Application of sod1 as a biomarker in preparation of a drug for improving sensitivity of head and neck squamous carcinoma to afatinib

Abstract

The application discloses application of SOD1 as a biomarker in preparation of a drug for improving sensitivity of head and neck squamous cell carcinoma to afatinib, and effectively screens an afatinib drug efficacy marker SOD1 through pharmacogenomics analysis, and verifies that head and neck squamous cell carcinoma with low expression of SOD1 has higher sensitivity to afatinib through in-vivo and in-vitro experiments, and combination of the SOD1 inhibitor and the afatinib can be used for precise medication of head and neck squamous cell carcinoma patients in clinic, and has great application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of SOD1 as a biomarker in the preparation of drugs for improving the sensitivity of head and neck squamous cell carcinoma to afatinib. Background Technology

[0002] Head and neck cancer is the sixth most common type of cancer worldwide, including epithelial malignant tumors originating from the sinuses, nasal cavity, oral cavity, pharynx, and larynx. Globally, there are over 550,000 new cases and over 300,000 deaths annually, with over 95% of head and neck cancers being squamous cell carcinoma. Currently, surgery, radiotherapy, and chemotherapy remain the main treatments for head and neck squamous cell carcinoma, but these can cause damage to head and neck organs and functions, leading to a decline in patients' quality of life. In recent years, with advancements in sequencing and multi-omics analysis technologies, targeted therapy specifically targets key molecules and their regulated signal transduction pathways in tumor development and progression, demonstrating high efficacy and low toxicity, and has become the mainstream treatment for many cancer types. However, the genetic characteristics of head and neck squamous cell carcinoma show that it is predominantly characterized by tumor suppressor gene mutations, and the current situation faces a severe lack of targeted drug therapy strategies.

[0003] Afatinib is a potent and irreversible dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases. Previous reports have demonstrated the efficacy of afatinib in recurrent or metastatic HNSCC after platinum-based therapy failure, and it has been used as second-line treatment for patients with recurrent or metastatic head and neck squamous cell carcinoma. However, in a clinical trial (LUX-Head & Neck 2) evaluating afatinib as adjuvant therapy in patients with primary unresected, locally advanced, intermediate- to high-risk head and neck squamous cell carcinoma, afatinib was not shown to have a significant advantage over placebo in disease-free survival. This may be due to the lack of effective biomarkers. Therefore, precise biomarkers are urgently needed to guide the clinical application of afatinib.

[0004] Afatinib is a potent and irreversible dual inhibitor of epidermal growth factor receptor EGFR and human epidermal growth factor receptor 2 HER2 tyrosine kinase. Although data from the phase III clinical trial LUX-Head&Neck1, published in 2014, showed that afatinib significantly delayed tumor growth in patients who had failed previous treatment regimens compared to chemotherapy, demonstrating a 20% reduction in the risk of disease progression, this clinical study aimed to evaluate the efficacy and safety of afatinib compared to methotrexate in patients with recurrent and / or metastatic head and neck squamous cell carcinoma. However, no significant difference was observed in overall survival between the afatinib and chemotherapy groups, with median survival of 6.8 months and 6.0 months, respectively. Furthermore, the Lux-Head&Neck2 clinical trial, published in JAMA Oncology in 2019, aimed to evaluate whether afatinib improved disease-free survival in patients with head and neck squamous cell carcinoma after intensive chemoradiotherapy. This study enrolled 617 patients who were randomly assigned (2:1) to receive either afatinib or placebo for 18 months, or until disease recurrence, unacceptable adverse events, or patient withdrawal. Results showed that afatinib treatment after chemoradiotherapy did not improve disease-free survival, and compared to placebo, it resulted in more adverse events in patients with primary, intermediate- to high-risk head and neck squamous cell carcinoma. This clinical study ultimately did not recommend the use of afatinib after chemoradiotherapy. Therefore, although afatinib is currently used as a second-line treatment for patients with recurrent or metastatic head and neck squamous cell carcinoma, its clinical efficacy is limited due to the lack of clearly defined and effective biomarkers.

[0005] Therefore, there is an urgent need to find an effective biomarker to guide precise medication use for patients with afatinib in the clinical treatment of head and neck squamous cell carcinoma. Summary of the Invention

[0006] The purpose of this invention is to address the current shortcomings of afatinib in clinical use for head and neck squamous cell carcinoma, which lacks effective biomarkers and thus results in poor clinical efficacy.

[0007] To achieve the above objectives, the present invention provides the use of SOD1 as a biomarker in the preparation of a drug for improving the sensitivity of head and neck squamous cell carcinoma to afatinib.

[0008] This invention provides the application of SOD1 as a biomarker in the preparation of drugs for improving the sensitivity of head and neck squamous cell carcinoma to afatinib.

[0009] Preferably, the drug comprises an SOD1 inhibitor.

[0010] Preferably, the SOD1 inhibitor comprises siRNA that binds to SOD1 mRNA.

[0011] Preferably, the siRNA is selected from any one of si-SOD1-1, si-SOD1-2, and si-SOD1-3, and the base sequences of si-SOD1-1, si-SOD1-2, and si-SOD1-3 are as follows:

[0012] si-SOD1-1:

[0013] Chain of Justice: 5'-CCUCACUUUAAUCCUCUAUTT-3';

[0014] Antonym chain: 5'-AUAGAGGAUUAAAGUGAGGTT-3';

[0015] si-SOD1-2:

[0016] Chain of Justice: 5'-CGAUGUGUCUAUUGAAGAUTT-3';

[0017] Antonym chain: 5'-AUCUUCAAUAGACACAUCGTT-3';

[0018] si-SOD1-3:

[0019] Chain of Justice: 5'-GGGCAAAGGUGGAAAUGAATT-3';

[0020] Antonym chain: 5'-UUCAUUUCCACCUUUGCCCTT-3'.

[0021] Preferably, the head and neck squamous cell carcinoma includes at least one of oral squamous cell carcinoma, nasopharyngeal carcinoma, and laryngeal carcinoma.

[0022] The present invention also provides a pharmaceutical composition for preparing a treatment for head and neck squamous cell carcinoma, the pharmaceutical composition comprising an SOD1 inhibitor and afatinib.

[0023] Preferably, the SOD1 inhibitor is a siRNA that binds to SOD1 mRNA.

[0024] The present invention also provides a kit for detecting the sensitivity of head and neck squamous cell carcinoma to afatinib treatment, characterized in that the kit contains at least an antibody that specifically binds to the SOD1.

[0025] Preferably, the kit is selected from any one of qPCR kits, immunoblotting kits, flow cytometry kits, and ELISA kits.

[0026] This invention also provides the use of afatinib in the preparation of a drug for treating head and neck squamous cell carcinoma, in which SOD1 is expressed at low levels.

[0027] The beneficial effects of this invention are:

[0028] This invention, through pharmacogenomics analysis, for the first time discovered that SOD1 can serve as a biomarker for afatinib in the treatment of head and neck squamous cell carcinoma. In vivo and in vitro experiments verified that head and neck squamous cell carcinoma with low SOD1 expression has higher sensitivity to afatinib. The combined use of SOD1 inhibitors and afatinib can improve the therapeutic sensitivity of head and neck squamous cell carcinoma to afatinib, thereby guiding precise clinical medication for head and neck squamous cell carcinoma and showing broad application prospects in the field of head and neck squamous cell carcinoma treatment. Attached Figure Description

[0029] Figure 1 This is a graph showing the efficacy results of afatinib against a subset of nine head and neck squamous cell carcinoma cells.

[0030] Figure 2 The figure shows the results of the expression validation experiment of 10 pharmacodynamic biomarkers in two groups of cells, one sensitive to afatinib and the other resistant to it.

[0031] Figure 3 The figure shows the experimental results of Western Blot analysis of SOD1 expression levels in primary tumor cells PDC_51 and commercially available head and neck squamous cell carcinoma cell line HN13 after knocking down SOD1 expression using siRNA.

[0032] Figure 4 This is a schematic diagram showing the relative cell viability of each group of cells after 72 hours of treatment with 1 μM afatinib following knockdown of SOD1 expression in primary tumor cells PDC_51 and the commercially available head and neck sclerocarcinoma cell line HN13.

[0033] Figure 5 This study investigated the growth of xenografts of head and neck squamous cell carcinoma with low SOD1 expression in PDX_3 tumors after treatment with afatinib or a solvent control. Figure 5 The 'a' represents a schematic diagram illustrating the growth size of the transplanted tumor; Figure 5 'b' represents a graph showing the relationship between the growth time and the size of the transplanted tumor, based on statistical analysis.

[0034] Figure 6 This study compares the growth of xenografts in SOD1-highly expressed head and neck squamous cell carcinoma (PDX_9) after treatment with afatinib or a solvent control. Figure 6 The 'a' represents a schematic diagram illustrating the growth size of the transplanted tumor; Figure 5 'b' represents a graph showing the relationship between the growth time and the size of the transplanted tumor, based on statistical analysis. Detailed Implementation

[0035] The technical solution of the present invention is further described below with reference to the accompanying drawings and examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and instruments adopted in the present invention are conventional reagents, methods and instruments in the art.

[0036] The commercialized cell line HN13 of head and neck squamous cell carcinoma in the present invention is purchased from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences.

[0037] The mice used for constructing the PDX model in the present invention are female BALB / c-nu nude mice, 6-8 weeks old, purchased from the Laboratory Animal Department of Shanghai Institute of Planned Parenthood Research (Animal License No.: SCXK (Shanghai) 2018-0006). The mice are raised in separate cages under SPF-grade conditions. The room temperature is maintained at 18-25°C, the relative humidity is 40%-60%, the special mouse cages, bedding, feed and drinking water are autoclaved at 121°C for 30 minutes, and the bedding is replaced at least once a week.

[0038] Example 1 Construction of a patient-derived xenograft model for head and neck squamous cell carcinoma

[0039] For the corresponding patients for whom the patient-derived xenograft (PDX) model is constructed in the present invention, their clinical information is recorded, including basic data (gender, age, smoking and alcohol history, etc.), clinicopathological diagnosis (tumor size and location, TNM stage, HPV infection status), previous treatment history (surgery, radiotherapy and chemotherapy status), prognostic information such as recurrence and metastasis, etc. Tumor, paracancerous and blood samples of the patients are collected. Pathohistomorphological identification and genetic information identification are performed on the tumor samples.

[0040] After the tumor tissue is surgically resected, the color, shape and texture of the tissue are observed, the necrotic tissue is removed, and the central part of the lesion is selected for sampling. Since head and neck squamous cell carcinoma generally grows in contaminated sites such as the oral cavity and nasal mucosa, the sample needs to be sterilized with 0.05% sodium hypochlorite before being inoculated and transplanted into mice, and quickly washed with PBS containing 1% penicillin-streptomycin double antibody for 30 seconds. The peripheral tissue of the tissue sample is gently scraped off, and the tumor is cut into 1-2mm 3 sized small pieces. Under sterile conditions, the patient tissue is transplanted into regions with abundant blood supply and lymph nodes of immunodeficient mice (such as bilateral axillae) to construct a subcutaneous PDX model, or inoculated into the bilateral submandibular spaces of animals to construct an orthotopic PDX model. To improve the inoculation success rate, Matrigel can be mixed with the patient's tumor tissue before inoculation, and the tissue from each patient is inoculated into 3-5 mice. The growth trajectory of the PDX model is tracked 1-2 weeks after model construction, when the tumor volume exceeds 800mm 3If the tumor volume does not show significant growth within two weeks, the transplanted tumor is passaged sequentially. Generally, the model is considered to be stable after passage to three or more generations. The constructed PDX model is used for subsequent in vivo experiments to conduct relevant validation experiments.

[0041] Example 2: Construction of primary tumor cells derived from patients with head and neck squamous cell carcinoma

[0042] After head and neck squamous cell carcinoma tumor tissue was transported back to the laboratory at 4°C, the outer packaging was sterilized, and the tissue was transferred to the cell culture room through a sterile transfer chamber. The tissue was then separated and minced using sterile microscissors and microforceps. A tissue digestion solution was prepared (1 mg / ml type IV collagenase, 200 U / ml hyaluronidase, 200 U / ml DNase type I enzyme, 1× phenol red-free trypsin), with a volume approximately 5-10 times the tumor volume.

[0043] After centrifuging the minced tissue at 1500 rpm for 3 minutes, resuspend it in tissue digestion solution, add it to a tissue dissociation tube, and place it in a Miltenyi tissue processor. Select the appropriate dissociation mode according to the tissue's toughness. After crushing the tissue, place it on a shaker at 37°C for 30-60 minutes, shaking the dissociation tube up and down every 10 minutes to mix the tissue and prevent clumping, which would affect digestion. Disintegration is stopped when the digestion solution becomes turbid and no obvious tissue remains. Neutralize the digestion with culture medium containing 10% fetal bovine serum, and collect undigested clumps and cell suspensions after passing the solution through 100μm and 40μm filters respectively. Centrifuge at 1500 rpm for 3 minutes. If significant red blood cell contamination is observed, add 1 ml of red blood cell lysis buffer, let stand for 3 minutes, then dilute with 10 ml of PBS and centrifuge at 1500 rpm for 3 minutes.

[0044] The supernatant was discarded, and the cells were resuspended in 5 ml of complete culture medium. The complete culture medium formulation was: high-glucose DMEM medium:DMEM / F12 medium = 3:1, insulin 5 μg / ml, amphotericin B 250 ng / ml, gentamicin 10 μg / ml, cholera toxin 0.1 nM, EGF 0.125 ng / ml, hydrocortisone 25 ng / ml, and ROCK inhibitor Y-2763 210 μM. The solution was sterilized using a 0.22 μm sterile filter and stored at 4°C for 2 months. The cell suspension was continuously cultured in a 37°C, 5% CO2 humidified incubator. Patient-Derived Cells (PDCs) were successfully constructed by passaged more than 5 times and maintaining high cell proliferation activity. The constructed PDCs were used for subsequent in vitro experiments to conduct relevant validation experiments.

[0045] Example 3: Pharmacogenomic analysis predicted that afatinib had high sensitivity against head and neck squamous cell carcinoma with low SOD1 expression.

[0046] This invention performed pharmacogenomics analysis on afatinib, identifying multiple characteristics associated with its efficacy. Based on biological function, 10 potential candidate biomarkers were selected, as shown in Table 1. Subsequently, this invention selected six independent primary tumor cell lines that had not undergone drug screening and three commercially available cell lines to form a validation cell set for afatinib efficacy testing.

[0047] Table 1 Potential biomarkers for the efficacy of afatinib

[0048] Predictive markers Drug Name p-value CDC34 Afatinib 8.24E-06 PARP8 Afatinib 0.000102472 MMP15 Afatinib 0.000109508 IFNE Afatinib 0.000141838 MAP2K2 Afatinib 0.000229808 SOD1 Afatinib 0.000379786 SLC35A5 Afatinib 0.000404167 UQCRFS1 Afatinib 0.000476895 EXOC7 Afatinib 0.001760195 CELF1 Afatinib 0.020082807

[0049] Experimental results: such as Figure 1 As shown, relative cell survival curves were obtained after the afatinib efficacy test. The cells were divided into afatinib-sensitive group (HSC3, PDC_55, PDC_54) and drug-resistant group (TU686, HN13, PDC_51, PDC_52, PDC_56, PDC_53).

[0050] like Figure 2 As shown, the present invention subsequently detected the expression of 10 pharmacodynamic biomarkers in the above 9 cell lines. The 10 pharmacodynamic biomarkers were SLC35A5, CELF1, EXOC7, IFNE, PAPR8, SOD1, UQCRFS1, CDC34, MAP2K2, and MMP15. The results showed that low SOD1 expression was significantly correlated with the sensitivity of afatinib (P < 0.05), while the other 9 pharmacodynamic biomarkers did not show significant correlations. Therefore, SOD1 has the potential to be used as a pharmacodynamic biomarker in head and neck squamous cell carcinoma.

[0051] The afatinib used in this application was purchased from Selleck, CAS number: 850140-72-6, molecular formula: C 24 H 25 CIFN5O3.

[0052] Superoxide dismutase (SOD) is a class of enzymes that catalyze the conversion of superoxide radicals into oxygen and hydrogen peroxide. SOD activity depends on a specific catalytic metal ion, which can be manganese (MNSOD), iron (FeSOD), nickel (NiSOD), or copper (Cu / ZnSod). All mammals possess three subtypes of SOD. Human SOD1 is a dimeric protein, 32 kDa, with each monomer composed of 153 amino acids (15.8 kDa), and its gene is located on chromosome 21 (locus 21q22). SOD1 is responsible for regulating superoxide levels caused by the mitochondrial intermembrane space, cytoplasm, and peroxisomes. In addition to its role as an antioxidant enzyme, several novel functions of the SOD1 protein have been reported, including nuclear gene transcriptional activation following exposure to oxidative stress, regulation of RNA metabolism, and inhibition of respiration in the glucose-sensing pathway. Some regions of SOD1 may be inherently disordered. Due to the lack of a defined ordered structure, structural flexibility is considered a functional advantage of SOD1, potentially enabling the protein to interact with various chaperones, such as other proteins, membranes, and nucleic acids, making SOD1 a multifunctional protein. Tumor cells are known to have high levels of reactive oxygen species (ROS), increasing their need for antioxidants. SOD1 is overexpressed in various cancers, and its inhibition leads to impaired mitochondrial function in breast cancer. SOD1 overexpression can activate the unfolded protein response (UPRMT), a key player in cellular protective responses and maintains appropriate ROS levels for tumor cells. Furthermore, knockdown or inhibition of SOD1 expression suppresses the growth of non-small cell lung cancer and hematological malignancies, indicating that SOD1 overexpression is crucial for tumorigenesis. Notably, studies have shown that inhibiting SOD1 can induce tumor cell apoptosis, suggesting that SOD1 is a potential novel target for cancer therapy. In head and neck squamous cell carcinoma, few studies have revealed the function of SOD1 in tumorigenesis, development, and resistance to afatinib treatment. Limited research shows that in nasopharyngeal carcinoma, disrupting SOD1 activity can inhibit cell growth and increase lipid accumulation. This application aims to verify the correlation of low SOD1 expression in head and neck squamous cell carcinoma.

[0053] Example 4: In vitro experiments verify that afatinib has higher sensitivity against SOD1-low-expressing head and neck phospholipid cancer cells.

[0054] siRNA design and synthesis: The target sequence of siRNA was synthesized by Gemma Gene Biotechnology Co., Ltd. The specific siRNA sequence is shown in Table 2.

[0055] Table 2 siRNA sequences

[0056] siRNA sense(5'-3') antisense (5'-3') si-SOD1-1 CCUCACUUUAAUCCUCUAUTT AUAGAGGAUUAAAGUGAGGTT si-SOD1-2 CGAUGUGUCUAUUGAAGAUTT AUCUUCAAUAGACACAUCGTT si-SOD1-3 GGGCAAAGGUGGAAAUGAATT UUCAUUUCCACCUUUGCCCTT si-NC UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT

[0057] siRNA transfection: One day before transfection, seed target cells into each well of a plate and add 2.5 mL of antibiotic-free growth medium to each well. Remove the growth medium from the cells and add 1.5 mL of fresh serum-free growth medium. Add 100 pmol of small interfering RNA to 250 μL of serum-free growth medium and mix gently. Dilute with 5 μL of transfection reagent in 250 μL of growth medium and incubate at room temperature for 5 min. Mix the diluted siRNA and transfection reagent from the previous two steps and incubate at room temperature for 20 min. Add the mixture to the wells containing cells seeded the day before. Incubate the cells at 37°C for 5-6 h. Replace with serum-containing medium and incubate for 24-48 h before proceeding with other post-transfection assays. Detect protein expression levels after 3-4 days of culture.

[0058] Western blotting: After cell lysis, BCA protein was quantified, followed by the addition of SDS loading buffer and denaturation at 95°C for 10 minutes. Then, 10-50 μg protein samples were subjected to SDS-PAGE electrophoresis. After electrophoresis, the proteins on the PAGE gel were transferred to a PVDF membrane using a transfer system and incubated with primary and secondary antibodies, respectively. Antibodies used included Anti-SOD1 and anti-GAPDH, to detect the knockdown efficiency of SOD1 in head and neck squamous cell carcinoma.

[0059] Experimental results: such as Figure 3 As shown, compared with the control group, the expression of the target gene in the siRNA treatment group was significantly reduced.

[0060] Cell proliferation assay: Head and neck squamous cell carcinoma cells were seeded at a density of 3000 cells / well in 96-well plates and incubated overnight. Subsequently, siRNA transfection was performed, followed by treatment with 1 μM afatinib. Cell proliferation was assessed using a Cell Counting Kit-8 after 72 hours.

[0061] Experimental results: such as Figure 4 As shown, compared with control cells, afatinib significantly inhibited the proliferation of SOD1 knockdown cells in PDC_51 and HN13, indicating that head and neck squamous cell carcinoma cells with low SOD1 expression are more sensitive to afatinib.

[0062] Example 5: In vivo tumor transplantation experiments demonstrate that afatinib has higher sensitivity against head and neck squamous cell carcinoma with low SOD1 expression.

[0063] Two head and neck squamous cell carcinoma PDX models (PDX_3 and PDX_9) were selected to evaluate the in vivo therapeutic effect of afatinib on head and neck squamous cell carcinoma with different SOD1 expression levels. Sixteen progeny models were constructed from each PDX model, and the tumor volume was increased to 100-200 mm².3 Then, 10-16 mice were selected and divided into two groups: an afatinib treatment group and a solvent control group.

[0064] The dosing regimen is as follows:

[0065] ① Afatinib treatment group: oral administration, 25 mg / kg, in a solvent of 5% DMSO + 40% PEG 300 + 5% Tween 80 + 50% ddH2O, administered daily.

[0066] ② Solvent control group: Oral administration, corresponding drug solvent 5% DMSO + 40% PEG300 + 5% Tween80 + 50% ddH2O, administered daily.

[0067] Continuously measure the tumor volume and body weight of the model until the tumor volume reaches 1000-1500 mm. 3 .

[0068] Experimental results: such as Figure 5 As shown, PDX_3 is a low SOD1 expression model, with an expression level of SOD1 FKPM = 28.07. In the PDX_3 model, the afatinib treatment group significantly inhibited tumor growth and the tumors were significantly smaller compared to the solvent control group. Figure 6 As shown, PDX_9 is a model with high SOD1 expression, with an expression level of SOD1FKPM = 170.37. In the PDX_9 model, the afatinib treatment group had no significant effect on tumor growth compared to the solvent control group. This indicates that the head and neck squamous cell carcinoma xenograft PDX_3, with low SOD1 expression, is sensitive to afatinib, while the head and neck squamous cell carcinoma xenograft PDX_9, with high SOD1 expression, is resistant to afatinib. In the PDX model, an SOD1FKPM below 28.07 is defined as low SOD1 expression.

[0069] In some embodiments, the present invention also provides a kit for determining the sensitivity of head and neck squamous cell carcinoma patients to afatinib. This kit contains at least an antibody that specifically binds to SOD1, and the sensitivity of head and neck squamous cell carcinoma to afatinib can be determined by detecting the expression level of SOD1 in head and neck squamous cell carcinoma tissue. The kit is selected from any one of qPCR kits, Western blot assay kits, flow cytometry kits, and ELISA kits.

[0070] This invention provides the application of SOD1 as a biomarker in the preparation of drugs to improve the sensitivity of head and neck squamous cell carcinoma to afatinib. In vivo and in vitro experiments have confirmed that head and neck squamous cell carcinoma with low SOD1 expression has higher sensitivity to afatinib. The combination of SOD1 inhibitors and afatinib for low SOD1 expression can improve the therapeutic sensitivity of head and neck squamous cell carcinoma to afatinib, enabling precise drug use in clinical practice and thus improving the therapeutic effect of afatinib on head and neck squamous cell carcinoma.

[0071] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The use of SOD1 inhibitors in the preparation of drugs for improving the sensitivity of head and neck squamous cell carcinoma to afatinib, characterized in that, The SOD1 inhibitor comprises siRNA that binds to SOD1 mRNA, wherein the siRNA is selected from any one of si-SOD1-1, si-SOD1-2, and si-SOD1-3, and the base sequences of si-SOD1-1, si-SOD1-2, and si-SOD1-3 are as follows: si-SOD1-1: Chain of Justice: 5' CCUCACUUUAAUCCUCUAUTT3'; Antonym chain: 5'AUAGAGGAUUAAAGUGAGGTT3'; si-SOD1-2: Chain of Justice: 5' CGAUGUGUCUAUUGAAGAUTT3'; Antonym chain: 5' AUCUUCAAUAGACACAUCGTT3'; si-SOD1-3: Chain of Justice: 5' GGGCAAAGGUGGAAAUGAATT3'; Antonym chain: 5' UUCAUUUCCACCUUUGCCCTT3'.

2. The application as described in claim 1, characterized in that, The head and neck squamous cell carcinomas include at least oral squamous cell carcinoma or laryngeal carcinoma.

3. A pharmaceutical composition for preparing a treatment for head and neck squamous cell carcinoma, characterized in that, The pharmaceutical composition comprises an SOD1 inhibitor and afatinib, wherein the SOD1 inhibitor comprises siRNA that binds to SOD1 mRNA, and the siRNA is selected from any one of si-SOD1-1, si-SOD1-2, and si-SOD1-3, and the base sequences of si-SOD1-1, si-SOD1-2, and si-SOD1-3 are as follows: si-SOD1-1: Chain of Justice: 5' CCUCACUUUAAUCCUCUAUTT3'; Antonym chain: 5'AUAGAGGAUUAAAGUGAGGTT3'; si-SOD1-2: Chain of Justice: 5' CGAUGUGUCUAUUGAAGAUTT3'; Antonym chain: 5' AUCUUCAAUAGACACAUCGTT3'; si-SOD1-3: Chain of Justice: 5' GGGCAAAGGUGGAAAUGAATT3'; Antonym chain: 5' UUCAUUUCCACCUUUGCCCTT3'.

4. The application of SOD1-specific binding antibodies in the preparation of a kit for detecting the sensitivity of head and neck squamous cell carcinoma to afatinib treatment, characterized in that, The antibody is used to detect the expression level of SOD1 in head and neck squamous cell carcinoma tissue, wherein low SOD1 expression indicates sensitivity to afatinib treatment.

5. The application as described in claim 4, characterized in that, The kit is selected from any one of the following: immunoblotting detection kit, flow cytometry analysis kit, and ELISA kit.

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