JP2008546720A - Regulation of EGFR signaling by regulating sphingosine kinase signaling - Google Patents

Abstract

The present invention relates generally to methods for modulating EGFR-mediated cellular functional activity, and agents useful therefor. More specifically, the present invention relates to a method of modulating agonist-induced EGFR-mediated cell proliferation by modulating upstream intracellular sphingosine kinase signaling. The methods of the invention are particularly useful for the treatment and / or prevention of conditions characterized by abnormal EGFR-mediated cell function, particularly abnormal EGFR-mediated cell proliferation.

Description

The present invention relates generally to methods for modulating EGFR-mediated cellular functional activity, and agents useful therefor. More specifically, the present invention relates to methods of modulating agonist-induced EGFR-mediated cell proliferation by modulating upstream intracellular sphingosine kinase signaling. The methods of the invention are particularly useful for the treatment and / or prevention of conditions characterized by abnormal EGFR-mediated cell function, particularly abnormal EGFR-mediated cell proliferation.

BACKGROUND OF THE INVENTION In this specification, details of the reference lists of publications cited by the authors are listed in alphabetical order at the end of the specification.

  References to any prior art herein are not, and should be taken as, approval or any form of suggestion that the prior art forms part of common general knowledge in Australia. is not.

  Separate signaling pathways have been shown to integrate estrogen (E2) action and directly affect its function, including normal mammalian development and breast cancer growth. Estrogens have been known for decades to associate with growth factor networks that exhibit increased growth promoting activity in breast cancer cells. The majority of growth factors and their receptors have been described to interact with estrogen signaling, in which the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases are those in human cancer Is of particular interest due to its clinical involvement (Bange et al., 2001, Nat Med, 7: 548-552); Levin, 2003, Mol Endocrinol, 17: 309-317 Patent Document 2)). Indeed, aberrant expression and activation of EGFR is often observed in various tumors, particularly breast and ovarian tumors, which correlate with poorer patient prognosis (Keen and Davidson, 2003, Cancer, 97: 825-833 (non-patent document 3); Roskoski, Jr., 2004, Biochem Biophys Res Commun, 319: 1-11 (non-patent document 4)). Moreover, up-regulation of EGFR signaling is thought to be an important mechanism conferring anti-estrogen resistance in breast cancer resulting in unsuccessful endocrine therapy (Ali and Coombes, 2002, Nat Rev Cancer, 2: 101-112 (Non-patent document 5); Nicholson et al., 2003, Breast Cancer Res Treat, 80 Suppl 1, S29-S34 (non-patent document 6)).

  A variety of evidence suggests that the interaction between EGFR and estrogen signaling can occur at various levels. Estrogens primarily act on the nuclear estrogen receptor (ER), which leads to the regulation of gene expression, traditionally thought to be the genomic action of estrogens. Many of these estrogen responsive genes are actually the major signaling molecules involved in EGFR signaling (Levin, 2003, supra). Alternatively, EGFR signaling can activate ER in the absence of estrogen (Picard et al., 1997, Biochem Soc Trans, 25: 597-602). In addition, recent studies have provided evidence for an interaction between EGFR and the membrane-associated form of ER (mER) in a G protein-dependent manner (Filardo et al., 2000, Mol Endocrinol, 14: 1649 -1660 (8); Razandi et al., 2003, J Biol Chem, 278: 2701-2712 (9)), by other well-supported G protein-coupled receptor (GPCR) ligands A model of EGFR transactivity by estrogen similar to that induced is suggested (Gschwind et al., 2001, Oncogene, 20: 1594-1600 (Non-patent Document 10)). However, mER has not yet been identified at the molecular level. mER is derived from an alternative transcript associated with itself or nuclear ER (Razandi et al., 1999, Mol Endocrinol, 13: 307-319), or GPCR family (eg GPR30 ) (Filardo et al., 2000, supra) or may be part of the PCR-ER complex (Razandi et al., 2003, supra). Thus, the actual nature of mER remains unclear and the mechanisms based on estrogen-induced EGFR transactivity remain largely unknown.

  Given the very serious problems breast cancer poses to both women and society in general, especially the increased prevalence of breast cancer that is resistant to anti-estrogen therapy, we will elucidate the intracellular mechanisms that support these cell defects, Thereby, there is an ongoing need to develop new and alternative treatment measures. Since first described by Ullrich and collaborators (Prenzel et al. 1999, Nature, 402: 884-888), transactivation of EGFR by GPCR ligands is important for cell signaling It is considered as a simple model. Some GPCR ligands include lysophosphatidic acid, thrombin, angiotensin II and endothelin-1, which have been shown to transactivate EGFR leading to activation of survival or cell division pathways ( Gschwind et al. 2001, supra). However, in the work leading up to the present invention, surprisingly and unexpectedly, sphingosine kinase is an estrogen non-genome for criss-cross transactivation of EGFR via the S1P receptor. It has been found to play a critical role in mediating signal transduction. Thus, sphingosine kinase mediates the interaction between the three transmembrane events induced by estrogen, S1P and EGF. These findings now show abnormal EGFR-mediated cellular functions by targeting upstream signaling molecules rather than EGFR, or estrogen or estrogen receptor molecules in the case of estrogen-induced EGFR activation, In particular, it is promoting the development of alternative treatment means for abnormal cell proliferation.

Bange et al., 2001, Nat Med, 7: 548-552 Levin, 2003, Mol Endocrinol, 17: 309-317 Keen and Davidson, 2003, Cancer, 97: 825-833 Roskoski, Jr., 2004, Biochem Biophys Res Commun, 319: 1-11 Ali and Coombes, 2002, Nat Rev Cancer, 2: 101-112 Nicholson et al., 2003, Breast Cancer Res Treat, 80 Suppl 1, S29-S34 Picard et al., 1997, Biochem Soc Trans, 25: 597-602 Filardo et al., 2000, Mol Endocrinol, 14: 1649-1660 Razandi et al., 2003, J Biol Chem, 278: 2701-2712 Gschwind et al., 2001, Oncogene, 20: 1594-1600 Razandi et al., 1999, Mol Endocrinol, 13: 307-319 Prenzel et al. 1999, Nature, 402: 884-888

SUMMARY OF THE INVENTION Throughout this specification and the appended claims, unless the context requires otherwise, the terms “comprise” and “comprises” and “comprising” etc. Variant is meant to include a specified integer or stage, or a group of integers or stages, but is not meant to include any other integer or stage, or a group of integers or stages. Understood.

  This specification contains nucleic acid sequence information created using the program PatentIn Version 3.1, which is shown after the list of references in this specification. Each nucleic acid sequence is identified in the sequence listing by a numerical heading <201> followed by a sequence identifier (eg, <201> 1, <201> 2, etc.). The length of each nucleic acid sequence, the type of sequence (such as DNA) and the biological origin are indicated by the information provided in <211>, <212> and <213> in the numeric headings, respectively. Nucleic acid sequences referred to herein are identified by the heading SEQ ID NO: followed by a sequence identifier (eg, SEQ ID NO: 1, SEQ ID NO: 2, etc.). The sequence identifier referred to in this specification correlates with the information provided in the numeric header field <400> in the sequence listing and the subsequent sequence identifier (eg, <400> 1, <400> 2, etc.). Yes. That is, SEQ ID NO: 1 detailed in this specification correlates with the sequence indicated as <400> 1 in the sequence listing.

  One aspect of the invention relates to a method of modulating EGFR-mediated cellular functional activity, comprising modulating the function of sphingosine kinase-mediated signaling in a cell, by down-regulating the sphingosine kinase signaling Downregulates EGFR-mediated intracellular signaling.

  In another aspect, a method of modulating agonist-induced EGFR-mediated cellular functional activity is provided, the method comprising modulating the function of sphingosine kinase-mediated signaling in a cell, downregulating the sphingosine kinase Down-regulate agonist-induced EGFR-mediated intracellular signaling.

  In yet another aspect, a method of modulating agonist-induced EGFR-mediated cell proliferation is provided, the method comprising modulating the function of sphingosine kinase-mediated signaling in a cell, wherein said sphingosine kinase signaling is Down-regulate agonist-induced EGFR-mediated intracellular signaling.

  Yet another aspect of the present invention provides a method of down-regulating agonist-induced EGFR-mediated neoplastic cell proliferation, which down-regulates the function of sphingosine kinase-mediated signaling in cells. Including.

  Yet another aspect of the invention provides a method of down-regulating agonist-induced EGFR-mediated neoplastic cell proliferation, which down-regulates the function of sphingosine kinase-1 mediated signaling in cells. Including that.

  A further aspect of the invention relates to a method for the treatment and / or prevention of a condition characterized by abnormal, undesirable or otherwise inappropriate EGFR-mediated cellular activity, said method comprising sphingosine in a cell Administering an effective amount of the agent to the mammal for a time and under conditions sufficient to modulate the function of kinase-mediated signaling, and downregulating said sphingosine kinase signaling to cause EGFR-mediated intracellular Down-regulate signaling.

  In another aspect, there is provided a method for the treatment and / or prevention of a condition characterized by abnormal, undesirable or otherwise inappropriate agonist-induced EGFR-mediated cellular activity, the method comprising: Administering an effective amount of the agent to the mammal for a time and under conditions sufficient to modulate the function of sphingosine kinase mediated signaling in the cell, and down-regulating said sphingosine kinase signaling Down-regulate inducible EGFR-mediated intracellular signaling.

  In yet another further aspect, there is provided a method for the treatment and / or prevention of a condition characterized by abnormal, undesirable or otherwise inappropriate agonist-induced EGFR-mediated cell proliferation, The method comprises administering to a mammal an effective amount of an agent for a time and under conditions sufficient to modulate the function of sphingosine kinase mediated signaling in a cell, and downregulating said sphingosine kinase signaling Down-regulates EGFR-mediated intracellular signaling.

  Yet another further aspect of the invention provides a method for the treatment and / or prevention of estrogen-induced breast cell malignancies, which down-regulates the function of sphingosine kinase-mediated signaling in cells. Administering an effective amount of the agent to the mammal for a time and under conditions sufficient to downregulate the EGFR-mediated intracellular signaling by downregulating the sphingosine kinase signaling.

  In another aspect, the present invention relates to sphingosine kinase mediated in the manufacture of a medicament for the treatment and / or prevention of conditions characterized by abnormal, undesirable or otherwise inappropriate EGFR mediated cellular activity. With respect to the use of agents that can modulate the functional activity of sexual signaling, EGFR-mediated intracellular signaling is downregulated by downregulating the sphingosine kinase signaling.

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

  Yet another aspect of the invention relates to a modulating agent as defined above when used in the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the identification of the existence of a signaling cross-mechanism that results in upregulation of EGFR-mediated cell signaling. This finding therefore facilitates the development of alternative means of treating abnormal EGFR-mediated cellular functions, which is particularly relevant in terms of treating cancers such as anti-estrogen resistant breast cancer. In addition, the decision that sphingosine kinase is a significant upstream mediator of EGFR-related cell signaling is a specific target for therapeutic interventions other than EGFR itself, or in the case of breast cancer estrogens. provide.

  Accordingly, one aspect of the present invention relates to a method of modulating EGFR-mediated cellular functional activity, the method comprising modulating the function of sphingosine kinase-mediated signaling in a cell, down-regulating said sphingosine kinase signaling Down-regulate EGFR-mediated intracellular signaling.

  “EGFR” should be understood as referring to any form of epidermal growth factor receptor. For example, it should be understood to include any isoform resulting from alternative splicing of EGFR mRNA, or a functional mutant or polymorphic variant of EGFR. Without limiting the present invention to any one theory or mode of action, EGFR is a mediator of epithelial growth factor (EGF) or similarly transforming growth factor-α (TGF-α) biological signals. . It is a transmembrane domain and a membrane protein with tyrosine kinase activity in the cytoplasmic domain; the extracellular domain binds to EGF and is highly glycosylated. Binding of EGF to its receptor results in induction of tyrosine kinase activity and dimer formation, resulting in autophosphorylation. Next, stimulation of cellular DNA synthesis and cell proliferation occurs. Internalization of the EGF receptor complex occurs after binding EGF.

  More specifically, provided is a method of modulating agonist-induced EGFR-mediated cellular functional activity, the method comprising modulating the function of sphingosine kinase-mediated signaling in a cell, said sphingosine kinase signaling Downregulates agonist-induced EGFR-mediated intracellular signaling.

  “Agonist-induced” EGFR-mediated cellular functional activity should be understood as a reference to EGFR-mediated cellular functional activity that is initially induced by the action of molecules that bind to receptors other than EGFR receptors. For example, in the context of estrogen-induced breast cancer, the final signal that leads to cell proliferation is mediated through the EGFR molecule, whereas the molecule that initiates a cascade of steps leading to EGFR stimulation is directed to the estrogen receptor Estrogen binding. In this specific example, the primary “agonist” is estrogen. Without limiting the present invention to any one theory or mode of action, elucidation of the concept of agonist-induced EGFR-stimulated cellular function is only facilitated through the identification of the existence of a cross-activation mechanism. This allows the design of alternative treatment methods.

  “EGFR-mediated cellular functional activity” should be understood as a reference to any one or more functional activities that a cell may perform as a result of EGFR stimulation. Preferably, this functional activity is cell proliferation.

  According to this preferred embodiment, a method of modulating agonist-induced EGFR-mediated cell proliferation is provided, the method comprising modulating the function of sphingosine kinase-mediated signaling in a cell, wherein said sphingosine kinase signaling Downregulates agonist-induced EGFR-mediated intracellular signaling.

  A “cell” in the context of the present invention should be understood as a reference to any form or type of cell, regardless of the origin of the cell. For example, the cells may be natural normal or abnormal cells, or frozen / thawed either in vitro or in vivo, or genetically, biochemically or otherwise modified. It may be manipulated, modified, or otherwise treated either in vitro or in vivo, such as a cell (eg, including cells that are the result of fusion of two different cell types). Preferably, the cell is a neoplastic cell. By “neoplastic cell” is meant a cell that exhibits uncontrolled proliferation. The neoplastic cell may be a benign cell or a malignant cell. Preferably the cell is malignant. In one particular aspect, the neoplastic cell is a malignant cell and its growth forms a solid tumor such as a malignant cell derived from the breast, colon, stomach, lung, brain, bone, esophagus, or pancreas.

  Thus, there is most preferably provided herein a method for down-regulating agonist-induced EGFR-mediated neoplastic cell proliferation, which down-regulates the function of sphingosine kinase-mediated signaling in cells. Including that.

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

  Even more preferably, the agonist is estrogen and the cell is a malignant breast cell or an anti-estrogen resistant malignant breast cell.

  “Sphingosine kinase” should be understood as a reference to all forms of this protein and functional derivatives thereof. This includes, for example, any isoform resulting from alternative splicing of the sphingosine kinase mRNA of interest, or functional mutants or polymorphic variants of these proteins. For example, this definition extends to the isoforms sphingosine kinase-1 and sphingosine kinase-2, wherein the sphingosine kinase is preferably sphingosine kinase-1.

  Accordingly, preferably provided herein is a method of down-regulating agonist-induced EGFR-mediated neoplastic cell proliferation, which down-regulates the function of sphingosine kinase-1 mediated signaling in cells Including doing.

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

  Even more preferably, the agonist is estrogen and the cell is a malignant breast cell or an anti-estrogen resistant malignant breast cell.

  “Sphingosine kinase-mediated signaling” should be understood as a reference to intracellular signaling pathways that utilize sphingosine kinase or functional derivatives thereof. Sphingosine kinase is a key regulatory enzyme in the activity of the sphingosine kinase signaling pathway and functions to produce the endogenous sphingolipid mediator sphingosine-1-phosphate. Still further, without limiting the invention in any way, sphingosine kinase was found to form part of a novel signaling system that mediates EGFR transactivation. In particular in connection with estrogen-induced breast cancer, estrogen stimulates sphingosine kinase activation and sphingosine-1-phosphate release, and estrogen sphingosine-1-phosphate leads to EGFR transactivation in a matrix metalloproteinase-dependent manner A specific GPCR, Edg3, can be activated. Thus, these findings identify the role of sphingosine kinase in mediating the interaction between the three transmembrane events induced by estrogen, S1P and EGF, and new information between the three individual ligand receptor systems Clarifying the transmission mechanism, this mechanism has been termed “cross” activation.

  Modulating the “function” of sphingosine kinase-mediated signaling is as a reference to regulating the level of sphingosine kinase activity present in any given cell, as opposed to the concentration of sphingosine kinase itself. Should be understood. Although a decrease in the intracellular concentration of sphingosine kinase generally correlates with a decrease in the level of sphingosine kinase functional activity observed in the cell, those skilled in the art also believe that this decrease in activity level is an absolute intracellular sphingosine kinase. It will be understood that it can be achieved by means other than simply reducing the concentration. For example, one is a means to reduce the half-life of sphingosine kinase, a means to sterically hinder the binding of this molecule to its substrate, or a means to competitively inhibit the binding of functional sphingosine kinase to its substrate May be used.

  It should also be understood that the regulation of sphingosine kinase-mediated signaling, particularly its downregulation, does not necessarily mean that the activity of this signaling pathway needs to return to basal levels. Rather, this level need only change compared to the pre-processing level. Thus, the methods of the invention may be applied to reduce uncontrolled cell growth at least until such time as it can be completely stopped. This adjustment may be temporary or long-term depending on the requirements of the particular situation.

Thus, modulation of the “activity” of sphingosine kinase-mediated signaling should be understood as a reference to either up-regulating or down-regulating signaling mechanisms. A preferred method is to down-regulate subject signaling in the context of patients exhibiting uncontrolled cell growth, but up-regulate sphingosine kinase signaling, eg, transformation of subject cells. There may be certain circumstances where it is desirable to facilitate analysis of cell line models or promote rapid expansion of cell proliferation without necessarily inducing it. Any adjustment form may be achieved by any suitable means, including:
(I) modulating the absolute levels of components of a sphingosine kinase-mediated signaling pathway, such as sphingosine kinase and / or sphingosine-1-phosphate. Thereby, more or less both of these molecules are available for activity and / or to interact with downstream targets such as Edg-3.
(Ii) actuate or antagonize components of sphingosine kinase-mediated signaling pathways such as sphingosine kinase and / or sphingosine-1-phosphate. Thereby, the functional effect of any one or more of these molecules is increased or decreased. For example, increasing the half-life of sphingosine kinase may be achieved by increasing the overall level of sphingosine kinase activity without actually requiring an increase in the absolute intracellular concentration of sphingosine kinase. Similarly, partial antagonism of sphingosine kinase or sphingosine-1-phosphate may cause these molecules to bind to components that induce some steric hindrance, for example in connection with their binding to downstream targets. However, it is not necessary to eliminate the signal transduction effect that they provide, but they may act to reduce the signal transduction effect. This may therefore provide a means to down-regulate sphingosine kinase mediated signaling without necessarily down-regulating the absolute concentration of components of this pathway.

Means for achieving this goal in terms of achieving up-regulation or down-regulation of sphingosine kinase-mediated signaling are well known to those of skill in the art and include, but are not limited to, the following.
(I) Introducing a nucleic acid molecule encoding a component of the sphingosine kinase signaling pathway or a functional equivalent or derivative thereof into the cell to upregulate the ability of the cell to express the component of the sphingosine kinase mediated pathway To do.
(Ii) introducing into the cell a proteinaceous or non-proteinaceous molecule that regulates the transcriptional and / or translational control of the gene. This gene directly or indirectly directs the expression of any sphingosine kinase signaling pathway component, in particular sphingosine kinase or sphingosine-1-phosphate or a functional part thereof, or a sphingosine kinase-mediated signaling pathway component There may be several other genes that regulate.
(Iii) introducing the expression product (in either active or inactive form) of one or more components of the sphingosine kinase-mediated signaling pathway, or a functional derivative, homolog or mimetic thereof, into the cell. .
(Iv) introducing a proteinaceous or non-proteinaceous molecule that functions as an antagonist of sphingosine kinase or sphingosine-1-phosphate.
(V) introducing into the cell a proteinaceous or non-proteinaceous molecule that modulates the expression and / or function of sphingosine kinase or sphingosine-1-phosphate receptor.
(Vi) any one or more constituent sphingosines, such as N'N'-dimethylsphingosine (a sphingosine kinase chemical inhibitor), DL-threo-dihydrosphingosine or SphK ^G82D (mutant sphingosine kinase dominant negative) Introducing proteinaceous or non-proteinaceous molecules that function as antagonists to the expression products of the kinase signaling pathway.
(Vii) introducing a proteinaceous or non-proteinaceous molecule that functions as an agonist for the expression product of a sphingosine kinase-mediated signaling pathway.

  The proteinaceous molecules described above may be derived from any suitable source, such as natural, recombinant, or synthetic sources, such as fusion proteins or molecules identified after screening of natural products, for example. Including. A non-proteinaceous molecule can be, for example, a reference to a nucleic acid molecule, or can be a molecule from a natural source, such as a natural product screen, or a chemically synthesized molecule. Also good. The present invention contemplates small molecules that can act as analogs, or agonists or antagonists, of components of the sphingosine kinase signaling pathway.

  A chemical agonist need not necessarily be derived from a component of a sphingosine kinase-mediated signal transduction pathway product, but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to meet certain physicochemical properties. Antagonists block the components of sphingosine kinase-mediated signaling pathways from performing their normal biological functions, such as molecules that interfere with activation or that interfere with downstream functions of other activated molecules And any compound that can be inhibited or otherwise prevented. Antagonists include monoclonal antibodies, dominant negative sphingosine kinase mutants, and antisense nucleic acids that prevent transcription or translation of genes or mRNAs that are components of sphingosine kinase-mediated signaling pathways in mammalian cells. Regulation of expression may also be achieved by using molecules suitable for use in antigens, RNA (particularly siRNA), ribosomes, DNAzymes, RNA aptamers, antibodies or cosuppression. The proteinaceous and non-proteinaceous molecules mentioned in points (i) to (vii) above are collectively referred to herein as “regulatory agents”.

  Screening for regulatory agents as defined above involves contacting the agent with a cell containing the sphingosine kinase gene (or any other gene encoding a component of the sphingosine kinase signaling pathway) or a functional equivalent or derivative thereof. And screening for regulation of sphingosine kinase protein production or functional activity, regulation of expression of nucleic acid molecules encoding sphingosine kinase, or regulation of cellular target activity or expression of downstream sphingosine kinase. It can be achieved by any one of several suitable methods, without limitation. Detection of such modulation can be achieved by utilizing techniques such as Western blotting, electrophoretic migration shift assays, and / or readout of a reporter of sphingosine kinase activity such as luciferase, CAT.

  It should be understood that the sphingosine kinase gene or functional equivalent or derivative thereof may be naturally present in the cell under test or may be transfected into the host cell for the purpose of the test. . In addition, naturally occurring or transfected genes may be constitutively expressed, thereby screening agents that down regulate sphingosine kinase activity, either at the nucleic acid or expression product level, among others. This gene may require activity, thereby providing, inter alia, a useful model for screening for agents that upregulate sphingosine kinase expression. . In addition, as long as the sphingosine kinase nucleic acid molecule is transfected into the cell, the molecule may contain the complete sphingosine kinase gene or simply contain a portion of the gene, such as a portion that controls the expression of the sphingosine kinase product. You may go out. For example, the sphingosine kinase promoter region may be transfected into the cell to be tested. In this regard, if only the promoter is utilized, detecting modulation of the activity of the promoter can be accomplished, for example, by linking the promoter to a reporter gene. For example, the promoter may be linked to a luciferase or CAT reporter, and regulation of gene expression can be detected via regulation of fluorescence intensity or CAT reporter activity, respectively.

  In another example, the subject of detection may be a downstream sphingosine kinase regulatory target (eg, sphingosine-1-phosphate) rather than sphingosine kinase itself. Yet another example includes a sphingosine kinase binding site linked to a minimal reporter. For example, modulation of sphingosine kinase activity can be detected by screening for modulation of breast cell proliferation ability. This is an example of an indirect system where regulation of sphingosine kinase expression itself is not a detection target. Rather, the regulation of the molecules that sphingosine kinase controls expression is monitored.

  These methods are mechanisms for performing high-throughput screening of putative regulatory agents such as proteinaceous or non-proteinaceous agents, including synthetic libraries, combinatorial libraries, chemical libraries, and natural libraries I will provide a. These methods also include an agent that binds to either the sphingosine kinase nucleic acid molecule or its expression product itself, or an agent that regulates the expression of an upstream molecule that the upstream molecule subsequently regulates sphingosine kinase expression or the activity of the expression product. Promote detection. Thus, these methods provide a mechanism for detecting agents that directly or indirectly modulate sphingosine kinase expression and / or activity.

  The agent utilized in accordance with the method of the present invention may be in any suitable form. For example, proteinaceous agents may be glycosylated or glycosylated to varying degrees, may be phosphorylated or dephosphorylated, and / or amino acids, lipids, carbohydrates or others It may include a series of other molecules used, linked, bound, or otherwise associated with a protein, such as a peptide, polypeptide or protein. Similarly, the non-proteinaceous molecule of interest may also be in any suitable form. Both proteinaceous and non-proteinaceous agents described herein may be linked, bound, or otherwise associated with any other proteinaceous or non-proteinaceous molecule. . For example, in one aspect of the invention, the agent is associated with a molecule that allows its targeting to a local region.

  The proteinaceous and non-proteinaceous molecules of interest may act directly or indirectly to modulate sphingosine kinase expression or sphingosine kinase expression product activity. When the molecule is associated with a sphingosine kinase nucleic acid molecule or expression product, it acts directly to modulate expression or activity, respectively. The molecule acts indirectly when associated with a molecule other than a sphingosine kinase nucleic acid molecule or expression product, and the other molecule directly or indirectly directs the expression or activity of the sphingosine kinase nucleic acid molecule or expression product, respectively. To adjust. Thus, the methods of the present invention encompass the control of sphingosine kinase nucleic acid molecule expression or expression product activity through the introduction of a cascade of regulatory steps.

  The term “expression” refers to the transcription and translation of a nucleic acid molecule. “Expression product” refers to the product produced from transcription and translation of a nucleic acid molecule. “Modulation” should be understood as a reference to up-regulation or down-regulation.

  A “derivative” of a molecule described herein (eg, sphingosine kinase, sphingosine-1-phosphate or other proteinaceous or non-proteinaceous agent) is a fragment, moiety from a natural or non-natural source , Including some or variants. Non-natural sources include, for example, recombinant or synthetic sources. By “recombinant source” is meant that the source of the cells from which the molecule of interest is recovered has been genetically modified. For example, genetic modifications may be made to increase or otherwise enhance the production rate and production by a particular cell source. The part or fragment includes, for example, the active region of the molecule. Derivatives may be derived from amino acid insertions, deletions or substitutions. Derivatives by amino acid insertion include amino-terminal and / or carboxy-terminal fusions as well as insertions within the sequence of one or more amino acids. Variants of the inserted amino acid sequence are those in which one or more amino acid residues are introduced at a given site in the protein, but random insertion is also possible using a suitable screen of the resulting product . Variants that are deleted are characterized by the removal of one or more amino acids from the sequence. An amino acid variant that is substituted is one in which at least one residue in the 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 detailed above.

  Derivatives also include fragments having specific epitopes or portions of the complete protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, sphingosine kinase or a derivative thereof can be fused to a molecule to facilitate its introduction into the cell. Analogs of molecules contemplated herein include, but are not limited to, modifications to side chains, insertion of unnatural amino acids and / or their derivatives during the synthesis of peptides, polypeptides or proteins, and crosslinkers And the use of other methods impose structural constraints on proteinaceous molecules or their analogs.

  Derivatives of nucleic acid sequences that can be utilized in accordance with the methods of the invention may be derived from one or more nucleotide substitutions, deletions and / or additions, including fusions with other nucleic acid molecules as well. . Derivatives of nucleic acid molecules utilized in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co-suppression, and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include modified variants.

  A “variant” of sphingosine kinase or sphingosine-1-phosphate should be understood to mean a molecule that exhibits at least some of the functional activity of the variant sphingosine kinase or sphingosine-1-phosphate form. Mutations can take any form and may be natural or non-natural. Mutant molecules are molecules that exhibit altered functional activity.

  “Homologs” are derived from species other than those processed according to the methods of the invention. For example, a species other than the species to be treated is similar to sphingosine kinase or sphingosine-1-phosphate naturally produced by the subject to be treated and has a suitable functional characteristic of sphingosine kinase or sphingosine-1-phosphate. Homologues may arise when it is decided to produce a form.

  Chemical and functional equivalents (mimetics) may be any of the molecules of interest that may be derived from any source as chemically synthesized or identified through a screening process such as screening of natural products. Should be understood as molecules exhibiting one or more functional activities. For example, chemical or functional equivalents can be designed and / or identified utilizing well-known methods such as combinatorial chemistry or recombinant library high-throughput screening, or subsequent natural product screening.

  For example, a library containing small organic molecules may be screened, and organic molecules having a majority of specific parent substituents are used. General synthetic schemes may follow published methods (eg, Bunin BA, et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 4708-4712; DeWitt SH, et al. (1993 Proc. Natl. Acad. Sci. USA, 90: 6909-6913). Briefly, in each successive synthesis step, a subset of tubes so that one of a plurality of different selected substituents yields all possible permutations of the different substituents employed in the production of the library. Is added to each of the selected subsets of tubes in the array. One suitable permutation strategy is outlined in US Pat. No. 5,763,263.

  There is currently wide interest in using combinatorial libraries of random organic molecules to search for biologically active compounds (see, eg, US Pat. No. 5,763,263). Ligands discovered by screening this type of library can be useful in mimicking or blocking natural ligands or in interfering with natural ligands of biological targets. In this regard, for example, they may be used as a starting point for the development of sphingosine kinase and / or sphingosine-1-phosphate analogs that exhibit properties such as more potent pharmacological effects. Sphingosine kinase and / or sphingosine-1-phosphate or a functional part thereof may be used in accordance with the present invention in combination libraries formed by various solid phase or liquid phase synthesis methods (see, eg, US Pat. No. 5,763,263 and references cited therein). By using techniques such as those disclosed in US Pat. No. 5,753,187, millions of new chemical and / or biological compounds can be routinely screened in less than a few weeks. Of the majority of identified compounds, only those that exhibit suitable biological activity are further analyzed.

  For high-throughput library screening methods, oligomer library compounds or small molecule library compounds capable of specifically interacting with a selected biological agent, such as a biomolecule, macromolecular complex or cell, are The screening is performed using a combinatorial library device that is easily selected by those skilled in the art from the scope of known methods as described above. In such a method, each member of the library is screened for the ability to specifically interact with the selected agent. In practicing this method, biological agents are collected in tubes containing compounds and are allowed to interact with individual library compounds within 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 function-based or non-function-based method for detecting a substance.

  In addition to screening for molecules that mimic the activity of sphingosine kinase and / or sphingosine-1-phosphate, up- or down-regulate the functional activity of sphingosine kinase and / or sphingosine-1-phosphate with respect to the regulation of EGFR-mediated cell proliferation In order to do so, it may also be desirable to identify and utilize molecules that function agonistically or most preferably antagonistically to sphingosine kinase and / or sphingosine-1-phosphate. The use of such molecules is described in more detail below. So long as the molecule of interest is proteinaceous, the molecule may be derived from natural or recombinant sources, including, for example, a fusion protein, or may follow, for example, the screening methods described above. Non-proteinaceous molecules may be, for example, chemical or synthetic molecules identified or generated according to the methodologies specified above. Thus, the present invention contemplates the use of chemical analogs of sphingosine kinase and / or sphingosine-1-phosphate that can act as agonists or antagonists. Chemical agonists are not necessarily 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 physicochemical properties of sphingosine kinase and / or sphingosine-1-phosphate. An antagonist may be any compound that can block, inhibit or otherwise prevent the normal biological function of sphingosine kinase and / or sphingosine-1-phosphate. Antagonists include monoclonal antibodies specific for sphingosine kinase and / or sphingosine-1-phosphate, or portions of sphingosine kinase and / or sphingosine-1-phosphate.

  Analogs of agonists or antagonists of sphingosine kinase and / or sphingosine-1-phosphate or sphingosine kinase and / or sphingosine-1-phosphate contemplated herein include, but are not limited to, side chains Modification, the insertion of unnatural amino acids and / or their derivatives during peptide, polypeptide or protein synthesis, and the use of crosslinkers and other methods impose structural constraints on their analogs. The particular form that such modifications can take depends on whether the molecule of interest is proteinaceous or non-proteinaceous. The nature and / or suitability of a particular modification can be routinely measured by those skilled in the art.

For example, examples of side chain modifications contemplated by the present invention include reductive alkylation that reacts with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidate; acylation with acetic anhydride; cyanide Carbamoylation of amino groups using acid salts; Trinitrobenzylation of amino groups using 2,4,6-trinitrobenzenesulfonic acid (TNBS); Acylation of amino groups using succinic anhydride and tetrahydrophthalic anhydride And modification of the amino group such as pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ .

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

  Carboxyl groups may be modified by carbodiimide activation via formation of O-acylisourea followed by derivatization to the corresponding amide, for example.

  Sulfhydryl groups can be carboxymethylated with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides Formation of mercury derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercury compounds; carbamoyl using cyanate at alkaline pH It can be modified by a method such as

  Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide, or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or halogenated sulfenyl. On the other hand, tyrosine residues may be modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

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

  Examples of the introduction of unnatural amino acids and derivatives during protein synthesis are norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenyl This includes, but is not limited to, the use of glycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1.

Crosslinking agents include, for example, bifunctional imide esters having a (CH ₂ ) _n spacer group where n = 1 to n = 6, glutaraldehyde, homobifunctional crosslinking agents such as N-hydroxysuccinimide ester, and N— Heterobifunctional reagents that usually contain an amino reactive moiety such as hydroxysuccinimide and a reactive moiety specific for another group can be used to stabilize the 3D conformation.

  The method of the invention contemplates modulation of EGFR-mediated cellular activity, particularly cell proliferation, both in vitro and in vivo. A preferred method is to treat an individual in vivo, but nevertheless the method of the present invention is an in vitro model for the analysis of cell proliferation, such as uncontrolled cell proliferation, in an in vitro environment. It should be understood that it may be desirable to be applied to provide In another example, the application of the method of the invention to an in vitro environment may be extended to provide a readout mechanism for screening techniques such as those described above. That is, molecules identified utilizing these screening techniques can be evaluated to observe the extent and / or nature of their functional effects on hyperglycemia-induced endothelial cell function.

  A preferred method is to down-regulate EGFR-mediated cell growth, particularly agonist-induced EGFR-mediated cell growth (eg, to down-regulate the progression of uncontrolled cell growth), but subject function It should be understood that there may be circumstances where it is desired to up-regulate activity, for example, to promote the occurrence of rapid but controlled cell growth.

  In a related aspect, as detailed above, the present invention is applied to mammals to modulate EGFR-mediated functional activity in vivo.

  A further aspect of the invention relates to the use of the invention in connection with the treatment and / or prevention of disease states. Without limiting the present invention to any one theory or mode of action, the development of new methods to target cancer is indispensable, with an increasing incidence of cancer (especially breast cancer) in society. . In particular, the diversity of cancers in terms of the mechanisms underlying cancer initiation is not yet fully understood, but is now widely recognized. Thus, the development of a series of methods that can more narrowly target abnormal populations of cells has progressed significantly compared to traditional treatment methods that have directed arbitrarily rapidly dividing cells to death. I will provide a. The method of the present invention significantly contributes to this purpose in facilitating a means to down-regulate undesirable agonist-induced EGFR-mediated cellular functional activity, which is agonist-induced EGFR-mediated undesirable cells Of particular importance in the context of proliferation. Thus, the methods of the invention are particularly useful for use in the treatment of primary and secondary malignancies such as those associated with solid tumors of the breast, colon, stomach, lung, brain, bone, esophagus and pancreas. However, you are not limited to them. A preferred method is to down-regulate uncontrolled cell growth in the subject, but up-regulation of cell growth is also in certain circumstances that promote wound healing, angiogenesis or other healing processes. May be expected.

  Accordingly, yet another aspect of the present invention relates to a method of treating and / or preventing a condition characterized by abnormal, undesirable or other inappropriate EGFR-mediated cellular activity, the method comprising sphingosine in a cell Administering an effective amount of the agent to the mammal for a time and under conditions sufficient to modulate the function of kinase-mediated signaling, and downregulating said sphingosine kinase signaling to cause EGFR-mediated intracellular Down-regulate signaling.

  More specifically, provided are methods for treating and / or preventing conditions characterized by abnormal, undesirable or otherwise inappropriate agonist-induced EGFR-mediated cellular activity, comprising: Agonist induction by down-regulating the sphingosine kinase signaling comprising administering to the mammal an effective amount of the agent for a time and under conditions sufficient to modulate the function of sphingosine kinase-mediated signaling in the cell Down-regulates sex-mediated EGFR-mediated intracellular signaling.

  Even more specifically, a method of treating and / or preventing a condition characterized by abnormal, undesirable or otherwise inappropriate agonist-induced EGFR-mediated cell proliferation is provided, the method comprising: Administering an effective amount of the agent to the mammal for a time and under conditions sufficient to modulate the function of sphingosine kinase-mediated signaling in the cell, and down-regulating said sphingosine kinase signaling Down-regulate mediated intracellular signaling.

  “Abnormal, undesirable, or otherwise inappropriate” cell growth is overactive cell growth, physiologically normal cell growth that is inappropriate in unnecessary, or insufficient cells It should be understood as a reference to proliferation. Preferably, the inappropriate cell growth is uncontrolled cell growth.

  Preferably, the cell growth is uncontrolled neoplastic cell growth. Most preferably, the malignant cells are from breast, colon, stomach, lung, brain, bone, esophagus, pancreas, ovary, or uterus.

  According to these aspects of the invention, abnormal EGFR-mediated cell proliferation is agonist-induced.

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

  Thus, preferably, a method is provided for treating and / or preventing estrogen-induced breast cell malignancies, which method has sufficient time to down-regulate the function of sphingosine kinase-mediated signaling in the cell. And administering an effective amount of an agent to the mammal under conditions to downregulate EGFR-mediated intracellular signaling by downregulating the sphingosine kinase signaling.

  The methods of the present invention preferably facilitate reducing, slowing or otherwise inhibiting the growth of the subject. “Reduce, slow or otherwise inhibit” should be understood as a reference to inducing or promoting partial or complete inhibition of cell proliferation.

  Subjects for treatment or prevention are generally humans, primates, livestock animals (eg sheep, cows, horses, donkeys, pigs), pets (eg dogs, cats), laboratory test animals (eg Mammals such as, but not limited to, mice, rabbits, rats, guinea pigs, hamsters), captured wild animals (eg, foxes, deer) and the like. Preferably the mammal is a human or primate. Most preferably, the mammal is a human. Although the present invention is illustrated utilizing a mouse model, this is not intended to be limiting with respect to application to other species of the method of the present invention, particularly humans.

  As used herein, “treatment” and “prevention” should be considered in the broadest context. The term “treatment” does not necessarily imply that a mammal is treated until total recovery. Similarly, “prevention” does not necessarily mean that the subject will not eventually contract a disease state. Thus, treatment and prevention includes ameliorating the symptoms of a particular condition, or preventing or otherwise reducing the risk of developing a particular condition. The term “prevention” may be considered as reducing the severity or onset of a particular condition. “Treatment” may also reduce the current severity.

  Administration of an agent (including sphingosine kinase or a functional derivative or mimetic thereof, or sphingosine kinase nucleic acid molecule) [referred to herein as a “modulating agent”] is in any convenient form in the form of a pharmaceutical composition. Can be done with good means. A pharmaceutical composition of a modulating agent is intended to exhibit therapeutic activity when administered in an amount depending on the particular case. The amount of change depends, for example, on the human or animal and the modulating agent selected. A wide range of dosages are applicable. Considering the patient, for example, from about 0.1 mg to about 1 mg of the modulating agent may be administered per kilogram body weight per day. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, divided doses can be administered daily, weekly, monthly, or at other appropriate time intervals, or the dose can be reduced proportionally as indicated by the urgency of the situation You may let them. The modulating agent may be conveniently administered by oral, intravenous (if water-soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository route or by implantation (eg, using a sustained release molecule). May be administered. The modulatory agent should be administered in the form of an acid addition salt or a pharmaceutically acceptable non-toxic salt such as, for example, a metal complex with zinc, iron, etc. (considered as a salt for purposes of this application) Can do. Examples of such acid addition salts include hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, Examples include ascorbate and tartrate. If the active ingredient must be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrant such as alginic acid; and a lubricant such as magnesium stearate.

  The administration routes include respiratory administration, intratracheal administration, nasopharyngeal administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intracranial administration, intradermal administration, intramuscular administration, intraocular administration, intrathecal administration, cerebrum This includes but is not limited to intracereberally, intranasal, infusion, oral, rectal, IV infusion patches and transplants.

  In another aspect, the present invention relates to sphingosine kinase mediated in the manufacture of a medicament for the treatment and / or prevention of conditions characterized by abnormal, undesirable or otherwise inappropriate EGFR mediated cellular activity. With respect to the use of agents that can modulate the functional activity of sexual signaling, EGFR-mediated intracellular signaling is downregulated by downregulating the sphingosine kinase signaling.

  More specifically, the cellular activity is cell proliferation.

  Preferably, the cell growth is uncontrolled neoplastic cell growth, and even more preferably malignant cell growth. Most preferably, the malignant cells are from breast, colon, stomach, lung, brain, bone, esophagus, pancreas, ovary, or uterus.

  In accordance with these aspects of the invention, abnormal EGFR-mediated cell proliferation is agonist-induced.

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

  In yet another aspect, the present invention relates to a pharmaceutical composition comprising a modulating agent as defined above and one or more pharmaceutically acceptable carriers and / or diluents. A modulatory agent refers to an active ingredient.

  Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile aqueous solutions or dispersions for cream or topical application Other suitable forms may be used. 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 surfactants. Prevention of microbial action can be accomplished 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 achieved in the composition by the use of absorption delaying agents such as aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by mixing the required amount of active compound in a suitable solvent with the various other ingredients listed above and, if necessary, then filter sterilization. Generally, dispersions are prepared by mixing the various sterilized active ingredients in a sterile vehicle containing 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 method of preparation is a vacuum drying technique and freezing that yields a powder of the active ingredient plus any additional desired ingredients from the solution that have been pre-sterilized by filtration. It is a drying technique.

  If the active ingredients are adequately protected, they can be administered orally, for example, with an inert diluent or an absorbable edible carrier, or they can be enclosed in hard or soft shell gelatin capsules. Or may be squeezed into tablets or taken directly with dietary food. For therapeutic oral 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, etc. Good. Such compositions and preparations should contain at least 1% by weight of active ingredient. The proportions of the compositions and preparations can of course vary and may conveniently be from about 5 to about 80% of the unit weight. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. According to the present invention, preferred compositions or preparations are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

  Tablets, troches, pills, capsules and the like may also contain the ingredients listed below. Binders such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sucrose, lactose that may be added Or a sweetening agent such as saccharin, or a flavoring agent such as peppermint, winter green oil or cherry flavor. When the dosage unit form is a capsule, it may contain, in addition to the above types of materials, a liquid carrier. Various other materials may be present as coating agents or otherwise to 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 flavoring such as cherry or orange flavor. 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 may be incorporated into sustained-release preparations and formulations.

  The pharmaceutical composition can also include a gene molecule, such as a vector, capable of transfecting the target cells, the vector carrying a nucleic acid molecule encoding a regulatory agent. The vector may be, for example, a viral vector.

  Yet another aspect of the invention relates to a modulating agent as defined above when used in the method of the invention.

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

Example 1
Sphingosine kinase is a material and method for mediating “cross-cross” transactivation of epidermal growth factor receptor by estrogen Cell culture and transfection Human breast cancer MCF-7 cells (EFα + / β +; ATCC HTB-22) Cultured in Dulbecco's modified Eagle medium (CSL Biosciences, Parkville, Australia) without phenol red with 10% fetal bovine serum (FBS). A stably transfected MCF-7 cell line that overexpresses SphK ^WT and SphK ^G82D constructs and SphK ^WT , SphK ^G82D or empty vector alone has already been described (Pitson et al., 2000b, J Biol Chem, 275: 33945-33950; Sukocheva et al., 2003, Mol Endocrinol, 17: 2002-2012).

下記の18-merのホスホチオエートオリゴヌクレオチドは、Geneworks（Australia）によって合成された。
EDG-3アンチセンス、

EDG-3センス、

。
トランスフェクションに関しては、Lipofectamine-2000試薬（Invitrogen）を使用し、MCF-7細胞は、実験の前日に、50,000細胞/ウェルの密度で6ウェルプレートに播種した。トランスフェクションの36〜48時間後、標的遺伝子の発現レベルを、RT-PCRおよび/またはウェスタンブロットによって検出した。 Experiments with siRNA and antisense oligonucleotides
Chemically synthesized siRNA duplexes containing 3'-fluorescein modifications were purchased from Qiagen-Xeragon (Germantown, MD). The siRNA target sequence was as follows.

The following 18-mer phosphothioate oligonucleotides were synthesized by Geneworks (Australia).
EDG-3 antisense,

EDG-3 sense,

.
For transfection, Lipofectamine-2000 reagent (Invitrogen) was used and MCF-7 cells were seeded in 6-well plates at a density of 50,000 cells / well the day before the experiment. 36-48 hours after transfection, the expression level of the target gene was detected by RT-PCR and / or Western blot.

およびSphK1（アンチセンス）

SphK2（センス）

およびSphK2（アンチセンス）

Edg-3（センス）

およびEdg-3（アンチセンス）

。
増幅産物は、エチジウムブロマイドで染色された1.5%アガロース中で電気泳動を行い視覚化した。画像は、UVitec Gelドキュメンテーションシステムに記録させた。 Reverse transcriptase polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from cell cultures using TRIzol (Invitrogen). Single stranded cDNA was synthesized from 1 mg total RNA in a total volume of 20 ml using Omniscript reverse transcriptase (Qiagen) and oligo-dT primer (Geneworks). SphK1, SphK2 and EdgG-3 were amplified with a PTC-100 Programmable Thermal Controller (MJ Research, Inc) along with an internal GAPDH control. The primers used for amplification were as follows.
SphK1 (sense)

And SphK1 (antisense)

SphK2 (sense)

And SphK2 (antisense)

Edg-3 (sense)

And Edg-3 (antisense)

.
Amplified products were visualized by electrophoresis in 1.5% agarose stained with ethidium bromide. Images were recorded in a UVitec Gel documentation system.

Immunoblot analysis Cells were harvested and 50 mM Tris / HCl (pH 7.4), 10% glycerol, 0.05% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na ₃ VO ₄ , 10 mM NaF, 1 mM EDTA and protease inhibition Lysis was performed by sonication in a solubilization buffer containing the agent (Roche Molecular Biochemicals). Aliquots of cell lysate were separated by 8-12% SDS-PAGE and transferred to Hybond-P membrane (Amersham). The membrane was then examined with the appropriate antibody according to the manufacturer's standard method. Immune complexes were detected using an enhanced chemiluminescence PLUS kit (Amersham Pharmacia Biotech). Densitometry was performed on Typhoon 9410 using the Image Quant program.

Immunofluorescence microscopy analysis
MCF-7 cells were seeded on 8-well chamber slides (Lab-Tech) coated with fibronectin and cultured for 48 hours. After stimulation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated with monoclonal anti-Edg-3 (1: 100) and polyclonal anti-EGFR (1: 100) antibodies, and indirect immunofluorescence was conjugated with a fluorescent probe (Alexa Fluor ⁴⁸⁸ or Fluor ⁵⁹⁴ ). Detection was with a secondary antibody. Fluorescently stained cells were imaged by epifluorescence microscopy on an Olympus BX-51 microscope equipped with a device camera (Photometrics) coupled with a Cool Snap FX charge. Images were analyzed using V ++ software (Digital Optics Ltd. Auckland New Zealand).

SphK activity and S1P release assay
SphK activity was determined as previously described (Xia et al., 1998, Proc Natl Acad Sci USA, 95: 14196-14201), 5 mM D-erythro-sphingosine dissolved in 0.1% Triton X-100, and [γ32P ] Cell fractions were routinely measured by incubating with ATP for 30 minutes at 37 ° C. Enzyme activity was defined as the amount of S1P formation (pmol) / min / mg protein. To measure extracellular S1P release, cells were metabolically labeled with [3H] serine (5 μCi / ml) for 48 hours. After 30 minutes of E2 stimulation, the medium was collected and lipids were extracted by adding chloroform: methanol: acetic acid (10: 10: 1, v / v). [3H] S1P is then separated by thin layer chromatography and quantified by scintillation spectrometry and recovered into total extracellular lipids as previously described (Xia et al., 1998, supra). Normalized by radioactivity.

result
S1P-stimulated activation of EGFR in MCF-7 cells
The effect of S1P on MCF-7 cells was tested. FIG. 1A shows that tyrosine phosphorylation of EGFR increased in a time-dependent manner in MCF-7 cells in response to 1 mmol / L S1P. EGFR phosphorylation peaked at 10 minutes, but thereafter a decrease was still clearly seen 240 minutes after treatment. In parallel, ERF1 / 2, the major downstream signal molecule of EGFR, was also phosphorylated in a time-dependent manner similar to phosphorylation of EGFR (FIG. 1A). The concentration response of S1P showed an EC ₅₀ of about 5 nmol / L for both EGFR and ERK1 / 2 phosphorylation, with maximum phosphorylation observed at about 500 nmol / L (FIG. 1B). Taken together, these results indicate the ability of S1P to induce EGFR activation in breast cancer cells.

E2 and S1P induced EGFR transactivation through a common signaling pathway
We explored whether S1P can mimic E2 in stimulating EGFR transactivation in MCF-7 cells. E2 treatment of MCF-7 cells resulted in rapid tyrosine phosphorylation of EGFR and ERK1 / 2 activation similar to that observed in S1P treated cells (FIG. 2A). E2-induced and S1P-induced EGFR transactivation was blocked by the Gi-specific inhibitor pertussis toxin (PTX). PTX also significantly inhibited ERK1 / 2 phosphorylation in response to either E2 or S1P stimulation (FIG. 2A). In contrast, EGF-stimulated EGFR and ERK1 / 2 phosphorylation was not inhibited by PTX (FIG. 2A).

  Whether tyrosine kinase activity is required for E2-induced or S1P-induced EGFR transactivation because EGFR transactivation depends on its internal tyrosine kinase activity (Prenzel et al., 1999, supra) Analysis was performed. In the presence of AG1478, the specific EGFR tyrosine kinase inhibitor E2-induced and S1P-induced EGFR transactivation was completely abolished (FIG. 2A). The activation of ERK1 / 2 was significantly inhibited. As a control, AG1478 induces complete inhibition of EGF-stimulated EGFR phosphorylation, supporting the specific effect of AG1478 on EGFR activity in MCF-7 cells.

  Src family kinases have been suggested to play a signaling role in GPCR-mediated transactivation of EGFR (Gschwind et al., 2001, supra). To evaluate the role of Src, cells were pretreated with PP2 (100 nmol / L), an Src inhibitor, for 30 minutes before stimulation. As shown in FIG. 2A, both EGFR transactivation stimulated with E2 and S1P was significantly inhibited by PP2 treatment.

  Depletion of heparin-binding EGF (HB-EGF) by matrix metalloproteinase (MMP) activity is also recognized as an important mechanism for mediating EGFR transactivation by GPCR ligands (Prenzel et al., 1999, supra). Therefore, we analyzed whether HB-EGF shedding was involved in E2-induced or S1P-induced EGFR transactivation. First, MCF-7 cells were subjected to an acid wash step to reduce irritation due to background autocrine, and then pretreated with MMP inhibitors o-phenanthroline or GM6001. E2-induced and S1P-induced EGFR transactivation and ERK1 / 2 activation were blocked by these two MMP inhibitors (FIG. 2B). In contrast, MMP inhibitors did not affect EGFR's own intrinsic kinase activity and EGF's ability to induce EGFR autophosphorylation. Furthermore, depletion of HB-EGF products from cultures by EGF neutralizing antibodies resulted in significant inhibition of E2-induced and S1P-induced EGFR transactivation (FIG. 2B). The efficiency and specificity of the neutralizing antibody was shown by inhibition of EGFR tyrosine phosphorylation stimulated by EGF. These data further suggest that HB-EGF shedding and E2 or S1P treated cells are involved in the process of EGFR transactivation.

SphK1 activation is involved in E2-induced EGFR transactivation
Since S1P could mimic the effects of E2-stimulated EGFR transactivation and E2 could stimulate S1P production by SphK1 activation, E2-induced EGFR transactivation activated SphK1 It was postulated that it could be mediated through. To test this hypothesis, we used stably transfected MCF-7 cell lines that overexpress wild-type SphK1 (SphK1 ^WT ), dominant negative SphK1 (SphK1 ^G82D ), or empty vector alone, respectively. It has previously been shown that basal SphK activity in cells transfected with SphK1 ^WT is approximately 10-fold higher than control cells (Sukocheva et al., 2003, supra). E2 stimulation resulted in a rapid increase in SphK activity that was approximately 2-fold above basal levels in both SphK1 ^WT transfected cells and control MCF-7 cells. In contrast, cells transfected with SphK1 ^G82D showed basal SphK activity similar to control cells, but E2-stimulated SphK activity disappeared completely. Interestingly, tyrosine phosphorylation of EGFR and ERK1 / 2 activation stimulated by E2 increased in cells transfected with SphK1 ^WT , but the stimulatory effect of E2 disappeared in SphK1 ^G82D transfectants ( Figure 3A). Significant differences between these transfected cell lines in either total EGFR expression levels measured by Western blot analysis (Figure 3A), or cell surface expression levels of EGFR measured by flow cytometry (Figure 3B) Absent. In contrast, neither EGF nor S1P stimulated EGFR phosphorylation was significantly affected by SphK1 ^G82D . Thus, these results suggest a specific role for SphK activity in E2-induced EGFR transactivation.

Two mammalian SphK isoforms (ie, SphK1 and SphK2) have been cloned, both isoforms accounting for the overall cellular SphK activity (Kohama et al., 1998, J Biol Chem, 273: 23722-23728; Liu et al., 1999, Mol Biol Cell, 10: 1179-1190). Knock down each SphK isoform gene expression in MCF-7 cells using siRNA strategies to identify which isoforms (or both) are involved in EGFR transactivation and a role for endogenous SphK Brought it down. Treatment of cells with SphK1-specific siRNA reduced SphK1 mRNA to 86 ± 11% and inhibited basal SphK activity to 48 ± 12% compared to cells treated with scrambled siRNA (Fig. 4A). Furthermore, the increased SphK activity in response to E2, EGF or S1P was almost completely lost by SphK1 siRNA (FIG. 4B). In conclusion, SphK1 siRNA induced E2-induced EGFR tyrosine phosphorylation and significant inhibition of ERK1 / 2 activity to the same extent as shown in cells transfected with SphK1 ^G82D (FIG. 4C). Again, in ^line with observations of experiments with SphK1 ^G82D , EGF and S1P-induced activation of EGFR and ERK1 / 2 was not significantly affected by SphK1 siRNA. Interestingly, SphK2-specific siRNA effectively knocked down SphK2 expression and inhibited basal SphK activity by about 50%, but the extent of SphK activity increase induced by E2, EGF or S1P Did not change (FIGS. 4A and B). Similarly, SphK2 siRNA had no significant inhibitory effect on EGFR tyrosine phosphorylation or ERK1 / 2 activation in response to E2, S1P or EGF stimulation (FIG. 4C). Taken together, these data suggest a critical role for SphK1, but not SphK2, in mediating EGFR transactivation by E2 stimulation.

The S1P receptor, Edg-3, is required for SphK1-dependent transactivation of EGFR by E2
Exploration was undertaken to determine the role of S1P and its receptor in SphK1-dependent EGFR transactivation by E2. The analysis was first directed to whether S1P is released by SphK1 activation in response to E2 stimulation in cells. As shown in FIG. 5A, S1P levels were significantly higher (172 ± 22%) in conditioned medium (CM) recovered from E2-stimulated MCF-7 cells than untreated cells. Furthermore, S1P levels increased up to approximately 5-fold in CM from cells transfected with SphK1 ^WT at both basal and stimulated levels compared to control MCF-7 cells. In CM from cells transfected with SphK1 ^G82D , no increase in S1P levels was detected after E2 stimulation (Figure 5A), indicating that SphK1 activation results in production of S1P and release from cells stimulated with E2. Show. Similarly, CM from cells treated with E2 showed a substantially greater ability to stimulate EGFR tyrosine phosphorylation compared to CM from untreated cells (FIG. 5B). Furthermore, CM from cells transfected with SphK1 ^WT treated with E2 had further stimulatory effect on tyrosine phosphorylation of EGFR. In contrast, we were unable to detect any stimulation of EGFR phosphorylation in CM from cells transfected with Eph-treated SphK1 ^G82D , indicating that cellular SphK activity or S1P release was It suggests that it leads to CM-induced EGFR activation. In addition, CM-induced EGFR activation is completely blocked by PTX (FIG. 5B) and further supports the critical involvement of S1P and its GPCR in the E2-stimulated transactivation pathway.

  To evaluate the potential role of Edg-3 in E2 signaling, the effect of E2 on Edg-3 activation was directly tested. Most other GPCRs like Edg-3 undergo internalization following ligand binding and receptor activation (Liu et al., 1999, supra). Therefore, Edg-3 internalization was assessed by immunofluorescence microscopy analysis. The results in FIG. 6 show that in MCF-7 cells, Edg-3 was rapidly internalized after 2 minutes of stimulation with E2 (10 nmol / L) and remained there for about 30 minutes. As a control, treatment of cells with 100 nmol / L S1P induced a rapid and transient Edg-3 internalization in the same pattern observed after E2 stimulation. To further solidify evidence for Edg-3 involvement in E2-mediated EGFR transactivation, an antisense strategy was used to knock down endogenous Edg-3 expression. Cells transfected with Edg-3 antisense oligonucleotide reduce Edg-3 mRNA and protein levels to 61 ± 17% and 68 ± 12%, respectively, compared to Edg-3 sense transfectants (Figs. 7A and B). Notably, Edg-3 antisense induced significant inhibition of EGFR tyrosine phosphorylation in cells in response to either E2 or S1P stimulation, while EGF-induced EGFR autophosphorylation was retained (Figure 7B). Taken together, these results suggest a critical role for the S1P receptor Edg-3 in mediating E2 signaling that transactivates 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 should be understood that the invention includes all such variations and modifications. The invention also includes, individually or collectively, all of the steps, features, compositions and compounds mentioned or indicated herein, and any combination of any two or more steps or features and all. Includes combinations.

List of references

It is an image showing that S1P transactivates EGFR. MCF-7 cells were stimulated with (A) 1 mmol / L S1P for a predetermined time or (B) S1P with increasing concentration for 15 minutes. Cell lysates were subjected to SDS-PAGE and phosphorylation of EGFR and ERK1 / 2 was analyzed with phosphate specific antibodies. The bar graph shows three combined experimental values expressed as a percentage of the control value (mean ± SD). Activity density values were normalized by total protein level. FIG. 3 is an image showing that S1P mimics E2 that induces EGFR transactivation in MCF-7 cells. Serum-depleted MCF-7 cells are pre-cultured (A) with PTX (100 ng / ml) for 16 hours or AG1478 (50 mmol / L) and PP2 (100 nmol / L) for 1 hour, or (B) Pretreatment with MMP inhibitory phenanthroline (20 mmol / L), GM6001 (50 nmol / L) or EGF neutralizing antibody (15 mg / ml) for 1 hour, then 10 nmol / L E2, 1 mmol / L S1P or 25 ng / ml EGF Stimulated for 15 minutes. Cell lysates were then analyzed by Western blotting and the extent of phosphorylated EGFR and ERK1 / 2 was quantified as in FIG. Data are mean ± SD. ^* P <0.01; † p <0.05, treated vs. untreated. It is an image showing the effect of SphK activity on E2-induced EGFR transactivation. (A) SphKl ^WT, the SphKl ^G82D or vector alone overexpression stably transfected MCF-7 cells that were stimulated 15 minutes with 10nmol / L E2,1mmol / L S1P or 25 ng / ml EGF. Cell lysates were then analyzed by Western blotting and the extent of phosphorylated EGFR and ERK1 / 2 was quantified as in FIG. Data are mean ± SD. ^* P <0.01; † p <0.05, SphK1 ^WT or SphK1 ^G82D versus vector alone. (B) Flow cytometry profile shows the cell surface expression level of EGFR in the transfected MCF-7 cell line. It is an image showing that SphK1 but not SphK2 leads to E2-induced EGFR transactivation. MCF-7 cells were transfected with siRNA specific for SphK1, SphK2 or scrambled control RNA as described in “Materials and Methods”. 24 hours after transfection, the following experiments were performed: (A) SphK1 and SphK2 mRNA levels were determined by RT-PCR and quantified by ratio to GAPDH mRNA levels. (B) MRNA-7 cells transfected with siRNA were stimulated with 10 nmol / L E2, 1 mmol / L S1P or 25 ng / ml EGF for 15 minutes. Next, SphK activity was assessed and (C) phosphorylated EGFR and ERK1 / 2 levels were analyzed as in FIG. Data are mean ± SD. ^* P <0.01; † p <0.05, SphK1 or SphK2 siRNA versus control. FIG. 2 is an image showing that E2-induced EGFR transactivation is mediated by S1P release from MCF-7 cells. The (A) extracellular S1P levels in MCF-7 cells transfected overexpressing SphKl ^WT, SphKl ^G82D or vector alone was determined after stimulation for 15 minutes at 10 nmol / L E2. Data are mean ± SD. (B) MCF-7 cells were stimulated for 15 minutes with conditioned medium from transfected MCF-7 cells pretreated with or without PTX for 16 hours and treated with or without E2. . Next, the level of phosphorylated EGFR was determined as in FIG. It is an image of Edg-3 internalization of E2 stimulation in MCF-7 cells. After stimulation with 10 nmol / L estrogen or 100 nmol / L S1P for 2 or 30 minutes, MCF-7 cells were immunofluorescently stained with antibodies against Edg-3 (upper panel) or EGFR (middle panel). Images of Edg-3 (red) and EGFR (green) staining were superimposed on the lower panel. It is an image showing the effect of Edg-3 on E2-induced EGFR transactivation. MCF-7 cells were transfected with Edg-3 antisense or sense as described in “Materials and Methods”. The following studies were performed 24 hours after transfection: (A) Edg-3 mRNA levels were measured by RT-PCR and quantified by ratio to GAPDH mRNA levels. (B) Cells transfected with Edg-3 sense or antisense were stimulated with 10 nmol / L E2, 1 mmol / L S1P or 25 ng / ml EGF for 15 minutes, and the extent of phosphorylated EGFR and ERK1 / 2 is Analysis was performed as shown in FIG. Data are mean ± SD. ^* P <0.01; † p <0.05, Edg-3 antisense vs. sense. 1 is a schematic diagram showing a model for E2-induced EGFR transactivation through activation of the SphK1 / S1P signaling pathway.

Claims (44)

  A method of modulating EGFR-mediated cellular activity, comprising modulating sphingosine kinase-mediated signaling function in a cell, wherein EGFR-mediated intracellular signaling is downregulated by down-regulating sphingosine kinase signaling The way to control down. Abnormal, undesirable or otherwise in a mammal, comprising administering to the mammal an effective amount of the agent for a time and under conditions sufficient to modulate the function of sphingosine kinase-mediated signaling in the cell A method for the treatment and / or prevention of a condition characterized by inappropriate EGFR-mediated cellular activity comprising:
A method wherein the EGFR-mediated intracellular signaling is downregulated here by downregulating sphingosine kinase signaling.   The method according to claim 1 or 2, wherein the sphingosine kinase is human sphingosine kinase.   4. The method according to claim 3, wherein the sphingosine kinase is sphingosine kinase 1 or 2.   5. The method according to any one of claims 1 to 4, wherein the EGFR-mediated cellular activity is agonist-induced EGFR-mediated cellular activity.   6. The method of claim 5, wherein the agonist-induced EGFR-mediated cell functional activity is cell proliferation.   7. The method of claim 6, wherein the cell proliferation is neoplastic cell proliferation.   8. The method of claim 7, wherein the neoplastic cell is a malignant cell derived from breast, colon, stomach, lung, brain, bone, esophagus, pancreas, ovary, or uterus.   9. The method of claim 8, wherein the neoplastic cell is a malignant breast cell or an anti-estrogen resistant malignant breast cell.   10. The method according to any one of claims 5 to 9, wherein the agonist is estrogen.   The regulation is upregulation of sphingosine kinase activity level and the upregulation is sphingosine kinase or a functional equivalent, derivative or homolog, or sphingosine kinase expression product or functional derivative, homolog, analog, equivalent Alternatively, the method according to claim 1 or 2, which is achieved by introducing a nucleic acid molecule encoding a mimetic into a cell.   The modulation is upregulation of sphingosine kinase activity levels, and the upregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an agonist of the sphingosine kinase expression product, The method according to claim 1 or 2.   The modulation is down-regulation of sphingosine kinase activity level, and the down-regulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an antagonist of the sphingosine kinase expression product, The method according to any one of claims 1 to 10.   14. The method of claim 13, wherein the antagonist is a sphingosine kinase substrate competitor.   15. A method according to claim 14, wherein the substrate is ATP.   15. A method according to claim 14, wherein the substrate is sphingosine.   17. The method of claim 16, wherein the agent is N, N-dimethylsphingosine or DL-threo-dihydrosphingosine. ^17. The method of claim 16, wherein the agent is a sphingosine kinase dominant negative such as SphK ^G82D .   14. The method of claim 13, wherein the antagonist downregulates sphingosine kinase enzyme activation.   20. The method of claim 19, wherein the agent alters sphingosine kinase related phosphorylation events.   20. The method of claim 19, wherein the agent alters sphingosine kinase lipid composition. Functional activity of sphingosine kinase-mediated signaling in the manufacture of a medicament for the treatment and / or prevention of conditions characterized by abnormal, undesirable or otherwise inappropriate EGFR-mediated cellular activity in mammals Use of an agent capable of regulating
Use herein wherein the EGFR-mediated intracellular signaling is downregulated by downregulating the sphingosine kinase signaling.   23. Use according to claim 22, wherein the sphingosine kinase is human sphingosine kinase.   24. Use according to claim 23, wherein the sphingosine kinase is sphingosine 1 or 2.   25. Use according to any one of claims 22 to 24, wherein the inappropriate EGFR-mediated cellular activity is agonist-induced EGFR-mediated cellular activity.   26. Use according to claim 25, wherein the inappropriate cellular activity is cell proliferation.   27. Use according to claim 26, wherein the cell proliferation is uncontrolled neoplastic cell proliferation.   28. Use according to claim 27, wherein the uncontrolled neoplastic cell proliferation is malignant cell proliferation.   29. Use according to claim 28, wherein the malignant cells are derived from breast, colon, stomach, lung, brain, bone, esophagus, pancreas, ovary or uterus.   30. Use according to any one of claims 25 to 29, wherein the agonist is estrogen.   31. Use according to any one of claims 22 to 30, wherein the condition is an estrogen-induced breast cell malignancy or an anti-estrogen resistant breast cell malignancy.   The regulation is upregulation of sphingosine kinase activity level and the upregulation is sphingosine kinase or a functional equivalent, derivative or homolog, or sphingosine kinase expression product or functional derivative, homolog, analog, equivalent 23. Use according to claim 22, which is achieved alternatively by introducing into the cell a nucleic acid molecule encoding the mimetic.   The modulation is upregulation of sphingosine kinase activity levels, and the upregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an agonist of the sphingosine kinase expression product, 23. Use according to claim 22.   The modulation is down-regulation of sphingosine kinase activity level, and the down-regulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an antagonist of the sphingosine kinase expression product, 32. Use according to any one of claims 22 to 31.   35. Use according to claim 34, wherein the antagonist is a sphingosine kinase substrate competitor.   36. Use according to claim 35, wherein the substrate is ATP.   36. Use according to claim 35, wherein the substrate is sphingosine.   38. Use according to claim 37, wherein the agent is N, N-dimethylsphingosine or DL-threo-dihydrosphingosine. ^38. Use according to claim 37, ^{wherein the} agent is a sphingosine kinase dominant negative such as SphK ^G82D .   35. Use according to claim 34, wherein the antagonist down regulates sphingosine kinase enzyme activation.   35. Use according to claim 34, wherein the agent alters sphingosine kinase related phosphorylation events.   35. Use according to claim 34, wherein the agent alters sphingosine kinase lipid composition.   A modulatory agent capable of modulating EGFR-mediated cellular activity, and one or more pharmaceutically acceptable carriers and / or when used according to the method of any one of claims 1-21 A pharmaceutical composition comprising a diluent.   An agent that modulates EGFR-mediated cellular functional activity when used according to any one of claims 1-21.

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