A polypeptide, a nucleic acid molecule, a cell and its application in breast cancer

Abstract

The present invention discloses a polypeptide, a nucleic acid molecule, a cell and their application in breast cancer, and relates to the technical field of breast cancer treatment. The present invention provides a polypeptide targeting cGAS phosphorylation, which can significantly inhibit the phosphorylation of cGAS in triple-negative breast cancer (TNBC), thereby inhibiting the metastasis of cancer, and can be used for the treatment of breast cancer (especially TNBC). In addition, the present invention also provides a nucleic acid molecule, a vector, a cell and a drug encoding the above polypeptide, which has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of breast cancer treatment, and in particular to a polypeptide, a nucleic acid molecule, a cell and applications thereof in breast cancer. Background Art

[0002] The first peptide drug for humans was insulin extracted from the pancreas of animals. More than 30 years later, Robert Bruce Merrifield synthesized the first biologically active peptide, and since then the research on peptide drugs has begun to develop rapidly. Compared with chemical small molecule drugs, peptide drugs have stronger biological activity, smaller dosage, lower toxic side effects and significant efficacy, so they have attracted the attention and favor of new drug developers. At present, peptide drugs used for the prevention, treatment and diagnosis of diseases mainly include peptide vaccines, anti-tumor peptides, antiviral peptides, peptide-guided drugs, cytokine mimetic peptides, antibacterial active peptides, diagnostic peptides and other medicinal small peptides.

[0003] Breast cancer is the most common malignancy in women worldwide and can be divided into three subtypes, including estrogen receptor (ER)+, human epidermal growth receptor 2 (Her2)+, and triple-negative breast cancer (TNBC) [1]. Globally, the incidence of TNBC is on the rise, accounting for nearly 20% of all breast cancers, and its mortality rate is as high as 83% compared with other subtypes [2]. Although clinical efficacy has improved with the development of new therapies and cytotoxic chemotherapy, TNBC remains an aggressive, metastatic, and significantly resistant breast cancer subtype [3, 4]. As breast cancer cells become increasingly resistant to anticancer drugs, researchers are looking for new approaches to treat breast cancer. In this regard, peptide therapy has become a viable treatment option for solid tumors, especially breast cancer. Peptides are an attractive treatment option because of their precise attachment to the surface of tumor cells, low molecular weight, and low toxicity to normal cells [5].

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide, a nucleic acid molecule, a cell and their application in breast cancer to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a polypeptide, the amino acid sequence of which has at least 90% identity with the sequence shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a protein comprising: a plurality of the above polypeptides connected in series.

[0009] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned polypeptide or the above-mentioned protein.

[0010] In a fourth aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0011] In a preferred embodiment of the present invention, the vector is a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0012] In a fifth aspect, the present invention provides a cell comprising the above-mentioned polypeptide, the above-mentioned protein, the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0013] In a sixth aspect, the present invention provides the use of the polypeptide or the above-mentioned protein in the preparation of a drug for preventing or treating breast cancer.

[0014] In a seventh aspect, the present invention provides the use of a polypeptide that inhibits the phosphorylation of cyclic guanine nucleotide adenine nucleotide synthetase (cGAS) in breast cancer cells in the preparation of a drug for preventing or treating breast cancer.

[0015] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or the above-mentioned protein.

[0016] The present invention has the following beneficial effects:

[0017] The present invention found that in various TNBC mouse models, nuclear cGAS initiated lung metastasis regardless of its cGAMP synthase activity. In contrast, protein kinase Cα (PKCα) directly phosphorylates cGAS at serine 120 to promote its nuclear translocation. Nuclear β-catenin hijacked by cGAS induces cell-autonomous metastasis and spread. Based on this, after screening, the present invention provides a polypeptide targeting cGAS phosphorylation, which can significantly inhibit the phosphorylation of cGAS in TNBC, thereby inhibiting the metastasis of cancer, and can be used for the treatment of breast cancer (especially TNBC).

[0018] In addition, the present invention also provides nucleic acid molecules, vectors, cells and drugs encoding the above polypeptides, which have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1This is an experimental result diagram showing that PKCα phosphorylates cGAS at serine 120 to promote its nuclear translocation;

[0021] Figure 2 This is a figure showing the research results that cGAS phosphorylated at serine 120 can induce cell autonomous metastasis and spread;

[0022] Figure 3 This is the result of predicting the spatial structure of a peptide using fingerprint coding technology;

[0023] Figure 4 is the HPLC chart of the purified SP-19 peptide;

[0024] Figure 5 is the MS graph of the purified SP-19 peptide;

[0025] Figure 6 The results of adding the antagonist peptide SP-19 to the complementing cell line (cGAS) and the cGAS gene knockout cell line (sgcGAS) on the migration of TNBC cells;

[0026] Figure 7 Results of animal model experiments ((A) Picric acid staining of the whole lung; (B) Statistical results of metastatic nodules in the lungs under different treatments; (C) H&E staining of paraffin sections. The scale bar in (A) is 1 cm; the scale bar in (C) is 300 μm));

[0027] Figure 8 cGAS KO and cGAS KO Figure 3. Detection results of SUM159 (A) and LM2 (B) cell lines expressing cGAS. DETAILED DESCRIPTION

[0028] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0029] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0030] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. The chain may be straight or branched, may contain modified amino acids, and / or may be interspersed with non-amino acids. The term also encompasses amino acid chains that have been modified naturally or by insertion; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that a polypeptide may exist as a single chain or in an associated chain.

[0031] Cyclic GMP-AMP synthase (cGAS).

[0032] In a first aspect, the present invention provides a polypeptide, the amino acid sequence of which has at least 90% identity with the sequence shown in SEQ ID NO.1.

[0033] SEQ ID NO. 1: SRAGSCRQRGARCSTKPRP.

[0034] The inventors found that in various TNBC mouse models, nuclear cGAS initiated lung metastasis and was independent of its cGAMP synthase activity. In contrast, protein kinase Cα (PKCα) directly phosphorylates cGAS at serine 120 to promote its nuclear translocation. Nuclear β-catenin hijacked by cGAS induces cell-autonomous metastasis and spread. Based on this, after screening, the present invention provides a polypeptide targeting cGAS phosphorylation, which can significantly inhibit the phosphorylation of cGAS in TNBC, thereby inhibiting the metastasis of cancer, and can be used for the treatment of breast cancer (especially TNBC).

[0035] Its amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO.1, or any point value between the above values.

[0036] The sequence alignment method used for the above sequence identity can be Protein Blast alignment using the Blast website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi); the same results can be obtained using other sequence alignment methods or tools well known in the art.

[0037] In addition, those skilled in the art know and can easily implement simple amino acid substitution, deletion or addition operations in the sequence shown in SEQ ID NO.1 and maintain that the mutated amino acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (etc.) identity with the amino acid shown in SEQ ID NO.1, and its function includes inhibiting the phosphorylation of cGAS in TNBC. Therefore, such polypeptides with at least 90% amino acid identity should also fall within the scope of protection of the present invention.

[0038] In a second aspect, the present invention provides a protein, comprising: a plurality of the above polypeptides connected in series. The proteins connected in series also have the function of a single polypeptide and have the effect of inhibiting the phosphorylation of cGAS in TNBC.

[0039] In a preferred embodiment of the present invention, the number of tandem polypeptides is 1 to 20;

[0040] In a preferred embodiment of the present invention, two adjacent polypeptides are connected via a linker.

[0041] In a preferred embodiment of the present invention, the linker is selected from PEG compounds, (GS) n , GGS, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGG or GGSGGGGSGGGGSGGGGSGGGGSGG; n is 1-9.

[0042] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned polypeptide or the above-mentioned protein.

[0043] In the case where the present invention provides the above amino acid sequence, those skilled in the art can easily obtain the nucleic acid sequence encoding the above polypeptide or protein based on the degeneracy of the codons. For example, corresponding nucleotide mutations can be made on the nucleic acid sequence encoding the polypeptide or protein to obtain the nucleic acid sequence encoding the above polypeptide or protein. This is easy to achieve for those skilled in the art.

[0044] In a fourth aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0045] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can introduce the target DNA fragment directly or indirectly (such as packaging into a virus) into a host cell by transformation, transfection or transduction to express the target gene.

[0046] One type of vector is a plasmid, which is a circular double-stranded DNA molecule into which the target DNA fragment can be connected. Another type of vector is a viral vector, which can package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0047] A person skilled in the art can also transcribe the nucleic acid sequence of the present invention into RNA by in vitro transcription, and further transfect, transduce or transform the RNA into a host cell to express the antibody of the present invention or its functional fragment to exert the biological efficacy of the present invention.

[0048] In a preferred embodiment of the present invention, the vector is a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0049] In a fifth aspect, the present invention provides a cell comprising the above-mentioned polypeptide, the above-mentioned protein, the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0050] In a preferred embodiment of the present invention, the cell is a mammalian cell;

[0051] In a preferred embodiment of the present invention, the mammalian cells are human cells, mouse cells, and rat cells;

[0052] In a preferred embodiment of the present invention, the mammalian cells are selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, Hela cells, MDCK cells, Hep-2 cells and Per6 cells.

[0053] In a preferred embodiment of the present invention, the human cells are primary human blood cells (PBMC);

[0054] In a preferred embodiment of the present invention, the cell is an immune cell;

[0055] In a preferred embodiment of the present invention, the immune cells are T cells or B cells.

[0056] In other embodiments, the cell may also be a bacterium or a fungus, and the bacterium may be Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis;

[0057] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0058] The above cells include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, without considering the number of passages. The progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations.

[0059] In a preferred embodiment of the present invention, the yeast is selected from at least one of the following genera: Dekkera, Brettanomyces, Hansenula, Kluyveromyces, Pichia, Candida, Kluyveromyces Kluyveromyces ), Debaryomyces spp. Debaryomyces ), Kazachstania , Wickerhamomyces , Lindnera , Zygotorulaspora , Zygosaccharomyces ( Zygosaccharomyces ), Rhodosporidium Rhodosporidium ) and Schizosaccharomyces spp. ( Schizosaccharomyces ).

[0060] The above cells are prepared by transforming the recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and the foreign gene carried can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0061] In a preferred embodiment of the present invention, the cell refers to at least one of a resting cell of a recombinant cell, a living cell of a recombinant cell, a dead cell of a recombinant cell, and a cell fragment of a recombinant cell.

[0062] Dormant cells, also known as resting cells, are a special cell state. In this state, cells do not grow and reproduce, but still contain various enzyme systems and have oxidation and fermentation capabilities. Under suitable conditions, dormant cells can resume growth. The characteristics of dormant cells include: a. Cells maintain growth potential: Although in a dormant state, these cells can re-enter the cell cycle and restore their proliferation ability when given appropriate stimulation. b. Strong specificity: Dormant cells have strong specificity in the reaction and can improve the substrate conversion rate. c. Not easy to be contaminated by foreign bacteria: Due to its characteristics, dormant cells can reduce the inhibition of bacterial growth and enzyme synthesis by products during use.

[0063] Killed recombinant cells include but are not limited to cells inactivated by heat, pressure, radiation, etc.

[0064] Cell disruption products refer to: including but not limited to, cell disruption products obtained by changing the permeability of cell membranes through ultrasonic, mechanical, chemical, biological and other means, resulting in leakage of cell contents.

[0065] In a preferred embodiment of the present invention, the dead cells are selected from at least one of a dead cell precipitate and a dead cell-free supernatant. The dead cell-free supernatant refers to the "exudate contents" remaining after removing the outer shell of the dead cells (or bacteria).

[0066] In a sixth aspect, the present invention provides the use of the polypeptide or the above-mentioned protein in the preparation of a drug for preventing or treating breast cancer.

[0067] Breast cancer includes but is not limited to: estrogen receptor (ER)+, human epidermal growth receptor 2 (Her2)+, and triple-negative breast cancer (TNBC).

[0068] In a preferred embodiment of the present invention, breast cancer is selected from triple-negative breast cancer;

[0069] In a preferred embodiment of the present invention, the application includes any of the following application modes:

[0070] (1) Inhibit the phosphorylation of cyclic guanine nucleotide adenine nucleotide synthetase (cGAS) in breast cancer cells through peptides or proteins;

[0071] (2) Inhibit the migration of breast cancer cells;

[0072] (3) Eliminate or partially eliminate metastatic nodules in the lungs.

[0073] The above-mentioned inhibition of phosphorylation of cyclic guanine nucleotide adenine nucleotide synthetase (cGAS) in breast cancer cells refers to: inhibiting the phosphorylation of Ser 120 of cGAS.

[0074] The above-mentioned inhibition of breast cancer cell migration includes but is not limited to: inhibition of breast cancer cell migration to the lungs.

[0075] In a seventh aspect, the present invention provides the use of a polypeptide that inhibits the phosphorylation of cyclic guanine nucleotide adenine nucleotide synthetase (cGAS) in breast cancer cells in the preparation of a drug for preventing or treating breast cancer.

[0076] In a preferred embodiment of the present invention, the polypeptide is a polypeptide having an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO.1, or a polypeptide composed of a plurality of polypeptides connected in series.

[0077] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0078] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of a filler, a disintegrant, a lubricant, a flavoring agent, a binder, a suspending agent and a flavor.

[0079] Pharmaceutically acceptable excipients include but are not limited to: pharmaceutically acceptable carriers, auxiliary substances or solvents. Pharmaceutically acceptable excipients include various organic or inorganic carriers and / or auxiliary materials because they are commonly used for pharmaceutical purposes, especially for solid pharmaceutical preparations. Examples include: excipients, such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders, such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropyl pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants, such as starch, hydrolyzed starch, carboxymethyl cellulose, carboxymethyl cellulose calcium salt, hydroxypropyl starch, ethylene glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants, such as magnesium stearate, talc, sodium lauryl sulfate; flavors, such as citric acid, menthol, glycine , orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers, such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0080] In a preferred embodiment of the present invention, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections or sprays.

[0081] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as a solution, a suspension and a gel usually containing a liquid carrier, such as water and / or a pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include a pH adjusting agent, an emulsifier or a dispersant, a buffer, a preservative, a wetting agent, a gelling agent (such as methylcellulose), a dye and / or a flavoring agent, such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be adjusted by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be adjusted by a pharmaceutically acceptable thickener such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

[0082] In a preferred embodiment of the present invention, the drug is formulated for oral or injection administration.

[0083] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or the above-mentioned protein.

[0084] In an optional embodiment, the above-mentioned pharmaceutical composition further comprises the above-mentioned pharmaceutically acceptable excipients.

[0085] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0086] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0087] Example 1

[0088] This example provides an example of influencing cGAS nuclear translocation.

[0089] (1) Construction of PKCα gene knockout SUM159 and LM2 cell lines

[0090] Determine the forward and reverse sgRNA targeting the cGAS gene: F-CACCGAAGTGCGACTCCGCGTTCAG, R-AAACCTGAACGCGGAGTCGCACTTC. The double-stranded oligo was connected to the lenti-V2 vector cut with enzymes. Then the plasmid connected with sgRNA was transfected into SUM159 or LM2 cells. After 48 hours of transfection, the cells were screened with puro antibiotics and then cultured for monoclonal growth. After the monoclonal cells grew to a certain extent, a certain proportion of cells were collected for gene sequencing and immunoblot protein detection, and the PKCα-KO SUM159 or LM2 monoclonal cell lines were screened, expanded, and frozen for subsequent use.

[0091] (2) Construction of complementation cell lines (PKCα-expressing lentivirus-transfected SUM159 and LM2 cell lines with PKCα gene knockout).

[0092] HEK-293T cells were seeded into a 6 cm cell culture dish and cultured overnight at 37 °C and 5% carbon dioxide. When the cell density reached 70-80%, transfection was performed. In a 1.5 mL sterile centrifuge tube, 300 µL of opti-MEM medium was added, and then 4 µg of plasmid DNA mixture was added, including 1 µg of psPAX2, 1 µg of pMD2.G, and 2 µg of PKCα. The mixture was gently flicked on the bottom of the test tube with a finger to mix, and the mixture was allowed to stand at room temperature for 5 min. 10 µL of 1 mg / mL PEI was added, and the test tube was flicked to mix, and the mixture was allowed to stand at room temperature for 15-20 min. The HEK-293T cells were replaced with a new medium, and the above transfection mixture was slowly dripped in, and the mixture was gently shaken horizontally to mix. The cells were cultured at 37 °C, and the medium was changed after 6 hours, and the cells were cultured overnight. 48 hours after transfection, the virus solution was collected, and fresh medium was added, and the cells were cultured overnight. The collected virus solution was filtered through a 0.45 µM membrane and then infected with PKCα-KO SUM159 or LM2 cells. After 48 hours of virus infection, blasticidin was added to screen the cells. After 3-5 days, all cells in the control group died and the stable cell line was constructed. Cell samples were collected to detect PKCα protein expression.

[0093] Western blot experiments: cells were harvested and lysed in lysis buffer (RIPA) (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% NP-40; 1% Triton X-100; 0.1% SDS; 1% sodium deoxycholate; Roche protease inhibitors) and then sonicated for 30 seconds. The cell lysate was heated at 95°C for 10 minutes and subjected to SDS-PAGE analysis.

[0094] The results of studies showing that PKCα phosphorylates cGAS at serine 120 to promote its nuclear translocation are as follows.

[0095] Immunofluorescence localization experiment. Cells were fixed with 4% paraformaldehyde (pH 7.4) for 20-30 minutes at room temperature. Fixed cells were permeabilized with 0.1% Triton X-100 for 30 minutes, incubated with blocking solution (5% BSA + PBS + 0.1% Tween20) for 2 hours, and then incubated with primary antibody (rabbit monoclonal anti-GAS (E5V3W)) in a humidified chamber at 4°C overnight. Cells were washed three times with PBS and then incubated with secondary antibody (goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor™ Plus 555) for 2 hours at room temperature. Cells were washed three times with PBS and stained with DAPI (1:600) for 5 minutes at room temperature. Cells were washed three times with PBS, stained with DAPI (1:600) for 5 minutes, washed twice with PBS for 20-30 minutes each time, and then antifade reagent was added and mounted, and then photographed and counted using ImageJ.

[0096] like Figure 1 As shown, PKCα deficiency significantly reduced cGAS phosphorylation at S120 and cGAS nuclear localization, whereas PKCα restoration (PKCα knockout SUM159 cells transfected with lentivirus expressing PKCα) reversed these effects ( Figure 1 A in the figure is the result of Western experiment. Figure 1 Figure B is the result of immunofluorescence localization experiment. Figure 1 Figure C in Figure 2 shows the statistical results of cGAS fluorescence localization and PKCα protein expression after PKCα knockout and restoration. These data indicate that PKCα directly phosphorylates cGAS at serine 120, thereby promoting its nuclear translocation.

[0097] (3) Construction of complementing cell lines (PKCα gene knockout SUM159 and LM2 cell lines transfected with lentivirus expressing cGAS or cGAS S120A or cGAS S120D).

[0098] According to the method shown in step (2), cGAS or cGAS S120A or cGAS S120D lentivirus was collected and then infected with PKCα-KO SUM159 or LM2 cells. After 48 hours of virus infection, blasticidin was added to screen the cells. After 3-5 days, all cells in the control group died and the stable cell line was constructed. Cell samples (containing cGAS or cGASS120A or cGAS S120D lentivirus) were collected to detect the expression of cGAS or cGAS S120A or cGAS S120D protein (Western blot).

[0099] (4) Construction of an MCF7 cell line stably expressing cGAS S120D

[0100] The collected cGAS S120D lentivirus was used to infect MCF7 cells. After 48 hours of viral infection, blasticidin was added to screen the cells. After 3-5 days, all cells in the control group died and the stable cell line was constructed. Cell samples were collected and the expression of cGAS S120D protein was detected by cell migration assay (transwell method). The migration treatment time was: 200μl of cell suspension was inoculated in the upper chamber, and the 24-well plate containing the chamber was placed at 37°C and 5% CO2 for 24 hours.

[0101] The results of studies showing that cGAS phosphorylated at serine 120 can induce cell autonomous metastasis and spread are as follows Figure 2 As shown, the migratory ability of PKCα-deficient SUM159 cells was significantly rescued by re-expression of PKCα or the cGAS S120D mutant, but not WT cGAS or the cGAS S120A mutant ( Figure 2 Figure 2 (A), indicating that PKCα kinase phosphorylation of cGAS is very important for cGAS-mediated cancer metastasis. In addition, we also attempted to study the effect of cGAS S120D on low metastatic breast cancer cells, such as the MCF7 cell line with low cGAS expression (Figure 2 (B). Interestingly, overexpression of cGAS S120D significantly improved the migration ability of MCF7 cells (Figure 2 (C, D). Through these studies, the inventors found that PKCα directly phosphorylates cGAS at serine 120 to promote its nuclear translocation. Nuclear cGAS induces cell-autonomous metastatic spread.

[0102] Example 2

[0103] This example uses fingerprinting technology to predict that the spatial structure of a polypeptide is rich in diversity, the structure is flexible, and it is easy to bind to the enzyme, thereby blocking a phosphorylation site of the endogenous protein ( Figure 3 ) is the result of fingerprint decoding technology prediction. An antagonist peptide (or polypeptide) containing cGAS phosphorylation site was screened out and named peptide SP-19. Its amino acid sequence is: Ser-Arg-Ala-Gly-Ser-Cys-Arg-Gln-Arg-Gly-Ala-Arg-Cys-Ser-Thr-Lys-Pro-Arg-Pro (serine-arginine-alanine-glycine-serine-cysteine-arginine-glutamine-arginine-glycine-alanine-arginine-cysteine-serine-threonine-lysine-proline-arginine-proline), SRAGSCRQRGARCSTKPRP.

[0104] Example 3

[0105] The method for solid phase synthesis of peptide SP-19 in Example 2 and the method for crude product analysis, purification and mass spectrometry analysis are as follows:

[0106] In this example, the peptide solid phase synthesis method designed by Merrifield was used to synthesize the antagonist peptide SP-19 of the present invention. The specific steps are as follows:

[0107] In this study, we refer to the peptide solid phase synthesis method designed by Merrifield, and according to the designed peptide sequence, extend from the carboxyl end to the amino end one by one until the full length of the designed peptide is synthesized. First, the carboxyl group of the first amino acid at the carboxyl end of the peptide chain to be synthesized is tightly bound to the insoluble polymer resin, and the amino protecting group adsorbed on the first amino acid of the solid phase resin is removed; then, the next amino acid is connected to initiate a condensation reaction to form an amide bond. After washing, deprotection, end-capping and washing steps, the peptide chain is extended again one amino group at a time. Repeat the above condensation, washing, deprotection, end-capping and washing operations, and connect the next amino acid until the designed complete peptide chain is obtained. Finally, the synthesized polypeptide is cut off from the resin, and after purification and freeze-drying, the polypeptide to be prepared is obtained.

[0108] The detailed synthesis process is as follows:

[0109] (1) The extension of the SRAGSCRQRGARCSTKPRP peptide chain (starting from the carboxyl end Pro and extending to the amino end one by one) condenses Arg. According to the calculation formula: feed amount = resin weight × molar mass of Arg × Fmoc-pro-wang Resin resin substitution degree (0.3 mmol / g) × 2 times feed amount, the feed amount of Fmoc-Arg-OH per 1 g of resin is calculated to be 0.238 g, the amount of condensation agent TBTU (321.1 g / mol × 0.3 mmol / g × 2 times feed = 0.193 g), the amount of NMM added is 0.1 mL, the amount of DMF added is 10 mL, the temperature of the condensation reaction is 35°C, and the reaction time is 40 min. After the condensation reaction, the liquid is filtered off by suction, and 10 mL of DMF is added to wash the resin. After repeated washing 6 times, it is filtered and dried. Pick about 20 resins and test them with Kaiser Test. If the solution does not appear blue and the resin is transparent, it means that Fmoc-Arg-OH has been condensed on the resin. Add 10 mL of a mixture of hexahydropyridine and 40 mL of dimethylformamide (V:V=1:4) to the resin and react for 20 minutes to remove the amide protecting group. Then, elute with 10 mL of DMF, MeOH and DCM respectively, for a total of three times. After filtering and drying, the Arg@Fmoc-Arg-OH connection product is obtained. Pick about 20 resins and test them with Kaiser Test. If the solution appears blue, prepare to condense the next amino acid.

[0110] According to the above-mentioned operation steps for condensing Arg, the remaining 17 amino acids (Pro, Lys, Thr, Ser, Cys, Arg, Ala, Gly, Arg, Gln, Arg, Cys, Ser, Gly, Ala, Arg and Ser) were condensed in sequence to obtain the fully protected resin polypeptide of SRAGSCRQRGARCSTKPRP.

[0111] (2) Add DCM to wash the resin, then add ether to wash twice, drain, and vacuum dry for 2 h.

[0112] (3) Cutting:

[0113] The obtained peptide-resin was placed in an eggplant-shaped bottle, and 20 mL of cleavage reagent (95% trifluoroacetic acid, TFA) was added and reacted for 3 hours. 10 mL of ice-cold anhydrous ether was added to the cleavage solution and precipitated. Centrifuge at 5000 rpm for 5 min and discard the supernatant. Repeat three times, and dry the precipitate with a vacuum pump after centrifugation to obtain 0.95 g of crude peptide SRAGSCRQRGARCSTKPRP.

[0114] (4) Analysis and purification of crude product

[0115] Prepare buffer (Solution A): 100% acetonitrile containing 0.1% TFA and (Solution B): water containing 0.1% TFA, 4 liters each. Install the C18 column correctly on the high performance liquid chromatography (HPLC) system according to the instructions. Column temperature, 37 degrees; Column model: SKU: WAT054275, Symmetry C18 Column, 100Å, 5 µm, 4.6 mm X 250 mm, 1 / pk; Manufacturer: WATERS.

[0116] The chromatographic column was adjusted as necessary, and 5% of solution A and 95% of solution B were flushed at a flow rate of 40 mL / min for 15 min. The column was then equilibrated with 50% mixed solution (containing 0.1% TFA) for 15 min, and the synthesized peptide was injected into the column through the injection valve at a flow rate of 1.0 mL / min. The peptide was linearly eluted with 5% to 40% solution A and 95% to 60% solution B for 25 min. The entire analytical purification process was monitored by a UV detector with a wavelength of 220 nm. The crude peptide was purified using a reverse chromatographic column, and the prepared liquid was concentrated into a small volume, frozen in a refrigerator, and then placed in a freeze dryer to dry into a solid powder to obtain the pure SRAGSCRQRGARCSTKPRP peptide.

[0117] (5) Mass spectrometry analysis

[0118] Take a small amount of the synthesized sample, dissolve the sample and matrix (4-hydroxy-α-cinnamic acid) in a methyl cyanide solution containing 0.1% TFA (the volume ratio of methyl cyanide and 0.1% TFA is 1:1). Then, take 0.5 μL of the solution, place it in a metal sample pan, air-dry it at room temperature, and then detect its molecular weight by mass spectrometry (using insulin B peptide as the standard).

[0119] Example 4

[0120] The present invention performs HPLC analysis and mass spectrometry identification on the pure SP-19 separated and obtained in Example 3:

[0121] HPLC analysis: Take the pure SP-19 peptide, dissolve it in 1 ml of double distilled water, and filter it through a 0.22 um diameter filter membrane for analysis. Liquid phase conditions: chromatographic analysis column (C18 column); column temperature, 37 degrees; column model: SKU: WAT054275, SymmetryC18 Column, 100Å, 5 µm, 4.6 mm X 250 mm, 1 / pk; manufacturer: WATERS. Mobile phase A: 100% acetonitrile containing 0.1% TFA; mobile phase B: water containing 0.1% TFA, flow rate: 1.0 mL / min, detection wavelength 220nm. Injection volume 20ul, running time 20 min. Retention time tR=7.045 min, purity is 98.03%, the results are as follows Figure 4 shown.

[0122] MS mass spectrometry analysis: detector voltage 1.5 kV, CDL temperature 250°C. The detection result of the antagonist peptide SP-19 prepared in Example 1 is: [M+2H] / 2 + =1038.1, [M+3H] / 3 + =692.3, [M+4H] / 4 + =519.5, [M+5H] / 5 + =415.8. After conversion, the actual molecular weight measured by mass spectrometry is consistent with the theoretical molecular weight of antagonist peptide SP-19, which is 2074.36. The mass spectrometry analysis is consistent with the prediction, and it can be inferred that the product is antagonist peptide SP-19. The results are as follows Figure 5 shown.

[0123] Example 5

[0124] This example provides a migration experiment of TNBC cells after treatment with the antagonist peptide SP-19.

[0125] (1) Construction of cGAS gene knockout SUM159 or LM2 cell lines KO

[0126] Determine the forward and reverse sgRNA targeting the cGAS gene: F-CACCGAAGTGCGACTCCGCGTTCAG, R-AAACCTGAACGCGGAGTCGCACTTC. The double-stranded oligo was connected to the lenti-V2 vector. Then the plasmid connected with sgRNA was transfected into SUM159 or LM2 cells. After 48 hours of transfection, the cells were screened with puro antibiotics and then cultured for monoclonal growth. After the monoclonal cells grew to a certain extent, a certain proportion of cells were collected for gene sequencing and immunoblot protein detection, and the cGAS-KO SUM159 monoclonal cell line was screened, expanded, and frozen for subsequent use.

[0127] (2) Construction of complementation cell lines (cGAS knockout SUM159 or LM2 cell lines transfected with cGAS-expressing lentivirus) KO +cGAS.

[0128] HEK-293T cells were seeded into a 6 cm cell culture dish and cultured overnight at 37 °C and 5% carbon dioxide. When the cell density reached 70-80%, transfection was performed. In a 1.5 mL sterile centrifuge tube, 300 µL of opti-MEM medium was added, and then 4 µg of plasmid DNA mixture was added, including 1 µg of psPAX2, 1 µg of pMD2.G, and 2 µg of cGAS. The mixture was gently flicked on the bottom of the test tube with a finger to mix, and the mixture was allowed to stand at room temperature for 5 min. 10 µL of 1 mg / mL PEI was added, and the test tube was gently flicked to mix, and the mixture was allowed to stand at room temperature for 15-20 min. The HEK-293T cells were replaced with a new medium, and the above transfection mixture was slowly dripped in, and the mixture was gently shaken horizontally to mix. The cells were cultured at 37 °C, and the medium was changed after 6 hours, and the cells were cultured overnight. 48 hours after transfection, the virus solution was collected, and fresh medium was added, and the cells were cultured overnight. The collected virus solution was filtered through a 0.45 µM membrane and then infected with cGAS-KO SUM159 or LM2 cells. After 48 hours of virus infection, blasticidin was added to screen the cells. After 3-5 days, all cells in the control group died and the stable cell line was constructed. Cell samples were collected to detect cGAS protein expression (detection method is the same as in Example 1).

[0129] cGAS KO and cGAS KO The results of the SUM159 (A) and LM2 (B) cell lines with cGAS are shown in the figure. Figure 8 The results showed that cGAS can be restored after transfection with cGAS lentivirus. KO Expression of cGAS in cell lines.

[0130] (3) Migration experiment:

[0131] The cell growth status was observed, and the cGAS gene knockout SUM159 cell line, the complemented cell line in step (2), and the wild-type triple-negative breast cancer cell line SUM159 were used as experimental groups, respectively. The complemented cell line in step (2) was set up with two experimental groups, for a total of four experimental groups. Only one of the complemented cell lines was added with 10 μM of the antagonist peptide SP-19 of the present invention and treated for 24 hours. The other three experimental groups were added with 10 μM of the control peptide scramble of the antagonist peptide SP-19 of the present invention and cultured for 24 hours.

[0132] Take the above cell lines with good growth status and resuspend them in serum-free medium. Control the cell density to 25w / ml. Take 500μl of complete medium containing 10% FBS and add it to the lower chamber of the 24-well plate. Use tweezers to place the Transwell chamber in the 24-well plate. Inoculate 200μl of cell suspension in the upper chamber and place the 24-well plate containing the chamber at 37℃ and 5% CO. 2 The cells were cultured under the same conditions for 12 hours. Then the Transwell chamber was taken out, the culture medium was removed, and the cells in the chamber were gently wiped with a cotton swab soaked in PBS. The chamber was fixed with 4% paraformaldehyde fixative for 30 minutes. The fixative was discarded, the inside and outside of the chamber were washed once with PBS, the chamber was placed in crystal violet staining solution for staining for 10 minutes, the chamber was taken out, the inside and outside of the chamber were washed 3 times with PBS, and after proper air drying, the qualitative study was observed under a microscope, and 6-8 fields of view were taken and photographed, and the average value was counted and averaged using ImageJ for quantitative study.

[0133] Similarly, the cGAS gene knockout LM2 cell line, the complemented cell line in step (2), and the wild-type triple-negative breast cancer cell line LM2 were used as experimental groups, wherein the complemented cell line in step (2) was set up with two experimental groups, a total of 4 experimental groups, only one of the complemented cell lines was treated with 10 μM of the antagonist peptide SP-19 of the present invention for 24 hours, and the remaining three experimental groups were not added with 10 μM of the antagonist peptide SP-19 of the present invention and cultured for 24 hours. According to the above method, cell culture, staining, and qualitative study under microscope observation were performed, and 6-8 fields of view were taken for photographing and then counted and averaged using ImageJ for quantitative study.

[0134] The results are as follows Figure 6 As shown, the addition of antagonist peptide SP-19 to the complement cell line (cGAS) significantly inhibited the migration of TNBC cells, which was almost equivalent to the inhibitory effect in the cGAS knockout cell line (sgcGAS). The inhibition rates in SUM159 and LM2 cells were 77.34% and 78.81%, respectively.

[0135] Example 6

[0136] In this example, the antagonist peptide SP-19 was used to treat a nude mouse model of breast cancer lung metastasis to observe the lung metastasis of TNBC in vivo.

[0137] Cultivation of TNBC cell line LM2 (cGAS complement cell line constructed in step (2) of Example 5) KO +cGAS), and when the cells grew to the logarithmic phase, the cells were collected and resuspended in PBS. The resuspended cells were injected intravenously into female BALB / c nude mice aged four to six weeks (4 x 10 67 days later, the antagonist peptide SP-19 of the present invention (10 mg / kg per mouse) was injected into the mice by tail vein injection. The control group was scramble, which was injected twice a week for 4 consecutive weeks. After the mice were killed, their lungs were separated and imaged. Scramble is a polypeptide sequence composed of random arrangement and combination of the amino acid types of cGAS: GIKSRTLEKGKPRVLSA.

[0138] The results are as follows Figure 7 As shown, peptide SP-19 treatment almost completely eliminated metastatic nodules in the lungs, which was similar to what was observed in the lungs of mice injected with cGAS knockout LM2 cells. Therefore, targeting cGAS phosphorylation is a potential therapeutic strategy for the treatment of metastatic TNBC. ((A) Picric acid staining of whole lung; (B) H&E staining of paraffin sections. Scale bar in (A) is 1 cm; scale bar in (C) is 300 μm).

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0140] References:

[0141] 1.Katz, H. and M. Alsharedi, Immunotherapy in triple-negative breast cancer. 2017.35(1): p. 13.

[0142] 2. Deepak, KGK, et al., Tumor microenvironment: Challenges and opportunities in targeting metastasis of triple negative breast cancer. Pharmacol Res, 2020.153: p. 104683.

[0143] 3.Pantelidou, C. and O. Sonzogni, PARP Inhibitor Efficacy Depends on CD8(+) T-cell Recruitment via Intratumoral STING Pathway Activation in BRCA- Deficient Models of Triple-Negative Breast Cancer. 2019.9(6): p. 722-737.

[0144] 4. Zong, Y. and M. Pegram, Research advances and new challenges in overcoming triple-negative breast cancer. Cancer Drug Resist, 2021.4(3): p.517-542.

[0145] 5.Karami Fath, M., et al., Anti-cancer peptide-based therapeutic strategies in solid tumors. Cellular&Molecular Biology Letters, 2022.27(1): p.33.

Claims

1. A polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. An isolated nucleic acid molecule, characterized in that It encodes the polypeptide according to claim 1.

3. A carrier, characterized in that It comprises the nucleic acid molecule according to claim 2.

4. A cell, characterized in that It comprises the polypeptide according to claim 1, the nucleic acid molecule according to claim 2 or the vector according to claim 3.

5. Use of the polypeptide according to claim 1 in preparing a drug for treating breast cancer, characterized in that: The application includes any of the following application modes: (1) Inhibiting the phosphorylation of cyclic guanine nucleotide adenine nucleotide synthetase (cGAS) in breast cancer cells by using the polypeptide; (2) Inhibit the migration of breast cancer cells; (3) Eliminate or partially eliminate metastatic nodules in the lungs.

6. The use according to claim 5, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

7. A pharmaceutical composition, characterized in that It comprises the polypeptide according to claim 1.

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