Use of uchl1 inhibitors in the preparation of medicaments for the treatment of head and neck squamous cell carcinoma

Abstract

The application relates to application of a UCHL1 inhibitor in preparation of a head and neck squamous cell carcinoma treatment drug and belongs to the technical field of biological medicines. It is found that the expression level of UCHL1 in positive tumor tissues of head and neck squamous cell carcinoma is obviously higher than that in negative tumor tissues of head and neck squamous cell carcinoma, and the expression level of UCHL1 is related to a PD1-PDL1 path. In vitro experiments find that UCHL1 regulates the expression of PDL1 and the phosphorylation level of ERK at the protein level, and the in vitro cell experiment results are consistent with the protein quantitative analysis in tumor tissue samples. The expression of UCHL1 is closely related to the resistance of head and neck squamous cell carcinoma tumor cells to NK cell killing, and the small molecule inhibitor LDN57444 of UCHL1 can significantly promote the killing effect of NK cells on tumor cells, and the combination of the small molecule inhibitor PD0325901 of MEK can significantly increase the killing toxicity of NK cells on head and neck squamous cell carcinoma tumor cells caused by LDN57444. The application provides a new thought and theoretical basis for the mechanism of HPV-related head and neck squamous cell carcinoma and UCHL1 as a PDL1 immune blocking treatment combined drug target.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of UCHL1 inhibitors in the preparation of therapeutic drugs for head and neck squamous cell carcinoma. Background Technology

[0002] Head and neck cancer (HNC) is a type of malignant tumor originating from the mucosal epithelium of the upper respiratory and digestive tract, such as the oral cavity, oropharynx, larynx, or hypopharynx. Over 95% of HNC pathological types are squamous cell carcinoma (SCC), characterized by a high recurrence rate, a high incidence of second primary cancer, and poor survival prognosis. Infection with high-risk human papillomavirus (HPV) is considered one of the major risk factors for head and neck squamous cell carcinoma (HNSCC). Despite advancements in surgery, radiotherapy, and chemotherapy, the prognosis and quality of life for patients with head and neck squamous cell carcinoma (HNSCC) still require improvement. In the past decade, immunotherapy has emerged as a significant treatment option for cancer, gaining widespread acceptance. Immunotherapy is currently a hot research topic in the treatment of HNSCC, with main research directions including the use of immune checkpoint inhibitors (ICIs), cancer vaccines, and chimeric antigen receptor T-cell therapy. Among these, immune checkpoint inhibitors are one of the most widely used immunotherapy methods. These drugs restore the anti-tumor activity of T cells by inhibiting tumor cells' utilization of immune checkpoints of the immune system.

[0003] Currently, immunotherapies targeting HPV-related HNSCC, including those targeting programmed death receptor 1 / ligand 1 (PD-1 / PDL1), are available. However, it's noteworthy that the clinical response rate to this immunotherapy for HPV-positive HNSCC is only around 20%, and up to 30% of patients die within 5 years due to tumor recurrence. Considering these factors, there is an urgent need to supplement clinical treatment strategies with new targets for HPV-related HNSCC to improve the response rate of immune checkpoint blockade therapy. Numerous neoadjuvant immunotherapy modalities exist, including single-agent immunotherapy, dual-agent immunotherapy, immunotherapy combined with chemotherapy, immunotherapy combined with targeted therapy, and immunotherapy combined with radiotherapy. Immunotherapy has brought tremendous breakthroughs and successes to cancer treatment, fundamentally changing the way tumors are treated and enabling many cancer patients to achieve better treatment outcomes. However, not all patients benefit from current immunotherapies; therefore, continued in-depth research is needed to elucidate the unique molecular characteristics of HNSCC and potential novel immune targets.

[0004] Ubiquitin-terminal carboxyl-terminal hydrolase L1 (UCHL1), also known as PGP9.5, is a 223-amino acid protein with a molecular weight of approximately 24.8 kDa. It is an abundant neuroprotein. Although its role in neurodegenerative diseases has been demonstrated, the specific function of UCHL1 in the pathogenesis of many diseases remains unclear. UCHL1 is absent in most normal tissues but exhibits heterogeneous expression in numerous non-neurogenic tumor tissues, including breast cancer, prostate cancer, colorectal cancer, gastric cancer, head and neck cancer, and pancreatic ductal carcinoma. The exact biological function of UCHL1 within tumors remains controversial, but increasing findings suggest that UCHL1 is an important regulator in the development and progression of human malignant tumors.

[0005] Ubiquitination and deubiquitination are involved in protein homeostasis and various cellular biological processes. Numerous oncoproteins and tumor suppressor factors have been shown to be degraded via the ubiquitin-protease system, and studies have demonstrated a close link between ubiquitination and deubiquitination and the stability of immune checkpoints and the regulation of tumor immune activity. UCHL1, as a member of the ubiquitin C-terminal hydrolase family within the ubiquitin-protease system, has been shown to possess certain immunomodulatory capabilities in some studies. For example, some studies have found that UCHL1 regulates lipopolysaccharide (LPS)-induced inflammatory responses through the NF-κB and MAPK signaling pathways; and in studies related to non-small cell lung cancer, UCHL1 has been found to regulate PDL1 expression through the Akt-P65 signaling axis, and cancer cells with high UCHL1 expression exhibit T-cell suppressive activity. Pro-inflammatory factors INF-γ and TNF-α can upregulate the expression level of UCHL1 in mesenchymal stromal cells (MSCs), and the upregulated UCHL1 negatively regulates the immunosuppressive ability and survival rate of MSCs. In one study, UCHL1 was even shown to act as a positive regulator of macrophage polarization.

[0006] Currently, immunotherapy has made significant progress in advanced hepatocellular carcinoma (HNSCC), primarily through achieving more durable responses and better safety profiles. Although immunotherapy for HNSCC has yielded beneficial outcomes for some patients, most exhibit primary resistance, and the overall response rate is low. Even a small number of patients with relapsed or metastatic (R / M) HNSCC who received immunotherapy experienced rapid disease progression and reduced survival. Therefore, not all patients can benefit from current immunotherapies, necessitating further in-depth research to elucidate the unique molecular characteristics of HNSCC and potential novel immune targets. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a new therapeutic target for head and neck squamous cell carcinoma in the prior art.

[0008] To address the aforementioned technical problems, this invention provides the application of a UCHL1 inhibitor in the preparation of a therapeutic drug for head and neck squamous cell carcinoma. Through cell experiments, clinical tumor sample analysis, and bioinformatics analysis, this invention revealed that the expression level of UCHL1 in positive head and neck squamous cell carcinoma tumor tissues was significantly higher than that in negative head and neck squamous cell carcinoma tumors. OPAL multicolor immunohistochemical staining results showed a significant correlation between UCHL1 and cell subtypes in the immunosuppressive microenvironment of head and neck squamous cell carcinoma tumors. In vitro experiments showed that knockdown or overexpression of UCHL1 did not affect the biological behaviors of cells such as migration, proliferation, and cell morphology, but it did regulate the expression of PDL1 and the phosphorylation level of ERK at the protein level; and the results of in vitro cell experiments were consistent with the quantitative protein analysis in head and neck squamous cell carcinoma tumor tissue samples. UCHL1 expression is closely related to the resistance of head and neck squamous cell carcinoma (HNSCC) tumor cells to NK cell killing. Although the use of the UCHL1 small molecule inhibitor LDN57444 cannot increase the sensitivity of tumor cells to the chemotherapy drug cisplatin and radiotherapy, it can significantly promote the killing effect of NK cells on head and neck squamous cell carcinoma tumor cells. Combined with the MEK small molecule inhibitor PD0325901, it can significantly increase the killing toxicity of NK cells on head and neck squamous cell carcinoma tumor cells induced by LDN57444. This provides new ideas and theoretical basis for the pathogenesis of HPV-related HNSCC tumors and for UCHL1 to be used as a target for combined drugs in PDL1 immunotherapy.

[0009] The first objective of this invention is to provide the use of a UCHL1 inhibitor in the preparation of a therapeutic agent for head and neck squamous cell carcinoma.

[0010] Furthermore, the UCHL1 inhibitor reduces UCHL1 expression or inhibits UCHL1 activity.

[0011] Furthermore, the head and neck squamous cell carcinoma treatment drug reduces the expression of mitogen-activated protein kinase kinase or inhibits the activity of mitogen-activated protein kinase kinase.

[0012] Furthermore, the head and neck squamous cell carcinoma treatment drug inhibits the expression of the PDL1 immune checkpoint.

[0013] A second objective of this invention is to provide a therapeutic target for head and neck squamous cell carcinoma, wherein the therapeutic target for head and neck squamous cell carcinoma is UCHL1.

[0014] The third objective of this invention is to provide an application of a combination of a UCHL1 inhibitor and a mitogen-activated protein kinase inhibitor in the preparation of a therapeutic drug for head and neck squamous cell carcinoma.

[0015] A fourth objective of this invention is to provide a therapeutic agent for head and neck squamous cell carcinoma, said therapeutic agent comprising a UCL1 inhibitor.

[0016] Furthermore, the UCHL1 inhibitor includes LDN57444. LDN57444 is a reversible competitive proteasome inhibitor. It works by binding to the active site of UCHL1 and regulating its enzymatic activity, thereby sensitizing head and neck squamous cell carcinoma cells to cytotoxic immune cell killing, blocking the immune escape pathways of tumor cells, and thus enhancing the body's immune surveillance and clearance of tumor cells.

[0017] Furthermore, the head and neck squamous cell carcinoma treatment includes an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor inhibits the expression of PDL1.

[0018] Furthermore, the head and neck squamous cell carcinoma treatment drug includes a mitogen-activated protein kinase inhibitor.

[0019] Furthermore, the mitogen-activated protein kinase inhibitor includes PD0325901.

[0020] The beneficial effects of this invention are:

[0021] This invention reveals that UCHL1 regulates the expression of PDL1 and phosphorylated ERK at the protein level, and that UCHL1 expression levels are closely related to the resistance of head and neck squamous cell carcinoma (HNSCC) tumor cells to NK cell killing. The small molecule inhibitor LDN57444, a UCHL1 inhibitor, significantly promotes the killing effect of NK cells on HNSCC tumor cells. Combined with the small molecule inhibitor mitogen-activated protein kinase kinase (MEK), PD0325901, it significantly increases the cytotoxicity of LDN57444-induced NK cell killing on HNSCC tumor cells. This invention provides new insights and theoretical basis for the pathogenesis of HPV-related HNSCC tumors and for UCHL1 to serve as a target for combined drug therapy with PDL1 immunotherapy. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0023] Figure 1 The heterogeneity of UCHL1 expression in transcriptome data of 33 pan-cancer diseases was measured using the Wilcoxon test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0024] Figure 2The analysis included: A) Differential expression of UCHL1 transcriptional water and protein levels in cancerous and adjacent tissues of head and neck squamous cell carcinoma patients in multiple cohorts; B) Differential expression of UCHL1 transcriptome levels in cancerous and adjacent tissues analyzed using the GEO database of head and neck squamous cell carcinoma; C) Differential expression and localization of protein in cancerous and adjacent tissues of head and neck squamous cell carcinoma patients analyzed by immunohistochemistry (IHC) staining; D) Differential expression of UCHL1 in head and neck squamous cell carcinoma tissue microarrays analyzed by ICH and the differences between HPV-positive and HPV-negative patients in the Suzhou cohort (E) and Heidelberg cohort (F). *P<0.05, **P<0.01;

[0025] Figure 3 The study included: correlation analysis of UCHL1 transcriptional levels with the abundance of tumor-infiltrating immune cell subtypes in HNSCC; differential analysis of the abundance of tumor-infiltrating immune cell subtypes between high and low UCHL1 transcriptome expression groups in the TCGA-HNSC(A) and GEO(GSE65858)(B) databases; association analysis of UCHL1 transcriptome levels with the abundance of tumor-infiltrating immune cell subtypes in the TCGA-HNSC(C) and GEO(GSE65858)(D) databases; E. unsupervised consensus clustering to divide the samples into four groups (A, B1, B2, B3) with different immune cell aggregation characteristics; F. differences in UCHL1 transcriptome expression levels among the four groups, with group A showing a significantly lower UCHL1 transcriptome expression level compared to the other three groups; statistical methods: Wilcoxon test, *P<0.05, **P<0.01, ***P<0.001;

[0026] Figure 4This study analyzed the differences in immune molecules and clinical prognosis among patients grouped by unsupervised clustering of UCHL1-related immune cell subtypes: A. Heatmap of transcriptomic expression levels of 37 immune checkpoint genes among the four groups; B. Analysis of differences in overall survival among the four groups in different cohorts; Analysis of differences in overall survival between group A and group B1 in the TCGA-HNSC(C) and GEO(GSE65858)(D) cohorts; E. Analysis of the top 50 differentially expressed genes between groups A and B1 with significant prognostic value; F. Heatmap analysis of the top 25 differentially expressed proteins between groups A and B1 using the TCPA proteomics database; G represents CASPASE7CLEAVEDD198, LCK, PDL1, STAT3_pY705, and P38MAP in groups A and B1. A violin plot comparing K protein expression levels; H represents the correlation analysis of UCHL1 and PDL1 expression levels at the transcriptomic level; I represents the correlation analysis of UCHL1 and PDL1 expression levels at the transcriptomic level in HPV-positive and HPV-negative tumors in the TCGA-HNSC cohort; J represents the difference in UCHL1 transcriptomic levels in HPV-positive and HPV-negative tumor tissues in the TCGA-HNSC cohort; Statistical methods: BH test with multiple correction, Wilcoxon test, *P<0.05, **P<0.01, ***P<0.001; H. Correlation analysis of UCHL1 and PDL1 expression levels at the transcriptomic level: Spearman correlation analysis, *P<0.05, **P<0.01, ***P<0.001;

[0027] Figure 5 This is a correlation analysis diagram of four groups of immune cells with HPV and UCHL1; where A and B are heatmaps of immune cell clusters A, B1, B2, and B3 in HPV-positive and HPV-negative samples, respectively; OS Kaplan-Meier survival curves of groups A, B1, B2, and B3 in HPV-positive (C) and HPV-negative (E) samples, and the UCHL1 transcriptome expression levels among the four groups; box plots comparing UCHL1 transcriptome expression levels in HPV-positive (D) and HPV-negative (F) samples; box plots of GO functional enrichment analysis (G) and KEGG pathway enrichment analysis (H); statistical methods: Log-rank (Mantel-Cox) test and Wilcoxon test, B and H were corrected for multiple tests, *P<0.05, **P<0.01, ***P<0.001;

[0028] Figure 6This image shows the results of Opal multiplex fluorescence immunohistochemical staining analysis of tumor-infiltrating immune cell subtypes in head and neck squamous cell carcinoma tissue. A shows the design of an 8-color panel antibody including CD4, CD8, CD19, CD68, CD163, FoxP3, UCHL1, and Pan-CK; B shows typical images of UCHL1 immunohistochemical staining and multiplex fluorescence staining; C shows a high-power magnified image of Opal multiplex fluorescence immunohistochemical staining; D shows the comparison and analysis of tumor-infiltrating immune cell differences between UCHL1 high and low expression groups (Mean and SEM); two-sided unpaired Mann-Whitney U test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0029] Figure 7 The role of UCHL1 in the cytotoxicity of NK cells against head and neck squamous cell carcinoma cells; AB. The correlation between UCHL1 expression and PDL1 expression protein and mRNA levels after overexpression and knockdown; The effect of UCHL1 small molecule inhibitors on the sensitivity of tumor cells to the chemotherapy drugs cisplatin (C) and radiotherapy (D); E. The effect of NK cells on the clonogenic ability of four types of head and neck squamous cell carcinoma cells; F. The cytotoxic effect of NK cells on FaDu and Detroit562 cells; G. The cytotoxic effect of NK cells on Detroit562 cells with knockdown of UCHL1 expression; H. The effect of NK cells on the clonogenic ability of Detroit562 cells with knockdown of UCHL1 expression; I. The effect of UCHL1 small molecule inhibitors on N The role of K cells in inhibiting tumor clonogenesis; the effect of J. UCHL1 small molecule inhibitors on NK cell cytotoxicity, statistical analysis was performed using unpaired t-test. *P<0.05, **P<0.01, ***P<0.001, where A represents the expression levels of UCHL1 and PDL1 proteins in different cells, B represents the mRNA level, pCMV6-Entry represents the control group; pCMV6-UCHL1 represents the group overexpressing UCHL1 protein, si-NC represents the control group; si-UCHL1 represents the group knocking down UCHL1 protein, IR represents radiation therapy; 2Gy represents the radiation dose, and Combi represents the combined treatment group.

[0030] Figure 8The study investigated the reversibility of MEK small molecule inhibitors in resisting NK cell killing by UCHL1-induced tumor cells. Specifically, the effects of MEK inhibitor PD0325901 on the expression levels of UCHL1 and PDL1 were examined. The effects of MEK inhibitor PD0325901 on the clonogenic ability of four head and neck squamous cell carcinoma lines were also examined. The combined use of UCHL1 small molecule inhibitor LDN57444 and MEK inhibitor PD0325901 on the clonogenic ability of Detroit562 cells was also examined. The combined use of UCHL1 small molecule inhibitor LDN57444 and MEK inhibitor PD0325901 on the killing effect on NK-92MI tumor cells Cal27(F) and Detroit562(G) was also examined. Statistical analysis was performed using unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0032] Example 1: Differential expression analysis of UCHL1 transcriptome levels in pan-cancer and head and neck squamous cell carcinoma

[0033] First, the expression levels of UCHL1 in the TPM format transcriptome data of 33 tumors from TCGA were extracted and processed. The UCHL1 transcriptome expression data were then processed using log2(TPM+1), and differential expression analysis was performed on the cancer tissues and adjacent normal tissues from various cancers. The results were then visualized. The results showed that UCHL1 expression was heterogeneous across the 33 cancers, with varying expression levels across different cancers. Differential expression of UCHL1 was observed between cancer tissues and adjacent normal tissues in many cancers. Furthermore, in HNSCC, the expression level of UCHL1 in tumor tissues was significantly higher than that in adjacent normal tissues. Figure 1 ).

[0034] Analysis of transcriptome data from nine cohorts in the GEO database revealed that UCHL1 was significantly higher in head and neck squamous cell carcinoma tissues than in adjacent normal tissues. Figure 2 A). Immunohistochemical staining revealed that UCHL1 protein was expressed in both the nucleus and cytoplasm, and the expression level in the adjacent normal tissue was significantly higher than that in the adjacent normal tissue. Figure 2 B). Analysis of the Suzhou and Heidelberg cohorts revealed that HPV-positive patients had significantly higher UCHL1 protein expression levels than HPV-negative patients. Figure 2 CF).

[0035] CIBERSORTx calculations were performed on the transcriptome data of TCGA-HNSC and GEO(GSE65858) tumor tissues to measure the infiltration degree of 22 immune cells in head and neck squamous cell carcinoma tissues. The CIBERSORTx data of TCGA-HNSC and GEO(GSE65858) were grouped according to the median value of UCHL1 transcriptome expression to compare the differences in the infiltration of 22 immune cells between samples with high and low UCHL1 expression. The results showed that, in the TCGA-HNSC CIBERSORTx data, the high UCHL1 expression group had significantly higher infiltration levels of plasma cells, Treg cells, and resting dendritic cells compared to the low expression group; while the infiltration levels of CD8 T cells, activated CD4 memory T cells, activated NK cells, M1 macrophages, and neutrophils were significantly lower. Figure 3 A). In the GEO (GSE65858) CIBERSORTx data, the high expression group of UCHL1 showed significantly higher infiltration levels of naive B cells (B cell natives) and Treg cells (T cells regulatory); and significantly lower infiltration levels of CD8 T cells (CD8 T cells) and M1 macrophages (M1). Although there was a significant difference in the number of naive CD4 T cells (CD4 native) between the high and low expression groups of UCHL1 in the GEO (GSE65858) CIBERSORTx data, the overall infiltration level was lower. This indicates that the high transcriptome expression group of UCHL1 resulted in a higher infiltration level of immunosuppressive immune cells compared to the low expression group. Figure 3 B).

[0036] To further determine the correlation between UCHL1 and the infiltration levels of 22 immune cells in HNSC, estimated immune matrix scores were calculated from transcriptome data of TCGA-HNSC and GEO (GSE65858) tumor tissues. Spearman correlation analysis was performed on the transcriptome expression level of UCHL1 with CIBERSORTx immune cell infiltration data and estimated immune matrix scores, and the results were visualized. The results showed that in TCGA-HNSC data, the transcriptome expression level of UCHL1 was positively correlated with plasma cells, Treg cells, and M0 macrophages; and negatively correlated with CD8 T cells, activated CD4 memory T cells, activated NK cells, M1 macrophages, neutrophils, and the estimated immune abundance score (ImmuneScore). Figure 4 A). In GEO (GSE65858) data, the transcriptomic expression level of UCHL1 was positively correlated with naive B cells, naive CD4 T cells, and Treg cells; and negatively correlated with CD8 T cells, M1 macrophages, and resting dendritic cells. Figure 3 (C, D) All of the above data are statistically significant. This further validates the previous analysis and once again confirms that high expression of the UCHL1 transcriptome is significantly positively correlated with immunosuppressive immune cell subtypes infiltrating tumors.

[0037] To further analyze the relationship between UCHL1 and the immune microenvironment, the TCGA-HNSC and GEO (GSE65858) datasets were integrated and normalized. CIBERSORTx analysis was performed on the integrated datasets, extracting data from 10 immune cell types that showed significant correlation with UCHL1 and high infiltration levels. Unsupervised, consistent clustering was then performed and integrated with the estimated immune matrix score data, resulting in a heatmap visualization. Furthermore, the differences in UCHL1 transcriptome expression levels across the four groups were compared. The results showed that unsupervised consistent clustering of immune cell infiltration data mainly divided the samples into four groups: Group A, which mainly clustered samples with high infiltration of CD8 T cells, activated CD4 memory T cells, and M1 macrophages; Group B1, which mainly clustered samples with high infiltration of M0 macrophages; Group B2, which mainly clustered samples with high infiltration of resting dendritic cells; and Group B3, which mainly clustered samples with high infiltration of plasma cells. Among the four groups, samples with higher estimated immune abundance scores mainly clustered in Group A, but there was no significant clustering of clinical characteristic status data (including HPV status, sex, stage, TN classification, and age) among the four groups. Figure 3 E). Compared to the other three groups, group A had the lowest UCHL1 transcriptome expression level, which was statistically significant compared to all other groups. Figure 3 F).

[0038] To further determine the differences in the immune microenvironment among groups A, B1, B2, and B3, we extracted the transcriptome expression levels of 37 immune checkpoint genes and grouped the samples into four groups: A, B1, B2, and B3. We also visualized the data using heatmaps. The results showed that, compared to the other three groups, samples with high expression levels of immune checkpoint genes were mostly clustered in group A. Specifically, samples with high expression of classic immune checkpoint genes such as PDCD1 (PD1), CD274 (PDL1), CTLA4, LAG3, and TIGIT were primarily clustered in group A. Figure 4 A). Based on the analysis in the previous step and here, tumor-toxic immune cells and immune checkpoint genes are mostly enriched in group A, and group A can be defined as "tumor-toxic immune cell enrichment type".

[0039] To further analyze the differences in clinical outcomes among the patients in each group, overall survival (OS) was compared among the four groups. Results showed that in the integrated data from TCGA-HNSC, GEO (GSE65858), and individual cohort analyses, the OS of group B1 was lower than that of the other three groups, and there was a statistically significant difference in survival outcomes between group A and group B1. Figure 4 (BD). To further explore the molecular differences between the two groups of patients with different immunophenotypic profiles, groups A and B1, which showed significant differences in survival prognosis, were extracted for further analysis. Using the limma software package, we identified genes with differential expression between the two groups and performed unsupervised consensus clustering on the top 50 genes with the most significant differences, dividing them into two groups (a and b). Heatmap visualization was also performed. Figure 4 E). The results showed that the differentially expressed genes in groups A and B1 were mostly immune-related genes. Samples with high expression of differentially expressed genes in the transcriptome were mainly enriched in gene group a, which highly overlapped with group A. Samples with low expression of differentially expressed genes in the transcriptome were mainly enriched in gene group b, which highly overlapped with group B1. Figure 4 E). After analyzing the transcriptomic data of groups A and B1, in order to further predict the differences in proteomics between groups A and B1, the TCPA proteomics database was used to extract protein expression levels for groups A and B1, perform correlation difference analysis, extract the top 25 differentially expressed proteins for heatmap plotting, and extract 5 proteomics data points for violin plot visualization to visually display the differences in protein expression between groups A and B1. Figure 4 F). The results showed that, on the differential protein expression heatmap between A and B1, the expression levels of CASPASE7, CLEAVEDD198, LCK, PDL1, STAT3_pY705, and P38MAPK proteins in group A were significantly higher than those in group B1, and this was statistically significant. Figure 4 Based on the above, we believe that PDL1 and UCHL1 may be highly correlated. Therefore, we performed a correlation analysis on the transcriptomic data of UCHL1 and PDL1. The results showed that: in TCGA-GEO integrated data, TCGA-HNSC and GEO (GSE65858), the transcriptomic levels of PDL1 and UCHL1 were all negatively correlated, and all were statistically significant. Figure 4 G). This significant relationship differed between HPV-negative and HPV-positive patients, and UCHL1 transcriptome levels were significantly higher in HPV-positive tumors than in HPV-negative tumors in the TCGA-HNSC cohort, consistent with protein level results collected from our cohort. Figure 4 G).

[0040] To eliminate the influence of human papillomavirus (HPV) infection status on the characteristic differences between groups, we separated and extracted HPV-negative and positive samples from the TCGA-GEO integrated data set, grouping them into A, B1, B2, and B3. We then plotted immune infiltrating cell heatmaps, Kaplan-Meier survival curves, and box plots of UCHL1 transcriptome expression levels in each group for both HPV-negative and positive samples. We also compared the UCHL1 transcriptome expression levels in HPV-positive and negative samples and plotted box plots. The results showed that the clustering patterns of highly expressed 10 immune cell types among the four groups of HPV-positive and negative samples remained similar to those in the TCGA-GEO integrated data set. Figure 5 A, B), but compared with HPV-negative samples, the survival prognosis of group A in HPV-positive samples was significantly better than that of group B1. Figure 5 CD). In HPV-positive samples, there was no significant difference in UCHL1 transcriptome expression levels among the four groups ( Figure 5 D), but in HPV-negative samples, the expression level of UCHL1 transcriptome in group A was significantly lower than that in the other three groups, and all were statistically significant. Figure 5 EF).

[0041] After obtaining the differentially expressed gene lists for the two groups, we extracted the top 20 genes with significant differences and performed GO functional enrichment analysis (Gene Ontology) and KEGG pathway enrichment analysis (Kyoto Encyclopedia of Genes and Genomes). The results showed that GO functional enrichment analysis (Gene Ontology) revealed that, in cellular components (CC), the main enrichment areas were regulation of T cell activation, response to virus, defense response to virus, defense response to symbiont, lymphocyte-mediated immunity, and response to interferon-gamma. In molecular functions (MF), the main enrichment areas were the external side of the plasma membrane, plasma membrane signaling receptor complex, T cell receptor complex, alpha-beta T cell receptor, and lysosomal granules. In biological processes (BP), the main enrichment areas were cytokine receptor binding and cytokine activity. Activities such as endopeptidase activity, G protein-coupled receptor binding, and chemokine activity. Figure 5G. In KEGG pathway enrichment analysis (Kyoto Encyclopedia of Genes and Genomes), the main enrichments were in cytokine-cytokine receptor interaction, PD-L1 expression and PD-1 checkpoint pathway in cancer, Th1 and Th2 cell differentiation, hematopoietic cell lineage, Chagas disease, T cell receptor signaling pathway, Th17 cell differentiation, influenza A, and chemokine signaling pathway, etc. Figure 5 H).

[0042] To further determine the relationship between UCHL1 protein and the subtypes of infiltrating immune cells in tumors, we performed Opal multiplex immunohistochemical staining on collected head and neck squamous cell carcinoma tissue microarray samples. FFPE sections were then subjected to multiplex immunohistochemical staining targeting human CD4, CD8, CD19, CD68, CD163, FoxP3, UCHL1, and Pan-CK. Figure 6 We divided tumor tissues into low-expression and high-expression UCHL1 groups based on UCHL1 expression levels and analyzed the differences in immune cell subtypes between the two groups. The results showed that the high-expression UCHL1 group had significantly higher levels of B cells (CD19+), macrophages (CD163+), Tregs (FoxP3+), and M2 type macrophages (CD68+CD163+) than the low-expression UCHL1 group. Figure 6 D). The results indicate that UCHL1 expression is closely related to the cellular subtypes of tumor immunosuppression, suggesting that UCHL1 is involved in the oncogenic molecular mechanism of immunosuppression and immune tolerance caused by HPV infection.

[0043] Example 2: The role and molecular mechanism of UCHL1 in NK cell killing escape in head and neck squamous cell carcinoma cells.

[0044] To verify the effect of UCHL1 on cell behavior, we constructed FaDu cell lines with transiently high UCHL1 expression and Detroit562 cell lines with transiently low UCHL1 expression using transient plasmid transfection and siRNA small interference transfection. The results showed that, in terms of protein expression, FaDu cells transiently transfected with the UCHL1 plasmid showed significantly higher UCHL1 expression in a short time compared to FaDu cells transfected with the empty vector plasmid and primary FaDu cells; Detroit562 cell lines transfected with siRNA showed significantly suppressed UCHL1 expression in a short time compared to Detroit562 cells transfected with siRNA and primary Detroit562 cells. The short-term overexpression and knockdown of UCHL1 cell models were successfully established. Figure 7 A).

[0045] In the FaDu cell line with short-term UCHL1 overexpression, PDL1 expression at the protein level increased with increasing UCHL1 levels. However, at the mRNA level, PDL1 expression decreased with increasing UCHL1 levels. In the Detroit562 cell line with short-term UCHL1 knockdown, PDL1 expression decreased with decreasing UCHL1 levels, both at the protein and transcriptomic levels. These results suggest that in head and neck squamous cell carcinoma cell lines, PDL1 protein expression may be positively correlated with UCHL1 protein expression, but this positive correlation between mRNA-level PDL1 and UCHL1 expression only exists in Detroit562 cells with high UCHL1 expression. Figure 7 B).

[0046] To verify whether targeting UCHL1 could sensitize current traditional cancer treatments, we used a cell colony formation assay to examine the effects of the UCHL1 small molecule inhibitor LDN57444 on cisplatin and radiotherapy on tumor cells. We found that inhibiting UCHL1 did not improve the effects of traditional treatments for head and neck squamous cell carcinoma. Figure 7 C, D).

[0047] To investigate the impact of UCHL1 on tumor cell immune escape, we co-cultured NK92MI cells with four tumor cell lines to mimic the killing effect of NK cells on tumor cells, further exploring the correlation between UCHL1 and tumor cell immune escape. The results showed that NK92MI cell cytotoxicity was positively correlated with the ratio of NK cells to tumor cells, while the UCHL1-overexpressing cell line Detroit562 exhibited significant tolerance to NK92MI cell killing. Figure 7 E). In vitro cytotoxicity assays confirmed a significant difference in the cytotoxic effects of FaDu (which does not express UCHL1) and Detroit562 (which highly expresses UCHL1) on NK92-MI cells. Figure 7F). Knocking down UCHL1 expression in Detroit562 cells increases the cytotoxicity of NK-92MI cells against tumor cells. Figure 7 GH). The same conclusion was also found when using the UCHL1 small molecule inhibitor LDN57444 in Detroit562 cells, which are most resistant to NK-92MI killing, so inhibiting UCHL1 expression or activity can significantly increase the killing toxicity of NK-92MI cells to tumor cells.

[0048] Example 3: Treatment of head and neck squamous cell carcinoma with a combination of UCHL1 small molecule inhibitor and MEK small molecule inhibitor.

[0049] During LDN57444 treatment, p-ERK protein expression showed significant changes in the Detroit562 cell line, which highly expresses UCHL1. To further explore the specific regulatory mechanism of UCHL1 on PDL1, we used the MEK inhibitor PD0325901 to treat four HNSCC cell lines for a short period of 24 hours and screened Detroit562 cells, which highly express UCHL1, and CAL27 cells, which do not express UCHL1, for a long period of 96 hours. Western blotting was used to observe changes in the protein expression levels of PDL1 and UCHL1. The results showed that PDL1 protein expression decreased in both the short-term (24 hours) and long-term (96 hours) MEK inhibitor treatments, but the UCHL1 protein expression level in Detroit562 cells did not change significantly. Figure 8 A, B). Cell colony formation assays showed that Detroit 562 cells, which highly expressed UCHL1, were most resistant to the MEK inhibitor PD0325901. Figure 8 C, D). CFA experiments showed that the combination of two small molecule inhibitors significantly inhibited Detroit562 cell colony formation, suggesting that targeted inhibition of UCHL1 activity can increase tumor sensitivity to MEK inhibitors. Figure 8 E). In vitro cytotoxicity experiments showed that MEK small molecule inhibitors could reverse the resistance of UCHL1-induced tumor cell NK cell killing. Figure 8 F, G).

[0050] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of UCHL1 inhibitors in the preparation of therapeutic drugs for head and neck squamous cell carcinoma, characterized in that, The head and neck squamous cell carcinoma is caused by HPV infection, and the UCHL1 inhibitor is LDN57444.

2. The application according to claim 1, characterized in that, The UCHL1 inhibitor reduces UCHL1 expression or inhibits UCHL1 activity.

3. The application according to claim 2, characterized in that, The therapeutic drugs for head and neck squamous cell carcinoma reduce the expression of mitogen-activated protein kinase kinase or inhibit the activity of mitogen-activated protein kinase kinase.

4. The application of a combination of UCHL1 inhibitors and mitogen-activated protein kinase inhibitors in the preparation of therapeutic drugs for head and neck squamous cell carcinoma, characterized in that... The head and neck squamous cell carcinoma is head and neck squamous cell carcinoma caused by HPV infection, the UCHL1 inhibitor is LDN57444, and the mitogen-activated protein kinase inhibitor is PD0325901.

5. A treatment drug for squamous cell carcinoma of the head and neck, characterized in that, The treatment for head and neck squamous cell carcinoma includes a UCHL1 inhibitor and a mitogen-activated protein kinase inhibitor. The head and neck squamous cell carcinoma is head and neck squamous cell carcinoma caused by HPV infection. The UCHL1 inhibitor is LDN57444, and the mitogen-activated protein kinase inhibitor is PD0325901.

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