Application of AKT1-K189 / 276 / 297Ufm protein in cancer diagnosis and treatment
By using jugation modification of AKT1-K189/276/297Ufm protein as a diagnostic biomarker and therapeutic target, the problem of insufficient sensitivity and specificity of existing diagnostic biomarkers for breast cancer and other cancers is solved, enabling rapid and accurate diagnosis and effective treatment.
Patent Information
- Application Number
- CN202511381436.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2025-12-19
AI Technical Summary
Existing diagnostic biomarkers for breast cancer, lung cancer, and pancreatic cancer lack sufficient sensitivity and specificity, making early detection difficult. Furthermore, the phosphorylation mechanism of AKT1 is unclear, resulting in a lack of effective therapeutic targets.
Using ursylation modification of AKT1-K189/276/297Ufm protein as a diagnostic biomarker, diagnostic products are developed by substances that specifically recognize and bind to AKT1-K189/276/297Ufm protein, and therapeutic products are developed by substances that inhibit AKT1-K189/276/297Ufm protein, including antibodies, oligonucleotide expression vectors, and small molecule compounds, to target and degrade or inhibit the activity of AKT1-K189/276/297Ufm protein.
It has improved the diagnostic accuracy of cancers such as breast cancer, provided new therapeutic targets, promoted the rapid and accurate diagnosis of cancers such as breast cancer, and laid the theoretical foundation for the development of new drugs.
Smart Images
Figure CN121164641A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of AKT1-K189 / 276 / 297Ufm protein in products for cancer diagnosis and 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] Breast cancer is one of the most common malignant tumors in women. The molecular mechanisms of its development and progression remain unclear, and breast cancer is prone to drug resistance and recurrence. Currently, X-rays and ultrasound are commonly used methods for breast cancer screening, but they are ineffective at detecting early lesions, requiring further confirmation through cytological or pathological examination. However, existing breast cancer biomarkers generally suffer from low sensitivity and poor specificity. This problem also exists in the detection, prevention, and treatment of lung and pancreatic cancer. Therefore, there is an urgent need to develop new diagnostic biomarkers and therapeutic targets.
[0004] Human AKT1 protein (also known as protein kinase B, PKB), encoded by the AKT1 gene (Genebank NC_000014.9), is an important protein kinase. Studies have shown that phosphorylation of threonine at position 308 (T308p) and serine at position 473 (S473p) in AKT1 are essential for its full activation, and these phosphorylations are significantly increased in various tumor cells, thereby enhancing the pro-cancer effect of AKT1. However, the molecular mechanisms underlying T308p and S473p in AKT1 remain unclear. Post-translational modifications of proteins play important roles in protein conformation, subcellular localization, activity, and stability. However, justinization, as an important type of post-translational modification, requires further investigation regarding whether and how it affects AKT1 phosphorylation. Currently, there are no reported studies on the role of AKT1 justinization in AKT1 activation and in breast cancer and other tumors. Summary of the Invention
[0005] To address the shortcomings of existing technologies, particularly the low sensitivity and poor specificity of current tumor markers, this invention proposes a method where the unique E3 ligase UFL1 catalyzes the juxtaposition of lysine residues 189 / 276 / 297 in AKT1 (AKT1-K189 / 276 / 297Ufm protein), thereby promoting the formation of AKT1-T308p and AKT1-S473p and the complete activation of AKT1. Therefore, the AKT1-K189 / 276 / 297Ufm protein has significant potential applications in the preparation of products for the detection, prevention, or treatment of cancer.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides the use of a substance for detecting AKT1-K189 / 276 / 297Ufm protein in the preparation of diagnostic or auxiliary diagnostic products for cancer, wherein the AKT1-K189 / 276 / 297Ufm protein is an AKT1 protein with lysine residues at positions 189, 276, and 297 lysine-treated. The use includes any one of the following: (a1) Screening of active drugs for cancer treatment; (a2) Diagnostic reagents for cancer diagnosis or auxiliary diagnosis.
[0007] The detection reagent is used to detect the expression level of AKT1-K189 / 276 / 297Ufm protein.
[0008] The cancers mentioned include breast cancer, lung cancer, and pancreatic cancer.
[0009] In a second aspect, the present invention provides a product comprising substances for detecting AKT1-K189 / 276 / 297Ufm protein, the product being used for the preparation of a product for the detection, prevention, and / or treatment of cancer.
[0010] In one or more embodiments, the cancer includes cancers such as breast cancer, lung cancer, and pancreatic cancer.
[0011] In one or more embodiments, the AKT1-K189 / 276 / 297Ufm protein is used as a biomarker in the preparation of products for detecting cancer occurrence and metastasis.
[0012] In one or more embodiments, the product includes a product for detecting UAKT1-K189 / 276 / 297Ufm at the protein level and a product for quantitatively detecting AKT1-K189 / 276 / 297Ufm protein; The product is selected from one or more of reagents, kits, test strips, and chips.
[0013] The product contains a substance specific to the AKT1-K189 / 276 / 297Ufm protein, and the substance specific to the AKT1-K189 / 276 / 297Ufm protein includes one or more of the following: b1), b2), and b3). b1) Antibodies that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins; b2) Ligand proteins or peptides that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins; b3) Non-protein compounds that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins.
[0014] In one or more embodiments, the substance specific to AKT1-K189 / 276 / 297Ufm protein is suitable for one or more of the following methods: immunoblotting, immunoprecipitation, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISA), colloidal gold immunoassay, immunochemiluminescence, fluorescence immunochromatography, immunohistochemistry, mass spectrometry, high-performance liquid chromatography (HPLC), capillary gel electrophoresis, flow cytometry, and proteomics.
[0015] In one or more embodiments, the test sample of the product includes one or more of tumor tissue, body fluids, and cells isolated from breast cancer patients.
[0016] In one or more embodiments, the antibody comprises an antibody that specifically recognizes AKT1-K189 / 276 / 297Ufm protein generated in mice, rats, guinea pigs, chickens, rabbits, pigs, monkeys, and any other organism using any AKT1 short peptide or protein containing K189 / 276 / 297Ufm modified.
[0017] Thirdly, the present invention provides the use of AKT1-K189 / 276 / 297Ufm protein as a target in the preparation of products for the prevention and / or treatment of cancer; Preferably, the product is a drug or a test reagent; Preferably, the product is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of the following: targeted chimeras (PROTAC) and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm proteins, and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 of the justolysis system and the protein kinase AKT1, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the jussylation system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
[0018] Preferably, the PROTAC refers to one or more protein degradation targeting chimeras that target the degradation of AKT1-K189 / 276 / 297Ufm proteins.
[0019] Preferably, the PROTAC derivative technology refers to one or more autophagy-lysosome targeting chimeras that target the degradation of AKT1-K189 / 276 / 297Ufm proteins, such as AUTOTAC (Autophagy-Targeting Chimera), ATTEC (Autophagosome-Tethering Compound), and LYTAC (Lysosome-Targeting Chimaeras).
[0020] Preferably, the molecular gel finger enhances the interaction between AKT1-K189 / 276 / 297Ufm protein and E3 ubiquitin ligase, thereby promoting the degradation of AKT1-K189 / 276 / 297Ufm protein by the proteasome pathway and one or more small molecules.
[0021] AKT1 has two homologous proteins, AKT2 and AKT3. All three are highly conserved in both amino acid sequence and structural domains. Therefore, they have the same or similar functions.
[0022] UFL1-mediated jussification modification is applicable to AKT1, AKT2, and AKT3.
[0023] The amino acid sequences of AKT1, AKT2, and AKT3 are highly similar, and the juxtaposition sites K189, K276, and K297 in AKT1 are highly conserved in AKT2 and AKT3. Figure 6 As shown.
[0024] Fourthly, this invention provides the application of AKT1-K189 / 276 / 297Ufm protein as a target in screening products for the prevention and / or treatment of cancer; Preferably, the product is a drug or a test reagent.
[0025] Fifthly, the present invention provides the application of a substance that inhibits the expression of AKT1-K189 / 276 / 297Ufm protein in the preparation of a product, wherein the product has any one or more of the following functions: d1) Inhibits cancer growth; d2) Inhibits cancer metastasis; d3) Cancer treatment; Preferably, the substance that inhibits the expression of AKT1-K189 / 276 / 297Ufm protein is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of targeted chimeras and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm protein and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 of the justolysis system and the protein kinase AKT1, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the jussylation system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
[0026] Sixthly, this invention provides the application of AKT1-K189 / 276 / 297Ufm protein in constructing cell and / or animal models of cancer. Preferably, the cancer is breast cancer, lung cancer, or pancreatic cancer; Preferably, the cell lines used in the cell model are MDA-MB-231 and MDA-MB-468 breast cancer cells.
[0027] In a seventh aspect, the present invention provides a product whose active ingredient includes a substance for inhibiting the expression level of AKT1-K189 / 276 / 297Ufm protein; The product is used to prepare for the detection, prevention, and / or treatment of cancer; The product is a drug or a test reagent.
[0028] The cancers mentioned include breast cancer, lung cancer, and pancreatic cancer.
[0029] Preferably, the product is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of the following: targeted chimeras (PROTAC) and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm proteins, and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 of the justolysis system and the protein kinase AKT1, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the jussylation system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
[0030] Eighthly, the present invention provides a method for treating cancer, the method utilizing AKT1-K189 / 276 / 297Ufm protein.
[0031] One or more of the above technical solutions have the following advantages or beneficial effects: This invention demonstrates for the first time at the cellular level and in mouse models that lysine reversion at positions 189 / 276 / 297 of human AKT1 (AKT1-K189 / 276 / 297Ufm) is essential for the complete activation of AKT1 and for AKT1 to promote breast cancer growth and metastasis. Therefore, AKT1-K189 / 276 / 297Ufm protein can serve as a novel diagnostic marker for the occurrence and metastasis of breast cancer and other cancers, improving the accuracy of predicting metastasis in breast cancer and other cancers, and making the diagnosis of breast cancer and other cancers faster and more accurate. AKT1-K189 / 276 / 297Ufm protein also provides a new molecular target for the treatment of breast cancer and other cancers, laying a theoretical foundation for the development of novel drugs for the treatment of breast cancer and other cancers. Attached Figure Description
[0032] 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.
[0033] Figure 1Example 1 describes the detection of the interaction between UFL1 and AKT1 at the cellular level. Specifically, A and B describe the detection of the interaction between UFL1 and AKT1 in MDA-MB-231 breast cancer cells using co-immunoprecipitation (CO-IP) and Western blotting; C describes the detection of the interaction between UFL1 and AKT1 in A549 lung cancer cells using CO-IP and Western blotting; and D describes the detection of the interaction between UFL1 and AKT1 in PANC-1 pancreatic cancer cells using CO-IP and Western blotting. Figure 2 The experimental data for UFL1 promoting AKT1 in Example 2 are shown below. A represents the ukolization level of AKT1 in shSC, shUFL1#1, and shUFL1#2 DMA-MB-231 breast cancer cells; B represents the ukolization level of AKT1 in shSC, shUFL1#1, and shUFL1#2 DMA-MB-468 breast cancer cells. Figure 3 This section describes the identification of AKT1 ursylation sites in Example 3. In this section, A represents lysine residues in the AKT1 protein that have undergone ursylation in HEK293T cells, and the ursylation of these lysine residues affects the phosphorylation of serine at position 473 of AKT1, i.e., AKT1-S473p; PH refers to the PH (pleckstrinhomology) domain of AKT1; KD refers to the kinase domain of AKT1; and Reg refers to the regulatory domain of AKT1. B represents the ursolic domain); B represents the ursolic level of wild-type AKT1 (AKT1-WT) and AKT1 (AKT1-K189 / 276 / 297R) with lysine at position 189 / 276 / 297 mutated to arginine; C represents the ursolic level of wild-type AKT1 (AKT1-WT) and AKT1 (AKT1-K189 / 276 / 297R) with lysine at position 189 / 276 / 297 mutated to arginine in MDA-MB-468 cells. Figure 4The effect of lysine eurypylation at positions 189 / 276 / 297 of AKT1 on AKT1 activity (i.e., the protein levels of AKT1-S473p and AKT1-T308p) in Example 4 is shown in Figure A, where A represents the S473p and T308p levels of Flag-AKT1-WT or Flag-AKT1-K189 / 276 / 297R in DMA-MB-231 cells that stably express Flag-AKT1-WT or Flag-AKT1-K189 / 276 / 297R; and B represents the S473p and T308p levels of Flag-AKT1-WT or Flag-AKT1-K189 / 276 / 297R in DMA-MB-468 cells that stably express Flag-AKT1-WT or Flag-AKT1-K189 / 276 / 297R. Figure 5 This image shows the regulatory effect of AKT1 lysine eurypylation at positions 189 / 276 / 297 (AKT1-K189 / 276 / 297Ufm) on breast cancer growth and metastasis in Example 5. A shows the volume of tumors formed by injecting shSC cells, shAKT1 cells, shAKT1+AKT1-WT cells, and shAKT1+AKT1-K189 / 276 / 297R cells into the fat pads of female NOD-SCID mice; B shows the growth curves of these tumors; and C shows the lung metastasis of these tumors. Figure 6 The amino acid sequences of AKT1, AKT2 and AKT3 of this invention and the sequence alignment results of the three are shown. Detailed Implementation
[0034] Human AKT1 protein (also known as protein kinase B, PKB), encoded by the AKT1 gene (Genebank NC_000014.9), is an important protein kinase. Studies have shown that phosphorylation of threonine at position 308 (T308p) and serine at position 473 (S473p) in AKT1 are essential for its full activation, and these phosphorylations are significantly increased in various tumor cells, thereby enhancing the pro-cancer effect of AKT1. However, the molecular mechanisms underlying T308p and S473p in AKT1 remain unclear. Post-translational modifications of proteins play important roles in protein conformation, subcellular localization, activity, and stability. However, justinization, as an important type of post-translational modification, requires further investigation regarding whether and how it affects AKT1 phosphorylation. Currently, there are no reported studies on the role of AKT1 justinization in AKT1 activation and in breast cancer and other tumors.
[0035] In one typical embodiment, the present invention provides the use of a substance for detecting AKT1-K189 / 276 / 297Ufm protein in the preparation of cancer diagnostic or auxiliary diagnostic products, wherein the AKT1-K189 / 276 / 297Ufm protein is an AKT1 protein with lysine residues at positions 189, 276, and 297 lysine-treated.
[0036] The application includes any of the following: a1) Screening of active drugs for cancer treatment; a2) Diagnostic reagents for cancer diagnosis or auxiliary diagnosis; Preferably, the cancers include breast cancer, lung cancer, and pancreatic cancer; Preferably, the detection reagent is used to detect the expression level of AKT1-K189 / 276 / 297Ufm protein.
[0037] In another typical embodiment, the present invention provides a product comprising substances for detecting AKT1-K189 / 276 / 297Ufm protein, the product being used for the preparation of a product for the detection, prevention, and / or treatment of cancer.
[0038] In one or more embodiments of this implementation, the AKT1-K189 / 276 / 297Ufm protein is used as a biomarker to prepare products for detecting cancer occurrence and metastasis.
[0039] In one or more embodiments of this implementation, the product includes a product for detecting UAKT1-K189 / 276 / 297Ufm at the protein level and a product for quantitatively detecting AKT1-K189 / 276 / 297Ufm protein; In one or more embodiments of this implementation, the product is selected from one or more of reagents, kits, test strips, and chips.
[0040] In one or more embodiments of this implementation, the product contains a substance specific to the AKT1-K189 / 276 / 297Ufm protein, wherein the substance specific to the AKT1-K189 / 276 / 297Ufm protein includes one or more of the following: b1), b2), and b3). b1) Antibodies that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins; b2) Ligand proteins or peptides that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins; b3) Non-protein compounds that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins.
[0041] In one or more embodiments of this implementation, the substance specific to the AKT1-K189 / 276 / 297Ufm protein is suitable for one or more of the following methods: immunoblotting, immunoprecipitation, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISA), colloidal gold immunoassay, immunochemiluminescence, fluorescence immunochromatography, immunohistochemistry, mass spectrometry, high-performance liquid chromatography (HPLC), capillary gel electrophoresis, flow cytometry, and proteomics.
[0042] In one or more embodiments of this implementation, the test sample of the product includes one or more of tumor tissue, body fluids, and cells isolated from breast cancer patients.
[0043] In one or more embodiments of this implementation, the antibody comprises an antibody that specifically recognizes AKT1-K189 / 276 / 297Ufm protein produced in mice, rats, guinea pigs, chickens, rabbits, pigs, monkeys, and any other organism using any AKT1 short peptide or protein modified with K189 / 276 / 297Ufm.
[0044] In another typical embodiment, the present invention provides the use of AKT1-K189 / 276 / 297Ufm protein as a target in the preparation of products for the prevention and / or treatment of cancer.
[0045] Preferably, the product is a drug or an experimental reagent. The experimental reagent is intended for basic research and can be used to construct relevant cell or animal models for basic research into the pathogenesis of diseases such as breast cancer.
[0046] Preferably, the product is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of the following: targeted chimeras (PROTAC) and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm proteins, and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 of the justolysis system and the protein kinase AKT1, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the jussylation system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
[0047] Preferably, the PROTAC refers to one or more protein degradation-targeting chimeras that target and degrade AKT1-K189 / 276 / 297Ufm proteins. A PROTAC is a hybrid bifunctional small molecule compound containing two different ligands: an E3 ubiquitin ligand and a ligand that binds to the intracellular target protein. The two ligands are linked by a linker, forming a "target protein ligand-linker-E3 ligand" polymer. PROTAC forcibly shortens the distance between the target protein and the intracellular E3 ubiquitin ligand, utilizing the ubiquitin-proteasome pathway to specifically degrade the target protein.
[0048] Preferably, the PROTAC derivative technology refers to one or more autophagy-lysosome targeting chimeras that target the degradation of AKT1-K189 / 276 / 297Ufm proteins, such as AUTOTAC (Autophagy-Targeting Chimera), ATTEC (Autophagosome-Tethering Compound), and LYTAC (Lysosome-Targeting Chimaeras).
[0049] Preferably, the molecular glue refers to one or more small molecules that enhance the interaction between AKT1-K189 / 276 / 297Ufm protein and E3 ubiquitin ligase, thereby promoting the degradation of AKT1-K189 / 276 / 297Ufm protein via the proteasome pathway. The molecular glue is typically a single small molecule that promotes interaction between the target protein or E3 ubiquitin ligase by altering their conformation, without requiring a specific linker chain or a high-affinity ligand for the target protein.
[0050] In another typical embodiment, the present invention provides the application of AKT1-K189 / 276 / 297Ufm protein as a target in screening products for the prevention and / or treatment of breast cancer; In one or more embodiments of this implementation, the product is a drug or a test reagent.
[0051] In another typical embodiment, the present invention provides the application of a substance that inhibits the expression of AKT1-K189 / 276 / 297Ufm protein in the preparation of a product, wherein the product has any one or more of the following functions: d1) Inhibits cancer growth; d2) Inhibits cancer metastasis; d3) Cancer treatment; Preferably, the substance that inhibits the expression of AKT1-K189 / 276 / 297Ufm protein is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of targeted chimeras and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm protein and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 of the justolysis system and the protein kinase AKT1, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the jussylation system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
[0052] In another typical embodiment, the present invention provides the application of AKT1-K189 / 276 / 297Ufm protein in constructing cell and / or animal models of breast cancer. Preferably, the cell lines used in the cell model are MDA-MB-231 and MDA-MB468 breast cancer cells.
[0053] In another typical embodiment, the present invention provides a product whose active ingredient includes a substance for inhibiting the expression level of AKT1-K189 / 276 / 297Ufm protein; The product is used to prepare for the detection, prevention, and / or treatment of breast cancer; The product is a drug or a test reagent.
[0054] Preferably, the product is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of the following: targeted chimeras (PROTAC) and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm proteins, and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 of the justolysis system and the protein kinase AKT1, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the jussylation system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
[0055] In another typical implementation, the present invention provides a method for treating cancer, the method utilizing the AKT1-K189 / 276 / 297Ufm protein.
[0056] Another typical implementation method is to extend the above implementation method to lung cancer and pancreatic cancer, etc.
[0057] Another typical implementation extends the above implementation to the eurythmic modification of other lysine residues on AKT1, including the eurythmic modification of lysines at positions 189, 276 and 297 of AKT1.
[0058] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0059] Example 1, Co-immunoprecipitation (Co-IP), sodium dodecyl sulfate polyacrylamide (SDS-PAGE) electrophoresis, and Western blotting experiments. Cancer cells were lysed using cell lysis buffer, centrifuged, and the supernatant was collected to determine protein concentration. IgG or specific antibody was added to the supernatant, followed by incubation with immunoprecipitation beads overnight. The immunoprecipitation beads were washed three times with cell lysis buffer, and the precipitate was retained, added to loading buffer, and boiled at 100°C for 8 minutes. The samples were then subjected to SDS-PAGE electrophoresis, transfer to a membrane, blocking, antibody incubation, and gel imaging to obtain results showing an interaction between UFL1 and AKT1. Figure 1 As shown in A, UFL1 interacts with AKT1, AKT2, and AKT3. For example... Figure 1As shown in the BE, UFL1 and AKT1 interact in MDA-MB-231 and MDA-MB-468 breast cancer cells.
[0060] Example 2, 2.1, Constructing Cell Lines shUFL1 cells: The shUFL1#1 or shUFL1#2 plasmids with UFL1 knockdown were packaged with virus, and then breast cancer cells were infected with the virus and screened for puromycin to obtain shUFL1#1 or shUFL1#2 breast cancer cells with UFL1 knockdown.
[0061] 2.2 Immunoprecipitation (Co-IP), Sodium Dodecyl Sulfonate Polyacrylamide (SDS-PAGE) Electrophoresis, and Western Blot Experiments Control group and shUFL1#1 or shUFL1#2 breast cancer cells were lysed using cell lysis buffer, centrifuged, and the supernatant was collected to determine protein concentration. Specific AKT1 antibody was added to the supernatant, followed by incubation with immunoprecipitation beads overnight. The immunoprecipitation beads were washed three times with cell lysis buffer, and the precipitate was retained, added to loading buffer, and boiled at 100°C for 8 minutes. The samples were then subjected to SDS-PAGE electrophoresis, transfer to a membrane, blocking, antibody incubation, and gel imaging to obtain the final AKT1 jugation modification results. Figure 1 As shown in A, knocking down UFL1 significantly reduced AKT1 jugation modification in MDA-MB-231 cells; Figure 1 As shown in B, knocking down UFL1 significantly reduced the jugation modification of AKT1 in MDA-MB-468 cells.
[0062] Example 3, 3.1, Constructing Cell Lines Flag-AKT1-WT cells: The Flag-AKT1-WT plasmid expressing wild-type (WT) AKT1 was packaged with virus, and then breast cancer cells were infected with the virus and selected by G418 to obtain Flag-AKT1-WT breast cancer cells that stably express wild-type (WT) AKT1.
[0063] Flag-AKT1-K189 / 276 / 297R cells: The Flag-AKT1-K189 / 276 / 297R plasmid expressing mutant AKT1 was packaged with virus, and then breast cancer cells were infected with the virus and selected by G418 to obtain Flag-AKT1-K189 / 276 / 297R breast cancer cells that stably express mutant AKT1.
[0064] 2.2, Transient transfection Plasmids expressing wild-type (WT) or mutant Flag-AKT1, HA-UFL1, and Myc-UFM1 were transiently transfected into HEK293T cells using the transfection reagent lipo3000. Cells were collected 24 hours later for subsequent experiments.
[0065] 3.3 Immunoprecipitation (Co-IP), Sodium Dodecyl Sulfonate Polyacrylamide (SDS-PAGE) Electrophoresis, and Western Blot Experiments Flag-AKT1-WT and Flag-AKT1-K189 / 276 / 297R breast cancer cells or HEK293T cells were lysed using cell lysis buffer, centrifuged, and the supernatant was collected to determine protein concentration. Flag antibody was added to the supernatant, followed by incubation with immunoprecipitation beads overnight. The immunoprecipitation beads were washed three times with cell lysis buffer, and the precipitate was retained, added to loading buffer, and boiled at 100°C for 8 minutes. The samples were then subjected to SDS-PAGE electrophoresis, transfer to a membrane, blocked, incubated with antibody, and developed using a gel imaging system to obtain the final results of AKT1 jugation modification. Figure 3 As shown in A, in HEK293T cells, both the K189 / 276 / 297R single mutation and the K198 / 276 / 297R triple mutation reduced AKT1 jugation and S473p modification, while other mutations did not affect AKT1 jugation or phosphorylation; Figure 3 As shown in B and C, K189 / 276 / 297R significantly reduced AKT1 jugation in MDA-MB-231(B) and MDA-MB-468(C) breast cancer cells.
[0066] Example 4, Co-immunoprecipitation (Co-IP), sodium dodecyl sulfate polyacrylamide (SDS-PAGE) electrophoresis, and Western blotting experiments. Flag-AKT1-WT and Flag-AKT1-K189 / 276 / 297R breast cancer cells were lysed using cell lysis buffer, centrifuged, and the supernatant was collected to determine protein concentration. Flag antibody was added to the supernatant, followed by incubation with immunoprecipitation beads overnight. The immunoprecipitation beads were washed three times with cell lysis buffer, and the precipitate was retained, added to loading buffer, and boiled at 100°C for 8 minutes. The samples were then subjected to SDS-PAGE electrophoresis, transfer to a membrane, blocked, incubated with antibody, and visualized using a gel imaging system to obtain the final results of AKT1 phosphorylation modification. Figure 4 As shown in Figure A, the K189 / 276 / 297R single mutation significantly reduced the protein levels of AKT1-T308p and AKT1-S473p in MDA-MB-231 cells; Figure 4As shown in A, the K189 / 276 / 297R single mutation significantly reduced the protein levels of AKT1-T308p and AKT1-S473p in MDA-MB-468 cells.
[0067] Example 5 5.1, Constructing Cell Lines shSC cells: The shSC plasmid was packaged with virus, and then breast cancer cells were infected with the virus and screened for puromycin to obtain shSC breast cancer cells that could not knock down AKT1.
[0068] shAKT1 cells: The shAKT1 plasmid with knocked-down AKT1 was packaged with virus, and then breast cancer cells were infected with the virus and screened for puromycin to obtain shAKT1 breast cancer cells with knocked-down AKT1.
[0069] shAKT1+AKT1-WT cells: The AKT1-WT plasmid was packaged with virus, and then shAKT1 breast cancer cells were infected with the virus and selected by G418 to obtain shAKT1+AKT1-WTMDA-MB-231 breast cancer cells with replenished AKT1.
[0070] shAKT1+AKT1-K189 / 276 / 297R cells: The AKT1-K189 / 276 / 297R plasmid was packaged with virus, and then shAKT1 breast cancer cells were infected with the virus and selected by G418 to obtain shAKT1+AKT1-K189 / 276 / 297RMDA-MB-231 breast cancer cells with replenished AKT1-K189 / 276 / 297R.
[0071] 5.2, Experiment on mice bearing fat pad tumors 1x10 6 Several shSC, shAKT1, shAKT1+AKT1-WT, and shAKT1+AKT1-K189 / 276 / 297R breast cancer cells were suspended in a mixture of 50 μL serum-free DMEM and 50 μL matrix gel. The cells were injected into the second left mammary gland tissue of female NOD-SCID mice. Tumor volume was measured every two days starting from day 30. On day 51, the mice were euthanized, and the lungs were perfused. The tumors were dissected and photographed. The dissected lungs were fixed, paraffin-embedded, sectioned, stained with H&E, and photographed. Figure 5As shown in AC, knockdown of AKT1 significantly inhibited the growth of breast tumors and lung metastasis. This inhibitory effect could be effectively compensated by overexpressed AKT1-WT, but not by overexpressed AKT1-K189 / 276 / 297R. These experiments demonstrate that the promotion of breast cancer growth and metastasis by AKT1 protein depends on its jugation modification.
[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The use of a substance for detecting AKT1-K189 / 276 / 297Ufm protein in the preparation of diagnostic or auxiliary diagnostic products for cancer, wherein the AKT1-K189 / 276 / 297Ufm protein is an AKT1 protein with lysine residues at positions 189, 276 and 297 lysine-treated.
2. The application according to claim 1, wherein the application includes any one of the following: a1) Screening of active drugs for cancer treatment; a2) Diagnostic reagents for cancer diagnosis or auxiliary diagnosis; Preferably, the cancers include breast cancer, lung cancer, and pancreatic cancer; Preferably, the detection reagent is used to detect the expression level of AKT1-K189 / 276 / 297Ufm protein.
3. A product characterized in that, The product contains substances for detecting AKT1-K189 / 276 / 297Ufm proteins, and the product is used to prepare for the detection, prevention, and / or treatment of cancer.
4. The product according to claim 3, characterized in that, The products include those for detecting UAKT1-K189 / 276 / 297Ufm at the protein level and those for quantitatively detecting AKT1-K189 / 276 / 297Ufm protein; Preferably, the product is selected from one or more of reagents, kits, test strips, and chips.
5. The product according to claim 3, characterized in that, The product contains a substance specific to the AKT1-K189 / 276 / 297Ufm protein, and the substance specific to the AKT1-K189 / 276 / 297Ufm protein includes one or more of the following: b1), b2), and b3). b1) Antibodies that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins; b2) Ligand proteins or peptides that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins; b3) Non-protein compounds that specifically recognize and bind to AKT1-K189 / 276 / 297Ufm proteins.
6. Application of AKT1-K189 / 276 / 297Ufm protein as a target in the preparation of products for the prevention and / or treatment of cancer; Preferably, the product is a drug or a test reagent; Preferably, the product is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of targeted chimeras and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm protein and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 and protein kinase AKT1 in the jujutosis system, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the justinization system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
7. Application of AKT1-K189 / 276 / 297Ufm protein as a target in screening products for the prevention and / or treatment of cancer; Preferably, the product is a drug or a test reagent.
8. The application of a substance that inhibits the expression of AKT1-K189 / 276 / 297Ufm protein in the preparation of a product, wherein the product has any one or more of the following functions: d1) Inhibits cancer growth; d2) Inhibits cancer metastasis; d3) Cancer treatment; Preferably, the substance that inhibits the expression of AKT1-K189 / 276 / 297Ufm protein is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of targeted chimeras and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm protein and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 and protein kinase AKT1 in the jujutosis system, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the justinization system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.
9. Application of AKT1-K189 / 276 / 297Ufm protein in the construction of cell and / or animal models of cancer. Preferably, the cancer is breast cancer, lung cancer, or pancreatic cancer; Preferably, the cell lines used in the cell model are MDA-MB-231 and MDA-MB-468 breast cancer cells.
10. A product whose active ingredient includes a substance for inhibiting the expression level of AKT1-K189 / 276 / 297Ufm protein; The product is used to prepare for the detection, prevention, and / or treatment of cancer; The product is a drug or a testing reagent; Preferably, the cancers include breast cancer, lung cancer, and pancreatic cancer; Preferably, the product is selected from: c1) Oligonucleotide or oligonucleotide expression vectors that reduce AKT1-K189 / 276 / 297Ufm protein levels; c2) Proteins or peptides that inhibit the activity of UAKT1-K189 / 276 / 297Ufm protein; c3) Small molecule compounds that inhibit the function of AKT1-K189 / 276 / 297Ufm proteins; c4) One or more of targeted chimeras and molecular gels that target the degradation of AKT1-K189 / 276 / 297Ufm protein and their derivatives; c5) Proteins or peptides that inhibit the interaction between the E3 ligase UFL1 and protein kinase AKT1 in the jujutosis system, thereby reducing the level of AKT1-K189 / 276 / 297Ufm protein. c6) Small molecule compounds that reduce the level of AKT1-K189 / 276 / 297Ufm protein by inhibiting the interaction between the E3 ligase UFL1 of the justinization system and the protein kinase AKT1. Preferably, the expression vector includes a eukaryotic expression vector, which includes one or more of plasmid expression vectors and viral expression vectors.