WO2008078109A2

WO2008078109A2 - Medicament

Info

Publication number: WO2008078109A2
Application number: PCT/GB2007/005010
Authority: WO
Inventors: Mohamed K. El-Tanani; Karl J. Hale; Soraya Manaviazar; Patrick G. Johnston
Original assignee: Queens University of Belfast
Current assignee: Queens University of Belfast
Priority date: 2006-12-22
Filing date: 2007-12-24
Publication date: 2008-07-03
Anticipated expiration: 2009-06-22
Also published as: GB0625783D0; WO2008078109A3

Abstract

There is disclosed the use of (-)-Agelastatin and / or an agelastatin analogue in the treatment of metastatic disease, pharmaceutical compositions comprising Agelastatin and other active agents, for example chemotherapeutic agents and an assay for the identification of modulators of metastasis and / or invasive cancer.

Description

Medicament

FIELD OF INVENTION

The present application relates to (-)-Agelastatin A and/or an agelastatin analogue for use in the treatment of metastatic disease, methods of treatment and pharmaceutical compositions for the same.

BACKGROUND

Metastasis is a complex process of genetic and phenotypic change which ultimately results in tumor cell dissemination and the formation of secondary tumors.

To date drug research has focused on finding agents to cure primary cancers. However, significant mortality of cancer deaths worldwide result when such drugs fail and the cancer metastasizes and the cancerous cells spread through the body via the blood and lymph.

The adhesive glycoprotein osteopontin (OPN) has been implicated in neoplastic transformation, cancer progression and metastasis. OPN is transcriptionally regulated by Wnt signalling factors including Tcf-4 and β- catenin. β-Catenin is commonly mutated in a number of cancers and can form an oncogenic complex with Tcf-4. It has been previously shown that β-Catenin, in contrast with Tcf-4, is highly expressed in primary breast tumors highly expressing OPN. Previous studies have shown that in benign rat mammary epithelial cells, (Rama 37) low expression of osteopontin levels is accompanied with high expression of Tcf-4. However, in the metastatic Rama 37 Met DNA (C9) cell line high levels of osteopontin are observed alongside fairly low levels of Tcf-4. Both these cell lines can be used as a model of malignant transformation and metastasis.

Currently no good broad-spectrum anti-metastatic drugs exist that have proven effectiveness. The introduction of a powerful new anti-metastatic drug would constitute a major medical breakthrough. The clinical introduction of a dual-action, broad-spectrum, antitumour drug that could simultaneously inhibit cancer cell growth and function as a potent anti- metastatic drug would be of even greater significance.

SUMMARY OF THE INVENTION

The brominated orodin alkaloid (-)-Agelastatin A has previously been shown to potently retard the growth of tumour cell lines. The present inventors have determined that agelastatin inhibits cell growth by inhibiting cell cycle progression. However, in addition, they have determined that agelastatin also acts as an anti-metastatic drug and / or inhibits invasion by cancer cells by inhibiting OPN-mediated malignant transformation.

Without wishing to be bound by theory, this mode of action is thought in part to_fbe via WNT signalling. It has previously been shown that increased levels of β-Catenin protein are associated with a decrease in Tcf-4 protein; a phenomenon that has been ascribed to different locations of β-Catenin within cells. The inventors propose that the downregulation of β-Catenin by agelastatin may be due to an ability to promote enhanced degradation of the β-Catenin protein; however, it might also be due to the small molecule causing reduced levels of the protein to be translated. The ability of a small molecule to reduce both osteopontin and β-catenin levels and simultaneously increase Tcf-4 expression provides a therapy for the treatment of metastatic cancer.

Accordingly, there is provided Agelastatin A or an Agelastatin analogue for the treatment of metastatic disease and / or invasive cancer.

Metastatic disease and / or invasion occurs when primary cancer cells spread from the place where the cancer has started to other parts of the body and this results in secondary tumours i.e. tumours which have spread from a previous or original tumor. This is very common in the later stages of cancer. Changes which allow for metastatic disease include modulation of a tumour cells adhesion, migration and invasion characteristics.

In order to spread and disseminate throughout the body, cells of a solid tumour must be able to accomplish the following tasks:

(i) detach from neighbouring cells,

(ii) break through supporting membranes,

(iii) burrow through other tissues until they reach a lymphatic or blood vessel, and

(iv) migrate through the lining of that vessel.

A range of diagnostic tests including; blood tests for tumour specific serum markers, biopsy with a fine needle or imaging techniques can be used to determine whether invasion / metastasis has occurred. In particular embodiments the invention relates to the use of agelastatin A or an agelastatin analogue in the preparation of a medicament for the treatment of metastic disease and / or invasive cancer. Suitably, the invention relates to the use of agelastatin A or an agelastatin analogue in subjects diagnosed to have a cancer and have a high serum osteopontin or high OPN expression in the primary tumor.

Metastatic disease includes metatastic tumours of the breast, colon, lung, prostate, melanoma, adrenals, liver, brain, lymph nodes, bones or ovary. Typically prostate cancer metastasizes to the bones, colon cancer metastasizes to the liver and, in women, stomach cancer metastasizes to the ovary.

In specific embodiments the invention relates to breast cancer metastasis.

According to a second aspect of the present invention there is provided a method of treating metastastic disease and / or invasive cancer comprising the step of providing a therapeutically effective amount of agelastatin A and / or an agelastatin analogue to a subject in need thereof.

As would be understood by those of skill in the art, it may be advantageous to provide chemotherapeutic agents in combination with agelastatin A or agelastatin analogues in the treatment of metastatic disease and / or invasive cancer.

Suitably there is provided a method of providing a therapeutically effective amount of agelastatin A and / or an agelastatin analogue and another agent, for example, a chemotherapeutic agent to a subject in need thereof. Accordingly, in a third aspect of the invention, there is provided a pharmaceutical composition comprising (i) a chemotherapeutic agent and (ii) (-)-agelastatin A and/or an agelastatin analogue.

In a fourth aspect, the invention comprises the use of (i) a chemotherapeutic agent and (ii) (-)-agelastatin A and/or an agelastatin analogue in the preparation of a medicament for simultaneous, separate or sequential use in the treatment of metastastic disease.

In a fifth aspect of the present invention there is provided a kit for the treatment of metastastic disease, said kit comprising:

(a) a chemotherapeutic agent and

(b) (-)-agelastatin A and/or an agelastatin analogue

(c) instructions for the administration of (a) and (b) separately, sequentially or simultaneously.

Chemotherapeutic Agents

In the invention, any suitable cancer chemotherapeutic agent may be used. For example, the chemotherapeutic agent may be selected from the group comprising alkylating agents; antimetabolites, including thymidylate synthase inhibitors, nucleoside analogs; platinum cytotoxic agents; topoisomerase inhibitors; antimicrotubule agents; anthracyclines; plant alkaloids; GTPase inhibitors; antiangiogenesis inhibitors; matrix metalloprotease inhibitors; cell cycle kinase inhibitors such as cyclin dependent kinase and cyclin inhibitors; Wnt signaling inhibitors; E2F transcription factor inhibitors; histone deacetylase inhibitors; AKT kinase or ATPase (e.g Heat Shock Protein 90 or kinesin or Trap-1) inhibitors. Examples of thymidylate synthase inhibitors which may be used in the invention include 5-FU, MTA and TDX. An example of an antimetabolite which may be used is tomudex (TDX). Examples of platinum cytotoxic agents which may be used include cisplatin and oxaliplatin.

Chemotherapeutic agents which may be used in the present invention in addition or instead of the specific agents recited above, may include alkylating agents; alkyl sulfonates; aziridines; ethylenimines; methylamelamines; nitrogen mustards; nitrosureas; anti-metabolites; folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti- adrenals; folic acid replenishers; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; ionidamine; mitoguazone; mitoxantrone.

In a particular embodiment of the invention, the chemotherapeutic agent is a topoisomerase inhibitor.

Any suitable topoisomerase inhibitor may be used in the present invention. In a particular embodiment, the topoisomerase inhibitor is a topoisomerase I inhibitor, for example a camptothecin. A suitable topoisomerase I inhibitor, which may be used in the present invention is irenotecan (CPT-11) or its active metabolite SN-38. CPT-11 specifically acts in the S phase of the cell cycle by stabilizing a reversible covalent reaction intermediate, referred to as a cleavage or cleavage complex and may also induces G₂-M cell cycle arrest. Suitably the chemotherapeutic agents may be selected from docetaxel (Taxotere), capecitabine (Xeloda) and 5-FU.

Specific combinations of chemotherapeutic agents which may be advantageously employed in the invention with agelastatin or an agelastatin analogue are: (i) Avastin and VEGFR, (ii) Cetximels and EGFR1 , and (iii) Herceptin and Her-2

Where reference is made to specific chemotherapeutic agents, it should be understood that analogues including biologically active derivatives and substantial equivalents thereof, which retain the antitumour activity of the specific agents, may be used.

In embodiments of the invention, (-)-agelastatin or an analogue of agelastatin may be provided at concentrations which inhibit invasion and / or metastasis by a cancer cell, but less than the concentration which results in inhibition of the cell cycle. In particular embodiments, (-)- agelastatin may be provided to cells to be treated at concentrations less than 1 μM, less than 0.5 μM, most preferably less than or equal to 10 nM.

The medicaments, methods, compositions and kits disclosed herein may be particularly useful in the treatment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery.

(-)-Agelastatin A may be suitably obtained from extracts of the Axinellid sponge Agelas dendromoφha . (-)-Agelastatin A has been identified as a potential anticancer agent and shown to potently inhibit the growth of murine and human cancer cell lines at low drug concentrations. However, the mechanisms by which this agent acts have not yet to be delineated and there has been no prior suggestion that this agent might also have anti-metastatic effects.

Suitably an agelastatin analogue can have a general structure I

or can be a pharmaceutically acceptable salt thereof;

wherein

A, X, Y and Z can be independently selected from C, O, N, S or Se; the bonds C-A, X-Y, Y-Z, and Z^C independently are either a single or double bond;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ groups may be independently substituted by one or more H, Ci-ioalkyl, Ci-iocycloalkyl, aryl, substituted aryl, optionally substituted alkylaryl, halogen, haloalkyl, OR⁹, SR⁹, OH, NO₂, CN, NH₂, NHR⁹, N(R⁹)₂, NHOR⁹, NHCONHR⁹, NHCONR⁹ ₂, NR⁹COR¹⁰, NHCO₂R⁹, CO₂R⁹, CO₂H, COR⁹, CONHR⁹, CONR⁹ ₂, S(O)₂R⁹, S(O)R⁹, SONH₂, SO₂NHR⁹, NHS(O)₂R⁹ groups, or an optionally substituted heterocyclic group; the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ groups can also be Si(R¹⁰)₃. When the R³, R⁴, R⁵, R⁶, R⁷, R⁸ are taken together in the pairs identified, they may also be =0, =C(R⁹)₂, =S, =NR⁹, =Se.

Suitably in formula I₁ n in (X)_n may be any number of atoms that will produce a partially unsaturated or fully saturated three to eight membered ring system containing zero to three heteroatoms.

Suitably in formula I₁ R³, R⁴, R⁵, R⁶, R⁷, R⁸ groups may be a fused aryl or heterocyclic ring system that is optionally substituted.

In particular embodiments when X and Y are absent, Z can be selected from C, N, S or Se.

In embodiments when Z is C, R⁷, R⁸ can be taken together as =0, =C(R⁹)₂, =S, =NR¹¹, or =Se.

In embodiments when Z is N, R⁷, R⁸ can be taken together as =C(R¹¹)₂.

In embodiments when Z is S or Se, R⁷, R⁸ can be taken together as =0 or as (=O)₂.

With regard to the C-A, X-Y, Y-Z, and Z^¹C bonds of formula I, when these are a double bond and A, B, X, Y, and Z are one of the atoms previously specified, the number of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ groupings must again satisfy normal chemical valency rules with regard to whether they are present or absent. These groups may be independently substituted by H, C_1-I0 alkyl, d-iocycloalkyl, aryl, substituted aryl, optionally substituted alkylaryl, halogen, haloalkyl, OR⁹, SR⁹, OH, NO₂, CN, NH₂, NHR⁹, NR⁹2, NHCOR⁹, NHCONHR⁹, NHCONR⁹ ₂, NR⁹COR⁹, NHCO₂R⁹, CO₂R⁹, CO₂H (R⁹ excluded), COR, CONHR, CONR⁹ ₂, S(O)₂R⁹, S(O)R⁹, SONH₂, SO₂NHR⁹, NHS(O)₂R⁹, or an optionally substituted heterocyclic group; the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ groups can also be Si(R¹⁰)₃ where R¹⁰ is alkyl, cycloalkyl or aryl. Moreover, when the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ groups are taken together in the pairs identified, they may be =O, =CH₂, =S, =NH, or =Se.

Agelastatin or analogues thereof may be provided as a salt, preferably as a pharmaceutically acceptable salt. The methods by which suitable salts may be made would be clear to those of skill in the art. Examples of such salts include, but are not limited to, those derived from organic acids, mineral acids and the like.

Suitable organic acids include, but are not limited to, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, succinic acid, cumaric acid.maleic acid, oxalic acid , benzoic acid, salicylic acid, phenylacetic acid, mandelic acid, methanesulfonic acid, benzenesulphonic acid, and p-toluenesulfonic acid.

Mineral acids include, but are not limited to, hydrochloride and sulfuric acid.

Suitably inorganic salts, for example, hydroxides, bicarbonates, carbonates, and alkoxides of ammonia, lithium, sodium, potassium, calcium, aluminium, iron, magnesium, zinc and the like may be used in the formation of salts of agelastatin or agelastatin analogues as described herein. Salts may also be formed with suitable non-toxic organic bases such as arginine and lysine, but may include mono-, di- trihydroxylalkylamines, and mono-di-, and trialkylamines, and the like. Suitably agelastatin A or an analogue thereof may be provided in a solvated, hydrated or polymorphic form.

Agelastatin A or an agelastatin analogue may be synthetically produced. Suitably agelastatin or suitable analogues may be obtained using the processes as provided in WO 2004/106343.

The assay used by the inventors to determine the effect of agelastatin A on metastatic disease is considered to be novel and inventive. Accordingly there is provided an assay to determine agents which inhibit metastatic disease and / or invasive cancer comprising the steps:

- providing at least one cell to be tested,

- determining the expression of Tcf-4, β-catenin protein or OPN protein in said cell(s),

- providing a modulator to be tested to said cell(s), and

- determining the expression of Tcf-4, β-catenin protein or OPN in said cell(s) following provision of the modulator to be tested to the cell(s), and

- wherein when said modulator to be tested (i) enhances expression of Tcf-4;

(ii) reduces β-catenin protein expression, or

(iii) inhibits OPN protein expression, the modulator is an agent which inhibits metastatic disease and / or invasive cancer.

Preferably in the determining steps, expression of Tcf-4, β-catenin protein expression and OPN protein is determined using techniques including, Western blotting, antibody detection or the like. Suitably a cell to be tested may be from a cell line selected from R37 OPN pBK CMV, C9, MDA-MB-231 , MDA-MB-435S, or RAMA 37 cells wherein said RAMA 37 cells have been treated with RNAi capable of suppressing Tcf-4 activity.

Suitably expression of Tcf-4 may be enhanced by a 3, preferably a 4, more preferably at least a 5 fold increase compared to non-invasive cells.

Suitably expression β-catenin may be reduced by between 50-60% compared to non-invasive cells.

Suitably expression of OPN protein may be reduced by 10-30% compared to invasive cells.

Treatment

Treatment" or "therapy" includes any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.

Suitably a method of treatment may comprise the simultaneous, sequential or separate, administration to said subject of an effective amount of agelastatin A or an agelastatin analogue.

A therapeutically effective amount may be administered in any suitable manner, including orally, parentarally, for example subcutaneously, intramuscularlarly, or intravenously, or topically. Pharmaceutical Compositions

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention may comprise, in addition to active ingredients, e.g (-)-Agelastatin A and/or an agelastatin analogue, a pharmaceutically acceptable excipient, ie. a carrier which is compatible with other ingredients of the composition, a carrier, buffer stabiliser or other materials well known to those skilled in the art.

Such materials may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such as polyvinylpyrrolidone ; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates; chelating agents; tonicifiers; and surfactants.

The pharmaceutical compositions may also contain one or more further active compounds selected as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect the activity of the composition of the invention.

Suitable compounds include, for example, Active Hexose Correlated Compound AHCC and multivitamin.

Suitable compositions may be provided for oral, rectal, topical, parenteral or other form of administration. Pharamaceutical compositions may be provided in the form of a tablet, sachets, lozenges, capsule, liquid (e.g. syrup) or injection. Suitably tablets may be made by compression or moulding techniques as would be known in the art.

Administration

(-)-Agelastatin A and/or an agelastatin analogue and/or chemotherapeutic agent(s) may be administered via microspheres, microcapsules, liposomes, other microparticulate delivery systems. For example, active ingredients may be entrapped within microcapsules which may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. For further details, see Remington: the Science and Practice of Pharmacy, 21^st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.

Sustained-release preparations may be used for delivery of active agents. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e. g. films, suppositories or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl- methacrylate), or poly (vinylalcohol)), polylactides (U. S. Pat. No. 3, 773, 919), copolymers of L-glutamic acid andy ethyl-Lglutamate.non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D- (-)-3-hydroxybutyric acid.

(-)-Agelastatin A and/or an agelastatin analogue may be administered simultaneously, separately or sequentially with a chemotherapeutic agent. Where administered separately or sequentially, they may be administered within any suitable time period e. g. within 1 , 2, 3, 6, 12, 24, 48 or 72 hours of each other. In preferred embodiments, they are administered within 6, preferably within 2, more preferably within 1 , hours of each other. Most preferably (-)-Agelastatin A and/or an agelastatin analogue may be administered within 20 minutes with a chemotherapeutic agent.

Kits

The invention further extends to a pharmaceutical kit comprising (-)- Agelastatin A and/or an agelastatin analogue and a chemotherapeutic agent for combination therapy by simultaneous, sequential or separate administration of (-)-Agelastatin A and/or an agelastatin analogue and chemotherapeutic agent, optionally with instructions for the administration of (a) and (b) separately, sequentially or simultaneously.

Dose

(-)-Agelastatin A and/or an agelastatin analogue and chemotherapeutic agents of and for use in the invention are suitably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual dosage regimen will depend on a number of factors including the condition being treated, its severity, the method of administration, the patient being treated, for example the subjects, body weight, age, medical condition, the agents being used, and will be at the discretion of the physician.

An appropriate therapeutically effective amount can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.

In one embodiment, (-)-Agelastatin A and/or an agelastatin analogμe and chemotherapeutic agents are administered in doses which produce a synergistic effect.

The term "synergistic" in the context of the present invention is used to indicate that (-)-Agelastatin A and/or an agelastatin analogue and the chemotherapeutic agent are present in a ratio such that the cytotoxic activity of the combination is greater than that of either component alone or of the additive activity that would be predicted for the combinations based on the activities of the individual components.

The combined medicament thus preferably produces a synergistic effect when used to treat tumour cells.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise. Embodiments of the invention will now be described in the following non- limiting examples with reference made to the accompanying drawings in which:

Figure ΛA illustrates Western blots showing OPN, Tcf-4 and β-catenin protein levels in R37, R37-OPN-pBK-CMV and C9 cells. Cell lysates were diluted and 15 μg loaded onto a SDS 10% (w/w) polyacrylamide gel as indicated. Specific proteins were detected using antibodies to OPN, Tcf-4, β-catenin and β-actin. Bands were quantified using densitometric analysis and normalized against β-actin - densitometric analysis showed that treatment with (-)-agelastatin A resulted in the following; OPN protein expression was reduced by 93% in R37 OPN pBK CMV and 60% in C9 cells, β-catenin expression was reduced by 98% in R37 OPN pBK CMV and 50% in C9 cells. Tcf-4 protein expression was increased 3 fold in R37 OPN pBK CMV and 1.5 fold in C9 cells;

Figure 1B illustrates Western blots showing OPN protein levels in MDA- MB435s cells. Cell lysates were diluted and 15 μg loaded onto a SDS 10% (w/w) polyacrylamide gel as indicated. Specific proteins were detected using antibodies to OPN and β-actin. Bands were quantified using densitometric analysis and normalized against β-actin - densitometric analysis showed that treatment with (-)-agelastatin A in MDA-MB-435S cells resulted in a 83% reduction in OPN protein expression:

Figure 1C illustrates the effect of (-)-agelastatin A upon osteopontin (OPN) promoter-linked luciferase activity. Rama 37 cells were cotransfected with the OPN promoter luciferase reporter and with various Wnt-signalling proteins in expression vectors (β-catenin, Tcf-4 and LeM). Results are shown as mean ± standard deviation of fold induction of luciferase activity of three independent experiments;

Figure 2A illustrates the effect of varying concentrations of (-)-agelastatin A on cell proliferation in Rama 37 cells. 1x10⁵ cells were seeded in 6-well plates and cell numbers were counted at regular time intervals. Results of the mean from three independent experiments are shown;

Figure 2B illustrates the effect of varying concentrations of (-)-agelastatin A on cell proliferation in C9 cells. 1x10⁵ cells were seeded in 6-well plates and cell numbers were counted at regular time intervals. Results of the mean from three independent experiments are shown;

Figure 2C illustrates the effect of varying concentrations of (-)-agelastatin A on cell proliferation in MDA-MB-231 cells. 1x10⁵ cells were seeded in 6- well plates and cell numbers were counted at regular time intervals. Results of the mean from three independent experiments are shown;

Figure 2D illustrates the effect of varying concentrations of (-)-agelastatin A on cell proliferation in MDA-MB-435S cells. 1x10⁵ cells were seeded in 6-well plates and cell numbers were counted at regular time intervals. Results of the mean from three independent experiments are shown;

Figure 3Λ illustrates the ability of cell lines to adhere to a laminin-treated surface was assessed over a 30 min period and the number of adherent cells quantified. Results of the mean ± standard deviation from three independent experiments are shown;

Figure 3B illustrates a soft agar assay was carried out to assess the ability of stably transfected cell lines to grow in an anchorage independent environment. The colony number was assessed after 5 days. Results of the mean ± standard error from three independent experiments are shown;

Figure 3C illustrates the migratory potential of R37, R37-pBK-CMV and R37-OPN was determined using Boyden chambers without Matrigel. The number of cells that migrated through the filter after 48 hrs was determined by staining and scanning using a digital imaging system. Results of the mean ± standard deviation from three independent experiments are shown;

Figure 3D illustrates the invasive potential of R37, R37-pBK-CMV and R37-OPN was determined using Matrigel-coated filters (500 μg/ml) in Boyden chambers. The number of cells that invaded through the filter after 48 hrs was determined by staining and scanning using a digital imaging system. Results of the mean ± standard deviation from three independent experiments are shown;

Figure 3E illustrates the ability of cell lines to adhere to a laminin-treated surface was assessed over a 30 min period and the number of adherent cells quantified. Results of the mean ± standard deviation from three independent experiments are shown;

Figure 3F illustrates a soft agar assay was carried out to assess the ability of cell lines to grow in an anchorage independent environment. The colony number was assessed after 5 days. Results of the mean ± standard deviation from three independent experiments are shown;

Figure 3G The invasive potential of MDA-MB-435s cells through Matrigel was determined using modified Boyden chambers. Results are the mean ± standard deviation represented as a percentage of control from three independent experiments.

Figure AA illustrates Western blots showing OPN protein levels in Rama 37 (R37) and R37 Tcf-4 siRNA cells. Cell lysates were diluted and 15 μg loaded onto a SDS 10% (w/w) polyacrylamide gel as indicated. Specific proteins were detected using antibodies to OPN and β-actin. Bands were quantified using densitometric analysis and normalized against β-actin:

Figure 4B illustrates the ability of transfected cell lines to adhere to a laminin-treated surface was assessed over a 30 min period and the number of adherent cells quantified. Results of the mean ± standard error from three independent experiments are shown; and

Figure 4C illustrates a soft agar assay was carried out to assess the ability of stably transfected cell lines to grow in an anchorage independent environment. The colony number was assessed after 5 days. Results of the mean ± standard error from three independent experiments are shown.

Figure 5 illustrates the effect of (-)-agelastatin A on progression of MDA- MB-435S cells through the cell cycle and expression of cell cycle regulatory proteins - A Flow cytometry profile demonstrating accumulation of MDA-MB-435s cells in G2-phase of the cell cycle following exposure to 1μM (-)-agelastatin A for 72 hours - B Western blot demonstrating concentration dependent increase in cyclin B1 expression and corresponding decrease in cyclin D1 and cyclin E following exposure of the cells to (-)-agelastatin A for 72 hours. Densitometric analysis showed that treatment with (-)-agelastatin A resulted in the following; cyclin B1 protein expression was increased in a concentration dependent manner by 29, 38, 61 and 107%, following treatment with 0.01 , 0.1 , 0.5 and 1μM (-)- agelastatin A respectively. Cyclin D1 and cyclin E protein expression was decreased by a maximum of 37% and 78% respectively following treatment with 1μM (-)-agelastatin A - C Western blot demonstrating similar effect of (-)-agelastatin A on expression of cyclin B1 and cyclin D1 in C9 cells. Densitometric analysis showed that treatment with (-)- agelastatin A resulted in the following; cyclin B1 protein expression was increased in a concentration dependent manner by 5 and 13%, following treatment with 0.5 and 1μM (-)-agelastatin A respectively. Cyclin D1 protein expression was decreased by 66 and 50%, following treatment with 0.5 and 1 μM (-)-agelastatin A respectively.

DETAILED DESCRIPTION

The inventors have investigated the effects of (-)-agelastatin A on OPN- mediated malignant transformation using a parental benign Rama 37 (R37) mammary epithelial cell model and two subclones rendered malignant/invasive by stable transformation with OPN (R37 OPN pBK CMV cells) or OPN inducing DNA fragments from a metastatic breast tumour Met-DNA (R37 Met DNA cells) (C9). Treatment by (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced β-catenin protein expression and reduced anchorage-independent growth, adhesion and invasion in R37 OPN pBK CMV and C9 cell lines. Moreover, similar effects were observed in MDA- MB-231 and MDA-MB-435s human breast cancer cell lines treated with (- )-agelastatin A. Suppression of Tcf-4 by RNA interference (siRNA) induced malignant/invasive transformation in parental benign Rama 37 cells. This effect was reversed by treatment with (-)-agelastatin A. This study demonstrated that (-)-agelastatin A upregulates Tcf-4, represses OPN expression and lead to inhibition of OPN-mediated malignant cell invasion, adhesion and colony formation in vitro. MATERIALS AND METHODS

(-)-Agelastatin A. (-)-Agelastatin A was chemically synthesized as known in the art, dissolved in DMSO at a concentration of 1 mM and stored at - 20⁰C. Aliquots of this solution were subsequently diluted and made up to the appropriate concentration prior to treatment of cells.

Cell Lines and Cell Culture. The Rama 37 and the C9-Met-DNA permanently transfected Rama 37 cells were obtained and cultured as described previously (Dunnington DJ, Hughes CM, Monaghan P, Rudland PS. Phenotypic instability of rat mammary tumor epithelial cells. J Natl Cancer Inst 1983 Dec;71 (6): 1227-40. and Oates AJ, Barraclough R, Rudland PS. The identification of metastasis-related gene products in a rodent mammary tumour model. Biochem Soc Trans 1996 Aug;24(3):353S).

The pBK CMV vector containing OPN was permanently transfected into Rama 37 cells in this laboratory. Breast cancer cells MB-MDA-231 and MB-MDA 435s were obtained from the European Collection of Cell Cultures (ECACC) (Wiltshire, UK). All cell lines were maintained in a humidified atmosphere of 95% (v/v) air and 5% (v/v) CO₂ at 37°C in routine medium (RM) (Dulbecco's Modified Eagles Medium (DMEM) (Sigma, Poole, UK) containing 10% (v/v) foetal calf serum (FCS), 100 μg/ml penicillin and 100 μg/ml streptomycin (Gibco BRL, Paisley, UK).

Treatment of Cells with (-)-Agelastatin A. Prior to treatment with (-)- agelastatin A, cells were grown overnight in routine media. The next day (-)-agelastatin A in DMSO was added to a final concentration of 10 nM in routine media unless otherwise specified. Cells were treated for 48 hours prior to assaying.

Cell Growth Assays. Cell growth assays were carried out by plating out 1 x 10⁵ cells in one well of a six well plate. At time points of 3, 6, 12, 24, 48, 72 and 96 hours cells were removed by trypsination and counted using a haemocytometer. All assays were carried out in triplicate.

Western Blotting for Proteins. For the detection of proteins 15 μg of whole cell lysates was electrophoresed through 10% (w/v) polyacrylamide, 1 % (w/v) SDS gels. Proteins were transferred from the gels by blotting onto a nitrocellulose membrane (Millipore Corporation, Watford, UK). The membranes were blocked with 0.02 M Tris-HCI (pH 7.0), 0.9% (w/v) NaCI, 0.1% (v/v) Tween 20 containing 5% (w/v) Marvel, for 1 hour. Monoclonal antibodies to OPN (1/500) (Developmental Studies Hybridoma Bank, Iowa City, IA), Tcf-4 (1/500) (Upstate, Cambridge, UK), β-catenin (1/1000) (Santa Cruz Biotechnology, California, USA) or β-actin (1/5000) (Sigma) were added and incubated overnight at 4⁰C. Bound antibodies were located by a further incubation with 1 :2500 horseradish peroxidase- conjugated rabbit antimouse IgG (for anti-OPN/Tcf-4/β-actin) (Dako, Ely, UK) or 1 :2500 horseradish peroxidase-conjugated donkey antimouse IgG (for anti-β-catenin) (Santa Cruz Biotechnology), visualized with Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and exposed to Kodak XAR5 film (Sigma). Densitometry data was obtained using a digital imaging system (Syngene, Genetool, Cambridge, UK).

Cell Adhesion Assay. The adhesive ability of cells was determined as previously described (Moye VE, Barraclough R, West C, Rudland PS. Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines. Br J Cancer 2004 May 4;90(9):1796-802). Briefly, cells were plated out at a known density (2 x 10⁵ cells per dish) in a 24 well plate in conditioned media and allowed to adhere to laminin coated plates for 15 minutes at 37⁰C, 5% CO₂. Cells were washed with PBS, fixed with methanol and stained with crystal violet (Sigma). The stain was released by 10 % (v/v) acetic acid and cell adhesion was determined by measuring the optical density at 570 nm and compared to controls. Three independent experiments were carried out in triplicate to ensure reproducibility.

Soft Agar Assays. Anchorage independent growth was determined as described previously (El-Tanani MK, Campbell FC, Crowe P, et al. BRCA1 suppresses osteopontin-mediated breast cancer. J Biol Chem 2006 Sep 8;281(36):26587-601). Briefly, 5 ml of 1.6% (w/v) agarose was plated in a 100-mm diameter tissue culture dish and allowed to harden. Cells were removed by trypsinization and resuspended at 1 x 10⁶ cells/ml in routine medium. To the agar 9 ml of routine media (containing (-)-agelastatin A for treated samples) and 1 ml of cells was added. The plates were incubated at 37⁰C in 5% (v/v) CO₂ for 5-7 days and stained with 1 ml of 0.2 % (w/v) crystal violet. The plates were scanned for colonies and counted using a digital imaging system (Syngene).

Matrigel Invasion Assays. Biocoat 250 μg/ml Matrigel invasion chambers (diameter 6.4 mm) (Falcon, Oxford, UK) were used to assess the invasiveness of cells, as described previously (19). Briefly, 1x10⁶ cells were resuspended in 100 μl of serum-free DMEM (containing (-)- agelastatin A for treated samples) and added to the cell culture inserts of the upper invasion chambers. A chemoattractant, 5μg of rat fibronectin (Gibco-BRL, U.K.) per ml in DMEM and 10% (v/v) FCS were added to the lower chambers. The cultures were incubated at 37°C in 5% (v/v) CO₂ atmosphere and allowed to invade through the matrix and the pores (8μm) of the attached lower membrane for 48h. Following incubation the upper surfaces of the filters were wiped clean of cells and the filters were fixed with methanol and stained by Gurr's eosin and methylene blue, according to the manufacturer's instructions (BDH Laboratory Supplies, Pool, and UK). The chambers were then treated with 10% (v/v) acetic acid to release the stain and the absorbance was measured at 650 nm using a microtiter plate reader (Molecular Devices, ThermoMax, CA, USA).

OPN Promoter Studies. Plasmids. Expression vectors for human Tcf-4 and LeM in the pcDNA3 were gifts from Prof. H. Clevers (University of Utrecht, Utrecht, Holland). The expression vector for human β -catenin in pcDNA3 was a gift from Prof. B. Vogelstein and Dr K. Kinzler (John Hopkins University, Baltimore, MD). Rat genomic DNA was donated by Dr A Ridall, Department of Basic Sciences, University of Texas Houston- Health Science Center, Houston and was used as template for isolation of the OPN promoter, as described (El-Tanani M, Fernig DG, Barraclough R, Green C, Rudland P. Differential modulation of transcriptional activity of estrogen receptors by direct protein-protein interactions with the T cell factor family of transcription factors. J Biol Chem 2001 Nov 9;276(45):41675-82.).

Transient Transfections. Rama 37 cells cultured in routine medium were harvested and seeded in 24-well plates at 2x10⁵ cells well in 1 ml of serum free medium. After 24 h the cells were cotransfected using

Lipofectamine™ and PLUS™ Reagent (Invitrogen) with predetermined amounts of the following, where indicated: 25 ng of Tcf-4 expression vector; 12.5 ng of β-catenin and LeM expression vectors; and 75 ng of OPN promoter. The control expression vector pRL Renilla (Promega) at 2.5 ng was used as a control expression vector. The cells were incubated for a further 48 hours (with (-)-agelastatin A for treated samples) and harvested in 300 μl of Reporter Lysis Buffer (Promega), and firefly luciferase and control Renilla luciferase were simultaneously assayed, as described in the Dual-Luciferase^® Reporter Assay System (Promega) according to the manufacturers instructions. Data was analyzed by calculating mean fold activation of 10 nM (-)-agelastatin A treated cells compared with the average of untreated control.

RNA Interference (siRNA). RNA interference was carried out using the Super RNAi™ library (Cancer Research UK). Briefly, the 19-mer sequences from the Tcf-4 gene were converted into pairs of complementary 59-mer hairpin oligonucleotides. The complementary 59- mer oligonucleotides targeting the Tcf-4 gene were annealed and ligated into the pRETROSUPER vector and transfected into competent DH5« bacteria. Glycerol stocks of transformants were prepared with the well containing bacteria with the short interfering RNA (siRNA) construct targeting the Tcf-4. DNA from the siRNA construct was isolated using the Qiagen mini-prep system (Qiagen, Crawley, UK) and confirmed by DNA sequencing. The three siRNA oligonucleotide sequences for Tcf-4, designed according to the human mRNA sequence (GenBank™ accession number BC125085) [GenBankl were as follows: 5¹- GCCCGTCCAGGAACTATGG-3', 5¹ CC ATTAC AGCACCTCTTCC-3¹, and 5^I-GGAGGCCTCTTCACAGTAG-3^I. This DNA was subsequently used for stable transfection into Rama 37 cells (with Lipofectamine™ and PLUS™ Reagent), with selection by puromycin (Sigma).

Statistical Treatment of Results. All biological experiments were performed at least 3 times. The mean and standard error were calculated and p values equal or less than 0.05 were considered significant as calculated using the Student's t-test. RESULTS

Effect of (-)-Agelastatin A on Protein Expression.

The effect of (-)-agelastatin A on the expression of OPN and various Wnt signaling proteins (Fig.1 A, B) was assessed by Western blotting. (-)- Agelastatin A treatment suppressed OPN and β -catenin expression but promoted Tcf-4 protein expression. Treatment of cells with 10 nM (-)- agelastatin A suppressed OPN expression by 10% (data not shown), whereas the reduction of OPN was maximal (93% reduction in R37 OPN pBK CMV and 60% in C9 cells) when cells were treated with 1 μM (-)- agelastatin A (Fig.1 A). Notable decreases in β -catenin expression (R37 OPN pBK CMV: 98% and C9 cells: 50%). A 3 fold was noted increase in Tcf-4 protein expression in R37 OPN pBK CMV cells and a 1.5 fold in C9 cells were detected at concentrations as low as 10 nM (Fig.1 A). Treatment of MDA-MB-435S cells with (-)-agelastatin A reduced osteopontin protein expression by 83% (Fig.1 B).

Effect of (-)- Agelastatin A on Transcriptional Regulators of the OPN Promoter.

To further assess the effects of (-)-agelastatin A on transcriptional regulators acting at the level of the OPN promoter, co-transfection experiments were performed (Fig. 1C). In untreated Rama 37 cells, co- transfection of an OPN promoter luciferase construct (OPN-luc) with β- catenin in an expression vector significantly enhanced the luciferase activity of the OPN reporter construct by an additional 10 fold. Co- transfection of OPN-luc with Tcf-4 in an expression vector resulted in a decrease in OPN promoter activity by 58%. Treatment of cells co- transfected by OPN-luc and the various transcriptional regulators with (-)- agelastatin A further reduced luciferase activity and β-catenin, Tcf-4 or LeM , and significantly reduced the luciferase activity of the OPN promoter by 92%, 80%, 67% and 70% respectively (Fig. 1C).

Effect of (-)-Agelastatin A on Growth of Rama 37, C9, MDA-MB-231 and MDA-MB -435s Mammary Cells.

Growth of rat benign mammary (Rama 37) cells and the metastatic C9 subclone was assessed with varying concentrations of (-)-agelastatin A (Fig. 2 A, B). The growth of both Rama 37 and C9 cells when treated with (-)-agelastatin A, at concentrations of 10 nM and 100 nM was similar to that of untreated cells. However, at a concentration of 1 μM, there was virtually no growth of either Rama 37 or C9 cells. At a mid-point concentration of 500 nM, the growth of Rama 37 (Fig. 2A) and C9 (Fig. 2B) cells slowed by around 60% in both cell types, compared to untreated cells or those exposed to 10 nM concentrations of the drug (Fig. 2 A,B). (- )-Agelastatin A treatment inhibited growth of MDA-MB-231 and MDA-MB- 435s cells in a dose-dependent manner similarly in both cell lines (Fig. 2C, D)

Effect of (-)- Agelastatin A on Cell Adhesion, Colony Formation, Migration and Invasion.

Various biological assays have been developed to assess the metastatic potential of cells in vitro. In this current study, all of the biological assays employed revealed that (-)-agelastatin A could significantly reduce the metastatic potential of cells (Fig 3).

A key feature of OPN-mediated metastasis is the adhesion of tumor cells to extracellular matrix components. Rama 37 cells have previously been proven to adhere at low levels and, in this study, although (-)-agelastatin A reduced adhesion; it did not do so significantly (p=0.217). However, in OPN pBK CMV cells a reduction of 46% was observed on treatment with (- )-agelastatin A (p=0.046). In the case of the metastatic C9 cells (the most adherent cell line), treatment with (-)-agelastatin A at 1OnM significantly reduced the adhesive ability of the cells by 47% (p=0.002) (Fig. 3A). There was no further decrease in adhesition with treatment with 0.5 or 1 μM concentrations of (-)- agelastatin A (Fig. 3A).

The ability of tumor cells to grow in soft agar correlates with the tumorigenic potential of cells through anchorage independent growth. In this study, Rama 37 cells showed low levels of anchorage independent growth, and (-)-agelastatin A had no significant effect on colony formation (p-value 0.074). On the other hand, in both R37 OPN pBK CMV and C9 cells, which contain high levels of osteopontin, a high rate of proliferation was observed in soft agar assays. Moreover, in R37 OPN pBK CMV and C9 cells, treatment with (-)-agelastatin A at 1OnM concentration, significantly reduced colony formation in soft agar by 81% and 48% respectively (p-values 0.048 and 0.016) (Fig. 3B).

The benign Rama 37 cell line has previously been shown to be non- metastatic and is therefore unable to invade or migrate to distant sites. Treatment of this cell line with (-)-agelastatin A showed no significant effect on the migratory or invasive potential of these cells (p= 0.127 and 0.260). In R37 cells, permanently transfected with OPN, and proven to be invasive in vitro (19), (-)-agelastatin A (1OnM) significantly reduced the migration and invasive ability of these cells by 51% and 40% respectively (p-values 0.039 and 0.028 respectively). In C9 cells, which are metastatic in both an in vitro and in vivo animal setting (20), treatment with (-)- agelastatin A at 1OnM concentration very significantly reduced their migratory and invasive potential by 49% and 61% respectively (p=0.011 and 8.47E-04) (Fig. 3C, D).

In vitro biological assays were also carried out to investigate the effect of (- )-agelastatin A on the metastatic potential of human breast cancer cells, as represented by adhesion and colony formation (Fig. 3). Treatment of MDA-MB-231 with 10 nM (-)-agelastatin A significantly reduced adhesion by 43% (p = 0.0014). Likewise treatment of MDA-MB-435S cells with (-)- agelastatin A significantly reduced adhesion in a concentration dependent manner with a maximal reduction of 56% achieved after treatment with

1μM (-) -agelastatin A (p=2.64E-.5) (Fig 3E). (-)-Agelastatin A treatment at 1OnM also inhibited anchorage independent growth in MDA-MB-231 and MDA-MB-435S cells by 37% and 25% (p=0.085 and 0.050 respectively; Fig. 3F) and reduced invasion of MDA-MB-435s by 36% (p=0.034 & p= 0.015) (Fig. 3G).

RNA Interference.

To investigate the specific role of Tcf-4 and OPN transcription, OPN protein expression and OPN-mediated neoplastic transformation through treatment with (-)-agelastatin A, RNA interference strategies were used. Stable transfection of Rama 37 cells with a Tcf-4 pSUPER RNAi expression vector impeded Tcf-4 protein expression and was associated with increased OPN protein levels (Fig. 4A).

Treatment of Tcf-4 siRNA stable transfectants with (-)-agelastatin A partially reversed the effects of Tcf-4 siRNA and induced a 3 fold increase of Tcf-4 protein expression accompanied by a 63% decrease of in OPN protein expression (Fig. 4A). Rama 37 cells stably transfected with Tcf-4 siRNA showed increased adhesion and anchorage independent growth over that of parental Rama 37 cells (Fig 4B, C). Treatment of Rama 37 - Tcf-4 siRNA stable transfectants with (-)-agelastatin A 10 nM, reduced adhesion by 74% (p-value 0.006). In soft agar assays this trend was repeated. The Tcf-4 siRNA Rama 37 cells showed an increase in anchorage independent growth by 31 fold (p= 0.016) compared to Rama 37 cells. This is an increase comparable to C9 cells. Treatment with (-)- agelastatin A significantly reduced this effect by a further 55% (p=0.016).

Effects of (-)-agelastatin A on cell cycle progression The effect of (-)-agelastatin A on progression of MDA-MB-435s cells through the cell cycle was determined by propidium iodide staining and analysis by flow cytometry (24) following 72 hours of exposure to (-)-agelastatin A. Treatment with (-)-agelastatin A induced an accumulation of cells in the G₂ phase of the cell cycle, increasing the proportion of cells in G₂ ranging from 18% in untreated cultures to a maximum of 40% in cells treated with 1 μM (-)-agelastatin A (Fig 5A). The increase in the proportion of cells in G₂ was sustained up to 96 hours post treatment with (-)-agelastatin A. There was a corresponding decline in the population of cells in Gi phase ranging from 54% in untreated cells to 31% in cells treated with 1μM (-)-agelastatin A. There was also some evidence of increased cell death accompanying the G₂/M arrest indicated by the appearance of a sub-Gi peak in the cell cycle profiles, with a 6% increase in the percentage of sub-Gi cells.

Since (-)- agelastatin A induced alterations in cell progression through the G₂M phase, western blotting was used to assess the effect of (-)- agelastatin A on the expression of cyclin B1 which regulates transition through the G₂ checkpoint. It was found that (-)-agleastatin A treatment correlated with a significant concentration dependent increase in the expression of cyclin B1 protein in MDA-MB-435s cells, consistent with the known rise in cyclin B1 levels during the G₂ phase. In accordance with the subsequent decline in the number of cells in G1 phase, a significant decrease in expression of cyclin D1 and cyclin E after treatment with (-)- agelastatin A (Fig 5B) was also detected. A similar effect on (-)- agelastatin A on cyclin D1 and B1 expression in C9 cells was also observed (Fig 5C).

To confirm the anticancer effects in vitro in human cancer cells the effect of (-)-agelastatin A was investigated in MDA-MB-231 and MDA-MB-435S human breast cancer cells, both of which are highly metastatic. Only

MDA-MB-435S cells overexpress osteopontin. The ability of (-)-agelastatin A to repress OPN protein expression in MDA-MB-435S cells is consistent with data obtained for rat mammary cells.

Osteopontin promoter studies were carried out to investigate the effects of (-)-agelastatin A on the osteopontin promoter. Data from these studies is consistent with the western blot analyses (Fig. 1 A, B), which revealed that (-)-agelastatin A can increase Tcf-4 protein levels whilst reducing β- catenin and OPN. It is thought that Tcf-4 inhibits OPN transcription while β-catenin co-activates OPN transcription. Thus, (-)-agelastatin A is thought to modulate Wnt signaling molecules that influence OPN transcription and may influence OPN-mediated neoplastic transformation.

The data obtained in this study on the affect of (-)-agelastatin A on cell growth is consistent with previous studies which show that (-)-agelastatin A can inhibit tumor cell growth in vitro.

Comparisons of the growth of human cancer cells with rat mammary cells indicate that (-)-agelastatin A is a more potent inhibitor of cell growth in human cancer cells. In rat mammary cells, at a concentration of 10 nM, growth was similar to that of untreated cells (Fig. 3). However in both the MDA-MB-231 and MDA-MB-435s cell lines, growth was decreased at this concentration. This suggests that in this study human cancer cells are more sensitive to the effects of (-)-agelastatin A at lower concentrations than rat cells. Further studies are currently underway in this laboratory to understand how (-)-agelastatin A can downregulate both osteopontin and β-catenin by using microarray technology. Such methodology might also shed valuable light on why Tcf-4 levels rise when osteopontin and β- catenin is downregulated by (-)-agelastatin A.

This study shows that (-)-agelastatin A inhibits OPN-mediated effects on adhesion, colony formation, migration and invasion in this in vitro model of metastasis processes in all cell types tested. Notably, (-)-agelastatin A exerted its biological effects in each of these assays at low concentrations of 10 nM. At this concentration only a 10% reduction in osteopontin was reported, however this is a sufficient enough reduction to reduce the invasive potential of these cells. This indicates that (-)-agelastatin A can function as a powerful anti-invasive / anti-metastatic agent even at quite low drug concentrations, for example 10 nM.

An siRNA approach further confirmed that (-)-agelastatin A modulates its effects through the Wnt signalling pathway. Data obtained that Tcf-4 siRNA increases osteopontin protein expression and is consistent with previous work conducted by the inventors which demonstrate that inhibition of Tcf-4 can enhance the expression of OPN. Interestingly this effect can be reversed through treatment with (-)-agelastatin A.

Synthetic (-)-agelastatin A used in this study was shown to inhibit OPN protein expression, in part through its potent downregulatory effects on the Wnt signaling pathway, although precisely how (-)-agelastatin A exerts its effects on Wnt signaling remains unclear. It is known that the epidermal growth factor (EGF) can induce osteopontin gene expression in cancer. Activation of the epidermal growth factor receptor has also been shown to activate β -catenin expression in liver carcinomas. It is hypothesized that the epidermal growth factor receptor (EGFR) may be one possible link between osteopontin and the Wnt signaling pathway identified in this study.

The fact that (-)-agelastatin A can potently down-regulate β-catenin expression within metastatic cancer cell lines (at 10 nM concentration) means that it can not only halt tumour cell growth and proliferation, it can also combat metastasis, since it can simultaneously down-regulate key proteins involved in metastatic spread via osteopontin. Moreover, because β-catenin controls transcription from the multidrug resistance 1 (MDR1 ) gene, this means that (-)-agelastatin A will be far less susceptible to encountering drug-resistance problems (unlike the majority of existing antitumour drugs), and potentially it will render tumours much more susceptible to effects of these existing drugs, and potentially be useful both as a single agent and as a combination therapy.

Various modifications may be made to the invention herein described without departing from the scope thereof.

Claims

1. (-)-Agelastatin A or an agelastatin analogue for use in the treatment of metastatic disease and / or invasive cancer.

2. The use of (-)-agelastatin A or an agelastatin analogue in the preparation of a medicament for the treatment of metastatic disease and / or invasive cancer.

3. Use of (i) a chemotherapeutic agent and (ii) (-)-agelastatin A and/or an agelastatin analogue for simultaneous, separate or sequential use in the treatment of metastatic disease and / or invasive cancer.

4. The use of as claimed in claim 2 or claim 3 wherein metastatic disease is defined by a tumour and a high serum osteopontin or high OPN expression.

5. The use as claimed in any one of claims 2 to 4 wherein the metastatic disease is selected from metastatic tumours of the breast, colon, lung, prostate, melanoma, adrenals, liver, brain, lymph nodes, bones or ovary.

6. A method of treating metastatic disease and / or invasive cancer comprising the step of providing a therapeutically effective amount of agelastatin A and / or an agelastatin analogue to a subject in need thereof.

7. The method of claim 6 wherein metastatic disease is defined by a tumour and a high serum osteopontin or high OPN expression.

8. The method of claim 6 or claim 7 wherein metastatic disease includes metastatic tumours of the breast, colon, lung, prostate, melanoma, adrenals, liver, brain, lymph nodes, bones or ovary.

9. The method of any one of claims 6 to 8 comprising the step of simultaneously, separately or sequentially providing a therapeutically effective amount of a second agent in addition to a therapeutically effective amount of agelastatin A and / or an agelastatin analogue to a subject for the treatment of metastatic disease and / or invasive cancer.

10. The method as claimed in claim 9 wherein the second agent is selected from a chemotherapeutic agent.

11. A pharmaceutical composition comprising (i) a chemotherapeutic agent and (ii) (-)-agelastatin A and/or an agelastatin analogue.

12. A pharamaceutic composition as claimed in claim 11 wherein ) (-)- agelastatin A and/or an agelastatin analogue is provided at a concentration which inhibits tumour cell metastasis and / or invasion, but which does not inhibit cell cycle progression.

13. A kit for the treatment of metastatic disease and / or invasive cancer, said kit comprising:

(a) a chemotherapeutic agent and (b) (-)-agelastatin A and/or an agelastatin analogue

14. An assay to determine candidate inhibitory agents of metastatic disease and / or invasive cancer comprising the steps: - providing at least one cell to be tested,

- providing a modulator to be tested to said cell(s), and - determining the expression of at least one of Tcf-4, β-catenin protein and OPN in said cell(s) following provision of the modulator to be tested to the cell(s),

- wherein when said modulator to be tested

(i) enhances expression of Tcf-4; (ii) reduces β-catenin protein expression, or

(iii) inhibits OPN protein expression, the modulator is a candidate inhibitory agent of metastatic disease and / or invasive cancer.