BICD2 protein S192 site o-glcnac glycosylation modification as pdac gemcitabine drug resistance marker and its application

Abstract

The application discloses BICD2 protein S192 site O-GlcNAc glycosylation modification as a PDAC gemcitabine drug resistance marker and application thereof, and belongs to the technical field of biological diagnosis and biological medicine. It is found by the application that in gemcitabine-resistant pancreatic ductal adenocarcinoma cells and organoids, OGT catalyzes O-GlcNAc glycosylation modification of the S192 site of the BICD2 protein, the modification promotes phosphorylation of the S102 site of the BICD2 protein, enhances the combination of the BICD2 protein with RanBP2 and DCTN1, accelerates G2 / M cell cycle conversion, drives tumor proliferation and gemcitabine drug resistance, and is significantly related to poor prognosis of patients. The application also provides a kit containing an antibody specifically recognizing the modification, and application of an inhibitor targeting the modification in reversing gemcitabine drug resistance drugs, thereby providing a brand-new biomarker and treatment target for clinical diagnosis and treatment of PDAC.

Description

Technical Field

[0001] This invention belongs to the fields of biological diagnostics and biomedicine, and in particular relates to the O-GlcNAc glycosylation modification of the S192 site of the BICD2 protein as a gemcitabine resistance marker in pancreatic ductal adenocarcinoma (PDAC) and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is the most malignant digestive system tumor, with a 5-year overall survival rate of less than 10%, and is projected to become the second leading cause of cancer-related deaths worldwide by 2030. PDAC has an insidious onset, with no specific symptoms in the early stages. More than 80% of patients are diagnosed at a locally advanced stage or with distant metastases, thus losing the opportunity for radical surgery; even among patients who undergo surgical resection, the recurrence rate remains as high as 80%.

[0003] Gemcitabine is the first-line standard chemotherapy drug for the clinical treatment of pancreatic dysplasia of the liver (PDAC). Whether used as monotherapy or in combination with albumin-bound paclitaxel, it remains a core treatment option for patients with advanced PDAC. However, the clinical efficacy of gemcitabine is severely limited by inherent and acquired tumor resistance, with an objective response rate of less than 20%. Most resistant patients experience disease progression and death within 12 months. Currently, gemcitabine resistance remains a major obstacle in the treatment of pancreatic cancer. Reported mechanisms of gemcitabine resistance include nucleoside transport abnormalities, apoptosis regulation imbalances, and DNA damage repair defects. However, there are currently no effective clinical interventions targeting these mechanisms, nor are there any approved targeted drugs to reverse gemcitabine resistance in PDAC. This is a core bottleneck in the clinical treatment of PDAC. Currently, there is a lack of reliable biomarkers that can effectively predict the gemcitabine response and prognosis of PDAC patients.

[0004] BICD2 is a highly conserved cell cycle regulatory adaptor protein that regulates nuclear membrane rupture, spindle assembly, and mitosis during the G2 / M phase by binding to nuclear porin RanBP2 and dynein activator DCTN1. Existing research has only reported that phosphorylation modification of BICD2 is involved in chemotherapy resistance in some tumors, but its role in PDAC is completely unreported, and no studies have revealed the function of O-GlcNAcylation modification of BICD2 in tumorigenesis, development, and chemotherapy resistance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide O-GlcNAc glycosylation modification at the S192 site of BICD2 protein as a gemcitabine resistance marker for pancreatic ductal adenocarcinoma (PDAC) and its application, providing a novel biomarker and therapeutic target for the clinical diagnosis and treatment of PDAC.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A biomarker for gemcitabine resistance or prognostic assessment in pancreatic ductal adenocarcinoma, wherein the biomarker is a protein with O-GlcNAc glycosylation modification at serine position 192 of the BICD2 protein.

[0007] Preferably, the markers also include O-GlcNAc transferase and / or proteins with phosphorylated serine residue 102 of the BICD2 protein.

[0008] The present invention also provides a kit for evaluating gemcitabine resistance in pancreatic ductal adenocarcinoma or for assessing the prognosis of patients with pancreatic ductal adenocarcinoma after gemcitabine treatment, the kit comprising an antibody that specifically recognizes O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein.

[0009] Preferably, the kit further includes OGT-specific quantitative PCR primers or OGT protein-specific antibodies for detecting OGT expression levels, and / or antibodies for detecting phosphorylation levels at the S102 site of the BICD2 protein.

[0010] Preferably, the antibody is a rabbit polyclonal antibody or monoclonal antibody that specifically recognizes the BICD2 protein that is O-GlcNAc glycosylated at the S192 site; The kit also includes a positive control and a negative control. The positive control is a recombinant BICD2 protein modified with O-GlcNAc at the S192 site, and the negative control is a wild-type recombinant BICD2 protein without O-GlcNAc modification.

[0011] The present invention also provides a method for screening candidate drugs for the treatment of gemcitabine-resistant pancreatic ductal adenocarcinoma, comprising the following steps: S1: Contact the candidate compound with cells expressing BICD2 protein modified by O-GlcNAc glycosylation at S192 site; S2: Detect the O-GlcNAc glycosylation level of BICD2 protein at S192 site in the cells, or the binding level of BICD2 to RanBP2 / DCTN1, or the cell cycle arrest in the G2 / M phase. S3: If the candidate compound can reduce the O-GlcNAc glycosylation level, disrupt the binding of BICD2 to RanBP2 / DCTN1, or induce G2 / M phase arrest, then the candidate compound is a candidate drug.

[0012] This invention also provides the application of an inhibitor that targets and inhibits the O-GlcNAc glycosylation modification of the S192 site of the BICD2 protein in the preparation of drugs for treating pancreatic ductal adenocarcinoma and reversing gemcitabine resistance.

[0013] Preferably, the inhibitor is at least one of the following: a small molecule inhibitor targeting O-GlcNAc modification at the BICD2 S192 site, a nucleic acid inhibitor, a neutralizing antibody or nanobody, or a gene editing reagent.

[0014] More preferably, the nucleic acid inhibitor is a siRNA or shRNA targeting BICD2, and the sequence of the siRNA is 5'-GGAGCUCACACUACAUGUU-3'.

[0015] Preferably, the inhibitor blocks the phosphorylation activation of BICD2 at the S102 site and its binding to the RanBP2 / DCTN1 complex by inhibiting the O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein, thereby blocking the progression of pancreatic cancer cells in the G2 / M phase and reversing gemcitabine resistance.

[0016] Compared with the prior art, the present invention has the following beneficial effects: In gemcitabine-resistant PDAC cells and patient-derived organoids, OGT expression levels and overall O-GlcNAc glycosylation levels were significantly upregulated. Mechanistic studies of this invention revealed that OGT directly binds to and catalyzes O-GlcNAc glycosylation of the BICD2 protein at serine position 192 (S192). This modification further promotes BICD2 phosphorylation, enhances its interaction with RanBP2 and DCTN1, and accelerates the G2 / M cell cycle transition, thereby driving tumor proliferation and gemcitabine resistance. Importantly, analysis of clinical tissue samples showed that high levels of OGT and O-GlcNAcylated BICD2 in PDAC patient tumor tissues were significantly associated with poor response to gemcitabine treatment, shortened overall survival (OS), and reduced disease-free survival (DFS).

[0017] Based on this, this invention reveals for the first time that high expression of OGT and O-GlcNAc glycosylated BICD2 protein is strongly correlated with gemcitabine resistance and poor prognosis in PDAC. O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein has clinical application value as a biomarker for assessing gemcitabine resistance and prognosis in PDAC patients. Furthermore, modification targeting this site can effectively reverse gemcitabine resistance in PDAC, providing a novel biomarker and therapeutic target for the clinical diagnosis and treatment of PDAC. Attached Figure Description

[0018] Figure 1Images of wild-type and gemcitabine-resistant pancreatic cancer cells and pancreatic cancer organoids in Example 1 of this invention are shown under a bright-field microscope; wherein, PANC-1 and AsPC-1 are wild-type pancreatic cancer cells, PANC-1-Gem-R and AsPC-1-Gem-R are gemcitabine-resistant pancreatic cancer cells, Human organoids are wild-type pancreatic cancer organoids, and Organoids-Gem-R are gemcitabine-resistant pancreatic cancer organoids.

[0019] Figure 2 This is a comparison of the expression capabilities of key glycolytic enzymes in WT and Gem-R cells and organoids in Example 1 of the present invention; wherein, AsPC-1 and PANC-1 are wild-type pancreatic cancer cells, AsPC-1-R and PANC-1-R are Gem-R cells, Organoids are wild-type pancreatic cancer organoids, and Organoids-R are gemcitabine-resistant pancreatic cancer organoids.

[0020] Figure 3 This is an electrophoresis image showing the levels of OGT and O-GlcNAc glycosylated proteins in WT and Gem-R cells and organoids in Example 1 of the present invention; wherein, AsPC-1 and PANC-1 are wild-type pancreatic cancer cells, AsPC-1-R and PANC-1-R are Gem-R cells, Organoids are wild-type pancreatic cancer organoids, and Organoids-R are gemcitabine-resistant pancreatic cancer organoids.

[0021] Figure 4 This is an electrophoresis image showing the levels of OGT and O-GlcNAc glycosylated proteins in 12 pancreatic cancer tissue samples from Example 1 of the present invention; where N represents gemcitabine-sensitive cancer tissue samples and R represents gemcitabine-sensitive cancer tissue samples.

[0022] Figure 5 This is an immunofluorescence staining image of OGT and O-GlcNAc glycosylated proteins in gemcitabine-sensitive and drug-resistant samples in Example 1 of the present invention.

[0023] Figure 6 This is a scatter plot showing the correlation between the expression levels of OGT and O-GlcNAc glycosylated proteins in gemcitabine-resistant samples in Example 1 of this invention.

[0024] Figure 7 This is a protein electrophoresis image of Gem-R cells co-transfected with FLAG-OGT and His-BICD2 in Example 1 of the present invention.

[0025] Figure 8 This is an immunofluorescence staining image of OGT and BICD2 in Gem-R cells in Example 1 of the present invention.

[0026] Figure 9 This is a graph showing the LC-MS / MS analysis results of the O-GlcNAcylation modification site of BICD2 in Example 1 of the present invention.

[0027] Figure 10 This is an electrophoresis diagram of Western Blot detection of BICD2 phosphorylation level downregulated by the S192 site mutation in BICD2 in Example 1 of the present invention; wherein, P-BICD2 represents phosphorylated BICD2 protein.

[0028] Figure 11 These are representative images of multiplex immunofluorescence co-staining of OGT, O-GlcNAc glycosylation, BICD2, and DCTN1 in a pancreatic cancer tissue microarray in Example 2 of this invention.

[0029] Figure 12 This is a comparison of Kaplan-Meier survival curves for high and low expression of O-GlcNAc glycosylation in Example 2 of the present invention; where DFS represents disease-free survival and OS represents overall survival.

[0030] Figure 13 This is a comparison of Kaplan-Meier survival curves for OGT high and low expression in Example 2 of the present invention; where DFS represents disease-free survival and OS represents overall survival.

[0031] Figure 14 This is a comparison of Kaplan-Meier survival curves for high and low expression of O-GlcNAc glycosylated BICD2 in Example 2 of the present invention; where DFS represents disease-free survival and OS represents overall survival.

[0032] Figure 15 Immunofluorescence staining images of O-GlcNAc glycosylated BICD2, OGT, and DCTN in gemcitabine-sensitive and drug-resistant samples in Example 2 of this invention.

[0033] Figure 16 This is a comparison of Kaplan-Meier survival curves for PDAC patients with high and low expression of BICD2 S192 O-GlcNAc glycosylation in Example 3 of the present invention; where DFS represents disease-free survival and OS represents overall survival.

[0034] Figure 17 This is the ROC curve of predicting gemcitabine resistance in PDAC patients by the O-GlcNAc glycosylation level at the BICD2 S192 site in Example 3 of the present invention. Detailed Implementation

[0035] This invention provides a biomarker for gemcitabine resistance or prognostic assessment in pancreatic ductal adenocarcinoma, wherein the biomarker is a protein with O-GlcNAc glycosylation modification at serine position 192 of the BICD2 protein; preferably, the biomarker also includes O-GlcNAc transferase and / or a protein with phosphorylation modification at serine position 102 of the BICD2 protein.

[0036] This invention also provides a kit for evaluating gemcitabine resistance in pancreatic ductal adenocarcinoma or assessing the prognosis of gemcitabine treatment in patients with pancreatic ductal adenocarcinoma. The kit includes an antibody that specifically recognizes O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein. Preferably, the kit further includes OGT-specific quantitative PCR primers or OGT protein-specific antibodies for detecting OGT expression levels, and / or an antibody for detecting phosphorylation levels at the S102 site of the BICD2 protein. More preferably, the antibody is a rabbit polyclonal or monoclonal antibody that specifically recognizes BICD2 protein with O-GlcNAc glycosylation at the S192 site. More preferably, the kit further includes a positive control and a negative control, wherein the positive control is a recombinant BICD2 protein with O-GlcNAc modification at the S192 site, and the negative control is a wild-type recombinant BICD2 protein without O-GlcNAc modification. The detection can be performed at the protein level, such as using specific antibodies via Western blot, immunohistochemistry, or enzyme-linked immunosorbent assay (ELISA); or at the mRNA level, such as using quantitative PCR or high-throughput sequencing. Preferably, the kit also includes protein lysis buffer, washing buffer, chromogenic solution, and stop solution. When using this kit, if the O-GlcNAc glycosylation level at the S192 site of the BICD2 protein in the tumor tissue of a PDAC patient is higher than a set threshold, the patient can be identified as having a high risk of gemcitabine resistance and a poor prognosis. The product can also be a diagnostic reagent or a gene sequencing chip.

[0037] The present invention also provides a method for screening candidate drugs for treating gemcitabine-resistant pancreatic ductal adenocarcinoma, comprising the following steps: S1, contacting cells expressing BICD2 protein modified by O-GlcNAc glycosylation at site S192 with a candidate compound; S2, detecting the O-GlcNAc glycosylation level of the BICD2 protein at site S192 in the cells, or the binding level of BICD2 to RanBP2 / DCTN1, or the cell cycle arrest at G2 / M phase; S3, if the candidate compound can reduce the O-GlcNAc glycosylation level, disrupt the binding of BICD2 to RanBP2 / DCTN1, or induce G2 / M phase arrest, then the candidate compound is considered a candidate drug.

[0038] This invention also provides the application of an inhibitor targeting and inhibiting O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein in the preparation of a medicament for treating pancreatic ductal adenocarcinoma and reversing gemcitabine resistance; preferably, the inhibitor is at least one of a small molecule inhibitor, a nucleic acid inhibitor, a neutralizing antibody or nanobody, or a gene editing reagent targeting O-GlcNAc modification at the S192 site of BICD2; more preferably, the nucleic acid inhibitor is siRNA or shRNA targeting BICD2, wherein the siRNA sequence is 5'-GGAGCUCACACUACAUGUU-3'; preferably, the inhibitor blocks phosphorylation activation of BICD2 at the S102 site and its binding to the RanBP2 / DCTN1 complex by inhibiting O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein, thereby inhibiting the progression of pancreatic cancer cells to the G2 / M phase and reversing gemcitabine resistance. Preferably, the medicament further includes a pharmaceutically acceptable carrier, diluent, or excipient, and the dosage form of the medicament is an injection, tablet, capsule, or sustained-release formulation.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] The English abbreviations used in this invention are explained below: Gem-R: Gemcitabine resistance; OGT: O-GlcNAc transferase; FLAG: A commonly used protein tag consisting of eight amino acids (DYKDDDDK). O-GlcNAc glycosylation: refers to OGT-mediated O-GlcNAc glycosylation of proteins, i.e., BICD2 proteins that undergo O-GlcNAc glycosylation modification; PDAC: Pancreatic ductal adenocarcinoma.

[0041] Example 1 Construction of gemcitabine resistance model and detection of OGT / O-GlcNAc glycosylation level 1. Experimental Materials This embodiment was approved by the Ethics Committee of Taizhou Hospital Affiliated to Zhejiang University School of Medicine (Ethics No.: K20220404). Before sample collection, all patients signed written informed consent forms, and the study strictly adhered to the Declaration of Helsinki and relevant ethical guidelines.

[0042] This study included 92 human pancreatic tissue samples, including: (1) 12 pairs of pancreatic cancer tissues and adjacent non-tumor tissues, used to detect OGT / O-GlcNAc glycosylation expression, metabolic enzyme analysis and liquid chromatography-tandem mass spectrometry (LC-MS / MS) metabolomics analysis; (2) tissue microarrays (TMA) containing 80 pairs of pancreatic cancer tissues and corresponding adjacent normal pancreatic tissues, used for multiplex immunofluorescence staining of O-GlcNAc glycosylation, OGT, BICD2, RanBP2 and DCTN1.

[0043] Human PANC-1 cells (RRID: CVCL_0480) and AsPC-1 cells (RRID: CVCL_0152) were purchased from the Cell Bank of the Chinese Academy of Sciences (March 2022). Cells were cultured in RPMI-1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in a humidified incubator at 37°C and 5% CO2. Mycoplasma contamination was routinely assessed every six months, and cell lines were identified by short tandem repeat (STR) analysis.

[0044] Plasmid, siRNA, and lentiviral manipulation: FLAG-OGT, His-BICD2, and BICD2 mutants (S182A, S192A) were constructed using the pcDNA3.1(+) vector and verified by Sanger sequencing. shRNA / siRNA targeting OGT / BICD2 and a negative control were transfected using Lipofectamine 2000. Stable cell lines were selected using puromycin and G-418. Detailed primer, antibody, and sequence information are shown in Tables 1-3.

[0045] Table 1. List of siRNA sequences used in Example 1

[0046] Table 2 List of antibodies used in Example 1

[0047] Table 3. List of primers used in Example 1

[0048] Identification of O-GlcNAc Glycosylation Sites and Antibody Synthesis: The protein complex obtained from anti-FLAG immunoprecipitation was sent to Shanghai Applied Protein Technology Co., Ltd. for LC-MS / MS (Liquid Chromatography-Tandem Mass Spectrometry) analysis to identify OGT-interacting proteins and O-GlcNAc glycosylation sites. Ser102 phosphorylated peptide antigen was synthesized and used to immunize New Zealand rabbits. The titer was detected by ELISA, and after affinity purification, a qualified rabbit polyclonal antibody for human BICD2 Western blot was obtained.

[0049] 2. Statistical Analysis Data are expressed as mean ± standard deviation (SD) of at least three independent trials. Statistical analysis was performed using SPSS 24.0 (SPSS Inc.) or GraphPad Prism 10.0. Normality was assessed using the D'Agostino-Pearson test. When data were normally distributed (as determined by the D'Agostino-Pearson test), differences between groups were assessed using Student's t-test; if variances were unequal (as determined by the F-test), Welch correction was used. If data were not normally distributed, nonparametric tests (Mann-Whitney test) were used. Differences among multiple groups were analyzed using two-way ANOVA. Survival curves were plotted using the Kaplan-Meier method, and Cox proportional hazards regression was used to determine the effects of biomarkers on overall survival (OS) and disease-free survival (DFS). A p-value <0.05 was considered statistically significant.

[0050] 3. Research Results 3.1. Elevated levels of O-GlcNAc glycosylated protein and OGT in gemcitabine-resistant pancreatic cancer. To investigate the metabolic basis of gemcitabine resistance, gemcitabine-resistant pancreatic cancer cell lines (PANC-1 and AsPC-1) and pancreatic cancer organoid models were established. Wild-type (WT) pancreatic cancer cells and patient-derived pancreatic cancer organoids were cultured with stepwise increasing concentrations of gemcitabine over approximately six months. See also Figure 1 ,pass Figure 1 Under bright-field microscopy, the Gem-R cell line exhibited mesenchymal transition, increased cell volume, loose arrangement, increased nuclear-cytoplasmic ratio, and enhanced cytoplasmic granularity. Gem-R organoids showed increased diameter, rougher boundaries, uneven internal translucency, and thickened marginal cell layers, and remained structurally stable without disintegration after drug treatment. At the end of the six-month treatment period, the established Gem-R cell line showed 300 to 700 times higher drug resistance than WT cells, while the Gem-R organoids showed approximately 40 times higher resistance.

[0051] The proliferative capacity of WT and Gem-R cells was compared. Results showed that Gem-R pancreatic cancer cell lines (PANC-1-R and AsPC-1-R) exhibited significantly enhanced proliferative capacity and colony-forming potential; EdU staining further confirmed the high proliferative activity of Gem-R cells. Furthermore, cell cycle analysis revealed an increased proportion of G2 / M phase cells in Gem-R cells, indicating a high proliferative state. To quantify the relative glycolytic metabolism of Gem-R cells and organoids, glucose uptake, lactate release, ATP production, oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) were further measured. Results showed that, compared with the control group, Gem-R cell lines and organoids exhibited significantly higher glucose uptake, lactate secretion, and ATP production. Compared with WT cells, both Gem-R cells and organoids showed increased ECAR and decreased OCR.

[0052] Key glycolytic enzymes in wild-type and Gem-R cells and organoids were analyzed. See also Figure 2 The results showed increased expression of hexokinase (HK), glucose-6-phosphate isomerase (GPI), glutamine-fructose-6-phosphoamyltransferase (GFAT), and OGT in PANC-1-Gem-R and AsPC-1-Gem-R cells. These findings suggest enhanced activity of the hexosamine biosynthesis pathway (HBP) driven by increased glucose uptake and enhanced OGT-mediated O-GlcNAc glycosylation in Gem-R cells. See also Figure 3 The results showed that, compared with the WT counterpart, the Gem-R cell line and organoids had higher levels of OGT and O-GlcNAc glycosylated proteins. Analysis was performed on 12 pancreatic cancer tissue samples (6 gemcitabine-sensitive and 6 gemcitabine-resistant). See [link to relevant documentation]. Figure 4 The results showed that in tumors with gemcitabine resistance, the levels of OGT and O-GlcNAc glycosylated proteins mediated by OGT were significantly increased.

[0053] To assess the metabolomic profiles of gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer samples, LC-MS / MS-based metabolomics analysis was performed to quantify metabolite levels in gemcitabine-sensitive / resistant pancreatic cancer cells and tissue samples. Results showed that glycolytic intermediates accumulate in gemcitabine-resistant pancreatic tumors. Specifically, levels of dihydroxypyruvate phosphate and glyceraldehyde-3-phosphate (upstream metabolites of pyruvate and lactate) were significantly elevated in resistant cells, along with a substantial increase in pyruvate and lactate levels. Further investigation was conducted to determine whether enhanced glucose uptake in Gem-R cells led to elevated O-GlcNAc glycosylation. Results showed that in Gem-R cells, the level of O-GlcNAc glycosylation of total protein increased with increasing glucose concentration. Immunofluorescence staining for OGT and O-GlcNAc glycosylation was performed on tissue sections from 12 pancreatic cancer samples; see [link to relevant documentation]. Figure 5 The results showed that in gemcitabine-resistant cancer tissue samples, the levels of OGT and O-GlcNAc glycosylated proteins were significantly increased, and there was a strong correlation between the expression levels of OGT and O-GlcNAc glycosylated proteins. (See also...) Figure 6 The results showed that both OGT and O-GlcNAc glycosylated proteins were significantly upregulated in drug-resistant cases.

[0054] 3.2 OGT promotes O-GlcNAc glycosylation of BICD2 through direct binding. To elucidate the mechanistic basis of OGT biological function, Gem-R cells were transfected with the FLAG-OGT plasmid, and OGT-interacting proteins were purified for LC-MS / MS analysis. BICD2 (a adaptor protein involved in cell cycle regulation) was identified as a potential OGT binding chaperone. After co-transfection of FLAG-OGT and His-BICD2 into Gem-R cells, immunoprecipitation was performed using anti-FLAG or anti-His antibodies. (See [link to relevant documentation]). Figure 7 , Figure 7 The electrophoresis results confirmed the interaction between the two proteins, OGT and BICD2. See also... Figure 8 Immunofluorescence staining showed that OGT and BICD2 were partially co-localized in Gem-R cells.

[0055] Structurally, the OGT comprises a tetrapeptide repeat (TPR), a nuclear localization signal (NLS), and a catalytic domain (CAT). Based on protein structure modeling and molecular docking results, three truncated OGT constructs were built: a catalytic core region (TPR: 1-486), a nuclear localization region (NLS: 487-900), and a catalytic domain (CAT: 901-1046) to locate the interacting domains. Co-IP analysis showed that the full-length OGT and the NLS region (487-900) could bind to BICD2, while the TPR (CC: 1-486) ​​and CAT (901-1046) domains could not, indicating that the NLS region is crucial for this interaction. BICD2 is a cell cycle regulatory protein composed of coiled-coil domains (CC1: 1-269; CC2: 267-537; CC3: 538-808). Truncated analysis shows that the absence of CC2 (ΔCC2), rather than ΔCC1 or ΔCC3, eliminates the binding with OGT, indicating that the CC2 domain mediates this interaction.

[0056] Given that OGT is the only enzyme catalyzing O-GlcNAc glycosylation of proteins, we further investigated whether BICD2 undergoes this modification. His-BICD2 protein was immunoprecipitated and enriched from Gem-R cells transfected with the His-BICD2 construct, followed by Western blot analysis using an anti-O-GlcNAc antibody. The results confirmed that BICD2 undergoes O-GlcNAc glycosylation, with a stronger level of modification observed in gemcitabine-resistant cell lines, and this modification increased in a glucose concentration-dependent manner. Furthermore, treatment with the specific OGT inhibitor OSMI-1 or OGT knockdown significantly inhibited BICD2 O-GlcNAc glycosylation. Conversely, OGT overexpression or treatment with the O-GlcNAc enzyme inhibitor PugNAc significantly enhanced this modification. These findings confirm that BICD2 O-GlcNAc glycosylation is directly regulated by OGT.

[0057] See Figure 9 LC-MS / MS analysis revealed two evolutionarily conserved potential O-GlcNAc glycosylation sites (S182 and S192) on BICD2. Site-directed mutagenesis to alanine (eliminating the hydroxyl group required for modification) showed that S192A (instead of S182A) significantly reduced O-GlcNAc glycosylation of BICD2. Further investigation demonstrated that S192 modification is regulated by OGT and glucose availability. These results reveal glycosylation modification of the BICD2 protein, identify the OGT-binding domain of BICD2, and identify the O-GlcNAc glycosylation site S192 on BICD2, providing a molecular basis for targeting BICD2 O-GlcNAc glycosylation.

[0058] 3.3 OGT-induced O-GlcNAc glycosylation of BICD2 promotes its phosphorylation and binding to RanBP2 and DCTN1. To further elucidate the molecular mechanism by which OGT and O-GlcNAc glycosylation regulate gemcitabine resistance and promote cell proliferation in pancreatic cancer cells, transcriptome sequencing was performed on Sh-NC and Sh-OGT Gem-R cells to identify differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis showed that, compared with Gem-R cells, OGT knockdown Gem-R cells were significantly enriched in pathways related to cell division and cell cycle progression, including DNA replication, nuclear plaque, and cell cycle. This result is consistent with the observed role of OGT / O-GlcNAc glycosylation in promoting cell cycle progression. Given that cell cycle progression is tightly regulated by cyclins and cyclin-dependent kinases (CDKs), changes in the expression of these genes were further examined. Quantitative PCR and Western blot showed that in Gem-R cells, OGT deletion or OSMI-1 treatment downregulated the expression of Cyclin-B1 and CDK1, while the levels of Cyclin-A2 and CDK2 did not change significantly. Conversely, in WT cells, OGT overexpression or PugNAc treatment upregulated Cyclin-B1 and CDK1 without affecting Cyclin-A2 or CDK2. These results strongly support the specific role of OGT and O-GlcNAc glycosylation in regulating the G2 / M transition rather than other phases of the cell cycle.

[0059] To validate these conclusions in clinical samples, this study performed transcriptome sequencing on 12 human pancreatic cancer tissues (6 gemcitabine-sensitive and 6 drug-resistant) and screened for differentially expressed genes (DEGs). Functional enrichment analysis again showed that cell division and cell cycle-related pathways were the core pathways with significant enrichment. Detection of cyclins and cyclin-dependent kinases (CDKs) showed that the expression levels of cyclin B1, CDK1, cyclin A2, and CDK2 were significantly upregulated in drug-resistant tissues, with cyclin B1 and CDK1 showing the most significant upregulation. These data indicate that gemcitabine-resistant pancreatic cancer exhibits an abnormally accelerated cell cycle, and that OGT and O-GlcNAc glycosylation primarily affect the G2 / M phase transition.

[0060] Given that BICD2's core biological function is primarily to interact with nuclear porin RanBP2 during the G2 phase of cell division, and that this protein can bind dynein and dynactin through its N-terminal CC1 domain and part of its intermediate CC2 domain, thereby assisting both in anchoring to the nuclear envelope, this study found that OGT-mediated O-GlcNAc glycosylation occurs at the S192 site of the BICD2 protein's CC1 domain. Based on this result, we further investigated whether OGT and O-GlcNAc glycosylation regulate the interaction between BICD2 and RanBP2, and the recruitment process of dynein.

[0061] First, this study used immunofluorescence assays to detect the co-localization of BICD2 with RanBP2 and DCTN1 in G2 phase (CDK1-positive) cells. The results showed that the co-localization level of BICD2 with RanBP2 / DCTN1 was significantly enhanced in Gem-R cells compared to WT cells. Subsequently, co-immunoprecipitation (Co-IP) experiments using His-BICD2-transfected cells confirmed that the binding ability of BICD2 with RanBP2 / DCTN1 was stronger in Gem-R cells. Knockdown of OGT in Gem-R cells significantly weakened the co-localization and binding efficiency of BICD2 with RanBP2 / DCTN1 in G2 phase; while overexpression of OGT had the opposite effect.

[0062] Gem-R cells expressing His-BICD2 were treated with the OGT inhibitor OSMI-1 and the O-GlcNAc activator PugNAc, respectively, yielding consistent results: OSMI-1 inhibited and PugNAc promoted the interaction between His-BICD2 and RanBP2 / DCTN1. The study also found that this binding interaction increased with increasing glucose concentration. Notably, the S192 mutation significantly disrupted the binding ability of BICD2 to RanBP2 / DCTN1, and this process was regulated by OGT expression levels. In summary, OGT-mediated O-GlcNAc glycosylation modification plays a crucial role in regulating the interaction between BICD2 and nuclear porin RanBP2 during the G2 phase of cell cycle, thereby affecting the recruitment process of dynein / DCTN1.

[0063] Previous studies have confirmed that the binding of BICD2 to RanBP2 and DCTN1 is regulated by the phosphorylation state of BICD2. Therefore, this embodiment further investigates whether BICD2 glycosylation modification affects its own phosphorylation level, and whether the effect of BICD2 glycosylation in promoting its binding to RanBP2 and DCTN1 is achieved by upregulating BICD2 phosphorylation. First, a specific antibody targeting the BICD2 phosphorylation site (S102) was prepared, and the specificity of the antibody was verified. Compared with WT cells, the BICD2 phosphorylation level in Gem-R cells was significantly increased. Subsequently, the BICD2 phosphorylation level of Gem-R cells cultured at different glucose concentrations was detected, and the results showed that the degree of BICD2 phosphorylation increased in a dose-dependent manner with increasing glucose concentration.

[0064] In addition, Gem-R cells with different OGT expression levels were examined: treatment with OGT overexpression or PugNAc significantly upregulated BICD2 phosphorylation levels; while knockdown of OGT or OSMI-1 intervention significantly reduced BICD2 phosphorylation levels.

[0065] Further verification using immunoprecipitation (IP) experiments showed that the S192 mutation in BICD2 (which inhibits glycosylation modification) simultaneously downregulates BICD2 phosphorylation levels. See [link to relevant documentation]. Figure 10 The results show that BICD2 glycosylation modification can regulate its autophosphorylation.

[0066] Finally, the co-regulatory effects of O-GlcNAc glycosylation and phosphorylation of BICD2 on its binding to RanBP2 and DCTN1 were investigated. The results showed that knockdown of OGT or mutation of the O-GlcNAc modification site S192 significantly inhibited the binding of BICD2 to RanBP2 and DCTN1; similarly, mutation of the BICD2 phosphorylation site S102 also blocked this binding. In cells expressing the S102 phosphorylation site mutant BICD2, knockdown or overexpression of OGT altered the glycosylation level of BICD2 but did not affect its phosphorylation level or regulate its binding ability to RanBP2 and DCTN1. These findings indicate that the regulatory role of OGT and O-GlcNAc glycosylation in BICD2 recruitment of RanBP2 and DCTN1 depends on the mediation of BICD2 phosphorylation.

[0067] In summary, OGT-mediated O-GlcNAc glycosylation of BICD2 at S192 promotes phosphorylation of BICD2 at S102, thereby regulating the binding of BICD2 to RanBP2 and DCTN1 and affecting cell cycle progression.

[0068] Example 2 The OGT-BICD2-O-GlcNAc glycosylation-RanBP2-DCTN1 regulatory axis is associated with poor prognosis in pancreatic cancer patients. To assess the clinical relevance of the results in Example 1, multiplex immunofluorescence staining was performed on a tissue microarray (TMA) containing paired human pancreatic cancer samples from 80 patients using specific antibodies against O-GlcNAc glycosylation, OGT, BICD2, and RanBP2. The clinicopathological characteristics of the enrolled patients are detailed in Table 4. See also... Figure 11 The results showed that O-GlcNAc glycosylation and OGT expression levels were significantly increased in pancreatic cancer tissues, especially in gemcitabine-resistant samples. Furthermore, a strong positive correlation was observed between O-GlcNAc glycosylation and OGT expression in both pancreatic cancer and normal pancreatic tissues. See also Figure 12 and 13 The results showed that patients with high O-GlcNAc glycosylation and OGT expression had significantly worse disease-free survival (DFS) and overall survival (OS).

[0069] Table 4 Baseline data of patients with pancreatic ductal adenocarcinoma

[0070] When BICD2 total protein expression was measured alone, no significant difference was observed in the gemcitabine-resistant patient group; however, O-GlcNAc glycosylated BICD2 (co-expression of BICD2 and O-GlcNAc glycosylation) was significantly elevated in pancreatic cancer tissues, and the upregulation was more pronounced in gemcitabine-resistant cases. Furthermore, its expression pattern was highly consistent with that of O-GlcNAc glycosylation and OGT. See also Figure 14 The results showed that high levels of O-GlcNAc glycosylation BICD2 were associated with poor patient prognosis.

[0071] For further analysis of co-expression patterns, see [link to relevant documentation]. Figure 15 The results showed that O-GlcNAc glycosylated BICD2 significantly co-localized with both OGT and DCTN1, which was more pronounced in gemcitabine-resistant cases. Quantitative assessment using triple-positive staining of O-GlcNAc glycosylation, BICD2, and DCTN1 revealed enhanced interaction between O-GlcNAc glycosylated BICD2 and DCTN1 in pancreatic cancer tissues, particularly in treatment-resistant samples, and was closely associated with adverse clinical outcomes. These clinical sample studies confirm that the OGT-BICD2-O-GlcNAc glycosylation-RanBP2 / DCTN1 regulatory axis plays a crucial biological role in pancreatic cancer progression and chemotherapy resistance.

[0072] Example 3 Preparation and validation of the PDAC gemcitabine resistance / prognostic assessment kit The kit composition of this embodiment is as follows: 1) Rabbit anti-human BICD2 S192 site O-GlcNAc glycosylation polyclonal antibody; The preparation method and concentration of the specific antibody against the O-GlcNAc glycosylation modification of serine at position 192 of the human BICD2 protein are as follows: ① Antigen peptide design and synthesis: Using the S192 site of the human BICD2 protein (NM_001003800.2) as the core, a 15aa specific glycosylated antigenic peptide was synthesized: Amino acid sequence: KQVSVLRQN-pS-VEFEG (pS=S192 site O-GlcNAc glycosylation modification), with a cysteine ​​(C) added at the C-terminus for coupling with KLH; at the same time, the corresponding non-glycosylated peptide was synthesized for cross-adsorption purification.

[0073] ② Immunogen preparation: The O-GlcNAc glycosylated peptide at the S192 site was chemically coupled to keyhole hemocyanin (KLH) to serve as an immunogen.

[0074] ③ Animal immunization: SPF-grade female New Zealand rabbits (6-8 weeks old, weighing 2.0-2.5 kg) were selected. The basic immunization was performed using Freund's complete adjuvant emulsified immunogen, 100 μg / rabbit, injected subcutaneously at multiple points on the back. Every 2 weeks, Freund's incomplete adjuvant emulsified immunogen was used for booster immunization, for a total of 3 booster immunizations.

[0075] ④ Serum collection and titer testing: Blood was collected from the marginal ear vein 7-10 days after the last immunization, and serum was separated. The antibody titer was detected by indirect ELISA using glycosylated peptides as the coating antigen. A titer ≥1:100000 was considered acceptable.

[0076] ⑤ Specific affinity purification: Serum was purified using an S192 glycosylated peptide affinity chromatography column. Cross-reactive antibodies were first removed by a non-glycosylated peptide column, and then the glycosylated peptide column was used for specific adsorption and elution to obtain a specific polyclonal antibody that recognizes only the O-GlcNAc glycosylation site of BICD2 at S192.

[0077] Antibody concentration: Quantified by BCA protein, the antibody storage concentration is 1 mg / mL; Western blot working dilution ratio: 1:1000; Immunohistochemistry / immunofluorescence working dilution ratio: 1:200; Specificity verification This antibody recognizes only BICD2 protein with O-GlcNAc glycosylation at the S192 site, and does not recognize unmodified wild-type BICD2 or S192A mutant protein, and has no cross-reactivity.

[0078] 2) HRP-labeled goat anti-rabbit secondary antibody; 3) Positive control (BICD2 recombinant protein modified with O-GlcNAc at S192 site); 4) Negative control (wild-type BICD2 recombinant protein); 5) Washing solution, color developing solution, and stop solution.

[0079] The kit described in this example was used to detect 80 PDAC tissue microarrays. See [link to kit]. Figure 16 and Figure 17 The results showed that patients with high expression of O-GlcNAc glycosylation at BICD2 S192 site had significantly shorter overall survival and disease-free survival than those with low expression, and were significantly positively correlated with gemcitabine resistance phenotype, demonstrating that the kit in this embodiment can effectively assess gemcitabine resistance and treatment prognosis in PDAC patients.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biomarker for gemcitabine treatment resistance or prognostic assessment in pancreatic ductal adenocarcinoma, characterized in that, The marker is a protein in which serine at position 192 of the BICD2 protein is modified by O-GlcNAc glycosylation.

2. The marker according to claim 1, characterized in that, The markers also include O-GlcNAc transferase and / or proteins with phosphorylated serine residue 102 of the BICD2 protein.

3. A kit for evaluating gemcitabine resistance in pancreatic ductal adenocarcinoma or for assessing the prognosis of patients with pancreatic ductal adenocarcinoma after gemcitabine treatment, characterized in that, The kit includes an antibody that specifically recognizes the O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein.

4. The reagent kit according to claim 3, characterized in that, The kit also includes OGT-specific quantitative PCR primers or OGT protein-specific antibodies for detecting OGT expression levels, and / or antibodies for detecting phosphorylation levels at the S102 site of the BICD2 protein.

5. The reagent kit according to claim 3, characterized in that, The antibody is a rabbit polyclonal or monoclonal antibody that specifically recognizes the BICD2 protein that is O-GlcNAc glycosylated at the S192 site. The kit also includes a positive control and a negative control. The positive control is a recombinant BICD2 protein modified with O-GlcNAc at the S192 site, and the negative control is a wild-type recombinant BICD2 protein without O-GlcNAc modification.

6. A method for screening candidate drugs for treating gemcitabine-resistant pancreatic ductal adenocarcinoma, characterized in that, Includes the following steps: S1: Contact the candidate compound with cells expressing BICD2 protein modified by O-GlcNAc glycosylation at S192 site; S2: Detect the O-GlcNAc glycosylation level of BICD2 protein at S192 site in the cells, or the binding level of BICD2 to RanBP2 / DCTN1, or the cell cycle arrest in the G2 / M phase. S3: If the candidate compound can reduce the O-GlcNAc glycosylation level, disrupt the binding of BICD2 to RanBP2 / DCTN1, or induce G2 / M phase arrest, then the candidate compound is a candidate drug.

7. Application of inhibitors targeting and inhibiting O-GlcNAc glycosylation modification at the S192 site of BICD2 protein in the preparation of drugs for treating pancreatic ductal adenocarcinoma and reversing gemcitabine resistance.

8. The application according to claim 7, characterized in that, The inhibitor is at least one of the following: a small molecule inhibitor targeting O-GlcNAc modification at the BICD2 S192 site, a nucleic acid inhibitor, a neutralizing antibody or nanobody, or a gene editing reagent.

9. The application according to claim 8, characterized in that, The nucleic acid inhibitor is a siRNA or shRNA that targets BICD2, and the sequence of the siRNA is 5'-GGAGCUCACACUACAUGUU-3'.

10. The application according to claim 7, characterized in that, The inhibitor inhibits the O-GlcNAc glycosylation modification at the S192 site of the BICD2 protein, blocks the phosphorylation activation of BICD2 at the S102 site and its binding to the RanBP2 / DCTN1 complex, thereby arresting the G2 / M phase progression of pancreatic cancer cells and reversing gemcitabine resistance.

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