8 A 202,4 A 8,5 A 16,5 A A

A 105,1 A 108,6 A 127,9 A 293,4 P A

A 103,3 A 42,5 A 108,6 A 42,5 A A

A 11,3 A 9,8 A 15,8 A 16,4 A A KJ U)

M 450,3 P 383,9 P 395,5 P 163,2 P A

A 238,4 A 117,3 A 338,9 A 210,1 A A

A 130,6 P 19,3 A 73 A 41,8 A A

P 479,6 P 105,6 P 640,9 P 410,7 P A

A 23,1 A 6,3 A 22,9 A 11,2 A A

P 1431 ,4 P 635 P 597,1 P 1189,1 P

80

P 413 P 701 ,1 P 238 P 502,7 P

P 281 ,9 P 374,1 P 30,3 A 206,6 P

P 1271 ,6 P 766,2 P 1292,3 P 1029,8 P

P 98,7 A 92 A 15,3 A 61,7 A

P 2291 ,3 P 2828,9 P 584,7 P 698,8 P

A 705,4 A 810,5 P 168,2 A 163,5 A

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Claims

1. A method for diagnosing a bone disorder in a host comprising determining transcription of a gene in a sample of a host, said gene having a coding portion which includes DNA having at least 90% identity to the DNA of a bone metastasis marker as set forth in Table 1 , whereby said transcription indicates a bone disorder in the host.

2. The method of claim 1 , wherein transcription is determined by detecting the presence of an altered level of RNA transcribed from said gene.

3. The method of claim 1 , wherein transcription is determined by detecting the presence of an altered level of DNA complementary to the RNA transcribed from said gene.

4. The method of claim 1 , wherein transcription is determined by detecting the presence of an altered level of an expression product of said gene.

5. The method according to any of claims 1 to 4, wherein said bone metastasis marker is ITGBL1.

6. The method according to any of claims 1 to 5, wherein said bone disorder is a bone metastasis or a bone defect.

7. Use of at least one bone metastasis marker as set forth in Table 1 , or a fragment thereof, for diagnosing a bone disorder.

8. The use according to claim 7, wherein said bone metastasis marker is ITGBL1.

9. The use according to claim 7 or 8, wherein said bone disorder is a bone metastasis or a bone defect.

10. A diagnostic composition comprising at least one compound selected in the group of : bone metastasis marker genes and bone metastasis marker polypeptides as set forth in Table 1 , and modulating compounds thereof.

11. A modulating compound of a bone metastasis marker selected in Table 1.

12. The modulating compound of claim 11 , wherein said bone metastasis marker is ITGBL1.

13. A pharmaceutical composition comprising at least one modulating compound according to claims 11 or 12 and, optionally, a pharmaceutically acceptable carrier.

14. Use of at least one modulating compound according to claim 11 or 12 for the manufacture of a pharmaceutical composition for preventing and/or treating bone disorders.

15. A method for the treatment of a patient having need to modulate a bone metastasis marker comprising: administering to the patient a therapeutically effective amount of: at least one modulating compound of claim 11 or 12, or a pharmaceutical composition of claim 13.

16. A method for selecting a modulating compound useful for preventing and/or treating bone disorders, comprising: a) testing the ability of a candidate compound to modulate the biological activity of a bone metastasis marker in vitro and/or in vivo; and b) if said candidate compound modulates said biological activity, selecting said compound.

17. The method of claim 16, further comprising purifying said compound.