Application of CCNB1 phosphorylation inhibition tool in preparation of medicine for preventing and / or treating triple negative breast cancer

Abstract

The invention discloses application of a CCNB1 phosphorylation inhibition tool in preparation of a medicine for preventing and / or treating triple negative breast cancer, and particularly relates to application of a CCNB1 protein S128 site phosphorylation inhibitor in preparation of a medicine for preventing and / or treating triple negative breast cancer. The phosphorylation inhibitor refers to a tool or means used in the process of blocking or weakening protein phosphorylation, particularly a gene editing system, and the 128 serine Ser coded by a CCNB1 gene is mutated into alanine Ala which is recorded as S128A in a target cell. Compared with a traditional small molecule drug, the gene editing tool accurately intervenes phosphorylation modification at the gene level, has the advantages of being clear in mechanism, high in targeting performance, high in safety and the like, is suitable for basic research and potential clinical transformation of TNBC targeted therapy, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of CCNB1 phosphorylation inhibitors in the preparation of medicaments for the prevention and / or treatment of triple-negative breast cancer. Background Technology

[0002] Breast cancer is currently the most common malignant tumor worldwide. Triple-negative breast cancer (TNBC) accounts for approximately 10.0% to 20.8% of all breast cancer pathological types. It is a bioheterogeneous disease characterized by aggressive clinical presentation and poor prognosis. Although novel treatments such as PARP inhibitors, immune checkpoint inhibitors, and antibody-drug conjugates (ADCs) are bringing breakthroughs in the treatment of advanced TNBC, chemotherapy remains the core of systemic therapy. It is noteworthy that the efficacy of chemotherapy is still not entirely satisfactory, and current treatment strategies are limited by strict indication criteria, highlighting the urgent need to explore new therapeutic targets for TNBC.

[0003] MAPK1 (also known as ERK2) is a key effector kinase in the MEK / ERK signaling pathway. Its activation is regulated by multiple upstream signals and is closely related to key physiological processes such as cell proliferation, migration, and survival. Studies have shown that MAPK1 plays a crucial role in tumor invasion and metastasis in various cancers, including gastric cancer, bladder cancer, pancreatic cancer, cervical cancer, and breast cancer. Traditionally, it is believed that MAPK1 primarily regulates gene expression and coordinates cellular responses through nuclear translocation, followed by phosphorylation of multiple transcription factors. However, this indirect regulatory mechanism cannot fully explain the rapid proliferation effect induced by MAPK1 in different tumor environments. It remains unclear whether MAPK1 can directly phosphorylate functional effector proteins, thereby mediating more immediate and specific biological responses.

[0004] CCNB1 (Cyclin B1, G2 / Mitotic-Specific Cyclin-B1) is a key regulator of the G2 / M phase transition in the cell cycle, and its high expression in TNBC has been reported and is associated with poor prognosis. However, current research on CCNB1 mainly focuses on its transcriptional regulation at the expression level, while other functions remain unclear. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing the use of a .CCNB1 phosphorylation inhibitor tool in the preparation of medicaments for the prevention and / or treatment of triple-negative breast cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of a .CCNB1 phosphorylation inhibitor tool in the preparation of medicaments for the prevention and / or treatment of triple-negative breast cancer.

[0008] Furthermore, the phosphorylation inhibition tool functions through at least one of the following mechanisms:

[0009] (1) Inhibit MAPK1-mediated phosphorylation modification of CCNB1 protein at site S128;

[0010] (2) Reduces the kinase activity of MAPK1 protein on CCNB1 protein.

[0011] Furthermore, the phosphorylation inhibition tool is selected from small molecule inhibitors, antibodies, peptides, or gene editing systems.

[0012] Furthermore, the phosphorylation inhibition tool is a gene editing system used to mutate serine (Ser) at position 128 of the CCNB1 gene to alanine (Ala) in target cells.

[0013] Secondly, the present invention provides a CCNB1 protein mutant, wherein the CCNB1 protein mutant mutates the 128th serine (Ser) of the wild-type CCNB1 protein to alanine (Ala), denoted as S128A.

[0014] Thirdly, the present invention provides the use of the above-mentioned CCNB1 protein mutant in the preparation of a drug for treating triple-negative breast cancer.

[0015] Fourthly, the present invention provides a pharmaceutical composition comprising the above-mentioned CCNB1 protein mutant.

[0016] Fifthly, the present invention provides a method for screening candidate drugs for the prevention or treatment of triple-negative breast cancer, comprising:

[0017] The candidate compound was brought into contact with a reaction system containing the MAPK1 kinase domain and the S128 site of the CCNB1 protein, and the phosphorylation level of the CCNB1 protein S128 site was detected. If the candidate compound could significantly reduce the phosphorylation level of the CCNB1 protein S128 site, the compound was determined to be a candidate drug for the treatment of triple-negative breast cancer.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention constructs an S128A mutant to simulate the non-phosphorylated state of the CCNB1 protein, clarifying the key role of inhibiting phosphorylation at the S128 site of the CCNB1 protein in the prevention and / or treatment of malignant progression of TNBC. This target is novel and specific.

[0020] This invention provides a gene-editing tool that targets and regulates phosphorylation modification at the S128 site of the CCNB1 protein, and applies it to the treatment of TNBC. After gene editing intervention, CCNB1 loses its function of binding to CDK1 to form a complex, thereby significantly inhibiting the proliferation of TNBC cells and achieving the goal of tumor suppression. The gene-editing tool of this invention also showed significant anti-tumor effects in a mouse model. Compared with traditional small molecule drugs, the gene-editing tool of this invention precisely intervenes in phosphorylation modification at the gene level, and has advantages such as a clear mechanism, strong targeting, and high safety. It is suitable for basic research and potential clinical translation of TNBC targeted therapy, and has good application prospects.

[0021] In summary, this invention not only deepens the understanding of metabolic heterogeneity in TNBC, but also provides a novel solution to overcome the current challenges of targeted therapy, demonstrating significant scientific value and promising prospects for clinical translation. Attached Figure Description

[0022] Figure 1CCNB1 was identified as a MAPK1 substrate regulating tumor growth using TurboID-based proximity labeling. (A) Schematic diagram of the experimental procedure. MAPK1 was fused with mini-TurboID and expressed via the CMV promoter in MDA-MB-231 and MDA-MB-468 cells. After biotin supplementation, neighboring proteins were biotinylated, captured by streptavidin magnetic beads, and identified by mass spectrometry (MS). (B) SDS-polyacrylamide gel electrophoresis analysis showed biotinylated proteins enriched by streptavidin magnetic beads in MDA-MB-231 and MDA-MB-468 cells. The red box indicates the significantly enriched region. (C) Venn diagram showing the MAPK1 interacting proteins identified in MDA-MB-231 and MDA-MB-468 cells. (D) Protein-protein interaction (PPI) analysis of these 59 overlapping proteins revealed a functional interaction network in which CCNB1 serves as a potential core regulatory protein. (E) Gene set enrichment analysis (GSEA) of breast cancer samples stratified by MAPK1 expression level. GSEA analysis of differentially expressed genes between the MAPK1 high-expression and low-expression groups revealed significant enrichment of cell cycle-related pathways. (F) Kaplan-Meier survival analysis showing CCNB1 expression in breast cancer patients. (G) Box plot comparing CCNB1 expression levels in invasive breast cancer (BRCA) tissues and adjacent normal tissues. Data sourced from TCGA. Figures (H–I) show the growth curves of MDA-MB-231 and MDA-MB-468 cells transduced with sh-NC, sh1-CCNB1, or sh2-CCNB1. (J–K) Representative images and quantitative analysis of colony formation experiments of MDA-MB-231 and MDA-MB-468 cells after CCNB1 gene knockdown. (LM) EdU incorporation assay and quantitative analysis showed reduced DNA synthesis after CCNB1 silencing. Red: EdU-positive cells; Blue: DAPI. (N–O) flow cytometry analysis and cell cycle distribution quantification showed that CCNB1-deficient MDA-MB-231 and MDA-MB-468 cells exhibited G2 / M phase arrest.

[0023] Figure 2Functional significance of CCNB1 protein S128 phosphorylation. (A–B) CCK-8 cell proliferation assays: proliferation of MDA-MB-231(A) and MDA-MB-468(B) cells expressing different CCNB1 variants (wild-type, knockdown, S128A mutant, or S128D mutant). (C–D) Colony formation assays of MDA-MB-231(C) and MDA-MB-468(D) cells with specified CCNB1 variants. (E–F) Quantitative analysis of colony numbers in (C) and (D). (G–I) In vivo xenografting assays of MDA-MB-231 cells: representative tumor images (G), tumor weight (H), and growth curves (I). (J–K) Ki67 immunohistochemical staining results (J) and quantitative analysis (K) of xenograft tumors.

[0024] Figure 3 Rescue experiments revealed that MAPK1 promotes tumor growth by phosphorylating CCNB1 at the S128 site. (A–B) CCK-8 assays in MDA-MB-231(A) and MDA-MB-468(B) cells showed that overexpression of phosphorylated mimic CCNB1-S128D rescued MAPK1 knockdown-induced proliferation defects. (C–D) Colony formation assays in MDA-MB-231(C) and MDA-MB-468(D) cells showed different treatment effects. (E–F) Quantitative analysis of colony numbers corresponding to (C) and (D). (G–I) In vivo xenograft experiments using MDA-MB-231 cells: representative tumor images (G), tumor weight (H), and tumor growth curves (I). (J–K) Ki67 immunohistochemical staining (J) and quantitative analysis (K) of xenograft tumors. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the technical field to which this application pertains. The following definitions are supplementary to those definitions in the art and relate to this application, but are not extrapolated to any relevant or unrelated situation, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing of this application, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0027] As previously stated, this invention provides the use of a phosphorylation inhibition tool at the S128 site of the CCNB1 protein in the preparation of medicaments for the prevention and / or treatment of triple-negative breast cancer. The phosphorylation inhibition tool is capable of inhibiting MAPK1-mediated phosphorylation modification at the S128 site of the CCNB1 protein or reducing the kinase activity of the MAPK1 protein on the CCNB1 protein.

[0028] In some implementations, the phosphorylation inhibition tool refers to a tool or means used to block or reduce protein phosphorylation, such as small molecule inhibitors, antibodies, peptides, nucleic acid interfering agents, or gene editing systems.

[0029] In some embodiments, the phosphorylation inhibition tool is a gene editing system, specifically a cell line or animal model expressing a non-phosphorylated mutant (S128A) is constructed using a gene editing system to regulate CCNB1 function. As demonstrated in the examples below, the phosphorylation inhibition tool of the present invention can significantly inhibit the proliferation of TNBC cells and significantly slow tumor progression in xenograft tumor models.

[0030] The following describes preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0031] Example 1: Investigating potential downstream targets of MAPK1 in regulating TNBC

[0032] Proteins that interact with or are adjacent to MAPK1 were identified using TurboID-based proximity labeling-based mass spectrometry. Figure 1 A and Figure 1 (B in the original text). Ultimately, a total of 59 proteins interacting with MAPK1 were identified in MDA-MB-231 and MDA-MB-468 cells. Figure 1 (C in the original text). Subsequent protein-protein interaction analysis of these 59 overlapping proteins revealed that CCNB1 may be a core regulatory protein (C in the original text). Figure 1 To further explore the potential mechanisms by which MAPK1 regulates TNBC growth, we performed gene set enrichment analysis (GSEA) using RNA sequencing data from TCGA. The results showed that differentially expressed genes associated with MAPK1 were mainly enriched in cell cycle-related pathways (D). Figure 1 (E in the text).

[0033] Subsequently, the role of CCNB1 in the progression of transurethral breast cancer (TNBC) was explored in depth. TCGA data revealed that CCNB1 mRNA expression levels were significantly elevated in TNBC patients compared to adjacent normal tissues. Survival analysis further showed a significant correlation between upregulated CCNB1 expression and shortened overall survival (p=0.026). Figure 1 F and Figure 1 (G in the text). A series of phenotypic evaluations were performed on the CCNB1 knockdown MDA-MB-231 and MDA-MB-468 cell lines. Cell proliferation assays (including flow cytometry and colony formation assays) showed that the cell proliferation rate of CCNB1 knockdown was significantly slowed down (G in the text). Figure 1 HL in. EdU labeling experiments showed that DNA synthesis levels decreased after CCNB1 silencing ( Figure 1 M and Figure 1 The O in the middle). Flow cytometry analysis and cell cycle distribution quantification showed that both MDA-MB-231 and MDA-MB-468 cells with knockdown of CCNB1 exhibited G2 / M phase arrest. Figure 1 N and Figure 1 (P in the middle).

[0034] Example 1: Construction of stable cell lines expressing CCNB1 (WT / S128A / S128D) with exogenous plasmids in MDA-MB-231 and MDA-MB-468 cell lines and detection of cell proliferation capacity.

[0035] The protein and primer sequences involved in this invention are as follows:

[0036] amino acid sequence

[0037] WT (SEQ ID NO.1):

[0038] MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPVKEEKLSPEPILVDTASP SPMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKV

[0039] S128A(SEQ ID NO.2):

[0040] MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTASP A PMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKV

[0041] S128D(SEQ ID NO.3):

[0042] MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTASP D PMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKV

[0043] Nucleotide sequence

[0044] WT (SEQ ID NO.4):

[0045] atggcgctccgagtcaccaggaactcgaaaattaatgctgaaaataaggcgaagatcaacatggcaggcgcaaagcgcgttcctacggcccctgctgcaacctccaagcccggactgaggccaagaacagctcttggggacattggtaacaaagtcagtgaacaactgcaggccaaaatgcctatgaagaaggaagcaaaaccttcagctactggaaaagtcattgataaaaaactaccaaaacctcttgaaaaggtacctatgctggtgccagtgccagtgtctgagccagtgccagagccagaacctgagccagaacctgagcctgttaaagaagaaaaactttcgcctgagcctattttggttgatactgcctctcca agcccaatggaaacatctggatgtgcccctgcagaagaagacctgtgtcaggctttctctgatgtaattcttgcagtaaatgatgtggatgcagaagatggagctgatccaaacctttgtagtgaatatgtgaaagatatttatgcttatctgagacaacttgaggaagagcaagcagtcagaccaaaaatacctactgggtcgggaagtcactggaaacatgagagccatcctaattgactggctagtacaggttcaaatgaaattcaggttgttgcaggagaccatgtacatgactgtctccattattgatcggttcatgcagaataattgtgtgcccaagaagatgctgcagctggttggtgtcactgccatgtttattgcaagcaaatatgaagaaatgtaccctccagaaattggtgactttgcttttgtgactgacaacacttatactaagcaccaa atcagacagatggaaatgaagattctaagagctttaaactttggtctgggtcggcctctacctttgcacttccttcggagagcatctaagattggagaggttgatgtcgagcaa catactttggccaaatacctgatggaactaactatgttggactatgacatggtgcactttcctccttctcaaattgcagcaggagctttttgcttagcactgaaaattctggata atggtgaatggacaccaactctacaacattacctgtcatatactgaagaatctcttcttccagttatgcagcacctggctaaaatgtagtcatggtaaatcaaggacttacaaagcacatgactgtcaagaacaagtatgccacatcgaagcatgctaagatcagcactctaccacagctgaattctgcactagttcaagatttagccaaggctgtggcaaaggtgtaa

[0046] S128A(SEQ ID NO.5):

[0047] atggcgctccgagtcaccaggaactcgaaaattaatgctgaaaataaggcgaagatcaacatggcaggcgcaaagcgcgttcctacggcccctgctgcaacctccaagcccggactgaggccaagaacagctcttggggacattggtaacaaagtcagtgaacaactgcaggccaaaatgcctatgaagaaggaagcaaaaccttcagctactggaaaagtcattgataaaaaactaccaaaacctcttgaaaaggtacctatgctggtgccagtgccagtgtctgagccagtgccagagccagaacctgagccagaacctgagcctgttaaagaagaaaaactttcgcctgagcctattttggttgatactgcctctcca GCAccaatggaaacatctggatgtgcccctgcagaagaagacctgtgtcaggctttctctgatgtaattcttgcagtaaatgatgtggatgcagaagatggagctgatccaaacctttgtagtgaatatgtgaaagatatttatgcttatctgagacaacttgaggaagagcaagcagtcagaccaaaaatacctactgggtcgggaagtcactggaaacatgagagccatcctaattgactggctagtacaggttcaaatgaaattcaggttgttgcaggagaccatgtacatgactgtctccattattgatcggttcatgcagaataattgtgtgcccaagaagatgctgcagctggttggtgtcactgccatgtttattgcaagcaaatatgaagaaatgtaccctccagaaattggtgactttgcttttgtgactgacaacacttatactaagcaccaa atcagacagatggaaatgaagattctaagagctttaaactttggtctgggtcggcctctacctttgcacttccttcggagagcatctaagattggagaggttgatgtcgagcaa catactttggccaaatacctgatggaactaactatgttggactatgacatggtgcactttcctccttctcaaattgcagcaggagctttttgcttagcactgaaaattctggata atggtgaatggacaccaactctacaacattacctgtcatatactgaagaatctcttcttccagttatgcagcacctggctaaaatgtagtcatggtaaatcaaggacttacaaagcacatgactgtcaagaacaagtatgccacatcgaagcatgctaagatcagcactctaccacagctgaattctgcactagttcaagatttagccaaggctgtggcaaaggtgtaa

[0048] S128D(SEQ ID NO.6):

[0049] atggcgctccgagtcaccaggaactcgaaaattaatgctgaaaataaggcgaagatcaacatggcaggcgcaaagcgcgttcctacggcccctgctgcaacctccaagcccggactgaggccaagaacagctcttggggacattggtaacaaagtcagtgaacaactgcaggccaaaatgcctatgaagaaggaagcaaaaccttcagctactggaaaagtcattgataaaaaactaccaaaacctcttgaaaaggtacctatgctggtgccagtgccagtgtctgagccagtgccagagccagaacctgagccagaacctgagcctgttaaagaagaaaaactttcgcctgagcctattttggttgatactgcctctcca GATccaatggaaacatctggatgtgcccctgcagaagaagacctgtgtcaggctttctctgatgtaattcttgcagtaaatgatgtggatgcagaagatggagctgatccaaacctttgtagtgaatatgtgaaagatatttatgcttatctgagacaacttgaggaagagcaagcagtcagaccaaaatacctactgggtcgggaagtcactggaaacatgagagccatcctaattgactggctagtacaggttcaaatgaaattcaggttgttgcaggagaccatgtacatgactgtctccattattgatcggttcatgcagaataattgtgtgcccaagaagatgctgcagctggttggtgtcactgccatgtttattgcaagcaaatatgaagaaatgtaccctccagaaattggtgactttgcttttgtgactgacaacacttatactaagcaccaaatcagacagatggaaatgaagattctaagagctttaaactttggtctgggtcggcctctacctttgcacttccttcggagagcatctaagattggagaggttgatgtcgagcaacatactttggccaaatacctgatggaactaactatgttggactatgacatggtgcactttcctccttctcaaattgcagcaggagctttttgcttagcactgaaaattctggataatggtgaatggacaccaactctacaacattacctgtcatatactgaagaatctcttcttccagttatgcagcacctggctaagaatgtagtcatggtaaatcaaggacttacaaagcacatgactgtcaagaacaagtatgccacatcgaagcatgctaagatcagcactctaccacagctgaattctgcactagttcaagatttagccaaggctgtggcaaaggtgtaa

[0050] Mutant primer

[0051]

[0052] Mutants mimicking different CCNB1 (S128) phosphorylation states were expressed in the MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cell lines, and cell cycle and proliferative behavior were analyzed in the cell models. The specific steps are as follows:

[0053] Cell Culture: Frozen MDA-MB-231, MDA-MB-468, and HEK293T cells were removed from liquid nitrogen and immediately placed in a 37°C water bath. Gently agitate to thaw rapidly. After thawing, transfer the cell suspension to centrifuge tubes containing culture medium, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in complete DMEM medium, transfer to culture dishes, mix well, and incubate at 37°C in a 5% CO2 cell culture incubator. When the cells reach 80% confluence, passage them by digesting with 0.25% trypsin for 2 min, transferring the cell suspension to centrifuge tubes, centrifuging at 1000 rpm for 3 min, discarding the supernatant, resuspending the cells in complete DMEM medium, transfer to culture dishes, and passage at a 1:4 ratio. Cells are passaged twice before use.

[0054] Lentiviral packaging and transfection of MDA-MB-231 and MDA-MB-468 cells: Healthy HEK293T cells were seeded into 100 mm dishes and transfected when cell coverage reached 90%. Lentiviral packaging plasmids pMD2G and psPAX2, along with the target vector, were added in a 1:3:4 ratio (11 μg DNA total) to 1 mL of Opti-MEM medium. After mixing, 22 μL of transfection reagent was added and gently pipetted to mix. After standing at room temperature for 15 min, the mixture was added dropwise to HEK293T culture dishes to a final volume of 10 mL. After 6 h of culture, the medium was replaced with 10 mL of DMEM complete medium. After 24 h, 4 mL of DMEM complete medium was added again. At 48 h of culture, the supernatant was collected, filtered through a 0.45 µm filter to remove impurities, and the lentiviral solution was stored at -80 °C. MDA-MB-231 and MDA-MB-468 cells were seeded into six-well plates to achieve a coverage of approximately 30%. The obtained lentivirus solution was added, and after 48 hours of incubation, the medium was replaced with normal medium. After another 48 hours of infection, medium containing 2 μg / mL Puromycin was added, and selection was performed continuously for 15 days. Cells without viral infection were used as controls. The entire drug screening process was completed when all control cells died during drug screening, and the surviving cells were identified as stable cell lines.

[0055] The CCK8 cell viability assay was used to detect cell proliferation: Cells in good growth condition were digested with trypsin, and the cell suspension was collected. After cell counting, the cell suspension was diluted to 5000 cells per 100 μL and seeded into 96-well plates with 6 replicates per group. After gentle mixing, the plates were incubated in a cell culture incubator. After 12, 24, 36, 72, and 96 hours of culture, 10 μL of CCK8 was added to each well, and the plates were incubated for another 3 hours at 37°C with 5% CO2. The absorbance was read at 450 nm using a microplate reader, and the proliferation capacity of each group was calculated. Each experiment was independently repeated three times. The relative cell proliferation rate was calculated as follows: Using the OD value of the control group transfected with the empty vector as a baseline (set as 100% proliferation rate), the relative proliferation percentage of each experimental group was calculated using the following formula: Relative proliferation rate (%) = (Experimental group OD value / Control group OD value) × 100%. Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used to test the significance of differences between groups, with p < 0.05 considered statistically significant. The final results were used to generate line plots using Graphpad Prism 9 software. Error bars represent the range of variation, and significant differences are marked. The results are shown below. Figure 2 As shown in AF.

[0056] In summary, the experimental results show that restoring CCNB1 to the phosphorylated state (S128D) further enhanced cell proliferation, while restoring CCNB1 to the dephosphorylated state (S128A) resulted in a decrease in cell proliferation compared to the NC group. This indicates that phosphorylation modification of CCNB1 can enhance the malignant behavior of triple-negative breast cancer cells.

[0057] Example 2: Validating the effect of CCNB1(S128) phosphorylation on TNBC tumor progression using a nude mouse xenograft tumor model.

[0058] CDX mouse model construction: Six-week-old female Balb / c nude athymic nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Before the experiment, the mice were acclimatized for one week in the SPF-grade barrier environment of the experimental animal platform. The rearing conditions were: room temperature 26℃, humidity 60%; 12-hour light / dark cycle; and free access to water and food. Sufficient MDA-MB-231 cells were cultured. Cells in the logarithmic growth phase were digested with trypsin, resuspended in pre-chilled PBS, and the cell suspension density was adjusted to 8 × 10⁻⁶. 7 Cells / mL. 12.5 μL (1 × 10⁻⁶ cells / mL) was injected into the fat pad of the fourth mammary gland in mice. 6The mice were fed individual tumor cells and their growth, activity, and tumor formation were closely monitored. When the tumor was visible to the naked eye, the mice's weight and tumor volume were measured every two days. All experimental procedures were performed in accordance with animal welfare and ethical review requirements. The animal experimental procedures received ethical approval under SYSU-IACUC-2025-000042.

[0059] Evaluation of the effect of CCNB1(S128) phosphorylation modification on tumor growth in vivo: Six-week-old female Balb / c nude athymic nude mice were randomly divided into groups of six. Three types of stable MDA-MB-231 cells expressing CCNB1 (shNC), CCNB1(S128A), and CCNB1(S128D) were injected into the fat pad of the fourth mammary gland of each group. All groups were seeded with 1×10⁻⁶ cells. 6 Each cell.

[0060] Tumor growth was observed, and nude mice were weighed every two days to measure and record the longest and shortest diameters of the tumors. On day 20 after cell inoculation, mice were humanely sacrificed, and the tumors were dissected, photographed, and measured. The volume of tumors in all mice did not exceed 2000 mm². 3 Tumor volume (mm) 3 = Major axis (mm) × Minor axis (mm) 2 ×0.52.

[0061] Actual images of tumors in mice at the end of 20 days are shown below. Figure 2 As shown in Figure G. In the analysis of tumor volume changes in each group of mice, to clarify the final therapeutic effect of the intervention, statistical comparisons were only performed on the tumor volume data at the experimental endpoint (i.e., the last measurement). The tumor volume data at this time point are expressed as mean ± standard error, and one-way ANOVA was used to test for overall inter-group differences. A p < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. The final results were generated using Graphpad Prism 9 software. Error bars in the figure represent the range of variation, and significant differences are marked. The results of the tumor volume change curves in each group of mice are shown in Figure G. Figure 2 As shown in Figure I, the tumor growth rate in mice inoculated with complemented CCNB1 (S128D) cells is accelerated, while the tumor growth rate in mice inoculated with complemented CCNB1 (S128A) cells is significantly reduced. This indicates that gene editing tools that mimic the non-phosphorylated state of the CCNB1 protein using the S128A mutant can significantly slow tumor progression in xenograft tumor models.

[0062] Subsequently, a rescue experiment was conducted on phosphorylated mimic CCNB1-S128D to investigate whether MAPK1 promotes tumor growth by phosphorylating CCNB1 at the S128 site.

[0063] CCK-8 and colony formation assays showed that MAPK1 knockdown significantly inhibited the proliferation and colony formation abilities of MDA-MB-231 and MDA-MB-468 cells. However, overexpression of phosphorylated mimic CCNB1-S128D effectively rescued the proliferation and colony formation defects caused by MAPK1 knockdown. Figure 3 The in vivo xenograft experiment using MDA-MB-231 cells further confirmed that phosphorylated mimic CCNB1-S128D could reverse the inhibitory effect of MAPK1 knockdown on tumor growth (AF). Figure 3 In addition, Ki67 immunohistochemical staining showed that overexpression of CCNB1-S128D could effectively eliminate the inhibitory effect of MAPK1 knockdown on tumor proliferation (GI). Figure 3 (JK in the data). These data indicate that expression of phosphorylated mimic CCNB1-S128D is sufficient to rescue the inhibitory effect of MAPK1 knockdown on tumor cell proliferation and tumor growth.

[0064] The above description of the embodiments is only for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. Use of CCNB1 phosphorylation inhibitors in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer.

2. The use according to claim 1, characterized in that, The phosphorylation inhibition tool functions through at least one of the following mechanisms: (1) Inhibit MAPK1-mediated phosphorylation modification of CCNB1 protein at site S128; (2) Reduces the kinase activity of MAPK1 protein on CCNB1 protein.

3. The use according to claim 1, characterized in that, The phosphorylation inhibition tool is selected from: small molecule inhibitors, antibodies, peptides, or gene editing systems.

4. The use according to claim 3, characterized in that, The phosphorylation inhibition tool is a gene editing system used to mutate serine Ser to alanine Ala at position 128 of the CCNB1 gene in target cells.

5. A CCNB1 protein mutant, characterized in that, The CCNB1 protein mutant mutates the 128th serine (Ser) of the wild-type CCNB1 protein to alanine (Ala), denoted as S128A.

6. Use of the CCNB1 protein mutant as described in claim 5 in the preparation of a medicament for treating triple-negative breast cancer.

7. A pharmaceutical composition, characterized in that, It includes the CCNB1 protein mutant as described in claim 5.

8. A method for screening candidate drugs for the prevention or treatment of triple-negative breast cancer, characterized in that, The method is specifically as follows: The candidate compound was brought into contact with a reaction system containing the MAPK1 kinase domain and the S128 site of the CCNB1 protein, and the phosphorylation level of the CCNB1 protein S128 site was detected. If the candidate compound could significantly reduce the phosphorylation level of the CCNB1 protein S128 site, the compound was determined to be a candidate drug for the treatment of triple-negative breast cancer.

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