WO2011110683A2 - Glce as a target for anti-tumour therapy - Google Patents
Glce as a target for anti-tumour therapy Download PDFInfo
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- WO2011110683A2 WO2011110683A2 PCT/EP2011/053743 EP2011053743W WO2011110683A2 WO 2011110683 A2 WO2011110683 A2 WO 2011110683A2 EP 2011053743 W EP2011053743 W EP 2011053743W WO 2011110683 A2 WO2011110683 A2 WO 2011110683A2
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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Definitions
- the present invention refers to the inhibition of GLCE (D-glucuronyl-C5- epimerase) (GenBank Acc No. NM_015554) for the treatment of diseases, particularly hyperproliferative and/or infectious diseases.
- GLCE D-glucuronyl-C5- epimerase
- Heparan sulfate is a negatively charged polysaccharide present at the cell surface that mediates the selective binding and cellular internalization of circulating soluble and insoluble extracellular ligands including growth factors and viral proteins.
- HS is composed of repeating disaccharide units of D-glucuronic acid (GIcA) or L-iduronic acid (IdoA) both of which may be 20-sulfated, and of unsubstituted, N-acetylated, or N-, 30- or 6O-sulfated glucosamine (GlcN).
- Typical HS chains contain relatively short segments of modified sequences represented by IdoA-GlcNS derivatives of different sulfation content dispersed among large sections of unmodified (GlcA-GlcNAc) units (Esko 2002).
- HS is synthesized through a series of steps whose regulation is still poorly understood.
- a key modification of the nascent polysaccharide chain is carried out in the Golgi apparatus by the GLCE (D-glucuronyl C5-epimerase) enzyme which converts GIcA (glucuronic acid) into IdoA (iduronic acid).
- GLCE is a rate limiting enzyme of HS biosynthesis and the modification this enzyme introduces in the HS chain is irreversible.
- GIcA epimerization releases the conformational constraints of the polysaccharide chain allowing the access of ligands to specific regions of HS (Ferro 990).
- IdoA residues are more frequently 2-O-sulphated than GIcA residues resulting in the appearance of clustered, negatively charged domains along the glycosaminoglycan chain. IdoA residues are invariably present in the HS sequences that bind soluble proteins and viruses (Casu 2001 ). Transgenic mice lacking GLCE are not viable (Li 2003) and modulation of the enzyme activity affects embryonic body axis formation (Ghiselli 2005a). The available information on the biology of GLCE corroborates the idea that the enzyme introduces a key modification in the HS structure that is necessary for the biological activity of the polysaccharide.
- GLCE expression is activated through the -catenin-TCF4 transactivation signaling pathway that is disregulated in many tumors.
- the expression of GLCE correlates with the degree of activation of the ⁇ - catenin-TCF4 pathway.
- GLCE might be an antitumor target and, in particular, that the inhibition of GLCE expression and/or enzymatic activity might lead to a regression of tumor growth.
- the prior art does not answer the question of how GLCE expression contributes (if at all) to tumorigenesis.
- GLCE inhibitors may be regarded as novel therapeutic approach in medicine, particularly to the treatment of cancer and/or infectious diseases such as viral infections. More particularly, the inventors have found that inhibition of GLCE effects at the same time cell growth, cell adhesion and angiogenesis.
- a first aspect of the present invention refers to a GLCE inhibiting agent for use a medicament.
- the inhibitor may be used for the treatment of hyperproliferative diseases and/or infectious diseases.
- the GLCE inhibitor may be used for the treatment of hyperproliferative diseases selected from cancer, e.g. a non-metastatic or metastatic cancer, or a drug refractory, e.g. multi-drug resistant cancer.
- the cancer may be bone, brain, breast, cervix, colon, gastric, liver, lung, pancreas, ovarian, renal, pancreas, prostate, stomach, soft tissue, bone marrow or skin cancer, e.g. melanoma or a lymphoma.
- cancer is a breast cancer, a colon cancer or a skin cancer.
- the hyperproliferative disease may also be a vascular disease such as artherosclerosis or restenosis.
- the infectious disease may be selected from viral, bacterial and/or parasitic infections.
- the infectious disease is a viral infection by e.g. hepatitis C virus, herpes simplex virus, respiratory syncytial virus, human papillomavirus and foot- and mouth-disease virus.
- the GLCE inhibiting agent may be a substance which acts as an anti- metastatic, anti-angiogenic and/or antiproliferative agent.
- the agent may inhibit metastasis formation, tumor angiogenesis (tubulogenesis), and/or p53-independent cell growth. Further, the agent may reduce or reverse drug resistance, e.g. multi-drug resistance of tumor cells.
- the GLCE inhibiting agent may be selected from inhibitors of GLCE acting on the nucleic acid level, e.g. inhibiting GLCE transcription and/or translation.
- the GLCE inhibiting agent may be an inhibitor of GLCE acting on the protein level, e.g. by binding to GLCE and thereby reducing its activity.
- the inhibitor of a GLCE nucleic acid may be a GLCE gene expression inhibitor, preferably selected from nucleic acid effector molecules directed against GLCE mRNA such as RNAi molecules or precursors or templates thereof, antisense molecules or ribozymes.
- RNAi molecules are RNA molecules or RNA analogs capable of mediating an interference of a target mRNA molecule.
- RNAi molecules may be siRNA molecules (short interfering RNA molecules) which are short double- stranded RNA molecules with a length of preferably 18-30 nucleotides and optionally at least one 3' overhang.
- RNAi molecules may be shRNA molecules (short hairpin RNA molecules) having a length of e.g. 14-50 nucleotides.
- the RNAi molecules may comprise ribonucleotide analogs in order to enhance the stability against degradation.
- the invention also encompasses precursors of RNAi molecules, i.e. RNA molecules which are processed by cellular mechanisms into active RNAi molecules.
- the invention encompasses DNA templates of RNAi molecules or precursors thereof, wherein the templates are operatively linked to an expression control sequence.
- the RNAi molecules have sufficient complementarity to the GLCE mRNA to allow specific degradation thereof, thereby inhibiting GLCE expression.
- the nucleic acid inhibitor molecule may be an antisense molecule, i.e. an antisense RNA, DNA or nucleic acid analog molecule which blocks translation of GLCE mRNA by binding thereto and preventing translation.
- Antisense molecules may be single-stranded and have preferably a length of 14-30 nucleotides. Antisense molecules directed against the translation initiation site of GLCE mRNA are preferred.
- the GLCE nucleic acid inhibitor may be a ribozyme.
- Ribozymes are enzymatic RNA molecules which catalyze specific cleavage of RNA, e.g. hammerhead ribozymes.
- the GLCE inhibiting agent is an siRNA molecule which is a double-stranded nucleic acid molecule of 18-30, preferably of 19-25 nucleotides or nucleotide analogs which optionally has at least one 3' overhang of 1-3, preferably of two nucleotides.
- the siRNA molecule may comprise nucleotide analogs, i.e. base, sugar and/or backbone modified ribonucleotides.
- the siRNA molecule is specifically directed against mammalian GLCE mRNA, particularly against human GLCE mRNA. Specific embodiments of preferred siRNA molecules have a sense strand selected from:
- the GLCE inhibitor acts on the protein level.
- the inhibitor may be selected from an antibody specific for GLCE or an antigen-binding fragment thereof, an aptamer directed against GLCE, a mutated form of the GLCE ligand or an inhibitor of GLCE ligand binding.
- the GLCE inhibitor is an antibody.
- the antibody may be selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody, or a fragment thereof.
- a host animal e.g. a mouse or rabbit
- a GLCE antigen may be immunized with a GLCE antigen, optionally together with an adjuvant to increase the immunological response.
- a monoclonal antibody may be prepared by using known techniques including, but not limited to the hybridoma technique developed by Kohler and Millstein.
- Chimeric antibodies may be obtained from monoclonal antibodies by replacing non-human constant regions by appropriate human constant regions.
- Humanized antibodies may be obtained by replacing non-human framework regions in the variable antibody domains by appropriate human sequences.
- Human antibodies may be obtained from host animals, e.g. mice, comprising a xenogenic human immune system.
- Recombinant antibodies may be obtained by phage display and affinity maturation of given antibody sequences.
- Recombinant antibodies may be single chain antibodies, bispecific antibodies etc.
- Antibody fragments which contain at least one binding site for GLCE may be selected from Fab fragments, Fab' fragments, F(ab') 2 fragments or single chain Fv fragments. Aptamers directed against GLCE may be obtained by affinity selection of nucleic acid and/or peptidic sequences according to known protocols.
- Mutated GLCE ligands may be selected from cellular interaction partners of GLCE which have been modified by mutation, e.g. substitution, deletion and/or addition of amino acid residues.
- interactions partners are 2-O-sulfotransferase (GenBank Acc No. AB024568) or calcium modulating ligand, CAML (GenBank Acc No. NM_001745).
- Inhibitors of GLCE ligand binding may be selected from negatively charged polymers or oligosaccharides.
- examples of such inhibitors are suramin or peptide dendrimers or oligosaccharides obtained by chemical or enzymatic degradation of heparin or heparin sulphate, e.g. low-molecular weight heparins.
- a further aspect of the invention is a pharmaceutical composition which comprises as an active agent at least one GLCE inhibitor as described above together with a pharmaceutically acceptable carrier, diluent and/or adjuvant.
- the pharmaceutical composition may be formulated e.g. as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions etc.
- composition may be administered systemically or locally.
- Suitable routes may, e.g. include oral, rectal, transmucosal, intestinal, intranasal, intraocular or pulmonal administration or parenteral delivery including intramuscular, subcutaneous, intrathecal, intravenous or intraperitoneal injection or infusion.
- the pharmaceutical composition comprises the active agent in an effective dose sufficient to achieve its intended purpose. Determination of an effective dose can be carried out by the skilled person. For example, the effective dose may be estimated from cell culture assays and animal models. Usual dosages for administration in human medicine may range from e.g. 0.01- 2000 mg/day, commonly from 0.1-1000 mg/day and typically from 1-500 mg/day.
- the pharmaceutical composition is preferably for use in medicine, e.g. in human or veterinary medicine.
- a method of treating a subject suffering from a hyperproliferative disease and/or an infective disease comprising administering to a subject, particularly to a human subject, a pharmaceutically effective amount of a GLCE inhibiting agent or a pharmaceutical composition comprising at least one GLCE inhibiting agent as an active ingredient.
- the hyperproliferative disease is preferably cancer, including metastatic cancer as described above.
- the GLCE inhibiting agent may be administered as a monotherapy or in combination with an additional therapy, e.g. with an additional anti-cancer or antiviral therapy.
- Additional anti-cancer therapy may include chemotherapy, immunotherapy, gene therapy, radiation therapy, surgery or on any combinations thereof.
- the GLCE inhibiting agent may be co- administered with at least one further anti-cancer agent, e.g. a chemotherapeutic agent.
- the anti-cancer agent may, for example, be selected from antimetabolites, DNA-fragmenting agents, DNA-crosslinking agents, intercalating agents, protein synthesis inhibitors, Topoisomerase 1 and 2 inhibitors, microtubule-directed agents, kinase inhibitors, hormones and hormone antagonists, anti-tumor antiboides, or any combination thereof.
- the anti-cancer agent is selected from platinum compounds such as cis-platin, carboplatin or oxaiiplatin.
- the GLCE inhibiting agent may be administered in combination with an additional therapy directed against the infectious agent, particularly together with at least one further antiviral agent.
- the antiviral agent may be selected from a protease inhibitor, a polymerase inhibitor, an integrase inhibitor, an entry inhibitor, an assembly/secretion inhibitor, a translation inhibitor, an immunostimulant or any combination thereof.
- a further aspect of the present invention is directed to a pharmaceutical composition or kit which comprises at least one GLCE inhibiting agent as described above in combination with at least one further medicament, e.g. with at least one further anti-cancer agent and/or with at least one further antiviral agent as described above.
- Still a further aspect of the present invention is a method for diagnosing an hyperproliferative disease and/or infectious disease comprising the step of measuring the expression, abundance and/or amount of GLCE in a sample from a subject suffering or suspected to suffer from said disease.
- the diagnostic method particularly allows monitoring the course of the disease by carrying out a plurality of measuring steps, particularly in the case, where a subject undergoes a therapy, in particular therapy with a GLCE inhibiting agent as described above.
- the sample may be a body fluid sample, e.g. blood, serum, plasma, sputum, saliva, lymph fluid etc. or a tissue sample, e.g. a tissue section or biopsy.
- the determination of GLCE may be carried out on the nucleic acid level, e.g. by determining GLCE mRNA and/or on the protein level, e.g. by determining GLCE protein and/or GLCE activity.
- a determination on the nucleic acid level may involve the use of a hybridization protocol, wherein a hybridization probe complementary to GLCE transcripts or any nucleic acid derived therefrom, e.g. cDNA, is employed.
- the determination may comprise nucleic acid amplification according to known protocols, such as PCR.
- Determination of GLCE on the protein level may comprise immunological methods involving the use of anti-GLCE antibodies or fragments thereof or determination of GLCE enzymatic activity, e.g. as described in Ghiselli et al., 2005b, supra, the content of which is herein incorporated by reference.
- the present invention also refers to a diagnostic composition or kit comprising a detection reagent for GLCE, particularly for use in the method as described above.
- the present invention also refers to a method of screening for an anti- hyperproliferative agent or anti-pathogen agent, e.g. antiviral agent comprising the steps of determining if a test compound is an inhibitor of GLCE.
- the screening method may, for example, comprise contacting of GLCE with a test compound and determining binding of the test compound to GLCE and/or determining the activity of GLCE in presence of the test compound compared to a control, e.g. in the absence of the test compound.
- the screening may be carried out in a cell-free or cellular system.
- the use of recombinant cells or non-human organisms transfected with GLCE and preferably overexpressing GLCE is preferred.
- test compounds may be selected from polypeptides, e.g antibodies or antibody fragments, aptamers, peptidic compounds or non-peptidic low molecular weight organic molecules (e.g. having a molecular weight of up to 2000 Da).
- a test compound which is identified as a GLCE inhibitor in a screening method as described above may be a suitable candidate agent for the treatment of hyperproliferative diseases and/or infectious diseases.
- the screening method may also be used for identification of cellular interaction partners of GLCE, e.g. polypeptides capable of forming multimeric complexes with GLCE.
- An example of such an interaction partner of GLCE is CAML (calcium-modulating ligand, GenBank Acc No. NM_001745).
- Figure 1 siRNA mediated downregulation of GLCE transcript level in tumoral cells.
- MCF7 human breast carcinoma cells were plated in 6-wells plate at 25% confluence and transfected 8 h later with 0 to 62 ng/ml of GLCE-siRNA or non-silencing siRNA oligonucleotide with sequence that has no homology to any known mammalian gene, henceforth identified as scRNA. After 40 h, the transfection was repeated using the same amount of GLCE-siRNA. At 96 h post-plating, the medium was removed, the cells washed in PBS and dissolved in Triazol DNA-RNA extracting reagent. After reverse transcription of total mRNA, gene-specific transcript levels were assessed by real-time PCR on a Roche Light-cycler 480.
- Figure 2 GLCE knock-down inhibits P-selectin and fibronectin mediated attachment of highly metastatic melanoma cells.
- B16F10 highly metastatic murine melanoma cells were seeded in 6-wells plates at 20% confluence and after 8 h transfected with 20 ng/ml GLCE- siRNA or scRNA (control). The transfection was repeated at 48 h post-plating and the cells harvested by mild trypsinization 40 h later. The cells were incubated and suspended in DMEM containing 1 % BSA. For the cell adhesion experiments, 96-wells plates were coated with Fibronectin (0.33 ng/ml) or P-Selectin (10 ng/ml) by overnight incubation at 4°C. In the morning the proteins were removed and the plates washed twice with PBS.
- FIG. 3 GLCE knock-down prevents tubulogenesis.
- ECV304 human endothelial cells were seeded in 6-wells plates at 20% confluence and after 8 h transfected with 20 ng/ml GLCE-siRNA or scRNA (control). The transfection was repeated 40 h later and the cells harvested by mild trypsinization 96 h post-plating.
- 96-well plates were coated with Matrigel by overnight incubation at 4°C.
- GLCE-siRNA or scRNA transfected cells resuspendent in DMEM/1 % BSA were then pipetted into the wells (10,000 cells).
- a group of cells also received 10 ng/ml of human recombinant BMP2.
- the plates were placed in a 5% C0 2 incubator at 37°C and examined periodically under a light inverted microscope. Representative results are shown.
- Figure 4 GLCE knock-down inhibits cell growths in a p53-independent fashion.
- HCT116 and SW480 human colon carcinoma cells were plated in 24-well plates at 20% confluence and transfected with 40 ng/ml GLCE-siRNA or scRNA (control) 8 h later. The treatment was repeated after 40 h and the cells harvested by mild trypsinization at 96 h post-plating. Resuspended cells were fixed in ice-cold methanol and DNA-stained with Propidium Iodide.
- Figure 6 Identification of GLCE interacting molecules.
- a yeast two-hybrid system was used to screen a Clontech Matchmaker pre- transformed human fetal brain cDNA library cloned in in pACT2 vector.
- AH 09 yeast was first transformed with the bait vector harboring the full length GLCE cDNA cloned in pGBKT7 plasmid, and grown on SD medium lacking tryptophan (SD-Trp) agar plates. Growth of the transformed yeast on SD-Trp/X- -Gal plates, confirmed that the reporter system is not activated by GLCE.
- SD-Trp SD medium lacking tryptophan
- the mating mixture was then plated onto SD-Ade/-His/-Leu/-Trp plates and grown at 30°C for 5 days. Positive colonies were rerankedd onto SD-Ade/-His/-Leu/-Trp/X-p-Gal plates and blue colonies used for the isolation of cDNAs encoding the interacting proteins. Eleven colonies were isolated and E.coli DH5-a amplified cDNA on ampicillin plates, was sequenced. The transcript of seven of the colonies matched that of the full length (calcium modulating ligand, GenBank Acc No. NM_001745) gene product.
- RNA antisense oligonucleotides targeting human or murine GLCE transcripts were obtained from Qiagen and had the following sequence:
- Annealed double-stranded siRNAs were transfected into cells using Hyperfect (Qiagen) transfection vehicle following the manufacturer's guidelines to optimize the transfection vehicle-to-siRNA mixing ratio.
- Example 1 siRNA-mediated down regulation of GLCE transcript level in tumoral cells.
- siRNA-mediated GLCE-expression knockdown MCF7 human breast carcinoma cells were used in these experiments. The cells were transfected with an equimolar mixture of four siRNA oligonucleotides targeting different region of the GLCE coding sequence. This protocol was adopted to limit potential off-target effects of the transfected siRNAs that would change the expression of other genes or have cytotoxic effects.
- siRNA non- silencing siRNA oligonucleotide
- the effect of the antisense oligonucleotides was monitored by monitoring by real-time PCR the transcript level of the targeted gene as well as that of two house-keeping genes such as ⁇ -actin and 18S rRNA subunit, whose expression has been shown not to change in a variety of experimental conditions.
- the effect of siRNA on GLCE expression was computed as percent reduction of GLCE transcript level compared to that of that of cells transfected with an equimolar amount of scRNA.
- Optimal downregulation of GLCE expression could be achieved by transfecting cells with GLCE-siRNA 8 h after plating, followed by a second transfection 40 h later with the same amount of siRNA. Using this protocol maximal GLCE expression knockdown was reached between 60 and 96 h after plating. The dose-response curves showed there is a positive correlation between the dose of siRNA employed (ranging between 0 to 60 ng/ml), the suppression of GLCE expression and the inhibition of cell growth pointing to a causal relationship between GLCE transcript level and cell proliferation rate ( Figure 1 ).
- Example 2 GLCE knockdown inhibits P-selectin- and fibronectin-mediated attachment of highly metastatic melanoma cells.
- Example 3 GLCE knockdown prevents tubulogenesis, an index of angiogenic activity of human endothelial cells.
- Angiogenesis involves the sprouting of new capillaries from tumor vessels that is essential for the sustainment with oxygen and nutrients of the growing tumor mass (Folkman 2007).
- the new vessel also provide the means for cancerous cells to migrate through the bloodstream to distant organs where can metastasize.
- Angiogenesis relies on the intrinsic ability of endothelial cells to migrate and aggregate to form tube-like structures. This spontaneous ability of endothelial cells to self-organize into vessels is enhanced by a number of growth factors and cytokines among which VEGF (vascular endothelial growth factor) and Angiomodulin are the best studied. For optimal angiogenetic activity many of these factors require the presence of HS.
- GLCE knockdown inhibits cell growth in a p53-independent fashion. Because GLCE-knockdown inhibited cell growth of several tumor cell lines including colon carcinoma cells, we investigated whether this effect was p53- dependent and if apoptosis was involved. Two human colon carcinoma cell lines, HCT116 (p53 proficient) and SW480 (p53 deficient), were investigated ( Figure 4). The distribution of cells at different stages of the cell cycle was analyzed in cell transfected with GLCE-siRNA or scRNA. Because of the defective p53-dependent checkpoint, a large fraction of SW480 cells were found in S and G2/M phase compared to p53-proficient HCT116 cells which were mostly maintained in G1 state.
- Example 5 GLCE-knockdown inhibits cell growth of cisplatin refractive colon carcinoma cells.
- first-line oncological treatments depends among other, upon a functional p53 system. Due to the high frequency of p53 mutations in human cancer compounded by the development of multidrug resistance, clinical efficacy of chemotherapy is poor especially in patients requiring long treatment. Oncological treatment also elicits strong side-effects that are dose-related.
- Cisplatin is a DNA- damaging antitumor compounds displaying clinical activity against a wide variety of solid tumors (Sedletska 2005). p53-proficent HCT116 colon carcinoma cells display a strong sensitivity to the apoptotic activity of cisplatin (Figure 5).
- Ghiselli G, Farber SA D-glucuronyl C5-epimerase acts in dorso-ventral axis formation in zebrafish.
- Ghiselli G, Agrawal A The human D-glucuronyl C5-epimerase gene is transcriptionally activated through the beta-catenin-TCF4 pathway. Biochem J 2005b, 390: 493-499.
- Cisplatin is a DNA-damaging antitumour compound triggering multifactorial biochemical responses in cancer cells: importance of apoptotic pathways. Curr Med Chem Anticancer Agents 2005, 5: 251-265.
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Abstract
The present invention refers to the inhibition of GLCE (D-gIucuronyl-C5- epimerase) for the treatment of diseases, particularly hyperproliferative and/or infectious diseases.
Description
GLCE as a target for anti-tumour therapy
The present invention refers to the inhibition of GLCE (D-glucuronyl-C5- epimerase) (GenBank Acc No. NM_015554) for the treatment of diseases, particularly hyperproliferative and/or infectious diseases.
Heparan sulfate (HS) is a negatively charged polysaccharide present at the cell surface that mediates the selective binding and cellular internalization of circulating soluble and insoluble extracellular ligands including growth factors and viral proteins.
HS is composed of repeating disaccharide units of D-glucuronic acid (GIcA) or L-iduronic acid (IdoA) both of which may be 20-sulfated, and of unsubstituted, N-acetylated, or N-, 30- or 6O-sulfated glucosamine (GlcN). Typical HS chains contain relatively short segments of modified sequences represented by IdoA-GlcNS derivatives of different sulfation content dispersed among large sections of unmodified (GlcA-GlcNAc) units (Esko 2002).
HS is synthesized through a series of steps whose regulation is still poorly understood. A key modification of the nascent polysaccharide chain is carried out in the Golgi apparatus by the GLCE (D-glucuronyl C5-epimerase) enzyme which converts GIcA (glucuronic acid) into IdoA (iduronic acid). GLCE is a rate limiting enzyme of HS biosynthesis and the modification this enzyme introduces in the HS chain is irreversible. GIcA epimerization releases the conformational constraints of the polysaccharide chain allowing the access of ligands to specific regions of HS (Ferro 990). IdoA residues are more frequently 2-O-sulphated than GIcA residues resulting in the appearance of clustered, negatively charged domains along the glycosaminoglycan chain. IdoA residues are invariably present in the HS sequences that bind soluble proteins and viruses (Casu 2001 ). Transgenic mice lacking GLCE are not viable (Li 2003) and modulation of the enzyme
activity affects embryonic body axis formation (Ghiselli 2005a). The available information on the biology of GLCE corroborates the idea that the enzyme introduces a key modification in the HS structure that is necessary for the biological activity of the polysaccharide.
We have reported (Ghiselli 2005b) that GLCE expression is activated through the -catenin-TCF4 transactivation signaling pathway that is disregulated in many tumors. In a set of human colon carcinoma cell lines, the expression of GLCE correlates with the degree of activation of the β- catenin-TCF4 pathway.
The publication, however, does not indicate that GLCE might be an antitumor target and, in particular, that the inhibition of GLCE expression and/or enzymatic activity might lead to a regression of tumor growth. Thus, in summary, the prior art does not answer the question of how GLCE expression contributes (if at all) to tumorigenesis.
The present inventors carried out experiments for knocking out GLCE expression in cell models of tumor growth, metastasis and angiogenesis and assessment whether the tumorigenic behaviour was altered. Results show that GLCE inhibition may inhibit/reduce tumorigenesis on different levels. Thus, GLCE inhibitors may be regarded as novel therapeutic approach in medicine, particularly to the treatment of cancer and/or infectious diseases such as viral infections. More particularly, the inventors have found that inhibition of GLCE effects at the same time cell growth, cell adhesion and angiogenesis.
Thus, a first aspect of the present invention refers to a GLCE inhibiting agent for use a medicament. Particularly, the inhibitor may be used for the treatment of hyperproliferative diseases and/or infectious diseases. More particularly, the GLCE inhibitor may be used for the treatment of hyperproliferative diseases selected from cancer, e.g. a non-metastatic or metastatic cancer, or a drug refractory, e.g. multi-drug resistant cancer. The
cancer may be bone, brain, breast, cervix, colon, gastric, liver, lung, pancreas, ovarian, renal, pancreas, prostate, stomach, soft tissue, bone marrow or skin cancer, e.g. melanoma or a lymphoma. More particularly, cancer is a breast cancer, a colon cancer or a skin cancer. The hyperproliferative disease may also be a vascular disease such as artherosclerosis or restenosis. The infectious disease may be selected from viral, bacterial and/or parasitic infections. Particularly, the infectious disease is a viral infection by e.g. hepatitis C virus, herpes simplex virus, respiratory syncytial virus, human papillomavirus and foot- and mouth-disease virus.
The GLCE inhibiting agent may be a substance which acts as an anti- metastatic, anti-angiogenic and/or antiproliferative agent. For example, the agent may inhibit metastasis formation, tumor angiogenesis (tubulogenesis), and/or p53-independent cell growth. Further, the agent may reduce or reverse drug resistance, e.g. multi-drug resistance of tumor cells.
The GLCE inhibiting agent may be selected from inhibitors of GLCE acting on the nucleic acid level, e.g. inhibiting GLCE transcription and/or translation. Alternatively, the GLCE inhibiting agent may be an inhibitor of GLCE acting on the protein level, e.g. by binding to GLCE and thereby reducing its activity.
The inhibitor of a GLCE nucleic acid may be a GLCE gene expression inhibitor, preferably selected from nucleic acid effector molecules directed against GLCE mRNA such as RNAi molecules or precursors or templates thereof, antisense molecules or ribozymes.
RNAi molecules are RNA molecules or RNA analogs capable of mediating an interference of a target mRNA molecule. RNAi molecules may be siRNA molecules (short interfering RNA molecules) which are short double- stranded RNA molecules with a length of preferably 18-30 nucleotides and optionally at least one 3' overhang. Further, RNAi molecules may be shRNA molecules (short hairpin RNA molecules) having a length of e.g. 14-50
nucleotides. Optionally, the RNAi molecules may comprise ribonucleotide analogs in order to enhance the stability against degradation. The invention also encompasses precursors of RNAi molecules, i.e. RNA molecules which are processed by cellular mechanisms into active RNAi molecules. Further, the invention encompasses DNA templates of RNAi molecules or precursors thereof, wherein the templates are operatively linked to an expression control sequence. The RNAi molecules have sufficient complementarity to the GLCE mRNA to allow specific degradation thereof, thereby inhibiting GLCE expression.
In a further embodiment, the nucleic acid inhibitor molecule may be an antisense molecule, i.e. an antisense RNA, DNA or nucleic acid analog molecule which blocks translation of GLCE mRNA by binding thereto and preventing translation. Antisense molecules may be single-stranded and have preferably a length of 14-30 nucleotides. Antisense molecules directed against the translation initiation site of GLCE mRNA are preferred.
In a further embodiment, the GLCE nucleic acid inhibitor may be a ribozyme. Ribozymes are enzymatic RNA molecules which catalyze specific cleavage of RNA, e.g. hammerhead ribozymes.
In an especially preferred embodiment, the GLCE inhibiting agent is an siRNA molecule which is a double-stranded nucleic acid molecule of 18-30, preferably of 19-25 nucleotides or nucleotide analogs which optionally has at least one 3' overhang of 1-3, preferably of two nucleotides. The siRNA molecule may comprise nucleotide analogs, i.e. base, sugar and/or backbone modified ribonucleotides. The siRNA molecule is specifically directed against mammalian GLCE mRNA, particularly against human GLCE mRNA. Specific embodiments of preferred siRNA molecules have a sense strand selected from:
(i) S'-CUCGAUGAUAACAGUAUGGUA-S" (SEQ ID No 1 ) (ϋ) 5'-UGGAAACUUAUUACAAAUCUA-3 (SEQ ID No 2)
(iii) 5'-CUGGGCCUAUCUGUUAACAAA-3' (SEQ ID No 3),
(iv) 5'-CACAGUAAAUAUGUACUUGUA-3' (SEQ ID No 4), or
(v) a nucleotide sequence which has an identity degree of at least 85%, at least 90% or at least 95% to one any one of the sequences SEQ
ID No 1 to 4.
In a further embodiment of the invention, the GLCE inhibitor acts on the protein level. In this embodiment, the inhibitor may be selected from an antibody specific for GLCE or an antigen-binding fragment thereof, an aptamer directed against GLCE, a mutated form of the GLCE ligand or an inhibitor of GLCE ligand binding.
Preferably, the GLCE inhibitor is an antibody. The antibody may be selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody, or a fragment thereof.
For the production of antibodies, a host animal, e.g. a mouse or rabbit, may be immunized with a GLCE antigen, optionally together with an adjuvant to increase the immunological response. A monoclonal antibody may be prepared by using known techniques including, but not limited to the hybridoma technique developed by Kohler and Millstein. Chimeric antibodies may be obtained from monoclonal antibodies by replacing non-human constant regions by appropriate human constant regions. Humanized antibodies may be obtained by replacing non-human framework regions in the variable antibody domains by appropriate human sequences. Human antibodies may be obtained from host animals, e.g. mice, comprising a xenogenic human immune system. Recombinant antibodies may be obtained by phage display and affinity maturation of given antibody sequences. Recombinant antibodies may be single chain antibodies, bispecific antibodies etc.
Antibody fragments which contain at least one binding site for GLCE may be selected from Fab fragments, Fab' fragments, F(ab')2 fragments or single chain Fv fragments. Aptamers directed against GLCE may be obtained by affinity selection of nucleic acid and/or peptidic sequences according to known protocols.
Mutated GLCE ligands may be selected from cellular interaction partners of GLCE which have been modified by mutation, e.g. substitution, deletion and/or addition of amino acid residues. Examples of such interactions partners are 2-O-sulfotransferase (GenBank Acc No. AB024568) or calcium modulating ligand, CAML (GenBank Acc No. NM_001745).
Inhibitors of GLCE ligand binding may be selected from negatively charged polymers or oligosaccharides. Examples of such inhibitors are suramin or peptide dendrimers or oligosaccharides obtained by chemical or enzymatic degradation of heparin or heparin sulphate, e.g. low-molecular weight heparins. A further aspect of the invention is a pharmaceutical composition which comprises as an active agent at least one GLCE inhibitor as described above together with a pharmaceutically acceptable carrier, diluent and/or adjuvant. The pharmaceutical composition may be formulated e.g. as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions etc. Depending on the specific disorder to be treated, the composition may be administered systemically or locally. Suitable routes may, e.g. include oral, rectal, transmucosal, intestinal, intranasal, intraocular or pulmonal administration or parenteral delivery including intramuscular, subcutaneous, intrathecal, intravenous or intraperitoneal injection or infusion.
The pharmaceutical composition comprises the active agent in an effective dose sufficient to achieve its intended purpose. Determination of an effective dose can be carried out by the skilled person. For example, the effective
dose may be estimated from cell culture assays and animal models. Usual dosages for administration in human medicine may range from e.g. 0.01- 2000 mg/day, commonly from 0.1-1000 mg/day and typically from 1-500 mg/day.
The pharmaceutical composition is preferably for use in medicine, e.g. in human or veterinary medicine. Thus, it is provided a method of treating a subject suffering from a hyperproliferative disease and/or an infective disease comprising administering to a subject, particularly to a human subject, a pharmaceutically effective amount of a GLCE inhibiting agent or a pharmaceutical composition comprising at least one GLCE inhibiting agent as an active ingredient. The hyperproliferative disease is preferably cancer, including metastatic cancer as described above. The GLCE inhibiting agent may be administered as a monotherapy or in combination with an additional therapy, e.g. with an additional anti-cancer or antiviral therapy. Additional anti-cancer therapy may include chemotherapy, immunotherapy, gene therapy, radiation therapy, surgery or on any combinations thereof. For example, the GLCE inhibiting agent may be co- administered with at least one further anti-cancer agent, e.g. a chemotherapeutic agent. The anti-cancer agent may, for example, be selected from antimetabolites, DNA-fragmenting agents, DNA-crosslinking agents, intercalating agents, protein synthesis inhibitors, Topoisomerase 1 and 2 inhibitors, microtubule-directed agents, kinase inhibitors, hormones and hormone antagonists, anti-tumor antiboides, or any combination thereof. Preferably, the anti-cancer agent is selected from platinum compounds such as cis-platin, carboplatin or oxaiiplatin.
For the treatment of infectious diseases, the GLCE inhibiting agent may be administered in combination with an additional therapy directed against the infectious agent, particularly together with at least one further antiviral agent. The antiviral agent may be selected from a protease inhibitor, a polymerase inhibitor, an integrase inhibitor, an entry inhibitor, an assembly/secretion
inhibitor, a translation inhibitor, an immunostimulant or any combination thereof.
Thus, a further aspect of the present invention is directed to a pharmaceutical composition or kit which comprises at least one GLCE inhibiting agent as described above in combination with at least one further medicament, e.g. with at least one further anti-cancer agent and/or with at least one further antiviral agent as described above. Still a further aspect of the present invention is a method for diagnosing an hyperproliferative disease and/or infectious disease comprising the step of measuring the expression, abundance and/or amount of GLCE in a sample from a subject suffering or suspected to suffer from said disease. The diagnostic method particularly allows monitoring the course of the disease by carrying out a plurality of measuring steps, particularly in the case, where a subject undergoes a therapy, in particular therapy with a GLCE inhibiting agent as described above.
The sample may be a body fluid sample, e.g. blood, serum, plasma, sputum, saliva, lymph fluid etc. or a tissue sample, e.g. a tissue section or biopsy. The determination of GLCE may be carried out on the nucleic acid level, e.g. by determining GLCE mRNA and/or on the protein level, e.g. by determining GLCE protein and/or GLCE activity. A determination on the nucleic acid level may involve the use of a hybridization protocol, wherein a hybridization probe complementary to GLCE transcripts or any nucleic acid derived therefrom, e.g. cDNA, is employed. In addition, the determination may comprise nucleic acid amplification according to known protocols, such as PCR. Determination of GLCE on the protein level may comprise immunological methods involving the use of anti-GLCE antibodies or fragments thereof or determination of GLCE enzymatic activity, e.g. as described in Ghiselli et al., 2005b, supra, the content of which is herein incorporated by reference.
The present invention also refers to a diagnostic composition or kit comprising a detection reagent for GLCE, particularly for use in the method as described above. Finally, the present invention also refers to a method of screening for an anti- hyperproliferative agent or anti-pathogen agent, e.g. antiviral agent comprising the steps of determining if a test compound is an inhibitor of GLCE. The screening method may, for example, comprise contacting of GLCE with a test compound and determining binding of the test compound to GLCE and/or determining the activity of GLCE in presence of the test compound compared to a control, e.g. in the absence of the test compound. The screening may be carried out in a cell-free or cellular system. For cellular screening methods, the use of recombinant cells or non-human organisms transfected with GLCE and preferably overexpressing GLCE is preferred.
The test compounds may be selected from polypeptides, e.g antibodies or antibody fragments, aptamers, peptidic compounds or non-peptidic low molecular weight organic molecules (e.g. having a molecular weight of up to 2000 Da). A test compound which is identified as a GLCE inhibitor in a screening method as described above may be a suitable candidate agent for the treatment of hyperproliferative diseases and/or infectious diseases.
The screening method may also be used for identification of cellular interaction partners of GLCE, e.g. polypeptides capable of forming multimeric complexes with GLCE. An example of such an interaction partner of GLCE is CAML (calcium-modulating ligand, GenBank Acc No. NM_001745). Further, the present invention is explained in more detail by the following Figures and Examples.
Figure Legends
Figure 1 : siRNA mediated downregulation of GLCE transcript level in tumoral cells.
MCF7 human breast carcinoma cells were plated in 6-wells plate at 25% confluence and transfected 8 h later with 0 to 62 ng/ml of GLCE-siRNA or non-silencing siRNA oligonucleotide with sequence that has no homology to any known mammalian gene, henceforth identified as scRNA. After 40 h, the transfection was repeated using the same amount of GLCE-siRNA. At 96 h post-plating, the medium was removed, the cells washed in PBS and dissolved in Triazol DNA-RNA extracting reagent. After reverse transcription of total mRNA, gene-specific transcript levels were assessed by real-time PCR on a Roche Light-cycler 480. β-actin transcript level was used to normalize GLCE transcript values. Data (mean ± SD, n=3) were plotted as percent of the GLCE transcript level in GLCE-siRNA-treated cells as compared to that of scRNA-treated cells.
Figure 2: GLCE knock-down inhibits P-selectin and fibronectin mediated attachment of highly metastatic melanoma cells.
B16F10 highly metastatic murine melanoma cells, were seeded in 6-wells plates at 20% confluence and after 8 h transfected with 20 ng/ml GLCE- siRNA or scRNA (control). The transfection was repeated at 48 h post-plating and the cells harvested by mild trypsinization 40 h later. The cells were incubated and suspended in DMEM containing 1 % BSA. For the cell adhesion experiments, 96-wells plates were coated with Fibronectin (0.33 ng/ml) or P-Selectin (10 ng/ml) by overnight incubation at 4°C. In the morning the proteins were removed and the plates washed twice with PBS. Residual binding sites of the plates were saturated with 1 % BSA by incubating 1 h at 37°C. After washing, GLCE-siRNA or scRNA transfected cells (5,000/well) were pipetted into the wells and the plates placed in a 5% C02 incubator at 37°C. After 1 h non-adherent cells were removed by
aspiration and washing twice with PBS. The adhering cells were fixed in methanol, stained with crystal violet, and visualized under a light inverted microscope. The bars represent the Mean number of attached cells/mm2 ± SD (n=3).
Figure 3: GLCE knock-down prevents tubulogenesis.
ECV304 human endothelial cells were seeded in 6-wells plates at 20% confluence and after 8 h transfected with 20 ng/ml GLCE-siRNA or scRNA (control). The transfection was repeated 40 h later and the cells harvested by mild trypsinization 96 h post-plating. For the tube-like formation experiments, 96-well plates were coated with Matrigel by overnight incubation at 4°C. GLCE-siRNA or scRNA transfected cells resuspendent in DMEM/1 % BSA were then pipetted into the wells (10,000 cells). A group of cells also received 10 ng/ml of human recombinant BMP2. The plates were placed in a 5% C02 incubator at 37°C and examined periodically under a light inverted microscope. Representative results are shown.
Figure 4: GLCE knock-down inhibits cell growths in a p53-independent fashion.
HCT116 and SW480 human colon carcinoma cells, were plated in 24-well plates at 20% confluence and transfected with 40 ng/ml GLCE-siRNA or scRNA (control) 8 h later. The treatment was repeated after 40 h and the cells harvested by mild trypsinization at 96 h post-plating. Resuspended cells were fixed in ice-cold methanol and DNA-stained with Propidium Iodide. A) Cell-cycle analysis was performed on a Coulter cytofluorimeter and data gated and analyzed using the WinMDI software. Note the lack of apoptotic cells in the pre-G1 area. B) Results were plotted as percent distribution of cell is G1 , S and G2/M phase. C) Real-time PCR quantitation of GLCE, p53 and BAX transcript level, β-actin normalized results are plotted. The bar represent the percent value of the transcript level in GLCE-siRNA compared to scRNA transfected cells.
Figure 5: GLCE knock-down inhibits cell growth of cis-platin with reactive colon carcinoma cells. HCT116 and SW480 human colon carcinoma cells were plated at 20% confluence in 24-well plates and transfected with 40 ng/ml GLCE-siRNA or scRNA (control) 8 h later. 40 h later, cells were re-transfected with the same dose of siRNA or scRNA and incubated at the same time with increasing concentration of cisplatin as indicated. The experiment terminated after additional 24 h incubation. The plates were washed in ice-cold PBS and cell density assessed by staining with crystal-violet. Points are the mean ± SD (n=3).
Figure 6: Identification of GLCE interacting molecules.
A yeast two-hybrid system was used to screen a Clontech Matchmaker pre- transformed human fetal brain cDNA library cloned in in pACT2 vector. For the identification of GLCE-interacting proteins, AH 09 yeast was first transformed with the bait vector harboring the full length GLCE cDNA cloned in pGBKT7 plasmid, and grown on SD medium lacking tryptophan (SD-Trp) agar plates. Growth of the transformed yeast on SD-Trp/X- -Gal plates, confirmed that the reporter system is not activated by GLCE. AH109-GLCE transformants were mated with the Y187 yeast pre-transformed human fetal brain cDNA library in pACT2 vector. The mating mixture was then plated onto SD-Ade/-His/-Leu/-Trp plates and grown at 30°C for 5 days. Positive colonies were restrecked onto SD-Ade/-His/-Leu/-Trp/X-p-Gal plates and blue colonies used for the isolation of cDNAs encoding the interacting proteins. Eleven colonies were isolated and E.coli DH5-a amplified cDNA on ampicillin plates, was sequenced. The transcript of seven of the colonies matched that of the full length (calcium modulating ligand, GenBank Acc No. NM_001745) gene product. The interaction between GLCE and CAML was confirmed by co-transforming AH109 yeast with the GLCE bait vector together with full length CAML cDNA cloned in pGADT7 vector, followed by
selection on plates. p53-T4 and p53-lamin interactions were used respectively as positive and negative interaction controls.
Examples
Material
Small-interfering RNA antisense oligonucleotides (siRNA) targeting human or murine GLCE transcripts were obtained from Qiagen and had the following sequence:
Human-specific GLCE-siRNAs (sense sequence):
a) 5'-CUCGAUGAUAACAGUAUGGUA-3' (SEQ ID NO:1 )
b) 5'-UGGAAACUUAUUACAAAUCUA-3' (SEQ ID NO:2)
c) 5'-CUGGGCCUAUCUGUUAACAAA-3' (SEQ ID NO:3)
d) 5'-CACAGUAAAUAUGUACUUGUA-3' (SEQ ID NO:4)
Mouse-specific GLCE-siRNAs (sense sequence):
a) 5'-GGCUUUAUGUAUUCUUUAA-3' (SEQ ID NO:5)
b) 5'-GGGCCUAUCUUCUAACGAA-3' (SEQ ID NO:6)
c) 5'-CAUUUGUCUCAGCAAUAAA-3' (SEQ ID NO:7)
d) 5'-CGUGAGCACACCAUUAAA-3' (SEQ ID NO:8)
Annealed double-stranded siRNAs were transfected into cells using Hyperfect (Qiagen) transfection vehicle following the manufacturer's guidelines to optimize the transfection vehicle-to-siRNA mixing ratio.
Example 1 siRNA-mediated down regulation of GLCE transcript level in tumoral cells.
Preliminary experiments were carried out to identify the optimal conditions for siRNA-mediated GLCE-expression knockdown. MCF7 human breast
carcinoma cells were used in these experiments. The cells were transfected with an equimolar mixture of four siRNA oligonucleotides targeting different region of the GLCE coding sequence. This protocol was adopted to limit potential off-target effects of the transfected siRNAs that would change the expression of other genes or have cytotoxic effects. As negative control, non- silencing siRNA oligonucleotide (scRNA) with sequence that has no homology to any known mammalian gene was used at the same concentration level as the silencing gene-specific siRNA. The effect of the antisense oligonucleotides was monitored by monitoring by real-time PCR the transcript level of the targeted gene as well as that of two house-keeping genes such as β-actin and 18S rRNA subunit, whose expression has been shown not to change in a variety of experimental conditions. The effect of siRNA on GLCE expression was computed as percent reduction of GLCE transcript level compared to that of that of cells transfected with an equimolar amount of scRNA.
Optimal downregulation of GLCE expression could be achieved by transfecting cells with GLCE-siRNA 8 h after plating, followed by a second transfection 40 h later with the same amount of siRNA. Using this protocol maximal GLCE expression knockdown was reached between 60 and 96 h after plating. The dose-response curves showed there is a positive correlation between the dose of siRNA employed (ranging between 0 to 60 ng/ml), the suppression of GLCE expression and the inhibition of cell growth pointing to a causal relationship between GLCE transcript level and cell proliferation rate (Figure 1 ).
Example 2 GLCE knockdown inhibits P-selectin- and fibronectin-mediated attachment of highly metastatic melanoma cells.
The ability to migrate through the bloodstream to distant tissues to form metastasis, is an hallmark of tumoral cells. Cancer cells migrates in the
bloodstream attached to circulating platelets and upon reaching destination extravasate by crossing the endothelial barrier. P-selectin, an adhesion protein, mediates cancer cell interaction with platelets and endothelial cells (Borsig 2008). In the subendothelium, homing of the tumor cells to the metastatic site is mediated by extracellular matrix proteins among which fibronectin plays a key role (Kaplan 2006). Agents that prevent interaction of tumoral cells with P-selecting and fibronectin may prevent tumor extravasion and metastasis in vivo. It has been suggested that tumor cell HS is involved in this process. We have obtained evidence (Figure 2) that knockdown of GLCE expression severely inhibits the ability of the highly metastatic B16F10 melanoma cells to adhere to both P-selectin and fibronectin.
Example 3 GLCE knockdown prevents tubulogenesis, an index of angiogenic activity of human endothelial cells.
Angiogenesis involves the sprouting of new capillaries from tumor vessels that is essential for the sustainment with oxygen and nutrients of the growing tumor mass (Folkman 2007). The new vessel also provide the means for cancerous cells to migrate through the bloodstream to distant organs where can metastasize. Angiogenesis relies on the intrinsic ability of endothelial cells to migrate and aggregate to form tube-like structures. This spontaneous ability of endothelial cells to self-organize into vessels is enhanced by a number of growth factors and cytokines among which VEGF (vascular endothelial growth factor) and Angiomodulin are the best studied. For optimal angiogenetic activity many of these factors require the presence of HS. We have obtained evidence (Figure 3) that GLCE expression knockdown inhibits the ability of ECV304 human endothelial cells to organize in tube-like structures both in the absence and the presence of angiogenetic factors such as the bone morphogenetic protein 2 (BMP2) (Langenfeld 2004).
Example 4
GLCE knockdown inhibits cell growth in a p53-independent fashion. Because GLCE-knockdown inhibited cell growth of several tumor cell lines including colon carcinoma cells, we investigated whether this effect was p53- dependent and if apoptosis was involved. Two human colon carcinoma cell lines, HCT116 (p53 proficient) and SW480 (p53 deficient), were investigated (Figure 4). The distribution of cells at different stages of the cell cycle was analyzed in cell transfected with GLCE-siRNA or scRNA. Because of the defective p53-dependent checkpoint, a large fraction of SW480 cells were found in S and G2/M phase compared to p53-proficient HCT116 cells which were mostly maintained in G1 state. Knockdown of GLCE expression inhibited G1 -to-S phase transition both in HCT116 and SW480 cells suggesting the effect was not dependent upon an active p53-pathway. No apoptosis was observed in GLCE-siRNA treated cells further suggesting p53 (a key mediator of apoptosis) was not the cause of the reduced cell number. The gene transcript level analysis showed that GLCE expression had been specifically affected whereas p53 and BAX (the terminal mediator of the p53 apoptotic pathway) expression was unchanged. These data are consistent with the idea that suppression of GLCE expression causes retardation of cell cycle progression in a p53-independent manner.
Example 5 GLCE-knockdown inhibits cell growth of cisplatin refractive colon carcinoma cells.
The efficacy of first-line oncological treatments (chemotherapy and radiotherapy) depends among other, upon a functional p53 system. Due to the high frequency of p53 mutations in human cancer compounded by the development of multidrug resistance, clinical efficacy of chemotherapy is poor especially in patients requiring long treatment. Oncological treatment also elicits strong side-effects that are dose-related. Cisplatin is a DNA- damaging antitumor compounds displaying clinical activity against a wide
variety of solid tumors (Sedletska 2005). p53-proficent HCT116 colon carcinoma cells display a strong sensitivity to the apoptotic activity of cisplatin (Figure 5). Knockdown of GLCE expression in these cells does not add to the effect of cisplatin which is well evident even at the lowest dose tested. By converse, p53-deficient SW480 cells are significantly less sensitive to cisplatin-induced apoptosis. Consistent with the idea that GLCE- knockdown affect cell growth in a p53-independent fashion, GLCE-siRNA enhanced the effect of cisplatin and caused a shift toward lower level of the dose of cisplatin needed to achieve maximal effect.
Example 6
Identification of GLCE binding molecules
We have shown previously that the epimerase enzymatic activity is effectively inhibited by knockdown of GLCE expression (Ghiselli 2005a). However GLCE expression downregulation potentially has additional important effects beside reducing the enzymatic activity. Previous studies had shown that GLCE can engage in the formation of multimeric protein complexes with intracellular proteins such as 2-O-sulfotransferase (a sulfotransferase involved in HS sulfation) (Pinhal 2001 ).
Using a different experimental approach, we have found that GLCE also strongly interacts with CAML (Figure 2), a protein that plays a crucial role in intracellular calcium signaling (Holloway 1998). Whereas the functional significance of GLCE interaction with other proteins has yet to be fully understood, the available results make clear that the suppression of GLCE protein level will effect both the enzymatic activity as well as those functions of GLCE that depend on the interaction with other proteins. A corollary of this idea is that the biological effect of specifically targeting GLCE expression (through the use of siRNA or other antisense-based techniques), cannot be predicted solely on the basis of reduced GlcA-to-ldoA conversion - and hence the production of less reactive HS. Rather because of the distinct functions GLCE will play as enzyme as opposed to protein-protein interactor,
and also because GLCE expression is controlled by complex signaling and gene transactivation pathways, GLCE expression is an additional relevant target for the development of new therapeutic agents.
List of references
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Claims
1 . GLCE (D-glucuronyl-C5-epimerase) inhibiting agent for use as a medicament.
2. GLCE inhibiting agent for use as a medicament for the treatment of hy- perproliferative diseases and/or infectious diseases.
3. The GLCE inhibiting agent of claim 1 or 2, which is a anti-metastatic, an- ti-angiogenic and/or anti-proliferative agent.
4. The GLCE inhibiting agent of any one of claims 1 to 3 which is selected from
(a) an inhibitor of GLCE on the nucleic acid level, or
(b) an inhibitor of GLCE on the protein level.
5. The GLCE inhibiting agent of any one of claims 1 to 4, wherein the inhibitor of GLCE on nucleic acid level is a GLCE gene expression inhibitor, preferably selected from nucleic acid effector molecules directed against GLCE mRNA, such as an RNAi molecule, a precursor or a template thereof, an antisense molecule or a ribozyme.
6. The GLCE inhibiting agent of claim 5, wherein the siRNA molecule is a double-stranded nucleic acid molecule of 18 to 30, preferably of 9 to 25 nucleotides or nucleotide analogs, which optionally has at least one 3' overhang of 1 to 3, preferably of 2 nucleotides.
7. The GLCE inhibiting agent of claim 5 or 6, wherein the siRNA molecule is directed against mammalian GLCE mRNA, particularly against human GLCE mRNA.
8. The GLCE inhibiting agent of claim 6 or 7, wherein the siRNA molecule has the sense strand selected from:
(i) 5'-CUCGAUGAUAACAGUAUGGUA-3' (SEQ ID No 1), (ii) 5'-UGGAAACUUAUUACAAAUCUA-3' (SEQ ID No 2), (iii) 5'-CUGGGCCUAUCUGUUAACAAA-3' (SEQ ID No 3), (iv) 5'-CACAGUAAAUAUGUACUUGUA-3' (SEQ ID No 4), or
(v) a nucleotide sequence which has an identity degree of at
85%, at least 90% or at least 95% to one any one of the sequences SEQ ID No 1 to 4.
9. The GLCE inhibiting agent of any one of claims 1 to 4, wherein the inhibitor of the GLCE on protein level is selected from an antibody specific for GLCE or an antigen-binding fragment thereof, an aptamer directed against GLCE, a mutated form of the GLCE ligand or an inhibitor of GLCE-ligand binding.
10. The GLCE inhibiting agent of claim 9, wherein the antibody specific for GLCE or the antigen-binding fragment thereof is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody or a fragment thereof.
11. The GLCE inhibiting agent of claim 9 or 10, wherein the antibody or the antigen-binding fragment thereof specifically binds mammalian, particularly human GLCE.
12. The GLCE inhibiting agent of claim 1 or 2, which is for use in a cancer disease including metastasis, or a vascular disease such as atherosclerosis or restenosis.
13. The GLCE inhibiting agent of claim 12, wherein the cancer disease is selected from the group consisting of bone, brain, breast, cervix, colon, gastric, liver, lung, pancreas, ovarian, renal, pancreas, prostate, stomach, soft tissue, bone marrow or skin cancer, or lymphoma, particularly breast cancer, skin cancer and colon cancer.
14. The GLCE inhibiting agent of claim 12 or 13 in combination with an additional anticancer therapy.
15. The GLCE inhibiting agent of claim 14, wherein the additional anticancer therapy is selected from chemotherapy, radiation therapy, surgery, immunotherapy, gene therapy or combinations thereof.
16. The GLCE inhibiting agent of any one of claims 12 to 15 in combination with at least one further anti-cancer agent, e.g. a chemotherapeutic agent.
17. The GLCE inhibiting agent according to claim 16, wherein the at least one further ant-cancer agent is selected from antimetabolites, DNA- fragmenting agents, DNA-crosslinking agents, intercalating agents, protein synthesis inhibitors, Topoisomerase 1 and 2 inhibitors, microtubule-directed agents, kinase inhibitors, hormones and hormone antagonists, anti-tumor antiboides, or any combination thereof.
18. The GLCE inhibiting agent of claim 16 or 17, wherein the chemotherapeutic agent is selected from platinum compounds, preferably cisplatin, carboplatin or oxaliplatin.
19. The GLCE inhibiting agent of claim 2, wherein the viral infections are selected from DNA and/or RNA virus infections, in particular infections by e.g. hepatitis C virus, herpes simplex virus, respiratory syncytial virus, human papillomavirus and foot- and mouth-disease virus.
20. The GLCE inhibiting agent of claim 19 in combination with at least one further antiviral agent.
21. The GLCE inhibiting agent according to claim 20, wherein the at least one further antiviral agent is selected from a protease inhibitor, a polymerase inhibitor, an integrase inhibitor, an entry inhibitor, an assembly/secretion inhibitor, a translation inhibitor, an immunostimulant or any combination thereof.
22. A method of treating a subject suffering from a hyperproliferative disease and/or an infectious disease comprising administering to a subject a pharmaceutically effective amount of a compound according to any one of claims 1-21.
23. The method of claim 22, wherein the hyperproliferative disease is a cancer disease including metastatic cancer.
24. A pharmaceutical composition or kit comprising as an active agent at least one GLCE inhibiting agent as defined in any one of claims 1-13, together with a pharmaceutically acceptable carrier, diluent and/or adjuvant.
25. The composition or kit of claim 24, wherein the at least one GLCE inhibiting agent is in combination with at least one further ant-cancer agent as defined in any one of claims 16 to 18 and /or with at least one further antiviral agent as defined in claim 20 or 21.
26. The composition or kit of claim 24 or 25 for use in medicine.
27. A method for diagnosing an hyperproliferative disease and/or an infectious disease comprising the step of determining the expression, abundance and/or amount of GLCE in a sample of a subject suffering or suspected to suffer from said disease.
28. The method of claim 27, wherein the subject undergoes a therapy, in particular a therapy with a GLCE inhibiting agent as defined in any one of claims 1 to 11.
29. A method of screening for an antihyperproliferative agent and/or an antipathogenic agent comprising the steps of determining, if a test compound is an inhibitor of GLCE.
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EP2307440A4 (en) * | 2008-07-01 | 2012-12-19 | Zacharon Pharmaceuticals Inc | Heparan sulfate inhibitors |
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