Application of DHODH-CHKalpha-TRIM28 in screening medicines for inhibiting proliferation or growth of colorectal cancer cells

Abstract

The invention belongs to the technical field of biological medicines, and provides application of DHODH-CHKalpha-TRIM28 in screening of medicines for inhibiting proliferation or growth of colorectal cancer cells. The prognosis of colorectal cancer patients with high coexpression of DHODH and CHKalpha is poor; dual targeting inhibition of DHODH and CHKalpha can synergistically inhibit proliferation or growth of colorectal cancer cells. DHODH is competitively combined with an F242 / N243 structural domain of CHK alpha, so that TRIM28 is spatially hindered from approaching a K244 residue on the CHK alpha, and the ubiquitination of the CHK alpha at the K244 site mediated by the TRIM28 and the subsequent proteasome degradation process of the CHK alpha are inhibited. The CHKalpha stability increase mediated by DHODH drives the accumulation of carcinogenic phosphatidylcholine, and promotes the proliferation of colorectal cancer cells and the growth of tumors. Flurmide and MN58b are jointly used for dual targeting of DHODH and CHKalpha, and growth of colorectal cancer is synergistically inhibited. It is revealed that the DHODH-CHKalpha-TRIM28 axis is a key metabolic susceptible pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of DHODH-CHKα-TRIM28 in screening drugs that inhibit the proliferation or growth of colorectal cancer cells. Background Technology

[0002] The increasing global burden of colorectal cancer, particularly its rising incidence in younger populations (with 2 million new cases in 2022, representing a 15% annual increase), underscores the urgent need for mechanistic-based treatment strategies [1,2]. While early-stage colorectal cancer can achieve a five-year survival rate of approximately 80% through surgical resection and multimodal therapy, metastatic cases remain largely incurable due to acquired resistance and tumor heterogeneity [3-6]. This treatment dilemma necessitates a deeper understanding of oncogenic drivers, especially those that control cancer metabolic plasticity [7-10].

[0003] Targeted metabolism dependence has become a strategic paradigm for the treatment of colorectal cancer [11-13]. Among them, dihydroorotate dehydrogenase (DHODH) exhibits a unique duality: as a mitochondrial flavoprotein that catalyzes the fourth step of de novo pyrimidine synthesis (dihydrouracil → orotate conversion), it drives the nucleotide biosynthesis necessary for tumor proliferation

[14] . In addition to this classic function, DHODH also plays a non-enzymatic role in redox homeostasis (clearing mitochondrial ROS) and stabilizing the Wnt / β-catenin signaling pathway through β-catenin deubiquitination [15-17]. This pleiotropic nature makes DHODH a core metabolic hub connecting anabolic metabolism and pro-survival signaling pathways. Clinically, DHODH inhibitors (buquina, leflunomide) have shown dual effects: although they can effectively block pyrimidine synthesis in preclinical models, their monotherapy efficacy is limited by compensatory salvage pathways (28% response rate in phase II trials) and dose-limiting cytotoxicity [18-20]. Recent evidence suggests that these limitations stem from a poorly understood mechanism—DHODH's "part-time" function may activate alternative survival pathways, thereby counteracting the inhibition of its enzymatic activity. This finding underscores the necessity of a paradigm shift: rather than simply inhibiting DHODH, strategically targeting its interactome may disrupt oncogenic networks while circumventing drug resistance. Current research has severely neglected the protein-protein interactions of DHODH and its ability to utilize lipid metabolism pathways. Summary of the Invention

[0004] To address the current lack of understanding of how DHODH coordinates phosphatidylcholine reprogramming through post-translational regulation, this invention provides the application of DHODH-CHKα-TRIM28 in screening drugs that inhibit the proliferation or growth of colorectal cancer cells.

[0005] This invention is achieved by the following technical solution: the application of DHODH-CHKα-TRIM28 in screening drugs that inhibit the proliferation or growth of colorectal cancer cells, wherein dihydroorotate dehydrogenase (DHODH) and choline kinase α (CHKα) are highly co-expressed in colorectal cancer and are significantly associated with poor prognosis in patients; dual-targeted inhibition of DHODH and CHKα can synergistically inhibit the proliferation or growth of colorectal cancer cells.

[0006] Furthermore, the targeted inhibition of DHODH refers to inhibiting the direct interaction between the DHODH protein and CHKα. Specifically, DHODH competitively blocks the recognition and binding of the E3 ubiquitin ligase TRIM28 to the adjacent K244 residue on CHKα by binding to the F242 / N243 domain of CHKα, thereby inhibiting the TRIM28-mediated CHKα ubiquitination-proteasome degradation pathway. Inhibiting DHODH can remove this competitive barrier, restore TRIM28's ubiquitination and degradation of CHKα, and ultimately reduce the protein expression level of CHKα.

[0007] The drug that targets and inhibits DHODH is leflunomide, and the drug that targets and inhibits CHKα is MN58b.

[0008] This invention experimentally demonstrates that DHODH competitively binds to the F242 / N243 domain of CHKα. This binding spatially prevents the E3 ubiquitin ligase TRIM28 from approaching the adjacent K244 residue on CHKα, thereby inhibiting TRIM28-mediated polyubiquitination of CHKα at the K244 site and subsequent proteasomal degradation. Increased CHKα stability mediated by DHODH drives the accumulation of carcinogenic phosphatidylcholine, which in turn promotes the proliferation and growth of colorectal cancer cells. Clinical sample analysis shows that high co-expression of DHODH and CHKα is closely associated with poor patient prognosis.

[0009] This invention further reveals that TRIM28 is a key E3 ubiquitin ligase regulating CHKα protein homeostasis in colorectal cancer cells, with K244 being the key site for TRIM28-mediated CHKα ubiquitination. DHODH competitively binds to CHKα, physically shielding the K244 site of CHKα from TRIM28-mediated ubiquitination degradation, thereby establishing a trigonometric (DHODH–CHKα–TRIM28) regulatory axis. This axis establishes DHODH as a competitive stabilizer against TRIM28-dependent CHKα protein degradation.

[0010] This invention fills a gap in our understanding of how DHODH coordinates phosphatidylcholine metabolic reprogramming through post-translational regulation. This mechanism helps explain metabolic adaptation in colorectal cancer. By analyzing the DHODH-CHKα-TRIM28 axis, this invention expands therapeutic strategies from single enzyme inhibition to systemic intervention on key metabolic protein interaction networks. Attached Figure Description

[0011] Figure 1 A comparison of DHODH expression in colorectal cancer tissues and adjacent normal tissues, and a semi-quantitative H-score analysis result. Figure 2 To verify the experimental results regarding the role of the DHODH gene in the proliferation of colorectal cancer cells and tumor growth in vivo; Figure 3 The effect of DHODH overexpression on phosphatidylcholine metabolism in colorectal cancer cells; In the figure: (A) Non-targeted metabolomics analysis showed that differentially expressed metabolites were significantly enriched in the phosphatidylcholine metabolic pathway. (B) Targeted quantitative verification showed that DHODH overexpression led to an increase in intracellular phosphatidylcholine levels. (C) The CHKα inhibitor MN58b could reverse the phosphatidylcholine accumulation caused by DHODH overexpression; Figure 4 Figure showing the effect of changes in DHODH expression levels on CHKα protein and mRNA expression. Figure 5 The results of co-immunoprecipitation (Co-IP) of DHODH and CHKα to verify their direct interaction are shown in the figure. Figure 6 Figure showing the effect of DHODH on the half-life of CHKα protein in a cyclohexane (CHX) tracking experiment; Figure 7 Figure showing the results of treatment with the proteasome inhibitor MG132 to rescue CHKα degradation; Figure 8 The figure shows the inhibitory effect of DHODH overexpression on CHKα ubiquitination levels in the presence of MG132. Figure 9 The diagram shows the predicted interaction interface and key binding residues between DHODH and CHKα proteins based on molecular docking. Figure 10 The diagram shows the results of the DHODH key residue mutant (R326A / K272A) disrupting its binding with CHKα and reducing the stability of CHKα; Figure 11 The image shows the results of identifying TRIM28 as a novel CHKα interacting protein using affinity purification-mass spectrometry. Figure 12 The image shows the results of the immunoprecipitation verification of CHKα and TRIM28. Figure 13 The figure shows the results of upregulation of CHKα protein levels after TRIM28 knockdown while mRNA levels remained unchanged. Figure 14 Figure showing the results of the TRIM28 knockdown experiment, which showed that knocking down TRIM28 could prolong the half-life of CHKα protein. Figure 15 Figure showing the results of MG132 treatment to reverse the stabilization of CHKα protein induced by TRIM28 knockdown; Figure 16 The diagram shows the results of in vitro ubiquitination experiments demonstrating that TRIM28 has E3 ligase activity against CHKα. Figure 17 The figure shows the results of prolonged CHKα protein half-life after TRIM28 knockdown; Figure 18 The results of mass spectrometry identification of the major ubiquitination modification sites (K244 and K325) of CHKα; Figure 19 The result of the CHKαK244R mutant inhibiting TRIM28-mediated ubiquitination modification; Figure 20 The figure shows the results of enhanced CHKα protein stability in CHKα K244R mutant cells; Figure 21 Figure showing the results of the cyclohexane tracking experiment demonstrating the prolonged half-life of CHKα protein in CHKα K244R mutant cells; Figure 22 The figure shows the verification results of DHODH overexpression weakening the binding of TRIM28 to CHKα; Figure 23 The verification results for knocking down DHODH to enhance the binding of TRIM28 to CHKα are shown in the figure. Figure 24 The figure shows the results of restoring CHKα protein expression by simultaneously knocking down TRIM28 in DHODH knockdown cells; Figure 25 The image shows a comparison of CHKα expression in colorectal cancer tissues and adjacent normal tissues, along with the results of semi-quantitative H-score analysis. Figure 26 Comparative diagram of CHKα expression analysis in nude mouse xenograft models; Figure 27 Figure showing the correlation analysis results of DHODH and CHKα expression levels in colorectal cancer tissue; Figure 28 The survival analysis curves for patients stratified according to the co-expression status of DHODH and CHKα are shown; where DLCL indicates low expression of both, DLCH indicates low DHODH / high CHKα, DHCL indicates high DHODH / low CHKα, and DHCH indicates high expression of both. Figure 29 The image shows the results of the MTT assay demonstrating the synergistic inhibition of colorectal cancer cell proliferation by the combined use of leflunomide and MN58b. Figure 30 The diagram shows the results of a clonogenic assay demonstrating that the combined use of flumectide and MN58b has a stronger inhibitory effect on the long-term proliferation of cells. Figure 31 The figure shows the results of significant inhibition of tumor growth in the animal model group treated with a combination of flumectide and MN58b. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited by them are incorporated herein by reference. Equivalent techniques of the specific embodiments described herein, which can be understood by those skilled in the art through routine experiments, are included in this application. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the instruments and equipment used in the following embodiments are standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0014] I. Materials and Methods 1. Immunohistochemical analysis Tissue microarray construction: All colorectal tissue specimens were double-verified by pathologists to confirm the histopathological diagnosis. Tumor regions and adjacent normal mucosa were marked on hematoxylin-eosin stained sections according to WHO classification criteria. Using a precision tissue array, 2.0 mm cores were extracted from donor paraffin blocks and transferred to recipient paraffin blocks preheated for 30 min. The TMA structure contained 32 cores arranged in an 8×4 grid, with randomized placement to mitigate batch effects. After assembly, the TMA blocks were baked at 60 °C for 2 h to ensure core adhesion, and then sectioned using a rotary microtome to a thickness of 4 μm. Sections were mounted on positively charged slides and stored at 4 °C until immunohistochemical treatment.

[0015] Protein detection: Paraffin-embedded xenograft specimens underwent sequential dewaxing and gradient ethanol hydration. Antigen retrieval was performed using the EDTA-based high-pressure epitope retrieval method, consisting of 5 min of pressurization followed by 8 min of sustained heating. Non-specific binding sites were blocked for 1 h at room temperature using 10% normal goat serum. After three PBST washes, sections were incubated with primary antibody at 4°C for 16 h, followed by incubation with Max Vision Mouse / Rabbit at 37°C for 1 h. Colorimetric development was performed using the DAB substrate reaction, followed by counterstaining with hematoxylin. Images and data were acquired using the ScanScope slide scanning system.

[0016] 2. Cell Culture, Construction of DHODH Overexpression and Knockdown Cell Lines: The human colorectal cell line used in this invention was purchased from Procell Life Sciences and identified by STR analysis. The cell line was cultured in RPMI-1640 medium at 37°C and 5% CO2. To construct a DHODH overexpression cell model, the complete coding sequence of human DHODH (cDNA, NCBI Reference Sequence: NM_001361.4) was cloned into the Ubi-MCS-CMV-GFP-IRES-Puro lentiviral vector to generate DHODH-OE particles. Conversely, the validated DHODH-targeting shRNA (5'-CGGTCCGGGATTTATCCAAACTCGAGTTTGAGTGTGTAAATCCCGGATTTTTTT-3') was packaged into the pLKO.1-puro lentiviral vector. All vectors were commercially synthesized by Genephamm.

[0017] 3. Cell proliferation experiment MTT method: cells are placed at a density of 3 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL in 96-well plates and pre-cultured for 24 h. After treatment, 20 μL of MTT solution was added to each well and incubated for 4 h. Formazan crystals were dissolved in 100 μL of DMSO containing 10 mM HCl and 0.1% SDS. The absorbance was measured at 490 nm using a multi-reader.

[0018] Soft agar colony formation assay: Base agar was immobilized in 6-well plates. Cells suspended in the top agar were seeded onto the base agar. After 14 days of incubation, colonies were fixed with 4% paraformaldehyde, stained with 0.05% crystal violet / 20% methanol, and destained with PBS. Colonies larger than 50 μm in diameter were counted using the ImageJ Colony Counter plugin.

[0019] 4. Animal Experiments: Female BALB / c-nu / nu mice were purchased from GemPharmatech. Mice were housed under specific pathogen-free conditions with free access to sterile food and water. For the xenograft model, SW480 cells stably overexpressing DHODH were suspended in 100 μL of a 1:1 mixture of Matrigel and PBS and subcutaneously injected into the right groin of the mice. Similar injections of SW480 control cells were performed. Mice were randomly assigned to the experimental groups. Starting from day 7 post-injection, tumor size was measured three times weekly using digital calipers. Tumor volume was calculated using a formula.

[0020] On day 20 post-inoculation, after palpable tumors formed, mice were treated with the following drugs via intraperitoneal injection: Group 1: leflunomide; Group 2: MN58b; Group 3: leflunomide and MN58b in combination; Control group: carrier solution. Administered every four days for a 16-day pretreatment period. On day 35, all mice were euthanized via intraperitoneal injection of sodium pentobarbital, followed by cervical dislocation as secondary confirmation of death. Tumors were carefully excised, weighed, and photographed under standardized light conditions. The tumor tissue was then flash-frozen in liquid nitrogen for subsequent molecular analysis. Animal health scores were monitored weekly throughout the study. A strict humane endpoint was defined as a tumor burden exceeding 15% of body weight or the appearance of significant tumor ulceration.

[0021] 5. qRT-PCR analysis: Total RNA was extracted from 5 × 10⁻⁶ cells using a total RNA extraction system. 6 Total RNA was extracted from cells. RNA integrity was verified by NanoDropOneC spectrophotometry, and samples with A260 / A280 = 1.8–2.1 and A260 / A230 ≥ 2.0 were retained. For cDNA synthesis, 600 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase. qRT-PCR was performed on an ABI 7500 real-time PCR system using SYBR1 Green PCR MasterMix, with three replicates per sample. Each 20 μL reaction volume contained: 10 μL 2×Master Mix, 0.5 μL primers (Table 1), 1 μL cDNA, and the volume was adjusted to 20 μL with nuclease-free H2O. Relative mRNA expression levels were calculated using the 2(-ΔΔCt) method, with GAPDH as an internal control.

[0022] Table 1: Gene-specific primers for DHODH, CHKA, and GAPDH used in real-time quantitative PCR analysis 6. Western Blotting and Immunoprecipitation: Cells were lysed in pre-chilled RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with a protease inhibitor cocktail added before use). The lysis buffer was centrifuged at 16,000 × g for 30 min at 4 °C, and the supernatant was collected. 30 μg of protein sample was taken and separated using a 10% SDS-PAGE gel, with the pre-stained protein molecular weight standard used as a reference. The protein was transferred to a pre-activated methanol-based 0.45 μm PVDF membrane using a semi-dry transfer method at 25 °C for 1 hour. After transfer, the membrane was blocked with TBST buffer containing 5% skim milk powder for 1 hour, followed by incubation with the corresponding primary antibody at 4 °C overnight. The primary antibodies used included rabbit anti-DHODH (1:2000), rabbit anti-CHKα (1:500), and rabbit anti-TRIM28 (1:500). The following day, after washing, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1:10,000) at room temperature for 2 hours. Development was performed using ECL chemiluminescent substrate, and signals were acquired via an imaging system. GAPDH (1:50,000) was used as an internal control to correct for the loading volume. For immunoprecipitation, 2 μg of the corresponding primary antibody was added to the clarified cell lysate, and the mixture was incubated at 4°C for 16 hours. Protein A / G magnetic beads were then added, and incubation continued for another 4 hours. The magnetic beads were washed 5 times with lysis buffer, and then 2×Laemmli loading buffer (containing 4% SDS, 20% glycerol, 120mM Tris-HCl pH 6.8 and 0.02% bromophenol blue, with 10% β-mercaptoethanol added before use) was added. The protein was eluted by heating at 95°C for 5 minutes, followed by immunoblotting analysis.

[0023] 7. CHKα Ubiquitination Assay: Cells were co-transfected with HA-Ub plasmid and target vector for 24 h, then treated with 10 μM M G132 for 6 h to stabilize ubiquitinated proteins. Cells were collected in ice-cold PBS and lysed in modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, with 1× protease inhibitor cocktail, 10 mM N-ethylmaleimide, and 1 mM PMSF added immediately before use). The lysates were sonicated (30% amplitude, 3×10 sec pulses) and centrifuged at 4 °C, 16,000×g for 30 min, and the supernatant was collected. For immunoprecipitation, 500 μg of total protein lysis buffer was incubated overnight at 4 °C with 2 μg of CHKα antibody-pre-coupled Protein A / G magnetic beads by rotation. The magnetic beads were then washed five times with lysis buffer, and proteins were eluted by heating at 95°C for 5 minutes with 2×Laemmli loading buffer. The eluted samples were separated in a 4–12% Bis-Tris gradient gel (Invitrogen NW04120BOX) and transferred to a PVDF membrane. Ubiquitination was detected using the following antibodies: primary antibody: anti-HA antibody; secondary antibody: HRP-labeled anti-mouse IgG. Chemiluminescence signals were quantified using Image Lab 6.1 (Bio-Rad) and normalized using the CHKα level in the input lysis buffer as an internal control.

[0024] 8. Molecular docking: The three-dimensional structures of target proteins DHODH and CHKα were obtained from a protein database. Before docking, water molecules were removed and hydrogen was added using AutoDock Tools 1.5.7. The processed protein structures were uploaded to the HDOCK server for docking simulation. The resulting docking complex was analyzed using PyMOL 2.5.2 and Discovery Studio Visualizer 20.1 to visualize and annotate the interacting amino acids at the binding interface.

[0025] 9. Cell Transfection: Plasmids of wild-type DHODH, its point mutation variant (R326A / K272A), and wild-type CHKα were cloned into the pcDNA3.1(+) vector, commercially synthesized and supplied by Genepharm (Shanghai, China). 24 hours before transfection, SW480 and HT29 colorectal cancer cells were transfected at 5 × 10⁶ cells / well. 5Cells were seeded at a density of [number] cells per well in 6-well plates, and cell viability was confirmed to be >95% using trypan blue staining. The transfection complex was prepared as follows: 2 mL of serum-free RPMI-1640 basal medium (Gibco, 11875-093) was added to 10 μL of Lipofectamine 3000 (Invitrogen), and mixed at a volume ratio of 1:5 (lipid:DNA), strictly following the manufacturer's recommended procedures. The transfection complex and cells were co-incubated at 37°C and 5% CO2 for 8 hours. The transfection mixture was then removed and replaced with complete medium containing 10% fetal bovine serum (FBS, Gibco 10099-141) to mitigate potential cytotoxicity caused by the lipid carrier.

[0026] 10. Statistical Analysis: All experimental data are expressed as mean ± standard deviation of three independent biological replicates. GraphPad Prism 9.4.1 was used for data visualization and to create box plots with superimposed data points. Normality and homogeneity of variance of the data were verified before parametric analysis. SPSS 26.0 was used for statistical comparisons, employing one-way ANOVA and post-hoc t-tests. The significance level was defined as: p<0.05, p<0.01, p<0.001.

[0027] II. Experimental Results and Analysis 1. DHODH overexpression as an independent prognostic indicator for colorectal cancer: DHODH expression in 173 colorectal cancer (CRC) specimens and matched adjacent normal tissues (ANTs) was evaluated using immunohistochemistry (IHC). Semi-quantitative H-score analysis showed that DHODH was mainly located in the cytoplasm, and its expression level in CRC tissues was significantly higher than that in ANTs. Figure 1 Cox regression analysis showed that DHODH overexpression was significantly associated with poorer overall survival (Table 2). These results suggest that cytoplasmic DHODH overexpression may serve as a biomarker for colorectal cancer progression and a potential therapeutic target.

[0028] Table 2: Analysis of prognostic factors in colorectal cancer patients based on Cox regression model 2. DHODH promotes colorectal cancer cell proliferation and accelerates tumor growth in vivo: To elucidate the role of DHODH in colorectal cancer development, DHODH was overexpressed in HT29 and SW480 cells, and DHODH was knocked down in HCT8 and SW620 cells to construct cell models with functional gain and loss of function for subsequent functional validation. Cellular function experiments showed that DHODH plays a key regulatory role in the proliferation of colorectal cancer cells, and MTT and colony formation assays quantitatively confirmed this phenotype. Figure 2 AD). Further in vivo xenotransplantation experiments showed that tumor growth in mice inoculated with DHODH-overexpressing cells was significantly accelerated (AD). Figure 2 EG), histopathological analysis also indicated an elevated Ki-67 proliferation index in tumors that highly expressed DHODH ( Figure 2 H). In summary, this invention demonstrates through multi-level experiments that DHODH has oncogene function and can promote the progression of colorectal cancer by enhancing its proliferative capacity.

[0029] 3. DHODH regulates phosphatidylcholine metabolism by affecting CHKα ubiquitination: Non-targeted metabolomics analysis of DHODH-overexpressing and control colorectal cancer cells revealed that differentially expressed metabolites were significantly enriched in the phosphatidylcholine metabolic pathway. Figure 3 A) The intracellular phosphatidylcholine content in DHODH-overexpressing cells was significantly higher than that in the control group. Figure 3 B). Pharmacological inhibition of choline kinase α (CHKα) using MN58b can effectively reverse the accumulation of phosphatidylcholine caused by DHODH overexpression. Figure 3 (C) indicates a functional association between DHODH and CHKα. Further experiments showed that DHODH overexpression upregulated CHKα protein levels without affecting its transcription; conversely, knockdown of DHODH reduced CHKα expression, suggesting a post-transcriptional regulatory mechanism. Figure 4 Immunoprecipitation experiments confirmed a direct physical interaction between DHODH and CHKα in both HT29 and SW480 cells. Figure 5 Protein turnover experiments showed that DHODH can stabilize CHKα protein. Figure 6 The proteasome inhibitor MG132 can block CHKα degradation (); Figure 7 Furthermore, DHODH overexpression significantly reduced CHKα ubiquitination levels. Figure 8 This indicates that DHODH protects CHKα by inhibiting the ubiquitin-proteasome pathway. Molecular docking and structural analysis identified a key binding interface between the F242 / N243 residues on CHKα and the R326 / K272 residues on DHODH. Figure 9Mutating these sites to alanine disrupts the formation of the DHODH-CHKα complex and leads to decreased CHKα protein stability. Figure 10 The above results suggest that DHODH, as an upstream regulatory node, maintains the protein stability of CHKα and regulates the phosphatidylcholine metabolic pathway by directly binding to and inhibiting the ubiquitination and degradation of CHKα.

[0030] 4. TRIM28 regulates the protein stability of CHKα by ubiquitinizing the key site K244: Through affinity purification combined with mass spectrometry analysis, TRIM28 was identified as a novel CHKα interacting protein in colorectal cancer cells. Figure 11 And further verification was performed using an immunoprecipitation experiment. Figure 12 Knocking out TRIM28 in colorectal cancer cells significantly increased CHKα protein levels but did not affect its mRNA expression. Figure 13 Actinomycete ketone tracking experiments showed that TRIM28 deletion prolonged the half-life of CHKα protein. Figure 14 Treatment with the proteasome inhibitor MG132 can reverse CHKα stabilization induced by TRIM28 knockdown. Figure 15 This indicates that TRIM28 is involved in the proteasome-dependent CHKα degradation pathway. In vitro ubiquitination analysis confirmed that TRIM28 possesses E3 ubiquitin ligase activity for CHKα. Figure 16 Its knockout significantly enhances the stability of CHKα protein. Figure 17 Mass spectrometry analysis identified K244 and K325 as the major ubiquitination sites on CHKα. Figure 18 CHKα-K244R mutations reduce CHKα ubiquitination in HT29 and SW480 cells. Figure 19 Enhanced protein stability Figure 20 and prolonged half-life ( Figure 21 The study confirmed that K244 is a key residue in TRIM28-mediated ubiquitination. These results indicate that TRIM28, as a novel E3 ubiquitin ligase, affects protein homeostasis in colorectal cancer cells by targeting CHKα to regulate its ubiquitination and degradation.

[0031] 5. DHODH competitively binds to CHKα and inhibits their interaction with TRIM28: To elucidate the molecular regulatory relationship between DHODH and TRIM28, immunoprecipitation experiments revealed that overexpression of DHODH in HT29 and SW480 cells significantly weakened the interaction between TRIM28 and CHKα. Figure 22 Knocking down DHODH promotes the binding of the two. Figure 23 Further knockout of TRIM28 in DHODH-knockdown cells restored CHKα protein expression levels. Figure 24 This suggests that DHODH and TRIM28 compete for control over CHKα stability. Structural analysis indicates that DHODH directly binds to CHKα, spatially hindering TRIM28's access to its E3 ligase recognition domain, thus creating competitive inhibition.

[0032] 6. Dual targeting of DHODH and CHKα effectively inhibits colorectal cancer growth in vitro and in vivo: Immunohistochemical analysis showed that the expression level of CHKα in CRC tissue was significantly higher than that of ANTs ( Figure 25 This result was further validated in a nude mouse xenograft model. Figure 26 Furthermore, in colorectal cancer samples, the expression of DHODH and CHKα showed a significant positive correlation. Figure 27 Compared with other subgroups, patients with high co-expression of DHODH and CHKα had shorter overall survival. Figure 28 This highlights its clinical prognostic significance. This synergistic effect is supported by previous biochemical evidence: DHODH can stabilize CHKα protein by inhibiting the ubiquitin-proteasome pathway, thereby maintaining its oncogenic signaling activity and synergistically driving tumor progression. To evaluate the potential of targeted therapy along this pathway, the effects of inhibiting DHODH and CHKα on colorectal cancer proliferation were investigated in in vitro and in vivo models. In vitro experiments showed that treatment with leflunomide (a DHODH inhibitor) or MN58b (a CHKα inhibitor) alone significantly inhibited cell proliferation, and the combination of the two drugs showed a synergistic inhibitory effect. Figure 29 , Figure 30 In xenograft models, monotherapy inhibited tumor growth, while combination therapy resulted in more significant tumor growth inhibition. Figure 31 Dual targeting of DHODH and CHKα effectively inhibited colorectal cancer proliferation both in vitro and in vivo, suggesting that simultaneously blocking this metabolic regulatory axis has potential therapeutic value.

[0033] This invention reveals a novel regulatory axis between pyrimidine biosynthesis and phospholipid metabolism in colorectal cancer, where DHODH can stabilize CHKα by competitively antagonizing TRIM28-mediated ubiquitination. CHKα, as the rate-limiting enzyme in phosphatidylcholine biosynthesis, is responsible for catalyzing the phosphorylation of choline to phosphocholine—a key step in membrane phospholipid synthesis [21-22]. It is highly expressed in malignant tumors and drives tumorigenesis by promoting phosphatidylcholine synthesis to maintain the membrane formation and signal transduction required for cancer cell proliferation [23-25]. This invention identifies for the first time TRIM28—a multifunctional E3 ubiquitin ligase—as a key negative regulator of CHKα protein stability. TRIM28 belongs to the TRIM protein family, which is widely involved in various cellular processes such as protein degradation, DNA repair, and immune signaling

[25] . Notably, TRIM28 has been recognized as an important regulator of oncoprotein turnover, and its substrate specificity is often regulated by competitive protein interactions [26-27].

[0034] The results of this study show that DHODH binds directly to CHKα via its key residues F242 / N243. This interaction spatially hinders TRIM28's recognition of the K244 site on CHKα, thereby inhibiting multi-site ubiquitination of CHKα and its subsequent proteasome degradation. DHODH overexpression thus exacerbates the accumulation of phosphatidylcholine and promotes the proliferation and growth of colorectal cancer cells. This result not only identifies TRIM28 as a novel E3 ubiquitin ligase regulating CHKα protein homeostasis but also reveals a previously unknown regulatory interface connecting pyrimidine metabolism and lipid synthesis. Although the enzymatic role of DHODH in pyrimidine synthesis has been well reported, we highlight its non-canonical function as a post-translational regulator of lipid metabolism. The structural competition between DHODH and TRIM28 introduces a new paradigm: metabolic enzymes can "double" as protein stabilizers, thereby extending their oncogenic effects beyond substrate catalysis. This mechanism may partially explain the limited efficacy of DHODH inhibitors in clinical trials—even with the inhibition of nucleotide synthesis, compensatory stabilization of lipid metabolism pathways may still maintain tumor survival. Clinically, DHODH and CHKα expression are strongly positively correlated in CRC, and their co-expression is significantly associated with poor patient prognosis, suggesting that this regulatory axis has important translational medical significance. Patients with dual high expression of DHODH / CHKα have significantly shorter overall survival, indicating that this interaction drives a more aggressive tumor phenotype. Furthermore, leflunomide and MN58b have both shown synergistic effects in inhibiting tumor growth in in vitro and in vivo experiments, providing preclinical evidence for dual targeting of DHODH and CHKα, and laying a theoretical foundation for the combined use of nucleotide synthesis inhibitors and choline metabolism modulators in the treatment of colorectal cancer.

[0035] In summary, this invention elucidates a novel mechanism by which DHODH regulates lipid metabolism reprogramming in colorectal cancer. DHODH stabilizes CHKα by competitively antagonizing TRIM28-mediated ubiquitination, thereby establishing a functional link between pyrimidine biosynthesis and phosphatidylcholine synthesis, thus creating a target for therapeutic intervention. The DHODH-CHKα-TRIM28 axis not only deepens our understanding of cancer metabolic plasticity but also provides a theoretical basis for combined targeted therapies. Combining DHODH inhibitors with therapies targeting CHKα can simultaneously interfere with nucleotide and phospholipid metabolism, potentially overcoming the compensatory survival pathways commonly found in single-target therapies.

[0036] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

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Claims

1. The application of DHODH-CHKα-TRIM28 in screening drugs that inhibit the proliferation or growth of colorectal cancer cells, characterized by: The dihydroorotate dehydrogenase DHODH and choline kinase α (CHKα) are highly co-expressed in colorectal cancer and are associated with poor patient prognosis; dual-targeted inhibition of DHODH and CHKα synergistically inhibits the proliferation or growth of colorectal cancer cells.

2. The application according to claim 1, characterized in that: The targeted inhibition of DHODH refers to intervening in the direct binding between DHODH and CHKα, thereby relieving the competitive barrier of DHODH to the interaction between TRIM28 and CHKα, enhancing the binding of TRIM28 to CHKα, promoting the ubiquitination of CHKα at the K244 site and its subsequent proteasome degradation, and ultimately reducing the stability and expression level of CHKα protein.

3. The application according to claim 1, characterized in that: The drug that targets and inhibits DHODH is leflunomide, and the drug that targets and inhibits CHKα is MN58b.

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