Application of SNIP1 protein 301 site as target spot in preparation or screening of tri-negative breast cancer resisting drugs

Abstract

The invention belongs to the technical field of biotechnology and gene therapy, and particularly relates to application of SNIP1 protein 301 site as a target spot in preparation or screening of anti-triple-negative breast cancer drugs. According to the invention, the SNIP1 protein is found to be a non-histone substrate of lysine methyltransferase KMT5A, and the SNIP1 protein promotes growth, invasion and lung metastasis of breast cancer cells through KMT5A-mediated K301 monomethylation; therefore, the SNIP1 protein can be used as a target for screening anti-tumor drugs, and the screened drugs for inhibiting methylation of the K301 site of the SNIP1 protein can be used for resisting tumors; the invention further provides a polypeptide specifically bound with the K301 site of the SNIP1 protein, the polypeptide is bound with the K301 site of the SNIP1 protein, methylation of the SNIP1 is inhibited, finally, growth, proliferation, invasion and metastasis of tumors can be inhibited, and the polypeptide can be used as a novel targeted anti-tumor drug.

Description

[0001] This case is a divisional application of the original application, the original application patent number: CN2022103996108; the original application patent name: application of SNIP1 as a target in preparation or screening of antitumor drugs; the original application date: April 16, 2022. TECHNICAL FIELD

[0002] The application belongs to the technical field of new targets for precise cancer treatment and drugs, and specifically relates to application of SNIP1 as a target in preparation or screening of antitumor drugs. BACKGROUND

[0003] Breast cancer is the most common type of cancer that affects women's life and health. Triple-negative breast cancer (TNBC) accounts for about 15% of all invasive breast cancers. Triple-negative breast cancer (TNBCs) is a special type of breast cancer, which has a higher risk of recurrence, metastasis and death. Therefore, in-depth study of the regulation mechanism and target of tumor invasion and metastasis has important clinical significance.

[0004] Smad nuclear interaction protein 1 (SNIP1) is a newly cloned nuclear protein that plays a regulatory role by inhibiting the TGF-β signaling pathway. It is expressed in different types of cells, including the brain, placenta and kidney. The N-terminal nuclear localization signal (NLS) domain of SNIP1 binds to the C / H1 domain of transcriptional coactivators CBP and p300, competing for binding of Smad4 and p65 / RelA to CBP / p300. Overexpression of SNIP1 inhibits the transactivation of Smad4 and p65 / RelA by preventing their interaction with p300 through the NLS domain. On the contrary, SNIP1 prevents proteasomal degradation of C-myc by stabilizing it and connecting the C-myc / p300 complex, and enhances the transcriptional activity of C-myc by binding to its N-terminal through its C-terminal. Therefore, SNIP1 may be an important endogenous regulator of multiple transcriptional pathways that require CBP / p300 coactivators.

[0005] In breast cancer, including triple-negative breast cancer (TNBC), the epigenetic landscape is dysregulated, partly due to abnormal patterns of post-translational modifications (PTMs).

[0006] In the research of the present application, it is found for the first time that lysine methyltransferase KMT5A (also known as SET8, PR-Set7 / 9, SETD8,) can interact with SNIP1, methylate the K301 site of non-histone SNIP1, promote the formation of c-MYC / p300 complex and the transcriptional activation of c-MYC target, enhance the key oncogenic pathway hippo signaling pathway, and thus promote TNBC metastasis. Therefore, the SNIP1 protein can be used as a target for screening of anti-tumor drugs; and the present application provides a polypeptide specifically binding to the K301 site of the SNIP1 protein, which binds to the K301 site of the SNIP1 protein, inhibits the methylation of SNIP1, and finally can inhibit tumor growth, proliferation and invasion and metastasis, and can be used as a new targeted anti-tumor drug. SUMMARY

[0007] In order to solve the above technical problems, the present application provides an application of SNIP1 as a target in the preparation or screening of an anti-tumor drug, which specifically comprises the following contents:

[0008] In the first aspect, the present application provides an application of human SNIP1 protein as a target in the preparation or screening of an anti-tumor drug, which inhibits the methylation of human SNIP1 protein, or which inhibits / silences the expression of human SNIP1 protein, or which prevents lysine methyltransferase KMT5A from binding to human SNIP1 protein; the amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO. 1.

[0009] Preferably, the application refers to taking human SNIP1 protein as an action object to screen drugs to find drugs that prevent lysine methyltransferase KMT5A from binding to human SNIP1 protein as tumor treatment drugs; the amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO. 1.

[0010] Preferably, the tumor is breast cancer.

[0011] Preferably, the breast cancer is triple-negative breast cancer.

[0012] In the second aspect, the present application provides an application of isolated human SNIP1 protein gene as a target for RNA interference of a drug or preparation on tumor cells in the screening of a tumor treatment drug, which refers to taking the isolated human SNIP1 protein gene as an action object to screen drugs to find drugs that inhibit the methylation of K301 site of SNIP1 gene encoded protein as tumor treatment drugs.

[0013] Preferably, the tumor is breast cancer.

[0014] Preferably, the breast cancer is triple negative breast cancer.

[0015] In a third aspect, the present application provides an anti-tumor polypeptide drug, the polypeptide comprising the following amino acid sequence: HPSCSKQHAVFQY, the polypeptide drug being capable of inhibiting the methylation of SNIP1 protein at K301 site of tumor cells.

[0016] Preferably, the tumor is breast cancer.

[0017] Preferably, the breast cancer is triple negative breast cancer.

[0018] In a fourth aspect, the present application provides a monoclonal antibody drug for treating tumors, the heavy chain or light chain variable region of the monoclonal antibody comprising amino acid residues capable of specifically binding to K301 site of SNIP1.

[0019] Preferably, the tumor is breast cancer.

[0020] Preferably, the breast cancer is triple negative breast cancer.

[0021] In a fifth aspect, the present application provides a method for screening anti-triple negative breast cancer drugs or anti-triple negative breast cancer metastasis drugs by taking human SNIP1 as a target protein, the method comprising the following steps:

[0022] (1) determining the domain of human SNIP1 protein interacting with KMT5A;

[0023] (2) using bioinformatics, determining the active center according to the domain of human SNIP1 protein interacting with KMT5A, and setting the active pocket;

[0024] (3) using bioinformatics, performing docking of compounds or polypeptides in a small molecule compound library or a polypeptide library;

[0025] (4) performing activity screening according to the docking results;

[0026] The amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO. 1.

[0027] In a fifth aspect, the present application provides a method for screening anti-triple negative breast cancer drugs or anti-triple negative breast cancer metastasis drugs by taking isolated human SNIP1 protein as a drug or preparation, the method being:

[0028] (1) expressing and preparing SNIP1 protein and lysine methyltransferase KMT5A, and labeling the SNIP1 protein with biotin;

[0029] (2) evaluating the drug to be evaluated, the biotin-labeled SNIP1 protein and 3H-AdoMet is added into the buffer, then lysine methyltransferase KMT5A is added, the reaction is carried out for 30-150 min, then a selective inhibitor of lysine methyltransferase KMT5A is added to stop the reaction, the methylation level of the K301 site of the SNIP1 protein is determined, a positive control is not added with the polypeptide drug to be evaluated, a blank control is not added with KMT5A and the polypeptide drug to be evaluated, and the method for determining the methylation level is as follows: streptavidin-modified SPA beads are added into the above reaction solution, and then the SPA signal is detected by using a MicroBeta scintillation counter after the reaction, and the signal value represents the methylation degree.

[0030] (3) The methylation level of the K301 site of the SNIP1 protein in the positive control group is compared with the methylation level of the K301 site of the SNIP1 protein in the drug group to be evaluated, and if there is a significant difference between the two, it is proved that the drug to be evaluated has the efficacy of inhibiting tumor growth or tumor metastasis.

[0031] The beneficial effects of the present application are as follows: ① Lysine methyltransferase KMT5A (also known as SET8, PR-Set7 / 9, SETD8,) is found to be able to interact with SNIP1 for the first time, to cause the methylation of the K301 site of non-histone SNIP1, and to promote the formation of a c-MYC / p300 complex and the transcriptional activation of a c-MYC target, to enhance the key oncogenic pathway hippo signaling pathway, thereby promoting TNBC metastasis, and therefore, the SNIP1 protein can be used as a target for the screening of anti-tumor drugs; ② It is found that the inhibition of the methylation of the K301 site of SNIP1 or the inhibition of the interaction between KMT5A and SNIP1 can significantly inhibit tumor growth, proliferation and invasion and metastasis, and therefore, the K301 site of SNIP1 can be used as a target for the screening of anti-tumor drugs; ③ A polypeptide is provided, which can bind to the K301 site of SNIP1, thereby inhibiting the methylation of SNIP1, ultimately inhibiting tumor growth, proliferation and invasion and metastasis, and can be used as an anti-tumor drug; ④ The polypeptide has the effect of targeting SNIP1 protein, and can be used as a lead molecule to increase the content of a drug or a drug-loaded carrier (such as a nanomaterial, a liposome, etc.) in SNIP1 positive cells, and then a pharmaceutically acceptable excipient or adjuvant is added to prepare a novel more effective targeted anti-cancer drug; ⑤ A method for screening an anti-triple negative breast cancer drug or an anti-triple negative breast cancer metastasis drug by taking human SNIP1 as a target protein is provided, which can realize the in vitro screening of an anti-triple negative breast cancer drug or an anti-triple negative breast cancer metastasis drug, and provides a new target drug for the treatment of triple negative breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 SNIP1 methylation plays a pro-cancer function;

[0033] Figure 2for SNIP1 interaction with KMT5A at Lys301 site and methylation;

[0034] Figure 3 for KMT5A-mediated SNIP1 K301 methylation to activate Hippo / YAP signaling pathway;

[0035] Figure 4 KMT5A-mediated SNIP1 K301 methylation activates Hippo / YAP signaling;

[0036] Figure 5 KMT5A-mediated SNIP1 K301 methylation promotes TNBC metastasis through activating YAP signaling;

[0037] Figure 6 SNIP1 K301 methylation disrupts its interaction with KAT2A and releases SNIP1 inhibition of KAT2A HAT activity;

[0038] Figure 7 KMT5A-mediated SNIP1 K301 methylation promotes c-MYC-dependent recruitment of KAT2A to MARK4 promoter and activates MARK4 transcriptional activity;

[0039] Figure 8 KMT5A catalytic activity reduction combined with YAP signaling inhibition can stop triple-negative breast cancer progression. DETAILED DESCRIPTION

[0040] Embodiments of the present application will be described in detail with reference to the drawings, in which the following examples are provided as illustrative only and therefore should not be considered as limiting the scope of the present application. Wherever specific conditions are not indicated in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Wherever the reagents or instruments are not indicated by the manufacturer, they are all conventional products that can be obtained commercially.

[0041] The cell lines MCF10A, T47D, MDA-MB-453, BT474, MCF7, BT549, MDA-MB-231 and HEK-293T cell lines used in the present application were purchased from China National Cell Repository (Beijing). The 4T1 cell line was obtained from American Type Culture Collection (ATCC). All human cell lines were STR DNA fingerprinted by Shanghai Boying Applied Biological Technology Co., Ltd. (Shanghai, China) before the experiment. T47D, MDA-MB-453, BT474, MCF7, BT549, MDA-MB-231 and HEK-293T cells were cultured in DMEM medium (Gibco TMMCF10A cells were maintained in DMEM / F12 (Invitrogen) supplemented with 20 ng / mL epidermal growth factor, 5% horse serum, 0.5 μg / mL hydrocortisone, 10 μg / mL insulin, 100 ng / mL cholera toxin, and 100 μg / mL penicillin-streptomycin. 4T1 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin-streptomycin. All cell lines were cultured at 37 °C and 5% CO2. Mycoplasma contamination was detected using LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich).

[0042] The patient specimens of the present application were 100 formalin-fixed paraffin-embedded primary tumors and matched metastatic lymph nodes obtained from breast cancer patients who underwent surgery at Zhejiang Provincial People's Hospital from January 2010 to December 2015. None of these patients received any chemotherapy or radiotherapy before surgery.

[0043] The methods described in the following examples:

[0044] Chromatin immunoprecipitation (ChIP) was performed using Plus Enzymatic Chromatin IP Kit (Magnetic Beads, Cell Signaling Technology, 9005S): Cells were cross-linked with 1% formaldehyde in RT PBS for 10 min, quenched with 2.5 M glycine for 5 min at RT, and washed with PBS three times. Nuclei were prepared, chromatin was incubated with micrococcal nuclease for 20 min at 37 °C, followed by an appropriate amount of sonication. The supernatant was incubated with 3 g of anti-C-myc, anti-KAT2A, anti-SNIP1, or non-specific rabbit IgG at 4 °C for 12-16 h for immunoprecipitation. After 30 μL ChIP-Grade Protein G magnetic beads were incubated at 4 °C for 2 h, they were washed with low-salt buffer three times and high-salt buffer once, for 5 min each time. Chromatin was eluted with ChIP elution buffer at 65 °C with gentle vortex mixing (1200 rpm) for 30 min, and cross-linking was reversed by 5 M NaCl and proteinase K at 65 °C overnight. The samples were incubated with RNase at 37 °C for 1 h. The CHIP DNA was purified and quantified by real-time quantitative PCR (qPCR). The gene expression level was normalized to the β-actin expression level. The data were finally expressed as a percentage of input DNA.

[0045] Rechip detection was performed using the rechip-it kit (Active Motif). The precipitated chromatin from the first ChIP reaction was eluted with 100 μΐ, of diluted rechip-it elution buffer for 30 min at RT, and the eluted precipitate was desalted using the desalting column provided in the kit. The second ChIP was performed with 30 μΐ, of Protein G magnetic beads, 90 μΐ, of desalted chromatin, and 3 μg of secondary antibody. Next, the second precipitate was washed, eluted, and reverse cross-linked from the first chip. DNA was obtained by phenol and phenol / chloroform extraction and subjected to real-time PCR evaluation. PCR primer sequences are available upon request.

[0046] Total cells were lysed in IP lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 5 mM NaF, and 1% Triton X-100) supplemented with complete protease inhibitor cocktail (Roche) for 30 min at 4°C. Centrifugation, SDS-PAGE separation, and immunoblot analysis. To detect methylation of SNIP1 in vivo, cells were lysed in IP buffer and supplemented with complete protease inhibitor cocktail, followed by sonication and centrifugation at 12000 rpm for 15 min at 4°C. The supernatant was incubated with SNIP1 antibody or HA antibody for 12 h at 4°C. The immunoprecipitate was washed with IP buffer six times, then boiled in lx SDS-loading buffer for immunoblot analysis.

[0047] To detect protein interactions, cells were lysed in IP buffer, and the supernatant was immunoprecipitated with the indicated antibodies, slowly shaken overnight at 4°C on a rotating shaker, and then mixed with Pierce TM protein a / G Magnetic Beads (Invitrogen) for 1 h at room temperature. Washed five times with wash buffer (25 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.5), then eluted, boiled in lx SDS-loading buffer, and prepared for immunoblot analysis. To detect SNIP1 -associated proteins, approximately 4 x 106 8 MDA-MB-231 cells overexpressing Flag-SNIP1 or Flag-GFP control were collected in IP lysis buffer supplemented with complete protease inhibitors for detection. Equal amounts of cell lysates, including flag-tagged SNIP1, C-myc, or Flag-GFP control proteins, were incubated with anti-FLAG (Sigma-Aldrich) and Pierce TMProtein A / G Magnetic Beads (Invitrogen) immunoprecipitation. Beads were washed with wash buffer and bound proteins were eluted after mixing for 10 minutes at RT with elution buffer (0.1 M glycine, pH 2.0). After magnetic separation of the beads, the saved supernatant containing the target antigen was immediately neutralized with 1 M Tris HC1, pH 8.5, followed by boiling, SDS-PAGE solubilization, and silver staining. Bands were excised, subjected to LC-MS / MS sequencing, and data analysis.

[0048] HEK-293T cells transfected with 6xhis-tagged SNIP1 were lysed at 4°C for 30 minutes in a buffer containing 3 mM MgCl2, 300 mM KCl, 5% glycerol, 0.5% NP-40, imidazole 10 mM, 1 mM Na2VO4, 20 mM sodium fluoride, 1 mM sodium pyrophosphate, 25 mM beta glycerophosphate, cocktail, and 1 x complete EDTA-free protease inhibitors (Roche). The lysate was then sonicated and centrifuged at 4°C and 21,000 g for 20 minutes. The supernatant was then loaded onto a pre-equilibrated cobalt resin (Thermo Scientific) column. The loaded resin was then washed with equilibration / wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 7.6). After 2 washes, the his-tagged SNIP1 protein was eluted with elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 7.6), and fractions were collected and dialyzed against PBS. The purified recombinant protein was further tested.

[0049] pGEX-4T-1-SET8 and pET-28a(+)-SNIP1 were transformed into E. coli BL21 to produce recombinant GST-conjugated SET8 and histidine (His)-conjugated SNIP1, respectively. Five milliliters of bacterial culture were grown overnight at 37°C, 150 rpm in a shaker flask, and inoculated into 500 ml of ampicillin- or kanamycin-resistant Luria-Bertani broth (LB). The cells were grown at 37°C until the logarithmic phase (optical density of 0.8) was reached; 0.25 mM IPTG was added to induce protein expression, and the culture was incubated at 16°C with continuous shaking for 16 h. After induction, the culture was centrifuged at 5000 rpm for 10 min at 4°C, the supernatant was discarded, and the pellet was resuspended in 30 mL of lysis buffer (1 x PBS, 500 mM NaCl, 1% Triton X-100, 0.5 mg / mL lysozyme, 1 x EDTA-free protease inhibitor cocktail) and sonicated in ice. The lysate was centrifuged at 15000 rpm for 15 min at 4°C. The supernatant was incubated with the corresponding volume of 50% glutathione Sepharose 4B beads slurry (GE Healthcare) or Ni-NTA magnetic agarose beads (Qiagen) for 8 h at 4°C, following the end-to-end mixing instructions. The glutathione beads were washed three times with lysis buffer and then stored in lysis buffer at 4°C, and the sample was eluted with elution buffer (10 mM glutathione, Tris-HCl pH 8.0, 50 mM). The nickel beads were washed three times with 50 mM Tris-HCl (pH 8.0) containing 20 mM imidazole (1 mL per time) and the sample was eluted with Tris containing 250 mM imidazole.

[0050] GST-SET8 and 6xHis-SNIP1 proteins were purified using glutathione beads and Ni-NTA agarose beads, respectively, as described above, following the manufacturer's procedure. In summary, 1 pg of GST-tagged methyltransferase was incubated with 2.5 pg of His-tagged hatching matrix in 50 pL of reaction buffer (50 mM Tris-HCl (pH 8.0), 10% glycerol, 20 mM potassium chloride, 5 mM MgCl2, 1 mM DTT, 1 mM PMSF) supplemented with 2 pg of Ci S-adenosyl-L-[methyl-3H] methionine (Amersham Biosciences) for 2 h at 30°C. The reaction was resolved by SDS-PAGE Coomassie staining (Expedeon, InstantBlue) and then exposed to x-ray film for final analysis.

[0051] KAT2A HAT assay in vitro was performed using the HAT kit from Active Motif.

[0052] Use Lipofectamine according to the instructions. TM pGL3.0 basic with wild-type MARK4 promoter and promoter with mutant c-Myc binding site were co-transfected or not with 3000 transfection reagent (Thermo Fisher). pRL Renilla luciferase control reporter vector (Promega) was used as negative control. Promega E1960 The reporter system was used for dual-luciferase assays 48 hours after co-transfection. SET8, SNIP1, c-Myc, and KAT2A cDNAs were amplified by RT-PCR using MDA-MB-231 cells. These cDNAs were then sequenced and subcloned into pcDNA3.3 or pLVX-Puro vectors (Clontech). SET8 or SNIP1 truncated constructs were generated by PCR using SET8 or SNIP1-pcDNA3.3 as templates and then inserted into pcDNA3.3. Wild-type cDNA was cloned into the pGEX-4T-1 vector to construct GST-SNIP1. SNIP1 mutants (K301R, K325R, and K342R) were generated by PCR-based site-directed mutagenesis (Invitrogen). The MARK4 promoter, ranging from -2000 to +200 relative to the transcription start site, was cloned into the pGL3-basic luciferase reporter vector and used as a template for subcloning and generating a series of MARK4 promoter deletions. A MARK4 promoter with a disrupted potential c-Myc binding site was constructed using polymerase chain reaction-based site-directed mutagenesis.

[0053] SET8 or SNIP1 sgRNA sequences were designed using the MIT online tool (http: / / crispr.mit.edu). shRNA sequences were purchased from GeneChem (Shanghai, China). DNA and packaging plasmids were transfected into HEK293 cells. At 48 and 72 h after transfection, the supernatant was filtered through a 0.22 μm membrane (Millipore) and concentrated. MDA-MB-231 and BT-549 cells were infected with lentivirus expressing the shRNA or shGFP control with 8 μg / mL polyamine. Selected infected cells were treated with 5 μg / mL puromycin 48–72 h after infection. Multiple monoclonal shRNAs were screened by western blotting and RT-PCR analysis. For transient transfection, plasmids were transfected using Lipofectamine 3000 (Invitrogen) reagent according to the manufacturer's instructions.

[0054] 5×10 4Cells were suspended in medium without fetal bovine serum and plated on Matrigel (BD Biosciences) on transwell inserts (8 pm pore size, 5 mm diameter, Corning). The inserts were incubated in 500 pL of medium with 10% fetal bovine serum (Invitrogen) for 16-24 hours. To assess the invasive ability of the cells, the top of the membrane was gently wiped with a cotton swab to remove non-invasive cells, the migrated or invaded cells were fixed with methanol, stained with crystal violet, and counted under a light microscope.

[0055] Lungs were dissected, fixed in 4% paraformaldehyde, and paraffin-embedded. Four step sections (100 microns each) were obtained from each lung. Sections were stained with H&E and scanned using a Scanscope XT digital slide scanner (Aperio Technologies). Digital images of lung sections were used to analyze metastatic burden. Immunohistochemistry (IHC) was performed on patient tissues with anti-SET8 (1 :500), anti-SNIP1 k301me1 (1 :500), anti-MARK4 (1 : 100) antibodies. Each sample was assigned an intensity score for data staining (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) and proportion of stained cells (0 = 0%, 1 = 1-25%, 2 = 25-50%, 3 = 5-75%, 4 = 75-100%). Negative control slides were performed without primary antibody. Nuclear staining was scored as negative (0) when <10% of tumor cells showed positive expression. Three individuals blinded to clinical parameters scored the stained tissues.

[0056] Total RNA was extracted and purified using Qiagen RNeasy Mini Kit (Valencia, CA, USA). RNA quality was assessed using an Agilent 2100 Bioanalyzer prior to sequencing. Poly(A)+ RNA libraries were prepared according to the Illumina protocol. Libraries were sequenced on the Illumina HiSeqX Ten platform. Differential expression was filtered by fold change (fold change > 2.0; P < 0.05). KEGG pathway analysis of differentially expressed genes was performed using DAVID software (https: / / david.ncifcrf.gov / ). P values for KEGG pathways were calculated using Fisher’s exact or Chi-square test with FDR, respectively. Significance was selected with a threshold of P < 0.05 and FDR < 0.25.

[0057] Four- to five-week-old pathogen-free female BALB / c and athymic nude mice were purchased from Shanghai Slaccas and maintained in a mouse-specific pathogen-free (SPF) facility for 1 week prior to injection to acclimate the mice to the new environment. Independent experiments were performed with 5-8 mice per group. MDA-MB-231 cells (1 x 10 5) or its derivatives were suspended in 100 μL PBS and injected into the tail vein of nude mice; MDA-MB-231 cells (5 x 10 5 ) or 4T1 cells (5 x 10 5 ) or its derivatives were suspended in 50 μL PBS, mixed with matrigel (1:1), and then injected into the mammary fat pad of mice in situ (5-8 mice per group) according to the established protocol. Mice were sacrificed 25 days after tail vein injection and 28 days after mammary fat pad injection. Tumor growth was determined by non-blind weekly caliper measurement and tumor volume calculation formula (width x length2x π) / 6. Lung metastasis was detected by bioluminescence imaging (BLI). All lung lobes were removed and the number of metastatic nodules on the lung surface was counted. In terms of in vivo drug treatment, 7 days after inoculation, tumor-bearing mice were injected intraperitoneally with vehicle or veraprofene (50 mg / kg) every day. Veraprofene was dissolved in DMSO (100 mg / mL) and stored at -80°C. Working solutions were prepared in fresh PBS before use, and 21 days after injection, luciferin was injected, and primary / metastatic tumors were detected by BLI using IVIS100 (Caliper Life Sciences, Hopkinton, MA, USA). The final animal survival rate was analyzed. Independent experimental groups with corresponding survival analysis under the same conditions were also performed. Mice were monitored daily and euthanized according to the guidelines of the UCSD Institutional Animal Care and Use Committee when criteria were met. Survival data were analyzed using the log-rank test. Data collected from at least two independent experiments showed similar results. All mouse studies were performed according to protocols approved by the Animal Protection and Use Committee of the Chinese Academy of Medical Sciences.

[0058] All experiments were repeated three times, and similar results were obtained in each test. Statistical analysis data were expressed as mean ± S.D. or mean ± s.e.m. A p-value < 0.05 indicates that the difference is statistically significant. All P values are from at least three independent experiments, each with three technical replicates. The sample size was not determined in advance using statistical methods.

[0059] Example 1 SNIP1 promotes oncogenic functions

[0060] 1. Methylation of SNIP1 at Lys301

[0061] (1) To determine important non-histone proteins regulated in a methylation-dependent manner, mass spectrometry (MS)-based screening was performed on cell lysates from two TNBC cell lines (i.e., BT549 and MDA-MB-231) using specific pan-lysine methylation antibodies, and the results are shown in FIG. 1A. Figure 1(2) Several SNIP1-derived peptides were identified, including methylation modifications of three nearby evolutionarily conserved lysine residues (K301, K325, and K342) in the FHA (281-344aa) domain. Figure 1 b and c; K301 methylation was detected in both cell lines. (3) SNIP1 mutants were generated by replacing the above lysine residues in the FHA domain with arginine. This lysine (K)-arginine (R) substitution prevents methylation but maintains a positive charge, thereby mimicking the unmethylated form of the protein. The results showed that only the SNIP1 K301R mutant was not methylated, while the other mutants were not methylated. Figure 1 As shown in d, Lys301 is the SNIP1 methylation site in FHA. (4) LC-MS / MS showed that K301 is monomethylated. Figure 1 (5) In cells treated with the global histone methylation inhibitor 3-deazacytidine a, SNIP1 monomethylation was confirmed using a Kme1-specific antibody. In addition, the Lys301 site is highly conserved and evolutionarily conserved in SNIP1 proteins from different species, indicating the functional specificity of lysine in SNIP1. In summary, SNIP1 is methylated at Lys301.

[0062] SNIP1 methylation may promote the growth and metastasis of TNBC both in vitro and in vivo

[0063] To reveal the potential biological function of monomethylation within its FHA region, a methylation-deficient variant of SNIP1 (K301R) was ectopically re-expressed in SNIP1 knockout MDA-MB-231 and BT549 cell lines (MDA-MB-231 / sgSNIP1 and BT549 / sgSNIP1). The results showed that SNIP1 knockout reduced tumor growth in vivo (compared with the control group (sgC)). Figure 1 f and g), which significantly downregulated Ki67 expression in primary tumor tissues ( Figure 1 h and i); while wild-type SNIP1 (SNIP1 WT ) rather than SNIP1 K301R The mutant significantly promoted tumor growth ( Figure 1 f and g) and increased Ki67 expression ( Figure 1 In addition, SNIP1 gene knockout significantly inhibited breast cancer cell invasiveness, tumor metastasis to the lungs, and the formation of metastatic lung nodules in vitro ( Figure 1 jm) and prolong animal survival time ( Figure 1 Similarly, recombinantly expressed SNIP1 WTRather than SNIP1 K301R The mutant restored cell invasion and tumor lung metastasis ( Figure 1 jm), and shortened animal survival time ( Figure 1 Chinese n).

[0064] Together, these findings demonstrate that SNIP1 methylation may promote TNBC growth and metastasis both in vitro and in vivo.

[0065] Example 2 SNIP1 interacts with KMT5A and is methylated by KMT5A at Lys301

[0066] To identify the physiological upstream methyltransferase (or methyltransferases) that mediate SNIP1 K301 monomethylation, co-immunoprecipitation (co-IP) analysis and LC-MS / MS were performed to capture SNIP1 interacting proteins from MDA-MB-231 cells. Figure 2 In (a), the methyltransferase KMT5A is the only lysine methyltransferase among SNIP1-interacting proteins. This suggests that KMT5A methylates SNIP1. A strong physical interaction between endogenous SNIP1 and KMT5A was observed in MDA-MB-231 and BT549 cells ( Figure 2 Subsequently, HA-SNIP1 was also detected in anti-Flag-KMT5A immunoprecipitates from HEK-293T cells transfected with the indicated plasmids ( Figure 2 In addition, SNIP1 expression was downregulated by GST-KMT5A, and vice versa, indicating an interaction between SNIP1 and KMT5A ( Figure 2 These results indicate that SNIP1 directly interacts with KMT5A.

[0067] The interaction between SNIP1 and KMT5A suggests that methylation regulation may exist. For example, anti-ubiquitin-methyl antibodies ( Figure 2 As demonstrated in Figure f), the methylated lysine (K) level of SNIP1 was significantly reduced in KMT5A-knockout MDA-MB-231 and BT549 cells. Considering that SNIP1 is monomethylated at Lys301 in the FHA domain that binds to KMT5A, K301 is a KMT5A methylation site. A mutant SNIP1 was generated by replacing the K301 residue and two other lysine residues (K325 and K342) in the FHA domain with arginine (R); Figure 2As shown in Figure g, in vivo methylation analysis confirmed that only replacement of K301 with R significantly affected SNIP1 lysine methylation by KMT5A when detected by anti-ubiquitin methylation antibodies. Furthermore, in vitro methylation analysis showed that only K301R (but not K325R and K342R) showed a reduced methylation signal ( Figure 2 This suggests that KMT5A acts as a SNIP1 methyltransferase, mediating the methylation of K301 on SNIP1.

[0068] The wild type of KMT5A (KMT5A WT ) or catalytically inactive mutants (KMT5A R336G ) were co-transfected with HA-SNIP1 into HEK-293T cells. WT Different, KMT5A R336G Significantly reduced the level of monomethyl K301 (K301me1) of HA-SNIP1 ( Figure 2 Similar results were observed in another independent in vitro methylation assay ( Figure 2 This methylation was further confirmed by testing with an anti-SNIP1 K301me1 antibody ( Figure 2 These results confirm a direct interaction between KMT5A and SNIP1, leading to monomethylation of lysine 301 in SNIP1 by KMT5A.

[0069] Example 3 KMT5A-mediated SNIP1 methylation activates Hippo / YAP signaling

[0070] To elucidate the functional significance of SNIP1 methylation, RNA sequencing analysis was performed to compare the gene expression profiles in MDA-MB-231 / sgSNIP1 cells with those in sgRNA-resistant HA-SNIP1 WT or HA-SNIP1 K301R Re-expression of mutants. A total of 632 down-regulated genes and 50 up-regulated genes were identified through re-expression (raw data can be obtained through Bioproject ID: PRJNA797682); WT Compared with the control group, sgRNA antagonized SNIP1 K301R Mutants (fold change > 2, P < 0.05) ( Figure 3 KEGG pathway analysis of 632 down-regulated genes identified the Hippo signaling pathway as the most important pathway ( Figure 3(b) 57 YAP target genes were found in various cell types, including breast cancer cells. Among them, 40 genes (also identified as YAP-regulated downstream genes) were downregulated in MDA-MB-231 / sgSNIP1 cells, and these genes were SNIP1-resistant to sgRNA compared with the control group. K301R Mutant ( Figure 3 c and d), while the levels of YAP target genes were significantly reduced; WT Compared with the group, the SNIP1K301 methylation mimic mutant (SNIP1 K301M ) in MDA-MB-231 / sgSNIP1 cells, the levels of YAP target genes were significantly increased ( Figure 3 (e)

[0071] like Figure 3 As shown in c, SNIP1 K301R It also downregulated the expression of MAP / microtubule affinity regulating kinase 4 (MARK4), which can phosphorylate Hippo core components MST1 / 2 or SAV; in addition, SNIP1 K301R It disrupts the complex formation between MST / SAV and LATS, thereby affecting YAP / TAZ activity. As an effective activator of YAP / TAZ activity, MARK4 inhibits YAP / TAZ phosphorylation to promote YAP / TAZ nuclear localization, ultimately inducing transcriptional activation in breast cancer. K301R The expression of MAR4, p-MST2, p-SAV, upregulated p-YAP and slightly increased p-LATS1 expression levels, and effectively altered the target genes of the Hippo kinase cascade and Hippo / YAP signaling pathway, namely CTGF and CYR6127 ( Figure 3 These results suggest that SNIP1 K301 methylation regulates the Hippo / YAP signaling pathway by modulating MARK4 expression and subsequently modifying the phosphorylation of MST2, SAV, LATS1, and YAP.

[0072] Example 4: KMT5A mediates SNIP1 K301 methylation, activates the YAP signaling pathway, and promotes TNBC metastasis

[0073] To investigate the role of KMT5A-dependent SNIP1 methylation in the regulation of TNBC metastasis, HA-SNIP1 WT or HA-SNIP1 K301MMutants were re-expressed in MDA-MB-231 / sgKMT5A and BT549 / sgKMT5A cells. Further experiments showed that, in MDA-MB-231 and BT549 cells, SNIP1 K301M overexpression (mimicking SNIP1 K301 methylation status, not SNIP1 WT) rescued KMT5A loss-mediated loss of MARK4 expression and transcriptional activity, YAP phosphorylation and expression of its target genes Figure 4 , consistent with these in vitro results, ectopic expression of SNIP K301M , but not SNIP1 WT , restored KMT5A depletion-mediated suppression of proliferation and invasion in vitro Figure 4 , d, f), and tumor growth and lung metastasis in vivo Figure 4 , e, g), and prolonged animal survival time Figure 4 , h). Taken together, these results indicate that KMT5A-dependent SNIP1 methylation promotes TNBC tumor and lung metastasis by activating MARK4 transcription and subsequent Hippo / YAP signaling pathway modification.

[0074] Example 5 SNIP1 K301 methylation disrupts its interaction with KAT2A

[0075] Tandem affinity purification combined with mass spectrometry analysis showed that c-MYC interacts with endogenous SNIP1 and KAT2A Figure 5 , a). Co-immunoprecipitation and immunoblotting analysis as shown in Figure 5 , b and c, where SNIP1 further binds to KAT2A and c-MYC, indicating that SNIP1 / KAT2A / c-MYC forms a complex in breast cancer cells. In addition, the results of the study showed that SNIP1 WT mutants have a stronger association with KAT2A protein than SNIP1 K301R , but not with c-MYC Figure 5 , d). However, SNIP1 K301 methylation significantly inhibited this interaction Figure 5 , e). In addition, GST pull-down results analysis showed that His-KAT2A was pulled down by purified GST-SNIP1 but not by GST alone, and GST-SNIP1-K301M significantly impaired this combination Figure 5 , f), indicating that SNIP1 methylation disrupts the physical interaction between SNIP1 and KAT2. In SNIP1 WT , SNIP1 K301R , or SNIP1 K301MKAT2A HAT activity was measured in the presence of. Similarly, recombinant SNIP1 WT exhibited a strong inhibitory effect, recombinant SNIP1 K301R exhibited a reduced inhibitory effect, but SNIP1 K301M could completely rescue it Figure 5 in g). Moreover, in vitro observations indicated that SNIP1 directly binds to KAT2A, suggesting that KMT5A-mediated SNIP1 K301 methylation can play a role in modulating KAT2A HAT activity. In the presence of BSA and empty vector (EV), KAT2A exhibited dose-dependent HAT activity; this was abolished in the presence of SNIP1 WT alone Figure 5 in h). In contrast, in the presence of KMT5A WT but not the inactive kinase activity mutant KMT5A R336G KAT2A HAT activity was largely rescued Figure 5 in h). These data suggest that KMT5A-mediated SNIP1 K301 methylation releases its interaction with KAT2A, ultimately leading to the activation of KAT2A enzymatic activity.

[0076] Example 6 SNIP1 K301 methylation promotes c-MYC-dependent recruitment of KAT2A to MARK4 promoter and activates MARK4 transcriptional activity

[0077] SNIP1 strongly inhibits Smad / p65-dependent transcriptional activity by competing with Smad4 and p65 / RelA for binding to the coactivator p300. Immunoprecipitation and western blotting of KAT2A showed that overexpression of c-MYC interfered with the interaction between KAT2A and SNIP1 Figure 6 in a). Conversely, SNIP1 overexpression also inhibited KAT2A-c-MYC interaction Figure 6 in b). These data suggest that SNIP1 can be a candidate modifier of MARK4 transcriptional activity by competing with c-MYC for binding to KAT2A. Importantly, KMT5A WT but not KMT5A R336G could methylate SNIP1 K301, promoting Flag-c-MYC binding to His-KAT2A; KMT5A WT significantly impaired the association between HA-SNIP1 and His-KAT2A Figure 6 in c). Moreover, re-expression of KMT5A WT but not KMT5AR336G significantly enhanced the interaction between endogenous c-MYC and KAT2A, SNIP1 K301 methylation, MARK4 expression and Hippo / YAP target gene CTGF expression; meanwhile, the binding of endogenous SNIP1 to KAT2A was significantly reduced, KMT5A WT re-expression also reduced YAP phosphorylation Figure 6 d). Furthermore, expression of KMT5A WT (not KMT5A R336G ) upregulated MARK4 mRNA expression in MDA-MB-231 / sgKMT5A and BT549 / sgKMT5A cells Figure 6 e). Overexpression of KMT5A WT (not KMT5A R336G ) significantly enhanced MARK4 transcriptional activity in HEK-293T cells Figure 6 f). Moreover, in MDA-MB-231 / sgKMT5A and BT549 / sgKMT5A cells, re-expression of KMT5A WT (not KMT5A R336G ) promoted KAT2A binding to the MARK4 promoter Figure 6 g, h). These findings suggest that KMT5A-mediated SNIP1 K301 methylation releases KAT2A to facilitate KAT2A HAT activity and c-MYC / KAT2A complex formation, and c-MYC / KAT2A complex recruitment to the MARK4 promoter. MARK4 expression is ultimately inhibited in a kinase-dependent manner to suppress the Hippo kinase cascade.

[0078] Example 7 KMT5A catalytic activity combined with YAP signaling inhibition can prevent triple-negative breast cancer progression

[0079] UNC0379, a selective inhibitor of KMT5A, which has been evaluated for progression and metastasis of hepatocellular carcinoma and ovarian cancer, was used to treat MDA-MB-231 and 4T1 cells. The results showed that UNC0379 or verteporfin had a modest inhibitory effect on cell proliferation in vitro and tumor growth in vivo compared to the control group Figure 7 a-c); then, UNC0379 and verteporfin combination therapy was more effective than single-agent therapy in cell proliferation and tumor growth Figure 7 a-c). In addition, we also determined whether the two small molecule inhibitors synergize in cell invasion in vitro and lung metastasis in vivo. Compared with single-agent therapy, the combined use of UNC0379 and verteporfin significantly inhibited cell invasion Figure 7Middle d); In vivo lung metastasis regimen was consistent with the combined treatment regimen of verteporfin and / or KMT5A depletion ( Figure 7 e). Seven days after cell inoculation, transplanted tumors were treated daily with verteporfin (50 mg / kg, ip) or / and UNC0379 (25 mg / kg, ip) for 14 days; tumor metastasis was monitored 21 days after implantation. Figure 7 fh), tumor metastasis was significantly slowed down and animal survival was prolonged by combined treatment ( Figure 7 These data suggest that the KMT5A / SNIP1 / MARK4 axis may comprise a clinically targetable vulnerability in TNBC driven by aberrant Hippo / YAP signaling.

[0080] Example 8 KMT5A expression is significantly associated with activated Hippo / YAP signaling in triple-negative breast cancer

[0081] The expression levels of KMT5A, SNIP1K301me1, and MARK4 were detected in 100 clinical TNBC species. Figure 8 As shown in Figure a, KMT5A, SNIP1K301me1, and MARK4 were significantly co-expressed in TNBC tissues. According to the protein expression quantification analyzed by immunohistochemical staining, KMT5A was significantly positively correlated with SNIP1K301me1 and MARK4, as shown by the chi-square test ( Figure 8 (b) KMT5A and MARK4 expression was significantly correlated with pathological grade; in addition, SNIP1K301me1 and MARK4 levels were associated with N stage, and SNIP1K301me1 was somewhat correlated with AJCC stage, indicating that KMT5A-mediated SNIP1 methylation may enhance the malignant metastasis of TNBC. Kaplan-Meier analysis showed that in TNBC patients, the co-expression of KMT5A / SNIP1K301me1 or KMT5A / MARK4 high expression levels was positively correlated with poor prognosis ( Figure 8 In addition, we detected the expression of KMT5A, SNIP1K301me1, and MARK4 in normal breast specimens and tumor tissues, and the levels of these proteins were significantly upregulated in tumor tissues ( Figure 8 Meanwhile, we also detected the expression of KMT5A and SNIP1K301me1 in tissues at different tumor stages and found that the expression of KMT5A and SNIP1K301me1 was closely related to the progression of TNBC ( Figure 8 Finally, the expression of KMT5A, SNIP1K301me1, and MARK4 was significantly upregulated in metastatic LNs compared with primary TNBC tumors ( Figure 8Figure 5. KMT5A-mediated SNIP1 methylation is significantly associated with activated MARK4 kinase cascade in TNBC. Figure 6. KMT5A-mediated SNIP1 methylation is significantly associated with activated MARK4 kinase cascade in TNBC.

[0082] Example 9. Screening method of anti-triple negative breast cancer drug

[0083] Method 1: molecular docking

[0084] (1) determining the domain of human SNIP1 protein interacting with KMT5A;

[0085] (2) using bioinformatics, determining the active center according to the domain of human SNIP1 protein interacting with KMT5A, and setting the active pocket;

[0086] (3) using bioinformatics, docking the compounds or polypeptides in the small molecule compound library or polypeptide library;

[0087] (4) performing activity screening according to the docking results;

[0088] The amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO. 1.

[0089] Method 2:

[0090] (1) expressing and preparing SNIP1 protein and lysine methyltransferase KMT5A, and labeling SNIP1 protein with biotin;

[0091] (2) adding the drug to be evaluated, biotin-labeled SNIP1 protein and 3 H-AdoMet into the buffer, then adding lysine methyltransferase KMT5A, reacting for 30-150 min, adding a selective inhibitor of lysine methyltransferase KMT5A to stop the reaction, and determining the methylation level of the K301 site of SNIP1 protein, wherein the positive control does not add the polypeptide drug to be evaluated, the blank control does not add KMT5A and the polypeptide drug to be evaluated, and the method for determining the methylation level is as follows: adding streptavidin-modified SPA beads to the above reaction solution, detecting the SPA signal after reaction using a MicroBeta scintillation counter, and the signal value represents the degree of methylation;

[0092] (3) comparing the methylation level of the K301 site of SNIP1 protein in the positive control group and the methylation level of the K301 site of SNIP1 protein in the drug to be evaluated group, and if there is a significant difference between the two, it is proved that the drug to be evaluated has the efficacy of inhibiting tumor growth or tumor metastasis.

[0093] Example 10. Polypeptide drug against triple negative breast cancer

[0094] A polypeptide with an amino acid sequence of HPSCSKQHAVFQY is synthesized by solid phase synthesis.

[0095] The polypeptide can specifically bind to the K301 site of the SNIP1 protein, prevent the binding of the KMT5A enzyme to the SNIP1 protein, inhibit the methylation of the K301 site of the SNIP1 protein, and inhibit the growth, proliferation and invasion and metastasis of triple-negative breast cancer, and can be used as an antitumor drug.

[0096] In the present study, the SNIP1 protein is studied in detail, and it is found that the SNIP1 protein is a non-histone substrate of lysine methyltransferase KMT5A, which promotes breast cancer cell growth, invasion and lung metastasis through K301 monomethylation mediated by KMT5A; therefore, the SNIP1 protein can be used as a target for screening of antitumor drugs, and can be used for screening of antitumor drugs. The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of human SNIP1 protein as a target in the preparation or screening of anti-tumor drugs, wherein the drug inhibits methylation at site 301 of the human SNIP1 protein, or the drug prevents the binding of lysine methyltransferase KMT5A to the human SNIP1 protein; the amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO.

1.

2. A method for screening anti-triple-negative breast cancer drugs or anti-triple-negative breast cancer metastasis drugs using human SNIP1 as a target protein, characterized in that: The method comprises the following steps: (1) Determine the domain of human SNIP1 protein that interacts with KMT5A; (2) Using bioinformatics, the active center was determined based on the domain structure of the interaction between human SNIP1 protein and KMT5A, and the active pocket was defined; (3) Using bioinformatics to dock compounds or peptides in a small molecule compound library or peptide library; (4) Activity screening based on the docking results; The amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO.

1.

3. A method for screening anti-triple-negative breast cancer drugs or anti-triple-negative breast cancer metastasis drugs using isolated human SNIP1 protein as a drug or preparation, characterized in that: The method is: (1) Expression and preparation of SNIP1 protein and lysine methyltransferase KMT5A, and labeling of SNIP1 protein with biotin; (2) The drug to be evaluated, biotin-labeled SNIP1 protein and 3 H-AdoMet is added to a buffer solution, and then lysine methyltransferase KMT5A is added. The reaction is allowed to proceed for 30-150 minutes. A selective inhibitor of lysine methyltransferase KMT5A is added to terminate the reaction, and the methylation level at the K301 site of the SNIP1 protein is measured. A positive control is performed without the addition of the peptide drug to be evaluated, and a blank control is performed without the addition of KMT5A and the peptide drug to be evaluated. The method for measuring the methylation level is as follows: streptavidin-modified SPA beads are added to the above reaction solution, and after the reaction, the SPA signal is detected using a MicroBeta scintillation counter, where the signal value represents the degree of methylation. (3) Compare the methylation level of the K301 site of the SNIP1 protein in the positive control group with the methylation level of the K301 site of the SNIP1 protein in the drug group to be evaluated. If there is a significant difference between the two, it proves that the drug to be evaluated has the effect of inhibiting tumor growth or tumor metastasis.

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