Application of Pitavastatin calcium in preparation of drugs for targeting TRAPPC4 to enhance ferroptosis sensitivity and treat head and neck squamous cell carcinoma

Abstract

The invention discloses an application of Pitavastatin calcium in preparation of a medicine for targeting TRAAPPC4 (TRAPPC4) so as to enhance ferroptosis sensitivity and treat head and neck squamous cell carcinoma. Through whole genome CRISPR / Cas9 screening, it is found that TRAPPC4 is a key factor of ferroptosis resistance in HNSCC. Through virtual screening and experimental verification, it is identified that the Pitavastatin calcimum is an inhibitor of the TRAPPC4. The Pitavastatin calcium and the RSL3 are combined for use, so that the ferroptosis effect can be synergistically enhanced, and the tumor growth can be remarkably inhibited. The invention provides a novel medicine application of Pitavastatin calcium, namely the Pitavastatin calcium is used for preparing a medicine or a medicine composition which is targeted to TRAPPC4 and is used for treating head and neck squamous cell carcinoma.

Description

Technical Field

[0001] This invention relates to the fields of tumor treatment, biomedicine and targeted drug development, and in particular to the application of Pitavastatin calcium in the preparation of drugs for targeting TRAPPC4 to enhance ferroptosis sensitivity and for the treatment of head and neck squamous cell carcinoma. Background Technology

[0002] Head and neck squamous cell carcinoma (HNSCC) originates from the mucosal epithelium of the oral cavity, pharynx, and larynx. It is the seventh most common cancer worldwide, with nearly one million new cases annually. Smoking, alcohol consumption, and infection with high-risk human papillomavirus (HPV) are the main causes. Due to its occult location and lack of early symptoms, HNSCC is often diagnosed at an advanced stage, resulting in poor treatment outcomes and low survival rates. Despite advancements in various treatment methods in recent years, including surgery, radiotherapy, chemotherapy, targeted therapy, and immune checkpoint inhibitors, the overall survival rate for patients with advanced HNSCC has not significantly improved over the past decade. HPV-negative HNSCC, in particular, responds poorly to existing therapies and has an even worse prognosis, thus necessitating new therapeutic targets and effective treatment strategies.

[0003] Ferroptosis is a non-apoptotic programmed cell death process induced by iron-catalyzed lipid peroxidation, characterized by mitochondrial shrinkage, cristae loss, and intracellular reactive oxygen species accumulation. Increasing research indicates that induced ferroptosis can inhibit growth and overcome drug resistance in various tumors, thus it is considered a novel antitumor strategy with potential clinical applications.

[0004] In recent years, drug reuse strategies have attracted widespread attention. Applying marketed drugs to the treatment of new diseases can not only significantly shorten the research and development cycle but also help promote rapid clinical translation. However, there are currently no studies reporting the use of lipid-lowering drugs such as Pitavastatin calcium to modulate ferroptosis sensitivity or as a treatment for head and neck squamous cell carcinoma.

[0005] Therefore, exploring new regulators of ferroptosis and combining them with drug reuse strategies to develop novel ferroptosis-based treatments is an important scientific issue in solving the precision treatment of HNSCC. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an application of Pitavastatin calcium in the preparation of drugs for targeting TRAPPC4 to enhance ferroptosis sensitivity and for the treatment of head and neck squamous cell carcinoma. This drug can enhance ferroptosis sensitivity by intervening in TRAPPC4 and is used to treat head and neck squamous cell carcinoma.

[0007] To solve the above-mentioned technical problems, one technical solution adopted by the present invention is to provide the application of Pitavastatincalcium in the preparation of a drug for targeting TRAPPC4 to enhance ferroptosis sensitivity.

[0008] To solve the above-mentioned technical problems, another technical solution adopted by the present invention is to provide the application of Pitavastatincalcium in the preparation of a drug for targeting TRAPPC4 to enhance ferroptosis sensitivity and treating head and neck squamous cell carcinoma.

[0009] In a preferred embodiment of the present invention, the combined use of Pitavastatin calcium and the ferroptosis inducer RSL3 has shown to enhance ferroptosis sensitivity and inhibit tumors in in vitro and in vivo models.

[0010] Furthermore, the Pitavastatin calcium is used in combination with RSL3 at a molar ratio of 1:2.

[0011] In a preferred embodiment of the present invention, the tumor is HPV-negative head and neck squamous cell carcinoma.

[0012] In a preferred embodiment of the present invention, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0013] This invention, through genome-wide CRISPR / Cas9 screening, identified TRAPPC4 as a key factor in ferroptosis resistance in HNSCC. Validation in cell lines, PDO, and CDX models showed that inhibiting TRAPPC4 significantly enhanced the antitumor effect of the ferroptosis inducer RSL3. Through virtual screening and experimental validation, Pitavastatin calcium was identified as an inhibitor of TRAPPC4.

[0014] The beneficial effects of this invention are: this invention discovers TRAPPC4 as a new target for the regulation of ferroptosis in HNSCC; it provides a new use for Pitavastatin calcium, expanding its clinical application; and it provides a combination therapy strategy that significantly enhances ferroptosis sensitivity and inhibits tumors. Attached Figure Description

[0015] Figure 1 This is a schematic diagram showing the cell viability of various HNSCC cell lines after 12 hours of treatment with different concentrations of RSL3. Figure 2 This is a schematic diagram illustrating the process by which CRISPR / Cas9 screening discovered TRAPPC4 as a ferroptosis resistance gene; Figure 3 This is a schematic diagram of the TRAPPC4 expression analysis process in the HNSCC cell line; Figure 4 This is a schematic diagram of the TRAPPC4 expression analysis process in the PDO model; Figure 5 This is a schematic diagram illustrating the process of analyzing the antitumor effect of TRAPPC4 expression on RSL3 in the CDX model; Figure 6 This is a schematic diagram illustrating the process of identifying Pitavastatin calcium as an inhibitor of TRAPPC4 through virtual screening and experimental verification. Figure 7 This is a schematic diagram illustrating the analytical process of how Pitavastatin calcium combined with RSL3 showed significant synergistic antitumor effects in in vitro and in vivo models. Detailed Implementation

[0016] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0017] Example 1: To elucidate the mechanism of ferroptosis resistance in HNSCC, the sensitivity of five HNSCC cell lines to RSL3 was first assessed, and it was found that LIU-LSC-1 cells had the highest resistance to RSL3.

[0018] Experimental steps: 1) Five HNSCC cell lines (LIU-LSC-1, FaDu, CAL27, TU212, TU177) were seeded in 96-well plates, with approximately 10,000 cells per well, and cultured at 37°C and 5% CO2 for 24 hours until the cells reached the logarithmic growth phase.

[0019] 2) Next, cells were treated with different concentrations of RSL3 for 12 hours. After treatment, 10 µL of CCK-8 reagent was added, and the cells were cultured for another 1-2 hours. Then, the absorbance (OD value) of each well was measured using a microplate reader at a wavelength of 450 nm. Cell viability was calculated based on the measured OD values, and a curve showing the relationship between cell viability and RSL3 concentration was plotted.

[0020] 3) Use GraphPad Prism software to fit the curve and calculate the IC50 value. The results are as follows: Figure 1 As shown, LIU-LSC-1 cells exhibited the highest resistance to RSL3.

[0021] Example 2: Whole-genome CRISPR-Cas9 screening revealed that TRAPPC4 is a key mediator of ferroptosis resistance in HNSCC.

[0022] Experimental steps: 1. Whole-genome CRISPR / Cas9 screening 1) Cell line selection: The LIU-LSC-1 cell line was used for whole-genome CRISPR / Cas9 screening.

[0023] 2) Viral infection: LIU-LSC-1 cells were transfected with the GeCKO v2 CRISPR library (containing approximately 20,000 sgRNAs) at an MOI (infection titer) of 0.3.

[0024] 3) Screening and amplification: After transfection, puromycin is used to select transduced cells, and the screening continues for 7 days to ensure that only successfully transduced cells survive.

[0025] 2. Screening for ferroptosis resistance genes 1) RSL3 treatment: After screening, cells continued to be treated with RSL3 for 7 days to induce ferroptosis and select resistant cells.

[0026] 2) gDNA extraction: Genomic DNA was extracted from the treated cells using standard gDNA extraction methods.

[0027] 3) PCR amplification: PCR amplification is performed using specific primers for the sgRNA region to confirm the insertion of sgRNA in the genome and to perform subsequent high-throughput sequencing.

[0028] 3. Data Analysis and Candidate Gene Screening 1) RRA score: The selected genes are scored using the Robust Ranking Aggregation (RRA) method, and ranked according to the score. Genes ranked higher are considered to be key regulators of ferroptosis resistance.

[0029] 2) Gene ranking and statistics: The top five candidate genes included POLR3H, TRAPPC4, PSMG4, LSM11, and LONP1. The RRA score and corresponding sgRNA count changes for each gene were recorded and analyzed.

[0030] 3) Changes in sgRNA counts: This section shows the changes in sgRNA counts for these five candidate genes during the screening process. TRAPPC4, in particular, showed the most significant exhaustion effect.

[0031] 4. Analysis of TRAPPC4-targeted sgRNA depletion RSL3-induced cell exhaustion: After treatment, cells were subjected to RSL3, and changes in TRAPPC4-targeting sgRNA were detected. The degree of sgRNA depletion was analyzed using qPCR or NGS techniques.

[0032] 5. Expression analysis of TRAPPC4 in normal and tumor tissues of HNSCC 1) Tissue Sample Collection: Collect normal and tumor tissue samples from HPV-negative HNSCC patients. Sample sources should meet ethical requirements, and all patients should sign informed consent forms.

[0033] 2) IHC staining: TRAPPC4 was stained by immunohistochemistry (IHC). TRAPPC4 was labeled with a specific antibody, and the sections were standardized for staining and development.

[0034] 3) Quantitative analysis of H-score: The H-score of IHC staining was calculated using quantitative analysis methods to further evaluate the correlation between TRAPPC4 upregulation and tumors.

[0035] Results: Genome-wide CRISPR-Cas9 screening was performed using the human GeCKO v2A library, which targets 19,052 protein-coding genes and 1,864 miRNAs. LIU-LSC-1 cells transduced with this library were treated with RSL3 or DMSO for 7 days. Figure 2 A is a flowchart of genome-wide CRISPR / Cas9 screening in LIU-LSC-1 cells to identify genes regulating ferroptosis. Figure 2 B represents the ranking of the selected genes based on the RRA score, with the top five candidate genes marked. Figure 2 C represents the relative change in sgRNA counts for the top five candidate genes. As shown in the figure, TRAPPC4 showed the largest fold change. Figure 2 D is a linear graph showing the depletion of TRAPPC4-targeting sgRNAs after RSL3 treatment. Notably, TRAPPC4 is among the top five candidate genes, and all sgRNAs targeting this gene were significantly reduced after RSL3 treatment. Figure 2 (BD), indicating that it plays a key role in resisting ferroptosis. Figure 2 E and Figure 2 F represents the representative IHC staining (E) and H-score quantitative analysis (F) of TRAPPC4 in normal and tumor tissues of HNSCC, respectively. IHC analysis of clinical samples consistently showed that the level of TRAPPC4 in tumors was significantly higher than that in adjacent normal tissues. Figure 2 EF).

[0036] Example 3: TRAPPC4 is a key protein in HNSCC cells that resist ferroptosis.

[0037] Experimental steps: 1. Cell Culture and Transfection HNSCC cell lines LIU-LSC-1 and TU177 were cultured in DMEM (high glucose) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2. To knock out TRAPPC4, gene knockout was performed using a CRISPR / Cas9 system. Two pairs of TRAPPC4-specific sgRNAs (sgTRA-1 and sgTRA-2) were designed and transduced into LIU-LSC-1 cells via viral infection. Forty-eight hours post-transfection, stable transduced cells were selected using puromycin, and TRAPPC4 expression was verified by Western blot. TRAPPC4 overexpression experiments were performed using the LV-TRAPPC4 vector transfected into TU177 cells, and TRAPPC4 expression was detected by Western blot after infection.

[0038] 2. RSL3 treatment and cell viability assay After cell seeding, RSL3 (0.4 µM or 4 µM) was added to different treatment groups for 12 hours. Cell viability was determined by the CCK-8 assay. After 12 hours of RSL3 treatment, the percentage of cell viability was assessed.

[0039] 3. Measurement of MDA and L-ROS levels After cell treatment, intracellular MDA and L-ROS levels were measured using an MDA assay kit and a Lipid ROS (L-ROS) detection kit. All experiments were performed according to the kit instructions.

[0040] 4. Observation of mitochondrial morphology After processing, the ultrastructural changes of mitochondria within the cells were observed using transmission electron microscopy (TEM). Cells were fixed using conventional electron microscopy methods, then embedded and sectioned for microscopic observation of mitochondrial morphology.

[0041] Results: In this embodiment, two independent TRAPPC4 knockout LIU-LSC-1 cell lines were constructed using CRISPR-Cas9. Figure 3A shows the Western blot analysis of TRAPPC4 expression in the cells shown. Figure 3 B represents the cell viability of LIU-LSC-1 cells after 12 hours of RSL3 treatment. Figure 3E and C represent intracellular MDA (C) and L-ROS (D) levels, and representative mitochondrial morphology images (E), respectively, after 12 hours of treatment with 4 µM RSL3. TRAPPC4 deficiency significantly enhanced RSL3 sensitivity, increased MDA levels, and promoted lipid peroxidation (Fig. 3B-D). Transmission electron microscopy showed that mitochondria were severely damaged in TRAPPC4-deficient cells after RSL3 exposure. Figure 3 E). Figure 3 F shows the Western blot analysis of TRAPPC4 expression in the TU177 cells shown. G shows the cell viability of the TU177 cells shown after 12 hours of RSL3 treatment. H and J show the intracellular MDA (H) and L-ROS (I) levels, and representative mitochondrial morphology images (J), respectively, after 12 hours of treatment with 0.4 µM RSL3. Conversely, overexpression of TRAPPC4 in TU177 cells conferred resistance to RSL3, thereby reducing the accumulation of MDA and lipid ROS and alleviating mitochondrial ultrastructural damage. Figure 3 In summary, these findings suggest that TRAPPC4 is a key mediator of ferroptosis resistance in HNSCC and highlight its potential as a therapeutic target to enhance tumor sensitivity to ferroptosis inducers.

[0042] Example 4: TRAPPC4 has anti-ferroptosis effects in organoid models.

[0043] Experimental steps: 1. Organoid (PDO) Culture and Transfection 1) Organoid construction: HNSCC tumor tissue (derived from patients) was collected and cut into small pieces, washed twice, and then treated with Cancer Organoid Basal Medium (BioGenous, Jiangsu, China). Non-epithelial components (such as muscle, fat, etc.) were removed, and the tumor tissue was cut into small pieces of 0.5-2 mm³.

[0044] 2) Digestion and Cell Separation: Add the tumor tissue to the tumor tissue digestion solution (BioGenous) and digest for 20-30 minutes at 37°C with gentle shaking (50-100 rpm). After digestion, add FBS to stop digestion and gently pipette to separate the tissue.

[0045] 3) Cell assessment and culture: Filter the suspension through a 100 μm cell filter, centrifuge, and wash twice. Remove red blood cells using red blood cell lysis buffer, ensuring cell viability exceeds 90% and the total cell count is >5 × 10⁻⁶.5 Subsequently, the cells were resuspended and seeded into organoid culture ECMs for 3D culture at a density of 8,000-30,000 cells / 20-30 μL.

[0046] 2. Lentiviral transfection and TRAPPC4 expression 1) TRAPPC4 knockdown and overexpression: TRAPPC4 gene knockdown and overexpression were performed using lentivirus vectors. TRAPPC4 was knocked down (shTRA-1, shTRA-2) or overexpressed (LvTRA) in organoids using shRNA or lentivirus vectors, respectively. After transfection, TRAPPC4 expression levels were detected using Western blot (e.g., ...). Figure 4 (As shown in B and F).

[0047] 2) Screening after transfection: Obtain stable transduced organoid models through appropriate screening methods (such as puromycin screening).

[0048] 3) Drug treatment RSL3 treatment: Before treatment, organoids were seeded in 96-well plates and treated with DMSO or RSL3 (10 μM) for 48 hours.

[0049] Cell viability assay: Cell viability was assessed using the CellTiter-Glo® 3D Cell Viability Assay Kit (Promega, Madison, WI). Cell viability changes were measured 48 hours after RSL3 treatment, following the manufacturer's instructions.

[0050] 4) Microscopic observation: After 48 hours of treatment, the morphological changes of the organoids were observed using an optical microscope and representative images were taken.

[0051] 3. Immunohistochemical staining 1) Tissue processing and IHC staining: Normal and tumor tissues were collected and routinely embedded in paraffin. HNSCC tissues and organoid samples were stained with CK5 / 6, TP63, and TRAPPC4 using IHC staining, labeled with the corresponding antibodies, and observed and photographed under an optical microscope.

[0052] 2) Staining score: Based on the results of IHC staining, the expression of TRAPPC4 was quantitatively analyzed, and the H-score was used to statistically analyze each group.

[0053] Results: This example successfully constructed a PDO model for HNSCC tumor specimens and stratified them according to high or low expression of endogenous TRAPPC4. Figure 4A shows a representative IHC image of CK5 / 6, TP63, and TRAPPC4 staining in HNSCC tissue and PDOs. Figure 4 B represents the assessment of TRAPPC4 expression levels in organoids transfected with control, TRAPPC4 knockdown, or lentiviral vectors using Western blot. Figure 4 CE represents representative images and relative cell viability statistics (E) of TRAPPC4 knockdown or control PDOs after treatment with DMSO (C) or RSL3 (10 μM, D) for 48 hours. In TRAPPC4-overexpressing PDOs, lentivirus-mediated TRAPPC4 knockdown significantly inhibited organoid growth and made organoids more sensitive to RSL3-induced ferroptosis, as evidenced by decreased organoid viability and enhanced 4-HNE staining after treatment. Figure 4 BE). Figure 4 FH represents representative images and relative cell viability statistics (H) of TRAPPC4 overexpressing or control PDOs after treatment with DMSO (F) or RSL3 (10 μM, G) for 48 hours. Conversely, in TRAPPC4-low-expressing PDOs, TRAPPC4 overexpression significantly enhanced organoid growth and viability while reducing sensitivity to RSL3-induced lipid peroxidation. Figure 4 B, Figure 4 In summary, these results indicate that TRAPPC4 plays a key inhibitory role in ferroptosis in the PDO model of HNSCC, and its depletion significantly enhances the therapeutic effect of ferroptosis inducers.

[0054] Example 5: TRAPPC4 has an anti-ferroptosis effect in the CDX model.

[0055] Experimental steps: 1. Animal model establishment and tumor transplantation 1) Mouse source: BALB / c-nude and NOD / SCID mice were purchased from GemPharmatech (Nanjing, China). All animal experiments complied with the guidelines of the Animal Experiment Ethics Committee of Anhui Medical University and have obtained ethical approval.

[0056] 2) Cell transplantation: 5 × 10⁶ LIU-LSC-1 cells (transfected with sgNTC or sgTRAPPC4-1) or TU177 cells (transfected with Lv or LvTRAPPC4) 6 Cells were subcutaneously inoculated into the right axilla of 6-week-old male BALB / c nude mice. Five mice were in each group.

[0057] 3) Drug treatment: Starting from day 14 (for tumors derived from LIU-LSC-1 cells) or day 10 (for tumors derived from TU177 cells) after transplantation, RSL3 (40 mg / kg) was injected intraperitoneally every other day for a total of 8 times (RSL3 group); the control group was given a solvent (Vehicle).

[0058] 2. Tumor growth monitoring and assessment 1) Tumor volume measurement: Measure the tumor volume every two days using calipers and calculate the tumor volume using the formula: Volume = (Length × Width²) / 2. Record the tumor growth curve.

[0059] 2) Tumor weight: At the end of the experiment, the mice were sacrificed and the tumors were collected, weighed and statistically analyzed.

[0060] 3. Assessment of cell viability and lipid peroxidation MDA Levels: The relative MDA levels in tumor tissue were detected using an MDA assay kit to assess the extent of lipid peroxidation.

[0061] 4. Immunohistochemical analysis 1) Tissue sectioning and staining: At the endpoint, tumor tissue was removed, paraffin-embedded, and sectioned. Immunohistochemical staining for TRAPPC4, Ki-67, and 4-HNE was performed using specific antibodies.

[0062] 2) Quantitative analysis: Representative images were captured using a microscope, and quantitative analysis was performed on the staining of Ki-67 and 4-HNE. Ki-67 was used to detect cell proliferation, and 4-HNE was used to detect lipid peroxidation markers.

[0063] Results: To elucidate the functional role of TRAPPC4 in ferroptosis resistance, a CDX model was first established in this example by subcutaneously injecting LIU-LSC-1 cells expressing either control sgRNA (sgNTC) or TRAPPC4-targeting sgRNA (sgTRAPPC4-1) into BALB / c nude mice (5 × 10⁶ cells per mouse). 6 (cells). Starting from day 14 post-inoculation, mice were intraperitoneally injected with RSL3 (40 mg / kg, intraperitoneal injection) every other day until day 28, and the mice were sacrificed on day 30. Figure 5 A shows a schematic diagram of the subcutaneous xenograft model in nude mice and a timeline of RSL3 treatment. In this CDX model, both TRAPPC4 knockout and RSL3 monotherapy inhibited tumor growth, while TRAPPC4 knockout combined with RSL3 treatment significantly inhibited tumor progression. Figure 5 Tumor images with BD as the endpoint ( Figure 5 B) Tumor growth curve ( Figure 5C) and tumor weight ( Figure 5 D, left) and relative MDA level ( Figure 5 (D, right). Consistent with enhanced ferroptosis, tumors in the sgTRAPPC4-1 group showed a significant increase in MDA levels after RSL3 administration (Fig. 5D, right). IHC further demonstrated that, compared with other groups, RSL3-treated sgTRAPPC4-1 tumors had a significant reduction in Ki-67 positive cells and a significant increase in 4-HNE levels. Figure 5 E and F show representative IHC images (E) of TRAPPC4, Ki-67, and 4-HNE proteins in the xenograft tumors, and quantitative analysis of Ki-67 (F, left) and 4-HNE (F, right) expression, indicating enhanced lipid peroxidation and reduced proliferation. To validate the role of TRAPPC4, this example established a CDX model using TU177 cells stably overexpressing TRAPPC4 or a control vector, and treated them with either the vector or RSL3. Figure 5G shows a schematic diagram of the subcutaneous xenograft model in nude mice and the RSL3 treatment schedule. TU177 cells (Lv or LvTRAPPC4) were xenografted into mice and administered RSL3 (40 mg / kg, intraperitoneal injection) or a solvent every other day from day 10 to day 24. As expected, TRAPPC4 overexpression promoted tumor growth, reduced RSL3 sensitivity, and prevented the RSL3-induced decrease in MDA, 4-HNE, and Ki-67 levels, supporting its role in ferroptosis resistance. Figure 5 shows tumor images (H), tumor growth curves (I), tumor weight (J, left), and relative MDA levels (J, right) at the H-J endpoint. Figure 5 K and L are representative IHC images of TRAPPC4, Ki-67, and 4-HNE in the xenograft tumors shown (K), and quantitative analysis of Ki-67 (L, left) and 4-HNE (L, right) expression.

[0064] Example 6: Pitavastatin calcium is an inhibitor of TRAPPC4.

[0065] Experimental steps: 1. Virtual screening and compound library screening The HY-L035P drug repositioning compound library Plus (containing 5,035 compounds) was virtually screened using Glide SP docking to identify small molecules that may interact with the TRAPPC4 protein. Fine-tuning was then performed using Glide XP docking, ultimately selecting the top five compounds with the highest TRAPPC4 binding scores.

[0066] 2. Protein Expression and Western Blot Analysis The selected compounds were added to LIU-LSC-1 cells, and the cell lysates were extracted and analyzed by Western blot to assess the expression level of TRAPPC4 protein.

[0067] 3. CETSA (Cellular Thermal Shift Analysis) 1) Treat LIU-LSC-1 cells for 2 hours using 10 μM Pitavastatin Calcium (PTV) or DMSO. Subsequently, wash the cells with cold PBS and resuspend them in PBS containing protease inhibitors.

[0068] 2) Divide the cell lysate into equal portions and heat them gradually to 40-70°C, heating for 3 minutes at each temperature point. Perform three freeze-thaw cycles using liquid nitrogen, and separate the supernatant by centrifugation (20,000 g, 20 minutes, 4°C).

[0069] 3) Quantitative analysis of the supernatant was performed, and the thermal stability of TRAPPC4 was detected by Western blot.

[0070] 4. DARTS (Drug Affinity Response Target Stability Assay) 1) Incubate LIU-LSC-1 cell lysates (normalized to 1 mg / mL) with different concentrations of Pitavastatin Calcium (PTV) or DMSO at room temperature for 30 minutes.

[0071] 2) Then add streptomycin E (1:300), incubate for 30 minutes, and terminate the reaction by heating with SDS sample buffer.

[0072] 3) The stability of TRAPPC4 protein was analyzed using Western blot.

[0073] 5. SPR (Surface Plasmon Resonance) Analysis 1) The interaction between Pitavastatin Calcium (PTV) and TRAPPC4 protein was assessed using SPR analysis on a Biacore 8K system.

[0074] 2) TRAPPC4 protein was immobilized on the CM5 sensor chip at a concentration of 50 μg / mL, and the chip was activated using EDC / NHS chemical cross-linking.

[0075] 3) The binding affinity between PTV and TRAPPC4 was measured by passing different concentrations of PTV or DMSO through the chip. The data were fitted using a 1:1 Langmuir binding model to calculate the binding constant (Ka), dissociation constant (Kd), and equilibrium dissociation constant (KD).

[0076] 6. Cellular protein expression analysis In LIU-LSC-1 cells, the expression levels of TRAPPC4 and GPX4 proteins were analyzed by Western blot after treatment with PTV (10 μM) for 12 hours.

[0077] Results: Given that Example 2 has identified TRAPPC4 as a key regulator of ferroptosis resistance and a potential therapeutic target for head and neck squamous cell carcinoma (HNSCC), this example focuses on identifying small molecules capable of modulating its activity. However, there are currently no TRAPPC4 inhibitors. To fill this gap, this invention performed structure-based HTVS on 5,035 candidate compounds. Figure 6 A virtual screening was performed on TRAPPC4 (PDB: 2J3T) and the HY-L035P drug repositioning compound library Plus (containing 5,035 compounds). Based on molecular docking scores, the top five compounds were Walker-Voganan (trihydrochloride), VX-150, flavin adenine dinucleotide, peptide T, and PTV. Figure 6 B shows the top five small molecules that interact with TRAPPC4. C shows the 2D binding mode of PTV to TRAPPC4, illustrating the binding behavior. Preliminary validation experiments indicate that among these candidate compounds, only PTV significantly downregulated TRAPPC4 protein levels in LIU-LSC-1 cells. Figure 6 D represents a Western blot analysis of TRAPPC4 expression in LIU-LSC-1 cells after treatment with five drugs. To confirm the direct binding of PTV to TRAPPC4, a series of complementary targeting binding assays were also performed in this example. CETSA revealed that PTV enhanced the thermostability of TRAPPC4, indicating a direct binding between PTV and TRAPPC4. Figure 6Figure 6E shows the thermodynamic binding of TRAPPC4 to PTV (10 μM, 2 h) assessed using the CETSA method, and the results were validated by Western blot analysis. DARTS analysis confirmed this, indicating that PTV enhanced TRAPPC4's resistance to protease-mediated degradation. Figure 6F shows further Western blot analysis of TRAPPC4 expression after incubation with Pronase E in LIU-LSC-1 cell lysates at different concentrations of PTV. SPR analysis confirmed a direct binding interaction between PTV and TRAPPC4, with a dissociation constant (KD) of 4.71 µM. Figure 6G shows the binding affinity between PTV and TRAPPC4 measured by SPR. Functionally, PTV treatment significantly reduced the levels of TRAPPC4 and GPX4 proteins, similar to the effect of TRAPPC4 knockdown. Figure 6H shows the expression of TRAPPC4 and GPX4 proteins in LIU-LSC-1 cells after 12 hours of PTV (10 μM) treatment. These findings collectively indicate that PTV directly binds to and disrupts the stability of TRAPPC4, thereby inhibiting its downstream ferroptosis-inhibiting function.

[0078] Example 7: Pitavastatin calcium targets TRAPPC4 and synergizes with RSL3 to enhance ferroptosis treatment in HNSCC.

[0079] Experimental steps: 1. Synergistic therapy experimental design (in vitro experiments) 1) Cell culture: LIU-LSC-1 cells were seeded into 96-well plates and cultured to the logarithmic growth phase.

[0080] 2) Drug treatment: Cells were treated with RSL3 (10 μM) and Pitavastatin Calcium (PTV, 20 μM) separately or in combination. The treatment time was 48 hours, and DMSO was used as a control group.

[0081] 3) Synergistic effect detection: The synergistic effect of combined treatment with different concentrations of RSL3 and PTV was measured by ZIP analysis. Heatmaps were plotted, the δ value of the synergistic effect was calculated, and the concentration range with the strongest synergistic effect was marked.

[0082] 4) Cell viability assessment: Cell viability was measured using the CCK-8 reagent, and a statistical analysis graph of relative cell viability was plotted.

[0083] 2. PDO drug testing 1) PDO culture and drug treatment: PDO was seeded into 96-well plates and treated with RSL3 (10 μM) and PTV (20 μM) alone or in combination for 48 hours.

[0084] 2) Microscopic observation: Observe the morphological changes of the treated organoids using an optical microscope.

[0085] 3) Cell viability assay: The cell viability of organoids was assessed using the CellTiter-Glo® 3D Cell Viability Assay Kit (Promega).

[0086] 3. PDX model (in vivo experiment) 1) Tumor transplantation: LIU-LSC-1 cells or HNSCC PDX model (patient-derived xenograft tumor) were subcutaneously in 6-week-old male BALB / c nude mice. Five mice were in each group.

[0087] 2) Drug treatment: Starting from day 14 post-transplantation, mice were given intraperitoneal injections of RSL3 (40 mg / kg) and PTV (20 mg / kg) every other day until the end of the experiment.

[0088] 3) Tumor growth monitoring: Regularly measure tumor volume using the formula: Volume = (Length × Width²) / 2. Plot a tumor growth curve.

[0089] 4. Tumor Sample Collection and Analysis 1) Tumor tissue collection: At the end of the experiment, the mice were sacrificed, the tumors were removed and weighed.

[0090] 2) MDA level measurement: The MDA level in tumor tissue was measured using an MDA assay kit to assess the level of lipid peroxidation.

[0091] 3) Immunohistochemical analysis: 4) Collect tumor tissue, embed it in paraffin, and section it.

[0092] 5) IHC staining of TRAPPC4 and 4-HNE with specific antibodies was performed, and representative images were observed and captured.

[0093] Results: To evaluate the therapeutic significance of TRAPPC4 targeting, this example assessed the efficacy of PTV alone and in combination with RSL3. Combination index analysis revealed a significant synergistic effect between PTV and RSL3. Figure 7A is a heatmap showing the synergistic effect of PTV and RSL3 on LIU-LSC-1 cells. Red intensity reflects the strength of the synergistic effect; white boxes mark the concentration range with the greatest synergistic effect. In PDO, the combination of PTV and RSL3 significantly enhanced growth inhibition compared to either drug alone. Figure 7 B and C show representative images of PDOs treated with RSL3 (10 μM, 48 hours), PTV (20 μM, 48 hours), or a combination of PTV and RSL3 (B), respectively, and statistical analysis of PDO activity (C). In vivo validation using a PDX model showed that PTV monotherapy inhibited tumor growth, while combination therapy with RSL3 significantly enhanced tumor suppression. Figure 7 DE). In tumors receiving PTV-RSL3 combination therapy, MDA levels were significantly elevated, indicating enhanced ferroptosis (DE). Figure 7 F). Figure 7 DF represents the endpoints: tumor image (D), tumor growth curve (E), and tumor weight and MDA level (F). IHC analysis showed decreased TRAPPC4 and GPX4 expression after PTV treatment, and RSL3 combination therapy further enhanced 4-HNE staining, consistent with increased ferroptosis activity. Figure 7 G represents representative IHC images of TRAPPC4 and 4-HNE in tumor tissues of mice that received the specified treatment. In summary, these findings suggest that PTV is a novel TRAPPC4 target that not only inhibits TRAPPC4 expression but also amplifies the ferroptosis-based treatment response in HNSCC.

[0094] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The use of Pitavastatin calcium in the preparation of a drug for targeting TRAPPC4 to enhance ferroptosis sensitivity.

2. The use of Pitavastatin calcium in the preparation of a drug for targeting TRAPPC4 to enhance ferroptosis sensitivity and for the treatment of head and neck squamous cell carcinoma.

3. The application according to claim 1 or 2, characterized in that, The combination of Pitavastatin calcium and the ferroptosis inducer RSL3 showed that it enhanced ferroptosis sensitivity and inhibited tumors in in vitro and in vivo models.

4. The application according to claim 3, characterized in that, The Pitavastatin calcium was used in combination with RSL3 at a molar ratio of 1:

2.

5. The application according to claim 2 or 3, characterized in that, The tumor is an HPV-negative squamous cell carcinoma of the head and neck.

6. The application according to claim 1 or 2, characterized in that, The drug may also include pharmaceutically acceptable carriers or excipients.

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