Application of GLRX5 in preparation of medicine for enhancing immunotherapy of renal cell carcinoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of GLRX5 in preparation of a medicine for enhancing immunotherapy of renal cell carcinoma. GLRX5 is a tumor inhibition factor in renal cell carcinoma and a key factor for regulating and controlling the stability of renal cell carcinoma PD-L1, can inhibit proliferation and metastasis of renal carcinoma, specifically interacts with PD-L1, and inhibits immune escape of renal carcinoma by enhancing SPOP-mediated PD-L1 K48 connection ubiquitination and proteasome degradation, so that treatment of an immunosuppressor is enhanced; the molecular mechanism determines that the GLRX5 is used as a key coordination factor of an SPOP-PD-L1 degradation axis in a renal cell carcinoma pathogenesis, also reveals that the GLRX5-SPOP-PD-L1 axis is a key mechanism of RCC immune escape, and provides a new strategy for promoting PD-L1 degradation and reversing immunosuppression by enhancing the expression or activity of the GLRX5.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, specifically relating to the application of GLRX5 in the preparation of drugs that enhance the immunotherapy of renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is a malignant tumor originating from the epithelial cells of the renal tubules, accounting for more than 90% of all kidney cancer cases and is one of the most common types of malignant tumors of the urinary system. According to the World Health Organization, RCC ranks 13th among cancer-related causes of death worldwide, with men having a 1.5 times higher incidence rate and mortality rate than women. Alarmingly, its global incidence is increasing year by year. Clear cell renal cell carcinoma (ccRCC), as the most prevalent histological subtype (accounting for 70-80%), exhibits greater clinical invasiveness compared to papillary carcinoma (10-20%) and eosinophilic carcinoma (5%). Although partial or total nephrectomy remains the primary treatment for localized ccRCC, 20-40% of patients experience recurrence post-surgery. Metastatic cases often spread through the bloodstream to the perirenal tissues, liver, and lungs, but treatment options are very limited due to inherent resistance to radiotherapy and chemotherapy. Renal cell carcinoma (RCC), a common and aggressive tumor of the urinary system, faces multiple challenges in clinical treatment. Although treatment regimens, such as sunitinib, are widely used, drug resistance severely limits their efficacy. Furthermore, RCC has low sensitivity to radiotherapy and chemotherapy, an immunotherapy response rate of less than 30%, and lacks reliable biomarkers to identify potential responders. Therefore, elucidating the molecular pathogenesis of RCC and exploring novel molecular biomarkers to guide precision treatment strategies is of significant clinical importance.

[0003] The advent of immune checkpoint inhibitors (ICIs) targeting PD-1 / PD-L1 and CTLA-4 has revolutionized the treatment of ccRCC, but clinical responses vary. Current combination therapies for metastatic disease (immune checkpoint inhibitors combined with targeted drugs) have limitations, including poor efficacy, frequent immune-related toxicities, and a lack of universality. Although established biomarkers such as PD-L1 expression levels and tumor microenvironment (TME) characteristics can guide treatment decisions, their predictive accuracy remains insufficient due to tumor heterogeneity and dynamic regulatory mechanisms. Therefore, identifying novel predictive biomarkers and elucidating the mechanisms of action of PD-L1 / PD-1 pathways is crucial for optimizing immunotherapy efficacy and reducing treatment-related adverse events in cancer patients.

[0004] Glutathione oxoreductin-5 (GLRX5) is a key regulator of the transfer of iron-sulfur clusters (ISCs) to iron regulatory proteins (IRPs) in mitochondria, maintaining cellular iron homeostasis and mitochondrial metabolic integrity. GLRX5 deficiency disrupts heme biosynthesis, leading to sideroblastic anemia and nonketotic hyperglycinemia. Recent research evidence suggests that GLRX5 participates in tumorigenesis as an inhibitor of ferroptosis, offering a potential therapeutic target. Emerging evidence indicates that GLRX5 is an inhibitor of ferroptosis involved in tumorigenesis, providing a potential therapeutic target. Despite these advances, its role in renal cell carcinoma remains to be explored. Summary of the Invention

[0005] To address the above-mentioned problems, the present invention aims to provide an application of GLRX5 in the preparation of drugs that enhance the immunotherapy of renal cell carcinoma.

[0006] The technical content of this invention is as follows: This invention provides a substance for detecting the expression level of the biomarker GLRX5 (glutathione reductase 5), comprising at least one of the following applications: a1) Application in the preparation of products for predicting the immunotherapeutic efficacy, prognostic assessment, or auxiliary prediction of renal cell carcinoma; a2) Application in the preparation of products for predicting the immunotherapeutic efficacy and prognosis of renal cell carcinoma using immune checkpoint therapy; The substance used to detect the expression level of the biomarker GLRX5 includes reagents for detecting the expression level, content, or RNA expression level of GLRX5. The reagent includes antibodies, peptides, proteins, or nucleic acid molecules that bind to the GLRX5 protein; preferably, the antibody is a GLRX5 monoclonal antibody. The auxiliary diagnostic products for renal cell carcinoma metastasis include those for diagnosing the malignancy of renal cell carcinoma; The predictive aids include predicting the sensitivity of renal cell carcinoma patients to sunitinib. The products include chips, kits, or reagents for detecting GLRX5 on high-throughput sequencing platforms; The renal cell carcinoma includes clear cell renal cell carcinoma.

[0007] The present invention also provides a substance that promotes GLRX5 expression, including the use of at least one of the following: b1) Application in the preparation of products that inhibit the growth, proliferation, migration and / or invasion of renal cell carcinoma; b2) Application in the preparation of products that inhibit immune escape from renal cell carcinoma; b3) Use in the preparation of products for enhancing the sensitivity of renal cell carcinoma to PD-L1 targeted therapy; b4) Application in the preparation of drugs for promoting the degradation of PD-L1 protein; b5) Application in the preparation of drugs for enhancing T cell-mediated antitumor immune responses The T cells include CD8+ T cells.

[0008] The GLRX5 was expressed at low levels in renal cell carcinoma cells; The substances that promote GLRX5 expression include enhancers, overexpression plasmid vectors, and nanoparticles carrying GLRX5 gene mRNA. The products include pharmaceuticals or laboratory reagents; The product includes GLRX5 as an active ingredient; The GLRX5 promotes the degradation of PD-L1 and inhibits the growth, proliferation, migration and / or invasion of renal cell carcinoma by specifically interacting with PD-L1. The degradation of PD-L1 is ubiquitination-dependent. Furthermore, the GLRX5 promotes the ubiquitination degradation of PD-L1 by enhancing the ubiquitination modification of K48 linkage, thereby regulating the ubiquitination mode of PD-L1. Furthermore, GLRX5 inhibits tumor immune escape by enhancing SPOP-mediated PD-L1 K48 linker ubiquitination and proteasome degradation. The GLRX5 inhibits the malignant progression of renal cell carcinoma by enhancing T cell-dependent tumor suppression; specifically, GLRX5 promotes PD-L1 degradation, triggers T cell-mediated tumor suppression, and thus inhibits immune escape from renal cell carcinoma.

[0009] The drug that inhibits immune escape in renal cell carcinoma includes a PD-L1 monoclonal antibody (PD-L1 mAb), and the GLRX5 enhancer enhances the anti-tumor efficacy of the PD-L1 monoclonal antibody by increasing the expression level of GLRX5. This invention also provides an application of the GLRX5-SPOP-PD-L1 control axis as a target point, including at least one of the following: c1) Application in the preparation of products that inhibit the growth, proliferation, migration and / or invasion of renal cell carcinoma; c2) Application in the preparation of products that inhibit immune escape from renal cell carcinoma; c3) Use in the preparation of products for enhancing the sensitivity of renal cell carcinoma to PD-L1 targeted therapy; c4) Application in the preparation of drugs for promoting the degradation of PD-L1 protein; c5) Application in the preparation of drugs for enhancing T cell-mediated antitumor immune responses; The GLRX5-SPOP-PD-L1 regulatory axis is used as the target, and GLRX5 is used as the intervention target to interfere with the expression of SPOP and PD-L1.

[0010] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of GLRX5 or an enhancer thereof, and a pharmaceutically acceptable carrier; The pharmaceutical composition further includes a PD-L1 inhibitor, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody or its antigen-binding fragment; The pharmaceutically acceptable carriers include at least one of tablets, capsules, oral liquids, granules, suspensions, injections, powder for injection, pellets, sustained-release agents, controlled-release agents, and targeted formulations.

[0011] The beneficial effects of this invention are as follows: This invention provides a biomarker, GLRX5, which is a tumor suppressor in renal cell carcinoma (RCC) and a key factor regulating PD-L1 stability in RCC. It inhibits RCC proliferation and metastasis, specifically interacts with PD-L1, and suppresses immune escape in RCC by enhancing SPOP-mediated PD-L1 K48 linkage ubiquitination and proteasome degradation, thereby enhancing immunosuppressant therapy. This molecular mechanism establishes GLRX5 as a key coordinator of the SPOP-PD-L1 degradation axis in the pathogenesis of RCC, and the loss of GLRX5 drives the malignant progression of RCC. The combination of GLRX5 enhancers and PD-1 / PD-L1 inhibitors can significantly enhance their efficacy and can be used as sensitizers. Furthermore, this invention reveals that the GLRX5-SPOP-PD-L1 axis is a key mechanism for RCC immune escape, providing a novel strategy to promote PD-L1 degradation and reverse immunosuppression by enhancing GLRX5 expression or activity. Attached Figure Description

[0012] Figure 1 The expression levels and significance of GLRX5 in 33 types of cancer and adjacent normal tissues in the TCGA database were analyzed (A), the expression levels and significance of GLRX5 in 30 types of cancer and adjacent normal tissues in the PCAWG dataset were analyzed (B), and the pan-cancer correlation analysis of GLRX5 expression levels and OS was performed (C). Figure 2The study included analyses of GLRX5 mRNA expression differences between ccRCC and adjacent normal tissues in the TCGA database (A, B), correlations between GLRX5 expression levels and clinicopathological features of ccRCC patients (C, D), and correlations between GLRX5 expression levels and survival prognosis in ccRCC patients (E) (risk indicates HR > 1 and P < 0.05; protection indicates HR < 1 and P < 0.05; ns indicates P > 0.05); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NSP > 0.05; Figure 3 qPCR analysis was performed on 15 normal tissue samples and 50 RCC specimens (A), qPCR analysis was performed on 15 pairs of matched RCC tissues and adjacent non-tumor tissues (B), the expression level of GLRX5 protein in RCC and adjacent normal tissues (C), and representative IHC staining of GLRX5 in RCC and adjacent normal tissues at a scale bar of 100 μm (D). Figure 4 To construct stable GLRX5-overexpressing Caki-1 and 786O cell lines (A), a colony formation assay was performed to assess the colony formation ability of RCC cells (B), a transverse wall migration assay was performed to assess the migration ability of RCC cells (C), and a CCK8 proliferation assay was performed to determine the proliferation ability of RCC cells (D). Figure 5 To stabilize the construction of GLRX5-knockout Caki-1 and 786O cell lines (A), a colony formation assay was performed to assess the colony-forming ability of GLRX5-knockout RCC cells (B), a transverse wall migration assay was performed to assess the migration ability of GLRX5-knockout RCC cells (C), and a proliferation assay was performed to determine the proliferation ability of GLRX5-knockout RCC cells (D). Figure 6 To perform CoIP experiments using anti-GLRX5 antibody, the enriched proteins were separated by SDS-PAGE, stained with Coomassie Brilliant Blue (A), and their structures were visualized using PyMOL on GLRX5 and PD-L1 in the PDB database (B). IF assays were also performed to confirm the subcellular localization of GLRX5 and PD-L1 in RCC cells (C). Figure 7 To perform exogenous CoIP experiments (A, B) and endogenous CoIP analysis using anti-GLRX5 antibody, 293T cells were co-transfected with Flag-labeled GLRX5 plasmid and HA-labeled PD-L1 plasmid. Figure 8To verify PD-L1 expression levels in GLRX5 knockdown renal cell carcinoma (RCC) cells (A), Western blot analysis was performed on RCC cells transfected with the Flag-GLRX5 plasmid, using the proteasome inhibitor MG132, the autophagy inhibitor BafA1, or the lysosomal inhibitor CQ, respectively, to assess dynamic changes in PD-L1 expression (B). CHX experiments were performed in 293T cells co-transfected with the Flag-GLRX5 and HA-PD-L1 plasmids (C), and CHX experiments were performed in GLRX5 knockdown RCC Caki-1 cells (D). Figure 9 To assess the dose-dependent changes in HA-PD-L1 ubiquitination levels by co-transfecting 293T cells with HA-tagged PD-L1 plasmid and Flag-tagged GLRX5 plasmid at increasing doses, co-immunoprecipitation (CoIP) was used to evaluate the results (A). In 293T cells co-transfected with HA-tagged PD-L1 plasmid, Flag-tagged GLRX5 plasmid, and Myc-tagged ubiquitin (Ub) wild-type (WT) or lysine mutant, CoIP was used to investigate whether GLRX5 enhances specific ubiquitination types of PD-L1 (B). Endogenous co-immunoprecipitation (CoIP) was also performed in GLRX5-knockdown RCC Caki-1 and 786O cells (C, D). Figure 10 To perform co-immunoprecipitation experiments in Caki-1 cells overexpressing GLRX5, anti-PD-L1 antibody or IgG was used as a control. The enriched proteins were separated by SDS-PAGE and then stained with Coomassie Brilliant Blue (A) and the results of peptide mass spectrometry were obtained (B). Figure 11 Exogenous co-immunoprecipitation experiments were performed in 293T cells co-transfected with Myc-labeled SPOP plasmid and HA-labeled PD-L1 plasmid (A, B). Further transfection with different doses of Flag-labeled GLRX5 plasmid was performed in 293T cells co-transfected with Myc-labeled SPOP and HA-labeled PD-L1 plasmids, followed by co-immunoprecipitation experiments (C). Exogenous co-immunoprecipitation experiments were performed in GLRX5-knockdown 293T cells transfected with Myc-labeled SPOP and HA-labeled PD-L1 plasmids (D). Endogenous co-immunoprecipitation experiments were performed in GLRX5-knockdown RCC Caki-1 and 786O cells (E, F). Endogenous co-immunoprecipitation experiments were performed in GLRX5-overexpressing RCC Caki-1 and 786O cells (G, H). Finally, SPOP-knockdown RCC Caki-1 and 786O cells were also tested. Caki-1 and 786O cells were transfected with Flag-labeled GLRX5 plasmid or negative control (NC) plasmid, respectively, and then subjected to Western blot analysis with or without proteasome inhibitor MG132 (I, J). Figure 12 The flowchart for co-culturing T cells and tumor cells is shown in Figure (A). The cytotoxic activity of CD8+ T cells co-cultured with GLRX5-knocked RCC Caki-1 and 786O cells was assessed by lactate dehydrogenase (LDH) release assay (B). The survival rate of tumor cells after co-culturing CD8+ T cells with GLRX5-knocked RCC Caki-1 and 786O cells was detected by CCK8 assay (C). Figure 13 To knock down SPOP in GLRX5-overexpressing RCC Caki-1 and 786O cells, these tumor cells were co-cultured with CD8+ T cells, and lactate dehydrogenase (LDH) release assay was performed (A). To knock down SPOP in GLRX5-overexpressing RCC Caki-1 and 786O cells, these tumor cells were co-cultured with CD8+ T cells, and tumor cell viability was assessed by CCK8 assay (B). Figure 14 This diagram illustrates tumor formation in BALB / c mice. BALB / c mice (n = 3) were injected with Renca cells and then treated with either IgG2b or PD-L1 monoclonal antibody at specified time points (A). The image shows the tumor dissected at the end of the G experiment (B), the tumor weight measured two weeks after treatment (C), and the tumor growth curves measured at different time points (D). Detailed Implementation

[0013] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0014] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0015] The cells, culture medium, reagents, and methods used in this invention are as follows: 1. Cell Cultures: RCC cell lines (Caki-1, 786O, and Renca) and human embryonic kidney cell line 293T cells were purchased from the American Type Culture Collection (ATCC). Caki-1 and 293T cells were cultured in DMEM (Procell), while 786O and Renca cells were cultured in RPMI 1640 (Procell). All media were supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin / streptomycin (Haixing, Suzhou).

[0016] 2. Commercial Antibodies and Chemical Reagents: Anti-GLRX5 antibody (Catalog No. #18884-1-AP, ProteinTech), Anti-PD-L1 antibody (Catalog No. #M033179, Abmart), Anti-SPOP antibody (Catalog No. #12108-1-AP, ProteinTech), Anti-HA-tagged antibody (Catalog No. #51064-2-AP, ProteinTech), Anti-Flag-tagged antibody (Catalog No. #M20008, Abmart), Anti-Myc-tagged antibody (Catalog No. #2278, CST), Anti-ubiquitination-tagged antibody (Catalog No. #10201-2-AP, ProteinTech). nTech), anti-GAPDH antibody (catalog number #60004-1, ProteinTech), HA agarose (catalog number #A2095, Sigma-Aldrich), Myc agarose (catalog number #A7470, Sigma-Aldrich), Flag agarose (catalog number #A2220, Sigma-Aldrich), CHX agarose (catalog number #7418, Selleck), MG132 (catalog number #HY-13259, MCE), BafA1 (catalog number #HY-100558, MCE), and CQ (catalog number #HY-17589A, MCE).

[0017] 3. Western Blotting (protein blotting) Forty-eight hours after transfection, protein lysates were collected using RIPA lysis buffer (Cell Signaling Technology) containing protease inhibitors (Suzhou New Cell & Molecular Biotechnology Co., Ltd.). Quantitative analysis was performed using the Pierce method with bovine serum albumin (BSA) as a standard for normalization. Samples (30 μg / channel) were denatured in Laemmli buffer at 95°C for 10 min, separated on 12% SDS-PAGE gels (Bio-Rad) using Tris-glycine buffer (80V / 30 min stacking, 120V / 90 min separation). Protein transfer to PVDF membranes (Millipore) was performed using Tris-glycine-methanol buffer at 300 mA for 2 hours. The membranes were blocked with 5% bovine serum albumin, incubated overnight at 4°C with primary antibody (1:1000 dilution), and then incubated at room temperature for 1 hour with HRP-labeled secondary antibody (1:10000 dilution). After washing with TBST, the sample was developed using ECL substrate (Bio-Rad ChemiDoc) and imaged using the ChemiDoc system. GAPDH was used as an internal control for sample loading to standardize the sample.

[0018] 4. qPCR Total RNA was extracted using TRIzol reagent and its purity was verified. Reverse transcription was performed under standard conditions using PrimeScript RT premix (Takara). SYBR Green-based qPCR amplification was performed using TB Green Premix ExTaq II (Takara) on a QuantStudio 5 system (Applied Biosystems) with a 20 μL reaction volume. The thermal cycling program was: 95°C pre-denaturation for 30 seconds, followed by 40 cycles of 95°C denaturation for 5 seconds and 60°C annealing for 30 seconds. Melting curve analysis (65-95°C gradient) confirmed amplification specificity. All samples were analyzed triplicate using GAPDH as an internal reference gene. Relative quantification was calculated using the ΔΔCt method, and expression levels were standardized against the control group.

[0019] 5. Immunohistochemical detection methods The immunohistochemical assay procedure for renal cell carcinoma tissue specimens is as follows: Paraffin-embedded renal cell carcinoma tissue is sectioned into 4-μm segments, and dewaxing and hydration are performed according to standard procedures. The tissue sections are incubated with 3% hydrogen peroxide solution at room temperature for 10 min to inhibit endogenous peroxidase activity. Subsequently, non-specific binding sites are blocked with 5% bovine serum albumin (BSA) at 25°C for 30 min. The tissue sections are then placed in a humidified chamber at 4°C and incubated with a validated primary antibody (rabbit / mouse specific antibody) for 16-18 hours. After three washes with PBS with shaking, the sections are incubated with HRP-labeled homologous secondary antibody at 37°C for 60 min. 3,3'-diaminobenzidine is used for staining, and finally, microscopic observation and image acquisition are performed using an Olympus BX53 upright microscope system (Olympus Corporation, Japan).

[0020] 6. Immunofluorescence and confocal microscopy RCC cells were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by three 5 min washes with PBS. Permeabilization was performed using 0.1% Triton X-100 solution (room temperature, 15 min), followed by the same washing procedure. Non-specific binding sites were blocked with 5% bovine serum albumin (BSA) at 25°C for 60 min. The primary antibody was diluted 1:100 in blocking buffer and incubated overnight (14-16 hours) in a humidified chamber at 4°C. After washing with PBS, the cells were incubated with Alexa Fluor-labeled homologous secondary antibody at room temperature in the dark for 60 min. Finally, the cell nuclei were counterstained with DAPI (5 min dark imaging). Fluorescence images were acquired using an Olympus confocal microscope (Tokyo, Japan).

[0021] 7. Co-immunoprecipitation (CoIP) RCC and 293T cells were lysed in EBC lysis buffer (containing a 1× protease / phosphatase inhibitor mixture, Beyotime Biotech, Shanghai) on ice, followed by centrifugation at 12,000g for 10 min. 10% of the supernatant was mixed with 4× Laemmli buffer as an internal control. The remaining lysis buffer was incubated with 20 μL of anti-Flag affinity gel (Sigma-Aldrich, Germany), anti-HA affinity gel (Sigma-Aldrich, Germany), or anti-Myc affinity gel (Sigma-Aldrich, Germany) at 4°C with 45°C rotation for 12 h. The immune complexes were washed four times with NETN buffer (100 mM sodium chloride, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% NP-40), centrifuged at 4°C for 5 min each time. The precipitated complexes were resuspended in 1× SDS loading buffer, denatured at 95°C for 10 min, and then analyzed by Western blot.

[0022] 8. Protein-protein docking The crystal structure data of the target protein were obtained from the Protein Database (PDB; https: / / www.rcsb.org). Structural preprocessing (dehydration and chain separation) was performed using PyMOL (v2.5.2, Schrodinger). Molecular docking simulations were conducted using the GRAMM-X server (https: / / vakser.compbio.ku.edu / resources / gramm / grammx / ).LigPlot+ (v2.2.8), and the final visualization results were generated by PyMOL for interfacial interaction analysis.

[0023] 9. Cycloheximide (CHX) assay for protein stability. In the CHX experiment, RCC cells were seeded in 6 cm culture dishes (5 × 10⁶ cells per dish). 5 Cells were cultured for 24 hours and then treated. Novel protein synthesis was then inhibited with 200 μg / ml cyclohexylimide (CHX), and cell samples were collected at specified time points. Protein lysates were analyzed by Western blotting to assess the dynamic changes in PD-L1 degradation.

[0024] 10. CD8+ T cell isolation and co-culture Monocytes (PBMCs) were isolated from healthy donor peripheral blood using Ficoll-Paque density gradient centrifugation. CD8+ T cells were enriched using magnetically activated cell sorting technology (Miltenyi Biotec). Expanded cells were cultured in anti-CD3 / CD28 coated plates (3.5 μg / mL and 1 μg / mL, respectively) with 10 ng / mL IL-2 (PeproTech). Tumor cytotoxicity was assessed using a lactate dehydrogenase (LDH) release assay (CytoTox 96®, Promega), specifically by measuring fluorescence intensity at 490 nm after 24 hours of co-culture.

[0025] 11. Xenograft tumor model Rainer cells (5×10) 5 Dissolved in 100 μL PBS, the tumor was subcutaneously implanted into the back of 6-week-old BALB / c mice, who were randomly divided into experimental and control groups. Tumor volume was dynamically monitored using calipers. The tumor was considered controlled when the average tumor volume in the experimental group reached 100 mm². 3 PD-L1 monoclonal antibody was injected intraperitoneally. The animals were euthanized by cervical dislocation 14 days after the intervention. This experimental protocol was approved by the Institutional Animal Ethics Committee of Nanfang Hospital, Southern Medical University (IACUC-LAC-20250421-001).

[0026] 12. Statistics All statistical analyses were performed using SPSS software (version 25.0). Student's t-test was used for comparisons between groups, and one-way ANOVA was used for multi-group analyses. Statistical significance was defined as p < 0.05 using a two-tailed test.

[0027] Example 1 GLRX5 shows low performance in ccRCC. To investigate GLRX5 mRNA expression in tumors and adjacent normal tissues, analysis was performed using the UCSCxenaShiny online interactive tool, which contains data from 33 cancer types for pan-cancer correlation analysis.

[0028] like Figure 1 As shown in Figure A, pan-cancer analysis revealed significant differences in GLRX5 expression levels between tumors and adjacent normal tissues in most of the 33 cancer types. Notably, GLRX5 expression levels were significantly reduced in ccRCC.

[0029] Furthermore, using the UCSCxenaShiny platform, which integrates pan-cancer genome-wide analysis (PCAWG), we systematically compared the GLRX5 expression patterns in tumor tissues and adjacent normal tissues in various cancers. The analysis results are as follows: Figure 1As shown in Figure B, the expression level of GLRX5 in clear cell renal cell carcinoma (ccRCC) was significantly reduced compared with adjacent normal tissue.

[0030] To investigate the correlation between GLRX5 expression and pan-cancer clinical prognosis, survival analysis was performed using the UCSCxenaShiny platform. Patients were divided into low-expression and high-expression groups based on median GLRX5 mRNA levels, and its prognostic association with overall survival (OS) across various cancer types was systematically evaluated. Results are as follows: Figure 1 As shown in Figure C, GLRX5 mRNA expression is significantly correlated with survival outcomes. Specifically, patients with high expression in clear cell renal cell carcinoma (ccRCC) have better prognoses, while those with low expression show poorer clinical outcomes.

[0031] To validate these findings, analysis of the TCGA database showed that GLRX5 mRNA expression was significantly downregulated in tumor tissues compared to normal tissues. Figure 2 (A and B in the original text). Next, the correlation between GLRX5 and patient clinical characteristics was analyzed. As the disease stage of ccRCC patients progressed, the expression level of GLRX5 gradually decreased (…). Figure 2 (C). GLRX5 expression was also significantly reduced in patients with lymph node metastasis or distant metastasis (C). Figure 2 (D, E in the text). Kaplan-Meier survival analysis showed that patients with low GLRX5 expression had a poorer prognosis (D, E in the text). Figure 2 (F in the middle).

[0032] Example 2 GLRX5 expression is low in RCC tissue. To validate these findings, qPCR analysis was performed on 50 renal cell carcinoma tissues and 15 normal tissues. The results showed that GLRX5 expression in renal cell carcinoma tissues was significantly lower than that in normal tissues. Figure 3 In addition, qPCR results from 15 pairs of adjacent non-cancerous and cancerous tissues also showed decreased GLRX5 expression in RCC tissues (A). Figure 3 (B in the original text). Western blot analysis of 15 pairs of renal cell carcinoma tissues revealed that GLRX5 protein expression was significantly reduced in most cancer tissues. Figure 3 In addition, immunohistochemical (IHC) analysis showed that the GLRX5 protein level was significantly downregulated in renal cell carcinoma tissue compared with adjacent non-tumor tissue. Figure 3 (D in the middle).

[0033] Example 3 GLRX5 inhibits the proliferation and migration of RCCs. To further investigate whether GLRX5 affects the progression of renal cell carcinoma (RCC), and considering the low expression level of this gene in RCC, a GLRX5 overexpression plasmid was designed, and stable GLRX5 overexpression cell lines were established in Caki-1 and 786O cells. First, the expression level of GLRX5 was detected by Western blotting, confirming the successful establishment of a stable GLRX5-expressing renal cell carcinoma cell line. Figure 4 (A) To further verify whether GLRX5 overexpression affects the colony-forming ability of RCC cells, a colony-forming assay was performed. The results showed that GLRX5 overexpression significantly reduced the colony-forming ability of RCC cells (A). Figure 4 (B in the text). The results of the transpore migration assay showed that GLRX5 overexpression inhibited the migration ability of RCC cells (…). Figure 4 (C in the text). Similarly, CCK8 proliferation assays showed that GLRX5 overexpression significantly inhibited the proliferation of RCC cells (C). Figure 4 (D in the middle).

[0034] Subsequently, a stable GLRX5 knockdown RCC cell line was successfully established. Figure 5 (A) Clonogenic assays showed that GLRX5 knockdown significantly enhanced colony formation ability. Figure 5 (B in the text). Transwell migration assays showed that GLRX5 knockdown cells exhibited enhanced migration ability (…). Figure 5 C). The CCK8 proliferation assay further confirmed the enhanced proliferation activity after GLRX5 inhibition (C). Figure 5 (D in the text). These results collectively indicate that reducing GLRX5 expression promotes RCC progression by enhancing migration and proliferation.

[0035] Example 4 GLRX5 specificity and interaction with PD-L1 To investigate whether GLRX5 regulates the progression of renal cell carcinoma (RCC) through protein-protein interactions, co-immunoprecipitation (CoIP) experiments were performed in RCC Caki-1 cells using either anti-GLRX5 antibody or IgG. The enriched proteins were separated by SDS-PAGE and then visualized using Coomassie Brilliant Blue staining. Figure 6 (A) Mass spectrometry analysis of the 40-50 kDa bands specifically observed in the GLRX5 group revealed PD-L1 as the major interacting protein. Given that PD-L1 plays a crucial role as an immune checkpoint protein overexpressed in tumor cells and mediating immune escape, the interaction mechanism of GLRX5-PD-L1 was investigated. Protein-protein docking analysis showed multiple binding sites between GLRX5 and PD-L1, indicating a strong molecular interaction between the two. Figure 6(B in the text). Immunofluorescence experiments revealed different subcellular localization patterns: GLRX5 was mainly localized in the cytoplasm, while PD-L1 was distributed in both the nuclear and cytoplasmic regions. Figure 6 The C in the text). Exogenous co-immunoprecipitation assays using 293T cells confirmed the direct binding of GLRX5 to PD-L1 (C). Figure 7 (A and B in the original text). Consistent with this, endogenous co-immunoprecipitation experiments in renal cell carcinoma Caki-1 and 786O cells also verified the specific interaction between GLRX5 and PD-L1. Figure 7 (C and D in the text).

[0036] Example 5 GLRX5 promotes PD-L1 degradation by enhancing K48-linked ubiquitination modification. To determine the GLRX5-mediated PD-L1 regulatory mechanism, Western blot analysis was performed in GLRX5 knockout RCC cells, revealing a significant increase in PD-L1 protein levels. Figure 8 (A) Further investigation was conducted to determine whether GLRX5 regulates the PD-L1 degradation pathway. Treatment of GLRX5-overexpressing RCC cells with a proteasome inhibitor (MG132), an autophagy inhibitor (BafA1), and a lysosomal inhibitor (chloroquine, CQ) revealed that MG132 significantly reversed GLRX5-induced PD-L1 degradation, indicating the involvement of the proteasome pathway. Figure 8 (B in the original text). Cycloheximide (CHX) assays in 293T cells co-transfected with Flag-GLRX5 and HA-PD-L1 revealed that PD-L1 degradation was accelerated when GLRX5 was overexpressed, confirming GLRX5-mediated PD-L1 protein instability. Figure 8 (C in the text). Consistent with this, CHX treatment in GLRX5-deficient Caki-1 cells prolonged the half-life of the PD-L1 protein. Figure 8 (D in the text). Through co-transfection experiments of HA-PD-L1 with different doses of Flag-GLRX5 in 293T cells, a dose-dependent increasing trend in PD-L1 ubiquitination levels was observed. Figure 9 Based on the different lysine linkage sites, ubiquitination modifications can be classified into types such as K6, K11, K27, K29, K33, K48, and K63, among which K48 and K63 play a key role in proteasome degradation. Transfection experiments using HA-PD-L1, Flag-GLRX5, and ubiquitin mutants revealed a significant increase in ubiquitination levels at the K48 site (…). Figure 9 In GLRX5 knockdown renal cell carcinoma lines, PD-L1 ubiquitination levels were significantly reduced, primarily at the K48 site rather than the K63 site. Figure 9 (C and D in the text) to verify the regulatory role of GLRX5 in the PD-L1 ubiquitination mode.

[0037] Example 6 GLRX5 enhances SPOP-mediated PD-L1 ubiquitination-dependent degradation Protein ubiquitination is a dynamic process primarily regulated by E3 ubiquitin ligases and deubiquitinases (DUBs). Next, we will attempt to determine whether GLRX5 regulates PD-L1 ubiquitination via E3 ligases or deubiquitinases (DUBs).

[0038] After co-immunoprecipitation in GLRX5-overexpressing RCC cells, SDS-PAGE and Coomassie Brilliant Blue staining showed a distinct band at 40 kDa. Figure 10 (A) Through peptide abundance analysis, mass spectrometry identified SPOP as the major interacting protein (A). Figure 10 SPOP (B in the original text) is one of the most abundant proteins enriched by anti-PD-L1 antibodies. Exogenous co-immunoprecipitation experiments were performed in 293T cells transfected with HA-PD-L1 and Flag-SPOP to verify the direct interaction between PD-L1 and SPOP. Figure 11 (A and B in the text). When SPOP, PD-L1, and GLRX5 are co-transfected at concentration gradients, the binding affinity of SPOP to PD-L1 increases in a dose-dependent manner. Figure 11 In 293T cells, knockdown of GLRX5 significantly weakened the interaction between SPOP and PD-L1 (C). Figure 11 (D in the figure), this result was further validated by an endogenous CoIP assay in renal cell carcinoma (RCC) cells. Figure 11 In E and F of the study, PD-L1-SPOP interaction is regulated in a GLRX5-dependent manner. Conversely, overexpression of GLRX5 in RCC cells enhances the binding of SPOP to PD-L1. Figure 11 (G, H in). Given the role of SPOP in PD-L1 ubiquitination, SPOP-knockdown RCC cells were treated with MG132 after transfection with GLRX5. Western blot analysis showed that GLRX5-mediated PD-L1 degradation depends on SPOP ubiquitination. Figure 11 (I, J in the original text). These results collectively indicate that GLRX5 regulates the ubiquitination-dependent degradation of PD-L1 by modulating the interaction between SPOP and PD-L1.

[0039] Example 7 GLRX5 enhances T-cell-dependent tumor suppression Next, further experimental verification was conducted on GLRX5-mediated PD-L1 regulation. Peripheral blood mononuclear cells (PBMCs) were extracted from the blood of healthy individuals, CD8+ T cells were isolated, and tumor cells were co-cultured with CD8+ T cells to perform T cell-mediated tumor cytotoxicity experiments. Figure 12 (A in the original text). The results showed that after GLRX5 knockdown in tumor cells, CD8+ T cells exhibited weaker cytotoxicity and higher tumor cell survival; however, after blocking PD-1 / PD-L1 interaction with a PD-L1 monoclonal antibody, no significant change in cytotoxicity was observed with GLRX5 knockdown compared to shNC. Figure 12 (B and C in the text). Consistently, overexpression of GLRX5 enhanced cytotoxicity, while overexpression of GLRX5 in SPOP knockdown cells significantly reduced cytotoxicity. Figure 13 A in the figure). Tumor cell survival is consistent with these results ( Figure 13 (B in the text). To investigate whether GLRX5 can enhance T cell-mediated antitumor activity in vivo, a subcutaneous tumor model was established by inoculating Renca renal cell carcinoma cells into BALB / c mice. Figure 14 (A) In this experiment, a PD-L1 monoclonal blocking antibody (mAb) was used to block the PD-1 / PD-L1 signaling pathway, with an IgG isotype control (IgG2b) used as a control. Results showed that in a tumor-bearing mouse model, PD-L1 monoclonal antibody treatment significantly inhibited tumor growth compared to the IgG2b control group. Notably, overexpression of GLRX5 in the PD-L1 mAb treatment group enhanced the antitumor efficacy. Figure 14 (B, C, D in the text).

[0040] in conclusion: In response to the problems in existing technologies, such as the low sensitivity of renal cell carcinoma (RCC) to radiotherapy and chemotherapy, the easy development of drug resistance to first-line drug sunitinib, the less than 30% efficacy rate of PD-1 / PD-L1 immune checkpoint inhibitors as monotherapy, the lack of effective biomarkers to screen for beneficial patients, and the unclear regulatory mechanism of PD-L1 protein stability, which leads to the inability to effectively block immune escape.

[0041] This invention, validated using the TCGA dataset and clinical cohorts, confirms that GLRX5 is significantly downregulated in renal cell carcinoma (RCC). Decreased GLRX5 expression is closely associated with advanced tumor stage, increased histological grade, and metastatic progression, indicating its crucial role in malignant transformation. Functional studies show that GLRX5 overexpression inhibits RCC cell migration and proliferation, while knockdown promotes these oncogenic phenotypes. These results collectively establish the antitumor effect of GLRX5 in RCC.

[0042] This invention reveals a novel interaction between GLRX5 and PD-L1 in renal cell carcinoma, with GLRX5 knockdown significantly increasing PD-L1 protein levels. Mechanistic studies show that GLRX5 regulates PD-L1 stability through ubiquitination-mediated degradation. Proteomics analysis identified SPOP as the major E3 ubiquitin ligase regulating this process. Crucially, GLRX5 acts as a molecular enhancer of SPOP catalytic activity, significantly enhancing its ability to degrade PD-L1. Functional validation using a tumor-CD8+ T cell co-culture system demonstrates that GLRX5 enhances anti-tumor immunity by promoting PD-L1 degradation and subsequently inducing T cell-mediated tumor suppression.

[0043] In summary, this invention reveals that GLRX5 is a tumor suppressor in renal cell carcinoma (RCC) and a key factor regulating PD-L1 stability in RCC. It specifically interacts with PD-L1, inhibiting tumor immune escape by enhancing SPOP-mediated PD-L1 K48 linker ubiquitination and proteasome degradation. This molecular mechanism establishes GLRX5 as a key coordinator of the SPOP-PD-L1 degradation axis in RCC pathogenesis. The loss of GLRX5 drives malignant progression in RCC, providing a mechanistic basis for developing novel immunotherapeutic strategies targeting the GLRX5-SPOP-PD-L1 axis.

Claims

1. An application of a substance for detecting the expression level of the biomarker GLRX5, characterized in that, Includes at least one of the following: a1) Application in the preparation of products for predicting the immunotherapeutic efficacy, prognostic assessment, or auxiliary prediction of renal cell carcinoma; a2) Application in the preparation of products for predicting the immunotherapeutic efficacy and prognosis of renal cell carcinoma using immune checkpoint therapy.

2. The application of the substance for detecting the expression level of the biomarker GLRX5 according to claim 1, characterized in that, The substance used to detect the expression level of the biomarker GLRX5 includes reagents for detecting the expression level, content, or RNA expression level of GLRX5. The reagents include antibodies, peptides, proteins, or nucleic acid molecules that bind to the GLRX5 protein.

3. The application of the substance for detecting the expression level of the biomarker GLRX5 according to claim 1, wherein the product includes a chip, a kit, or a reagent for detecting GLRX5 using a high-throughput sequencing platform.

4. The application of the substance for detecting the expression level of the biomarker GLRX5 according to claim 1, wherein the predictive aid product includes a product for predicting the sensitivity of renal cell carcinoma patients to sunitinib.

5. The application of a substance that promotes GLRX5 expression, characterized in that, Includes at least one of the following: b1) Application in the preparation of products that inhibit the growth, proliferation, migration and / or invasion of renal cell carcinoma; b2) Application in the preparation of products that inhibit immune escape from renal cell carcinoma; b3) Use in the preparation of products for enhancing the sensitivity of renal cell carcinoma to PD-L1 targeted therapy; b4) Application in the preparation of drugs for promoting the degradation of PD-L1 protein; b5) Application in the preparation of drugs for enhancing T cell-mediated antitumor immune responses.

6. The application of the substance that promotes GLRX5 expression according to claim 5, characterized in that, The substances that promote GLRX5 expression include enhancers, overexpression plasmid vectors, and nanoparticles carrying GLRX5 gene mRNA.

7. An application of a GLRX5-SPOP-PD-L1 control axis as a target point, characterized in that, Includes at least one of the following: c1) Application in the preparation of products that inhibit the growth, proliferation, migration and / or invasion of renal cell carcinoma; c2) Application in the preparation of products that inhibit immune escape from renal cell carcinoma; c3) Use in the preparation of products for enhancing the sensitivity of renal cell carcinoma to PD-L1 targeted therapy; c4) Application in the preparation of drugs for promoting the degradation of PD-L1 protein; c5) Application in the preparation of drugs for enhancing T cell-mediated antitumor immune responses; The GLRX5-SPOP-PD-L1 regulatory axis is used as the target, and GLRX5 is used as the intervention target to interfere with the expression of SPOP and PD-L1.

8. The application of the substance that promotes GLRX5 expression according to claim 5 or the application of the GLRX5-SPOP-PD-L1 regulatory axis as a target according to claim 7, characterized in that, The T cells include CD8+ T cells.

9. The application of the substance that promotes GLRX5 expression according to claim 5 or the application of the GLRX5-SPOP-PD-L1 regulatory axis as a target according to claim 7, characterized in that, The products include pharmaceuticals or laboratory reagents.

10. A pharmaceutical composition, characterized in that, It includes a therapeutically effective amount of GLRX5 or an enhancer thereof, and a pharmaceutically acceptable carrier; The pharmaceutical composition further includes a PD-L1 inhibitor, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody or its antigen-binding fragment; The pharmaceutically acceptable carriers include at least one of tablets, capsules, oral liquids, granules, suspensions, injections, powder for injection, pellets, sustained-release agents, controlled-release agents, and targeted formulations.

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