WO2004108142A2 - Traitement d'etats lies au vieillissement - Google Patents

Traitement d'etats lies au vieillissement Download PDF

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WO2004108142A2
WO2004108142A2 PCT/GB2004/002383 GB2004002383W WO2004108142A2 WO 2004108142 A2 WO2004108142 A2 WO 2004108142A2 GB 2004002383 W GB2004002383 W GB 2004002383W WO 2004108142 A2 WO2004108142 A2 WO 2004108142A2
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cells
tep
activity
cancer
compound
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WO2004108142A3 (fr
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Ian Hampson
Lynn Hampson
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University of Manchester
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Victoria University of Manchester
University of Manchester
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Priority claimed from GB0313034A external-priority patent/GB0313034D0/en
Priority claimed from GB0322665A external-priority patent/GB0322665D0/en
Application filed by Victoria University of Manchester, University of Manchester filed Critical Victoria University of Manchester
Publication of WO2004108142A2 publication Critical patent/WO2004108142A2/fr
Publication of WO2004108142A3 publication Critical patent/WO2004108142A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/57595—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving intracellular compounds

Definitions

  • the present invention relates to the treatment, or limitation of tissue damage and especially such damage that may occur as a result of the ageing process.
  • the invention also concerns the diagnosis, and treatment (including prophylactic treatment) of, cancer and to a method for the identification of cancer targets involved in the process of tumour invasion
  • Tissue damage may arise as a result of physical damage (e.g. wounding), chemical damage and viral damage.
  • the damage can take a number of forais.
  • a consequence of hepatic damage can be the development of liver cirrhosis.
  • One form of damage that may occur in individuals, and particularly aged individuals, is the development of genetic abnormalities that may be observed microscopically as polyploidy. Genetic damage, and polyploidy in particular, may lead to the development of a number of diseases including cancer.
  • TIP-1 HTLVl Tax Interacting Protein-1
  • a method for the prevention of and treatment of age or damage related conditions comprising administering to a patient in need of such treatment an agent which is effective to (i) reduce cellular levels of TIP-1 protein or (ii) acts to inhibit, or alter the functional effects of TLP-1.
  • condition characterised by tissue damage we mean tissue damage that arises as a result of physical damage (e.g. wounding), chemical damage or viral damage and in particular damage or degeneration that is associated with aging. Such damage, and particularly viral damage, may lead to the development of a number of diseases including cancer.
  • the invention has been based on the inventors' findings relating to the effects of the TIP-1 protein, (i.e.HTLVl Tax interacting protein-1) on the development of genetically abnormal pre-cancerous cells and on cancer cell growth and motility.
  • TIP-1 protein i.e.HTLVl Tax interacting protein-1
  • TIP-1 we mean natural or synthetic homologues of the protein of the above-mentioned sequence, which have tumour-promoting activity in vivo and also active fragments thereof.
  • the inventors have found that the agents are particularly useful for preventing the development of cancers. Accordingly normal subjects (i.e. no detectable cancer) or subjects with pre malignant cells or particularly cancer prone subjects may be treated according to the invention.
  • the inventors have established, in cells expressing the HPV16 E6 protein that the PDZ domain TIP-1 protein is found associated with the virus E6 early protein ( Figure 1 A, C & D). Surprisingly they have found, unlike many other E6 targeted proteins (e.g. ⁇ 53), that this association of TIP-1 and HPV E6 does not cause degradation and hence reduction in the level of TLP-1 accumulated in the cell ( Figure 1 E & F). Furthermore they found that the expression of TIP-1 mRNA is altered in various tumours with expression usually being increased above expression levels found in corresponding normal tissues from the same patient ( Figure 8).
  • TIP-1 is known to interact with the cellular protein rhotekin, which in turn interacts with constitutively GTP activated RhoA (4). Interaction of these three proteins has been shown to produce a strong activation of serum response factor (SRF) transcription as measured by an increase in the SRF component of the c-fos serum response element (SRE) transcription (4).
  • SRF serum response factor
  • SRE serum response element
  • Rhotekin has been previously shown to be a negative regulator of intrinsic RhoA GTPase activity (5, 6) which has the effect of stabilising GTP bound RhoA.
  • RhoA is more readily activated and less readily deactivated in the presence of the HPV16 E6 protein and TIP-1.
  • RhoA kinases RhoA kinases
  • ROCK's are known to deactivate myosin light chain (MLC) phosphatase and to directly phosphorylate MLC's (7) .
  • MLC myosin light chain
  • Figure 4B Analysis of TLP-1 expression in the absence of E6 showed no detectable phospho-MLC expression whereas a signal was present in the presence of E6 ( Figure 4B).
  • E6 does not activate SRE signalling from endogenous GTPRhoA
  • the inventors realised that E6 may be interacting with TIP-1 which in turn interacts with rhotekin to promote GTP RhoA dependent activation of ROCK.
  • agents used according to the first and second aspects of the invention have utility in treating cancer and most particularly the prevention of metastases.
  • TIP-1 interacts with the cellular protein guanidine triphosphate exchange factor 16 (ARHGEF16, Accession NM_014448), which belongs to the GEF family of proteins ( Figure 1A & B).
  • GEFs are positive regulators of GTP activation of RhoA and most are oncogenes for example the Vav family of proteins function as GEFs activating Rho GTP'ase (9). Consistent with this finding the inventors have established that TIP-1 is capable of regulating both the activation status of endogenous RhoA and its downstream effects (ROCK activation).
  • RhoA mutants to transform NLH3T3 cells does not correlate with their ability to fransactivate c-fos SRE's but with their ability to bind with and activate the RhoA kinase ROCK(10).
  • Reynaud (4) indicated that TLP-1 could activate SRE transcription this study did not link this to transforming ability or to increased cell motility nor demonstrate the importance of TLP-1 in these physiological responses.
  • the inventors have shown that transfection of tumour cells with HPV 16 E6 produces increased cell motility and that this effect can be abrogated by antisense silencing of TIP-1 expression (See Figure 2).
  • TIP-1 activity may be regulated by alternative mechanisms other than interaction with viral oncoproteins in these cancers. For example up-regulation of expression, altered phosphorylation status, or interaction with other cellular proteins may play a role in controlling TLP-1 activity.
  • up-regulation of expression, altered phosphorylation status, or interaction with other cellular proteins may play a role in controlling TLP-1 activity.
  • over expression of the TIP-1 protein is sufficient to completely abrogate cell-cell contact inhibition in NLH 3T3 cells (see Figure 5).
  • TLP-1 expressing 3T3 cells had very high levels of phosphorylated MLC when compared to control cells.
  • Fluorescent activated cell sorter analysis of TLP-1 3T3 cells indicated that the ploidy of these cells had become unstable compared to control cells. Fluorescent microscopy visualisation of these cells with propidium iodide confirmed that many TIP-1 expressing cells had abnormal numbers and shapes of nuclei - an effect consistent with abnormal cytokinesis leading to polyploidy, genetic instability and eventually cancer. It is highly significant that increased numbers of cells with abnormal ploidy are found in many human tissues associated with the ageing process and that these cells are functionally compromised when compared to normal diploid cells (11). Indeed there are now in vivo models of pre-mature ageing which are characterised by the development of abnormal tissue ploidy (12).
  • agents capable of inhibiting the oncogenic, polyploidy inducing, ROCK activating effects of TLP-1 will inhibit the development of age or damage related abnormal polyploidy. This will in turn inhibit the development of many damage or age related diseases including cancer and thus form the basis of chemopreventative agents.
  • agents such as Y27632 may inhibit or suppress these age or damage related changes.
  • the invention may be applied to a wide range of cancers such as ovarian carcinoma, breast carcinoma, lung carcinoma, uterine carcinoma, cervical carcinoma and thyroid carcinoma. It may also be applicable to cancer prone conditions.
  • the invention is applicable particularly, but by no means exclusively, to pre-cancerous conditions and cancers caused by oncogenic viruses, e.g. transforming human papilloma viruses (HPVs) or human T-cell leukaemia virus type-I (HTLV-I).
  • HPVs transforming human papilloma viruses
  • HTLV-I human T-cell leukaemia virus type-I
  • TLP-1 activity Several classes of compound, maybe used as an agent according to the first or second aspects of the invention, to influence TLP-1 activity. These compounds include:
  • Agents according to the present invention may modulate the effects of TIP-1 on the activity and effects of GTP RhoA.
  • Preferred agents for use according to the invention are inhibitors of GTPRhoA or RhoA Kinase (ROCK). Such agents are known to the art. For instance, preferred examples of RhoA Kinase inhibitors are disclosed in Japanese patent application No. 2001-266055.
  • Y27632 A most preferred agent is Y27632.
  • the inventors have found that this compound is not only useful for treating cancer but is also surprisingly useful for preventing age or damage related changes in cell ploidy (e.g. preventing the development of cancer). Accordingly Y27632 may be advantageously used as a prophylactic. For instance, it may be given to subjects with a genetic disposition to developing cancer or even those facing environmental risk (e.g. people exposed to carcinogens). It may also be given to modulate the effects of wounding and the development of scarification and tissue fibrosis and wherever polyploidisation is a consequence of such damage.
  • TEP-l e.g. monoclonal or polyclonal
  • antibody fragments for example Fab fragments or intrabodies
  • ribozymes for example antisense oligonucleotides against TEP-l (see the Example 1).
  • a preferred TLP-1 antisense oligonucleotide is:
  • antisense oligonucletides that may be used according to the invention are modulators of Rl o family gene expression such as those disclosed in WO 00/17223 or US 6,410,323.
  • the compounds may be used to treat age related conditions such as cancer as a mono therapy (i.e. use of the compound alone or in combination with other compounds or treatments used in cancer therapy (e.g. chemotherapeutic agents, radiotherapy).
  • a mono therapy i.e. use of the compound alone or in combination with other compounds or treatments used in cancer therapy (e.g. chemotherapeutic agents, radiotherapy).
  • the medicaments used according to the invention may take a number of different forms depending, in particular on the manner in which the medicament is to be used.
  • the medicament may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, liposome or any other suitable form that may be administered to a person or animal.
  • the vehicle of the medicament of the invention should be one which is well tolerated by the subject to whom it is given and enables delivery of the agents to the effected site.
  • Therapy with the agents may be effected in a number of ways.
  • systemic administration may be required in which case the agent may be contained within a medicament, which may, for example, be ingested orally in the form of a tablet, capsule or liquid.
  • the medicament may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion).
  • the agents may also be administered by inhalation (e.g. intranasally). Alternatively the agents may be administered transdermally by way of patches.
  • the agent may also be incorporated within a slow or delayed release device.
  • a slow or delayed release device Such devices may, for example, be inserted under the skin and the agent may be released over weeks or even months.
  • the devices may be particularly advantageous when an agent is used which would normally require frequent administration (e.g. at least daily ingestion of a tablet or daily injection).
  • the amount of an agent required is determined by biological activity and bioavailability which in turn depends on the mode of administration, the physicochemical properties of the agent employed and whether the agent is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the agent within the subject being treated.
  • a daily dose of between O.Ol ⁇ g/kg of body weight and l.Og/kg of body weight of an agent according to first or second aspects of the invention may be used for the treatment of cancer depending upon which specific agent is used. More preferably the daily dose is between O.Olmg/kg of body weight and lOOmg/kg of body weight.
  • the required dose will be influenced by the route of administration.
  • the preferred dose may be lower than a suitable dose chosen for oral administration.
  • Daily doses may be given as a single administration (e.g. a daily tablet for oral consumption or as a single daily injection).
  • the agent used may require administration twice or more times during a day.
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3 or 4 hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses to a patient without the need to administer repeated doses.
  • a potential means of using protein or peptide agents according to the invention is to deliver proteins or peptides to the tumour site by means of gene therapy.
  • gene therapy may be used to modulate expression of TEP-l, by increased expression of enzyme(s) responsible for the degradation of TIP-1 or of a protein or peptide, which promotes the inactivation of TLP-1. Therefore according to a third aspect of the present invention there is provided a delivery system for use in a gene therapy technique, said delivery system comprising a DNA molecule encoding for a protein which directly or indirectly reduces TEP-l activity, said DNA molecule being capable of being transcribed to allow the expression of said protein and thereby treat cancer.
  • the delivery systems according to the first and second aspects of the invention are highly suitable for achieving sustained levels of a protein, which directly or indirectly reduces TLP-1 activity over a longer period of time than is possible for most conventional therapeutic regimes.
  • the delivery system may be used to induce continuous protein expression from cells of the tumour that have been transfected with the DNA molecule. Therefore, even if the protein has a very short half-life as an agent in vivo, therapeutically effective amounts of the protein may be continuously expressed from the treated tissue.
  • the delivery system of the invention may be used to provide the DNA molecule (and thereby the protein which is an active therapeutic agent) without the need to use conventional pha ⁇ naceutical vehicles such as those required in tablets, capsules or liquids.
  • the delivery system of the present invention is such that the DNA molecule is capable of being expressed (when the delivery system is administered to a patient) to produce a protein, which directly or indirectly has activity for reducing wild-type TLP- 1 activity.
  • directly we mean that the product of gene expression per se has the required activity.
  • indirectly we mean that the product of gene expression undergoes or mediates (e.g. as an enzyme) at least one further reaction to provide an agent effective for reducing the capacity of TEP-l to activate GTPRhoA dependent activation of ROCK's and thereby treating cancer.
  • the DNA molecule may encode for any of the compounds (i-ii) as defined above.
  • a preferred DNA molecule encodes an intrabody against TEP-l.
  • the DNA molecule may encode for any of the compounds (i-ii) as defined above.
  • the DNA molecule may be contained within a suitable vector to form a recombinant vector.
  • the vector may for example be a plasmid, cosmid or phage. Such recombinant vectors are higlily useful in the delivery systems of the invention for fraiisforrning cells with the DNA molecule.
  • Recombinant vectors may also include other functional elements.
  • recombinant vectors can be designed such that the vector will autonomously replicate in the cell. In this case elements, which induce DNA replication may be required in the recombinant vector.
  • the recombinant vector may be designed such that the vector and recombinant DNA molecule integrates into the genome of a cell. In this case DNA sequences, which favour targeted integration (e.g. by homologous recombination) are desirable.
  • Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.
  • the recombinant vector may also further comprise a promoter or regulator to control expression of the gene as required.
  • the DNA molecule may (but not necessarily) be one, which becomes incorporated in the DNA of cells of the subject being treated. Undifferentiated cells may be stably transformed leading to the production of genetically modified daughter cells (in which case regulation of expression in the subject may be required e.g. with specific transcription factors or gene activators). Alternatively, the delivery system may be designed to favour unstable or fransient transformation of differentiated cells in the subject being treated. When this is the case, regulation of expression may be less important because expression of the DNA molecule will stop when the transformed cells die or stop expressing the protein (ideally when the cancer has been treated or prevented).
  • the delivery system may provide the DNA molecule to the subject without it being incorporated in a vector.
  • the DNA molecule may be incorporated within a liposome or virus particle.
  • the "naked" DNA molecule may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake.
  • the DNA molecule may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
  • transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecule, viral vectors (e.g. adenovirus) and means of providing direct DNA uptake (e.g. endocytosis) by application of the DNA molecule directly to the cancer or prospective site of cancer, either topically or by injection.
  • the treatment according to the first, second or third aspects of the invention may be for the purposes of treating an existing cancer or may be a prophylactic treatment administered to a person believed to be at risk of developing such a cancer.
  • a method for the diagnosis of pre-cancer and cancer comprising analysing a cell sample from a patient for the presence of elevated levels of TEP-l mRNA or protein or the presence of mRNA or protein for TLP-1 induced proteins.
  • TLP-1 induced proteins we mean proteins that may be come inappropriately expressed as a consequence of TEP-l expression. Examples of such proteins are apparent from Figure 9 and include Rhotekin, GTPRhoA, ROCK, Myosin Light Chain (MLC) Kinase.
  • Tip-1 induced protein is phosphorylated MLC.
  • Diagnosis may be effected on a sample from a patient believed to be suffering from cancer, or alternatively to a patient believed to be at risk of developing cancer.
  • Diagnosis according to the invention may be effected in order to establish whether or not a patient is suffering from cancer and the metastatic potential of their disease. This diagnosis may be carried out in order to assess the suitability of a specified therapeutic regime for the treatment of a patient's disease.
  • the sample taken may, for instance, be a tissue biopsy, blood sample or swab.
  • TEP-l or TEP-l induced proteins may be earned out by assessing the level of expressed protein within the cell, or alternatively, by taking a measurement of the level of gene transcription. Protein may, for example, be assessed through the use of specific binding agents including polyclonal and monoclonal antibodies in techniques such as immuno-cytochen istry, immuno- precipitation or immuno-blotting (Western blotting).
  • a method of screening a compound to test whether or not the compound has efficacy for treating cancer comprising:
  • a method of screening a compound, to test whether or not the compound causes cancer comprising:
  • the screening methods of the fifth and sixth aspects of the invention are based upon the inventors realisation that inappropriate TEP-l expression and activity in defined cell types may be closely related to carcinogenesis and metastasis.
  • TEP-l expression is increased in some cancerous tissues compared to normal tissues. Accordingly any compound, identified according to the fifth aspect of the invention, that decreases the expression or activity of TEP-l or TEP-l induced proteins, is a good candidate as an agent for treating cancer. It will be appreciated that the pharmaceutical industry will be able to use the method according to the fifth aspect of the invention in high throughput screens to identify candidate medicaments for use in the treatment of cancer. Therefore according an seventh aspect of the present invention there is provided an anti-cancer agent identified by the method according to the fifth aspect of the invention The method according to the sixth aspect of the invention represents a good test for evaluating whether or not a test compound is carcinogenic.
  • any compound, identified according to the sixth aspect of the invention, that reduces the expression or activity of TEP-l or TLP-1 induced proteins is less likely to be carcinogenic.
  • the method may be used to screen compounds to assess whether or not they are safe to be used by the public. For instance cosmetics, foodstuffs, candidate therapeutic agents etc may all be tested to investigate whether or not they may cause cancer.
  • the method according to the sixth aspect of the invention may also be used in an environmental setting. For instance, the test may be used to evaluate whether or not effluent from a factory may contain carcinogenic compounds.
  • TEP-l or TEP-l induced proteins may be measured using a number of conventional techniques. For instance, labelled antibodies may be used in an immunoassay to evaluate protein levels in the cells or subject being tested. Alternatively a functional activity measuring TLP-1 activity may be employed.
  • cDNA may be generated from mRNA extracted from the tested cells or subject and primers designed to amplify test sequences used in a Polymerase Chain Reaction to amplify from cDNA.
  • TEP-l has a marked affect on the morphology of cells (e.g. See Figure 6). Although the inventors do not wish to be bound by any hypothesis, they believe the morphological changes may be caused by am alteration in the phosphorylation state of MLC or Actin. Therefore preferred methods according to the fourth, fifth or sixth aspects of the invention may involve monitoring the phosphorylation state of MLC or Actin.
  • Other preferred methods according to the fourth, fifth or sixth aspects of the invention may involve monitoring the morphological changes per se. These may be observable microscopically and/or histologically. Once such observable change is in the ploidy of the cells (See Figure 6). Inappropriate expression of TEP-l appears to increase disorganisation during mitosis and in particular causes cells to become polyploid. Therefore prefen-ed methods can monitor morphological changes and particularly the proportion of polyploid cells in a tested cell sample. A most prefereed way of monitoring polyploidy is be conducting a FACS analysis (see Figures 6 and 7). FACS analysis has the advantage that its is fast and automated and it is therefore easy to detect whether or not a screened compound increases or decreases polyploidy.
  • the motility assay may involve comparison of the morphology of control and E6 expressing cells and is based upon the inventors' observation that E6 cells produce more projections and appear more motile in culture.
  • the inventors adapted a method previously applied to endothelial cells (11) where cells are plated in oil, allowed to attach, the oil replaced with growth medium and the cells allowed to grow freely for approximately 18 hours.
  • the inventors adapted the known method by combining it with engineered E6 expressing tumour cells.
  • This motility assay represents a simple method for identifying protein targets or drugs that interfere with the process of cell movement since this is involved in the process of tumour invasion.
  • TEP-l protein expression may be silenced by transfection with an antisense TEP-l phosphorothioate oligonucleotide (e.g.5' TGTAGGACATCTCGAC 3' see -Seq ED No2 for location.) Transfected cells may be left for about 48 hours prior to carrying out the motility assay.
  • an antisense TEP-l phosphorothioate oligonucleotide e.g.5' TGTAGGACATCTCGAC 3' see -Seq ED No2 for location.
  • the motility assay represents an important aspect of the invention and according to an eight aspect of the invention there is provided a method of screening a compound to test whether or not the compound has efficacy for treating cancer, comprising:
  • a ninth aspect of the invention there is provided a method of screening a compound, to test whether or not the compound causes cancer, comprising:
  • Cells used according to the fifth, sixth, eight or ninth aspects of the invention may be from a number of sources.
  • the cells may be ex vivo samples but are preferably a cell-line that is either capable of expressing TEP-l or has been engineered to do so.
  • the cell-line is preferably eukaryotic and ideally a human cell line.
  • yeast may be used and even prokaryotic models that have been engineered to be capable of expressing TEP-l.
  • the cells may be cultured with a test compound for a predetermined length of time and then tested for levels of TEP-l or TEP-l induced protein expression or activity. When a subject is used (e.g.
  • test compound should be administered to the subject for a predetermined length of time and then a sample taken from the subject for testing TLP-1 or TEP-l induced protein expression or activity or the consequences of abnonnal Tip-1 expression such as abnormal ploidy.
  • the sample may for instance be blood or biopsy tissue.
  • preferred methods according to the fifth, sixth, eight or ninth aspects of the invention may be adapted such that a cell or subject is first treated with TLP-1 and then the cells are subsequently treated with a screened compound to test its ability to augment or inhibit the activity of TEP-l.
  • TEP-l treated cells have increased polyploidy. Therefore any test compound that has the ability to reduce TEP-l induced polyploidy is likely to have general anti-ageing properties and will also act to decrease fibrosis and scarring that can occur during the repair of tissue damage. Such compounds will thus inhibit the development of cancer and many human diseases. Conversely any compound that has the ability to increase TLP-1 induced polyploidy is likely to accelerate age related tissue damage and is likely to be carcinogenic.
  • Fig. 1 illustrates that TLP-1 Interacts With HPV16 E6 and GEF16 in:
  • Fig. 2. illustrates the results of Cell Motility Assays under the following conditions: (A) 40 x of C33A-V cells at time 0. No migration; (B) 40x of C33A-V cells at 18 h showing - Minimal migration; (C) 250x of C33A-V cells at 18 h - Compact cell morphology and maintenance of cell boundary; (D) 40x of C33A-E6 cells at time 0 - No migration; (E) 40x of C33A-E6 cells at 18 h - Extensive migration; (F) 400x of C33A-E6 cells at 18 h - Cell spreading and complete disruption of the cell boundary;
  • Fig. 3 represents the effect of Antisense Inhibition of TLP-1 Expression in ploidy
  • the photographs represent data for: C33A-E6 cells transfected with fluorescein labelled scramble and antisense TLP-1 ODN's showing equal uptake of both ODN types; and Northern blot of RNA isolated from C33AE6 cells treated with (1) antisense TEP-l ODN and (2) Scramble ODN probed with labelled TEP-l cDNA.
  • the ribosomal bands (R) are shown as a loading control.
  • Antisense TEP-l ODN induces a six-fold reduction in TEP-l transcript expression compared to scramble ODN.
  • Fig. 4 illustrated analysis of SRF and RhoA Activation in C33A and C33AE6 cells in which:
  • Fig. 5 illustrates the effects of Constitutive Expression of TEP-l in NLH/3T3 Cells in which:
  • Fig 6. illustrates the effects of TEP-l on the Ploidy of NLH/3T3 Cells in which:
  • NTH/3T3 cells and C33A cells had the typical NEH/3T3 profile whereas
  • TEP10 cells adopted a profile more characteristic of C33A tumour cells with far higher numbers of polyploid cells with abnormal nuclei.
  • B-F Fluorescence and phase microscopy of propidium iodide and toluidine blue stained
  • B-D Monoclonal TLP10 cells. Abnormal and multinucleated cells are arrowed which confirms the FACS results
  • E C33A cells. Multinucleated, abnormal cells are arrowed
  • F Fluorescence and phase microscopy of vector transfected monoclonal NLH/3T3 cells. Normal mononuclear cells shown. (B,C,E and F, 400x ;
  • FIG. 7 illustrates that Y27632 Inhibits the Ability of TLP-1 to Induce Abnormal Nuclei in TLP-10 3T3 cells that were seeded onto glass cover slips and grown in the presence and absence of lO ⁇ M Y27632 in which: (A) Shows the cells in the presence of Y27632; and (B) in the absence visualised by fluorescence microscopy analysis of Propidium iodide stained cells. The results clearly show that Y27632 had inhibited the development of polyploid TEP-10 3T3 cells.
  • Fig. 8 shows a TEP-l Probe of Matched Pair Human Tumour cDNA Array in which:
  • FIG. 1 32 P labelled TEP-l probe of a tumour arcay showing cDNA from normal tissue on the left and tumour on the right from the same patient, of 250 matched pairs of normalised total cDNA's from normal (Left hand column dot in Figure 7 (A) and tumour tissue (Right hand column dot Figure 7 (A)) from the same individual. (Clontech).
  • Figure 4 (B) represents the same blot probed with the housekeeping gene ubiquitin.
  • Fig 9 is a scheme illustrating interactions of TLP-1 which identifies new binding partners of Tip-1 and how these may interact with RhoA and it's effectors.
  • the Tip-1 protein interacts with the HPV16 E6 oncoprotein and the guanine exchange factor GEF16 (ARHGEF16, Accession NM_014448). Although many proteins targeted by E6 are subject to degradation (e.g. p53) TLP-1 is not degraded by its interaction with E6. Expression of the E6 protein in HPV negative cervical carcinoma cells induces increased levels of phosphorylated myosin light chains (MLC); increased capacity to GTP activate RhoA and a large increase in cell motility, which is prevented by the RhoA kinase (ROCK) inhibitor Y27632 and by antisense silencing of TLP-1 expression.
  • MLC phosphorylated myosin light chains
  • ROCK RhoA kinase
  • TLP-1 is a regulator of the effects of RhoA with a potential oncogenic, gain-of-fimction role in the control of cell growth, migration and cytokinesis in both viral and non- viral malignancies.
  • HPV16 E6 Interacts with and Does Not Degrade TIP-1 and TIP-1 Binds to GEF16.
  • Yeast two-hybrid screening with the HPV16 E6 protein as bait identified the HTLVl Tax interacting protein TEP-l (Accession No. NM_014604) as a binding partner.
  • TLP-1 has a class 1 PDZ domain (12) and further screening using the TEP-l protein as bait identified the guanine exchange factor 16 (GEF16, Accession No. BC051838)) as a TLP-1 binding protein.
  • Fig. 1A shows yeast, mating assays carried out between E6 and TLP-1 and TEP-l and GEF16, which confirm both of these interactions in yeast.
  • Fig. IB shows the C-tenninal PDZ domain binding site consensus (13) for previously identified TEP-l binding partners (4, 14-16) in addition to our observations with HPV16 E6, GEF16 and HPV18 E6 (13).
  • Fig. 1C shows a Northern blot of HPV negative human C33A cervical carcinoma cells stably transfected with the type 16 E6 protein and parent vector (C33A-E6 and C33A-Vector control cell lines (17)) before and after transient transfection with TEP-l cDNA in pcDNA3.1 V5His.
  • This demonstrates that there is no difference in the level of endogenous TEP-l RNA between C33A-V and C33A-E6 cells and that the transient transfection of TLP-1 produced equivalent levels of ectopically expressed TLP-1 RNA in both cell types.
  • Identical transfected cultures were used for the co-immuneprecipitation of transiently expressed V5 tagged TLP-1 protein (Fig.
  • Fig. 2F is a 400x magnification of the C33A-E6 cell boundary and demonstrates complete disruption of the circular edge.
  • Fig. 2G and 21 show the extent of migration obtained with Y27632 treated cells after 18 hours.
  • Fig. 21 is a 250x magnification of the cell circle boundary and
  • Fig. 2H is a 400x magnification of Y27632 treated C33A-E6 cells.
  • C33AE6 cells have Higher Levels of phosphorylated MLC than C33A cells.
  • Activated ROCK is known to directly phosphorylate myosin light chains (MLC) and to inactivate MLC phosphatase (7).
  • MLC myosin light chains
  • Fig. 4B demonstrate that C33AE6 cells have higher levels of phosphorylated MLC than C33A-V cells, a result which is consistent with increased ROCK activity in these cells.
  • FIG. 2J shows that scramble treated C33A- E6 cells were still highly motile whereas antisense TLP-1 treated cells were non- migratory (Fig. 2K).
  • Fig. 2L is a 250x magnification of antisense TEP-l C33A-E6 cells and demonstrates maintenance of the circle boundary and that the morphology now resembles that of C33A (Fig. 2C) and Y27632 treated C33A-E6 cells (Fig.'s 2G, 2H and 21).
  • Neither antisense TEP-l nor scramble ODN's had any effect on the motility of C33A-V cells (Fig. 2 M, N and O).
  • SRE SEAP based assay is an indirect means of assessing steady state levels of activated RhoA thus we used the EZ-Detect activated RhoA detection method to directly measure the levels of activated RhoA in C33A-E6 and C33A-V cells.
  • Fig. 4C confirms the results of the SRE assay in that there is no difference in the steady state levels of activated RhoA in these cells.
  • pre- treatment of cell lysates with an excess of GDP deactivates and an excess of GTP activates endogenous RhoA.
  • Fig. 5D shows a Western blot, which confinns expression of the V5 tagged TEP-l protein in both polyclonal and monoclonal G418 selected NEH/3T3 cells.
  • Polyclonal TLP-1 and vector transfected NLH/3T3's were inoculated at the same density, grown for nine days and the cells fixed and stained with toluidine blue.
  • TEP-l 3T3 polyclonal cells did not form colonies in soft agar colony forming assays.
  • Y27632 Restores Contact Growth Inhibition to TIP-1 NIH/3T3 Cells.
  • the above experiment was repeated in the presence of Y27632, which completely abrogated the growth of non-contact inhibited foci in TLP-1 expressing cells (Fig. 5A).
  • growth assays were carried out on TEP-l transfected and vector transfected polyclonal cells plus and minus Y27632 (added at days 3, 6 and 8). After a total of 10 days in culture TEP-l transfected 3T3 cells grow to a higher density than vector transfected control cells - an effect which can be prevented by treatment with Y27632 (Fig. 5B). It can be seen that there is little change in the cell density of control vector cells between days 6 and 10 whereas TLP-1 transfected cells continued to expand over this period. Y27632 treatment of TEP-l cells clearly restores contact growth inhibition to these cells.
  • TIP-1 NIH/3T3 Cells Have Increased Levels of Phosphorylated MLC.
  • a marked up-regulation in the level of phosphorylated MLC was observed in both polyclonal and monoclonal TLP-1 3T3 cells whereas this was undetectable in vector control and parental 3T3 cells (Fig. 5E).
  • Fig. 6 A shows two profiles of different 3T3 vector control monoclonal cell lines which both have the classical proportions of Go/Gl, S and G2/M phase expected for nonnal NTH/3T3 cells.
  • the TLP-1 clone (clone 10 refened to as TLP10 3T3 cells) shown is typical of other TLP-1 transfected monoclones and has a FACS profile indistinguishable from C33A tumour cells.
  • FIG. 6B, 6C and 6D are toluidine blue and propidium iodide stained TEP-l 3T3 cells viewed by fluorescence and phase microscopy which show gross nuclear abnormalities of number, size and shape (arrowed) compared to vector transfected cells (Fig. 6F).
  • Fig. 6E shows C33A tumour cells with abnonnal nuclei clearly visible.
  • Figure 7A & B shows that the development of abnonnal nuclei in TLP10 3T3 cells is inhibited by treatment of cells with Y27632.
  • the FACS profile ( Figure 7C) demonstrates that Y27632 treatment of TEP10 3T3 cells produces a profile indistinguishable from normal 3T3 cells ( Figure 6 A).
  • TIP-1 mRNA is Increased in a Variety of Human Tumours.
  • TLP-1 cDNA was probed a matched pair human tumour array with labelled TLP-1 cDNA.
  • Fig. 8 A clearly shows that the expression of Tip-1 RNA is up-regulated in the majority of breast carcinomas and some from uterus, lung, ovarian and thyroid when compared to normal tissue from the same patient.
  • Fig. 7B demonstrates the uniform signal obtained with a probe made from a selected housekeeping gene.
  • the PDZ protein TLP-1 interacts with the Rho effector rhotekin and is involved in Rho signaling to the serum response element. J Biol Chem, 275: 33962-33968., 2000.
  • RhoA Transformation mediated by RhoA requires activity of ROCK kinases. Cun Biol, 9: 136-145, 1999.
  • E6 protein of human papillomavirus type 16 functions as a transcriptional repressor in a mechanism independent of the tumor suppressor protein, p53. Virology, 205: 583-585, 1994.

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Abstract

L'invention concerne l'utilisation d'agents permettant de réduire les niveaux cellulaires de la protéine-1 Tax (TIP-1) de HTLV1 ou d'inhiber ou modifier les effets fonctionnels de TlP-1 afin de traiter, prévenir ou limiter des dommages tissulaires. Ces agents sont particulièrement utiles pour prévenir un dommage pouvant apparaître comme résultat du processus de vieillissement et dans le traitement du cancer. L'invention concerne également le diagnostic et le traitement (y compris le traitement prophylactique) du cancer, et une méthode permettant d'identifier des cibles cancéreuses impliquées dans le processus d'invasion tumorale.
PCT/GB2004/002383 2003-06-06 2004-06-04 Traitement d'etats lies au vieillissement Ceased WO2004108142A2 (fr)

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WO2009071880A3 (fr) * 2007-12-04 2009-11-05 Vibio Limited Marqueur de pronostic de cancer
WO2015023110A1 (fr) * 2013-08-12 2015-02-19 사회복지법인 삼성생명공익재단 Biomarqueur pour le diagnostic d'une maladie tumorale et utilisation associée

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BR9711154A (pt) * 1996-08-12 1999-08-17 Yoshitomi Pharmaceutical Agente farmac-utico contendo inibidor cinase rho
WO1999066946A1 (fr) * 1998-06-24 1999-12-29 Trustees Of The University Of Pennsylvania Compositions et procedes servant a provoquer une apoptose chez des cellules exprimant e6
US6410323B1 (en) * 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
EP1177796B1 (fr) * 1999-04-27 2011-04-27 Mitsubishi Tanabe Pharma Corporation Médicament pour la prévention ou le traitement des maladies du foie
AU2002321903A1 (en) * 2001-08-03 2003-02-24 Arbor Vita Corporation Molecular interactions in cells
CA2400996A1 (fr) * 2002-09-03 2004-03-03 Lisa Mckerracher Cyclohexanes substitues en positions 1 et 4
EP1452589A1 (fr) * 2003-02-28 2004-09-01 Migragen AG ADP-ribosyltransférase c3cer

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009071880A3 (fr) * 2007-12-04 2009-11-05 Vibio Limited Marqueur de pronostic de cancer
WO2015023110A1 (fr) * 2013-08-12 2015-02-19 사회복지법인 삼성생명공익재단 Biomarqueur pour le diagnostic d'une maladie tumorale et utilisation associée

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