WO2009155662A1

WO2009155662A1 - A tumour suppressor protein, caspase-2

Info

Publication number: WO2009155662A1
Application number: PCT/AU2009/000827
Authority: WO
Inventors: Sharad Kumar; Lien Ho; Loretta Dorstyn
Original assignee: Medvet Science Pty Ltd
Current assignee: Medvet Science Pty Ltd
Priority date: 2008-06-27
Filing date: 2009-06-26
Publication date: 2009-12-30
Anticipated expiration: 2010-12-27

Abstract

Methods of diagnosis, prognosis and predisposition assessment of cancer are described comprising detecting, in a suitable body sample from a subject, the presence or absence of at least one non¬ functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression. Also disclosed is a method and agent for treating or preventing cancer in a subject.

Description

A TUMOUR SUPPRESSOR PROTEIN, CASPASE-2

FIELD OF THE INVENTION

The present invention relates to the finding that caspase-2 has a tumour suppressor function. In one particular application, the invention provides a method of diagnosis or prognosis of cancer in a subject involving the determination of the presence or absence of at least one non-functional allele of caspase-2 gene.

BACKGROUND OF THE INVENTION

The caspase family of cysteine proteases play a key role in inflammation and the execution of apoptosis (reviewed in 1). The mammalian enzyme known as caspase-2 (originally designated Nedd-2 and sometimes known as ICH-I) is the most evolutionarily conserved of the mammalian caspases, and is a close homologue of the Caenorhabditis elegans caspase CED-3 and the caspase DRONC of Drosophila melanogaster; all of which contain a caspase recruitment domain (CARD) and are essential for most developmental cell deaths (2-7). However, while many in vitro studies have implied an important role for caspase-2 in DNA damage and stress-induced cell death (reviewed in 1, 10), caspase-2 knockout (KO) mice are nevertheless viable and show no overt phenotype (8, 9). Thus, and despite the fact that caspase-2 was one of the first mammalian caspases to be cloned, the physiological function of caspase-2 has remained enigmatic.

However, hitherto, there has been some indirect evidence to suggest that caspase-2 may play some role in cancer. For example, the human caspase-2 gene region is frequently affected/deleted in leukaemia (11), and caspase-2 and caspase-3 levels have been proposed as predictors of complete remission and survival in adults with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) (12, 13). Further, caspase-2 levels are known to be reduced in mantle cell lymphoma, childhood ALL (14, 15) and in some multiple myeloma cell lines (unpublished). Moreover, a recent report suggests that caspase-2 induces apoptosis in a pathway downstream of ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) following Chkl inhibition and γ-irradiation induced DNA damage (16). However, it has not, prior to the experimentation described hereinafter, been known whether the loss of caspase-2 in vivo can potentiate or facilitate tumourigenesis.

In the experimentation described hereinafter, cells from caspase-2 KO mice and a transgenic mouse model of cancer were used to assess what effect loss of caspase-2 would have on tumourigenesis and, further, to determine whether caspase-2 may play a role in tumour surveillance and/or inhibition. In particular, caspase-2 -I- mouse embryonic fibroblasts (MEFs) were transformed with the ElA and Ras oncogenes and their respective growth properties compared with caspase-2 +/+ cells transformed in a similar manner. Further, the mouse Eμ-myc lymphoma model (20) was used to assess whether the loss of caspase-2 predisposes animals to tumour development. As shown in the Example, the experimentation showed that ElAIRas transformed caspase-2 -I- cells grow significantly faster than caspase-2 +/+ cells, generate larger colonies in soft agar, and form more aggressive and accelerated tumours in nude mice. Further, the caspase-2 -/- MEFs showed an aberrant cell cycle response to radiation-induced damage and reduced expression of the targets of the tumour suppressor protein p53. Moreover, the Eμ-myc mice in caspase-2 -/- background were found to develop more rapid clonal pre- B or B cell lymphomas. These results therefore indicate that caspase-2 is a tumour suppressor protein which plays a role in protection against tumour development.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method of diagnosis or prognosis of cancer in a subject, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a loss or reduction of caspase-2 gene expression.

The method of the first aspect may be particularly suitable for the diagnosis or prognosis of a cancer selected from lymphomas, cancers induced by a gene selected from the group consisting of one or more of the oncogenes Myc, ElA and Ras, and/or the loss of the lymphoma suppressor gene Bim and/or the tumour suppressor gene p53.

In a second aspect, the present invention provides a method for assessing a subject's predisposition to cancer, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a loss or reduction of caspase-2 gene expression.

In a third aspect, the present invention provides a method for assisting the selection of a therapy for a cancer in a subject, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a loss or reduction of caspase-2 gene expression.

In a fourth aspect, the present invention provides a method of treating or preventing cancer in a subject, wherein said method comprises administering to said subject an agent for modulating the activity of caspase-2, optionally in combination with a pharmaceutically acceptable carrier.

In a fifth aspect, the present invention provides the use of an agent for modulating the activity of caspase-2, optionally in combination with a pharmaceutically acceptable carrier, for the treatment or prevention of cancer. In a sixth aspect, the present invention provides the use of an agent for modulating the activity of caspase-2 in the preparation of a pharmaceutical composition for treating or preventing cancer.

Further, in a seventh aspect, the present invention provides a pharmaceutical composition for treating or preventing cancer, said composition comprising an agent for modulating the activity of caspase-2 in combination with a pharmaceutically acceptable carrier.

Moreover, in an eighth aspect, the present invention provides a non-human animal model for cancer, wherein said model lacks a functional caspase-2 gene.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides: (a) Growth data for caspase-2 +1+ and -/-MEFs, (b) Growth oi ElAIRas transformed caspase-2 +1+ and -/- MEFs, (c, d) Soft-agar colony formation by caspase-2 +/+ and -/- MEFs and ElAIRas transformed caspase-2+/+ and -/- MEFs. In panels a, b and d, the results are shown as mean ± SEM from three independent experiments using MEFs isolated from three different embryos per genotype, and performed in triplicate. * indicates p<0.05;

Figure 2 shows results for primary non-transformed caspase-2 +1+ and -/- MEFs, and ElAIRas transformed caspase-2+/+ and -/-MEFs having been exposed to γ-radiation (10 Gy), vincristine (50 mg/ml) or cisplatin (30 μM) for the indicated times and apoptosis analysed by Annexin V staining. Results are mean ± SEM from three experiments. * p<0.05;

Figure 3 provides results showing that the lack of caspase-2 accelerates tumour formation in male athymic nude mice, (a) Representative mice subcutaneously injected with saline (LHS panels showing mice at day 15), 1 x 10⁶ ElAIRas transformed caspase-2+/+ (RHS panels showing mice at day 15), or 1 x 10⁶ ElAIRas transformed caspase-2-l- MEFs (lower RHS panels showing mice at day 10) in the hind flanks, (b) The data represent the average tumour volume for the ElAIRas transformed caspase- 2+1+ MEFs injected mice (n = 10) and ElAIRas transformed caspase-2-l- (8 different batches) injected mice (n = 8) and expressed as mean ± SEM. MEFs derived from four animals for each genotype were transformed with ElAIRas and injected in nude mice. Thus, 2-3 mice received cells derived from a single caspase-2+l+ or caspase-2-l- animal. p=1.9 x 10^"4. Note that all mice injected with ElAIRas transformed caspase-2-l- MEFs developed large tumours by day 10 and were euthanised before day 15;

Figure 4 provides results indicating that caspase-2 contributes to apoptosis induced by IR. (a) Apoptosis level of non-transformed and ElAIRas transformed caspase-2-l- MEFs following restoration of caspase-2 expression via transfection of caspase-2-GFP. Cells were γ-irradiated (IR) and then cultured for 48 or 120 h before apoptosis analysis. Results are expressed as mean ± SEM from three experiments, * indicates p<0.05. (b) Immunoblots showing levels of caspase-2 protein following transfection of caspase-2-/- MEFs and ElAIRas transformed caspase-2-/- MEFs with the caspase-2- GFP expression construct. Data from 2-3 separate transfection experiments are shown. *indicates a band that presumably represents caspase-2 from which GFP has been cleaved off. The relative positions of caspase-2-GFP (78 kD), caspase-2 (51 kD) and β-actin (42 kD) are indicated by arrowheads. The same blots were sequentially probed with a caspase-2 antibody and a β-actin antibody;

Figure 5 provides results showing the levels of expression of p53 and p53 target genes (iep21, puma and noxa) in caspase-2-/- (C2^~'^~) MEFs. WT (casp2^+l+) and caspase-2-/- (casp2~'^~) MEFs were grown in high glucose DMEM and RNA extracted at passages Pl, P4 and P8, as indicated. Levels oϊp53, p21, puma and noxa transcripts were measured in triplicate reactions using Real-Time qPCR and expressed relative to the internal control gene β-actin. The results shown are averages from a single experiment;

Figure 6 provides results demonstrating that the loss of caspase-2 accelerates lymphoma development induced by Eμ-Myc transgene. (a) Cumulative incidence of all tumours in mice of the indicated genotype. Lymphoma development was accelerated in Eμ-myc mice by the loss of one allele of caspase-2 or Bim (p<0.001). (b) Lymphoma development was further accelerated in Eμ-myc mice by the loss of both alleles of caspase-2 or Bim (p<0.001). (c) Peripheral blood white cell counts from mice with lymphoma following sacrifice, expressed as % mean ± SEM of non-granulocytes, granulocytes and lymphoblasts from total number of cells counted in Eμ-myc, caspase-2+l- and Bim+ι '-/Eμ-myc mice, (d) Peripheral blood white cell count from mice with lymphoma following sacrifice in Eμ-myc, caspase-2-/- and Bim-l-IEμ-myc mice. In (c) and (d), cells were counted from blood smears. * indicates p<0.001;

Figure 7 shows representative histology of lymph nodes, liver and lung from Eμ-myc (LHS) and caspase-2-l -/Eμ-myc (RHS) mice showing (arrows) increased lymphocytic infiltrates and prominent lymphoblasts in blood smear. Sections were stained with H&E. The Eμ-myc and caspase-2-l -/Eμ-myc mice show lymphocytic infiltrates characteristic of lymphoma;

Figure 8 provides results indicating that the loss of caspase-2 results in reduced apoptosis in lymphoma cells. Eμ-myc and caspase-2-l -lEμ-myc lymphoma cells were treated with; (a) zoledronic acid for 24h; (b) Cytochalasin D for 24h; (c) Vincristine for 24h; or (d) γ- irradiated and cultured for 48h. Apoptosis was determined by Annexin V staining using flow cytometry. Results are expressed as mean + SEM from 3-4 separate experiments. * p<0.05; Figure 9 shows results indicating that caspase-2 contributes to IR-induced apoptosis following Chkl inhibition, (a) Levels of apoptosis in caspase-2+/+ and -/-MEFs treated with γ radiation (10Gy) and/or Chkl inhibitor (G56976) for 48 h were assessed by Annexin V staining, (b) Levels of apoptosis in EIA/Ras transformed caspase-2+/+md -I- MEFs treated as in (a). The results in (a) and (b) are shown as mean ± SEM from 3-4 independent experiments with different batches of MEFs. * indicates p<0.05. Caspase activity in caspase-2+/+ and -/-, and EIA/Ras transformed caspase-2+l+ and -/-MEFs was assessed by cleavage of DEVD-AMC (c, d). Results are expressed as mean ± SEM relative fluorescence units (RFU) from three experiments using different batches of MEFs. * indicates p<0.05;

Figure 10 provides the results of an assessment of VDVADase activity in cells undergoing IR-induced apoptosis following Chkl inhibition. Caspase activity in (a) caspase-2+l+ and -/-, and (b) EIA/Ras transformed caspase-2+/+ and -/- MEFs, was assessed by cleavage of VDVAD-AMC substrate by cell lysates. Results (from three experiments) are expressed as mean ± SEM relative fluorescence units (RFU). *p<0.05; and

Figure 11 provides results showing that caspase-2 contributes to IR-induced cell cycle arrest, (a) and (b), cells stained positive for BrdU 24 h following γ-irradiation. Caspase-2+/+ or -/- MEFs, and EIA/Ras transformed caspase-2+/+and -I- MEFs were seeded onto glass coverslips and exposed to 10 Gy γ-irradiation. 24 h following γ-irradiation, cells were pulsed for 4 h with BrdU and BrdU +ve cells detected by immunostaining (a). Results in (b) are expressed as mean ± SEM from three experiments. * indicates p<0.005 for comparison between irradiated caspase-2+/+ versus -/- MEFs and EIA/Ras transformed caspase-2+/+ and -/- MEFs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention stems from the finding that caspase-2 has a tumour suppressor function and that the loss of either one or two caspase-2 gene alleles results in accelerated tumour potential. It is therefore considered that, for example, determining the caspase-2 genotype or the level or lack of caspase-2 expression in a tumour tissue sample, can be used to provide valuable information on the diagnosis or prognosis of cancer in a subject.

Thus, in a first aspect, the present invention provides a method of diagnosis or prognosis of cancer in a subject, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression. As used herein, the term "non-functional allele" refers to an allele of the caspase-2 gene that either can not be substantially expressed or, otherwise, encodes an inactive or substantially inactive caspase-2 enzyme (as determined by, for example, using a standard protease activity assay such as the Protease Fluorescent Detection Kit available from Sigma-Aldrich Inc., St Louis, MO, United States of America). A heterozygous subject, or heterozygous cells of a sample therefrom, therefore includes one non-functional caspase-2 allele and one functional caspase-2 allele (ie caspase-2 +/-), while a homozygous subject, or homozygous cells of a sample therefrom, includes either two non-functional caspase-2 alleles (ie caspase-2 -/-) or two functional caspase-2 alleles (ie caspase-2 +/+).

It will be understood by persons skilled in the art that functional caspase-2 alleles may encode, for example, any of the functional caspase-2 enzymes that have been previously reported, including the protein originally designated Nedd-2 (11) and the truncated variant forms of the protein originally designated ICH- 1_L and ICH-I₅ (5). It will also be understood by persons skilled in the art that a nonfunctional allele of caspase-2 gene may be the result of genetic mutation such as, for example, deletion of the allele, truncation of the allele, a missense mutation, a nonsense mutation, a frameshift mutation or a splice-site mutation.

It is considered that the tumour suppressor function of caspase-2 is a general tumour suppression function and, as such, the method of the first aspect is broadly applicable to the diagnosis and prognosis of cancer. Nevertheless, it is recognised that the method may be particularly suitable for the diagnosis or prognosis of a cancer selected from lymphomas (such as ALL, AML, mantle cell lymphoma, and childhood ALL) and, preferably, lymphomas induced by an oncogene other than Bcl2 and/or Ras. Moreover, the method may be particularly suitable for the diagnosis or prognosis of a cancer induced by an oncogene or the loss of a lymphoma suppressor gene (eg Bim) and/or a tumour suppressor gene (egp53).

The method may utilise any suitable body sample, such as a sample of blood, serum, urine, cheek cell, or tumour tissue (eg tissue biopsy). Preferably, the body sample used will be a sample of tumour tissue.

The presence or absence of at least one non-functional allele of the caspase-2 gene may be determined using any of the methods well known to persons skilled in the art.

Thus, the step of detecting the presence or absence of at least one non-functional allele of the caspase- 2 gene may comprise probing genomic DNA (eg by Southern blotting with appropriate DNA probes) or amplifying the caspase-2 gene or a chromosomal locus or region normally expected to include the caspase-2 gene (eg a locus or region at human chromosome 7q35) with suitable primer sequences. For amplification reactions, the primer sequences may be designed to amplify all caspase-2 gene sequences present in the sample, thereby necessitating the determination of the sequences of the amplified DNA (ie to determine whether the amplified sequences represent a non-functional and/or functional caspase-2 gene alleles), or may otherwise be designed to amplify, under appropriate conditions, only a caspase-2 gene sequence representing a non-functional allele (or a portion thereof). In an embodiment, a suitable amplification may be a multiplex reaction utilising two or more pairs of primer sequences designed to amplify two or more known non-functional caspase-2 gene alleles (or a portion thereof).

Alternatively, the step of detecting the presence or absence of at least one non-functional allele of the caspase-2 gene may comprise determining the presence or absence of functional caspase-2 enzyme (or messenger RNA encoding same) present in a body sample. For example, the presence or absence of caspase-2 protein may be determined by using standard immunoassay employing an antibody or fragment thereof (eg a monoclonal fragment or recombinant fragment such as an scFv fragment) which is capable of distinguishing between non-functional and functional variants of caspase-2 (ie the antibody or fragment thereof preferentially binds to functional caspase-2 or, otherwise, preferentially binds to one or more non-functional caspase-2 variants). Otherwise, any caspase-2 enzyme present in the sample may be isolated and thereafter assessed for molecular weight, amino acid sequence and/or protease activity to determine whether the caspase-2 is functional or non-functional.

The lack or reduction of caspase-2 gene expression may be determined using any of the methods well known to persons skilled in the art. A reduction of caspase-2 gene expression may be determined by, for example, comparison of a measured level of caspase-2 protein or mRNA from the sample against a similarly measured level from a normal tissue sample from the subject and/or similarly measured levels from equivalent tissue samples taken from normal (ie WT) subjects.

Such a lack or reduction of caspase-2 gene expression may arise, of course, from the presence of at least one non-functional allele of caspase-2 gene (eg a deleted caspase-2 allele or an allele that has been otherwise mutated such that it can no longer be expressed), but might also arise for other reasons such as, for example, a lack or deficiency of a transcription factor required for caspase-2 gene expression (ie a lack or deficiency of an activator of caspase-2 gene), abnormal expression (eg over- expression) of a repressor of caspase-2 gene expression, or altered protein stability. For a subject (or a sample therefrom) which is caspase-2 -I- and expresses no caspase-2 protein (eg where the caspase-2 alleles have been deleted or otherwise mutated such that they can no longer be expressed), the sample may simply be assessed by standard immunoassay using a specific anti-caspase-2 antibody (or fragment thereof); the lack of detection of any caspase-2 enzyme will thereby indicate that the subject (or sample therefrom) is caspase-2 -/-. It is also possible to determine the relative amount of caspase-2 present (eg relative to the caspase-2 amount present in equivalent samples from normal subjects) by, for example, immunoblotting of protein lysates, to identify a subject (or sample therefrom) that is caspase-2 +/-.

In the context of cancer prognosis, determining the presence of at least one non-functional caspase-2 gene allele (or a reduction of caspase-2 gene expression) is indicative of a poorer prognosis (without suitable intervention) than for a similar cancer from a subject, or sample thereof, having two functional caspase-2 gene alleles (ie caspase-2 +/+) and normal levels of expression of caspase-2 protein. Further, for a subject, or sample thereof, having two non-functional alleles of the caspase-2 gene (ie caspase-2 -/-) and/or a lack of caspase-2 gene expression, the prognosis of a cancer will be poorer (without suitable intervention) than the prognosis of a similar cancer from a subject, or a sample thereof, having one non-functional caspase-2 gene allele (ie a heterozygous caspase-2 +/- genotype) and/or a reduction of caspase-2 gene expression.

In a second aspect, the present invention provides a method for assessing a subject's predisposition to cancer, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression.

A subject determined as having at least one non-functional allele of caspase-2 gene (ie a subject that has a caspase-2 +/- or caspase-2 -I- genotype) is likely to have a greater predisposition to cancer (particularly, a cancer selected from: lymphomas).

In a third aspect, the present invention provides a method for assisting the selection of a therapy for a cancer in a subject (such as in a "personalised medicine" approach), said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one nonfunctional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression.

Preferably, the body sample will be a tumour tissue sample.

The method of the third aspect is particularly suitable for assisting in the selection of a chemotherapy for a cancer in a subject by, for example, providing information to identify chemotherapeutic agents that are unlikely to bring about a desirable therapeutic outcome (eg a slowing or diminishing of cancer growth or spread). In particular, it has been found that a tumour from a subject (or sample therefrom) that has at least one non-functional allele of caspase-2 gene (ie is caspase-2 +/- or caspase-2 -/-) is likely to show resistance to γ-irradiation and some chemotherapeutic agents. Accordingly, if operation of the method, and particularly the step of detecting the presence or absence of at least one non- functional allele of caspase-2 gene, reveals a caspase-2 +/- or caspase-2 -I- genotype, the method preferably further comprises a step of selecting a therapy for a cancer in the particular subject which does not include radiotherapy with γ-irradiation or chemotherapy with some chemotherapeutic agents or, at least, only utilises radiotherapy or chemotherapy of this type in a combination therapy with another cancer therapy (eg chemotherapy with a mitosis impairing agent, antiangiogenesis agent, apoptosis inducing agent, or an agent which modulates (eg activates) a downstream target of caspase- 2). Similarly, if operation of the method reveals a lack or reduction of caspase-2 gene expression, the method preferably further comprises a step of selecting a therapy for a cancer in the particular subject which does not include radiotherapy with γ-irradiation or chemotherapy with some chemotherapeutic agents or, at least, only utilises radiotherapy or chemotherapy of this type in a combination therapy with another cancer therapy.

In the method of the second or third aspects, the step of detecting the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression, may be as described above in relation to the method of the first aspect.

Preferably, the agent enhances the activity of caspase-2 (ie is a caspase-2 enhancing agent). Such an agent may provide caspase-2 to a subject (or tissue thereof) lacking caspase-2, or may simply increase the amount of endogenous caspase-2 present. Such an agent may be selected from caspase-2 (preferably including a native or heterologous nuclear localisation signal (NLS)), agents which enhance transcription or translation of the caspase-2 gene (eg a transcription factor associated with caspase-2 over-expression) and gene therapy agents such as expression vectors or oligonucleotides or other delivery systems (eg viral vectors such as retroviral or adenoviral vectors) containing a polynucleotide sequence encoding caspase-2 (preferably including a native or heterologous nuclear localisation signal (NLS)). Alternatively, the caspase-2 enhancing agent may enhance Ihe activity of endogenous caspase-2 in the subject. As used herein, the term "caspase-2 enhancing agent" is to be understood as including agents which mimic the activity of caspase-2 (eg functional fragments of caspase-2, peptide mimetics of the active domains of caspase-2, and small organic molecules which mimic caspase-2 activity).

Caspase-2 modulating agents for use in the method may be formulated into any suitable pharmaceutical composition or dosage form (eg compositions for oral, buccal, nasal, intramuscular and intravenous administration). Typically, such a composition will be administered to the subject in an amount which is effective to achieve a therapeutic effect, and may therefore provide between about 0.01 and about 100 μg/kg body weight per day of the caspase-2 modulating agent, and more preferably, provide from 0.05 and 25 μg/kg body weight per day of the caspase-2 modulating agent. A suitable composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.

Preferably, the cancer to be treated or prevented will be selected from: lymphomas and, preferably, lymphomas induced by an oncogene other than Bcl2 and/or Ras; a cancer selected from pancreatic cancers and, preferably, pancreatic cancers induced by an oncogene other than Bcl2 and/or Ras; and a cancer induced by an oncogene selected from the group consisting of Bcl2, Myc, ElA, Ras and Bim, or a cancer induced by an oncogene other than Bcl2 and/or Ras) than a caspase-2 +/+ subject.

The method of the fourth aspect may be used as a combination therapy wherein the method further comprises administering a chemotherapeutic agent such as a cytoskeletal disrupting agent, mitosis impairing agent, antiangiogenesis agent and/or apoptosis inducing agent (including, specifically, one or more of zoledronic acid, vincristine, cytochalasin D, paclitaxel, cisplatin and etoposide), and/or involve radiotherapy.

In a fifth aspect, the present invention provides the use of an agent for modulating the activity of caspase-2, optionally in combination with a pharmaceutically acceptable carrier, for the treatment or prevention of cancer.

In a related, sixth aspect, the present invention provides the use of an agent for modulating the activity of caspase-2 in the preparation of a pharmaceutical composition for treating or preventing cancer.

Moreover, it has been found that caspase-2 deficient mice can provide a model of accelerated ageing (30). This accelerated ageing can be readily observed by premature greying of hair in such mice. The present applicant has now found, surprisingly, that these mice also spontaneously develop pancreatic cancer.

Thus, in an eighth aspect, the present invention provides a non-human animal model for cancer, wherein said model lacks a functional caspase-2 gene. The model may be particularly suitable for the assessment of agents or therapies for the treatment or prevention of age-related cancer such as a pancreatic cancer.

It is also to be understood that the present invention relates to kits for use in the methods of the first, second or third aspects, wherein said kits may comprise a caspase-2 protein or functional fragments thereof and/or an anti-caspase-2 antibody. Additionally, or alternatively, such kits may comprise oligonucleotide probes for hybridisation assays or oligonucleotide primers for polynucleotide amplification-based assays.

The invention is hereinafter described by way of the following, non-limiting examples and accompanying figures.

EXAMPLES

Example 1 Loss of caspase-2 enhances tumour development

Materials and Methods

Proliferation of caspase-2 +/+ and -/- MEFs

Primary MEFs were derived from WT and caspase-2 -I- embryos at day 13.5 as previously described (17, 18). MEFs were grown and maintained in high glucose DME medium and routinely passaged just prior to reaching confluence. Proliferation of caspase-2 -/- MEFs was assayed by doubling time experiment and PROMEGA Proliferation Assay cell titre 96 Aqueous ONE kit (Promega Corporation, Madison, WI, United States of America). Similar results were obtained for both assays. For the doubling time experiment, a specific number (1x10⁴) of cells were seeded and viable cells counted at various time points up to 72 hrs with trypan blue. Colony forming assay was performed by resuspending cells (1 x 10⁴) in 0.3% agar in DMEM +10% foetal calf serum (FCS) and overlaid on 0.6% agar in the same medium in 35mm culture dishes. The dishes were incubated at 37⁰C in 5% CO₂ for 14 days. Colonies were stained in 0.1% toluidine blue in 1% formaldehyde and counted.

Transformation of caspase-2 +/+ and -/- MEFs

Caspase-2 +/+ and caspase-2 -/- MEFs were transfected with pWZL-H retroviral vector containing the ElAIRas oncogenes (obtained from Scott Lowe, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, United States of America)(19, 20). BOSC-23 cells (obtained from Warren Pear, Rockefeller University, NY, United States of America) were transiently transfected with the retroviral vector using lipofectamine (Invitrogen Corporation, Carlsbad, CA, United States of America). Viral supernatants were collected 24 and 48 hrs after transfection and cleared by filtration. 2ml of filtered viral supernatant was added to caspase-2 +/+ and -/- MEFs at 80% confluency and incubated for 24 hrs. Infected cell populations were selected by culture in hygromycin B to eliminate uninfected cells.

In vivo tumour growth and histological analysis

Suspension of pWZL-H ElAIRas transformed caspase-2 +/+ and -/- MEFs (1 x 10⁶ in 200μl sterile PBS) were injected subcutaneously into both hind flanks of male 6-8 wks old Balbc athymic nude mice, and tumours were allowed to develop for up to 20 days. Tumours were then measured externally with a caliper, in two dimensions on indicated days. Tumour volumes were calculated from the equation V= (LxW²) x 0.5, where L is length and W is width (21). Blood and tissue samples were collected on animals for histological examination, and tumours were collected in 10% buffered neutral formalin, and then paraffin processed, sectioned and stained with haematoxylin and eosin.

Animal studies

Eμ-myc transgene, Bim and caspase-2 KO mice (obtained from Professor Jerry Adams (The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia) and athymic Balbc mice were used in these studies. Eμ-myc transgenic mice have been backcrossed onto the C57BL/6J background for more than 20 generations (22). Bim -I- and caspase-2 -/- mice have been backcrossed to C57BL/6J mice for twelve or eight generations, respectively. All mice were rederived and then Eμ- myc males were crossed with caspase-2 -/- or Bim+/- females. Then, caspase-2 +/- Eμ-myc male offspring were crossed with caspase-2 -/- females to generate caspase-2 -/- Eμ-myc mice. Similarly, Bim +/- Eμ-myc male offspring were crossed with Bim -I- females to generate Bim -I- Eμ-myc mice. The Eμ-myc transgene and the KO alleles were detected by PCR. The myc transgene was always bred through the male because differences in lymphoma onset have been observed between offspring from Eμ-myc males versus offspring from Eμ-myc females (ie many Eμ-myc females develop lymphoma while rearing their offspring and are therefore poor mothers (23)).

Mice of each genotype were monitored daily for tumour development. Rate and incidence of lymphoma in cohorts of recipient mice were compared by log-rank test and Kaplan-Meier analysis using the GraphPad Prism 4® software (GraphPad Software, Inc., La Jolla, CA, United States of America). Cause of death was attributed to Eμ-myc lymphoma if a combination of the following features were noted: presence of an enlarged spleen and/or lymph nodes, histologic evidence of invasive lymphoma, and a lymphocyte count >20 x 10⁶/ml. Tumours, peripheral blood, and tissue samples were collected after euthanisation of sick mice. Lymphoma cells from tumours were cultured in DMEM with 10% FBS supplemented with 50 μM β-mercaptoethanol and 100 μM asparagine for further studies. All mice used for breeding were censored from the analysis at the time of mating to exclude any effect of breeding on tumour development. Pre-B or B cell lymphomas were assessed by surface staining of IgM positive or negative cells by flow cytometry (24). Analysis of the expression of p53 target genes

Total RNA was isolated from MEF using TRIzol® reagent (Invitrogen). cDNA was synthesised from lμg RNA using High capacity cDNA reverse transcription kit (Applied Biosystems Inc., Carlsbad, CA, United States of America) and random primers. Real-Time PCR was performed on a Rotor-Gene 3000 (Corbett Research Pty Ltd, Sydney, NSW, Australia) using RT² Real-Time SYBR Green / ROX PCR MasterMix (SuperArray Bioscience Inc., Frederick, MD, United States of America) as per the manufacturer's instructions. The following primer sets were used:

p21 AGTGTGCCGTTGTCTCTTCG (SEQ ID NO: 1 ) and ACACCAGAGTGCAAGACAGC (SEQ ID NO: 2)

p53 CTCACTCCAGCTACCTGAAGA (SEQ ID NO: 3) and AGAGGCAGTCAGTCAGTCTGAGTCA (SEQ NO: 4)

Puma ATGCCTGCCTCACCTTCATCT (SEQ ID NO: 5) and AGCACAGGATTCACAGTCTGGA (SEQ ID NO: 6)

Noxa ACTGTGGTTCTGGCGCAGAT (SEQ ID NO: 7) and TTGAGCACACTCGTCCTTCAA (SEQ ID NO: 8)

β-actin GATCATTGCTCCTCCTGAGC (SEQ BD NO: 9) and AGTCCGCCTAGAAGCACTTG (SEQ ID NO: 10).

Reactions were performed in triplicate and the mRNA expression levels normalised against the internal control gene β-actin using the ΔΔC_T method.

Cell death analysis

Caspase-2 +/+ and -/- MEFs, or lymphoma cells derived from Eμ-myc, Caspase-2-/- Eμ-myc and Bim-/- Eμ-myc mice were incubated with various drugs at indicated concentrations. Apoptosis was determined by Annexin V (Roche Applied Science, Penzburg, Germany) and propidium iodide staining using flow cytometry (25). Where indicated, transformed and nontransformed caspase-2+/+ and caspase-2 -/-MEFs were irradiated with γ-irradiation (10 Gy) with or without 1 μM Go6976 (Calbiochem, San Diego, CA, United States of America) and incubated for 48 h prior to staining with Annexin V. Caspase assays

Caspase assays were carried out essentially as previously described (26, 27), using VDVAD-AMC (California Peptide Research Inc., Napa, CA, United States of America) and DEVD-AMC (Enzyme System Products, Livermore, CA, United States of America) as substrates. Fluorescence was analysed on a FLUOstar Optima Luminescence Spectrometer (BMG Labtech, Durham, NC, United States of America; excitation 355 nm, emission 460 nm). Results from independent experiments were expressed as relative fluorescence units.

Caspase-2 knock-in

A caspase-2-GFP construct (2μg) was transfected into non-transformed and transformed caspase-2-l- MEFs with lipofectamine (Invitrogen). After 48 hours, the cells were γ-irradiated (10Gy) and apoptosis assessed at indicated times by Annexin V staining using flow cytometry. As overexpression of caspase-2 leads to apoptosis, dead cells (presumably expressing very high levels of caspase-2) were washed out prior to irradiating cells. Lysates from transfected cells were immunoblotted using a caspase-2 antibody (9) and a β-actin antibody (Sigma-Aldrich).

BrdU incorporation

1 x 10⁵ MEFs or MEFs transformed with ElAIRas were seeded on coverslips in 6 cm dishes and exposed to 10Gy γ-radiation 16h later. Following 24 hours, cells were pulsed with 30μM BrdU for 4h. Cells were washed and fixed in cold 70% ethanol, hydrolysed in 2M HCl in PBS, blocked in 1%BSA in PBS for Ih, and incubated with anti-BrdU (BD Pharmingen Inc., San Diego, CA, United States of America) overnight at 4°C. After washing, cells were incubated with mouse Alexa 488 antibody for Ih and counterstained with Hoechst dye (Roche Diagnostics Inc., Indianapolis, IN, United States of America). Coverslips were mounted in Antifade solution (1% propylgalate, 87% glycerol) and fluorescence images captured using an epifluorescence microscope (model BX51, Olympus) and a camera (U0CMAD3/CVM300, Olympus). Due to the loss of some cells through apoptosis induced by γ-irradiation, the results shown in Figure 11 provides an approximate % of cells in S phase.

Statistical analysis

Statistical analyses were performed with Student's /-test unless stated otherwise. Where appropriate, the results are expressed as mean ± SEM. The p values of <0.05 were considered significant. Results

Growth properties of caspase-2-/- MEFs

The growth potential of caspase-2 +/+ and -/- MEFs was assessed by doubling time experiment over a period of 72 hrs. It was consistently found that caspase-2 -I- MEFs had a significantly higher growth rate compared to caspase-2 +/+ MEFs (Figure Ia). To avoid potential selection of cells with higher growth rates, in most of the experiments, MEFs at passages 2-4 were used. It was noted that following replating, caspase-2 -I- MEFs appeared to require somewhat longer recovery periods than the matched caspase-2 +/+ MEFs. It was then tested to determine whether increased growth rate of caspase-2 -I- cells lead to their transformation, by analysing their ability to form colonies in soft agar. It was found that both caspase-2 +/+ and -/- MEFs were unable to do so (Figure Ic).

Caspase-2 -I- MEFs transform readily with ElA and Ras oncogenes

Given that caspase-2 deficiency leads to increased proliferation and some resistance to apoptosis induced by specific agents (Figure 2), testing was conducted to assess whether the loss of caspase-2 can enhance oncogenic transformation of MEFs. Accordingly, caspase-2 +/+ and caspase-2 -I- MEFs were transformed with a pWZL-H ElAI Ras retroviral vector. It was evident that the growth rates of E IAI Ras transformed caspase-2 -I- MEFs were significantly higher than those of transformed caspase- 2 +/+ MEFs (Figure Ib). Further, El Al Ras transformed caspase-2 +/+ MEFs formed fewer colonies and at a slower rate compared to the rapid formation of larger colonies by the El Al Ras transformed caspase-2 -I- MEFs in soft agar (Figures Ic and Id; data not shown). These results indicate that the loss of caspase-2 facilitates cell transformation.

ElAIRas transformed caspase-2 -I- cells are highly tumourigenic in nude mice To evaluate the tumourigenic properties of ElAIRas transformed caspase-2 +/+ and caspase-2 -I- MEFs, an experiment was conducted to determine their ability to grow in vivo in immunocompromised mice. When injected subcutaneously (sc) in male athymic nude mice, ElAIRas transformed caspase-2 -I- MEFs, from all eight different batches of MEFs tested, produced rapid and aggressive tumours with all animals developing large tumours within 10 days (Figure 3a and 3b). During this period, ElAIRas transformed caspase-2 +1+ MEFs from multiple batches produced small or non-detectable tumours (Figures 3a and 3b). The average tumour size for caspase-2 -I- MEFs at day 10 was approximately 3 times the average tumour size for caspase-2 +1+ MEFs at day 15 (Figure 3b). Although caspase-2 +/+ and caspase-2 -I- MEFs were similarly transformed by the pWZLH ElAIRas retroviral vector, the delay in the tumour formation and size of tumours produced by transformed caspase-2 +/+ MEFs compared to transformed caspase-2 -I- MEFs suggests that the expression of caspase-2 is required for the inhibition of tumourigenesis. Caspase-2-l- and ElAI Ras transformed caspase-2 -I- MEFs showed significantly reduced sensitivity to apoptosis induced by various cytotoxic drugs and γ-irradiation when compared to caspase-2 +/+ and E IAI Ras transformed caspase-2 +1+ MEFs respectively (Figure 2). Re-expression of caspase-2 in primary and E IAI Ras transformed caspase-2 -I- MEFs resulted in increased levels of γ-irradiation induced apoptosis (Figure 4), suggesting that the levels of caspase-2 may determine the sensitivity to apoptosis in caspase-2 -I- MEFs.

Caspase-2 may play a role in regulating the function of tumour suppressor protein p53 As mentioned above, caspase-2 has been suggested to be involved in p53 dependent apoptosis (1, 9, 29). To investigate this, an analysis was conducted to determine whether the loss of caspase-2 in MEFs leads to reduced expression of p53 and its target genes, including p21, puma and noxa, which are all induced by the expression of p53 in response to cell damage (the protein p21 is involved in the arresting of the cell cycle, while puma and noxa are involved in the induction of apoptosis). It was found that, at various passages, MEFs derived from caspase-2 KO mice showed reduced expression of p53 target genes, compared to WT MEFs, even though p53 expression itself was similar in KO and WT cells (Figure 5). This observation suggests that loss of caspase-2 leads to reduced transcription by p53 in MEFs. Thus, caspase-2 has implications in regulating p53 - a key tumour suppressor gene.

Lack of caspase-2 leads to rapid onset of lymphoma in Eμ-mvc mice

To further test whether loss of caspase-2 results in increased susceptibility to tumour formation in vivo, the Eμ-myc mouse model was used. It was found that Eμ-myc transgenic mice develop spontaneous lymphomas with a mean onset of illness at 16 weeks (23). When caspase-2 deficiency (ie caspase-2 -I- or caspase-2 +/-) was combined with the Eμ-myc transgene, an accelerated occurrence of tumours, compared to Eμ-myc transgenic mice alone, was observed (Figure 6). Interestingly, the loss of one caspase-2 allele was sufficient to accelerate tumour formation; the median lifespan of 12 caspase-2 +/- Eμ-myc mice was 12 weeks and over 90% of the mice were terminally ill before 50% of caspase-2 +/+ Eμ-myc mice were affected (Figure 6a). Caspase-2 -I- Eμ-myc mice had a median lifespan of 8 weeks and became terminally ill within 4 weeks (Figure 6b). Histological examination showed lymphocytic infiltrates in organs including lung, liver and lymph nodes (Figure 7). As in Eμ- myc and caspase-2 +/+ Eμ-myc mice, the tumours induced by Myc in caspase-2 deficient mice were either B cell lymphomas or pre-B lymphomas (Table 1). Table 1 Frequency of pre-B and B lymphomas induced by the Eμ-myc transgene

Lymphomas were typed by flow cytometry of surface immunofluorescence; pre-B cell lymphoma (B220⁺, CD19⁺, IgM^") and B cell lymphoma (B220^', CD 19^", IgM⁺) and expressed as % pre-B or B lymphoma from total number of lymphomas collected from of each genotype. The frequency of B lymphoma in either Eμ-Myc, Bim^+/'Eμ-Myc or Bini'Εμ-Myc versus Caspase-2^+/~Eμ-Myc or Caspase-T^/Εμ-Myc mice was not significant.

Loss of the BH3-only protein Bim results in early onset of tumourigenesis in Eμ-myc mice (24). Thus, the experiment used Bim +/- and Bim -I- Eμ-myc mice as positive controls; Bim +/- Eμ-myc mice had a median lifespan of 13 weeks (Figure 6a). The Bim -I- Eμ-myc mice median lifespan of 11 weeks was slightly longer than caspase-2 -I- Eμ-myc mice (Figure 6b). Analysis of peripheral blood showed that there were significantly higher numbers of lymphocytes, particularly the immature lymphoblasts in Bim^'1' Eμ-myc compared to that of caspase-2 -I- Eμ-myc and Eμ-7/iyc mice (Figures 6c and 6d). This indicated that Bim-/- Eμ-myc mice had increased leukemia compared to caspase-2 -I- Eμ-myc or Eμ- myc mice. These observations indicate that, similar to Bim studies, the loss of caspase-2 accelerates morbidity in Em-myc mice.

Primary lymphoma cells from caspase-2 -I- Eμ-myc mice were assessed for their susceptibility to apoptosis induced by various agents. As previously reported for Eμ-myc lymphoma cells (18), caspase-2 -I- Eμ-myc tumour cells show a high rate of spontaneous apoptosis in culture. Despite high levels of cell death, caspase-2 +/- and -/- lymphoma cells showed reduced rates of apoptosis induced by cytotoxic agents, when compared to lymphoma cells from Eμ-myc mice (Figure 8). These results indicate that caspase-2 contributes to the apoptosis of tumour cells in response to cytotoxic stimuli. Loss of the DNA-damage induced checkpoint control in caspase-2 -I- cells

Caspase-2 has been suggested to be involved in p53 dependent apoptosis (1, 9, 29), which could partly explain its tumour suppressor function. However, the growth properties of caspase-2 deficient MEFs suggest that it also contributes to cell cycle control. Recent studies using zebrafish and siRNA mediated knockdown in cell lines suggest that caspase-2 is required in the checkpoint kinase 1 (Chkiy-suppressed apoptotic pathway in p53-deficient cells (16). Thus, to test the potential role of caspase-2 in Chkl pathway, caspase-2 +/+ and -/- MEFs were used along with ElAIRas transformed caspase-2 +/+ and-/- MEFs in experiments similar to Sidi et al. (16); cells were treated with the Chkl inhibitor Go6976 (which is also known to inhibit PKC) and γ-irradiation. It was found that caspase-2 -I- MEFs were significantly more resistant to apoptosis than the caspase-2 +/+ MEFs treated by either agent alone, as assessed by Annexin V staining and caspase activity measurements (Figures 9a-d and Figure 10). Interestingly, a reduced sensitivity to apoptosis was observed in caspase-2 -I- and ElAIRas transformed caspase-2 -I- MEFs when treated with Gό'6976 and γ-irradiation together (Figure 9). These results suggest that caspase-2 has an important role in apoptosis induced by both Chkl- dependent and -independent pathways, and when Chkl is inhibited, apoptosis induced by double- strand DNA breaks becomes even more dependent on caspase-2. It is important to note that in both the caspase-2 +/+ and caspase-2 -I- cells, caspase activity determined using the two substrates, VDVAD- AMC and DEVD-AMC, was proportional to the number of apoptotic cells. This observation suggests that VDVAD is not a specific substrate of caspase-2 (Figures 9c and 4d, and Figure 10).

In a further experiment, a test was conducted to assess whether caspase-2 deficiency results in an abnormal response to double-strand DNA breaks. It was found that following γ-irradiation a significantly larger proportion of caspase-2 -I- and ElAIRas transformed caspase-2 -I- MEFs were cycling, as assessed by BrdU staining, compared to caspase-2 +/+ cells (Figure 11). Cell cycle analysis also indicated that caspase-2 -I- cells fail to arrest properly following γ-irradiation (data not shown). These results indicate that caspase-2 deficiency results in abnormal checkpoint regulation following DNA damage.

Discussion

Despite being one of the first caspases to be discovered, the physiological function of caspase-2 has remained enigmatic. The experimentation described herein indicates that caspase-2 is a tumour suppressor. In particular, it has been shown that the loss of caspase-2 enhances oncogenic potential both in vitro and in vivo. The only other caspase that may have some role in preventing cell transformation is caspase-8; that is, a recent study suggests that following continuous growth in culture, SV40 T antigen immortalised caspase-8 -I- MEFs become transformed more readily and show increased tumourigenic potential in nude mice than caspase-8 +/+ cells (31). However, while these findings suggest that the loss of caspase-8 may contribute to the rate of cell transformation, they are very different from those described herein with respect to caspase-2. Primary MEFs from caspase-2 -I- mice show higher rates of proliferation and are transformed more readily by ElAlRas than the caspase-2 +/+ MEFs. This suggests that the loss of caspase-2 leads to some deregulation of the cell cycle in vitro, even before oncogenic transformation. Interestingly, it was found that caspase-2 -I- MEFs contain reduced levels of p21 transcript, when compared to passage matched WT MEFs. As reduced levels of p21 mRNA expression are indicative of reduced p53 function, it is suggested that caspase-2 -I- MEFs in culture have a tendency to lose p53 function. This may contribute to aberrant growth and apoptotic response observed in caspase-2 -I- MEFs. Reduced p53 function in caspase-2 -I- MEFs may also facilitate their transformation by ElAlRas. That lack of caspase-2 promotes cell transformation was further validated by accelerated onset of tumours seen in athymic nude mice injected with ElAlRas transformed caspase-2 -I- MEFs.

In vivo, accelerated onset of lymphomas was observed in caspase-2+l- Eμ-myc and caspase-2-/- Eμ- myc mice compared to caspase-2 +/+ Eμ-myc mice, suggesting that even the loss of one allele of caspase-2 results in accelerated Myc-induced lymphomagenesis. As shown previously (24), and in the control study herein, loss of a single allele ofBim is also sufficient for increased lymphomagenesis in Eμ-myc mice. The accelerated Myc-induced lymphomagenesis in Bim+/- Eμ-myc and Bim-I- Eμ-myc mice is consistent with Bim's key role as a regulator of lymphoid and myeloid homeostasis (32-34). However, how the loss of caspase-2 leads to accelerated lymphomagenesis in Eμ-myc mice remains to be explored. Nevertheless, as caspase-2 levels have been proposed as a predictor of remission and survival in various forms of adult leukaemia and its levels are also reduced in certain other cancers (12-15), the results described herein indicate an important role for caspase-2 in tumour suppression.

The results also show that loss of caspase-2 leads to resistance/delay in apoptosis induced by DNA damage. In γ-irradiated cells, this resistance was further enhanced by the inhibition of Chkl; which supports recent findings suggesting that caspase-2 is involved in an apoptotic pathway downstream of ATM and ATR following Chkl inhibition (16). Using the same Chkl inhibitor (which is also known to inhibit PKC), the results showed a significant resistance to apoptosis in γ-irradiated caspase-2 -I- and ElAlRas transformed caspase-2 -I- MEFs. These results suggest that following DNA double- strand breaks, caspase-2 is required for apoptosis downstream of ATM/ATR when Chkl is suppressed. The study by Sidi et al. (16) also suggested that the "Chkl -suppressed" caspase-2 dependent pathway acts independently of the mitochondrial pathway of apoptosis. In response to stress signalling, caspase-2 has been shown to be activated upstream of mitochondria, and consistent with an initiator function, its activation can occur without processing (26, 35-37).

Further, unlike other caspases, caspase-2 has a unique ability to localise to the nucleus (37-39). The predicted cell cycle function of caspase-2 is consistent with its nuclear localisation. In preliminary experiments, it was noticed that compared to their WT counterparts, the late passage caspase-2 -I- MEFs and ElAIRas transformed caspase-2 -I- MEFs have an increased tendency to acquire chromosomal aberrations and become aneuploid. This observation suggests that the lack of caspase-2 promotes genetic instability, presumably due to deregulation of cell cycle checkpoints. Although not validated in this experimentation, the increased genetic instability of caspase-2 -I- MEFs in culture may explain the loss of p53 function in late passage MEFs. Since tumourigenesis is often a consequence of defective apoptosis and cell cycle control, the results described herein showing that the loss of caspase-2 results both in resistance to apoptosis and loss of DNA damage-induced cell cycle regulation, indicates that caspase-2 is a tumour suppressor. The tumour suppressor role of caspase-2 might therefore explain its reduced expression or loss in many cancers (12-15). Caspases are generally regarded as the downstream effectors of apoptosis, and caspase activation as the point of no return in apoptotic signalling. As such, the role of caspase-2 as a tumour suppressor appears to be unique among mammalian caspases.

Although a preferred embodiment(s) of the present invention has been described in the foregoing detailed description, it will be understood that the invention is not limited to the embodiment(s) disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. REFERENCES

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Claims

1. A method of diagnosis or prognosis of cancer in a subject, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression.

2. The method of claim 1, wherein the detecting step comprises determining the presence or absence of at least one non-functional allele of the caspase-2 gene.

3. The method of claim 1, wherein determining the presence of at least one non-functional caspase-2 gene allele, or a reduction of caspase-2 gene expression, is indicative of a poor prognosis.

4. A method for assessing a subject's predisposition to cancer, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression.

5. The method of claim 4, wherein the detecting step comprises determining the presence or absence of at least one non-functional allele of the caspase-2 gene.

6. The method of claim 4 or 5, wherein determining the presence of at least one non-functional allele of caspase-2 gene indicates a predisposition to cancer.

7. A method for assisting the selection of a therapy for a cancer in a subject, said method comprising the step of detecting, in a suitable body sample from said subject, the presence or absence of at least one non-functional allele of caspase-2 gene or, otherwise, a lack or reduction of caspase-2 gene expression.

8. The method of claim 7, wherein the detecting step comprises determining the presence or absence of at least one non-functional allele of the caspase-2 gene.

9. The method of any one of claims 1 to 8, wherein the sample is a tumour tissue sample.

10. A method of treating or preventing cancer in a subject, wherein said method comprises administering to said subject an agent for modulating the activity of caspase-2, optionally in combination with a pharmaceutically acceptable carrier.

11. The method of claim 10, wherein the agent enhances the activity of caspase-2.

12. The method of claim 11, wherein the agent is selected from caspase-2 (preferably including a native or heterologous nuclear localisation signal (NLS)), agents which enhance transcription or translation of the caspase-2 gene and gene therapy agents.

13. The method of claim 12, wherein the agent is selected from expression vectors or oligonucleotides or viral delivery systems containing a polynucleotide sequence encoding caspase-2 (preferably including a native or heterologous nuclear localisation signal (NLS)).

14. The method of any one of claims 10 to 13, wherein the method further comprises administering a chemotherapeutic agent and/or radiotherapy.

15. The method of any one of claims 1 to 14, wherein the cancer is selected from lymphomas.

16. The method of claim 15, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), mantle cell lymphoma, and childhood ALL.

17. The method of any one of claims 1 to 14, wherein the cancer is selected from lymphomas induced by an oncogene other than Bcl2 and/or Ras.

18. The method of any one of claims 1 to 14, wherein the cancer is selected from cancers induced by a gene selected from the group consisting of one or more of the oncogenes Myc, ElA and Ras, and/or the loss of the lymphoma suppressor gene Bim and/or the tumour suppressor gene p53.

19. The use of an agent for modulating the activity of caspase-2, optionally in combination with a pharmaceutically acceptable carrier, for the treatment or prevention of cancer.

20. The use of an agent for modulating the activity of caspase-2 in the preparation of a pharmaceutical composition for treating or preventing cancer.

21. A pharmaceutical composition for treating or preventing cancer, said composition comprising an agent for modulating the activity of caspase-2 in combination with a pharmaceutically acceptable carrier.

22. A non-human animal model for cancer, wherein said model lacks a functional caspase-2 gene.