Application of substance for inhibiting USP8 gene expression in preparation of medicine for treating triple negative breast cancer

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to application of a substance for inhibiting USP8 gene expression in preparation of a medicine for treating triple negative breast cancer. The triple-negative breast cancer is limited in clinical treatment means and mainly depends on chemotherapy, but drug resistance is easily generated, and prognosis is poor. Bioinformatics analysis finds that the USP8 gene is remarkably and highly expressed in the triple negative breast cancer, and finds that the development of the triple negative breast cancer can be effectively intervened by inhibiting the expression of the USP8 gene in cells and in-vivo tumors, so that a new way is opened up for the treatment of the triple negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of new uses of drugs, and more specifically, relates to the use of substances that inhibit the expression of USP8 gene in the preparation of drugs for treating triple-negative breast cancer. Background Art

[0002] Breast cancer is the most common female malignant tumor globally, and triple-negative breast cancer is the most aggressive subtype among them. Compared with luminal or HER2-type breast cancers with unique targeted therapeutic molecular markers, triple-negative breast cancer is a type of breast cancer lacking effective therapeutic targets. Recent genome-wide association and biological studies have found that the Hippo signal is often abnormal in triple-negative breast cancer samples. Manipulating the Hippo pathway can effectively change the biological behavior of triple-negative breast cancer, making it a promising target for the treatment of triple-negative breast cancer.

[0003] The biological connection between triple-negative breast cancer and the Hippo pathway has been established for decades. For example, YAP expression is elevated in triple-negative breast cancer and is associated with poor overall survival. As an important co-activator of the Hippo pathway, YAP can also cooperate with the AP-1 family member Fra-1 to initiate the oncogenic transcriptional program in triple-negative breast cancer cells, and YAP deletion can hinder the oncogenic process in MMTV-PyMT model mice. Although the inhibitory kinase cascade continues to function, the cause of YAP overactivation remains uncertain. Recent studies have shown that ubiquitination modification may regulate the activity and stability of YAP in the tumorigenesis of triple-negative breast cancer. The results suggest that inhibiting deubiquitination (USP) genes may become a treatment strategy for triple-negative breast cancer. However, there are many genes in the USP gene family, and it is necessary to further study which specific USP genes are related to the development of triple-negative breast cancer. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of substances that inhibit the expression of USP8 gene in the preparation of drugs for treating triple-negative breast cancer to solve the above technical problems.

[0005] The present invention provides the use of substances that inhibit the expression of USP8 gene in the preparation of drugs for treating triple-negative breast cancer.

[0006] Through bioinformatics analysis, the present invention found that the USP8 gene is significantly highly expressed in triple-negative breast cancer. Therefore, on the one hand, the present invention proved that inhibiting the USP8 gene can slow down the development of triple-negative breast cancer by silencing the expression of the USP8 gene, and on the other hand, verified that inhibiting the expression of the USP8 gene in cells and in vivo tumors can improve the development of triple-negative breast cancer through the chemical drug action of the USP8 gene target inhibitor, which provides a new idea for the treatment of triple-negative breast cancer.

[0007] Furthermore, the drug uses a small molecule compound or siRNA that targets and inhibits the USP8 gene as the active ingredient.

[0008] Furthermore, the small molecule compound is DUB-IN-2.

[0009] Furthermore, the siRNA is selected from one of siUSP8#1 and siUSP8#2. The sense strand sequence of siUSP8#1 is shown as SEQ ID NO.1, the antisense strand sequence is shown as SEQ ID NO.2, the sense strand sequence of siUSP8#2 is shown as SEQ ID NO.3, and the antisense strand sequence is shown as SEQ ID NO.4.

[0010] Furthermore, when the drug uses a small molecule compound that targets and inhibits the USP8 gene as the active ingredient, the active ingredient further includes paclitaxel.

[0011] Furthermore, the molar ratio of the active ingredients DUB-IN-2 and paclitaxel is 98 - 102:0.5 - 1.5.

[0012] Furthermore, the drug further includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include any one or more of non-toxic fillers, stabilizers, diluents, and adjuvants.

[0014] Beneficial effects:

[0015] The present invention first reveals the key role of the USP8 gene in triple-negative breast cancer. Through a combination of bioinformatics analysis and experimental verification, it is clarified that silencing USP8 can effectively improve the treatment of triple-negative breast cancer, providing theoretical support for targeted therapy and a new idea for the treatment of triple-negative breast cancer. Description of the drawings

[0016] Figure 1 It is a schematic diagram of the expression and prognostic correlation of USP8 in triple-negative breast cancer. Among them, A is a Kaplan-Meier survival analysis chart, B is a positive correlation analysis chart between USP8 and the YAP gene in TCGA data, and C is a gene enrichment analysis chart of siUSP8.

[0017] Figure 2 It is a result chart of the change of YAP target genes after knocking down USP8. Among them, A is a volcano chart of gene expression after knocking down USP8, and B is a heat map of gene expression after knocking down USP8.

[0018] Figure 3Verification diagrams for knocking down the USP8 gene. Among them, A is the Western blot detection diagram after knocking down the USP8 gene in BT549 cells, B is the Western blot detection diagram after knocking down the USP8 gene in MDA-MB-231 cells, C is the qRT-PCR detection diagram after knocking down the USP8 gene in BT549 cells, D is the qRT-PCR detection diagram after knocking down the USP8 gene in MDA-MB-231 cells, E is the statistical chart of CCK8 detection results after knocking down the USP8 gene in BT549 cells, and F is the statistical chart of CCK8 detection results after knocking down the USP8 gene in MDA-MB-231 cells.

[0019] Figure 4 Diagrams of the effects of knocking down the USP8 gene on triple-negative breast cancer cells. Among them, A is the diagram of cell migration and invasion results, B is the EdU staining result diagram, and C is the scratch assay result diagram.

[0020] Figure 5 Diagrams of the results of DUB-IN-2 treatment. Among them, A is the diagram of USP8 protein expression in cells after treatment with different concentrations of DUB-IN-2 for 8 h, B is the statistical chart of the relative mRNA expression levels of the USP8 gene in BT549 cells after treatment with DMSO and different concentrations of DUB-IN-2 for 8 h, C is the statistical chart of the relative mRNA expression levels of USP8 in MDA-MB-231 cells after treatment with DMSO and different concentrations of DUB-IN-2 for 8 h, D is the diagram of the detection results of BT549 cell viability under the action of DMSO and different concentrations of DUB-IN-2, and E is the diagram of the detection results of MDA-MB-231 cell viability under the action of DMSO and different concentrations of DUB-IN-2.

[0021] Figure 6 Diagrams of the results of DUB-IN-2 inhibiting triple-negative breast cancer cells. Among them, A is the diagram of the detection results of cell migration and invasion after treatment with DMSO and different concentrations of DUB-IN-2 for 12 h, B is the detection diagram of the proliferation ability after treatment with DMSO and different concentrations of DUB-IN-2 for 12 h, and C is the healing detection diagram after treatment with DMSO and different concentrations of DUB-IN-2.

[0022] Figure 7 Diagrams of the results after injecting DUB-IN-2 after tumor-bearing. Among them, A is the tumor picture, B is the statistical chart of tumor volume, and C is the statistical chart of tumor weight.

[0023] Figure 8Graph for detecting the viability of triple-negative breast cancer cells with USP8 inhibited after chemotherapy. Among them, A is the statistical graph of cell viability of BT549 cells treated with different concentrations of PTX and different concentrations of PTX combined with 2 μM DUB-IN-2 for 24 h, B is the statistical graph of cell viability of MDA-MB-231 cells treated with different concentrations of PTX and different concentrations of PTX combined with 2 μM DUB-IN-2 for 24 h, C is the statistical graph of cell viability of BT549 cells treated with DMSO, 20 nM PTX and 20 nM PTX + 2 μM DUB-IN-2 for 24 h, and D is the statistical graph of cell viability of MDA-MB-231 cells treated with DMSO, 20 nM PTX and 20 nM PTX + 2 μM DUB-IN-2 for 24 h.

[0024] Figure 9 Graph for detecting the results of chemotherapy of triple-negative breast cancer cells with USP8 inhibited. Among them, A is the detection result graph of cell migration and invasion ability after treatment with DMSO, 20 nM PTX and 20 nM PTX + 2 μM DUB-IN-2 for 24 h, B is the detection graph of cell proliferation ability after treatment with DMSO, 20 nM PTX and 20 nM PTX + 2 μM DUB-IN-2 for 24 h, and C is the cell scratch detection graph after treatment with DMSO, 20 nM PTX and 20 nM PTX + 2 μM DUB-IN-2.

[0025] Figure 10 Graph for the combined treatment results of the tumor-bearing experiment. Among them, A is the representative photo graph of the tumor, B is the quantification graph of the tumor tissue weight, and C is the statistical graph of the volume change during the development of the tumor tissue. Detailed implementation mode

[0026] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0027] The CAS number of DUB-IN-2 is: 924296-19-5.

[0028] Example 1

[0029] I. Experimental materials and settings.

[0030] 1. Cell lines and cell culture: Human triple-negative breast cancer cells BT549, MDA-MB-231, and human embryonic kidney cells HEK-293T were all purchased from the American Type Culture Collection. BT549 cells were cultured in RPMI-1640 medium. MDA-MB-231 and HEK-293T cells were cultured in Dulbecco's Modified Eagle Medium. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture by mass fraction. They were cultured at 37 °C and passaged and trypsinized according to the recommendations of ATCC. Short tandem repeat (STR) analysis was performed using the PowerPlex 21 system to verify all cell lines included in this study.

[0031] 2. Nude mouse rearing: BALB / c female nude mice were reared in a SPF-class barrier environment, using individually ventilated cages (IVC), maintaining a temperature of 25 °C, humidity of 50%, and a 12-hour light cycle. Feed and bedding were both sterilized by high temperature / irradiation, and sterile drinking water was provided.

[0032] 3. Construction of USP8 knockdown cell lines: In the siUSP8#1 and siUSP8#2 groups, siUSP8#1 and siUSP8#2 targeting USP8 were transiently transfected into triple-negative breast cancer cells. The sequences of siUSP8#1 and siUSP8#2 are shown in Table 1. siControl was the negative control, and its sequence is shown in Table 1. Cell detection was performed 48 hours after transfection.

[0033] Table 1: siRNA sequences

[0034]

[0035] Note: TT overhangs were added to the 3′ ends of the above siRNAs.

[0036] II. Experimental methods.

[0037] 1. Public clinical data analysis: USP8 RNA-seq data in triple-negative breast cancer tumors were available from the Genomic Data Commons data portal. (https: / / portal.gdc.cancer.gov / ) The collected data were analyzed and calculated using GraphPad Prism 8.0. For the CORDENONSI YAP CONSERVED SIGNATURE gene set, it was used and downloaded from the GSEA Molecular Signatures Database. Heatmap plotting was performed using Xiantao Online (https: / / www.xiantao.love / ). The relationship between USP8 expression and clinical prognosis was analyzed using the KM-Plot database (https: / / kmplot.com).

[0038] 2. RNA Sequencing and Analysis: Gene Set Enrichment Analysis (GSEA) was performed using the GSEA program provided by the Broad Institute. We used GSEA to evaluate the relative enrichment of YAP positive regulatory genes in the siControl and siUSP8 groups. Enrichment analysis was performed using the Hallmark gene set and KEGG pathways of Metascape, which enabled us to explore the pathways associated with differentially expressed genes. The OmicStudio tool was used to generate volcano plots for differentially expressed genes.

[0039] 3. RNA Extraction and Real-Time Quantitative PCR: Total RNA was extracted using Trizol, reverse transcribed into cDNA, and gene-specific real-time quantitative PCR was performed on the cDNA using a 7500 Real-Time Fluorescence Quantitative PCR System (Applied Biosystems, Singapore). The 36B4 gene was used as an internal reference, and the gene expression levels were detected by qPCR.

[0040] 4. Western Blotting: Cells were lysed with RIPA buffer. Then, protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk at room temperature for 1 hour. After washing three times with PBST, the membrane was incubated with the corresponding primary antibodies: Anti-β-Actin (Proteintech, 20536-1-AP) and Anti-USP8 (Proteintech, 27791-1-AP) overnight at 4°C. After washing three times with PBST, the membrane was incubated with secondary antibodies: HRP-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime, A0208), HRP-labeled Goat Anti-Mouse IgG (H+L) (Beyotime, A0216) for 1 hour. The blots were visualized using an ECL detection kit.

[0041] 5. Cell Viability Assay: The CCK8 method was used to detect cell viability. 4000 cells were collected and re-seeded into 96-well plates, repeated three times. For drug sensitivity analysis, 5000 cells were seeded into 96-well plates, repeated three times and incubated overnight. The CCK8 detection kit was used to detect cell viability.

[0042] 6. Edu Assay: The cell proliferation was evaluated using an EDU assay kit. Cells were seeded into 96-well plates, incubated overnight after experimental treatment, and then incubated with 50 μM EdU for 2 hours. The cells were fixed with 4% paraformaldehyde. The staining procedure was performed according to the manufacturer's instructions. Nucleic acids were stained with Hoechst33342, and the cytoplasm was stained by Merge. The cell proliferation rate was calculated using ImageJ.

[0043] 7. Scratch Assay: Cells were seeded into 12-well plates and subjected to experimental treatment. When the cells reached 100% confluence, a straight line was scratched on the cell monolayer using a 200 μl yellow pipette tip, and the scratch area was measured at the designated time points.

[0044] 8. Transwell Assay: In the cell migration experiment, 50,000 cells were suspended in serum-free medium and seeded into the upper chamber without matrix; in the cell invasion experiment, 50,000 cells were suspended in serum-free medium and seeded into the upper chamber coated with Matrigel. The lower chamber was filled with medium containing 20% fetal bovine serum. After incubation for 12 hours, the cells in the lower chamber were fixed with methanol, stained with 0.2% crystal violet, and photographed for observation.

[0045] 9. Tumor-bearing Experiment: 4×10 6 BT549 cells were suspended in 200 μl of PBS and subcutaneously injected into the mammary fat pads of 4-week-old nude mice. When the average tumor volume reached 100 mm 3 , the experiment of injecting drugs was started. In the DMSO group, mice were intraperitoneally injected with 1 mg / kg of DMSO every 3 days; in the DUB-IN-2 group, mice were intraperitoneally injected with 1 mg / kg of DUB-IN-2 every 3 days; in the PTX group, mice were intraperitoneally injected with 10 mg / kg of paclitaxel (PTX) every 3 days; in the PTX + DUB-IN-2 group, mice were intraperitoneally injected with 1 mg / kg of DUB-IN-2 and 10 mg / kg of PTX every 3 days. The tumor weight and volume were measured every 7 days, and the mice were sacrificed on the 35th day to end the experiment. All experimental procedures involving mice were conducted in accordance with the guidelines approved by the Animal Care Committee of Xinxiang Medical University.

[0046] 10. Statistical Data: The statistical analysis used in this study included the t-test and Pearson correlation coefficient applied to the publicly available data. The data were expressed as the mean ± standard deviation (SD) of the mean. *, **, and *** indicated P < 0.05, P < 0.01, and P < 0.001, respectively.

[0047] III. Experimental Results.

[0048] 1. USP8 is a key regulator of the Hippo signaling pathway in triple-negative breast cancer: Further explore the impact of USP8 on the prognosis of triple-negative breast cancer patients from the Kmplot database. The results are as Figure 1 shown. Through Kmplot analysis, it was found that high expression of USP8 was associated with a low survival rate in triple-negative breast cancer patients. GSEA analysis of triple-negative breast cancer data in the TCGA database revealed a positive correlation between USP8 and YAP-related genes. Moreover, the deletion of USP8 inhibited the activity of the Hippo signaling pathway.

[0049] The results of RNA sequencing after knocking out USP8 in BT549 cells are as Figure 2 shown. The deletion of USP8 inhibited some classical Hippo downstream target genes, including CTGF, CYR61, and SLIT2. When USP8 was deleted, the expression of Hippo target genes in BT549 cells was inhibited. In summary, USP8 is considered a positive regulator of the Hippo signaling pathway in triple-negative breast cancer.

[0050] 2. Depletion of USP8 inhibits the proliferation of human triple-negative breast cancer cells: As Figure 3 shown, USP8 was effectively silenced by two independent siRNAs, and the deletion of USP8 inhibited the proliferation of BT549 and MDA-MB-231 cells. As Figure 4 shown, inhibiting USP8 could inhibit the migration and invasion abilities of BT549 and MDA-MB-231 cells. The deletion of USP8 reduced the number of Edu-positive cells in BT549 and MDA-MB-231 cells, and it was found that the deletion of USP8 inhibited the cell migration of BT549 and MDA-MB-231.

[0051] 3. DUB-IN-2 inhibits the proliferation of human triple-negative breast cancer cells: As Figure 5 shown, DUB-IN-2 has an inhibitory effect on USP8. DUB-IN-2 effectively inhibited the proliferation of BT549 and MDA-MB-231 cells. As Figure 6 shown, DUB-IN-2 could inhibit the migration and invasion abilities of BT549 and MDA-MB-231 cells. The number of Edu-positive cells in BT549 and MDA-MB-231 cells decreased after treatment with DUB-IN-2. This inhibitor also slowed down the wound closure rate of BT549 and MDA-MB-231 cells. A xenograft mouse model was also used to evaluate the effect of DUB-IN-2. As Figure 7 shown, DUB-IN-2 inhibited the potential of breast cancer cells to occur.

[0052] 4. Inhibition of USP8 can enhance the sensitivity of triple-negative breast cancer cells to chemotherapeutic drugs: Chemotherapy, especially paclitaxel, is the mainstay of treatment for triple-negative breast cancer patients in clinical practice. Paclitaxel is the first-line chemotherapeutic drug for triple-negative breast cancer patients. However, a significant number of triple-negative breast cancer patients develop drug resistance, leading to treatment failure. Therefore, the combination of the small molecule inhibitor DUB-IN-2 and paclitaxel was designed to explore its effect on the triple-negative breast cancer phenotype. As Figure 8 shown, the use of DUB-IN-2 decreased the IC50 value of paclitaxel and enhanced the inhibitory effect of paclitaxel on the cell proliferation of BT549 and MDA-MB-231. As Figure 9 shown, the combined use of paclitaxel and DUB-IN-2 inhibited the migration and invasion ability of triple-negative breast cancer cells. The EdU assay showed that the number of RU-positive cells in breast cancer cells treated with paclitaxel and DUB-IN-2 decreased. In the scratch assay, the inhibitory effect of the combination treatment on the wound closure rate of breast cancer cells was more significant. Finally, paclitaxel and DUB-IN-2 were combined in a xenograft mouse model to explore their effect on breast cancer development. As Figure 10 shown, the combined use of paclitaxel and DUB-IN-2 could better inhibit tumor growth in vivo. The above data indicate that inhibition of USP8 enhances the sensitivity of triple-negative breast cancer cells to chemotherapeutic drugs.

[0053] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. To avoid repetition, the preferred embodiments of the present invention are described.

[0054] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0055] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. Application of substances that inhibit USP8 gene expression in the preparation of drugs for the treatment of triple-negative breast cancer.

2. Use of the substance for inhibiting USP8 gene expression according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The drug uses small molecule compounds or siRNA that target and inhibit USP8 gene as effective ingredients.

3. Use of the substance for inhibiting USP8 gene expression according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The small molecule compound is DUB-IN-2.

4. Use of the substance for inhibiting USP8 gene expression according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The siRNA is selected from one of siUSP8#1 and siUSP8#2, the sense chain sequence of siUSP8#1 is shown in SEQ ID NO.1, and the antisense chain sequence is shown in SEQ ID NO.2, the sense chain sequence of siUSP8#2 is shown in SEQ ID NO.3, and the antisense chain sequence is shown in SEQ ID NO.

4.

5. Use of the substance for inhibiting USP8 gene expression according to claim 3 in preparing a drug for treating triple-negative breast cancer, characterized in that: When the drug uses the small molecule compound DUB-IN-2 that targets and inhibits the USP8 gene as an active ingredient, the active ingredient also includes paclitaxel.

6. Use of the substance for inhibiting USP8 gene expression according to claim 5 in preparing a drug for treating triple-negative breast cancer, characterized in that: The molar ratio of DUB-IN-2 to paclitaxel is 98-102:0.5-1.

5.

7. Use of the substance for inhibiting USP8 gene expression according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. Use of the substance for inhibiting USP8 gene expression according to claim 7 in the preparation of a drug for treating triple-negative breast cancer, characterized in that: The auxiliary materials include any one or more of fillers, stabilizers, diluents and adjuvants.

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