Application of ferroptosis inhibitor ANAPC11 as a biomarker for triple-negative breast cancer

Abstract

This invention discloses the application of ferroptosis inhibitor ANAPC11 as a biomarker for triple-negative breast cancer. Specifically, this invention discloses the use of the ANAPC11 protein, its encoding gene, or detection reagents thereof for the preparation of diagnostic reagents or kits, which are used for the diagnosis or auxiliary diagnosis of triple-negative breast cancer, as well as for the monitoring of treatment efficacy, prognostic assessment, and / or drug sensitivity prediction of triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biomedicine. Specifically, this invention relates to the application of ferroptosis inhibitor ANAPC11 as a biomarker for triple-negative breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide. Among all subtypes of breast cancer, triple-negative breast cancer (TNBC) is characterized by negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for approximately 15%-20% of all breast cancers. Due to the lack of these three key receptors, TNBC patients cannot benefit from endocrine therapy or HER2-targeted therapy, and current clinical treatment remains primarily chemotherapy. However, TNBC exhibits high heterogeneity, strong invasiveness, a very high risk of early recurrence, and poor prognosis. Therefore, the search for novel biomarkers for diagnosis and prognostic assessment, and the exploration of potential therapeutic approaches, are urgent needs in the field of breast cancer research.

[0003] Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation, discovered in recent years. It differs significantly from apoptosis, necrosis, and autophagy in its morphological, biochemical, and genetic characteristics. Studies have shown that inducing ferroptosis in tumor cells has become a highly promising strategy for killing drug-resistant tumor cells, especially those insensitive to chemotherapy. However, tumor cells often escape death by upregulating ferroptosis-inhibiting genes or enhancing antioxidant capacity (such as increasing glutathione (GSH) synthesis and enhancing the ability to scavenge lipid peroxides). In tumor-associated neoplasia (TNBC), disrupting the cellular ferroptosis defense mechanism and increasing its sensitivity to ferroptosis inducers represents a new direction for improving the therapeutic efficacy of TNBC.

[0004] Currently, molecular diagnostics for TNBC primarily relies on immunohistochemical negative assays for the three receptors, lacking more precise biomarkers that can indicate tumor progression and metabolic characteristics. Furthermore, although ferroptosis is considered a breakthrough in TNBC treatment, clinically, there is still a lack of indicators to predict patient sensitivity to ferroptosis therapy. Summary of the Invention

[0005] This invention delves into the expression of ANAPC11 in TNBC and its mechanism of inhibiting ferroptosis, which not only helps in the development of new diagnostic and prognostic biomarkers, but also provides new theoretical basis and potential targets for targeted therapy against the ferroptosis pathway.

[0006] In a first aspect, the present invention provides the use of the ANAPC11 protein, its encoding gene, or a detection reagent thereof for the preparation of diagnostic reagents or kits, said diagnostic reagents or kits being used for: (1) Diagnosis or auxiliary diagnosis of triple-negative breast cancer, (2) Monitoring the efficacy of treatment for triple-negative breast cancer. (3) Prognostic assessment of triple-negative breast cancer, and / or (4) Drug sensitivity prediction for triple-negative breast cancer.

[0007] In another preferred embodiment, the drug sensitivity prediction for triple-negative breast cancer includes predicting the sensitivity of triple-negative breast cancer to ferroptosis inducers.

[0008] In another preferred embodiment, the efficacy monitoring or prognostic assessment of triple-negative breast cancer includes the resistance of triple-negative breast cancer cells to ferroptosis.

[0009] In another preferred embodiment, the detection reagent is selected from the group consisting of antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips), protein chips, or combinations thereof.

[0010] In another preferred embodiment, the detection reagent is a reagent selected from the following detection methods: RT-qPCR, digital PCR, in situ hybridization (ISH / FISH), transcriptome sequencing (RNA-seq), serum / plasma detection, circulating tumor DNA (ctDNA) detection, or a combination thereof.

[0011] In another preferred embodiment, the ANAPC11 protein comprises the full-length ANAPC11 protein or a fragment of the ANAPC11 protein.

[0012] In another preferred embodiment, the ANAPC11 protein, whose encoding gene is derived from mammals, more preferably from primates and humans.

[0013] In another preferred embodiment, the diagnosis is tissue sample testing, circulating tumor DNA (ctDNA) testing, plasma testing, or serum testing.

[0014] In another preferred embodiment, the ANAPC11 protein is coupled with or carries a detectable marker.

[0015] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0016] In another preferred embodiment, the ANAPC11 protein further includes derivatives of the ANAPC11 protein.

[0017] In another preferred embodiment, the derivatives of the ANAPC11 protein include modified ANAPC11 protein, protein molecules with amino acid sequences homologous to and having the activity of natural ANAPC11 protein, dimers or polymers of ANAPC11 protein, and fusion proteins containing the amino acid sequence of ANAPC11 protein.

[0018] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to the natural ANAPC11 protein and has the activity of the natural ANAPC11 protein" means a protein molecule whose amino acid sequence has ≥85% homology with the ANAPC11 protein, preferably ≥90% homology, more preferably ≥95% homology, and most preferably ≥98% homology; and has the activity of the natural ANAPC11 protein.

[0019] In a second aspect, the present invention provides the use of an ANAPC11 antagonist for the preparation of a medicament or composition for the prevention or treatment of triple-negative breast cancer.

[0020] In another preferred embodiment, the ANAPC11 antagonist is selected from the group consisting of: (a) Substances that reduce or inhibit the activity of ANAPC11; (b) Substances that reduce the expression or stability of ANAPC11.

[0021] In another preferred embodiment, the prevention or treatment of triple-negative breast cancer includes improving the prognosis of triple-negative breast cancer.

[0022] In another preferred embodiment, the drug or composition is used to reduce the resistance of triple-negative breast cancer to ferroptosis or to increase the sensitivity of triple-negative breast cancer to ferroptosis.

[0023] In another preferred embodiment, the ANAPC11 antagonist is selected from: antibodies, peptides, shRNA, dsRNA, miRNA, siRNA, antisense oligonucleotides, PROTAC, compounds, or combinations thereof.

[0024] In another preferred embodiment, the ANAPC11 antagonist is selected from: anti-ANAPC11 antibodies, antisense oligonucleotides, siRNAs or dsRNAs targeting ANAPC11, small molecule inhibitors of ANAPC11, or competitive peptides.

[0025] In another preferred embodiment, the ANAPC11 antagonist is a small molecule inhibitor targeting the RING finger domain of ANAPC11.

[0026] In another preferred embodiment, the ANAPC11 antagonist is a polypeptide that interferes with the binding of ANAPC11 to other subunits of the APC / C complex (such as ANAPC2).

[0027] In another preferred embodiment, the ANAPC11 antagonist is a CRISPR / Cas9 reagent used to knock out or modify the ANAPC11 gene.

[0028] In another preferred embodiment, the drug or composition further includes a chemotherapeutic agent, a ferroptosis inducer, an immunosuppressant, or a combination thereof.

[0029] In another preferred embodiment, the chemotherapeutic agent is paclitaxel, doxorubicin, cyclophosphamide, or a combination thereof.

[0030] In another preferred embodiment, the ferroptosis inducer is selected from RSL3, ML162, FIN56, cystinase, or a combination thereof.

[0031] In another preferred embodiment, the immunosuppressant is a PD-1 / PD-L1 inhibitor.

[0032] A third aspect of the present invention provides a method for detecting triple-negative breast cancer, comprising the steps of: a) Prepare test samples for the subjects; and b) Detect the level of ANAPC11 protein or its encoding gene in the test sample and compare the results with the reference value. If the level of ANAPC11 protein or its encoding gene is significantly higher than the reference value, it indicates that the subject has triple-negative breast cancer or a poor prognosis of triple-negative breast cancer, or a higher probability of poor prognosis and shortened survival of triple-negative breast cancer than the normal population.

[0033] In another preferred embodiment, “significantly higher than” in the detection step (b) means that the ratio of the level E1 of the ANAPC11 protein or its encoding gene in the test sample to the reference value E0 is ≥1.2, preferably ≥1.5, more preferably ≥2, and even more preferably ≥3.

[0034] In another preferred embodiment, the test sample is a tissue sample or a serum sample.

[0035] In another preferred embodiment, the reference value in detection step (b) is the level of ANAPC11 protein or its encoding gene in the same sample from a normal population.

[0036] In another preferred embodiment, the anti-ANAPC11 antibody is a monoclonal antibody or a polyclonal antibody.

[0037] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0038] A fourth aspect of the present invention provides a method for predicting sensitivity to ferroptosis inducers, comprising the steps of: i) Prepare test samples for the subjects; and ii) Detect the level of ANAPC11 protein or its encoding gene in the test sample and compare the results with the reference value. If the level of ANAPC11 protein or its encoding gene is significantly higher than the reference value, it indicates that the subject is more likely to have resistance to ferroptosis inducers than the normal population. If the level of ANAPC11 protein or its encoding gene is significantly lower than the reference value, it indicates that the subject is more sensitive to ferroptosis inducers than the normal population.

[0039] In another preferred embodiment, “significantly higher than” in the detection step (ii) means that the ratio of the level C1 of the ANAPC11 protein or its encoding gene in the test sample to the reference value C0 is ≥1.2, preferably ≥1.5, more preferably ≥2, and even more preferably ≥3.

[0040] In another preferred embodiment, “significantly lower than” in the detection step (ii) means that the ratio of the level C1 of the ANAPC11 protein or its encoding gene in the test sample to the reference value C0 is ≤0.9, preferably ≤0.8, more preferably ≤0.6, and even more preferably ≤0.5.

[0041] In another preferred embodiment, the test sample is a tissue sample or a serum sample.

[0042] In another preferred embodiment, the reference value in the detection step (ii) is the level of ANAPC11 protein or its encoding gene in the same sample from a normal population.

[0043] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0044] In another preferred embodiment, the subject has cancer, preferably a triple-negative breast cancer patient.

[0045] In another preferred embodiment, the method is a method for predicting the sensitivity of triple-negative breast cancer to ferroptosis inducers.

[0046] A fifth aspect of the present invention provides a method for screening potential therapeutic agents for the prevention and / or treatment of triple-negative breast cancer, comprising the steps of: (1) In the test group, in the culture system, in the presence of the test compound, cells expressing ANAPC11 protein were cultured for a period of time T1, and the level of ANAPC11 protein or its encoding gene in the culture system of the test group was detected L1. Furthermore, in a control group where the test compound was absent and all other conditions were identical, the levels of ANAPC11 protein or its encoding gene (L2) in the culture system of the control group were measured; and (2) Compare the L1 and L2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for the prevention and / or treatment of triple-negative breast cancer; If L1 is significantly lower than L2, it indicates that the tested compound is a potential therapeutic agent for the prevention and / or treatment of triple-negative breast cancer.

[0047] In another preferred embodiment, “significantly lower than” means L2 / L1 ≥ 1.2, more preferably ≥ 1.5, and even more preferably ≥ 2.

[0048] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0049] A sixth aspect of the present invention provides a composition comprising: (a) A first active ingredient, wherein the first active ingredient is an ANAPC11 antagonist; and (b) A second active ingredient, wherein the second active ingredient is selected from the group consisting of chemotherapeutic drugs, ferroptosis inducers, immunotherapeutic drugs, or combinations thereof.

[0050] In another preferred embodiment, the composition further includes a pharmaceutically acceptable carrier or excipient.

[0051] In another preferred embodiment, the chemotherapeutic agent is paclitaxel, doxorubicin, cyclophosphamide, or a combination thereof.

[0052] In another preferred embodiment, the ferroptosis inducer is selected from RSL3, ML162, FIN56, cystinase, or a combination thereof.

[0053] In another preferred embodiment, the immunosuppressant is a PD-1 / PD-L1 inhibitor.

[0054] The present invention also provides the use of the compositions described in the sixth aspect of the invention for the preparation of medicaments for the prevention and / or treatment of tumors.

[0055] In another preferred embodiment, the tumor is an ANAPC11-positive tumor.

[0056] In another preferred embodiment, the tumor is a tumor that has antibodies against ferroptosis.

[0057] In another preferred embodiment, the tumor is triple-negative breast cancer.

[0058] The present invention also provides a medicine box, the medicine box comprising: (a) A first formulation containing an ANAPC11 antagonist and a pharmaceutically acceptable carrier; (b) A second formulation comprising a second active ingredient and a pharmaceutically acceptable carrier, the second active ingredient being selected from the group consisting of chemotherapeutic agents, ferroptosis inducers, immunotherapeutic agents, or combinations thereof; and (c) The instruction manual, which describes a method of using the first and second formulations in combination to treat tumors.

[0059] In another preferred embodiment, the first formulation and the second formulation are independent of each other.

[0060] In another preferred embodiment, the first and second formulations are lyophilized or liquid formulations.

[0061] In another preferred embodiment, the dosage forms of the first and second formulations are injections or gastrointestinal administration formulations.

[0062] In another preferred embodiment, the first formulation is applied before, during, or after the application of the second formulation.

[0063] The present invention also provides a method for treating tumors in a subject in need, comprising the steps of: Administer an ANAPC11 antagonist to the recipient; and optionally a second drug selected from the group consisting of chemotherapeutic agents, ferroptosis inducers, immunotherapeutic agents, or combinations thereof.

[0064] In another preferred embodiment, the subject suffers from a tumor.

[0065] In another preferred embodiment, the subject has a tumor, preferably a tumor that is sensitive to ferroptosis and has a poor prognosis, such as breast cancer (e.g., triple-negative breast cancer, HR+ breast cancer or HER2+ breast cancer with endocrine therapy resistance), ovarian cancer, lung adenocarcinoma, or pancreatic cancer.

[0066] In another preferred embodiment, the object is a mammal, more preferably a rodent (such as a mouse or rat) or a human.

[0067] The present invention also provides the use of ANAPC11 as a biomarker for the auxiliary diagnosis, drug sensitivity prediction and / or prognostic detection of triple-negative breast cancer.

[0068] In another preferred embodiment, the use of ANAPC11 is provided as a biomarker for detecting ferroptosis resistance in triple-negative breast cancer cells.

[0069] The present invention also provides a diagnostic kit comprising: a detection reagent for ANAPC11 protein or its encoding gene, and a label or instruction manual.

[0070] In another preferred embodiment, the detection reagent is selected from the group consisting of antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips), protein chips, or combinations thereof.

[0071] In another preferred embodiment, the label or instructions specify that the kit is for the detection of triple-negative breast cancer.

[0072] In another preferred embodiment, the label or instructions state the following: if the level of ANAPC11 protein or its encoding gene in the subject's test sample is significantly higher than the reference value, it indicates that the subject has triple-negative breast cancer or a poor prognosis for triple-negative breast cancer, or that the probability of a poor prognosis and shortened survival for triple-negative breast cancer is higher than that of the general population.

[0073] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0074] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0075] Figure 1 The results showed that ANAPC11 was highly expressed in triple-negative breast cancer tissues. (AB) Analysis of ANAPC11 expression levels in the FUSCC-TNBC and TCGA-TNBC databases revealed that ANAPC11 expression levels in cancerous tissues were higher than those in adjacent normal tissues. (C) ANAPC11 expression levels in triple-negative breast cancer tissues were significantly higher than those in HR+ and HER2+ breast cancers. (D) Immunoblotting analysis of specimens from three pairs of clinical triple-negative breast cancer patients showed that ANAPC11 was highly expressed in cancerous tissues. (E) Immunohistochemical staining of specimens from three pairs of triple-negative breast cancer patients showed that ANAPC11 was highly expressed in cancerous tissues.

[0076] Figure 2The results showed that high expression of ANAPC11 was associated with poor prognosis in triple-negative breast cancer. (AB) Analysis of patient survival in an online database indicated a poor prognosis in the ANAPC11 high-expression group. (C) Analysis of ANAPC11 expression levels in patients at different stages in the TCGA database indicated that ANAPC11 was associated with malignant stage. (DG) CCK-8 cell proliferation assays were performed to detect cell proliferation in two triple-negative breast cell lines (SUM159PT and MDA-MB-231) after knockdown (DE) / overexpression (FG) of ANAPC11, suggesting that ANAPC11 promotes TNBC cell proliferation and plays an oncogene role. (HI) Plate clone assays in SUM159PT and MDA-MB-231 cells showed that ANAPC11 promotes TNBC cell proliferation. (JM) Nude mouse tumorigenesis assays were performed to verify in vivo that ANAPC11 promotes TNBC progression by analyzing tumor growth curves and tumor weight.

[0077] Figure 3 The results showed that ANAPC11 inhibits ferroptosis. (A) Ferroptosis-related drug sensitivity tests demonstrated that ANAPC11 expression inhibits the sensitivity of TNBC cells to Erastin. (B) PCR results indicated that ANAPC11 is positively correlated with the expression of ferroptosis-inhibiting genes and negatively correlated with the expression of ferroptosis-promoting genes. (CD) MDA and GSH (classic ferroptosis experiments) experiments demonstrated that knocking down ANAPC11 increased cellular lipid peroxidation and weakened antioxidant capacity; overexpression of ANAPC11 decreased cellular lipid peroxidation and enhanced antioxidant capacity. (EG) Nude mouse tumorigenesis experiments verified in vivo that ANAPC11 inhibits tumor ferroptosis. Detailed Implementation

[0078] Through extensive and in-depth research, this invention has discovered new diagnostic biomarkers for triple-negative breast cancer, which can be used for the diagnosis or auxiliary diagnosis, prognostic evaluation, drug sensitivity prediction, or treatment of triple-negative breast cancer.

[0079] This invention discovered that the expression level of ANAPC11 in triple-negative breast cancer tissues is higher than that in normal tissues. The expression level of ANAPC11 is significantly correlated with disease progression, prognosis, and sensitivity to ferroptosis inducers. High expression of ANAPC11 can aid in the diagnosis of triple-negative breast cancer and predicts a poor prognosis and resistance to ferroptosis inducers. Therefore, ANAPC11 can be used as a diagnostic biomarker for the auxiliary diagnosis, drug sensitivity prediction, or prognostic assessment of triple-negative breast cancer. This invention was completed based on these findings.

[0080] This invention provides the application of ANAPC11 as a specific diagnostic biomarker for TNBC. Its expression in TNBC tissues is significantly higher than in adjacent normal tissues and other breast cancer subtypes (HR+, HER2+), enabling precise identification of TNBC. Furthermore, its high expression is significantly correlated with poor prognosis and malignant clinical stage, providing a reliable molecular indicator for prognostic assessment of TNBC patients. This invention reveals a novel mechanism by which ANAPC11 exerts its oncogene function in TNBC: by inhibiting ferroptosis, a non-apoptotic cell death pathway, it promotes TNBC cell proliferation, colony formation, and tumorigenic capacity in vivo. This invention clarifies the function of ANAPC11 as a ferroptosis inhibitor, revealing its regulatory role in the expression profile of ferroptosis-related genes by regulating lipid peroxidation (MDA) and antioxidant capacity (GSH), thereby improving the theory of the ferroptosis defense network in TNBC cells. This invention provides a technical solution for predicting the sensitivity of TNBC cells to ferroptosis drugs. By detecting the expression level of ANAPC11, it predicts the response of tumor cells to ferroptosis inducers, providing a theoretical basis and experimental support for personalized treatment and precision medication of TNBC. This invention also provides the application of ANAPC11 as a potential therapeutic target, demonstrating that knocking down ANAPC11 expression can effectively enhance lipid peroxidation in tumor cells, induce ferroptosis, and inhibit tumor growth, thereby providing a starting point for the development of novel targeted drugs against TNBC.

[0081] ANAPC11 ANAPC11 (Anaphase-Promoting Complex Subunit 11) is the core catalytic subunit of the late-phase promoting complex / cycle body (APC / C), belonging to the RING finger class of E3 ubiquitin ligases. Current research mainly focuses on the regulatory role of ANAPC11 in the cell cycle, promoting target protein degradation through ubiquitination modification, thereby regulating the process of mitosis. Although a few studies suggest aberrant expression of ANAPC11 in certain malignant tumors, the specific expression profile of ANAPC11 in triple-negative breast cancer, its effects on TNBC cell proliferation and in vivo tumorigenesis, especially its molecular link with the ferroptosis regulatory pathway, remains unclear. Most existing techniques treat breast cancer as a whole or focus only on its general regulatory mechanisms on the cell cycle of other cancer cells. This invention, however, reveals for the first time that ANAPC11 has a significantly higher expression profile in triple-negative breast cancer (TNBC), a highly malignant subtype, than in HR+ and HER2+ breast cancer, and establishes a direct and clear correlation between ANAPC11 and the clinical prognosis (poor prognostic indicators) of TNBC patients. This invention also reveals that ANAPC11 can inhibit ferroptosis by regulating lipid peroxidation or GSH levels, thereby leading to TNBC resistance to chemotherapy or drug sensitivity induction.

[0082] The full-length sequence of ANAPC11 in this invention is as follows: In this invention, the terms "ANAPC11," "ANAPC11 protein," or "ANAPC11 polypeptide" are used interchangeably and all refer to proteins or polypeptides having the amino acid sequence of human ANAPC11 protein. These proteins or polypeptides may be isolated.

[0083] As used in this article, "isolated" means that a substance has been separated from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their natural state.

[0084] As used herein, "isolated ANAPC11 protein or polypeptide" means that the ANAPC11 polypeptide is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify the ANAPC11 protein using standard protein purification techniques. A substantially pure polypeptide produces a single master band on a non-reducing polyacrylamide gel.

[0085] The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides, with recombinant polypeptides being preferred. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptides of the present invention can be glycosylated or non-glycosylated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0086] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the natural protein of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0087] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0088] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0089] The full-length human ANAPC11 nucleotide sequence or fragments thereof of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0090] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0091] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0092] Currently, the DNA sequence encoding the protein of the present invention (or its fragments, derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0093] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0094] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or the ANAPC11 protein coding sequence, and methods for generating the polypeptides of the present invention via recombinant technology.

[0095] The polynucleotide sequence of this invention can be used to express or produce recombinant ANAPC11 peptides using conventional recombinant DNA techniques. Generally, the following steps are involved: (1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the human ANAPC11 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0096] In this invention, in addition to Western blotting (WB) and immunohistochemistry (IHC), the detection or evaluation of ANAPC11 levels can also be achieved using the following methods: Nucleic acid level detection: (1) RT-qPCR / digital PCR: The mRNA transcription level of ANAPC11 is detected instead of the protein level detection, which serves as the basis for diagnosis and prognosis; (2) In situ hybridization (ISH / FISH): The expression of ANAPC11 mRNA is directly located and quantified on tissue sections; (3) Transcriptome sequencing (RNA-seq): The expression value of ANAPC11 is extracted using whole transcriptome data for bioinformatics evaluation.

[0097] Liquid biopsy techniques: (1) Serum / plasma detection: Detects the expression level of ANAPC11 in circulating tumor cells (CTC) or exosomes in peripheral blood. This method is non-invasive and is an important alternative to tissue biopsy; (2) Circulating tumor DNA (ctDNA) detection: Detects the copy number variation (CNV) or promoter methylation level of the ANAPC11 gene.

[0098] As used herein, the terms "antagonist" and "inhibitor" have the same meaning, referring to substances that interact with the ANAPC11 protein, especially inhibitors, that can be screened using the protein of this invention through various conventional screening methods.

[0099] This invention relates to inhibitors of the ANAPC11 protein (including antibodies, antisense nucleic acids, small molecule compounds, and other inhibitors), which, when administered therapeutically, inhibit the expression and / or activity of the ANAPC11 protein, thereby reducing ferroptosis resistance in triple-negative breast cancer. These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes.

[0100] Inhibitors that can be used in this invention include: antibodies against ANAPC11 (such as ANAPC11 neutralizing antibodies), inhibitory mRNAs, antisense RNAs of ANAPC11 nucleic acids, siRNAs, shRNAs, small molecule compounds, and inhibitors of ANAPC11 activity. Typical ANAPC11 inhibitors include inhibitory miRNAs, siRNAs, shRNAs, and ANAPC11 neutralizing antibodies.

[0101] Other technical approaches to suppressing ANAPC11 functionality include: Gene editing technology: Using CRISPR / Cas9 technology to directly knock out or modify the ANAPC11 gene, thereby completely blocking its function.

[0102] Other nucleic acid interference techniques: transient knockdown using siRNA (small interfering RNA); blocking ANAPC11 synthesis at the translational level using ASO (antisense oligonucleotide).

[0103] Protein degradation technology (PROTAC): Design a protein degradation targeting consortium (PROTAC) for ANAPC11, which utilizes the cell's own ubiquitin-lysosome system to degrade the ANAPC11 protein.

[0104] Small molecule inhibitors or competitive peptides: Develop small molecule inhibitors targeting the RING finger domain of ANAPC11 to block its E3 ubiquitin ligase activity; design peptides to interfere with the binding of ANAPC11 to other subunits of the APC / C complex (such as ANAPC2), thereby disrupting the integrity of the complex.

[0105] As used herein, the term "sample" or "sample" refers to material specifically associated with a subject from which specific information relating to the subject can be determined, calculated, or inferred. A sample may consist wholly or partially of biological material from the subject. A sample may also be material that has been in contact with the subject in a manner that allows testing of the sample to provide information relating to the subject. A sample may also be material that has been in contact with other materials, not belonging to the subject, but which enable subsequent testing of the first material to determine information relating to the subject; for example, a sample may be a cleaning solution for a probe or scalpel. A sample may be a source of biological material other than that in contact with the subject, as long as those skilled in the art can still determine information relating to the subject from the sample.

[0106] As used herein, the term "expression" includes the production of mRNA from a gene or gene segment, and includes the production of proteins encoded by RNA or a gene or gene segment, as well as the appearance of detection substances associated with expression. For example, the binding of cDNA, ligand-binding ligands (such as antibodies) to genes or other oligonucleotides, proteins, or protein fragments, and the chromogenic portion of the ligand-binding ligand are all included within the scope of the term "expression." Therefore, an increase in the density of the upper half-spot in immunoblotting such as Western blotting also falls within the scope of the biologically molecular-based term "expression."

[0107] As used herein, the term "reference value" refers to a value that is statistically relevant to a particular outcome when compared with the results of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of studies comparing the expression of the ANAPC11 protein with known clinical outcomes. Some such studies are shown in the Examples section of this document. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by taking into account circumstances and outcomes that are particularly relevant to the patient's medical history, genetics, age, and other factors.

[0108] This invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific to the human ANAPC11 protein. Here, "specificity" means that the antibody can bind to the human ANAPC11 gene product or fragment. Preferably, it refers to antibodies that can bind to the human ANAPC11 gene product or fragment but do not recognize or bind to other unrelated antigen molecules. Antibodies in this invention include molecules that can bind to and inhibit the human ANAPC11 protein, as well as antibodies that do not affect the function of the human ANAPC11 protein. This invention also includes antibodies that can bind to modified or unmodified forms of the human ANAPC11 gene product.

[0109] The invention includes not only complete monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have mouse antibody binding specificity but still retain the antibody portion derived from humans.

[0110] The antibodies of the present invention can be prepared using various techniques known to those skilled in the art. For example, purified human ANAPC11 gene product or its antigenic fragments can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human ANAPC11 protein or its antigenic fragments can be used to immunize animals to produce antibodies. The antibodies of the present invention can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., In Monoclonal Antibodies and TCell Hybridomas, Elsevier, NY, 1981). The various antibodies of the present invention can be obtained using fragments or functional regions of the human ANAPC11 gene product through conventional immunoassay techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a peptide synthesizer. Antibodies that bind to the unmodified form of the human ANAPC11 gene product can be produced by immunizing animals with the gene product generated in prokaryotic cells (e.g., E. coli); antibodies that bind to the post-translational modified form (e.g., glycosylated or phosphorylated proteins or peptides) can be obtained by immunizing animals with the gene product generated in eukaryotic cells (e.g., yeast or insect cells).

[0111] This invention also provides a pharmaceutical composition containing a safe and effective amount of an ANAPC11 protein antagonist and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as tablets and capsules can be prepared using conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram to 10 milligrams per kilogram of body weight per day.

[0112] The term "pharmaceutical composition" refers to a mixture containing a therapeutically effective amount of one or more of the said compounds and their pharmaceutically acceptable tautomers, solvates, hydrates, or salts, along with other pharmaceutically acceptable carriers. The purpose of preparing the said compounds into a pharmaceutical composition is to facilitate administration to the therapeutic subject.

[0113] The term "therapeutic effective amount" refers to the amount of drug that can achieve a therapeutic effect in the body of the treated individual. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the route of administration, the excipients used, and the combination with other drugs.

[0114] According to one aspect of the present invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an ANAPC11 antagonist.

[0115] The compositions of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0116] According to certain embodiments of the present invention, the pharmaceutical composition comprises a first active ingredient and a second active ingredient, wherein the first active ingredient is an ANAPC11 antagonist and the second active ingredient is a chemotherapeutic drug, a ferroptosis inducer, an immunotherapy drug, or a combination thereof.

[0117] According to certain embodiments of the present invention, the molar concentration ratio of the first active ingredient and the second active ingredient is about 1:5000-100:1, for example 1:4000-10:1, 1:3000-10:1, 1:2000-10:1, 1:1000-10:1, 1:100-10:1, 1:100-10:1, 1:10-10:1, 1:10-10:1.

[0118] Microcapsules containing the pharmaceutical compositions of the present invention can be used for sustained-release administration of the active ingredients of the present invention. Sustained-release formulations of the active ingredients of the present invention can be prepared from lactide / glycolide polymers (PLGA), which have good biocompatibility and broad biodegradability. The degradation products of PLGA, lactic acid and glycolic acid, are rapidly eliminated by the human body. Moreover, the degradation capacity of this polymer can be extended from several months to several years depending on its molecular weight and composition (Lewis, “Controlled release of bioactive agents from lactide / glycolide polymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41)).

[0119] When using the pharmaceutical composition, a safe and effective amount of the active ingredient of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the usual dose is 0.01–300 mg, preferably 0.5–100 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0120] According to one aspect of the present invention, the present invention provides a method for preparing the pharmaceutical composition, the method comprising mixing the first active ingredient and the second active ingredient with a pharmaceutically acceptable excipient.

[0121] The term "excipient" refers to a pharmaceutically acceptable ingredient that does not have any pharmacological activity and is commonly used in pharmaceutical techniques for preparing granular and / or solid oral dosage forms and / or liquid injectable dosage forms. Excipients may act as carriers, diluents, solubilizers, absorption enhancers, stabilizers, or adjuvants in the preparation of pharmaceutical compositions, and other functions. Excipients useful in the preparation of pharmaceutical compositions are generally safe, non-toxic, and acceptable for medical and pharmaceutical use. As used in this specification, "excipient" or "pharmaceutically acceptable excipient" includes one or more such excipients.

[0122] According to certain embodiments of the present invention, the pharmaceutical composition is used to treat cancer.

[0123] According to certain embodiments of the present invention, the tumor is selected from: lung cancer, stomach cancer, liver cancer, kidney tumor, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or combinations thereof.

[0124] According to certain embodiments of the present invention, the cancers include breast cancer, ovarian cancer, lung adenocarcinoma, and pancreatic cancer.

[0125] According to certain embodiments of the present invention, the total content of the first active ingredient and the second active ingredient accounts for 1-100% of the pharmaceutical composition, for example 1-99.5%, 1-99%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-99.5%, 10-99%, 10-9 0%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-20%, 20-100%, 20-99.5%, 20-99%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 30-100%, 30-99.5%, 30-99% %, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-100%, 40-99.5%, 40-99%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-99.5%, 50-99%, 50-90%, 50-80%, 50-70% 50-60%, 60-100%, 60-99.5%, 60-99%, 60-90%, 60-80%, 60-70%, 70-100%, 70-99.5%, 70-99%, 70-80%, 80-100%, 80-99.5%, 80-99%, 80-90%, 90-100%, 90-99.5%, or 90-99%.

[0126] According to certain embodiments of this application, the total content of the first active ingredient and the second active ingredient accounts for approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, and 47% of the pharmaceutical composition. 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%.

[0127] Synergistic effect: When two or more drugs are used together, if their actions are aligned and they enhance each other, this is called a synergistic effect. The total effect exceeds the sum of the effects of each drug used alone. In other words, the combined effect of two drugs is greater than the efficacy of either drug alone, and greater than the additive effect of the two drugs.

[0128] The term "about" can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined. In this application, when "about" is used to modify a numerical value, it means that the value can fluctuate within a range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0129] The term "regulation" includes treatment, prevention, or intervention.

[0130] The term "treatment" refers to the administration of the medicine of this invention to a subject requiring treatment, with the aim of curing, alleviating, improving, reducing, or influencing the disease, symptoms, or predisposition of the subject. Subjects of treatment according to this invention include mice, rabbits, monkeys, humans, and other mammals.

[0131] The pharmaceutical compositions of the present invention comprise the medicament (active ingredient) of the present invention within a safe and effective range, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.001-1000 mg of the active ingredient per dose, preferably 0.05-300 mg of the active ingredient per dose, and more preferably 0.5-200 mg of the active ingredient per dose.

[0132] The active ingredient of this invention and its pharmacologically acceptable salt can be formulated into various preparations containing, within a safe and effective range, the active ingredient of this invention or its pharmacologically acceptable salt, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. The safe and effective range of the active ingredient is determined based on the age, condition, and course of treatment of the patient.

[0133] "Pharmacologically acceptable excipients or carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmacologically acceptable excipients or carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.).

[0134] When administering the compositions of the present invention, they can be taken orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), or topically. They can be prepared into any pharmaceutically permissible dosage form, including but not limited to tablets, oral preparations, granules, injections, liposomes, targeted drug delivery injections, pills, capsules, granules, powders, suppositories, powders, ointments, patches, injection solutions, solutions, suspensions, sprays, lotions, drops, liniments, etc. The pharmaceutical compositions can be prepared as dry powders and mixed with sterile water or buffer solutions to form a solution before administration. The pH of the buffer solution is typically 3-11, preferably 5-9, and more preferably 7-8.

[0135] The present invention also provides the use of ANAPC11 protein, or its encoding gene, or its detection reagent for the preparation of diagnostic reagents or kits, said diagnostic reagents or kits for: (1) diagnosing or assisting in the diagnosis of triple-negative breast cancer, (2) monitoring the efficacy of triple-negative breast cancer treatment, or (3) evaluating the prognosis of triple-negative breast cancer.

[0136] Ferroprelation is a controlled form of cell death caused by the accumulation of iron-dependent lipid peroxides. GPX4, SLC7A11 (System Xc-), and FSP1 are known as inhibitors of ferroptosis. TNBC cells exhibit high sensitivity to ferroptosis. The core regulatory mechanisms of ferroptosis include: the GPX4 (glutathione peroxidase 4)-mediated antioxidant pathway, the SLC7A11-mediated cysteine / glutamate transport pathway, and the FSP1 (ferroptosis inhibitor 1)-mediated coenzyme Q10-dependent pathway. Ferroprelation in TNBC cells can be induced using Erastin (inhibiting System Xc-) or RSL3 (directly inhibiting GPX4) to kill tumor cells. Although GPX4 and SLC7A11 are classic inhibitors of ferroptosis, their specific expression in TNBC tissues is not always highly linearly correlated with patient prognosis, and drugs targeting these targets often have severe systemic toxicity when used in vivo. Furthermore, while inducers such as Erastin are effective in vitro, the response of different individuals with TNBC to these drugs varies greatly.

[0137] In evaluating the inhibitory effect of ferroptosis, this invention can replace MDA and GSH with the following equivalent indices: (1) Alternative for lipid peroxidation detection: Flow cytometry using C11-BODIPY 581 / 591 fluorescent probes was used to detect the degree of lipid peroxidation in cell membranes. (2) Alternative for iron ion detection: Intracellular free ferrous ions (Fe2+) were detected using FerroOrange or FeRhonox-1 probes. 2+ (3) Detection of downstream effector genes: Detect changes in the mRNA levels of key marker genes of ferroptosis such as PTGS2, CHAC1 or TFRC. (4) Observation of mitochondrial morphology: Observe typical morphological features of ferroptosis such as smaller mitochondria and increased membrane density through transmission electron microscopy.

[0138] This application discloses a kit containing a therapeutically effective amount of the therapeutic agent or pharmaceutical composition. According to some embodiments of this application, the kit further contains one or more other therapeutic agents. According to some embodiments of this application, the kit further contains instructions for use. According to some embodiments of this application, the kit further contains a device for a corresponding administration method, such as, but not limited to, a needle.

[0139] According to one aspect of the present invention, a medicine box is provided, the medicine box comprising the first formulation and the second formulation.

[0140] According to one aspect of the invention, the invention provides a method for treating tumors in a subject in need, comprising the steps of: administering an ANAPC11 antagonist and optionally a metromor death inducer to the subject in need.

[0141] The beneficial effects of this invention are: 1. Improved the accuracy of triple-negative breast cancer diagnosis and subtype differentiation capabilities. This invention, through the detection of ANAPC11 expression levels in its technical solution, can effectively distinguish TNBC tissue from normal tissue. Furthermore, the expression level of ANAPC11 in TNBC is significantly higher than in other subtypes such as HR+ and HER2+. Figure 1 (As shown in C), this provides a highly specific molecular diagnostic biomarker for clinical use, solving the problem of the lack of specific diagnostic indicators for TNBC in existing technologies.

[0142] 2. Provides a reliable prognostic stratification and assessment tool for TNBC patients. This invention demonstrates that ANAPC11 is a powerful prognostic indicator by establishing a correlation model between ANAPC11 expression levels and patient survival and malignancy stage. This allows physicians to perform early prognostic risk stratification based on patients' molecular characteristics, thereby developing more targeted postoperative follow-up and intensive treatment plans.

[0143] 3. It has opened up a new therapeutic pathway targeting the ferroptosis pathway in TNBC. This invention breaks through the traditional understanding that ANAPC11 is limited to cell cycle regulation in existing technologies, revealing for the first time its function of inhibiting ferroptosis. By inhibiting ANAPC11, GSH consumption and MDA lipid peroxidation product accumulation in TNBC cells can be directly caused, inducing ferroptosis. The discovery of this new mechanism provides a novel therapeutic target and theoretical basis for solving the problems of drug resistance and lack of targeted drugs in TNBC.

[0144] 4. Accurate drug sensitivity prediction for ferroptosis-inducing drugs was achieved. The technical solution of this invention can effectively predict the sensitivity of TNBC cells to ferroptosis inducers such as Erastin by monitoring the expression level of ANAPC11. This helps to screen potential sensitive populations before clinical medication, avoids the toxic side effects of blind chemotherapy, and promotes the precision medicine process for triple-negative breast cancer.

[0145] 5. Significantly inhibits tumor progression and has good clinical translational potential. Both in vivo and in vitro experiments demonstrated that intervening in ANAPC11 expression significantly inhibited TNBC cell proliferation, colony formation, and tumor growth rate and weight in nude mice. This approach achieves a dual effect of "cell cycle inhibition" and "ferroptosis induction" by inhibiting a single target, demonstrating excellent prospects for anti-tumor applications.

[0146] Unless otherwise stated in this application or obviously contradicted by the context, the terms “a,” “an,” “the,” “the,” and “at least one,” and similar designations used in the context of describing this application (including the claims) are to be interpreted to cover both the singular and plural. Unless otherwise stated in this application or obviously contradicted by the context, the terms “comprising,” “having,” “including,” and “containing” used in this application are to be interpreted as open-ended terms (i.e., “including but not limited to”). Unless otherwise stated in this application or obviously contradicted by the context, all methods described in this application may be performed in any suitable order as understood by those skilled in the art.

[0147] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0148] Example 1. siRNA targeting ANAPC11 and its knockdown efficiency We designed siRNA targeting the ANAPC11 gene and tested its knockdown efficiency and effectiveness.

[0149] siRNA knockdown efficiency screening: After the designed siRNA was synthesized, it was transfected into triple-negative breast cancer cells MDA-MB-231 and SUM159PT, and the cellular RNA was extracted. After reverse transcription, the knockdown efficiency of the ANAPC11 gene was detected by qPCR.

[0150] The following are some representative siRNAs designed: Table 1

[0151] Based on the initial screening results, three siRNAs that showed knockdown effects on the ANAPC11 gene were re-screened.

[0152] Based on the results of the secondary screening, the siRNAs with the best knockdown effects, 01 and 02, were named siANAPC11-1 (SEQ ID NO.2) and siANAPC11-2 (SEQ ID NO.3), respectively, for subsequent experiments.

[0153] Example 2. Application of ANAPC11 as a biomarker in the diagnosis and prognostic assessment of TNBC 1. Expression profile validation 1.1 Database Analysis: Differential expression analysis was performed in the FUSCC-TNBC and TCGA-TNBC databases ( Figure 1 AC).

[0154] Steps: Download TCGA-BRCA transcriptome HTSeq data, patient clinicopathological data, and survival data from USCS Xena (http: / / xena.ucsc.edu). Download FUSCC-TNBC transcriptome and clinical data from The National Omics Data Encyclopedia (NODE) ​​(http: / / www.biosino.org / node / project / detail / OEP000155). Perform survival analysis using the R packages "survival" and "survminer", differential analysis using the R package "limma", and plot Kaplan-Meire curves using the R package "ggsurvplot".

[0155] Results: Analysis of ANAPC11 expression levels in the FUSCC-TNBC and TCGA-TNBC databases revealed that ANAPC11 expression levels in cancerous tissues were higher than those in adjacent normal tissues, and that ANAPC11 expression levels in triple-negative breast cancer tissues were significantly higher than those in HR+ and HER2+ breast cancer tissues.

[0156] 1.2 Clinical specimen testing: Cancerous tissue and adjacent tissue were collected from patients with TNBC (specimens obtained from patients who underwent surgery / puncture at the Department of Breast Surgery, First Affiliated Hospital of China Medical University and signed informed consent forms). The samples were analyzed by Western blotting. Figure 1 D) and IHC ( Figure 1 E) Verify the differential expression of ANAPC11.

[0157] 1) Western Blot assay Procedure: Cellular proteins were extracted in 1% Triton lysis buffer (1% Triton X-100, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 μg / mL aprotinin) and quantified using the BCA method. Cell lysates were subjected to SDS-PAGE for protein electrophoresis and transferred to a PVDF membrane. 5% skim milk was prepared using tris-buffered saline Tween-20 (TBST) buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% Tween-20). The PVDF membrane was blocked by immersing it in 5% skim milk at room temperature for 1 hour. The PVDF membrane was then cut to the pre-designed size and incubated overnight at 4°C with the appropriate primary antibody. The following day, the membrane was washed with TBST for 10 minutes each time for a total of 4 times. It was then incubated at room temperature with the corresponding secondary antibody diluted at a ratio of 1:10000 for 60 minutes. The membrane was washed again with TBST for 10 minutes each time for a total of 4 times. Finally, luminescence was generated using the ultrasensitive luminescent solution.

[0158] Results: Immunoblotting was performed on specimens from three pairs of clinical triple-negative breast cancer patients, and the results showed that ANAPC11 was highly expressed in cancer tissues.

[0159] 2) Immunohistochemistry (IHC) Procedure: After fixing the tissue in 10% formalin, embed it in paraffin and section it serially. Bake the sections in a 70℃ oven for 1 hour. After baking, allow the temperature to drop slightly before dewaxing and hydration. Following the order of xylene and alcohol (100%, 100%, 95%, 85%, 75%), place the slide rack in the oven for 3 minutes, agitate for 2-3 minutes, rinse with tap water for 5 minutes, and then immerse in distilled water. Fill the slide container with 1x sodium citrate retrieval solution and perform antigen retrieval using autoclaving. After naturally cooling to room temperature, place the slide in distilled water. Rinse twice with PBS, then treat with solution A for 10 minutes. Rinse three times with PBS, then treat with solution B for 1 hour. Add 200 μL of primary antibody diluted in PBS to each slide and incubate overnight at 4℃. The next day, rinse three times with PBS, then treat with solution C for 10 minutes. Rinse three times with PBS, then treat with solution D for 20 minutes. After rinsing twice with PBS, add 200 μL of DAB to each slide and develop under a microscope. Once the staining intensity is suitable, rinse with tap water for 10 minutes and place in a slide holder containing distilled water. After drying the slides, stain with hematoxylin for 1 minute for nuclear counterstaining, then rinse with tap water for 10 minutes. Following the sequence of 75%, 85%, 95%, 100%, 100% alcohol, and 100%, 100% xylene, place the slide holders one at a time, shaking for 3 minutes each time. Finally, leave the slides in xylene and quickly drop resin onto the tissue, then cover with a coverslip. Staining is considered positive when cells show a brownish-yellow color. Classification is based on the proportion and intensity of positive cells: less than 10% positive cells with light staining are considered negative; more than 10% positive cells with deep staining are considered positive. This assessment was performed separately by two independent pathologists.

[0160] Results: Immunohistochemical staining of three pairs of triple-negative breast cancer patients showed that ANAPC11 was highly expressed in cancer tissues.

[0161] 2. Diagnostic Plan This embodiment provides a method for detecting the expression level of ANAPC11 in clinical specimens, which can be used to assist in the diagnosis and prognosis of triple-negative breast cancer.

[0162] Sample collection: Obtain breast tissue specimens to be tested (including surgically removed specimens or puncture biopsy specimens).

[0163] Expression detection: The protein expression level of ANAPC11 in tissues was detected by immunohistochemical staining (IHC) or Western blotting. (The steps are the same as those in Part 1 for IHC and Western Blot.) By comparing cancerous tissue with adjacent tissue, if ANAPC11 shows significantly high expression in cancerous tissue (deep protein band or high proportion and intensity of IHC-positive cells), it suggests a malignant lesion. This can be further compared with HR+ and HER2+ breast cancer subtypes (…). Figure 1 C) If the ANAPC11 expression level is significantly elevated, it can be used as a molecular basis for auxiliary identification of triple-negative breast cancer subtype.

[0164] 3. Prognostic assessment Steps: Use the R package "ggsurvplot" to plot the Kaplan-Meire curve.

[0165] Results: In both the PAM50 and St. Gallen breast cancer classification criteria, high ANAPC11 expression was associated with poor prognosis in TNBC patients. High ANAPC11 expression was positively correlated with shorter overall survival and higher clinical stage of malignancy. Specifically, Figure 2 (AB) Analysis of patient survival in online databases suggests that patients with high ANAPC11 expression have a poor prognosis; (C) Analysis of ANAPC11 expression levels in patients at different stages in the TCGA database suggests that ANAPC11 is associated with malignant staging.

[0166] The expression intensity of ANAPC11 was scored. TNBC patients with high ANAPC11 expression were classified as a high-risk prognostic group and required more intensive postoperative follow-up and chemotherapy.

[0167] Example 3. ANAPC11 as a scheme for predicting TNBC's sensitivity to ferroptosis drugs 1. Verification of the ferroptosis inhibition mechanism 1.1 Drug susceptibility screening: Cells with different ANAPC11 levels were treated with Erastin, and the effect on drug susceptibility was confirmed by cell viability curves.

[0168] Procedure: MDA-MB-231 and SUM159PT cell lines were constructed for ANAPC11 overexpression, knockdown, and control (si-ANAPC11#1 and si-ANAPC11#2 were transient knockdown groups using siANAPC11-1 and siANAPC11-2 from Example 1, respectively; siNC was the corresponding control group; OE-ANAPC11 was the ANAPC11 overexpression group, and Vector was the corresponding control group). Cells were cultured to the logarithmic growth phase. After cell digestion and counting, cells were seeded into 96-well plates and cultured overnight until fully adherent. Cells in each group were treated with culture medium containing gradient concentrations of Erastin, with blank and negative controls included, and replicates for each group. After 24–48 h of further culture, CCK-8 reagent was added for incubation, and the OD value at 450 nm was measured using a microplate reader. Cell viability was calculated, dose-response curves were fitted, and the sensitivity and IC50 of cells with different ANAPC11 levels to Erastin were compared. 50 difference.

[0169] Results: Ferroplasmosis-related drug sensitivity tests demonstrated that ANAPC11 expression inhibited the sensitivity of TNBC cells to Erastin. Figure 3 A).

[0170] 1.2 Gene expression regulation: Key genes for ferroptosis were detected by PCR.

[0171] Steps: RNA extraction and quantitative real-time PCR (qRT-PCR) RNA was extracted using an RNA extraction kit (Shanghai Promega Eastep Super Total RNA Extraction Kit). After digestion and centrifugation of adherent cells, 1 mL of PBS was added for resuspending, followed by centrifugation at 1500 rpm for 10 minutes, and the supernatant was discarded. 300 μL of RNA lysis buffer was added to the cell pellet, mixed, and then 300 μL of RNA dilution buffer was added. The mixture was incubated for 5 minutes. Centrifugation was performed at 14000 rpm for 5 minutes, and 600 μL of the supernatant was collected and transferred to a centrifuge column. 300 μL of anhydrous ethanol was added, mixed, and centrifuged at 14000 rpm for 1 minute, and the filtrate was discarded. 600 μL of RNA washing buffer was added, and the mixture was centrifuged at 14000 rpm for 1 minute, and the filtrate was discarded. 50 μL of prepared DNase I (10) was added to the center of the adsorption module. Add 5 μL of DNase I buffer, 5 μL of DNase I, and 40 μL of nuclease-free water. Incubate at room temperature for 15 minutes. Add 600 μL of RNA washing buffer and wash twice, 140 ml each time. Centrifuge at 100g for 1 minute. Place the centrifuge column in the collection tube, add 20 μL of nuclease-free water to the center of the adsorption module, incubate at room temperature, centrifuge, and collect the liquid in the eluent tube. Measure the absorbance at 260 nm using a NanoDrop ND-100 spectrophotometer for quantification. The PrimeScript RT kit (Vazyme, R333) was used for mRNA reverse transcription. Real-time quantitative PCR was performed using the SYBR Premix Ex Taq II kit (Vazyme, Q711) on an AppliedBiosystems 7500 Real-Time PCR System. The conditions were 95℃ for 30 s, then 95℃ for 10 s, 60℃ for 30 s for 40 cycles, followed by a cycle of 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. 2^(- The relative expression level was calculated using the Ct method, with the GAPDH transcript serving as an internal control.

[0172] Results: PCR results showed that ANAPC11 was positively correlated with the expression of ferroptosis-inhibiting genes and negatively correlated with the expression of ferroptosis-promoting genes. This confirms that ANAPC11 can promote the expression of anti-ferroptosis genes and inhibit the expression of pro-ferroptosis genes. Figure 3 B).

[0173] 1.3 Metabolic Indicator Detection: Procedures were followed according to the instructions for the GSH and MDA detection kits. This verified that ANAPC11 prevents lipid peroxidation by maintaining redox balance. Figure 3 CD).

[0174] step: (1) MDA detection Triple-negative breast cancer cells were collected, washed twice with pre-chilled PBS, and then lysed in 200 μL of RIPA lysis buffer (containing 1% PMSF) for 30 min on ice. After centrifugation at 12000 rpm for 15 min, the supernatant was collected as the protein sample. Protein concentration was determined using the BCA method and adjusted to a uniform concentration. 100 μL of the protein sample was added to 200 μL of MDA detection working solution (containing 0.37% TBA and 15% TCA), mixed well, and incubated at 95°C for 60 min. After cooling on ice for 10 min, the sample was centrifuged at 12000 rpm for 10 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance (OD value) was measured at 532 nm using a microplate reader. The MDA content (nmol / mg prot) of the sample was calculated based on a standard curve plotted using tetraethoxypropane (MDA standard).

[0175] (2) GSH detection Triple-negative breast cancer cells were divided into 5×10 4 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured to 80% confluence. The culture medium was discarded, and the cells were washed twice with pre-chilled PBS. 200 μL of lysis buffer (PBS containing 1% NP-40) was added to each well, and the cells were lysed on ice for 15 min. The lysate was collected using a cell scraper into a 1.5 mL centrifuge tube. The cells were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected as the total protein extract. 50 μL of the supernatant was added to 150 μL of freshly prepared DTNB chromogenic solution (0.2 mM dissolved in 0.1 M PBS, pH 8.0), mixed well, and reacted at room temperature in the dark for 30 min. The absorbance (OD value) was measured at 412 nm using a microplate reader. The GSH content of the sample was calculated based on the standard curve (0–100 μM GSH gradient concentration), and the final data were normalized to μmol GSH / mg total protein.

[0176] Results: The experiment showed that knocking down ANAPC11 led to MDA accumulation, decreased GSH levels, increased cellular lipid peroxidation, and weakened antioxidant capacity, thereby inducing ferroptosis. Figure 3 CD).

[0177] 1.4 Verification of intracellular ferroptosis: Procedure: 4-6 week old BALB / c nude mice were selected and acclimatized for 1 week in an SPF-grade animal facility. Stable triple-negative breast cancer cells MDA-MB-231, transfected with ANAPC11 virus, were resuspended in PBS to a concentration of 1×10⁻⁶. 7 Take 100 μL of suspension (containing 1×10 cells / mL) 6 (cells) were injected subcutaneously into the right axilla of nude mice, forming a uniform wheal. After inoculation, mice were divided into experimental groups (e.g., the siANAPC11-1 knockdown group from Example 1, sh-ANAPC11) and control groups (empty vector group, control), with 6 mice in each group. Starting from day 7 post-inoculation, the long diameter (L) and short diameter (W) of the tumor were measured three times a week using calipers, and the result was calculated using the formula V = (L × W) 2 ) / 2 to calculate tumor volume (mm) 3 The tumor size reached 50mm. 3 Subsequently, some mice were intraperitoneally injected with Ferrostatin-1 (20 mg / kg) every two days. When the average tumor volume in the control group reached 2000 mmHg... 3 If the maximum diameter is >20 mm, nude mice are euthanized by CO2 asphyxiation. Subcutaneous tumor tissue is completely dissected, and the wet weight (g) is recorded. A portion of the tissue is fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned for H&E staining and immunohistochemical analysis.

[0178] Results: Ferroprelation-related markers were detected in tumor tissues of nude mice, demonstrating that ANAPC11 inhibited tumor ferroptosis, and that inhibition of ANAPC11 induced ferroptosis in vivo. Figure 3 EG).

[0179] 2. ANAPC11 as a regimen for predicting the drug sensitivity of TNBC to ferroptosis. This embodiment provides a method for predicting the sensitivity of TNBC patients to ferroptosis-inducing drugs (such as Erastin) by detecting ANAPC11 levels.

[0180] Technical principle: ANAPC11 inhibits ferroptosis by regulating intracellular antioxidant capacity. The higher its expression level, the stronger the cell's resistance to ferroptosis inducers.

[0181] Implementation steps of the plan (in conjunction with the appendix) Figure 3 ): Detection: Baseline expression of ANAPC11 in the patient's tumor cells was determined.

[0182] Predictive assessment: If ANAPC11 levels are low, the patient is predicted to have high sensitivity to ferroptosis inducers (such as Erastin), and ferroptosis-related treatments should be considered first. If ANAPC11 levels are significantly high ( Figure 3 If A), it is predicted that the patient has resistance to simple ferroptosis inducers, and a combination therapy regimen should be considered.

[0183] Example 4. ANAPC11 as a therapeutic target for screening or preparation of anti-TNBC drugs. This embodiment proposes a method to induce TNBC cell death and inhibit tumor progression by inhibiting ANAPC11 expression or activity.

[0184] Interference sequences targeting ANAPC11 (such as shRNA, siRNA) or small molecule inhibitors are used.

[0185] 1. Proliferation assay (CCK-8): MDA-MB-231 and SUM159PT cells with transient knockdown and overexpression of ANAPC11 were constructed respectively. Knockdown group (si-ANAPC11#1, si-ANAPC11#2) and its control group (siNC), and overexpression group (OE-ANAPC11) and its control group (Vector) were set up.

[0186] Steps: CCK8 proliferation curve Cell proliferation capacity was analyzed using 96-well plates 24 hours after cell treatment. The treated cells were seeded in 96-well plates with an appropriate cell number / 100 μL of culture medium containing FBS and double antibiotics. The absorbance was measured daily to assess cell growth and proliferation.

[0187] Results: Cell proliferation was examined in two triple-negative mammary cell lines (SUM159PT and MDA-MB-231) after knockdown (DE) / overexpression (FG) of ANAPC11. The results showed that the expression level of ANAPC11 was directly proportional to the cell proliferation rate. Figure 2 DG), ANAPC11 promotes TNBC cell proliferation, suggesting that it plays an oncogene role.

[0188] 2. Clonogenesis experiment: Observe long-term proliferation capacity and verify its promoting effect on cell population dependence.

[0189] Procedure: ANAPC11 overexpressing, knockdown, and control cells in logarithmic growth phase were collected, trypsinized, and single-cell suspensions were prepared. Cells were counted and seeded into culture plates. Cells were cultured in a cell culture incubator for 10-14 days, with the culture medium replaced every 3 days. When visible cell clones appeared in the culture plates, the culture medium was discarded, cells were washed with PBS, fixed with fixative, and stained with crystal violet. The staining solution was discarded, excess stain was washed off with PBS, and the cells were air-dried. The number of clones with ≥50 cells was counted under a microscope. The colony formation rate was calculated, and the number and size of clones in each group were compared to assess the regulatory effect of ANAPC11 on long-term cell proliferation and population dependence.

[0190] Results: Knockdown of ANAPC11 in vitro significantly reduced the proliferation and colony-forming ability of TNBC cells (SUM159PT and MDA-MB-231), indicating that ANAPC11 promotes TNBC cell proliferation and inhibiting ANAPC11 inhibits TNBC cell proliferation. Figure 2 HI).

[0191] 3. In vivo tumorigenesis: The modified cells were seeded subcutaneously into nude mice, and their volume and weight were measured to verify their tumor-promoting effect in vivo.

[0192] Procedure: 4-6 week old BALB / c nude mice were selected and acclimatized for 1 week in an SPF-grade animal facility. Stable triple-negative breast cancer cells MDA-MB-231, stably transfected with ANAPC11 virus, were divided into three groups: an ANAPC11 knockdown group (using siANAPC11-1 and siANAPC11-2 from Example 1 to knock down ANAPC11 expression, named sh-ANAPC11#1 and sh-ANAPC11#2 respectively), an ANAPC11 overexpression group (OE-ANAPC11), and a control group. The cells were resuspended in PBS to a concentration of 1×10⁻⁶. 7 Take 100 μL of suspension (containing 1×10 cells / mL) 6 (cells) were injected subcutaneously into the right axilla of nude mice, forming a uniform wheal. After inoculation, mice were divided into experimental groups (e.g., ANAPC11 knockdown group) and control groups (empty vector group), with 6 mice in each group. Starting from day 7 post-inoculation, the long diameter (L) and short diameter (W) of the tumor were measured three times a week using calipers, and the result was calculated using the formula V = (L × W) 2 ) / 2 to calculate tumor volume (mm) 3 When the average tumor volume in the control group reached 2000 mm... 3 If the maximum diameter is >20 mm, nude mice are euthanized by CO2 asphyxiation. Subcutaneous tumor tissue is completely dissected, and the wet weight (g) is recorded. A portion of the tissue is fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned for H&E staining and immunohistochemical analysis.

[0193] Results: In a nude mouse tumorigenesis model, inhibiting ANAPC11 expression significantly slowed tumor growth and reduced tumor weight. This in vivo validation demonstrated that ANAPC11 promotes TNBC progression. Figure 2 JM).

[0194] The results show: (1) In vitro: After knocking down ANAPC11, the proliferation ability of TNBC cells (such as SUM159PT and MDA-MB-231) decreased significantly and the colony formation ability was weakened.

[0195] (2) In vivo: In a nude mouse tumor model, inhibiting ANAPC11 expression significantly slowed tumor growth and reduced tumor weight.

[0196] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an ANAPC11 protein, its encoding gene, or a detection reagent thereof for the preparation of a diagnostic reagent or kit, said diagnostic reagent or kit being used for: (1) Diagnosis or auxiliary diagnosis of triple-negative breast cancer, (2) Monitoring the efficacy of treatment for triple-negative breast cancer. (3) Prognostic assessment of triple-negative breast cancer, and / or (4) Drug sensitivity prediction for triple-negative breast cancer.

2. The use as described in claim 1, characterized in that, The drug sensitivity prediction for triple-negative breast cancer includes predicting the sensitivity of triple-negative breast cancer to ferroptosis inducers.

3. The use as described in claim 1, characterized in that, The detection reagents are selected from the following group: antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips), protein chips, or combinations thereof.

4. Use of an ANAPC11 antagonist for the preparation of a medicine or composition for the prevention or treatment of triple-negative breast cancer.

5. The use as described in claim 4, characterized in that, The drug or composition is used to reduce the resistance of triple-negative breast cancer to ferroptosis or to increase the sensitivity of triple-negative breast cancer to ferroptosis.

6. The use as described in claim 4, characterized in that, The ANAPC11 antagonist is selected from: antibodies, peptides, shRNA, dsRNA, miRNA, siRNA, antisense oligonucleotides, PROTAC, compounds, or combinations thereof.

7. A method for detecting triple-negative breast cancer, characterized in that, Including the following steps: a) Prepare test samples for the subjects; and b) Detect the level of ANAPC11 protein or its encoding gene in the test sample and compare the results with the reference value. If the level of ANAPC11 protein or its encoding gene is significantly higher than the reference value, it indicates that the subject has triple-negative breast cancer or a poor prognosis of triple-negative breast cancer, or a higher probability of poor prognosis and shortened survival of triple-negative breast cancer than the normal population.

8. A method for predicting sensitivity to ferroptosis inducers, characterized in that, Including the following steps: i) Prepare test samples for the subjects; and ii) Detect the level of ANAPC11 protein or its encoding gene in the test sample and compare the results with the reference value. If the level of ANAPC11 protein or its encoding gene is significantly higher than the reference value, it indicates that the subject is more likely to have resistance to ferroptosis inducers than the normal population. If the level of ANAPC11 protein or its encoding gene is significantly lower than the reference value, it indicates that the subject is more sensitive to ferroptosis inducers than the normal population.

9. A method for screening potential therapeutic agents for the prevention and / or treatment of triple-negative breast cancer, characterized in that, Including the following steps: (1) In the test group, in the culture system, in the presence of the test compound, cells expressing ANAPC11 protein were cultured for a period of time T1, and the level of ANAPC11 protein or its encoding gene in the culture system of the test group was detected L1. Furthermore, in a control group where the test compound was absent and all other conditions were identical, the level L2 of ANAPC11 protein or its encoding gene in the culture system of the control group was detected; and (2) Compare the L1 and L2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for the prevention and / or treatment of triple-negative breast cancer; If L1 is significantly lower than L2, it indicates that the tested compound is a potential therapeutic agent for the prevention and / or treatment of triple-negative breast cancer.

10. A composition, characterized in that, The composition comprises: (a) A first active ingredient, wherein the first active ingredient is an ANAPC11 antagonist; and (b) A second active ingredient, wherein the second active ingredient is selected from the group consisting of chemotherapeutic drugs, ferroptosis inducers, immunotherapeutic drugs, or combinations thereof.

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