Use of snip1 as a target in the preparation or screening of antitumor drugs

Abstract

The application belongs to the technical field of biotechnology and gene therapy, and particularly relates to application of SNIP1 protein as a target in preparation or screening of an antitumor drug. The application finds that the SNIP1 protein is a non-histone substrate of lysine methyltransferase KMT5A, which promotes breast cancer cell growth, invasion and lung metastasis through KMT5A-mediated K301 monomethylation; therefore, the SNIP1 protein can be used as a target for screening of an antitumor drug, and a drug obtained by screening and inhibiting methylation of the K301 site of the SNIP1 protein can be used for antitumor; and the application provides a polypeptide specifically combined with the K301 site of the SNIP1 protein, the polypeptide is combined with the K301 site of the SNIP1 protein, inhibits methylation of the SNIP1 protein, and finally can inhibit tumor growth, proliferation, invasion and metastasis, and can be used as a new targeted antitumor drug.

Description

Technical Field

[0001] The present invention belongs to the field of new targets and drug technologies for precision cancer medicine, and specifically relates to the use of SNIP1 as a target in the preparation or screening of anti-tumor drugs. Background Art

[0002] Breast cancer is the most common type of cancer, affecting women's lives and health. Triple-negative breast cancer (TNBC) accounts for approximately 15% of all invasive breast cancers. Triple-negative breast cancers (TNBCs) are a specialized type of breast cancer with a higher risk of recurrence, metastasis, and mortality. Therefore, in-depth research into the mechanisms and targets regulating tumor invasion and metastasis is of great clinical significance.

[0003] Smad nuclear interaction protein 1 (SNIP1) is a newly cloned nuclear protein that regulates TGF-β signaling by inhibiting it. It is expressed in various cell types, including the brain, placenta, and kidney. The N-terminal nuclear localization signal (NLS) domain of SNIP1 binds to the C / H1 domains of the transcriptional coactivators CBP and p300, competing for the 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 via the NLS domain. Conversely, SNIP1 enhances C-myc transcriptional activity by stabilizing C-myc, preventing proteasomal degradation, and ligating the C-myc / p300 complex, binding to the N-terminus of C-myc through its C-terminus. Therefore, SNIP1 may be an important endogenous regulator of multiple transcriptional pathways that require the CBP / p300 coactivator.

[0004] In breast cancer, including triple-negative breast cancer (TNBC), the epigenetic landscape is dysregulated, in part due to aberrant patterns of post-translational modifications (PTMs).

[0005] The present study first discovered that the lysine methyltransferase KMT5A (also known as SET8, PR-Set7 / 9, SETD8) can interact with SNIP1, methylating the K301 site of the non-histone protein SNIP1. This promotes the formation of the c-MYC / p300 complex and the transcriptional activation of c-MYC targets, enhancing the key oncogenic pathway, the hippo signaling pathway, and thus promoting TNBC metastasis. Therefore, the SNIP1 protein can be used as a target for screening anti-tumor drugs. Furthermore, the present invention provides a polypeptide that specifically binds to the K301 site of the SNIP1 protein. The polypeptide binds to the K301 site of the SNIP1 protein, inhibiting SNIP1 methylation, ultimately inhibiting tumor growth, proliferation, invasion, and metastasis, and can be used as a new targeted anti-tumor drug. Summary of the Invention

[0006] In response to the above technical problems, the present invention provides an application of SNIP1 as a target in the preparation or screening of anti-tumor drugs, which specifically includes the following contents:

[0007] In a first aspect, the present invention provides the use of human SNIP1 protein as a target in the preparation or screening of anti-tumor drugs, wherein the drug inhibits the methylation of human SNIP1 protein, or the drug inhibits / silences the expression of human SNIP1 protein, or the drug prevents the binding of lysine methyltransferase KMT5A to human SNIP1 protein; the amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO.1.

[0008] Preferably, the application refers to using human SNIP1 protein as the target of action 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.

[0009] Preferably, the tumor is breast cancer.

[0010] Preferably, the breast cancer is triple-negative breast cancer.

[0011] In the second aspect, the present invention provides the use of an isolated human SNIP1 protein gene as a drug or preparation for RNA interference targeting tumor cells in screening tumor therapeutic drugs. The use refers to using the isolated human SNIP1 protein gene as the object of action to screen drugs to find drugs that inhibit methylation at K301 of the protein encoded by the SNIP1 gene as tumor therapeutic drugs.

[0012] Preferably, the tumor is breast cancer.

[0013] Preferably, the breast cancer is triple-negative breast cancer.

[0014] In a third aspect, the present invention provides an anti-tumor polypeptide drug, wherein the polypeptide comprises the following amino acid sequence: HPSCSKQHAVFQY, and the polypeptide drug can inhibit the methylation of the K301 site of the SNIP1 protein in tumor cells.

[0015] Preferably, the tumor is breast cancer.

[0016] Preferably, the breast cancer is triple-negative breast cancer.

[0017] In a fourth aspect, the present invention provides a monoclonal antibody drug for treating tumors, wherein the heavy chain or light chain variable region of the monoclonal antibody comprises an amino acid residue that can specifically bind to the K301 site of SNIP1.

[0018] Preferably, the tumor is breast cancer.

[0019] Preferably, the breast cancer is triple-negative breast cancer.

[0020] In a fifth aspect, the present invention provides a method for screening an anti-triple-negative breast cancer drug or an anti-triple-negative breast cancer metastasis drug using human SNIP1 as a target protein, the method comprising the following steps:

[0021] (1) Determine the domain of human SNIP1 protein that interacts with KMT5A;

[0022] (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;

[0023] (3) Using bioinformatics to dock compounds or peptides in a small molecule compound library or peptide library;

[0024] (4) Activity screening based on the docking results;

[0025] The amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO.1.

[0026] In a fifth aspect, the present invention provides 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, the method comprising:

[0027] (1) Expression and preparation of SNIP1 protein and lysine methyltransferase KMT5A, and labeling of SNIP1 protein with biotin;

[0028] (2) The drug to be evaluated, biotin-labeled SNIP1 protein and 3H-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.

[0029] (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.

[0030] The beneficial effects of the present invention are: ① It was discovered for the first time that lysine methyltransferase KMT5A (also known as SET8, PR-Set7 / 9, SETD8) can interact with SNIP1, causing methylation of the K301 site of the non-histone SNIP1, and promoting the formation of the c-MYC / p300 complex and the transcriptional activation of the c-MYC target, thereby enhancing the key oncogenic pathway hippo signaling pathway, thereby promoting TNBC metastasis. Therefore, the SNIP1 protein can be used as a target for screening anti-tumor drugs; ② The present invention found that by inhibiting the methylation of the K301 site of SNIP1, or inhibiting the interaction between KMT5A and SNIP1, tumor growth, proliferation, invasion and metastasis can be significantly inhibited. Therefore, the K301 site of SNIP1 can be used as a target for anti-tumor drugs. screening; ③ The present invention provides a polypeptide that can bind to the K301 site of SNIP1, thereby inhibiting the methylation of SNIP1, and ultimately inhibiting tumor growth, proliferation, invasion and metastasis, and can be used as an anti-tumor drug; ④ The polypeptide of the present invention has the effect of targeting SNIP1 protein, and can be used as a lead molecule to increase the content of drugs or drug-loaded carriers (such as nanomaterials, liposomes, etc.) in SNIP1-positive cells, and then add pharmaceutically acceptable excipients or adjuvants to prepare new and more effective targeted anti-cancer drugs; ⑤ The present invention provides a method for screening anti-triple-negative breast cancer drugs or anti-triple-negative breast cancer metastasis drugs with human SNIP1 as the target protein, which can realize the in vitro screening of anti-triple-negative breast cancer drugs or anti-triple-negative breast cancer metastasis drugs, and provide new target drugs for the treatment of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SNIP1 methylation plays a pro-oncogenic role;

[0032] Figure 2SNIP1 interacts with KMT5A at Lys301 and is methylated;

[0033] Figure 3 KMT5A-mediated SNIP1 K301 methylation activates the Hippo / YAP signaling pathway;

[0034] Figure 4 KMT5A-mediated SNIP1 K301 methylation activates Hippo / YAP signaling;

[0035] Figure 5 KMT5A-mediated SNIP1 K301 methylation promotes TNBC metastasis by activating YAP signaling;

[0036] Figure 6 Methylation of SNIP1 K301 disrupts its interaction with KAT2A and releases the inhibitory effect of SNIP1 on KAT2AHAT activity;

[0037] Figure 7 KMT5A-mediated SNIP1 K301 methylation promotes c-MYC-dependent recruitment of KAT2A to the MARK4 promoter and activates MARK4 transcriptional activity;

[0038] Figure 8 Reduction of KMT5A catalytic activity combined with inhibition of YAP signaling prevents triple-negative breast cancer progression. DETAILED DESCRIPTION

[0039] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0040] The cell lines MCF10A, T47D, MDA-MB-453, BT474, MCF7, BT549, MDA-MB-231 and HEK-293T used in the present invention were purchased from the China National Cell Line Resource Center (Beijing). The 4T1 cell line was obtained from the American Type Culture Collection (ATCC). All human cell lines were subjected to STR DNA fingerprinting at Shanghai Boyi Applied Biotechnology Co., Ltd. (Shanghai, China) before the start of the experiment. T47D, MDA-MB-453, BT474, MCF7, BT549, MDA-MB-231 and HEK-293T cells were cultured in the presence of 10% fetal bovine serum (Gibco TM, 10100147) and 100 U / mL penicillin-streptomycin. MCF10A cells were cultured 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 the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich).

[0041] The patient specimens of the present invention 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 between January 2010 and December 2015. None of these patients received any chemotherapy or radiotherapy before surgery.

[0042] The methods described in the following examples:

[0043] Chromatin immunoprecipitation (ChIP) Plus Enzymatic Chromatin IP Kit (MagneticBeads, Cell Signaling Technology, 9005S): Cells were cross-linked with 1% formaldehyde in PBS at room temperature for 10 minutes, quenched with 2.5 M glycine at room temperature for 1 minute, and washed three times with PBS. Nuclei were prepared, and chromatin was incubated with micrococcal nuclease for 20 minutes at 37°C, followed by sonication as appropriate. Supernatants were incubated with 3 μg of anti-C-myc, anti-KAT2A, anti-SNIP1, or nonspecific rabbit IgG at 4°C for 12–16 hours before immunoprecipitation. 30 μL of ChIP-Grade Protein G magnetic beads were incubated at 4°C with rotation for 2 hours, followed by three washes with low-salt buffer and one wash with high-salt buffer (5 minutes each). Chromatin was eluted with ChIP elution buffer at 65°C for 30 minutes with gentle vortexing (1200 rpm). Cross-links were reversed with 5 M NaCl and proteinase K at 65°C overnight. Samples were incubated with RNase at 37°C for 1 hour. ChIP DNA was purified and quantified using real-time quantitative PCR (qPCR). Gene expression levels were normalized to β-actin expression levels. Data were analyzed and expressed as a percentage of input DNA.

[0044] ReChIP assays were performed using the ReChIP-it kit (Active Motif). Chromatin precipitated from the first ChIP reaction was eluted with 100 μL of diluted ReChIP-it elution buffer at room temperature for 30 min, and the eluted precipitate was then desalted using the desalting columns provided in the kit. A second ChIP assay was performed using 30 μL of Protein G magnetic beads, 90 μL of desalted chromatin, and 3 μg of secondary antibody. Next, the second precipitate was washed, eluted, and reverse crosslinked to the first ChIP. DNA was obtained by phenol and phenol / chloroform extraction and evaluated by real-time PCR. PCR primer sequences are available upon request.

[0045] Total cells were incubated 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 a complete protease inhibitor cocktail (Roche) at 4°C for 30 minutes. The cells were centrifuged, separated by SDS-PAGE, and analyzed by immunoblotting. To detect SNIP1 methylation in vivo, cells were lysed in IP buffer supplemented with a complete protease inhibitor cocktail, followed by sonication and centrifugation at 12,000 rpm at 4°C for 15 minutes. The supernatant was incubated with SNIP1 antibodies or HA antibodies at 4°C for 12 hours. The immunoprecipitates were washed six times with IP buffer, boiled in 1× SDS-loading buffer, and analyzed by immunoblotting.

[0046] To detect protein interactions, cells were lysed in IP buffer, and the supernatants were immunoprecipitated with the indicated antibodies, shaken slowly on a rotary shaker at 4°C overnight, and then incubated with Pierce TM Protein a / G Magnetic Beads (Invitrogen) were mixed and incubated at room temperature for 1 hour. They were washed five times with wash buffer (25 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.5), eluted, and boiled in 1× SDS loading buffer to prepare samples for immunoblotting analysis. For detection of SNIP1-related proteins, approximately 4×10 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 lysate, including flag-tagged SNIP1, C-myc, or Flag-GFP control protein, were incubated at 4°C for appropriate times with anti-FLAG (Sigma-Aldrich) and Pierce TMImmunoprecipitation was performed using protein A / G Magnetic Beads (Invitrogen). The beads were washed with wash buffer, and bound proteins were eluted with elution buffer (0.1 M glycine, pH 2.0) at room temperature with mixing for 10 minutes. After magnetic separation of the beads, the retained supernatant containing the target antigen was immediately neutralized with 1 M Tris HCl, pH 8.5 neutralization buffer, then boiled, resolved by SDS-PAGE, and silver stained. Bands were excised and subjected to LC-MS / MS sequencing and data analysis.

[0047] HEK-293T cells transfected with 6× his-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, 10 mM imidazole, 1 mM Na2VO4, 20 mM sodium fluoride, 1 mM sodium pyrophosphate, 25 mM β-glycerophosphate, and a 1× complete EDTA-free protease inhibitor cocktail (Roche). The lysate was then sonicated and centrifuged at 21,000 g for 20 minutes at 4°C. 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 washing twice, 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 multiple fractions were collected and dialyzed against PBS. The purified recombinant protein was further tested.

[0048] Recombinant GST-conjugated SET8 and histidine (His)-conjugated SNIP1 were transformed into Escherichia coli BL21 cells using pGEX-4T-1-SET8 and pET-28a(+)-SNIP1, respectively. Five milliliters of bacterial culture were grown overnight at 37°C at 150 rpm in a shake flask and inoculated into 500 ml of Luria-Bertani broth (LB) containing ampicillin or kanamycin. Cells were cultured at 37°C until they reached logarithmic phase (optical density 0.8). Protein expression was induced by the addition of 0.25 mM IPTG, and the cells were shaken continuously at 16°C for 16 hours. After induction, the culture was centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the microspheres were resuspended in 30 ml of lysis buffer (1× PBS, 500 mM NaCl, 1% Triton X-100, 0.5 mg / mL lysozyme, and 1× EDTA-free protease inhibitors) and sonicated on ice. The lysate was centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was incubated with the corresponding volume of 50% glutathione Sepharose 4B bead slurry (GE Healthcare) or Ni-NTA magnetic agarose beads (Qiagen) at 4°C for 8 hours and mixed end-to-end according to the manufacturer's instructions. The glutathione beads were washed three times with lysis buffer and then stored in lysis buffer at 4°C. 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 / time), and the sample was eluted with Tris wash buffer containing 250 mM imidazole.

[0049] GST-SET8 and 6×His-SNIP1 proteins were purified using glutathione beads and Ni-NTA agarose beads, respectively, as described above, according to the manufacturer's procedures. In summary, 1 μg of GST-tagged methyltransferase was incubated with 2.5 μg of His-tagged substrate in 50 μL of reaction buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM PMSF) supplemented with 2 μg of CiS-adenosyl-L-[methyl-3H]methionine (Amersham Biosciences) for 2 h at 30°C. Reactions were resolved by SDS-PAGE with Coomassie staining (Expedeon, InstantBlue) and then exposed to X-ray film for final analysis.

[0050] The KAT2A HAT in vitro assay was performed using the HAT kit from Active Motif.

[0051] 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 assayed for dual luciferase activity 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.

[0052] 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.

[0053] 5×10 4Cells were suspended in culture medium without fetal bovine serum and plated with Matrigel (BD Biosciences) on an upper chamber membrane (8 μm pore size, 5 mm diameter, Corning). The inserts were incubated in 500 μL of culture medium containing 10% fetal bovine serum (Invitrogen) for 16-24 hours. To assess cell invasion, non-invasive cells were removed by gently wiping the top of the membrane with a cotton swab. Migrated or invasive cells were fixed with methanol, stained with crystal violet, and counted under a light microscope.

[0054] Mouse lungs were necropsied, fixed with 4% paraformaldehyde, and embedded in paraffin. Four sections (100 mm each) were obtained from each lung group. The 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) of patient tissues was performed using anti-SET8 (1:500), anti-SNIP1k301me1 (1:500), and anti-MARK4 (1:100) antibodies. Each sample was assigned a score of staining intensity (0 = no staining, 1 = weak staining, 2 = mild staining, and 3 = strong staining) and the proportion of stained cells (0 = 0%, 1 = 1-25%, 2 = 25-50%, 3 = 5-75%, and 4 = 75-100%). Negative control images were obtained without primary antibody. Nuclear staining was scored as negative (0) when <10% of tumor cells showed positive expression. Three people who were blinded to clinical parameters scored the stained tissues.

[0055] Total RNA was extracted and purified using the Qiagen RNeasy Mini Kit (Valencia, CA, USA). RNA quality was assessed using an Agilent 2100 Bioanalyzer before sequencing. Poly(A)+ RNA libraries were prepared according to the Illumina protocol. Libraries were sequenced on the Illumina HiSeqX Ten platform. Fold change was filtered (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 tests and FDR, respectively. Significance was determined using P < 0.05 and FDR < 0.25 as thresholds.

[0056] Pathogen-free female BALB / c and athymic nude mice, 4-5 weeks old, were purchased from Shanghai Slaccas and maintained in a specific pathogen-free (SPF) facility for 1 week before injection to allow the mice to acclimate to the new environment. 5-8 mice were used in each group for independent experiments. MDA-MB-231 cells (1×10 5) or its derivatives were suspended in 100 μL PBS and injected into the tail vein of nude mice; MDA-MB-231 cells (5×10 5 ) or 4T1 cells (5×10 5 ) or its derivatives were suspended in 50 μL of PBS and mixed with streptavidin (1:1). The mixture was then orthotopically injected into the mammary fat pad of mice according to the established protocol (5-8 mice per group). Mice were sacrificed 25 days after tail vein injection and 28 days after mammary fat pad injection. Tumor growth was determined by unblinded peritoneal caliper measurement and tumor volume calculated using the formula (width × length² × π) / 6. Lung metastases were detected by bioluminescence imaging (BLI). All lung lobes were excised, and the number of macroscopic metastatic nodules on the lung surface was counted. For in vivo drug treatment, 7 days after inoculation, tumor-bearing mice received daily intraperitoneal injections of vehicle or verteporfin (50 mg / kg). Verteporfin was dissolved in DMSO (100 mg / mL) and stored at -80°C. Working solutions were prepared in fresh PBS before use. 21 days after injection, fluorescein was injected, and primary and metastatic tumors were detected using BLI using an IVIS 100 (Caliper Life Sciences, Hopkinton, MA, USA). Animal survival was finally analyzed. Separate experimental groups under identical conditions performed corresponding survival analyses. Mice were monitored daily and euthanized after reaching mortality criteria according to the guidelines of the UCSD Institutional Animal Care and Use Committee. Survival data were analyzed using the log-rank test. Data pooled from at least two independent experiments showed similar results. All mouse studies were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences.

[0057] All experiments were repeated three times, with similar results. Statistical analysis: Data are presented as mean ± SD or mean ± SEM. A p-value < 0.05 indicated statistical significance. All P values ​​are from at least three independent experiments, each with three technical replicates. No statistical methods were used to predetermine sample size.

[0058] Example 1 SNIP1 promotes oncogenic function

[0059] 1. SNIP1 is methylated at Lys301

[0060] (1) To identify important non-histone proteins that are regulated in a methylation-dependent manner, a mass spectrometry (MS)-based screening of cell lysates from two TNBC cell lines (i.e., BT549 and MDA-MB-231) was performed using a specific pan-lysine methylation antibody. 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 As shown in 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.

[0061] SNIP1 methylation may promote the growth and metastasis of TNBC both in vitro and in vivo

[0062] 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).

[0063] Together, these findings demonstrate that SNIP1 methylation may promote TNBC growth and metastasis both in vitro and in vivo.

[0064] Example 2 SNIP1 interacts with KMT5A and is methylated by KMT5A at Lys301

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Example 3 KMT5A-mediated SNIP1 methylation activates Hippo / YAP signaling

[0069] 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)

[0070] 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.

[0071] Example 4 KMT5A mediates SNIP1 K301 methylation to activate the YAP signaling pathway and promote TNBC metastasis

[0072] To investigate the role of KMT5A-dependent SNIP1 methylation in the regulation of TNBC metastasis, HA-SNIP1 WT or HA-SNIP1 K301MThe mutant was re-expressed in MDA-MB-231 / sgKMT5A and BT549 / sgKMT5A cells. Further experiments showed that SNIP1 K301M Overexpression (mimicking the methylation status of SNIP1K301, but not SNIP1WT) rescued the loss of MARK4 expression and transcriptional activity, YAP phosphorylation, and its target gene expression induced by KMT5A deficiency. Figure 4 These in vitro findings are consistent with the findings that SNIP K301M Ectopic expression of (instead of SNIP1 WT ) restored KMT5A depletion-mediated inhibition of proliferation and invasion in vitro ( Figure 4 d, f), and in vivo tumor growth and lung metastasis ( Figure 4 e, g), and KMT5A depletion reduced the survival time of animals ( Figure 4 Taken together, these results suggest that KMT5A-dependent SNIP1 methylation promotes TNBC tumorigenesis and lung metastasis by activating MARK4 transcription and subsequent modification of the Hippo / YAP signaling pathway.

[0073] Example 5: Methylation of SNIP1 K301 disrupts its interaction with KAT2A

[0074] Tandem affinity purification combined with mass spectrometry analysis showed that c-MYC interacts with endogenous SNIP1 and KAT2A ( Figure 5 Immunoprecipitation and immunoblotting analysis were performed as in (a). Figure 5 As shown in b and c, SNIP1 further binds to KAT2A and c-MYC, indicating that SNIP1 / KAT2A / c-MYC forms a complex in breast cancer cells. WT In contrast, SNIP1 K301R The mutant was more strongly associated with KAT2A protein but not with c-MYC ( Figure 5 However, SNIP1 K301 methylation significantly inhibited this interaction ( Figure 5 Furthermore, analysis of GST knockdown results showed that His-KAT2A was knocked down by purified GST-SNIP1 but not by GST alone, and GST-SNIP1-K301M significantly weakened this combination ( Figure 5 f), indicating that SNIP1 methylation disrupts the physical interaction between SNIP1 and KAT2. WT SNIP1 K301R or SNIP1 K301MThe HAT activity of KAT2A was determined in the presence of WT It has a strong inhibitory effect on it, recombinant SNIP1 K301R The inhibitory effect on it was weakened, but SNIP1 K301M It can be completely rescued ( Figure 5 In addition, in vitro observations showed that SNIP1 directly binds to KAT2A, indicating that KMT5A-mediated SNIP1 K301 methylation may play a role in regulating the HAT activity of KAT2A. In the presence of BSA and empty vector (EV), KAT2A exhibited dose-dependent HAT activity; this was not the case with SNIP1. WT Abolition when it exists alone ( Figure 5 In contrast, in KMT5A WT KMT5A is not a kinase-inactive mutant. R336G In the presence of , KAT2AHAT activity was largely rescued ( Figure 5 These data suggest that KMT5A-mediated methylation of SNIP1 K301 releases its interaction with KAT2A, ultimately leading to activation of KAT2A enzymatic activity.

[0075] Example 6: SNIP1 K301 methylation promotes c-MYC-dependent recruitment of KAT2A to the MARK4 promoter and activates MARK4 transcriptional activity

[0076] SNIP1 strongly inhibits Smad / p65-dependent transcriptional activity by competing with Smad4 and p65 / RelA for binding to the coactivator p300. Immunoprecipitation of KAT2A and western blotting showed that overexpression of c-MYC interfered with the interaction between KAT2A and SNIP1. Figure 6 In contrast, SNIP1 overexpression also inhibited the KAT2A-c-MYC interaction ( Figure 6 These data suggest that SNIP1 may be a candidate modifier of MARK4 transcriptional activity by competing with c-MYC for KAT2A binding. WT Instead of KMT5A R336G It can methylate SNIP1 K301 and promote the binding of Flag-c-MYC and His-KAT2A; KMT5A WT Significantly impaired the relationship between HA-SNIP1 and His-KAT2A ( Figure 6 In addition, KMT5A was re-expressed in MDA-MB-231 / sgKMT5A and BT549 / sgKMT5A. WT 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.

[0077] Example 7 KMT5A catalytic activity combined with YAP signaling inhibition can prevent triple-negative breast cancer progression

[0078] UNC0379, a selective inhibitor of KMT5A that 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 monotherapy in cell proliferation and tumor growth Figure 7 a-c). Furthermore, we also determined whether the two small molecule inhibitors synergize in cell invasion in vitro and lung metastasis in vivo. Compared to monotherapy, 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.

[0079] Example 8 KMT5A expression is significantly associated with activated Hippo / YAP signaling in triple-negative breast cancer

[0080] 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 8These data confirm that KMT5A-mediated SNIP1 methylation is significantly associated with activated MARK4 kinase cascade signaling in TNBC.

[0081] Example 9 Screening Method for Anti-Triple Negative Breast Cancer Drugs

[0082] Method 1: Molecular docking

[0083] (1) Determine the domain of human SNIP1 protein that interacts with KMT5A;

[0084] (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;

[0085] (3) Using bioinformatics to dock compounds or peptides in a small molecule compound library or peptide library;

[0086] (4) Activity screening based on the docking results;

[0087] The amino acid sequence of the human SNIP1 protein is shown in SEQ ID NO.1.

[0088] Method 2:

[0089] (1) Expression and preparation of SNIP1 protein and lysine methyltransferase KMT5A, and labeling of SNIP1 protein with biotin;

[0090] (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.

[0091] (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.

[0092] Example 10 Polypeptide drug against triple-negative breast cancer

[0093] Solid phase synthesis of a polypeptide with the amino acid sequence HPSCSKQHAVFQY;

[0094] 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, inhibit the growth, proliferation, invasion and metastasis of triple-negative breast cancer, and can be used as an anti-tumor drug.

[0095] This study conducted a detailed investigation of the SNIP1 protein and found that SNIP1 is a non-histone substrate of the lysine methyltransferase KMT5A. It promotes breast cancer cell growth, invasion, and lung metastasis through KMT5A-mediated monomethylation of K301. Therefore, SNIP1 protein can be used as a target for anti-tumor drug screening. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. An anti-tumor polypeptide drug, characterized in that: The amino acid sequence of the polypeptide is: HPSCSKQHAVFQY, and the polypeptide drug can inhibit the methylation of the K301 site of the SNIP1 protein in tumor cells; the tumor is triple-negative breast cancer.

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