Application of the tumor marker CKB with protein kinase function in the preparation of tumor diagnostic products

Abstract

The present invention relates to the use of the tumor marker CKB, which functions as a protein kinase, in the preparation of tumor diagnostic products, belonging to the field of oncology medicine. The product primarily comprises reagents for detecting tumor marker expression levels, including phosphorylation of CKB T133 and phosphorylation of GPX4S104. Based on the mechanisms of tumor metabolism, the present invention identifies these tumor metabolism markers. By detecting marker expression levels, early diagnosis of tumors can be achieved. Furthermore, testing can be performed using venous peripheral blood samples, significantly reducing the psychological burden and physical damage to the subjects.

Description

Technical Field

[0001] The present invention relates to the application of a tumor marker CKB having a protein kinase function in the preparation of tumor diagnostic products, and belongs to the field of tumor medicine. Background Art

[0002] As we all know, cancer has become the leading cause of threat to the lives and health of the Chinese people. According to the 2022 National Cancer Report released by the National Cancer Center, the overall crude incidence and mortality rates of cancer in my country continued to rise between 2000 and 2016, reflecting the heavy actual cancer burden in my country. The key to cancer treatment lies in early detection and diagnosis so that appropriate treatment measures can be taken. Currently, cancer can only be completely cured if it is treated early. If the tumor progresses to the middle or late stages, the tumor tissue has expanded or metastasized, even if various treatments are used, it is difficult to cure the disease. Conversely, if the tumor is detected in the early or relatively early stages and before metastasis occurs, and timely and appropriate treatment is given, a considerable number of patients can achieve satisfactory treatment results, or even achieve a complete cure.

[0003] Currently, tumor diagnosis is divided into five levels: ① Clinical diagnosis: a presumptive diagnosis based on clinical symptoms, signs, and imaging studies, with reference to the underlying disease progression pattern; ② Surgical diagnosis: a diagnosis based solely on the naked eye appearance of the tumor during surgery or various endoscopic examinations, without pathological confirmation; ③ Physical and chemical diagnosis: a diagnosis consistent with clinical presentation and supported by positive physical and chemical examinations, such as X-rays, ultrasound, CT, and MRI, or measurements of carcinoembryonic antigen and alpha-fetoprotein; ④ Cytopathological diagnosis: a diagnosis based on various types of exfoliated cells and aspirate cytology; and ⑤ Histopathological diagnosis: a diagnosis based on pathological analysis of tissue sections obtained through core needle aspiration. Within these five levels, diagnostic reliability increases, with level 5 being the most reliable. However, the above methods cannot achieve early detection and diagnosis of tumors. More often than not, the optimal treatment period is missed after the tumor has developed to a certain extent, or even when the patient develops serious symptoms and signs. This makes subsequent treatment extremely passive. In addition, pathological diagnosis with better diagnostic results requires puncture to obtain cells or tissue slices, which has a significant mental and physical damage and negative impact on the patient.

[0004] Because the mechanisms of tumor occurrence and development are very complex, there are many deficiencies in related research, which seriously hinders the development of new methods for the diagnosis and treatment of tumors. Metabolism is a key factor affecting the occurrence and development of tumors. During the occurrence and development of tumors, the metabolic pattern of tumor cells will change, which is called "metabolic reprogramming." As one of the important characteristics of tumors, metabolic reprogramming meets the three key needs of tumor cells: the energy required for sustained growth, sufficient biomacromolecules for the synthesis of nucleic acids, proteins, and lipids, and the maintenance of redox homeostasis. Only by understanding and recognizing the mechanisms of tumor metabolism more comprehensively and systematically can we find biomarkers for tumor metabolism, break through the current bottleneck in our research on tumor diagnosis, and truly achieve early diagnosis of tumors from the perspective of metabolic abnormalities.

[0005] In tumor cells, mutations in metabolic enzyme genes or changes in expression levels can lead to significant changes in the content of intracellular metabolites. This "classical" metabolic reprogramming plays a key role in tumor progression. However, in recent years, increasing evidence has shown that some metabolic enzymes and metabolic small molecules can also participate in various important life processes of tumor cells through "non-classical" functions that are not dependent on metabolic pathways, thereby promoting the occurrence and development of tumors. Activation of receptor protein kinases is common in various cancers, however, the relationship between receptor protein kinase activation and ferroptosis is still unclear.

[0006] We found that activation of the insulin-like growth factor 1 (IGF1) receptor (IGF1R) in hepatocellular carcinoma (HCC) cells stabilizes the expression of glutathione peroxidase 4 (GPX4), a key inhibitory regulator of ferroptosis, in a creatine kinase B (CKB)-dependent manner. Akt activated by IGF1R signaling binds to CKB and phosphorylates it at T133. This phosphorylation reduces creatine binding to CKB, thereby decreasing CKB's metabolic activity and enabling CKB to interact with GPX4. Importantly, CKB, a protein kinase, phosphorylates GPX4 at S104, adjacent to the chaperone-mediated autophagy (CMA) target motif in GPX4. GPX4 S104 phosphorylation prevents HSC70 from binding to GPX4, thereby abrogating CMA-mediated GPX4 degradation, alleviating the effects of cystine deprivation and SLC7A11 inhibitor-induced ferroptosis, and promoting tumor growth in mice. Furthermore, GPX4 levels were positively correlated with phosphorylation levels of CKB T133 and GPX4 S104 in human HCC specimens and correlated with poor prognosis in HCC patients. These findings reveal a key mechanism by which tumor cells combat ferroptosis through receptor protein kinase activation and non-metabolic functions of CKB to enhance GPX4 stability and highlight the potential of targeting CKB protein kinase activity for cancer diagnosis and therapy. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a tumor marker for preparing an early diagnosis product for tumors. By detecting the expression level of the tumor marker, the early diagnosis of the tumor in the subject can be performed.

[0008] The present invention provides the use of a tumor marker CKB having a protein kinase function in the preparation of a tumor diagnostic product, wherein the tumor marker is one or more of CKB T133 phosphorylation and GPX4 S104 phosphorylation;

[0009] Among them, CKB T133 phosphorylation refers to the phosphorylation at position 133 of the amino acid sequence of CKB, and GPX4 S104 phosphorylation refers to the phosphorylation at position 104 of GPX4; among them, the NCBI sequence number of CKB is: NP_001814.2, and the NCBI sequence number of GPX4 is: NP_001354761.1.

[0010] The product includes a reagent for detecting the expression level of a tumor marker in a sample.

[0011] The reagents include a primary antibody and a secondary antibody.

[0012] The first antibody is the CKB T133 phosphorylation antibody CKB pT133 or the GPX4 S104 phosphorylation antibody GPX4pS104.

[0013] The second antibody is homologous to the first antibody and is labeled with horseradish peroxidase.

[0014] The detection method of the product is Elisa or Western Blot.

[0015] The sample can be venous peripheral blood, paracancerous tissue, or tumor tissue. After collecting venous peripheral blood, it must be placed on ice for 15 minutes and then centrifuged at 3500 rpm for 15 minutes to obtain serum or plasma for testing or storage at -80°C. Samples that cannot be tested immediately must be stored at -80°C and thawed on ice before testing.

[0016] The product is primarily a test kit. The kit includes reagents for detecting tumor marker expression levels, as well as one or more substances selected from the group consisting of a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent. The instructions describe how to use the kit for testing and how to use the test results to assess tumor progression and select treatment options. The kit components can be packaged in aqueous or lyophilized form.

[0017] The diagnostic product can be used for the early, middle or late diagnosis of the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, bone sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Based on the mechanism of tumor metabolism, the present invention has found tumor markers targeting tumor metabolism. By detecting the expression level of tumor markers, early diagnosis of tumors can be achieved;

[0020] 2. The test can be completed by collecting venous peripheral blood samples, which greatly reduces the psychological burden and physical damage of the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The changes in GPX4 protein levels in Huh7 and HCCLM3 under IGF1 stimulation;

[0022] Figure 2 Immunoprecipitation analysis of liver cancer cell lines showed that CKB can interact with GPX4 under IGF1 stimulation;

[0023] Figure 3 To analyze which inhibitors can block the interaction between CKB and GPX4 by immunoprecipitation;

[0024] Figure 4 For autoradiography and immunoprecipitation analysis, in vitro phosphorylation of CKB T133 by AKT;

[0025] Figure 5 To analyze the effect of CKB T133 phosphorylation on the interaction between CKB and GPX4 by immunoprecipitation;

[0026] Figure 6Immunoblot analysis of GPX4 protein levels in Huh7 (top) and HCCLM3 (bottom) cells expressing constitutively activated IGF1R (IGF1R-CA) and the CKB T133A mutant cell line treated with CHX (100 μg / mL) for the indicated times; GPX4 expression (right) using tubulin as a marker; **P < 0.001 by two-tailed Student's t-test;

[0027] Figure 7 For autoradiography and immunoprecipitation analysis, in vitro phosphorylation of GPX4S104 by CKB T133 under the action of AKT;

[0028] Figure 8 For immunoprecipitation analysis, phosphorylation of CKB T133 under IGF1 stimulation can phosphorylate GPX4 S104;

[0029] Figure 9 For immunoblotting analysis, GPX4 protein expression in the presence or absence of IGF1 treatment was assessed using tubulin as a marker;

[0030] Figure 10 For immunoblotting analysis, GPX4 expression in the presence or absence of mutation of the GPX4 S104A site and with or without IGF1 treatment was investigated using tubulin as a marker;

[0031] Figure 11 Purified SFB-CKB protein on streptavidin affinity beads with or without His-AKT was used for in vitro kinase assays; the beads were washed five times with PBS and incubated with GST-GPX4 protein in the presence of ATP. The beads were removed, and GST-GPX4 was incubated with His-HSC70 for GST pμLldown assays.

[0032] Figure 12 For immunoprecipitation analysis, the interaction of GPX4 with HSC70 or LAMP2A was investigated with or without mutation of the GPX4 S104A site and with or without IGF1 treatment;

[0033] Figure 13 For immunoprecipitation analysis, the interaction of GPX4 with HSC70 or LAMP2A was investigated with or without mutation of the CKB T133A site and with or without IGF1 treatment;

[0034] Figure 14 For immunoblotting analysis, GPX4 expression was assessed under conditions expressing constitutively activated IGFR (IGFR-CA), various GPX4 mutants, and different CHX treatment times, using tubulin as a marker.

[0035] Figure 15 Analysis of the GPX4 amino acid sequence showed that GPX4 S104 is adjacent to the CMA target motif (97NVKFD101) of GPX4;

[0036] Figure 16 Huh7 cells and the indicated cell lines knocked in with CKB T133A (left) or GPX4 S104A (right) mutants were treated with cysteine ​​depletion and IGF1 (100 ng / mL) for 24 h, and the Lipid ROS-positive cell rate was determined. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0037] Figure 17 Huh7 cells and the indicated cell lines knocked in with CKB T133A (left) or GPX4 S104A (right) mutants were treated with cysteine ​​depletion and IGF1 (100 ng / mL) for 24 h, and cell death rates were determined. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0038] Figure 18 Huh7 cells and the indicated cell lines knocked in with CKB T133A (left) or GPX4 S104A (right) mutants were treated with Erastin and IGF1 (100 ng / mL) for 24 h, and the Lipid ROS-positive cell rate was determined. Data are mean ± SD, **P < 0.01 (two-tailed t-test);

[0039] Figure 19 Huh7 cells and the indicated cell lines knocked in with CKB T133A (left) or GPX4 S104A (right) mutants were treated with Erastin and IGF1 (100 ng / mL) for 24 h, and cell death rates were determined. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0040] Figure 20 Huh7 cells and the indicated cell lines knocked in with CKB T133A (left) or GPX4 S104A (right) mutants were treated with sulfasalazine and IGF1 (100 ng / mL) for 24 h, and cell viability was measured. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0041] Figure 21 Huh7 cells and the indicated cell lines knocked in with CKB T133A (left) or GPX4 S104A (right) mutants were treated with Erastin and IGF1 (100 ng / mL) for 24 h, and cell viability was measured. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0042] Figure 22 The indicated cell lines of Huh7 and HCCLM3 cells and the GPX4 S104D mutant were treated with cysteine ​​depletion and IGF1 (100 ng / mL) for 24 h, and the Lipid ROS-positive cell rate (left) and cell death rate (right) were measured, respectively. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0043] Figure 23 The indicated cell lines of Huh7 and HCCLM3 cells and the GPX4 S104D mutant were treated with Erastin and IGF1 (100 ng / mL) for 24 h, respectively. The Lipid ROS-positive cell rate (left) and cell death rate (right) were measured, respectively. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0044] Figure 24 Huh7 cells and the indicated cell lines with different mutants were treated with cysteine ​​depletion (left) and erastin (right) for 24 h, and the Lipid ROS-positive cell rate was determined. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0045] Figure 25 Huh7 cells and the indicated cell lines with different mutants were treated with cysteine ​​depletion (left) and erastin (right) for 24 h, and the cell death rate was determined. Data are mean ± SD, **P < 0.01 (two-tailed t test);

[0046] Figure 26 The results of mouse tumor size detection are shown in Table 1. **P < 0.001 by two-tailed t test.

[0047] Figure 27 For immunohistochemistry experiments on mouse tumor samples;

[0048] Figure 28 Immunohistochemistry experiments of human liver cancer samples (top) and statistical graphs of the indicated IHC staining scores (bottom), **P < 0.0001 by two-tailed t-test;

[0049] Figure 29 Immunohistochemistry experiment (top) and correlation analysis (bottom) of human liver cancer samples;

[0050] Figure 30 is the survival curve of CKB T133 phosphorylation level;

[0051] Figure 31 Survival curve of GPX4 S104 phosphorylation level. DETAILED DESCRIPTION

[0052] In order to make the purpose and technical solution of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings. The experimental methods described in the following examples are all conventional methods unless otherwise specified; if no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; the reagents and materials described are all commercially available unless otherwise specified.

[0053] The tumor diagnostic product of the present invention is a kit, which contains reagents for detecting the expression levels of CKB T133 phosphorylation and GPX4 S104 phosphorylation. Each reagent includes a first antibody and a second antibody. The first antibody is the CKB T133 phosphorylation antibody CKB pT133 and the GPX4 S104 phosphorylation antibody GPX4 pS104. The second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0054] The kit also includes a container, instructions, a positive control, a negative control, a buffer, an adjuvant, and a solvent. The instructions describe how to use the kit for testing and how to use the test results to assess tumor progression and select treatment options. The kit components can be packaged in an aqueous medium.

[0055] The kit can be used for the early, middle or late diagnosis of the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, bone sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0056] The detection method of the kit is Western Blot.

[0057] Western Blot, also known as protein immunoblotting experiment, is a technique commonly used to separate and identify proteins in research. It uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane. The membrane is then incubated with specific antibodies against the target protein. During the membrane washing process, unbound antibodies are washed away, leaving only antibodies bound to the target protein, and finally the bound antibodies are detected by developing film or fluorescent scanning. Since the antibody only binds to the target protein, generally only a clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the location and intensity of specific reactions, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the strong specificity and high sensitivity of immunity, Western blot analysis can detect as low as 1ng of target protein. This method is widely used in molecular biology, biochemistry, immunogenetics and other molecular biology fields. The specific detection steps are as follows:

[0058] 1. Sample Preparation

[0059] 1) Sample collection: Collect venous peripheral blood, place it on ice for 15 minutes, and centrifuge it at 3500 rpm for 15 minutes to obtain serum for testing;

[0060] 2) Make ice and prepare ice boxes;

[0061] Prepare cell lysis buffer: Determine the required lysis buffer according to the number of cells: 50 μL / well of a six-well plate;

[0062] Lysis buffer formula: 100 μL Biyuntian lysis buffer + 1 μL protease inhibitor cocktail + 1 μL PMSF;

[0063] 3) Prepare cells as needed: Remove culture medium, wash three times with 1x PBS (to remove serum from the culture medium), and add an appropriate amount of lysis buffer to each well of a 6-well plate. Quickly scrape cells with a cell scraper and transfer to a 1.5 mL tube. Place on ice for 20 minutes, shake to mix, and place on ice again for 10 minutes.

[0064] 4) Centrifuge at 12000g, 4°C for 15 min and collect the supernatant into another 1.5 mL tube;

[0065] 5) Take 2.5 μL of sample and dilute it with 22.5 μL of triple-distilled water for protein concentration determination by BCA assay;

[0066] 6) Add 5× Loading Buffer (2.5 mL Buffer / 10 mL protein) to the remaining sample and cook at 95°C for 10 min, shaking once during the cooking process.

[0067] 7) Load the sample directly onto the gel or aliquot and store at -80℃ for long-term storage.

[0068] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0069] 1) Prepare separation gel (5 mL / gel);

[0070] 2) Carefully inject the separating gel, leaving about 2 cm of space (below the red border of the gel preparation rack) for the stacking gel. Cover the top layer with deionized water and let it stand for about 30 minutes.

[0071] 3) Prepare stacking gel (2 mL / gel);

[0072] 4) Inject the concentrated gel into the upper end of the separation gel, taking care to avoid air bubbles;

[0073] 5) Insert the comb and wait for the stacking gel to solidify (there should be a clear boundary between the gel and the comb, and the solidification time of the separating gel should be greater than 2 hours). Wash the well with double-distilled water to remove gel fragments, and then dry with filter paper.

[0074] 6) Place the gel in the electrophoresis tank and add 1× electrophoresis buffer to both the upper and lower tanks (do not reuse more than 3 times);

[0075] 7) Sample loading: Take 5 μL of prestained marker from the marker well and add an appropriate amount of 1× loading buffer to equal the total volume of the sample well. The sample loading volume is generally 15-25 μL. First, heat the well at 95°C for 5-10 minutes, shaking once, then quickly centrifuge and load the sample onto the gel. Add an equal volume of 1× loading buffer to the well without sample.

[0076] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the stacking gel, increase the current to 100-120V. The electrophoresis time is determined by the size of the target protein and the position of the marker. Generally, it is sufficient to allow the target protein to run to two-thirds of the separation gel.

[0077] 3. Membrane Transfer

[0078] 1) Cut the gel according to the marker and the position of the target band (make sure to mark the cutting corner of the gel), and immerse the eluted gel in transfer buffer for 15 minutes;

[0079] 2) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it in transfer buffer along with four 3mm filter papers and a sponge for 15 minutes;

[0080] 3) Preparation of "sandwich biscuits":

[0081] Follow the following sequence: fiber pad—filter paper—PVDF membrane—gel—filter paper—fiber pad;

[0082] Note: Align each item you add to ensure there are no bubbles;

[0083] 4) Transfer: Transfer time is determined according to 1)

[0084] One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0085] 4. Membrane Blocking and Antibody Incubation

[0086] 1) After transfer, wash the membrane with 10 mL of 1× TBS at room temperature for 10 minutes.

[0087] 2) Incubate 5 mL of milk powder blocking solution at room temperature for 2 hours or at 4°C with gentle shaking overnight. Since milk powder is difficult to dissolve, it should be prepared at least 1 hour in advance.

[0088] 3) Wash the membrane three times with 10 mL of TBS / T, each time for 5 minutes;

[0089] 4) Add 5 mL of primary antibody dilution buffer (antibody diluted according to the instructions), shake gently at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (to inhibit bacterial growth) at 5 μL / mL primary antibody solution, and store at 4°C (uncommon antibodies can be stored at -20°C for long-term storage). It can be reused.

[0090] 5) Wash the membrane three times with 10 mL of TBS / T, each time for 5 minutes;

[0091] 6) Add secondary antibody (usually 1:2000 dilution) and gently shake at room temperature for 1 hour;

[0092] 7) Wash the membrane three times with 10 mL of TBS / T, each time for 5 minutes.

[0093] 5. Development and fixation (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibodies)

[0094] 1) Color development: First, lay out plastic wrap, then lay more plastic wrap on absorbent paper. Pour water, developer (do not use if the color darkens), and fixer into separate trays. Mix 2.5mL of ECL-A and 2.5mL of ECL-B and protect from light. Bring the ECL mixture, membrane, etc. into the darkroom. Close the door, lock it, and pull back the cloth. Pour the ECL mixture into a small box. Lightly blot the membrane with absorbent paper and place it in the ECL mixture, shaking it at room temperature for 5 minutes (to ensure that the ECL spreads evenly across the membrane). After blotting with absorbent paper, place it on plastic wrap, wrap it face down, and place it on a clip. Cut X-film (note that you can only handle the edges of the X-film with your hands) and place it on the membrane, using the cut corner as a marker (note: when removing the film, turn the light to minimum and do not face the light; face away from the light). Adjust the exposure time based on the brightness of the bands. Generally, you can start with a 2-minute exposure. Observe the depth of the bands and then determine the optimal exposure time.

[0095] 2) Development and fixing: Take out the X-film and place it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it with water once and then place it in the fixing solution for more than 5 minutes.

[0096] Finally, the target protein band is obtained on the PVDF membrane. By comparing with the size of the protein marker, the size and position of the target protein can be determined (CKB protein size is 42KD, GPX4 protein size is 17KD), and the expression result of the target protein can be obtained.

[0097] Diagnostic analysis of test results: If protein bands of the following sizes appear on the PVDF membrane: CKB T133 phosphorylation or GPX4 S104 phosphorylation, it indicates that the subject may have invasive cancer, be in the advanced stage of invasive cancer, or have a poor prognosis.

[0098] The tumor markers of the present invention were obtained through the following studies:

[0099] 1 Materials and Methods

[0100] 1.1 Materials used:

[0101] (1) Cell type:

[0102] Huh7 cells (human hepatocellular carcinoma cell line) were obtained from ATCC, and HCCLM3 cells (human hepatocellular carcinoma cell line) were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0103] (2) Athymic nude mice are BALB / c athymic nude mice.

[0104] (3) Patient samples:

[0105] Tissue samples were obtained from the First Affiliated Hospital of Zhejiang University and the Affiliated Hospital of Qingdao University. Human HCC tissue specimens and adjacent adjacent tissues were stained with immunohistochemistry (IHC) to compare protein expression between tumor and normal tissue samples. Staining of tissue sections was quantitatively scored based on the percentage of positive cells and staining intensity.

[0106] (4) The shRNA sequences used for gene knockdown are as follows:

[0107] CKB: 5'-ACCTCATGCCTGCCCAGAAAT-3';

[0108] GPX4: 5'-GTGAGGCAAGACCGAAGTAAA-3'.

[0109] 1.2 Test methods

[0110] (1) Protein level detection:

[0111] First, the protein is extracted from the cells, and then the corresponding antibody is used to react with the protein to produce an antigen-antibody reaction, and the expression and function of the specific target protein are determined by substrate color development.

[0112] The protein level detection systems used in the present invention include immunoprecipitation and immunoblotting.

[0113] Antibodies used: HA (ab18181) (for IP), c-SRC (ab16885), c-SRC pY418 (ab4816), CKB (ab151579) (for WB), HSC70 (ab154415), LAMP2A (ab18528) were purchased from Abcam (Cambridge, United Kingdom); AKT (#4685), AKT pS473 (#4060), HA (#3724) (for WB), ERK1 / 2 (#4695), pERK1 / 2 (#4370), c-Jun (#9165), c-Jun pS73 (#3270) were purchased from Cell Signaling Technology (Danvers, MA). Normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027), GST (sc-138), tubμLin (sc8035) were purchased from Santa Cruz Biotechnology. [γ32P] ATP was purchased from PerkinElmer. SP600125, U0126, SU6566, CHX, ATP, streptavidin magnetic beads, anti-Flag M2 agarose beads, EDTA-free protease inhibitor cocktail, Erastin, IGF1, sμLfasalazine(SAS), 3XFlagpeptides, mouse anti-Flag(F1804), rabbit anti-Flag(F7425)and anti-His (SAB1305538) was purchased from Sigma-Aldrich (St. Louis, MO). MK-2206, hygromycin (400053), puromycin (540222), and G418 (345810) were purchased from EMD Biosciences (San Diego, CA). GPX4 (GTX54095) antibody (for IP and WB) was purchased from Genetex (Irvine, CA). Antibodies against CKB (NBP2-59462) (for WB) and GPX4 Antibody (MAB5457) (for WB) were purchased from Novus Biologicals (Littleton, CO).Glutathione agarose and antibodies against CKB (PA5-117870) (for IP), GPX4 Antibody (PA5-102521) (for IHC), 4Hydroxynonenal (4-HNE) (BS-6313R) and PTGS2 (TA805286) were purchased from Thermo Fisher Scientific. Ni-NTA agarose was purchased from Qiagen. CKB pT133 blocking peptide (LSSRVR-pT-GRSIRG) and GPX4 pS104 blocking peptide (VKFDMF-pS-KICVNG) were purchased from Selleck-Chem. CKB pT133- and GPX4 pS104-specific antibodies were purchased from Signalway Biotechnology (Pearland, TX). .

[0114] (2) Cell activity detection

[0115] The viable cells were measured using Cell Counting Kit-8 (CCK-8, Dojindo). 4 Cells were seeded into clear-bottom 96-well plates and treated with different doses of sulfadiazine (SAS) or erastin for 4 h. The cells were then treated with 10 μL CCK-8 reagent (100 μL culture medium per well) for 1 h. The absorbance of the cells at a wavelength of 450 nm was measured using a Flustar Omega microplate analyzer (BMG Labtech).

[0116] (3) Cell death detection

[0117] Quantification of cell death was confirmed by propidium iodide staining. Briefly, cells were seeded into 12-well plates at a confluence density of 50%. The next day, cells were treated with different reagents or cysteine-depleted medium for a certain period of time. Cells, including floating dead cells, were collected and stained with 5 μg / mL propidium iodide. The percentage of propidium iodide-positive dead cells was analyzed by flow cytometry (FACS).

[0118] (5) Lipid peroxidation detection

[0119] Cells were seeded into 12-well plates at a confluence density of 50% and treated with different reagents or cysteine-free medium for the indicated time on the next day. They were then stained with 5 μM BODIPY-C11 (Invitrogen) at 37°C for 30 min and analyzed by flow cytometry. Lipid ROS-positive cells were defined as cells with FITC fluorescence greater than 99% of that of the unstained sample.

[0120] (5) In vitro kinase assay

[0121] Purified GST-AKT was incubated with His-CKB (200 ng) proteins purified from different bacteria in kinase buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 50 mM MgCl2, 1 mM Na3VO4, 1 mM DTT, 5% glycerol, 0.5 mM ATP and 10 μCi [γ-32P]ATP) at 25°C for 1 h. The reaction was then terminated by adding SDS-PAGE loading buffer and heating to 100°C for 5 min. Finally, the mixture was subjected to SDS-PAGE or autoradiography analysis.

[0122] (6) Mouse tumor sample acquisition and analysis

[0123] One million Huh7 cells expressing recombinant CKB mutants or Huh7 cells were suspended in 20 μL of DMEM medium containing 33% matrix and injected into the livers of 6-week-old male BALB / c nude mice (n = 6 / group). Animals were euthanized 28 days after injection and examined for tumor growth.

[0124] The tumor tissue in the liver was surgically dissected, fixed with 4% formaldehyde, and embedded in paraffin. The formation and phenotype of the tumor were determined by histological analysis using H&E staining and immunohistochemical staining. The tumor volume was calculated using the formula: V = 1 / (2a 2 b) (V, volume; a, shortest diameter; b, longest diameter).

[0125] 2 Results and Analysis

[0126] 2.1IGF1 induces AKT-mediated CKB T133 phosphorylation, which promotes the binding of CKB to GPX4, thereby upregulating the stability of GPX4.

[0127] Time course experiments showed that IGF1 treatment significantly increased GPX4 protein levels ( Figure 1 To determine the regulatory mechanism, we performed immunoprecipitation analysis of GPX4 and found that IGF1 induced the interaction between GPX4 and CKB ( Figure 2 ).

[0128] To elucidate the mechanism by which IGF1 induces the binding of CKB to GPX4, we pretreated Huh7 cells with U0126, SU6656, SP600125, and MK-2206, which inhibited IGF1-induced activation of ERK, c-Src, JNK, and AKT, respectively. Notably, only MK-2206 blocked the IGF1-induced interaction between CKB and GPX4 ( Figure 3 ).

[0129] Mutation of T133 to alanine (A) abolished purified AKT-mediated CKB phosphorylation, which was also detected by a specific validated anti-CKB pT133 antibody ( Figure 4 ).

[0130] Recombinant expression of CKB T133A abolished IGF1-induced CKB binding to GPX4 in HCC cells, whereas the phosphorylation-mimicking mutant of CKB T133D could interact with GPX4 in the absence of IGF1 treatment ( Figure 5 ).

[0131] Notably, expression of CKB T133A reduced the half-life of GPX4 and inhibited IGF1-enhanced GPX4 protein ( Figure 6 ).

[0132] 2.2CKB-mediated GPX4 S104 phosphorylation promotes GPX4 protein stability.

[0133] Given that tumor cells can confer non-canonical functions as protein kinases on metabolic enzymes through post-translational modifications, we performed an in vitro phosphorylation assay by incubating purified CKB with purified GPX4 in the presence of purified active AKT. The results showed that CKB WT, but not CKB T133A, phosphorylates GPX4 on the conserved serine (S)104 residue ( Figure 7 ).

[0134] In HCC cells, IGF1 induces GPX4 S104 phosphorylation, while CRISPR / Cas9 knock-in expression of GPX4 S104A and CKB T133A in HCC cells inhibits GPX4 S104 phosphorylation ( Figure 8 ). Expression of CKB R292H inhibited IGF1-enhanced GPX4 expression and reduced the half-life of GPX4 ( Figure 9 GPX4 S104A has a shortened half-life and IGF1 has no promoting effect on its protein expression ( Figure 10 ).

[0135] 2.3CKB-mediated GPX4 S104 phosphorylation promotes stable GPX4 expression by blocking GPX4 binding to HSC70 and CMA-mediated GPX4 degradation.

[0136] To test whether phosphorylation of S104 in GPX4 alters the local conformation of the protein, thereby preventing its binding to HSC70, we performed a GST pμLldown assay. The results showed that purified GST-GPX4 bound to purified His-HSC70, and this interaction was abolished by CKB-mediated phosphorylation of S104 in GPX4 in the presence of active AKT ( Figure 11 ).

[0137] IGF1 treatment of HCC cells reduced GPX4 binding to HSC70 and LAMP2A, and this reduction was largely attributable to GPX4 S104A ( Figure 12 ) and CKB T133A( Figure 13 ). In addition, mutation of the CMA target motif (N97A / V98A) of GPX4, which disrupts the binding of GPX4 to HSC70, rescued the impaired GPX4 S104A protein expression and its stability upon IGFR activation ( Figure 14 Analysis of the GPX4 amino acid sequence showed that GPX4 S104 is adjacent to the CMA target motif (97NVKFD101) of GPX4 ( Figure 15 ).

[0138] IGF1R-induced and CKB-mediated GPX4 S104 phosphorylation inhibits ferroptosis

[0139] Cysteine ​​depletion or treatment of Huh7 and LM3 HCC cells with erastin reduced their activity and correspondingly increased lipid ROS levels. IGF1 treatment alleviated these effects, while knock-in expression of CKB T133A or GPX4 S104A in HCC cells abolished the rescue effect induced by IGF1 treatment. As expected, IGF1 treatment ameliorated cell death and decreased cell viability induced by cysteine ​​depletion and erastin, and these IGF1-induced effects were attenuated after knock-in expression of CKB T133A or GPX4 S104A ( Figure 16-21 ).

[0140] The role of CKB-mediated GPX4 S104 phosphorylation in suppressing ferroptosis was further revealed by expressing phosphorylation-mimicking GPX4 S104D or CKB T133D mutants, which suppressed cysteine ​​depletion- and erastin-induced ferroptosis. Figure 22, 23). Furthermore, expression of GPX4 S104A in HCC cells attenuated this protective effect induced by CKB T133D ( Figure 24 , 25).

[0141] 2.5CKB-mediated GPX4 S104 phosphorylation promotes tumor growth and is positively correlated with the clinical aggressiveness of HCC.

[0142] To determine the role of CKB-mediated GPX4 S104 phosphorylation in tumor growth, we injected Huh7 cells expressing IGF1R-CA (with or without knock-in expression of CKB T133A or GPX4 S104A) subcutaneously or orthotopically into glandular nude mice. Figure 26 ) Through immunohistochemical (IHC) analysis of tumor tissue, it was found that tumor growth was inhibited to a certain extent, GPX4 expression was reduced, and the expression of ferroptosis marker proteins prostaglandin peroxidase synthase 2 (PTGS2) and 4-hydroxynonenal (4-HNE) was enhanced ( Figure 27 ).

[0143] Next, we performed immunohistochemical analysis on 90 human HCC specimens and their adjacent normal tissues. Compared with normal tissues, the phosphorylation levels of CKB T133 and GPX4 S104 were increased, the total GPX4 level was decreased, and the levels of PTGS2 and 4-HNE were decreased in tumor specimens ( Figure 28 ). In addition, in HCC specimens, GPX4 levels were positively correlated with the phosphorylation levels of CKB T133 and GPX4 S104, and negatively correlated with PTGS2 and 4-HNE ( Figure 29 ).

[0144] Patients whose tumors had high levels of CKB T133 phosphorylation (47 patients) or GPX4 S104 phosphorylation (49 patients) had shorter survival than those whose tumors had low levels of protein phosphorylation ( Figure 30 , 31). These results support the role of CKB-regulated GPX4 phosphorylation in human HCC clinically and reveal a relationship between CKB-mediated GPX4 S104 phosphorylation and enhanced GPX4 stability, ferroptosis, and clinical aggressiveness of HCC.

[0145] Activation of receptor protein kinases, including IGFR, frequently occurs in various cancers, including HCC. However, whether receptor protein kinase activation plays a role in regulating ferroptosis in cancer cells remains unclear. We demonstrate that IGF1R activation induces GPX4 expression, which is dependent on the atypical function of the metabolic enzyme CKB. CKB was originally described as a metabolic enzyme that catalyzes ATP-dependent creatine phosphorylation, which is crucial for energy buffering in tissues with fluctuating energy demands. 18-21 In this study, we found that IGF1R signaling activates AKT to bind to CKB and phosphorylate CKB at T133. Phosphorylated CKB reduces its creatine binding, thereby reducing CKB's metabolic activity and enabling it to interact with GPX4. Importantly, GPX4-associated CKB acts as a protein kinase and phosphorylates GPX4 at S104. The CKB R292H ATP-binding-deficient mutant, but not the CKB C283S protein-binding-deficient mutant, lost the ability to phosphorylate GPX4, further demonstrating that CKB uses ATP as a phosphate donor to phosphorylate GPX4. Phosphorylation of GPX4 S104, adjacent to the CMA recognition sequence in GPX4, prevents HSC70 from binding to GPX4, thereby abrogating CMA-mediated GPX4 degradation. IGF1 treatment attenuates cysteine ​​deprivation- and erastin-induced lipid ROS production and cell death; expression of CKB T133A or GPX4 S104A abrogates these effects. Furthermore, expression of CKB T133A or GPX4 S104A reduces tumor growth and significantly exacerbates the ferroptosis and tumor growth inhibitory effects of sulfasalazine. Analysis of human hepatocellular carcinoma specimens and adjacent normal tissues revealed that tumor cells exhibit reduced ferroptosis compared with normal cells, with a corresponding upregulation of CKB T133 and GPX4 S104 phosphorylation. These findings suggest that oncogenic signaling induces tumor-specific regulation to eliminate lipid peroxides and counteract ferroptosis by conferring noncanonical functions on CKB. Metabolic enzymes, such as phosphoenolpyruvate carboxylic acid kinase 1 (PCK1), choline kinase α2, 6-phosphofructo-2-kinase (PFKFB3), phosphoglycerate kinase 1 (PGK1), ketohexokinase (KHK) isoform A (KK-A), and pyruvate kinase M2 (PKM2), act as protein kinases to regulate a broad spectrum of important cellular activities.

[0146] The finding that CKB protein kinase activity is dependent on GPX4 and ferroptosis in tumor cells, and that CKB pT133A or GPX4 pS104 levels are positively correlated with shorter survival in HCC patients, highlights the potential of targeting CKB protein kinase activity for the treatment of human cancers. This finding emphasizes the importance of oncogenic signaling in reprogramming the metabolic and non-metabolic functions of metabolic enzymes in combating ferroptosis.

[0147] Therefore, inhibiting CKB expression specifically blocks the positive regulatory effect of AKT on CKB phosphorylation, inhibiting CKB from exerting its non-classical function to suppress tumor development and progression. While not affecting CKB's gluconeogenic metabolic enzyme function, maximally inhibiting its non-classical function can inhibit tumor development and progression to the greatest extent. This invention will provide new ideas and effective solutions for the development of anti-tumor drugs targeting lipid metabolic reprogramming and for the early diagnosis of tumors.

Claims

1. Use of a reagent for detecting a marker in the preparation of a liver cancer diagnostic product, characterized in that: The marker is one or more of T133-phosphorylated CKB and S104-phosphorylated GPX4.

2. The use according to claim 1, characterized in that: The product includes reagents for detecting the expression level of biomarkers in a sample.

3. The use according to claim 2, characterized in that: The reagents include a primary antibody and a secondary antibody.

4. The use according to claim 3, characterized in that: The primary antibodies include the CKB T133 phosphorylation antibody CKBpT133 or the GPX4 S104 phosphorylation antibody GPX4 pS104.

5. The use according to claim 4, characterized in that: The secondary antibody is homologous to the primary antibody and is labeled with horseradish peroxidase.

6. The use according to claim 2, characterized in that: The detection method of the product is Elisa or Western Blot.

7. The use according to claim 2, characterized in that: The sample is venous peripheral blood, paracancerous tissue or tumor tissue.

8. The use according to any one of claims 2 to 6, characterized in that: The product includes a kit.

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