Application of agents that silence or inhibit the LPCAT1 gene in inducing ferroptosis in tumor cells

Abstract

The present invention provides the use of an agent for silencing or inhibiting the LPCAT1 gene in inducing ferroptosis in tumor cells. The present invention finds that LPCAT1 plays a key role in ferroptosis resistance, which increases membrane phospholipid saturation through the Lands cycle and reduces the level of polyunsaturated fatty acids on the cell membrane, thereby protecting cells from membrane damage caused by phospholipid peroxidation and inhibiting ferroptosis. Therefore, an agent for silencing or inhibiting the LPCAT1 gene can induce ferroptosis in cells, thereby acting as a cell ferroptosis inducer. In addition, the enhancement of tumorigenicity of tumor cells in vivo is closely related to the upregulation of LPCAT1 and the emergence of ferroptosis resistance. Ferropoptosis inducers combined with LPCAT1 inhibition can synergistically trigger ferroptosis and inhibit tumor growth, thereby treating tumors. The present invention shows that LPCAT1-mediated membrane phospholipid remodeling promotes ferroptosis escape by increasing phospholipid saturation, reveals the important role of LPCAT1 in ferroptosis escape, and provides new targets and strategies for tumor treatment targeting ferroptosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of an agent for silencing or inhibiting LPCAT1 gene in inducing ferroptosis of tumor cells. Background Art

[0002] Ferroptosis is a form of non-apoptotic cell death driven by iron-dependent lipid peroxidation. The essence of ferroptosis is the metabolic disorder of intracellular lipid peroxides. When lipid peroxides cannot be metabolized by antioxidant defense systems such as GSH reduction reactions catalyzed by glutathione peroxidase (GPX4), it may lead to excessive accumulation of lipid peroxides on the cell membrane and subsequent cell membrane collapse and rupture, thereby triggering cell death. Increasing evidence shows that ferroptosis plays a key role in tumor biology and cancer treatment. On the one hand, some compounds or therapeutic agents have been shown to provide new ways to treat cancers that are refractory to traditional therapies by inducing ferroptosis in tumor cells; on the other hand, in some cases, intracellular ferroptosis defense pathways may be used by cancer cells, enabling them to escape the fate of ferroptosis and promote the occurrence and development of tumors. Therefore, the development of inducers or sensitizers targeting ferroptosis has great potential in tumor treatment.

[0003] The occurrence and execution of ferroptosis are closely related to the peroxidation of the iron-catalyzed substrate, namely phospholipids containing polyunsaturated fatty acids (PUFAs) (PUFA-PLs). Mammalian cell membrane phospholipids are mainly divided into three types: SFA-PLs, MUFA-PLs and PUFA-PLs. Among them, PUFA-PLs are more likely to form peroxyl radicals in an enzymatic or non-enzymatic form, which propagate throughout the membrane through a chain reaction, leading to irreversible membrane damage and cell death. Promoting the incorporation of PUFAs into membrane phospholipids can promote ferroptosis; conversely, increasing the number of less oxidative MUFAs in membrane phospholipids can effectively resist ferroptosis. Therefore, the enzymes and pathways that control the metabolism of PUFAs and MUFAs play a key role in determining the sensitivity of cells to ferroptosis. For example, the membrane remodeling enzyme ACSL4 can activate the conversion of PUFA to PUFA-CoA, and LPCAT3 is responsible for the incorporation of PUFA-CoA into PLs5. In contrast, ACSL3 activates the conversion of MUFA to MUFA-CoA, and the phospholipid modification enzymes MBOAT1 / 2 (transcriptionally upregulated by sex hormone receptors) incorporate MUFA-CoA into PLs to antagonize PUFA activation and thus protect cells from ferroptosis.

[0004] Downregulation of peroxidized PUFA-PL levels in cancer cells is associated with ferroptosis escape and enhanced tumor growth, but the specific mechanism remains poorly understood. Therefore, finding key molecules that can regulate the dynamic remodeling of cell membrane phospholipids to mediate the sensitivity of tumor cells to ferroptosis is crucial for tumor prevention and treatment strategies targeting ferroptosis. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an agent for silencing or inhibiting the LPCAT1 gene for use in inducing ferroptosis in tumor cells. The LPCAT1 gene can be used as a new target for tumor treatment targeting ferroptosis.

[0006] To achieve the above object, the present invention adopts the following technical solution.

[0007] In a first aspect, the present invention provides the use of an agent for silencing or inhibiting LPCAT1 gene expression in the preparation of a ferroptosis inducer.

[0008] The second aspect of the present invention provides the use of an agent for silencing or inhibiting the expression of the LPCAT1 gene in the preparation of a drug for inducing ferroptosis of tumor cells.

[0009] The third aspect of the present invention provides the use of an agent for silencing or inhibiting LPCAT1 gene expression in the preparation of a tumor cell ferroptosis sensitizer.

[0010] A fourth aspect of the present invention provides the use of an agent for silencing or inhibiting LPCAT1 gene expression in the preparation of a ferroptosis inducer sensitizer.

[0011] A fifth aspect of the present invention provides the use of an agent for silencing or inhibiting LPCAT1 gene expression in combination with a ferroptosis inducer in the preparation of a drug for inducing ferroptosis of tumor cells.

[0012] In some embodiments, the ferroptosis inducer comprises at least one of RSL3 and erastin.

[0013] In some embodiments, the agent for silencing or inhibiting LPCAT1 gene expression can reduce the content of phospholipids containing saturated fatty acids in the cell membrane and increase the content of phospholipids containing polyunsaturated fatty acids in the cell membrane.

[0014] In a sixth aspect, the present invention provides a drug for preventing and treating tumors, the drug comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises an agent for silencing or inhibiting LPCAT1 gene expression and a ferroptosis inducer.

[0015] In some embodiments, the ferroptosis inducer comprises at least one of RSL3 and erastin.

[0016] In a seventh aspect, the present invention provides a method for inducing cell ferroptosis in vitro, comprising the following steps: (1) constructing cells with silenced or inhibited LPCAT1 gene and culturing them in vitro; (2) treating the cell culture system with a ferroptosis inducer.

[0017] In some embodiments, the cell is a tumor cell.

[0018] In some embodiments, the ferroptosis inducer comprises at least one of RSL3 and erastin.

[0019] In some embodiments, the final concentration of the ferroptosis inducer in the culture system can be the concentration of the IC50 corresponding to the cells. For example, the final concentration of RSL3 in the 293FT cell culture system is 1.5 μM, and the final concentration in the U-2OS cell culture system is 5 μM. The final concentration of erastin in the 293FT cell culture system is 3 μM, and the final concentration in the U-2OS cell culture system is 10 μM.

[0020] The present invention found that LPCAT1 plays a key role in ferroptosis resistance. It increases membrane phospholipid saturation through the Lands cycle and reduces the level of polyunsaturated fatty acids on the cell membrane, thereby protecting cells from membrane damage caused by phospholipid peroxidation and inhibiting ferroptosis. Therefore, agents that silence or inhibit the LPCAT1 gene can induce ferroptosis in cells, thereby acting as cell ferroptosis inducers to induce ferroptosis in tumor cells.

[0021] In addition, the enhancement of tumorigenicity of tumor cells in vivo is closely related to the upregulation of LPCAT1 and the emergence of ferroptosis resistance. Ferroptosis inducers combined with LPCAT1 inhibition can synergistically trigger ferroptosis and inhibit tumor growth, thereby treating tumors. Therefore, agents that silence or inhibit LPCAT1 gene expression can serve as ferroptosis sensitizers for tumor cells.

[0022] The present invention shows that LPCAT1-mediated membrane phospholipid remodeling promotes ferroptosis escape by increasing phospholipid saturation, revealing the important role of LPCAT1 in ferroptosis escape. The present invention provides a new target and strategy for tumor treatment targeting ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results show that continuous RSL3 / erastin treatment leads to saturation of membrane phospholipids.

[0024] Figure 2 These results suggest that RSL3 / erastin-resistant cells tend to incorporate SFA into membrane phospholipids.

[0025] Figure 3 The research results show that LPCAT1 can increase the saturation of membrane phospholipids.

[0026] Figure 4 These are the results of the study that LPCAT1 knockout makes cells sensitive to ferroptosis.

[0027] Figure 5These are the results of the study that LPCAT1 inhibits ferroptosis in a dependent manner by exogenous or endogenous SFA.

[0028] Figure 6 The results suggest that LPCAT is upregulated to escape ferroptosis and thus promote tumorigenesis.

[0029] Figure 7 The results of the study showed that inhibiting LPCAT1 can inhibit tumor growth. DETAILED DESCRIPTION

[0030] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0031] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0033] The term "at least one" used in the present invention refers to one or more than one. The character " / " generally indicates that the objects associated with each other are in an "or" relationship.

[0034] As an important part of the dynamic remodeling of membrane phospholipids, the Lands cycle introduces long polyunsaturated acyl chains into phospholipids by erasing (deacylation) and rewriting (reacylation) the acyl groups of phospholipids. This process determines the biophysical properties of the cell membrane, thereby affecting its biological processes. As an important component of the cell lipid bilayer, phospholipids are a functional barrier between the cell contents and the surrounding environment. Since cells dynamically reshape membrane phospholipids in response to changes in the external environment, thereby maintaining continuous growth and coping with external stress, different tissues and cell types present different phospholipid compositions. Recently, a large number of studies have shown that the initiation and execution of ferroptosis are closely related to membrane phospholipid peroxidation.

[0035] Phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), are composed of two hydrophobic fatty acyl chains and a hydrophilic head group. The length and saturation of membrane phospholipid acyl chains show great diversity, which determines the biophysical properties of cell membranes and affects membrane-related biological processes. Hydrogen atoms at the diallyl position in polyunsaturated fatty acid (PUFA) chains can be replaced by hydroxyl radicals, leading to lipid peroxidation and inducing membrane damage and ferroptosis. Several antioxidant proteins or pathways have been shown to regulate ferroptosis by inhibiting the generation of lipid peroxidation. For example, glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides (PUFA-OOH) into non-toxic lipid alcohols (PUFA-OH), while ferroptosis suppressor protein 1 (FSP1), dihydrolactate dehydrogenase (DHODH), and GTP cyclohydrolase-1 (GCH1) inhibit ferroptosis by capturing lipid peroxides. Interestingly, Lands' cycle is not only involved in the rapid replacement of phospholipid fatty acyl chains, but also in the repair of peroxidized chains of cell membrane phospholipids, a process that involves the replacement of oxidized acyl chains to maintain membrane integrity.

[0036] LPCAT1: lysophosphatidylcholine acyltransferase 1.

[0037] In some embodiments, conventional techniques in the art can be used to silence or inhibit the expression of the LPCAT1 gene, such as CRISPR / cas9, transfection, etc.

[0038] In some embodiments, the expression of LPCAT1 gene is silenced by transfection. LPCAT siRNA was transfected into 293FT and U2OS cells according to the instructions of Invitrogen 3000 (Invitrogen, Carlsbad, CA, USA). The specific siRNA sequence information is shown in Table 1 below:

[0039] Table 1

[0040]

[0041] The following describes the invention in conjunction with specific embodiments.

[0042] Example 1 Continuous RSL3 / erastin treatment leads to saturation of membrane phospholipids

[0043] In order to explore the regulatory mechanism of membrane phospholipid peroxidation during ferroptosis, we detected the degree of membrane lipid peroxidation in RSL3- / erastin-resistant 293FT and U-2OS cell lines, which were constructed by continuous treatment with RSL3 (reference Yang, W Setal. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317-331 (2014)) or erastin (reference Dolma, S., Lessnick, SL, Hahn, WC & Stockwell, BR Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3, 285-296 (2003)). Under RSL3 / erastin treatment, the membrane lipid peroxidation levels of both RSL3-resistant 293FT / U-2OS cells were much lower than those of parental cells, as shown by the overlap ratio of Liperfluo fluorescence and membrane Dil signal ( Figure 1 Similar results were observed in erastin-resistant 293FT / U-2OS cells: RSL3 / erastin treatment induced reduced levels of membrane lipid peroxidation in erastin-resistant 293FT / U-2OS cells relative to corresponding parental cells ( Figure 1 ab). These findings suggest that ferroptosis-resistant cells acquire resistance to membrane lipid peroxidation through long-term RSL3 / erastin treatment.

[0044] Unexpectedly, RSL3 / erastin-resistant cells showed increased levels of oxidized DCFDA compared with parental cells, regardless of whether they were treated with RSL3 / erastin ( Figure 1 c) and GSH levels were reduced (data omitted). However, in parental cells and RSL3 / erastin-resistant cells, Fe 2+ The content ( Figure 1 d) There was no significant difference in the expression of various lipid peroxidation scavengers (including FSP1, GCH1, DHODH, LPCAT3 and ASCL4) ( Figure 1 e) These findings further suggest that RSL3 / erastin-resistant cells rely on membrane phospholipid remodeling to resist lipid peroxidation induced by ferroptosis inducers.

[0045] We then used untargeted lipidomics to assess the phospholipid composition of membrane fractions in parental and RSL3 / erastin-resistant cells. A total of 190 phospholipids from five major classes, including PC, PE, PS, PI, and PG, were detected. Compared with parental cells, the levels of phospholipids containing saturated fatty acids (SFA) were significantly increased, while the levels of phospholipids containing PUFA were significantly decreased in RSL3- / erastin-resistant cells (FDR-corrected P < 0.05; Figure 1 fj). Statistical analysis showed that SFA-containing PC and PE were mainly 16:0 and 18:0 diacyl phospholipids, which were dominant and at higher levels in the RSL3 / erastin-resistant cell line (FDR-corrected P < 0.05; Figure 1 fj). In addition, the levels of 20:4 and 22:4 diacylphospholipids were significantly decreased in the RSL3 / erastin-resistant cell line (FDR-corrected P < 0.05; Figure 1 fi). By the proportion of phospholipid SFA, MUFA and PUFA in the cells shown ( Figure 1 j) and quantity ( Figure 1 k) We also found that the acyl chain composition of RSL3- / erastin-resistant cells was significantly saturated compared with control cells ( Figure 1 jk). Taken together, these results suggest that membrane phospholipid remodeling based on increased SFA-PL levels and decreased PUFA-PL levels may be involved in RSL3- / erastin resistance.

[0046] Figure 1In the figure, a: Immunofluorescence staining of cells treated with RSL3 (293FT: 1.5 μM, U-2OS: 5 μM; 10 h) or erastin (293FT: 3 μM, U-2OS: 10 μM; 10 h) using Liperfluo (20 μM) and Dil (5 μM) fluorescence signals. Scale bar, 10 μm or 2 μm. b: Quantification of the cell membrane Liperfluo fluorescence intensity normalized to the Dil fluorescence intensity in the indicated cells. c: Relative ROS levels in cells detected by DCFHDA probe. d: Ferro-Orange staining was used to assess the intracellular ferrous ion levels in the indicated cells after treatment with RSL3 (293FT: 1.5 μM, U-2OS: 5 μM; 10 h) or erastin (293FT: 3 μM, U-2OS: 10 μM; 10 h). e: Immunoblot analysis of GPX4, FSP1, GCH1, DHODH, LPCAT3, and ACSL4 expression in RSL3-treated cells (293FT: 1.5 μM, U-2OS: 5 μM; 10 h) or erastin-treated cells (293FT: 3 μM, U-2OS: 10 μM; 10 h). GAPDH was used as an internal control. fi: Scatter plots showing changes in the levels of the indicated phospholipids in RSL3 / erastin-resistant cells compared with parental cells. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG). FC, fold change. Cutoff: FC threshold = 2, P < 0.05. jk: The proportion (j) and amount (k) of phospholipid SFA, MUFA, and PUFA in the indicated cells. Data are presented as mean ± SD, n = 3 biologically independent samples in ek and n = 6 biologically independent samples in ad. Statistical analysis for fi was performed using unpaired two-tailed Student's t test, and for bd and k, one-way ANOVA followed by Dunnett's test. NS: not statistically significant.

[0047] Example 2 RSL3 / erastin-resistant cells tend to incorporate SFA into membrane phospholipids

[0048] To further investigate the mechanism of membrane phospholipid remodeling in RSL3 / erastin-resistant cells, cells were treated with PUFAs such as a-linolenic acid (ALA, 18:3, n-3) and arachidonic acid (AA, 20:4, n-6), and non-PUFAs such as saturated palmitic acid (PA, C16:0) and monounsaturated oleic acid (OA, C18:1) at 5 μM for 10 h, and non-targeted lipidomics analysis was performed. Treatment with free fatty acids (FFAs) alone or in combination with PA / OA / ALA / AA significantly altered the corresponding membrane phospholipid composition in parental cells and RSL3 / erastin-resistant cells within 10 h (data omitted). These findings are consistent with previous studies reporting that the phospholipid composition of cell membranes is rapidly regulated by extracellular fatty acids. However, in both parental cells and RSL3 / erastin-resistant cells, treatment with FFAs alone did not affect the levels of other types of FFAs and related metabolites such as acylcarnitines ( Figure 2 c), indicating that exogenous FFA treatment does not alter fatty acid uptake, de novo synthesis, elongation, or catabolism.

[0049] Notably, we observed that PA treatment alone significantly increased the levels of PA-containing phospholipids (PL-PA) in the membranes of RSL3- / erastin-resistant cells relative to parental cells ( Figure 2 However, there was no significant change in the levels of OA-PLs in RSL3 / erastin-resistant cells treated with OA alone compared with the parental cells treated with OA ( Figure 2 ab). However, the PUFA treatment alone, including ALA and AA, significantly downregulated the levels of ALA- / AA-containing acyl chain phospholipids on the membrane (PL-ALA and PL-AA, respectively) ( Figure 2 ab). These results indicate that RSL3-erastin-resistant cells are more inclined to incorporate SFA into membrane phospholipids. Importantly, in RSL3-erastin-resistant cells treated with ALA / AA / PA / OA in combination, the tendency of ALA / AA to be incorporated into phospholipids was significantly lower than that in the ALA- / AA alone-treated group (data omitted), indicating that SFA is more easily incorporated into the membrane phospholipids of RSL3-erastin-resistant cells than PUFA.

[0050] To further verify that RSL3- / erastin-resistant cells preferentially incorporate SFA into the cell membrane, we applied a fatty acid analogue for studying the dynamic metabolic process of lipids, that is, adding an alkynyl group (FA-alkyne) to the end of the fatty acid and combining it with an isotope-labeled click tag. The analogue was detected by coupling azide 488 to reflect the incorporation efficiency ( Figure 2d). After 10 h of treatment with FA-alkyne alone, the membranes of RSL3 / erastin-resistant cells showed lower fluorescence intensity of ALA- / AA-alkyne-488, while the fluorescence intensity of PA-alkyne-488 was higher than that of parental cells ( Figure 2 These data confirm that SFAs are preferentially incorporated into the membranes of RSL3 / eastin-resistant cells.

[0051] Following the aforementioned FFA-induced phospholipid remodeling, pretreatment with ALA / AA alone increased the level of RSL3- / erastin-induced membrane lipid peroxidation in parental cells, whereas pretreatment with PA or OA alone reduced the level of membrane lipid peroxidation ( Figure 2 h). However, PA / OA pretreatment alone significantly reduced RSL3- / erastin-induced lipid peroxidation and ferroptosis in RSL3- / erastin-resistant cells. Figure 2 i shows the cell viability assay after 24 h of treatment with different FFAs and RSL3 (5 μM) or erastin (10 μM). In summary, our results indicate that RSL3 / erastin-resistant cells tend to incorporate SFAs into membrane phospholipids, thereby inhibiting lipid peroxidation and ferroptosis.

[0052] Figure 2 In ab: fold changes in the levels of acyl chain fatty acids in the phospholipids of the indicated cells. c: heat map showing the content of metabolites detected by metabolomics. d: click chemistry reaction between click-tagged fatty acid analogs and azide 488. eg: quantification (e) and representative images (f, g) of the fluorescence intensity of azide 488 normalized to the fluorescence intensity of integrin β1 after treatment with PA-alkyne (5 μM, 10 h) or AA-alkyne (5 μM, 10 h). Scale bar, 10 μm or 2 μm. h: quantification of membrane Lipefluo fluorescence intensity (normalized to Di1 fluorescence intensity) induced by RSL3 (5 μM, 10 h) in the indicated cells under different FAs treatment. i: cell viability assay after treatment with different FFAs and RSL3 (5 μM) or erastin (10 μM) for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples in ac and i, n = 6 biologically independent samples in eh. Statistical analysis was performed using unpaired two-tailed Student's t test and one-way ANOVA followed by Dunnett's test, a, b, e, h, i. NS: not statistically significant.

[0053] Example 3

[0054] LPCAT1 is upregulated in RSL3- / erastin-resistant cells

[0055] In order to explore the key factors affecting SFA incorporation into membrane phospholipids, RNA sequencing analysis (RNA-seq) was performed on RSL3 / erastin-resistant cells and corresponding parental cells. The results showed that compared with parental cells, 10 genes were significantly upregulated in RSL3 / erastin-resistant 293FT and U-2OS cells ( Figure 3 a).

[0056] In addition, we found that among these 10 genes, silencing LPCAT1 (a key enzyme involved in lipid remodeling pathway via Lands' cycle) had the most significant inhibitory effect on PA-alkyne incorporation into membrane phospholipids of RSL3- / erastin-resistant cells, while promoting the incorporation of ALA-alkyne and AA-alkyne ( Figure 3 bd). Immunoblotting (IB) analysis further showed that the expression of LPCAT1 was increased in RSL3 / erastin-resistant cells compared with parental cells ( Figure 3 e) Taken together, these results suggest that upregulation of LPCAT1 promotes membrane phospholipid remodeling induced by RSL3 / erastin treatment.

[0057] LPCAT1 increases the saturation of membrane phospholipids

[0058] To further determine the role of LPCAT1 in membrane phospholipid remodeling during ferroptosis, we established LPCATI1-transduced 293FT / U-2OS cells ( Figure 3 f). Lipidomic analysis showed that overexpression of LPCAT1, but not the active site mutant (LPCAT1 / mu,H135A), significantly increased the level of SFA in the membrane phospholipids of 293FT / U-2OS cells, while decreasing the level of PUFA ( Figure 3 gi). These results suggest that the LPCAT activity of LPCAT1 plays a key role in increasing membrane phospholipid saturation. At the same time, lipidomic analysis showed that SFA-containing PCs / Pes were the main phospholipid components with increased membrane phospholipid saturation levels in LPCAT1-overexpressing cells ( Figure 3 Similarly, in RSL3- / erastin-resistant cells with CRISPR / cas9 knockout of LPCAT1, the levels of SFA-containing PC / PEs were reduced, but the levels of PUFA-PLs were increased ( Figure 3 jk).

[0059] To further investigate the function of LPCAT1 in promoting membrane phospholipid saturation, we evaluated the incorporation efficiency of FFA-alkyne in LPCAT1 cells. The results showed that after 10 h of FFA-alkyne treatment, the fluorescence intensity of PA-alkyne-488 was significantly increased in LPCAT1-overexpressing U-2OS cells but not in the LPCAT1 enzyme-active mutant (LPCAT1 / mu, H135A), while the fluorescence intensity of AA / ALA-alkyne was significantly decreased. In contrast, the fluorescence intensity of PA-alkyne-488 was decreased, but the fluorescence intensity of AA / ALA-alkyne-488 was significantly increased in LPCAT1-KO RSL3- / erastin-resistant cells compared with control cells ( Figure 3 l). In addition, RSL3- / erastin treatment further enhanced the efficiency of PA-alkyne incorporation mediated by LPCAT1, while after LPCAT1 knockout, PA-alkyne incorporation after RSL3- / erastin treatment almost disappeared (data omitted). In conclusion, LPCAT1 overexpression can mediate SFA incorporation into membrane phospholipids and increase membrane phospholipid saturation, and this effect will be further enhanced under lipid peroxidation stimulation conditions.

[0060] Figure 3 a: Venn diagram showing the intersection of upregulated transcripts in four RNA-seq datasets: 293FT-RSL3-R vs. 293FT-P, 293FT-Erastin-R vs. 293FT-P, U-2OS-RSL3-R vs. U-2OS-P, and U-2OS-Erastin-R vs. U-2OS-P. Fold change ≥ 2.5, P < 0.05. bd: Heatmaps showing the incorporation of FA-alkynes into the membranes of the indicated cells. ef: Immunoblot analysis of LPCAT1 in the indicated cells. GAPDH was used as an internal control. gh, jk: Scatter plots showing the changes in the levels of different phospholipids in the indicated cells compared with parental cells. FC: Fold change. Cutoff: FC threshold = 2, P < 0.05. i: The proportion (upper) and amount (lower) of SFA, MUFA, and PUFA in the phospholipids of the indicated cells. l: Representative images (left) and quantification (right) of the fluorescence intensity of azide 488 normalized to that of integrin β1 after treatment with PA-alkyne (5 μM, 10 h) or AA-alkyne (5 μM, 10 h). Scale bar, 10 μm or 2 μm. Data are presented as mean ± SD, n = 3 biologically independent samples in bk and n = 6 biologically independent samples in l. Unpaired two-tailed Student's t test was used in a, g, h, jk, and Dunett's test was used in i, l. NS: not statistically significant.

[0061] Example 4 LPCAT1 knockout sensitizes cells to ferroptosis

[0062] After verifying the effect of LPCAT1 on membrane lipid remodeling ( Figure 3 ), we further found that RSL3 / erastin-induced lipid peroxidation was increased in LPCAT1-KO RSL3 / erastin-resistant cells but decreased in LPCAT1-overexpressing cells ( Figure 4 ab). However, LPCAT1 dysregulation did not affect the expression of GPX4, GCH1, FSP1, DHODH, LPCAT3, and ASCL4, the cellular levels of oxidized DCFDA and GSH, and Fe 2+ content( Figure 4 c). These results indicate that LPCAT1-mediated reduction of membrane lipid peroxidation is through the membrane phospholipid remodeling pathway, independent of other signaling pathways associated with ferroptosis. Consistent with the above results, after 24 h of RSL3 / erastin treatment, the iodinated acetone (PI) fluorescence signal in 292FT / Vector or U-2OS / Vector cells was significantly increased, but the PI fluorescence signal in LPCAT1-overexpressing cells and RSL3-resistant cells was still low, and the PI fluorescence signal could be reversed by downregulating LPCAT1 (data omitted). In addition, LPCAT1 overexpression reduced the induction effect of RSL3 / erastin treatment on cell ferroptosis, while knockout or knockdown of LPCAT1 enhanced these effects ( Figure 4 dg). RSL3- / erastin-treated LPCAT1-KO cells exhibit typical morphological features of ferroptosis, such as membrane blebbing phenotype and disappearance of mitochondrial cristae ( Figure 4 h), indicating that LPCAT1 is involved in regulating ferroptosis. Meanwhile, LPCAT1-KO increased the ferroptosis sensitivity of RSL3 / erastin-resistant cells, while LPCAT1 overexpression enhanced the resistance of 293FT / U-2OS normal cells to various ferroptosis inducers, including ML210, FIN56, sorafenib, and sulfasalazine ( Figure 4 jk). Importantly, in LPCAT1-KO cells, only the ferroptosis inhibitors ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and iron chelator (DFO), but not the apoptosis inhibitor Z-VAD-FMK, the necrosis inhibitor necrostatin-1 (NEC-1), and the pyroptosis inhibitor VX765, could counteract the induction of ferroptosis (Figure 41). Together, these results suggest that knockout of LPCAT1 sensitizes cells to ferroptosis.

[0063] Figure 4In, ab: Quantification of membrane Lipefluo fluorescence intensity (normalized by Di1 fluorescence intensity) of the indicated cells after treatment with RSL3 or erastin. c: Immunoblotting analysis showing the expression of GPX4, FSP1, GCH1, DHODH, LPCAT3, and ACSL4 in the indicated cells. GAPDH was used as an internal reference. de: Cell viability assay of the indicated cells treated with RSL3 or erastin for 24 h. fg: Left: Viability assay of the indicated cells treated with RSL3 or erastin for 24 h; right: Immunoblotting analysis of LPCAT1 expression in the indicated cells. GAPDH was used as an internal reference. h: Representative images of cells treated with RSL3 for 24 h. i: Transmission electron microscopy images showing mitochondrial cristae of cells treated with RSL3 for 24 h. Scale bars, 400 nm (left) and 50 nm (right). jk: Cell viability analysis of U-2OS (h) or U-2OS-RSL3-R (i) cells treated with the indicated ferroptosis inducers ML210 (2μM), FIN56 (5μM), sorafenib (10μM), and sulfasalazine (500μM) for 24h. l: Cell viability analysis of RSL3 (20μM) plus the indicated cell death inhibitors frrostatin-1 (fer1, 2μM), Liproxstatin-1 (lip1, 0.2μM), deferoxamine (DFO, 100μM), necrostatin-1 (nec1, 10μuM), Z-VAD-FMK (10μuM), and VX765 (10μM). Data are expressed as mean ± SD, n = 3 biologically independent samples in c-1, n = 6 biologically independent samples in ab. Unpaired two-tailed Student's t test was used in b and j, one-way ANOVA followed by Dunn't test was used in a, k, and 1, and two-way ANOVA followed by Tukey's test was used in dg. NS: not statistically significant.

[0064] Example 5 LPCAT1 inhibits ferroptosis in a manner dependent on exogenous or endogenous SFA

[0065] Next, we observed that LPCAT1 overexpression could reverse ferroptosis induced by downregulation of GPX4, FSP1, GCH1, or DHODH or upregulation of LPCAT3 ( Figure 5 ad). This suggests that LPCAT1-mediated inhibition of ferroptosis is independent of GPX4, FSP1, GCH1, DHODH, and LPCAT3.

[0066] To further investigate the source of SFAs involved in LPCAT1 remodeling and ferroptosis inhibition, we examined endogenous SFAs and exogenous SFAs. We found that TOFA, an inhibitor of acetyl-CoA carboxylase (ACC), induced ferroptosis in LPCAT1-overexpressing cells in a dose-dependent manner. Figure 5 e), indicating that the ability of LPCAT1 to inhibit ferroptosis depends on endogenously synthesized SFAs. Although silencing SCD1 rendered control cells more susceptible to ferroptosis, consistent with existing reports, neither silencing SCD1 nor treatment with the SCD1 inhibitor CAY10566 significantly altered cell viability in LPCAT1-overexpressing cells ( Figure 5 fi). These results suggest that LPCAT1 prevents ferroptosis by incorporating SFAs, but not MUFAs, into membrane phospholipids.

[0067] In addition to de novo synthesis, dietary SFAs are also an important source of the SFA pool. In addition, we found that the supply of exogenous PA abolished TOFA- and ferroptosis-inducing agent-induced ferroptosis in LPCAT1-KO ferroptosis-resistant cells in a dose-dependent manner ( Figure 5 jk), and enhanced the anti-ferroptosis capacity of tumor spheroids formed by LPCAT1-overexpressing HCT-116 cells and FaDu cells (data omitted). Collectively, these results suggest that both endogenous SFAs synthesized de novo and exogenously provided SFAs contribute to LPCAT1-mediated inhibition of ferroptosis ( Figure 5 l).

[0068] Figure 5Middle, ad: Left: Cell viability assay after 24 h of RSL3 treatment. Right: Immunoblotting analysis of the expression of LPCAT1, GPX4, FSP1, GCH1, and DHODH in the indicated cells. GAPDH was used as an internal control. e: The indicated cells were pretreated with TOFA at the indicated concentrations for 24 h in dialyzed fetal bovine serum (DFBS) medium and then treated with RSL3 for 24 h with or without ferrostatin-1 for cell viability assay. f: Immunoblotting analysis of SCD1 expression in the indicated cells. α-tubulin was used as an internal control. gh: The indicated cells were treated with RLS3 / Erastin in DFBS medium and then subjected to cell viability assay. i: The indicated cells were pretreated with CAY10566 at the indicated concentrations for 24 h in DFBS medium and then treated with RSL3 for 24 h with or without ferrostatin-1 for cell viability assay. l: Model showing how LPCAT1 inhibits ferroptosis using endogenous or exogenous SFA. Data are presented as mean ± SD, n = 3 biologically independent samples in ak. Statistical analysis was performed using one-way ANOVA followed by Dunnett's test in e and gk; two-way ANOVA followed by Tukey's test in ad. NS: not statistically significant.

[0069] Example 6 LPCAT is upregulated to escape ferroptosis and promote tumorigenesis

[0070] The Cancer Genome Atlas (TCGA) database analysis showed that LPCAT1 was upregulated in most tumor types ( Figure 6 a). In addition, we observed that high expression of LPCAT1 was positively correlated with ferroptosis resistance in 16 different cancer cell lines (data omitted). Silencing LPCAT1 significantly increased the sensitivity of these cancer cell lines to ferroptosis (data omitted), further indicating a key role of LPCAT1 in ferroptosis inhibition. Figure 6 b: Immunoblotting analysis of LPCAT1 expression in the indicated tumors of PAAD and LIHC patients and in cells isolated from the corresponding tumors.

[0071] Subsequently, we explored the relationship between LPCAT1-mediated ferroptosis escape and tumorigenesis and progression in patient-derived pancreatic cancer cell lines (PDPA) and hepatocellular carcinoma cell lines (PDLC) ( Figure 6 ).and Figure 3 and Figure 5 The results were consistent. PDPA and PDLC cell lines with high LPCAT1 expression had higher phospholipid saturation and stronger resistance to ferroptosis ( Figure 6cd). In addition, in PDPA and PDLC tumor cell xenograft models, LPCAT1-high-expressing cells, including PDPA#1, PDLC#2, and PDLC#3, rapidly developed subcutaneous tumors in mice ( Figure 6 e). Surprisingly, LPCAT1 low-expressing cells, including PDPA#2 and PDLC#1, initially seemed unable to colonize mice, but after a period of latency, we observed large tumor nodules ( Figure 6 e). We isolated tumor cells from three mice constructed with LPCAT1 low-expressing cells PDPA#2 and PDLC#1 after the incubation period and found that compared with parental cells, tumor cells isolated from tumors formed by PDPA#2 (PDPA#2 / T-ICs) and tumor cells isolated from tumors formed by PDLC#1 (PDLC#1 / T-ICs) showed increased LPCAT1 expression ( Figure 6 f). In addition, PDPA#2 / T-ICs and PDLC#1 / T-ICs also showed higher phospholipid saturation and were more resistant to ferroptosis induction compared with parental cells ( Figure 6 These results suggest that LPCAT1 is essential for tumor growth in vivo.

[0072] To further determine whether LPCAT1 affects tumor development by promoting ferroptosis escape in vivo, we used the doxycycline-inducible Tet-on system to establish LPCAT1 knockdown PDPA#1, PDLC#2, and PDLC#3 cell lines, as well as LPCAT1 overexpression PDPA#2, PDPA#3, and PDLC#1 cell lines ( Figure 6 j-1). The addition of DOX at week 3 resulted in a dramatic downregulation of LPCAT1, and at week 12 we observed a significant inhibition of PDPA#1 subcutaneous tumor growth ( Figure 6 j); however, discontinuation of DOX or addition of Fer-1 at week 9 abolished the tumor growth inhibition at week 12 ( Figure 6 j). In contrast, both DOX additions resulted in upregulation of LPCAT1 expression, and Fer-1 treatment significantly accelerated the formation of PDPA#2 subcutaneous tumors at 12 weeks ( Figure 6 k). However, we found that after adding ferroptosis inducer (IKE) at week 4, we observed that PDPA#3 subcutaneous tumor growth was significantly inhibited at week 12, and the addition of DOX at week 4 to upregulate LPCAT1 expression or discontinuation of IKE at week 12 significantly reversed the tumor growth inhibition induced by IKE (Figure 61). Similar results were obtained in our subcutaneous tumor model using PDLC cells (data omitted). In summary, upregulation of LPCAT1 can escape ferroptosis and promote tumor development.

[0073] Figure 6 In the figure, a: The mRNA expression level of LPCAT1 in pan-cancer tissues was analyzed using the TCGA database. b: Immunoblotting analysis of LPCAT1 expression in the indicated tumors of PAAD and LIHC patients and cells isolated from the corresponding tumors. GAPDH was used as an internal reference. c: LC-MS analysis of membrane phospholipids with different FAs (SFA, MUFA, and PUFA). d: Cell viability assay after 24 h of treatment with IKE (2 μM) and IKE (2 μM) + Fer1 (4 μM). e: Tumor growth curves of the indicated xenograft models. N = 5 in each group. f: Immunoblotting analysis of LPCAT1 expression in the indicated cells. GAPDH was used as an internal reference. gh, LC-MS analysis of membrane phospholipids with different FAs (SFA, MUFA, and PUFA). i: Cell viability assay after 24 h of treatment with IKE (2 μM) or IKE (2 μM) plus Fer1 (4 μM). jl: Upper figure: Immunoblotting analysis of LPCAT1 expression in the indicated cells. GAPDH was used as an internal reference. Lower panel: Tumor growth curves of the xenograft models shown. N = 5 per group. Data are presented as mean ± SD, n = 3 biologically independent samples in bd and fI. Statistical analysis was performed using unpaired two-tailed Student's t test in a and one-way ANOVA followed by Dunnett's test in cd, gl. NS: not statistically significant.

[0074] Example 7 Inhibition of LPCAT1 can inhibit tumor growth

[0075] To verify the therapeutic potential of LPCAT1 in inhibiting tumor growth in vivo, we established two mouse models: a cell line-derived xenograft model (CDX) and a patient-derived xenograft model (PDX). In the CDX model, mice transplanted with LPCAT1-overexpressing PDLC#1 cells in the liver showed higher tumor growth rate, lower levels of 4-HNE and MDA, and shorter survival time compared with the control group ( Figure 7 ac). In contrast, mice transplanted with LPCAT1-silenced PDLC#2 cells had significantly reduced tumor growth rate, higher levels of 4-HNE and MDA in the liver, and longer survival time compared with mice transplanted with control PDLC#2 cells ( Figure 7 ac). In addition, in PDX models, we found that IKE treatment alone or in combination with AAV-si-LPCAT1 produced potent antitumor effects in mice with LIHC#1 (low expression of LPCAT1) ( Figure 7d). However, in LHC#2 (highly LPCAT1-expressing) mice, AAV-si-LPCAT1 alone significantly inhibited tumor growth, while combined treatment with IKE had a superior antitumor effect than AAV-si-LPCAT1 or IKE alone, accompanied by increased 4-HNE and MDA levels ( Figure 7 ej). In conclusion, inhibition of LPCAT1 expression can induce ferroptosis and thus inhibit tumor growth.

[0076] Figure 7 a: Representative images of tumors. Relative changes in bioluminescent signals of intraperitoneal tumors in NSG mice after Dox treatment (right) were observed in tumor-bearing NSG mice inoculated with the indicated cells at the indicated times in response to Dox treatment (left). N = 6 mice per group. b: Staining scores of MDA (left) and 4-HNE (right) in the indicated xenograft tumors. c: Kaplan-Meier survival of mice after intraperitoneal inoculation of the indicated tumors. N = 6 mice per group. d: Schematic diagram of the orthotopic patient-derived xenograft model. ef: Representative immunohistochemical images of LIHC#1- and LIHC#3 xenograft tumors (scale bar: 40 μM). gh: Kaplan-Meier survival of mice inoculated intraperitoneally with LIHC#1 and LIHC#3 xenograft tumors. N = 6 mice per group. ij: Staining scores of LPCAT1, MDA, 4-HNE, and cleaved-caspase 3. Data are presented as mean ± SD, n = 6 biologically independent samples in ac, ej. Statistical analysis Unpaired two-tailed Student's t test was used in b, one-way ANOVA followed by Duntt's test was used in ac and gh, and two-way ANOVA followed by Tukey's test was used in 1-j. NS: not statistically significant.

[0077] In summary, LPCAT1-mediated membrane phospholipid remodeling promotes ferroptosis escape by increasing phospholipid saturation. Our results reveal a key ferroptosis escape pathway and suggest that inhibition of LPCAT1 may be an effective strategy for targeting ferroptosis in cancer therapy.

[0078] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of an agent for silencing or inhibiting LPCAT1 gene expression in combination with a ferroptosis inducer in the preparation of a drug for treating liver cancer, characterized in that: The reagent for silencing or inhibiting LPCAT1 gene expression is siRNA as shown in SEQ ID NO.1; the ferroptosis inducing agent is IKE.

2. A drug for preventing and treating liver cancer, characterized in that: The drug consists of active ingredients and pharmaceutically acceptable excipients; the active ingredients are reagents for silencing or inhibiting LPCAT1 gene expression and ferroptosis inducers, the reagents for silencing or inhibiting LPCAT1 gene expression are siRNAs as shown in SEQ ID NO.1; the ferroptosis inducer is IKE.

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