Application of AURKA-NCOA4 interaction inhibitor in preparation of medicine for reversing ferroptosis resistance

Abstract

The invention discloses application of an AURKA-NCOA4 interaction inhibitor in preparation of a medicine for reversing ferroptosis resistance, and relates to the technical field of biological medicines. According to the application disclosed by the invention, the fact that the AURKA regulates ferroptosis through a non-cell cycle way is revealed for the first time, and the AURKA-NCOA4 interaction inhibitor can fundamentally reverse the iron homeostasis imbalance of drug-resistant cells by relieving competitive inhibition of the AURKA on NCOA4, restoring the autophagy degradation capability of ferritin FTH1 and reconstructing an iron ion release channel in the cells. The AURKA-NCOA4 interaction inhibitor provided by the invention can break through the core molecule bottleneck of ferroptosis resistance, and provides a new target for overcoming drug resistance. Reverse hepatoma sorafenib drug-resistant cell ferroptosis resistance is achieved by inhibiting interaction of AURKA and NCOA4, the drug-resistant cell responds to the killing effect of sorafenib and other ferroptosis inducers again, the problem of clinical drug resistance of sorafenib is solved, the drug treatment window phase is prolonged, and the application prospect is broad. And a new solution is provided for treatment failure caused by resistance of the drug-resistant cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of AURKA-NCOA4 interaction inhibitors in the preparation of drugs for reversing ferroptosis resistance. Background Technology

[0002] Treatment for advanced liver cancer primarily involves systemic therapy. Although immunotherapy has made rapid progress in recent years and has gradually become a first-line treatment for advanced liver cancer, sorafenib, as the first targeted therapy drug used to treat advanced liver cancer, is still widely used as a first-line treatment due to its rich clinical experience and reliable efficacy. In particular, sorafenib is the main treatment option for patients who are not suitable for immunotherapy or whose immunotherapy has failed.

[0003] However, sorafenib has significant limitations in clinical efficacy, with only about 30% of patients benefiting from it, and this population typically develops resistance within 6 months. Primary or acquired resistance not only limits its therapeutic efficacy, but the development of resistance is also closely related to tumor recurrence, further restricting treatment options for patients with advanced liver cancer.

[0004] Ferroprelation is a programmed cell death process dependent on iron ions, caused by the accumulation of lipid peroxides and lipid reactive oxygen species within cells. Ferroprelation activation has been identified as one of the core mechanisms of sorafenib efficacy, but clinical resistance suggests that tumor cells can evade feroprelation by activating feroprelation resistance mechanisms. Studies have found the activation of certain feroprelation suppressor genes in sorafenib-resistant cells; knocking down or inhibiting these genes significantly improves sorafenib treatment sensitivity, further indicating that feroprelation inhibition may be one of the key factors leading to sorafenib resistance.

[0005] As a core regulator of the cell cycle, AURKA regulates the cell cycle by controlling centrosome maturation, spindle assembly, and chromosome segregation, thus maintaining genome stability. However, the mechanism of action of AURKA in liver cancer cells remains unclear.

[0006] Currently, sorafenib has limited efficacy in treating liver cancer, as it induces ferroptosis resistance. There is an urgent need to develop a new intervention strategy to overcome this treatment bottleneck. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide the use of AURKA-NCOA4 interaction inhibitors in the preparation of drugs for reversing ferroptosis resistance.

[0008] To address the above problems, the present invention proposes the following technical solution:

[0009] This invention provides the use of AURKA-NCOA4 interaction inhibitors in the preparation of medicaments for reversing ferroptosis resistance.

[0010] Furthermore, the inhibitor promotes the degradation of FTH1 protein by blocking the binding of AURKA protein to NCOA4 protein.

[0011] Furthermore, the inhibitor blocks the binding of AURKA protein to NCOA4 protein by targeting the Ser186, Ser234, or Ser492 phosphorylation sites of NCOA4.

[0012] Furthermore, the inhibitor is an AURKA selective inhibitor.

[0013] Furthermore, the inhibitor is Alisertib or CD532.

[0014] Furthermore, the drug is used to treat sorafenib-resistant liver cancer.

[0015] The present invention also provides the use of AURKA selective inhibitors in the preparation of medicaments for enhancing the ferroptosis sensitivity of hepatocellular carcinoma cells, wherein the ferroptosis sensitivity is achieved by promoting the degradation of FTH1 protein.

[0016] The present invention also provides a pharmaceutical composition for treating liver cancer, comprising an AURKA-NCOA4 interaction inhibitor, said pharmaceutical composition being able to reverse ferroptosis resistance.

[0017] Furthermore, the pharmaceutical composition comprises an AURKA selective inhibitor.

[0018] Furthermore, the pharmaceutical composition also contains a ferroptosis inducer.

[0019] Iron ions are a key factor in ferroptosis, and ferritin autophagy, which releases free iron ions through the degradation of ferritin, is a crucial process influencing ferroptosis levels. As a core mediator of ferroptosis, NCOA4 binds to FTH1, mediating the autophagic degradation of ferritin to release free ferrous ions and maintain intracellular iron homeostasis. Given the role of ferritin autophagy in regulating iron homeostasis, regulating NCOA4-mediated ferritin autophagy flux can alter sensitivity to ferroptosis. Therefore, factors affecting the binding of these two molecules can influence the process of ferroptosis, thereby reversing ferroptosis resistance.

[0020] This invention reveals that AURKA can directly bind to NCOA4 and competitively inhibit its binding to FTH1, thereby blocking ferrophagy and supporting the view that the binding of NCOA4 and FTH1 can enhance the sensitivity to ferroptosis. Furthermore, we found that AURKA directly binds to and phosphorylates serine sites on NCOA4, thus affecting the binding of NCOA4 and FTH1. Mutations at serine sites S186, S334, and S492 on NCOA4 reduced the level of serine phosphorylation by AURKA, decreased the binding of NCOA4 to AURKA, and increased the binding to FTH1, indicating that phosphorylation of NCOA4 affects the binding ability to FTH1. In this invention, AURKA phosphorylates NCOA4 at sites S186, S334, and S492A. AURKA and FTH1 competitively bind to NCOA4. When NCOA4 is phosphorylated by AURKA, the binding between NCOA4 and AURKA decreases, the competition between AURKA and FTH1 decreases, thereby increasing the binding between NCOA4 and FTH1 and enhancing ferroptosis.

[0021] Compared with the prior art, the technical effects achieved by the present invention include:

[0022] This invention reveals for the first time that AURKA regulates ferroptosis through non-cell cycle pathways, filling a gap in the AURKA kinase-ferroptosis regulatory mechanism. The AURKA-NCOA4 interaction inhibitor, by relieving AURKA's competitive inhibition of NCOA4, restores the autophagic degradation capacity of ferritin FTH1 and rebuilds intracellular iron ion release channels, fundamentally reversing the iron homeostasis imbalance in drug-resistant cells. The AURKA-NCOA4 interaction inhibitor provided by this invention can overcome the core molecular bottleneck of ferroptosis resistance, offering a new target for overcoming drug resistance.

[0023] This invention reverses ferroptosis resistance in sorafenib-resistant liver cancer cells by inhibiting the interaction between AURKA and NCOA4. The resistant cells then respond again to the killing effect of ferroptosis inducers such as sorafenib, solving the clinical problem of sorafenib resistance, prolonging the therapeutic window, and providing a new solution for treatment failure caused by resistance in drug-resistant cells. Attached Figure Description

[0024] Figure 1 The Co-IP / mass spectrometry analysis results show that NCOA4 is an interacting protein of AURKA. In the figure, (A) shows the KEGG pathway enrichment analysis of proteins that bind to AURKA detected by mass spectrometry (MS). (B) Partially shows the protein spectrum results of AURKA binding, indicating that NCOA4 may bind to it.

[0025] Figure 2The results of Co-IP and immunofluorescence co-localization show a direct interaction between AURKA and NCOOA4. In the figure, (A) shows endogenous Co-IP in HepG2 and Huh7 cells, with AURKA and NCOOA4 binding, and IgG antibody as a negative control. (B) shows exogenous Co-IP in HEK293T cells co-transfected with Flag-labeled AURKA and HA-labeled NCOOA4 plasmids (48h), showing their binding, with IgG antibody as a negative control. (C) shows immunofluorescence co-localization of AURKA and NCOOA4; the green FITC channel shows AURKA, the red 594 channel shows NCOOA4, and the blue DAPI channel shows the cell nucleus. (D) Quantitative analysis of AURKA and NCOOA4 co-localization using immunofluorescence.

[0026] Figure 3 Knockdown of NCOA4 can reverse the increase in ferrophagy levels caused by AURKA knockdown. In the figure, (A) shows the expression levels of FTH1 and NCOA4 proteins in hepatocellular carcinoma cells after 48 h of AURKA knockdown and / or NCOA4 silencing by Western blot. (B) shows the colocalization of ferritin and lysosomal marker LAMP1 after 36 h of AURKA knockdown and / or NCOA4 silencing by immunofluorescence colocalization analysis. The red fluorescence 594 channel shows ferritin, the green fluorescence FITC channel shows LAMP1, and the blue fluorescence DAPI shows the cell nucleus.

[0027] Figure 4 Flow cytometry results showing that NCOA4 knockdown could reverse the elevated ferrophagy levels induced by AURKA knockdown. In the figure, (AB) show the ROS levels (A) and lipid peroxidation levels (B) of liver cancer cells after 48 h of AURKA knockdown and / or NCOA4 silencing, as determined by flow cytometry. (***: p < 0.001, ****: p < 0.0001, data from three replicates, shown as mean ± standard deviation.)

[0028] Figure 5 The results show that FTH1 degradation was inhibited in drug-resistant cell lines. In the figure, (A) shows the FTH1 protein degradation in sorafenib-sensitive and drug-resistant cell lines after treatment with the protein synthesis inhibitor cyclohexylimide (CHX, 20 μg / mL) for 0, 3, 6, 9, and 12 h. (B) shows the quantitative analysis of FTH1 protein degradation in sensitive and drug-resistant cell lines (ImageJ band grayscale analysis method), normalized with the initial 0 h as the baseline. (*: p < 0.05, **: p < 0.01, data from three replicate experiments.)

[0029] Figure 6 Knockdown of AURKA can restore FTH1 degradation in drug-resistant cell lines. In the figure, (A) shows the FTH1 protein degradation after AURKA knockdown in sensitive, drug-resistant, and drug-resistant cell lines (CHX, 20 μg / mL), as detected by Western blot. (B) shows the changes in ferrous ions after AURKA knockdown in sensitive, drug-resistant, and drug-resistant cell lines, as detected by Ferro Orange fluorescent probe, after no treatment with sorafenib (10 μM) or 2 h of treatment. HepG2: 40× objective; Huh7: 20× objective.

[0030] Figure 7 The results showed that overexpression of FTH1 could reverse ferroptosis induced by AURKA knockdown. In the figure, (AB) shows the ROS (A) and lipid peroxidation (B) levels in hepatocellular carcinoma cells after 48 h of AURKA knockdown and / or FTH1 overexpression, as detected by flow cytometry. (C) shows cell viability after 48 h of AURKA knockdown and / or FTH1 overexpression, as detected by CCK-8 assay. (D) shows the effect of AURKA knockdown on the protein expression levels of DMT1 (divalent metallotransferrin 1) and TFRC1 (transferrin receptor 1), as detected by Western blot. (E) shows the effect of AURKA knockdown on FTH1 mRNA expression, as detected by RT-qPCR. (ns: p≥0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, data from three replicate experiments, shown as mean ± standard deviation.)

[0031] Figure 8 The Co-IP results show that AURKA and FTH1 competitively bind to NCOA4. In the figure, (A) shows the effect of endogenous Co-IP on the binding ability of AURKA knockdown to NCOA4 and AURKA, FTH1 in HepG2 and Huh7 cells. (B) shows the change in the binding ability of NCOA4 and AURKA, FTH1 in HEK293T cells after co-transfection with HA-NCOA4, Myc-FTH1, and Flag-AURKA or Flag-VEC (empty vector) plasmids for 48 h in exogenous Co-IP experiments. (C) shows the effect of exogenous Co-IP on HEK293T cells co-transfected with HA-NCOA4. 383-522 After 48 hours, NCOA4 mutants, Myc-FTH1, and Flag-AURKA plasmids were used. 383-522 The binding status with AURKA and FTH1.

[0032] Figure 9The results show that AURKA inhibitors can enhance NCOA4-FTH1 binding and promote ferrophagy. In the figure, (A) shows the endogenous Co-IP analysis of the binding capacity of NCOA4 to AURKA and FTH1, the NCOA4 serine phosphorylation level, and the AURKA phosphorylation level in HepG2 and Huh7 cells after treatment with the AURKA selective inhibitor ALS (1.0 μM) or CD532 (200 nM) for 36 h. (B) shows the FTH1 protein expression level in liver cancer cells after treatment with different concentrations of ALS (0, 0.1, 0.5, 1.0 μM) for 36 h by Western blot. (C) shows the FTH1 protein expression level after treatment with different concentrations of CD532 (HepG2: 0, 50, 100, 200 nM; Huh7: 0, 100, 200, 500 nM) for 36 h by Western blot. (DE) represents the FTH1 protein expression level detected by Western blot after transfection with siNC or siNCOA4 for 24 h followed by treatment with ALS (1.0 μM) (D) or CD532 (200 nM) (E) for 36 h.

[0033] Figure 10 AURKA inhibitors can increase ferrous ion levels, thereby promoting ferroptosis. In the figure, (AB) shows the fluorescence intensity of the FerroOrange probe (Ex: 543nm / Em: 580nm) of hepatocellular carcinoma cells transfected with siNC or siNCOA4 for 24 h, and then treated with ALS (1.0μM) (A) or CD532 (200nM) (B) for 12 h (detected by a multi-mode microplate reader). (CD) shows the cell viability of hepatocellular carcinoma cells after pretreatment with DFO (40μM) for 12 h, and then treated with ALS (1.0μM) (C) or CD532 (200nM) (D) for 24 h (detected by CCK-8 assay). (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, data from three replicate experiments, shown as mean ± standard deviation.)

[0034] Figure 11 Mutations at serine sites on NCOA4 can alter its binding affinity to AURKA and FTH1. In the figure, (A) is a Venn diagram showing predictions of possible serine phosphorylation sites on NCOA4 from the UniProt and PhosphoSitePlus databases, revealing three common phosphorylation sites: Ser186, Ser234, and Ser492. (B) shows the results of an exogenous Co-IP experiment analyzing HEK293T cells co-transfected with Myc-FTH1, Flag-AURKA, and HA-NCOA4. WT Or the phosphorylation-inactivating mutant HA-NCOA4 S186A Or HA-NCOA4S234A HA-NCOA4 S492A The binding affinity of NCOA4 to AURKA and FTH1 and the level of serine phosphorylation of NCOA4 were determined after 48 hours of plasmid exposure.

[0035] Figure 12 The effect of combining an AURKA inhibitor with sorafenib on drug-resistant cells. In the figure, (AB) shows the lipid peroxidation level in sorafenib-resistant cells after 36 h of treatment with the AURKA inhibitor ALS (1.0 μM) (A) or CD532 (200 nM) (B) in combination with sorafenib (HepG2 SR: 8 μM, Huh7 SR: 6 μM) as detected by flow cytometry. (CD) shows the cell viability of drug-resistant cells after 48 h of treatment with ALS (1.0 μM) (C) or CD532 (200 nM) (D) in combination with sorafenib (HepG2 SR: 8 μM, Huh7 SR: 6 μM) and the ferroptosis inhibitor Fer-1 (5 μM) as detected by CCK-8 assay. (**: p < 0.01, ***: p < 0.001, ****: p < 0.0001, data from three replicate experiments, shown as mean ± standard deviation.)

[0036] Figure 13 The effect of combining AURKA inhibitors with RSL3 on drug-resistant cells. In the figure, (AB) shows the lipid peroxidation level in wild-type sensitive cells after 36 h of treatment with the AURKA inhibitor ALS (1.0 μM) (A) or CD532 (200 nM) (B) combined with RSL3 (HepG2: 0.5 μM, Huh7: 0.1 μM) as detected by flow cytometry. (CD) shows the cell viability of cells after 48 h of treatment with ALS (1.0 μM) (C) or CD532 (200 nM) (D) combined with RSL3 (HepG2: 0.5 μM, Huh7: 0.1 μM) and the ferroptosis inhibitor Fer-1 (5 μM) as detected by CCK-8 assay. (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, data from three replicate experiments, shown as mean ± standard deviation.)

[0037] Figure 14To construct and validate sorafenib-resistant cell lines. In the figure, (A) shows the cell proliferation activity of wild-type sensitive cells (WT) and drug-resistant cells (SR) after 48 h of treatment with different concentrations of sorafenib (Srf) as detected by the CCK-8 assay, where the HepG2 concentration gradient was 0, 1, 3, 6, 9, 12, 16, 20 μM, and the Huh7 concentration gradient was 0, 1, 2, 4, 8, 12, 16, 20 μM. (B) shows the colony formation assay to detect the colony formation of wild-type sensitive cells and drug-resistant cells after 72 h of treatment with the same concentration of sorafenib. (C) shows the colony formation area calculated using ImageJ software. (ns: P≥0.05, *: P<0.05, ***: P<0.001, data are expressed as mean ± standard deviation, three independent experiments per group.)

[0038] Figure 15 Iron death was inhibited in sorafenib-resistant cell lines. In the figure, (A) shows the fluorescence intensity of ROS labeled with the DCFH-DA probe (0.5 μM) in sensitive (WT) and resistant (SR) cells after treatment with gradient concentrations of Srf (sorafenib, HepG2: 0, 3, 6, 9, 12 μM; Huh7: 0, 2, 4, 6, 8 μM) for 36 h using fluorescence microscopy. (B) shows the changes in ROS levels in sensitive and resistant cells after treatment with different concentrations of sorafenib for 36 h using flow cytometry. (C) shows the fluorescence intensity of lipid peroxidation labeled with the BODIPY-C11 probe (1 μM) in sensitive and resistant cells after treatment with gradient concentrations of sorafenib (HepG2: 0, 3, 6, 9, 12 μM; Huh7: 0, 2, 4, 6, 8 μM) for 36 h using fluorescence microscopy. Green represents the oxidized state, and red represents the reduced state. (D) Flow cytometry was used to quantitatively analyze changes in lipid peroxidation levels in sensitive and resistant cells after treatment with different concentrations of sorafenib for 36 h. Flow cytometry results were normalized with the 0 μM group as a reference. (ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. Data were obtained from three independent replicates and are presented as mean ± standard deviation.)

[0039] Figure 16 This is the result of an in vivo experiment showing that the combination of an AURKA inhibitor and a ferroptosis inducer significantly inhibited tumor growth. In the figure, (A) is a schematic diagram of the experimental design: a subcutaneous tumorigenesis model of Huh7 cells, with 2 × 10^7 cells injected subcutaneously into each animal, 5 animals / group, on day 8 (tumor volume ≈ 100 mm). 3(A) Daily administration began on day 22. Dosing regimens were as follows: Control group: normal saline (daily gavage); ALS monotherapy group: 20 mg / kg (daily gavage); RSL3 monotherapy group: 20 mg / kg (daily intraperitoneal injection); ALS+RSL3 combination group: ALS 20 mg / kg (daily gavage) + RSL3 20 mg / kg (daily intraperitoneal injection); Srf (sorafenib) monotherapy group: 30 mg / kg (daily gavage); ALS+Srf combination group: ALS 20 mg / kg + Srf 30 mg / kg (daily gavage). (B) Tumor tissue excised from nude mice on day 22 after cell inoculation. (C) Changes in tumor growth volume in the six groups from day 8 to day 22 after cell inoculation (measured every 2 days, calculated using the formula: V = 0.5 × major axis × minor axis²). (D) Weight of tumors excised from nude mice on day 22 (in mg). (E) Changes in body weight of nude mice in the six groups from day 1 to day 20 of cell inoculation (measured and recorded every 4 days). (**: p < 0.01, ***: p < 0.001, ****: p < 0.0001, n = 5). Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0041] 1. Experimental Materials

[0042] 1.1 Liver cancer tissue samples

[0043] This study used 134 paraffin-embedded hepatocellular carcinoma (HCC) specimens for survival analysis. All specimens were obtained from the specimen bank of Sun Yat-sen University Cancer Center, collected between January 2005 and December 2013, and pathologically confirmed as HCC, excluding metastatic HCC and other malignant liver tumors. Prior to specimen collection, none of the patients had received surgical resection, interventional embolization, radiotherapy, chemotherapy, or molecular targeted therapy to ensure homogeneity of baseline treatment status. Patients signed written informed consent authorizing the use of their biosamples for scientific research. This study protocol was independently reviewed and approved by the Ethics Committee of Sun Yat-sen University Cancer Center (Ethics No.: SL-B2022-724-01).

[0044] The cell lines involved were: HepG2, Hep3B, and SNU449, which were kindly donated by Professor Chen Minshan of Sun Yat-sen University Cancer Center; Huh7 and PLC / PRF / 5 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai); and HEK293T was purchased from ATCC.

[0045] All reagents and kits used in the following experiments were commercially available.

[0046] Experimental Methods: All animal experimental protocols in this study adhered to the ethical guidelines for laboratory animal welfare and were approved by the Laboratory Animal Ethics Committee of Sun Yat-sen University Cancer Center (ethics number: L102042023050L). Four-week-old female BALB / c-Nude mice (SPF grade) were used in the experiment. These mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd., and housed in a barrier environment at the Animal Experiment Center of Sun Yat-sen University, with free access to irradiated sterilized feed and acidified drinking water throughout the experiment. A 7-day acclimatization period was implemented before the experiment to eliminate the effects of transportation stress.

[0047] The AURKA knockdown combined with sorafenib or RSL3 experimental protocol: Log-growing wild-type (WT) and AURKA knockdown (AURKA-KD) Huh7 cells were used. The cells were resuspended in PBS and counted. 2×10^7 cells were injected subcutaneously into each BALB / c-Nude mouse (Matrix gel:PBS cell suspension = 1:2). The mice were observed every 3 days after injection. Drug intervention was started when the tumor volume reached about 100 mm3. Mice that successfully developed the tumor model were randomly divided into six groups (n=5 / group): (1) WT cell + sorafenib treatment group (30mg / kg, daily gavage); (2) WT cell + RSL3 treatment group (20mg / kg, daily intraperitoneal injection); (3) WT cell + saline control group (equal volume gavage); (4) AURKA-KD cell + sorafenib treatment group; (5) AURKA-KD cell + RSL3 treatment group; (6) AURKA-KD cell + saline control group. The dosage and administration method of the latter three groups were the same as those of the first three groups. Intervention was performed at a fixed time every day for 15 consecutive days. The long diameter (L) and short diameter (W) of the tumor were measured and recorded every 2 days using an electronic vernier caliper, according to the formula V=0.5×L×W. 2 Tumor volume was calculated. Mice were euthanized by cervical dislocation on day 22 of subcutaneous tumor formation. The tumor tissue was completely dissected and its weight was recorded. The tumor tissue was then fixed with 4% paraformaldehyde for 24 hours, followed by paraffin embedding and sectioning for subsequent immunohistochemical staining.

[0048] AURKA inhibitor combined with sorafenib or RSL3 experimental protocol: Log-growing wild-type Huh7 cells were injected subcutaneously into BALB / c-Nude mice, with 2×10^7 cells injected per mouse (resuspended in matrix gel:PBS cell suspension at a ratio of 1:2). Observation was performed every 3 days after injection. Drug intervention was started when the tumor volume reached approximately 100 mm3. Mice that successfully developed the model were randomly divided into six groups (n=5 / group): (1) saline control group (gavage daily); (2) ALS monotherapy group (20mg / kg, gavage daily); (3) RSL3 monotherapy group (20mg / kg, intraperitoneal injection daily); (4) ALS+RSL3 combination group (ALS: 20mg / kg, gavage daily; RSL3: 20mg / kg, intraperitoneal injection daily); (5) Sorafenib monotherapy group (30mg / kg, gavage daily); (6) ALS+Sorafenib combination group (ALS: 20mg / kg + Sorafenib: 30mg / kg, gavage daily). Medication was administered at fixed times daily starting on day 8 after cell inoculation. Tumor volume was measured and recorded every 2 days, and mouse weight was measured and recorded every 4 days. On day 22 after inoculation, mice were euthanized by cervical dislocation, and the tumor tissue was completely dissected and its weight recorded.

[0049] Cell culture: The human hepatocellular carcinoma cell lines HepG2, Hep3B, SNU449, Huh7, and PLC / PRF / 5, the tool cell line HEK293T, and the drug-resistant cell lines HepG2 SR and Huh7 SR (wild-type cells induced by gradient sorafenib treatment) used in this study were all cultured in DMEM high-glucose medium as the basal medium, supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin antibiotics. They were cultured routinely in an incubator at a constant temperature of 37℃ and a concentration of 5% CO2.

[0050] In this invention, the nucleotide sequences of AURKA and NCOA4 knocked down are as follows:

[0051] siAURKA-1:5'-GGACCUGUUAAGGCUACAG-3';

[0052] siAURKA-2: 5'-CCAGCGCAUUCCUUUGCAA-3';

[0053] siNCOA4: 5'-UGAACAGGUGGACCUUAUUUA-3.

[0054] The primer pairs involved in this invention are as follows:

[0055]

[0056]

[0057] In this invention, primer synthesis and sequencing were outsourced to Guangzhou Ruibo Biotechnology Co., Ltd., and the experimental procedures involved in the examples, such as RT-qPCR, co-immunoprecipitation (Co-IP), and Western blot, were all routine procedures.

[0058] The drug-resistant cells and their construction method involved in this invention are as follows: HepG2 and Huh7 liver cancer cells were continuously induced using a gradient concentration escalation method. After approximately 6 months of continuous induction culture, sorafenib-resistant cell lines HepG2 SR and Huh7 SR were successfully constructed. The half-maximal inhibitory concentration (IC50) of sorafenib in the drug-resistant and parental sensitive cells was detected using the CCK-8 assay. The results showed that the IC50 of sorafenib in the drug-resistant cells was significantly higher than that in the sensitive cells. Specifically, the IC50 value of HepG2 SR increased from 7.11±0.9 μM in the parental cells to 15.06±1.49 μM, and the IC50 value of Huh7 SR increased from 4.56±0.44 μM in the parental cells to 11.66±1.21 μM. Figure 14 A). Further plating colony formation assays were used to verify the stability of the resistance phenotype. The results showed that, under the same concentration of sorafenib treatment, the colony formation ability of the susceptible strains was significantly inhibited, while the colony formation ability of the resistant strains was not significantly affected. Figure 14 These results (BC) demonstrate the successful construction of drug-resistant strains.

[0059] In this embodiment of the invention, drug-resistant and drug-sensitive strains were treated with different concentrations of sorafenib for 36 h. The DCFH-DA fluorescent probe and the BODIPY-C11 fluorescent probe were used to detect ferroptosis-related indicators: ROS level and lipid peroxidation level, respectively. Fluorescence microscopy and flow cytometry results showed that, compared with the drug-sensitive strain, the drug-resistant strain had a significantly lower ROS level after treatment with the same concentration of sorafenib. Figure 15 AB) and lipid peroxidation level ( Figure 15 No significant changes were observed in CD (corticotropic enzyme activity). This result confirms that ferroptosis is inhibited in sorafenib-resistant cells.

[0060] Example 1

[0061] This embodiment uses co-immunoprecipitation combined with mass spectrometry (Co-IP / MS) to detect proteins interacting with AURKA in wild-type sensitive Huh7 cells. After rigorous screening, approximately 2000 potential interacting proteins were identified, including known interacting proteins such as NPM1 and HNRNPK. KEGG enrichment analysis of the detected proteins revealed that AURKA can regulate the ferroptosis signaling pathway (…). Figure 1A). Analysis of mass spectrometry data revealed that the nuclear receptor coactivator 4 (NCOA4) has a potential binding affinity to AURKA. Figure 1 B). NCOA4, a key regulatory protein of ferritinophagy, mediates the autophagic degradation of ferritin by binding to the ferritin heavy chain FTH1, promoting the release of free ferrous ions in the cell, and thereby activating the ferroptosis cascade. Knockdown of NCOA4 can inhibit ferritin degradation, reduce the level of free ferrous ions, and thus inhibit ferroptosis.

[0062] Furthermore, in this embodiment, Co-IP and Western blot experiments were performed in HepG2 and Huh7 cells, and the results showed that AURKA interacts with NCOA4. Figure 2 A). Subsequently, HEK293T cells were transfected with Flag-labeled AURKA and HA-labeled NCOA4 plasmids, respectively, and the results also showed that the two could bind directly. Figure 2 B). Furthermore, immunofluorescence co-localization assays also showed that AURKA and NCOA co-localize within cells (B). Figure 2 CD), all of the above support the existence of a direct interaction between AURKA and NCOA4.

[0063] Further Western blot results showed that knocking down AURKA significantly reduced FTH1 protein expression levels. Figure 3 A) suggests that AURKA knockdown may promote ferrophagy. Further immunofluorescence colocalization analysis revealed that AURKA knockdown significantly increased the spatial colocalization of ferritin and the lysosomal marker LAMP1. Figure 3 B), and the above changes were reversed after NCOA4 gene silencing (siNCOA4). Figure 3 (AB) indicates that knocking down AURKA can significantly promote NCOA4-mediated ferrophagy.

[0064] Flow cytometry results showed that knockdown of AURKA-induced ROS accumulation and elevated lipid peroxidation levels could be effectively reversed by knockdown of NCOA4. Figure 4 (AB) This result indicates that AURKA inhibits ferroptosis in liver cancer cells by negatively regulating the NCOA4-mediated ferrophagy pathway.

[0065] This embodiment further investigated the degradation kinetics of FTH1 in drug-resistant and drug-sensitive cells and its effect on ferrous ions. The results showed that, compared to the sorafenib-sensitive cell line, the degradation rate of FTH1 protein in the drug-resistant line was significantly reduced. Figure 5(AB) suggests that ferroautophagy-mediated FTH1 degradation is inhibited in drug-resistant strains.

[0066] By specifically knocking down AURKA, which is highly expressed in drug-resistant strains, the degradation of FTH1 can be effectively accelerated. Figure 6 A), indicating that AURKA overexpression may be a key molecular mechanism for inhibiting ferrophagy. Further analysis using the FerroOrange fluorescent probe to detect intracellular free ferrous ions revealed significant ferrous ion accumulation in sensitive strains 2 hours after sorafenib treatment, while no significant change in ferrous ion levels was observed in resistant strains; however, in AURKA knockdown resistant strains, sorafenib could re-induce an increase in ferrous ion levels (A). Figure 6 B). The above multidimensional evidence suggests that AURKA inhibits the degradation of FTH1 mediated by the key autophagy protein NCOA4, thereby hindering the release of ferrous ions and ultimately leading to the formation of the ferroptosis resistance phenotype in drug-resistant strains.

[0067] To further clarify that AURKA inhibits ferroptosis by regulating the FTH1 protein, this invention validated this through gene function complementation experiments and multidimensional comparative analysis. First, FTH1 was overexpressed in AURKA-knockdown liver cancer cells. Flow cytometry analysis revealed that FTH1 overexpression could reverse the ROS (reactive oxygen species) induced by AURKA knockdown. Figure 7 A) and elevated lipid peroxidation levels ( Figure 7 B); CCK-8 assays showed that FTH1 overexpression could reverse the inhibitory effect of AURKA knockdown on cell growth. Figure 7 C) confirmed the specific regulatory role of the AURKA-FTH1 signaling axis in the ferroptosis phenotype. Further Western blot analysis revealed that knockdown of AURKA had no significant effect on the expression levels of the other two proteins involved in ferrous ion metabolism, DMT1 (divalent metallotransferrin 1) and TFRC1 (transferrin receptor 1). Figure 7 D). Furthermore, qRT-PCR results showed that AURKA knockdown had no significant effect on FTH1 mRNA levels. Figure 7 E), combined with previously observed changes in the FTH1 protein degradation rate ( Figure 6 A) indicates that AURKA maintains FTH1 protein stability by inhibiting the NCOA4-dependent ferrophagy pathway, rather than through transcriptional regulation, thereby reducing ferritin degradation and ferrous ion release, ultimately inhibiting ferroptosis. The following sections will analyze endogenous interactions, exogenous overexpression, and functional validation of truncated mutants.

[0068] This invention knocks down AURKA in liver cancer cells. Endogenous Co-IP experiments showed increased binding of NCOA4 to FTH1, while the NCOA4-AURKA interaction level decreased synchronously. Figure 8A) indicates that the binding of AURKA to NCOA4 directly regulates its binding ability to FTH1. To further verify this competitive effect, we exogenously co-transfected HEK293T cells with HA-NCOA4, Myc-FTH1, and Flag-AURKA plasmids. We found that AURKA overexpression significantly inhibited the binding of NCOA4-FTH1, while the interaction between NCOA4 and AURKA increased ( Figure 8 B). Since the binding of the 383-522 amino acid domains of NCOA4 to FTH1 is crucial, we constructed NCOA4... 383-522 Functional validation was performed on truncated mutants. HA-labeled NCOOA4 was co-transfected into HEK293T cells. 383 -522 The Co-IP results for FTH1 labeled with MYC and AURKA labeled with Flag show: NCOOA4 383-522 The molecular weight of the truncated mutation is approximately between 15-25 kDa. Figure 8 C), and the truncated form still retains the ability to bind to AURKA and FTH1. This result supports the interaction between AURKA and the 383-522 amino acid domain of NCOOA4, resulting in spatial overlap between this region and the FTH1 binding site.

[0069] The above multidimensional interaction studies show that AURKA competitively binds to the NCOOA4 and FTH1 interaction domains (aa383-522), forming a steric hindrance effect that interferes with the binding of NCOOA4 and FTH1, ultimately leading to the inhibition of ferrophagy and ferroptosis.

[0070] Example 2

[0071] This embodiment uses the AURKA selective inhibitors ALS (Alisertib) and CD532 for functional intervention. Endogenous Co-IP experiments showed that ALS and CD532 treatment significantly reduced AURKA autophosphorylation levels and simultaneously decreased NCOA4 serine phosphorylation levels, while also reducing the binding of NCOA4 and AURKA and promoting NCOA4-FTH1 interaction. Figure 9 A), this result supports the view that AURKA competitively binds to NCOOA4 with FTH1, which depends on its kinase activity. Western blot analysis further showed that ALS and CD532 treatment reduced FTH1 protein levels (A). Figure 9 BC), and the above effects can be reversed by knocking down NCOA4 (BC). Figure 9 DE). This further verified that AURKA regulates the phosphorylation state of NCOA4 through kinase activity, thereby affecting its binding to FTH1 and inhibiting ferrophagy.

[0072] Further detection using ferrous ion-specific probes showed that inhibitor treatment significantly increased the level of free ferrous ions in liver cancer cells, and this effect could be reversed after NCOA4 silencing. Figure 10 (AB) indicates that AURKA inhibitors can promote ferrous ion release by activating NCOA4-dependent ferrophagy. Notably, treatment with the iron chelator DFO significantly reversed the inhibitory effect of ALS or CD532 on the proliferation of liver cancer cells. Figure 10 CD). The above results further confirm that AURKA regulates iron metabolism (rather than other pathways) in a kinase-dependent manner, thereby mediating the inhibition of ferroptosis.

[0073] Example 3

[0074] Using bioinformatics prediction and point mutation functional analysis, we systematically elucidated the AURKA-mediated NCOA4 phosphorylation sites and their molecular regulatory mechanisms. First, by integrating phosphoproteomics data from the UniProt website and the PhosphoSitePlus database, three potential NCOA4 serine phosphorylation sites were screened: Ser186, Ser234, and Ser492. Figure 11 A). To further determine the impact of these three phosphorylation sites, we performed point mutations at these three serine sites to construct serine→alanine (S→A) phosphorylation inactivation mutants: S186A, S234A, and S492A. Co-IP results showed that the mutations at the S186A, S234A, and S492A sites significantly reduced the serine phosphorylation level of NCOA4, while also reducing the binding of NCOA4 and AURKA and significantly restoring the binding of NCOA4-FTH1. Figure 11 B). The above results demonstrate that AURKA mediates phosphorylation modification of key serine sites (Ser186 / 234 / 492) of NCOA4 in a kinase-dependent manner, competitively inhibiting NCOA4-FTH1 binding through steric hindrance, thereby suppressing ferritin autophagy, reducing ferrous ion release, and antagonizing Fenton-dependent ferroptosis. This discovery provides an important theoretical basis for targeting AURKA-ferrophagy in the treatment of liver cancer and a key theoretical basis for developing small molecule inhibitors (such as competitive peptides or allosteric modulators) targeting NCOA4 phosphorylation sites.

[0075] Example 4

[0076] In this embodiment, sorafenib-resistant cells were treated with a combination of the selective AURKA inhibitor ALS or CD532 and sorafenib. The results showed that the level of lipid peroxidation induced by sorafenib was significantly increased. Figure 12AB). Further CCK-8 experiments showed that both ALS and CD532 significantly enhanced the sensitivity of resistant strains to sorafenib, and this effect could be reversed by the ferroptosis inhibitor Fer-1 (AB). Figure 12 These results demonstrate that AURKA inhibitors significantly improve the sensitivity of resistant cells to sorafenib by enhancing ferroptosis.

[0077] Further treatment with sorafenib-resistant cells using the selective AURKA inhibitors ALS or CD532 in combination with RSL3 showed that both ALS and CD532 significantly enhanced RSL3-induced lipid peroxidation levels. Figure 13 AB). CCK-8 assay results confirmed that the combined group significantly enhanced the inhibitory effect on cell proliferation compared to the RSL3 monotherapy group. Figure 13 CD), and this effect can also be significantly reversed by Fer-1, supporting the idea that AURKA inhibitors combined with ferroptosis inducers can produce better anti-tumor effects.

[0078] Example 5

[0079] To evaluate the clinical translational potential of an AURKA inhibitor combined with a ferroptosis inducer, this embodiment established a Huh7 cell subcutaneous tumorigenesis model in nude mice (n=5 / group), and the tumor volume was allowed to grow to approximately 100 mm. 3 (On day 8 post-inoculation), tumor-bearing mice were randomly divided into the following 6 groups: saline control group (daily gavage), ALS monotherapy group (20 mg / kg, daily gavage), RSL3 monotherapy group (20 mg / kg, daily intraperitoneal injection), ALS+RSL3 combination group (ALS: 20 mg / kg, daily gavage; RSL3: 20 mg / kg, daily intraperitoneal injection), sorafenib monotherapy group (30 mg / kg, daily gavage), and ALS+sorafenib combination group (ALS: 20 mg / kg + sorafenib: 30 mg / kg, daily gavage). Mice were sacrificed on day 22. Figure 16 A). Experimental results showed that the tumor growth rate in the ALS combined with RSL3 or ALS combined with sorafenib treatment groups was significantly slower than that in the RSL3 monotherapy group or the sorafenib monotherapy group. Figure 16 BD). Furthermore, compared to the single-drug groups, there was no significant difference in body weight change in mice in the ALS combined with RSL3 or sorafenib groups. Figure 16 E) indicates that the combination therapy regimen has a good safety window.

[0080] The above preclinical in vitro and in vivo experimental data confirm that the combination of AURKA selective inhibitors and ferroptosis inducers exhibits synergistic antitumor effects in both chemotherapy-resistant and chemotherapy-sensitive liver cancer models.

[0081] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0082] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of AURKA-NCOA4 interaction inhibitors in the preparation of drugs for reversing ferroptosis resistance.

2. The use as described in claim 1, characterized in that, The inhibitor promotes the degradation of FTH1 protein by blocking the binding of AURKA protein to NCOA4 protein.

3. The use as described in claim 1, characterized in that, The inhibitor blocks the binding of AURKA protein to NCOOA4 protein by targeting the Ser186, Ser234, or Ser492 phosphorylation sites of NCOOA4.

4. The use as described in claim 1, characterized in that, The inhibitor is a selective inhibitor of AURKA.

5. The use as described in claim 4, characterized in that, The inhibitor is either Alisertib or CD532.

6. The use as described in claim 5, characterized in that, The drug is used to treat sorafenib-resistant liver cancer.

7. Use of AURKA selective inhibitors in the preparation of medicaments for enhancing ferroptosis sensitivity in hepatocellular carcinoma cells, wherein said ferroptosis sensitivity is achieved by promoting the degradation of FTH1 protein.

8. A pharmaceutical composition for treating liver cancer, characterized in that, Containing an AURKA-NCOA4 interaction inhibitor, the pharmaceutical composition is able to reverse ferroptosis resistance.

9. The pharmaceutical composition according to claim 8, characterized in that, It contains a selective inhibitor of AURKA.

10. The pharmaceutical composition according to claim 8, characterized in that, It also contains ferroptosis inducers.

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