Application of Deptor as a target in the preparation of drugs for enhancing radiosensitivity of head and neck cancer radiotherapy

Abstract

The present invention relates to the application of Deptor as a target in the preparation of a drug for enhancing the radiosensitivity of head and neck cancer radiotherapy. The present invention has conducted in-depth research on the correlation between Deptor and radiotherapy resistance of head and neck cancer, and found that the decrease in intracellular Deptor level caused by radiotherapy is one of the key reasons for the generation of radiotherapy resistance of head and neck cancer. By overexpressing Deptor or maintaining or increasing the Deptor protein level through a Deptor enhancer, the generation of radiotherapy resistance of head and neck cancer can be effectively reduced and the radiotherapy sensitivity can be improved. Through the analysis of clinical head and neck cancer patients, the present invention found that Deptor can be used as an independent factor for predicting the radiotherapy efficacy and evaluating the prognosis of clinical head and neck cancer patients. It has important practical significance for solving the problems of clinical efficacy differences among individuals with head and neck cancer and the blank of regression effect / prognosis evaluation, and for better realizing precise treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to the application of Deptor as a target in the preparation of a drug for enhancing the radiosensitivity of head and neck cancer radiotherapy. Background Art

[0002] Malignant tumors (cancers) are one of the main causes of death, an important obstacle to extending lifespan in countries around the world, and a major public health problem seriously threatening the health of people in countries around the world. In 2015, 4.292 million people in China were newly diagnosed with malignant tumors, and the mortality rate of malignant tumors accounted for 23.91% of all deaths.

[0003] The occurrence of malignant tumors is related to genetics and gene mutations caused by lifestyle and environmental factors, such as: smoking, obesity, unreasonable diet, lack of exercise, alcoholism, hormones, viral infections, etc. The occurrence and development of malignant tumors are related to the activation of oncogenes, the inactivation of tumor suppressor genes, as well as the regulation of non-coding RNAs, epigenetics, and stem cells, which in turn affect downstream signaling pathways. Currently, the main treatment methods for tumors include: comprehensive treatments such as surgery, chemotherapy, radiotherapy, biotherapy, and immunotherapy.

[0004] Head and neck tumors include three major parts: neck tumors, otolaryngology tumors, and oral and maxillofacial tumors. Neck tumors belong to general surgery in comprehensive hospitals, and the more common one is thyroid tumors; common otolaryngology tumors include laryngeal cancer, paranasal sinus cancer, etc.; common oral and maxillofacial tumors are various oral cancers, such as tongue cancer, gingival cancer, buccal cancer, etc. Therefore, the number of primary sites and pathological types of tumors occurring in the head and neck ranks first among all body tumors. More than 90% of head and neck tumors are squamous cell carcinomas. In the past 10 years, the incidence of head and neck squamous cell carcinoma (SCCHN) has increased significantly globally, especially in women.

[0005] In recent years, driven by continuous technological progress, radiotherapy-based comprehensive treatment is increasing and has become the main treatment method for head and neck cancer, while the utilization rate of surgical resection is also decreasing. In fact, compared with resection after radiotherapy, chemotherapy after radiotherapy has similar survival outcomes, but the quality of life has been greatly improved. Nevertheless, the prognosis of head and neck cancer is still very poor, and its overall survival rate is usually less than 50% after 3 - 5 years of treatment.

[0006] In addition, current radiotherapy decisions are usually based on clinical factors, surgical difficulty, and patient willingness, rather than tumor molecular characteristics or radiosensitivity. Therefore, tumors may be ineffective for radiotherapy due to re-resistance. At the same time, this not only leads to the possibility that patients may not benefit from radiotherapy and produce unbearable side effects, but also causes overtreatment in terms of resources. Understanding its mechanism or discovering some biomarkers of radioresistance will help provide new drug target information for future treatment, overcome radioresistance, make head and neck cancer cells sensitive to radiation, and reduce recurrence and metastasis after radiotherapy. Summary of the invention

[0007] The purpose of the present invention is to solve the problem of tolerance during radiotherapy of head and neck cancer in the prior art, thereby providing a new therapeutic target Deptor, which can effectively improve the sensitivity of head and neck cancer during radiotherapy and reduce the generation of tolerance by maintaining or increasing the expression level of Deptor in the body. And by detecting the expression of Deptor in the body, the radiotherapy tolerance of head and neck cancer can be predicted, providing auxiliary diagnostic basis for the formulation of later treatment plans and prognosis evaluation.

[0008] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0009] The first aspect of the present invention provides the use of Deptor protein and / or Deptor enhancer in the preparation of a drug for promoting radiosensitization of head and neck cancer.

[0010] Preferably, the Deptor enhancer includes but is not limited to an agent for maintaining the stability of Deptor and an agent for promoting overexpression of Deptor.

[0011] Preferably, the agent for maintaining the stability of Deptor is selected from the compounds shown in the following structure:

[0012] .

[0013] Preferably, the agent promoting Deptor overexpression is selected from a vector containing Deptor mRNA sequence.

[0014] Preferably, the Deptor mRNA sequence is shown as SEQ ID NO: 1.

[0015] Preferably, the head and neck cancer is selected from one or more of hypopharyngeal cancer, nasopharyngeal cancer, thyroid cancer, laryngeal cancer, and oral cancer.

[0016] Preferably, the radiotherapy is selected from photon beam radiotherapy.

[0017] Preferably, the photon radiotherapy is selected from one or more of X-ray radiotherapy and gamma-ray radiotherapy.

[0018] The second aspect of the present invention provides the use of a reagent for detecting the expression level of Deptor in the preparation of a product for detecting the radiosensitivity of head and neck cancer and / or predicting the curative effect.

[0019] Preferably, the reagent for detecting the expression level of Deptor includes primers for detecting the expression level of the Deptor gene and / or a reagent for detecting the content of the Deptor protein.

[0020] Preferably, the primers for detecting the expression level of the Deptor gene are selected from at least one of the following primer pairs:

[0021] Primer pair 1: The upstream sequence is as shown in SEQ ID NO: 2, and the downstream sequence is as shown in SEQ ID NO: 3;

[0022] Primer pair 2: The upstream sequence is as shown in SEQ ID NO: 4, and the downstream sequence is as shown in SEQ ID NO: 5;

[0023] Primer pair 3: The upstream sequence is as shown in SEQ ID NO: 6, and the downstream sequence is as shown in SEQ ID NO: 7;

[0024] Primer pair 4: The upstream sequence is as shown in SEQ ID NO: 8, and the downstream sequence is as shown in SEQ ID NO: 9;

[0025] Primer pair 5: The upstream sequence is as shown in SEQ ID NO: 10, and the downstream sequence is as shown in SEQ ID NO: 11;

[0026] Primer pair 6: The upstream sequence is as shown in SEQ ID NO: 12, and the downstream sequence is as shown in SEQ ID NO: 13;

[0027] Primer pair 7: The upstream sequence is as shown in SEQ ID NO: 14, and the downstream sequence is as shown in SEQ ID NO: 15;

[0028] Primer pair 8: The upstream sequence is as shown in SEQ ID NO: 16, and the downstream sequence is as shown in SEQ ID NO: 17;

[0029] Primer pair 9: The upstream sequence is as shown in SEQ ID NO: 18, and the downstream sequence is as shown in SEQ ID NO: 19;

[0030] Primer pair 10: The upstream sequence is as shown in SEQ ID NO: 20, and the downstream sequence is as shown in SEQ ID NO: 21.

[0031] Preferably, the reagent for detecting the content of Deptor protein is selected from Deptor monoclonal antibody and / or Deptor polyclonal antibody.

[0032] Preferably, the reagent for detecting the content of Deptor protein is selected from DEPTOR / DEPDC6 (D9F5) (Cell Signaling Technology, CST, catalog number 11816).

[0033] Preferably, the head and neck cancer is selected from one or more of hypopharyngeal cancer, nasopharyngeal cancer, thyroid cancer, laryngeal cancer, and oral cancer.

[0034] Preferably, the radiotherapy is selected from photon beam radiotherapy.

[0035] Preferably, the photon beam radiotherapy is selected from one or more of X-ray radiotherapy and γ-ray radiotherapy.

[0036] The non-lysosomal protein degradation process mediated by the ubiquitin-proteasome system is an important mechanism for the body to regulate intracellular protein levels and function. Through a large number of studies, the present invention found that radiotherapy treatment can lead to a decrease in the expression level of Deptor in head and neck cancer cells. By overexpressing Deptor or using a Deptor enhancer, the intracellular Deptor level reduced due to radiotherapy can be effectively restored. At the same time, it was also found that the restoration of Deptor after radiotherapy induced by the Deptor enhancer is related to proteasome-mediated protein degradation. By inhibiting protein degradation, the intracellular Deptor level can be effectively maintained. When Deptor in head and neck cancer cells is knocked out, it will promote the generation of radiotherapy tolerance in cells. However, when Deptor is overexpressed or a Deptor enhancer is used to maintain or increase the intracellular Deptor expression level, the emergence of tolerance in head and neck cancer cells during radiotherapy can be effectively reduced, thereby improving radiotherapy sensitivity.

[0037] In addition, through the analysis of clinical head and neck cancer patients, it was found that the level of Deptor expression in the body is closely related to the treatment effect and prognosis of the patients. Specifically, high expression of Deptor is significantly correlated with tumor regression, and is also closely related to a longer overall survival (OS) and progression-free survival (PFS). This means that Deptor can be used as an independent factor for predicting the radiotherapy efficacy and evaluating the prognosis of clinical head and neck cancer patients. For patients with low Deptor expression, methods such as using a Deptor enhancer in combination with radiotherapy can be adopted to stabilize the expression of Deptor, thereby improving the radiotherapy sensitivity and prognosis of the patients, and providing a more personalized and scientific treatment plan for the treatment of clinical head and neck cancer patients.

[0038] The present invention has the following technical effects compared with the prior art:

[0039] (1) The present invention has conducted in-depth research on the correlation between Deptor and radiotherapy tolerance in head and neck cancer, and found that the decrease in intracellular Deptor level caused by radiotherapy is one of the key reasons for the generation of radiotherapy tolerance in head and neck cancer. Overexpressing Deptor or maintaining or increasing the Deptor protein level through a Deptor enhancer can effectively reduce the generation of radiotherapy tolerance in head and neck cancer and improve radiotherapy sensitivity.

[0040] (2) The present invention has clarified the specific mechanism of action in the process of reducing radiotherapy tolerance in head and neck cancer mediated by Deptor, and clarified the related targets and pathways that interact with it, laying a theoretical foundation for further in-depth research and development in the future.

[0041] (3) Through the analysis of clinical head and neck cancer patients, the present invention found that Deptor can be used as an independent factor for predicting the radiotherapy efficacy and evaluating the prognosis of clinical head and neck cancer patients. It has important practical significance for solving the problems of differences in clinical efficacy among individuals with head and neck cancer and the blank of regression effect / prognosis evaluation, and provides a new direction for better realizing precision treatment. It provides a new drug treatment target for humans to overcome head and neck cancer, and thus provides a new direction for subsequent drug research and development, clinical treatment, etc., and has extremely high social value and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the results of detecting the effect of XR on endogenous and exogenous Deptor by WB.

[0043] Figure 2 Schematic diagram of the results of detecting the effect of a Deptor enhancer on Deptor after XR by WB.

[0044] Figure 3 Schematic diagram of the results of detecting the effect of a Deptor enhancer on Deptor after XR after CHX treatment by WB.

[0045] Figure 4 Schematic diagram of the results of detecting ubiquitinated Deptor at K48 by Co-IP and WB.

[0046] Figure 5 Schematic diagram of the results of the flat-plate cloning experiment of the 2D Fadu cell model.

[0047] Figure 6 Schematic diagram of the treatment of XR combined with a Deptor enhancer in two different modes in the conditional 3D cell sphere culture model.

[0048] Figure 7Schematic diagram of the effect of XR combined with a Deptor enhancer on tumor spheroid formation on Day 4 in a conditional 3D cell spheroid culture model.

[0049] Figure 8 Schematic diagram of the effect of XR combined with a Deptor enhancer on tumor spheroid formation on Day 1 in a conditional 3D cell spheroid culture model.

[0050] Figure 9 Schematic diagram of the flow cytometry results of the effect of XR combined with a Deptor enhancer on cell apoptosis after 72 h of cell treatment.

[0051] Figure 10 Schematic diagram of the quantitative results of the effect of XR combined with a Deptor enhancer on cell apoptosis after 72 h of cell treatment.

[0052] Figure 11 Schematic diagram of the results of the effect of a Deptor enhancer on tumor mass in mice under radiotherapy treatment.

[0053] Figure 12 Schematic diagram of the tumor mass in mice under radiotherapy treatment with a Deptor enhancer.

[0054] Figure 13 Schematic diagram of the results of detecting the Deptor knockout efficiency in cells by WB.

[0055] Figure 14 Schematic diagram of the results of the effect on cell survival rate in a 2D plate cloning experiment under different doses of radiotherapy treatment in Deptor-knockout cells.

[0056] Figure 15 Schematic diagram of the cell survival status in a 2D plate cloning experiment under 0 Gy radiotherapy treatment in Deptor-knockout cells.

[0057] Figure 16 Schematic diagram of the cell survival status in a 2D plate cloning experiment under 3 Gy radiotherapy treatment in Deptor-knockout cells.

[0058] Figure 17 Schematic diagram of the cell survival status in a 2D plate cloning experiment under 6 Gy radiotherapy treatment in Deptor-knockout cells.

[0059] Figure 18 Schematic diagram of the cell survival status in a 2D plate cloning experiment under 0 Gy radiotherapy treatment in Deptor-knockout cells obtained using different sgRNAs.

[0060] Figure 19Schematic diagram of cell survival status in the 2D plate cloning experiment under 3 Gy radiotherapy treatment in cells with Deptor knocked out using different sgRNAs.

[0061] Figure 20 Schematic diagram of the results of the ratio of the number of clones (KO / NC) in the 2D plate experiment under different doses of radiotherapy in cells with Deptor knocked out.

[0062] Figure 21 Schematic diagram of the results of the effect of Deptor enhancer on the cell survival rate in the 2D plate cloning experiment of Deptor-knockout cells under XR treatment.

[0063] Figure 22 Schematic diagram of the cell survival status in the 2D plate cloning experiment of Deptor-knockout cells under XR treatment with Deptor enhancer.

[0064] Figure 23 Schematic diagram of the results of the analysis of the expression level of Deptor in patients with head and neck cancer using the Oncomine and TCGA databases.

[0065] Figure 24 Schematic diagram of the MRI images of patients with hypopharyngeal cancer before and after radiotherapy and chemotherapy and the corresponding immunohistochemical results of Deptor before treatment.

[0066] Figure 25 Schematic diagram of the results of the overall survival and progression-free survival of patients with high and low expression of Deptor analyzed by Kaplan-Meier.

[0067] Figure 26 Schematic diagram of the results of the PFS survival factors of patients with hypopharyngeal cancer analyzed by univariate and multivariate survival analysis.

[0068] Figure 27 Schematic diagram of the results of the OS survival factors of patients with hypopharyngeal cancer analyzed by univariate and multivariate survival analysis. Detailed implementation manners

[0069] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the 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.

[0070] Unless otherwise specified, cell lines such as Fadu and Detroit 562 listed in the context of the present invention were purchased from the American Type Culture Collection (ATCC), stored in our laboratory, cultured according to the ATCC guidelines, and all cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan) and verified for mycoplasma contamination using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen and used for subsequent experiments. All reagents used in the present invention were commercially available. For the use of clinical specimens, informed consent forms were signed with the patients, and the relevant procedures and methods complied with the requirements of medical ethics and Good Clinical Practice. The experimental methods used in the present invention, such as DNA extraction, whole-genome sequencing, primer design, vector construction, co-immunoprecipitation, immunohistochemistry, Western blot, flow cytometry, cell experiments, animal experiments, etc., were all conventional methods and techniques in the art.

[0071] Representative results from biological experiment replicates were presented in the context figures, and data were shown as mean±SD and mean±SEM as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 20.0 software. The t-test or analysis of variance was used to compare the mean differences between two or more groups. A p < 0.05 was considered a significant difference.

[0072] Example 1 Effect of radiotherapy on the level of Deptor in cells

[0073] (1) Using the mRNA of Deptor (sequence shown in SEQ ID NO: 1) as a template, reverse-transcribe it into cDNA, and then amplify it by PCR;

[0074] (2) Digest, purify the obtained amplification product and then ligate it to a plasmid, and then transform it into competent Escherichia coli cells for identification;

[0075] (3) After correct plasmid identification, expand the corresponding Escherichia coli, obtain a large number of bacteria, and then use an endotoxin-free plasmid extraction kit to obtain the target plasmid;

[0076] (4) Functionally transfect the target plasmid and lentivirus into 293T cells, collect the culture medium after culturing for 48 h, and filter the cell debris to obtain the virus solution;

[0077] (5) Inoculate Fadu cells in the logarithmic growth phase into a culture dish. After they are completely adherent, discard the original culture medium, add the virus solution and polybrene to promote virus infection. After 24 h, discard the virus solution, replace it with fresh culture medium, and continue culturing for 24 h;

[0078] (6) After 48 h of infection, discard the culture medium, use puromycin to screen the stable transfected cell line overexpressing Deptor, and identify it by qPCR or WB;

[0079] (7) Respectively take the above-mentioned Fadu cells overexpressing Deptor and wild-type Fadu cells, perform radiation treatment (6 Gy) with X-rays, harvest cell samples at 0, 8, 12, and 24 hours after treatment, and detect the expression levels of endogenous Deptor and exogenous Flag-Deptor in the cells by Western Blot.

[0080] The detection results are as Figure 1 shown. The results show that after radiotherapy, the expression level of Deptor in Fadu cells will be significantly reduced, and for both endogenous Deptor and exogenous Flag-Deptor, they show a decreasing trend in a time-dependent manner within a certain period of time.

[0081] Furthermore, investigate the effect of whether the intracellular Deptor level can be restored by exogenous drug stimulation. The specific steps are as follows:

[0082] (1) Take Fadu cells in the logarithmic growth phase and divide them into two groups. Group 1 is added with compound 1 (100 nM) with the following structural formula, and group 2 is added with an equal volume of solvent DMSO as a control; ;

[0083] (2) Respectively perform radiation treatment (6 Gy) on the cells in group 1 and group 2, and harvest cell samples at 0, 5 min, 4 h, 8 h, 12 h, and 24 h after treatment, and detect the changes in the expression level of Deptor in them by WB.

[0084] The results are as Figure 2 shown. The results show that after radiation treatment with X-rays, the expression level of Deptor in Fadu cells shows a time-dependent decreasing trend within a certain period of time, which is consistent with the above results; and after adding compound 1, it can significantly improve the decrease in the intracellular Deptor level caused by radiation, maintain the intracellular stability of Deptor, and even increase the expression level of Deptor, that is, it can be used as an enhancer of Deptor.

[0085] To investigate the mechanism by which the above-mentioned Deptor enhancer maintains the stability of intracellular Deptor expression, cycloheximide (CHX) was used for tracking analysis. That is, 2 hours before the start of radiotherapy, CHX was added to inhibit the ribosomal protein synthesis function in cells, and then the time-course after radiotherapy was examined. The experiment was carried out by the method of Western Blot, and the results are as Figure 3 shown. The results showed that after radiotherapy, compared with the DMSO control group, the increase in Deptor after treatment with Compound 1 was mainly due to the increase in the half-life of Deptor.

[0086] Example 2 Effect of stabilizing the intracellular Deptor expression level on the radiosensitivity of head and neck cancer cells

[0087] To verify whether the restoration of Deptor after radiotherapy induced by the Deptor enhancer is dependent on proteasome-mediated protein degradation, HA-Ubiquitin (K48 site-specific) was further transfected into the Fadu Flag-Deptor stable cells (the construction method is shown in Example 1), and then the CO-IP immunoprecipitation experiment was carried out. The specific steps are as follows:

[0088] (1) Reagent preparation: Cell lysis buffer: Dilute 10× cell lysis buffer to 1× with ddH 2 O and add protease inhibitors and phosphatase inhibitors; Moderate salt concentration TBS: Weigh 6 g of NaCl and add it to 1 L of TBS solution; 1× loading buffer: Dilute 5× loading buffer to 1× with ddH2O for later use;

[0089] (2) Protein extraction: Log-phase Fadu cells (Group 1 was treated with 100 nM of Compound 1, Group 2 was treated with an equal volume of DMSO, and the two groups of cells were respectively treated with X-ray radiation (6 Gy)) were digested and collected at different time points, washed twice with PBS to remove residual culture medium; Add an appropriate amount of cell lysis buffer; Lyse on ice for 1 h; Centrifuge at 15000×g at 4℃ for 15 min, and take the supernatant for later use; Determine the protein concentration with a BCA protein concentration detection kit; Reserve 30 μL of the protein solution as the total protein sample, add 7.5 μL of loading buffer (5×), and incubate in a water bath at 95℃ for 5 min for later use;

[0090] (3) Primary antibody incubation:

[0091] ① If the target protein carries a Flag tag, take 30 μL of M2 anti-flag agarose, add 1 mL of pre-cooled TBS solution, then centrifuge at 5000×g for 1 min at 4°C, discard the supernatant, then add the protein solution, and invert overnight at 10 - 12 rpm on a vertical shaker at 4°C;

[0092] ② If the target protein does not carry a Flag tag, add the IP-grade antibody of the target protein (1 μg antibody : 1 mg protein) to the protein solution, and invert overnight at 10 - 12 rpm on a vertical shaker at 4°C. The next day, add 50 μL of Protein GPLUS-Agarose to each sample and incubate at 10 - 12 rpm on a vertical shaker at 4°C for 3 - 4 h.

[0093] (4) Wash the beads: Centrifuge the above samples at 3000×g for 5 min at 4°C, carefully aspirate the supernatant, taking care not to aspirate the beads; add 1 mL of medium-salt-concentration TBS, wash at medium speed on a vertical shaker at 4°C for 5 min, then centrifuge at 3000×g for 5 min at 4°C, carefully discard the supernatant, and repeat at least 5 times;

[0094] (5) Add 40 μL of 1×loading buffer to each sample, incubate in a 95°C water bath for 5 min. For IP Flag, then detect ubiquitination of K48-linked Deptor by IB.

[0095] The detection results are as Figure 4 shown. The results show that at 12 h after radiotherapy, the expression of ubiquitinated K48-linked Deptor reaches a peak. Although the expression decreases afterwards, it still persists until 24 h, which is a unidirectional ubiquitin-degradation process; this process can be blocked by a Deptor enhancer, thereby maintaining the expression of ubiquitinated and non-ubiquitinated Deptor at K48. It is thus presumed that the recovery of Deptor after radiotherapy induced by the Deptor enhancer is related to proteasome-mediated protein degradation. By inhibiting protein degradation, the intracellular level of Deptor can be effectively maintained, thereby enhancing the sensitivity of tumor cells to radiotherapy.

[0096] To verify this, the following experiments were conducted:

[0097] Inoculate exponentially growing Fadu cells at a density of 1000 cells per well in a 6-well plate; after 24 hours, perform radiotherapy (XR) or radiotherapy (XR) + drug (Compound 1, 100 nM) intervention; continue culturing for 10 - 14 days; discard the culture medium, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol, wash the remaining methanol with water, and cell clones can be observed; under a microscope, only when the number of cells > 50 is counted as a valid clone, count the total number of clones and the cell survival fraction in each group. The calculation method of the cell survival fraction is as follows: Cell survival fraction = (number of clones in the intervention group / number of clones in the control group) × 100%.

[0098] The experimental results are as Figure 5 shown. The results show that after using the Deptor enhancer Compound 1, the number of colony-forming units of Fadu cells after radiotherapy can be significantly reduced, cell proliferation can be inhibited, and the sensitivity to radiotherapy can be increased.

[0099] Subsequently, a 3D cell sphere formation experiment was used to verify the effects of Deptor and the Deptor enhancer on the radiosensitivity of cells. The specific steps are as follows:

[0100] Take exponentially growing Fadu cells with mCherry fluorescence in 2D adherent growth, digest them, filter them through a mesh to form a single-cell suspension, count the cells, and inoculate them in a low-adhesion 6-well plate at a density of 1000 - 4000 cells per well. Place the culture dish in an incubator at 37°C and 5% CO2, and treat the cells according to the experimental plan (the treatment method is shown in Figure 6 ), supplement 500 µL of culture medium every 3 - 4 days, culture for 14 days, observe the number and diameter of tumor spheres under an inverted fluorescence microscope, measure the number and diameter of the spheres using ImageJ software, and count the number of valid tumor spheres with a diameter > 75 µm in each group.

[0101] The experimental results are as Figures 7 - 8 shown. The results show that whether it is on Day 4 ( Figure 7 ) or Day 1 ( Figure 8)Treat the cells. Compared with the single-treatment groups of XR or Compound 1, the combination of XR and Compound 1 can more significantly inhibit the number of tumor spheroids formed by Fadu cells. At the same time, we calculated the Q value to analyze the synergistic effect of the combination of XR and Compound 1. Q value = EAB / [EA + EB(1 - EA)] (EA, EB, and EAB represent the inhibition rates of Compound 1, XR, and Compound 1 + XR respectively: Q < 0.85, 0.85 ≤ Q ≤ 1.15, or Q > 1.15 represent antagonism, additive effect, or synergistic effect respectively. It is worth noting that whether on Day 4 or Day 1, Compound 1 (5 or 10 nM) can synergistically increase the inhibition rate of XR on tumor spheroid formation (all Q > 1.15).

[0102] Subsequently, the role of Deptor in the radiosensitivity of tumor cells was verified by apoptosis experiments, and the results are as Figures 9 - 10 shown. The results showed that in Fadu and Detroit 562 cells, Compound 1 significantly increased apoptosis induced by XR. In Fadu cells, the average apoptosis ratio of cells increased from 23.29% to 37.78%; in Detroit 562 cells, the average apoptosis ratio of cells increased from 13.61% to 19.77%. However, single treatment with Compound 1 had almost no effect on cell apoptosis. We also calculated the combined effect of XR and Compound 1 in promoting cell apoptosis using the Q value, and in both cells, all Q > 1.15.

[0103] Furthermore, in vivo experiments were conducted to clarify the anti-tumor effect of Deptor and its enhancer in promoting radiotherapy on head and neck cancer cells. The specific steps are as follows:

[0104] (1) Take 2 - 3 female Balb / c nude mice aged 4 - 6 weeks, inoculate Fadu cells (1 - 5×106 per mouse) subcutaneously on their backs, and wait for the tumor to grow to an appropriate size;

[0105] (2) Remove the above tumors, process them into several tumor pieces about 5 mm 3 in size for standby, pay attention to removing necrotic tissues, and select parts with good blood supply;

[0106] (3) Take another 60 female Balb / c nude mice aged 4 - 6 weeks, randomly divide them into 4 groups, numbered as Group 1 - 4, 15 mice per group. After anesthesia, transplant the tumor pieces subcutaneously on the back to establish a xenograft model, which can reduce the tumor heterogeneity;

[0107] Intervention began 7 days after transplantation. Group 1 was the blank control group, without drug treatment or radiotherapy; Group 2 was given Compound 1 (1 mg / kg·day, once every other day) without radiotherapy; Group 3 received radiotherapy (1 Gy / day, once every other day) without drug treatment; Group 4 received alternating intervention with Compound 1 (1 mg / kg·day) and radiotherapy (1 Gy / day); the total treatment cycle for each group was 2 weeks;

[0108] (5)The mice were sacrificed after 1 month, and the tumor masses were collected.

[0109] The experimental results were as Figures 11 - 12 shown. The results showed that the single treatment group of Compound 1 had no significant effect on the tumor growth of mouse tumors (P>0.05). Consistent with the in vitro experiment, compared with the single XR treatment group, Compound 1 combined with XR could significantly reduce the tumor volume. It is worth noting that the tumor inhibition rates of XR combined with Compound 1 were 86.63% respectively, while the tumor inhibition rate of the single XR group was 51.21%. In addition, the combination of Compound 1 and XR also had a synergistic effect (Q = 1.46).

[0110] Based on the above experimental results, it can be clearly seen that the decrease in Deptor expression level is one of the key reasons for the generation of radiotherapy tolerance in head and neck cancer. By increasing the intracellular Deptor protein expression level or using enhancers to stabilize or increase the Deptor level, the sensitivity of head and neck cancer cells to radiotherapy can be effectively improved, and the generation of radiotherapy tolerance can be reduced. The recovery of Deptor after radiotherapy induced by the Deptor enhancer is related to proteasome-mediated protein degradation. By inhibiting protein degradation, the intracellular Deptor level can be effectively maintained. For this, other active ingredients (Ixazomib, Bortezomib, etc.) with certain inhibitory effects on proteasome were further selected to repeat the above cell and in vivo experiments, and the results were similar to those of Compound 1 (not shown in the figure), that is, they could effectively maintain or even increase the intracellular Deptor level, prevent the degradation of Deptor during radiotherapy, and promote the sensitivity of head and neck cancer cells to radiotherapy.

[0111] Example 3 Effect of Deptor knockout on radiotherapy tolerance of head and neck cancer cells

[0112] In order to explore whether Deptor deletion would affect the radiotherapy tolerance process, the following experiment was carried out:

[0113] (1)Using the Crispr cell knockout technique, a stable cell line with Deptor knockout was constructed in Fadu cells (the sgRNA1 sequence used was as shown in SEQ ID NO: 22; the sgRNA2 sequence was as shown in SEQ ID NO: 23); and the efficiency was verified by WB (see Figure 13 );

[0114] (2) Perform a 2D cell colony formation assay using the knocked-out Fadu cells: Seed the cells in the logarithmic growth phase at 1000 cells / well in a 6-well plate; after 24 hours, perform radiotherapy or radiotherapy + drug intervention; continue culturing for 10 - 14 days; discard the medium, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol, and wash the remaining methanol with water to observe cell colonies; under a microscope, only colonies with >50 cells are counted as a valid clone. Count the total number of colonies and the cell survival fraction in each group. The calculation method for the cell survival fraction is as follows: Cell survival fraction (Survival fraction) = number of colonies in the intervention group / number of colonies in the control group × 100%.

[0115] The results are as Figures 14 - 19 shown. In the figure, the Mock group is wild-type Fadu cells without Deptor knockout treatment; the NC group is Fadu cells transfected with a blank vector. The results show that after moderate-dose radiotherapy (3 Gy) in cells with Deptor knocked out, the radiotherapy tolerance is significantly increased. When treating cells with Deptor knocked out with a higher dose (6 Gy), the cells will still improve the radiotherapy tolerance.

[0116] By calculating the ratio of the number of colonies in the DEPTOR KO group to the NC group in each radiotherapy dose group, at 0 Gy, DEPTOR KO / NC > 1. Combining the above data, it can be seen that knocking out DEPTOR itself can promote the proliferation of Fadu cells. After radiotherapy, DEPTOR KO / NC = 3 - 4, which is consistent with the radiotherapy tolerance caused by knocking out DEPTOR in the cell survival experiment (see Figure 20 ).

[0117] Subsequently, in wild-type and Deptor-knocked-out Fadu cells, after treatment with the Deptor enhancer compound 1 (100 nM), perform a 2D plate colony assay. The results are as Figures 21 - 22 shown. The results show that in Deptor wild-type cells, the Deptor enhancer has an obvious radiotherapy sensitizing effect, while in Deptor-deficient cells, the Deptor enhancer almost loses the ability to sensitize radiotherapy.

[0118] The above experiments together show that Deptor is very closely related to the generation of radiotherapy tolerance in head and neck cancer cells. Knocking out Deptor can promote the generation of radiotherapy tolerance in head and neck cancer cells. While overexpressing Deptor, or using a Deptor enhancer to maintain or increase the expression level of Deptor in cells, can effectively reduce the appearance of tolerance in head and neck cancer cells during radiotherapy.

[0119] Study on the Correlation between Deptor Expression Level and Radiotherapy Tolerance and Prognosis in Head and Neck Cancer Patients

[0120] By using the Oncomine Cancer Microarray database to analyze the overall expression level of Deptor in different tumors, and at the same time using the Oncomine and TCGA databases to analyze the difference in the expression level of Deptor between normal head and neck tissues and head and neck squamous cell carcinoma. It was found that the expression level of Deptor in head and neck squamous cell carcinoma was significantly lower than that in normal tissues, indicating that the low expression of Deptor may be related to the occurrence and development of head and neck squamous cell carcinoma (see Figure 23 ).

[0121] The above results together indicate that the Deptor gene plays an important role in head and neck cancer, and its low expression is closely related to radiotherapy tolerance. To further verify this conclusion, biopsy specimens of hypopharyngeal cancer patients from the First Affiliated Hospital of Sun Yat-sen University (N = 63) were collected, and these patients subsequently received standard radical radiotherapy. Then, immunohistochemistry was used to detect the expression level of Deptor in these hypopharyngeal cancer tissues. Among them, 28 patients had high expression of Deptor (cut-off ≥ 12), and 35 patients had low expression of Deptor (cut-off < 12). After combined radiotherapy and chemotherapy, according to the RECIST 1.1 solid tumor evaluation criteria, 15, 16, 24, and 16 patients achieved complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), respectively. CR + PR was defined as objective response (OR), which represents the sensitivity of the tumor to radiotherapy and chemotherapy.

[0122] Clinical analysis showed that among 27 hypopharyngeal cancer patients with low expression of DEPTOR, 8 achieved OR (13.3%), and among 33 hypopharyngeal cancer patients with high expression of DEPTOR, 23 achieved OR (38.3%). At the same time, the correlation of clinical parameters was analyzed, and it was found that high expression of DEPTOR was related to OR (P = 0.002) and better prognosis (P = 0.002), as shown in Table 1 below.

[0123] Table 1 Correlation between DEPTOR Expression and Clinical Parameters of Samples before Radiotherapy and Chemotherapy

[0124]

[0125]

[0126] Subsequently, MRI images of hypopharyngeal cancer patients before and after chemoradiotherapy were collected, and RECIST 1.1 was used as the evaluation criterion for solid tumors. The results are as Figure 24 shown. It can be seen from the MRI images that generally, the tumors of patients with high expression of Deptor regressed better.

[0127] By analyzing the survival of hypopharyngeal cancer patients who received a standardized radiotherapy regimen, it was found that high expression of DEPTOR was associated with a longer overall survival (OS) (P<0.001) and progression-free survival (PFS) (P<0.001) (see Figure 25 ).

[0128] In the univariate survival analysis, induction chemotherapy (OS P =0.044), concurrent chemotherapy (OS P =0.022), smaller tumor size (OS P =0.03), OR after chemoradiotherapy (OS P=0.028; PFS P =0.018), and high expression of DEPTOR (OS P=0.028; PFS P <0.001) were associated with a longer overall survival or progression-free survival. In the multivariate survival analysis, high expression of DEPTOR was associated with a longer OS (HR=0.18) and PFS (HR=0.09) (see Figures 26 - 27 ). The above experiments indicate that Deptor is an independent prognostic factor.

[0129] Based on the above results, the present invention clearly reveals the role and mechanism of Deptor in radiotherapy resistance of head and neck cancer. It was found that radiotherapy treatment led to a decrease in the expression level of Deptor in head and neck cancer cells. By overexpressing Deptor or using a Deptor enhancer, the intracellular Deptor level reduced due to radiotherapy could be effectively restored, preventing the degradation of Deptor during radiotherapy; at the same time, it was also found that the restoration of Deptor after radiotherapy induced by the Deptor enhancer was related to proteasome-mediated protein degradation. By inhibiting protein degradation, the intracellular Deptor level could be effectively maintained. When Deptor was knocked out in head and neck cancer cells, it promoted the generation of radiotherapy tolerance in cells, while overexpressing Deptor or using a Deptor enhancer to maintain or increase the intracellular Deptor expression level could effectively reduce the emergence of radiotherapy tolerance in head and neck cancer cells during radiotherapy.

[0130] In addition, through the analysis of clinical head and neck cancer patients, it was found that the level of Deptor expression in the body is closely related to the treatment effect and prognosis of the patients. Specifically, high expression of Deptor is significantly correlated with tumor regression, and is also closely related to longer overall survival (OS) and progression-free survival (PFS). This means that Deptor can be used as an independent factor for predicting the radiotherapy efficacy and evaluating the prognosis of clinical head and neck cancer patients. For patients with low Deptor expression, methods such as using Deptor enhancers in combination with radiotherapy can be adopted to stabilize the expression of Deptor, thereby improving the radiotherapy sensitivity and prognosis of the patients, and providing a more personalized and scientific treatment plan for the treatment of clinical head and neck cancer patients.

[0131] The above specific implementation part specifically introduces the analysis 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 idea of the present invention, rather than a limitation on the relevant content. Without departing from the principle of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also fall within the protection scope of the present invention. Sequence Listing <110> The First Affiliated Hospital of Sun Yat-sen University <120> Application of Deptor as a Target in the Preparation of Drugs for Enhancing the Sensitivity of Head and Neck Cancer to Radiotherapy <160> 23 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1227 <212> DNA <213> Artificial Sequence <400> 1 atggaggagg gcggcagcac tggcagtgct ggcagtgaca gcagcaccag cgggagtggc 60 ggggcgcagc aaagggagct ggagcgcatg gctgaggtct tggtcaccgg ggaacagcta 120 cggctcaggc tgcacgaaga aaaggttatt aaagatagac gtcatcatct caagacctac 180 ccaaactgtt ttgtcgcaaa agaactgatt gactggctga ttgaacacaa agaggcttct 240 gacagagaga cggcaattaa actcatgcag aaattagcag accggggcat tattcaccat 300 gtgtgtgatg agcataagga attcaaggat gtcaaactct tctaccgctt tagaaaggat 360 gacggcacct tcccattgga taatgaagtg aaggccttta tgagaggaca gaggctatat 420 gaaaagctga tgagccctga aaacacactc ctgcagccca gggaggagga aggggtcaag 480 tatgagcgca ccttcatggc atctgaattc ctggactggc tggttcagga aggtgaggcc 540 accacgagga aagaggcaga gcagctttgc caccggctta tggagcatgg catcatccag 600 catgtgtcca acaagcaccc atttgtggac agcaatcttc tctaccagtt cagaatgaac 660 ttccggcgga ggcgaagact gatggagctg ctcaatgaaa agtccccctc ctcccaggaa 720 actcatgaca gtcccttctg cctgaggaag cagagccatg acaatcggaa atctaccagc 780 tttatgtcag tgagccccag caaggagatc aagatcgtgt ctgcagtgag gagaagcagc 840 atgagcagct gtggcagcag cggctacttc agcagcagcc ccaccctcag cagcagcccc 900 cctgtgctct gcaaccccaa gtccgtgctg aagagacctg tcacctctga ggaactcctt 960 actcccgggg ctccgtatgc aaggaagaca ttcacgattg ttggtgacgc ggttggctgg 1020 ggttttgtgg tgcgaggaag taagccatgc cacatccagg ctgtagaccc cagtggccct 1080 gcagccgcag caggaatgaa ggtctgtcag tttgtcgtct ctgtcaacgg gctcaatgtc 1140 ctgcatgtag actaccggac cgtgagcaat ctgattctga cgggcccacg gacgattgtc 1200 atggaagtca tggaggagtt agagtgc 1227 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ttgtcgtctc tgtcaacggg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tgtcgctgtt tgggctagag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ggtgcgagga agtaagccat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gtcgctgttt gggctagaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 taaaaccatg gaggagggcg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gcgctcatac ttgacccctt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 actgatccga gcacccaaac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 aggtgcgctc atacttgacc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 catgtgtcca acaagcaccc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 agcagctcca tcagtcttcg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 ccacatccag gctgtagacc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 caggacattg agcccgttga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 agcgacatgc taaagtcccc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 ggaatttcct ctgcacgctg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 cgtgctgaag agacctgtca 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 cttcctcgca ccacaaaacc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 ggttttgtgg tgcgaggaag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 aggacattga gcccgttgac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 tctctagccc aaacagcgac 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <400> 21 tccctaacta cccccaacca 20 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <400> 22 caccgtcgca aaagaactga ttgac 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <400> 23 caccggccgc acggccctaa aacca 25

Claims

1. Use of Deptor enhancer in the preparation of a drug for enhancing radiosensitization in hypopharyngeal cancer radiotherapy; the Deptor enhancer is selected from reagents for maintaining the stability of Deptor; The reagent for maintaining the stability of Deptor is selected from the compounds shown in the following structures: 。 2. Use of a reagent for detecting the expression level of Deptor in the preparation of a product for detecting the radiosensitivity and / or predicting the curative effect of hypopharyngeal cancer radiotherapy, characterized in that the reagent for detecting the expression level of Deptor includes primers for detecting the expression level of the Deptor gene; the primers for detecting the expression level of the Deptor gene are selected from at least one pair of the following primer pairs: Primer pair 1: The upstream sequence is as shown in SEQ ID NO: 2, and the downstream sequence is as shown in SEQ ID NO: 3; Primer pair 2: The upstream sequence is as shown in SEQ ID NO: 4, and the downstream sequence is as shown in SEQ ID NO: 5; Primer pair 3: The upstream sequence is as shown in SEQ ID NO: 6, and the downstream sequence is as shown in SEQ ID NO: 7; Primer pair 4: The upstream sequence is as shown in SEQ ID NO: 8, and the downstream sequence is as shown in SEQ ID NO: 9; Primer pair 5: The upstream sequence is as shown in SEQ ID NO: 10, and the downstream sequence is as shown in SEQ ID NO: 11; Primer pair 6: The upstream sequence is as shown in SEQ ID NO: 12, and the downstream sequence is as shown in SEQ ID NO: 13; Primer pair 7: The upstream sequence is as shown in SEQ ID NO: 14, and the downstream sequence is as shown in SEQ ID NO: 15; Primer pair 8: The upstream sequence is as shown in SEQ ID NO: 16, and the downstream sequence is as shown in SEQ ID NO: 17; Primer pair 9: The upstream sequence is as shown in SEQ ID NO: 18, and the downstream sequence is as shown in SEQ ID NO: 19; Primer pair 10: The upstream sequence is as shown in SEQ ID NO: 20, and the downstream sequence is as shown in SEQ ID NO: 21.

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