CN118109588A - Use of NPRL as target in cancer radiotherapy sensitization - Google Patents

Abstract

The invention relates to an application of NPRL as a target in cancer radiotherapy sensitization. The invention discovers NPRL that the nuclear penetration of the NPRL is one of key factors for causing the tolerance of radiotherapy, and simultaneously defines the specific action mechanism of the radiotherapy. While strategies using intranuclear deubiquitination inhibition can inhibit NPRL's 2 interaction with WDR24, maintain NPRL's 2 interaction with DEPDC5, and do not change over time, thereby blocking GATOR's invasion of GATOR1, ultimately resulting in NPRL2 locking onto lysosomal membranes with GATOR1 complex members. By detecting the expression level of NPRL < 2 > in the nucleus, reasonable evaluation of radiotherapy curative effect and prognosis can be realized; meanwhile, intervention of the deubiquitinase inhibitor can effectively inhibit nuclear entering behavior of NPRL so as to reduce radiation therapy tolerance and improve benefit of radiation therapy. The invention provides a potential strategy for colorectal cancer radiotherapy tolerance intervention scheme, and has important practical significance for clinically preparing an accurate treatment scheme for colorectal cancer.

Description

Application of NPRL2 as a target in cancer radiosensitization

Technical Field

The invention belongs to the field of biomedicine and relates to the application of NPRL2 as a target in cancer radiotherapy sensitization.

Background technique

With the increase in morbidity and mortality, malignant tumors (cancer) have long become a major public problem that seriously threatens the health of people in my country and other countries in the world. Colorectal cancer usually adopts the TNM staging system of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual. T describes the size of the primary tumor and the infiltration of surrounding tissues, N describes the involvement of surrounding and regional lymph nodes, and M describes distant metastasis. According to different stages, the treatment of colorectal cancer mainly adopts comprehensive treatment with surgery as the main treatment, and combines radiotherapy and chemotherapy to reduce tumor recurrence and metastasis. Colon cancer in stages I and II is mainly treated with colon cancer resection (84%), while two-thirds of stage III (some high-risk stage II) colon cancer patients use colorectal cancer resection combined with chemotherapy to reduce recurrence. Stage I rectal cancer uses proctocolectomy or proctocolectomy as the conventional treatment, and some high-risk patients are treated with radiotherapy or chemotherapy. Patients with II and III rectal cancer use neoadjuvant chemoradiotherapy combined with surgery and/or postoperative adjuvant chemotherapy. The 5-year survival rate of colorectal cancer is 65%, and the 10-year survival rate is 58%.

Locally Advanced Rectal Cancer (LARC) refers to rectal cancer with clinical stage T3/T4 or N+. According to the 2018 NCCN Rectal Cancer Guidelines, the standard principle of LARC treatment is: neoadjuvant chemoradiotherapy + surgery + adjuvant chemotherapy. Among them, there are two main radiotherapy options: one is long-course radiotherapy: 45-50Gy/25-28F, 5 days a week; the other is short-course radiotherapy: 25Gy/5F. Long-course radiotherapy is usually preferred, and short-course radiotherapy will only be selected after multidisciplinary consultation to reduce the tumor stage or when the toxicity of long-course radiotherapy is too high.

The Neoadjuvant Rectal Score (NAR) is a scoring system that weights clinical T stage (cT), pathological T stage (ypT) and N stage (ypN) after neoadjuvant chemoradiotherapy. In the CAO/ARO/AIO-04 Phase III clinical trial, it was confirmed that this indicator can be used to replace disease-free survival (DFS) as a new clinical research observation endpoint after neoadjuvant chemoradiotherapy for rectal cancer, which helps to judge the efficacy of neoadjuvant chemoradiotherapy. For the evaluation of the efficacy of neoadjuvant chemoradiotherapy for LARC, the TRG grading (tumor regression grade, TRG) is currently used more frequently. That is, after neoadjuvant chemotherapy, the postoperative tumor tissue specimen is sliced and H-E stained. After chemoradiotherapy, cells often have necrosis, ulceration, inflammatory cell infiltration, fibrosis, cell foamy degeneration and other changes. The proportion of residual tumor cells and the proportion of fibrosis infiltration in the tumor tissue are evaluated, and then the effect of neoadjuvant chemoradiotherapy is graded and evaluated to determine the degree of tumor regression.

Colorectal cancer mostly uses disease-free survival (DFS), overall survival (OS), and progression-free survival (PFS) as the endpoints of clinical research. Whether TRG can indicate clinical prognosis has always been controversial, because malignant tumors that are sensitive to radiotherapy and chemotherapy often proliferate rapidly, indicating a high degree of malignancy, and does not mean that a low TRG grade will eventually produce a survival benefit. In general, although it cannot be a key indicator for predicting prognosis, it can determine the sensitivity of neoadjuvant chemoradiotherapy and help screen patients who are tolerant to neoadjuvant chemoradiotherapy for possible intervention. Many studies have also shown that different responses to CRC treatment may be related to different genetic variations or gene expressions in tumor cells. Therefore, the combination of clinical parameters and molecular biomarkers can better predict and judge the effect of neoadjuvant therapy and the prognosis of CRC patients. However, there are few related genes or biomarkers that can predict the response to radiotherapy tolerance. Therefore, it is urgent to clarify the relevant mechanisms of colorectal cancer radiotherapy tolerance and find reliable genes or biological targets that can effectively predict the radiotherapy sensitivity of CRC patients.

Summary of the invention

The purpose of the present invention is to solve the problem in the prior art that CRC patients are prone to radiotherapy tolerance during the treatment process and cannot predict and evaluate the treatment effect well, thereby providing a key target NPRL2 for detecting and evaluating the sensitivity of cancer radiotherapy. By detecting the expression of NPRL2 in cells, especially in the nucleus, the radiotherapy tolerance can be predicted; at the same time, the nuclear entry process of NPRL2 can be inhibited by deubiquitinase inhibitors to improve the sensitivity to radiotherapy, thereby providing auxiliary diagnostic basis for the formulation of later treatment plans and prognosis evaluation.

In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

The first aspect of the present invention provides the use of NPRL2 in preparing a product for detecting the sensitivity of cancer radiotherapy.

Preferably, the cancer is selected from colorectal cancer; most preferably, the colorectal cancer is selected from locally advanced colorectal cancer.

A second aspect of the present invention provides use of a reagent for detecting NPRL2 expression level in the preparation of a product for detecting cancer radiotherapy sensitivity.

Preferably, the NPRL2 expression level is specifically the NPRL2 expression level in the cell nucleus.

Preferably, the cancer is selected from colorectal cancer; most preferably, the colorectal cancer is selected from locally advanced colorectal cancer.

Preferably, the reagent for detecting the expression level of NPRL2 includes primers for detecting the expression level of NPRL2 gene and/or a reagent for detecting the content of NPRL2 protein.

Preferably, the primers for detecting the expression level of the NPRL2 gene are selected from at least one of the following primer pairs:

Primer pair 1: the upstream primer sequence is as shown in SEQ ID NO: 1, which is 5'-CGAGGCTGTCTCTGAC AAGT-3', and the downstream primer sequence is as shown in SEQ ID NO: 2, which is 5'-GGGTGGAACTCGCTGAAGAA-3';

Primer pair 2: The upstream primer sequence is shown in SEQ ID NO: 3, which is 5’-TCGAGGCTGTCTCTGA CAAG-3’, and the downstream primer sequence is shown in SEQ ID NO: 4, which is 5’-ACGAAGCCCAGGTTGAAGAG-3’.

Preferably, the reagent for detecting the NPRL2 protein content is selected from NPRL2 monoclonal antibody and/or NPRL2 polyclonal antibody.

Preferably, the reagent for detecting the NPRL2 protein content is selected from NPRL2 (F-3) FITC (Santa Cruz Biotechnology, sc-376986), NPRL2 (D8K3X) Rabbit mAb (Cell Signaling Technology, 37344), and NPRL2 Polyclonal antibody (Proteintech, 10157-1-AP).

The third aspect of the present invention provides the use of a deubiquitinase inhibitor in the preparation of a product for inhibiting NPRL2 from entering the cell nucleus of a cell.

Preferably, the deubiquitinase inhibitor is selected from one or more of PT33, bortezomib, ixazomib and carfillizomib.

Preferably, the cells are selected from tumor cells; more preferably, the cells are selected from colorectal cancer cells; most preferably, the cells are selected from locally advanced colorectal cancer cells.

Preferably, the cells have been subjected to radiation therapy.

The fourth aspect of the present invention provides a kit for detecting cancer radiotherapy sensitivity, comprising a reagent for detecting the expression level of NPRL2; and one or more of a PCR enzyme, a PCR buffer, dNTPs, and a fluorescent substrate.

Preferably, the NPRL2 expression level is specifically the NPRL2 expression level in the cell nucleus.

Preferably, the cancer is selected from colorectal cancer; most preferably, the colorectal cancer is selected from locally advanced colorectal cancer.

Preferably, the cancer has been treated with radiation therapy.

Preferably, the reagent for detecting the expression level of NPRL2 includes primers for detecting the expression level of NPRL2 gene and/or a reagent for detecting the content of NPRL2 protein.

Preferably, the primers for detecting the expression level of the NPRL2 gene are selected from at least one of the following primer pairs:

Primer pair 1: the upstream primer sequence is as shown in SEQ ID NO: 1, which is 5'-CGAGGCTGTCTCTGAC AAGT-3', and the downstream primer sequence is as shown in SEQ ID NO: 2, which is 5'-GGGTGGAACTCGCTGAAGAA-3';

Primer pair 2: The upstream primer sequence is shown in SEQ ID NO: 3, which is 5’-TCGAGGCTGTCTCTGA CAAG-3’, and the downstream primer sequence is shown in SEQ ID NO: 4, which is 5’-ACGAAGCCCAGGTTGAAGAG-3’.

Preferably, the reagent for detecting the NPRL2 protein content is selected from NPRL2 monoclonal antibody and/or NPRL2 polyclonal antibody.

Preferably, the reagent for detecting the NPRL2 protein content is selected from NPRL2 (F-3) FITC (Santa Cruz Biotechnology, sc-376986), NPRL2 (D8K3X) Rabbit mAb (Cell Signaling Technology, 37344), and NPRL2 Polyclonal antibody (Proteintech, 10157-1-AP).

It should be understood that, in the context of the present invention, unless otherwise specified, the primers and/or primer pairs refer to PCR primers used to synthesize the cDNA chain of the NPRL2 gene in PCR, so as to detect the expression level of the NPRL2 gene mRNA. In addition to the primers and/or primer pairs listed in the present invention, those skilled in the art are fully capable of designing corresponding primers and/or primer pairs according to the gene sequence of NPRL2 using conventional methods and means in the art including but not limited to molecular biology, and screening the designed primers and/or primer pairs by conventional experimental means. In addition to designing primers to detect the expression level of NPRL2, those skilled in the art can also detect the expression level of NPRL2 in cells and nuclei by means of WB, IHC, etc. through reagents such as antibodies according to conventional methods and equipment in the art, as long as the specific detection of the expression level of NPRL2 can be achieved.

Radiotherapy uses the biological effects of ionizing radiation to directly target DNA, mainly causing DNA double-strand breaks (DSBs), that is, both strands of the double helix are damaged. DSBs are the most serious form of DNA damage, and if not repaired or repaired abnormally, they can directly lead to cell death. Ionizing radiation mainly kills malignant tumor cells through this mechanism, controls the growth of tumor cells, improves the local control rate of tumors, and then treats malignant tumors and improves the survival rate of tumor patients. DNA damage response refers to the process in which when DNA in cells is damaged, it is recognized by various receptor proteins and then activates cell cycle checkpoints (mainly occurring in G1/S and G2/M phases, and there are also cell cycle checkpoints in S phase), causing cell cycle arrest, cell division inhibition, and preventing the replication of harmful DNA, thereby giving cells enough time to repair DNA damage. DNA damage response is conducive to maintaining the stability of the genome.

Deubiquitination is mediated by deubiquitinating enzymes (DUBs), which decompose and release ubiquitin from target proteins. In this process, cells regulate the abundance and localization of deubiquitinating enzymes in cells through transcription, translation, and post-translational modification, thereby affecting their activity. Since protein ubiquitination plays an important role in regulating DNA repair, deubiquitinating enzymes can remove ubiquitin and naturally play an important role in regulating DNA repair.

Radiation-induced DNA double-strand breaks are usually repaired by error-prone non-homologous end joining (NHEJ) or error-free homologous recombination (HR). Both repair methods require the recruitment of a large number of cytokines to the break region to protect and isolate the break region and regulate DNA repair, and these processes are regulated by ubiquitination/deubiquitination. Although the effect of enhanced deubiquitination activity on the NHEJ or HR signaling pathway is still unknown, nuclear deubiquitinating enzymes are still potential therapeutic targets for improving the efficacy of radiotherapy by inhibiting the repair of radiation-induced DNA double-strand breaks. Current studies have pointed out that the dynamic balance between ubiquitination status and deubiquitination function in tumor cells is conducive to DNA damage repair, ultimately leading to tolerance to genotoxic anti-tumor treatment regimens. DUBs play a vital role in the normal life activities of the body. Changing the function of DUBs or abnormal DUBs function will induce the occurrence and development of various diseases, especially in cancer.

The present invention combines cell-level analysis with clinical verification to find that colorectal cancer radiotherapy mediates the improvement of the deubiquitination ability in the nucleus of tumor cells, and high ubiquitination levels in the nucleus of tumor cells often indicate better radiotherapy efficacy and better prognosis for patients with locally advanced rectal cancer. Based on this important judgment, in-depth research has found that radiotherapy can induce NPRL2, one of the members of GATOR1, to enter the nucleus and undergo nuclear translocation. After entering the nucleus, NPRL2 interacts with the carboxyl terminus of HERC2 containing the HECT domain, competitively inhibiting the interaction between HERC2 and BRCA1, thereby maintaining the stability and activity of BRCA1. The nuclear entry behavior of NPRL2 after radiation induction is regulated by GATOR2. Specifically, the radiation-induced GATOR2 complex or its subunits strip NPRL2 from the GATOR1 complex on the surface of the lysosome and release it into the cell nucleus. Deubiquitinase inhibitors can block the behavior of GATOR2 complex stripping NPRL2, lock NPRL2 in lysosomes, and maintain the integrity of GATOR1 complex in lysosomal membrane after radiation, prevent the interaction between NPRL2 and HERC2, and finally make the ubiquitination of BRCA1 by HERC2 in the nucleus no longer negatively regulated. At the same time, the present invention found through clinical analysis that the increase of radiation-induced nuclear localization of NPRL2 is related to radiotherapy tolerance and poor prognosis in patients with locally advanced rectal cancer receiving new radiotherapy, and the level of NPRL2 in the nucleus of tumor cells in patients with locally advanced rectal cancer receiving new adjuvant chemoradiotherapy, especially the level after radiotherapy, is significantly positively correlated with worse radiotherapy efficacy and poor prognosis. Therefore, it can be clearly stated that the increase of nuclear localization of NPRL2 induced by radiation is an important factor in radiation promoting HR-based DNA double-strand break repair and leading to radiotherapy tolerance.

In general, the present invention found that NPRL2 nuclear entry is one of the key factors causing radiotherapy tolerance, and clarified its specific mechanism of action, that is, radiotherapy-induced NPRL2 can be separated from GATOR1 with the assistance of GATOR2. Using the strategy of nuclear deubiquitinase inhibition, it was found that deubiquitinase inhibitors after radiotherapy can inhibit the interaction between NPRL2 and WDR24, maintain the interaction between NPRL2 and DEPDC5, and do not change over time, thereby hindering the invasion of GATOR2 into GATOR1, and ultimately causing NPRL2 to be locked on the lysosomal membrane as a member of the GATOR1 complex. By detecting the expression level of NPRL2 in the nucleus of the subject's cells, a reasonable evaluation of the radiotherapy efficacy and prognosis of the subject can be achieved; at the same time, the intervention of deubiquitinase inhibitors can effectively inhibit the nuclear entry of NPRL2, so as to reduce radiotherapy tolerance and enhance the benefit of radiotherapy to the subject. The present invention proposes a potential strategy for the intervention scheme of colorectal cancer radiotherapy tolerance, which has important practical significance for formulating a "precision treatment" scheme for colorectal cancer in clinical practice.

Compared with the prior art, the present invention has the following technical effects:

(1) The present invention has found through a large number of studies and screening that NPRL2 is a gene that is extremely related to colorectal cancer radiotherapy tolerance. In clinical research analysis and in vitro and in vivo functional analysis, it was found that a higher expression level of NPRL2 in the cell nucleus is significantly positively correlated with a worse radiotherapy efficacy and poor prognosis, which can be used to predict colorectal cancer radiotherapy tolerance and sensitivity, and provide an early assessment for patients to receive preoperative treatment. Deubiquitinase inhibitors can significantly inhibit NPRL2 from entering the cell nucleus, reduce the nuclear expression level, thereby reducing radiotherapy tolerance and improving the treatment effect.

(2) The present invention reveals the correlation between the NPRL2 gene and colorectal cancer radiotherapy tolerance, which has important practical significance for solving the problem of inter-individual differences in clinical efficacy and blanks in regression effect/prognosis assessment, and better realizing precision treatment. It provides a new drug treatment target for humans to conquer colorectal cancer, thereby providing a new direction for subsequent drug development, clinical treatment, etc., and has extremely high social value and market application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the silver staining results showing the interaction with HERC2 before and after radiotherapy.

FIG2 is a schematic diagram of the results of verifying the interaction between HERC2 and NPRL2.

FIG3 is a schematic diagram of the results of verifying the co-localization of NPRL2 into the nucleus and HERC2.

FIG4 is a schematic diagram of the analysis results of the binding region between HERC2 and NPRL2.

FIG5 is a schematic diagram showing the results of WB showing the effect of PT33 on the nuclear entry of radiation-induced NPRL2.

FIG6 is a schematic diagram showing the results of IF showing the effect of deubiquitinase inhibitors on the nuclear entry of radiation-induced NPRL2.

FIG. 7 is a schematic diagram showing the process results of the effect of deubiquitinase inhibitors on the nuclear translocation of radiation-induced NPRL2 and its interaction with HERC2.

FIG8 is a schematic diagram showing the results of IF showing the effect of deubiquitinase inhibitors on the localization of NPRL2 after radiation.

FIG. 9 is a schematic diagram showing the interaction results between GATOR1 and GATOR2 after radiotherapy.

FIG. 10 is a schematic diagram showing the time effect results of the interaction between GATOR1 and GATOR2 after radiotherapy.

FIG11 is a schematic diagram showing the effect of WDR24 deficiency on NPRL2 localization after radiotherapy.

FIG. 12 is a schematic diagram showing the comparison results of the nuclear levels of NPRL2 before and after neoadjuvant therapy.

FIG13 is a schematic diagram of the IHC scores and representative images of the nuclear levels of NPRL2 before and after neoadjuvant therapy.

FIG. 14 is a schematic diagram showing the effects of 5-FU and OXA on NPRL2 localization.

FIG15 is a schematic diagram showing the relationship between the nuclear level of NPRL2 and TRG in LARC patients before and after radiotherapy.

FIG16 is a schematic diagram showing the effect of NPRL2 nuclear level on TRG distribution in LARC patients before and after radiotherapy.

FIG17 is a schematic diagram showing the relationship between nuclear NPRL2 levels and prognosis in LARC patients before treatment.

FIG18 is a schematic diagram showing the relationship between nuclear NPRL2 levels and prognosis in LARC patients after treatment.

FIG19 is a schematic diagram of the results of univariate/multivariate regression analysis of OS in LARC patients (N=151, selected for analysis of cases before treatment).

FIG20 is a schematic diagram of the results of univariate/multivariate regression analysis of DFS in LARC patients (N=151, selected for analysis of cases before treatment).

FIG21 is a schematic diagram of the results of univariate/multivariate regression analysis of OS in LARC patients (N=102, selected for analysis of cases after treatment).

FIG22 is a schematic diagram of the results of univariate/multivariate regression analysis of DFS in LARC patients (N=102, selected for analysis of cases after treatment).

Detailed ways

In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

In the absence of special instructions, the cell lines such as HCT116 listed in the context of the present invention are cultured according to the prior art, and all cell lines are identified by short tandem repeat analysis of China Type Culture Collection (Wuhan), and PCR detection kit (Shanghai Biothrive Sci) is used to verify the presence of mycoplasma contamination, and frozen in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention are all commercially available. For the use of clinical specimens, informed consent forms were signed with patients, and relevant procedures and methods met the requirements of medical ethics and the quality management standards for drug clinical trials. The experimental methods used in the present invention, such as DNA extraction, whole genome sequencing, primer design, immunohistochemistry, Western blot, cell experiments, etc. are all conventional methods and techniques in the art.

Representative results from repeated biological experiments were presented in the context figures, and the data were presented as mean ± SD and mean ± SEM as specified in the figure. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 8.0 or SPSS22.0 software. Conventional medical statistical methods such as t-test, chi-square test, and analysis of variance were used to compare the mean differences between two or more groups. p < 0.05 was considered a significant difference.

Example 1

Studies have shown that the dynamic balance between the ubiquitination state and deubiquitination function in tumor cells is conducive to DNA damage repair, which ultimately leads to tolerance to genotoxic anti-tumor treatment regimens. Radiotherapy can mediate the improvement of the deubiquitination ability in the nucleus of tumor cells. High ubiquitination levels in the nucleus of tumor cells often indicate better radiotherapy efficacy and better prognosis in patients with locally advanced rectal cancer. Inhibition of deubiquitinase can achieve better nuclear deubiquitination inhibition ability, thereby reversing the generation of radiotherapy tolerance. HERC2 is an important regulatory factor in the process of deubiquitinase inhibitors inhibiting homologous recombination repair (HR). In order to further clarify which factors realize nuclear regulation to affect deubiquitinase inhibitors to promote HERC2 to play an important role in inhibiting HR, the process of HERC2 regulation was analyzed. First, the similarities and differences of HCT116 interacting proteins with HERC2 before and after radiotherapy were analyzed by Co-IP-silver staining-mass spectrometry (MS). The nuclear/cytoplasmic protein extraction method is as follows:

(1) Digest and collect the cells, wash them twice with PBS, carefully discard the supernatant, and try to aspirate as much of the supernatant as possible.

(2) Depending on the number of cells, add CER I, 200 μL per 2 × 10 ⁶ cells, and lyse on ice for 10 min.

(3) Add 11 μL of CER II, vortex for 5 seconds, and lyse on ice for 1 minute.

(4) Centrifuge at 16,000 × g for 5 min at 4°C. The supernatant is the cytoplasmic protein and should be aspirated for later use. The precipitate contains the cell nucleus.

(5) Add 100 μL of NER to the precipitate, blow off the precipitate, vortex for 15 seconds, and lyse on ice for 40 minutes, vortexing every 10 minutes.

(6) Centrifuge at 16,000 × g for 5 min at 4°C. The supernatant contains nuclear protein and should be aspirated for later use.

The specific steps of the silver staining experiment are as follows:

(1) Solution preparation: Fixative: add 25 mL of ethanol, 5 mL of acetic acid, and 20 mL of _ddH2O in sequence; 30% ethanol: add 5 mL of anhydrous ethanol to ₃₅ mL of _ddH2O ; Silver stain sensitizer: add 0.5 mL of silver stain sensitizer (100×) to 45.5 mL of _ddH2O . Use within 2 hours; Silver solution: add 0.5 mL of silver solution (100×) to 45.5 mL of ddH2O. Use within 2 hours; Silver stain developer: add 10 mL of silver stain basic developer (5×) to 40 mL of _ddH2O , then add 25 μL of silver stain developer acceleration solution (2000×). Use within 20 minutes; Silver stain stop solution: add 2.5 mL of silver stain stop solution (20×) to 47.5 mL of _ddH2O . Use within the same day after preparation.

(2) SDS-PAGE: First, perform gel electrophoresis on the protein sample according to conventional methods in the art.

(3) Fixation: After electrophoresis, place the gel in about 50 mL of fixative solution and shake on a vertical shaker at 60 rpm for 20 min at room temperature.

(4) Ethanol washing: discard the fixative, add 50 mL of 30% ethanol, and shake on a vertical shaker at 60 rpm for 10 min at room temperature.

(5) Water washing: discard 30% ethanol, add 50 mL ddH ₂ O, and shake on a vertical shaker at 60 rpm for 10 min at room temperature.

(6) Sensitization: Discard the water, add 50 mL of silver staining sensitization solution, and shake on a vertical shaker at 60 rpm for 2 min at room temperature.

(7) Silver staining: discard the sensitizing solution, add 50 mL of silver solution, and shake on a vertical shaker at 60 rpm for 1 min at room temperature.

(8) Water washing: discard the silver solution, add 50 mL of ddH ₂ O, and shake on a vertical shaker at 60 rpm at room temperature for 1 min, but not more than 1.5 min.

(9) Color development: Discard the water, add 50 mL of silver staining solution, and shake on a vertical shaker at 60 rpm at room temperature for 3-10 min until the expected ideal protein band appears. The reaction must be observed and stopped at any time.

(10) Termination: Discard the color development solution, add 50 mL of silver staining stop solution, and shake on a vertical shaker at 60 rpm for 10 min at room temperature. It is normal for gas to be generated during termination, and the gas generated is carbon dioxide.

(11) Water washing: discard the stopping solution, add ddH ₂ O, and shake on a vertical shaker at 60 rpm for 5 min at room temperature.

(12) Storage: The sample can be stored in ddH ₂ O or prepared into a dry gel using appropriate methods.

The results are shown in Figure 1. The results showed that after radiotherapy, a specific band with a molecular weight between 40-50kDa appeared. The band gel blocks were dug out for mass spectrometry detection and Co-IP+WB method verification one by one, and it was found that the binding degree of HERC2 and NPRL2 was significantly enhanced after radiotherapy (see Figure 2). NPRL2 is a member of the GATOR1 complex and is usually distributed in the cytoplasm. Analysis found that after radiotherapy, NPRL2 can enter the nucleus and co-localize with HERC2 in the nucleus (see Figure 3).

In order to explore the interaction region between HERC2 and NPRL2, several truncations were designed for interaction analysis based on the results of HERC2, and the results are shown in Figure 4. The results show that the binding domain is the HECT domain located at the carboxyl terminus (amino acids 4421-4834). Previous reports have described that the functional region of HERC2 binding to BRCA1 and other proteins is located in the HECT domain at the C-terminus. Therefore, it is speculated that NPRL2 can competitively bind to the ubiquitination catalytic region of HERC2.

Furthermore, in order to study the effect of deubiquitinase inhibitor treatment on NPRL2 nuclear entry after radiotherapy, HCT116 cells were treated with deubiquitinase inhibitor PT33 ((1E,6E)-4-(3-bromo-4-hydroxy-5-methoxybenzylidene)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,6-diene-3,5-dion e, CAS No. 2099022-84-9), and the cells were detected by WB or IF. The specific steps of the IF experiment are as follows:

(1) Reagent preparation: Blocking solution: first prepare 0.3% Triton-X 100 (v/v) with PBS, then measure 950 μL of 0.3% Triton-X-PBS and add 50 μL of normal goat serum; Antibody diluent: weigh 0.01 g of BSA and dissolve it with 1 mL of 0.3% Triton-X-PBS; Primary antibody: prepare the primary antibody with antibody diluent according to the antibody instructions; Secondary antibody: prepare fluorescent secondary antibody with antibody diluent at a ratio of 1:1000, and keep it away from light. Note that different species of secondary antibodies require different wavelengths of fluorescence; DAPI: prepare DAPI to 1 ug/mL with PBS.

(2) Cell treatment: HCT116 cells in the logarithmic growth phase were seeded at 1000 cells/well in a 6-well plate. Radiotherapy or radiotherapy plus drug intervention was performed 24 hours later. The intervention process was performed in a glass-bottom culture dish. After the intervention, the culture medium was discarded and the cells were washed three times with PBS.

(3) Fixation: Discard PBS, add 1 mL of paraformaldehyde, and fix at room temperature for 20 min.

(4) Washing: discard the paraformaldehyde, add 1 mL of PBS, and wash at room temperature for 5 min on a vertical shaker at 60 rpm. Repeat 3 times.

(5) Blocking: discard PBS, add blocking solution, and block at 37°C for 1 h.

(6) Primary antibody incubation: discard the blocking solution, add the primary antibody, cover the glass surface, and incubate at 4°C overnight. Note that when detecting two proteins at the same time, primary antibodies of different species should be used.

(7) Washing: Recover the primary antibody (can be reused 2-3 times), add 1 mL of PBS, and wash at room temperature for 5 min on a vertical shaker at 60 rpm. Repeat 3 times.

(8) Secondary antibody incubation: From this step onwards, all operations must be performed in a dark environment. Discard PBS, add the secondary antibody, cover the glass surface, and incubate at room temperature in a dark environment for 1 h.

(9) Washing: discard the secondary antibody, add 1 mL of PBS, and wash at room temperature in a vertical shaker at 60 rpm in the dark for 5 min. Repeat 3 times.

(10) Nuclear staining: discard PBS, add DAPI, cover the glass surface, and incubate at room temperature for 2 min away from light.

(11) Washing: discard DAPI, add 1 mL of PBS, and wash at room temperature for 5 min on a vertical shaker at 60 rpm in the dark. Repeat 3 times.

(12) Observe and take photos using a laser confocal microscope.

The specific steps of WB are as follows: HCT116 cells in the logarithmic growth phase were inoculated into a 6-well plate at 1000/well; radiotherapy or radiotherapy + drug intervention was performed 24 hours later; the original culture medium was discarded 24 hours after intervention, PBS was added for washing twice, cell lysis solution (including PMSF) was added, and lysis was carried out on ice for 30 minutes. The cells were scraped off with a cell scraper, transferred to a sterile centrifuge tube, centrifuged at 4°C and 12000rpm for 10 minutes, and the supernatant was taken to obtain the cell sample for WB experiment.

The experimental results are shown in Figures 5-6. The results show that after irradiation, the WB results of the DMSO-treated group showed that the NPRL2 nuclear entry effect was very obvious, and the IF results showed that NPRL2 was obviously separated from certain aggregation points in the cytoplasm, which was called diffuse distribution, and part of it entered the nucleus. However, after PT33 treatment, NPRL2 remained in the cytoplasm without dispersion or detachment, indicating that PT33 can inhibit the nuclear entry process of NPRL2 induced by radiation.

The above results have shown that NPRL2 can only interact with HERC2 in the nucleus after being induced by radiotherapy to enter the nucleus. The role of the deubiquitinase inhibitor PT33 is to restrict NPRL2 in the nucleus. By doing so, further verification at the cellular level showed that PT33 can indeed hinder the binding process of NPRL2 and HERC2 (see Figure 7). From the above, it can be clearly seen that radiotherapy can induce NPRL2 to enter the nucleus in colorectal cancer cells, allowing it to interact with HERC2 in the nucleus and maintain its important role in the DNA damage repair process.

Example 2

The GATOR1 complex includes multiple members including NPRL2, which has the function of activating Rag GTPase and inhibiting the localization of mTORC1 in lysosomes. Under normal circumstances, NPRL2 is mainly distributed in the cytoplasm to participate in regulating the activity of the mTORC1 pathway, and is concentrated on the lysosome. The above-mentioned embodiment found that after radiotherapy, the distribution of NPRL2 was diffuse and gradually entered the nucleus. Further analysis found that deubiquitinase inhibitors would inhibit the diffusion-nuclear entry process, causing NPRL2 to be locked on the lysosome (LAMP2 indicates lysosomal membrane localization) (see Figures 6 and 8). The GATOR2 complex is composed of members such as WDR24 and Seh1L. The complex has the function of negatively regulating GATOR1. This explores whether GATOR2 is involved in regulating the nuclear entry process of NPRL2.

First, overexpression of genes such as WDR24 and Seh1L was performed in CRC cells. The specific steps are as follows:

(1) A plasmid carrying the WDR24 or Seh1L gene was constructed according to conventional methods in the art.

(2) Take 1.5 mL of Opti-MEM medium, add 30 μL of Lipofectamine 3000, mix gently, and let stand for 5 min.

(3) Take another 1.5 mL of Opti-MEM medium, add the plasmid to be transfected and 40 μL of P3000 and mix well.

(4) After 5 minutes, the two were mixed, allowed to stand for 20 minutes, and then added to HCT116 cells.

The interaction between different proteins was detected by Co-IP+WB, and the results are shown in Figure 9. The results showed that when no intervention factors were applied, NPRL2 only bound to DEPDC5, not to the WDR24 subunit, while WDR24 interacted with She1L, indicating that GATOR1 and GATOR2 existed independently without intervention. However, after radiotherapy, the interaction between NPRL2 and GATOR2 subunits WDR24 and She1L was enhanced, and the interaction with DEPDC5 was weakened. It can be clearly seen that radiotherapy rapidly promoted the invasion of GATOR2 into GATOR1, and some subunits of GATOR2 interacted with some subunits of GATOR1 to dissociate the GATOR1 complex, among which NPRL2 was interacting with some subunits of GATOR2.

Further studies have found that as time passes after radiotherapy, the interaction between NPRL2 and WDR24 gradually weakens, but the interaction between NPRL2 and DEPDC5 does not recover over time (see Figure 10). Based on this, it can be inferred that the entire or partial members of GATOR2 can dissociate NPRL2 from GATOR1 after radiotherapy, providing a structural basis for its nuclear entry, and the detached NPRL2 then enters the nucleus to function, which can also be seen in Figure 6.

Furthermore, we used gene interference to knock down WDR24, an important regulatory factor of GATOR2, in order to observe the effect of GATOR2 destruction on NPRL2 (GATOR1) induced by radiotherapy. The specific steps are as follows:

(1) A plasmid carrying shWDR24 (the sequence is shown in SEQ ID NO: 5, CTTCATGAAGTGCTTTGACCT) was constructed according to conventional methods in the art.

(2) Take 1.5 mL of Opti-MEM medium, add 30 μL of Lipofectamine 3000, mix gently, and let stand for 5 min.

(3) Take another 1.5 mL of Opti-MEM medium, add the plasmid to be transfected and 40 μL of P3000 and mix well.

(4) After 5 minutes, the two were mixed, allowed to stand for 20 minutes, and then added to HCT116 cells.

(5) After 48 hours of transfection, discard the original culture medium, add PBS to wash twice, add cell lysis buffer (containing PMSF), and lyse on ice for 30 minutes. Scrape the cells with a cell scraper, transfer to a sterile centrifuge tube, centrifuge at 4°C and 12,000 rpm for 10 minutes, and take the supernatant to obtain the cell sample.

The above cell samples were taken for IF, and it was found that after the loss of WDR24, the localization of NPRL2 on the lysosome would not be affected by radiation, and the nuclear entry process would naturally be significantly inhibited (see Figure 11). This conclusion is consistent with the above, that is, NPRL2 must undergo the invasion of GATOR1 to leave the lysosome.

Example 3

The above experiments confirmed at the cellular level that NPRL2 is induced to enter the nucleus after radiotherapy. This example analyzes clinical samples to clarify whether NPRL2 also plays a key role in actual treatment.

(1) Pre-radiotherapy biopsy samples of locally advanced rectal cancer (LARC) from the Sun Yat-sen University Cancer Center were selected; for each selected case, the patient completed standard neoadjuvant chemoradiotherapy, followed by radical surgery, survived for more than 1 month after surgery, and had complete pathological data. In neoadjuvant therapy, all patients received conventional long-course radiotherapy (2 Gy/time/day, 5 times/week, 5 weeks, total dose of 50 Gy) and concurrent fluorouracil-based chemotherapy.

The tumor regression after neoadjuvant therapy was evaluated by TRG defined by the eighth edition of the AJCC grading system: grade 0 (complete response): no viable cancer cells remained; grade 1 (moderate response): single or small clusters of cancer cells remained; grade 2 (mild response): residual cancer foci, interstitial fibrosis; grade 3 (poor response): only a few or no cancer cells disappeared, large pieces remained. Patient tissue samples were staged according to the eighth edition of the AJCC grading system TNM stage and followed up regularly. Overall survival (OS): from the time of preoperative chemoradiotherapy to the time of death due to any cause or the last follow-up; progression-free survival (PFS): from the time of preoperative chemoradiotherapy to the time of the first disease progression, death due to any cause or the last follow-up. Paraffin sections were stored in a refrigerator at 4°C.

(2) The fresh biopsy tissue was embedded in paraffin and sealed. The paraffin specimens were stored at room temperature, and the cut white slices were stored in a refrigerator at 4°C. During the experiment, the paraffin slices were taken out, dewaxed with xylene, hydrated with gradient alcohol, removed peroxidase in the tissue with 0.3% _H2O2 solution, repaired with _citrate solution by microwave, incubated with primary antibody (1:200) and corresponding species secondary antibody, developed with DAB and hematoxylin, differentiated with hydrochloric acid and alcohol, dehydrated with gradient alcohol, permeabilized with xylene twice, and sealed with neutral gum.

IHC was used to detect the nuclear NPRL2 levels in tumor samples of patients with locally advanced rectal cancer (LARC) before (N=151) and after (N=102) neoadjuvant therapy. Overall, the results showed that the nuclear NPRL2 levels of LARC patients after neoadjuvant therapy were significantly higher than those before neoadjuvant therapy (see Figure 12, ***p<0.001).

Subsequently, paired tissues (N=69) with both before and after treatment and residual tumor cells after treatment were selected from the above clinical samples to further verify the situation of the same patient before and after treatment, and the results are shown in Figure 13. The results showed that, consistent with the above, after neoadjuvant therapy, the trend of NPRL2 nuclear level upregulation was very obvious.

Then, the effect of chemotherapy on NPRL2 nuclear entry was verified at the cellular level. The results of IF showed that the chemotherapy drugs 5-fluorouracil (5-FU) and oxaliplatin (OXA) treatment could not induce NPRL2 nuclear entry in CRC cells (HCT116) (see Figure 14). Based on this, it can be clearly seen that the driving force for NPRL2 nuclear entry is mainly the radiotherapy tolerance process.

Furthermore, the relationship between the nuclear level of NPRL2 before and after radiotherapy and the radiotherapy effect was clarified in clinical samples, and the results are shown in Figures 15-16. The results showed that before radiotherapy (N = 151), the nuclear level of NPRL2 in the TRG-1-3 combined group was significantly higher than that in the TGR-0 group (pathological complete remission) (see Figure 15), and in terms of case distribution, the high and low levels of NPRL2 in the nucleus had no significant effect on the distribution of TRG (see Figure 16). After radiotherapy (N = 102), the nuclear level of NPRL2 in the TRG-1 group was significantly lower than that in the TRG-2 or TRG-3 group (see Figure 15), and the cases with high TRG grades included more cases with high nuclear levels of NPRL2, and relatively fewer cases with low nuclear levels of NPRL2 (see Figure 16). The above results indicate that during clinical radiotherapy, the induction of NPRL2 into the nucleus may be an important cause of radiotherapy tolerance in LARC patients.

After clarifying the relationship with the efficacy of radiotherapy, it is necessary to further analyze the relationship between the nuclear level of NPRL2 before and after radiotherapy and prognosis. Survival analysis of clinical samples found that high levels of nuclear NPRL2 before radiotherapy were significantly associated with shorter overall survival time (p = 0.006), and the comparison results of the five-year survival rate were: 65.4% for high levels vs. 85.2% for low levels (see Figure 17). The high level of nuclear NPRL2 after radiotherapy was more significantly correlated with short overall survival time (p < 0.001), and the comparison results of the five-year survival rate were: 37.9% for high levels vs. 89.0% for low levels; and the high level of nuclear NPRL2 after radiotherapy was significantly correlated with shorter disease-free survival time (p = 0.013), and the comparison results of the five-year survival rate were: 21.5% for high levels vs. 30.6% for low levels (see Figure 18).

Finally, univariate and multivariate prognostic analysis of various clinical pathological parameters was performed. The results showed that the high level of nuclear NPRL2 before radiotherapy significantly prolonged DFS and OS in the other univariate and multivariate survival analyses, except for the multivariate survival analysis of OS. The high level of nuclear NPRL2 after radiotherapy significantly prolonged DFS and OS in the univariate and multivariate survival analysis, and was more significant than that before radiotherapy (see Figures 19-22). This shows that the high level of nuclear NPRL2 can indicate a poor prognosis, especially after treatment, and its nuclear level may directly reflect the DNA damage repair after radiotherapy.

Based on the above clinical analysis results, it is clear that radiotherapy does promote the nuclear entry of NPRL2, significantly increasing its nuclear level. Higher nuclear NPRL2 levels (especially in patients after radiotherapy) are associated with worse treatment effects, stronger tumor progression and shorter survival time. Clinical analysis further confirmed that high nuclear levels of NPRL2 in colorectal cancer are one of the key factors for radiotherapy tolerance.

The present invention combines clinical analysis with verification at the cellular level and finds that radiotherapy for colorectal cancer mediates the improvement of the deubiquitination ability in the nucleus of tumor cells. High ubiquitination levels in the nucleus of tumor cells often indicate better radiotherapy efficacy and better prognosis for patients with locally advanced rectal cancer. Based on this important judgment, in-depth research has found that radiotherapy can induce NPRL2, one of the members of GATOR1, to enter the nucleus and undergo nuclear translocation. After entering the nucleus, NPRL2 interacts with the carboxyl terminus of HERC2 containing the HE CT domain, competitively inhibiting the interaction between HERC2 and BRCA1, thereby maintaining the stability and activity of BRCA1. The nuclear entry behavior of NPRL2 after radiation induction is regulated by GATOR2. Specifically, the radiation-induced GATOR2 complex or its subunits strip NPRL2 from the GATOR1 complex on the surface of the lysosome and release it into the cell nucleus. Deubiquitinase inhibitors can block the behavior of GATOR2 complex stripping NPRL2, lock NPRL2 in lysosomes, and maintain the integrity of GATOR1 complex in lysosomal membrane after radiation, preventing the interaction between NPRL2 and HERC2, and finally making the ubiquitination of BRCA1 by HERC2 in the nucleus no longer negatively regulated. At the same time, the present invention found through clinical analysis that the increase of radiation-induced nuclear localization of NPRL2 is related to radiotherapy tolerance and poor prognosis in patients with locally advanced rectal cancer receiving new radiotherapy, and the level of NPRL2 in the nucleus of tumor cells of patients with locally advanced rectal cancer receiving new adjuvant chemoradiotherapy, especially the level after radiotherapy, is significantly positively correlated with worse radiotherapy efficacy and poor prognosis. Therefore, it can be clearly stated that the increase of nuclear localization of NPRL2 induced by radiation is an important factor in radiation promoting HR-based DNA double-strand break repair and leading to radiotherapy tolerance.

In general, the present invention found that NPRL2 nuclear entry is one of the key factors causing radiotherapy tolerance, and clarified its specific mechanism of action, that is, radiotherapy-induced NPRL2 can be separated from GATOR1 with the assistance of GATOR2. Using the strategy of nuclear deubiquitinase inhibition, it was found that deubiquitinase inhibitors after radiotherapy can inhibit the interaction between NPRL2 and WDR24, maintain the interaction between NPRL2 and DEPDC5, and do not change over time, thereby hindering the invasion of GATOR2 into GATOR1, and ultimately causing NPRL2 to be locked on the lysosomal membrane as a member of the GATOR1 complex. By detecting the expression level of NPRL2 in the nucleus of the subject's cells, a reasonable evaluation of the radiotherapy efficacy and prognosis of the subject can be achieved; at the same time, the intervention of deubiquitinase inhibitors can effectively inhibit the nuclear entry of NPRL2, so as to reduce radiotherapy tolerance and enhance the benefit of radiotherapy to the subject. The present invention proposes a potential strategy for the intervention scheme of colorectal cancer radiotherapy tolerance, which has important practical significance for formulating a "precision treatment" scheme for colorectal cancer in clinical practice.

The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims (10)

1. Use of nprl2 for the preparation of a product for detecting sensitivity to cancer radiotherapy.
2. 2. The use according to claim 1, wherein the cancer is selected from colorectal cancer.
3. 3. Use of an agent that detects NPRL levels of expression in the manufacture of a product for detecting sensitivity to cancer radiation.
4. 4. The use according to claim 1, wherein the NPRL2 expression level is in particular the intracellular NPRL2 expression level.
5. 5. The use according to claim 1, wherein the cancer is selected from colorectal cancer.
6. 6. Use of a deubiquitinase inhibitor for the preparation of a product for inhibiting NPRL a 2 from entering the nucleus of a cell.
7. 7. The use according to claim 6, wherein the deubiquitinase inhibitor is selected from one or more of PT33, bortezomib, ai Shazuo meters, carfilzomib.
8. 8. The use according to claim 7, wherein the cells are selected from tumor cells.
9. 9. The use according to claim 7, wherein the cells are subjected to a radiation therapy treatment.
10. 10. A kit for detecting sensitivity of radiation therapy for cancer comprising reagents for detecting NPRL expression levels; and one or more of PCR enzymes, PCR buffers, dNTPs, fluorogenic substrates.