Application of combination of PPARalpha agonist and NPC1L1 inhibitor in tumor treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a PPARalpha agonist combined with an NPC1L1 inhibitor in tumor treatment. Specifically, it is proved for the first time that systemic PKM2 deficiency can cause increase of the total cholesterol level and promote growth of homologous allograft tumors, and the phenomenon is partially attributed to increase of the expression level of intestinal NPC1L1. In addition, in breast cancer cells, PKM2 gene knockout not only weakens the activity of a PPARalpha signal channel, but also improves the expression of NPC1L1. Fenofibrate and Ezetimibe can be used for cooperatively inhibiting proliferation and metastasis of tumor cells in vitro and in vivo, so that the Fenofibrate and Ezetimibe have important clinical significance and social value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of PPARα agonists combined with NPC1L1 inhibitors in tumor treatment. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the harsh metabolic environment of oxygen and nutrient restriction, cancer cells adapt to this variable environment by cleverly balancing endogenous biosynthesis and exogenous lipid uptake, thereby promoting their proliferation. This subtle change is a double-edged sword, but it also provides a promising therapeutic target for cancer treatment.

[0004] Pyruvate kinase (PK) is a key enzyme in the glycolysis pathway, responsible for catalyzing the final step of the reaction. Four PK isoforms (L, R, M1, and M2) have been identified in mammalian cells. Although PKM is universally expressed in various tissues, the ratio of M1 to M2 varies among different tissues. PKM2 is mainly expressed in tissues such as the lung, intestine, kidney, and brain, and is the predominant form in proliferating cells, including cancer cells. The small molecule compound TEPP-46, an activator of PKM2, promotes the formation of its tetramer and may help prevent complications of diabetes and its kidney disease. The role of PKM2 in cancer development and progression has been extensively studied and was once considered a potential target for cancer therapy. However, recent studies have shown that PKM2 deficiency exacerbates breast tumor formation in a mouse model of breast cancer. In addition, PKM2-deficient mice also exhibit hepatic steatosis and the development of advanced spontaneous hepatocellular carcinoma (HCC). We have also previously reported that systemic PKM2 deficiency leads to elevated serum total cholesterol levels and promotes the growth of homogeneous xenografts.

[0005] Cholesterol is a crucial lipid, not only a major component of cell membranes but also a precursor to the synthesis of steroid hormones, sex hormones, and vitamin D. Serum cholesterol levels in the human body are primarily regulated through de novo synthesis in the liver and absorption in the intestines. Ezetimibe, a promising cholesterol-lowering drug, reduces cholesterol absorption by inhibiting the activity of Niemann-Pick C1-like 1 (NPC1L1) and has been approved for the treatment of hypercholesterolemia. Notably, NPC1L1, in addition to its role in cholesterol transport, is also involved in the transport of vitamin E, vitamin K, and saturated fatty acids. Clinical trials have explored the combined use of Ezetimibe with other cholesterol-lowering drugs. For example, compared to placebo or other oral monotherapy, the combination of Ezetimibe and bempedoic acid significantly reduced LDL cholesterol levels. In addition to treating hypercholesterolemia, inhibiting NPC1L1 expression via Ezetimibe or genetic methods can improve hepatic insulin resistance and steatosis in Zucker's obese fat rats (ZOF) and mice fed a high-fat diet (HFD). Due to the relatively low expression level of NPC1L1 in tumor tissues, research on its impact on cancer remains limited.

[0006] Peroxisome proliferation-activated receptors (PPARs) are members of the nuclear receptor superfamily and play a crucial role as ligand-activated transcription factors. Currently, three closely related PPAR isoforms have been identified: α, β / δ, and γ. Among them, PPARα regulates the expression of key genes involved in lipid and lipoprotein metabolism and is considered a key regulator of lipid oxidation. Natural ligands for PPARα include unsaturated and saturated fatty acids and their derivatives. Furthermore, insulin-mediated phosphorylation modulates the transcriptional activity of PPARα. Therefore, PPARα agonists can improve insulin sensitivity in obese and insulin-resistant rodents. PPARα is primarily expressed in metabolically active cells with strong fatty acid oxidation capabilities, such as kidney, heart, and liver cells. However, it is also involved in the development and progression of various cancers, thus possessing great potential as a target for cancer therapy. Fenofibrate, a third-generation cellulose acid, has multiple effects as a PPARα agonist. It is widely used not only to treat hypercholesterolemia and hyperlipidemia but also to manage diabetes and cardiovascular disease. The combination therapy of Fenofibrate and Ezetimibe can improve the overall atherosclerotic lipid profile in patients with mixed hyperlipidemia and type II diabetes. However, the potential benefits of this combination therapy in cancer treatment require further investigation. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided the application of PPARα agonists combined with NPC1L1 inhibitors in tumor treatment. Specifically, this invention is the first to demonstrate that systemic PKM2 deficiency leads to elevated total cholesterol levels and promotes the growth of allogeneic xenograft tumors, a phenomenon partly attributed to increased intestinal NPC1L1 expression. Furthermore, in breast cancer cells, PKM2 gene knockout not only weakens the activity of the PPARα signaling pathway but also enhances NPC1L1 expression. Fenofibrate and Ezetimibe synergistically inhibit tumor cell proliferation and metastasis both in vitro and in vivo. Based on the above research findings, this invention was completed.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0009] A first aspect of the invention provides the use of a PPARα agonist in combination with an NPC1L1 inhibitor in any one or more of the following:

[0010] a) Synergistically inhibit tumor cell proliferation or prepare products that synergistically inhibit tumor cell proliferation;

[0011] b) Synergistically induce endoplasmic reticulum stress and death in tumor cells or prepare products that synergistically induce endoplasmic reticulum stress and death in tumor cells;

[0012] c) Synergistically activate IRE1α / XBP1 signaling in tumor cells or prepare products that synergistically activate IRE1α / XBP1 signaling in tumor cells;

[0013] d) Synergistically inhibit tumor growth or prepare products that synergistically inhibit tumor growth;

[0014] e) Prepare products for tumor treatment.

[0015] In a second aspect, the present invention provides a composition wherein the active ingredients of the composition comprise at least the above-mentioned PPARα agonist and NPC1L1 inhibitor.

[0016] The composition has any one or more of the following uses:

[0017] a) Inhibits tumor cell proliferation;

[0018] b) Induces endoplasmic reticulum stress and cell death in tumor cells;

[0019] c) Activate IRE1α / XBP1 signaling in tumor cells;

[0020] d) Inhibit tumor growth;

[0021] e) Tumor treatment.

[0022] A third aspect of the present invention provides a method for treating a tumor, the method comprising administering to a subject the above-described PPARα agonist in combination with an NPC1L1 inhibitor or the above-described composition.

[0023] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0024] The above-mentioned technical solutions have been experimentally proven to produce a strong synergistic anti-tumor effect by simultaneously targeting PPARα and NPC1L1. Specifically, systemic PKM2 knockout increases serum total cholesterol levels and promotes the growth of homogeneous xenograft tumors, a phenomenon that may be related to increased NPC1L1 expression. Furthermore, the above-mentioned technical solutions also found that PKM2 knockout weakens the activity of the PPARα signaling pathway and increases the expression level of NPC1L1 in breast cancer cells. The increased NPC1L1 expression may be partly due to the activation of Nrf1 / 2. At the cellular level, the combined use of fenofibrate and ezetimibe significantly induced cytoplasmic vacuolation and cell death in breast cancer cells, while the IRE1α / XBP1s pathway was significantly activated. At the animal level, fenofibrate and ezetimibe synergistically inhibited tumor growth in vivo.

[0025] The results of the above technical solutions demonstrate the potential application value of simultaneously targeting PPARα and NPC1L1 in the treatment of tumors (such as breast cancer). Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 HFD promotes an increase in total cholesterol levels caused by systemic knockout or inhibition of the PKM2 gene.

[0028] (A) PKM2 after HFD fl / fl and PKM2 - / - Changes in mouse body weight. (B) Measurement of serum total cholesterol levels in (A). (C) Measurement of total cholesterol levels in liver tissue in (A). (D) Measurement of serum total cholesterol levels in mice after two weeks of specific treatment. (E) PKM2 mice fed a high-fat diet for 8 weeks. fl / fl and PKM2 - / - IHC staining of PKM2, PKM1, and NPC1L1 in mouse small intestinal tissue. (F) PKM2 mice fed a high-fat diet for 8 weeks. fl / fl and PKM2 - / -IHC staining of PKM2, PKM1, PKLR, and NPC1L1 in mouse liver tissue. RD: normal diet; HFD: high-fat diet. Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's t-test, with *P < 0.05, **P < 0.01, and ***P < 0.001, and ns indicating no significance.

[0029] Figure 2 Ezetimibe alleviated the increase in total cholesterol levels and the growth of xenograft tumors caused by systemic PKM2 gene knockout.

[0030] (A) Measure PKM2 after specified treatment - / - or PKM2 fl / fl Circulating cholesterol levels in mice. Transplantation of (B,C)py8119WT(B) or PKM2 KO(C) cell lines into systemic PKM2... - / - or PKM2 fl / fl Tumor growth curves in mice after treatment with different drugs. (D) shows the weight of xenograft tumors in groups (B and C). (E) shows the anatomical morphology of xenograft tumors in groups (B and C). (F) shows the relative LDL-C levels in mice in groups (B and C). 100 μl of plasma was extracted from mice in groups B and C before death for LDL-C analysis. Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's t-test; *P < 0.05, **P < 0.01, and ***P < 0.001, ns indicates no significance.

[0031] Figure 3 PKM2 KO increased the expression of NPC1L1 in breast cancer cells.

[0032] (A) Volcano plot showing differentially expressed genes between PKM2 KO and control cells. (B) q-RTPCR analysis of NPC1L1 mRNA levels in the PKM2KO breast cancer cell line. (C) Western blot analysis of NPC1L1, PPARα, and PKM2 protein levels in the PKM2 KO breast cancer cell line. (D) Inhibitory effect of Ezetimibe on Cal51 WT and PKM2 KO cells. (E, F) NPC1L1 mRNA (E) and protein (F) levels after treatment of Cal51 cells with different doses of Ezetimibe. (G, H) ChIP-qPCR analysis of Nrf1 (G) and Nrf2 (H) enrichment levels at the NPC1L1 promoter in Cal51 WT and PKM2 KO cells. IgG was used as a negative control. The enrichment of Nrf1 / 2 in the NPC1L1 promoter region was normalized to the IgG control. (I) Western blot analysis of NPC1L1 protein levels after Cal51 treatment with ML385 (an Nrf2 inhibitor). Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's t-test, with *P < 0.05, **P < 0.01, and ***P < 0.001, and ns indicating no significance. Figure 4(A) Significantly enriched pathways obtained after gene set enrichment analysis of the Cal51 PKM2 KO cell line. (B) Cell growth curves of Cal51 WT and PKM2 KO cells after treatment with a specified dose of Fenofibrate. (C, D) mRNA (C) and protein (D) levels of NPC1L1 in Cal51 cells after treatment with different doses of Fenofibrate. (E) Inhibitory effect of Ezetimibe, Fenofibrate, or combined treatment on cell proliferation. Cells were treated with the specified drugs for 24 h, and the treatment group and control group were normalized. (F) Synergistic effect score curves of Cal51 WT and PKM2 KO cells after treatment with Fenofibrate and / or Ezetimibe. Synergistic effect score was calculated using SynergyFinder 2.0. (G) Representative synergistic effect score plot of Cal51 WT and PKM2 KO cells in (F). (H) Synergistic effect score of MCF7, MDA-MB-231, HCT116 and BT29 cells after treatment with Fenofibrate and / or Ezetimibe. (I) Representative morphological images of Cal51 cells in DMSO, Fenofibrate, Ezetimibe and combination therapy groups. (J) Changes in reactive oxygen species (ROS) after treatment of Cal51 cells with Fenofibrate, Ezetimibe and combination therapy. (KL) Flow cytometry analysis of the effects of Fenofibrate, Ezetimibe and combination therapy on apoptosis of Cal51 cells. (K) shows the flow cytometry analysis of Cal51 cells after 18 h of corresponding treatments, and (L) shows the apoptosis rate of Cal51 cells after 18 h of corresponding treatments.

[0033] Figure 5(A) Heatmap showing different gene expression patterns in the DMSO, Fenofibrate, Ezetimibe, and combination therapy groups, synergistic ER stress induced by Fenofibrate and Ezetimibe. (B) Volcano plot showing differentially expressed genes between the Fenofibrate and DMSO groups. (C) Volcano plot showing differentially expressed genes between the Ezetimibe and DMSO groups. (D) Volcano plot showing differentially expressed genes between the combination therapy group and DMSO. (E, F) Pathway enrichment analysis of Ezetimibe and DMSO (E) or combination therapy and DMSO (F) in Cal-51 cells. The numbers in the bar charts represent the number of differentially expressed genes enriched in that pathway. (G) Venn plot showing upregulated and downregulated genes in the Fenofibrate, Ezetimibe, and combination therapy groups relative to the DMSO group. (H) Volcano plot showing differentially expressed genes between the combination therapy group and the Ezetimibe group. (I) Signal pathways that were significantly enriched after GSEA enrichment analysis of the combined drug administration group and the Ezetimibe group using Hallmark gene set.

[0034] Figure 6 Fenofibrate and Ezetimibe synergistically activate the IRE1α / XBP1 signaling pathway. (A) Western blot analysis of protein expression levels of IRE1α, XBP1s, PERK, and ATF6 in Cal51 cells after treatment with DMSO, Fenofibrate, Ezetimibe, and a combination thereof. (B) Heatmap showing the ChIP-seq signal distribution of XBP1s at the transcription start site (TSS) in Cal51 cells, extending 2.0 kb on each side. Comprehensive cross-sectional view of XBP1s ChIP-seq signals in different groups within ±2.0 kb of the peak center. (C) Cal51 cells treated with DMSO, Fenofibrate, Ezetimibe, or a combination thereof for 8 h. (D) XBP1s peak diagrams of DNAJB9, HSPA5, MYC, and XBP1 gene regions under different drug treatment conditions. (E) Venn plot shows upregulated and downregulated genes whose RNA seq data overlap with XBP1s ChIP-seq peaks. (F) qRT-PCR detection of mRNA levels of the genes shown. Combined drug treatment of Cal51 cells significantly upregulated the mRNA levels of the genes shown, and these regions were enriched with a large number of XBP1s.

[0035] Figure 7 Fenofibrate and Ezetimibe synergistically inhibit tumor growth in vivo.

[0036] (A) Synergistic effect score curve of 4T1 and Py8119 cells after combined drug treatment. SynergyFinder 2.0 was used to calculate the synergistic effect score. (BC) are representative synergistic effect scores of 4T1 and Py8119 in (A). (D) Tumor growth curve of Py8119 cells under specified treatments. (E) Tumor weight under different drug treatments. (F) Tumor images under different drug treatments. (G) Ki67 immunohistochemical staining of tumors. Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's test, *P<0.05, **P<0.01 and ***P<0.001, ns indicates no significance.

[0037] Figure 8 PKM2 KO can alleviate HFD-induced lung metastasis of 4T1 cells in mice.

[0038] (A) Bioluminescence intensity at different time points after injection of 4T1 cells in mice of the HFD and RD groups. Mice were fed with HFD or RDs for 2 weeks and then injected with 1x10 cells via the tail vein. 5 (A) Stable luciferase-expressing 4T1 cells. (B) Representative images of the RD and HFD groups. (C) Survival curves of mice in (A). (D) Western blot analysis of PKM1 and PKM2 protein expression levels in 4T1 PKM2 KO and WT cells. (E) Bioluminescence intensity of mice over a period of time after injection of 4T1 WT or PKM2 KO cells. (F) Survival curves of mice under different treatments in (E). Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's t-test, *P<0.05, **P<0.01 and ***P<0.001, ns indicates no significance.

[0039] Figure 9 PKM2 KO cells show increased sensitivity to Ezetimibe.

[0040] (AC) Western blot analysis of PKM1 and PKM2 expression levels in MCF7 cells (A), MDA-MB-231 cells (B), and Cal51 cells (C). (D) Colony formation results of Cal51WT and PKM2 KO cells treated with different doses of Ezetimibe. (E) Quantification of the number of single colonies in (D). Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's t test, with *P < 0.05, **P < 0.01, and ***P < 0.001, and ns indicating no significance.

[0041] Figure 10 HMGCR inhibitors and Ezetimibe can synergistically inhibit Cal51 cell proliferation.

[0042] Figure 11 (A) Synergistic effect of Fenofibrate and Ezetimibe on MCF710A cell proliferation. (B) Cell morphology of MCF10A cells treated with Fenofibrate (100µm) in combination with Ezetimibe (50µm) at different time points. (C) Cell morphology of MCF10A cells treated with specific drug concentrations at different time points after reduction. (D) Cell morphology of Cal51 cells treated with specific drug concentrations at different time points after reduction.

[0043] Figure 12 Fenofibrate and Ezetimibe synergistically induced apoptosis in (A) Cal51 cells after 10 h of corresponding treatments, by flow cytometry; (B) apoptosis rate of Cal51 cells after 10 h of corresponding treatments. Results are expressed as mean ± SD. Significance was determined using a two-tailed unpaired Student's t-test; *P < 0.05, **P < 0.01, and ***P < 0.001, ns indicated no significance.

[0044] Figure 13 Fenofibrate and Ezetimibe synergistically induce ER stress. (A) GSEA pathway enrichment analysis using the Reactome gene set in the Fenofibrate and DMSO groups. (B) KEGG pathway enrichment analysis in the Ezetimibe and DMSO groups. (C) KEGG pathway enrichment analysis in the combined drug group and DMSO group. (D) Significantly enriched pathways in the combined drug group and DMSO treatment group. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0046] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0047] In a typical embodiment of the present invention, the use of a PPARα agonist in combination with an NPC1L1 inhibitor in any one or more of the following is provided:

[0048] a) Synergistically inhibit tumor cell proliferation or prepare products that synergistically inhibit tumor cell proliferation;

[0049] b) Synergistically induce endoplasmic reticulum stress and death in tumor cells or prepare products that synergistically induce endoplasmic reticulum stress and death in tumor cells;

[0050] c) Synergistically activate IRE1α / XBP1 signaling in tumor cells or prepare products that synergistically activate IRE1α / XBP1 signaling in tumor cells;

[0051] d) Synergistically inhibit tumor growth or prepare products that synergistically inhibit tumor growth;

[0052] e) Prepare products for tumor treatment.

[0053] The molar ratio of PPARα agonist to NPC1L1 inhibitor is 2-12:1-3; further including 6-12:1-3; 4-8:1-3; and 2-6:1-2.

[0054] The PPARα agonist includes, but is not limited to, miRNAs or chemical mimics of said miRNAs or analogs of said miRNAs; it can also be a promoter or lentivirus that upregulates PPARα expression, as well as small molecule compounds or oligonucleotides, such as small molecule compounds that can perform post-translational modifications of proteins, and also includes compound agonists or promoters. In one specific embodiment of the present invention, the PPARα agonist includes fenofibrate, which is a highly effective PPARα agonist.

[0055] The NPC1L1 inhibitors include, but are not limited to, RNA interference molecules or antisense oligonucleotides targeting the NPC1L1 encoding gene, small molecule inhibitors, shRNA, siRNA, substances for lentiviral infection or gene knockout, and specific antibodies against NPC1L1 itself or its upstream and downstream molecules, such as anti-NPC1L1 antibodies, and may also include compound inhibitors. In one specific embodiment of the invention, the NPC1L1 inhibitor includes ezetimibe, which is a highly effective NPC1L1 inhibitor.

[0056] The product may be a drug or a general experimental reagent for non-medical purposes. The general experimental reagent can be used for basic research to explore the mechanism of tumor development and progression.

[0057] It should be noted that the term "tumor" is used in this invention as is known to those skilled in the art, and includes benign tumors and / or malignant tumors. A benign tumor is defined as the excessive proliferation of cells that cannot form an invasive, metastatic tumor in the body. Conversely, a malignant tumor is defined as cells with various cellular and biochemical abnormalities that can cause systemic disease (e.g., tumor metastasis in distant organs).

[0058] In another specific embodiment of the present invention, the medicament of the present invention can be used to treat malignant tumors. Examples of malignant tumors that can be treated with the medicament of the present invention include solid tumors and hematologic malignancies. Solid tumors can be tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as the thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin (such as melanoma), ureter, vagina, and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and Wilms' tumor. Furthermore, malignant tumors include primary tumors in the said organs and corresponding secondary tumors (tumor metastases) in distant organs. Hematologic malignancies can include, for example, aggressive and painless forms of leukemia and lymphoma, namely non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkin's disease, multiple myeloma, and T-cell lymphoma. It also includes myelodysplastic syndromes, plasmacytoma, tumor-like syndromes, cancers of unknown primary location, and AIDS-related malignancies. No specific limitations are set here.

[0059] According to the present invention, when the product is a drug, the drug may further include at least one inactive pharmaceutical ingredient.

[0060] The inactive ingredient of the drug may be a pharmaceutically acceptable carrier that can be determined by a person skilled in the art to meet clinical standards. It may be any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delayers, and the like. Typically, the nature of the carrier depends on the specific route of administration. For example, parenteral preparations often contain an injectable fluid as a vehicle, which may be a pharmaceutically and physiologically acceptable fluid, such as water, saline, balanced salt solution, glucose solution, glycerin, etc. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate. No specific limitations are made here.

[0061] In another specific embodiment of the invention, the drug of the invention can be administered into the body by known means, such as intravenous systemic delivery. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the invention can vary considerably depending on a variety of factors, such as target cells, biological type or tissue, the general condition of the subject to be treated, route of administration, manner of administration, etc.

[0062] In another specific embodiment of the present invention, the drug can be applied to humans and non-human mammals, including mice, rats, guinea pigs, cattle, sheep, cats, dogs, horses, monkeys, orangutans, etc., without being specifically limited here.

[0063] In another specific embodiment of the present invention, a composition is provided, wherein the active ingredients of the composition include at least the above-mentioned PPARα agonist and NPC1L1 inhibitor.

[0064] The composition has any one or more of the following uses:

[0065] a) Inhibits tumor cell proliferation;

[0066] b) Induces endoplasmic reticulum stress and cell death in tumor cells;

[0067] c) Activate IRE1α / XBP1 signaling in tumor cells;

[0068] d) Inhibit tumor growth;

[0069] e) Tumor treatment.

[0070] Specifically, the definition and scope of tumors in the aforementioned uses are as described above, and therefore will not be repeated here.

[0071] The molar ratio of PPARα agonist to NPC1L1 inhibitor is 2-12:1-3; further including 6-12:1-3; 4-8:1-3; and 2-6:1-2.

[0072] The PPARα agonist includes, but is not limited to, miRNAs or chemical mimics of said miRNAs or analogs of said miRNAs; it can also be a promoter or lentivirus that upregulates PPARα expression, as well as small molecule compounds or oligonucleotides, such as small molecule compounds that can perform post-translational modifications of proteins, and also includes compound agonists or promoters. In one specific embodiment of the present invention, the PPARα agonist includes fenofibrate, which is a highly effective PPARα agonist.

[0073] The NPC1L1 inhibitors include, but are not limited to, RNA interference molecules or antisense oligonucleotides targeting the NPC1L1 encoding gene, small molecule inhibitors, shRNA, siRNA, substances for lentiviral infection or gene knockout, and specific antibodies against NPC1L1 itself or its upstream and downstream molecules, such as anti-NPC1L1 antibodies, and may also include compound inhibitors. In one specific embodiment of the invention, the NPC1L1 inhibitor includes ezetimibe, which is a highly effective NPC1L1 inhibitor.

[0074] In another specific embodiment of the present invention, a method for tumor treatment is provided, the method comprising administering to a subject a therapeutically effective amount of the above-mentioned PPARα agonist combined with an NPC1L1 inhibitor or the above-mentioned combination.

[0075] In this invention, the term "therapeuticly effective amount" refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dosage and time period. A therapeutically effective amount of an agent may eliminate, reduce, delay, minimize, or prevent adverse effects of a disease.

[0076] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.

[0077] Example

[0078] Materials and methods

[0079] cell lines

[0080] Human breast cancer cell lines MCF-7, MDA-MB-231, and Cal51 were purchased from ATCC and cultured in DMEM (Gibco) medium containing 10% fetal bovine serum (FBS, ExCell) and 1% penicillin-streptomycin (P / S, Gibco). Mouse-derived cell lines Py8119 and 4T1 were purchased from ATCC and cultured in F12K / DMEM and RPMI 1640 medium, respectively, containing 10% FBS and 1% P / S. Cells were cultured in a 37°C, 5% CO2 humidified incubator. Cell lines were identified by short tandem repeat atlases and routine mycoplasma contamination tests were performed.

[0081] Antibodies and drugs

[0082] Information on antibodies and drugs is shown in Table 1 below.

[0083] Table 1 Antibody and Drug Information

[0084]

[0085] Apoptosis detection

[0086] Cells (8×10) 5 Cells were seeded in 6-well plates, with 2 mL of DMEM added to each well. Cells were then treated with Fenofibrate, Ezetimibe, and a combination of Fenofibrate and Ezetimibe. After culturing at 37°C for 8 h, cells were digested, centrifuged, and washed twice with cold PBS. Apoptosis was measured using Annexin V / APC and propidium iodide (PI) staining (BestBio, BB-4101). Flow cytometry analysis was performed using a CytoFLEX S flow cytometer (Backman). Data analysis was performed using FlowJo (version 10.8.1).

[0087] RNA-seq analysis

[0088] Cells were collected and washed twice with cold PBS, then processed using the Sparkeasy Cell RNA Extraction Kit (SparkJade). For RNA-seq analysis, samples were sent to Novogene Biotechnology Co., Ltd. (China) and sequenced using the Illumina platform. Sequencing reads were aligned to the human genome (hg38) using HISAT2. Duplicate reads and reads with low map quality were removed using the Sambamba tool (v0.7.1). Reads were counted using feature counting (subread package, v2.0.1) to measure gene expression. Differentially expressed genes were identified using DESeq2 (v1.38.3) in R, requiring at least a two-fold change and an adjusted p-value less than 0.05. GSEA analysis was performed on all detected genes with p < 0.05 in the four samples using a marker gene set.

[0089] Chromatin immunoprecipitation sequencing (ChIP-seq)

[0090] Cells were fixed with 1% formaldehyde for 15 minutes. Cross-linked cells were decross-linked with 0.125M glycine for 5 minutes at room temperature. Cross-linked cells were lysed with a series of buffers. Cell nuclei were resuspended in 0.5 ml nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, 1 mM PMSF, and 1x protease inhibitor) and incubated on ice for 10 minutes. Subsequently, chromatin was sonicated in an ice-water bath using a Branson Sonifier 450. Equal volumes of total DNA and antibody were used for chromatin immunoprecipitation. Eluted DNA fragments were purified using AMPure XP Beads (Beckman). ChIP-seq libraries were used according to the manufacturer's instructions. Ultra TMIIDNALibrary Prep Kit for The library was prepared using (NEB, E7645S). Sequencing was performed using an Illumina HiSeq 4000.

[0091] Cell proliferation assay

[0092] Cell proliferation analysis was performed using the Cell Counting Kit-8 (CCK8, Beyotime) according to the manufacturer's instructions. Cells were seeded at a density of 5,000-8,000 cells / well in 96-well plates and cultured for 3 days in the presence of a single drug or a combination of drugs. CCK-8 reagent was added to each well, and the cells were incubated for 2 hours. Absorbance was measured at 45 nm. SynergyFinder (version 3.0) was used to quantify the degree of synergistic effect between the two drugs.

[0093] Immunohistochemical (IHC) staining

[0094] Fresh tumor tissue was fixed in 10% formalin solution, embedded in paraffin, and sectioned. After heating at 56°C for half an hour, the tissue sections were hydrated using xylene and ethanol. Antigen retrieval was performed with 10 mM citrate buffer, followed by blocking with goat serum for one hour. The tissue sections were incubated overnight at 4°C with Ki67 antibody (TA500265, ZSGB-BIO). Then, the tissue sections were incubated at room temperature for 20 minutes with streptavidin-conjugated HRP secondary antibody (PV-9000, ZSGB-BIO). Expression signal intensity was detected using a DAB detection kit. Slides were reverse stained with hematoxylin and then covered with coverslips using Cytoseal™ XYL (xylene-based loading medium).

[0095] Animal experiments and mouse models

[0096] All animal experiments were conducted in accordance with the protocols approved by the Ethics Committee of Qilu Medical College of Shandong University, and complied with the ethical regulations for animal research.

[0097] Allogeneic tumor transplantation experiment

[0098] Xenograft tumor experiments were conducted using approximately 6-week-old female C57BL / 6 mice. Six Py8119 cells (1 x 10^6 cells) were injected into the mammary fat pads of the inguinal region of each mouse. Mice were randomly assigned to four groups. Mice were orally administered daily with either Fenofibrate (100 mg / kg, suspended in corn oil), Ezetimibe (50 mg / kg, suspended in corn oil), or a combination of Fenofibrate and Ezetimibe (100 mg / kg + 50 mg / kg, suspended in corn oil). Tumor size was measured using calipers, and tumor volume was calculated using the formula 1 / 2 × L × W^2.

[0099] Statistics and reproducibility

[0100] Statistical tests were performed using unpaired two-tailed Student's t-tests and / or ANOVA. Unless otherwise stated, all experiments were repeated at least three times. The N number is indicated in the legend. P < 0.05 (*) was considered statistically significant.

[0101] Research Results

[0102] In a mouse model, PKM2 is involved in high-fat diet-induced lung metastasis of breast cancer cells.

[0103] The incidence of obesity and dyslipidemia is rising sharply worldwide, profoundly impacting cancer morbidity and mortality. Western dietary patterns are considered a core contributing factor to this health crisis. This study aimed to evaluate the effects of a high-fat diet (HFD) on lung metastasis of breast cancer cells. Six-week-old female BALB / c mice were used in the experiment and divided into a normal diet (RD) group and a high-fat diet (HFD) group. After two weeks of feeding, 3x10 mg of high-fat diet was injected into the mice via tail vein. 5 4T1 cells. The results showed that, starting from day 11, a high-fat diet significantly promoted the growth of lung tumors. Figure 8 Consistent with this, the overall survival rate in the HFD group was significantly lower (A, B). Figure 8 C). To elucidate the effect of PKM2 on HFD-induced lung metastases, we specifically knocked out PKM2 in 4T1 cells using the CRISPR Cas9 system. Figure 8 D). Experimental results showed that PKM2 knockout effectively slowed the growth of lung tumors and prolonged the overall survival of mice. Figure 8 (E, F). Furthermore, PKM2 knockout also weakened the negative effects of a high-fat diet on tumor growth and overall survival, highlighting that the cell-autonomous effects of PKM2 are more important in promoting lung metastasis of tumor cells than the tumor microenvironment alterations induced by a high-fat diet. Inhibition of NPC1L1 expression effectively alleviated the increase in total cholesterol levels and significantly inhibited the growth of in vivo allogeneic xenografts induced by systemic PKM2 gene knockout.

[0104] To further elucidate the role of PKM2 in lipid homeostasis in vivo, we constructed a systemic PKM2 gene knockout mouse model. fl / fl Compared to mice, PKM2 - / - Mice fed HFD showed a significant increase in body weight gain and serum total cholesterol levels. Figure 1 A, B). Furthermore, with PKM2 fl / fl Compared to mice, PKM2 - / - After mice were fed HFD for eight weeks, the total cholesterol content in their livers also showed an increasing trend. Figure 1 C). To further clarify the function of PKM2, we subsequently selected wild-type mice and fed them a regular diet and a high-fat diet, while also administering TEPP-46 via gavage. This was in contrast to the PKM2... - / - Similar findings were observed in mice; TEPP-46 exacerbated the high-fat diet-induced increase in serum total cholesterol. Figure 1 D). NPC1L1 is a key sterol sensor responsible for intestinal cholesterol absorption; its deficiency can lead to a 69% reduction in cholesterol absorption. Our study shows that PKM2 deficiency leads to enhanced NPC1L1 expression in the small intestine, but not in the liver, and PKM2... - / - Mice fed a high-fat diet for eight weeks exhibited severe hepatic steatosis. Figure 1 E, F). We also found that Ezetimibe can effectively alleviate PKM2. - / - Increased cholesterol in mice ( Figure 2 A). To simulate the effect of hyperlipidemia on tumor growth and elucidate the antitumor effect of Ezetimibe, we fed mice HFD for two weeks, followed by allogeneic xenograft experiments using wild-type or PKM2 KO Py8119 cells. The results showed that although intracellular PKM2 is crucial for tumor growth ( Figure 2 Systemic PKM2 deficiency, leading to altered microenvironment, still promotes the growth of wild-type and PKM2 KO Py8119 allogeneic xenografts. Administration of Ezetimibe to tumor-bearing mice significantly reduced the growth of both wild-type and PKM2 KOPy8119-derived tumors. Figure 2 Ezetimibe (BE) may be due to its ability to lower circulating cholesterol levels. Figure 2 F). In summary, NPC1L1 is a key factor regulating the growth of xenograft tumors induced by systemic PKM2 gene knockout.

[0105] PKM2 knockout increases NPC1L1 expression in breast cancer cells.

[0106] Given the crucial role of PKM2's cell-autonomous function in tumor growth, we systematically investigated the potential functions of PKM2 using breast cancer cells as an example. To this end, we constructed several PKM2 KO breast cancer cell lines (…). Figure 9 AC), and transcriptomic (RNA-seq) analysis was performed in Cal51 cells. We observed a significant increase in NPC1L1 expression levels in PKM2 KO cells (AC). Figure 3 A), real-time quantitative PCR and Western blot analysis corroborated this finding. Figure 3(B, C). Given that PKM2 KO can induce NPC1L1 expression, we further evaluated the effect of Ezetimibe on cell proliferation. The results showed that PKM2 KO cells were more sensitive to Ezetimibe treatment compared to control cells. Figure 3 D and Figure 9 (D, E). Furthermore, we noted that Ezetimibe can induce the expression of NPC1L1 ( Figure 3 E, F). Besides being an inhibitor of NPC1L1, Ezetimibe is also a potent activator of nuclear factor-E2 p45-associated factor 2 (Nrf2). Our previous study found that PKM2KO can promote Nrf1 cleavage and activation. Both Nrf1 and Nrf2 belong to the CNC transcription factor family and regulate gene expression through antioxidant response elements (AREs). By screening transcription factor binding motifs, we identified a potential ARE motif in the NPC1L1 promoter region (…). Figure 3 G). To investigate whether PKM2 knockout increases NPC1L1 expression by activating Nrf1 / 2, we performed chromatin immunoprecipitation combined with quantitative PCR (ChIP-qPCR) experiments. The results showed that Nrf1 was slightly enriched in the promoter region of NPC1L1 in control cells, and this trend was significantly enhanced with PKM2 knockout. Simultaneously, Nrf2 enrichment was also evident in control cells, and the enrichment was further enhanced after PKM2 knockout. Figure 3 GH). Furthermore, Nrf2 inhibitors reduced the protein level of NPC1L1 (GH). Figure 3 I). These data indicate that PKM2 KO can induce the expression of NPC1L1 in breast cancer cells by activating Nrf1 / 2.

[0107] Fenofibrate and Ezetimibe synergistically inhibit cancer cell proliferation.

[0108] Gene feature enrichment analysis (GSEA) results showed that PPARα-mediated lipid metabolism was decreased in Cal51-PKM2 KO cells. Figure 4 A), while the protein level of PPARα did not change significantly. Figure 3 C). Fenofibrate plays a crucial role in lowering circulating cholesterol and triglyceride levels, and it also exhibits significant anti-tumor effects. To investigate the effect of fenofibrate on breast cancer cell proliferation, we performed a CCK-8 assay. Figure 4As shown in Figure D, different concentrations of Fenofibrate had little effect on the proliferation of wild-type and PKM2 KO Cal51 cells during the first three days. Only with prolonged treatment could Fenofibrate effectively inhibit the proliferation of both wild-type and PKM2 KO Cal51 cells. Figure 4 B). It has been reported that Fenofibrate specifically reduces small intestinal cholesterol absorption in a PPARα-dependent manner by inhibiting NPC1L1 expression and negatively impacts the Ezetimibe-sensitive pathway in vivo. However, another study showed that PPARα and Fenofibrate can positively regulate NPC1L1 expression, and PGC1α can further stimulate PPARα / RXRα-mediated NPC1L1 promoter activity in HepG2 cells. We observed that NPC1L1 significantly increased at both the transcriptional and translational levels after Fenofibrate treatment. Figure 4 (C, D) Given that Fenofibrate can positively regulate NPC1L1 expression, we hypothesized that the combined use of Ezetimibe and Fenofibrate might produce a synergistic inhibitory effect on cell proliferation. Experimental observations revealed that Fenofibrate treatment for 24 hours had no significant effect on cell proliferation; only high concentrations of Ezetimibe showed an inhibitory effect on cell proliferation after 24 hours of treatment. Figure 4 However, when low-dose Ezetimibe was combined with Fenofibrate, cell proliferation was significantly inhibited, suggesting a possible synergistic effect between the two drugs in inhibiting cancer cell proliferation. To test our hypothesis, we treated wild-type and PKM2 KO Cal51 cells with different doses of Fenofibrate and / or Ezetimibe and calculated the synergistic index over four days. The results showed that wild-type cells exhibited a strong synergistic effect in all four days, reaching the maximum synergistic score on day four. However, the synergistic effect was not significant in PKM2 KO cells, which may be due to the inhibition of PPARα signaling pathway activity by the absence of PKM2 (…). Figure 4 F, G). As inhibitors of HMG-CoA reductase (HMGCR), statins are the first-line treatment for hypercholesterolemia. However, the combined use of statins and zetimibe only shows an additive effect. Figure 10 To verify whether the observed synergistic effect was limited to Cal51 cells, we also investigated two other human breast cancer cell lines and two other human colon cancer cell lines. Figure 4H). As expected, we observed a synergistic effect in all four cell lines, suggesting that the combined application of Fenofibrate and Ezetimibe can be broadly applied to different types of cancer cells. Notably, this synergistic effect was not observed in immortalized human mammary epithelial cells MCF10A. Figure 11 A). Although the combined use of high concentrations of Fenofibrate and Ezetimibe can impair the activity of MCF10A ( Figure 11 B), but moderate concentrations of the combined drug did not significantly affect the proliferation of MCF10A cells, but could induce the death of Cal51 cells. Figure 11 C). Given that the combined treatment induces cytoplasmic vacuolation in Cal51 cells and significantly increases reactive oxygen species (ROS) levels ( Figure 4 Following this, we performed apoptosis analysis. The results showed that Fenofibrate alone had no significant effect on apoptosis, while Ezetimibe slightly induced apoptosis. However, when Fenofibrate was used in combination with Ezetimibe, its apoptosis-promoting effect was significantly enhanced, reaching approximately 15% and 80% apoptosis rates at 10 h and 18 h, respectively. Figure 12 as well as Figure 4 In summary, the synergistic effect of Fenofibrate and Ezetimibe can effectively inhibit the proliferation of cancer cells in vitro.

[0109] Fenofibrate and Ezetimibe synergistically induce extreme endoplasmic reticulum stress and cell death.

[0110] To further investigate the mechanism by which Fenofibrate and Ezetimibe synergistically induce cell death, we performed RNA-seq to compare the gene expression profiles of Cal-51 cells treated with Fenofibrate or Ezetimibe alone and in combination with the DMSO control group. Figure 5 A). The results showed that the effect of fenofibrate alone on gene expression was relatively limited, leading to significant upregulation of only 93 genes and downregulation of only 9 genes (|log2FC|≥2, p.adjust<0.05). Notably, most of the genes related to cholesterol biosynthesis showed an upregulation trend. Figure 5 B). Reactome pathway enrichment analysis further revealed that cholesterol biosynthesis and SREBP activation were the most enriched pathways in the Fenofibrate-treated group, which may be related to the negative feedback mechanism induced by lipid deprivation. Figure 13A). In contrast, Ezetimibe had a relatively significant effect on gene expression, significantly upregulating 367 genes and downregulating 105 genes. Similar to Fenofibrate, Ezetimibe also upregulated genes in the steroid biosynthesis pathway ( Figure 5 C and Figure 13 B), and both induced the expression of NPC1L1. Figure 5 B, C). However, compared to the Fenofibrate-treated group, the Ezetimibe-treated group was enriched in different pathways, such as unfolded protein response (UPR) and IREα / XBP1s activation, suggesting that it induced endoplasmic reticulum stress. Figure 5 E). Surprisingly, compared with the DMSO control group, the combined treatment with Fenofibrate and Ezetimibe resulted in a wider range of gene expression changes, including upregulation of 2057 genes and downregulation of 1450 genes (E). Figure 5 D). Notably, the UPR and IREα / XBP1s activation pathways also dominated in the Fenofibrate plus Ezetimibe treatment group, and involved more genes than in the Ezetimibe-only treatment group. Figure 5 F and Figure 13 (C, D) indicates that Fenofibrate exacerbates Ezetimibe-induced endoplasmic reticulum stress. Furthermore, compared to the Ezetimibe-only treatment group, the combined treatment with Fenofibrate and Ezetimibe significantly reduced the expression of mitochondrial-encoded proteins and tRNAs. Figure 5 These changes (G, H) may explain the enrichment of apoptosis and hypoxia pathways, as well as the inhibition of oxidative phosphorylation pathways. Figure 5 I). In summary, Fenofibrate and Ezetimibe synergistically induce endoplasmic reticulum stress and cell death, revealing their potential application value in anticancer therapy.

[0111] Fenofibrate and Ezetimibe synergistically activate IRE1α / XBP1 signaling.

[0112] UPR is regulated by three major endoplasmic reticulum (ER) sensors: activated transcription factor 6 (ATF6), double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and inositol-required enzyme 1 (IRE1). To investigate which branch specifically induced ER stress in the co-treatment of Fenofibrate and Ezetimibe, we assessed the expression of ATF6, PERK, and IRE1α under different treatment conditions. The results showed that only IRE1α showed a significant increase in the co-treatment of Fenofibrate and Ezetimibe. Figure 6 A). When the pathway is activated, IRE1α cleaves an intron of the X-box binding protein 1 (XBP1) precursor mRNA, resulting in a frameshift mutation and the generation of transcriptionally active XBP1 (XBP1s), which in turn initiates the expression of ER stress-related genes. We observed that Ezetimibe moderately induced the expression level of XBP1s, while the combined treatment of Fenofibrate and Ezetimibe significantly increased its expression level. Figure 6 A) indicates that Fenofibrate enhances the activation of the IRE1α / XBP1s axis by Ezetimibe. These findings are consistent with our previous RNA-seq analysis. To investigate the distribution pattern of XBP1s in cells treated with Fenofibrate in combination with Ezetimibe, we performed ChIP-seq and identified more than 1000 XBP1s binding sites in Cal-51 cells. Figure 6 (B, C). However, although the combination of Fenofibrate and Ezetimibe strongly induced XBP1s expression, we did not observe a significant difference compared to Ezetimibe alone. Figure 6 This result indicates that XBP1s target genes are specific and limited in number, and an appropriate amount of XBP1s protein can cover all target genes. Association analysis of ChIP-seq and RNA-seq data revealed that in the Fenofibrate combined with Ezetimibe group and the Ezetimibe alone group, XBP1s ChIP-seq enriched approximately 19.3% (201 / 1043) and 4% (40 / 981) of upregulated genes, respectively. Figure 6 (D) This indicates that the increase in XBP1s induced by the combination therapy can effectively activate gene expression. qRT-PCR further validated this conclusion; for example, Ezetimibe increased the expression levels of DNAJB9, ERN1, and MYC, while the combined treatment of Fenofibrate and Ezetimibe significantly increased the expression levels of these genes. Figure 6 F). In summary, Fenofibrate and Ezetimibe can synergistically activate IRE1α / XBP1 signaling, thereby inducing endoplasmic reticulum stress and cell death.

[0113] Fenofibrate and Ezetimibe synergistically inhibit tumor growth in vivo.

[0114] Similar to the synergistic inhibitory effect of Fenofibrate and Ezetimibe on the proliferation of human cancer cells in vitro, we also observed a synergistic effect of these two drugs in mouse breast cancer 4T1 and Py8119 cell lines. Figure 7 To further investigate the antitumor effects of Fenofibrate and Ezetimibe in vivo, we conducted a tumor xenograft experiment using Py8119 breast cancer cells in C57BL / 6 mice. The results showed that neither Fenofibrate nor Ezetimibe alone effectively inhibited the growth of xenograft tumors. However, when Fenofibrate and Ezetimibe were used in combination, we observed a significant inhibitory effect on tumor growth. Figure 7 DF). To further validate this finding, we performed Ki67 IHC staining, and the results showed that treatment with Fenofibrate combined with Ezetimibe significantly reduced Ki67 expression levels (DF). Figure 7 G), thus further confirming the synergistic effect of these two drugs in inhibiting tumor growth in vivo.

[0115] In summary, this invention reveals the potential role of NPC1L1 as a downstream gene of PKM2 in breast cancer treatment. Fenofibrate and Ezetimibe demonstrate the potential to synergistically inhibit breast cancer cell growth by simultaneously targeting PPARα and NPC1L1.

[0116] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The use of PPARα agonists in combination with NPC1L1 inhibitors in any one or more of the following: a) Synergistically inhibit tumor cell proliferation or prepare products that synergistically inhibit tumor cell proliferation; b) Synergistically induce endoplasmic reticulum stress and death in tumor cells or prepare products that synergistically induce endoplasmic reticulum stress and death in tumor cells; c) Synergistically activate IRE1α / XBP1 signaling in tumor cells or prepare products that synergistically activate IRE1α / XBP1 signaling in tumor cells; d) Synergistically inhibit tumor growth or prepare products that synergistically inhibit tumor growth; e) Prepare products for tumor treatment.

2. The application as described in claim 1, characterized in that, The molar ratio of the PPARα agonist to the NPC1L1 inhibitor is 2-12:1-3.

3. The application as described in claim 1, characterized in that, The PPARα agonist includes miRNA or a chemical mimic of the miRNA or an analog of the miRNA; a promoter or lentivirus that upregulates PPARα expression; and small molecule compounds or oligonucleotides, as well as compound agonists or promoters; further, the PPARα agonist includes fenofibrate.

4. The application as described in claim 1, characterized in that, The NPC1L1 inhibitors include RNA interference molecules or antisense oligonucleotides targeting the NPC1L1 encoding gene, small molecule inhibitors, shRNA, siRNA, substances for lentiviral infection or gene knockout, and specific antibodies against NPC1L1 itself or its upstream and downstream molecules, and also include compound inhibitors; furthermore, the NPC1L1 inhibitors include ezetimibe.

5. The application as described in claim 1, characterized in that, The product is a pharmaceutical or non-pharmaceutical test reagent intended for use in basic research.

6. The application as described in claim 1, characterized in that, When the product is a drug, the drug also includes at least one inactive pharmaceutical ingredient.

7. A composition, characterized in that, The active ingredients of the composition include at least a PPARα agonist and an NPC1L1 inhibitor; Furthermore, the composition has any one or more of the following uses: a) Inhibits tumor cell proliferation; b) Induces endoplasmic reticulum stress and cell death in tumor cells; c) Activate IRE1α / XBP1 signaling in tumor cells; d) Inhibit tumor growth; e) Tumor treatment.

8. The composition according to claim 7, characterized in that, The molar ratio of PPARα agonist to NPC1L1 inhibitor is 2-12:1-3.

9. The composition according to claim 7, characterized in that, The PPARα agonist includes miRNA or a chemical mimic of the miRNA or an analog of the miRNA; a promoter or lentivirus that upregulates PPARα expression; and small molecule compounds or oligonucleotides, as well as compound agonists or promoters; further, the PPARα agonist includes fenofibrate.

10. The composition according to claim 7, characterized in that, The NPC1L1 inhibitors include RNA interference molecules or antisense oligonucleotides targeting the NPC1L1 encoding gene, small molecule inhibitors, shRNA, siRNA, substances for lentiviral infection or gene knockout, and specific antibodies against NPC1L1 itself or its upstream and downstream molecules, and also include compound inhibitors; furthermore, the NPC1L1 inhibitors include ezetimibe.

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