WO2006135967A1

WO2006135967A1 - Modulation of egfr signalling by modulation of sphingosine kinase signalling

Info

Publication number: WO2006135967A1
Application number: PCT/AU2006/000865
Authority: WO
Inventors: Pu Xia; Olga Sukocheva; Mathew Alexander Vadas
Original assignee: Medvet Science Pty. Ltd.
Priority date: 2005-06-21
Filing date: 2006-06-21
Publication date: 2006-12-28
Also published as: CA2612640A1; EP1910523A4; JP2008546720A; EP1910523A1

Abstract

The present invention relates generally to a method of modulating EGFR-mediated cellular functional activity and agents useful for same. More particularly, the present invention relates to a method of modulating agonist induced EGFR-mediated cellular proliferation by modulating upstream intracellular sphingosine kinase signalling. The method of the invention is useful, inter alia, in the treatment and/or prophylaxis of conditions characterised by aberrant EGFR-mediated cellular functioning, in particular aberrant EGFR-mediated cellular proliferation.

Description

MODULATION OF EGFR SIGNALLING BY MODULATION OF SPHINGOSINE KINASE SIGNALLING

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Distinct signalling pathways have been demonstrated to orchestrate estrogen (E2) action and directly affect its function including normal mammary development and breast cancer growth, oestrogen has been known for decades to be coupled with growth factor networks exhibiting the enhanced growth-promoting activity in breast cancer cells. A large number of growth factors and their receptors have been illustrated to interact with oestrogen signalling, among which the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases are of particular interest due to their critical involvement in human cancer (Bange et al, 2001 , Nat Med, 7:548-552; Levin, 2003, MoI Endocrinol, 17:309-317). Indeed, aberrant expression and activation of EGFR is frequently observed in various tumors, especially of the breast and ovary, where it correlates with a poorer patient prognosis (Keen and Davidson, 2003, Cancer, 97:825-833; Roskoski, Jr., 2004, Biochem Biophys Res Commun, 319: 1-11). In addition, up-regulation of EGFR signalling has been considered as an important mechanism that confers anti-estrogen resistance of breast cancer resulting in failure of endocrine therapy (AIi and Coombes, 2002, Nat Rev Cancer, 2:101-112; Nicholson et al., 2003, Breast Cancer Res Treat, 80 Suppl 1, S29-S34).

Multiple lines of evidence have suggested that the interaction of EGFR with oestrogen signalling can occur at various levels, oestrogen primarily acts on nuclear estrogen receptors (ER) leading to regulation of gene expression, which was traditionally deemed the genomic action of oestrogen. Many of these oestrogen-responsive genes are indeed key signalling molecules that participate in EGFR signalling (Levin, 2003, supra). Alternatively, EGFR signalling is capable of activating ER in the absence of oestrogen (Picard et al, 1997, Biochem Soc Trans, 25:597-602). In addition, recent studies provide evidence showing an interaction between EGFR and a membrane-associated form of ER (mER) in a G protein-dependent manner (Filardo et al., 2000, MoI Endocrinol, 14:1649- 1660; Razandi et al, 2003, J Biol Chem, 278:2701-2712), and suggest a model of EGFR transactivation by oestrogen similar to that induced by other well-documented G protein coupled receptor (GPCR) ligands (Gschwind et al., 2001, Oncogene, 20:1594-1600). However, the mER has not yet been molecularly identified. It has been variously suggested that mER may be derived from the same or an alternative transcript related to nuclear ER (Razandi et al., 1999, MoI Endocrinol, 13:307-319), or belongs to a GPCR family (e.g., GPR30) (Filardo et al, 2000, supra), or may be a part of GPCR-ER complex (Razandi et al, 2003. supra). Therefore, the true nature of mER is still undefined and the mechanism underlying oestrogen induced transactivation of EGFR remains largely unknown.

In light of the extremely serious problem which breast cancer presents to both women and society in general, in particular the increasing incidence of breast cancers which are resistant to anti-estrogen therapy, there is an ongoing need to elucidate the intracellular mechanisms which underpin these cellular defects and to thereby develop new and alternative means of treatment. Since first described by Ullrich and colleagues (Prenzel et al. 1999, Nature, 402:884-888), the transactivation of EGFR by GPCR ligands has been considered an important model of cellular signal transduction. Several GPCR ligands, including lysophosphatidic acid, thrombin, angiotensin II and endothelin-1, have been documented to transactivate EGFR, leading to activation of survival or mitogenic pathways (Gschwind et al. 2001, supra). In work leading up to the present invention, however, it has been surprisingly and unexpectedly found that sphingosine kinase plays a critical role in mediating oestrogen non-genomic signalling for the criss-cross transactivation of EGFR via SlP receptors. Accordingly, sphingosine kinase mediates the interaction between three membrane spanning events induced by oestrogen, SlP and EGF. These findings have now facilitated the development of an alternative means of treating aberrant EGFR-mediated cellular functioning, in particular aberrant cellular proliferation, by targetting an upstream signalling molecule rather than EGFR or, in the case of oestrogen-induced EGFR activation, the oestrogen or oestrogen receptor molecule.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The subject specification contains nucleotide sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <201> followed by the sequence identifier (eg, <210>l, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1 , SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>l, <400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as <400>l in the sequence listing.

One aspect of the present invention is directed to a method of modulating EGFR-mediated cellular functional activity, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling.

In another aspect, there is provided a method of modulating agonist induced EGFR- mediated cellular functional activity, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates said agonist induced EGFR-mediated intracellular signalling. In yet another aspect, there is provided a method of modulating agonist induced EGFR- mediated cellular proliferation, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates said agonist induced EGFR-mediated intracellular signalling.

Still yet another aspect of the present invention provides a method of downregulating agonist induced EGFR-mediated neoplastic cell proliferation, said method comprising downregulating the functioning of sphingosine kinase mediated signalling in said cell.

Yet still another aspect of the present invention provides a method of downregulating agonist induced EGFR-mediated neoplastic cell proliferation, said method comprising downregulating the functioning of sphingosine kinase- 1 mediated signalling in said cell.

A further aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate EGFR-mediated cell activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling.

In another aspect there is provided a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate agonist induced EGFR-mediated cell activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates said agonist induced EGFR-mediated intracellular signalling.

In yet another further aspect there is provided a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate agonist induced EGFR-mediated cellular proliferation, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulate EGFR-mediated intracellular signalling.

Still another further aspect of the present invention provides a method for the treatment and/or prophylaxis of an oestrogen induced breast cell malignancy, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling.

In another aspect the present invention relates to the use of an agent capable of modulating the functional activity of sphingosine kinase mediated signalling in the manufacture of a medicament for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate EGFR-mediated cell activity wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling.

In yet another aspect the present invention relates to a pharmaceutical composition comprising a modulatory agent as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents.

Yet another aspect of the present invention relates to modulatory agents, as hereinbefore defined, when used in the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an image depicting that. SlP transactivates EGFR. MCF -7 cells were stimulated with (A) 1 mmol/L SlP for the indicated time or (B) an increasing concentration of SlP for 15 min. Cell lysates were subjected to SDS-PAGE, and phosphorylation of EGFR and ERKl/2 was analysed by phospho-specific antibodies. The bar graphs represent three combined experiments expressed in percent of the control value (the mean ± SD). The density values of activities were normalised by total protein levels.

Figure 2 is an image depicting that. SlP mimics E2 to induce EGFR transactivation in MCF-7 cells. Serum-starved MCF-7 cells were (A) preincubated for 16 hrs with PTX (100 ng/ml), or for 1 hr with AG1478 (50 mmol/L), and PP2 (100 nmol/L), or (B) pretreatment for 1 hr with the MMP inhibitor phenanthroline (20 mmol/L), GM6001 (50 nmol/L), or the EGF neutralising antibodies (15 mg/ml), followed by stimulation with 10 nmol/L E2, 1 mmol/L SlP or 25 ng/ml EGF for 15 min. Cell lysates were then analysed by Western blotting and the extent of phosphorylated EGFR and ERKl/2 was quantified as in Figure 1. Data are mean ± SD. *p < 0.01; f p < 0.05, pretreated versus nonpretreated.

Figure 3 is an image depicting the effect of SphK activity on E2-induced EGFR transactivation. (A) Stably transfected MCF-7 cells overexpressing SphKl^, SphKl^G82D or vector alone were stimulated with 10 nmol/L E2, 1 mmol/L SlP or 25 ng/ml EGF for 15 min. Cell lysates were then analysed by Western blotting and the extent of phosphorylated EGFR and ERKl/2 was quantified as in Figure 1. Data are mean ± SD. *p < 0.01 ; f p < 0.05, SphKl^WT or SρhKl^G82D versus vector alone. (B) Flow cytometry profiles show the cell-surface expression levels of EGFR in the transfected MCF-7 cell lines.

Figure 4 is an image depicting that SphKl, but not SphK2, is responsible for the E2- induced EGFR transactivation. MCF-7 cells were transfected with siRNA specifically for SphKl, SphK2 or scramble control RNA as described in 'Material and Methods'. After 24 hrs transfection, the following experiments were conducted: (A) Levels of SphKl and

SphK2 mRNA were determined by RT-PCR and quantified by the ratio to GAPDH mRNA level. (B) The siRNA-transfected MCF-7 cells were stimulated with 10 nmol/L E2, 1 mmol/L SlP or 25 ng/ml EGF for 15 min. SphK activity was then assayed, and (C) levels of phosphorylated EGFR and ERK1/2 were analysed as in Figure 1. Data are mean ± SD. *p < 0.01; f p < 0.05, SphKl or SphK2 siRNA versus control.

Figure 5 is an image depicting that E2-induced EGFR transactivation is mediated by SlP release from MCF-7 cells. (A) Extracellular SlP levels were determined in transfected MCF-7 cells overexpressing SphKl , SphKl or vector alone after stimulation with 10 nmol/L E2 for 15 min. Data are mean ± SD. (B) MCF-7 cells were pretreated with or without PTX for 16 hrs and stimulated for 15 min with conditioned medium derived from the transfected MCF-7 cells treated with or without E2. Levels of phosphorylated EGFR were then determined as in Figure 1.

Figure 6 is an image of E2 stimulating Edg-3 internalisation in MCF-7 cells. After stimulation for 2 min or 30 min with 10 nmol/L estrogen or 100 nmol/L SlP, MCF-7 cells were immunofluorescently stained with antibodies against Edg-3 (upper panels) or EGFR (middle panels). The images of Edg-3 (red) and EGFR (green) staining are merged in bottom panels.

Figure 7 is an image depicting the effect of Edg-3 on E2-induced EGFR transactivation. MCF-7 cells were transfected with Edg-3 antisense or sense as described in 'Material and Methods'. After 24 hrs transfection, the following studies were performed: (A) Levels of Edg-3 mRNA were determined by RT-PCR and quantified by the ratio to GAPDH mRNA level. (B) The Edg-3 sense or antisense-transfected cells were stimulated with 10 nmol/L E2, 1 mmol/L S IP or 25 ng/ml EGF for 15 min, and the extent of phosphorylated EGFR and ERK1/2 was analysed as in Figure 1. Data are mean ± SD. *ρ < 0.01; fp < 0.05, Edg-3 antisense versus the sense.

Figure 8 is a schematic representation of a model for E2-induced EGFR transactivation through activation of the SphK 1 /S 1 P signalling pathway. DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the identification of the existence of a crisscross mechanism of signalling which leads to the upregulation of EGFR-mediated cell signalling. Accordingly, this finding facilitates the development of alternative means of treating aberrant EGFR-mediated cellular functioning, this being particularly relevant in terms of treating cancers such as anti-oestrogen resistant breast cancers. Further, the determination that sphingosine kinase is a crucial upstream mediator of EGFR-related cell signalling provides a specific target, other than the currently utilised EGFR itself, or in the case of breast cancer oestrogen, for therapeutic intervention.

Accordingly, one aspect of the present invention is directed to a method of modulating EGFR-mediated cellular functional activity, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling.

Reference to "EGFR" should be understood as a reference to all forms of the epidermal growth factor receptor. For example, it should be understood to include reference to any isoforms which arise from alternative splicing of EGFR mRNA or functional mutants or polymorphic variants of EGFR. Without limiting the present invention to any one theory or mode of action, EGFR is the mediator of the biological signal of epidermal growth factor (EGF) and also of transforming growth factor-α (TGF-α). It is a membrane protein with a single transmembrane domain, and a tyrosine kinase activity in the cytoplasmic domain; the extracellular domain, which binds EGF, is highly glycosylated. Binding of EGF to the receptor leads to the induction of tyrosine kinase activity, and formation of dimers, as a result of which autophosphorylation occurs; stimulation of cell DNA synthesis and cell proliferation follows. Internalization of the EGF-receptor complex occurs subsequent to binding EGF.

More particularly, there is provided a method of modulating agonist induced EGFR- mediated cellular functional activity, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates said agonist induced EGFR-mediated intracellular signalling.

Reference to "agonist induced" EGFR-mediated cellular functional activity should be understood as a reference to EGFR-mediated cellular functional activity which is initially induced by the actions of a molecule which couples to a receptor other than the EGFR receptor. For example, in the context of oestrogen induced breast cancers, although the final signal which leads to cellular proliferation is mediated via the EGFR molecule, the molecule which commences the cascade of steps leading to EGFR stimulation is the binding of oestrogen into the oestrogen receptor. In this particular example, the subject "agonist" is oestrogen. Without limiting the present invention to any one theory or mode of action, it is only through the identification of the existence of a criss-cross activation mechanism that the elucidation of the notion of agonist induced EGFR stimulated cellular functioning has been facilitated and the design of alternative methods of treatment thereby rendered possible.

Reference to "EGFR-mediated cellular functional activity" should be understood as a reference to any one or more of the functional activities which a cell is capable of performing as a result of EGFR stimulation. Preferably, said functional activity is cellular proliferation.

According to this preferred embodiment, there is provided a method of modulating agonist induced EGFR-mediated cellular proliferation, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates said agonist induced EGFR-mediated intracellular signalling.

It should be understood that reference to a "cell" in the context of the present invention is a reference to any form or type of cell, irrespective of its origin. For example, the cell may be a naturally occurring normal or abnormal cell or it may be manipulated, modified or otherwise treated either in vitro or in vivo such as a cell which has been freezed/thawed or genetically, biochemically or otherwise modified either in vitro or in vivo (including, for example, cells which are the result of the fusion of two distinct cell types). Preferably, the cell is a neoplastic cell. By "neoplastic cell" is meant a cell exhibiting uncontrolled proliferation. The neoplastic cell may be a benign cell or a malignant cell. Preferably the cell is malignant. In one particular embodiment, the neoplastic cell is a malignant cell the proliferation of which would form a solid tumour such as a malignant cell derived from the breast, colon, stomach, lung, brain, bone, oesophagus or pancreas.

There is therefore most preferably provided a method of do wnregulating agonist induced EGFR-mediated neoplastic cell proliferation, said method comprising downregulating the functioning of sphingosine kinase mediated signalling in said cell.

Preferably, said neoplastic cell is a malignant cell derived from the breast, colon, stomach, lung, brain, bone, oesophagus, pancreas, ovary or uterus.

Still more preferably, said agonist is oestrogen and said cell is a malignant breast cell or an anti-oestrogen resistant malignant breast cell.

Reference to "sphingosine kinase" should be understood as reference to all forms of this protein and to functional derivatives thereof. This includes, for example, any isoforms which arise from alternative splicing of the subject sphingosine kinase mRNA or functional mutants or polymorphic variants of these proteins. For example, this definition extends to the isoforms sphingosine kinase- 1 and sphingosine kinase-2, although said sphingosine kinase is preferably sphingosine kinase-1.

There is therefore preferably provided a method of downregulating agonist induced EGFR- mediated neoplastic cell proliferation, said method comprising downregulating the functioning of sphingosine kinase-1 mediated signalling in said cell. Preferably, said neoplastic cell is a malignant cell derived from the breast, colon, stomach, lung, brain, bone, oesophagus, pancreas, ovary or uterus.

Reference to "sphingosine kinase mediated signalling" should be understood as a reference to the intracellular signalling pathway which utilises sphingosine kinase or functional derivatives thereof. Sphingosine kinase is a key regulatory enzyme in the activity of the sphingosine kinase signalling pathway and functions to generate the endogenous sphingolipid mediator sphingosine- 1 -phosphate. Still further, and without limiting the present invention in any way, sphingosine kinase has been found to form part of a novel signalling system which mediates EGFR transactivation. In the specific context of oestrogen-induced breast cancers, oestrogen stimulates activation of sphingosine kinase and the release of sphingosine- 1 -phosphate, by which oestrogen is able to activate a sphingosine- 1 -phosphate specific GPCR, Edg3, leading to EGFR transactivation in a matrix metalloprotease-dependent manner. These findings therefore define a role for sphingosine kinase in mediating the interaction between three membrane spanning events induced by oestrogen, SlP and EGF, and reveal a novel signal transduction mechanism between three individual ligand-receptor systems, this mechanism having been termed "criss-cross" activation.

Reference to modulating the "functioning" of sphingosine kinase mediated signalling should be understood as a reference to modulating the level of sphingosine kinase activity which is present in any given cell as opposed to the concentration of sphingosine kinase, per se. Although a decrease in the intracellular concentration of sphingosine kinase will generally correlate to a decrease in the level of sphingosine kinase functional activity which is observed in a cell, the person skilled in the art would also understand that decreases in the level of activity can be achieved by means other than merely decreasing absolute intracellular sphingosine kinase concentrations. For example, one might utilise means of decreasing the half life of sphingosine kinase, sterically hindering the binding of this molecule to its substrate or competitively inhibiting the binding of functional sphingosine kinase to its substrate.

It should also be understood that reference to modulation of sphingosine kinase mediated signalling, in particular its down-regulation, does not necessarily mean that the activity of this signalling pathway need be returned to baseline levels. Rather, the level need only be one which is changed relative to the pretreatment level. Accordingly, the method of the present invention may be applied to at least reduce uncontrolled cellular proliferation until such time as it can be entirely ceased. The subject modulation may be transient or long term, depending on the requirements of the particular situation.

Accordingly, modulation of the "activity" of sphingosine kinase mediated signalling should be understood as a reference to either up-regulating or down-regulating the signalling mechanism. Although the preferred method is to down-regulate the subject signalling in the context of a patient exhibiting uncontrolled cellular proliferation, there may be certain circumstances where it is desirable to up-regulate sphingosine kinase signalling, for example to facilitate the analysis of a cell line model or to facilitate the rapid expansion of a cellular population without necessarily inducing transformation of the subject cells. Either form of modulation may be achieved by any suitable means and include:

(i) modulating absolute levels of the components of the sphingosine kinase mediated signalling pathway, such as sphingosine kinase and/or sphingosine- 1 -phosphate, such that either more or less of these molecules are available for activation and/or to interact with downstream targets such as Edg-3.

(ii) agonising or antagonising the components of the sphingosine kinase mediated signalling pathway, such as sphingosine kinase and/or sphingosine- 1 -phosphate, such that the functional effectiveness of any one or more of these molecules is either increased or decreased. For example, increasing the half life of sphingosine kinase may achieve an increase in the overall level of sphingosine kinase activity without actually necessitating an increase in the absolute intracellular concentration of sphingosine kinase. Similarly, the partial antagonism of sphingosine kinase or sphingosine-1 -phosphate, for example by coupling these molecules to components that introduce some steric hindrance in relation to their binding to downstream targets, may act to reduce, although not necessarily eliminate, the effectiveness of the signalling which they provide. Accordingly, this may provide a means of down- regulating sphingosine kinase mediated signalling without necessarily down- regulating the absolute concentrations of the components of this pathway.

In terms of achieving the up or down-regulation of sphingosine kinase mediated signalling, means for achieving this objective would be well known to the person of skill in the art and include, but are not limited to:

(i) introducing into a cell a nucleic acid molecule encoding a sphingosine kinase signalling pathway component or functional equivalent or derivative thereof in order to up-regulate the capacity of said cell to express the sphingosine kinase mediated pathway component;

(ii) introducing into a cell a proteinaceous or non-proteinaceous molecule which modulates transcriptional and/or translational regulation of a gene, wherein this gene may be any sphingosine kinase signalling pathway component, in particular sphingosine kinase or sphingosine-1 -phosphate or functional portion thereof, or some other gene which directly or indirectly modulates the expression of the components of sphingosine kinase mediated signalling pathways;

(iii) introducing into a cell one or more of the sphingosine kinase mediated signalling pathway component expression products (in either active or inactive form) or a functional derivative, homologue or mimetic thereof;

(iv) introducing a proteinaceous or non-proteinaceous molecule which functions as an antagonist of sphingosine kinase or sphingosine-1-phosphate; (v) introducing into a cell a proteinaceous or non-proteinaceous molecule which modulates the expression and/or function of sphingosine kinase or sphingosine- 1- phosphate receptors;

(vi) introducing a proteinaceous or non-proteinaceous molecule which functions as an antagonist to any one or more components of the sphingosine kinase signalling pathway expression product such as N'N'-dimethylsphingosine (sphingosine kinase chemical inhibitor), DL-threo-dihydrosphingosine or SphK^{G 2D} (mutant sphingosine kinase dominant negative).

(vii) introducing a proteinaceous or non-proteinaceous molecule which functions as an agonist of the sphingosine kinase mediated signalling pathway expression product.

The proteinaceous molecules described above may be derived from any suitable source such as natural, recombinant or synthetic sources and includes fusion proteins or molecules which have been identified following, for example, natural product screening. The reference to non-proteinaceous molecules may be, for example, a reference to a nucleic acid molecule or it may be a molecule derived from natural sources, such as for example natural product screening, or may be a chemically synthesised molecule. The present invention contemplates analogues of the sphingosine kinase signalling pathway components or small molecules capable of acting as agonists or antagonists.

Chemical agonists may not necessarily be derived from the components of the sphingosine kinase mediated signalling pathway product but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to meet certain physiochemical properties. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing components of the sphingosine kinase mediated signalling pathway from carrying out their normal biological function, such as molecules which prevent activation or else prevent the downstream functioning of activated molecules. Antagonists include monoclonal antibodies, dominant-negative sphingosine kinase mutants and antisense nucleic acids which prevent transcription or translation of the genes or mRNA of components of the sphingosine kinase mediated signalling pathway in mammalian cells. Modulation of expression may also be achieved utilising antigens, RNA (particularly siRNA), ribosomes, DNAzymes, RNA aptamers, antibodies or molecules suitable for use in cosuppression. The proteinaceous and non-proteinaceous molecules referred to in points (i)-(vii), above, are herein collectively referred to as "modulatory agents".

Screening for the modulatory agents hereinbefore defined can be achieved by any one of several suitable methods including, but in no way limited to, contacting a cell comprising the sphingosine kinase gene (or any other gene which encodes a component of the sphingosine kinase signalling pathway) or functional equivalent or derivative thereof with an agent and screening for the modulation of sphingosine kinase protein production or functional activity, modulation of the expression of a nucleic acid molecule encoding sphingosine kinase or modulation of the activity or expression of a downstream sphingosine kinase cellular target. Detecting such modulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of sphingosine kinase activity such as luciferases, CAT and the like.

It should be understood that the sphingosine kinase gene or functional equivalent or derivative thereof may be naturally occurring in the cell which is the subject of testing or it may have been transfected into a host cell for the purpose of testing. Further, the naturally occurring or transfected gene may be constitutively expressed - thereby providing a model useful for, inter alia, screening for agents which down regulate sphingosine kinase activity, at either the nucleic acid or expression product levels, or the gene may require activation - thereby providing a model useful for, inter alia, screening for agents which up regulate sphingosine kinase expression. Further, to the extent that a sphingosine kinase nucleic acid molecule is transfected into a cell, that molecule may comprise the entire sphingosine kinase gene or it may merely comprise a portion of the gene such as the portion which regulates expression of the sphingosine kinase product. For example, the sphingosine kinase promoter region may be transfected into the cell which is the subject of testing. In this regard, where only the promoter is utilised, detecting modulation of the activity of the promoter can be achieved, for example, by ligating the promoter to a reporter gene. For example, the promoter may be ligated to luciferase or a CAT reporter, the modulation of expression of which gene can be detected via modulation of fluorescence intensity or CAT reporter activity, respectively.

In another example, the subject of detection could be a downstream sphingosine kinase regulatory target (for example, sphingosine- 1 -phosphate), rather than sphingosine kinase itself. Yet another example includes sphingosine kinase binding sites ligated to a minimal reporter. For example, modulation of sphingosine kinase activity can be detected by screening for the modulation of the proliferative capacity of a breast cell. This is an example of an indirect system where modulation of sphingosine kinase expression, /^r se, is not the subject of detection. Rather, modulation of the molecules which sphingosine kinase regulates the expression of, are monitored.

These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as the proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the sphingosine kinase nucleic acid molecule or expression product itself or which modulate the expression of an upstream molecule, which upstream molecule subsequently modulates sphingosine kinase expression or expression product activity. Accordingly, these methods provide a mechanism of detecting agents which either directly or indirectly modulate sphingosine kinase expression and/or activity.

The agents which are utilised in accordance with the method of the present invention may take any suitable form. For example, proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules used, linked, bound or otherwise associated with the proteins such as amino acids, lipid, carbohydrates or other peptides, polypeptides or proteins. Similarly, the subject non-proteinaceous molecules may also take any suitable form. Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound or otherwise associated with any other proteinaceous or non-proteinaceous molecules. For example, in one embodiment of the present invention, said agent is associated with a molecule which permits its targeting to a localised region.

The subject proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the expression of sphingosine kinase or the activity of the sphingosine kinase expression product. Said molecule acts directly if it associates with the sphingosine kinase nucleic acid molecule or expression product to modulate expression or activity, respectively. Said molecule acts indirectly if it associates with a molecule other than the sphingosine kinase nucleic acid molecule or expression product which other molecule either directly or indirectly modulates the expression or activity of the sphingosine kinase nucleic acid molecule or expression product, respectively. Accordingly, the method of the present invention encompasses the regulation of sphingosine kinase nucleic acid molecule expression or expression product activity via the induction of a cascade of regulatory steps.

The term "expression" refers to the transcription and translation of a nucleic acid molecule. Reference to "expression product" is a reference to the product produced from the transcription and translation of a nucleic acid molecule. Reference to "modulation" should be understood as a reference to up-regulation or down-regulation.

"Derivatives" of the molecules herein described (for example sphingosine kinase, sphingosine- 1 -phosphate or other proteinaceous or non-proteinaceous agents) include fragments, parts, portions or variants from either natural or non-natural sources. Non- natural sources include, for example, recombinant or synthetic sources. By "recombinant sources" is meant that the cellular source from which the subject molecule is harvested has been genetically altered. This may occur, for example, in order to increase or otherwise enhance the rate and volume of production by that particular cellular source. Parts or fragments include, for example, active regions of the molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above.

Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, sphingosine kinase or derivative thereof may be fused to a molecule to facilitate its entry into a cell. Analogs of the molecules contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogs.

Derivatives of nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. The derivatives of the nucleic acid molecules utilised in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

A "variant" of sphingosine kinase or sphingosine- 1 -phosphate should be understood to mean molecules which exhibit at least some of the functional activity of the form of sphingosine kinase or sphingosine- 1 -phosphate of which it is a variant. A variation may take any form and may be naturally or non-naturally occurring. A mutant molecule is one which exhibits modified functional activity.

By "homologue" is meant that the molecule is derived from a species other than that which is being treated in accordance with the method of the present invention. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of sphingosine kinase or sphingosine-1 -phosphate which exhibits similar and suitable functional characteristics to that of the sphingosine kinase or sphingosine- 1- phosphate which is naturally produced by the subject undergoing treatment.

Chemical and functional equivalents (mimetics) should be understood as molecules exhibiting any one or more of the functional activities of the subject molecule, which functional equivalents may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening. For example chemical or functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening.

For example, libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used. A general synthetic scheme may follow published methods (eg., Bunin BA, et al. (1994) Proc. Natl. Acad. Set USA, 91:4708-4712; DeWitt SH, et al. (1993) Proc. Natl. Acad. ScL USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an array, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library. One suitable permutation strategy is outlined in US. Patent No. 5,763,263.

There is currently widespread interest in using combinational libraries of random organic molecules to search for biologically active compounds (see for example U.S. Patent No. 5,763,263). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occurring ligands of a biological target. In the present context, for example, they may be used as a starting point for developing sphingosine kinase and/or sphingosine-1 -phosphate analogues which exhibit properties such as more potent pharmacological effects. Sphingosine kinase and/or sphingosine-1 -phosphate or a functional part thereof may according to the present invention be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein). By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent. In practising the method, a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction. Preferably, the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.

In addition to screening for molecules which mimic the activity of sphingosine kinase and/or sphingosine-1 -phosphate , it may also be desirable to identify and utilise molecules which function agonistically or, most preferably, antagonistically to sphingosine kinase and/or sphingosine-1 -phosphate in order to up or down-regulate the functional activity of sphingosine kinase and/or sphingosine-1 -phosphate in relation to modulating EGFR- mediated cellular proliferation. The use of such molecules is described in more detail below. To the extent that the subject molecule is proteinaceous, it may be derived, for example, from natural or recombinant sources including fusion proteins or following, for example, the screening methods described above. The non-proteinaceous molecule may be, for example, a chemical or synthetic molecule which has also been identified or generated in accordance with the methodology identified above. Accordingly, the present invention contemplates the use of chemical analogues of sphingosine kinase and/or sphingosine-1 -phosphate capable of acting as agonists or antagonists. Chemical agonists may not necessarily be derived from sphingosine kinase and/or sphingosine-1 -phosphate but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of sphingosine kinase and/or sphingosine-1 -phosphate. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing sphingosine kinase and/or sphingosine-1 -phosphate from carrying out its normal biological functions. Antagonists include monoclonal antibodies specific for sphingosine kinase and/or sphingosine-1 -phosphate or parts of sphingosine kinase and/or sphingosine-1 -phosphate.

Analogues of sphingosine kinase and/or sphingosine-1 -phosphate or of sphingosine kinase and/or sphingosine-1 -phosphate agonistic or antagonistic agents contemplated herein include, but are not limited to, modifications to side chains, incorporating unnatural amino acids and/or derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the analogues. The specific form which such modifications can take will depend on whether the subject molecule is proteinaceous or non-proteinaceous. The nature and/or suitability of a particular modification can be routinely determined by the person of skill in the art.

For example, examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy- 5 -phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1.

TABLE 1

Non-conventional Code Non-conventional Code amino acid amino acid α-aminobutyπc acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-niethylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl- -aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(I -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3 -indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-niethylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl-Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

The method of the present invention contemplates the modulation of EGFR-mediated cellular activity, in particular proliferation, both in vitro and in vivo. Although the preferred method is to treat an individual in vivo it should nevertheless be understood that it may be desirable that the method of the invention may be applied in an in vitro environment, for example to provide an in vitro model for the analysis of cellular proliferation, such as uncontrollable cellular proliferation. In another example the application of the method of the present invention to an in vitro environment may extend to providing a readout mechanism for screening technologies such as those hereinbefore described. That is, molecules identified utilising these screening techniques can be assayed to observe the extent and/or nature of their functional effect on hyperglycaemia- induced endothelial cell functioning.

Although the preferred method is to downregulate EGFR-mediated cellular proliferation, in particular agonist induced EGFR-mediated cellular proliferation (for example in order to downregulate the progression of uncontrolled cellular proliferation), it should be understood that there may also be circumstances in which it is desirable to upregulate the subject functional activity, for example to facilitate an episode of rapid but controlled cellular proliferation.

In a related aspect, and as detailed hereinbefore, the present invention is applied to a mammal in order to modulate EGFR-mediated functional activity in vivo.

A further aspect of the present invention relates to the use of the invention in relation to the treatment and/or prophylaxis of disease conditions. Without limiting the present invention to any one theory or mode of action, with the ever growing incidence of cancer (in particular, breast cancer) in society, the development of new methods of treating cancers has become imperative. In particular, it is now widely recognised, although not yet fully understood, that there is diversity among cancers in terms of the mechanisms underpinning their onset. Accordingly, the development of a range of methods which are able to more narrowly target aberrant populations of cells provides a significant step forward relative to traditional treatment methods which have been directed to killing any rapidly dividing cell. The method of the present invention contributes significantly to this objective in that it facilitates a means of downregulating unwanted agonist induced EGFR-mediated cellular functional activity, this being particularly important in the context of agonist induced EGFR-mediated unwanted cellular proliferation. Accordingly, the method of the present invention is particularly useful, but in no way limited to, use in the treatment of primary and secondary malignancies such as those associated with solid tumours of the breast, colon, stomach, lung, brain, bone, oesophagus and pancreas. Although the preferred method is to down-regulate uncontrolled cellular proliferation in a subject, up-regulation of cell growth may also be desirable in certain circumstances such as to promote wound healing, angiogenesis or other healing process.

Accordingly, yet another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate EGFR-mediated cell activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling.

More particularly, there is provided a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate agonist induced EGFR-mediated cell activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates said agonist induced EGFR-mediated intracellular signalling. Still more particularly, there is provided a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate agonist induced EGFR-mediated cellular proliferation, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulate EGFR-mediated intracellular signalling.

Reference to "aberrant, unwanted or otherwise inappropriate" cellular proliferation should be understood as a reference to over active cell growth, to physiologically normal cell growth which is inappropriate in that it is unwanted or to insufficient cell growth. Preferably, said inappropriate cell growth is uncontrolled cell proliferation.

Preferably, said cellular proliferation is uncontrolled neoplastic cell proliferation. Most preferably, said malignant cell is derived from the breast, colon, stomach, lung, brain, bone, oesophagus, pancreas, ovary or uterus.

In accordance with these aspects of the present invention, said aberrant EGFR-mediated cellular proliferation is agonist induced.

In a most preferred embodiment, said condition is an oestrogen induced breast cell malignancy or an anti-oestrogen resistant breast cell malignancy.

There is therefore preferably provided a method for the treatment and/or prophylaxis of an oestrogen induced breast cell malignancy, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR-mediated intracellular signalling. The method of the present invention preferably facilitates the subject proliferation being reduced, retarded or otherwise inhibited. Reference to "reduced, retarded or otherwise inhibited" should be understood as a reference to inducing or facilitating the partial or complete inhibition of cell proliferation.

The subject of the treatment or prophylaxis is generally a mammal such as but not limited to human, primate, livestock animal (eg. sheep, cow, horse, donkey, pig), companion animal (eg. dog, cat), laboratory test animal (eg. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (eg. fox, deer). Preferably the mammal is a human or primate. Most preferably the mammal is a human. Although the present invention is exemplified utilising a murine model, this is not intended as a limitation on the application of the method of the present invention to other species, in particular, humans.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis including amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.

Administration of the agent (including sphingosine kinase or functional derivative or mimetic thereof or sphingosine kinase nucleic acid molecule) [herein referred to as "modulatory agent"], in the form of a pharmaceutical composition, may be performed by any convenient means. The modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal^, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant.

More particular, said cell activity is cellular proliferation.

Preferably, said cellular proliferation is uncontrolled neoplastic cell proliferation and even more preferably malignant cell proliferation. Most preferably, said malignant cell is derived from the breast, colon, stomach, lung, brain, bone, oesophagus, pancreas, ovary or uterus.

In yet another aspect the present invention relates to a pharmaceutical composition comprising a modulatory agent as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents. Said modulatory agents are referred to as the active ingredients.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and > propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule encoding a modulatory agent. The vector may, for example, be a viral vector.

Yet another aspect of the present invention relates to modulatory agents, as hereinbefore defined, when used in the method of the present invention.

The present invention is further described by reference to the following non-limiting examples.

EXAMPLE 1

SPHINGOSINE KINASE MEDIATES 'CROSS-CROSS' TRANSACTIVATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR BY ESTROGENS

Materials; and Methods

Cell Culture and Transfection.

The human breast cancer MCF-7 cells (ERα+/β+; ATCC HTB-22) were cultured in phenol red-free Dulbecco's modified Eagle's medium (CSL Biosciences, Parkville, Australia) containing 10% Fetal Bovine Serum (FBS). Constructs of SphK^ and SphK^G82D and stably transfected MCF-7 cell lines overexpressing SphK^¹, SphK^G82D or empty vector alone were described previously (Pitson et al, 2000b, J Biol Chem, 275:33945-33950; Sukocheva et al, 2003, MoI Endocrinol, 17:2002-2012).

Experiments with siRNA and Antisense oligonucleotides.

Chemical synthesized siRNA duplexes with 3 '-Fluorescein modification were purchased from Qiagen-Xeragon (Germantown, MD). The siRNA targeted sequences were: AAGAGCTGCAAGGCCTTGCCC (SEQ ID NO: 1) (SphKl),

AACCTCATCCAGACAGAACGA (SEQ ID NO:2) (SphK2), and AATTCTCCGAACGTGTCACGT (SEQ ID NO:3)(for a scrambled control siRNA). The following 18-mer phosphothioate oligonucleotides were synthesized by Geneworks (Australia): EDG-3 antisense, 5'-CGGGAGGGCAGTTGCCAT-S ' (SEQ ID NO:4); EDG- 3 sense, 5'-ATGGCAACTGCCCTCCCG-S' (SEQ ID NO:5). For transfection,

Lipofectamine-2000 reagent (Invitrogen) was used and MCF-7 cells were seeded into 6-well plates at a density of 50,000 cells per well the day before the experiment. After 36-48 h transfection, the targeted gene expression levels were detected by RT-PCR and/or Westernblot. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis.

Total RNA was extracted from cell cultures using TRIzol (Invitrogen). First-strand cDNA was synthesised from lmg total RNA using Omniscript reverse transcriptase (Qiagen) and oligo-dT primer (Geneworks), in a 20ml total volume. SphKl, SphK2 and EdgG-3 were amplified on a PTC-100 Programmable Thermal Controller (MJ Research, Inc) with an internal GAPDH control. The primers used to amplify were: SphKl (sense) 5'-TTGAACCATTATGCTGGCTATGA (SEQ ID NO:6) and SphKl (antisense) 5'- GCAGGTGTCTTGGAACCC(SEQ ID NO.-7); SphK2 (sense) 5'- GCTCAACTGCTCACTGTTGC (SEQ ID NO:8) and SphK2 (antisense) 5'- GCAGGTCAGACACAGAACGA (SEQ ID NO:9); Edg-3 (sense) 5'- GCCCTCTCGTGGATTTTGG (SEQ ID NO-.10) and Edg-3 (antisense) 5'- CGCATGGAGACGATCAGTTG (SEQ ID NO: 11). The amplified products were visualized by electrophoresis on 1.5% agarose stained with ethidium bromide. Images were captured on UVitec Gel documentation system.

Immiinoblot analysis.

Cells were harvested and lysed by sonication in lysis buffer containing 50 mM Tris/HCl (pH 7.4), 10% glycerol, 0.05% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na₃VO₄, 10 mM NaF, 1 mM EDTA and protease inhibitors (Roche Molecular Biochemicals). Aliquots of cell lysates were resolved by 8-12% SDS-PAGE and transferred to Hybond-P membranes (Amersham). The membranes were then probed with appropriate antibodies according to manufacturer's standard method. The immunocomplexes were detected with an enhanced chemiluminescence PLUS kit

(Amersham Pharmacia Biotech). Densitometry was performed on a Typhoon 9410 Mode Imager using the Image Quant program. Immunofluorescence microscopic analysis.

MCF-7 cells were seeded onto fibronectin coated 8-well chamber slides (Lab-Tech) and cultured for 48 hours. After stimulation, cells were fixed with 4% paraformaldehyde and permeabilised in 0.1% Triton X-IOO. The cells were then incubated with monoclonal anti- Edg-3 (1:100) and polyclonal anti-EGFR (1:100) antibodies and indirect immunofluorescence detected by incubation with the fluorophore (Alexa Fluor⁴⁸⁸ or Fluor⁵⁹⁴) coupled secondary antibodies. The fluorescence stained cells were imaged by epifluorescent microscopy on an Olympus BX-51 microscope equipped with a Cool Snap FX charge-coupled device camera (Photometries). Images were analysed with V++ software (Digital Optics Ltd. Auckland New Zealand).

Assays of SphK activity and SlP release.

SphK activity was routinely determined by incubating the cytosolic fraction with 5 mM D- erythro-sphingosine dissolved in 0.1% Triton X-100 and [γ32P]ATP for 30 min at 37⁰C as described previously (Xia et ah, 1998, Proc Natl Acad Sci USA, 95:14196-14201). The enzyme activity was defined as the amount of SlP formation (pmol)/min/mg protein. To determine extracellular SlP release, cells were metabolically labelled with [3H] serine (5μCi/ml) for 48h. After E2 stimulation for 30 min, the media was collected and lipids were extracted by adding chloroform: methanol: acetic acid (10:10:1, v/v). [3H]SlP was then resolved by thin-layer chromatography, quantified by scintillation spectrometry, and normalized by radioactivity recovered in total extracellular lipids as described previously (Xia et al., 1998, supra).

Results

SlP stimulates activation of EGFR in MCF-7 cells.

The effect of S IP in MCF-7 cells was examined. Figure IA shows that in response to 1 mmol/L SlP, tyrosine phosphorylation of EGFR increased in a time-dependent manner in MCF-7 cells. EGFR phosphorylation peaked at 10 min decreasing thereafter but was still evident at 240 min post treatment. In parallel, ERK1/2, key downstream signalling molecules of EGFR₅ were also phosphorylated in a similar time-dependent pattern to the EGFR phosphorylation (Figure IA). The concentration response for SlP showed an approximate EC₅0 of 5 nmol/L for phosphorylation of both EGFR and ERK 1/2, and maximum phosphorylation was observed at about 500 nmol/L (Figure IB). Collectively, these results demonstrate an ability of SlP to induce EGFR activation in the breast cancer cells.

E2 and SlP induced EGFR transactivation through a common signalling pathway .

It was sought to determine whether SlP could mimic E2 in the stimulation of EGFR transactivation in MCF-7 cells. Treatment of MCF-7 cells with E2 resulted in a rapid tyrosine phosphorylation of EGFR and ERK 1/2 activation similar to that observed in the SlP-treated cells (Figure 2A). Both E2- and SlP- induced transactivation of EGFR was blocked by pertussis toxin (PTX), a Gi specific inhibitor. PTX also significantly inhibited the phosphorylation of ERK 1/2 in response to either E2 or SlP stimulation (Figure 2A). In contrast, EGF-stimulated phosphorylation of EGFR and ERK 1/2 were not inhibited by PTX (Figure 2A).

As transactivation of EGFR relies on its internal tyrosine kinase activity (Prenzel et ah, 1999, supra), an analysis was performed as to whether the tyrosine kinase activity is required for E2 or S IP-induced EGFR transactivation. In the presence of AG 1478, a specific EGFR tyrosine kinase inhibitor, both E2 and S IP-induced EGFR transactivation were abolished completely (Figure 2A). The activation of ERKl/2 was also significantly inhibited. As a control, AG 1478 induced a complete inhibition of EGF-stimulated EGFR phosphorylation, supporting a specific effect of AG1478 on EGFR activity in MCF-7 cells.

Src family kinases have been suggested to play a signalling role in GPCR-mediated transactivation of EGFR (Gschwind et ah, 2001, supra). To evaluate the role of Src, cells were pretreated with the Src inhibitor, PP2 (100 nmol/L), for 30 min prior to stimulation. As shown in Figure 2A, both E2 and S IP-stimulated EGFR transactivation were significantly inhibited by PP2 treatment.

Shedding of heparin-binding EGF (HB-EGF) upon matrix metalloprotease (MMP) activation has also been recognized as an important mechanism in mediating EGFR transactivation by GPCR ligands (Prenzel et ah, 1999, supra). Therefore, an analysis was performed as to whether HB-EGF shedding was involved in E2 or S IP-induced EGFR transactivation. Firstly, MCF-7 cells were subjected to an acid wash step to reduce background autocrine stimulation and then pre-treated with the MMP inhibitors, o- phenanthroline or GM6001. Both E2 and S 1 P-induced EGFR transactivation as well as ERKl/2 activation were blocked by these two MMP inhibitors (Figure 2B). In contrast, the MMP inhibitors had no effect on the intrinsic kinase activity of the EGFR itself and the capacity of EGF to induce auto-phosphorylation of EGFR. Furthermore, depletion of HB- EGF production from the culture media by EGF neutralizing antibodies resulted in a significant inhibition of both E2- and S IP-induced EGFR transactivation (Figure 2B). Efficiency and specificity of the neutralizing antibodies were demonstrated by the inhibition of EGF-stimulated EGFR tyrosine phosphorylation. These data further suggest that HB-EGF shedding and release from the E2 or S IP-treated cells are involved in the process of EGFR transactivation.

SphKl activation is involved in E2-induced EGFR transactivation,

As SlP was able to mimic the effect of E2 stimulated EGFR transactivation, and that E2 was capable of stimulating SlP production upon SphKl activation, it was hypothesised that the E2-induced EGFR transactivation could be mediated via SphKl activation. To test this hypothesis, stably transfected MCF-7 cell lines overexpressing wild-type SphKl (SρhKl^wτ), dominant-negative SphKl (SphKl^G82D) or empty vector alone, respectively, were used. It has previously been demonstrated that the baseline SphK activity in SphKl ^w transfected cells is about 10-fold higher than in control cells (Sukocheva et ah, 2003, supra). E2 stimulation resulted in a rapid increase in SphK activity of approximately 2-fold above the basal level in both SphKl^WT transfected and control MCF-7 cells. In contrast, the SphKl^G82D transfected cells had a similar basal SphK activity to the control cells, whereas E2-stimulated SphK activity was completely abolished. Interestingly, while E2- stimulated tyrosine phosphorylation of EGFR and activation of ERK1/2 were enhanced in SphKl^WT transfected cells, the stimulatory effects of E2 were abrogated in the SphKl^G82D transfectants (Figure 3A). There are no significant differences between these transfected cell lines in either total EGFR expression levels determined by Western blot analysis (Figure 3A) or the cell surface expression levels of EGFR as measured by flow cytometry (Figure 3B). In contrast, neither EGF nor S IP-stimulated EGFR phosphorylation were significantly influenced by SphKl^G82D. These results thus suggest a specific role for SphK activity in the E2-induced EGFR transactivation.

Two mammalian SphK isoforms (i.e., SphKl and SphK2) have been cloned and both isoforms account for total cellular SphK activity (Kohama et ah, 1998, J Biol Chem, 273:23722-23728; Liu et al., 1999, MoI Biol Cell, 10:1179-1190). In order to define which isoform (or both) is responsible for the transactivation of EGFR as well as the role for endogenous SphK, a siRNA strategy was used to knock down each SphK isoform gene expression in MCF-7 cells. Treatment of cells with SphKl -specific siRNA resulted in a suppression of SphKl mRNA by 86±11%, and an inhibition of basal SphK activity by 48±12%, compared to cells treated with a scramble siRNA (Fig. 4A). Additionally, the elevated SphK activities in response to E2, EGF or SlP were almost completely abolished by SphKl siRNA (Figure 4B). Consequently, the SphKl siRNA induced a significant inhibition of E2-induced EGFR tyrosine phosphorylation and ERKl/2 activity to a similar extent shown in the SphKl^G82D transfected cells (Figure 4C). Again, consistent with the observations from the experiments with SphKl^G82D, neither EGF nor S IP-induced activation of EGFR and ERKl/2 were significantly influenced by SphKl siRNA. Interestingly, although SphK2-specific siRNA effectively knocked down SphK2 expression and inhibited basal SphK activity by about 50%, the extent of increased SphK activity induced by E2, EGF or SlP was not changed (Figure 4A and B). Correspondingly, SphK2 siRNA had no significant inhibitory effect on EGFR tyrosine phosphorylation or ERKl/2 activation in response to E2, S IP or EGF stimulation (Figure 4C). Taken together, these data suggest a critical role for SphKl, but not SphK2, in mediating E2-stimulated transactivation of EGFR.

SlP receptor, Edg-3, is required for the SphKl -dependent transactivation of EGFR by E2.

It was sought to determine the role for SlP and its receptors in the SphKl -dependent EGFR transactivation induced by E2. Analysis was first directed to whether SlP is released upon SphKl activation in cells responding to E2 stimulation. As shown in Figure 5 A, SlP levels were significant higher (172±22%) in conditioned media (CM) collected from the E2-stimulated MCF-7 cells than that from unstimulated cells. In addition, SlP levels were increased by an approximate 5 -fold in CM from SphKl transfected cells at both baseline and stimulated levels, compared to the control MCF-7 cells. No increased SlP level was detected after E2 stimulation in CM from the SphKl^G82D transfected cells (Figure 5A)₅ indicating that SphKl activation is responsible for SlP production and release from the E2-stimulated cells. Correspondingly, CM derived from the E2 -treated cells exhibited substantially greater capacity to stimulate EGFR tyrosine phosphorylation compared with the CM from untreated cells (Figure 5B). Moreover, CM from the E2- treated SphKl^WT transfected cells had a further stimulatory effect on the tyrosine phosphorylation of EGFR. In contrast, we could not detect any stimulation of EGFR phosphorylation by CM derived from the E2-treated SphKl ^G82D transfected cells, suggesting that the cellular SphK activity or the release of SlP is required for the CM- induced EGFR activation. In addition, the CM-induced activation of EGFR was completely blocked by PTX (Figure 5B), further supporting a critical involvement of SlP and its GPCR in the E2-stimulated transactivation pathway.

To evaluate a potential role of Edg-3 in E2 signalling, the effect of E2 on Edg-3 activation was directly examined. Edg-3 like most other GPCR undergoes internalization following ligand binding and activation of the receptor (Liu et ah, 1999, supra). Therefore, Edg-3 internalisation was assessed by immuno-fluorescence microscopic analysis. The results in Figure 6 shows that in MCF-7 cells, Edg-3 was rapidly internalised following a 2 min stimulation with E2 (10 nmol/L) and remained for about 30 min. As a control, treatment of cells with 100 nmol/L SlP induced a rapid and transient internalisation of Edg-3 in a similar pattern to that observed following E2 stimulation. To further strengthen the evidence for the involvement of Edg-3 in E2 -mediated EGFR transactivation, the antisense strategy was used to knock down endogenous Edg-3 expression. Cells transfected with Edg-3 antisense oligonucleotides resulted in decreases in both Edg-3 mRNA and protein levels by 61±17% and 68±12%, respectively, in comparison to the Edg-3 sense transfectants (Figure 7A and B). Remarkably, Edg-3 antisense induced a significant inhibition of EGFR tyrosine phosphorylation in cells responding to either E2 or SlP stimulation, whereas EGF-induced EGFR auto-phosphorylation was retained (Figure 7B). Taken together, these results suggest a critical role for the S IP receptor Edg-3 in mediating E2 signalling that transactivates the EGFR.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. A method of modulating EGFR-mediated cellular activity, said method comprising modulating the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR- mediated intracellular signalling.

2. A method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate EGFR- mediated cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the functioning of sphingosine kinase mediated signalling in said cell wherein downregulating said sphingosine kinase signalling downregulates EGFR- mediated intracellular signalling.

3. The method according to claim 1 or 2 wherein said sphingosine kinase is human sphingosine kinase.

4. The method according to claim 3 wherein said sphingosine kinase is sphingosine kinase 1 or 2.

5. The method according to any one of claims 1-4 wherein said EGFR-mediated cellular activity is agonist induced EGFR-mediated cellular activity.

6. The method according to claim 5 wherein said agonist induced EGFR-mediated cellular functional activity is cellular proliferation.

7. The method according to claim 6 wherein said cellular proliferation is neoplastic cell proliferation.

8. The method according to claim 7 wherein said neoplastic cell is a malignant cell derived from the breast, colon, stomach, lung, brain, bone, oesophagus, pancreas, ovary or uterus.

(

9. The method according to claim 8 wherein said neoplastic cell is a malignant breast cell or an anti-oestrogen resistant malignant breast cell.

10. The method according to any one of claims 5-9 wherein said agonist is oestrogen.

11. The method according to claim 1 or 2 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by introducing into said cell a nucleic acid molecule encoding sphingosine kinase or functional equivalent, derivative or homologue thereof or the sphingosine kinase expression product or functional derivative, homologue, analogue, equivalent or mimetic thereof.

12. The method according to claim 1 or 2 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an agonist of the sphingosine kinase expression product.

13. The method according to any one of claims 1-10 wherein said modulation is downregulation of the level of sphingosine kinase activity and said downregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an antagonist of the sphingosine kinase expression product.

14. The method according to claim 13 wherein said antagonist is a sphingosine kinase substrate competitor.

15. The method according to claim 14 wherein said substrate is ATP.

16. The method according to claim 14 wherein said substrate is sphingosine.

17. The method according to claim 16 wherein said agent is N,N-dimethylsphingosine or DL-threo-dihydrosphingosine.

18. The method according to claim 16 wherein said agent is a sphingosine kinase dominant negative such as SphK^G82D.

19. The method according to claim 13 wherein said antagonist down-regulates sphingosine kinase enzyme activation.

20. The method according to claim 19 wherein said agent modifies sphingosine kinase related phosphorylation events.

21. The method according to claim 19 wherein said agent modifies sphingosine kinase lipid composition.

22. Use of an agent capable of modulating the functional activity of sphingosine kinase mediated signalling in the manufacture of a medicament for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate EGFR-mediated cell activity, wherein downregulating said sphingosine kinase signalling downregulates EGFR- mediated intracellular signalling.

23. Use according to claim 22 wherein said sphingosine kinase is human sphingosine kinase.

24. Use according to claim 23 wherein said sphingosine kinase is sphingosine 1 or 2.

25. Use according to any one of claims 22-24 wherein said inappropriate EGFR- mediated cell activity is agonist induced EGFR-mediated cell activity.

26. Use according to claim 25 wherein said inappropriate cell activity is cellular proliferation.

27. Use according to claim 26 wherein said cellular proliferation is uncontrolled neoplastic cell proliferation.

28. Use according to claim 27 wherein said uncontrolled neoplastic cell proliferation is malignant cell proliferation.

29. Use according to claim 28 wherein said malignant cell is derived from the breast, colon, stomach, lung, brain, bone, oesophagus, pancreas, ovary or uterus.

30. Use according to any one of claims 25-29 wherein said agonist is oestrogen.

31. Use according to any one of claims 22-30 wherein said condition is an oestrogen induced breast cell malignant or an anti-oestrogen resistant breast cell malignancy.

32. Use according to claim 22 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by introducing into said cell a nucleic acid molecule encoding sphingosine kinase or functional equivalent, derivative or homologue thereof or the sphingosine kinase expression product or functional derivative, homologue, analogue, equivalent or mimetic thereof.

33. Use according to claim 22 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an agonist of the sphingosine kinase expression product.

34. Use according to any one of claims 22-31 wherein said modulation is downregulation of the level of sphingosine kinase activity and said downregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an antagonist to the sphingosine kinase expression product.

35. Use according to claim 34 wherein said antagonist is a sphingosine kinase substrate competitor.

36. Use according to claim 35 wherein said substrate is ATP.

37. Use according to claim 35 wherein said substrate is sphingosine.

38. Use according to claim 37 wherein said agent is N,N-dimethylsphingosine or DL- threo-dihydrosphingosine.

39. Use according to claim 37 wherein said agent is a sphingosine kinase dominant negative such as SphK ^82D.

40. Use according to claim 34 wherein said antagonist down-regulates sphingosine kinase enzyme activation.

41. Use according to claim 34 wherein said agent modifies sphingosine kinase related phosphorylation events.

42. Use according to claim 34 wherein said agent modifies sphingosine kinase lipid composition.

43. A pharmaceutical composition comprising a modulatory agent capable of modulating EGFR-mediated cellular activity and one or more pharmaceutically acceptable carriers and/or diluents when used in accordance with the method of any one of claims 1-21.

44. An agent, which agent modulates EGFR-mediated cellular functional activity when used in accordance with any one of claims 1-21.