KR102492241B1 - Peptide interfering a dimerization of KITENIN and use thereof - Google Patents

Abstract

The present invention relates to a peptide inhibiting KITENIN dimer formation and a use thereof. The peptide of the present invention includes an amino acid sequence represented by SEQ ID NO: 3, and suppresses KITENIN dimer formation, thereby exhibiting effects of suppressing tumorigenicity, invasiveness, and metastasis of cancer.

Description

Peptide interfering a dimerization of KITENIN and use thereof {Peptide interfering a dimerization of KITENIN and use thereof}

The present invention relates to a peptide that inhibits KITENIN dimer formation and uses thereof, and more specifically, to a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 and inhibits KITENIN dimer formation and uses thereof.

Protein oligomerization can be defined as the arrangement of monomeric units of a protein into homo-oligomers or hetero-oligomers. Oligomerization creates protein structures that allow specificity and diversity of cellular functions, such as mediating gene expression and regulating the activity of enzymes, ion channels, and receptors (1). Oligomerization, both in homo-oligomers and in hetero-oligomers, is regulated by ligands, temperature and other proteins, and provides sites for allosteric regulation (2). Protein stability can be improved by forming large assemblies through oligomerization without increasing genome size (3-5). Thus, many membrane-associated proteins (MAPs) and soluble proteins form homo-oligomeric complexes in cells (5, 6). Homologous oligomers most often exist as dimers and tetramers, which are about four times more common than heterogeneous oligomers (7). Exposure of the hydrophobic interface of dimeric proteins is known to cause structural changes that can lead to protein destabilization and degradation via proteasome or autophagy pathways (8-10). Thus, oligomerization is a very promising therapeutic strategy for the treatment of diseases involving oligomeric proteins (11), and a better understanding of the molecular aspects of protein oligomerization is needed (12).

Vangl protein has an important function in neural tube formation during embryonic development, and mutations in the Vangl1 and Vangl2 genes cause neural tube defect cranial splitting (13). In previous studies, Vangl1, a membrane-associated atypical tetraspanin with a long intracellular C-terminal domain (renamed by the present inventors as KITENIN [KAI1 C-terminal interaction tetraspanin]) binds to the C-terminus of KAI1, leading to colorectal cancer ( CRC) has been reported to act as a protein that enhances metastasis (14). CT-26 mouse colon cancer cells overexpressing KITENIN showed increased invasiveness and tumorigenicity and early liver metastasis due to KITENIN gain-of-function (KITENIN-GOF). The functional KITENIN complex acts as an executor in relation to cell motility, controlling CRC cell invasion and contributing to metastasis promotion (15). KITENIN levels also positively correlate with advanced stage of CRC (15) and lymph node metastasis (16). The presence of the KITENIN/ErbB4-Dvl2-c-Jun axis, an unconventional EGFR-independent signal of EGF, also mediates increased CRC cell invasion and shows a low response to cetuximab ( 17, 18). The KITENIN axis also plays an important role in colorectal carcinogenesis within the adenomatous polyposis coli-loss-associated environment (19). Therefore, these reports suggest that the KITENIN axis is a molecular target for the development of therapeutic agents that block the malignant progression of CRC. However, except for expression and post-translational modification analysis of KITENIN's biochemical properties, no studies have been conducted on how KITENIN stability is regulated and which molecules are closely involved in this regulation.

Accordingly, the present inventors investigated the biochemical properties that regulate the stability of KITENIN. As a result, it was confirmed that by identifying biochemical features that maintain the structural integrity of KITENIN, it was possible to develop a new therapeutic agent for cancer patients expressing higher KITENIN levels by targeting KITENIN, which led to the completion of the present invention. It became.

On the other hand, documents described with reference numbers in this specification are as follows.

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One object of the present invention is to provide a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 and inhibiting KITENIN dimer formation.

Another object of the present invention is to provide a polynucleotide coated with the peptide, a recombinant vector containing the polynucleotide, and a transformant transformed with the recombinant vector.

Another object of the present invention is to provide a composition for treating or improving cancer containing the peptide.

Another object of the present invention is to provide a composition for inhibiting invasion and metastasis of cancer containing the peptide.

Another object of the present invention is to provide a method for treating cancer in a subject or inhibiting cancer invasion and metastasis in a subject by administering the composition to the subject.

In one aspect, the present invention provides a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 and inhibiting KITENIN dimer formation.

In the present invention, KAI1-C-terminal interaction tetraspin (KITENIN) is a protein that binds to the C-terminus of KAI1 to increase cancer invasiveness and tumorigenicity and activate early cancer metastasis.

The present inventors found that KITENIN increases the invasiveness and tumorigenicity of cancer and activates early cancer metastasis by forming homodimerization. In addition, it was found that the activity of KITENIN was increased while being stabilized by Myo10. Therefore, a method of inhibiting the above activity of KITENIN is proposed in the present invention.

The peptide of the present invention includes an amino acid sequence represented by SEQ ID NO: 3, wherein the amino acid sequence of SEQ ID NO: 3 is derived from the 466th to 470th amino acid sequence of (SEQ ID NO: 1) constituting the full length of KITENIN. sequence or a sequence derived from the 223rd to 227th amino acid sequence of the amino acid sequence (SEQ ID NO: 2) constituting the C-terminal domain of KITENIN.

As one specific example, when the C-terminal domain, the N-terminal domain, and the C-terminal and N-terminal domains of KITENIN were deleted from the amino acid sequence of SEQ ID NO: 1 constituting the full length of KITENIN, respectively, and then transduced and expressed When the C-terminal domain of KITENIN was deleted, dimer of KITENIN was not detected. Accordingly, the dimerization site of KITENIN was identified as the C-terminal domain of KITENIN, and the C-terminal domain of KITENIN was composed of the amino acid sequence of SEQ ID NO: 2.

As another specific example, as a result of experiments on several peptides in which the C-terminal domain of KITENIN was divided into 10-mer or less, a peptide composed of the 462nd to 469th amino acid sequence of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 1 It was confirmed that the peptide consisting of the 463rd to 471st amino acid sequence of the amino acid sequence of and the peptide consisting of the 467th to 474th amino acid sequence of SEQ ID NO: 1 inhibited dimer formation of KITENIN and effectively inhibited cell invasion. In addition, it was confirmed that the peptide composed of the 463rd to 471st amino acid sequences of SEQ ID NO: 1 among the above amino acid sequences inhibits KITENIN dimer formation and inhibits cell invasion most effectively.

The 462nd to 469th amino acid sequence of SEQ ID NO: 1 is the same as the 219th to 226th amino acid sequence of SEQ ID NO: 2, and is represented by SEQ ID NO: 6 herein.

The 463rd to 471st amino acid sequence of SEQ ID NO: 1 is the same as the 220th to 228th amino acid sequence of SEQ ID NO: 2, and is represented by SEQ ID NO: 7 herein.

The 467th to 474th amino acid sequence of SEQ ID NO: 1 is the same as the 224th to 231st amino acid sequence of SEQ ID NO: 2, and is represented by SEQ ID NO: 8 herein.

Accordingly, the peptide of the present invention has the effect of inhibiting the formation of KITENIN dimer, thereby reducing the stability of KITENIN and inhibiting the activity of KITENIN.

As one specific embodiment, the peptide of the present invention may be the amino acid sequence represented by SEQ ID NO: 3.

In one specific example, the amino acid sequence consisting of SEQ ID NOs: 6 to 8 inhibited KITENIN dimer formation and effectively inhibited cell invasion, and among the amino acid sequences, the amino acid sequence consisting of SEQ ID NO: 7 inhibited KITENIN dimer formation and inhibited cell invasion most effectively. The amino acid sequence common to these amino acid sequences with excellent effects is SDE, and it can be expected to have the above-described effects when parts of the front and rear sequences including the SDE are included in the amino acid sequence of SEQ ID NO: 7. Therefore, the amino acid sequence represented by SEQ ID NO: 3 is the minimum amino acid sequence capable of inhibiting KITENIN dimer formation and cell invasion.

In another specific embodiment, the peptide of the present invention includes the amino acid sequence of SEQ ID NO: 3, and the 222nd amino acid of the first amino acid from the 222nd amino acid of the amino acid sequence of SEQ ID NO: 2 in front of the amino acid sequence of SEQ ID NO: 3 from and/or from the 228th amino acid to the 281st amino acid of the amino acid sequence of SEQ ID NO: 2 to the rear of the amino acid sequence of SEQ ID NO: 3, and/or one or more from the 228th amino acid. there is.

As one specific example, the peptide of the present invention is an amino acid sequence added to the amino acid sequence represented by SEQ ID NO: 3, and the 222nd amino acid to 1 of SEQ ID NO: 2 in the front (5' direction) of the amino acid sequence of SEQ ID NO: 3 Among the amino acids, one or more amino acids from the 222nd amino acid may be additionally included. An example in which one or more amino acids are further included from the 222nd amino acid is the 222nd amino acid, the 221st and 222nd amino acids, the 220th to 222nd amino acids, the 219th to 222nd amino acids, and the 218th to 218th amino acids of the amino acids of SEQ ID NO: 2. It may be the 222nd, 217th to 222nd, 216th to 222nd, or 215th to 222nd amino acids, or the 1st to 222nd amino acids.

As another specific example, the peptide of the present invention is an amino acid sequence added to the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 2 in the rear (3' direction) of the amino acid sequence of SEQ ID NO: 3 Among the 228th to 281st amino acids, one or more amino acids from the 228th amino acid may be further included. An example in which one or more amino acids are further included from the 228th amino acid is the 228th amino acid, the 228th and 229th amino acids, the 228th to 230th amino acids, the 228th to 231st amino acids, and the 227th to 232nd amino acids of SEQ ID NO: 2. It may be an amino acid or the like or the 228th to 281st amino acids.

As another specific example, the peptide of the present invention is an amino acid sequence added to the amino acid sequence represented by SEQ ID NO: 3, and the 222nd sequence of SEQ ID NO: 2 forward (5' direction) of the amino acid sequence of SEQ ID NO: 3 One or more amino acids from the 222nd amino acid of the amino acid to the 1st amino acid are further included, and 228 of the 228th amino acid to the 281st amino acid of the amino acid sequence of SEQ ID NO: 2 to the rear (3' direction) of the amino acid sequence of SEQ ID NO: 3 One or more amino acids from the th amino acid may be further included. In this example, the 222nd amino acid, the 221st and 222nd amino acids, the 220th to 222nd amino acids, the 219th to 222nd amino acids, and the 218th to 218th amino acids of SEQ ID NO: 2 of SEQ ID NO: 2 are forward of the amino acid sequence of SEQ ID NO: 3. 222nd, 217th to 222nd, 216th to 222nd, or 215th to 222nd amino acids, etc., or the 1st to 222nd amino acids; 228th amino acid, 228th and 229th amino acids, 228th to 230th amino acids, 228th to 231st amino acids, and 227th to 232nd amino acids of SEQ ID NO: 2 to the rear (3' direction) of the amino acid sequence of SEQ ID NO: 3 etc., or amino acids such as the 228th to 281st amino acids.

As another specific example, the peptide of the present invention may be the amino acid sequence represented by SEQ ID NO: 2, that is, the amino acid sequence constituting the C-terminal domain (KITEIN-CTD) of KITENIN.

As another specific aspect, the peptide of the present invention may be any one amino acid sequence of SEQ ID NOs: 5 to 6.

In addition, the peptide of the present invention is a concept including a functional variant thereof. "Functional variant" refers to any similar sequence in which some amino acid substitution occurs at an amino acid position that does not affect the properties of the peptide of the present invention that inhibits the formation of homodimers of KITENIN.

The peptide of the present invention inhibits the formation of homodimers of KITENIN, increases the binding between KITENIN and RACK1, and serves to degrade KITENIN protein in an autophagy-dependent manner. Accordingly, the peptide effectively inhibits oncogenic KITENIN in vitro and in vivo, and has an effect of reducing the stability of KITENIN protein, thereby inhibiting tumorigenicity, invasiveness, and metastasis of cancer.

The peptide of the present invention may also be combined with a cell-permeable material. These cell-permeable materials may include any material that penetrates cells and delivers the peptide of the present invention to KITENIN. As an example, the cell-permeable material may be any one or more selected from the group consisting of peptides and compounds.

The peptides include polyarginine, Hph-1, Tat, SPACE (skin penetration and cell entering peptide), TD1 (transdermal peptide-1), DLP (dermis localizing peptide), LP-12 (linear peptide-12 mer), penetratin, and tumor homing peptides such as EGFR-1 and EGFR-2.

The compound includes cholic acid, oleic acid, and derivatives thereof.

As one specific example, a peptide including the amino acid sequence represented by SEQ ID NO: 7 or a peptide in which the peptide and a cell-permeable material are bound were mixed and used as KDIP (KITENIN Dimerization-Interfering Peptide).

As another aspect, the present invention provides a polynucleotide encoding the peptide.

The "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides that exists in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

The polynucleotide includes not only a nucleotide sequence encoding the peptide, but also a sequence complementary to the sequence. The complementary sequences include sequences that are substantially complementary as well as sequences that are perfectly complementary.

In addition, the polynucleotide may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides. A polynucleotide encoding the amino acid sequence is also construed to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when the nucleotide sequence and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art, It may be a sequence exhibiting at least 90% homology or at least 95% homology.

As another aspect, the present invention provides a recombinant vector containing the polynucleotide and a transformant transformed with the recombinant vector.

In the present invention, "vector" means a self-replicating DNA molecule used to transfer clonal genes (or other fragments of clonal DNA).

In the present invention, "recombinant vector" refers to a plasmid, viral vector or other medium known in the art capable of expressing an inserted nucleic acid in a host cell, and is compared to conventional expression vectors known in the art. Polynucleotides encoding the peptides of the invention may be operably linked. The recombinant vector is generally operably linked to an origin of replication capable of proliferating in a host cell, one or more expression control sequences (eg, promoter, enhancer, etc.) for controlling expression, a selective marker, and an expression control sequence. It may contain polynucleotides encoding the peptides of the invention. A transformant may be one transformed by the recombinant vector.

Preferably, the transformant is a recombinant vector containing a polynucleotide encoding the peptide of the present invention by methods known in the art, such as, but not limited to, transient transfection, microinjection, transfection Transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran- mediated transfection, polybrene-mediated transfection -mediated transfection), electroporation (electropora tion), gene gun (gene gun), and can be obtained by introducing into a host cell by other known methods for introducing nucleic acid into the cell.

In another aspect, the present invention provides a composition for preventing, treating, or improving cancer, or a composition for inhibiting invasion and metastasis of cancer, comprising the peptide as an active ingredient.

As described above, the peptide of the present invention inhibits the formation of homodimers of KITENIN, increases the binding between KITENIN and RACK1, and degrades KITENIN protein in an autophagy-dependent manner. Therefore, the peptide effectively inhibits oncogenic KITENIN in vitro and in vivo, and effectively reduces the stability of the KITENIN protein to inhibit tumorigenicity, invasiveness, and metastasis of cancer, thereby providing a composition for treating or improving cancer, and cancer cell invasion and It can be usefully used as a composition for inhibiting metastasis.

In the present invention, "treatment" may include without limitation any action that improves or benefits symptoms caused by cancer by using the composition of the present invention.

In addition, in the present invention, "prevention" or "improvement" may include without limitation any action that blocks symptoms caused by cancer by using the composition of the present invention, or suppresses or delays the symptoms.

The cancers include colorectal cancer, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, lung cancer, brain tumor, rectal cancer, stomach cancer, kidney cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, pancreatic cancer, head cancer It may be cervical cancer or squamous cell carcinoma, but is not limited thereto. Preferably, the cancer is colorectal cancer, breast cancer, liver cancer, esophageal cancer, lung cancer, brain tumor, rectal cancer, gastric cancer, bile duct cancer, saccular cancer, pancreatic cancer, head and neck cancer or squamous cell carcinoma.

When the composition for preventing, treating or improving cancer or inhibiting cancer invasion and metastasis of the present invention is used as a pharmaceutical composition, a pharmaceutically acceptable carrier, diluent or excipient is added in addition to the peptide as an active ingredient It can be formulated in various forms such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injections of sterile injection solutions according to conventional methods according to each purpose of use. It can be used and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical, and the like. Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like. In addition, the composition of the present invention may further include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. Formulated by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

Oral liquid preparations may include suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. can

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. The suppositories are Witepsol, Macrogol, and Tween 61. Cacao butter, laurin fat, glycerogeratin and the like can be used. Meanwhile, conventional additives such as solubilizers, tonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives may be included in the injection.

The formulation may be prepared by conventional mixing, granulation, or coating methods, and contains an active ingredient in an amount effective for medical treatment, specifically, inhibition of metastasis and invasion of cancer or treatment of cancer.

At this time, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health condition. , depending on the type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. can be determined The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

For example, the dosage may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., so the dosage is not limited to the scope of the present invention in any way.

A preferred dosage of the peptide of the present invention varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art.

In addition, since the pharmaceutical composition of the present invention inhibits cancer metastasis and invasion and exhibits cancer treatment effects, the pharmaceutical composition of the present invention can be used alone, but is combined with other anti-cancer therapies such as radiation therapy to increase treatment efficiency. It may be used in combination, but is not limited thereto.

For example, the pharmaceutical composition of the present invention may be administered in combination with other anticancer agents. The anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, lesta urtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide , dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomabtucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan , gemcitabine, doxifluridine, femetrexed, tegafur, capecitabine (5-Fu), gimeracin, oteracil, azacytidine, methotrexate, uracil, cytarabine, fluoururacil, flu Dagabine, enocitabine, flutamide, capecitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine , Etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pirarubicin, a Clarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melpharan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol , leucovorin, tretonin, exmestane, aminoglutesimide, anagrelide, navelbine, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicaluta At least one selected from the group consisting of mead, lomustine, vorinostat, entinostat, and carmustine may be used, but is not limited thereto.

When the composition for preventing, treating, or improving cancer or inhibiting cancer invasion and metastasis of the present invention is used as a food composition, in addition to the peptide as an active ingredient, an ingredient commonly added during food preparation, for example, It may further include proteins, carbohydrates, fats, nutrients, seasonings and flavors.

Examples of such carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared as a drink, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc. may be further included in addition to the peptide of the present invention. .

The food composition of the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the food composition may be used with other foods or food additives in addition to active ingredients, and may be appropriately used according to conventional methods. there is. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the food composition may be used according to the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control, However, it is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.

The type of food is not particularly limited, and examples thereof include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream, various soups, beverages, tea, and drinks. , alcoholic beverages and vitamin complexes.

In another aspect, the present invention provides a method for inhibiting dimer formation of KITENIN, or inhibiting metastasis and invasion of cancer, or treating cancer, comprising administering the composition of the present invention to a subject in need thereof. provides a way

The method for inhibiting dimer formation of KITENIN of a peptide containing the amino acid sequence represented by SEQ ID NO: 3, which is an active ingredient of the composition, inhibiting cancer metastasis and invasion, and treating or preventing cancer according to the method is as described above.

The term "subject" of the present invention refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, It means any animal including mouse, rat, rabbit or guinea pig, and by administering the composition of the present invention to a subject, cancer metastasis and invasion can be inhibited or cancer can be effectively treated or improved.

The term "administration" of the present invention means providing a predetermined substance to a patient by any suitable method. The administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration may be administered, but is not limited thereto.

In addition, the composition of the present invention may be administered by any device capable of transporting an active substance to a target cell. Preferred administration modes and preparations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. Injections are formulated with aqueous solvents such as physiological saline and IV, vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), and non-aqueous solvents such as alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g., ascorbic acid, sodium hydrogensulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.) to prevent deterioration, emulsifiers, buffers to control pH, A pharmaceutical carrier such as a preservative (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

The peptide of the present invention inhibits KITENIN dimer formation, increases the binding between KITENIN and RACK1, and serves to degrade KITENIN protein in an autophagy-dependent manner. Therefore, the peptide effectively inhibits oncogenic KITENIN in vitro and in vivo, and has an effect of reducing the stability of KITENIN protein, thereby inhibiting tumorigenicity, invasiveness, and metastasis. Therefore, the peptide of the present invention will be very useful as an anti-cancer therapeutic agent for suppressing cancer development, invasion, and metastasis.

1 is a diagram showing that KITENIN-Myc and KITENIN-V5 form a homodimer according to an embodiment of the present invention. (A) HEK293T cells were transfected with a plasmid encoding KITENIN-myc, purified Myc-tagged KITENIN-specific eluate was prepared, and SDS-PAGE and Coomassie blue staining were performed. Arrows indicate proteins identified by peptide mass fingerprinting (PMF) analysis showing that KITENIN-Myc interacts with endogenous KITENIN to form homodimers. (B) Shows the results of Western blot and co-immunoprecipitation (co-IP) analysis in Caco2 cells co-transfected with KITENIN-Myc or KITENIN-V5.
FIG. 2 is a diagram showing that KITENIN-Myc and KITENIN-V5 form a homodimer together with FIG. 1 . Caco2 cells (A) and HCT116 cells (B) were transfected with KITENIN-V5 and KITENIN-myc plasmids, followed by incubation for 48 hours, followed by immunoprecipitation (IP) with anti-V5 or anti-Myc antibodies, respectively It was the result of analysis through immunoblotting with the indicated antibody. The IgG band was used as a control for antibody dose in the immunoprecipitation assay. Each protein level was investigated through immunoblot analysis using whole cell lysate (WCL).
Figure 3 is a diagram showing a schematic diagram of the GST tagging domain deletion KITENIN mutation. Full length, DC (1-240), DN (110-524), DNDC (110-240).
Figure 4 shows the results of analysis on an SDS-PAGE gel showing the comparative sizes of GST fusion proteins after each mutant for the GST tagging domain deletion KITENIN mutant was expressed in E. coli, immobilized on glutathione-Sepharose beads ( black arrowheads, right panel). 10% of the lysate used for pulldown was used to show input.
Figure 5 is a picture showing the result of homodimerization of KITENIN (Homodimerization) occurring in the cell membrane. (A) Caco2 cells were transfected with empty vector (EV), KITENIN-Myc, and KITENIN-V5 for 48 hours, and the intracellular fraction was treated with cytoplasmic and membrane fractions, followed by analysis by immunoprecipitation (IP). to be. Tubulin and Na ⁺ /K ⁺ -ATPase were used as cytoplasmic and membrane markers, respectively. W; whole cell lysate, C; cytoplasm, M; membrane. (B) It is a picture showing the result of detecting the intracellular distribution of KITENIN homodimer by in situ proximity ligation assay. Caco2 cells transfected with KITENIN-Myc and KITENIN-HA were grown on cover slips, and red fluorescent dots represent KITENIN homodimers.
Figure 6 is a schematic diagram showing two possible models of homodimerization of KITENIN.
7 is a result of a co-IP (co-Immunoprecipitation) experiment to confirm whether KITENIN dimerization occurred in cis (on the same cell surface) or trans (across different cells) formation, and KITENIN homodimers were cis It is a picture that represents something that only exists in form. HEK-293T cells were transfected with plasmids encoding KITENIN-Myc or KITENIN-V5 as follows: cells transfected with empty vector (lane 1), cells co-transfected with plasmids KITENIN-Myc and KITENIN-V5 (lane 2), cells transfected with each plasmid alone but mixed for co-culture (lane 3), cells transfected with KITENIN-Myc (lane 4) or cells transfected with KITENIN-V5 (lane 5). These cells were immunoprecipitated with anti-Myc or anti-V5 antibodies, respectively, and analyzed with the indicated antibodies.
Figure 8 is a picture showing the result of reducing the expression of KITENIN-CTD dimerization of KITENIN. (A) Caco2 cells co-expressing KITENIN-V5/Myc were transfected with increasing doses of KITENIN-CTD-HA, and after 48 h of incubation, Caco2 cells were immunoprecipitated with anti-Myc antibody and assayed by the indicated antibodies. This is the detected result. (B) Caco2 cells co-expressing KITENIN-V5/Myc were transfected with KITENIN, KITENIN and NTD (1-240) and KITENIN and CTD (110-524) for 48 hours and subjected to in vitro transwell invasion assay to be. The figures shown in (A) and (B) represent three independent experiments. Histograms represent infiltrating cells counted in four selected areas and displayed as bar graphs (mean ± SEM, n = 3).
Figure 9 is a picture showing the inhibitory effect of KITENIN dimerization by the defined intracellular C-terminal region of KITENIN. (A) Schematic depiction of the effect of the intracellular C-terminal region of KITENIN on deletion mutants of KITENIN and dimerization of KITENIN. The intracellular KITENIN C-terminal domain was consecutively deleted at about 40 amino acid intervals at the C-terminal end (524 aa). (B) Caco2 cells co-expressing KITENIN-Myc and KITENIN-V5 were transfected with empty vector (EV), or wild-type KITENIN (WT), CTD and consecutively deleted KITENIN mutants (1-339, 1-394, 1- 449, 1-487), each cell lysate was immunoprecipitated with an anti-V5 antibody and then immunoblotted with an anti-Myc antibody to detect KITENIN dimers to be.
Figure 10 is a picture showing the effect of the intracellular C-terminal region of KITENIN on cell motility. Invasion assay observed after transfection of Caco2/KITENIN-V5 cells with HA-tagged deletion mutants of KITENIN (WT, CTD, 1-339, 1-394, 1-449, 1-487) for 48 hours. Photographs and histograms (mean ± SEM, n = 3, ^** P < 0.01, ^*** P < 0.001).
11 is a diagram listing seven peptide sequences classified within the C-terminal region (449-487) of KITENIN in the design of stapled KITENIN-targeting peptides.
12 is a diagram showing the inhibitory effect of KITENIN dimerization on CTD or each of the seven peptides. Caco2 cells co-expressing KITENIN-Myc and KITENIN-V5 were transfected with empty vector (EV), CTD or each of the 7 labeled peptides, immunoprecipitated with anti-V5 antibody followed by immunoblotting with anti-Myc antibody did
13 is a diagram showing the cell movement inhibitory effect of CTD or each of the seven peptides. Photographs and histograms of invasion assays observed after transfection of Caco2 cells co-expressing KITENIN-Myc and KITENIN-V5 with empty vector (EV), CTD, or each of the 7 labeled peptides (mean ± SEM, n = 3 , ^** P<0.01, ^*** P<0.001).
14 is a diagram showing the design of variant 463-471 peptides with various cell-penetrating or tumor-homing peptides.
15 is a diagram comparing the dimerization inhibitory effect of KITENIN on variant 463-471 peptides having various cell-permeable or tumor-homing peptides. Caco2 cells co-expressing KITENIN-Myc and KITENIN-V5 were treated with the variant 463-471 peptides having various cell-permeable or tumor-homing peptides for 24 hours, and each cell lysate was immunoprecipitated with an anti-V5 antibody KITENIN dimers were detected by immunoblotting with an anti-Myc antibody.
16 is a diagram comparing inhibitory effects on cell motility between variant 463-471 peptides having various cell-permeable or tumor-homing peptides. After transfecting Caco2/KITENIN-V5 cells with KITENIN-Myc and culturing for 24 hours, the cells were treated with the mutant 463-471 peptide having various cell penetrating or tumor homing peptides for 24 hours and invasion assay was performed. and histograms (mean ± SEM, n = 3, ^** P < 0.01, ^*** P < 0.001).
17 is a picture showing the levels of KITENIN transcripts after forced expression of KITENIN-WT, KITENIN-CTD or KITENIN-NTD. Caco2 cells were transfected with the empty vector, WT KITENIN-HA, KITENIN-NTD-HA (1-240 aa) or KITENIN-CTD-HA (110-524 aa), and after 48 h cells were transfected with RT- PCR analysis was applied.
18 is a picture showing that KDIP reduces the amount of KITENIN protein but not the expression of KITENIN. (A) Caco2/KITENIN-V5 cells were transfected with KITENIN-myc. After 24 hours, cells were treated with KDIP (1 μM) for the indicated times and intracellular fractions were treated as cytosolic and membrane fractions. Relative KITENIN expression in scrambled peptide (scr-pep) or KDIP-treated Caco2 cells was analyzed by the 2-ΔΔ method using Q-PCR. (B) Data represent mean ± standard deviation (n = 3).
19 is a picture showing that KDIP directly inhibits the formation of KITENIN homodimers and reduces upregulated cell invasion by overexpressed KITENIN. Caco2 cells were co-transfected with KITENIN-V5/Myc and treated with or without KDIP or scrambled peptide (scr), immunoprecipitated with an anti-V5 antibody and detected by the indicated antibodies (B). Caco2 cells were transfected with empty vector or KITENIN-V5, treated with scrambled peptide or KDIP, and subjected to in vitro transwell invasion assay (B). Histograms represent infiltrating cells counted and plotted as bar graphs in four selected areas (mean±SEM, n=4, ^** P < 0.01).
20 is a picture showing that the formation of KITENIN monomers and dimers is reduced by KDIP treatment. KITENIN-GST and KITENIN-His were expressed in bacteria, purified in vitro, mixed with KDIP and run on a native SDS-PAGE gel (A). Protein sizes are indicated by arrowheads. After GST pulldown, KITENIN-GST attached to streptavidin was detected by anti-His antibody (B).
21 is a diagram showing that degradation of KITENIN after KDIP treatment requires an intact KDIP sequence (463-471 peptide, RACK1 binding site) in KITENIN. Caco2 cells were transfected with KITENIN-V5/Myc or Δ463-471 KIT-V5/Myc and treated with KDIP for 24 h, immunoprecipitated with anti-V5 antibody followed by immunoreactive V5-tagging and Myc-tagging (tagging) Protein was investigated (A). The effect of forced expression of Δ463-471 KITENIN on the effect of KDIP on cell invasion is shown (B). After 24 hours of KDIP treatment, cells were subjected to invasion assay. Photographs and histograms of infiltration assays were obtained as in (B) (mean ± SEM, n = 4). Asterisks ( ^* P<0.05) indicate significant differences between groups. NS means no significant difference between groups.
Figure 22 is a picture showing that RACK1 binds specifically to KITENIN. Caco2/KITENIN-V5 cells were transfected with RACK1-GFP for 48 hours, immunoprecipitated with an anti-GFP antibody, and immunoprecipitated with an anti-V5 antibody. This is the result of detecting KITENIN bound to RACK1 by blotting. Proteins in whole cell lysates (WCL) were detected with the indicated antibodies.
Figure 23 is a picture showing that RACK1 specifically binds to KITENIN, Caco2 cells were transfected with KITENIN-V5 and / or RACK-GFP and 24 hours later, the cells were treated with KDIP (1 uM) for 24 hours This is the result of immunoprecipitation with an anti-GFP antibody. Proteins in whole cell lysates (WCL) were detected with the indicated antibodies.
24 is a diagram showing that RACK1 specifically binds to KITENIN. Caco2 cells were transfected with KITENIN-V5 or Δ463-471-KITENIN-V5 and/or RACK1-GFP for 48 hours and immunized with an anti-GFP antibody. This is the result of detecting the interaction between Δ463-471 KITENIN and RACK1 by precipitation. Proteins in whole cell lysates (WCL) were detected with the indicated antibodies.
Figure 25 is a picture confirming the change in the effect of KDIP on KITENIN degradation according to the expression state of RACK1. Caco2 cells were transfected with KITENIN-V5 and / or RACK1-GFP, and after KDIP (1 μM, 24 hours) treatment These are the results detected by the indicated antibodies (A) and invasion assay (B). Pictures and histograms of the infiltration analysis were shown in FIG. 21 .
Figure 26 is a diagram confirming the change in the effect of KDIP on KITENIN degradation according to the expression status of RACK1. Caco2 cells were transfected with KITENIN-V5 and / or si-RACK1, and after KDIP (1 μM, 24 hours) treatment These are the results detected by the indicated antibodies (A) and invasion assay (B). A photograph and histogram of the infiltration analysis were shown in FIG. 21 .
Figure 27 shows Caco2 cells co-transfected with WT KITENIN-V5, KITENIN-CTD-HA and/or RACK1-GFP for 48 hours and analyzed by immunoblotting with the indicated antibodies (A) or by invasion assay (B). The picture showing the results shows that RACK1 influences the effect of KITENIN-CTD on KITENIN degradation and cell motility inhibition. Photographs and histograms of the infiltration assay were shown in FIG. 21 (mean ± SEM, n = 3, ^** P <0.01).
Figure 28 shows Caco2 cells co-transfected with WT KITENIN-V5, KITENIN-CTD-HA and/or RACK1-siRNA for 48 hours and analyzed by immunoblotting with the indicated antibodies (A) or by invasion assay (B). The picture showing the results shows that RACK1 influences the effect of KITENIN-CTD on KITENIN degradation and cell motility inhibition. Photographs and histograms of the infiltration assay were shown in FIG. 21 (mean ± SEM, n = 3, ^** P <0.01).
29 is a diagram showing changes in the amount of KITENIN protein according to RACK1 expression. Caco2/KITENIN-V5 cells were transfected with empty vector or RACK1-GFP for 48 h, treated with cycloheximide (CHX, 20 ng/ml) for the indicated time points (hours) and examined by immunoblot analysis.
Figure 30 shows the lysosomal-autophagy pathway dependent pattern of degradation of KITENIN after KDIP treatment. Caco2 cells transfected with KITENIN-myc for 48 hours were treated with KDIP (1 μM) for 24 hours. Cells were re-treated with MG132 (10 μM) and A1 (Bafilomycin A1, 1 μM), CQ (chloroquine, 100 μM), or 3-MA (1 mM) for 4 and 12 h, respectively, prior to harvesting the cells during this experiment. did The amount of KITENIN protein was confirmed with an anti-myc antibody.
31 is a picture showing the results of KITENIN degradation in a lysosome-autophagy pathway dependent manner after expression of KITENIN-CTD. Caco2 cells were either transfected with empty vector or co-transfected with KITENIN and/or KITENIN-CTD for 48 hours. During this experiment, cells were incubated with MG132 (10 μM) and A1 (Bafilomycin A1, 1 μM), CQ (chloroquine, 100 μM) or 3-MA (1 mM) for 4 h and 3-MA (1 mM), respectively, before harvesting the cells. It was treated again for 12 hours. The amount of KITENIN protein was confirmed with an anti-KITENIN antibody.
32 is a picture showing that KDIP stimulates the autophagic degradation of KITEINN. Caco2/KITENIN-V5 cells were treated with KDIP for 24 hours and evaluated by immunoblot analysis using anti-KITENIN, anti-p62 or anti-LC3B antibodies (A). LC3B and p62 were used as markers of autophagy. Caco2/KITENIN-V5 cells were treated with KDIP and/or 3-MA for 24 hours, and changes in LC3B expression were examined by immunoblot analysis (B). After treatment with Rapamycin or KDIP (5M) in Caco2/KITENIN-V5 cells (C), autophagic vacuoles were stained with Cyto-ID green dye.
33 is a diagram showing the results of identification of KITENIN-binding proteins showing quantitatively changed patterns after KDIP treatment. Caco2/KITENIN-V5 cells were treated with scr-peptide or KDIP, immunoprecipitated with anti-V5 antibody, and the eluate was separated on an SDS-PAGE gel and stained with Coomassie blue to obtain a peptide mass fingerprint (PMF). ) is the result of analysis (A). Arrows indicate proteins whose binding to KITENIN was weakened after KDIP. Changes in KITENIN-binding protein identified in KITENIN-transfected cells (B). Expression patterns of eEF2 and Myo10 identified by PMF analysis in Caco2/KITENIN-V5 cells were examined. This is the reduced expression pattern of KITENIN binding protein after KDIP treatment (C). Caco2/KITENIN-V5 cells were treated with KDIP followed by immunoprecipitation with anti-V5 antibodies and immunoblotting with the indicated antibodies.
34 is a picture showing changes in the binding profile of KITENIN and RACK1 by knockdown of the identified KITENIN binding proteins, eEF2 and Myo10. Caco2 cells were transfected with KITENIN-V5 and/or RACK1-GFP under knockdown of eEF2 (A) or Myo10 (B) via siRNA transfection and treated with KDIP (1 μM) for 24 h. Cells were immunoprecipitated with anti-GFP antibody (RACK1) and immunoblotted with anti-V5 antibody (KITENIN).
35 is a picture showing the results of Myo10 mediating the inhibitory effect of KDIP on KITENIN level and cell invasion. Caco2 cells were transfected with empty vector or KITENIN-V5 under knockdown of eEF2 via siRNA transfection and treated with KDIP (1 μM) for 24 hours. Cells were analyzed by immunoblot analysis to examine KITENIN levels (A) or invasion assay (B). Photographs and histograms of invasion analysis were shown in Figure 21 (mean ± SEM, n = 4, ^* P <0.05, ^** P <0.01, ^*** P <0.001).
FIG. 36, along with FIG. 35, is a diagram showing the results of Myo10 mediating the inhibitory effect of KDIP on KITENIN level and cell invasion. Caco2 cells were transfected with empty vector or KITENIN-V5 under knockdown of Myo10 via siRNA transfection and treated with KDIP (1 μM) for 24 hours. Cells were analyzed by immunoblot analysis to examine KITENIN levels (A) or invasion assay (B). Photographs and histograms of infiltration assays are shown in Figure 21 (mean ± SEM, n = 4, ^* P <0.05, ^** P <0.01, ^*** P <0.001).
37 is a diagram confirming that Myo10 binds to the transmembrane portion of KITENIN. Bacterially expressed proteins from GST-tagged KITENIN deletion constructs were purified, incubated with cell lysates containing Myo10 and pulled down. Bound protein complexes were eluted and analyzed by SDS-PAGE followed by immunoblotting. Interaction was investigated by detecting the blot with anti-Myo10 antibody. The positive bands are KITENIN full length and ΔC (KITENIN without intracellular C-terminal region), ΔN (KITENIN without intracellular N-terminal region) and ΔNΔC (KITENIN -terminal and C-terminal without intracellular N). region, but with transmembrane regions) constructs, but not CTD (intracellular C-terminal regions without membrane regions) constructs. Arrows indicate the titrated size of each protein.
38 is a picture showing that the C-terminal FERM domain of Myo10 interacts with the transmembrane portion of KITENIN. A schematic representation of the Myo10-HA deletion mutant was shown. Open boxes represent each functional domain of Myo10. Three (left panel) or two (right panel) deletion mutants of HA-Myo10 were expressed in Caco2/KITENIN-V5 cells, and cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-HA antibody. to detect the interaction between KITENIN and HA-tagged Myo10. Each Myo10 deletion mutant that binds to KITENIN is indicated by an arrow.
39 is a picture showing that Myo10 acts as a regulator of KITENIN expression. Caco2/KITENIN-V5 cells were transfected with si-KITENIN (A) or si-Myo10 (B) for 48 hours and analyzed by immunoblot analysis using the indicated antibodies. Expression of Myo10 was not affected by knockdown of KITENIN (A), but expression of KITENIN was almost absent in the knockdown state of Myo10 (B).
40 is a picture showing that the formation of KITENIN dimers is almost stopped under knockdown of Myo10. Caco2 cells co-transfected with KITENIN-V5 and/or KITENIN-Myc were transfected with si-Myo10 for 48 hours, immunoprecipitated with anti-V5 antibodies and assayed with the indicated antibodies.
41 is a picture showing that Myo10, which was reduced after KDIP treatment, was recovered after MG132 pretreatment, but not by treatment with a lysosome inhibitor or an autophagy inhibitor. Caco2 cells transfected with KITENIN-V5 were treated with KDIP (1 μM) for 24 hours. During this experiment, cells were re-treated with MG132 (10 μM) and chloroquine (100 μM) or 3-MA (1 mM) for 4 and 12 hours, respectively, before harvesting the cells. The amounts of Myo10 and KITENIN proteins were confirmed by immunoblot analysis.
42 is a picture showing that Myo10 was reduced after transfection with KITENIN-CTD. Caco2 cells expressing empty vector or KITENIN-V5 were transfected with CTD-HA and treated with KDIP for 24 hours. Cells were analyzed with the indicated antibodies.
43 is a picture showing the effect of MG132 on inhibition of cell invasion by KDIP. Caco2 cells were transfected with empty vector or KITENIN-V5 and treated with KDIP (1 μM, 24 hours). During this experiment, cells were harvested and treated again with MG132 (10 M) for 12 hours prior to invasion assay. A photograph and histogram of the infiltration assay were shown in Figure 21.
44 is a picture showing that the levels of Myo10 and KITENIN were further decreased after treatment with KDIP under the expression of Nrdp1. Caco2/KITENIN cells were transfected with empty vector (EV) or Nrdp1 and then treated with KDIP (1 M). The protein levels of Myo10 and KITENIN were checked after treatment with cycloheximide (CHX, 20ng/ml) at indicated times.
45 is a picture showing that the E3-ligase (ligase) Nrdp1 is associated with Myo10 ubiquitination in KDIP-treated Caco2 cells. Caco2 cells were transfected with KITENIN and Nrdp1 or ubiquitin and treated with scrambled peptide or KDIP (1 μM) for 24 hours. Cell products immunoprecipitated with anti-Myo10 antibody were separated and transferred on a 6% SDS-polyacrylamide gel and then immunoblotted with anti-ubiquitin antibody.
Figure 46 is a picture showing that the E3-ligase (ligase) Nrdp1 mediates the effect of KDIP treatment on Myo10 degradation and reduced cell invasion. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5 under knockdown of Nrdp1 via siRNA transfection and treated with KDIP (1 M) for 24 hours. Cells were analyzed by immunoblot analysis to perform Myo10 and KITENIN levels (A) or invasion assay (B). Photographs and histograms of infiltration assays are shown in Figure 21 (mean ± SEM, n = 4, ^* P <0.05, ^** P <0.01, ^*** P <0.001).
47 is a schematic diagram showing how Myo10 stabilizes KITENIN homodimers and the effect of KDIP on the degradation and stability of KITENIN dimers. Here, Myo10 acts as an effector that selectively binds to KITENIN and stabilizes dimerization to regulate the oncogenic activity of KITENIN. Through downregulation of Myo10, KDIP exhibits more specific antitumor activity in cancer cells expressing higher levels of KITENIN, RIM, and RACK1 interaction motifs.
48 is a picture showing that KDIP treatment reduces cell invasion of KITENIN-overexpressing CT-26 cells in an in vivo mouse model of CRC. Cell invasion was compared in CT-26 cells transfected with KITENIN and treated with scr-peptide or KDIP (1 μM) (A). 7×10 ⁴ KITENIN-transformed Caco2 cells (Caco2/KITENIN) or KITENIN-transformed CT-26 cells expressing iRFP (CT-26/iRFP/KITENIN) were suspended in a medium containing 0.2% BSA and then , cells were treated with scrambled peptide (1 μM) or KDIP at the indicated concentrations (up to 10 μM) for 24 h and invasion assay was performed (B). 48 shows three independent experiments. A photograph and histogram of the infiltration analysis were shown in FIG. 21 . Based on these results, the IC ₅₀ (half-maximal inhibitory concentration) of KDIP for cell invasion was obtained. Asterisks indicate Caco2/KITENIN-V5 cells (scr-peptide-treated versus KDIP-treated, ^** P<0.01; ^*** P<0.001) or CT-26/iRFP/KITENIN cells (scr-peptide-treated versus KDIP-treated, ^# P<0.05, ^## P<0.01).
49 is a diagram illustrating the schedule of animal experiments for syngeneic tumor models of BALB/c mouse and CT-26 mouse colon adenocarcinoma cell lines to investigate whether KDIP has an antitumor effect in vivo.
50 is a diagram showing that intravenous injection of KDIP significantly reduces tumor size in a syngeneic mouse tumor model. BALB/c mice were inoculated subcutaneously with 1×10 ⁶ CT-26/KITENIN-V5 cells. After tumors had grown for 1 week, peptides were administered intravenously every other day for 14 days. Mice were sacrificed on day 17, and images of tumors and tumor weights in different treatment groups were obtained (mean ± SEM) (A). Line graphs of tumor growth and individual body weights of CT-26/KITENIN-tumor mice after intravenous injection of vehicle, scr-peptide or KDIP (1 and 5 mg/kg, mpk) are shown as mean ± SEM. shown (B).
51 is a diagram illustrating a schedule of animal experiments for an intra-splenic liver metastasis model to investigate whether KDIP has an anti-metastatic effect in vivo.
52 is a picture showing that intravenous injection of KDIP significantly reduced colorectal liver metastasis in an intrasplenic liver metastasis model. An experimental liver metastasis model was prepared by splenectomy after intrasplenic inoculation into syngeneic mice stably expressing CT-26/iRFP/KITENIN-V5 cells. For 2 weeks, mice were intravenously injected with KDIP (5 mg/kg) or scrambled peptide dissolved in 0.1% DMSO 3 times a week. For metastasis evaluation, total fluorescence emitted from liver nodules expressing iRFP was measured and counted (A), or metastatic tumor growth was counted as nodules that migrated to the liver surface and a metastatic score was calculated by multiplying by size (B). Metastatic scores or total fluorescence are presented as mean ± SEM. Asterisks indicate significant differences between indicated groups ( ^* P<0.05). Line graphs of individual weights after intravenous injection of scr-peptide or KDIP (5 mg/kg, mpk) were presented as mean ± SEM (C).
53 is a picture showing that the levels of Myo10, KITENIN, their down signals, and phospho-c-Met decreased, but the levels of KAI1 were recovered in KDIP-treated regressed tumor tissues. Expression levels of Myo10, eEF2, KITENIN, phospho-ERK, phospho-c-Jun, KAI1 and phospho-c-Met were compared in metastatic liver collected from two groups, KDIP (5 mg/kg) versus scramble peptide (scr-pep). Nodules were examined by immunoblot analysis (A). The quantitative amount of each protein was measured by densitometry and expressed as an arbitrary score (mean ± SEM, n = 6) (B). Asterisks indicate significant differences between indicated groups ( ^* P<0.05, ^** P<0.01, NS: not significant).

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Materials and Methods

1-1. Cell culture and reagents

Cell lines were purchased from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and regularly tested for mycoplasma contamination. CT-26-WT-iRFP-Neo cells were purchased from Imanis Life Sciences (Rochester). The cells were cultured in RPMI-1640 medium or DMEM containing 10% fetal bovine serum (GenDEPOT), 100 units/mL penicillin, and 100 μg/mL streptomycin (Corning) at 37° C. in a humid atmosphere containing 5% CO ₂ . Cells were passaged before reaching confluence. Brefeldin A, chloroquine, MG132, 3-MA, rapamycin and cycloheximide (Sigma) were injected at the indicated concentrations.

1-2. Plasmids and siRNAs

Expression constructs were prepared by a PCR-based method: V5 tag, Myc tag, HA tag KITENIN, GST tag KITENIN deletion mutant, D463-KITENIN and His tag KITENIN. All constructs were confirmed by sequencing. pEGFRP-N1-RACK1 was provided by Anna Huttenlocher (Addgene plasmid #41088). All siRNAs used for gene silencing were purchased from Santa Cruz Biotechnology. Each consisted of a mixture of several sequences, eliminating sequence-specific diversity.

1-3. Acrylamide gel staining and PMF analysis

Coomassie brillant blue G 250 staining was performed for visualization of proteins separated by SDS-PAGE. Specifically, the gel was fixed for 30 minutes in a fixation solution (30% ethanol, 2% phosphoric acid aqueous solution) on a shaking platform. The fixed gel was washed with a washing solution (2% aqueous phosphoric acid solution) and then equilibrated in an equilibration solution (18% ethanol, 15% ammonium sulfate, 2% aqueous phosphoric acid solution) for 30 minutes. Protein bands visualized by staining were punched for MALDI-TOF mass spectrometry after in-gel digestion. All degraded peptides were verified by PMF (Peptide Mass Fingerprinting) analysis (Genomine, Korea).

1-4. Antibodies and immunoprecipitation reactions

Antibodies against the following proteins were purchased from the indicated suppliers: Na ⁺ /K ⁺ ATPase, GFP and tubulin (Santa Cruz Biotech); c-Jun, pc-Jun, ERK, p-ERK, c-MET, pc-MET and Cell Signaling Technol (GST); V5, Myc (MBL); HA and b-actin (Sigma); KITENIN (Atlas); His and RACK1, LC3, p62, Myo10 and eEF2 (Abcam). These were used with appropriate secondary antibodies (Thermo). For transiene transfection analysis, 293T, Caco2 and HCT116 cells were transfected with various plasmids and acquired for immunoblot analysis 48 h post transfection. Most assays using stable cell lines used mixed polyclonal cells to rule out clonal variation. Cellular proteins were isolated, transferred, and immunoblotted as previously described (14). Cell lysates of 293T, Caco2 and HCT116 cells were used for immunoprecipitation experiments as described in a previous paper (15).

1-5. Subcellular fractionation

Cytoplasmic and membrane fractions were prepared with an intracellular protein fractionation kit (Thermo Scientific) according to the manufacturer's instructions. Each fraction was analyzed by SDS-PAGE and probed for KITENIN by tagging antibodies to V5 and Myc. Fraction purity was assessed by probes for Na ⁺ -K ⁺ ATPase for tubulin and membrane proteins for the cytoplasm.

1-6. Cell invasion assay

Cell invasion was measured using a transwell transfer device as described in a previous paper (14). Specifically, the cultured cells were loaded on top of a 24-well invasion chamber assay plate (Costar). Conditioned DMEM medium containing 10 μg/ml fibronectin (Calbiochem) and 1% FBS was added to the lower chamber as a chemoattractant. Cells were stained after 16 or 48 hours (HCT116) incubation. Cells on the top surface of the filter were wiped off with a cotton swab and migrated cells on the bottom surface were counted in four random squares of 0.5 mm × 0.5 mm for each filter. The results are presented as the mean ± SEM of cell numbers per field for at least three independent experiments.

1-7. In situ In situ proximity ligation assay (PLA)

Control-Caco2/EV and Caco2/KITENIN-Myc/HA cells were embedded in 1% agarose and fixed overnight with 10% neutral buffered formalin solution. After fixation, cells were stained overnight at 4°C with a primary antibody against the tagging of KITENIN. After that, secondary antibodies for mouse and rabbit antibodies (Duolink ^® in situ proximity ligation assay Probes, Sigma-Aldrich) were added. A brightfield detection system (Duolink ^® Brightfield in situ detection reagents, Sigma-Aldrich, Darmstadt, Germany) was applied to the slides according to the manufacturer's protocol (Duolink proximity ligation assay reagents Brightfield Protocol. Courtesy: https://www.sigmaaldrich. com/content/dam/sigmaaldrich/docs/promo_NOT_INDEXED/General_Information/1/duolink-pla-brightfield-protocol-msig.pdf). The density of glomerular signals was analyzed using confocal microscopy.

1-8. Pulldown assay

GST-KITENIN or its derivatives were immobilized on glutathione-sepharose beads. Myc-tagged KITENIN was incubated with GST-tagged KITENIN or a derivative thereof immobilized on glutathione-sepharose as indicated. Bound protein complexes were eluted and analyzed by SDS-PAGE followed by IB. To detect KITENIN homodimerization in vitro, buffer [50 mM Tris.Cl (pH 7.5), 1 mM dithiothreitol (DTT), 4% (v/v) glycerol, 0.1 mg/ml BSA, 5 mM MgCl ₂ , 1 mM ATP and 50 mM NaCl], GST-KITENIN pulled down and Ni-NTA purified KITENIN-His or KDIP (5M) were incubated at 4°C for 2 hours. After incubation, the beads were thoroughly washed using wash buffer (pull-down buffer containing 300 mM NaCl), and the bead-bound proteins were resolved on SDS-PAGE followed by immunoblot analysis using the indicated antibodies.

1-9. RT-PCR and Q-PCR

RNA preparation and reverse transcription were performed as described in a previous paper (14). The exponential phase of RT-PCR was set to 30 cycles to enable quantitative comparison of various cDNAs developed in the same reaction with the GeneAmp PCR system (Eppendorf). Real-time PCR was performed using Rotor-gene (Qiagen). Reactions were performed in triplicate in three independent experiments. Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control for variability in expression levels and analyzed using the 2- ^DDCT method as previously described.

1-10. Autophagosome staining

To determine whether KDIP induces autophagy in KITENIN-expressing Caco2 cells, cells were grown on coverslips for 24 hours. Cells were washed with PBS and then stained with Cyto-ID green fluorescence reagent (Enzo Life Sciences, Plymouth Meeting, PA) for 1 hour at 37° C. in a cell culture incubator. Cells were washed with PBS and mounted with Vectashield mounting medium (Vector Lab) containing nuclear DAPI. Cells were imaged by confocal microscopy.

1-11. In vivo tumor growth and liver metastasis model

All animal experiments were performed in accordance with the guidelines of the Animal Protection Committee of the Research Institution, College of Medicine, Chonnam National University, and all experimental protocols were approved by the committee (CNU IACUC-H-2019-3).

The in vivo effect of KDIP on colorectal tumorigenesis was investigated using a CT-26 cell/syngeneic mouse model, as the syngeneic mouse tumor model was reported to be useful for testing the anticancer effect of candidate substances (21, 22) Male Balb/c mice (5 weeks old) were purchased from DaMul Science (Korea) and adapted for 1 week before subcutaneous injection of syngeneic CT-26/KITENIN cells (1 × 10 ⁶ ) into the back. Tumor volume was calculated using the following equation: V = 1/2 × a × b2 , where a and b are the longest and shortest diameters of the tumor (in mm), respectively. To confirm the effect of KDIP, tumor volume was measured every other day for 19 days. All mice were sacrificed after 30 days, and subcutaneous tumor grafts were surgically excised and weighed.

For liver metastasis analysis, 5-6 week old Balb/c mice were purchased from OrientBio, Inc. (Seongnam, Korea) and housed in metal cages with free access to water and food. A syngeneic mouse model of colorectal liver metastasis was established by administering tumor cells into the portal system via intrasplenic injection (23). Specifically, CT-26/KITENIN-iRFP expressing cells (2 × 10 ⁵ cells/mouse) were administered to the spleen. Splenectomy was performed 15 minutes after CT-26 cell injection. Mice were randomly divided into two groups: scrambled peptide (0.1% DMSO) and KDIP (5 mg/ml). Each peptide was intravenously injected 6 times for 2 weeks 7 days after tumor cell inoculation. Metastatic tumor nodules >1.0 mm in diameter in the liver were counted microscopically and scored as 0 (total no metastases), 1 (tumor size >1 mm), 2 (tumor size >5 mm), and 3 (tumor size >10) according to nodule size. mm). The metastasis score was multiplied by the number of nodules and the score. Fluorescence images of iRFP-expressing liver nodules were taken using a Fluorescence-labeled Organism Bioimaging Instrument (FOBI; Cellgentek, Korea), and total fluorescence emitted from the liver nodules was measured and compared between groups.

1-12. statistics

Experimental results were tested for statistical significance using ANOVA followed by Tukey HSD post hoc test or Student's t test. All statistical tests were two-sided and a P -value of less than 0.05 was considered statistically significant. Statistical analysis was performed using PASW Statistics 20 software (SPSS, IBM Company, Chicago, IL).

Experiment result

2-1. The intracellular C-terminal region of KITENIN contributes to the homodimerization and stability of the KITENIN protein.

The KITENIN complex plays an important role in CRC progression and metastasis ((15,17,19), but little is known about how proteins maintain structural integrity and stability. Considering that it forms a stable structure (1, 3-5), we first investigated whether the KITENIN protein forms a homo-oligomer complex.

To identify proteins interacting with KITENIN, first, the total protein content of KITENIN-myc overexpressing 293T cells was immunoprecipitated with a Myc-Tag antibody, and the precipitated proteins were separated by size using SDS-PAGE. The 130 kDa band was isolated and identified as KITENIN by peptide mass fingerprint (PMF) (see Fig. 1(A)). Since the band size was about twice that of the KITENIN monomer, it was assumed to be a homodimerized form of KITENIN. Then, Western blot and co-immunoprecipitation (co-IP) analysis were performed on cells transfected with two different KITENIN tagging constructs. Immunoblots using antibodies against the V5 or myc epitope, respectively, confirmed the co-expression of KITENIN monomers in the transfected cells (see FIG. 1(B)). The interaction between KITENIN-V5 and KITENIN-myc was observed in HEK293T cells (see FIG. 1(B)), but also in CRC cell lines such as Caco2 and HCT116 cells (see FIGS. 2(A) and (B)). To identify the domains participating in dimer binding, each domain of the KITENIN protein was expressed using the GST bacterial expression system. The KITENIN protein was divided into four truncated forms: full-length, deleted C-terminal domain (DC; CTD), deleted N-terminal domain (DN; NTD), deleted N and C-terminal domains (DNDC; TM, transmembrane domain) (see Figure 3). The lysate of 293T cells expressing full-length KITENIN-myc and the purified GST fusion cleaved form were pulled down with glutathione-sepharose beads and then reacted with myc antibody to detect the KITENIN dimerization site did When the KITENIN C-terminal region was deleted, the protein was not detected by the myc antibody, indicating that the KITENIN dimer occurred through the intracellular C-terminal domain (KITENIN-CTD, see Fig. 4). In order to determine where KITENIN dimerizes in cells, IP of the cell membrane fraction was performed. It was observed that KITENIN, a protein that spans the cell membrane, formed a dimer in the cell membrane (see FIG. 5(A)). Dimerization spots could also be observed as red fluorescence under a confocal microscope using a proximity ligation assay (see FIG. 5(B)). To confirm whether this dimerization of KITENIN occurred in cis (on the same cell surface) or trans (across different cells) formation, co-IP experiments were performed. Two Caco2 cell populations were transfected with KITENIN-V5 or KITENIN-myc. Under co-culture conditions, KITENIN-myc and KITENIN-V5 proteins did not interact with each other. On the other hand, a reciprocal interaction between KITENIN-myc and KITENIN-V5 was observed under co-expression conditions (see FIGS. 6 and 7). Therefore, it was confirmed that the KITENIN homodimer exists only in the cis form.

Next, since it was observed that dimerization occurs through the intracellular C-terminal region, whether the expression of KITENIN-CTD affects dimerization of KITENIN was investigated. Interestingly, KITENIN dimerization was reduced after ectopic overexpression of KITENIN-CTD (see Fig. 8(A)). In addition, the increased cell invasion caused by the expression of wild-type KITENIN (KITENIN-GOF) was restored to a level similar to that of the empty vector after co-expression of KITENIN-CTD. In contrast, the increased cell invasion caused by KITENIN-GOF was not affected by the co-expression of KITENIN's N-terminal region (NTD) (see Fig. 8(B)). These results suggested that ectopic expression of KITENIN-CTD interfered with KITENIN homodimerization at the protein level. Overall, these results suggest that KITENIN homodimers are important for the stability and oncogenic function of KITENIN proteins and that inhibition of KITENIN-GOF by KITENIN-CTD may be through dysregulation of homodimerization. That is, KITENIN-CTD can down-regulate homodimerization by competitive interactions with other full-length KITENIN molecules, but also destroy homodimers through regulation of unidentified molecules that maintain the stability of KITENIN dimers, thereby reducing homodimerization. You can control your anger down.

2-2. Identification of candidate peptide sequences in KITENIN-CTD that interfere with KITENIN dimerization

Since overexpression of KITENIN-CTD inhibited KITENIN dimerization, the present inventors next attempted to identify a responsible region within CTD using serial deletion constructs. Enforced expression of the three deletion constructs (1-339, 1-394, 1-449) resulted in increased cell invasion compared to wild-type KITENIN, whereas the 1-487 construct reduced cell invasion similar to KITENIN-CTD . These results indicate that the 449-487 region of KITENIN may be responsible for the inhibitory effect on dimerization and cell invasion (see FIGS. 9(A) and 9(B)). To narrow the region, the 449-487 region of KITENIN was split into 10-mers or less to design several peptides and fused to a cell-permeable peptide sequence (YARVRRRGPRR; CPP) derived from the human transcription factor Hph-1 (Table 1 and Figure 1). 10) (24, 25), and the inhibitory effect of each peptide on KITENIN dimerization was tested. As shown in FIGS. 12 and 13 , the 463-471 peptide sequence was most effective in inhibiting dimerization and cell invasion due to KITENIN overexpression. Therefore, this peptide sequence was named KDIP (KITENIN dimerization-interfering peptide). Various other CPPs or tumor-homing sequences were attached to KDIP to find the most suitable peptide-linker for KDIP's effect on efficient cell permeability, dimer interference and invasion inhibition (see Table 2 and Figure 14) experimented. As a result, KITENIN dimerization was inhibited to a similar degree by KDIP-CPP or tumor homing peptide, but KDIP-CPP showed the highest inhibitory effect on cell invasion (p<0.001) (see FIGS. 15 and 16). Accordingly, the KDIP peptide derived from KITENIN-CTD could effectively inhibit dimer formation and cell invasion caused by KITENIN overexpression.

sequence number location KITENIN-CTD 4 449-456 peptide PTLQYDKD 5 455-462 peptide KDRWLSTQ 6 462-469 peptide QWRLVSDE 7 463-471 peptide WRLVSDEAV 8 467-474 peptide SDEAVTNG 9 473-480 peptide NGLRDGIV 10 480-487 peptide VFVLKCLD

name order sequence number CPP
(Cell penetration peptide) Hph-1 YARVRRRGPRR-WRLVSDEAV 11 TAT(47-57) YGRKKRRQRRR-GFLG-WRLVSDEAV 12 Penetratin RQIKIWFQNRRMKWKK-GFLG-WRLVSDEAV 13 Tumor homing peptide EGFR-1 KCCYSL-GFLG-WRLVSDEAV 14 EGFR-2 AEFLR-GFLG-WRLVSDEAV 15

2-3. KDIP reduces homodimerization and stability of KITENIN protein

Next, it was confirmed whether the effect of KITENIN-CTD overexpression on KITENIN level and dimerization level was at the transcriptional or protein level. As a result, it was found that the level of the KITENIN transcript was almost the same after forced expression of KITENIN-WT or KITENIN-NTD and after forced expression of KITENIN-CTD (FIG. 17), indicating that dimerization of KITENIN by KITENIN-CTD and This means that a decrease in protein amount can be considered an effect on protein levels. As with KITENIN-CTD, KDIP treatment reduced KITENIN in the plasma membrane fraction, with the greatest reduction at 48 hours independent of the KITENIN transcript used (FIG. 18). Also, as in the case of KITENIN-CTD, KDIP treatment reduced KITENIN homodimerization and cell invasion by KITENIN overexpression (FIG. 19). Additionally, the effect of KDIP on dimerization of KITENIN by SDS-PAGE under non-reducing/non-denaturing conditions was reconfirmed. As a result, it was found that the 130-kDa KITENIN homodimer band and the 70-kDa KITENIN monomer band were also reduced after KDIP treatment (see FIG. 20(A)). In addition, dimerization of KITENIN proteins was directly reduced by KDIP treatment when two purified KITENIN proteins with different tags were pulled down in vitro (see Fig. 20(B)). Therefore, we hypothesized that the reduction of KITENIN dimers after KDIP treatment could be the result of KITENIN degradation after exposure of specific regions important for protein stability. Dimerization of the KITENIN protein may prevent exposure of this region through structural disruption.

Next, to confirm whether the 463-471 peptide sequence is required for increased KITENIN degradation after KDIP treatment, a mutant KITENIN construct (D463-471-KITENIN, Fig. 21 (A) left) was prepared. D463-471-KITENIN formed homodimers like WT-KITENIN, but the effect of KDIP on dimerization and KITENIN protein reduction was not observed (Fig. 21 (A) right). In addition, overexpression of D463-471-KITENIN increased cell invasion similarly to overexpression of WT-KITENIN, but the inhibitory effect of KDIP on the increased cell invasion by D463-471-KITENIN was not observed (FIG. 21(B) ). Thus, the 463-471 peptide sequence is required for continuous degradation of KITENIN after KDIP treatment, and degradation of KITENIN dimers after KDIP resulted in increased inhibition of cell invasion due to overexpressed KITENIN.

2-4. KDIP treatment accelerates the binding of RACK1 to KITENIN and degradation of KITENIN.

KITENIN (Vangl1) is a membrane protein and its membrane localization is essential for its role in maintaining polarity as a component of the PCP pathway (26, 27). RACK1 is known to maintain PCP signaling and membrane localization of Vangl2 (28). As a result, it was found that RACK1 acts as an adapter protein for molecules involved in downstream signaling of the KITENIN complex, which can control the levels of Dvl2 and KITENIN through autophagy-dependent degradation (23). To confirm whether RACK1 directly interacts with KITENIN, an IP experiment using an antibody against GFP was performed in Caco2 cells co-expressing RACK1-GFP and KITENIN-V5, and it was found that RACK1 binds to KITENIN (FIG. 22 ). Interestingly, when RACK1-GFP was overexpressed and then treated with KDIP, KITENIN binding to RACK1 increased and KITENIN protein decreased (FIG. 23). It could be inferred that after KDIP treatment, KITENIN dimerization was broken, resulting in a structural change in which the region masked by dimerization was exposed and the binding of RACK1 to KITENIN increased. We hypothesized that the 463-471 amino acid sequence of KITENIN is a masked area involved in KITENIN dimer maintenance and tested by co-IP using D463-471-KITENIN-V5 and GFP-RACK1. Unlike WT-KITENIN, D463-471-KITENIN did not bind to RACK1 (FIG. 22). Collectively, these results indicate that the 463-471 peptide sequence in KITENIN is required for the binding of RACK1 to KITENIN, and that this site is structurally hidden when KITENIN is in a dimerized form, preventing its binding to RACK1. Therefore, the present inventors named the 463-471 aa sequence of CTD as the RACK1-interacting motif (RIM).

Next, it was confirmed whether RACK1 is required for KITENIN degradation by KDIP treatment or KITENIN-CTD transfection. As shown, KITENIN was further reduced by KDIP (Figs. 25(A) and 25(B)) or KITENIN-CTD (Figs. 27(A) and 27(B)) when RACK1 was overexpressed. Transfection of RACK1 alone did not alter KITENIN content. However, when cells were treated with KDIP or KITENIN-CTD in the presence of RACK1 overexpression, the KITENIN content was further reduced. These results indicate that KDIP treatment or transfection of KITENIN-CTD induces KITENIN degradation by increasing the binding between KITENIN and RACK1. When RACK1 was knocked down by treatment with RACK1-specific siRNA, KDIP (FIG. 27) or KITENIN-CTD (FIG. 29) attenuated the effect of reducing the amount of KITENIN protein. In contrast to the KITENIN content, the increased cell invasion by KITENIN was suppressed by the expression of RACK1 (Fig. 25(A) and (B)). When RACK1 was co-overexpressed, KDIP showed a greater inhibitory effect on cell invasion than when KITENIN was expressed alone (Fig. 25(A) and (B)), whereas under si-RACK1 treatment, KDIP inhibited cell invasion. No inhibitory effect was shown (Fig. 26(A) and (B)). Similarly, the reduced cell invasion by KITENIN-CTD was further accelerated by overexpression of RACK1 (Figure 27(A) and (B)), but si-RACK1 treatment blocked the inhibitory effect of KITENIN-CTD on cell invasion. (FIGS. 28(A) and (B)).

2-5. RACK1 promotes KITENIN degradation after KDIP treatment through the autophagic pathway.

RACK1 has been reported to bind to ribosomes and affect mRNA translation and protein synthesis (29). To rule out the possibility that RACK1 affects KITENIN protein synthesis, cells were pretreated with cycloheximide, a translation blocker, and KITENIN content was examined. As a result, it was found that degradation of KITENIN protein was accelerated by overexpressed RACK1 in cells pretreated with cycloheximide compared to EV-transfected control cells (FIG. 29). In most cases, proteins are broken down by proteolytic enzymes (peoteasomes) or lysosomes. To explain the pathway responsible for KITENIN degradation by KDIP or KITENIN-CTD, proteasome inhibitor MG132, lysosome inhibitor bafilomycin A1 (BFA1) and chloroquine ( After pretreatment with cloroquine (CQ) or autophagy inhibitor 3-MA, intracellular KITENIN content was examined. Degradation of KITENIN by KDIP (FIG. 30) or KITENIN-CTD (FIG. 31) was attenuated by BFA1, CQ, and 3-MA but not MG132, indicating that KDIP or KITENIN-CTD inhibited the lysosomal-autophagy pathway. It was suggested that it promotes the degradation of KITENIN. Moreover, the level of LC3, one of the characteristics of autophagy, increased after KDIP treatment (FIG. 32(A)), and this effect was reduced by treatment with the autophagy inhibitor 3-MA (FIG. 32(B)). In addition, to analyze the effect of KDIP treatment on autophagy induction, changes in autophagosomes after KDIP treatment were observed through cytoID staining. The formation of autophagosomes was increased by KDIP, similar to the effect of rapamycin, a positive control (FIG. 32(C)).

2-6. Myo10 prevents RACK1 from binding to RIM and maintains a stable KITENIN dimer state.

As we observed that increased binding of RACK1 to KITENIN caused degradation of KITENIN after KDIP treatment, when KITENIN exists as a homodimer, the interaction between RACK1 and KITENIN is hindered because the RACK1 interaction domain is structurally hidden or masked. hypothesized that If another KITENIN-interacting molecule maintaining the stability of the KITENIN dimer is regulated after KDIP treatment, the KITENIN dimer will be degraded after KDIP treatment and subsequently RIM will be exposed, whereby KITENIN is can be disassembled To test this possibility, KDIP-treated Caco2 cells were immunoprecipitated with KITENIN antibody, separated by SDS-PAGE, and the bands that changed after KDIP treatment were excised and protein sequencing was performed. In this way, the present inventors identified Myosin-X (Myosin10 or Myo10), one of several actin-based motor molecules of the myosin superfamily (30), and a member of the GTP-binding translation elongation factor family (which is essential for protein synthesis). 31), eEF2 (eukaryotic elongation factor 2) was confirmed (FIG. 33(A)). When the endogenous levels of the two proteins were confirmed in Caco2 cells transfected with KITENIN, the amount of Myo10 increased, but the amount of eEF2 did not increase (FIG. 33(B)). Also, after KDIP treatment in Caco2/KITENIN-V5 cells, the binding of KITENIN to eEF2 slightly decreased, but the binding of KITENIN to Myo10 was virtually eliminated (FIG. 33(C)). To confirm whether these two proteins can contribute to the stability of KITENIN, the two proteins were knocked down by treatment with Myo10 or eEF2-specific siRNA, and then the binding between RACK1 and KITENIN was examined. Interestingly, the interaction of RACK1 with KITENIN was not substantially affected by downregulation of eEF2 or Myo10 (FIG. 34(A) and (B)). However, binding of RACK1 to KITENIN was increased after KDIP treatment and this effect of KDIP was increased after knockdown of Myo10 (Fig. 34(B)) compared to knockdown of eEF2 (Fig. 34(A)). Consistently, KDIP further reduced the amount of KITENIN under eEF2-knockdown (Fig. 35(A) and (B)), whereas no additional inhibitory effect of KDIP on KITENIN levels was observed when Myo10 was knocked down (Fig. 35(A) and (B)). To confirm the contribution of the two proteins to the stability of KITENIN, we investigated whether knockdown of Myo10 or eEF2 by siRNA alters the effect of KDIP on the cell invasiveness of KITENIN transfected cells. As a result, it was found that the increased cell invasion caused by the overexpression of KITENIN was greatly reduced by the downregulation of these two proteins (FIGS. 35 and 36). However, the inhibitory effect of KDIP on the increased cell invasiveness due to overexpressed KITENIN was still evident when eEF2 was knocked down (FIG. 35) but not with Myo10 knockdown (FIG. 36).

2-7. Myo10 interacts with the transmembrane portion of KITENIN and stabilizes the KITENIN homodimer.

Myo10 has a motor or head domain with a nucleotide binding site and an actin binding site; an IQ or neck domain that binds three calmodulin molecules; a C-terminal tail domain followed by a single α-helical region and a coiled-coil region presumably involved in dimerization; three PEST sequences conferring sensitivity to specific proteases; three pleckstrin homology (PH) domains; a myosin tail homology 4 (MyTH4) domain that binds to microtubules; and band 4.1, Ezrin, Radixin, Merlin (FERM) domains (30, 32). Myo10 is located at the tip of filopodia, which are actin-rich, finger-like protrusions found at the leading edge of cells (32), guiding cell migration, wound healing, adhesion to the extracellular matrix, and chemoattractants. , believed to be involved in neuronal growth-cone path finding, embryonic development and tumorigenesis (33).

We hypothesized that Myo10 levels were regulated after KDIP treatment and that this change was essential for the effect of KDIP on KITENIN degradation. Therefore, the present inventors attempted to confirm whether the regulation of Myo10 affects the stability of KITENIN. First, which domain of Myo10 interacts with which part of KITENIN was tested. Interestingly, Myo10 bound to the transmembrane portion of KITENIN via its C-terminal FERM domain (FIG. 38) (FIG. 37). Since Myo10 associates with the basic actin cytoskeleton through its N-terminal head domain (30), these results indicate that Myo10 interacts with intracellular cytoplasmic loops within the transmembrane portion of KITENIN. Knockdown of KITENIN did not affect the level of Myo10, whereas knockdown of Myo10 reduced the level of KITENIN (FIG. 39), indicating that Myo10 acts as a regulator of KITENIN expression. In addition, knockdown of Myo10 reduced the formation of KITENIN dimers (FIG. 40), but enhanced the interaction between RACK1 and KITENIN and increased the degradation of KITENIN (FIG. 34(B)). Thus, degradation of KITENIN homodimers and subsequent degradation of KITENIN after KDIP treatment is caused by downregulation of Myo10.

2-8. Myo10 is reduced through proteasome degradation after KDIP treatment.

Degradation of KITENIN after treatment with KDIP was somewhat restored when pretreated with MG132, but completely restored when pretreated with an inhibitor of the lysosomal-autophagy pathway (FIG. 30), suggesting that an additional proteasome or lysosomal degradation pathway is involved. The purpose was to determine if there was an effect on the reduction of Myo10 by KDIP. For this experiment, cells were pretreated with the proteasome inhibitor MG132, the lysosomal inhibitor chloroquine (CQ) and the autophagy inhibitor 3MA. As a result, the decrease in Myo10 after KDIP treatment was recovered by pretreatment with MG132, but not by pretreatment with CQ or 3-MA (FIG. 41), and the degradation of KITENIN promoted by KDIP was somewhat reduced by MG132. found to be weakened. Since the cells were pretreated with MG132 for 4 hours before harvesting due to cytotoxicity, these results suggest that the reduction of Myo10 after KDIP treatment is an important event for KDIP-induced degradation of KITENIN. Interestingly, Myo10 was also decreased after transfection of KITENIN-CTD (FIG. 42). In vitro phenotypic analysis was performed using a cell invasion assay to verify the effect of MG132 on Myo10 reduction after KDIP treatment. In this experiment, pretreatment with the protease inhibitor MG132 did not affect cell invasion or the effect of KDIP in parental Caco2 cells, but blocked the inhibitory effect of KDIP on increased cell invasion in KITENIN-overexpressing Caco2 cells. (FIG. 43). These results indicate that KITENIN-CTD or KDIP induces degradation of KITENIN homodimers through downregulation of Myo10.

The E3-ligase Nrdp1 interacts with KITENIN-CTD within a functional KITENIN/ErbB4 complex and mediates proteasomal degradation of KITENIN-bound Dvl2, releasing c-Jun from the EGF-KITENIN/ErbB4-c-Jun axis. (34), it was confirmed that Nrdp1 is also involved in KDIP-induced proteasome degradation and downregulation of Myo10, which is also bound to the transmembrane portion of KITENIN (FIG. 37). First, after KDIP was regulated by the E3-ligase Nrdp1, to test the stability of Myo10, the amount of Myo10 was monitored after treatment with cycloheximide, a reagent that inhibits protein synthesis regardless of the presence of Nrdp1. . As a result, the expression of both KITENIN and Myo10 decreased after KDIP treatment. However, when Nrdp1 was transfected under these conditions, the levels of KITENIN and Myo10 were further reduced (FIG. 44). Next, the effect of Nrdp1 on Myo10 ubiquitination was investigated, and it was found that KDIP-induced Myo10 ubiquitination was significantly increased by forced expression of Nrdp1 (FIG. 45). As shown in FIG. 46 , these effects, such as suppression of increased cell invasion by forced expression of KITENIN and suppression of reduced expression of Myo10 and KITENIN observed after KDIP treatment, were restored by knockdown of Nrdp1. These results indicate that the E3-ligase Nrdp1 is also responsible for proteasome degradation of Myo10 after KDIP. These data can be interpreted as follows. After KDIP treatment, first, E3-ligase Nrdp1-mediated proteasome degradation of Myo10 was induced, and second, downregulated Myo10 resulted in degradation of KITENIN in a lysosomal-autophagy pathway dependent manner.

Overall, the results of the above experiments can be summarized as follows: Since KITENIN achieves stability by forming dimers in the cell membrane, the oncogenic function of KITENIN will also be an event in the cell membrane. Myo10 stabilized the cis conformation of the KITENIN homodimer by tying the two intracellular cytoplasmic loops of the transmembrane region of KITENIN. Downregulation of Myo10 after KDIP through proteasome degradation results in the loosening and disassembly of KITENIN dimers, exposing the RIM site and increasing the interaction of RACK1 with the exposed RIM, thereby inhibiting KITENIN in an autophagy-dependent manner. trigger disassembly (FIG. 47).

2-9. KDIP treatment inhibits tumor growth and liver metastasis of CRC in vivo

First, we investigated whether KDIP has an antitumor effect in vivo using syngeneic tumor models in BALB/c mouse and CT-26 mouse colon adenocarcinoma cell lines. Before examining the in vivo effect of KDIP, the effect of KDIP on the in vitro invasiveness of CT-26 cells was tested. Cell invasion assay was performed with CT-26/KITENIN-V5 cells stably expressing KITENIN-V5, and it was significantly reduced with increasing dose of KDIP compared to scrambled peptide (FIG. 48). For the in vivo experiment, CT-26/KITENIN-V5 cells were injected into the right back of BALB/c mice to form tumors, and KDIP was intravenously injected according to the timeline shown in FIG. 49 . Considering the inhibitory effect of KDIP on cell invasion of CT-26 cells, the initial test dose was set at 1 mg/kg, which corresponds to half of the maximum inhibitory concentration (IC ₅₀ ) of KDIP on cell invasion. In subsequent experiments, the dose of KDIP was increased to 5 mg/kg. KDIP was administered every other day through intravenous injection for 2 weeks from the 7th day after cell injection. After 2 weeks of treatment, mice were sacrificed and tumors analyzed. Tumor volume was significantly reduced by KDIP, and this effect depended on the dose of KDIP used (FIG. 50).

Next, the anti-tumor effect of KDIP was further tested using a mouse model of CRC liver metastasis. To create an experimental model, intrasplenic inoculation of cells stably expressing CT-26/KITENIN-iRFP was performed in syngeneic mice. After a subsequent splenectomy, they were allowed to recover for 2 weeks. For 2 weeks, mice were intravenously injected with KDIP every other day. Liver metastases were assessed by detecting total fluorescence emitted from tumor nodules expressing iRFP. The fluorescence of liver nodules was significantly decreased by increasing the dose of KDIP compared to that of the scrambled peptide group (FIGS. 51 and 52). In addition, as a result of analyzing the expression levels of KITENIN, Myo10, and eEF2 in the metastatic liver nodules collected from the two groups, it was found that the protein levels of Myo10, but not eEF2, were significantly reduced by KDIP (Fig. 53). As shown in FIG. 53, downstream signals (15, 17) such as KITENIN, phospho-ERK, and phospho-c-Jun were greatly reduced by KDIP. Since we reported that KITENIN expression was inversely correlated with the expression of KAI1 and other metastasis suppressor genes (14), we compared the expression levels of KITENIN and KAI1 in metastatic liver nodules collected from both groups. As a result, it was found that the decreased KAI1 expression in the scr-peptide injection group was significantly recovered in the KDIP injection group along with the degradation of KITENIN (FIG. 53). Also, the expression of phospho-c-MET was significantly decreased in the KDIP injection group compared to the scr-peptide injection group (FIG. 53).

These results positively support the in vitro results indicating the inhibition of KITENIN's oncogenic function by KDIP. As a result, KDIP specifically inhibits the dimerization of KITENIN and effectively inhibits the oncogenic activity of KITENIN.

Review

The peptide has a low molecular weight and good cellular uptake, and can bind specifically to tumor cells with low toxicity to normal tissues. Thus, they are ideal molecules for targeted cancer therapy (35). Similar to antibodies, certain peptides bind to target proteins as well as block signal transduction and function (36). In this experiment, KDIP, that is, KDIP, a short peptide that specifically inhibits KITENIN dimerization, effectively inhibits oncogenic KITENIN in vitro and in vivo, and reduces the stability of KITENIN protein, was introduced. In addition to siRNAs (21), mi-RNAs (22) and low molecular weight compounds (23) targeting functional KITENIN complexes, the present results are the first report on the inhibition of KITENIN's oncogenic function by peptides.

RACK1 was previously identified as a novel hypoxia-inducible factor-1a (HIF-1a)-interacting protein that regulates HIF-1a stability through competition with HSP90 and recruitment of the Elongin-C/B ubiquitin ligase complex. (37). Similarly, in this experiment, we found that the binding of RACK1 to KITENIN aa 463-471 (RIM) is essential for the degradation of KITENIN. Formation of stable KITENIN homodimers through ligation of the two intracellular cytoplasmic loops of the transmembrane domain may create a conformational hindrance that masks RIM binding to RACK1. Thus, KDIP treatment increased the interaction of RACK1 with exposed RIM, just as KDIP treatment caused down-regulation of Myo10 and induced loosening and disassembly of KITENIN dimer, exposing the RIM site. These increased interactions triggered degradation of KITENIN and reduction of KITENIN dimers in an autophagy-dependent manner. Therefore, based on the present results and previous observations that KITENIN has no receptor function (14, 15, 17), we suggest that KITENIN homodimers are indeed the structural and functional units for KITENIN's oncogenic function. When KITENIN acts as an enhancer of invasion in CRC cells, the KITENIN homodimer acts as a scaffolding protein to form a functional KITENIN complex that provides advantages such as structural hindrance to binding to RACK1, preventing protein degradation. protect

Myo10 (also called Myosin-X) is involved in cell-cell adhesion-associated signaling and cytoskeleton reorganization (30, 38). The anti-parallel coiled-coil protein structure of Myo10 is optimized for the movement of the actin bundle (39). This property is critical for Myo10's role as a fast filopodia transporter and possibly as an actin organizer that promotes filopodia formation (42). Both Myo10 and Myo1b are expressed at higher levels in prostate cancer cells with high metastatic potential and in metastatic prostate cancer tissues (42). Myo10 knockdown eliminates filopodia and reduces filopodia migration speed. In contrast, Myo1b knockdown increases the number of stress fibers but does not affect migration speed. This indicates that these molecular motions of myosin use actin as a walking track, but also directly affect actin organization and cell morphology, which may contribute to the metasatatic phenotype. Myo10 has also been reported to colocalize with and colocalize with vascular endothelial (VE)-cadherin in filopodia, indicating that Myo10 establishes connections between the actin cytoskeleton and VE-cadherin shown to allow transport of VE-cadherin along filopodia actin cables (43). Therefore, it has been proposed that trafficking of VE-cadherin along filopodia using Myo10 is a prerequisite for the formation of cell–cell junctions, a process that may be functionally important in endothelial repair and angiogenesis. Our current results showing the interaction of Myo10 with the transmembrane portion of KITENIN suggest that Myo10 establishes a link between the actin cytoskeleton and the atypical tetraspanin, KITENIN, allowing anchorage and transport of KITENIN to the plasma membrane. suggests that Thus, stabilization of KITENIN homodimers by Myo10 is a prerequisite for the formation of the oncogenic KITENIN complex, which triggers downstream signaling processes for increased cell invasiveness and metastatic spread.

KITENIN is an aggressive agent in the progression of various human cancers. Expression of KITENIN was significantly higher in human colon (15, 16), larynx (44), oral cavity squsamous (45) stomach (46), hepatocytes (47), and glioma (48) tumor tissues than in normal mucosa. showed a higher In this regard, KITENIN is a promising target for anti-metastatic therapy (23). As shown in FIGS. 29 and 30 , KDIP containing the consensus sequence of the CxGpPxFxC peptide had a specific effect on the KITENIN dimer and inhibited the downstream signaling of the KITENIN complex. After KDIP treatment, Myo10 was reduced through proteasomal degradation and the stability of KITENIN dimer was lost. Binding of RACK1 to RIM caused degradation of KITENIN in an autophagy-dependent manner. These results indicate that downregulation of Myo10 is responsible for the therapeutic action of KDIP on colorectal tumors with higher KITENIN expression through degradation of KITENIN dimers and consequent degradation of KITENIN protein. In addition, this peptide efficiently inhibited CRC tumor xenografts and liver metastasis of orthotopic CRC tumors in syngeneic mice. Interestingly, as in cultured CRC cells in which KITENIN overexpression reduced KAI1 expression (14), the reduced KAI1 expression in metastatic liver nodules collected from the control group was restored in the KDIP-injected group with KITENIN degradation (FIG. 53 (A) and (B)). In addition, the expression of phospho-c-MET was also decreased in KDIP-injected metastatic liver nodules, as in KAI1-reexpressing PC3 prostate cancer cells, which showed inhibition of integrin- and ligand-mediated activation of c-Met (49) (FIG. 53). . These expression changes of KAI1 and c-MET after KDIP treatment may have contributed to the suppression of colorectal liver metastasis by KDIP. Therefore, KDIP is a new drug that can be used in combination with existing anticancer therapies to block tumor development in CRC expressing higher levels of KITENIN.

In breast carcinoma, Myo10 is mainly expressed at the invasive margin of the carcinoma, and its expression correlates with the presence of TP53 mutations and poor prognosis. Also, transport of β-integrin by Myo10 to the filopodia tip is required for cell invasion (50). These findings suggest that Myo10 is required for breast cancer cell invasion in addition to prostate cancer cells. In addition, Myo10 was shown to have a direct effect on invadopodia to promote invasive growth and metastasis of breast tumors in a nude mouse model (51), but was also involved in lung adenocarcinoma metastasis (52). In the present invention, we found that the expression of Myo10 was increased in KITENIN transfected CRC cells and Myo10 further stabilized the KITENIN dimer. These reports and our present data suggest that Myo10 upregulation and stabilization of overexpressed KITENIN in KITENIN-overexpressing cancer tissues contribute to aggressive invasiveness and metastasis in mutant p53-induced cancers. Both proteins can serve as specialized translocation engines. Since downregulation of Myo10 is responsible for the therapeutic action of KDIP on colorectal tumors with higher KITENIN expression, we suggest that KDIP can be used as a dual blocker for the oncogenic function of both KITENIN and Myo10 proteins.

In summary, the essential function of the KITENIN homodimer is to maintain the stability of the cancer metastasis-promoting KITENIN protein to provide a docking site for a major interaction partner. We propose a new strategy for the development of specifically blocking peptide cancer therapeutics.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION OF SUNCHON NATIONAL UNIVERSITY <120> {Peptide interfering a dimerization of KITENIN and use thereof <130> PA-21-0302 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 524 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(524) <223> Full length of KITENIN protein <400> 1 Met Asp Thr Glu Ser Thr Tyr Ser Gly Tyr Ser Tyr Ser Ser His 1 5 10 15 Ser Lys Lys Ser His Arg Gln Gly Glu Arg Thr Arg Glu Arg His Lys 20 25 30 Ser Pro Arg Asn Lys Asp Gly Arg Gly Ser Glu Lys Ser Val Thr Ile 35 40 45 Gln Pro Pro Thr Gly Glu Pro Leu Leu Gly Asn Asp Ser Thr Arg Thr 50 55 60 Glu Glu Val Gln Asp Asp Asn Trp Gly Glu Thr Thr Thr Thr Ala Ile Thr 65 70 75 80 Gly Thr Ser Glu His Ser Ile Ser Gln Glu Asp Ile Ala Arg Ile Ser 85 90 95 Lys Asp Met Glu Asp Ser Val Gly Leu Asp Cys Lys Arg Tyr Leu Gly 100 105 110 Leu Thr Val Ala Ser Phe Leu Gly Leu Leu Val Phe Leu Thr Pro Ile 115 120 125 Ala Phe Ile Leu Leu Pro Pro Ile Leu Trp Arg Asp Glu Leu Glu Pro 130 135 140 Cys Gly Thr Ile Cys Glu Gly Leu Phe Ile Ser Met Ala Phe Lys Leu 145 150 155 160 Leu Ile Leu Leu Ile Gly Thr Trp Ala Leu Phe Phe Arg Lys Arg Arg 165 170 175 Ala Asp Met Pro Arg Val Phe Val Phe Arg Ala Leu Leu Leu Val Leu 180 185 190 Ile Phe Leu Phe Val Val Ser Tyr Trp Leu Phe Tyr Gly Val Arg Ile 195 200 205 Leu Asp Ser Arg Asp Arg Asn Tyr Gln Gly Ile Val Gln Tyr Ala Val 210 215 220 Ser Leu Val Asp Ala Leu Leu Phe Ile His Tyr Leu Ala Ile Val Leu 225 230 235 240 Leu Glu Leu Arg Gln Leu Gln Pro Met Phe Thr Leu Gln Val Val Arg 245 250 255 Ser Thr Asp Gly Glu Ser Arg Phe Tyr Ser Leu Gly His Leu Ser Ile 260 265 270 Gln Arg Ala Ala Leu Val Val Leu Glu Asn Tyr Tyr Lys Asp Phe Thr 275 280 285 Ile Tyr Asn Pro Asn Leu Leu Thr Ala Ser Lys Phe Arg Ala Ala Lys 290 295 300 His Met Ala Gly Leu Lys Val Tyr Asn Val Asp Gly Pro Ser Asn Asn 305 310 315 320 Ala Thr Gly Gln Ser Arg Ala Met Ile Ala Ala Ala Ala Arg Arg Arg 325 330 335 Asp Ser Ser His Asn Glu Leu Tyr Tyr Glu Glu Ala Glu His Glu Arg 340 345 350 Arg Val Lys Lys Arg Lys Ala Arg Leu Val Val Ala Val Glu Glu Ala 355 360 365 Phe Ile His Ile Gln Arg Leu Gln Ala Glu Glu Gln Gln Lys Ala Pro 370 375 380 Gly Glu Val Met Asp Pro Arg Glu Ala Ala Gln Ala Ile Phe Pro Ser 385 390 395 400 Met Ala Arg Ala Leu Gln Lys Tyr Leu Arg Ile Thr Arg Gln Gln Asn 405 410 415 Tyr His Ser Met Glu Ser Ile Leu Gln His Leu Ala Phe Cys Ile Thr 420 425 430 Asn Gly Met Thr Pro Lys Ala Phe Leu Glu Arg Tyr Leu Ser Ala Gly 435 440 445 Pro Thr Leu Gln Tyr Asp Lys Asp Arg Trp Leu Ser Thr Gln Trp Arg 450 455 460 Leu Val Ser Asp Glu Ala Val Thr Asn Gly Leu Arg Asp Gly Ile Val 465 470 475 480 Phe Val Leu Lys Cys Leu Asp Phe Ser Leu Val Val Asn Val Lys Lys 485 490 495 Ile Pro Phe Ile Ile Leu Ser Glu Glu Phe Ile Asp Pro Lys Ser His 500 505 510 Lys Phe Val Leu Arg Leu Gln Ser Glu Thr Ser Val 515 520 <210> 2 <211> 281 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(281) <223> C-terminal domain of KITENIN <400> 2 Arg Gln Leu Gln Pro Met Phe Thr Leu Gln Val Val Arg Ser Thr Asp 1 5 10 15 Gly Glu Ser Arg Phe Tyr Ser Leu Gly His Leu Ser Ile Gln Arg Ala 20 25 30 Ala Leu Val Val Leu Glu Asn Tyr Tyr Lys Asp Phe Thr Ile Tyr Asn 35 40 45 Pro Asn Leu Leu Thr Ala Ser Lys Phe Arg Ala Ala Lys His Met Ala 50 55 60 Gly Leu Lys Val Tyr Asn Val Asp Gly Pro Ser Asn Asn Ala Thr Gly 65 70 75 80 Gln Ser Arg Ala Met Ile Ala Ala Ala Ala Arg Arg Arg Asp Ser Ser 85 90 95 His Asn Glu Leu Tyr Tyr Glu Glu Ala Glu His Glu Arg Arg Val Lys 100 105 110 Lys Arg Lys Ala Arg Leu Val Val Ala Val Glu Glu Ala Phe Ile His 115 120 125 Ile Gln Arg Leu Gln Ala Glu Glu Gln Gln Lys Ala Pro Gly Glu Val 130 135 140 Met Asp Pro Arg Glu Ala Ala Gln Ala Ile Phe Pro Ser Met Ala Arg 145 150 155 160 Ala Leu Gln Lys Tyr Leu Arg Ile Thr Arg Gln Gln Asn Tyr His Ser 165 170 175 Met Glu Ser Ile Leu Gln His Leu Ala Phe Cys Ile Thr Asn Gly Met 180 185 190 Thr Pro Lys Ala Phe Leu Glu Arg Tyr Leu Ser Ala Gly Pro Thr Leu 195 200 205 Gln Tyr Asp Lys Asp Arg Trp Leu Ser Thr Gln Trp Arg Leu Val Ser 210 215 220 Asp Glu Ala Val Thr Asn Gly Leu Arg Asp Gly Ile Val Phe Val Leu 225 230 235 240 Lys Cys Leu Asp Phe Ser Leu Val Val Asn Val Lys Lys Ile Pro Phe 245 250 255 Ile Ile Leu Ser Glu Glu Phe Ile Asp Pro Lys Ser His Lys Phe Val 260 265 270 Leu Arg Leu Gln Ser Glu Thr Ser Val 275 280 <210> 3 <211> 5 <212> PRT <213> artificial sequence <220> <223> Peptide interfering a dimerization of KITENIN <400> 3 Val Ser Asp Glu Ala 1 5 <210> 4 <211> 8 <212> PRT <213> artificial sequence <220> <223> Peptide originated from 449th to 456th of KITENIN <400> 4 Pro Thr Leu Gln Tyr Asp Lys Asp 1 5 <210> 5 <211> 8 <212> PRT <213> artificial sequence <220> <223> Peptide originated from 455th to 462th of KITENIN <400> 5 Lys Asp Arg Trp Leu Ser Thr Gln 1 5 <210> 6 <211> 8 <212> PRT <213> artificial sequence <220> <223> Peptide interfering a dimerization of KITENIN and originated from 462th to 469th of KITENIN <400> 6 Gln Trp Arg Leu Val Ser Asp Glu 1 5 <210> 7 <211> 9 <212> PRT <213> artificial sequence <220> <223> Peptide interfering a dimerization of KITENIN and originated from 463th to 471th of KITENIN <400> 7 Trp Arg Leu Val Ser Asp Glu Ala Val 1 5 <210> 8 <211> 8 <212> PRT <213> artificial sequence <220> <223> Peptide interfering a dimerization of KITENIN and originated from 467th to 474th of KITENIN <400> 8 Ser Asp Glu Ala Val Thr Asn Gly 1 5 <210> 9 <211> 8 <212> PRT <213> artificial sequence <220> <223> Peptide originated from 473th to 480th of KITENIN <400> 9 Asn Gly Leu Arg Asp Gly Ile Val 1 5 <210> 10 <211> 8 <212> PRT <213> artificial sequence <220> <223> Peptide originated from 480th to 487th of KITENIN <400> 10 Val Phe Val Leu Lys Cys Leu Asp 1 5 <210> 11 <211> 20 <212> PRT <213> artificial sequence <220> <223> KITENIN dimerization-interfering peptide fused to Hph-1 <400> 11 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Trp Arg Leu Val Ser 1 5 10 15 Asp Glu Ala Val 20 <210> 12 <211> 24 <212> PRT <213> artificial sequence <220> <223> KITENIN dimerization-interfering peptide fused to TAT <400> 12 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Phe Leu Gly Trp 1 5 10 15 Arg Leu Val Ser Asp Glu Ala Val 20 <210> 13 <211> 29 <212> PRT <213> artificial sequence <220> <223> KITENIN dimerization-interfering peptide fused to Penetratin <400> 13 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 Gly Phe Leu Gly Trp Arg Leu Val Ser Asp Glu Ala Val 20 25 <210> 14 <211> 19 <212> PRT <213> artificial sequence <220> <223> KITENIN dimerization-interfering peptide fused to EGFR-1 <400> 14 Lys Cys Cys Tyr Ser Leu Gly Phe Leu Gly Trp Arg Leu Val Ser Asp 1 5 10 15 Glu Ala Val <210> 15 <211> 18 <212> PRT <213> artificial sequence <220> <223> KITENIN dimerization-interfering peptide fused to EGFR-2 <400> 15 Ala Glu Phe Leu Arg Gly Phe Leu Gly Trp Arg Leu Val Ser Asp Glu 1 5 10 15 Ala Val

Claims (14)

A pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 that inhibits KITENIN dimer formation. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the peptide is further bound to a cell-permeable material. The pharmaceutical composition for preventing or treating cancer according to claim 2, wherein the cell-permeable material is a peptide or a compound. The method of claim 3, wherein the peptide is polyarginine, Hph-1, Tat, SPACE (skin penetration and cell entering peptide), TD1 (transdermal peptide-1), DLP (dermis localizing peptide), LP-12 (linear peptide- 12 mer), penetratin, or a pharmaceutical composition for preventing or treating cancer, characterized in that it is a tumor homing peptide. The pharmaceutical composition for preventing or treating cancer according to claim 3, wherein the compound is cholic acid, oleic acid, or a derivative thereof. A pharmaceutical composition for inhibiting cancer invasion and metastasis, comprising as an active ingredient a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 that inhibits KITENIN dimer formation. The pharmaceutical composition for inhibiting invasion and metastasis of cancer according to claim 6, wherein the peptide is further bound to a cell-permeable material. The pharmaceutical composition for inhibiting invasion and metastasis of cancer according to claim 7, wherein the cell-permeable material is a peptide or a compound. The method of claim 8, wherein the peptide is polyarginine, Hph-1, Tat, SPACE (skin penetration and cell entering peptide), TD1 (transdermal peptide-1), DLP (dermis localizing peptide), LP-12 (linear peptide- 12 mer), penetratin, or a tumor homing peptide, characterized in that a pharmaceutical composition for inhibiting invasion and metastasis of cancer. The pharmaceutical composition for inhibiting invasion and metastasis of cancer according to claim 8, wherein the compound is cholic acid, oleic acid, or a derivative thereof. delete delete delete delete

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