# Patent

## Title
Inhibitors of metastasis

## Abstract

The present invention relates to methods and compounds for treating or preventing cancer. Methods and compositions provided include including inhibiting or suppressing the development, maintenance, and proliferation of cancers, including blocking or inhibiting cancer cell metastasis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/808,966, filed Apr. 5, 2013, which is herein incorporated by reference in its entirety.


FIELD OF THE INVENTION
This invention relates to compositions and formulations comprising antibodies, nucleic acid molecules, polynucleotides and peptides, and methods of their use for the prevention and treatment of metastatic cancers, especially for the reduction, blocking, or inhibition of cancer cell proliferation, metastasis and/or angiogenesis.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: BMRK_006_01US_SeqList_ST25.txt, date recorded: Apr. 4, 2014, file size 58 kilobytes).
BACKGROUND OF THE INVENTION
MARCKS protein (Myristoylated Alanine-Rich C Kinase Substrate) is a ubiquitous phosphorylation target of protein kinase C (PKC) (Li et al., Journal of Biological Chemistry 276; 40982 (2002)). MARCKS has three evolutionarily-conserved regions (Aderem et al., Nature 1988; 332:362-364; Thelen et al., Nature 1991; 351:320-322; Hartwig et al., Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996; 271:18797-18802): an N-terminus, a phosphorylation site domain (or PSD; also known as the effector domain), and a multiple homology 2 (MH2) domain. The N-terminus, an alpha-amino acid sequence comprising 24 amino acid residues with a myristic acid moiety attached via an amide bond to the N-terminal glycine residue is involved in binding of MARCKS to membranes in cells (Seykora et al., J Biol Chem 1996; 271:18797-18802) and possibly to calmodulin (Matsubara et al., J Biol Chem 2003; 278:48898-48902). This 24 amino acid sequence is known as the MANS peptide. MANS peptide and related peptides are disclosed in U.S. Pat. Nos. 7,265,088; 7,529,926; 7,544,772; 8,492,518; 8,501,911; 7,918,293,870; and 8,563,689; the entire contents of each of which are incorporated by reference in their entireties.
There is a need in the art for new, safe therapies directed to preventing, treating, and inhibiting cancer, including inhibiting cancer cell metastasis, cancer cell proliferation, tumor growth, and/or angiogenesis. The present invention addresses these and other needs.
BRIEF SUMMARY OF THE INVENTION
This invention relates to methods and compositions useful for preventing or treating cancer. In one embodiment, methods and compositions for inhibiting cancer cell metastasis, cancer cell proliferation, tumor growth, or angiogeneis are provided. In one embodiment, methods and compositions for preventing and inhibiting cancer cell metastasis, cancer cell proliferation, tumor growth, or angiogeneis are provided, comprising inhibition of myristoylated alanine-rich C-kinase substrate (MARCKS). In one embodiment, the compositions comprise MARCKS-inhibitory compounds including peptides, polypeptides, antibodies or fragments thereof, and nucleic acid molecules such as antisense polynucleotides, aptamers, small interfering RNA (siRNA), micro RNA (miRNA), and short-hairpin RNA (shRNA). “MARCKS-inhibitory nucleic acid molecules” as used herein refer to polynucleotides or nucleic acid molecules such as siRNA, miRNA, shRNA, or antisense polynucleotides, that reduce expression and/or function of MARCKS. In one embodiment, the compositions comprise one or more MARCKS-related peptides. In another embodiment, the MARCKS-related peptides correspond to the MH2 domain of MARCKS. In another embodiment, the peptides are myristoylated N-terminal sequence (MANS peptide, which is a 24 amino-acid fragment of MARCKS)-related peptides (i.e., “MANS-related peptides”). In a further embodiment, MANS-related peptides are selected from the group consisting of: MANS peptide; unsubstituted fragments of MANS which contain four or more amino acids and which comprise the same sequence found in the N-terminal amino acid sequence in MANS peptide; peptides comprising a sequence substantially identical to the sequence found in the MANS peptide or MANS peptide fragment; MANS peptide or fragments of MANS peptide with the identical or substantially identical amino acid sequence as the MANS peptide that are N-terminal myristoylated or N-terminal acylated with, for example, an acetyl group; and MANS peptide or fragments of MANS peptide with the identical or substantially identical amino acid sequence as the MANS peptide that are C-terminal chemically modified. In one embodiment, the MANS-related peptides are both N-terminal and C-terminal chemically modified. In one embodiment, the MARCKS-inhibitory compounds provided herein are antibodies or fragments thereof. In one embodiment, the antibody or fragment thereof inhibits the functions of the MARCKS protein. In another embodiment, the antibody or fragment thereof binds to the N-terminal of the MARCKS protein or the MH2 sequence of the MARCKS protein. We have found surprisingly that use of different types of MARCKS-inhibitory compounds exhibit an inhibitory effect on cancer cell migration in vitro and inhibit metastasis in vivo. In one embodiment, MARCKS-inhibitory compounds exhibit an inhibitory effect on migration of aggressive cancer cell lines. In one embodiment, the MARCKS-inhibitor compounds provided herein inhibit metastasis of cancer cells in a tumor in a mammal. In a further embodiment, the tumor is a solid tumor. In another embodiment, the tumor is a non-solid tumor. In one embodiment, the MARCKS-inhibitor compounds provided herein inhibit metastasis of cancer cells associated with a lymphoma or leukemia.
In one embodiment, MARCKS-inhibitory compounds comprise MARCKS-inhibitory polynucleotides or MARCKS-inhibitory nucleic acid molecules. In a further embodiment, the MARCKS-inhibitory compounds are antisense RNA, siRNA, shRNA, or microRNA polynucleotides that inhibit MARCKS expression and/or function. In one embodiment, the MARCKS-inhibitory compounds are mimics of proteins or polynucleotides that regulate MARCKS expression, such as mimics of miR21.
In one embodiment, the MARKS-inhibitory compounds are MARCKS-related or MANS-related peptides. In another embodiment, the MARCKS-related peptides correspond to the N-terminal myristoylated domain of MARCKS. Thus, in one embodiment, the MARCKS-related peptides are MANS-related peptides. In one embodiment, MANS-related peptides and certain chemically modified MANS-related peptides block migration of aggressive cancer cell lines. In one embodiment, MANS-related peptides are used to exert an inhibitory effect on metastasis of cancer cells. In one embodiment, MARCKS-inhibitory compounds exhibit blocking effects on metastasis of cancer cells in vivo. In vivo sites of inhibition of metastatic disease include at least lung tissue, heart tissue, spleen tissue, intestine tissue, and diaphragm tissue. In one embodiment, MANS-related peptides are used to treat or prevent cancer cell metastasis, cancer cell proliferation, tumor cell growth, or angiogenesis.
In one aspect, compositions and methods are provided for treating or preventing cancer comprising administration of a MARCKS-inhibitory compound to a cancer cell or to a cell that plays a role in the development, maintenance, proliferation, or metastasis of cancer cells. In one embodiment, a method is provided for inhibiting metastasis of a cancer cell comprising administration to the cancer cell of a metastasis-inhibiting amount of a MARCKS-inhibitory compound. In one embodiment, a method is provided for inhibiting metastasis of a cancer cell comprising administration to the cancer cell of a metastasis-inhibiting amount of a MANS-related peptide. In another embodiment, a method is provided for inhibiting metastasis of a cancer cell in a tumor comprising administration to the cancer cell of a metastasis-inhibiting amount of a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 231 (inclusive), SEQ ID NO: 234, and SEQ ID NO: 235; wherein the N-terminal and/or C-terminal amino acid of the peptide sequence is optionally chemically modified. In another embodiment, a method is provided for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a MANS-related peptide. In another embodiment, a method is provided for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 231 (inclusive), SEQ ID NO: 234, and SEQ ID NO: 235; wherein the N-terminal and/or C-terminal amino acid of the peptide sequence is optionally chemically modified.
In one embodiment, the N-terminal amino acid of the peptide is chemically modified by acylation of the N-terminal amino acid of the peptide in the form of an amide selected from the group consisting of:

    
    
        an amide of a C2 (acetyl) to C24 aliphatic carboxylic acid which may be linear, branched, saturated, or unsaturated,
        an amide of trifluoroacetic acid,
        an amide of benzoic acid, and
        an amide of a C1 to C24 aliphatic alkyl sulfonic acid; or
        the N-terminal amine group of the N-terminal amino acid of the peptide can be alkylated with a group selected from the group consisting of:
        a C1 to C24 aliphatic alkyl group,
        a linear 2-(C1 to C24 aliphatic alkyl)oxyethyl group,
        an omega-methoxy-poly(ethyleneoxy)n-ethyl group, where n is from 0 to 10.
    
    


In a further embodiment, the N-terminal amide is selected from the group consisting of acetyl and myristoyl.
In another embodiment, the C-terminal amino acid of the peptide is chemically modified by amide formation at the C-terminal carboxylic acid group of the C-terminal amino acid of the peptide in the form of an amide selected from the group consisting of:

    
    
        an amide of ammonia,
        an amide of a C1 to C24 aliphatic alkyl amine,
        an amide of a hydroxyl-substituted C2 to C24 aliphatic alkyl amine,
        an amide of a linear 2-(C1 to C24 aliphatic alkyl)oxyethylamine group, and
        an amide of an omega-methoxy-poly(ethyleneoxy)n-ethylamine group, where n is from 0 to 10.
    
    


In one embodiment, a method for inhibiting metastasis of a cancer cell, or for treating cancer, comprising administering a MANS-related peptide to the cancer cell or subject, respectively, is provided. In one embodiment, the peptide is selected from the group consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1); N-myristoyl-GAQFSKTAAKGEAAAERPGEAAV (SEQ ID No: 2); N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID No: 4); N-myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl-GAQFSKTAAKGEAAAERPGE (SEQ ID No: 11); N-myristoyl-GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl-GAQFSKTAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl-GAQFSKTAAKGEAA (SEQ ID No: 56); N-myristoyl-GAQFSKTAAKGEA (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl-GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFSKTAA (SEQ ID No: 121); N-myristoyl-GAQFSKTA (SEQ ID No: 137); N-myristoyl-GAQFSKT (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), N-myristoyl-GAQF (SEQ ID No: 211), N-acetyl-RGAQFSKTAAK (SEQ ID No: 234), N-acetyl-RGAQFSKTAAK-NH2 (SEQ ID No: 234), N-acetyl-RAKGE (SEQ ID NO: 235), and a combination thereof.
In one embodiment, the peptide is selected from the group consisting of N-acetyl-GAQFSKTAAK (SEQ ID No: 106; BIO-11006); N-myristoyl-AKGE (SEQ ID No: 219; BIO-91200); N-myristoyl-GAQFSKTAAK-NH2 (SEQ ID No: 106; BIO-11002); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106; BIO-11000); N-acetyl-GAQFSKTAA (SEQ ID No: 121; BIO-10901); N-myristoyl-GAQFSKTAAK (SEQ ID No: 121; BIO-10900); and N-acetyl-GAQFSKTAAK-NH2 (SEQ ID No: 106). In one embodiment, certain amino acids are present in d-configuration. For example, in one embodiment, the peptide is N-acetyl-GAQFS(d)KTAA(d)K (SEQ ID NO: 106; BIO-11037), in which the lysine (K) at positions 6 and 10 of the peptide are of d-configuration.
In some embodiments, MANS-related peptides exhibit properties that make them suitable for use in therapeutic applications, for example, in the treatment of cancers. For example, in one embodiment, certain MANS-related peptides disclosed herein exhibit enhanced solubility relative to MANS peptide or peptides other than MANS-related peptides. In another embodiment, certain MANS-related peptides provided herein exhibit longer half-lives in plasma than the MANS peptide or peptides other than MANS-related peptides.
In one embodiment, the MARCKS-related peptide exhibits reduced cancer cell migration. For example, in one embodiment, pretreatment of cancer cells with a MANS-related peptide (e.g., BIO-11006, BIO11002, BIO10901, BIO10900, BIO11000, or BIO-91200) may reduce migration of the cancer cells when cells are pretreated with from about 10 μm peptide to about 200 μm peptide; or pretreated with from about 20 μm to about 200 μm; or pretreated with from about 25 μm peptide to about 75 μm peptide. In one embodiment, the MARCKS-related peptide exhibits reduced cancer cell migration when administered at concentrations of about 1 μM to about 500 μM, about 5 μM to about 250 μM, or about 10 μM to about 200 μM. In one embodiment, the MARCKS-related peptide exhibits reduced cancer cell migration when administered at concentrations of about 1 μM, about 5 μM, about 10 μM, about 25 μM, about 50 μM, about 100 μM, about 150 μM, about 200 μM, or about 500 μM. In one embodiment, cancer cells are treated with the peptide in vitro to determine the effects of the peptide. In one embodiment, the cancer cells are derived from a patient. In a further embodiment, the cancer cells are treated with the peptide in vitro to determine if the patient is likely to respond to treatment with the peptide.
In one embodiment, the MARCKS-related peptide exhibits reduced cancer cell metastasis when administered to a patient at concentrations of about 0.01 mg/kg/day to about 10 mg/kg/day. In a further embodiment, the MARCKS-related peptide exhibits reduced cancer cell metastasis when administered to a patient at concentrations of about 0.1 mg/kg/day to about 5.0 mg/kg/day. In a yet further embodiment, the MARCKS-related peptide exhibits reduced cancer cell metastasis when administered to a patient at concentrations of about 0.5 mg/kg/day to about 2.5 mg/kg/day. For example, the MARCKS-related peptide exhibits reduced cancer cell migration when administered to a patient at concentrations of about 0.01, about 0.05, about 0.1, about 0.5, about 0.75, about 1.0, about 1.25, about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.5, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, or more mg/kg/day.
In one embodiment, the peptide is administered by inhalation of a liquid solution or suspension, or by inhalation of a dry powder formulation of the peptide. In another embodiment, the peptide is administered by injection of a liquid formulation or suspension of the peptide. In a further embodiment, the injection is into a primary tumor region, wherein the region contains the cancer cell. In a further embodiment, the cancer cell resides in a tumor in a mammal. In one embodiment, the tumor is a solid tumor. In another embodiment, the tumor is a non-solid tumor. In one embodiment, the MARCKS-inhibitor compounds provided herein inhibit metastasis of cancer cells associated with a lymphoma or leukemia. In another embodiment, the liquid formulation is isotonic. In another embodiment, the liquid formulation is buffered.
In one embodiment, the metastasis-inhibiting amount of the peptide is in the range from about 0.1 to about 100 micromoles per milliliter. In a further embodiment, the metastasis-inhibiting amount of the peptide is in the range from about 1 to about 10 micromoles per milliliter. In another embodiment, the peptide is in a formulation comprising an additional drug useful in the treatment of cancer, or is formulated for administration with an additional drug.
In one aspect, a method for treating or preventing cancer or inhibiting metastasis of a cancer cell in a mammal is provided, wherein the method comprises administering to said mammal a MARCKS-inhibitory compound. In one embodiment, the MARCKS-inhibitory compound is a polynucleotide or nucleic acid molecule that reduces expression or activity of MARCKS. In a further embodiment, the MARCKS-inhibitory polynucleotide is an antisense RNA, siRNA, shRNA, or miRNA. In one embodiment, the MARCKS-inhibitory polynucleotide is administered in an amount from about 10 nM to 10 μM, or from about 20 nM to about 500 nM, or from about 30 nM to about 300 nM, or from about 40 nM to about 200 nM, or from about 50 nM to about 100 nM. In one embodiment, the MARCKS-inhibitory polynucleotide is a mimic of an miRNA that regulates MARCKS expression. For example, in one embodiment, the MARCKS-inhibitory polynucleotide is a mimic of miR21. In one embodiment, the polynucleotides and nucleic acid molecules are administered together with a delivery agent such as a peptide, protein, lipid, sterol, polymer, transfection reagent, or any polynucleotide or nucleic acid delivery agent known in the art.
In one aspect, a method for treating or preventing cancer or inhibiting metastasis of a cancer cell in a mammal is provided, wherein the method comprises administering to said mammal a MANS-related peptide, wherein said peptide exhibits a migration index of at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8 at least about 2.9, at least about 3.0, or more, following pretreatment of non-small-cell lung carcinoma (NSCLC) cells. In a further embodiment, the MANS-related peptide is present at a concentration of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 μmolar of said peptide. In another embodiment, the migration period is 3, 4, 5, 6, 7, 8, 12, 15, 20, 21, 22, 23, or 24 hours. In one embodiment, the MANS-related peptide exhibits a migration index of at least about 1.5 following pretreatment of NSCLC cells with a concentration of 50 μmolar of said peptide and a migration period of about 12 hours. In another embodiment, the MANS-related peptide exhibits a migration index of at least about 2.0 following pretreatment of NSCLC cells with a concentration of at least about 100 μmolar of said peptide and a migration period of about 12 hours.
In one aspect, a method for treating or preventing cancer including cancer metastasis in a subject in need thereof is provided, the method comprising administering a MANS-related peptide to the subject at a dose of about 0.01 mg/kg/day to about 10 mg/kg/day. In a further embodiment, the MANS-related peptide is administered at concentrations of about 0.1 mg/kg/day to about 5.0 mg/kg/day. In a yet further embodiment, the MANS-related peptide is administered at concentrations of about 0.5 mg/kg/day to about 2.5 mg/kg/day. For example, the MANS-related peptide is administered at a dose of about 0.01, about 0.05, about 0.1, about 0.5, about 0.75, about 1.0, about 1.25, about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.5, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, or more mg/kg/day for the treatment or prevention of cancer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 displays a cell count field of a negative control after a migration time of 12 hours.
FIG. 2A displays a cell count field obtained after pretreatment with 50 μmolar of MANS peptide followed by a migration time of 12 hours.
FIG. 2B displays a cell count field obtained after pretreatment with 100 μmolar of MANS peptide followed by a migration time of 12 hours.
FIG. 3A displays a cell count field obtained after pretreatment with 50 μmolar of RNS peptide followed by a migration time of 12 hours.
FIG. 3B displays a cell count field obtained after pretreatment with 100 μmolar of RNS peptide followed by a migration time of 12 hours.
FIG. 4A displays a cell count field obtained after pretreatment with 50 μmolar of MANS-related peptide BIO-11002 followed by a migration time of 12 hours.
FIG. 4B displays a cell count field obtained after pretreatment with 100 μmolar of MANS-related peptide BIO-11002 followed by a migration time of 12 hours.
FIG. 5A displays a cell count field obtained after pretreatment with 50 μmolar of MANS-related peptide BIO-10901 followed by a migration time of 12 hours.
FIG. 5B displays a cell count field obtained after pretreatment with 100 μmolar of MANS-related peptide BIO-10901 followed by a migration time of 12 hours.
FIG. 6A displays a cell count field obtained after pretreatment with 50 μmolar of MANS-related peptide BIO-91200 followed by a migration time of 12 hours.
FIG. 6B displays a cell count field obtained after pretreatment with 100 μmolar of MANS-related peptide BIO-91200 followed by a migration time of 12 hours.
FIG. 7A graphically displays migrated cell numbers obtained 12 hours after pretreatment at 50 μmolar with MANS peptide, BIO11002, BIO10901, BIO91200, or RNS peptide, or no peptide (control).
FIG. 7B graphically displays migrated cell numbers obtained 12 hours after pretreatment at 100 μmolar with MANS peptide, BIO11002, BIO10901, BIO91200, or RNS peptide, or no peptide (control).
FIG. 8A graphically displays the migration index of aggressive human NSCLC cell line cells, wherein a higher value of the migration index signifies less migration after pretreatment with peptides of the invention at 50 μmolar followed by 12 hours of treatment according to the protocol.
FIG. 8B graphically displays the migration index of aggressive human NSCLC cell line cells, wherein a higher value of the migration index signifies less migration after pretreatment with peptides of the invention at 100 μmolar followed by 12 hours of treatment according to the protocol.
FIG. 9 graphically displays respective cell numbers (left panel) and migration index number (right panel) for MANS, RNS, BIO-11000, or BIO-11006, or control (no peptide) in experiments using 50 μmolar of the indicated peptide and the aggressive human NSCLC cell line.
FIG. 10 graphically displays respective cell numbers (left panel) and migration index number (right panel) for MANS, RNS, BIO-11000, BIO-11006, BIO-91200, or control (no peptide) in experiments using 100 μmolar of the indicated peptide and the aggressive human NSCLC cell line.
FIG. 11 shows the migrated cell numbers 12 hours after pretreatment of A549 cells with no peptide or with the indicated test peptide (MANS, RNS, BIO-11006, BIO-11000, BIO-11002, BIO-91200, or BIO-10901) at 10 μmolar (top left panel), 25 μmolar (top right panel) or 50 μmolar (bottom panel) of peptide.
FIG. 12 shows the average number of tumors per mouse in the left lung, right lung, heart, and diaphragm in animals treated with BIO-11006. BIO-11006 (100 μM in PBS) was administered once daily for 22 days, starting at 3 days post cancer cell inoculation, via intraperitoneal injection (50 μL) or inhalation (30 mins, Nebulizer Delivery System, Aeroneb Lab).
FIG. 13 shows the average number of tumors per mouse in the left lung, right lung, heart, and diaphragm in mice administered BIO-11006 (100 μM in PBS) by inhalation using a Nebulizer Delivery System (Aeroneb Lab) over 30 days, once daily beginning on Day 15 or on Day 4 relative to injection of human adenocarcinoma cells (PC-9).
FIG. 14 shows the total number of tumors in mice administered BIO-11006 (100 μM in PBS) by inhalation using a Nebulizer Delivery System (Aeroneb Lab) over 30 days, once daily beginning on Day 15 or on Day 4 relative to injection of PC-9 cells.
FIG. 15 depicts the number of metastatic nodules found in mice treated with every other day with vehicle control, aerosolized BIO-11006 beginning on day −1 or day +3 relative to A549 cancer cell injection, or aerosolized MANS beginning on day −1 or day +3 relative to A549 cancer cell injection. Aerosolized peptides (100 μM in PBS) were administered by inhalation using the Nebulizer Delivery System (Aeroneb Lab). *, p<0.05, statistically significant compared to control group; a, statistically non-significant in comparison among the groups.
FIG. 16 shows the level of MARCKS protein following administration of 100 nM MARCKS siRNA or control siRNA in PC9 cells.
FIG. 17 shows the level of MARCKS protein following administration of 100 nM MARCKS siRNA or control siRNA in A549 cells.
FIG. 18 shows the migration of PC9 cancer cells following treatment with 100 nMMARCKS siRNA or control siRNA.
FIG. 19 shows the migration of A549 cancer cells following treatment with 100 nM MARCKS siRNA or control siRNA.
FIG. 20 shows expression of MARCKS in PC9 cells by Western Blot following treatment with 50 nM negative control (HiPerfect vehicle; lane A) or 50 nM miR21 inhibitor (lane B).
FIG. 21 shows the migration of PC9 cancer cells following miR21 inhibition (50 nM or 100 nM mir-21 inhibitor) or miR21 activation (50 nM or 100 nM miR-21 mimic).



DETAILED DESCRIPTION OF THE INVENTION
Myristoylated alanine-rich protein kinase C substrate (MARCKS) protein has been implicated previously in multiple cell processes. For example, it has been shown that MARCKS protein is involved integrally in cellular secretion, degranulation, migration and gene expression. These studies were based on the ability of a peptide identical to the myristoylated N-terminal sequence of MARCKS protein (i.e., MANS peptide) to affect processes in disparate cell types when the cells were pre-treated with the MANS peptide prior to stimulation. In all of these instances, a missense control peptide (consisting of a random amino acid sequence of the amino acids of the MANS peptide, and which is referred to herein as the RNS peptide) was without effect relative to activity exhibited by MANS peptide.
In one embodiment, the compositions comprise MARCKS-inhibitory compounds such as any type of inhibitory compound known in the art including peptides, polypeptides, antibodies or fragments thereof, and polynucleotides or nucleic acid molecules, such as antisense polynucleotides, aptamers, small interfering RNA (siRNA), micro RNA (miRNA), and short-hairpin RNA (shRNA). In one embodiment, the compositions comprise one or more myristoylated alanine-rich C-kinase substrate (MARCKS)-related peptides. In another embodiment, the MARCKS-related peptides correspond to the MH2 domain of MARCKS. In another embodiment, the compositions comprise MARCKS-inhibitory peptides, including peptides corresponding to the N-terminal sequence.
In one embodiment, the MARCKS-inhibitory compound provided herein is an antibody. As used herein, the term “antibody” refers to a binding protein having at least one antigen-binding domain and includes monoclonal antibodies, polyclonal antibodies, and antibody fragments and/or variants, including recombinant polypeptides, fusion proteins, and immunoconjugates. Examples of antibody fragments of the invention include, but are not limited to, the Fab fragment, the Fc fragment, the Fv fragment, the dAb fragment, isolated CDR regions, F(ab′)2, bivalent fragments comprising two linked Fab fragments, and single chain Fv molecules (scFv). The skilled artisan will recognize that antibodies or fragments provided herein may be generated from any species including, but not limited to, mouse, rat, rabbit, primate, llama and human. The skilled artisan will further recognize that the antibodies or fragments provided herein may be chimeric, humanized, or fully human.
In one aspect, compositions and methods for treating or preventing cancer are provided. Methods for treating or preventing cancer disclosed herein include treating or preventing all aspects of cancer including, but not limited to, metastasis, tumor growth, cancer cell proliferation, and angiogenesis. In one embodiment, compositions and methods are provided for treating or preventing cancer comprising administration of a MARCKS-inhibitory compound to a cancer cell or to a cell that plays a role in the development, maintenance, proliferation, or metastasis of cancer cells, such as, for example, an endothelial cell.
In one aspect, compositions and methods for inhibiting the metastasis of cancer cells are provided, wherein the method comprises administering MARCKS-inhibitory compounds. In one embodiment, the MARCKS-inhibitory compounds are MARCKS-inhibitory peptides, antibodies or fragments thereof that bind to MARCKS or MARCKS peptides, or polynucleotides or nucleic acid molecules including antisense polynucleotides, aptamers, siRNA, miRNA, and shRNA that inhibit the functions of the MARCKS protein. In a further embodiment, the peptides are MANS-related peptides, wherein the peptides inhibit the metastasis of cancer cells. In one embodiment, MARCKS-inhibitory peptides that inhibit the metastasis of cancer cells are provided. In another aspect, methods of treating cancers using the compositions disclosed herein are provided. In one embodiment, the methods provided comprise contacting a cancer cell with a MARCKS-inhibitory peptide. In a further embodiment, the cancer cell is present in a tumor. In one embodiment, the MARCKS-inhibitory peptide is MANS-related peptide. As used herein, the term “MANS-related peptide” refers to MANS peptide or a peptide substantially identical to MANS; or a fragment of MANS peptide that contains at least four contiguous amino acids found in MANS peptide, or is substantially identical to a peptide containing at least 4 contiguous amino acids found in MANS peptide. Thus, MANS-related peptides are from 4 to 24 amino acids in length. As used herein, the term “substantially identical” means, with respect to comparison of the amino acid sequences of two peptides or comparison of the amino acid sequences of two peptide segments (e.g., segments of a reference peptide amino acid sequence), that the amino acid sequence of the peptides or segments of peptides have at least about 75% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or at least about 95% sequence identity. Preferably, the amino acid sequence of the peptides have at least about 80% sequence identity to MANS peptide or the MANS peptide fragment. In one embodiment, the MANS-related peptide may comprise a peptide from 4 to 24 amino acids in length that is identical to or substantially identical to MANS peptide and may further comprise one or more additional amino acids. For example, in one embodiment, the MANS-related peptide comprises from 4 to 24 contiguous amino acids identical to or substantially identical to MANS peptide and further comprises at least one N-terminal amino acid that is not present in MANS peptide such as, for example, arginine.
In one embodiment, the MARCKS-inhibitory peptide is chemically modified. In one embodiment, the MARCKS-inhibitory peptide is a MANS-related peptide that is acylated at the N-terminal position. In a further embodiment, the MANS-related peptide is acylated with an acetyl group at the N-terminal position. In another embodiment, the MANS-related peptide is myristoylated at the N-terminal position. In another embodiment, the MANS-related peptide is chemically modified at the C-terminal position. In a further embodiment, the MANS-related peptide is chemically modified at the C-terminal position by formation of an amide with an amine (e.g., ammonia). In another embodiment, the MANS-related peptide is chemically modified at both the N-terminal and C-terminal position. Table 1 lists peptides relevant to the current invention which are myristoylated at their N-terminal position but unsubstituted at their C-terminal position. Certain control peptides (RNS peptides) are listed in Tables 1 and 2, and are myristoylated. However, the RNS peptides are not considered to reside within the scope of the current invention.







TABLE 1







MANS-related peptides of the invention which are N-terminal myristoylated and


which can be further chemically modified at the C-terminal position as described herein










Peptide No.
N-myristoyl Amino Acid Sequences
SEQ ID No:






peptide 1
GAQFSKTAAKGEAAAERPGEAAVA
SEQ ID NO: 1
MANS


 


peptide 2
GAQFSKTAAKGEAAAERPGEAAV
SEQ ID NO: 2



 


peptide 4
GAQFSKTAAKGEAAAERPGEAA
SEQ ID NO: 4



 


peptide 7
GAQFSKTAAKGEAAAERPGEA
SEQ ID NO: 7



 


peptide 11
GAQFSKTAAKGEAAAERPGE
SEQ ID NO: 11



 


peptide 16
GAQFSKTAAKGEAAAERPG
SEQ ID NO: 16



 


peptide 22
GAQFSKTAAKGEAAAERP
SEQ ID NO: 22



 


peptide 29
GAQFSKTAAKGEAAAER
SEQ ID NO: 29



 


peptide 37
GAQFSKTAAKGEAAAE
SEQ ID NO: 37



 


peptide 46
GAQFSKTAAKGEAAA
SEQ ID NO: 46



 


peptide 56
GAQFSKTAAKGEAA
SEQ ID NO: 56



 


peptide 67
GAQFSKTAAKGEA
SEQ ID NO: 67



 


peptide 79
GAQFSKTAAKGE
SEQ ID NO: 79



 


peptide 92
GAQFSKTAAKG
SEQ ID NO: 92



 


peptide 106
GAQFSKTAAK
SEQ ID NO: 106



 


peptide 121
GAQFSKTAA
SEQ ID NO: 121



 


peptide 137
GAQFSKTA
SEQ ID NO: 137



 


peptide 154
GAQFSKT
SEQ ID NO: 154



 


peptide 172
GAQFSK
SEQ ID NO: 172



 


peptide 191
GAQFS
SEQ ID NO: 191



 


peptide 211
GAQF
SEQ ID NO: 211



 


Peptide 232
GTAPAAEGAGAEVKRASAEAKQAF
SEQ ID NO: 232
RNS









Table 2 lists MANS-related peptide fragments of the invention, which can be substituted or chemically modified at the N-terminal and/or C-terminal position. In one embodiment, these active fragments of MANS peptide may be myristoylated at the N-terminal position as are those in Table 1. In another embodiment, chemical modification at the C-terminal position comprises amidation, for example, formation of an amide with an amine, such as, for example, ammonia. Peptide 234 (SEQ ID NO: 234) is an N-terminal arginine-substituted peptide homolog of peptide 106 (SEQ ID NO: 106; RGAQFSKTAAK), which may be chemically modified at the N-terminus (e.g., N-terminal acetyl analog, Ac-RGAQFSKTAAK), and which also may be chemically modified at its N-terminus and its C-terminus (e.g., N-terminal acetyl-, —C-terminal amide with ammonia analog, Ac-RGAQFSKTAAK-NH2). Peptide 235 (SEQ ID NO: 235) is an N-terminal arginine-substituted peptide homolog of peptide 219, (SEQ ID NO: 219; RAKGE) which may be chemically modified at the N-terminus (e.g., N-terminal acetyl analog, Ac-RAKGE), and which also may be chemically modified at its N-terminus and its C-terminus (e.g., N-terminal acetyl-, —C-terminal amide with ammonia analog, Ac-RAKGE-NH2). Preferred N-terminal modifications or substitutions include myristoyl and acetyl groups as well as N-terminal arginine groups, N-terminal acetyl-arginine groups, and N-terminal myristoyl-arginine groups. Preferred C-terminal modification includes the amide group from ammonia.







TABLE 2







MARCKS-related peptide sequences which may be chemically modified at the N-


terminal and/or C-terminal position as described herein.










MANS peptide and active MANS-



Table 2
related peptide fragments



Peptide No.
Amino Acid Sequence
SEQ ID No:













peptide 1
GAQFSKTAAKGEAAAERPGEAAVA
SEQ ID NO: 1
MANS


 


peptide 2
GAQFSKTAAKGEAAAERPGEAAV
SEQ ID NO: 2



 


peptide 3
AQFSKTAAKGEAAAERPGEAAVA
SEQ ID NO: 3



 


peptide 4
GAQFSKTAAKGEAAAERPGEAA
SEQ ID NO: 4



 


peptide 5
AQFSKTAAKGEAAAERPGEAAV
SEQ ID NO: 5



 


peptide 6
QFSKTAAKGEAAAERPGEAAVA
SEQ ID NO: 6



 


peptide 7
GAQFSKTAAKGEAAAERPGEA
SEQ ID NO: 7



 


peptide 8
AQFSKTAAKGEAAAERPGEAA
SEQ ID NO: 8



 


peptide 9
QFSKTAAKGEAAAERPGEAAV
SEQ ID NO: 9



 


peptide 10
FSKTAAKGEAAAERPGEAAVA
SEQ ID NO: 10



 


peptide 11
GAQFSKTAAKGEAAAERPGE
SEQ ID NO: 11



 


peptide 12
AQFSKTAAKGEAAAERPGEA
SEQ ID NO: 12



 


peptide 13
QFSKTAAKGEAAAERPGEAA
SEQ ID NO: 13



 


peptide 14
FSKTAAKGEAAAERPGEAAV
SEQ ID NO: 14



 


peptide 15
SKTAAKGEAAAERPGEAAVA
SEQ ID NO: 15



 


peptide 16
GAQFSKTAAKGEAAAERPG
SEQ ID NO: 16



 


peptide 17
AQFSKTAAKGEAAAERPGE
SEQ ID NO: 17



 


peptide 18
QFSKTAAKGEAAAERPGEA
SEQ ID NO: 18



 


peptide 19
FSKTAAKGEAAAERPGEAA
SEQ ID NO: 19



 


peptide 20
SKTAAKGEAAAERPGEAAV
SEQ ID NO: 20



 


peptide 21
KTAAKGEAAAERPGEAAVA
SEQ ID NO: 21



 


peptide 22
GAQFSKTAAKGEAAAERP
SEQ ID NO: 22



 


peptide 23
AQFSKTAAKGEAAAERPG
SEQ ID NO: 23



 


peptide 24
QFSKTAAKGEAAAERPGE
SEQ ID NO: 24



 


peptide 25
FSKTAAKGEAAAERPGEA
SEQ ID NO: 25



 


peptide 26
SKTAAKGEAAAERPGEAA
SEQ ID NO: 26



 


peptide 27
KTAAKGEAAAERPGEAAV
SEQ ID NO: 27



 


peptide 28
TAAKGEAAAERPGEAAVA
SEQ ID NO: 28



 


peptide 29
GAQFSKTAAKGEAAAER
SEQ ID NO: 29



 


peptide 30
AQFSKTAAKGEAAAERP
SEQ ID NO: 30



 


peptide 31
QFSKTAAKGEAAAERPG
SEQ ID NO: 31



 


peptide 32
FSKTAAKGEAAAERPGE
SEQ ID NO: 32



 


peptide 33
SKTAAKGEAAAERPGEA
SEQ ID NO: 33



 


peptide 34
KTAAKGEAAAERPGEAA
SEQ ID NO: 34



 


peptide 35
TAAKGEAAAERPGEAAV
SEQ ID NO: 35



 


peptide 36
AAKGEAAAERPGEAAVA
SEQ ID NO: 36



 


peptide 37
GAQFSKTAAKGEAAAE
SEQ ID NO: 37



 


peptide 38
AQFSKTAAKGEAAAER
SEQ ID NO: 38



 


peptide 39
QFSKTAAKGEAAAERP
SEQ ID NO: 39



 


peptide 40
FSKTAAKGEAAAERPG
SEQ ID NO: 40



 


peptide 41
SKTAAKGEAAAERPGE
SEQ ID NO: 41



 


peptide 42
KTAAKGEAAAERPGEA
SEQ ID NO: 42



 


peptide 43
TAAKGEAAAERPGEAA
SEQ ID NO: 43



 


peptide 44
AAKGEAAAERPGEAAV
SEQ ID NO: 44



 


peptide 45
AKGEAAAERPGEAAVA
SEQ ID NO: 45



 


peptide 46
GAQFSKTAAKGEAAA
SEQ ID NO: 46



 


peptide 47
AQFSKTAAKGEAAAE
SEQ ID NO: 47



 


peptide 48
QFSKTAAKGEAAAER
SEQ ID NO: 48



 


peptide 49
FSKTAAKGEAAAERP
SEQ ID NO: 49



 


peptide 50
SKTAAKGEAAAERPG
SEQ ID NO: 50



 


peptide 51
KTAAKGEAAAERPGE
SEQ ID NO: 51



 


peptide 52
TAAKGEAAAERPGEA
SEQ ID NO: 52



 


peptide 53
AAKGEAAAERPGEAA
SEQ ID NO: 53



 


peptide 54
AKGEAAAERPGEAAV
SEQ ID NO: 54



 


peptide 55
KGEAAAERPGEAAVA
SEQ ID NO: 55



 


peptide 56
GAQFSKTAAKGEAA
SEQ ID NO: 56



 


peptide 57
AQFSKTAAKGEAAA
SEQ ID NO: 57



 


peptide 58
QFSKTAAKGEAAAE
SEQ ID NO: 58



 


peptide 59
FSKTAAKGEAAAER
SEQ ID NO: 59



 


peptide 60
SKTAAKGEAAAERP
SEQ ID NO: 60



 


peptide 61
KTAAKGEAAAERPG
SEQ ID NO: 61



 


peptide 62
TAAKGEAAAERPGE
SEQ ID NO: 62



 


peptide 63
AAKGEAAAERPGEA
SEQ ID NO: 63



 


peptide 64
AKGEAAAERPGEAA
SEQ ID NO: 64



 


peptide 65
KGEAAAERPGEAAV
SEQ ID NO: 65



 


peptide 66
GEAAAERPGEAAVA
SEQ ID NO: 66



 


peptide 67
GAQFSKTAAKGEA
SEQ ID NO: 67



 


peptide 68
AQFSKTAAKGEAA
SEQ ID NO: 68



 


peptide 69
QFSKTAAKGEAAA
SEQ ID NO: 69



 


peptide 70
FSKTAAKGEAAAE
SEQ ID NO: 70



 


peptide 71
SKTAAKGEAAAER
SEQ ID NO: 71



 


peptide 72
KTAAKGEAAAERP
SEQ ID NO: 72



 


peptide 73
TAAKGEAAAERPG
SEQ ID NO: 73



 


peptide 74
AAKGEAAAERPGE
SEQ ID NO: 74



 


peptide 75
AKGEAAAERPGEA
SEQ ID NO: 75



 


peptide 76
KGEAAAERPGEAA
SEQ ID NO: 76



 


peptide 77
GEAAAERPGEAAV
SEQ ID NO: 77



 


peptide 78
EAAAERPGEAAVA
SEQ ID NO: 78



 


peptide 79
GAQFSKTAAKGE
SEQ ID NO: 79



 


peptide 80
AQFSKTAAKGEA
SEQ ID NO: 80



 


peptide 81
QFSKTAAKGEAA
SEQ ID NO: 81



 


peptide 82
FSKTAAKGEAAA
SEQ ID NO: 82



 


peptide 83
SKTAAKGEAAAE
SEQ ID NO: 83



 


peptide 84
KTAAKGEAAAER
SEQ ID NO: 84



 


peptide 85
TAAKGEAAAERP
SEQ ID NO: 85



 


peptide 86
AAKGEAAAERPG
SEQ ID NO: 86



 


peptide 87
AKGEAAAERPGE
SEQ ID NO: 87



 


peptide 88
KGEAAAERPGEA
SEQ ID NO: 88



 


peptide 89
GEAAAERPGEAA
SEQ ID NO: 89



 


peptide 90
EAAAERPGEAAV
SEQ ID NO: 90



 


peptide 91
AAAERPGEAAVA
SEQ ID NO: 91



 


peptide 92
GAQFSKTAAKG
SEQ ID NO: 92



 


peptide 93
AQFSKTAAKGE
SEQ ID NO: 93



 


peptide 94
QFSKTAAKGEA
SEQ ID NO: 94



 


peptide 95
FSKTAAKGEAA
SEQ ID NO: 95



 


peptide 96
SKTAAKGEAAA
SEQ ID NO: 96



 


peptide 97
KTAAKGEAAAE
SEQ ID NO: 97



 


peptide 98
TAAKGEAAAER
SEQ ID NO: 98



 


peptide 99
AAKGEAAAERP
SEQ ID NO: 99



 


peptide 100
AKGEAAAERPG
SEQ ID NO: 100



 


peptide 101
KGEAAAERPGE
SEQ ID NO: 101



 


peptide 102
GEAAAERPGEA
SEQ ID NO: 102



 


peptide 103
EAAAERPGEAA
SEQ ID NO: 103



 


peptide 104
AAAERPGEAAV
SEQ ID NO: 104



 


peptide 105
AAERPGEAAVA
SEQ ID NO: 105



 


peptide 106
GAQFSKTAAK
SEQ ID NO: 106



 


peptide 107
AQFSKTAAKG
SEQ ID NO: 107



 


peptide 108
QFSKTAAKGE
SEQ ID NO: 108



 


peptide 109
FSKTAAKGEA
SEQ ID NO: 109



 


peptide 110
SKTAAKGEAA
SEQ ID NO: 110



 


peptide 111
KTAAKGEAAA
SEQ ID NO: 111



 


peptide 112
TAAKGEAAAE
SEQ ID NO: 112



 


peptide 113
AAKGEAAAER
SEQ ID NO: 113



 


peptide 114
AKGEAAAERP
SEQ ID NO: 114



 


peptide 115
KGEAAAERPG
SEQ ID NO: 115



 


peptide 116
GEAAAERPGE
SEQ ID NO: 116



 


peptide 117
EAAAERPGEA
SEQ ID NO: 117



 


peptide 118
AAAERPGEAA
SEQ ID NO: 118



 


peptide 119
AAERPGEAAV
SEQ ID NO: 119



 


peptide 120
AERPGEAAVA
SEQ ID NO: 120



 


peptide 121
GAQFSKTAA
SEQ ID NO: 121



 


peptide 122
AQFSKTAAK
SEQ ID NO: 122



 


peptide 123
QFSKTAAKG
SEQ ID NO: 123



 


peptide 124
FSKTAAKGE
SEQ ID NO: 124



 


peptide 125
SKTAAKGEA
SEQ ID NO: 125



 


peptide 126
KTAAKGEAA
SEQ ID NO: 126



 


peptide 127
TAAKGEAAA
SEQ ID NO: 127



 


peptide 128
AAKGEAAAE
SEQ ID NO: 128



 


peptide 129
AKGEAAAER
SEQ ID NO: 129



 


peptide 130
KGEAAAERP
SEQ ID NO: 130



 


peptide 131
GEAAAERPG
SEQ ID NO: 131



 


peptide 132
EAAAERPGE
SEQ ID NO: 132



 


peptide 133
AAAERPGEA
SEQ ID NO: 133



 


peptide 134
AAERPGEAA
SEQ ID NO: 134



 


peptide 135
AERPGEAAV
SEQ ID NO: 135



 


peptide 136
ERPGEAAVA
SEQ ID NO: 136



 


peptide 137
GAQFSKTA
SEQ ID NO: 137



 


peptide 138
AQFSKTAA
SEQ ID NO: 138



 


peptide 139
QFSKTAAK
SEQ ID NO: 139



 


peptide 140
FSKTAAKG
SEQ ID NO: 140



 


peptide 141
SKTAAKGE
SEQ ID NO: 141



 


peptide 142
KTAAKGEA
SEQ ID NO: 142



 


peptide 143
TAAKGEAA
SEQ ID NO: 143



 


peptide 144
AAKGEAAA
SEQ ID NO: 144



 


peptide 145
AKGEAAAE
SEQ ID NO: 145



 


peptide 146
KGEAAAER
SEQ ID NO: 146



 


peptide 147
GEAAAERP
SEQ ID NO: 147



 


peptide 148
EAAAERPG
SEQ ID NO: 148



 


peptide 149
AAAERPGE
SEQ ID NO: 149



 


peptide 150
AAERPGEA
SEQ ID NO: 150



 


peptide 151
AERPGEAA
SEQ ID NO: 151



 


peptide 152
ERPGEAAV
SEQ ID NO: 152



 


peptide 153
RPGEAAVA
SEQ ID NO: 153



 


peptide 154
GAQFSKT
SEQ ID NO: 154



 


peptide 155
AQFSKTA
SEQ ID NO: 155



 


peptide 156
QFSKTAA
SEQ ID NO: 156



 


peptide 157
FSKTAAK
SEQ ID NO: 157



 


peptide 158
SKTAAKG
SEQ ID NO: 158



 


peptide 159
KTAAKGE
SEQ ID NO: 159



 


peptide 160
TAAKGEA
SEQ ID NO: 160



 


peptide 161
AAKGEAA
SEQ ID NO: 161



 


peptide 162
AKGEAAA
SEQ ID NO: 162



 


peptide 163
KGEAAAE
SEQ ID NO: 163



 


peptide 164
GEAAAER
SEQ ID NO: 164



 


peptide 165
EAAAERP
SEQ ID NO: 165



 


peptide 166
AAAERPG
SEQ ID NO: 166



 


peptide 167
AAERPGE
SEQ ID NO: 167



 


peptide 168
AERPGEA
SEQ ID NO: 168



 


peptide 169
ERPGEAA
SEQ ID NO: 169



 


peptide 170
RPGEAAV
SEQ ID NO: 170



 


peptide 171
PGEAAVA
SEQ ID NO: 171



 


peptide 172
GAQFSK
SEQ ID NO: 172



 


peptide 173
AQFSKT
SEQ ID NO: 173



 


peptide 174
QFSKTA
SEQ ID NO: 174



 


peptide 175
FSKTAA
SEQ ID NO: 175



 


peptide 176
SKTAAK
SEQ ID NO: 176



 


peptide 177
KTAAKG
SEQ ID NO: 177



 


peptide 178
TAAKGE
SEQ ID NO: 178



 


peptide 179
AAKGEA
SEQ ID NO: 179



 


peptide 180
AKGEAA
SEQ ID NO: 180



 


peptide 181
KGEAAA
SEQ ID NO: 181



 


peptide 182
GEAAAE
SEQ ID NO: 182



 


peptide 183
EAAAER
SEQ ID NO: 183



 


peptide 184
AAAERP
SEQ ID NO: 184



 


peptide 185
AAERPG
SEQ ID NO: 185



 


peptide 186
AERPGE
SEQ ID NO: 186



 


peptide 187
ERPGEA
SEQ ID NO: 187



 


peptide 188
RPGEAA
SEQ ID NO: 188



 


peptide 189
PGEAAV
SEQ ID NO: 189



 


peptide 190
GEAAVA
SEQ ID NO: 190



 


peptide 191
GAQFS
SEQ ID NO: 191



 


peptide 192
AQFSK
SEQ ID NO: 192



 


peptide 193
QFSKT
SEQ ID NO: 193



 


peptide 194
FSKTA
SEQ ID NO: 194



 


peptide 195
SKTAA
SEQ ID NO: 195



 


peptide 196
KTAAK
SEQ ID NO: 196



 


peptide 197
TAAKG
SEQ ID NO: 197



 


peptide 198
AAKGE
SEQ ID NO: 198



 


peptide 199
AKGEA
SEQ ID NO: 199



 


peptide 200
KGEAA
SEQ ID NO: 200



 


peptide 201
GEAAA
SEQ ID NO: 201



 


peptide 202
EAAAE
SEQ ID NO: 202



 


peptide 203
AAAER
SEQ ID NO: 203



 


peptide 204
AAERP
SEQ ID NO: 204



 


peptide 205
AERPG
SEQ ID NO: 205



 


peptide 206
ERPGE
SEQ ID NO: 206



 


peptide 207
RPGEA
SEQ ID NO: 207



 


peptide 208
PGEAA
SEQ ID NO: 208



 


peptide 209
GEAAV
SEQ ID NO: 209



 


peptide 210
EAAVA
SEQ ID NO: 210



 


peptide 211
GAQF
SEQ ID NO: 211



 


peptide 212
AQFS
SEQ ID NO: 212



 


peptide 213
QFSK
SEQ ID NO: 213



 


peptide 214
FSKT
SEQ ID NO: 214



 


peptide 215
SKTA
SEQ ID NO: 215



 


peptide 216
KTAA
SEQ ID NO: 216



 


peptide 217
TAAK
SEQ ID NO: 217



 


peptide 218
AAKG
SEQ ID NO: 218



 


peptide 219
AKGE
SEQ ID NO: 219



 


peptide 220
KGEA
SEQ ID NO: 220



 


peptide 221
GEAA
SEQ ID NO: 221



 


peptide 222
EAAA
SEQ ID NO: 222



 


peptide 223
AAAE
SEQ ID NO: 223



 


peptide 224
AAER
SEQ ID NO: 224



 


peptide 225
AERP
SEQ ID NO: 225



 


peptide 226
ERPG
SEQ ID NO: 226



 


peptide 227
RPGE
SEQ ID NO: 227



 


peptide 228
PGEA
SEQ ID NO: 228



 


peptide 229
GEAA
SEQ ID NO: 229



 


peptide 230
EAAV
SEQ ID NO: 230



 


peptide 231
AAVA
SEQ ID NO: 231



 


Peptide 232
GTAPAAEGAGAEVKRASAEAKQAF
SEQ ID NO: 232
RNS


 


Peptide 233
GKASQFAKTA
SEQ ID NO: 233
RNS2


 


Peptide 234
RGAQFSKTAAK
SEQ ID NO: 234



 


Peptide 235
RAKGE
SEQ ID NO: 235









The MANS peptide is myristoylated (denoted as MA), and contains the 24 amino acid sequence MA-GAQFSKTAAKGEAAARPGEAAVA. Without wishing to be bound by theory, the peptide is theorized to interfere with the full length MARCKS protein attaching naturally to the cell membrane and interfering with phosphorylation of MARCKS protein by protein kinase C (PKC).
MANS peptide has been shown to provide a significant reduction in degranulation of goblet cells both in vitro and in vivo. MANS also affects the rate of migration of neutrophils and mesenchymal stem cells. Degranulation of human leukocytes is also inhibited by MANS. In one aspect of this invention, treatment of certain cancer cell lines with MANS-related peptides decreases migration of those cancer cell lines. In one embodiment, MANS-related peptides exhibit inhibition of metastasis of cancer cells. Thus, in one embodiment, the MANS-related peptides provided may be used to treat or prevent metastatic cancer in a subject in need thereof. In some embodiments, MANS-related peptides exhibit properties that make them suitable for use in therapeutic applications, for example, in the treatment of cancers. For example, in one embodiment, MANS-related peptides exhibit enhanced solubility relative to MANS peptide. In another embodiment, some MANS-related peptides exhibit longer half-lives in plasma than the MANS peptide or relative to peptides other than MANS-related peptides. In one embodiment, MANS-related peptides may be useful in the treatment of proliferation of cancer cells. For example, in one embodiment, MANS-related peptides may inhibit proliferation and or migration of cancer cells. In another embodiment, some MANS-related peptides may provide greater inhibition of proliferation and or migration of cancer cells at lower concentrations than the MANS peptide or than other MANS-related peptides.
In one aspect, the MANS-related peptide is selected from the group consisting of:









(SEQ ID No: 106; BIO-11000)



N-myristoyl-GAQFSKTAAK



 



(SEQ ID No: 106; BIO-11006)



N-acetyl-GAQFSKTAAK;



 



(SEQ ID No: 219; BIO-91200)



N-myristoyl-AKGE;



 



(SEQ ID No: 106; BIO-11002)



N-myristoyl-GAQFSKTAAK-NH2;



 



(SEQ ID No. 121; BIO-10901)



N-acetyl-GAQFSKTAA;



 



(SEQ ID No: 106; BIO-11026)



N-acetyl-GAQFSKTAAK-NH2;



 



(SEQ ID No: 106; BIO-11037)



N-acetyl-GAQFS(d)KTAA(d)K (Lys at positions



 



positions 6 and 10 of the peptide are of d-



 



configuration;



 



(SEQ ID No: 234; BIO-11027)



N-acetyl-RGAQFSKTAAK;



 



(SEQ ID No: 234; BIO-11028)



N-acetyl-RGAQFSKTAAK-NH2;



and



 



(SEQ ID NO: 235; BIO-91204)



N-acetyl-RAKGE.






In one aspect, peptides having 4 to 24 amino acids and which have amino acid sequences that are identical to or are substantially identical to amino acid sequences found in the MANS peptide can be useful in one or more aspects of this invention. These peptides are herein referred to as MANS-related peptides, and exemplary MANS-related peptides are listed in Table 2 as SEQ ID NOs: 1 to 231, 234, and 235. Table 2 also includes the amino acid sequence of the random sequence (RNS) peptide No. 232 (SEQ ID NO: 232), which is used as a control and to demonstrate that amino acid sequence order can be relevant to efficacy in this invention, as well as a second random sequence control peptide 233 (RNS2; SEQ ID NO: 233). Peptides 234 and 235 (SEQ ID NO: 234 and 235) are N-terminal arginine-substituted peptide homologs of peptides 106 and 219, respectively. The arginine can be acylated with, for example, an acetyl group or a myristoyl group.
In one embodiment, peptides which can be useful in the current invention can be selected from the group consisting of synthetic peptides having amino acid sequences listed in Table 2 (excluding random sequence peptides 232 and 233).
In another embodiment, peptides which can be useful in the current invention can be selected from peptides of amino acid sequences as listed in Table 2 (SEQ ID NO: 1 to 231 (inclusive), 234, and 235) and which are optionally N-terminal- and/or C-terminal-chemically modified.
Preferred independent N-terminal chemical modifications of the peptides listed in Table 2 include N-terminal amine group modification by acylation of the N-terminal amino acid of the peptide in the form of an amide selected from the group consisting of:

    
    
        an amide of a C2 (acetyl) to C24 aliphatic carboxylic acid which may be linear, branched, saturated, or unsaturated,
        an amide of trifluoroacetic acid,
        an amide of a benzoic acid, and
        an amide of a C1 to C24 aliphatic alkyl sulfonic acid; or
        the N-terminal amine group of the N-terminal amino acid of the peptide can be alkylated with a group selected from the group consisting of:
        a C1 (methyl) to C24 aliphatic alkyl group,
        a linear 2-(C1 to C24 aliphatic alkyl)oxyethyl group,
        an omega-methoxy-poly(ethyleneoxy)n-ethyl group, where n is from 0 to 10.
    
    


Preferred independent C-terminal chemical modifications of the peptides listed in Table 2 include amide formation at the C-terminal carboxylic acid group of the C-terminal amino acid of the peptide in the form of an amide selected from the group consisting of:

    
    
        an amide of ammonia,
        an amide of a C1 to C24 aliphatic alkyl amine,
        an amide of a hydroxyl-substituted C2 to C24 aliphatic alkyl amine,
        an amide of a linear 2-(C1 to C24 aliphatic alkyl)oxyethylamine group, and
        an amide of an omega-methoxy-poly(ethyleneoxy)n-ethylamine group, where n is from 0 to 10.
    
    


In addition, the C-terminal carboxylic acid group of the C-terminal amino acid of the peptide is optionally in the form of an ester selected from the group consisting of:

    
    
        an ester of a C1 to C24 aliphatic alkyl alcohol,
        an ester of a 2-(omega-methoxy-poly(ethyleneoxy)n)-ethanol group, where n is from 0 to 10, and
        an ester of a linear PEG-amine, the PEG component of molecular weight from 1,000 to 25,000 Daltons.
    
    


In one embodiment, aliphatic portions of groups such as carboxylic acid groups and sulfonic acid groups and alcohol and amino groups can comprise a ring of at least C3 (i.e., at least a cyclopropyl ring).
In one embodiment, the peptide can be N-terminally modified, for example, by an acetyl group or a myristoyl group, as an N-terminal amide, such as Acetyl-GAQFSKTAAK (N-terminal acetyl SEQ ID No: 106) and myristoyl-GAQFSKTAAK (N-terminal myristoyl SEQ ID No: 106), respectively. In another embodiment, the peptide can be C-terminally modified (for example by an amide with ammonia) such as GAQFSKTAAK-NH2 (SEQ ID No: 106 C-terminal amide). In another embodiment, the peptide can be N-terminally modified and C-terminally modified, for example as N-acetyl-peptide-C-amide (with ammonia) such as Acetyl-GAQFSKTAAK-NH2. (N-terminal acetyl SEQ ID No: 106 C-terminal amide) and Myristoyl-GAQFSKTAAK-NH2 (N-terminal myristoyl SEQ ID No: 106 C-terminal amide). These peptides can be used in the methods of this invention and to determine their ability to inhibit metastases of cancer cells.
In one embodiment, a peptide which may find use in this invention can be selected from the group of peptides which contain the amino acid sequence AKGE (SEQ ID No: 219). Such peptides include SEQ ID No: 1 through SEQ ID No: 54, SEQ ID No: 56 through SEQ ID No: 64, SEQ ID No: 67 through SEQ ID No: 75, SEQ ID No: 79 through SEQ ID No: 87, SEQ ID No: 93 through SEQ ID No: 100, SEQ ID No: 108 through SEQ ID No: 114, SEQ ID No: 124 through SEQ ID No: 129, SEQ ID No: 141 through SEQ ID No: 145, SEQ ID No: 159 through SEQ ID No: 162, SEQ ID No: 178 through SEQ ID No: 180, SEQ ID No: 198, SEQ ID No: 199, SEQ ID No: 219, and SEQ ID NO: 235. In one currently preferred embodiment, these peptides are myristoylated or acetylated at the N-terminal amino group.
In one embodiment, this invention discloses a method of attenuating the metastasis of a cancer cell toward an increasing concentration gradient of a chemotactic agent in a fluid or tissue, the method comprising treatment of said cancer cell with a migration-inhibiting amount of a migration-modulating peptide and incubation of said cell with said peptide to form a migration-inhibited cancer cell, wherein the peptide is a MANS-related peptide.
In one aspect, the migration-modulating peptide is selected from the group consisting of MANS-related peptides. In another aspect, the MANS-related peptide comprises the amino acid sequence GAQFSKTAAK (SEQ ID No: 106).
In another aspect, the MANS-related peptide is selected from the group consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID No: 1) N-myristoyl-GAQFSKTAAKGEAAAERPGEAAV (SEQ ID No: 2); N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID No: 4); N-myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID No: 7); N-myristoyl-GAQFSKTAAKGEAAAERPGE (SEQ ID No: 11); N-myristoyl-GAQFSKTAAKGEAAAERPG (SEQ ID No: 16); N-myristoyl-GAQFSKTAAKGEAAAERP (SEQ ID No: 22); N-myristoyl-GAQFSKTAAKGEAAAER (SEQ ID No: 29); N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID No: 37); N-myristoyl-GAQFSKTAAKGEAAA (SEQ ID No: 46); N-myristoyl-GAQFSKTAAKGEAA (SEQ ID No: 56); N-myristoyl-GAQFSKTAAKGEA (SEQ ID No: 67); N-myristoyl-GAQFSKTAAKGE (SEQ ID No: 79); N-myristoyl-GAQFSKTAAKG (SEQ ID No: 92); N-myristoyl-GAQFSKTAAK (SEQ ID No: 106); N-myristoyl-GAQFSKTAA (SEQ ID No: 121); N-myristoyl-GAQFSKTA (SEQ ID No: 137); N-myristoyl-GAQFSKT (SEQ ID No: 154); N-myristoyl-GAQFSK (SEQ ID No: 172), N-myristoyl-GAQFS (SEQ ID No: 191), N-myristoyl-GAQF (SEQ ID No: 211), N-acetyl-RGAQFSKTAAK (SEQ ID No: 234), N-acetyl-RGAQFSKTAAK-NH2 (SEQ ID No: 234), N-acetyl-RAKGE (SEQ ID NO: 235), and a combination thereof.
In another aspect, the MANS-related peptide is selected from the group consisting of:









(SEQ ID No: 1; MANS peptide)



N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA;



 



(SEQ ID No: 106; BIO-11000)



N-myristoyl-GAQFSKTAAK;



 



(SEQ ID No: 106; BIO-11006)



N-acetyl-GAQFSKTAAK;



 



(SEQ ID No: 106; BIO-11026)



N-acetyl-GAQFSKTAAK-NH2;



 



(SEQ ID No: 219; BIO-91200)



N-myristoyl-AKGE;



 



(SEQ ID No: 106; BIO-11002)



N-myristoyl-GAQFSKTAAK-NH2;



 



(SEQ ID No. 121; BIO-10901)



N-acetyl-GAQFSKTAA



 



(SEQ ID No: 234; BIO-11027)



N-acetyl-RGAQFSKTAAK



 



(SEQ ID No: 234; BIO-11028)



N-acetyl-RGAQFSKTAAK-NH2,



and



 



(SEQ ID NO: 235; BIO-91204)



N-acetyl-RAKGE.






In one aspect, the metastasis-inhibiting dose of a peptide of this invention can be in the range of about 0.01 mg/kg/day to about 10 mg/kg/day. In a further embodiment, the metastasis-inhibiting dose of a peptide is about 0.1 mg/kg/day to about 5.0 mg/kg/day. In a yet further embodiment, the metastasis-inhibiting dose of a peptide is about 0.5 mg/kg/day to about 2.5 mg/kg/day. For example, the metastasis-inhibiting dose of a peptide of this invention is about 0.01, about 0.05, about 0.1, about 0.5, about 0.75, about 1.0, about 1.25, about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.5, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, or more mg/kg/day.
In one embodiment, this invention provides a method for inhibiting metastasis of a cancer cell, wherein the administration is by oral, intravenous, intraperitoneal, intramuscular, inhalation, or suppository routes. In another embodiment, this invention discloses a method for inhibiting metastasis of a cancer cell, wherein the administration is by inhalation of a liquid solution or suspension or dry powder formulation of the MARCKS-inhibitory compound. In one embodiment, a method for treating cancer is provided, wherein a MARCKS-inhibitory compound is administered to a subject in need thereof by inhalation of a liquid solution or suspension or dry powder formulation of the MARCKS-inhibitory compound. For example, in one embodiment, a MANS-related peptide is administered to a subject in need thereof by inhalation of a liquid solution or suspension or dry powder formulation of the MANS-related peptide. In another embodiment, a MANS-related peptide is administered by intravenous, intraperitoneal, or intramuscular injection, or by oral or suppository administration.
In another aspect, this invention discloses a method for inhibiting metastasis of a cancer cell, or treating cancer in a subject in need thereof, wherein the MANS-related peptide is administered is by injection of a liquid formulation of the peptide, and wherein the liquid formulation is isotonic, and wherein the liquid or suspension formulation is buffered, and wherein the injection is systemic into a subject. In another embodiment, the injection is into the region of a tumor. In another embodiment, the injection is into the tumor.
In another aspect, this invention discloses a method for inhibiting metastasis of a cancer cell, wherein the cancer cell resides in a mammal. In one embodiment, a method for treating cancer is provided, wherein a MANS-related peptide is administered to the subject in need thereof, and wherein the subject has a tumor. The peptides of this invention can be formulated using one or more pharmaceutically acceptable excipients or ingredients to provide pharmaceutical compositions useful for administration to cancer cells, such as cancer cells in a primary tumor. Such compositions can be as solutions or suspensions in a liquid, especially in buffered solution, wherein phosphate buffer is useful, when administration by injection or by inhalation is useful. Isotonic solutions or suspensions are preferred embodiments.
It is anticipated that administration of an antibody, polynucleotide, nucleic acid molecule, or peptide composition of this invention in mammals such as canines, felines, and human patients, can be effective if done by injection into a primary tumor region (e.g., directly into a primary tumor, or into a margin of a primary tumor, or into a blood vessel feeding a primary tumor) in the mammal, wherein the injection is done at regular intervals (for example, every 1 to every 72 hours), optionally in combination with or separately with one or more other or additional chemotherapy drugs.
One or more additional therapeutic agents, including chemotherapeutic drugs and cancer specific antibodies, can be administered in addition to the MARCKS-inhibitory antibody, polynucleotide, nucleic acid molecule, or peptide formulation, before, during, or after the peptide administration. Exemplary chemotherapeutic drugs include, but are not limited to, carboplatin, cisplatin, oxaliplatin, cyclophosphamide, dacarbazine, temozolomide, gemcitabine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanadine, daunorubicin, doxurubicin, epirubicin, idarubicin, topotecan, irinotecan, etoposide, eniposide, colchicine, vincristine, vinblastine, vinorelbine, paclitaxel, and docetaxel. Exemplary cancer specific agents and antibodies include, but are not limited to, Afatinib, Aldesleukin, Alemtuzumab, Axitinib, Belimumab, Bevacizumab, Bortezomib, Bosutinib, Brentuximab vedotin, Cabozantinib, Canakinumab, Carfilzomib, Cetuximab, Crizotinib, Dabrafenib, Dasatinib, Denosumab, Erlotinib, Everolimus, Gefitinib, Ibritumomab tiuxetan, Ibrutinib, Imatinib, Ipilimumab, Lapatinib, Nilotinib, Obinutuzumab, Ofatumumab, Panitumumab, Pazopanib, Pertuzumab, Ponatinib, Regorafenib, Rituximab, Romidepsin, Ruxolitinib, Sipuleucel-T, Sorafenib, Temsirolimus, Tocilizumab, Tofacitinib, Tositumomab, Trametinib, Trastuzumab, Vandetanib, Vemurafenib, Vismodegib, Vorinostat, and Ziv-aflibercept.
Administration may be, for example, by inhalation as a liquid or dry powder aerosol or spray, such as into the airways of a patient with cancer, which spray may form a coating on a tissue containing a cancer cell, or as a liquid for injection into a fluid or tissue containing or in contact with a cancer cell prior to metastasis. Use of a mild surface active agent such as a phospholipid is contemplated to assist solubilization and transmembrane uptake of the MANS-related peptide. Administration may also be, for example, by a dry powder, preferably a nanoparticulate or a microparticulate powder, applicable by sprinkling onto a tissue containing a cancer cell. Addition of a microparticulate carbohydrate carrier to the preparation of the peptide will facilitate inhalation delivery of nanoparticulate peptide into airways and epithelial tissue areas.
A preferred method of application of the MARCKS-inhibitory antibody, polynucleotide, nucleic acid molecule, or peptide composition comprises injection into tissue in or proximal to a tumor, which tumor contains a cancer cell prior to metastasis.
As used herein, the phrase “effective amount” or “therapeutically effective amount” refers to a nontoxic but sufficient amount of the compositions used in the practice of the invention that is effective to achieve the desired effect, i.e., to inhibit metastasis and/or proliferation of a cancer cell, and/or to inhibit, treat, or prevent cancer in a subject in need thereof. Thus, the activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate, including, for example, a reduction and/or alleviation of the signs, symptoms, or causes of a cancer. A therapeutically effective amount of compound of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective intracellular concentration and local concentration in the tissue.
“Cancer” herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma), osteoclastoma, neuroendocrine tumors, mesothelioma, chordoma, synovioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, small cell lung carcinoma, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, Ewing's tumor, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic disease, heavy chain disease, neuroendocrine tumors, Schwanoma, and other carcinomas, head and neck cancer, myeloid neoplasias such as acute myelogenous leukemias, including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemias, myelodysplastic syndromes, and chronic myeloproliferative disorders, including chronic myelogenous leukemia, tumors of the central nervous system, e.g., brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer, primary liver cancer or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma), hematologic neoplasias and neoplastic-like conditions for example, Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursor cells, including B-cell acute lymphoblastic leukemia/lymphoma, and T-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T and NK cells, including peripheral T-cell leukemias, adult T-cell leukemia/T-cell lymphomas and large granular lymphocytic leukemia, osteolytic bone cancers, and bone metastasis.
The present invention is further illustrated by reference to the following Examples. However, it should be noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the invention in any way.
EXAMPLES
Example 1
Peptide Effects on Migration of Cancer Cell Lines
Protocol for a Migration Assay
CL1-5 cells, an aggressively metastatic cell line derived from a human adenocarcinoma, were cultured in RPMI 1640 media with 10% FBS, 37° C. at 95% Oxygen/5% CO2 until ready for use. Transwell plates (24-well, 8-um pore size; Costar, Cambridge, Mass., USA) were used to conduct the migration assay. The lower chambers of the Transwell plates were filled with 600 μl of basal media containing 10% FBS. The cells (1×105) were pretreated with 50 or 100 μM of the indicated test peptide for 30 min, and then suspended in 100 μl of basal media containing 1% BSA and added to the upper chamber, and the cells then were incubated at 37° C. for 12 hr. The cells on the upper surface of the filters were removed using cotton swabs, and the cells that migrated to the lower surface of the filters were washed, fixed, and stained with hematoxylin and counted under the microscope. The percent change in migration was determined by counting the number of cells that migrated to the lower surface of the filters. At least 3 separate microscopic fields were counted per membrane (n=4).
Alternative Transwell Method
Primary CL1-5 cells originated from human adenocarcinoma lung cancer. Transwell plates with 24-wells, 8-g pore size (Costar, Cambridge, Mass., USA) were used to conduct the migration assay. The lower chambers of the transwell plates were filled with 600 μL of basal media containing 10% FBS. The cells (1-2×105) were suspended in 100 μL of basal media containing 1% BSA. The test peptide at desired concentration was added to the upper chamber directly or after preincubation with cancer cells. The plates were then incubated at 37° C. for 12 hours. The cells on the upper surface of the filters were removed using cotton swabs. The cells that migrated to the lower surface of the filters were washed, fixed, and stained with hematoxylin and counted under the microscope. At least five separate microscopic fields are counted per membrane (n=3). The percent change in migration was determined by counting the number of cells that migrated to the lower surface of the filters.
The count means the actual number of cells which migrated to the lower chamber and counted by a light microscope at 40× magnification, presented as the mean number of cells in 10 randomly chosen fields for each treatment.
The “index” was calculated by dividing the number of cells that migrated in the presence of peptides by the number of cells that migrated randomly (control group). Index=control cell count/treated cells count. This calculation can reflect the extent that the migration of cells was blocked by 30 minute pretreatment with MANS-related peptides.
FIG. 1 displays a cell count field of a negative control after a migration time of 12 hours.
FIG. 2A displays a cell count field obtained after pretreatment with 50 μmolar of MANS peptide followed by a migration time of 12 hours.
FIG. 2B displays a cell count field obtained after pretreatment with 100 μmolar of MANS peptide followed by a migration time of 12 hours.
FIG. 3A displays a cell count field obtained after pretreatment with 50 μmolar of RNS peptide followed by a migration time of 12 hours.
FIG. 3B displays a cell count field obtained after pretreatment with 100 μmolar of RNS peptide followed by a migration time of 12 hours.
FIG. 4A displays a cell count field obtained after pretreatment with 50 μmolar of MANS-related peptide BIO-11002 (N-myristoyl-SEQ ID NO: 106-NH2) followed by a migration time of 12 hours.
FIG. 4B displays a cell count field obtained after pretreatment with 100 μmolar of MANS-related peptide BIO-11002 followed by a migration time of 12 hours.
FIG. 5A displays a cell count field obtained after pretreatment with 50 μmolar of MANS-related peptide BIO-10901 (N-acetyl-SEQ ID NO: 121) followed by a migration time of 12 hours.
FIG. 5B displays a cell count field obtained after pretreatment with 100 μmolar of MANS-related peptide BIO-10901 followed by a migration time of 12 hours.
FIG. 6A displays a cell count field obtained after pretreatment with 50 μmolar of MANS-related peptide BIO-91200 (N-myristoyl-SEQ ID NO: 219) followed by a migration time of 12 hours.
FIG. 6B displays a cell count field obtained after pretreatment with 100 μmolar of MANS-related peptide BIO-91200 followed by a migration time of 12 hours.
FIG. 7A displays migrated cell numbers obtained 12 hours after pretreatment at 50 μmolar with MANS peptide, RNS peptide, and MANS-related test peptides BIO-11002 (SEQ ID NO: 6), BIO-10901 (SEQ ID NO: 121), and BIO-9120 (SEQ ID NO: 219), and control (no peptide). For MANS peptide, the cell number was approximately 40; for RNS peptide, the cell number was approximately 95; for BIO-11002 (SEQ ID NO: 106), the cell number was approximately 50); for BIO-10901 (SEQ ID NO: 121), the cell number was approximately 60; and for BIO-91200 (SEQ ID NO: 219), the cell number was approximately 65. Each of the MANS-related peptides MANS, BIO-11002, BIO-10901, and BIO-91200 demonstrated significantly reduced migrated cell numbers of the aggressive human NSCLC cell line (CL1-5) relative to control and to RNS random sequence peptide.
FIG. 7B displays migrated cell numbers obtained 12 hours after pretreatment at 100 μmolar with MANS peptide, RNS peptide, and MANS-related test peptides BIO-11002 (SEQ ID NO: 106), BIO-10901 (SEQ ID NO: 121), and BIO-91200 (SEQ ID NO: 219), and control (no peptide). For MANS peptide, the cell number was approximately 20); for RNS peptide, the cell number was approximately 90; for BIO-11002 (SEQ ID NO: 106), the cell number was approximately 25; for BIO-10901 (SEQ ID NO: 121), the cell number was approximately 35; and for BIO-91200 (SEQ ID NO: 219), the cell number was approximately 40; for the control (no peptide), the cell number was approximately 90. Each of the MANS-related peptides MANS, BIO-11002, BIO-10901, and BIO-91200 demonstrated significantly reduced migrated cell numbers of the aggressive human NSCLC cell line (CL1-5) relative to control and to RNS random sequence peptide.
FIG. 8A displays the migration index of aggressive human NSCLC cell line cells, wherein a higher value of the migration index signifies less migration after pretreatment with a peptide or peptide composition of the invention at 50 μmolar followed by 12 hours of treatment according to the protocol. The following are the migration index values: control=1; MANS peptide=approximately 2.5; RNS peptide=approximately 1.1; BIO-11002=approximately 2.7; BIO-10901=approximately 1.75; and BIO-91200=approximately 1.7. Each of the MANS-related peptides MANS, BIO-11002, BIO-10901, and BIO-91200 demonstrated significantly increased migration index numbers relative to control and to RNS peptide.
FIG. 8B displays the migration index of aggressive human NSCLC cell line cells, wherein a higher value of the migration index signifies less migration after pretreatment with a peptide or peptide composition of the invention at 100 μmolar followed by 12 hours of treatment according to the protocol. The following are depicted migration index values: control=1; MANS peptide=approximately 4.3; RNS peptide=approximately 1; BIO-11002=approximately 3.8; BIO-10901=approximately 2.7; and BIO-91200=approximately 2.4. Each of the MANS-related peptides MANS, BIO-11002, BIO-10901, and BIO-91200 demonstrated significantly increased migration index numbers relative to control and to RNS peptide.
FIG. 9 graphically displays side-by-side respective cell numbers and migration index number results of 50 μmolar peptide pretreatment in experiments using the aggressive human NSCLC cell line (CL1-5). Control with no peptide pretreatment gives a cell number of approximately 105; and migration index number=1.0 Results after 50 μmolar pretreatment with MANS peptide followed by 12 hours according to the protocol gives a cell number of approximately 45; and migration index of approximately 2.5. Results after 50 μmolar pretreatment with RNS peptide gives a cell number of approximately 90; and migration index of approximately 1.3. Results after 50 μmolar pretreatment with BIO-11000 gives a cell number of approximately 70; and migration index of approximately 1.5. Results after 50 μmolar pretreatment with BIO-11006 give cell number of approximately 50; and migration index of approximately 2. The results demonstrate that higher values of migration index are associated with less migration, and less migration is associated with higher values of migration index. Each of the MANS-related peptides MANS, BIO-11000, and BIO-11006 demonstrated decreased migrated cell numbers and increased migration index numbers relative to control and to RNS peptide.
FIG. 10 graphically displays side-by-side respective cell numbers and migration index number results for 100 μmolar peptide pretreatment in experiments using the aggressive human NSCLC cell line (CL1-5). Control with no peptide pretreatment gives a cell number of approximately 105; and migration index number=1.0. Results after 100 μmolar pretreatment with MANS peptide followed by 12 hours according to the protocol to gives a cell number of approximately 35; and migration index of approximately 3.5. Results after 100 μmolar pretreatment with RNS peptide gives a cell number of approximately 95; and migration index of approximately 1.2. Results after 100 μmolar pretreatment with BIO-11000 gives a cell number of approximately 50; and migration index of approximately 2.3. Results after 100 μmolar pretreatment with BIO-11006 gives a cell number of approximately 50; and migration index of approximately 2.1. Results after 100 μmolar pretreatment with BIO-91200 gives a cell number of approximately 55; and migration index of approximately 2.1. The results demonstrate that higher values of migration index are associated with less cell migration, and less cell migration is associated with higher values of migration index. Each of the MANS-related peptides MANS, BIO-11000, BIO-11006, and BIO-91200 demonstrated decreased migrated cell numbers and increased migration index numbers relative to control and to RNS peptide.
Example 2
Inhibition of Lung Cancer Metastasis by MANS Peptide or BIO-11006 Using Orthotopic Lung Injection Xenograph Model
The metastatic activity of cancer cells in vivo after treatment with MANS peptide or BIO-11006 was assessed in the orthotopic lung injection xenograph model. After pretreatment with either PBS, or PBS+MANS, BIO-11006 (SEQ ID NO: 106), or the control RNS peptide at 100 μM for 4 hours, PC-9 cells were injected into the left lobe of the lung of nude mice. Seven days later, these mice were given systemic treatment with either PBS only (Con), RNS (additional control peptide), MANS or BIO-11006 at 50 μmoles per intraperitoneal injection once every three days. At 25 days (6 injections) post-seeding of these tumor cells, mice were sacrificed and the number of metastasized tumor nodules in the contralateral lung and other organs was counted. As shown in Table 3 below, MANS, RNS or BIO-11006-treated groups showed no difference in average size of the tumor at the site of injection compared to PBS-treated or to each other, suggesting that these treatments do not affect tumorigenesis. However, a significant decrease of metastatic nodules was noted in the contralateral lung and other organs in the MANS and BIO-11006-treated mice compared to the PBS- or RNS-treated groups; in fact, treatment with the MANS or BIO-11006 peptides essentially totally blocked all metastasis from the tumor to other lung sites as well as to other organs.







TABLE 3







The suppressive effect of MARCKS-related peptides on cancer


metastasis in vivo












No. of















Tumor
Metastatic















Size
Lung Tumors
Metastases (affected mice/total mice)














(mm)
Mean ± SE



Dia-


Group
Mean ± SE
(L't; R't)
Heart
Spleen
Intestines
phragm





PBS 
1.50 ± 0.26
 1.67 ± 0.29;
1/1
2/3
1/3
2/3


(n = 3)

5.67 ± 0.77






RNS 
1.72 ± 0.41
 1.00 ± 0.40;
3/4
2/4
1/4
3/4


(n = 4)

5.75 ± 2.01






MANS
1.48 ± 0.54
0;0
0/4
0/4
0/4
0/4


(n = 4)








BIO-
1.61 ± 0.37
0;0
0/2
0/2
0/2
0/2


11006








(n = 2)









These in vivo results support the concept that inhibition of MARCKS function by MANS-related peptides can reduce the metastatic spread of lung cancer cells in vivo.
Example 3
Peptide Effects on Migration of Cancer A549 Cell Lines
The human adenocarcinoma-derived alveolar epithelial cell line A549 (an invasive cell line) was obtained from the American Type Culture Collection (ATCC) and was cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin, in 75 cm2 tissue culture flasks. The cells reached confluence by the third day of culture at 37° C. in an atmosphere of 95% air and 5% CO2 and were maintained by serial passage.
The test peptides (MANS, RNS, BIO-11006, BIO-11000, BIO-11002, BIO-91200, and BIO-10901) were dissolved in PBS at a pH of 7.0; slow vortex mixing for about two hours aided solubility.
Transwell plates (24-well, 8-μM pore size; Costar, Cambridge, Mass., USA) were used to conduct the migration assay. The lower chambers of the transwell plates were filled with 600 μl of basal medium containing 10% FBS. The cells (1×105) were suspended in 100 μl of basal medium containing 1% BSA and added to the upper chamber, and the plates incubated at 37° C. with 5% CO2 for 12 hr in PBS (control), or the indicated test peptides at 10, 25 or 50 μM. The cells on the upper surface of the filters were removed using cotton swabs. The cells that migrated to the lower surface of the filters were washed, fixed, and stained with hematoxylin and counted under the microscope. The percent change in migration is determined by counting the number of cells that migrated to the lower surface of the filters. At least four separate microscopic fields are counted per membrane, with a total of three replicate experiments at each concentration. The statistics software “Prizm” was used for data analysis.
As shown in FIG. 11, treatment with each of the test peptides resulted in a reduced cell number after treatment relative to control (no peptide) or to RNS peptide. At 50 μm, each of the MANS-related peptides MANS, BIO-11006, BIO-11000, BIO-11002, BIO-91200, and BIO-10901 demonstrated decreased migrated cell numbers and increased migration index numbers relative to control and to RNS peptide. At lower concentrations, the effect of several MANS-related peptides was significantly different even in comparison to MANS peptide. In particular, at 25 μm, treatment with BIO-11006, BIO-11002, or BIO-91200 resulted in a significantly reduced cell numbers in comparison to control (no peptide) or control RNS peptide, as well as in comparison to MANS peptide (FIG. 11).
Taken together, the results of the studies showed that the MANS-related peptides can block migration of aggressive cancer cell lines, and that several different MANS-related peptides exhibited an effect on migration of at least three different cancer cell lines. The results of the studies also showed that at least two different MANS-related peptides were able to block or inhibit metastasis of cancer cells injected into mammals (i.e., mice).
Example 4
Effects of Peptide BIO-11006 in Lung Cancer Implantation
In this study, inhibition of lung cancer metastasis by peptide BIO-11006 in an orthotopic lung cancer implantation model in SCID mice assessed. Human adenocarcinoma cells (PC-9) (1-2×105) were suspended in 40 μL of PBS, pH 7.4 containing 0.5 mg/mL of MatrigelTM (BD Bioscience) and injected into the left lung of SCID mice (n=3) using a syringe with 29 gauge needle. BIO-11006 (100 μM, in PBS) was administered either (a) by i.p. injection (50 μL) once daily for 22 days starting at 3 days post cancer cell inoculation or (b) by aerosol inhalation using a Nebulizer Delivery System (Aeroneb Lab) over 30 min once daily for 22 days starting at 3 days post cancer cell inoculation. The results of the study are depicted in FIG. 12, which graphically displays the average number of tumors in the right lung, heart, and diaphragm as a function of the route of administration of the test compound (ip injection versus inhalation). Both i.p. and aerosol administration of BIO-11006 reduced cancer cell metastasis by more than 50% in the right lung and heart, and 100% in the diaphragm. Thus, both routes of administration had significant inhibitory effects on cancer cell metastasis.
Example 5
Inhibition of Lung Cancer Metastasis
In this study, inhibition of lung cancer metastasis by BIO-11006 was investigated in an orthotopic lung cancer implantation model in SCID mice. Human adenocarcinoma cells (PC-9) (1-2×105) were suspended in 40 μL of PBS containing 0.5 mg/mL of MatrigelTM (BD Bioscience) and injected into the left lung of SCID mice (n=3) using a 29 gauge needle. BIO-11006 was administered starting either 4 days or 15 days post cancer cell inoculation by aerosol using 100 μM solution in PBS by Nebulizer Delivery System (Aeroneb Lab) over 30 min once daily for 25 days post cancer cell inoculation. At end of the experiment, the mice were sacrificed and lungs, heart, and diaphragm tissues collected and number of tumor nodules in each tissue measured. The results of this experiment are presented graphically in FIGS. 13 and 14. FIG. 13 shows that when the treatment was started at 4 days post inoculation of cancer cells, the tumor metastasis was inhibited by 60-90% in the left lung, heart, and diaphragm, and by 100% in the right lung. When the treatment was initiated at 15 days post inoculation of cancer cells, the inhibition of tumor metastasis was about 50% in the lungs, heart, and diaphragm. FIG. 14 depicts the total number of tumor nodules found in all tissues when peptide treatment was started at 15 days post cancer cell inoculation or 4 days post cancer cell inoculation. As shown in FIG. 14, peptide treatment starting at 15 days post cancer cell inoculation resulted in reduced tumor nodules, and peptide treatment starting at 4 days post inoculation resulted in even further reduced tumor nodules.
Example 6
Anti-Metastatic Efficacy of Peptides
This example demonstrates the anti-metastatic efficacy of test compounds of the invention in SCID mice bearing human lung adenocarcinoma cells. Female NOD.CB17-Prkdcscid/NCrHsd, Mus musculus mice (Harlan, The Netherlands), housed in individually ventilated cages, were randomized into six groups of eight mice each. A549 cells (2.5×106) were injected into the mice via the tail vein. The control group received aerosolized vehicle PBS; while the groups 2 and 3 received the aerosolized test compound BIO-11006 either from −1 day before the cancer cells injection (group 2) or from +3 days after the cancer cell injection (group 3) on every other day for 7 weeks via nebulizer (Aeroneb Lab). Groups 4 and 5 received the aerosolized test compound MANS peptide either from −1 day before the cancer cell line injection (group 4) or from +3 days after the cell line injection (group 5) on every other day for 7 weeks via a nebulizer (Aeroneb Lab). For aerosol delivery, solution of each test compound (100 μM) was prepared in PBS, pH 7.0. For each treatment, 5 mL of test compound solution was aerosolized over 30 minutes into a chamber containing four mice at a time. The mice were monitored for body weight every other day for 7 weeks.
One group of mice (n=7) served as normal control. These were untreated naïve mice used to monitor the health status of mice during the study period. All animals from all groups were sacrificed on Day 53. The results are provided in Table 4 and FIG. 15. FIG. 15 compares number of metastatic nodules in lungs after respective administration of BIO-11006 and MANS peptide on Day 53 following A549 cell injection. Table 4 shows the number of metastatic nodules in the lungs, as well as the number of animals with focal tumor nodules and/or distant metastasis. Overall, the test peptides showed substantial inhibition of tumor metastasis (70-80%) in animals administered BIO-11006 or MANS peptide starting on day −1 or Day +3 relative to A549 cell injection (FIG. 15). Moreover, the number of metastatic nodules in treated mice was significantly reduced relative to vehicle control recipients, and none of the treated mice exhibited evidence of distant metastases (Table 4). There were few focal tumor nodules in 7⁄8 of mice treated with BIO-11006 peptide starting at either Day −1 or Day +3 relative to A549 cell injection.







TABLE 4







Metastatic nodules and distant metastasis


in mice treated with BIO-11006 or MANS










No. of metastatic




nodules in lungs


Group
Mean ± SEM
Remarks





Vehicle Control
 97 ± 21
Multi-focal tumor nodules in




all animals (8/8)




Evidence of distant metastasis in




diaphragm and sternum (2/8)


BIO-11006 peptide
 34 ± 14
Few focal tumor nodules in 7/8


(−1 Day treatment

animals


group)

No distant metastasis


BIO-11006 peptide
21 ± 6
Few focal tumor nodules in 7/8


(+3 Day treatment

animals


group)

No distant metastasis


MANS peptide
22 ± 7
Few focal tumor nodules in 4/8


(−1 Day treatment

animals


group)

No distant metastasis


MANS peptide
13 ± 4
Few focal tumor nodules in 7/8


(+3 Day treatment

animals


group)

No distant metastasis









Example 7
Anti-Metastatic Activity of MANS-Related Peptides in Murine Melanoma
In this study, the relative anti-metastatic activity of MANS-related peptides administered by four different routes is determined using a syngeneic mouse model. Murine melanoma cell line B16F10 2×106 cells in 200 μl of cell suspension in DMEM media is injected in the footpad or between the skin and cartilage on the dorsal side of the ear; or by intravenous, intramuscular, or intraperitoneal injection.
Two days after cell inoculation, animals are randomized and divided into groups consisting of n=10 in each group. Animals in different groups are administered with MANS-related peptide by intraperitoneal (ip), intravenous (iv), or intramuscular (im) routes respectively at a dose of about 6.25 mg/kg. In some groups, animals are treated by inhalation route (5 ml of 0.1 mM MANS-related peptide in PBS) using preclinical nebulizer (Aeroneb Lab; Aerogen). One group of mice serves as vehicle control and treated with PBS by im route. Peptides are administered every other day for 6 weeks.
Tumor scoring is done weekly. After 6 weeks of treatment, all animals are humanely sacrificed and the lymph nodes & other tissue samples are collected, fixed in formalin and subjected to histopathology to assess the presence of metastatic melanoma cells. Clinical toxic signs and symptoms are assessed during the 6 wee administration period. Tumor burden and mortalities are assessed at the end of the study period. The results of the study will show that MANS-related peptides inhibit tumor metastasis in a murine melanoma model.
Example 8
siRNA Knockdown of MARCKS in Cancer Cells
This study was conducted to determine the effects of siRNA knockdown of MARCKS expression on migration of cancer cells. PC-9 or A-549 human lung cancer cells were seeded in plastic wells and cultured until cells reached 70% confluence. Cells were then transfected with 100 nM of MARCKS siRNA or control siRNA (100 nM) from Ambion (Austin, Tex.) by using the DharmaFECT DuoTransfection reagent (Dharmacon, Lafayette, Colo.). After 72 hours, the cells were harvested and equivalent amounts of proteins separated by SDS/PAGE for immunoblot analysis using MARKS specific antibodies. Western blot analysis was performed to confirm siRNA-induced down regulation of endogenous MARCKS. As shown in FIGS. 16 and 17, MARCKS protein was knocked down by approximately 60% in PC9 cells (FIG. 16) and approximately 50% in A549 cells (FIG. 17) compared to cells treated with control siRNA.
The effects MARCKS knockdown by siRNA on cell migration were determined using a transwell assay. Following siRNA knockdown, PC-9 or A549 cells were cultured in RPMI 1640 medium with 10% FBS at 37° C., with 5% CO2. Transwell® plates (24-well, 8-um pore size) were used for the migration assays. The lower chambers contained 600 μl basal medium+10% FBS. Cells (1×105) were suspended in 100 μl of basal medium+1% BSA and added to the upper chamber; plates were incubated for 12 hr. Cells that migrated to the lower surface of the filters were stained with hematoxylin and counted. At least 3 separate microscopic fields were counted per membrane.
The results of the study are shown in FIGS. 18 and 19. Treatment with MARCKS siRNA significantly inhibited migration of both PC-9 and A549 cells. siRNA knockdown of MARCKS resulted in 90% reduction in cell migration for PC9 cell line (FIG. 18) and resulted in approximately 50% reduction in cell migration for A549 cell line (FIG. 19). Therefore, inhibition of MARCKS expression results in significant reduction in cancer cell migration.
Example 9
microRNA21 Inhibition Increases MARCKS and Enhances Cancer Cell Migration
MicroRNA 21 (miR21) regulates levels of MARCKS in cells. In these studies, PC9 human lung cancer cells were transfected with 50 nM or 100 nM of miR21 inhibitor or a mir21 mimic, or a vehicle-only negative control. Levels of MARCKS were measured 48 hours later by Western Blot. Treatment with the miR21 inhibitor (50 nM) increased levels of MARCKS in the cells by  ̃2.5 fold (FIG. 20). These values correlated with the migration capability of the cells when placed in migration chambers over a 12 hour period. Cells treated with the miR21 inhibitor at 50 or 100 nM showed enhanced migration correlating with increased levels of MARCKS, while cells treated with miR21 mimic showed decreased migration correlating with decreased levels of MARCKS (FIG. 21). Cells treated with HiPerfect vehicle control showed the same level of migration relative to untreated PC9 cells (FIG. 21). The results of the study showed that an increase in MARCKS expression via miR21 inhibition increases cancer cell migration. Moreover, activation of miR21 resulted in decreased migration of PC9 cells.
Taken together, the studies showed that cancer cell migration, and metastasis of cancer cells, can be inhibited by targeting MARCKS using several different means of MARCKS inhibition, including MANS-related peptides and inhibitory microRNAs, i.e., a miR21 mimic.
Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications cited herein are incorporated herein by reference for the purpose of disclosing and describing specific aspects of the invention for which the publication is cited.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

## Claims

1. A method for treating cancer comprising administering a Myristoylated Alanine-Rich C Kinase Substrate (MARCKS)-inhibitory peptide to a subject that has been diagnosed with cancer, in an effective amount to treat said cancer, wherein the peptide is selected from the group consisting of Ac-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-NH2 (SEQ ID NO: 106), Ac-RGAQFSKTAAK-NH2 (SEQ ID NO: 234), Ac-GAQFSKTAA-OH (SEQ ID NO: 121), Ma-RGAQFSKTAA-NH2 (SEQ ID NO: 234), and Ma-AKGE-OH (SEQ ID NO: 219), wherein Ma is myristoyl and Ac is acetyl.

2. The method of claim 1, wherein the method for treating cancer comprises inhibiting metastasis, cancer cell proliferation, tumor growth, or angiogenesis.

3. The method of claim 1, wherein the peptide is administered by inhalation of a liquid solution or suspension, or by inhalation of a dry powder formulation of the peptide.

4. The method of claim 1, wherein the peptide is administered by injection of a liquid formulation of the peptide.

5. The method of claim 1, wherein the peptide is administered to a subject by intravenous, intramuscular, or intraperitoneal injection or by oral or suppository administration.

6. The method of claim 5, wherein the injection is into a tumor containing the cancer cell.

7. The method of claim 5, wherein the injection is systemic.

8. The method of claim 6, wherein the tumor is a solid tumor.

9. The method of claim 6, wherein the tumor is a non-solid tumor.

10. The method of claim 1, wherein the peptide is administered in a dose from between about 0.01 mg/kg/day to about 10 mg/kg/day.

11. The method of claim 10, wherein the peptide is administered in a dose from between about 0.5 mg/kg/day to about 2.5 mg/kg/day.

12. A method for inhibiting metastasis of a cancer cell comprising administration to the cancer cell of a metastasis-inhibiting amount of a formulation of a peptide selected from the group consisting of Ac-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-NH2 (SEQ ID NO: 106), Ac-RGAQFSKTAAK-NH2 (SEQ ID NO: 234), Ac-GAQFSKTAA-OH (SEQ ID NO: 121), Ma-RGAQFSKTAA-NH2 (SEQ ID NO: 234), Ma-AKGE-OH (SEQ ID NO: 219), and a combination thereof), wherein Ma is myristoyl and Ac is acetyl.

13. The method of claim 12, wherein the formulation comprises an additional drug useful in the treatment of cancer.

14. A method for inhibiting metastasis of a cancer cell in a mammal comprising administering to said mammal a peptide, wherein said peptide exhibits a migration index of at least about 1.5 following pretreatment of non-small-cell lung carcinoma (NSCLC) cells with a concentration of 50 μmol of said peptide and a migration period of 12 hours.

15. A method for inhibiting metastasis of a cancer cell in a mammal comprising administering to said mammal a peptide, wherein said peptide exhibits a migration index of at least about 2.0 following pretreatment of non-small-cell lung carcinoma (NSCLC) cells with a concentration of 100 μmol of said peptide and a migration period of 12 hours.

16. The method of claim 1, further comprising administering a chemotherapy drug to the subject.

17. The method of claim 16, wherein the chemotherapy drug is selected from carboplatin, cisplatin, oxaliplatin, cyclophosphamide, dacarbazine, temozolomide, gemcitabine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanadine, daunorubicin, doxurubicin, epirubicin, idarubicin, topotecan, irinotecan, etoposide, eniposide, colchicine, vincristine, vinblastine, vinorelbine, paclitaxel, and docetaxel.

18. The method of claim 17, wherein the chemotherapy drug is cisplatin or carboplatin.

19. The method of claim 12, further comprising administering to the cancer cell a chemotherapy drug.

20. The method of claim 19, wherein the chemotherapy drug is selected from carboplatin, cisplatin, oxaliplatin, cyclophosphamide, dacarbazine, temozolomide, gemcitabine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanadine, daunorubicin, doxurubicin, epirubicin, idarubicin, topotecan, irinotecan, etoposide, eniposide, colchicine, vincristine, vinblastine, vinorelbine, paclitaxel, and docetaxel.

21. The method of claim 20, wherein the chemotherapy drug is cisplatin or carboplatin.

22. The method of claim 14, wherein the peptide is selected from the group consisting of Ac-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-NH2 (SEQ ID NO: 106), Ac-RGAQFSKTAAK-NH2 (SEQ ID NO: 234), Ac-GAQFSKTAA-OH (SEQ ID NO: 121), Ma-RGAQFSKTAA-NH2 (SEQ ID NO: 234), and Ma-AKGE-OH (SEQ ID NO: 219), wherein Ma is myristoyl and Ac is acetyl.

23. The method of claim 15, wherein the peptide is selected from the group consisting of Ac-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-OH (SEQ ID NO: 106), Ma-GAQFSKTAAK-NH2 (SEQ ID NO: 106), Ac-RGAQFSKTAAK-NH2 (SEQ ID NO: 234), Ac-GAQFSKTAA-OH (SEQ ID NO: 121), Ma-RGAQFSKTAA-NH2 (SEQ ID NO: 234), and Ma-AKGE-OH (SEQ ID NO: 219), wherein Ma is myristoyl and Ac is acetyl.