CN108404130B

CN108404130B - Application of DMAP1pY246 blocker in preparation of antitumor drugs

Info

Publication number: CN108404130B
Application number: CN201710074976.7A
Authority: CN
Inventors: 蒋玉辉; 王兴鹏; 黄文华; 李静婕; 胡斌
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2017-02-10
Filing date: 2017-02-10
Publication date: 2021-02-02
Anticipated expiration: 2037-02-10
Also published as: CN108404130A

Abstract

The invention relates to an application of a DMAP1pY246 blocker in preparation of an anti-tumor drug. The invention discovers that the important protooncogene c-Src can phosphorylate DMAP1Y246, and the phosphorylation enhances the antagonism of tumor cells to mitotic blockers, so that the phosphorylation sites can be used as targets to develop medicaments for treating the tumors which are clinically activated abnormally by the c-Src and have antagonism effect on the mitotic blockers; in addition, the present invention also demonstrates that the phosphorylation of DMAP1Y246 is closely related to the development and poor prognosis of pancreatic cancer, and thus a reagent for detecting the phosphorylation level of DMAP1Y246 can be used for diagnosis or prognosis of pancreatic cancer.

Description

Application of DMAP1pY246 blocker in preparation of antitumor drugs

Technical Field

The invention relates to the technical field of molecular biology and medicines, in particular to application of a DMAP1pY246 blocking agent in preparation of antitumor drugs.

Background

The protooncogene c-Src is a normal gene inherent in human or animal cells and plays an important role in regulating the growth, development, differentiation and other biological functions of cells. The c-Src and its expression product Src protein are non-receptor tyrosine protein kinases, which regulate and control cell behavior through complex network pathways and various signal pathways, and participate in the process of intracellular signal transduction.

DMAP1 is a DNMT 1-related protein. DNA methyltransferase 1(DNMT1) is a major member of the DNA methyltransferase (DNMT) family and plays a central role in maintaining and regulating tumor cell genome-wide and local methylation. DNMT1 is highly expressed in malignant tumors, causes methylation abnormality of gene DNA, particularly hypermethylation of cancer suppressor genes, and then causes relevant gene expression silencing, thereby causing malignant growth of cells. From a cell cycle perspective, it has been reported that the effect of DMAP1 on gene transcriptional regulation is correlated with the regulation of the G1/S and G2/M checkpoints; however, the potential effect of 'DMAP 1-DNA methylation-gene transcription' on cell mitosis has not been reported.

The Bub3 protein is a key mitotic Spindle checkpoint (Spindle assembly checkpoint) factor that recognizes centromeric elements and recruits other centromeric cleavage checkpoint molecules to the centromeric site. In addition to the classical functions on spindle checkpoint regulation, Bub3 is also involved in other cell biological activities such as DNA damage, gene transcription. Bub3 was also found to bind to other non-spindle checkpoint proteins, as the transcription factor TAp73 was reported to be involved in spindle checkpoint regulation. TAp73 is a p53 family member protein that has pro-apoptotic function through regulation of gene transcription, as opposed to the anti-apoptotic function of another family analogous member, Δ Np73(TA domain deletion); in this respect, whether Bub3 is involved in the transcriptional regulation function of the TAp73 gene remains to be elucidated.

In summary, there are no reports of the new functions of Bub3 in the process of 'gene transcription-expression' reactivation after mitotic arrest and the action of tumor cells against mitotic pressure. The research on the mechanism also provides an important molecular basis for the development of tumor drugs.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a blocking agent of DMAP1Y246 and a new application of a reagent for detecting the phosphorylation level of DMAP1Y246 based on the facts that important proto-oncogene c-Src can phosphorylate DMAP1 tyrosine 246 (DMAP1Y246), the phosphorylation enhances the antagonism of tumor cells to mitotic blocking agents and proves that the phosphorylation of DMAP1Y246 is closely related to the occurrence, development and poor prognosis of pancreatic cancer.

In a first aspect of the invention, there is provided the use of a DMAP1pY246 blocker in the preparation of a mitotic blocker for cells.

In a second aspect of the invention, the use of a DMAP1pY246 blocker in the preparation of an anti-tumor medicament is provided.

In one embodiment, the tumor is one in which c-Src is abnormally activated or has an antagonistic effect on mitotic blockers.

As an embodiment of the present invention, the tumor is pancreatic cancer.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a cell mitotic blocker and a DMAP1pY246 blocker.

Preferably, the pharmaceutical composition further comprises a pharmaceutically conventional carrier.

In a fourth aspect of the invention, there is provided the use of a reagent for detecting the phosphorylation level of DMAP1Y246 in the preparation of a pancreatic cancer diagnostic reagent or kit.

In a fifth aspect of the invention, there is provided the use of a reagent for detecting the phosphorylation level of DMAP1Y246 in the preparation of a pancreatic cancer prognostic reagent or kit.

As a specific embodiment, the prognosis is in particular prediction of survival.

As an example of the present invention, the reagent for detecting the phosphorylation level of DMAP1Y246 is a phosphorylated DMAP1Y 246-specific antibody.

The invention has the advantages that:

the invention discovers that important proto-oncogene c-Src can phosphorylate DMAP1(DNA methyltransferase 1(DNMT1) related protein) tyrosine 246, and the phosphorylation enhances the antagonism of tumor cells (pancreatic cancer cells) to mitotic blockers (paclitaxel), so that the phosphorylation sites can be used as targets to develop medicaments for treating the tumors which are clinically activated abnormally and have antagonism effect on the mitotic blockers. In addition, the present invention also demonstrates that the phosphorylation of DMAP1Y246 is closely related to the development and poor prognosis of pancreatic cancer, and thus a reagent for detecting the phosphorylation level of DMAP1Y246 can be used for diagnosis or prognosis of pancreatic cancer.

Drawings

FIG. 1: under mitotic arrest pressure, Bub3 bound to DMAP 1. A HPDE cells stably expressing Flag-Bub3 were synchronized to release 8 hours after either inter-cellular (I) or thymidine (2mM) double block by thymidine (2mM) double block, and cells were treated with nocodazole (200nM) for 16 hours to arrest the cell cycle in mitosis (M). Cells at interphase and mitotic phase were collected separately and subjected to immunoprecipitation with the aid of specific anti-Flag antibodies after lysis of the cells with lysis buffer. The immunoprecipitation assay results were analyzed by SDS polyacrylamide gel electrophoresis and Coomassie blue staining. B-thymidine (2mM) double block HPDE cells stably expressing Flag-Bub3 were released for 8 hours followed by treatment of the cells with nocodazole (200nM) for 16 hours to arrest the cell cycle in mitosis. Cells were collected, lysed and subjected to co-immunoprecipitation with an antibody against Bub 3. C silencing DMAP1 in HPDE cells by using a small interfering RNA (siRNA) method, collecting the cells, cracking the cells, and performing a co-immunoprecipitation experiment by using an anti-Bub 3 antibody. D treatment with thymidine (2mM) double block and nocodazole (200nM) blocked the cell cycle of pancreatic cancer cells PANC-1 and SW1990 at mitosis. The cells were subsequently treated with the c-Src inhibitor SU6656 (10. mu.M) for 1 hour. Cells were collected, lysed and subjected to co-immunoprecipitation with an antibody against Bub 3. E overexpresses continuously activated c-Src (Src Y527F) in HPDE cells, and treatment with thymidine (2mM) double block and nocodazole (200nM) blocks the cell cycle in mitosis. Cells were collected, lysed and subjected to co-immunoprecipitation with an antibody against Bub 3. F Flag-tagged Bub3 deletion mutant and HA-tagged DMAP1 co-transfected HPDE cells, were treated with thymidine (2mM) double block and nocodazole (200nM) to maintain mitotic arrest in the cells. The cells were collected, lysed and subjected to co-immunoprecipitation with the aid of anti-Flag antibodies.

FIG. 2: under mitotic arrest pressure, Bub3 bound to DMAP 1. A HPDE cells stably expressing Flag-Bub3 were synchronized to release 8 hours after either inter-cellular (I) or thymidine (2mM) double block by thymidine (2mM) double block, and cells were treated with nocodazole (200nM) for 16 hours to arrest the cell cycle in mitosis (M). The cells were collected, lysed and subjected to immunoprecipitation with an anti-Flag antibody. The results of the experiment were analyzed by mass spectrometry. B-D treatment with thymidine (2mM) double block and nocodazole (200nM) maintained the mitotic arrest of HPDE cells. The cells were collected, lysed and subjected to co-immunoprecipitation experiments with the aid of antibodies against Bub3 (B, C); or a co-immunoprecipitation experiment (D) with the aid of an antibody against DMAP 1. E silencing DNMT1 in HPDE cells by using a small interfering RNA (siRNA) method, collecting the cells, and performing a co-immunoprecipitation experiment by using an anti-Bub 3 antibody after lysis.

FIG. 3: p38 phosphorylated Bub3 serine 211 (Ser211) to facilitate binding of Bub3 to DMAP 1. In A-D and G, cells were arrested at either interphase (I) by thymidine (2mM) double block, or at mitotic phase (M) by thymidine (2mM) double block and nocodazole (200nM) treatment. A synchronizes HPDE cells in the interphase or mitotic phase. Cells were harvested, lysed and immunoprecipitation experiments were performed with anti-Bub 3 antibody and immunoprecipitated complexes were treated with calf intestinal alkaline phosphatase (CIP, 10 units). B synchronizes HPDE cells at an interphase or mitotic phase. The cells were subsequently treated with the AMPK inhibitor Compound C (10 μ M), the JNK inhibitor SP600125(20 μ M) and the p38 inhibitor SB203580(25 μ M), respectively (1 hour). Co-immunoprecipitation experiments were performed with the aid of antibodies against Bub 3. C synchronization of PANC-1 and SW1990 cells at either the interphase or mitotic phase. Cells were treated with the c-Src inhibitor SU6656 (10. mu.M) and the p38 inhibitor SB203580 (25. mu.M) (1 hour). Co-immunoprecipitation experiments were performed with the aid of antibodies against Bub 3. D synchronization of HPDE cells or PANC-1 cells at either the interphase or mitotic phase. The cells were collected, lysed and subjected to co-immunoprecipitation with an antibody against p 38. E-F in the presence of [ gamma-32P ] ATP was used to perform in vitro kinase assays using active His-P38 and GST-Bub3 recombinant proteins. The serine at Bub 3211 (Ser211) is an evolutionarily conserved site in each of the species listed. G expressed Flag-tagged wild-type Bub3 or Bub3 serine-specific mutants in HPDE cells. Cell cycle arrest after mitosis, cells were collected for co-immunoprecipitation experiments with anti-Flag antibodies. H purified His-tagged Bub3 protein (expressing wild-type Bub3 or Bub3 with a specific serine site mutation) was co-incubated with purified GST-tagged DMAP1 protein, while adding active p38 protein, followed by GST pull-down experiments.

FIG. 4: p38 phosphorylated Bub3 serine 211 (Ser211) to facilitate binding of Bub3 to DMAP 1. In A-D, cells were arrested at either interphase (I) by thymidine (2mM) double block, or at mitotic phase (M) by thymidine (2mM) double block and nocodazole (200nM) treatment. A synchronizes HPDE cells in the interphase or mitotic phase. Cells were harvested, lysed and subjected to co-immunoprecipitation with anti-Bub 3 antibody and treated with calf intestinal alkaline phosphatase (CIP, 10units) or calf intestinal alkaline phosphatase (10units)/Na₃VO₄The immunoprecipitated complex is processed. B synchronization of HPDE cells at either the interphase (I) or induction of mitotic arrest (6 hours arrest noted "S"; 16 hours arrest noted "L"). Immunoblotting experiments were performed with the antibodies shown in the figure, respectively. C synchronizes HPDE cells in the interphase or mitotic phase. The cells were subsequently treated with the AMPK inhibitor Compound C (10 μ M), the JNK inhibitor SP600125(20 μ M) and the p38 inhibitor SB203580(25 μ M), respectively (1 hour). Immunoblotting experiments were performed with the antibodies shown in the figure, respectively. Synchronization of HPDE cells the p38 inhibitor SB203580(25 μ M) was added to treat cells (1 hour) during the inter-cell or post-mitotic phase. Immunoblotting experiments were performed with the antibodies shown in the figure, respectively. E synchronization of HPDE cells after the G1 phase the cells were stimulated with TGF-. beta.for 1 hour. Co-immunoprecipitation experiments were performed with anti-p 38 antibody. Treatment of pancreatic cancer cells expressing wild-type Bub3 and Bub3 serine mutants with thymidine (2mM) double block and nocodazole (200nM) blocked the PANC-1 cell cycle in mitosis. The cells were subsequently treated with the c-Src inhibitor SU6656 (10. mu.M) for 1 hour. Co-immunoprecipitation experiments were performed with anti-Flag antibodies.

FIG. 5: c-Src phosphorylates DMAP1 tyrosine 246 (Tyr246) to inhibit Bub3 binding to DMAP 1. In A-D and E, cells were arrested at either interphase (I) by thymidine (2mM) double block, or at mitotic phase (M) by thymidine (2mM) double block and nocodazole (200nM) treatment. A synchronizes PANC-1 and SW1990 cells at either the interphase or mitotic phase. The cells were collected, lysed and subjected to co-immunoprecipitation with the aid of an antibody against c-Src. B in vitro kinase experiments were performed in the presence of [ gamma-32P ] ATP using active His-c-Src and GST-DMAP1 recombinant proteins. The DMAP 1246 tyrosine (Tyr246) is an evolutionarily conserved site in each of the species listed. C, cell cycle arrest of PANC-1 expressing Flag-tagged wild-type DMAP1 or DMAP1 specific site tyrosine mutant is carried out after mitosis, and cells are collected and subjected to co-immunoprecipitation experiment by using anti-Flag antibody. D PANC-1 cell lysates in mitotic arrest were incubated with purified GST-DMAP1, with the addition of active c-Src, followed by GST pull-down experiments. E His-Bub3 protein which is not phosphorylated or is phosphorylated by p38 was incubated with GST-DMAP1 protein which is not phosphorylated or is phosphorylated by c-Src, respectively, followed by GST pull-down experiments.

FIG. 6: c-Src phosphorylates DMAP1 tyrosine 246 (Tyr246) to inhibit Bub3 binding to DMAP 1. PANC-1 cells are synchronized during the interphase or mitotic phase. The cells were subsequently treated with the c-Src inhibitor SU6656 (10. mu.M) for 1 hour. Immunoblotting experiments were performed with the antibodies shown in the figure, respectively.

FIG. 7: the Bub3/DMAP1 complex inhibited anti-apoptotic gene transcription. A silences endogenous DMAP1 with small hairpin rna (shRNA) in SW1990 cells, followed by expression of shRNA interference resistant wild-type DMAP1(rDMAP1) or Tyr246 site mutated DMAP1(rDMAP 1Y 246F) in cells. Immunoblotting experiments were performed with the DMAP1 antibody. B silenced endogenous DMAP1 and Bub3 with small hairpin rna (shRNA) in SW1990 cells, followed by expression of shRNA interference resistant wild-type DMAP1(rDMAP1), wild-type Bub3 or (rBub3), DMAP1 with a mutation at position Tyr246 (rDMAP 1Y 246F) and Bub3 with a mutation at position Ser211 (r Bub3S211A) in cells. Immunoblotting experiments were performed with the antibodies shown in the figure, respectively.

FIG. 8: the Bub3/DMAP1 complex inhibited anti-apoptotic gene transcription. In fig. C-E, p <0.05 and p < 0.01. SW1990 cells expressing wild type DMAP1 and Tyr246 site mutant DMAP1 were treated with thymidine (2mM) double block and nocodazole (200nM) and released separately for the indicated time to allow the cells to enter G1 phase. Immunoblotting experiments were performed with antibodies to the G1 phase-specific marker protein CyclinD1 and the mitotic phase-specific marker protein CyclinB1, respectively. SW1990 cells expressing wild-type DMAP1(WT rMAP 1) and DMAP1Tyr246 point mutation (rMAP 1Y 246F) were released for 4 hours after treatment with thymidine (2mM) double block and nocodazole (200 nM). Hierarchical cluster expression of 4307 gene probes shows that the gene expression in wild-type DMAP1 cells and DMAP1Tyr246 point mutation cells is obviously different: the expression of the anti-apoptosis gene in DMAP1Tyr246 point mutation cells is obviously lower than that of wild cells; the expression level of the autophagy related gene is obviously increased in DMAP1Tyr246 point mutation cells. SW1990 cells expressing the corresponding vector C-D were released for the corresponding time after thymidine (2mM) double-block and nocodazole (200nM) treatment. After collecting the cells, the transcriptional activation of BCL2L1 and HMGA2 was detected by real-time quantitative PCR. The EAnnexin V/PI staining analyzed SW1990 apoptosis expressing the corresponding vector.

FIG. 9: TAp73 recruits the Bub3/DMAP1 complex to the promoter region of the target gene. Cells in B-F were arrested at either interphase (I) by thymidine (2mM) double block, or at mitotic phase (M) by thymidine (2mM) double block and nocodazole (200nM) treatment. Denotes p <0.05, denotes p < 0.01. A SW1990 cells expressing the corresponding vector were arrested in mitosis and cells were collected for co-immunoprecipitation with anti-Flag antibody. B SW1990 cells expressing the corresponding vector were arrested in interphase or mitotic phase and cells were collected for chromatin co-immunoprecipitation (ChIP) experiments. The ChIP primers contained the recognition sequences of TAp73 in the BCL2L1 and HMGA2 promoter regions, respectively. C SW1990 cells were arrested at interphase or mitotic phase and cells were collected for chromatin co-immunoprecipitation (ChIP) experiments. The ChIP primers contained the recognition sequences of TAp73 in the BCL2L1 and HMGA2 promoter regions, respectively. D the corresponding vector was expressed in SW1990 cells and the level of 5-mc in the promoter region of BCL2L1 was determined by pyrosequencing. The PCR primers contained the recognition sequence of TAp73 in the promoter region of BCL2L 1.

FIG. 10: TAp73 recruits the Bub3/DMAP1 complex to the promoter region of the target gene. Cells in A-H were arrested at either interphase (I) by thymidine (2mM) double block, or at mitotic phase (M) by thymidine (2mM) double block and nocodazole (200nM) treatment. Denotes p <0.05, denotes p < 0.01. After a silencing of DMAP1 in SW1990 cells, shRNA interference resistant wild-type DMAP1(rDMAP1) or a mutant DMAP1 at position Tyr246 (rDMAP 1Y 246F) was expressed in the cells. TAP73 was subsequently silenced in the cells and then subjected to real-time quantitative PCR. B SW1990 cells were arrested at interphase or mitotic phase and cells were harvested for chromatin co-immunoprecipitation (ChIP) experiments to detect recruitment of TAP73 at the promoter regions of BCL2L1 and HMGA 2. C-D silences TAP73 in SW1990 cells and blocks cells at interphase or mitotic phase, and cells were collected for chromatin co-immunoprecipitation (ChIP) experiments. The ChIP primers contained the recognition sequences of TAp73 in the BCL2L1 and HMGA2 promoter regions, respectively. E in SW1990 cells respectively expressing corresponding vectors, inducing cell cycle arrest in interphase or mitotic phase, collecting cells for chromatin co-immunoprecipitation (ChIP) experiment. F, corresponding vectors are respectively expressed in SW1990 cells, and the level of 5-mc of the BCL2L1 promoter region is detected by a pyrosequencing method. The PCR primers contained the recognition sequence of TAp73 in the promoter region of BCL2L 1.

FIG. 11: phosphorylation of DMAP1Y246 is essential for the development of pancreatic cancer. A is respectively 5 × 10⁶SW1990 cells expressing wild type rBub 3/wild rMAP 1, wild type rBub 3/rMAP 1Y246F or rBub3S 211A/rMAP 1Y246F were injected subcutaneously into nude mice. When the tumor volume reaches 200mm³Mice were initially injected intraperitoneally with paclitaxel (5mg/kg) to induce mitotic arrest. Each group had 8 mice, and at the end of the experiment, one representative experimental mouse was selected for each group. Tumors were dissected from each group of mice and photographed. B measuring the length (a) and the width (B) of the tumor of the experimental mouse by using a vernier caliper every week in the whole experimental period, and calculating the tumor volume V-ab according to the following formula²/2. After the experiment, the tumor weight of each group of experimental mice was weighed. C anti-DMAP 1pY246 antibody against pancreatic cancer containing 90 pairsThe tissue chips of clinical specimens and their corresponding paracancerous tissues were subjected to immunohistochemical staining. Clinical samples of pancreatic cancer were collected 8 and their corresponding paracarcinoma immunoblotting assays to examine the levels of DMAP1pY246, c-Src pY416 and c-Src in tumor and paracarcinoma tissues. D90 pancreatic cancer patients in the chip were scored for DMAP1pY246 levels by immunohistochemical staining: 0-5 points, 0 point representing no obvious positive coloration; 1 is a mark representing<10% of the area had light brown positive coloration; 2 points represent that 10 to 30 percent of the area has light brown positive coloration; 3 points represent that 31% -50% of the area has light brown positive coloration; 4 points represent that the area which is more than or equal to 50 percent has brown positive coloration; 5 points represent > 50% of the area with dark brown positive coloration. 0-2 points to low expression of DMAP1pY 246; 3-5 are classified as high expression of DMAP1pY 246. The relationship between DMAP1pY246 levels and survival rates of 90 pancreatic cancer patients in the chip was analyzed using tissue chip follow-up data and immunohistochemical staining, and Cox multifactorial analysis was performed using SPSS software.

FIG. 12: phosphorylated DMAP1Y246 antibody specificity was verified by specific blocking polypeptide treatment and immunohistochemical analysis. Pancreatic cancer tissue was immunostained with an antibody specific for DMAP1pY246 with or without the corresponding blocking polypeptide.

FIG. 13: schematic representation of the p38/Bub3/DMAP1 signaling axis action pattern in normal and pancreatic cancer cells. Mitotic arrest activates p 38. Activated p38 promoted phosphorylation of the Bub3 Ser211 site and further promoted interaction of Bub3 with DMAP1/DNMT 1. TAp73 recruited the Bub3/DMAP1 complex to the promoter region of the target gene. DMAP1/DNMT1 now represses transcription of the anti-apoptotic gene by regulating the level of methylation in the promoter region. c-Src inhibits the formation of the Bub3/DMAP1 complex by maintaining a higher level of phosphorylation of DMAP1Tyr 246. In normal cells, the p38/Bub3/DMAP1 signaling axis is activated rapidly once the cell is mitotically arrested due to the lower c-Src activity. Cells were more susceptible to apoptosis at this time (left panel). In tumor cells, abnormally activated c-Src leads to phosphorylation at the DMAP1Tyr246 site, thereby inhibiting p38/Bub3/DMAP1 signaling axis regulated apoptosis during mitotic arrest (right panel).

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1

1. Under the pressure of mitosis block, Bub3 can form a complex with DMAP1

To investigate whether Bub3 under mitotic arrest pressure was involved in other unknown cellular events in addition to its classical spindle checkpoint function, we stably expressed Flag-tagged Bub3 in immortalized human pancreatic ductal epithelial cells HPDE, followed by thymidine double block and nocodazole treatment to arrest the cell cycle in mitosis. Flow cytometry analysis and immunoblot detection of late G2 phase and mitotic phase specific marker phosphorylated (serine 10) histone H3 demonstrated the efficiency of cell mitotic arrest (figure 1A). After collection of the cells, we performed co-immunoprecipitation using agarose beads conjugated with Flag antibody (anti-Flag m2) and searched the protein with the Bub3 interaction by means of mass spectrometry. Mass spectrometry data show that DNA methyltransferase 1(DNMT1) -related protein DMAP1 can specifically bind to Bub3 during the mitotic arrest phase. Although binding was less than that of DMAP1, we found that when cells were mitotically arrested (fig. 2A), the interaction of DNMT1 with Bub3 was still somewhat enhanced compared to interphase (fig. 2B). In addition, mass spectrometry results showed that TAp73 also binds to Bub3 during mitotic arrest, a finding consistent with previous findings (fig. 2C). We carried out forward and reverse co-immunoprecipitation experiments with antibodies that specifically recognized Bub3 (FIG. 1B) and DMAP1 (FIG. 2D), respectively, and the experimental results showed that the Bub3/DMAP1 interaction was indeed significantly increased under mitotic arrest pressure. Further experimental results showed that silencing DNMT1 did not affect binding of Bub3 to DMAP1 (fig. 2E); however, the absence of DMAP1 inhibited the formation of the Bub3/DNMT1 complex (FIG. 1C). This result indicates that binding of Bub3 to DNMT1 during mitotic arrest is in the ligament of DMAP 1. In addition, we also examined the binding of Bub3 and DAMP1 to each other in pancreatic cancer cells PANC-1 and SW 1990. However, in tumor cells, the binding of Bub3 to DAMP1 to each other was only slightly increased in the presence of mitotic arrest pressure (fig. 1D), and thus some tumor-specific signaling pathways may be involved in the Bub3/DAMP1 complex formation process. It has been reported that the signal pathways associated with kinases such as PI3K, MEK/ERK, c-Src, etc. are abnormally activated during the development of pancreatic cancer. We treated pancreatic cancer cells PANC-1 and SW1990 with LY294002 (a PI3K inhibitor), U1026 (a MEK/ERK inhibitor) and SU6656 (a c-Src inhibitor), respectively. Mitosis arrest-induced binding of Bub3/DAMP1 was significantly increased only in the case where c-Src activity was inhibited (FIG. 1D). Thus c-Src can be considered as a negative regulator of Bub3/DAMP1 binding. To further demonstrate this result, we expressed constitutively activated c-Src (c-Src 527F) in pancreatic ductal epithelial cells HPDE, where c-Src activity was relatively low. Mitotic arrest-induced Bub3/DMAP1 interaction did decrease significantly following c-SrcY527F expression in HPDE (FIG. 1E). Subsequently, we constructed a series of deletion mutants of Bub3 in an attempt to find a region where Bub3 binds DMAP1 (fig. 1F). We expressed the deletion mutant of Bub3 (containing Flag tag) and wild-type DMAP1 (containing HA tag) in mitosis-blocked HPDE cells, respectively, and we found through co-immunoprecipitation experiments that Bub3 substantially lost its binding to DMAP1 when it lacked amino acids 197-306 or 203-328 (FIG. 1F). Therefore, the binding region between Bub3 and DMAP1 should be located between amino acids 203-306 of Bub 3.

2. p38 phosphorylated Bub3 serine 211 (Ser211) to facilitate binding of Bub3 to DMAP1

After demonstrating that Bub3 interacts with DMAP1 under mitotic arrest pressure and finding a specific binding region for Bub3 and DMAP1, we began to investigate the upstream regulatory signals of Bub3/DMAP1 binding under mitotic arrest. We found that binding of Bub3 to DMAP1 disappeared after treatment of the immunoprecipitated complexes with calf intestinal alkaline phosphatase (CIP); and use Na₃VO₄This was not the case with immunoprecipitated complexes (FIG. 3A and FIG. 4A). This result indicates that Bub3/DAMP1 binding is phosphorylation dependent. Serine/threonine kinases such as JNK, AMPK and p38 are activated in the presence of intracellular pressure signals, and they are also activated during mitotic arrest (figure 4B). We used SP600125(JNK inhibitor), Compondc (AMPK inhibitor) and SB20, respectively3580(p38 inhibitor) treated HPDE cells (fig. 4C). Mitotic arrest-induced binding of Bub3/DAMP1 was only specifically inhibited by SB203580 (FIG. 3B). Likewise, SB203580 largely blocks mitosis-arrest-induced Bub3/DAMP1 complex formation in pancreatic cancer cells PANC-1 and SW1990 in the presence of inhibited C-Src activity (FIG. 3C). Thus, the mitotic arrest effector kinase p38 is involved in the Bub3/DMAP1 process. Furthermore, we found that mitotic arrest promoted the binding of p38 to Bub3 in HPDE cells and PANC-1 cells by co-immunoprecipitation experiments (fig. 3D), which prompted us to further explore whether Bub3 is a substrate for p38 kinase. In vitro protein kinase experiments demonstrated that purified active p38 protein was able to phosphorylate purified recombinant Bub3 (autoradiography) (fig. 3E). At the same time, we performed immunoblotting experiments using antibodies against phosphorylated serine and found that p38 did phosphorylate the serine of Bub3 (fig. 3E). Scansite analysis predicted a series of potential sites in the amino acid sequence of Bub3 that could be phosphorylated by p38 (FIG. 3F). We have made single point mutations at these potential sites and found that Bub3 cannot be phosphorylated by p38 kinase only if the evolutionarily conserved serine at position 211 is mutated (Bub3S 211A). This was confirmed by autoradiography and immunoblotting experiments (using specific anti-Bub 3pSer-211 antibody) (FIG. 3F). Phosphorylation of Bub3 serine 211 (S211) was indeed inhibited by the p38 kinase inhibitor SB203580 in mitotic arrest (fig. 4D). To further explore whether Bub3S211 phosphorylation was dependent on p38 activity under mitotic pressure, we synchronized HPDE cells in G1 phase and stimulated the cells with the known p38 activator TGF- β. However, our findings showed that neither p38/Bub3 interaction nor Bub3S211 phosphorylation were significantly increased (FIG. 4E). Taken together, we believe that the activity of p38 under mitotic arrest pressure is an important prerequisite for phosphorylation of Bub3S 211. Subsequently, to investigate the effect of Bub3S211 phosphorylation on Bub3/DMAP1 binding, we expressed wild-type and mutant Bub3, respectively, in mitotically arrested HPDE cells. The co-immunoprecipitation experiment showed that the binding of Bub3/DMAP1 disappeared only when mutation of Bub3S211 occurred. Similarly, when c-Src activity is affectedUpon inhibition, mitotic arrest significantly promoted the binding of DMAP1 to wild-type Bub3 but not Bub3S211A in pancreatic cancer cells PANC-1 (fig. 3G). Subsequently, we performed the following experiments again: recombinant GST-DMAP1 protein was incubated with recombinant His-Bub3 or His-Bub3S211A, with or without purified, active p38 protein, respectively. By GSTpull-down experiments, we found that p38 promoted binding of wild-type Bub3 to DMAP1 but did not promote binding of the Bub3S211A mutant to DMAP1 (fig. 3H). Taken together, these results indicate that p 38-regulated phosphorylation of Bub3S211 under mitotic arrest is critical for its binding to DMAP 1.

3. c-Src phosphorylates DMAP1 tyrosine 246 (Tyr246) to inhibit Bub3 binding to DMAP1

Mitotic arrest resulted in a significant increase in the phosphorylation of Bub3S211 in pancreatic cancer cells PANC-1, a process unrelated to c-Src activity (fig. 4F). Therefore, the negative regulation of Bub3/DMAP1 by c-Src is independent of the phosphorylation level of Bub3S211, and the research on the molecular mechanism of Bub3/DMAP1 binding by c-Src is prompted. In both PANC-1 and SW1990 pancreatic cancer cells, c-Src formed a significant complex with DMAP1, but not with Bub3, throughout the cell cycle (FIG. 5A), suggesting that DMAP1 is highly likely to serve as a substrate for c-Src kinase. Based on Scansite analysis, we found multiple potential sites in the DMAP1 amino acid sequence that could be phosphorylated by c-Src (FIG. 5B). The c-Src phosphorylation of DMAP1 also disappears following the mutation of the evolutionarily conserved DMAP1 at tyrosine 246. This was confirmed by autoradiography and immunoblotting experiments (using specific anti-DMAP 1pY246 antibody) (fig. 5B). Meanwhile, the results of immunoblotting experiments showed that the level of DMAP1pY246 in PANC-1 cells was more constant throughout the cell cycle and decreased with the inhibition of c-Src (FIG. 6). When PANC1 cells underwent mitotic arrest, the DMAP1Y246F mutant bound more significantly to Bub3 than did the wild-type DMAP1 and other mutants of DMAP1 (fig. 5C), which is consistent with our previous binding of DMAP1 to Bub3 after inhibition of C-Src activity in pancreatic cancer cells (fig. 1D). Thus, c-Src regulated DMAP1 phosphorylation can block mitosis-arrest-induced Bub3/DMAP1 complex formation. Notably, expression of the DMAP1Y246F mutant did not affect mitosis-block-induced phosphorylation of Bub3S211 in PANC1 cells (fig. 5C). In addition, the results of the GSTpull-down experiments showed that the purified DMAP1 protein binds to Bub3 in cell extracts at the time of mitotic arrest, and this binding was significantly inhibited by c-Src in the system (FIG. 5D). In another GSTpull-down experiment, when we incubated purified DMAP1, bu 3, p38, and c-Src, DMAP1 was still able to form a complex with phosphorylated bu 3 even in the presence of c-Src (fig. 5E). These results indicate that c-Src mediated phosphorylation of DMAP1 does not directly hinder DMAP1/bu 3 interaction, and that there may be other undetected factors that compete with bu 3 for binding to phosphorylated DMAP1 during mitotic arrest.

4. Inhibition of anti-apoptotic gene transcription by the Bub3/DMAP1 complex

DMAP1 has been reported to interact with DNMT1 and thus be involved in gene transcription repression. We silenced endogenous DMAP1 in SW1990 cells and expressed RNA interference resistant wild-type rDMAP1 or rY246FDMAP1 in cells to investigate whether c-Src-mediated inhibition of mab 1 phosphorylation on Bub3/DMAP1 binding is associated with post-mitotic gene transcription (fig. 7A and 8A). First, we arrest the cell cycle in mitosis with thymidine and nocodazole. Release 1 hour, 2 hours, 4 hours and 6 hours after removal of nocodazole allowed the cells to enter G1 phase. SW1990 cells expressing wild type rDMAMAP 1 or rY246FDMAP1 after 4 hours of release were taken for cDNA microarray (cDNAmicrraray) analysis. We found that most of the genes with reduced expression were associated with autophagy or anti-apoptotic signals in SW1990 cells expressing rY246FDMAP 1; in contrast, elevated expression of many genes was associated with pro-apoptosis or maintenance of cell survival (fig. 8B). In view of the role of DMAP1 in gene transcription repression, we believe that in cells expressing DMAP1 mutant Y246F, those genes whose expression is down-regulated may serve as potential target genes for the Bub3/DMAP1 complex. Of these target genes, we chose two genes encoding anti-apoptotic proteins, HMGA2 and BCL2L1, as subjects to further explore the effect of the Bub3/DMAP1 complex on post-mitotic gene reactivation. By real-time quantitative PCR experiments, we found that the DMAP1Y246F mutation was able to significantly delay the reactivation of both HMGA2 and BCL2L1 genes within 4 and 6 hours after mitotic release (most cells were now in G1 phase) (fig. 8C and 8D), which is also consistent with our cdnmicroarray results. Our previous experimental results showed that phosphorylation of Bub3S211 is required for Bub3/DMAP1 binding (FIG. 3H and FIG. 5C), and thus it is highly likely that phosphorylation of Bub3S211 is involved in gene transcriptional regulation. To verify this, we silenced Bub3 in SW1990 and expressed RNA interference resistant wild-type rBub3 or rBub3S211A in cells (fig. 7B). We found that rBub3S211A significantly reversed the process of delayed gene reactivation mediated by DMAP1Y246F (fig. 8C). In addition, silencing of DNMT1 also inhibited the delay in gene reactivation by DMAP1Y246F (fig. 8D). The inhibition of postmitotic gene transcription by DMAP1Y246F prompted us to study its relationship to cell survival under mitotic arrest pressure. With the aid of PI/AnnexinV staining and cell flow analysis, we found that when SW1990 cells released from mitosis, the DMAP1Y246F mutation caused apoptosis in a proportion of the cells, whereas the Bub3S211A mutation or co-expression of the two genes HMGA2 and BCL2L1 reversed apoptosis (fig. 8E). From the above results, it is thought that under mitotic arrest pressure, the Bub3/DMAP1 complex constitutes a transcription repression regulator to regulate the transcription of anti-apoptotic genes, and highly activated c-Src in tumor cells can repress the action of the Bub3/DMAP1 complex against the transcription of anti-apoptotic genes.

5. TAp73 recruits the Bub3/DMAP1 complex to the promoter region of the target gene

Under mitotic arrest, Bub3 bound to TAp73 (fig. 2C), and the binding was not dependent on the phosphorylation state of Bub3S211 (fig. 9A). From this, we concluded that TAp73 might be involved in Bub3/DMAP 1-mediated gene transcription regulation. As shown in FIG. 10A, transcriptional repression of HMGA2 and BCL2L1 by DMAP1Y246F was also eliminated following TAp73 silencing, so TAp73 was indeed involved in transcriptional regulation of genes by Bub3/DMAP 1. By analysis, we found that the sequence of the HMGA2 promoter region 'tgcatgtgcttacacgcg' and the sequence of the BCL2L1 promoter region 'ggcatgcgccaccacgcc' were potential binding sites for the transcription factor TAp73 (fig. 10B). Chromatin co-immunoprecipitation experimental results confirmed that TAp73 can be enriched in the promoter regions of these two target genes during mitosis. In addition, at the time of mitotic arrest, both Bub3 (fig. 10C) and phosphorylated Bub3S211 (fig. 10D) of HMGA2 and BCL2L1 promoter regions were significantly increased in SW1990 cells, but the absence of TAp73 prevented recruitment of Bub3 and phosphorylated Bub3S211 in the promoter region. In SW1990 expressing rDMAP1Y246F, mitotic arrest also recruited more DMAP1 (fig. 10E) and DNMT1 (fig. 9B) to the target gene promoter region in a TAp73 dependent manner, but not in cells expressing only wild-type rDMAP1 or both rDMAP1Y246F and rBub3S211A (fig. 9B). This suggests that p 38-regulated phosphorylation of Bub3 is essential for the recruitment of the promoter region DMAP1/DNMT 1.DMAP1 phosphorylated at Tyr246 was not recruited to the target gene promoter region under mitotic arrest (fig. 9C). Next, we examined the effect of the Bub3/DMAP1/DNMT1 complex on the methylation of the promoter regions of the HMGA2 and BCL2L1 genes at the time of mitotic arrest (FIG. 9D). The results of DNA methylation experiments showed that the 5mc of the promoter region of the target gene (BCL2L1) was significantly increased in the Nokobazole-treated SW1990 cells expressing rMAP 1Y246F compared to rMAP 1Y246F/rBub3S 211A-expressing cells (FIG. 10F), which is consistent with the effect of these mutants on the transcriptional regulation of the target gene. Taken together, we believe that c-Src mediated phosphorylation of DMAP1Y246 at mitotic arrest prevents Bub 3-induced promoter-associated DMAP1/DNMT1 recruitment to inhibit DNA methylation in the relevant promoter region and thereby relieve gene transcription repression.

6. Phosphorylation of DMAP1Y246 is essential for the development of pancreatic cancer

Our previous results indicate that c-Src mediated phosphorylation of DMAP1Y246 under mitotic arrest can block the formation of the Bub3/DMAP1 complex and thus promote survival and proliferation of pancreatic cancer cells (FIG. 8E). To further confirm the role of DMAP1Y246 phosphorylation in pancreatic cancer development, we injected SW1990 pancreatic cancer cells expressing wild-type rBub 3/wild-type rDMAP1, wild-type rBub3/rDMAP1Y246F, and rBub3S211A/rDMAP1Y246F, respectively, subcutaneously into nude mice for tumor growth. When the tumor volume reaches 200mm³Mice were given an intraperitoneal injection of paclitaxel (5mg/kg, induction of pancreatic cancer cell mitotic arrest). Injection of wild type rD despite paclitaxel treatmentThe tumors of mice in the MAP1 cell group were still able to grow rapidly. In contrast, tumors from mice injected with rDMAP1Y246F mutant cells grew more slowly (fig. 11A and 11B), and this inhibitory effect was reversed by expression of the rBub3S211A mutant. The results of the above in vivo experiments indicate that the inhibitory effect of DMAP1Y246 phosphorylation on the Bub3/DMAP1 complex is crucial for the development of pancreatic cancer.

7. Phosphorylation of DMAP1Y246 is closely related to poor prognosis of pancreatic cancer

We have demonstrated that DMAP1Y246 phosphorylation blocks Bub 3-mediated transcriptional repression. Next we aimed to investigate whether the previous findings were clinically relevant. First, we examined the c-Src activity and DMAP1Y246 phosphorylation levels in 8 pairs of pancreatic cancer tissues and their corresponding paraneoplastic tissues by immunoblotting experiments. We found that DMAP1Y246 phosphorylation was positively correlated with C-Src/C-SrcpY416 phosphorylation (FIG. 11C). In addition, we collected 90 tumor tissues and corresponding paraneoplastic tissues from pancreatic cancer patients and examined the level of DMAP1Y246 phosphorylation by immunohistochemical staining. The specificity of the phosphorylated DMAP1Y246 antibody has been verified by its specific blocking polypeptide treatment and immunohistochemical analysis (fig. 12). The level of DMAP1Y246 phosphorylation in tumor tissues was significantly higher than in the corresponding paraneoplastic tissues (fig. 11C). Subsequently, we analyzed the relationship between DMAP1Y246 phosphorylation levels and survival in these 90 pancreatic cancer patients (fig. 11D). Median survival was longer and not achieved in patients with lower levels of DMAP1Y246 phosphorylation (total 29, on a DMAP1Y246 phosphorylation score scale of 0-2), while median survival was only 10.5 months in patients with high levels of DMAP1Y246 phosphorylation (total 61, on a DMAP1Y246 phosphorylation score scale of 3-5). Cox multifactor model analysis showed that DMAP1Y246 phosphorylation could be an independent predictor of pancreatic cancer patient survival (FIG. 11D). These results all indicate that phosphorylation of DMAP1Y246 is closely related to poor prognosis of pancreatic cancer.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

Use of a DMAP1pY246 blocker in the preparation of a mitotic blocker for a cell in which c-Src is abnormally activated or has an antagonistic effect on the mitotic blocker.
The application of DMAP1pY246 blocker in preparing antitumor drugs, wherein the tumor is the tumor with c-Src abnormal activation or mitosis blocker antagonistic effect.
3. The use of claim 2, wherein the neoplasm is pancreatic cancer.
4. Application of a reagent for detecting the phosphorylation level of DMAP1Y246 in preparation of a pancreatic cancer diagnostic reagent or kit.
5. Application of a reagent for detecting the phosphorylation level of DMAP1Y246 in preparation of pancreatic cancer prognostic reagent or kit.
6. The use according to claim 5, wherein the prognosis is in particular prediction of survival.
7. The use of any one of claims 4-6, wherein said reagent for detecting the level of phosphorylation of DMAP1Y246 is a phosphorylated DMAP1Y 246-specific antibody.