Application of CCN1 in preparation of medicine for enhancing ferroptosis sensitivity of tumor cells

Abstract

The invention relates to the technical field of biomedicine, in particular to application of CCN1 in preparation of a medicine for enhancing ferroptosis sensitivity of tumor cells. According to the application disclosed by the invention, the correlation between the CCN1 and the ferroptosis sensitivity of the tumor cells is defined, the CCN1 moves downwards under the induction of ferroptosis and enters mitochondria to participate in fatty acid oxidative metabolism, and the tumor cells of the CCN1 with mitochondrial localization are higher in ferroptosis sensitivity; and the high fatty acid can promote the anti-cancer effect of ferroptosis induced by CCN1. The invention focuses on signal transduction of mitochondrial ROS and determines a novel ferroptosis sensitivity regulatory factor CCN1; the invention reveals that the sensitivity of tumor cells to ferroptosis can be enhanced by enabling CCN1 to shift into mitochondria to participate in fatty acid oxidative metabolism under ferroptosis induction, and clarifies a new signal channel, and the ferroptosis-based tumor treatment effect can be enhanced by targeting the channel.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to application of CCN1 in preparing a drug for enhancing the ferroptosis sensitivity of tumor cells. Background Art

[0002] Ferroptosis is a new type of programmed death for tumor cells with RAS mutations proposed and named by Stockwell in 2012. Its main characteristics are lipid peroxidation overload caused by iron ion overload and unsaturated fatty acid peroxidation. Excessive lipid peroxidation products can lead to the destruction of cell biomembrane structure, thereby causing cell death (Dixon SJ et al., Ferroptosis: an iron-dependent form of nonapoptotic Cell death, 2012; Stockwell BR et al., Emerging mechanisms, physiological functions, and therapeutic applications, 2022). However, the signaling pathways involved in ferroptosis show significant heterogeneity and adaptability (Dai E et al., A guideline on the molecular ecosystem regulating ferroptosis, 2024). Classical ferroptosis can be induced and activated by small molecule compounds such as erastin and RSL3, among which erastin and RSL3 inhibit the antioxidant pathway by targeting carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), respectively (Dixon SJ et al., Ferroptosis: an iron-dependent form of nonapoptotic Cell death, 2012; Yang WS et al., Regulation of ferroptotic Cancer Cell death by GPX4, 2014). Currently, ferroptosis has become a promising therapeutic strategy for killing drug-resistant tumor cells (Hangauer M J et al., Drug-tolerant persister Cancer Cells are vulnerable to GPX4 inhibition, 2017; Viswanathan VS et al., Dependency of a therapy-resistant state of Cancer Cells on a lipid peroxidase pathway, 2017).In addition, ferroptosis activators can enhance the efficacy of immunotherapy and radiotherapy in various tumors through synergistic effects (Wang W et al., CD8(+)T Cells regulate tumor ferroptosis during Cancer immunotherapy, 2019; Li G et al., The role of ferroptosis in ionizing radiation-induced Cell death and tumor suppression, 2020).

[0003] Therefore, exploring new ferroptosis regulators is of great significance for advancing ferroptosis-based treatments. Summary of the invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide an application of CCN1 in the preparation of a drug for enhancing the sensitivity of tumor cell ferroptosis. Cell communication network factor 1 (CCN1) belongs to the CCN family of matricellular proteins. CCN1 in the matrix can participate in various biological processes such as cell adhesion, proliferation and apoptosis by interacting with membrane proteins such as integrins and heparan sulfate proteoglycans, thereby playing different roles in different types of tumors (Jun JI et al., The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in Cutaneous wound healing, 2012; Xie L et al., Aberrant activation of CYR61 enhancers in Colorectal Cancer development, 2019; K et al., Regulation of CyR61expression and release by 3-mercaptopyruvate sulfurtransferase in ColonCancer Cells, 2022). In addition, CCN1 has been shown to be involved in oxidative stress regulation and lipid metabolism (Wang Z et al., Extracellular vesicles in fatty liver promote a metastatic tumormicroenvironment, 2023;

[0005] Mooring M et al., Hepatocyte CYR61 polarizes profibrotic macrophages to orchestrate NASH fibrosis, 2023). The present invention clarifies the correlation between CCN1 and the sensitivity of tumor cells to ferroptosis. CCN1 is translocated into mitochondria under ferroptosis induction to participate in fatty acid β-oxidation (FAO) metabolism. Tumor cells with mitochondrial-localized CCN1 are more sensitive to ferroptosis; and high fatty acids can promote the anti-cancer effect of CCN1-induced ferroptosis. The present invention focuses on the signal transduction of mitochondrial ROS and identifies a new ferroptosis sensitivity regulator CCN1; it reveals that the translocation of CCN1 into mitochondria under ferroptosis induction to participate in FAO metabolism can enhance the sensitivity of tumor cells to ferroptosis, and clarifies a new signaling pathway that can enhance the effect of ferroptosis-based tumor treatment by targeting this pathway.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide an application of CCN1 in the preparation of a drug for enhancing the ferroptosis sensitivity of tumor cells, wherein the amino acid sequence of CCN1 is shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the drug is a drug that inhibits the growth of tumor cells by enhancing the ferroptosis sensitivity of tumor cells.

[0009] In one embodiment of the present invention, the drug is a drug that enhances the ferroptosis sensitivity of tumor cells through mitochondrial translocation of CCN1.

[0010] In one embodiment of the present invention, CCN1 achieves mitochondrial translocation via a mitochondrial targeting sequence;

[0011] The nucleotide sequence of the mitochondrial targeting sequence is shown in SEQ ID NO.2.

[0012] In one embodiment of the present invention, the drug is a drug that high fatty acids cooperate with mitochondrial translocation of CCN1 to enhance the ferroptosis sensitivity of tumor cells.

[0013] In one embodiment of the present invention, the drug is a drug that promotes fatty acid oxidation metabolism through mitochondrial translocation of CCN1 to enhance the ferroptosis sensitivity of tumor cells.

[0014] In one embodiment of the present invention, the drug contains a ferroptosis inducer.

[0015] In one embodiment of the present invention, the ferroptosis inducer is selected from one or both of RSL3 and erastin.

[0016] In one embodiment of the present invention, the drug contains IKE and / or cisplatin.

[0017] In one embodiment of the present invention, the tumor cells are lung adenocarcinoma cells or lung squamous cell carcinoma cells with high fatty acid and / or triglyceride levels (tumor cells with high fatty acid or triglyceride levels have higher sensitivity to ferroptosis).

[0018] In one embodiment of the present invention, H1299 cells were treated with RSL3 and erastin, and the expression of CCN1 was enhanced as the action time prolonged.

[0019] In one embodiment of the present invention, after CCN1 is knocked out, IKE has no effect of inhibiting tumor growth and inducing lipid peroxidation; after CCN1 is overexpressed, the effects of IKE on inhibiting tumor growth and inducing lipid peroxidation are enhanced.

[0020] In one embodiment of the present invention, after H1299 cells were treated with RSL3 and erastin to induce ferroptosis, CCN1 was translocated into mitochondria.

[0021] In one embodiment of the present invention, for H1299 cells in which CCN1 is knocked out, overexpression of mitochondrial-localized CCN1 can enhance the ferroptosis sensitivity of tumor cells.

[0022] In one embodiment of the present invention, palmitic acid is a common substrate for fatty acid oxidation; the simultaneous use of palmitic acid in cells treated with RSL3 and erastin promotes tumor cell ferroptosis.

[0023] Treatment of tumor cells with the fatty acid oxidation inhibitor ETO weakened the ferroptosis sensitivity of tumor cells.

[0024] Co-treatment of IKE, CDDP and high fatty acids inhibited tumor growth.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention focuses on the signal transduction of mitochondrial ROS and identifies a new ferroptosis sensitivity regulator CCN1; it reveals that the translocation of CCN1 into mitochondria under ferroptosis induction to participate in FAO metabolism can enhance the sensitivity of tumor cells to ferroptosis, and clarifies a new signaling pathway that can enhance the effect of ferroptosis-based tumor treatment by targeting this pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the screening process for candidate molecules that promote ferroptosis;

[0028] Figure 2 A is a schematic diagram of the results of candidate molecules that promote ferroptosis; Figure 2 B is the final confirmation diagram of multi-omics screening for ferroptosis-promoting screening factors;

[0029] Figure 3 A is the relationship between CCN1 protein expression and drug concentration after ferroptosis induction; Figure 3 B is a graph showing the changes in CCN1 protein expression over time after ferroptosis induction;

[0030] Figure 4 This is a diagram for efficiency verification of CCN1 gene knockout or overexpression;

[0031] Figure 5 A is a diagram showing the changes in cell ferroptosis sensitivity after knocking out CCN1; Figure 5 B is a graph showing changes in lipid peroxidation levels after cell ferroptosis induction after CCN1 knockout;

[0032] Figure 6 A is a diagram showing changes in cell ferroptosis sensitivity after overexpression of CCN1; Figure 6 B is a graph showing changes in lipid peroxidation levels after ferroptosis induction in cells overexpressing CCN1;

[0033] Figure 7 A is Ccn1 + / + 、Ccn1 + / - 、Ccn1 - / - Mouse embryo morphology and Ccn1 protein expression; Figure 7 B is a graph of MDA levels in mouse embryos after ferroptosis induction; Figure 7 C is a graph showing the expression of 4-HNE in mouse embryos after ferroptosis induction; Figure 7 D is the effect of CCN1 level on 4-HNE level after ferroptosis induction in CDX tumors; Figure 7 E is the effect of CCN1 level on MDA level after ferroptosis induction in CDX tumors;

[0034] Figure 8 Schematic diagram of immunoelectron microscopy showing the sublocalization changes of CCN1 after ferroptosis induction, scale bar: 200 nm;

[0035] Fig. 9 Schematic diagram of immunofluorescence showing the changes in CCN1 sublocalization after ferroptosis induction, scale bar: 5 μm;

[0036] Fig.10 Schematic diagram of the changes in CCN1 sublocalization with ferroptosis induction time;

[0037] Fig.11Schematic diagram of the change of CCN1 sublocalization with ferroptosis induction concentration;

[0038] Fig.12 The WB schematic diagram of the sublocalization changes of endogenous CCN1 after ferroptosis induction;

[0039] Fig.13 Schematic diagram of immunofluorescence showing the sublocalization changes of exogenous CCN1 after ferroptosis induction, scale bar: 5 μm;

[0040] Fig.14 Schematic diagram for construction of CCN1 exogenous expression vector;

[0041] Fig.15 This is a diagram showing the effect of MTS sequence changes on sub-localization changes after exogenous CCN1 ferroptosis induction;

[0042] Fig.16 The effect of sub-localization changes of CCN1 on cell ferroptosis sensitivity;

[0043] Fig.17 Schematic diagram of screening candidate proteins for mitochondrial localization of CCN1 interactions;

[0044] Fig.18 Schematic diagram for the identification of CCN1-interacting proteins for mitochondrial localization;

[0045] Fig.19 Schematic diagram of the changes in the interaction between CCN1 and ETFA after ferroptosis induction, scale bar: 5 μm;

[0046] Fig. 20 A is the efficiency verification diagram of CCN1 overexpression and ETFA knockout; Fig. 20 B is the effect of ETFA knockout on the ferroptosis sensitivity of cells overexpressing CCN1;

[0047] Fig.21 The lipid metabolite screening and KEGG pathway analysis diagram for CCN1 knockout after ferroptosis induction;

[0048] Fig. 22 This is a diagram showing the changes in the expression of fatty acids and acylcarnitines affected by CCN1 knockout after ferroptosis induction;

[0049] Fig.23 Schematic diagram of the isotope labeled metabolic flux pathway;

[0050] Fig.24 This is a diagram showing the effect of CCN1 knockout on the levels of FAO pathway metabolites after ferroptosis induction;

[0051] Fig.25 A is a graph showing the changes of FADH2:FAD over time after RSL3 induction; Fig.25B is a graph showing the changes of FADH2:FAD over time after erastin induction;

[0052] Fig.26 A is a graph showing the effect of CCN1 knockout on the sensitivity of cell ferroptosis to increased fatty acids; Fig.26 B is the effect of FAO inhibitor on the sensitivity of cell ferroptosis increased by fatty acids;

[0053] Fig. 27 This is a diagram to verify the efficiency of gene knockout in the FAO pathway;

[0054] Fig.28 The figure shows the effect of different protein knockouts on ferroptosis-induced FAO activation;

[0055] Fig.29 The figure shows the effect of CCN1 and CPT2 knockout on the conversion of CoA consumption to acyl-CoA;

[0056] Fig.30 This is a graph showing the effects of CCN1 and CPT2 knockout on FAO-enhanced ferroptosis sensitivity;

[0057] Fig.31 This is a diagram showing the changes in protein localization in the mitochondrial complex after ferroptosis induction;

[0058] Fig.32 Schematic diagram of CDX mouse modeling and treatment;

[0059] Fig.33 Graph showing the effects of different treatments on CDX tumor volume;

[0060] Fig.34 Schematic diagram of treatment of lung cancer metastasis mouse model;

[0061] Fig.35 This is a graph showing the effects of different feeds and treatments on the weight changes of mice;

[0062] Fig.36 This is a graph showing the effects of different treatments on lung metastatic cancer, scale: 2 mm;

[0063] Fig.37 A is the effect of different treatments on 4-HNE expression in lung metastatic cancer tissues; Fig.37 B is the effect of different treatments on MDA levels in lung metastatic cancer tissues; Fig.37 C is a graph showing the effects of different treatments on PDX tumor volume;

[0064] Fig.38 A is a graph showing the effects of different treatments on CDX tumor volume and CCN1 expression; Fig.38 B is a graph showing the BMI index of different patients; Fig.38C is the effect of BMI index on the sensitivity of primary lung cancer cells to ferroptosis; Fig.38 D is the effect of BMI index on FAO rate of primary lung cancer cells;

[0065] Fig.39 A is a schematic diagram of serum triglyceride concentrations in different patients; Fig.39 B is the effect of serum triglyceride concentration on the ferroptosis sensitivity of primary lung cancer cells within the same BMI index range; Fig.39 C is a schematic diagram of serum triglyceride concentrations at different BMI indices; Fig.39 D is the effect of similar serum triglyceride concentrations on the ferroptosis sensitivity of primary lung cancer cells;

[0066] Fig.40 This is a diagram showing the expression of CCN1 in lung cancer tissue microarray;

[0067] Fig.41 This is the correlation diagram between CCN1 and serum triglycerides in lung cancer tissue microarray;

[0068] Fig.42 The graph shows the expression and sublocalization of CCN1 protein in primary lung cancer cells at different serum triglyceride concentrations;

[0069] Fig.43 The figure shows the effect of different fatty acid treatments on CCN1 protein expression;

[0070] Among them, NS represents no significant difference, * represents P < 0.05, and ** represents P < 0.01. DETAILED DESCRIPTION

[0071] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0072] In the following examples, H1299, A549, HEK293T and LLC cell lines were purchased from Shanghai Fuheng Biotechnology Co., Ltd.; patient-derived primary lung cancer cells were isolated and cultured from fresh lung cancer tissues, and the cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0073] The reagents used in the cell and animal experiments were: DMSO (D1435, Sigma-Aldrich), ferrostatin-1 (Fer-1, SML0583, Sigma-Aldrich), deferoxamine (DFO, 1166003, Sigma-Aldrich), palmitic acid (P9767, Sigma-Aldrich), L-carnitine (C0158, Sigma-Aldrich), linolenic acid (L2376, Sigma-Aldrich), linoleic acid (L 1012, Sigma-Aldrich), palmitoylcarnitine (61251, Selleck), RSL3 (S8155, Selleck), erastin (S7242, Selleck), etomoxir (ETO, HY-50202A, MCE), arachidonic acid (HY-109590), IKE (S8877, Selleck), cisplatin (CDDP, HY-17394, MCE), doxycycline (HY-N0565, MCE) and vitamin E (HY-N0683, MCE). Cytoplasm and mitochondria were isolated using a mitochondrial extraction kit (SM0020, Solebo). Lenti CRISPR v2-based plasmid constructs were used to knock out CCN1: CCN1 KO1 plasmid and CCN1 KO2 plasmid. The pLVX plasmid was used to construct a plasmid expressing CCN1. Others are commercially available unless otherwise specified; the detection methods are conventional detection methods in the art unless otherwise specified.

[0074] In the present invention, the amino acid sequence of CCN1 is shown in SEQ ID NO.1, which is as follows:

[0075] MSSRIARALALVVTLLHLTRL

[0076] Example 1

[0077] This example provides an experimental method for using CCN1 as a target for improving the anti-tumor effect of ferroptosis.

[0078] (1) Animal experiments

[0079] Since CCN1 homozygous is lethal, to evaluate the role of CCN1 in ferroptosis, Ccn1 + / - Mice were crossbred together, and IKE (50 mg / kg) was intraperitoneally injected into female mice at day 16 of gestation for three consecutive days. Female mice at day 19 of gestation were euthanized and mouse embryos were isolated and cultured. Mouse embryonic fibroblasts (MEFs) were isolated and cultured, and genotype identification was performed at the same time. Ccn1 + / + and Ccn1 - / - Lipid peroxidation levels in mouse embryos.

[0080] To evaluate the effect of high-fat diet on IKE enhancement, C57BL / 6 (Jicui Yaokang) mice were divided into two groups, one group was fed with normal diet and the other group was fed with high-fat diet. After 8 weeks of continuous feeding, each mouse was subcutaneously injected with 2×10 6 LLC cells were used to construct a cell-derived xenograft (CDX) mouse model. Five days after cell injection, mice were intraperitoneally injected with IKE (50 mg / kg) every day, and vitamin E (2 mg / kg) or ETO (15 mg / kg) was injected intraperitoneally every two days for two weeks. 2 The tumor volume (L represents length and W represents width) was calculated.

[0081] To elucidate the importance of CCN1 in IKE-based tumor therapy, 1×10 7 A subcutaneous cell-derived xenograft (CDX) mouse model was constructed by inoculating wild-type and CCN1-overexpressing A549 cells in nude mice. Five days after cell injection, mice were intraperitoneally injected with IKE (50 mg / kg) every day for two weeks. Tumor tissues were collected after mice were euthanized at the end point.

[0082] To elucidate the importance of CCN1 and high-fat diet in cisplatin-based lung cancer therapy, C57BL / 6 (Jicui Yaokang) mice were divided into two groups, one group was fed with normal diet and the other group was fed with high-fat diet. After 8 weeks of continuous feeding, each mouse was injected with 1×10 5 LLC cells stably transfected with Dox-inducible CCN1 knockdown plasmid (Vision Bio, BVSH061406) were used to construct a mouse lung cancer metastasis model. At the same time, doxycycline (1 mg / mL) was added to the drinking water of the mice. One week after the injection of cells, the mice were intraperitoneally injected with cisplatin (5 mg / kg) twice a week for two weeks. At the end point, the mice were euthanized and the mouse lung tissues were collected. In order to construct a patient-derived xenograft (PDX) model, fresh lung cancer tissues were cut into 2-3 mm 3 Small pieces were implanted subcutaneously in NSG mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ, SBEFO) once the tumors reached a size larger than 1 cm 3 The size of the tumor was determined and the tumor was resected and implanted into the second generation NSG mice. The subcutaneous tumor tissue of the second generation mice was implanted into the third generation NSG mice fed with NCD or HFD for eight weeks, and cisplatin (5 mg / kg) was injected intraperitoneally twice a week. The relative growth rate of the tumor was calculated based on the tumor volume (expressed as % of the treatment group / control group).

[0083] To elucidate the importance of CCN1 in IKE-mediated cisplatin therapy, nude mice (BALB / c-nu, Spafol) were injected subcutaneously with 1×10 7 H1299 cells were used to construct a subcutaneous CDX model in nude mice. IKE (50 mg / kg), CDDP (5 mg / kg), and IKE (50 mg / kg) combined with CDDP (5 mg / kg) were injected intraperitoneally into mice, respectively. The mice were euthanized at the endpoint and the tumor tissues were collected.

[0084] All the above mouse experiments were approved by the Ethics Committee of Shanghai Chest Hospital.

[0085] (2) Western blotting (WB)

[0086] Immunoblotting was performed according to standard protocols. Proteins were extracted from whole cell lysates using Western and IP lysis buffer (Beyotime, P0013). For tissue proteins, tissues were mixed with Western and IP lysis buffer (Beyotime, P0013), homogenized on ice using a high-throughput tissue grinder (Wonbio), and then centrifuged. After boiling at 100°C for 10 min, proteins were separated by SDS-PAGE (10%) and transferred to polyvinylidene fluoride membranes (PVDF; Pall Life Sciences, BSP0161). They were then blocked with 5% skim milk powder (Beyotime, P0216) for 1 h. PVDF membranes were incubated with primary antibodies overnight at 4°C. The main antibodies include anti-CCN1 (Abcam, Ab230947 or Proteintech, 26689-1-AP), anti-GPX4 (Abcam, ab125066), anti-TUBB / β-tubulin (Servicebio, GB15140), anti-COX4 / COX IV (Cell Signaling Technology, 11967), anti-GOLM1 / GP73 (Proteintech, 15126-1-AP), anti-LMNB1 (Abcam, ab16048), anti-HA (Cell Signaling Technology, 3724), anti-TOMM20 (Cell Signaling Technology, 42406), anti-CPT1A (Proteintech, 15181-1-AP), anti-CPT2 (Proteintech, 26555-1-AP), anti-ACTB / β-actin (Servicebio, GB15001), anti-4-hydroxyenal (4-HNE, Abcam ab48506). After washing three times in PBS containing 0.1% tween 20, the membrane was incubated with secondary antibody (Cell Signaling Technology, 70074 or 7076) at room temperature for 1 hour. The bands were exposed using an enhanced chemiluminescence kit (Life-iLab, AP34L025).

[0087] (3) Immunofluorescence (IF)

[0088] Cells were cultured on 14 mm cell slides (NEST Biotechnology, 801010). They were fixed with paraformaldehyde fixative (Servicebio, G1101) for 20 minutes, permeabilized with 0.1% Triton X-100 (Solarbio, T8200) for 10 minutes, blocked with 1% goat serum (Boster, AR1009), and incubated with primary antibodies at 4°C overnight. The primary antibodies used for immunofluorescence were anti-CCN1 (Proteintech, 26689-1-AP), anti-COX4 (Cell Signaling Technology, 1967), anti-PDIA4 (Proteintech, 14712-1-AP), and anti-HA (Cell Signaling Technology, 3724 or Absin, abs137963). Alexa fluorescent conjugated secondary antibodies (Abcam, ab150077, ab150078, ab150113, ab150114) were used for incubation. DAPI (Servicebio, G1012) was used for nucleus staining. Images were acquired using a Keyence microscope system, and colocalization was detected using ImageJ software.

[0089] (4) Immunohistochemistry (IHC) and hematoxylin-eosin staining (H&E)

[0090] The tissues fixed with paraformaldehyde were embedded in paraffin and cut into 5 μm sections for immunohistochemical detection. After dewaxing and hydration, antigen retrieval was performed with sodium citrate antigen retrieval solution (Beyotime, P0081), and endogenous peroxidase was inactivated with endogenous peroxidase blocker (Beyotime, P0100A). Then, the sections were blocked with 5% goat serum (Boster, AR1009) and 1% bovine serum albumin (Sigma-Aldrich, 9048-46-8), and the primary antibody was incubated at 4°C overnight. The primary antibody used was anti-ccn1 antibody (Abcam, ab230947). DAB development kit (Absin, abs9210) was used for staining, and hematoxylin (Servicebio, G1001) was used for counterstaining. Hematoxylin-eosin (H&E) dye was purchased from Sevier (G1001 and G1004). The slides were stained with hematoxylin for 3 min, differentiated with hydrochloric acid and alcohol, and then soaked in water to turn blue. The slides were stained with eosin for 3 min, and then dehydrated and sealed.

[0091] (5) Proximity Ligation (PLA)

[0092] Cells were cultured on 14 mm cell slides (NEST Biotechnology, 801010). They were then fixed in paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes. After washing three times with PBS, the PLA experiment was performed using the Duolink in situ kit (Sigma-Aldrich, DUO92101), strictly following the instructions. To stain mitochondria, after the last amplification step of PLA, the slides were first washed twice with wash buffer B in the kit for 10 minutes each, and then washed once with wash buffer A for 1 minute each. The slides were then incubated with anti-COX4 antibody (Cell Signaling Technology, 1967) at 37°C for 40 minutes. Washed twice with wash buffer A for 2 minutes each, and incubated with Alexa Fluor 488-conjugated fluorescent secondary antibody (Abcam, ab150113) at 37°C for 40 minutes. Finally, the slides were washed again with wash buffer A and 0.01× wash buffer B, and nuclear staining and sealing were performed with a mounting medium containing DAPI. The primary antibodies used in this experiment included anti-CCN1 (Abcam, ab230947 or Santa Cruz Biotechnology, sc-374129) and anti-ETFA (Proteintech, 67661-1-Ig). All images were taken using a Keyence microscope system, and colocalization was detected using ImageJ software.

[0093] (6) Flow cytometry detection of cell death, lipid peroxidation and mitochondrial reactive oxygen species

[0094] Cells were stained with SYTOX Green (KeyGEN, KGA261), C11-BODIPY 581 / 591 (Invitrogen, D3861) or MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher Scientific, M36008) diluted in PBS for 15 min at 37°C. After washing three times with PBS, cells were resuspended in PBS and analyzed using BD FACSCanto II TM Flow cytometry was used for quantification and the results were analyzed using FlowJo v10.4 software.

[0095] (7) Cell viability assay

[0096] Cell viability was determined using the CellTiter-Glo luminescent cell viability assay (Promega, G7572). Cells were seeded in an opaque 96-well white plate (Jingan Bio, J09601) at a density of 8,000 cells per well. The culture plate was equilibrated at room temperature for 30 minutes, and then an equal volume of CellTiter-Glo reagent and cell culture medium were added to each well, mixed on a horizontal shaker for 2 minutes, and incubated at room temperature for 10 minutes. The luminescent signal was recorded using a BioTek Synergy H1 multimode reader (Agilent).

[0097] (8) MDA level detection

[0098] The level of MDA in tissues was detected using a kit (BC0025) from Solebio. Before using the kit, the tissues were ground on ice using a high-throughput tissue grinder (Wonbio).

[0099] (9) Enzyme-linked immunosorbent assay (ELISA)

[0100] The levels of FAD and FADH2 were determined by enzyme-linked immunosorbent assay (ELISA), and the kits were purchased from Mlbio (YJ624750 and YJ114780). The cells were resuspended in PBS and repeatedly frozen and thawed, and the supernatant was collected for detection after centrifugation at 1000g for 15 minutes.

[0101] (10) Immunoelectron microscopy (TEM)

[0102] The cells were collected and fixed with TEM fixative (Servicebio, G1124) and encapsulated in 2% agarose. The samples were hydrated with ethanol gradient, infiltrated, resin embedded and polymerized under low temperature UV light. The resin blocks were then cut into 70-80 nm slices and placed on 150 mesh nickel grids. Anti-CCN1 antibody (Proteintech, 26689-1-AP) was used for immunolabeling. 12 nm round black gold particles on the nickel grid indicate positive signals. Immuno-electron microscopy experiments were performed by Seewell.

[0103] (11) CPT2 and CCN1 activity detection

[0104] The direct activity of CPT2 and CCN1 was evaluated by measuring the conversion of palmitoyl carnitine to palmitoyl CoA, following the previous literature protocol (Mao Y et al., Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation, 2024). After protein enrichment with anti-CPT2 (Santa Cruz Biotechnology, sc-377294) and anti-CCN1 (Abcam, ab230947) antibodies, CPT2 and CCN1 proteins were captured with protein A / G magnetic beads. After washing the magnetic beads, a reaction system containing 2mM palmitoyl-L-carnitine (Sigma-Aldrich, 61251), 2mM CoA (Targetmol, T10857), 50mM Tris-HCl (pH 7.5; Beyotime, ST775), 120mM KCl (Beyotime, ST345) and 1mM EDTA (Beyotime, ST1303) was added. The reaction was carried out at 37°C for 1 hour. The reaction was terminated by adding 10 mM Ellman's reagent (DTNB; Targetmol, T15177), and the absorbance was measured at 410 nm using a BioTek Synergy Epoch2 Multimode Reader (Agilent) to quantify the remaining CoA. The percentage of CoA consumption was calculated by the remaining amount and the initial CoA amount, and was positively correlated with the CPT2 and CCN1 enzyme activities.

[0105] (12) Mitochondrial Isolation

[0106] Mitochondria were isolated using the Solebo Mitochondria Extraction Kit (SM0020) according to the instructions. Cells were collected using a cell scraper and washed with pre-cooled PBS. The cells were resuspended in lysis buffer and then homogenized using a Dounce grinder, centrifuged at 1000g and 4°C for 10 minutes, and centrifuged twice to remove the nuclei and residual cells. The supernatant was collected and centrifuged at 3500g for 10 minutes at 4°C. After centrifugation, the supernatant contained cytoplasmic components and the precipitate contained mitochondria. Subsequently, the mitochondrial precipitate was resuspended in washing buffer and centrifuged at 1000g for 5 minutes, followed by centrifugation at 12000g for 10 minutes to improve the purity of mitochondria. The cytoplasmic protein (supernatant) was obtained by centrifuging the supernatant containing cytoplasm at 12000g for 10 minutes.

[0107] (13) Blue-green native electrophoresis (BNE)

[0108] After the mitochondria were separated, the NativePAGE sample pretreatment kit (Thermo Fisher Scientific, BN2008) was used for pretreatment. According to the literature (Wittig I et al., Blue native PAGE, 2006), the sample buffer, anode buffer, cathode buffer B and cathode buffer B / 10 required for blue-green non-denaturing electrophoresis (BNE) were configured. The 4%-13% BNE gel used was from Shanghai Wansheng Haotian Biotechnology Co., Ltd. (GBN2001-413F). Anode buffer and cathode buffer B were used at the start of electrophoresis, with a constant voltage of 100V until the sample entered the gel. When the leading edge of the Coomassie brilliant blue band migrated to about one-third of the gel, cathode buffer B was replaced with cathode buffer B / 10, and electrophoresis was continued at 150V for 1.5h. Subsequently, the blue native PAGE gel strips were washed several times with distilled water, and soaked in 1% SDS for 15 minutes, and conventional WB was performed to further identify the components in the mitochondrial complex.

[0109] (14) Fatty acid oxidation rate detection

[0110] Cells were seeded in Seahorse XF cell culture microplates (Agilent) and cultured overnight at 37°C with 5% CO2. After 12 h of culture with or without 3 μM etomoxir (ETO; MedChemExpress, HY-50202A), the medium was replaced with the prepared experimental medium and then incubated at 37°C without CO2 for 1 h. Working solutions of compounds including 1.5 μM oligomycin, 1.0 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazine (FCCP), and 0.5 μM rotenone / antimycin A (Rot / AntA) were loaded into the XFe / XF96 sensor probe card (Agilent, 103793-100). Oxygen consumption rate (OCR) was measured using the Seahorse Extracellular Flux Analyzer XF96 (Agilent). The fatty acid oxidation rate was the difference between the maximum oxygen consumption rate with and without ETO treatment.

[0111] (15) Multi-omics and bioinformatics analysis

[0112] RNA-seq and TMT quantitative proteomics of short-term ferroptosis induction were performed by BGI and Oebiotech. RNA-seq data of long-term ferroptosis resistance and partial short-term ferroptosis induction were obtained from the GEO database. Proteomics to identify CCN1 interacting proteins was performed by Oebiotech using immunoprecipitation with anti-FLAG antibody (Cell Signaling Technology, 14793). Peptides were identified using ProteomeDiscover 2.1 and UniProt human database.

[0113] Non-targeted lipidomics was performed by the Instrumental Analysis Center of Shanghai Jiao Tong University using a Vanquish UHPLC system (ThermoFisher Scientific) and a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Raw data were acquired using Xcalibur software and processed using LipidSearch 4.2 to identify lipids. Gene ontology enrichment analysis was performed on the OeYi Cloud platform (https: / / cloud.oebiotech.cn / task / ). KEGG analysis of differential lipids was performed using MetaboAnalyst5.0 (https: / / www.metaboanalyst.ca / ).

[0114] exist 13 In C tracing experiments, cells were treated with 100 μM 1- 13 C palmitic acid (a carbon label; Scrbio, SCSI-310676) and 50 μM L-carnitine (Sigma-Aldrich, C0158) were incubated for 3 h. Subsequently, cells were quenched in liquid nitrogen and lipid extraction was performed. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) analysis with selected reaction monitoring (SRM) was performed using TSQ Altis (Thermo Fisher Scientific).

[0115] (16) Statistical methods

[0116] Statistical analysis was performed using GraphPad Prism software. All data are expressed as mean ± standard deviation (SD). The test methods used for statistical analysis were Student's t test, one-way ANOVA, and two-way ANOVA. P < 0.05 was considered statistically significant.

[0117] Example 2

[0118] Ferroptosis inducers promote CCN1 expression in lung cancer cells

[0119] To identify novel ferroptosis regulators, we used a combined RNA-seq and proteomics analysis to identify genes whose mRNA and protein expression increased under conditions of RSL3 and erastin-induced short-term ferroptosis (24 h) and long-term ferroptosis resistance (45 days). Figure 1 ). Using RNA-Seq, a total of 996 genes were identified whose expression was significantly altered under both RSL3-induced short-term ferroptosis and long-term ferroptosis resistance conditions. Similarly, erastin altered the expression of 961 genes ( Figure 2 A). Among the genes regulated by RSL3 (383 of 996; 38.5%) or erastin (348 of 961; 36.2%), a subset was upregulated after induction of both short-term ferroptosis and long-term ferroptosis resistance ( Figure 2 A). Subsequently, further screening by tandem mass tag (TMT) proteomics revealed that CCN1 was the only gene that was upregulated at both mRNA and protein levels after inducing short-term ferroptosis (24 h) and long-term ferroptosis resistance (45 days) ( Figure 2 B). The experimental results showed that in the lung adenocarcinoma cell line H1299, the expression level of CCN1 was upregulated by both RSL3 and erastin in a concentration- and time-dependent manner ( Figure 3 AB). These results indicate that CCN1 is associated with ferroptosis in lung cancer cells.

[0120] Example 3

[0121] CCN1 increases tumor sensitivity to ferroptosis

[0122] CCN1 was knocked out using sgRNA in NCI-H1299 cells and overexpressed in A549 cells ( Figure 4 CCN1 knockout leads to resistance of NCI-H1299 cells to ferroptosis, as evidenced by a reduction in the decrease in cell viability after treatment with ferroptosis inducers and an increase in lipid peroxidation ( Figure 5 AB); CCN1 overexpression caused A549 cells to produce higher levels of cell death and lipid peroxidation after treatment with ferroptosis inducers ( Figure 6 AB), and this death can be inhibited by the ferroptosis inhibitor Fer-1, proving that this death is ferroptosis. The above results indicate that CCN1 is a new type of ferroptosis-promoting factor in tumors.

[0123] Since Ccn1 - / - Therefore, IKE was injected intraperitoneally around day 19 of gestation in pregnant mice, and the pregnant mice were euthanized three days after the injection to obtain mouse embryos. - / - The fetus cannot be formed ( Figure 7 However, under IKE induction, the levels of lipid peroxidation products 4-HNE and MDA were significantly higher than those of Ccn1 + / + Fetal mice expressed lower levels ( Figure 7 BC), indicating that CCN1 is involved in the ferroptosis process.

[0124] The CDX model was constructed by subcutaneously injecting wild-type and CCN1-overexpressing A549 cells on both sides of nude mice. Five days after modeling, IKE was injected intraperitoneally for two weeks. The experiment found that under IKE induction, the MDA and 4-HNE levels of CCN1-overexpressing tumor tissues were higher than those of wild-type ( Figure 7 DE). This further illustrates that CCN1 is involved in ferroptosis and can increase the sensitivity of tumors to ferroptosis.

[0125] Example 4

[0126] Mitochondrial-localized CCN1 promotes ferroptosis sensitivity

[0127] CCN1 is generally considered to be a secreted protein that functions in the matrix. Immuno-electron microscopy and immunofluorescence results showed that under ferroptosis induction, intracellular CCN1 translocated into mitochondria ( Figure 8-9 ). Isolation of cell fractions revealed that RSL3 and erastin-induced CCN1 mitochondrial translocation was time- and concentration-dependent ( Figure 10-11 ). And this translocation can be blocked by ferroptosis inhibitors ( Figure 12-13 ), indicating that CCN1 translocation into mitochondria is necessary for ferroptosis. The potential mitochondrial targeting sequence (MTS) of CCN1 was then predicted by software ( Fig.14 ), and experimentally demonstrated that CCN1 indeed enters mitochondria through this targeting sequence ( Fig.15 By adding the MTS sequence of the classic mitochondrial protein COX4 to the N-terminus of the CDS sequence of CCN1, a mitochondrial-localized CCN1 overexpression plasmid was constructed and transfected into CCN1 knockout H1299 cells ( Fig.16 ). The experimental results showed that the expression of mitochondrial-localized CCN1 could rescue the ferroptosis level suppressed by CCN1 knockout ( Fig.16 ), these results indicate that mitochondrial-localized CCN1 is associated with ferroptosis.

[0128] The nucleotide sequence of the mitochondrial targeting sequence is shown in SEQ ID NO.2:

[0129] SEQ ID NO. 2: 5'-ATGTTGGCTACCAGGGTATTTAGCCTAGTTGGCAAGCGAGCAATTTCCACCTCTGTGTGTGTACGA-3'.

[0130] Example 5

[0131] Mitochondrial-localized CCN1 regulates ferroptosis through fatty acid oxidation

[0132] IP-mass spectrometry showed that CCN1 translocated into mitochondria interacted with ETFA ( Fig.17 ), cell viability assay screening found that only silencing of ETFA and two other genes could antagonize ferroptosis in cells ( Fig.18 ), among which only ETFA is a classic mitochondrial protein. PLA experiments further demonstrated the interaction between CCN1 and ETFA in mitochondria ( Fig.19 ETFA is a key gene in the FAO pathway. Knockout of ETFA can inhibit the ferroptosis level increased by CCN1 overexpression ( Fig. 20 AB), proving that ETFA is a downstream protein of CCN1. Lipid metabolomics also showed that under ferroptosis induction, the knockout of CCN1 affected the FAO metabolic pathway ( Fig.21 ). Under ferroptosis induction, the levels of some fatty acids decreased and the levels of acylcarnitine increased ( Fig. 22 ), proving that ferroptosis promotes FAO. Palmitic acid is the most common substrate for FAO, using 1- 13 C-labeled palmitic acid treatment induced ferroptosis in cells, and CCN1 knockout inhibited ferroptosis-induced 13 The levels of C-labeled FAO products increased ( Figure 23-24 ETFA mediates the conversion of FAD and FADH2 in FAO. Experimental results show that CCN1 knockout inhibits the conversion of FAD and FADH2 induced by ferroptosis ( Fig.25 AB). Treating cells with various fatty acids to activate FAO revealed that ferroptosis sensitivity increased and was inhibited by CCN1 knockout and FAO inhibitor ETO, demonstrating that FAO can promote ferroptosis and is dependent on CCN1 ( Fig.26 AB). These results indicate that CCN1 participates in ferroptosis through the FAO pathway.

[0133] After knocking out multiple key genes in the FAO pathway, the FAO rate was detected and it was found that the knockout of CPT1 and ETFA could completely block the ferroptosis-induced FAO activation, while the knockout of CCN1 and CPT2 could only partially inhibit it. Only when CCN1 and CPT2 were knocked out at the same time could the activation be completely blocked ( Figure 27-28 ). The cells were treated with palmitic acid to activate FAO. The experimental results showed that palmitic acid treatment increased the sensitivity of ferroptosis, and the increased ferroptosis level could only be partially inhibited by CCN1 and CPT2, indicating that the two may play similar functions in the FAO pathway and are independent of each other ( Fig.29 CPT2 mediates the conversion of acylcarnitine to acyl-CoA in the FAO pathway. In vitro experimental results show that only when CPT2 and CCN1 are knocked out at the same time can the conversion of acylcarnitine to acyl-CoA be completely blocked ( Fig.30). BNE results showed that under ferroptosis induction, CCN1 entered the mitochondria and formed two mitochondrial complexes with CPT2 and ETFA ( Fig.31 ), and knockout of CPT2 did not prevent CCN1 from entering these two complexes ( Fig.31 ). These results demonstrate that CCN1 and CPT2 play the same function in the FAO pathway and are independent of each other.

[0134] Example 6

[0135] FAO promotes ferroptosis in tumors through CCN1

[0136] C57 mice were divided into two groups and fed with normal diet or high-fat diet for 8 weeks, respectively. LLC cells were then injected subcutaneously to construct CDX models. One week after modeling, IKE was injected intraperitoneally for treatment. At the same time, antioxidant vitamin E or FAO inhibitor ETO ( Fig.32 ) and counted the tumor volume. The experimental results showed that IKE reduced the tumor volume, and the high-fat diet group had a better therapeutic effect, and this therapeutic effect could be inhibited by vitamin E and ETO ( Fig.33 ), demonstrating that FAO promotes the tumor therapeutic effect of IKE via ferroptosis.

[0137] C57 mice were divided into two groups and fed with normal diet and high-fat diet for 8 weeks, respectively. LLC cells were stably transfected with Dox-inducible Ccn1 knockdown plasmid, and the cells were injected into the tail vein of mice to construct a lung cancer metastasis model. Doxycycline was added to the drinking water to induce Ccn1 knockdown. One week after the injection of cells, cisplatin was injected intraperitoneally for treatment. After two weeks, the mice were euthanized and the lung tissues were collected ( Fig.34 The experimental results showed that a high-fat diet significantly increased the weight of mice, and cisplatin treatment did not affect the weight change ( Fig.35 ). A high-fat diet promoted the therapeutic effect of cisplatin, but knockdown of Ccn1 attenuated the efficacy of cisplatin in both the normal diet and high-fat diet groups ( Fig.36 Moreover, cisplatin treatment increased 4-HNE and MDA levels, which were synergistically enhanced by a high-fat diet and inhibited by Ccn1 knockdown ( Fig.37 AB).

[0138] NSG mice were fed normal diet and high-fat diet for 8 weeks, and fresh lung cancer tissues with high and low CCN1 expression were used to construct PDX models and the mice were treated with CDDP. The results showed that the PDX mice with high CCN1 expression and high-fat diet had the best tumor treatment effect, while the group with low CCN1 expression and normal diet had the worst treatment effect ( Fig.37 C).

[0139] The above results demonstrate that a high-fat diet promotes ferroptosis through CCN1 to treat tumors.

[0140] Example 7

[0141] Assessing the clinical and translational significance of CCN1 in tumors

[0142] Wild-type H1299 cells were injected subcutaneously into nude mice, and mice were treated with IKE, cisplatin, and IKE combined with cisplatin. The results showed that both IKE and cisplatin could promote the expression of CCN1 in tumor tissues, and the synergistic effect of the two in treating tumors was significant ( Fig.38 A). This suggests that tumors with high CCN1 expression are more likely to benefit from ferroptosis-based therapeutic strategies.

[0143] BMI index ≥ 25.0 indicates a tendency to be overweight and obese. Experimental results show that primary lung cancer cells with BMI ≥ 25.0 are more sensitive to ferroptosis and have higher FAO activation levels ( Fig.38 BD), indicating that overweight and obese people have a higher potential for FAO activation and are therefore more sensitive to ferroptosis. Triglycerides are one of the storage forms of fatty acids and can indicate fatty acid levels. Overweight people are often accompanied by hypertriglyceridemia. Primary lung cancer cells with BMI ≥ 25.0 were grouped according to serum triglyceride levels ( Fig.39 A) found that primary cells with high triglycerides had higher levels of ferroptosis ( Fig.39 B) However, when serum triglyceride levels were similar and BMI index was different, primary cells were equally sensitive to ferroptosis ( Fig.39 These results suggest that the fundamental reason for the different ferroptosis sensitivities caused by different BMI indexes is triglyceride levels, and therefore lung cancer patients with hypertriglyceridemia or high fatty acid levels are more likely to benefit from ferroptosis-based treatment strategies.

[0144] Next, tissue microarrays showed that the majority of tumor tissues had high expression of CCN1 in lung adenocarcinoma and lung squamous cell carcinoma ( Fig.40 ), and the CCN1 level in tumor tissue was correlated with serum triglyceride level ( Fig.41 In addition, it was found that the CCN1 protein level in primary cells with high triglycerides was also higher ( Fig.42 ), treatment of cells with different fatty acids promoted CCN1 expression ( Fig.43 Together, these findings suggest that CCN1 and FAO are essential for ferroptosis-based tumor treatment strategies.

[0145] In the above examples, 1) the sgRNA primers of the CCN1 KO1 plasmid are KO1-F (SEQ ID NO. 3) and KO1-R (SEQ ID NO. 4);

[0146] SEQ ID NO.3: 5'-CACCGAACTTGGGCTGTCCCAACCC-3';

[0147] SEQ ID NO.4: 5'-AAACGGGTTGGGACAGCCCAAGTTC-3';

[0148] 2) The sgRNA primers for CCN1 KO2 plasmid were KO2-F (SEQ ID NO.5) and KO2-R (SEQ ID NO.6);

[0149] SEQ ID NO.5: 5'-CACCGGGTTGGGACAGCCCAAGTTG-3';

[0150] SEQ ID NO.6: 5'-AAACCAACTTGGGCTGTCCCAACC-3';

[0151] 3) The Dox-inducible CCN1 knockdown plasmid uses pLKO-tet-on as a vector, and the shRNA primer sequence is shown in SEQ ID NO.7:

[0152] SEQ ID NO.7: 5'-CCGGGGAGTTAACGAGAAACAATGATCAAGAGTCATTGTTTCTCGTTAACTCCTTTT-3';

[0153] 4) CCN1 overexpression related plasmids were all based on pLVX vector (WT-CCN1-HA, Δ a.a 1-21 -CCN1-HA, CMΔ21, CMwt, WT-CCN1-FLAG);

[0154] WT-CCN1-HA (wild-type CCN1 overexpression vector insertion sequence): CCN1 CDS sequence, with HA tag added before the C-terminal terminator, as shown in SEQ ID NO.8:

[0155]

[0156] Δ a.a 1-21 -CCN1-HA (CCN1 overexpression vector insertion sequence with MTS removed): remove the first 21 amino acid sequences, add a start codon at the N-terminus, and add an HA tag before the TAA terminator at the C-terminus, as shown in SEQ ID NO.9:

[0157]

[0158] CMΔ21 (a CCN1 overexpression sequence localized in mitochondria and with the CCN1 MTS sequence removed): the first 21 amino acids of the CCN1 CDS sequence are removed, the MTS sequence of COX4 is added to the N-terminus, and the HA tag is inserted before the C-terminal terminator TAA, as shown in SEQ ID NO.10:

[0159]

[0160] CMwt (mitochondrial-localized CCN1 overexpression vector): insert the MTS sequence of COX4 at the N-terminus of the CCN1 CDS sequence, and insert the HA tag before the C-terminal terminator TAA, as shown in SEQ ID NO.11:

[0161]

[0162] WT-CCN1-FLAG (wild-type CCN1 overexpression vector insertion sequence): CCN1 CDS sequence, with a FLAG tag added before the C-terminal terminator, as shown in SEQ ID NO.12:

[0163]

[0164] The HA tag is shown in SEQ ID NO. 13: 5'-TACCCATACGACGTCCCAGACTACGCT-3'.

[0165] The FLAG tag is shown in SEQ ID NO. 14: 5'-GATTACAAGGACGACGATGACAAG-3'.

[0166] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. The use of CCN1 in the preparation of a drug for enhancing the sensitivity of tumor cell ferroptosis, characterized in that: The amino acid sequence of CCN1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The drug is a drug that inhibits the growth of tumor cells by enhancing the ferroptosis sensitivity of tumor cells.

3. The use according to claim 1, characterized in that: The drug is a drug that enhances the ferroptosis sensitivity of tumor cells through CCN1 translocation of mitochondria.

4. The use according to claim 3, characterized in that: CCN1 achieves mitochondrial translocation via a mitochondrial targeting sequence; The nucleotide sequence of the mitochondrial targeting sequence is shown in SEQ ID NO.

2.

5. The use according to claim 3, characterized in that: The drug is a drug that uses high fatty acids to coordinate CCN1 translocation in mitochondria to increase the sensitivity of tumor cells to ferroptosis.

6. The use according to claim 3, characterized in that: The drug is a drug that promotes fatty acid oxidation metabolism through mitochondrial translocation of CCN1 to increase the ferroptosis sensitivity of tumor cells.

7. The use according to claim 1, characterized in that: The medicine contains a ferroptosis inducer.

8. The use according to claim 7, characterized in that: The ferroptosis inducer is selected from one or both of RSL3 and erastin.

9. The use according to claim 1, characterized in that: The medicine contains IKE and / or cisplatin.

10. The use according to claim 1, characterized in that: The tumor cells are lung adenocarcinoma cells or lung squamous cell carcinoma cells with high fatty acid and / or triglyceride levels.

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