# Patent

## Title
Hedgehog pathway modulation and uses thereof for treating, preventing and/or diagnosing cancer

## Abstract

Methods, uses, agents and compositions useful for the diagnosis, prevention and/or treatment of invasive diseases such as cancer based on the modulation of the expression and/or activity of brother of CDON (BOC) are disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit, under 35 U.S.C. 119(e), of U.S. Provisional Patent Application Ser. No. 61/095,155 filed on Sep. 8, 2008, which is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The present invention generally relates to cancer, and more particularly to methods for the prevention, treatment, diagnostic and prognostic of invasive diseases, such as medulloblastoma.
SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form entitled [12810—267-seq listing_ST25.txt], created Sep. 3, 2009 having a size of 65 Ko. The computer readable form is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Neoplastic Diseases
The transformation of a normal cell into a malignant cell results, among other things, in the uncontrolled proliferation of the progeny cells, which exhibit immature, undifferentiated morphology, exaggerated survival and proangiogenic properties. It is also often associated with overexpression or constitutive activation of oncogenes not normally expressed by normal mature cells. Once a tumor has formed, cancer cells can leave the original tumor site and migrate to other parts of the body via the bloodstream and/or the lymphatic system by a process called metastasis. In this way, the disease may spread from one organ or part to another non-contiguous organ or part.
The increased number of cancer cases reported around the world is a major concern. Currently there are only a handful of treatments available for specific types of cancer and these treatments provide only limited efficacy. In order to be most effective, these treatments require: early detection of the malignancy, reliable assessment of its severity, methods of tracking possible metastasis, and readouts to monitor patients' response to treatment.
The Hedgehog Pathway
The seven transmembrane domain containing protein smoothened (SMO) serves as the key player for signal transduction of the Hedgehog (Hh) pathway. There are 3 Hh-related genes in vertebrates: sonic hedgehog (Shh), Indian Hedgehog and Desert Hedgehog. Hh is used here to encompass all three genes. However, the pathway's function is inhibited by a Hh-binding twelve transmembrane domain protein, patched (PTC), in the absence of Hh ligands. There are two homologs of patched in vertebrates (e.g., Ptch1 and Ptch2 in mouse, PTCH1 and PTCH2 in human), and one gene in Drosophila. As used herein, “Ptc” is used to designate the human gene or the mouse gene. The context specifies which species was used. It has recently been shown that the type I transmembrane proteins CDON (cell-adhesion-molecule-related/downregulated by oncogenes) and BOC (biregional CDON-binding protein; also known as “brother of CDON”) are also able to bind Hh (Okada, A et al., Nature 444 (7117): 369-373). In the presence of active Hh ligands, it is thought that binding of Hh to PTC and BOC/CDON complexes releases their inhibition of SMO, allowing SMO to signal downstream to Gli/Ci transcription factors. As transcription factors, Gli molecules can regulate the expression of Hh target genes by directly associating with a specific consensus sequence located in the promoter region of the target genes such as GLI1 itself, PTCH1 and HIP (McMahon, A P et al., Curr Top Dev Biol 2003, 53: 1-114). FIG. 1 shows a simplified diagram of Hh signaling in the presence of Hh. Hh proteins (sonic hedgehog (Shh), Indian hedgehog (Ihh) (not shown) and desert hedgehog (Dhh) (not shown)) are secreted molecules, functioning both on nearby and distant cells in developing tissues. Following translation, Hh proteins enter the secretory pathway and undergo autoprocessing and other post-translational modifications. Binding of Hh to PTC1 alone, in the absence of interaction with BOC/CDON complexes, results in lower levels of hedgehog pathway activation. Interaction of Hh with both PTC and BOC/CDON complexes results in increased hedgehog pathway activation. Other major components includes Su(Fu), REN, Costal2, Fused and recently Gas1. The C-terminal tails of BOC/CDON might convey other signals to cytoskeletal or integral membrane proteins that induce morphological changes in the cell and signal to the nucleus through unknown mechanisms.
CDON and BOC are type I transmembrane receptors consisting of four or five immunoglobulin (Ig) and two or three fibronectin type III (FNIII) repeats in the extracellular domain, and an intracellular domain with no identifiable motifs. This domain architecture is closely related to that of axon guidance receptors of the Robo and DCC (deleted in colorectal cancer) families (FIG. 2). Both CDON and BOC share a high degree of homology in their extracellular domains and are expressed during early stages of development of the central nervous system (Okada et al., 2006. Nature, 444: 369-373). CDON and BOC form complexes with each other in a cis fashion.
Cerebellum Development and Medulloblastoma
During cerebellum development, Granule Cell Precursors (GCP) undergo rapid proliferation in the External Germinal Layer (EGL) of the cerebellum. Expansion of the GCP is dependent of Sonic hedgehog secreted by Purkinje cells. After their final division, GCP differentiate into Granule cells (GC) and migrate through the Purkinje cell layer to form mature granule cells that reside in the internal granule cell layer (IGL).
Medulloblastoma is an embryonal neuroepithelial tumor of the cerebellum and the most common malignant brain tumor occurring in children (Reviewed in Rossi et al. 2008. Clin. Cancer. Res, 14: 971-976). The tumors develop in the cerebellum, in a part of the brain called the posterior fossa, but may spread to other parts of the brain including the spinal cord. Medulloblastomas may also spread to other parts of the body often through the cerebrospinal fluid (CSF), which surrounds and protects the brain and spinal cord.
It is common knowledge that a clinical diagnosis of medulloblastoma is based on a combination of symptoms and signs observed in the evolution of the disease in the patient. An earlier diagnosis, particularly during neurosurgical clinical examination, would greatly increase the chances of successful treatment and recovery. Currently, proper diagnosis of medulloblastoma involves an invasive procedure whereby a biopsy is performed and sample cells are removed from the tumour and subsequently analyzed. In addition, CSF can be examined for the presence of tumor cells to determine if the tumor has metastasized, which is usually a very poor prognostic (Reviewed in Rossi et al. 2008, supra).
Despite improved multimodal treatment regimens, approximately one-third of patients with medulloblastoma remain incurable and current treatments significantly damage long-term survivors. Although fewer than 500 children per year in the US are diagnosed with medulloblastoma, the outcome is almost invariably poor. Surgery with subsequent radiation or chemotherapy has increased survival to greater than 50%, but the current methods of treatment result in severe long-term side effects including mental retardation.
Medulloblastoma can be a very aggressive tumor. The treatment of Medulloblastoma consists of surgery to remove as much tumor mass as possible, followed by radiotherapy, sometimes also combined with chemotherapy. In spite of these very aggressive treatments, only about 50 to 70% of patients survive after 5 years. For the survivors, the intense radiotherapy often leads to severe side effects, such as permanent cognitive impairment. However, not all MB are equally aggressive and information which informs prognosis, such as risk-assessment by the presence or absence of a molecular marker, would be useful to help determine which type of treatment (less or more aggressive) is most appropriate for each patient.
After surgery and radiotherapy, Medulloblastoma patients are closely followed by MRI for potential relapse of the tumor. This is particularly important in patients in which total resection of the tumor was impossible, which constitute 30-40% of the total number of patients undergoing surgery. Here, having a rapid, simple and robust method to detect tumor relapse at early stages would be very useful.
In some cases, tumor cells might disseminate in the CSF and metastasize to the spinal cord and other parts of the brain. This is one of the most feared complications of MB. The sensitive detection of MB disseminating cells in the CSF or the detection of metastases at early stage would certainly improve treatment outcome.
To improve the outcome of those medulloblastoma patients with high-risk disease as well as the quality of life of all survivors following treatment, both novel therapies and improved tumor classification are required. Novel therapies will result from a greater understanding of the disease process and are likely to involve small molecules designed to target specific pathways, including hedgehog, that become dysregulated during oncogenesis. An improved tumor classification will incorporate an assessment of the molecular profiles of medulloblastomas with defined biological behaviors or of the status of cellular pathways that are potential targets for novel therapies.
Thus, there is a need for the identification of novel targets that could serve as biomarkers of early stages of cancer. There is a need for the identification of novel biomarkers for better characterization and classification of tumors. Also, there is a need for improved diagnostic and prognostic detection methods as well as novel anti-cancer treatments which can be administered to subjects either suffering from or who are at a high risk of developing cancer to prevent, inhibit or treat the disease and the spread thereof.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present inventors show herein that BOC deficiency is associated with lower proliferation of cerebellum cells, and that overexpression of BOC in these cells results in increased proliferation. They demonstrate that BOC is expressed/overexpressed in various tumors, including medulloblastoma tumors, and that mice having an homozygous deletion of the BOC gene (BOC−/− mice) show increased survival as compared to their heterozygous counterparts (BOC+/− mice) in a medulloblastoma mouse model. They also show that BOC expression can be correlated with poor or good outcome. Therefore, BOC may constitute a tumor marker for diagnosis applications, and agents that inhibit or decrease the expression and/or activity of BOC (BOC inhibitors) may be useful for inhibiting the growth of tumor cells and for the treatment of cancers such as medulloblastoma.
Accordingly, in an aspect, the present invention provides a method for preventing and/or treating cancer comprising administering to a subject in need thereof an effective amount of an inhibitor of biregional cell adhesion molecule-related/down-regulated by oncogenes (CDON) binding protein (BOC).
In another aspect, the present invention provides a use of a BOC inhibitor for preventing and/or treating cancer in a subject.
In another aspect, the present invention provides a use of a BOC inhibitor for the preparation of a medicament for preventing and/or treating cancer in a subject.
In another aspect, the present invention provides a BOC inhibitor for preventing and/or treating cancer.
In another aspect, the present invention provides a composition for preventing and/or treating cancer comprising a BOC inhibitor, and a pharmaceutically acceptable carrier.
In another aspect, the present invention provides a method for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: contacting said test compound with a BOC polypeptide, or a fragment thereof or variant thereof having BOC activity; and determining whether the activity of the BOC polypeptide, fragment or variant thereof, is decreased in the presence of said test compound, wherein a decrease in the activity of said BOC polypeptide, or a fragment thereof, in the presence of said test compound is indicative that said test compound may be used for the prevention and/or treatment of cancer.
In another aspect, the present invention provides a method for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: contacting said test compound with a cell expressing BOC; and determining whether the expression and/or activity of BOC is decreased in the presence of said test compound, wherein said decrease in the expression and/or activity of BOC in the presence of said test compound is indicative that said test compound may be used for the prevention and/or treatment of cancer.
In another aspect, the present invention provides a method for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: contacting said test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a BOC gene, operably linked to a second nucleic acid comprising a reporter gene encoding a reporter protein; and determining whether the reporter gene expression and/or reporter protein activity is decreased in the presence of said test compound; wherein said decrease in reporter gene expression and/or reporter protein activity is indicative that said test compound may be used for prevention and/or treatment of cancer.
In another aspect, the present invention provides a method for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: contacting said test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a gene whose expression is increased by BOC activity, operably linked to a second nucleic acid comprising a reporter gene encoding a reporter protein; and determining whether the reporter gene expression and/or reporter protein activity is decreased in the presence of said test compound; wherein said decrease in reporter gene expression and/or reporter protein activity is indicative that said test compound may be used for prevention and/or treatment of cancer.
In an embodiment, the above-mentioned gene whose expression is increased by BOC activity is glioma-associated oncogene homolog 1 (GLI-1).
In another aspect, the present invention provides a method for diagnosing a cancer or a predisposition to cancer in a first subject comprising: determining the expression and/or activity of BOC in a sample from said first subject; comparing said expression and/or activity to a corresponding reference expression and/or activity; and diagnosing said cancer or predisposition to cancer based on said comparison.
In an embodiment, the above-mentioned reference expression and/or activity corresponds to an expression and/or activity determined in a sample from a control subject known to not being predisposed to cancer and to not have cancer, and wherein a higher expression and/or activity in said sample from said first subject is indicative that said first subject has cancer or a predisposition to cancer.
In another embodiment, the above-mentioned reference expression and/or activity corresponds to an expression and/or activity determined in a sample from a control subject known to have a predisposition to cancer, and wherein a comparable or higher expression and/or activity in said sample from said first subject is indicative that said first subject has cancer or a predisposition to cancer.
In another aspect, the present invention provides a method for inhibiting the proliferation and/or differentiation of a cell having an active hedgehog pathway comprising contacting said cell with a BOC inhibitor.
In another aspect, the present invention provides a use of a BOC inhibitor for inhibiting the proliferation and/or differentiation of a cell having an active hedgehog pathway.
In another aspect, the present invention provides a use of a BOC inhibitor for the preparation of a medicament for inhibiting the proliferation and/or differentiation of a cell having an active hedgehog pathway.
In another aspect, the present invention provides a method for determining whether a tumor is amenable for treatment with a BOC inhibitor, said method comprising determining the expression of BOC in a sample from said tumor, wherein the expression of BOC in said sample is indicative that said tumor is amenable for treatment with a BOC inhibitor.
In an embodiment, the above-mentioned cell is a tumor cell.
In an embodiment, the above-mentioned cancer or tumor is associated with BOC expression and/or activity, in a further embodiment with an increase in BOC expression and/or activity (e.g., BOC overexpression).
In another embodiment, the above-mentioned cancer is a brain tumor, an ovary tumor, a breast tumor, a glioblastoma, a skin tumor, a meningioma, an astrocytoma, a liver tumor, a prostate carcinoma, a bladder tumor, a lung tumor, a lymph node lymphoma, a vascular endothelium hemangioma, a kidney carcinoma or a thyroid follicular adenoma.
In an embodiment, the above-mentioned subject is a human.
In an embodiment, the above-mentioned inhibitor inhibits the expression of BOC. In a further embodiment, the above-mentioned inhibitor is an antisense oligonucleotide (ASO), a RNA interference (RNAi), a short hairpin loop interfering RNA (ShRNA) or an antisense RNA Locked Nucleic Acid analogues (LNA).
In another embodiment, the above-mentioned inhibitor inhibits the activity of BOC. In a further embodiment, the above-mentioned inhibitor specifically binds to BOC. In yet a further embodiment, the above-mentioned inhibitor is an antibody, an antigen-binding fragment of an antibody, a peptide fragment of BOC, or a protein or peptide fragment binding to BOC.
In an embodiment, the above-mentioned sample is a CNS cell, tissue or fluid. In a further embodiment, the above-mentioned CNS fluid is cerebrospinal fluid.
In an embodiment, the above-mentioned expression of BOC is determined at the nucleic acid level. In a further embodiment, the above-mentioned determining the expression of BOC comprises determining the expression of a nucleic acid encoding the polypeptide comprising the sequence of SEQ ID NO: 2. In another embodiment, the above-mentioned determining the expression of BOC comprises determining the expression of a nucleic acid comprising the coding sequence of the nucleotide sequence of SEQ ID NO: 1.
In another embodiment, the above-mentioned expression of BOC is determined at the polypeptide level. In a further embodiment, the above-mentioned determining the expression of BOC comprises determining the expression of a polypeptide comprising the sequence of SEQ ID NO: 2.
In another aspect, the present invention provides a transgenic non human mammal, the nucleated cells of which comprise a transgene including a coding region encoding BOC operatively associated with a transcriptional regulatory element, wherein the non human mammal exhibits, relative to a wild-type non human animal, an elevated BOC expression level in cerebellum cells and/or cerebellum cell precursors.
In another aspect, the present invention provides a method for prognosis of a cancer patient, said method comprising: (a) detecting an expression and/or activity of BOC in a sample from said cancer patient, and (b) prognosing said cancer patient based on said detection; wherein the detection (i.e., presence) of BOC expression and/or activity in said sample is indicative that said subject has a poor prognosis, and wherein the lack of detection (i.e., absence) of BOC expression and/or activity in said sample is indicative that said subject has a good prognosis. In an embodiment, the above-mentioned sample is a tumor sample.
In another embodiment, the above-mentioned cancer is a cancer of the central nervous system (CNS). In a further embodiment, the above-mentioned cancer is a neuroectodermal tumor. In a further embodiment, the above-mentioned neuroectodermal tumor is a medulloblastoma.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
FIG. 1 shows a schematic representation of the Sonic hedgehog signalling pathway with the ligand SHH, the BOC/CDON receptors, the 12-transmembrane PTCH and the 7-transmembrane SMO proteins as well as one possible intracellular target, the transcription factor Gli;
FIG. 2 is a schematic representation of some of the domains of BOC and of other related receptors (CDON, iHog, Robo1, and DCC). Oval=an Ig domain; square=a FNIII domain; TM domain=a transmembrane domain;
FIG. 3 shows the expression of BOC in a developing cerebellum as measured by immunohistochemistry using an anti-BOC antibody on sagittal cerebellum sections from post-natal day 6 mouse. Anti-Lim-1 is used to detect Lim1, a marker of Granule Cell Precursors (GCP) and Purkinje cells (PJC). In External germinal layer (EGL): proliferating Granule Cell Precursors co-express BOC and Lim1 (right panels);
FIG. 4 shows a cerebellum analysis of BOC−/− mice compared to control BOC+/− mice. Photographs compare BOC−/-31  mice and control BOC−/-31  whole cerebellums (FIG. 4A) and cerebellum cross sections (FIG. 4B). Measurements of cerebellum weight (FIG. 4C) and cerebellum surfaces on brain sections (FIG. 4D) revealed that BOC−/− animals have a smaller cerebellum than control mice;
FIG. 5 shows apoptosis and proliferation measurements in developing cerebellum of BOC−/− and BOC+/− control mice at post-natal day 3. Cell proliferation was evaluated by BrdU incorporation (FIGS. 5A and 5B) and by immunostaining using the proliferation marker phosphohistone H3 (PhH3) (FIG. 5C) and apoptosis, by TUNEL assay (FIGS. 5D and 5E). No difference in the total number of apoptotic nuclei on matching cerebellum sections were observed between BOC−/− and BOC+/− animals (FIG. 5E). However, BOC−/− mice show a significant decrease in number of BrdU+ (FIG. 5B) and PhH3+ cells (FIG. 5C) in external granular layer, indicating a diminished proliferation compared to control BOC+/− mice. EGL: external germinal layer;
FIG. 6 shows a graph illustrating the cell autonomous decrease of GCP proliferation in the absence of BOC. The GCP derived from cerebellum of BOC−/− and BOC+/− mice were cultured in vitro in the presence of increasing concentration of Shh as indicated. The percentage of Ki-67 positive cells from 2 experiments was then measured and compared. While Boc null GCPs still respond to Shh stimulation, their proliferative response remains lower than wild type GCPs. Note that at high Shh doses, no difference is observed between BOC+/− and BOC−/− GCPs;
FIG. 7 shows a graph illustrating the Shh-dependent effect of overexpression of BOC on cell proliferation. GCP purified from post-natal day 4 rat cerebellum were co-electroporated with either a combination of BOC with a red fluorescence protein expressing plasmids (BOC) or with a control empty plasmid with a red fluorescence protein expressing plasmid (control). Proliferation was evaluated by measuring the percentage of Ki-67 positive cells among electroporated cells in absence or in presence of sonic hedgehog (Shh) and also in presence of Shh in combination with an inhibitor of Smo (SANT-1);
FIG. 8 presents the murine Ptc1+/− medulloblastoma model. FIG. 8A shows a mouse having a medulloblastoma tumor. Cerebellum sections showing the presence of a medulloblastoma tumor (FIG. 8B, right panel) compared to normal cerebellum (FIG. 8B, middle panel). FIG. 8C: Survival curves, Ptc1+/−: n=41, Ptc1+/+: n=21;
FIG. 9 shows the expression of BOC in a medulloblastoma tumor obtained from a BOC+/−; Ptc1+/− mouse. FIG. 9A shows BOC immunostaining on a medulloblastoma tumor using anti-BOC antibodies and showing that BOC is highly expressed in tumor tissue as compared to adjacent normal cerebellum (Cb). FIG. 9B shows BOC-promoter driven alkaline phosphatase (PLAP) reporter gene expression on an adjacent section of the same tumor. Note that BOC mutant mice were generated by targeting BOC with a genetrap cassette encoding β-galactosidase-neomycin fusion (β-gal) and human placental alkaline phosphatase (PLAP) reporter genes with the use of the ‘targeted trapping’ method (Friedel et al., 2005. Proc Natl Acad Sci USA 102: 13188-13193). FIG. 9C shows BOC mRNA levels in cortex (left bar), normal cerebellum (middle bar) and medulloblastoma tumor (right bar) as measured by quantitative PCR;
FIG. 10 shows BOC expression in the Ptc+/− medulloblastoma mice model. BOC is highly expressed in pre-malignant and malignant mouse medulloblastoma tumors. Anti-BOC immunohistochemistry on sections presenting different medulloblastoma malignancy stages, namely pre-malignant (upper panel), 60 days-old malignant (middle panel) and 220 days-old malignant (lower panel). All Ptc+/− mice develop premalignant lesions, but only 50% of the mice will develop malignant tumors (Oliver et al., Development 2005, 132: 2425);
FIG. 11 shows BOC protein level in normal adult mice cerebellum and in medulloblastoma tumors. BOC was detected by western blotting on 100 μg of total protein extracts. Medulloblastoma tumors (lanes 3-7) and normal cerebellum (lanes 1, 2 and 8) were obtained from BOC+/+; Ptc1+/−, BOC+/+; Ptc1+/−, BOC+/−; Ptc1+/−, BOC−/−; Ptc1+/− and BOC−/−; Ptc1+/+ mice as indicated. Anti-BOC antibody detects two major bands, corresponding to wild-type BOC and the mutant chimeric form of BOC (Boc-βgeo), respectively. Note that BOC mutant mice were generated by targeting BOC with a genetrap cassette encoding a β-galactosidase-neomycin fusion (βgeo) and human placental alkaline phosphatase (PLAP) reporter genes with the use of the ‘targeted trapping’ method (Friedel et al., 2005, supra). Ptc1-deficient mice were generated by replacing a part of exon 1 and exon 2 of the Ptc1 gene by the LacZ gene, so as to generate a chimeric Ptc protein. An anti-βgal antibody was used to confirm the specificity of the bands detected by anti-BOC;
FIG. 12 shows a graph of the survival of mice harboring different combinations of BOC and Ptc1 alleles. Survival analyses were performed on BOC+/−; Ptc1+/− (n=36, circles), BOC+/−; Ptc1+/− (n=35, squares), and BOC−/−; Ptc1+/− mice (n=21, diamonds). The absence of BOC is associated with an increase in survival;
FIG. 13 shows a tissues microarray analysis of different human medulloblastoma tumors. Slides containing thin sections of 73 human medulloblastoma tissue samples were immunostained with anti-BOC antibody. Example of BOC-positive (FIG. 13A) and BOC-negative (FIG. 13B) tumor tissues are presented. FIG. 13C shows a table summarizing the statistical analysis obtained from the tissues microarray analysis of 73 human medulloblastoma tumors;
FIG. 14 shows SAGE analysis performed on 19 medulloblastoma tumor tissues using a variety of markers including BOC (FIG. 14A). These “Digital Northern” results for Boc (SAGE tag: AGAACAGACC (SEQ ID NO: 14)) were obtained from SAGE Genie (Lash A E et al., 2000. Genome Res, 10(7): 1051-1060). By analyzing their specific gene signature, each medulloblastoma tumors were characterized into Shh, Wnt or other type of medulloblastoma. The analysis was performed for markers characterizing medulloblastoma Shh (e.g., Gli1, Igf1, CXCR4, patch1, sfrp1) and markers for medulloblastoma Wnt (e.g., tnc, epha4, Dkk1, Amhr2, Emx2). FIG. 14B shows a table summarizing the expression of BOC in Shh and Wnt medulloblastoma tumors;
FIGS. 15A and 15B show the nucleotide sequence of human BOC (SEQ ID NO: 1), with the coding sequence indicated in bold, and FIG. 15C shows the amino acid sequence of human BOC (SEQ ID NO: 2);
FIG. 16A shows the nucleotide sequence of human SHH (SEQ ID NO: 3), with the coding sequence indicated in bold, and FIG. 16B shows the amino acid sequence of human SHH (SEQ ID NO: 4), with the sequence of the mature polypeptide indicated in bold;
FIG. 17A shows the nucleotide sequence of the murine BOC nucleic acid used in the experiments described herein (SEQ ID NO: 10), and FIG. 17B shows the amino acid sequence of the murine BOC polypeptide used in the experiments described herein (SEQ ID NO: 11). The last 8 residues (GPVATMVS (SEQ ID NO: 12), underlined) in the amino acid sequence of FIG. 17B and the corresponding nucleotides encoding these residues (ggaccggtcgccaccatggtgagc (SEQ ID NO: 13), underlined) in the nucleotide sequence of FIG. 17A are not present in the native murine BOC;
FIGS. 18A and 18B shows an alignment of the amino acid sequences of human and murine BOC;
FIG. 19 shows a correlation between survival and BOC expression in human medulloblastoma samples (n=66). Patients having BOC positive medulloblastoma tumors have a worse prognosis than those having BOC negative tumors;
FIG. 20 shows direct ELISA performed with anti-BOC antibodies for detection. The anti-BOC antibodies recognize either the cytoplasmic tail of BOC (FIG. 20A) or its extracellular portion (FIG. 20B). ELISA quantifications were performed on extracts of COS7 cells transfected with a control plasmid (CTL, squares) or mouse BOC (mBOC, triangles). Saturation curves show that both antibodies (intracellular and extracellular) can specifically detect mBOC in cellular extracts;
FIG. 21 shows a direct-ELISA using anti-BOC antibodies for detection. Antibodies recognize either the cytoplasmic tail (FIGS. 21A and C) or the extracellular portion (FIGS. 21B and D) of BOC were used. ELISA quantifications were performed on cellular extracts (FIGS. 21A and B) or tissue-culture media harvested (FIGS. 21C and D) from COS7 cells transfected with a control plasmid (CTL, squares) or mouse BOC (mBOC, triangles). The linear range of detection is represented in each graph and shows that the anti-BOC antibody binding to the extracellular portion can detect mBOC both in cellular extracts and tissue-culture media, whereas the anti-BOC antibody recognizing the cytoplasmic region can only detect mBOC in cellular extracts;
FIG. 22 shows a direct-ELISA performed with anti-BOC antibodies which recognize either the extracellular portion of BOC (FIGS. 22A and B) or its cytoplasmic tail (FIG. 22C). ELISA quantifications were performed on cellular extracts (FIGS. 22A and C) or tissue-culture media (FIG. 22B) harvested from COS7 cells transfected with a control plasmid (CTL, squares), mouse BOC (mBOC, triangles), mouse Cdo (mCdo, inverted triangles) or human BOC (hBOC, diamonds). Each graph represents the linear range of detection, showing that both anti-BOC antibodies are specific for mBOC; and
FIG. 23 shows direct-ELISA performed with an anti-human BOC as detection antibody. ELISA quantifications were performed on cellular extracts from COS7 cells transfected with a control plasmid (CTL, squares), mouse BOC (mBOC, triangles) or human BOC (hBOC, inverted triangles). Saturation curve (FIG. 23A) and linear range of detection (FIG. 23B) both show that the anti-human BOC antibody is specific for hBOC.



DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Methods of Prevention/Treatment of Cancer
The invention described herein is based on the novel and unexpected observation that BOC expression is associated with increased cell proliferation, as well as with cancer development and/or progression. The present inventors have shown that overexpression of BOC in cerebellum cells results in an increase in their proliferation. The studies described herein also demonstrate that BOC−/− mice are less susceptible to tumor development as compared to their BOC-expressing counterparts, and that BOC is expressed and/or overexpressed in various human tumors, including medulloblastoma tumors. The present inventors have further shown that expression of Boc in medulloblastoma tumors is generally associated with a poor prognosis.
Accordingly, in a first aspect, the present invention provides a method for preventing and/or treating cancer comprising administering to a subject in need thereof an effective amount of a brother of CDON (BOC, also knows as biregional CDON-binding protein) inhibitor (i.e., an agent capable of inhibiting BOC expression and/or activity).
The present invention also provides a method for inhibiting the proliferation and/or differentiation of a cell (e.g., a tumor cell) having an active hedgehog pathway comprising contacting said cell with a BOC inhibitor.
In another aspect, the present invention provides a use of a BOC inhibitor for preventing and/or treating cancer in a subject.
The present invention also provides a use of a BOC inhibitor for the preparation of a medicament for preventing and/or treating cancer in a subject.
The present invention also provides a use of a BOC inhibitor for the preparation of a medicament for inhibiting the proliferation and/or differentiation of a cell (e.g., a tumor cell) having an active hedgehog pathway.
In another aspect, the present invention provides a BOC inhibitor, or a composition comprising said BOC inhibitor, and a pharmaceutically acceptable carrier, for preventing and/or treating cancer in a subject.
In an embodiment, the above-mentioned inhibitor blocks or inhibits paracrine Hedgehog signaling, for example by inhibiting the activation of Hedgehog signaling in the surrounding stroma by Hh ligand-secreting tumor cells. The activation of Hh signaling in the stroma surrounding the tumors has recently been shown to create a favorable environment for tumor growth (Theunissen J W and de Sauvage F J., Cancer Res. 2009, 69(15): 6007-10. Epub 2009 Jul. 28; Scales S J and de Sauvage F J. Trends Pharmacol Sci. 2009 30(6): 303-12. Epub 2009 May 13; Tian H et al., Proc Natl Acad Sci USA. 2009 106(11): 4254-9. Epub 2009 Feb. 25; Yauch R L et al., Nature. 2008, 455(7211): 406-10. Epub 2008 Aug. 27).
Combination of Active/Therapeutic Agents
In an embodiment, the above-mentioned prevention/treatment comprises the use/administration of more than one (i.e. a combination of) active/therapeutic agent (e.g., an agent capable of inhibiting BOC expression and/or activity). The combination of prophylactic/therapeutic agents and/or compositions of the present invention may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one prophylactic or therapeutic agent in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent may be administered to a patient before, concomitantly, before and after, or after a second active agent is administered. The agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time. In an embodiment, the one or more active agent(s) of the present invention is used/administered in combination with one or more agent(s) currently used to prevent or treat the disorder in question (e.g., an anticancer agent).
Dosage
The amount of the agent or pharmaceutical composition which is effective in the prevention and/or treatment of a particular disease, disorder or condition (e.g., cancer) will depend on the nature and severity of the disease, the chosen prophylactic/therapeutic regimen (i.e., compound, DNA construct, protein, cells), the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 1000 mg/kg of body weight/day will be administered to the subject. In an embodiment, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in a further embodiment of about 1 mg/kg to about 100 mg/kg, in a further embodiment of about 10 mg/kg to about 50 mg/kg, may be used. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial prophylactic and/or therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.
The terms “treat/treating/treatment” and “prevent/preventing/prevention” as used herein, refers to eliciting the desired biological response, i.e., a therapeutic and prophylactic effect, respectively. In accordance with the subject invention, the therapeutic effect comprises one or more of a decrease/reduction in tumor, a decrease/reduction in the severity of the cancer (e.g., a reduction or inhibition of metastasis development), a decrease/reduction in symptoms and cancer-related effects, an amelioration of symptoms and cancer-related effects, and an increased survival time of the affected host animal, following administration of the agent/composition of the invention. In accordance with the invention, a prophylactic effect may comprise a complete or partial avoidance/inhibition or a delay of cancer development/progression (e.g., a complete or partial avoidance/inhibition or a delay of metastasis development), and an increased survival time of the affected host animal, following administration of the agent that inhibits BOC expression and/or activity (or of a composition comprising the agent).
As such, a “therapeutically effective” or “prophylactically effective” amount of an agent capable of inhibiting the expression and/or activity of BOC, or a combination of such agents, may be administered to an animal, in the context of the methods of treatment and prevention, respectively, described herein.
As used herein, “inhibition” or “decrease” of BOC expression/level or activity refers to a reduction in BOC expression or activity of at least 10% as compared to reference (e.g., normal) expression or activity, in an embodiment of at least 20% lower, in a further embodiment of at least 30%, in a further embodiment of at least 40%, in a further embodiment of at least 50%, in a further embodiment of at least 60%, in a further embodiment of at least 70%, in a further embodiment of at least 80%, in a further embodiment of at least 90%, in a further embodiment of 100% (complete inhibition).
Neoplastic Disease and Cancer
By the term “neoplastic disease” or “invasive disease” is meant a disease associated with new growth of any body tissue. A neoplastic tissue according to the invention may retain some characteristics of the tissue from which it arises but has biochemical characteristics that are distinct from those of the parent tissue. The tissue formed due to neoplastic growth is a mutant version of the original tissue and appears to serve no physiologic function in the same sense as did the original tissue. It may be benign or malignant.
In one embodiment, the present invention relates to benign neoplastic disease. In another embodiment the present invention relates to malignant neoplastic disease. In specific embodiments, the malignant neoplastic disease is cancer.
Cancer is defined herein as a disease characterized by the presence of cancer cells which are characterized by two heritable properties: they and their progeny are able (1) to reproduce unrestrained in defiance of the normal restrains (i.e., they are neoplastic) and (2) invade and colonize territories normally reserved for other cells (i.e., they are malignant). Invasiveness of cancer cells usually implies an ability to break loose, enter the bloodstream or lymphatic vessels, and form secondary tumors, or metastases at the other distant sites in the body.
Cancer refers herein to a cluster of cancer or tumor cells showing over proliferation by non-coordination of the growth and proliferation of cells due to the loss of the differentiation ability of cells. The terms “cancer cell” and “tumor cell” are used interchangeably herein.
In an embodiment, the above-mentioned cancer/tumor is a brain tumor (e.g., brain ependynoma, a medulloblastoma), an ovary tumor (e.g., ovary carcinoma, endometriosis or adenocarcinoma), a breast tumor (e.g., breast fibroadenoma or carcinoma), a glioblastoma, a skin tumor (e.g., skin melanoma), a meningioma, an astrocytoma, a liver tumor (e.g., liver cholangiocarcinoma), a prostate carcinoma, a bladder tumor (e.g., bladder adenocarcinoma), a lung tumor, a lymph node lymphoma, a vascular endothelium hemangioma, a kidney carcinoma or a thyroid follicular adenoma.
In an embodiment, the above-mentioned cancer/tumor is associated with BOC expression and/or activity (e.g., BOC overexpression or increased/abnormal BOC activity). In another embodiment, the above-mentioned cancer is a cancer of the central nervous system (e.g., brain cancer). In a further embodiment, the above-mentioned cancer is a neuroectodermal tumor. In yet a further embodiment, the above-mentioned cancer is a medulloblastoma. In a further embodiment, the above-mentioned medulloblastoma is a medulloblastoma associated with a deregulation of the SHH pathway and/or of the Wnt pathway.
Regulation of BOC Expression or Activity
The regulation of BOC expression/level and/or activity could be achieved by various mechanisms, which among others could act at the level of (i) transcription (ii) translation (e.g., degradation of BOC mRNA using antisense oligonucleotides), (iii) post-translational modifications, e.g., glycosylation, sulfation, phosphorylation (e.g., kinase inhibitors), ubiquitination (iv) cellular localization, (v) shedding from the membrane, (vi) protein-protein interaction, for example by modulating expression or activity of a protein that binds to BOC or to a BOC ligand (e.g., overexpression of GAS-1, HIP-1 and/or CDON which bind to SHH and compete with BOC). These regulatory processes occur through different molecular interactions that could be modulated by a variety of compounds or modulators.
BOC Gene
As used herein the terms “BOC gene” or “BOC” refers to genomic DNA encoding sequences comprising those sequences referred to in GenBank by GeneID number 91653. The description of the various aspects and embodiments of the invention is provided with reference to exemplary BOC nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2). Such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to other BOC nucleic acids and polypeptides, such as BOC nucleic acid or polypeptide mutants/variants, splice variants of BOC nucleic acids, BOC variants from species to species or subject to subject. UNIPROT accession No. Q9BWV1 describes three BOC isoforms produced by alternative splicing. The isoform 1 (identifier: Q9BWV1-1, 1114 amino acids, SEQ ID NO: 2) has been designated the “canonical” sequence. All positional information refers to it. The sequence of isoform 2 (identifier: Q9BWV1-2) differs from the canonical sequence by the deletion of the amino acids at positions 1-299. The sequence of isoform 3 (identifier: □9BWV1-3) differs from the canonical sequence as follows: position 514: K→KQ (i.e. insertion of a glutamine residue). Natural variants in the coding sequence include sequences with an alanine (A) to valine (V) substitution at position 43 (rs34208374); a glycine (G) to arginine (R) substitution at position 309 (rs3814400); a threonine (T) to isoleucine (I) substitution at position 675 (rs61735687); a valine (V) to methionine (M) substitution at position 713; a lysine (K) to asparagine (N) substitution at position 883 (rs35536878); a proline (P) to serine (S) substitution at position 912 (rs3814404); and a glutamine (Q) to histidine (H) substitution at position 915 (rs3814405), as well as frameshift mutations at position 340 (rs35253657) and at position 1087 (rs35293574). The presence of these variants, if associated with an increase or decrease BOC expression and/or activity, may be used for diagnostic and/or prognostic applications, as described below. Natural variants in the non coding regions or coding sequence exist that may affect the function/activity and expression of BOC. Without being so limited, Table I below lists of known BOC variants in the coding and non coding regions of BOC.







TABLE I







Natural BOC variants




















Ambi-
Amino
AA co-





ID
Type
Chr: bP
Alleles
guity
Acid
ordinate
Class
Source
Validation



















rs9822391
5prime_utr
3: 112935113
G/C
S
—
—
snp
dbSNP, Affy
cluster,










GenomeWideSNP_6.0
freq,











hapmap


rs4682130
intronic
3: 112930138
C/G
S
—
—
snp
ENSEMBL:Watson,
cluster,










HGVbase, dbSNP,
freq,










ENSEMBL:Venter
doublehit,











hapmap


rs34208374
non_synon-
3: 112969432
C/T
Y
A/V
43 (2)
snp
dbSNP
—



ymous_coding










rs34174640
intronic
3: 112969704-
−/C

—
—
insertion
dbSNP
—




112969703









rs712528
intronic
3: 112989603
A/T
W
—
—
snp
HGVbase, dbSNP, TSC
cluster,











freq,











submitter,











hapmap


rs9819129
synonymous_coding
3: 112989763
C/T
Y
S
213 (3)
snp
dbSNP
cluster,











freq,











hapmap


rs3930154
intronic
3: 112991196
C/G
S
—
—
snp
ENSEMBL:Watson,
cluster,










HGVbase,
freq,










ENSEMBL:celera,
hapmap










dbSNP, TSC,











ENSEMBL:Venter,











Illumina_Human1M-











duoV3



rs16860765
synonymous_coding
3: 112991306
C/A
M
T
239 (3)
snp
dbSNP,
cluster,










Illumina_Human1M-
freq,










duoV3
hapmap


rs3814398
synonymous_coding
3: 112991312
C/T
Y
I
241 (3)
snp
HGVbase,
cluster,










ENSEMBL:celera,
freq,










dbSNP, Affy
hapmap










GenomeWideSNP_6.0,











Illumina_Human1M-











duoV3



rs3814399
synonymous_coding
3: 112991492
C/T
Y
A
301 (3)
snp
HGVbase, dbSNP, Affy
freq,










GenorneWideSNP_6.0,
hapmap










Illumina_Human1M-











duoV3



rs3814400
non_synon-
3: 112991514
G/A
R
G/R
309 (1)
snp
HGVbase, dbSNP
—



ymous_coding










rs11717833
intronic
3: 112991842
A/G
R
—
—
snp
dbSNP,
cluster,










Illumina_Human1M-
freq,










duoV3
doublehit,











hapmap


rs11710894
synonymous_coding
3: 112991959
C/T
Y
P
335 (3)
snp
dbSNP,
cluster,










Illumina_Human1M-
freq,










duoV3
hapmap


rs35253657
frameshift_coding
3: 112991974
C/−


340 (3)
deletion
dbSNP
—


rs7612497
intronic
3: 112992225
G/A
R
—
—
snp
dbSNP
—


rs13093741
synonymous_coding
3: 112993292
C/T
Y
L
435 (3)
snp
dbSNP,
cluster,










ENSEMBL:Venter
freq,











hapmap


rs13094203
synonymous_coding
3: 112993367
G/A
R
P
460 (3)
snp
dbSNP,
cluster,










ENSEMBL:Venter
freq,











hapmap


rs7615578
synonymous_coding
3: 112993412
C/T
Y
P
475 (3)
snp
dbSNP,
cluster,










Illumina_Human1M-
freq,










duoV3
hapmap


rs7615718
intronic
3: 112993561
C/T
Y
—
—
snp
dbSNP
—


rs7627584
intronic
3: 112996898
C/T
Y
—
—
snp
dbSNP
freq,











hapmap


GA021627
essentail_splice_site,
3: 112997106
T/A
W
—
—
snp
Illumina_Human1M-
—



intronic






duoV3



rs3814401
intronic
3: 112997141
G/A
R
—
—
snp
HGVbase, dbSNP,
cluster,










Illumina_Human1M-
freq,










duoV3
hapmap


rs775228
synonymous_coding
3: 112997554
A/G
R
K
579 (3)
snp
HGVbase,
cluster,










ENSEMBL:celera,
freq,










Illumina_CytoSNP12v1,
submitter,










dbSNP, TSC,
doublehit,










ENSEMBL:Venter,
hapmap










Illumina_Human1M-











duoV3



rs13066489
intronic
3: 112997725
T/G
K
—
—
snp
dbSNP,
—










ENSEMBL:Venter



rs3856720
splice_site, intronic
3: 112998090
T/C
Y
—
—
snp
ENSEMBL:celera,
cluster,










dbSNP, TSC,
freq,










Illumina_Human1M-
doublehit,










duoV3
hapmap


rs2649878
synonymous_coding
3: 112998265
A/G
R
P
661 (3)
snp
HGVbase,
cluster,










ENSEMBL:celera,
freq,










dbSNP, TSC,
doublehit,










Illumina_Human1M-
hapmap










duoV3



rs2650100
synonymous_coding
3: 112998268
G/A
R
S
662 (3)
snp
HGVbase, dbSNP, TSC
cluster, freq


rs34039703
intronic
3: 112998343-
−/C

—
—
insertion
dbSNP
—




112998342









rs11920382
intronic
3: 112998373
C/T
Y
—
—
snp
ENSEMBL:celera,
cluster










dbSNP



rs61735687
non_synon-
3: 112998674
C/T
Y
T/I
675 (2)
snp
dbSNP
—



ymous_coding










rs16860782
synonymous_coding
3: 112998792
A/G
R
A
714 (3)
snp
Affy GeneChip 500K
cluster,










Array, dbSNP, Affy
freq,










GenomeWideSNP_6.0,
hapmap










Illumina_Human1M-











duoV3



GA021628
essential_splice_site,
3:113000001
G/C
S
—
—
snp
Illumina_Human1M-
—



intronic






duoV3



GA021629
splice_site, intronic
3: 113000003
G/C
S
—
—
snp
Illumina_Human1M-
—










duoV3



rs63500461
intronic
3: 113002186-
−/T

—
—
insertion
dbSNP
—




113002185









rs5851891
intronic
3: 113002194-
−/T

—
—
insertion
dbSNP
—




113002193









rs63715961
intronic
3: 113002195-
−/T

—
—
insertion
dbSNP
—




113002194









rs35536878
non_synon-
3: 113002475
G/C
S
K/N
883 (3)
snp
dbSNP
cluster, freq



ymous_coding










rs55671299
intronic
3: 113002500
G/T
K
—
—
snp
dbSNP
—


rs41271349
intronic
3: 113003157
C/T
Y
—
—
snp
dbSNP
—


rs59291757
synonymous_coding
3: 113003234
G/A
R
P
902 (3)
snp
dbSNP
—


rs3814404
non_synon-
3: 113003262
C/T
Y
P/S
912 (1)
snp
HGVbase, dbSNP
—



ymous_coding










rs3814405
non_synon-
3: 113003273
G/T
K
Q/H
915 (3)
snp
HGVbase, dbSNP
cluster, freq



ymous_coding










rs41271353
intronic
3: 113003466
G/A
R
—
—
snp
dbSNP
—


rs41271351
intronic
3: 113003562
G/C
S
—
—
snp
dbSNP
—


rs34440754
intronic
3: 113003578-
−/A

—
—
insertion
dbSNP
—




113003577









rs775222
intronic
3: 113003595
G/C
S
—
—
snp
HGVbase, dbSNP
cluster,











freq,











hapmap


rs13084974
intronic
3: 113004123
C/G
S
—
—
snp
dbSNP,
—










ENSEMBL:Venter



rs41271355
intronic
3: 113004155
G/A
R
—
—
snp
dbSNP
—


rs34600669
synonymous_coding
3: 113004240
C/T
Y
D
995 (3)
snp
dbSNP
—


rs16860788
intronic
3: 113004421
C/T
Y
—
—
snp
dbSNP,
freq,










illumina_Human1M-
hapmap










duoV3



rs35293574
frameshift_coding
3: 113005623
G/−


1087 (1)
deletion
dbSNP
—


rs5022662
3prime_utr
3: 113005754
A/T
W
—
—
snp
dbSNP,
—










ENSEMBL:Venter



rs1553220
3prime_utr
3: 113005756-
−/A/T

—
—
mixed
HGVbase, dbSNP
—




113005755









rs5851892
3prime_utr
3: 113005764
A/−

—
—
deletion
dbSNP
—


rs34562339
3prime_utr
3: 113005766-
−/A

—
—
insertion
dbSNP
—




113005765









rs2399476
3prime_utr
3: 113005952
G/A
R
—
—
snp
HGVbase, dbSNP, TSC
cluster,











freq,











submitter,











doublehit


ENSSNP12397219
3prime_utr
3: 113006041
G/A
R
—
—
snp
ENSEMBL:Watson
—


GA003496
3prime_utr
3: 113006054
T/C
Y
—
—
snp
Illumina_Human1M-
—










duoV3



rs9826018
3prime_utr
3: 113006135
C/T
Y
—
—
snp
dbSNP
doublehit









SHH Gene

As used herein, the terms “SHH gene” or “SHH” refers to genomic DNA encoding sequences comprising those sequences referred to in GenBank by GeneID number 6469. The description of the various aspects and embodiments of the invention is provided with reference to an exemplary SHH nucleic acid (SEQ ID NO: 3) and polypeptide (SEQ ID NO: 4). Such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to other genes that express alternate SHH nucleic acids, such as mutant SHH nucleic acids, splice variants of SHH nucleic acids, SHH variants from species to species or subject to subject.
BOC Inhibitors
As used herein, the term “BOC inhibitor” includes any compound able to directly or indirectly affect the regulation of BOC by reducing for example the expression of BOC (i.e., transcription and/or the translation), or a BOC activity. It includes intracellular as well as extracellular BOC inhibitors. Without being so limited, such inhibitors include siRNA, antisense molecules, proteins, peptides, small molecules, antibodies, etc.
As used herein the terms “BOC activity” and “BOC function” refer to detectable (direct or indirect) enzymatic, biochemical or cellular activity attributable to BOC. Without being so limited, such activities include the binding of BOC to SHH (or to a peptide fragment of SHH), the activation of any subsequent step of the Hedgehog pathway including the transcription of Hh-targeted genes (e.g., Gli-1, PTC), and cell proliferation (which may be measured, for example, by an MTT assay, BrdU incorporation, Carboxyfluorescein succinimidyl ester (CFSE) dilution, or Ki-67 immunodetection).
BOC activity may also be measured by protein-protein binding assay using purified BOC and a purified BOC ligand (e.g., SHH).
BOC activity could also be indirectly measured by evaluating the level of expression of BOC (or of a nucleic acid/polypeptide whose expression is modulated by BOC activity), or a fragment thereof, in cells as well as in a biological sample (tissue, organ, fluid). BOC expression levels could be determined at the polypeptide and/or nucleic acid levels using any standard methods known in the art (see below). BOC activity could also be indirectly measured by evaluating the level of expression or activity of a reporter gene (e.g., luciferase, β-galactosidase, alkaline phosphatase, GFP) operably linked to a transcriptionally regulatory element normally associated with a BOC gene or with a gene whose expression is modulated by BOC activity (e.g., GLI-1) (Yao et al., Cell (2006) 125: 343-357).
Antisense Molecules
In an embodiment, the agent capable of inhibiting or reducing expression of BOC is an antisense molecule.
Generally, the principle behind antisense technology is that an antisense molecule hybridizes to a target nucleic acid and effects modulation of gene expression such as transcription, splicing, translocation of the RNA to the site of protein translation, translation of protein from the RNA. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi is a form of antisense-mediated gene silencing involving the introduction of dsRNA (typically of less than 30 nucleotides in length, and generally about 19 to 24 nucleotides in length) leading to the sequence-specific reduction of targeted endogenous mRNA levels, here the RNA transcript of the BOC gene. Such dsRNA are generally substantially complementary to at least part of an RNA transcript of the BOC gene. Another example of modulation of gene expression is the RNA analogue Locked Nucleic Acid (LNA). Other examples relate to double stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (sRNA), micro-RNA (miRNA). The use of single stranded antisense oligonucleotides (ASO) is also encompassed by the method of the present invention. Sequence-specificity makes antisense compounds extremely attractive as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of any one of a variety of diseases.
Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
As used herein “antisense molecule” is meant to refer to an oligomeric molecule, particularly an antisense oligonucleotide for use in modulating the activity or function of nucleic acid molecules encoding a BOC polypeptide (e.g., the polypeptide of SEQ ID NO: 2), ultimately modulating the amount of said BOC produced in tumor cells or in producer cells located in normal distal or surrounding tissues. This is accomplished by providing oligonucleotide molecules which specifically hybridize with one or more nucleic acids encoding BOC. As used herein, the term “nucleic acid encoding a BOC polypeptide” encompasses DNA encoding said polypeptide, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA (e.g., a nucleic acid comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO: 1). The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. The overall effect of such interference with target nucleic acid function is modulation of the expression of BOC. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
In the context of this invention, “hybridization” means hydrogen bonding between complementary nucleoside or nucleotide bases. Terms “specifically hybridizable” and “complementary” are the terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. Such conditions may comprise, for example, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 to 70° C. for 12 to 16 hours, followed by washing. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. Examples of modified nucleotides include a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate and a non-natural base comprising nucleotide.
Methods to produce antisense molecules directed against a nucleic acid are well known in the art. The antisense molecules of the invention may be synthesized in vitro or in vivo.
The antisense molecule may be expressed from recombinant viral vectors, such as vectors derived from adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, and the like. Such vectors typically comprises a sequence encoding an antisense molecule of interest (e.g., a dsRNA specific for BOC) and a suitable promoter operatively linked to the antisense molecule for expressing the antisense molecule. The vector may also comprise other sequences, such as regulatory sequences, to allow, for example, expression in a specific cell/tissue/organ, or in a particular intracellular environment/compartment. Methods for generating, selecting and using viral vectors are well known in the art.
Examples of antisense molecules inhibiting the expression of BOC, as well as methods for producing same, are described in Okada et al., 2006, supra. Also, Human BOC-specific siRNA are commercially available, for example from Santa Cruz Biotechnology, Inc (Cat. No. sc-72161, sc-72161-SH and sc-72161-V) and Origene (Cat. No. TR306384). Also, several providers (e.g., InvivoGen, Qiagen, Ambion, Inc.) offer custom-made antisense synthesis services.
Peptide Compound
By the “peptide fragment of a BOC” in the present context is meant any peptide fragment derived from the amino acid sequence set forth in SEQ ID NO: 2, fragments, variants thereof, or modified forms, capable of inhibiting the activity of BOC. In an embodiment, the above-mentioned fragment is the ectodomain of BOC (extracellular domain). In a further embodiment, the above-mentioned fragment is a fibronectin type III (FNIII) domain/repeat (e.g., FNIIIc).
In another embodiment, the agent of the invention is a peptide fragment of BOC corresponding to residues 694-802 of the amino acid sequence of murine Boc (GenBank accession No. NP—766094) (Okada et al., 2006, supra).
In an embodiment, the peptide fragment of BOC inhibiting BOC activity is a natural or synthetic contiguous amino acid sequence of at least 60 amino acids from the sequence set forth in SEQ ID NO: 2, or variants thereof.
In another embodiment, the “peptide fragment” of the invention is a peptide fragment derived from an interacting partner of BOC, such as SHH and CDON or other BOC-binding proteins having an inhibitory effect on BOC activity. In another embodiment, the “peptide fragment” of the invention is a peptide fragment from an interacting partner of a BOC ligand (e.g., SHH), such as GAS-1 and HIP-1, which inhibits the activation of BOC by its ligand.
A peptide fragment inhibiting BOC activity of the present invention may be prepared by conventional synthetic methods or recombinant DNA technologies. The methods for synthetic production of peptides are well known in art. Chemically modified amino acids are used for incorporation into compounds to enhance one or more properties, such as protease resistance, pharmacokinetics or affinity for its molecular target. Detailed descriptions as well as practical advice for producing synthetic peptides may be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant G. A. ed., Oxford University Press, 2002, or in Pharmaceutical Formulation: Development of Peptides and Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.
BOC Antibody
By “BOC antibody” or “anti-BOC” in the present context is meant an antibody capable of detecting (i.e. binding to) a BOC protein or a BOC protein fragment. In an embodiment, the above-mentioned antibody inhibits the biological activity of BOC. In another embodiment, the BOC protein fragment is an extracellular domain (ectodomain) of BOC. In another embodiment, the BOC protein fragment is an intracellular domain of BOC. In a further embodiment, the BOC protein fragment is a FNIII domain/repeat. In another embodiment, the BOC protein fragment is an extracellular domain of BOC that is shedded from the cell surface in the extracellular fluid.
By “BOC antibody” or “anti-BOC” in the present context is also meant an antibody capable of binding (e.g., specifically binding) to BOC, and inhibiting its biological activity (e.g., its activity of binding to SHH or its activity on cell proliferation).
As used herein, the term “anti-BOC” also refers to an antibody that specifically binds to (interacts with) a BOC polypeptide (e.g., the polypeptide of SEQ ID NO: 2) and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as a BOC polypeptide. The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.
In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology”, Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH Laboratories). The term antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g., Fab and Fab′ fragments) which inhibit or neutralize their respective interaction domains in Hyphen and/or are specific thereto.
Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c.), intravenous (i.v.) or intraperitoneal (i.p.) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups.
Animals may be immunized against the antigen (BOC polypeptide or a fragment thereof), immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with 1⁄5 to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293.
In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Antibodies/antibody fragments specifically binding to BOC are commercially available (from R&D SystemsTM, Cat. Nos. MAB2036, MAB20361, AF2036, AF2385, for example).
In an embodiment, the above-mentioned antibody blocks the interaction between BOC and an interacting partner, such as a BOC ligand (e.g., SHH). In a further embodiment, the above-mentioned antibody is an anti-SHH antibody. In a further embodiment, the above-mentioned antibody is anti-SHH monoclonal antibody clone 5E1 (Ericson et al., Cell (1996) 87: 661-673).
Non-Antibody-Based BOC Inhibitors
Inhibitors of BOC may also be in the form of non-antibody-based scaffolds, such as avimers (Avidia); DARPins (Molecular Partners); Adnectins (Adnexus), Anticalins (Pieris) and Affibodies (Affibody). The use of alternative scaffolds for protein binding is well known in the art (see, for example, Binz and Plückthun, 2005, Curr. Opin. Biotech. 16: 1-11).
Agents and Pharmaceutical Compositions
In another aspect, the present invention provides a BOC inhibitor for preventing and/or treating cancer. Such an agent may be, for example (i) an antisense compound against the sequence set forth in SEQ ID NO: 1 (BOC) which inhibits BOC expression; (ii) a peptide fragment capable of binding to a BOC polypeptide and inhibiting the activity of said polypeptide; (iii) a peptide fragment capable of inhibiting the activity of BOC; (iv) an antibody (e.g., polyclonal or monoclonal), natural or artificial variants, or antibody fragments, which specifically binds to BOC and blocks its activity; (v) a small molecule that inhibits the activity of BOC. In an embodiment, the antibody, natural or artificial variants, or antibody fragments specifically binds to an epitope located in the ectodomain of a BOC polypeptide (e.g., a FNIII domain), and inhibits its activity.
The invention also provides a pharmaceutical composition (medicament) comprising at least one agent of the invention (e.g., a BOC inhibitor), and a pharmaceutically acceptable diluent, carrier, salt or adjuvant. Such carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition may be adapted for the desired route of administration (e.g., oral, parental, intravenous, intramuscular, intraperitoneal, aerosol).
The invention also provides pharmaceutical compositions which comprise one or more agent(s) modulating BOC activity. Typically, the activity of BOC is reduced or inhibited. The invention also provides pharmaceutical compositions which comprise one or more agent(s) modulating BOC activity in combination with other anticancer treatment including chemotherapeutical agents, such as cyclopamine, CUR0199691, CisplatinTM OxaliplatinTM and their derivatives, cyclophosphamide compound (Cy), 13-cis retinoic acid (RA), histone deacetylase inhibitor (SAHA), nucleotide analogues (e.g., 5-fluoro uracyl, 5-AZA), kinase inhibitors, GDC-0449 (antagonist of Hedgehog signaling), etc.
The present invention also provides a kit or package comprising the above-mentioned agent or pharmaceutical compositions. Such kit may further comprises, for example, instructions for the prevention and/or treatment of cancer, containers, devices for administering the agent/composition, etc.
Screening Assays
Given the correlation between BOC expression/activity and cancer development/progression, compounds which are capable of decreasing BOC expression/activity may be used for the prevention and/or treatment of cancer. Therefore, the invention further relates to screening methods for the identification and characterization of compounds capable of decreasing BOC expression and/or activity, which may be used for the prevention and/or treatment of cancer.
The present invention also provides a method (e.g., an in vitro method) for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: (a) contacting said test compound with a BOC polypeptide, or a fragment thereof or variant thereof having BOC activity; and (b) determining the activity of the BOC polypeptide, fragment or variant thereof, in the presence or absence of said test compound; wherein a decrease in the activity of BOC in the presence of said test compound relative to the absence thereof is indicative that said test compound may be used for the prevention and/or treatment of cancer.
The present invention also provides a method (e.g., an in vitro method) for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: (a) contacting said test compound with a cell expressing BOC; and (b) determining the expression and/or activity of BOC in the presence or absence of said test compound; wherein a decrease in the expression and/or activity of BOC in the presence of said test compound relative to the absence thereof is indicative that said test compound may be used for the prevention and/or treatment of cancer.
The present invention also provides a method (e.g., an in vitro method) for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: (a) contacting said test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a BOC gene, operably linked to a second nucleic acid comprising a reporter gene encoding a reporter protein; and (b) determining whether the reporter gene expression and/or reporter protein activity is decreased in the presence of said test compound; wherein said decrease in reporter gene expression and/or reporter protein activity is indicative that said test compound may be used for prevention and/or treatment of cancer.
The present invention also provides a method (e.g., an in vitro method) for determining whether a test compound is useful for the prevention and/or treatment of cancer, said method comprising: (a) contacting said test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a gene whose expression is modulated by BOC activity (e.g., GLI-1), operably linked to a second nucleic acid comprising a reporter gene encoding a reporter protein; and (b) determining whether the reporter gene expression and/or reporter protein activity is decreased in the presence of said test compound; wherein said decrease in reporter gene expression and/or reporter protein activity is indicative that said test compound may be used for prevention and/or treatment of cancer.
The above-mentioned methods may be employed either with a single test compound or a plurality or library (e.g., a combinatorial library) of test compounds. In the latter case, synergistic effects provided by combinations of compounds may also be identified and characterized. The above-mentioned compounds may be used for prevention and/or treatment of cancer, or may be used as lead compounds for the development and testing of additional compounds having improved specificity, efficacy and/or pharmacological (e.g., pharmacokinetic) properties. In an embodiment the compound may be a prodrug which is altered into its active form at the appropriate site of action, (e.g., a cell, tissue or organ affected by cancer). In certain embodiments, one or a plurality of the steps of the screening/testing methods of the invention may be automated.
Such assay systems may comprise a variety of means to enable and optimize useful assay conditions. Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal BOC activity and stability (e.g., protease inhibitors), temperature control means for BOC activity and or stability, and detection means to enable the detection of a BOC activity reaction product. A variety of such detection means may be used, including but not limited to one or a combination of the following: radiolabelling (e.g., 32P, 14C, 3H), antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
The assay may be carried out in vitro utilizing a source of BOC which may comprise naturally isolated or recombinantly produced BOC, in preparations ranging from crude to pure. Recombinant BOC may be produced in a number of prokaryotic or eukaryotic expression systems, which are well known in the art. Such assays may be performed in an array format.
As noted above, the invention further relates to methods for the identification and characterization of compounds capable of decreasing BOC gene expression. Such a method may comprise assaying BOC gene expression in the presence versus the absence of a test compound. Such gene expression may be measured by detection of the corresponding RNA or protein, or via the use of a suitable reporter construct comprising one or more transcriptional regulatory element(s) normally associated with a BOC gene, operably-linked to a reporter gene.
A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since, for example, enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. “Transcriptional regulatory element” is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked. The expression of such a reporter gene may be measured on the transcriptional or translational level, e.g., by the amount of RNA or protein produced. RNA may be detected by for example Northern analysis or by the reverse transcriptase-polymerase chain reaction (RT-PCR) method (see for example Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA).
Protein levels may be detected either directly using affinity reagents (e.g., an antibody or fragment thereof (for methods, see for example Harlow, E. and Lane, D (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); a ligand which binds the protein) or by other properties (e.g., fluorescence in the case of green fluorescent protein) or by measurement of the protein's activity, which may entail enzymatic activity to produce a detectable product (e.g., with altered spectroscopic properties) or a detectable phenotype (e.g., alterations in cell growth/function). Suitable reporter genes include but are not limited to chloramphenicol acetyltransferase, β-D galactosidase, luciferase, or green fluorescent protein (GFP).
BOC levels (expression levels) could be determined using any standard methods known in the art. Non-limiting examples of such methods include Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunocytochemistry, immunohistochemistry, mass spectrometry, as well as methods to determine mRNA levels such as RT-PCR and northern analysis, real-time PCR, PCR, in situ hybridization and so on.
For example, a test compound may be added to a reaction mixture containing a purified BOC and a BOC ligand or a peptide fragment of a BOC ligand (e.g., Shh), and the binding between BOC and the BOC ligand is determined and compared to the binding when the mixture is incubated under similar conditions but without the test compound. A lower binding in the presence of the test compound is indicative that the test compound may be useful for inhibiting BOC activity and in turn for the prevention and/or treatment of cancer. The detection step (i.e. determination of the binding) could be monitored by any number of means including, but not limited to binding-dependent optical spectroscopy, fluorimetry, and radioactive label variation and could use various techniques such as Surface Plasmon resonance, FRET, yeast two hybrids, and alpha-screen.
Cell-based assays designed for identifying BOC inhibitors may comprise the addition of a test compound to cells stimulated by Shh and the determination of BOC expression and/or BOC activity observed when the cells are incubated under similar conditions but without the inhibitory compound. The detection step could be monitored by any number of means including by measuring cell proliferation (e.g., by MTT assay) or BOC downstream activation (e.g., expression of a gene (e.g., a reporter gene) operably linked to a transcription element normally associated with a gene whose expression is modulated by BOC activity (e.g., GLI-1)).
In another aspect, the present invention provides an agent that inhibits BOC expression or activity identified by the above-noted screening method.
Transgenic Non Human Mammal
As used herein, the terminology “transgenic non human mammal” refers to any non human mammal which harbors a nucleic acid sequence having been inserted into a cell and having become part of the genome of the mammal that develops from that cell. In one specific embodiment of the present invention, the genetic alteration of the transgenic non human mammal has been introduced in a germ-line cell, such that it enables the transfer of this genetic alteration to the offspring thereof. Such offspring, containing this genetic alteration are also transgenic non human mammals.
Techniques for the preparation of such transgenic mammals are well known in the art (e.g., a standard pronuclear microinjection; introduction of a transgene in embryonic stem (ES) cells; microinjecting the modified ES cells into blastocyst; or infecting a cell with a recombinant virus containing the transgene in its genome). Non-limiting examples of patents relating to a transgenic non human animal include U.S. Pat. Nos. 4,736,866; 5,087,571; 5,175,383; 5,175,384 and 5,175,385. Many animals may be used as host for the transgenes of the present invention, including all laboratory animals including mice, rats and rabbits. In a specific embodiment, the transgenic mammal is a mouse. In a more specific embodiment, the mouse strain is the C57BL/6J. Any other mouse strain however may be used in accordance with the present invention and identified as containing the BOC transgene. Other commonly used mouse strains for transgenic studies include C57Black, FVB, 129Sv, CD1 and ICR.
Diagnosis, Prognosis and Disease Monitoring Methods
The present inventors have shown in a mouse tumor model (medulloblastoma model) that BOC is overexpressed in 100% of medulloblastoma tumors analyzed compared to normal adjacent tissues, and thus that BOC may be used as a biological marker for the detection and characterization of tumors. Also, BOC overexpression was observed in preneoplasia, indicating that BOC is a marker of early stage invasive cancer and/or may be used to detect pre-malignant lesions.
The present inventors have also shown that BOC is expressed in human medulloblastoma tumor samples and that its high expression correlates with an increased risk for cancer progression.
Therefore, in another aspect, the invention relates to the diagnosis and prognostic of cancer, or of a susceptibility or predisposition to cancer. The invention thus provides a method for diagnosing cancer, or a predisposition to cancer, in a subject based on the expression and/or activity of BOC determined in a sample (e.g., a biopsy) from the subject. The expression and activity of BOC in the sample may be determined using the assays/methods described above.
In another aspect, the present invention provides a method for prognosis of a cancer patient, said method comprising: (a) determining the level and/or activity of BOC in a sample from said cancer patient, (b) comparing said level and/or activity to a corresponding reference level and/or activity; and (c) prognosing said cancer patient based on said comparison.
In another aspect, the present invention provides a method for prognosis of a cancer patient, said method comprising: (a) detecting an expression and/or activity of BOC in a sample (e.g., a tumor sample) from said cancer patient, and (b) prognosing said cancer patient based on said detection; wherein the detection (i.e., presence) of BOC expression and/or activity in said sample is indicative that said subject has a poor prognosis, and wherein the lack of detection (i.e., absence) of BOC expression and/or activity in said sample is indicative that said subject has a good prognosis.
In an embodiment, the above-mentioned prognosis is a good prognosis, wherein said good prognosis indicates that said individual has a high probability (e.g., at least 35%, 50%, 60%, 70% or 75%) of surviving after five, ten or fifteen years of initial diagnosis of cancer.
In an embodiment, the above-mentioned prognosis is a poor prognosis, and wherein said poor prognosis indicates that said individual has a low probability (e.g., less than 50% or 40%) of surviving after five, ten or fifteen years of initial diagnosis of cancer.
Poor prognosis may indicate that a tumor is relatively aggressive, while good prognosis may indicate that a tumor is relatively nonaggressive. Therefore, the invention provides for a method of determining a course of treatment of a cancer patient, comprising determining whether the level of expression of BOC correlates with the level of BOC expression in a reference sample representing a “good prognosis” expression level or a “poor prognosis” level; and determining a course of treatment, wherein if the expression correlates with the “poor prognosis” level, the tumor is treated as an aggressive tumor.
In another aspect, the present invention provides a method for performing a cancer follow-up or monitoring, for example to confirm the absence or removal of all tumor tissue following surgery, cancer chemotherapy and/or radiation therapy, or to monitor cancer chemotherapy and tumor reappearance. Such method comprising: (a) determining the expression and/or activity of BOC in a sample from said patient; (b) comparing said expression or activity to a corresponding expression and/or activity of BOC determined in a biological sample obtained from said patient at an earlier time (e.g., before surgery, just after surgery, at the start of treatment). For example, a decrease in said expression and/or activity relative to a corresponding expression and/or activity of BOC determined in a biological sample obtained from said patient at an earlier time (before surgery, at the start of treatment) is indicative that said patient is responsive to the treatment. Alternatively, an increase in BOC expression levels over time is indicative that the patient is not responsive to the treatment, or that this is a cancer recurrence (tumor reappearance).
In an embodiment, the method may comprise determining whether BOC activity and/or expression is modulated, e.g., upregulated or increased, relative to a control/reference activity or expression. In yet another embodiment, the control BOC expression or activity can be selected from an established standard, a corresponding BOC expression or activity determined in the subject (in a sample from the subject) at an earlier time; a corresponding BOC expression or activity determined in one or more control subject(s) known to not being predisposed to cancer, known to not having cancer, or known to have a good prognosis. In such cases, an increased or higher expression and/or activity in the sample from the subject relative to the control activity or expression is indicative that the subject has cancer, has a predisposition to cancer (e.g., has a higher risk of developing cancer and/or of experiencing cancer progression) or has a poor prognosis (e.g., lower survival probability, higher probability of cancer recurrence), while a comparable or lower expression or activity in a sample from the subject relative to the control expression or activity is indicative that the subject does not have cancer, is not predisposed to cancer or has a good prognosis (e.g., higher survival probability, lower probability of cancer recurrence).
In another embodiment, the reference/control BOC expression or activity is a corresponding expression or activity in a control subject known to have a predisposition to cancer, known to have cancer or known to have a poor prognosis. In such a case, a comparable or higher BOC expression and/or activity in a sample from the subject relative to the control expression or activity is indicative that the subject has cancer, has a predisposition to cancer or has a poor prognosis (e.g., lower survival probability, higher probability of cancer recurrence), while a lower expression or activity in a sample from the subject relative to the control expression or activity is indicative that the subject does not have cancer, is not predisposed to cancer or has a good prognosis (e.g., higher survival probability, lower probability of cancer recurrence).
In another embodiment, the reference/control BOC expression or activity is the average or median value obtained following determination of BOC expression or activity in a plurality of samples (e.g., samples obtained from several healthy and/or cancer patients).
BOC expression levels may in general be detected by either detecting mRNA from the cells and/or detecting expression products, such as polypeptides and proteins. Expression of the transcripts and/or proteins encoded by the nucleic acids described herein may be measured by any of a variety of known methods in the art. In general, the nucleic acid sequence of a nucleic acid molecule (e.g., DNA or RNA) in a sample can be detected by any suitable method or technique of measuring or detecting gene sequence or expression. Such methods include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase-PCR(RT-PCR), in situ PCR, SAGE, quantitative PCR (q-PCR), in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA hybridization platforms. For RNA expression, preferred methods include, but are not limited to: extraction of cellular mRNA and Northern blotting using labeled probes that hybridize to transcripts encoding all or part of one or more of the genes of this invention; amplification of mRNA expressed from one or more of the genes of this invention using gene-specific primers, polymerase chain reaction (PCR), quantitative PCR (q-PCR), and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the cells, which is then labeled and used to probe cDNAs or oligonucleotides encoding all or part of the genes of this invention, arrayed on any of a variety of surfaces; in situ hybridization; and detection of a reporter gene.
Methods to measure protein expression levels of selected genes of this invention, include, but are not limited to: Western blot, tissue microarray, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and assays based on a property of the protein including but not limited to DNA binding, ligand binding, or interaction with other protein partners. In a further embodiment, the BOC expression level is measured by immunohistochemical staining, and the percentage and/or the intensity of immunostaining of immunoreactive cells in the sample is determined.
Methods for normalizing the level of expression of a gene are well known in the art. For example, the expression level of a gene of the present invention can be normalized on the basis of the relative ratio of the mRNA level of this gene to the mRNA level of a housekeeping gene, or the relative ratio of the protein level of the protein encoded by this gene to the protein level of the housekeeping protein, so that variations in the sample extraction efficiency among cells or tissues are reduced in the evaluation of the gene expression level. A “housekeeping gene” is a gene the expression of which is substantially the same from sample to sample or from tissue to tissue, or one that is relatively refractory to change in response to external stimuli. A housekeeping gene can be any RNA molecule other than that encoded by the gene of interest that will allow normalization of sample RNA or any other marker that can be used to normalize for the amount of total RNA added to each reaction. For example, the GAPDH gene, the G6PD gene, the actin gene, ribosomal RNA, 36B4 RNA, PGK1, RPLP0, or the like, may be used as a housekeeping gene.
Methods for calibrating the level of expression of a gene are well known in the art. For example, the expression of a gene can be calibrated using reference samples, which are commercially available. Examples of reference samples include, but are not limited to: StratageneTM QPCR Human Reference Total RNA, ClontechTM Universal Reference Total RNA, and XpressRefTM Universal Reference Total RNA.
In an embodiment, the above-mentioned method comprises determining the level of a BOC nucleic acid (e.g., the nucleic acid of SEQ ID NO: 1) in the sample. In another embodiment, the above-mentioned method comprises determining the level of a BOC polypeptide (e.g., the polypeptide of SEQ ID NO: 2) in the sample. In an embodiment, the level of a BOC polypeptide is determined using an anti-BOC antibody. In a further embodiment, the anti-BOC antibody binds to the extracellular portion of BOC (e.g., the portion that is shedded from the cell surface in the extracellular fluid).
Alternatively, the above-mentioned diagnosis or prognosis may be performed based on the detection of BOC variants (e.g., the BOC variants described above), and more particularly BOC variants associated with either an increase or a decrease in BOC expression and/or activity. For example, the detection of a nucleic acid encoding a BOC variant (such as a nucleic acid comprising a polymorphism (e.g., SNP) in the coding region or a regulatory region) known to have increased BOC activity or known to provoke increased BOC expression in a sample from a subject may be used to determine that a subject has a cancer, is predisposed to (or at risk of developing) cancer, and/or has a poor cancer prognosis. Such BOC variants may be detected at the polypeptide or nucleic acid level using methods well known in the art, such as PCR-based methods as well as DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis and the like.
In an embodiment, the methods of diagnosis/prognostication noted above may be performed in conjunction with the therapeutic/prophylactic methods noted above, for preventing and/or treating cancer in a subject. Such a method thus comprises the diagnosis or prognostication of cancer in a subject and, in accordance with the diagnosis/prognosis, decreasing BOC levels in the subject (e.g., in a cell, tissue or organ of the subject) to prevent or treat cancer.
“Sample” or “biological sample” refers to any solid or liquid sample isolated from a live being. In a particular embodiment, it refers to any solid or liquid sample isolated from a human, such as a biopsy material, blood (e.g., plasma, serum or whole blood), saliva, synovial fluid, urine, amniotic fluid and cerebrospinal fluid. Such sample may be, for example, fresh, fixed (e.g., formalin-, alcohol- or acetone-fixed), paraffin-embedded or frozen prior to analysis of BOC expression level. In an embodiment, the above-mentioned sample is obtained from the central nervous system (e.g., a CNS cell, tissue or fluid). In a further embodiment, the CNS fluid is cerebrospinal fluid. In embodiments, the above-mentioned sample is a tumor cell/tissue sample (e.g., biopsy material).
The present invention also provides a kit or package comprising a reagent useful for determining BOC expression and activity (e.g., a ligand that specifically binds BOC polypeptide such as an anti-BOC antibody, or a ligand that specifically binds a BOC nucleic acid such as an oligonucleotide). Such kit may further comprise, for example, instructions for the prognosis and/or diagnosis of cancer, control samples, containers, reagents useful for performing the methods (e.g., buffers, enzymes), etc.
In an embodiment, the above-mentioned further comprises analyzing in the sample of the subject the activity and/or expression of at least another marker associated with medulloblastoma. Two main classes of medulloblastoma have been described, namely (a) medulloblastoma Shh generally associated with Gli1, Igf1, CXCR4, patch1 and/or sfrp1 expression and/or activity and (b) medulloblastoma Wnt, generally associated with tnc, epha4, Dkk1, Amhr2, and/or Emx2 expression and/or activity.
The above-mentioned methods may also be used for classifying or stratifying a subject into subgroups (based on BOC level and/or activity, for example or on a combination of BOC level and/or activity with that of another marker associated with medulloblastoma) having different phenotypes enabling a better characterization of cancer and eventually a better selection of treatment depending on the subgroup to which the subject belongs.
As used herein, a substantially similar level refers to a difference in the level of expression or activity between the level determined in a first sample (i.e. test sample) and the reference level which is about 10% or less; in a further embodiment, 5% or less; in a further embodiment, 2% or less.
As used herein, a “higher” or “increased” level refers to a level of expression or activity in a sample (i.e. test sample) which is at least 15% higher, in an embodiment at least 25% higher, in a further embodiment at least 40% higher; in a further embodiment at least 50% higher, in a further embodiment at least 100% higher (i.e. 2-fold), in a further embodiment at least 200% higher (i.e. 3-fold), in a further embodiment at least 300% higher (i.e. 4-fold), relative to the reference level (e.g., in a control sample, in corresponding normal adjacent tissue or alternatively, in the presence of an BOC inhibitor).
As used herein the term “subject” is meant to refer to any animal, such as a mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In a particular embodiment, it refers to a human.
A “subject in need thereof” or a “patient” in the context of the present invention is intended to include any subject that will benefit or that is likely to benefit from the decrease in the expression or activity of BOC. In an embodiment, a subject in need thereof is a subject diagnosed invasive disease overexpressing BOC.
As used herein, the term “a” or “the” means “at least one”.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
The present invention is illustrated in further details by the following non-limiting examples.
Example 1
BOC is Involved in Cerebellum Development
Expression of BOC in GCP During the Cerebellum Development
To explore the involvement of BOC in the cerebellum development, its expression was first examined through mouse cerebellum histological sections. Immunohistochemistry on sections of wild-type mice cerebelli showed a distribution of BOC labeling in the Granule Cell Precursors (GCP) (FIG. 3). BOC is expressed in proliferating GCP whereas CDON is not expressed in the cerebellum.
BOC−/− Mice Cerebellum Shows Less Cell Proliferation
Comparative analyses of size and weight of cerebellum from mice harbouring different BOC alleles were performed. Measurements have shown that mice completely lacking BOC gene (BOC−/− mice obtained from Dr S. K. McConnell at University of Stanford) possess significantly smaller cerebelli, both in term of weight and size, as compared to heterozygote BOC+/− or wild-type (BOC+/+) control groups (FIG. 4).
The reduction in size of BOC−/− cerebellum could be due either to a decrease in cell proliferation or to an increase of apoptosis. Sections of cerebellum obtained from BOC−/− and BOC+/− mice were analyzed and compared. GCP proliferation in the EGL was measured by immunostaining using an antibody specific for different cell proliferation markers such as phospho-histone H3 (PhH3) and BrdU. BOC−/− mouse cerebelli exhibited a significant decrease in the number of BrdU-positive cells as compared to control BOC+/− mice (FIGS. 5A and 5B). Similar results were obtained with another marker of cell proliferation, phospho-histone H3 (phH3) staining (FIG. 5C).
To measure the number of apoptotic cells, cerebellum sections obtained from BOC−/− and BOC+/− mice were stained with a marker of apoptosis, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) to detect DNA fragmentation. TUNEL-positive nuclei were counted on a series of cerebellum matching sections. No difference was observed in the number of TUNEL-positive cells on cerebellum sections of BOC−/− and BOC+/− mice (FIGS. 5D and 5E). The decrease in size of the cerebellum of BOC−/− mice is thus correlated with a decrease in cell proliferation of GCP.
Loss of BOC in GCP: Cell Autonomous Decrease of Cell Proliferation
GCP purified from the cerebellum of both BOC−/− and BOC+/− mice were cultured in vitro in the presence of increasing concentrations of Shh before labeling with an anti-Ki-67 (Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0), and is thus an excellent marker to determine the fraction of proliferating cells in a given cell population). GCP were extracted from newborn mice from post-natal age day 3 to 5. Cerebelli were dissected, chopped into small pieces and treated with trypsin. Cells were then dissociated using fire polished pipettes and spin in a 35%-65% PercollTM gradient. Pure GCP were then collected at the 35%-65% PercollTM interface and plated in a pre-coated PDL-coated dish. GCP were then treated with different concentrations of Shh and cultured for 48 h before being fixed and processed for Ki-67 immunostaining. Finally, the proportion of Ki-67-positive cell was analyzed. In the absence of Shh, the percentage of Ki-67-positive GCP cells was significantly reduced in GCP cells derived from BOC−/− mice as compared to those derived from BOC+/− mice (FIG. 6). In both cultures, increasing the concentration of Shh resulted in a dose-dependent increase in the number of Ki-67-positive cells. However, at 1 nM, 3 nM and 10 nM, a significant difference is still observed between GCP derived from BOC−/− and GCP derived from BOC+/− mice. These data show a cell autonomous decrease of GCP proliferation in the absence of BOC.
BOC Overexpression Increases GCP Proliferation in the Presence of Shh
GCP purified from post-natal day 4 rat cerebellum were co-electroporated with either a combination of a plasmid containing a nucleic acid encoding murine BOC with a red fluorescent protein (RFP)-expressing plasmids (BOC) (both genes under the control of the chicken beta-actin promoter) or with a control empty plasmid with a red fluorescent protein-expressing plasmid (control). Proliferation was evaluated by measuring the percentage of Ki-67-positive cells among electroporated cells in the presence or absence of Shh. In addition, the role of the Hh signalling protein Smoothened (Smo) was tested using an inhibitor of Smo (SANT-1) in combination with Shh. The percentage of Ki-67-positive cells stimulated by the presence of 10 nM of Shh was markedly increased in GCP overexpressing BOC as compared to control electroporated cells treated in parallel (FIG. 7). The addition of SANT-1 prevents this increase, suggesting that Smo plays a role in the BOC-mediated increase in cell proliferation.
Example 2
Detection of BOC in Medulloblastoma
The Ptc1+/− Mouse Medulloblastoma Animal Model
Activation of Sonic hedgehog signaling normally regulates granule cell precursor proliferation during cerebellum development. In humans, germline mutations of the Shh receptor gene PATCHED (PTCH) predispose to medulloblastoma. Mice with heterozygous patched mutation (Ptc1+/−) also develop medulloblastoma tumors (Goodrich et al., 1997, Science 277(5329):1109-13). Survival analysis of Ptc1+/− mice revealed that about 55% of these mice developed large medulloblastoma tumor and generally died at about 6 months of age (FIG. 8).
BOC is Expressed in Mouse Medulloblastoma Tumors
Using the Ptc1+/− mouse model, double-mutant mice in which one or both BOC alleles have been deleted were generated. BOC-promoter driven alkaline phosphatase (PLAP) reporter gene expression and BOC immunostaining using anti-BOC antibodies (R&D Systems, Minneapolis, Minn., USA), in a medulloblastoma tumor obtained from the Ptc+/−/BOC+/− mouse. FIG. 9A shows BOC immunostaining on a medulloblastoma tumor using anti-BOC antibodies and showing that BOC is highly expressed in tumor tissue as compared to adjacent normal cerebellum (Cb). FIGS. 9B and C shows BOC-promoter driven alkaline phosphatase (PLAP) reporter gene expression on an adjacent section of the same tumor. FIG. 9C shows BOC mRNA levels in cortex (left bar), normal cerebellum (middle bar) and medulloblastoma tumor (right bar) as measured by quantitative PCR. BOC is thus overexpressed in tumor tissue as compared to adjacent normal cerebellum (FIGS. 9A-9C), at both the protein and mRNA levels.
This observation was further confirmed by Western blot analysis of total proteins purified from medulloblastoma tumors derived from different mouse genotypes (FIG. 11). These data show that BOC is a good marker of medulloblastoma tumors. The increase in BOC protein expression correlates with an increase of the reporter gene PLAP (placental alkaline phosphatase, knocked into the BOC gene). Therefore, an increase in BOC expression at both the protein and nucleic acid levels was observed in medulloblastoma tumors.
BOC in Overexpressed in Preneoplasia
While about 55% of Ptc1+/− mice develop medulloblastoma at 3 to 6 months of age, most of them have regions of ectopic cell in their cerebellum at 8 weeks of age (P60). These cells have been described as remnants of the external germinal layer (EGL) and have been reported to represent a pre-neoplastic stage of tumorigenesis. Immunostaining using an anti-BOC antibody applied on cerebellum section of P60 Ptc1+/− mice revealed that BOC is also highly expressed in all of these pre-neoplasia (FIG. 10, upper panel). BOC therefore represents a marker at early stage of medulloblastoma tumors.
Example 3
BOC is Associated with the Development of Medulloblastoma Tumors
BOC is Involved in Medulloblastoma Tumorigenesis
Survival analyses were performed on BOC−/−; Ptc1+/− mice and compared to BOC+/−; Ptc1+/− and BOC+/−; Ptc1+/− mice. In both groups of Ptc1+/− mice, the percentage of survival was significantly reduced as compared to that observed in Ptc1+/+ mice (FIG. 12). However, a higher survival was observed in Ptc1+/− mice having an homozygous deletion of BOC (BOC) as compared to their heterozygous counterparts (BOC+/−) (FIG. 12).
Overexpression of BOC In Vivo Using Retroviral Infection of GCP
Retroviral vector-mediated delivery of cDNA to mice in vivo is an effective way to overexpress a gene in animals. Using this approach, BOC is overexpressed in GCP of young rats. The virus is administrated by stereotaxic injection directly in developing cerebellum to infect dividing GCP cells, which are the most numerous proliferating cell type in developing cerebellum. The development of medulloblastoma tumors is analyzed in these mice and compared to that of control mice (infected with a control retroviral vector).
Inhibition of BOC Expression in Mouse and Human Medulloblastoma-Derived Cell Lines
Inhibition of BOC expression in medulloblastoma-derived cell lines is performed using different RNA interfering (RNAi) compounds. Ptc1+/− (derived from Ptc1+/− mice medulloblastoma), Daoy (human), UW2 (human) and UW3 (human) cell lines are transfected with one of the 4 shRNA constructs. These shRNA are designed to include a hairpin of 21 base pair sense and antisense stem and a 6 base pair loop. Each hairpin sequence is cloned into the lentiviral vector (pLKO.1) and the sequence is verified (Seq B9: CCCATGAGAACAGACCAAGAT (SEQ ID NO: 5); Seq B10: CCCGTATACTATGGTGCCATT (SEQ ID NO: 6); Seq B11: CGACATTAAGATGCAGTGCTT (SEQ ID NO: 7); Seq C1: GAGGGAAACACAGCAGTCATT (SEQ ID NO: 8); Seq B12: CCTCTACAATGTCCAGGTGTT (SEQ ID NO: 9)); These multiple constructs are created to ensure adequate coverage of the BOC gene. Stable cell lines may be selected using the puromycin selectable marker. The efficiency of each shRNA is verified by Q-PCR. The effect of the inhibition of BOC is then measured by using a MTT Cell Proliferation Assay. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, first described by Mosmann (J. Immunol. Methods. 1983, 65(1-2): 55-63, incorporated by reference in its entirety) is based on the ability of active mitochondrial reductase enzymes from viable cells to cleave the tetrazolium rings of the pale yellow MTT. The cleavage reaction forms a purple formazan crystals which is largely impermeable to cell membranes resulting in its accumulation within healthy cells. Solubilization of the cells by addition of a detergent liberates and solubilizes the formazan crystals. The number of surviving cells is directly proportional to the level of the formazan product created. The color can then be quantified spectrophotometrically. The production of purple formazan in cells treated with a compound is measured relative to the production in control cells, and dose-response curves can be generated. This assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability, thus also allowing the evaluation of the cytotoxicity of tested compounds. The effect of the inhibition of BOC by the different shRNA constructs is tested in the Ptc1+/−, Daoy, UW2 and UW3 cell lines using the MTT Cell Proliferation Assay.
The shRNAs inhibiting cell proliferation are compounds that inhibit expression of BOC. These compounds could serve as a basis for the development of a drug for preventing and/or treating cancer, such as medulloblastoma.
Example 4
BOC in Human Tumors
BOC is Overexpressed in Human Medulloblastoma
A tissue microarray was constructed from 73 different human medulloblastoma tumor samples, as well as from positive and negative control samples (Ray A. et al., Clin Cancer Res. 2004. 10(22): 7613-20). For each patient, all pathological blocks and corresponding slides were obtained and reviewed by neuropathology for diagnostic accuracy and tissue adequacy. Representative tumor areas were identified, and between three and four cores were obtained for each tumor. Clinical data collected for each patient included age and metastatic disease status at presentation, sex, extent of surgical resection, chemotherapy use, radiotherapy use, progression-free survival, and overall survival. The latter was the primary end point for this study. Metastatic disease was defined as either the presence of malignant cells on cerebrospinal fluid (CSF) cytology (obtained between 7 and 14 days after surgery) or definite radiographic evidence of spread before the onset of chemotherapy or radiotherapy. Equivocal CSF specimens were considered positive if the next follow-up CSF sample within 2 weeks was cytologically positive. Clumping of spinal roots on magnetic resonance imaging was not considered positive. The arrays were assayed for immunohistochemical expression of BOC (representative examples of immunohistochemical stainings are illustrated at FIG. 13A (positive BOC staining) and FIG. 13B (negative BOC staining), and results analyzed for correlation between BOC expression and the factors listed above (e.g., sex, survival). FIG. 13C summarized the data obtained, which revealed that 38 tumors out of the 73 analyzed ( ̃52%) were positive for BOC expression.
A higher level of BOC in tissue microarray from human tumor samples as compared to that in a reference sample is indicative that modulation of BOC expression/activity may be useful for the treatment of medulloblastoma.
Tumor samples are also tested by tissues microarray with a series of antibodies for measuring the level of others markers characterizing medulloblastoma (e.g., Ptc, Shh, Gli-1, Igf1, CXCR4, sfrp1, tnc, epha4, Dkk1, Amhr2, Emx2). Statistical analyses also include characteristics such as age, sex, therapeutic treatment, presence of metastasis and survival of the patient.
Survival correlation analysis indicated that patients with medulloblastoma tumors positive for BOC have a tendency to show a lower overall survival probability over years than patients with BOC negative tumors (FIG. 19). These results indicate that the presence of tumors expressing BOC correlates with a worse prognosis for some patients.
BOC Expression and Gene Profiling for Human Medulloblastoma Classification
Analysis of BOC expression in a variety of human medulloblastoma tissue samples was performed by Serial Analysis of Gene Expression (SAGE) using the available database of the Cancer Genome Anatomy Project (Lash A E et al., 2000. Genome Res, 10(7): 1051-1060). Overexpression of BOC was reported in six medulloblastoma among 19 samples (about 32%) (FIGS. 14A and B). The analysis was also performed for markers characterizing medulloblastoma Shh (e.g., Gli1, Igf1, CXCR4, Ptc1, sfrp1) and markers for medulloblastoma Wnt (e.g., tnc, epha4, Dkk1, Amhr2, Emx2). Data analysis revealed that BOC was overexpressed in 80% (4/5) of medulloblastoma Shh and 100% (2/2) of medulloblastoma Wnt (FIG. 14B).
BOC Expression in Various Human Cancers
Analysis of BOC expression in a variety of human tumor tissue samples was also performed by SAGE. As shown in Tables II and III below, overexpression of BOC was detected in several tumor tissues including brain ependynoma, ovary tumor, medulloblastoma, breast tumor, skin tumor, glioblastoma, meningioma, astrocytoma, chondrosarcoma, colon adenocarcinoma, liver cholangiocarcinoma, prostate carcinoma, bladder tumor, lung tumor, lymph node lymphoma, vascular endothelium hemangioma, kidney carcinoma and thyroid follicular adenoma.







TABLE II







SAGE analysis of BOC expression in tumor tissue libraries











Total Tags
Tag
Tag per


SAGE Library
in library
counts
200,000













Brain ependymoma B R353
73822
30
81


Ovary adenocarcinoma B OVT-8
31987
10
62


Brain ependymoma B 239
46653
12
51


Brain medulloblastoma B 98-04-P494
43068
9
41


Brain medulloblastoma B H306
60454
10
33


Brain medulloblastoma B H876
67404
10
29


Breast fibroadenoma MD
55688
8
28


Ovary carcinoma CL A2780
21369
3
28


Brain ependymoma B R1023
122690
17
27


Breast carcinoma B BWHT18
50701
6
23


Skin Normal B hs0277
5269789
587
22


Skin Cancer B hs0279
5703298
610
21


Uterus normal B hs0195
3718463
373
20


Brain ependymoma B R510
120431
12
49


Breast carcinoma associated 
65091
6
18


myofibroblast CD10+ AP IDC7





Breast carcinoma associated 
81452
7
17


myoepithelium AP





DCIS6





Brain ependymoma B R510
84073
7
16


Skin Normal B hs0272
4397550
339
15


Foreskin Normal B hs0305
3940350
279
14


Brain ependymoma B R512
75379
5
13


Brain glioblastoma B GBM1062
59762
4
13


Brain medulloblastoma B H275
72318
5
13


Breast carcinoma associated 
73967
5
13


myofibroblast CD10+ AP T392303





Breast normal myoepithelium AP 
57222
4
13


myoepithelial1





Brain meningioma grade II B SF2366
48711
3
12


Skin Normal B hs0271
5807686
368
12


Skin Normal B hs0278
3042221
190
12


Brain ependymoma B R580
68614
4
11


Eye lens B UIH10
85898
5
11


Brain astrocytoma grade I B H1043
75922
4
10


Brain meningioma grade III B SF2366
59786
3
10


Brain glioblastoma control CL H247
60428
3
9


GallBladder Normal B HN
85706
4
9


Pancrease normal B 1
21520
1
9


Uterus Normal B hs0194
6452623
312
9


Brain medulloblastoma B 97-05-P312
74295
3
8


Brain meningioma grade I B 5F4271
69253
3
8


Brain normal thalamus B 1
24015
1
8


Skin Normal B hs0282
4411636
180
8


Brain astrocytoma grade III B 407
108312
4
7


Brain astrocytoma grade III B 584
103008
4
7


Brain ependymoma B 1394
56314
2
7


Brain ependymoma B R455
51825
2
7


Brain ependymoma B R582
52189
2
7


Brain fetal normal B 51
305546
12
7


Brain glioblastoma control B pooled
56428
2
7


Brain glioblastoma CL 28d
110624
4
7


Breast carcinoma associated stroma 
57049
2
7


B DCIS6





Breast carcinoma epithelium 
52342
2
7


CD44+ AP PE2





Breast normal epithelium CD44+ AP N1
56008
2
7


Breast normal stroma AP 1
79152
3
7


Breast normal stroma B IDC8
50485
2
7


Cartilage chondrosarcoma grade 2 
75019
3
7


CL UIFUO





Cartilage dedifferentiated chondrosarcoma 
108319
4
7


metastasis CL UIGPO





Esophagus normal B CN01
75634
3
7


Peritoneum normal B 13
53527
2
7


Skin Normal B hs0281
6517089
243
7


Spinal cord normal B 1
54785
2
7


Vascular endothelium normal breast 
57023
2
7


associated AP 1





Brain astrocytoma grade III B 828
99939
3
6


Brain ependymoma B 1150
62373
2
6


Brain glioblastoma CL 4d
115212
4
6


Breast carcinoma B IDC-4
64095
2
6


Breast carcinoma associated 
63539
2
6


myofibroblast CD10+ AP T112603





Breast normal myoepithelium 
63994
2
6


CD10+ AP N15





Brain astrocytoma grade III B 439
107824
3
5


Brain astrocytoma grade III B R927
107344
3
5


Brain astrocytoma grade II B H516
108116
3
5


Brain astrocytoma grade II B H518
116022
3
5


Brain astrocytoma grade II B H530
102439
3
5


Brain glioblastoma B H1110
68986
2
5


Brain glioblastoma CL stem cells
108414
3
5


Brain glioblastoma hypoxia CL H247
71765
2
5


Breast carcinoma B 95-259
39364
1
5


Breast carcinoma B IDC-3
68891
2
5


Breast carcinoma myoepithelium 
78785
2
5


X ITGB6+ AP





Breast normal FS NER
34565
1
5


Breast normal myoepithelium AP IDC7
69006
2
5


Breast normal myoepithelium 
76173
2
5


CD10+ AP N5





Skin Melanoma B hs0275
4128221
110
5


Skin normal B NS
36615
1
5


Brain astrocytoma grade III B H272
96059
2
4


Brain astrocytoma grade II B H501
128309
3
4


Brain glioblastoma B H1425C
88990
2
4


Brain normal cerebellum B BB542
40500
1
4


Breast carcinoma MD DCIS
40783
1
4


Breast normal epithelium AP 1
48729
1
4


Breast normal epithelium CD24+ AP N1
41551
1
4


Leukocytes normal B 1
47873
1
4


Ovary adenocarcinoma B OVT-6
41443
1
4


Retinal Pigment Epithelium normal 
90199
2
4


B 4PeriRPE





Skin cancer B hs0283
3692239
74
4


Brain astrocytoma grade III B H1055
109886
2
3


Brain astrocytoma grade III B R140
118733
2
3


Brain glioblastoma B R20
101053
2
3


Brain glioblastoma CL H54+EGFRvIII
56982
1
3


Brain medulloblastoma B 96-04-P019
52645
1
3


Brain meningioma grade I B SF4465
54647
1
3


Breast carcinoma B IDC-5
60451
1
3


Breast carcinoma epithelium 
52478
1
3


CD44+ AP ASC3





Breast normal epithelium AP Br N
50512
1
3


Breast normal organoid B
58181
1
3


Colon adenocarcinoma CL HCT116
55641
1
3


Embryonic stem cell H13 normal 
221101
4
3


p22 CL SHE15





Liver cholangiocarcinoma B K1
60319
1
3


Prostate carcinoma CL LNCaP
60250
1
3


Prostate normal B 2
64058
1
3


Prostate normal MD PR317
59277
1
3


Retina Pigment Epithelium normal B 1
53666
1
3


Thyroid normal B 001
115938
2
3


Vascular normal Cs control
51642
1
3


White blood cells monocyte depleted 
51634
1
3


mononuclear cells normal AP P1





Bone marrow normal AP CD34+/CD38+/lin+
81595
1
2


Brain medulloblastoma CL UW228-N
94937
1
2


Brain astrocytoma grade II B H563
88568
1
2


Brain normal leptomeninges B AL2
72473
1
2


Breast carcinoma MD LCIS
67834
1
2


Breast carcinoma associated stroma B IDC7
68024
1
2


Breast carcinoma epithelium AP DCIS6
72857
1
2


Breast carcinoma epithelium AP IDC7
73410
1
2


Breast carcinoma epithelium X MUC1+ AP
75147
1
2


Breast normal epithelium CD24+ AP N5
76469
1
2


Embryonic stem cell H9 normal 
401432
6
2


p38 CL SHES2





Embryonic stem cell HSF6 normal 
224488
3
2


p50 CL SHES9





GallBladder adenocarcinoma B HAIIb
66988
1
2


Heart normal B 1
83063
1
2


Lung normal B 1
88708
1
2


Lung tumor associated chronic 
98962
1
2


inflammation B UihMO





Lymph Node Lymphoma B 1
72008
1
2


Ovary endometriosis CL E12
76097
1
2


Skin cancer B hs0280
3618498
48
2


Vascular endothelium hemangioma B 146
75680
1
2


Bladder cancer B hs0241
3335801
17
1


Brain astrocytoma grade III B H970
106982
1
1


Brain astrocytoma grade II B H127
114489
1
1


Embryonic stem cell H14 normal 
212170
2
1


p22 CL SHE14





Embryonic stem cell H9 normal 
151735
1
1


p38 CL SHES1





Kidney carcinoma B D2
100281
1
1


Retina Macula normal B 4Mac
101417
1
1


Retina Peripheral normal B 2
105312
1
1


Retina macula normal B HMAC2
102417
1
1


Retina normal B 4cRet
103701
1
1


Skin cancer B has0284
3892507
38
1


Thyroid follicular adenoma B TT005
101578
1
1
















TABLE III







Summary of BOC expression in tumor tissues









Organ/Tissue
Tumor type
# of Boc positive












Brain
Brain ependynoma
11



Glioblastoma
11



Meningioma
4



Astrocytoma
16


Ovary
Ovary carcinoma
1



Ovary endometriosis
1



Ovary adenocarcinoma
2


Breast
Breast fibroadenoma
1



Breast carcinoma
19


Skin
Skin cancer
4



Skin melanoma
1


Liver
Liver cholangiocarcinoma
1


Prostate
Prostate carcinoma
1


Bladder
Bladder adenocarcinoma
1



Bladder cancer
1


Lung
Lung tumor
1


Lymph node
Lymph node lymphoma
1


Vascular endothelium
Vascular endothelium
1



hemangioma



Kidney
Kidney carcinoma
1


Thyroid gland
Thyroid follicular adenoma
1









Example 5
Detection of BOC Protein by Enzyme-Linked Immunosorbant Assay (ELISA)
An enzyme-linked immunosorbant assay (ELISA) that allows the specific detection of mouse BOC was developed (FIGS. 20 to 22). Two different antibodies, one targeting the extracellular (R&D Systems, Cat. No. AF2385) and one targeting the intracellular domain of mouse BOC (mouse Boc polyclonal rabbit antiserum generated against a Boc cytoplasmic domain synthetic peptide, RRTSESPGLESSWDPPYH (SEQ ID NO: 15), Okada et al., Nature (2006) 444: 369-373), can detect mouse BOC with high sensitivity (FIGS. 20 and 21), with a detection threshold of about 10 to 100 pg, depending on the type of sample analyzed. Moreover, these assays are specific for mouse BOC, with minimal or no cross-reaction with the highly-related homologous family member Cdo (mCdo) or the highly-conserved ortholog human BOC (hBOC; FIGS. 22A to C). Additionally, the antibody directed against the extracellular portion of BOC can detect BOC in the tissue culture media, while the antibody targeting the intracellular portion of BOC cannot (FIG. 21). These results indicate that a significant portion of BOC comprising an extracellular domain is shed from the cell surface. Similar observations were made by Western blots in different cell types tested (COS, 293 and NIH 3T3 cells).
Similarly to the assay described above, an ELISA was developed using an antibody (R&D Systems, Cat No. AF2036) that detects the extracellular domain of human BOC (hBOC; FIG. 23). This antibody had minimal cross-reaction with the highly-conserved ortholog mouse BOC (mBOC; FIG. 23). The detection threshold for this assay was between about 10 to 100 pg.
Example 7
Detection of BOC Protein and RNA Present in the Cerebrospinal Fluid and Blood
BOC protein could be present in the CSF as a shed extracellular domain of BOC or expressed by tumor cells detached from the tumor and found in the CSF. After CSF collection, the cells are separated from the liquid phase by centrifugation. Proteins and RNAs are isolated from the CSF cells. The presence of BOC protein in the CSF liquid phase (e.g., due to shed extracellular domain) and CSF cells (due to detached tumor cells) are determined by known techniques such as ELISA (as described above as well as any ELISA-related technologies), immunoprecipitation followed by Western blotting or quantitative mass spectrometry. The presence of BOC mRNA in CSF cells is determined by known techniques such as quantitative reverse-transcriptase PCR (qPCR).
In parallel, as brain tumor proteins are also sometimes found in the blood or expressed by tumor cells that have escaped from the tumor and entered the circulation (Hormigo, A. et al., Clin Cancer Res 12(19): 5698 (2006); Tanwar, M. K. et al., Cancer Res 62(15): 4364 (2002)), the presence of BOC protein in the blood at this late tumorigenic stage is determined. This is done by known techniques such as ELISA (as described above), Sandwich immunoprecipitation followed by Western blotting or quantitative mass spectrometry performed on blood serum. In addition, ELISA and qPCR are performed on cells isolated from the blood.
The measurement is used as a diagnostic tool for the tailor-made therapeutic approach applicable to each patient (e.g., patient stratification). For example, a higher level of BOC protein or RNA in the tested sample than in a reference sample may be indicative of a predisposition to medulloblastoma. A higher level of BOC protein or RNA may also be indicative of a need for a treatment targeting BOC activity, or a more aggressive treatment.

## Claims

1. A method for diagnosing a cancer or a predisposition to cancer in a first subject comprising:
(a) determining a level and/or activity of BOC in a sample from said first subject;
(b) comparing said level and/or activity to a corresponding reference level and/or activity; and
(c) diagnosing said cancer or predisposition to cancer based on said comparison,

wherein said cancer is a brain tumor, an ovary tumor, a breast tumor, a glioblastoma, a skin tumor, a meningioma, an astrocytoma, a liver tumor, a prostate carcinoma, a bladder tumor, a lung tumor, a lymph node lymphoma, a vascular endothelium hemangioma, a kidney carcinoma or a thyroid follicular adenoma; and
wherein a higher level and/or activity of said BOC in said sample from said first subject as compared to said reference level and/or activity is indicative that said first subject has cancer or a predisposition to cancer.

2. The method of claim 1, wherein said reference level and/or activity corresponds to a level and/or activity determined in a sample from a control subject known to not being predisposed to cancer and not having cancer.

3. The method of claim 1, wherein said brain tumor is a neuroectodermal tumor.

4. The method of claim 1, wherein said neuroectodermal tumor is a medulloblastoma.

5. The method of claim 1, wherein said sample is cerebrospinal fluid.

6. The method of claim 1, wherein the level of BOC is determined by determining the level of a nucleic acid encoding the polypeptide comprising the sequence of SEQ ID NO: 2.

7. The method of claim 6, wherein said nucleic acid comprises the coding sequence of the nucleotide sequence of SEQ ID NO: 1.

8. The method of claim 1, wherein the level of BOC is determined by determining the level of a polypeptide comprising the sequence of SEQ ID NO: 2, or of a fragment thereof.

9. The method of claim 8, wherein said fragment is an extracellular fragment.

10. The method of claim 1, wherein said cancer is a liver tumor.

11. The method of claim 1, wherein said cancer is an ovary tumor.

12. The method of claim 1, wherein said cancer is a breast tumor.

13. The method of claim 1, wherein said cancer is a glioblastoma.

14. The method of claim 1, wherein said cancer is a skin tumor.

15. The method of claim 1, wherein said cancer is a meningioma.

16. The method of claim 1, wherein said cancer is an astrocytoma.

17. The method of claim 1, wherein said cancer is a prostate carcinoma.

18. The method of claim 1, wherein said cancer is a bladder tumor.

19. The method of claim 1, wherein said cancer is a lung tumor.

20. The method of claim 1, wherein said cancer is a lymph node lymphoma.

21. The method of claim 1, wherein said cancer is a vascular endothelium hemangioma.

22. The method of claim 1, wherein said cancer is a kidney carcinoma.

23. The method of claim 1, wherein said cancer is a thyroid follicular adenoma.