WO2007056604A2 - Procédés et compositions servant à moduler la motilité cellulaire et à inhiber la métastase de tumeurs - Google Patents

Procédés et compositions servant à moduler la motilité cellulaire et à inhiber la métastase de tumeurs Download PDF

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WO2007056604A2
WO2007056604A2 PCT/US2006/044027 US2006044027W WO2007056604A2 WO 2007056604 A2 WO2007056604 A2 WO 2007056604A2 US 2006044027 W US2006044027 W US 2006044027W WO 2007056604 A2 WO2007056604 A2 WO 2007056604A2
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migration
pro
cell
molecule
tumor cell
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WO2007056604A3 (fr
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Garret M. Hampton
Jiyong Hong
Cynthia Collins
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IRM LLC
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IRM LLC
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the present invention generally relates to methods for identifying modulators of cell motility, and to therapeutic applications of such modulators. More particularly, the invention pertains to genes that play a role in promoting metastasis of tumor cells, and to methods of using such genes to identify novel compounds that inhibit tumor metastasis.
  • Cell migration is a fundamental biological process, necessary for the spatial distribution of developing cell types and tissues, wound healing, blood vessel development, immune responses and renewal of cell layers in tissues such as the skin, esophagus and colorectum.
  • the movements that constitute cell migration are complex, requiring the integration and transduction of diverse signaling cues with the mechanical processes of cell movement.
  • Tumor cell motility is a critical component of invasion and metastasis, but the regulation of this motility is still poorly understood.
  • the process of tumor metastasis is a multistage event involving local invasion and destruction of the extracellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location (Fidler et al., Adv. Cancer Res. 28:149-250, 1978; Liotta, et al., Cancer Treatment Res. 40:223-238, 1988; Nicolson, Biochim. Biophy. Acta 948:175- 224, 1988; and Zetter, N. Eng. J. Med. 322:605-612, 1990).
  • the invention provides methods for identifying an agent that modulates motility of a cell.
  • the methods involve (a) screening test compounds to identify modulating compounds which modulate a pro-migration molecule selected from the group consisting of MAP4K4, CDK7, FGFRl, DYRKlB and SERPINB3; and (b) testing the identified modulating compounds for ability to modulate motility of the cell.
  • the test compounds are screened for ability to modulate cellular level of the pro-migration molecule.
  • Some of the methods employ MAP4K4, CDK7, FGFRl, or DYRKlB, and the test compounds are screened for ability to modulate the kinase activity of the pro-migration molecule.
  • Some of the methods employ SERPINB3, and the test compounds are screened for ability to modulate the proteinase-inhibiting activity of SERPINB3.
  • Some of the methods are directed to identifying agents that inhibit motility of a tumor cell.
  • the invention provides methods for identifying agents that inhibit tumor metastasis. These methods entail (a) screening test compounds to identify modulating compounds which down-regulate a pro-migration molecule selected from the group consisting of MAP4K4, CDK7, FGFRl, DYRKlB and SERPINB3, and (b) testing the identified modulating compounds for ability to inhibit migration of a tumor cell. Some of the methods further involve testing the identified modulating compounds for ability to modulate migration of a non-tumor control cell. In some of the methods, the test compounds are screened for ability to down-regulate cellular level of the pro-migration molecule.
  • the methods employ MAP4K4, CDK7, FGFRl 5 or DYRKlB as the screening target, and the test compounds are screened for ability to inhibit the kinase activity of the pro-migration molecule.
  • Some other methods employ SERPINB3 as the screening target, and the test compounds are screened for ability to inhibit the proteinase-inhibiting activity of SERPINB3.
  • the employed pro-migration molecule is MAP4K4, CDK7, or FGFRl
  • the tumor cell is selected from the group consisting of an ovarian tumor cell, a breast tumor cell, a melanoma tumor cell, and a prostate tumor cell.
  • the employed pro-migration molecule is SERPINB3, and the tumor cell is selected from the group consisting of an ovarian tumor cell, a melanoma tumor cell, and a prostate tumor cell.
  • the pro-migration molecule employed is DYRKlB, and the tumor cell is selected from the group consisting of an ovarian tumor cell and a prostate tumor cell.
  • (b) entails comparing migration of a cultured tumor cell in the presence of a modulating compound to migration of the tumor cell in the absence of the modulating compound.
  • a significant inhibition of migration of the tumor cell in the presence of the modulating compound relative to migration of the tumor cell in the absence of the modulating compound identifies the modulating compound as an agent that inhibits tumor metastasis.
  • migration of the tumor cell is examined with a matrigel Boyden chamber assay.
  • migration of the tumor cell is examined by monitoring closure of a scratch in a culture of the tumor cell.
  • the invention provides methods for inhibiting tumor invasion and metastasis in a subject. These methods involve administering to the subject a pharmaceutical composition that contains an effective amount of an agent which down-regulates a pro-migration molecule.
  • the pro-migration molecule is selected from the group consisting of MAP4K4, CDK7, FGFRl, DYRKlB and SERPINB3.
  • Some of the methods are directed to inhibit metastasis of a tumor selected from the group consisting of an ovarian tumor, a breast tumor, a melanoma tumor, and a prostate tumor.
  • the employed agent down-regulates expression of a gene encoding the pro- migration molecule.
  • the agent used in these methods can be, e.g., an short interfering ElNA (siRNA), a microRNA (miRNA), and a synthetic hairpin RNA (shRNA), an anti-sense nucleic acid, or a complementary DNA (cDNA).
  • the employed agent is an antagonist antibody that specifically binds to the pro-migration molecule.
  • Some of these methods employ an antibody which specifically inhibits the kinase activity of MAP4K4, CDK7, FGFRl, or DYRKlB.
  • Some other methods employ an antibody which specifically inhibits the proteinase-inhibiting activity of SERPINB 3.
  • the antagonist antibody employed in these methods is a monoclonal antibody.
  • FIG. 1A-1C show the validation of the automated cell motility assay.
  • FIG. IA shows the temporal (0, 4, 8, 12 and 16hrs) migration of SKOV-3 cells in the presence and absence of controls.
  • siCON FITC-conjugated control siRNA
  • siRAC a sequence-specific siRNA targeting RAC
  • DMSO dimethyl sulfoxide
  • SRC c-Src family kinase inhibitor, Compound 43;
  • Fig. IB displays quantification of SKOV-3 migration. The degree of scratch closure ("migration score”) was determined by an automated algorithm, and plotted as a function of time. Low migration scores reflect migratory inhibition;
  • Fig. IA shows the temporal (0, 4, 8, 12 and 16hrs) migration of SKOV-3 cells in the presence and absence of controls.
  • siCON FITC-conjugated control siRNA
  • siRAC a sequence-specific siRNA targeting RAC
  • DMSO dimethyl sulfoxide
  • SRC c-Sr
  • FIG. 1C shows western blot demonstrating knock-down of the RAC protein by the RAC-specific siRNA used in Fig. IA, compared to a control siRNA (CON) and mock transfected cells (LIPO). The same blot was re-probed with anti-actin antibody to demonstrate equal loading.
  • Figure 2 schematically illustrates the SKOV-3 siRNA screen and the associated follow-up studies.
  • Figures 3A-3B show the identification and validation of pro- migration genes.
  • Fig. 3 A demonstrates validation of migratory inhibition by phenotypic and transcriptional analysis.
  • RT-PCR analysis is shown for each transcript, and the relative transcriptional knockdown was quantified using ImageJ software (from the NIH, Bethesda, MD); Fig.
  • 3B shows that four unique siRNA duplexes (three from the primary screen and one additional sequence) have similar effects on the migration of SKO V-3 cells, reducing motility by up to 75%, with a corresponding knockdown of MAP4K4 transcript, ranging from 64 to 94%.
  • Figure 4 shows that inhibition of MAP4K4 affects the motility of multiple carcinoma cell lines. Shown are the affects of the two most potent MAP4K4 siRNAs on the motility of SKOV-3, MDA-MB-231 (MDA-231), DU145, ES-2 and A2058 cell lines. A graphical representation of migratory inhibition relative to control siRNA is shown above an RT-PCR analysis of the MAP4K4 transcript in each of the cell lines.
  • Figure 5 shows that down-regulation of MAP4K4 decreases SKOV-3 cell invasion.
  • Transiently transfected cells were subjected to Boyden chamber assay of cell invasion through matrigel. The data were collected from 5 individual consecutive fields of view of each of 4 invasion chambers. Representative photomicrographs are shown beneath the graph.
  • Figure 6 displays phosphor- Western Blot graphs showing effect of siRNA knockdown of MAP4K4 on phosphorylation level of JNK, p38 or Erk MAP kinases.
  • Figure 7 shows inhibition by a JNK inhibitor of the scratch-closing ability of the SKOV-3 cell. Viability of the cells treated with the JNK inhibitor at different concentration, measured as a percentage of viability of control cells treated with DMSO, is also indicated in the figure. The viability data were the average from cells in quadruplicate wells.
  • Figures 8A-8B show that inhibition of FGFRl decreases SKOV-3 cell motility.
  • Fig. 8A depicts the quantitative results of FGFRl knockdown by 5 independent siRNAs, as compared to control siRNA transfected cells. RT-PCR was performed to assess transcriptional down-regulation, and quantified using ImageJ;
  • Fig. 8B demonstrates the effect of FGFRl inhibition on SKOV-3 cell invasion through matrigel. The data on migrating cell number was calculated as described for Figure 5.
  • FIG. 9 shows that inhibition of FGFRl affects the motility of multiple carcinoma cell lines. Shown in the figure are the effects of the two most potent FGFRl siRNAs on the motility of SKOV-3, MDA-MB-231 (MDA-231), DU145, ES-2 and A2058 cell lines. A graphical representation of migratory inhibition relative to control siRNA is shown above RT-PCR analysis of the MAP4K4 transcript in each of the cell lines.
  • Figures 10A-10B illustrate validation of the effects of FGFRl knockdown using small molecule inhibitors of the FGFRl kinase. Fig.
  • FIG. 1OA shows the quantitative effects of the FGFRl inhibitor, PD173074, the c-src inhibitor (compound 43) and a pan- kinase inhibitor, staurosporine.
  • This invention is predicated in part on the discoveries by the present inventors that several genes not previously known to be involved in cell motility promote migration of cells of a few cultured tumor cell lines.
  • the present inventors employed a genetic screen designed to identify components of cancer- associated cell migration using a precision engineered 384-well based wound healing device, coupled with automated microscopy.
  • the screen employed a large-scale siRNA library which comprises 10,996 siRNAs targeting human genes in a highly motile ovarian carcinoma cell line, SKO V-3.
  • Known modulators of cell migration were identified alongside other novel cell motility stimulators whose involvement in cell motility has not yet been known in the art.
  • pro-motility genes are MAP4K4 (NM_004834), FGFRl (NM_000604); CDK7 (NM_001799), DYRKlB (NM_004714) and SERPINB3 (NM__006919).
  • FGFR-I and MAP4k4 were also subject to additional examination. It was found that diminution of both transcripts inhibits the migration of several carcinoma cell lines of differing anatomic origins, and that abrogation by RNA interference or small molecule inhibition of FGFR-I inhibits cell invasion through matrigel. It was also found that MAP4K4 signals through c-Jun N terminal kinase, independent of AP-I activation and downstream transcription.
  • the invention provides methods for identifying modulators of cell motility, especially agents that inhibit tumor cell migration and tumor metastasis.
  • compounds e.g., siRNAs or small molecules
  • the invention further provides methods for inhibiting cell motility or migration in various therapeutic applications.
  • compounds which inhibit tumor cell migration can be employed for preventing tumor metastasis and for treating various forms of cancers in human or non- human subjects.
  • compounds which stimulate cellular migration and motility could also be useful in many therapeutic applications. For example, they can be useful for promoting wound healing and for preventing or treating neurodegenerative diseases.
  • agent includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” can be used interchangeably.
  • analog is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.
  • contacting has its normal meaning and refers to combining two or more molecules (e.g., a test agent and a polypeptide) or combining molecules and cells (e.g., a test agent and a cell).
  • Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container.
  • Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
  • a heterologous sequence or a “heterologous nucleic acid,” as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that, although being endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous nucleic acid.
  • homologous when referring to proteins and/or protein sequences indicates that they are derived, naturally or artificially, from a common ancestral protein or protein sequence.
  • nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology.
  • a "host cell” refers to a prokaryotic or eukaryotic cell into which a heterologous polynucleotide can be introduced.
  • the polynucleotide can be introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • pro-motility molecule or "pro-migration molecule” encompasses "pro-migration genes” and “pro-migration proteins.” It refers to genes or their encoded polypeptides identified by the present inventors that positively regulate cell motility and migration as illustrated in the Examples below. Unless otherwise noted, this term specifically refer to five genes and their encoded polypeptides. These five molecules are MAP4K4, FGFRl, CDK7, DYRKlB and SERPINB3.
  • MAP4K4 (NM_004834; SEQ ID NO: 1)
  • FGFRl (NM_000604; SEQ IDNO: 7)
  • CDK7 NM_001799; SEQ ID NO: 25
  • DYRKlB NM_004714; SEQ ID NO: 29
  • SERPINB3 NM_006919; SEQ ID NO: 35.
  • sequence identity in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • a “comparison window” refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math.
  • the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 95% or 99% or more identical to a reference polypeptide, e.g., a pro-motility molecule described herein, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.
  • a reference polypeptide e.g., a pro-motility molecule described herein, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.
  • nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to a reference nucleic acid, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters.
  • a "substantially identical" nucleic acid or amino acid sequence refers to a nucleic acid or amino acid sequence which comprises a sequence that has at least 90% sequence identity to a reference sequence using the programs described above (preferably BLAST) using standard parameters. The sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
  • a Boyden chamber assay is a system well known in the art for measuring the ability of cells to pass through an extracellular matrix of coated membranes, such as Matrigel (see, e.g., Kramer et al., Cancer Res 46:1980-1989, 1986; and Albrni et al., Cancer Res 47:3239-3245, 1987).
  • Such assay system is commercially available, e.g., the Matrigel transwell chamber assay system from Becton Dickinson & Co. (Franklin Lakes, NJ).
  • the matrigel Boyden chamber assays have been routinely employed by the skilled artisans in the art to study cell motility in general and tumor cell metastasis in particular. See, e.g., U.S. Pat. No. 5,935,850; Kunda et al., J. Cell Biol. 130: 725, 1995; and Parish et al., Int. J. Cancer 52:378-383, 1992.
  • metastasis refers to the invasion and migration of tumor cells away from the primary tumor site.
  • the invasion and metastasis of cancer is a complex process which involves changes in cell adhesion properties which allow a transformed cell to invade and migrate through the extracellular matrix (ECM) and acquire anchorage-independent growth properties.
  • ECM extracellular matrix
  • Metastatic disease occurs when the disseminated foci of tumor cells seed a tissue which supports their growth and propagation, and this secondary spread of tumor cells is responsible for the morbidity and mortality associated with the majority of cancers.
  • modulate with respect to a biological activity of a reference protein (e.g., a pro-migration molecule disclosed herein) or its fragment refers to a change in the expression level or other biological activities of the protein. For example, modulation may cause an increase or a decrease in expression level of the reference protein, enzymatic modification (e.g., phosphorylation) of the protein, binding characteristics (e.g., binding to another molecule), or any other biological (e.g., enzymatic), functional, or immunological properties of the reference protein.
  • enzymatic modification e.g., phosphorylation
  • binding characteristics e.g., binding to another molecule
  • any other biological e.g., enzymatic
  • the change in activity can arise from, for example, an increase or decrease in expression of one or more genes that encode the reference protein, the stability of an mRNA that encodes the protein, translation efficiency, or from a change in other biological activities of the reference protein.
  • the change can also be due to the activity of another molecule that modulates the reference protein (e.g., a kinase which phosphorylates the reference protein).
  • Modulation of a reference protein can be up-regulation (i.e., activation or stimulation) or down-regulation (i.e. inhibition or suppression).
  • the mode of action of a modulator of the reference protein can be direct, e.g., through binding to the protein or to genes encoding the protein, or indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates the reference protein.
  • the term "subject” includes mammals, especially humans. It also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.
  • a "variant" of a reference molecule refers to a molecule substantially similar in structure and biological activity to either the entire reference molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.
  • the pro-migration molecules identified by the present inventors provide novel targets to screen for modulators of cell motility, e.g., small molecule inhibitors. They are particularly suitable for screening test compounds to identify agents that inhibit tumor metastasis.
  • modulators of cell motility e.g., small molecule inhibitors. They are particularly suitable for screening test compounds to identify agents that inhibit tumor metastasis.
  • Various biochemical and molecular biology techniques or assays well known in the art can be employed to practice the present invention. Such techniques are described in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N " . Y-, Second (1989) and Third (2000) Editions; and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).
  • test compounds are typically first assayed for their ability to modulate (e.g., down-regulate) expression or other biological activities of a pro-migration molecule described herein ("the first assay step”). Modulating compounds thus identified are then subject to further screening for ability to modulate (e.g., inhibit) motility or migration of a cell (e.g., a tumor cell), typically in the presence of the pro-migration molecule (“the second testing step”). Depending on the pro-migration molecule employed in the method, modulation of different biological activities of the pro- migration molecule can be assayed in the first step. As detailed below, test agents can be assayed for binding to the pro-migration molecule.
  • the test agents can be assayed for activity to modulate expression of the pro-migration molecule, e.g., transcription or translation.
  • the test agents can also be assayed for activities in modulating cellular level or stability of the pro-migration molecule, e.g., post-translational modification or proteolysis. If the pro-migration molecule has a known biological or enzymatic function (e.g., kinase activity), the biological activity monitored in the first screening step can be the specific biochemical or enzymatic activity of the pro-migration molecule.
  • both the first assaying step and the second testing step either an intact pro-migration molecule, or a fragment thereof, may be employed.
  • Polynucleotide and amino acid sequences of the five pro-migration molecules disclosed herein are all known in the art.
  • accession numbers of 3 different transcript variants of human MAP4K4 are NM_004834 (SEQ ID NO:1); NMJ45686 (SEQ ID NO:3); and NM_145687(SEQ IUD NO:5).
  • the respective amino acid sequences encoded by the polynucleotide sequences are NP_004825 (SEQ ID NO:2); NP_663719 (SEQ ID NO:4); and NP_663720 (SEQ ID NO:6).
  • Human FGFRl polynucleotide sequences are shown in, e.g., accession numbers NM__000604 (SEQ ID NO:7); NM_015850 (SEQ ID NO:9); NM_023105 (SEQ ID NO: 11); NM_023106 (SEQ ID NO:13); NM_023107 (SEQ IDNO:15); NM_023108 (SEQ ID NO: 17); NM_023109 (SEQ ID NO: 19); NM_023110 (SEQ ID NO:21); and NM_023111 (SEQ ID NO:23).
  • NP_056934 SEQ ID NO:8
  • NP_000595 SEQ ID NO: 10
  • NP_075593 SEQ ID NO: 12
  • NP_075594 SEQ ID NO: 14
  • NP_075595 SEQ ID NO: 16
  • NP_075596 SEQ ID NO: 18
  • NP_075597 SEQ ID NO:20
  • NP_075598 SEQ ID NO:22
  • NP_075599 SEQ ID NO:24.
  • Examples of human CDK7 gene accession numbers are NM_001799 (SEQ ID NO:25) and AK130859 (SEQ ID NO:27).
  • accession numbers NP_001790 (SEQ ID NO:26) and AAM77799 (SEQ ID NO:28).
  • Polynucleotide sequences encoding human DYRKlB transcript variants are shown in, e.g., accession numbers NM_004714 (SEQ ID NO:29); NM_006483 (SEQ ID NO:31); and NM_006484 (SEQ ID NO:33). These sequences respectively encode human DYRKlB isoform polypeptide sequences with accession numbers of NP_004705 (SEQ ID NO:30); NP_006474 (SEQ ID NO:32); and NP_006475 (SEQ ID NO:34).
  • Human SERPINB3 is encoded by, e.g., polynucleotide sequence with accession number NM_006919 (SEQ ID NO.35) and the corresponding amino acid sequence with accession number NP_OO885O (SEQ ID NO:36). Any of these pro-migration molecule polynucleotide sequences or polypeptide sequences can be employed to screen test compounds for modulators of the pro-migration molecules.
  • non- human homologs of any of the pro-migration molecules described herein can also be employed to practice the screening methods of the invention.
  • mouse MAP4K4 NM_008696
  • FGFRl NM_201230
  • CDK7 NM_009874
  • DYRKlB DYRKlB
  • Homologs of these pro-migration molecules in many other non-human species have been similarly described in the art. Any of these non-human homologs can be used to screen test compounds for modulators.
  • polynucleotide sequences or polypeptide sequences that are substantially identical to the sequence of any of the pro-migration genes or polypeptides disclosed herein can also be employed in the screening methods of the invention.
  • analogs or functional derivatives of any of the pro-migration molecules described herein can also be used in the screening.
  • the fragments or analogs that can be employed in these assays usually retain one or more of the biological activities of the pro-migration molecule (e.g., kinase activity if the pro-migration molecule employed in the first assaying step is a kinase). Fusion proteins containing such fragments or analogs can also be used for the screening of test agents.
  • Functional derivatives of a pro-migration molecule usually have amino acid deletions and/or insertions and/or substitutions while maintaining one or more of the bioactivities and therefore can also be used in practicing the screening methods of the present invention.
  • a functional derivative of a given pro-migration molecule can be prepared from a pro-migration molecule by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art.
  • the functional derivative can be produced by recombinant DNA technology by expressing only fragments of a pro-migration molecule that retain one or more of their bioactivities.
  • Test compounds or agents that can be screened with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrrolidines, derivatives, structural analogs or combinations thereof.
  • Some test agents are synthetic molecules, and others natural molecules.
  • the test agents are nucleic acids. Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
  • the screening methods are directed to screening inhibitory polynucleotides for agents that specifically down-regulate expression or cellular level of a pro-migration molecule.
  • inhibitory polynucleotides include, e.g., short interfering RNAs (siRNAs), microRNAs (miRNAs), synthetic hairpin KNAs (shRNAs), anti-sense nucleic acids, and complementary DNAs (cDNAs).
  • Test agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion.
  • Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642.
  • Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field.
  • pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterif ⁇ cation, amidification to produce structural analogs.
  • Combinatorial libraries of peptides or other compounds can be fully randomized, with no sequence preferences or constants at any position. Alternatively, the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • Libraries of test agents to be screened with the claimed methods can also be generated based on structural studies of the pro-migration molecules discussed above or their fragments. Such structural studies allow the identification of test agents that are more likely to bind to the pro-migration molecules.
  • Test agents also include antibodies that specifically bind to a pro-migration molecule described herein. Some of the antibody agents antagonize (i.e., suppress) a biochemical activity (e.g., kinase) of the pro-migration molecule employed in the screening methods. Some other antibody agents agonize (i.e., stimulate) the biochemical activity of the pro-migration molecule.
  • the antibodies can be monoclonal or polyclonal. Such antibodies can be generated using methods well known in the art.
  • non-human monoclonal antibodies e.g., murine or rat
  • production of non-human monoclonal antibodies can be accomplished by, for example, immunizing the animal with a pro-migration molecule or its antigenic fragment (see, e.g., Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press, 3 rd ed. 5 2000).
  • a pro-migration molecule or its antigenic fragment see, e.g., Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press, 3 rd ed. 5 2000.
  • Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression.
  • humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci.
  • Human antibodies can be produced using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047.
  • the test agents are small molecule organic compounds, e.g., chemical compounds with a molecular weight of not more than about 500 or 1,000.
  • high throughput assays are adapted and used to screen for such small molecules.
  • Combinatorial libraries of small molecule test agents can be readily employed to screen for small molecule compound modulators of cell motility.
  • a number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1 :384-91.
  • Test agents can be screened for their ability to either up-regulate or down- regulate expression level or other biological activities of the pro-migration molecule in the first assay step.
  • test compounds are first screened to identify modulating compounds which modulate expression or cellular level of one of the pro- migration molecules described herein.
  • cell-based assays are used to identify agents that modify expression of genes encoding the pro-migration molecules.
  • each test compound can be contacted with a cell (e.g., a tumor cell).
  • Expression of at least one of the pro-migration molecules e.g., MAP4K4 is then measured in cells that have been treated with the compounds.
  • a modulating compound is identified if the level of expression of the gene in the cells that have been treated is up-regulated or down-regulated relative to the level of expression of the same gene in cells that have not been treated with the compound.
  • the level of expression of the gene can be detected by, for example, measuring the level of mRNA transcripts corresponding to or proteins encoded by the pro-migration molecule genes.
  • Standard detection techniques well known in the art for detecting RNA, DNA, proteins and peptides can readily be applied to detect expression levels of the pro- motility genes. Such techniques may include detection with nucleotide probes or may comprise detection of the protein by, for example, antibodies or their equivalent. Types of probe include cDNA, riboprobes, synthetic oligonucleotides and genomic probes.
  • endogenous levels of a pro-migration molecule can be directly monitored in cells normally expressing the pro-migration molecule (e.g., a tumor cell).
  • expression or cellular level of a pro-migration molecule can be examined in an expression system using cloned cDNA or genomic sequence encoding the pro-migration molecule.
  • modulation of expression of a pro-migration molecule is examined by monitoring expression of a reporter gene under the control of a transcription regulatory element of a pro-migration molecule.
  • modulation of expression of a pro-migration molecule gene is examined in a cell-based system by transient or stable transfection of an expression vector into cultured cell lines.
  • Assay vectors bearing transcription regulatory sequences (e.g., promoter) of a pro-migration molecule gene operably linked to reporter genes can be transfected into any mammalian host cell line for assays of promoter activity.
  • transcription regulatory sequences e.g., promoter
  • Constructs containing a transcription regulatory element of a pro- migration molecule gene that is operably linked to a reporter gene can be prepared using only routinely practiced techniques and methods of molecular biology (see, e.g., Sambrook et al. and Brent et al., supra).
  • Any readily transfectable mammalian cell line may be used to assay A pro-migration molecule promoter function or to express A pro-migration molecule, e.g., CHO, COS, HCTl 16, HEK 293, MCF-7, and HepG2 cell lines.
  • a pro-migration molecule e.g., CHO, COS, HCTl 16, HEK 293, MCF-7, and HepG2 cell lines.
  • the transcription regulatory elements in the expression vector induces transcription of the reporter gene by host RNA polymerases.
  • Reporter genes typically encode polypeptides with an easily assayable enzymatic activity that is naturally absent from the host cell.
  • Typical reporter polypeptides for eukaryotic promoters include, e.g., chloramphenicol acetyltransferase (CAT), firefly or Renilla luciferase, beta-galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP).
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • Binding of test agents to a pro-migration molecule can be assayed by a number of methods including e.g., labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168; and also Bevan et al., Trends in Biotechnology 13:115-122, 1995; Ecker et al., Bio/Technology 13:351-360, 1995; and Hodgson, Bio/Technology 10:973-980, 1992.
  • the test agent can be identified by detecting a direct binding to the pro-migration molecule, e.g., co-immunoprecipitation with the pro- migration molecule by an antibody directed to the pro-migration molecule.
  • the test agent can also be identified by detecting a signal that indicates that the agent binds to the pro- migration molecule, e.g., fluorescence quenching or FRET.
  • FRET fluorescence quenching
  • combinatory library based screening methods are used.
  • binding of a pro-migration molecule or fragment thereof to a test agent can be determined by, e.g., changes in fluorescence of either the pro-migration molecule or the test agents, or both.
  • Fluorescence may be intrinsic or conferred by labeling either component with a fluorophor.
  • Binding of a test agent to a pro-migration molecule provides an indication that the agent can be a modulator of the pro-migration molecule. It also suggests that the agent may modulate cell motility through, e.g., binding to and modulating the pro-migration molecule.
  • a test agent that binds to a pro-migration molecule can be further tested for ability to modulate motility of a cell, e.g., a tumor cell (i.e., in the second testing step outlined above).
  • a test agent that binds to a pro-migration molecule can be further examined to determine whether it modulates another biological activity (e.g., an enzymatic activity) of the pro-migration molecule.
  • another biological activity e.g., an enzymatic activity
  • the existence, nature, and extent of such modulation can be tested by an activity assay.
  • Such an activity assay can confirm that the test agent binding to the pro-migration molecule indeed modulates the pro-migration molecule. More often, such activity assays can be used independently to identify test agents that modulate activities of a pro-migration molecule (i.e., without first assaying their ability to bind to the pro-migration molecule).
  • test agents involve adding a test agent to a sample containing a pro-migration molecule in the presence or absence of other molecules or reagents which are necessary to test a biological activity of the pro-migration molecule (e.g., enzymatic activity if the pro-migration molecule is an enzyme), and determining an alteration in the biological activity of the pro-migration molecule.
  • test compounds are first screened for ability to modulate an enzymatic activity or another biochemical activity of the pro-migration molecule employed in the screen.
  • test compounds are first examined for ability to modulate the kinase activity of the pro-migration molecule (e.g., MAP4K4, FGFRl , DYRKlB, or CDK7).
  • the substrate to be used in the screening can be a molecule known to be enzymatically modified by the enzyme (e.g., a kinase), or a molecule that can be easily identified from candidate substrates for a given class of enzymes.
  • kinase substrates are available in the art. See, e.g., www.emdbiosciences.com; and www.proteinkinase.de.
  • a suitable substrate of a kinase can be screened for in high throughput format.
  • substrates of a kinase can be identified using the Kinase-Glo® luminescent kinase assay (Promega) or other kinase substrate screening kits (e.g., developed by Cell Signaling Technology, Beverly, Massachusetts).
  • FGFRl tyrosine kinase activity can be identified by monitoring their effect on FGFRl phosphorylation of a substrate (e.g., SNTl) in vitro.
  • a substrate e.g., SNTl
  • assays have been described in the art, e.g., Jin et al., Shi Yan Sheng Wu Xue Bao. 35:184- 90, 2002.
  • FGFRl tyrosine kinase activity may also be examined with in vivo assays as described in Mohammadi et al., Science 276:955-60, 1997; and Chen et al., Proc Natl Acad Sci USA. 101 :14479-84, 2004.
  • Kinase activity of CDK7 can be similarly monitored using assays known in the art (e.g., William et al., Arch Biochem Biophys. 314:99-106, 1994; and Rosales et al., Cell Physiol Biochem. 13:285-96, 2003).
  • test compounds can be screened for ability to modulate (e.g., inhibit) the serine/threonine kinase activity of DYRKlB.
  • Kinase assays for examining DYRKlB kinase activities are known in the art, e.g., using MBP or recombinant GST-HNFl as substrate described in Lim et al., J. Biol. Chem.
  • SERPINB3 inhibits cysteine proteinases such as cathepsin S, K, L, and papain. Test compounds can be screened for ability to modulate this biochemical activity of SERPINB3.
  • SERPINB3 e.g., GST-fused SERPINB3 polypeptide
  • cysteine proteinase e.g., papain or cathepsin L
  • test agents that modulate the pro-migration molecule are typically further tested for ability to modulate (e.g., inhibit) motility or migratory activity of a cell (e.g., a tumor cell).
  • This further testing step is often needed to confirm that their modulatory effect on the pro-migration molecule would indeed lead to modulation of motility or migration of a cell.
  • this screening step is performed in the presence of the pro-migration molecule on which the modulating agent acts.
  • this screening step is performed in vivo using cells that endogenously express the pro-migration molecule. As a control, effect of the modulating agents on the motility of a cell that does not express the pro-migration molecule can also be examined.
  • Some of the screening methods of the invention are directed to identifying agents that inhibit motility of tumor cells.
  • compounds which modulate (e.g., down-regulate) a pro-migration molecule are further tested for ability to inhibit motility or migration of a tumor cell.
  • compounds which down-regulate expression level of a pro-migration molecule can be further tested in order to confirm that such modulation can result in suppressed or reduced proliferation of a cell harboring the pro- migration molecule (e.g., a tumor cell such as SKOV-3 cell).
  • a test agent which inhibits an enzymatic activity of a pro-migration molecule is usually further examined for ability to modulate motility of a cell.
  • the modulating agents can be further screened for lack of significant effect on the motility or migration of a normal non-tumor control cell. This additional step could ensure that the agents identified with the screening methods of the invention are specific for tumor cells.
  • tumor cell lines can be used in this screening step of the methods of the present invention. These include the tumor cell lines described in the Examples herein, e.g., SKOV-3 (ovarian), ES-2 (ovarian), MDA-MB-231 (breast), A2058 (melanoma) and DU145 (prostate) cell lines. In addition, many other tumor or non-tumor cell lines can also be used to screen for compounds that specifically inhibit tumor cell migration and tumor metastasis.
  • tumor cell lines include human glioblastoma cell line U373 (ATCC); melanoma cell line SK-MEL-2; ovarian cancer cell line OVCAR-4; leukemia lines HL60 and RPMI- 8226; lung cancer cell lines NCI-H322M and NCI-H460; colon lines COLO 205 and HCC- 2998; brain tumor lines SF-539 and SNB-75; and breast cancer lines MCF7 and HS 578T (Monks et al... Anticancer Drug Des 12: 533-541, 1997; and Boyd and Paull, Drug Dev Res 34: 91-109, 1995).
  • ATCC human glioblastoma cell line U373
  • SK-MEL-2 melanoma cell line SK-MEL-2
  • ovarian cancer cell line OVCAR-4 ovarian cancer cell line OVCAR-4
  • leukemia lines HL60 and RPMI- 8226 lung cancer cell lines NCI-H322M and NCI-H
  • Non-tumor cell lines include, e.g., human embryonic kidney cell line (HEK293); human umbilical vein endothelial cell line (HUVEC); epithelial cell line MCF- 1OA (Soule et al., Cancer Res. 50: 6075-6086, 1990); colon cell line (CCD-18Co) and ovarian cell line (NOV-31 (Hirasawa et al., Cancer Research 62, 1696-1701, March 15, 2002).
  • HEK293 human embryonic kidney cell line
  • HAVEC human umbilical vein endothelial cell line
  • MCF- 1OA Soule et al., Cancer Res. 50: 6075-6086, 1990
  • colon cell line CCD-18Co
  • NOV-31 Hirasawa et al., Cancer Research 62, 1696-1701, March 15, 2002.
  • ATCC provides many tumor/normal cell line pairs that are used to elucidate the underlying causes of cancers. They can also be employed to
  • tumor/normal cell line pairs include non-small cell lung cancer cell line (ATCC No. CCL-256) and normal peripheral blood cell line ATCC No. CCL-256.1; adenocarcinoma cell line ATCC No. CRL- 5868 and normal peripheral blood cell line ATCC No. CRL-5957; malignant melanoma cell line ATCC No. CRL-1974 and normal cell line ATCC No. CRL-1980; basal cell carcinoma cell line ATCC No. CRL-7762 and normal skin cell line ATCC No. CRL-7761; colorectal adenocarcinoma cell line ATCC No. CCL-228 and normal lymph node cell line ATCC No. CCL-227; and giant cell sarcoma cell line ATCC No.
  • motility of cells can be assessed with a cell scratching device described in the Examples below. Coupled with automated high-speed microscopy, this system examines motility of cells by assaying a cell's ability to close a uniform wound ("scratch") in an in vitro culture of the cell. The ability to close a scratch by migration in a tissue culture plate is a well-established measure of a cell's migratory capacity.
  • modulation of cell motility by test compounds can be examined with a number of methods known to the skilled artisans.
  • the compounds can be examined for ability to prevent cells from crossing a barrier.
  • extracellular matrix coated membranes such as Matrigel.
  • One commonly used assay is the Boyden chamber invasion assay which measures the ability of the cell to pass through a matrix of basement membrane (BM) (see, e.g., Kramer et al., Cancer Res 46:1980-1989, 1986; and Albini et al., Cancer Res 47:3239-3245, 1987).
  • BM matrix of basement membrane
  • Such assay systems are commercially available, e.g., the Matrigel transwell chamber assay system from Becton Dickinson & Co. (Franklin Lakes, NJ) as described in the Examples below.
  • U.S. Pat. No. 5,935,850; and Kunda et al., J. Cell Biol. 130, 725, 1995) can also be used in the practice of the screening methods of the present invention.
  • Kunda et al. employs multiwell chemotaxis chambers overlaid with porosity polycarbonate filters. Migration of cells placed in the upper well toward the lower well was monitored by examining cells that passed through the filters. Cells that passed through the filters and adhered to the lower side were fixed in formalin, and counted under light microscopy. Additional assays for measuring motility of various tumor or non-tumor cells have also been described in the art. See, e.g., In Vitro.
  • the present invention provides novel methods and compositions for modulating cell motility and migratory activity.
  • the methods and compositions of the present invention find therapeutic applications in treating various clinical conditions or disease states by stimulating desired or inhibiting undesired cellular migration. Modulation of cell migratory activities is also useful for preventing or modulating the development of such diseases or disorders in a subject suspected of being, or known to be, prone to such diseases or disorders.
  • Some of the methods are directed to treat various diseases and disorders using therapeutic agents which inhibit cellular migration by down-regulating at least one of the pro-migration molecules. These agents include compounds that can be identified in accordance with the screening methods described above, e.g., small molecule compounds or antibodies (e.g., antagonist antibodies).
  • nucleic acid agents which down-regulate the pro-motility molecules such as anrisense nucleotides, ribozymes, double-stranded RNAs 5 and double-stranded small interference RNAs (siRNAs).
  • these agents can be employed to treat or prevent metastatic tumors.
  • such compounds are also useful for treating and/or preventing disorders associated with inflammation in a subject. Inflammatory disorders and ischemic diseases are characterized by inflammation associated with neutrophil migration to local tissue regions that have been damaged or have otherwise induced neutrophil migration and activation. By specifically inhibiting motility of certain immune or hematopoetic cells, these compounds can be used for preventing inflammation that is associated with immune cell migration and for treating preventing inflammatory disorders and ischemic diseases.
  • the tumor types include, e.g., biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
  • siRNAs corresponding to at least one of the disclosed pro-motility molecule genes are utilized to interfere with expression of at least one of the genes. Interference with the function and expression of endogenous genes by double-stranded RNA has been shown in various organisms such as C. elegans as described, e.g., in Fire et al., Nature, Vol. 391, pp. 806-811 (1998); drosophilia as described, e.g., in Kennerdell et al., Cell, Vol. 95, No. 7, pp. 1017-1026 (1998); and mouse embryos as described, e.g., in Wianni et al., Nat.
  • Double-stranded RNA can be synthesized by in vitro transcription of single-stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands.
  • Double-stranded RNA can also be synthesized from a cDNA vector construct in which the gene of interest is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal.
  • Double-stranded RNA corresponding to the pro-motility genes can be introduced into a cell (e.g., a tumor cell) by transfection of an appropriate construct.
  • Compounds which inhibit cellular migration can be used in a subject to treat or prevent cancer metastasis or inflammatory disorders alone or in combination with the administration of other therapeutic compounds for the treatment or prevention of these disorders.
  • anti-cancer and anti-inflammation drugs known in the art, e.g., as described in, e.g., Cancer Therapeutics: Experimental and Clinical Agents, Teicher (Ed.), Humana Press (1 st ed., 1997); and Goodman and Oilman's The Pharmacological Basis of Therapeutics, Hardman et al. (Eds.), McGraw-Hill Professional (10 th ed., 2001).
  • Compounds which up-regulate the pro-migration molecule and thereby stimulate cellular migration also find therapeutic applications. They can be used to aid tissue regeneration, e.g., in would healing and neuroregeneration.
  • the wound healing process involves a complex cascade of biochemical and cellular events to restore tissue integrity following an injury.
  • Agents which stimulate cell motility are useful for promoting wound healing by promoting cellular migration and thus remodeling.
  • these compounds can be employed for treating a wound to the dermis or epidermis, e.g., a burn or tissue transplant, injury to the skin.
  • tissue regeneration these compounds can be useful in the treatment of subjects having or at risk of developing neurodegenerative diseases.
  • Neurodegenerative disorders are typically associated with a progressive loss of neurons in the peripheral nervous system or in the central nervous system.
  • Examples of neurodegenerative disorders include chronic neurodegenerative diseases such as familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson's disease, Huntington's disease, familial and sporadic Alzheimer's disease, multiple sclerosis, and etc.
  • Symptoms of these disorders may be treated or alleviated by administering to a subject a compound which promotes neuron cell motility.
  • Other therapeutic compounds for the treatment or prevention of these disorders can be employed concurrently in these therapeutic applications of the present invention.
  • the cell motility-modulating compounds of the present invention can be directly administered under sterile conditions to the subject to be treated. They can be administered alone or as the active ingredient of a pharmaceutical composition.
  • Pharmaceutical compositions of the present invention typically comprise at least one active ingredient together with one or more acceptable carriers thereof.
  • Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject.
  • This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral.
  • the antitumor compound can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties.
  • compositions of the present invention include syrup, water, isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution, oils, glycerin, alcohols, flavoring agents, preservatives, coloring agents starches, sugars, diluents, granulating agents, lubricants, and binders, among others.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • concentration of therapeutically active compound in the formulation may vary from about 0.1-100% by weight.
  • Therapeutic formulations are prepared by any methods well known in the art of pharmacy.
  • the therapeutic formulations can be delivered by any effective means that can be used for treatment.
  • the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream.
  • parenteral administration antitumor agents of the present invention may be formulated in a variety of ways.
  • Aqueous solutions of the modulators may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art.
  • the compounds of the present invention may also be administered encapsulated in liposomes.
  • compositions may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • the therapeutic formulations can conveniently be presented in unit dosage form and administered in a suitable therapeutic dose.
  • a suitable therapeutic dose can be determined by any of the well known methods such as clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage. Except under certain circumstances when higher dosages may be required, the preferred dosage of an antitumor agent of the present invention usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day.
  • the preferred dosage and mode of administration of an antitumor agent can vary for different subjects, depending upon factors that can be individually reviewed by the treating physician, such as the condition or conditions to be treated, the choice of composition to be administered, including the particular antitumor agent, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration.
  • the quantity of an antitumor agent administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.
  • SKOV-3 (ATCC# HTB-77) and ES-2 (ATCC# CRL-1978) cells were cultured in McCoy's 5A medium
  • MDA-MB-231 (ATCC# HTB-26) and A2058 (ATCC# CRL-11147) cells were cultured in Dulbecco's Modified Eagle Medium
  • DU- 145 (ATCC# HTB-81) cells were cultured in RPMI medium. All tissue culture media and supplements were obtained from Invitrogen (Carlsbad, CA).
  • Assay hardware an automated 384-well plate-based cell scratch device was built.
  • the device included a machined aluminum holding block into which 384 orifices had been drilled wide enough to accommodate 12.5 ⁇ l pipette tips (Matrix Technologies, Hudson, NH, USA). Sterilized pipette tips were inserted into the holding block, which was placed in a holding block receiving area of the system to suspend the holding block on a vertical tracking arm of a translational mechanism of the system. The holding block was raised against a top plate to stabilize the tip position and prevent any movement upon scratching. 384-well clear bottom tissue culture plates (Greiner Bio-One, Frickenhausen, Germany) were placed on a level platform or container positioning component below the aluminum block.
  • the aluminum holding block was automatically lowered to a point at which the pipette tips touched the bottom of each of the 384 wells.
  • the holding block was raised up from the plate, and the container positioning component returned to the start position to allow plates to be manually switched by the user.
  • siRNAs Small interfering (si)RNAs were purchased from Dharmacon
  • the library is comprised of 10,996 siRNAs targeting 5,234 unique genes. Approx. 500 siRNAs in the collection are targeted to known and predicted human kinases as described; the remaining 10,500 siRNAs were designed to target specific families of genes which are considered pharmaceutically tractable, such as proteases, G-protein coupled receptors (GPCRs), cytokines and cytokine receptors, as well as other classes of genes, such as transcription factors, components of the cell cycle and apoptotic machinery.
  • GPCRs G-protein coupled receptors
  • cytokines and cytokine receptors as well as other classes of genes, such as transcription factors, components of the cell cycle and apoptotic machinery.
  • [0073J 384-well scratch assay Cells were plated at high density (4,000-5,000 cells per well) in media supplemented with 10% FBS. Cell density was calculated to result in >95% confluence at the time of scratching, accounting for the toxicity of the transfection reagent lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA). Cells were added to a siRNA/transfection reagent cocktail and deposited on the pre-plated siRNAs, resulting in reverse transfection, as described previously (Aza-Blanc et al., MoI. Cell 12:627-637, 2003). For small molecule experiments, compounds were added 12 hours prior to scratching at a final concentration of 0.5% DMSO.
  • Each well of the 384-well plate was photographed by a fluorescent microscope retooled by Q3DM Inc (Beckman Coulter, San Diego, CA, USA) to automate image capture. A 4x objective lens was used to capture a majority of the space within each well. Images were collated and quantitatively scored as described below. For display purposes, images were imported into ImageJ (downloaded from the NIH; http://rsb.info.nih.gov/ij/). DAPI- stained nuclei were encircled and the images inverted.
  • Cell viability Cells were plated into a "sister" set of 384-well siRNA assay plates and processed identically to the scratch plates. Viability was measured using Cell Titre GIo (Promega, Madison, WI, USA). The mean luminescent intensity of each plate was calculated, and the percent of the plate mean was calculated for each well. Small interfering RNAs or compounds resulting in an average percent mean of less than 90% were considered to negatively impact viability, and were eliminated from further study. [0075] Quantitative scoring method: Automated microscopic capture of the assay generates one grayscale image per well (4x magnification). Bright regions represent DAPI- stained nuclei (cells) and black regions represent background; pixel intensities vary.
  • the grayscale image is first converted into a binary black and white mask image, where cells are shown as white pixels and background in black pixels.
  • the presence of contaminants, such as small hairs, etc, show up as unusually large blocks of continuous white regions and can be identified and excluded from our analysis.
  • the initial scratch proceeds from left to right; however, on occasion, a scratch does not start or end beyond the left and right image borders. To avoid incorporating areas of unscratched, confluent cells, the left and right 25% of the original image are cropped.
  • a score close to 1 is assigned to cells with high motility, and a score close to 0 to those with low motility. Since the score is self normalized by cell density, it is comparable across wells and plates.
  • the vertical center of the scratch may vary from well to well; therefore the algorithm does not assume a fixed scratch location.
  • the above S score was iteratively calculated with every possible scratch center within a given range. Only the minimal possible S score is reported, and the corresponding location is the optimal guess of the scratch center.
  • the method only takes the width of the scratch window and a possible range of scratch center. It does not require any training data and is insensitive to variations in cell density. Analysis on some randomly selected wells showed good correlation between our S score and visual inspection.
  • wound healing assay designed to identify genes involved in promoting tumor cell metastasis.
  • the process of monolayer wound healing is a widely used measure of a cell's migratory capacity. Experimentally, this entails scratching the surface of a confluent cell monolayer with a pipette tip or other blunt instrument, and observing the movement of cells across the denuded surface to "heal” the wound.
  • This procedure was automated by developing a "scratch” device as described above.
  • the scratch device creates uniform wounds in confluent monolayers of cells cultured in 384-well microliter plates. Wound closure is monitored by automated CCD image capture of formaldehyde-fixed and DAPI- stained cells at a time when the wounds are typically "healed” in the presence of negative controls.
  • tumor cell with migratory potential are plated at high density in 384-well plates in which different siRNAs (or cDNAs) have been pre-plated.
  • siRNAs the cells are incubated for 48 hours, scratched, and incubated for a further 12 hours to allow cells to migrate.
  • cells are plated, grown to confluency, and molecules are added 12 hours prior to scratching. Following scratching, cells are incubated for 12 hours as above. Following the timed post-scratch incubation, cells are fixed with formaldehyde and stained with the nuclear stain DAPI.
  • Each well of the 384-well plate is then photographed by the Q3DM high content imaging microscope using a 4x objective to visualize a majority of the space of each well.
  • AU assays are conducted in duplicate to assess the reproducibility of the results.
  • Efficacy of the assay system was first tested by examining migration of a tumor cell line in the presence of known modulators of tumor migration.
  • the efficacy of siRNA-mediated migratory inhibition was assessed using a siRNA against the RhoGTPase Racl and compared to a sequence scrambled, FITC-conjugated siRNA control (Figure IA).
  • Racl is an enzyme which integrates pro-migratory signals with dynamic reorganization of the actin cytoskeleton (Ridely et al., Science 302:1704-1709, 2003).
  • the assay also included 2 compounds that target the c-Src kinase: SKI-606 (Golas et al, Cancer Research 65, 5358-5365, 2005) and Compound 43 (2-phenyl-aminoimidazo-[4,5- h]-isoquinolin-9-one; Goldberg et al, J. Medchem 46: 1337-1349, 2003).
  • the activated form of c-Src plays a central role in the motility and invasion of cancer cells, including ovarian cancer. Effects of these compounds on SKOV-3 migratory activity were compared to diluent (DMSO) alone. As shown in Figure IA, at cell densities ranging from 3,000 to 5,000 cells per well, cells migrated to close the wound typically within 12 hours. In contrast, the addition of Rac siRNA or Src inhibitor significantly inhibited wound closure in the same period of time.
  • FIG. 1C shows western blot demonstrating knock-down of the RAC protein by the RAC-specific siRNA used in Fig. IA, compared to a control siRNA (CON) and mock transfected cells (LIPO). Also shown in the figure are results of the same blot that was re-probed with anti-actin antibody to demonstrate equal loading.
  • Tire automated motility assay system described above was used to screen an siRNA library to identify genes that promote tumor cell motility.
  • the screening employed a pre-plated library of 10,996 siRNAs, targeting 5,234 genes, to identify inhibitors of cellular motility in SKOV-3 cells ( Figure 2).
  • the screen was performed in duplicate (approx. 22,000 wells), as described above, and quantitatively scored. Measurement of cell viability was performed in a set of duplicate siRNA library plates and the luminescence of each well was compared to the normalized mean well intensity of each 384-well plate.
  • control siRNAs Based on measurements from multiple controls that did not affect viability in this assay (i.e., control siRNAs), a cut-off of 0.9 (10% deviation from the plate mean) was adopted, below which siRNAs affecting migration may have resulted from arrested cell growth or cell death and . were therefore disregarded.
  • siRNAs targeting 23 genes 36 (74%) which target 17 genes yielded migratory phenotypes similar to that of the primary screen.
  • transcripts of 4 of these 17 genes were significantly diminished by both siRNAs, correlating precisely with the wounding phenotype ( Figure 3A).
  • These 4 genes are MAP4K4 (NM_004834), CDK7 (NMJ)01799), DYRKlB (NM_004714) and SERPINB3 (NM_006919).
  • FGFRl fibroblast growth factor receptor 1
  • Example 4 Further examination of MAP4K4 and FGFRl activities in tumor cell motility [0088] Two of the pro-migration molecules described above, MAP4K4 and FGFRl , were subject to additional studies.
  • the map kinase, MAP4K4 was chosen for further characterization for several reasons. First, siRNAs targeting MAP4K4 variably retarded the migration of all motile carcinoma cells tested, suggesting a central role for this kinase in cell migration. Second, MAP4K4-deficient mice exhibit specific defects in the migration of neural crest cells during early development (Xue et al, Development 128:1559-1572, 2001), further supporting a migratory role.
  • Figure 3B illustrates the quantitative effects of 4 independent siRNAs (three from the primary screen and one additional siRNA, hereafter termed si_l through si_4) on SKOV-3 migration.
  • the migratory inhibition which ranged from 50 to 66% relative to control siRNA-transfected cells, was consistent with the degree of transcript knockdown (Figure 3B), which ranged from 64 to 94%.
  • siRNAs si_l and si_2
  • si_l and si_2 The effect of the two most potent siRNAs (si_l and si_2) on other highly motile carcinoma cells, and their associated transcriptional inhibition, are depicted in Figure 4.
  • Migratory inhibition is evident in all 4 cell lines relative to control siRNA, with variably potency.
  • the variability reflects: (1) variable transfection efficiencies, (2) differences in the relative expression of the gene and, (3) the effects of wide-ranging cellular densities on the ability of the automated scoring algorithm to comparably score different cell types relative to SKOV-3.
  • transient MAP4K4 knockdown could affect cell invasion.
  • SKOV-3 cells were transfected using si 1 and si_2 and invasion monitored using a matrigel (Boyden) chamber assay. The results were compared to those obtained with SKOV-3 cells transfected with scrambled siRNA control. Invasion was inhibited by 76 and 52% with si_l and si_2, respectively relative to control transfected cells (Figure 5).
  • JNK c-Jun N-terminal kinase
  • siRNAs targeting FGFRl present in the original library were re-synthesized, and a small series of siRNAs targeting FGFRl from were purchased Dha ⁇ nacon Incorporated (Lafayette, CO).
  • a small series of siRNAs targeting FGFRl from were purchased Dha ⁇ nacon Incorporated (Lafayette, CO).
  • RT-PCR using primers specific for the FGFRl transcript was performed on cells trasfected with all 5 siRNAs under identical conditions to the scratch assay. All of the siRNAs diminished the FGFRl transcript by at least 74% (range 74-86%).
  • siRNAs were used in the Boyden chamber invasion assay to assess whether inhibition of FGFRl affected the invasive capacity of SKO V-3 cells. Both siRNAs had an appreciable affect in the assay, reducing the numbers of invading cells by ⁇ 20 and 30%, respectively, compared to controls (p ⁇ 0.05).
  • FGFRl, PDl 73074 assessed its effect in the scratch assay over a range of concentrations (from 3.3uM to 0.04uM). The effects were compared to a SRC inhibitor (described above), as well as a pan-kinase inhibitor, staurosporine, for reference. FGFRl inhibition reduced motility by at least 40%, even at concentrations as low as 4OnM. Notably, PD173074 concentrations as high as 3.3 ⁇ M did not appreciably affect cell viability (Figure 10A). The small molecules were used to assess SKO V-3 cell invasion in Boyden chambers as above. The results indicate that PD173074 at a concentration of 3 ⁇ M inhibited invasion by -50% ( Figure 10B).

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Abstract

Cette invention concerne l'identification de gènes qui favorisent la motilité et la migration de cellules tumorales, par exemple MAP4K4, CDK7, FGFR1, DYRK1B et SERPINB3. L'invention concerne également des procédés d'utilisation de la molécule favorisant la migration pour cribler des composés qui inhibent la métastase de tumeurs. Le procédé consiste à cribler d'abord les composés testés pour rechercher des modulateurs qui freinent l'une de ces molécules favorisant la migration (en ce qui concerne son niveau cellulaire ou ses activités enzymatiques) et à cribler ensuite encore les composés identifiés selon leur aptitude à inhiber la migration ou la motilité d'une cellule tumorale. L'invention concerne en outre des procédés et des compositions pharmaceutiques servant à inhiber la métastase de tumeurs chez un sujet.
PCT/US2006/044027 2005-11-09 2006-11-09 Procédés et compositions servant à moduler la motilité cellulaire et à inhiber la métastase de tumeurs Ceased WO2007056604A2 (fr)

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WO2012125124A1 (fr) * 2011-03-14 2012-09-20 Agency For Science, Technology And Research Anticorps anti-fgfr1 et traitement du cancer
EP3301176A1 (fr) * 2011-02-11 2018-04-04 The Rockefeller University Procédé d'identification d'acides nucléiques régulant la métastasation
EP3643782A1 (fr) * 2008-02-11 2020-04-29 Phio Pharmaceuticals Corp. Polynucléotides d'arni modifiés et leurs utilisations
EP3468564A4 (fr) * 2016-06-14 2020-07-29 Entos Pharmaceuticals Inc. Méthodes pour le diagnostic et le traitement du cancer métastatique
EP3516081A4 (fr) * 2016-09-23 2020-07-29 Memorial Sloan Kettering Cancer Center Déterminants de la réponse d'un cancer à l'immunothérapie
US11254940B2 (en) 2008-11-19 2022-02-22 Phio Pharmaceuticals Corp. Inhibition of MAP4K4 through RNAi

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WO2001062206A2 (fr) * 2000-02-22 2001-08-30 Mount Sinai School Of Medicine Of New York University Modulation de la migration, de l'invasion et de la metastase des cellules qui expriment la n-cadherine
WO2003072590A1 (fr) * 2002-02-20 2003-09-04 Sirna Therapeutics, Inc. Inhibition a mediation par interference d'arn de genes de map kinase

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3643782A1 (fr) * 2008-02-11 2020-04-29 Phio Pharmaceuticals Corp. Polynucléotides d'arni modifiés et leurs utilisations
US11254940B2 (en) 2008-11-19 2022-02-22 Phio Pharmaceuticals Corp. Inhibition of MAP4K4 through RNAi
US10301684B2 (en) 2011-02-11 2019-05-28 The Rockefeller University Treatment of angiogenesis disorders
EP3604534A1 (fr) * 2011-02-11 2020-02-05 The Rockefeller University Procédé d'identification d'acides nucléiques de régulation de la métastatisation
EP3301176A1 (fr) * 2011-02-11 2018-04-04 The Rockefeller University Procédé d'identification d'acides nucléiques régulant la métastasation
US12060619B2 (en) 2011-02-11 2024-08-13 The Rockefeller University Treatment of angiogenesis disorders
WO2012125124A1 (fr) * 2011-03-14 2012-09-20 Agency For Science, Technology And Research Anticorps anti-fgfr1 et traitement du cancer
US9333257B2 (en) 2011-03-14 2016-05-10 Agency For Science, Technology And Research FGFR1 antibodies and treatment of cancer
EP3468564A4 (fr) * 2016-06-14 2020-07-29 Entos Pharmaceuticals Inc. Méthodes pour le diagnostic et le traitement du cancer métastatique
US11236331B2 (en) 2016-06-14 2022-02-01 Entos Pharmaceuticals Inc. Methods for diagnosing and treating metastatic cancer
US12421514B2 (en) 2016-06-14 2025-09-23 Entos Pharmaceuticals Inc. Methods for diagnosing and treating metastatic cancer
EP3516081A4 (fr) * 2016-09-23 2020-07-29 Memorial Sloan Kettering Cancer Center Déterminants de la réponse d'un cancer à l'immunothérapie
US11332530B2 (en) 2016-09-23 2022-05-17 Memorial Sloan Kettering Cancer Center Determinants of cancer response to immunotherapy

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