POLR2A as a Biomarker for the Efficacy of Treating Tumors

Abstract

The present invention relates to the use of POLR2A as a biomarker for the efficacy of treating tumors. The inhibitory effects of histone deacetylase inhibitors and echinocandin antifungal drugs on tumor cells with low POLR2A expression levels are significantly higher than those on tumor cells with high POLR2A expression levels. In tumor cells with low POLR2A expression levels, there is hemizygous deletion of the POLR2A gene; and / or the expression product of the POLR2A gene has a lower expression level compared to the control; and / or there is hemizygous deletion of the TP53 gene. This compound can effectively predict the efficacy of histone deacetylase inhibitors in treating breast cancer, colon cancer, prostate cancer, and acute leukemia, providing a new method for the precise treatment of cancer and thus avoiding the blindness of chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an application for inhibiting POLR2A in tissues, which is mainly applicable to predicting the efficacy of histone deacetylase inhibitors and echinocandin antifungal drugs in the treatment of tumors. Background Art

[0002] TP53 is a tumor suppressor gene. In most tumors, it is inactivated due to mutation or deletion, resulting in poor tumor development and drug treatment effects. POLR2A is a gene in the adjacent region of TP53. The deletion of TP53 will also cause hemizygous deletion of POLR2A. A single copy of the POLR2A gene can maintain the survival of cancer cells. There are also genes such as SHBG, SENP3, TNFSF12-13, ATP1B2, CD68 between TP53 and POLR2A. However, tumor cells with a single copy of POLR2A are more sensitive to POLR2A inhibitors.

[0003] Epigenetics plays an important role in the occurrence and development of tumors and is related to a variety of malignant tumors. Epigenetic modifications include DNA modifications, histone modifications, and other aspects. Histone deacetylase inhibitors can inhibit the activity of histone deacetylases, affect the acetylation level of nucleosomal histones, regulate gene expression at the transcriptional level, and induce a series of biological effects such as cell apoptosis, differentiation, and regulation of the activation and inhibition of transcription factors.

[0004] Echinocandin antifungal drugs can inhibit the formation of fungal cell walls. By non-competitively inhibiting glucan synthase, it causes a lack of cell wall glucan during the growth of fungal cells, abnormal osmotic pressure, and ultimately fungal cell lysis. Currently, it is mainly used for the treatment of invasive aspergillosis. The applicant previously found that echinocandin antifungal drugs such as anidulafungin, caspofungin, micafungin, etc. have antitumor effects.

[0005] However, there are few reports on the application of predicting the tumor efficacy of inhibiting POLR2A in tissues. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of POLR2A as a biomarker for predicting the efficacy of histone deacetylase inhibitors and echinocandin antifungal drugs in the treatment of tumors.

[0007] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0008] The application of POLR2A as a biomarker for the efficacy of treating tumors.

[0009] The specific application is as follows:

[0010] In tumor cells with low POLR2A expression levels, loss of heterozygosity, or concomitant p53 hemizygous deletion, the expression level of POLR2A is low. In these cells, the inhibitory effects of histone deacetylase inhibitors and echinocandin antifungal drugs on cancer are significantly higher than those in tumor cells with high POLR2A expression levels.

[0011] Preferably, the tumors with low POLR2A expression levels, loss of heterozygosity, or concomitant p53 hemizygous deletion include one or more of breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, brain tumor, ovarian cancer, colorectal cancer, kidney cancer, melanoma, leukemia, gastric cancer, esophageal cancer, thyroid cancer, cervical cancer; further, the leukemia includes at least one of acute myeloid leukemia, acute lymphoblastic leukemia / lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia. Further, the tumors include one or more of breast cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, brain tumor, ovarian cancer, colorectal cancer.

[0012] The cancers described include any malignant tumor cells, such as solid tumors and hematological tumors.

[0013] Further, the tumor cells are human breast cancer cells MDA-MB-453 and human breast cancer cells MDA-MB-231.

[0014] Preferably, the histone deacetylase inhibitors include Vorinostat (SAHA), Belinostat (PXD101), Romidepsin (FK228, Depsipeptide), Panobinostat (LBH589), Givinostat (ITF2357), Mocetinostat (MGCD0103), Entinostat (MS-275), Quisinostat (JNJ-26481585) 2HCl, Pracinostat (SB939), Abexinostat (PCI-24781), Ricolinostat (ACY-1215), Tacedinaline (CI994), Fimepinostat (CUDC-907), Resminostat, Valproic acid (VPA), Tucidinostat, Chidamide, Domatinostat (4SC-202), Sodium valproate.

[0015] More preferably, the histone deacetylase inhibitor is one or more of Vorinostat (SAHA), Belinostat (PXD101), Romidepsin (FK228, Depsipeptide), or Panobinostat (LBH589).

[0016] Preferably, the echinocandin antifungal drugs include anidulafungin, caspofungin, micafungin, cilofungin, enfumafungin, arentefungin, echinocandin B, biapenem, CD101IV, rezafungin. Preferably selected from caspofungin, micafungin, anidulafungin or one of their semi-synthetic derivatives or one of their salts or one of their esters or one of their ester salts.

[0017] More preferably, the echinocandin antifungal drugs include anidulafungin, caspofungin and micafungin.

[0018] The structural formula of Vorinostat is shown in Formula I, and the molecular formula is: C 14 H 20 N 2 O 3 ;

[0019]

[0020] The structural formula of belinostat is shown in Formula II, and its molecular formula is: C 15 H 14 N 2 O 4 S;

[0021]

[0022] The structural formula of romidepsin is shown in Formula III, and its molecular formula is: C 24 H 36 N 4 O 6 S 2 ;

[0023]

[0024] The structural formula of panobinostat is shown in Formula IV, and its molecular formula is: C 21 H 23 N 3 O 2 ;

[0025]

[0026] The structural formula of anidulafungin is shown in Formula V, and its molecular formula is: C 58 H 73 N 7 O 17 ;

[0027]

[0028] The structural formula of caspofungin is shown in Formula VI, and its molecular formula is: C 52 H 88 N 10 O 15 ;

[0029]

[0030] The structural formula of micafungin is shown in Formula VII, and its molecular formula is: C 56 H 71 N 9 O 23 S;

[0031]

[0032] The present invention also provides the use of POLR2A as a biomarker for predicting the efficacy of combined use of histone deacetylase inhibitors and echinocandin antifungal drugs with other chemotherapeutic drugs in the treatment of tumors.

[0033] Preferably, the other chemotherapeutic drugs are one or more of cytotoxic anti-tumor drugs, epigenetic modification enzyme inhibitors, ubiquitin proteasome inhibitors, cyclin-dependent kinase inhibitors, immune checkpoint inhibitors, anti-apoptotic protein inhibitors, compensatory pathway inhibitors, anti-angiogenic drugs, and other kinase inhibitors.

[0034] Preferably, the cytotoxic anti-tumor drugs are at least one of drugs that affect DNA structure and function, drugs that affect nucleic acid biosynthesis, drugs that interfere with the transcription process and prevent RNA synthesis, and tyrosine kinase inhibitors.

[0035] Preferably, the drugs that affect DNA structure and function are at least one of cyclophosphamide, cisplatin, carboplatin, camptothecin drugs, irinotecan, topotecan, and podophyllotoxin derivatives;

[0036] Preferably, the drugs that affect nucleic acid biosynthesis are at least one of methotrexate, 5-FU, and capecitabine;

[0037] Preferably, the drugs that interfere with the transcription process and prevent RNA synthesis are at least one of doxorubicin, epirubicin, pirarubicin, aclarubicin, idarubicin, daunorubicin, and mitoxantrone.

[0038] Preferably, the tyrosine kinase inhibitors are at least one of imatinib, dasatinib, nilotinib, sunitinib, lapatinib, regorafenib, pazopanib, and ponatinib.

[0039] The present invention also provides the use of POLR2A as a biomarker for predicting the efficacy of combined use of histone deacetylase inhibitors and echinocandin antifungal drugs with non-drug therapies in the treatment of tumors.

[0040] Preferably, the non-drug therapies include chemotherapy, radiotherapy, immunotherapy, surgery, gene therapy, or inhibitory antibody therapy.

[0041] The following further explains the present invention:

[0042] The applicant's research found that in tumor cells with low POLR2A expression levels, loss of heterozygosity, or accompanied by p53 hemizygous deletion, the POLR2A expression level is low. In these cells, the inhibitory effects of histone deacetylase inhibitors and echinocandin antifungal drugs on cancer are significantly higher than those on tumor cells with high POLR2A expression levels. At the same time, histone deacetylase inhibitors and echinocandin antifungal drugs combined with other anti-tumor drugs can enhance the anti-tumor effects of other anti-tumor drugs. Especially when combined with other anti-tumor drugs such as cisplatin, doxorubicin, ponatinib, pazopanib, etc., the anti-tumor effects of these drugs can be enhanced, and these effects are more sensitive in tumor cells with POLR2A hemizygous deletion. When used in combination, the ratio of histone deacetylase inhibitors and echinocandin antifungal drugs to other anti-tumor drugs is 1:(0.01 - 100) according to the dose ratio, and it can be specifically determined according to the sensitivity of the drugs to different tumors.

[0043] This invention confirms that tumor cells with low POLR2A expression levels are more sensitive to protein deacetylase inhibitors and echinocandin antifungal drugs and can be used for precision treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Inhibitory effect curves (IC50 μmol / L, μM) of the histone deacetylase inhibitor vorinostat on human breast cancer cells MDA-MB-231 and MDA-MB-453.

[0045] Figure 2 : Inhibitory effect curves IC50 (μmol / L, μM) of the histone deacetylase inhibitor vorinostat combined with the anti-tumor drug cisplatin on human breast cancer cells MDA-MB-231 and MDA-MB-453.

[0046] Figure 3 : Inhibitory effect curves (IC50 μmol / L, μM) of the echinocandin antifungal drug anidulafungin on human breast cancer cells MDA-MB-231 and MDA-MB-453.

[0047] Figure 4 : POLR2A protein expression in MDA-MB-231 and MDA-MB-453 cells, and the effects of the histone deacetylase inhibitor vorinostat and the echinocandin antifungal drug anidulafungin on POLR2A protein expression.

[0048] Figure 5 : POLR2A siRNA silencing significantly down-regulated POLR2A expression in breast cancer cells MDA-MB-231.

[0049] Figure 6 : Down-regulating POLR2A expression significantly enhanced anidulafungin (ANI,Figure 6 A), Vorinostat (SAHA, Figure 6 B) Anti-breast cancer effect.

[0050] Figure 7 : Anidulafungin inhibits the growth of breast cancer 4T1 cells.

[0051] Figure 8 : Anidulafungin significantly inhibits the expression of POLR2A in breast cancer tissues (P < 0.05, n = 5); as Figure 8 A is the immunohistochemical image of POLR2A in the solvent group and anidulafungin-treated group, Figure 8 B is the statistical analysis chart of POLR2A expression level. Detailed implementation manners Detailed implementation manners:

[0053] The present invention will be described in detail below in conjunction with embodiments.

[0054] POLR2A serves as a biomarker for predicting the efficacy of histone deacetylase inhibitors and echinocandin antifungal drugs in the treatment of tumors.

[0055] Materials and methods

[0056] To prove that POLR2A can serve as a biomarker for predicting the efficacy of histone deacetylase inhibitors and echinocandin antifungal drugs in the treatment of tumors, the applicant will use different tumor cell lines and treat them with Vorinostat (Vorinostat, SAHA), Belinostat (Belinostat, PXD101), Romidepsin (Romidepsin, FK228, Depsipeptide) or Panobinostat (Panobinostat, LBH589), or in combination with anidulafungin, cisplatin, 5-fluorouracil, CTLA-4 inhibitor or PD-1 / PD-L1 inhibitor; anidulafungin, or in combination with vorinostat, belinostat, romidepsin, panobinostat, cisplatin, 5-fluorouracil, CTLA-4 inhibitor or PD-1 / PD-L1 inhibitor, etc. for 24 h to 72 h; CCK8 is used to detect cell viability, and Western Blot is used to detect the expression of POLR2A protein.

[0057] Implementation drugs: Vorinostat, Belinostat, Romidepsin, Panobinostat, Anidulafungin, Cisplatin or 5-fluorouracil are purchased from reagent companies.

[0058] Tumor cell lines: human breast cancer POLR2A neutral cells (MDA-MB-231, BT20, BT549), human breast cancer POLR2A loss of heterozygosity cells (MDA-MB-453, HCC-1599, HCC70, DU4475), human colon cancer POLR2A neutral cells (HT29, HCT116, SW480) and human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197), human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b) and human prostate cancer POLR2A loss cells (PC3, LNCap, VCap), human acute leukemia POLR2A neutral cells (U937, HL-60) and human acute leukemia POLR2A loss cells (MV-4-11).

[0059] Cell culture method: Conducted routinely. The above cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin and streptomycin). When the cell fusion reached 90%, trypsin digestion was used. When the cells shrank and became round and the cell gaps were obvious, digestion was immediately terminated with the medium. The cells were dispersed and blown evenly into a single suspended state, subcultured in flasks, and cultured in a cell incubator at 37°C with 5% CO 2 2. The subsequent experiments were completed using cells in the logarithmic growth phase.

[0060] Drug treatment method: The above cells were treated with different concentrations of histone deacetylase inhibitors, echinocandin antifungal drugs, or a combination of these two types of drugs, or in combination with other drugs for 24, 48, or 72 hours. Control groups (containing medium and cells, without drug treatment), experimental groups (containing medium and cells with different concentrations of drugs), solvent groups (containing medium and cells with DMSO, without drug treatment), and / or blank groups (only containing medium, without cells) were set up, with 4 - 6 replicates in each group. CCK8 was used to detect cell viability to evaluate the inhibitory effect of the drug on tumor cell growth; Western Blot was used to detect POLR2A protein expression.

[0061] POLR2A Silencing Experiment (POLR2A siRNA Experiment): Take out the lyophilized powder of POLR2A siRNA or Negative Control (NC) siRNA (5 nmol) (purchased from Guangzhou Ribobio Co., Ltd.), centrifuge the lyophilized powder instantaneously to the bottom of the centrifuge tube, add 250 μL of sterile DEPC water to prepare a siRNA stock solution with a concentration of 20 μmol / L, aliquot and store at -80 °C. When the cell density reaches 30 - 50%, cell transfection can be carried out. Add the transfection complex containing POLR2A siRNA or NC siRNA (prepared with ribo FECT TM CP Buffer and reagent) to high-glucose DMEM medium containing 1% FBS and add it to the cell plate so that the final concentration of the transfection complex is 50 nmol / L. Shake well, and incubate at 37 °C in a 5% CO 2 2 environment for 8 h, then replace it with complete medium and continue to culture for 14 h. Digest and centrifuge the cells, retain some cells for verification of transfection efficiency, and transfer the remaining cells to a 96-well plate at a density of 4000 cells / well. After culturing for 24 h, perform drug treatment with anidulafungin (ANI) or vorinostat (SAHA).

[0062] CCK8 Assay for Cell Viability: When the cell density in the culture flask is about 80%, digest and centrifuge the cells, discard the supernatant and resuspend the cells to make a cell suspension and count. Take a certain amount of the cell suspension into a new centrifuge tube, add complete medium to a cell density of 40 cells / μL to make a cell working solution. Add 100 μL of the cell working solution to each well of a 96-well plate, that is, seed 4000 cells per well. Incubate at 37 °C in a 5% CO2 environment for about 24 h. When the cell density reaches 60% - 70%, perform drug treatment. Dilute the drug stock solution with medium in a gradient dilution manner to prepare the required concentration of the drug solution for standby. Discard the original medium in all wells of the 96-well plate, add 100 μL of medium or the corresponding concentration of the drug solution to each well according to the grouping, and incubate at 37 °C in a 5% CO2 environment. After 24 h or 48 h of drug treatment, add 10 μL of CCK-8 to each well of the 96-well plate under light-proof conditions, shake gently, incubate at 37 °C in a 5% CO2 environment for 2 h, and measure the absorbance value at 450 nm on the machine. Perform at least 3 independent repeated experiments and calculate the half-maximal inhibitory concentration (IC50) of the drug.

[0063] Western blot assay: After washing and centrifuging the collected cells with PBS, lysis buffer was added, and the cells were lysed on ice bath for 30 min. Then, the supernatant was collected by centrifuging at 12,000 rpm at 4 °C for 15 min. After determining the protein concentration by Bradford method, 10 - 40 μg of protein was separated by 8 - 10% SDS-PAGE gel and transferred to PVDF membrane. The membrane was incubated overnight with POLR2A antibody (Santa Cruz, California, USA) and β-actin antibody (Beyotime, Jiangsu). After incubation with the corresponding secondary antibody, it was developed by MolecularImager ChemiDoc XRS System (Bio-Rad, Philadelphia, PA), and β-actin was used as an internal reference. The protein gray value was measured by Image J software to evaluate the protein expression level.

[0064] Immunohistochemistry assay: The tissue paraffin sections were baked at 80 °C to remove wax, and then immersed in xylene I, xylene II, absolute ethanol, 80% ethanol, and 50% ethanol for 10 min each. Subsequently, they were placed in ddH 2 O and washed 3 times, and then washed 3 times with PBS, 5 min each time. Dilute Tris-EDTA antigen retrieval solution with ddH 2 O to 1× and boil it in a water bath for standby. The sections were put into the boiling antigen retrieval solution and boiled in a water bath for 20 min, then taken out and cooled to room temperature naturally. Subsequently, they were washed 3 times with PBS, 8 min each time. After drying, 3% H 2 O 2To completely cover the tissue surface, let it stand at room temperature for 10 min, then wash it 3 times with PBS, 3 min each time. After drying, add 10% goat blocking serum to the section tissue area to completely cover the tissue surface, and block it at room temperature for 30 min. Dilute the POLR2A antibody (Santa Cruz, California, USA) with the primary antibody diluent according to the dilution ratio in the instruction manual, add the diluted primary antibody solution to the section tissue area to completely cover the tissue surface, place it in a wet box and incubate overnight at 4°C, then wash it 3 times with PBS, 8 min each time. Add the reaction enhancer solution to the section tissue area to completely cover the tissue surface, and let it stand at room temperature for 20 min. Wash it 3 times with PBS, 8 min each time. Add the enzyme-labeled goat anti-mouse / rabbit IgG polymer to the section tissue area to completely cover the tissue surface, and incubate it at room temperature for 60 min. Wash it 3 times with PBS, 8 min each time. After drying, add the DAB chromogenic solution (DAB substrate solution: concentrated DAB solution = 20:1) to the section tissue area to completely cover the tissue surface, observe the tissue color change under the microscope. When obvious brownish-yellow appears in some sections, rinse all the sections with a small stream of tap water for 5 min. Add hematoxylin stain to the section tissue area to completely cover the tissue surface, stain for 15 s, then rinse the section with a small stream of tap water for 5 min. Immerse the section successively in 50% ethanol, 80% ethanol, absolute ethanol, xylene II, and xylene I for 10 min each. After drying, add 1 drop of neutral resin to the section tissue area and cover it with a cover slip. Take tissue photos at magnifications of 200×, 400×, and 1000× under the microscope respectively. Analyze the data with Image J..

[0065] Establishment of a mouse breast cancer cell 4T1 xenograft tumor model: Digest the 4T1 cells in the logarithmic growth phase with trypsin and centrifuge. Resuspend all the cell precipitates with 1 mL of high-glucose DMEM to make a cell suspension. Inject 100 μL of 4T1 cells at 1×10 7 / mL subcutaneously into the flanks of Balb / c mice to construct a 4T1 xenograft tumor model. Set up an anidulafungin drug treatment group and a corn oil vehicle group, with 6 mice in each group. Starting from the 4th day after tumor formation (when the tumor size reaches 60 mm 3 ), give anidulafungin drug treatment (30 mg / kg, dissolved in corn oil) by intraperitoneal injection every other day. After 16 days of drug administration, stop the drug administration. Detect the expression of POLR2A in the xenograft tumor tissue of mouse breast cancer 4T1 cells by immunohistochemistry.

[0066] Mouse xenograft tumor model experiment: A mouse xenograft tumor model was established using POLR2A neutral tumor cells and POLR2A loss tumor cells, and they were treated with different concentrations of histone deacetylase inhibitors, echinocandin antifungal drugs, or a combination of these two types of drugs, or in combination with other drugs. The tumor volume or weight was detected to evaluate the inhibitory effect of the drugs on the growth of tumor cells; Western Blot or immunohistochemistry was used to detect the expression of POLR2A protein.

[0067] Predictive role of POLR2A in the efficacy of vorinostat and anidulafungin on breast cancer POLR2A neutral (neutral) cells MDA-MB-231 and human breast cancer POLR2A loss (heterozygous deletion) cells MDA-MB-453:

[0068] Experimental grouping:

[0069] Group 1: To detect the inhibitory effect of vorinostat and anidulafungin on the growth of human breast cancer cells MDA-MB-231 and MDA-MB-453. The final concentrations of vorinostat in MDA-MB-231 cells were 1.25, 2.5, and 5 μmol / L in sequence, while in MDA-MB-453 cells, they were 0.05, 0.1, and 0.5 μmol / L in sequence; the final concentrations of anidulafungin were 2.5, 5, and 10 μmol / L in sequence. A control group, a solvent DMSO group, and / or a blank control group were set up, with 4 - 6 replicate wells in each group, and the treatment time was 48 hours.

[0070] Group 2: To detect the inhibitory effect of the combined use of vorinostat, anidulafungin and cisplatin (in subsequent experiments, these two drugs will be combined, or with 5-fluorouracil, CTLA-4 inhibitor or PD-1 / PD-L1 inhibitor) on tumor cells. Separate single-drug treatment groups were set up: vorinostat (1, 2.5 μM), cisplatin (5, 10, 20 μM); combined treatment groups: vorinostat (1 μM) + cisplatin (5, 10, 20 μM) in MDA-MB-453 cells, vorinostat (2.5 μM) + cisplatin (5, 10, 20 μM) in MDA-MB-231 cells. A solvent DMSO and / or a blank control group were set up. The blank control group was only added with the corresponding volume of normal saline and treated for 48 hours respectively.

[0071] Detection method: CCK8 was used to detect cell viability to evaluate the inhibitory effect of the drugs on the growth of tumor cells; Western Blot was used to detect the regulatory effect of POLR2A protein expression.

[0072] Group 3: Mouse xenograft tumor model experiment. A mouse xenograft tumor model was established using POLR2A neutral breast cancer cells and POLR2A loss breast cancer cells. They were treated with different concentrations of histone deacetylase inhibitors, echinocandin antifungal drugs, or a combination of these two types of drugs, or combined with other drugs, and the tumor volume or weight was measured to evaluate the inhibitory effect of the drugs on the growth of tumor cells; POLR2A protein expression was detected by Western Blot or immunohistochemistry.

[0073] Group 4: In MDA-MB-231 cells (POLR2A neutral), siRNA was used to silence the expression of POLR2A. A control group, an siRNA silencing group, and a negative control group were set up, and at least 3 independent repeated experiments were carried out.

[0074] Western Blot was used to detect the regulatory effect on POLR2A protein expression.

[0075] Group 5: Detection of the inhibitory effect of vorinostat and anidulafungin on breast cancer cells in MDA-MB-231 cells (POLR2A neutral) with downregulated POLR2A expression by siRNA. Separate drug treatment groups were set up: vorinostat (0.3125, 0.625, 1.25, 2.5, 5 μM), anidulafungin (0.625, 1.25, 2.5, 5, 10 μM), with DMSO as the solvent and a blank control group. Only the corresponding volume of normal saline was added to the blank control group. Each group had 4 - 6 replicate wells, and the treatment time was 48 hours. At least 3 independent repeated experiments were carried out.

[0076] Detection method: CCK8 was used to detect cell viability to evaluate the inhibitory effect of the drugs on the growth of tumor cells.

[0077] Group 6: 4T1 cells were injected subcutaneously into Balb / c mice to construct a 4T1 xenograft tumor model. An anidulafungin drug treatment group and a corn oil solvent group were set up, with 6 mice in each group. Starting from the 4th day after tumor formation, anidulafungin drug treatment (30 mg / kg, dissolved in corn oil) was given intraperitoneally every other day. After 16 days of drug administration, the drug administration was stopped. Immunohistochemistry was used to detect the expression of POLR2A in the 4T1 cell xenograft tumor tissue of mouse breast cancer.

[0078] Results:

[0079] Result 1, as Figure 1As shown, vorinostat-treated MDA-MB-231 and MDA-MB-453 cells for 48 hours significantly reduced cell viability, showing obvious inhibitory and killing effects on tumor cells and presenting a dose-dependent manner; the inhibitory effect on MDA-MB-453 was significantly higher than that on MDA-MB-231 (n = 3 - 4) cells, and the IC50 of vorinostat on MDA-MB-231 was 6.5 times that of MDA-MB-453.

[0080] Result 2, as Figure 2 As shown, vorinostat combined with cisplatin-treated MDA-MB-231 and MDA-MB-453 cells for 48 hours significantly reduced cell viability, showing obvious inhibitory and killing effects on tumor cells and presenting a dose-dependent manner; the inhibitory effect of combined drug use on MDA-MB-453 was significantly higher than that on MDA-MB-231; in MDA-MB-231 cells, vorinostat combined with cisplatin could reduce the IC50 of cisplatin by 2.3 times; while in MDA-MB-453 cells, vorinostat combined with cisplatin reduced the IC50 by 7.5 times (n = 3 - 4, *P < 0.05, **P < 0.01 vs the non-combined drug group), and its enhancement of cisplatin drug sensitivity was 3.3 times that of MDA-MB-231 cells.

[0081] Result 3, as Figure 3 As shown, anidulafungin-treated MDA-MB-231 and MDA-MB-453 cells for 48 hours significantly reduced cell viability, showing obvious inhibitory and killing effects on tumor cells and presenting a dose-dependent manner; the inhibitory effect on MDA-MB-453 cells was significantly higher than that on MDA-MB-231 (n = 3 - 4) cells, and the IC50 of anidulafungin on MDA-MB-231 was 4 times that of MDA-MB-453.

[0082] Result 4, as Figure 4 As shown, the expression of POLR2A in POLR2A-neutral breast cancer cells MDA-MB-231 was significantly higher than that in POLR2A heterozygous deletion cells MDA-MB-453. The protein expression of POLR2A in MDA-MB-231 was about 2 times that of MDA-MB-453 (see Figure 4 A), and vorinostat ( Figure 4 C, D), anidulafungin ( Figure 4 B) could reduce the protein expression of POLR2A in the above cells.

[0083] Result 5, the sensitivity of anidulafungin combined with other drugs in human breast cancer POLR2A loss cells was significantly higher than that in human breast cancer POLR2A neutral cells.

[0084] Result 6, Vorinostat, anidulafungin, or the combination of these two drugs, or the combination with other drugs, showed significantly higher inhibitory effects on the growth of xenografts of human breast cancer POLR2A loss cells than on those of human breast cancer POLR2A neutral cells.

[0085] Result 7, as Figure 5 shown, POLR2A siRNA silencing significantly downregulated the expression of POLR2A in breast cancer cells MDA-MB-231.

[0086] Result 8, as Figure 6 shown, downregulation of POLR2A expression could significantly enhance the anti-breast cancer effects of anidulafungin (ANI, Figure 6 A) and vorinostat (SAHA, Figure 6 B). The cell viability of the POLR2A silencing group (POLR2A loss) was significantly lower than that of the non-silencing group (POLR2A neutral). The IC50 of anidulafungin for MDA-MB-231 POLR2A neutral (4.543 μM) was approximately 2 times that of the IC50 (2.218 μM). The IC50 of vorinostat for MDA-MB-231 POLR2A neutral (2.659 μM) was 2 times that of the IC50 of MDA-MB-231 POLR2A loss (1.308 μM). Anidulafungin group (ANI): # P < 0.05, ## P < 0.01, ### P < 0.001, vs MDA-MB-231 POLR2A neutral ; Vorinostat group (SAHA): & P < 0.05, &&P < 0.01 vs MDA-MB-231 POLR2A neutral ; n = 3.

[0087] Result 9, as Figure 7 shown, anidulafungin significantly inhibited the growth of breast cancer 4T1 cells. The tumor weight of the anidulafungin group was significantly lower than that of the control group (P < 0.05, n = 6). As Figure 8 shown by the results of immunohistochemistry, anidulafungin significantly inhibited the expression of POLR2A in breast cancer tissues (P < 0.05, n = 5). As Figure 8 A is the immunohistochemical image of POLR2A in the solvent group and the anidulafungin-treated group, Figure 8 B is the statistical analysis chart of the POLR2A expression level.

[0088] Conclusion: POLR2A heterozygous deletion or low expression is a predictive molecule for the sensitivity of histone deacetylase inhibitors such as vorinostat and echinocandin antifungal drugs such as anidulafungin to breast cancer.

[0089] The predictive role of POLR2A in the efficacy of vorinostat and anidulafungin against human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) and human colon cancer POLR2A neutral cells (HT29, HCT116, SW480):

[0090] Experimental grouping:

[0091] Group 1: Detect the inhibitory effects of vorinostat and anidulafungin on the growth of human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) and human colon cancer POLR2A neutral cells (HCT116, SW480). Set the drug concentrations according to the preliminary experiment, and set the control group, drug group, solvent DMSO and / or blank control group respectively. The treatment time is 24, 48 or 72 hours.

[0092] Group 2: Detect the inhibitory effects of vorinostat, anidulafungin, or the combination of these two drugs, or the combination with other drugs on tumor cells. Set the drug concentrations according to the preliminary experiment, and set the control group, drug group, solvent DMSO and / or blank control group respectively. The treatment time is 24, 48 or 72 hours.

[0093] Detection methods: Use CCK8 to detect cell viability and evaluate the inhibitory effects of drugs on tumor cell growth; use Western Blot to detect the regulatory effects of POLR2A protein expression.

[0094] Group 3: Mouse xenograft tumor model experiment. Establish mouse xenograft tumor models with POLR2A neutral colon cancer cells and POLR2A loss colon cancer cells, and treat them with different concentrations of histone deacetylase inhibitors, echinocandin antifungal drugs, or the combination of these two types of drugs, or the combination with other drugs. Detect the tumor volume or weight to evaluate the inhibitory effects of drugs on tumor cell growth; use Western Blot or immunohistochemistry to detect POLR2A protein expression.

[0095] Results:

[0096] Result 1: The inhibitory effect of vorinostat in human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) was significantly higher than that in human colon cancer POLR2A neutral cells (HCT116, SW480).

[0097] Result 2: The sensitivity of vorinostat in combination with other drugs in human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) was significantly higher than that in human colon cancer POLR2A neutral cells (HCT116, SW480).

[0098] Result 3: The inhibitory effect of anidulafungin in human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) was significantly higher than that in human colon cancer POLR2A neutral cells (HCT116, SW480).

[0099] Result 4: The sensitivity of anidulafungin in combination with other drugs in human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) was significantly higher than that in human colon cancer POLR2A neutral cells (HCT116, SW480).

[0100] Result 5: The protein expression of POLR2A in human colon cancer POLR2A loss cells (SW837, SNU283, SNU1197) was significantly lower than that in POLR2A neutral cells (HCT116, SW480), and vorinostat and anidulafungin could reduce the protein expression of POLR2A in the above cells.

[0101] Result 6: The inhibitory effect of vorinostat, anidulafungin, or the combination of these two drugs, or in combination with other drugs on the growth of xenografts in human colon cancer POLR2A loss cells was significantly higher than that on the inhibition of xenografts in human colon cancer POLR2A neutral cells.

[0102] Conclusion: POLR2A heterozygous deletion or low expression is a predictive molecule for the sensitivity of histone deacetylase inhibitors such as vorinostat and echinocandin antifungal drugs such as anidulafungin against colon cancer.

[0103] The predictive effect of POLR2A on the efficacy of vorinostat and anidulafungin in human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b) and human prostate cancer POLR2A loss cells (PC3, LNCap, VCap):

[0104] Experimental grouping:

[0105] Group 1: Detect the inhibitory effects of vorinostat and anidulafungin on the growth of human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b) and human prostate cancer POLR2A loss cells (PC3, LNCap, VCap). Set the drug concentrations according to the preliminary experiments, and set up control groups, drug groups, solvent DMSO and / or blank control groups respectively. The treatment time is 24, 48 or 72 hours.

[0106] Group 2: Detect the inhibitory effects of the combination of vorinostat and other drugs on tumor cells. Set the drug concentrations according to the preliminary experiments, and set up control groups, drug groups, solvent DMSO and / or blank control groups respectively. The treatment time is 24, 48 or 72 hours.

[0107] Detection method: Detect cell viability by CCK8 to evaluate the inhibitory effect of the drug on the growth of tumor cells; Detect the regulatory effect of POLR2A protein expression by Western Blot.

[0108] Group 3: Mouse xenograft tumor model experiment. Establish mouse xenograft tumor models with POLR2A neutral prostate cancer cells and POLR2A loss prostate cancer cells, and treat them with different concentrations of histone deacetylase inhibitors, echinocandin antifungal drugs, or the combination of these two types of drugs, or the combination with other drugs, and detect the tumor volume or weight to evaluate the inhibitory effect of the drug on the growth of tumor cells; Detect POLR2A protein expression by Western Blot or immunohistochemistry.

[0109] Results:

[0110] Result 1: The inhibitory effect of vorinostat on human prostate cancer POLR2A loss cells (PC3, LNCap, VCap) is significantly higher than that on human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b).

[0111] Result 2: The inhibitory effect of the combination of vorinostat and other drugs on human prostate cancer POLR2A loss cells (PC3, LNCap, VCap) is significantly higher than that on human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b).

[0112] Result 3: The inhibitory effect of anidulafungin on human prostate cancer POLR2A loss cells (PC3, LNCap, VCap) is significantly higher than that on human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b).

[0113] Result 4: The inhibitory effect of anidulafungin in combination with other drugs on human prostate cancer POLR2A loss cells (PC3, LNCap, VCap) was significantly higher than that on human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b).

[0114] Result 5: The expression of POLR2A in human prostate cancer POLR2A loss cells (PC3, LNCap, VCap) was significantly lower than that in human prostate cancer POLR2A neutral cells (DU145, 22Rv1, NCIH660, MDAPCa2b), and vorinostat and anidulafungin could reduce the protein expression of POLR2A in the above cells.

[0115] Result 6: The inhibitory effect of vorinostat, anidulafungin, or the combination of these two drugs, or the combination with other drugs on the growth of xenografts of human prostate cancer POLR2A loss cells was significantly higher than that on xenografts of human prostate cancer POLR2A neutral cells.

[0116] Conclusion: POLR2A heterozygosity deletion or low expression is a predictive molecule for the sensitivity of histone deacetylase inhibitors such as vorinostat and echinocandin antifungal drugs such as anidulafungin against prostate cancer.

[0117] Predictive effect of POLR2A on the efficacy of vorinostat and anidulafungin on human acute leukemia POLR2A neutral cells (U937, HL-60) and human acute leukemia POLR2A loss cells (MV-4-11):

[0118] Experimental grouping:

[0119] Group 1: To detect the inhibitory effect of vorinostat and anidulafungin on the cell growth of human acute leukemia POLR2A neutral cells (U937, HL-60) and human acute leukemia POLR2A loss cells (MV-4-11), set the drug concentrations according to the preliminary experiment, and set the control group, drug group, solvent DMSO and / or blank control group respectively, and the treatment time was 24, 48 or 72 hours.

[0120] Group 2: To detect the inhibitory effect of vorinostat combined with other drugs on tumor cells, set the drug concentrations according to the preliminary experiment, and set the control group, drug group, solvent DMSO and / or blank control group respectively, and the treatment time was 24, 48 or 72 hours.

[0121] Detection method: CCK8 was used to detect cell viability and evaluate the inhibitory effect of drugs on the growth of tumor cells; Western Blot was used to detect the regulatory effect on POLR2A protein expression.

[0122] Group 3, mouse xenograft tumor model experiment. Mouse xenograft tumor models were established using POLR2A neutral leukemia cells and POLR2A loss leukemia cells. They were treated with different concentrations of histone deacetylase inhibitors, echinocandin antifungal drugs, or the combination of these two types of drugs, or combined with other drugs, and the tumor volume or weight was measured to evaluate the inhibitory effect of drugs on the growth of tumor cells; Western Blot or immunohistochemistry was used to detect POLR2A protein expression.

[0123] Results:

[0124] Result 1, Vorinostat showed a significantly higher inhibitory effect on human acute leukemia POLR2A loss cells (MV-4-11) than on human acute leukemia POLR2A neutral cells (U937, HL-60).

[0125] Result 2, The combined administration of Vorinostat and other drugs showed a significantly higher inhibitory effect on human acute leukemia POLR2A loss cells (MV-4-11) than on human acute leukemia POLR2A neutral cells (U937, HL-60).

[0126] Result 3, Anidulafungin showed a significantly higher inhibitory effect on human acute leukemia POLR2A loss cells (MV-4-11) than on human acute leukemia POLR2A neutral cells (U937, HL-60).

[0127] Result 4, The combined administration of Anidulafungin and other drugs showed a significantly higher inhibitory effect on human acute leukemia POLR2A loss cells (MV-4-11) than on human acute leukemia POLR2A neutral cells (U937, HL-60).

[0128] Result 5, The expression of POLR2A in human acute leukemia POLR2A loss cells (MV-4-11) was significantly lower than that in human acute leukemia POLR2A neutral cells (U937, HL-60). Vorinostat and Anidulafungin could reduce the protein expression of POLR2A in the above cells.

[0129] Result 6, The inhibitory effect of Vorinostat, Anidulafungin, or the combination of these two drugs, or the combined administration with other drugs on the growth of human leukemia POLR2A loss cell xenografts was significantly higher than that on human leukemia POLR2A neutral cell xenografts.

[0130] Conclusion: Loss of heterozygosity or low expression of POLR2A is a predictive molecule for the sensitivity of histone deacetylase inhibitors such as vorinostat and echinocandin antifungal drugs such as anidulafungin against acute leukemia.

[0131] The above embodiments found that loss of heterozygosity or low expression of POLR2A can predict the inhibitory effect of drugs on colon cancer, breast cancer, prostate cancer, and acute leukemia. Tumor cells with hemizygous deletion or low expression of POLR2A are more sensitive, and the anti-tumor effect of the drugs is better, providing a new method for the precision treatment of cancer.

[0132] However, this patent is not limited to the above cancers, and this drug is also applicable to the treatment of other tumor cells with hemizygous deletion or low expression of POLR2A.

[0133] The content clarified in the above embodiments should be understood that these embodiments are only used to illustrate the present invention more clearly, rather than to limit the scope of the present invention. After reading the present invention, various equivalent forms of modification of the present invention by those skilled in the art all fall within the scope defined by the appended claims of this application.

Claims

1. Use of a reagent for detecting POLR2A expression level as a reagent for preparing a reagent for predicting the efficacy of tumor treatment, characterized in that, in tumor cells with low POLR2A expression level, loss of heterozygosity or accompanied by p53 hemizygous deletion, the expression level of POLR2A is low. In these cells, the inhibitory effect of histone deacetylase inhibitors and echinocandin antifungal drugs on cancer is significantly higher than that of tumor cells with high POLR2A expression level; the tumors with low POLR2A expression level, loss of heterozygosity or accompanied by p53 hemizygous deletion include breast cancer, prostate cancer, colon cancer and acute leukemia; the histone deacetylase inhibitor is Vorinostat (SAHA), and the echinocandin antifungal drug is anidulafungin.

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