Micromolecular targeted drug aiming at MSX1 phosphorylation site and application thereof in gastric cancer treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a small-molecule targeted drug aiming at an MSX1 phosphorylation site and application of the small-molecule targeted drug in gastric cancer treatment. According to the invention, the MSX1Ser136 site which can be used as a gastric cancer treatment target is found, and gastric cancer cell proliferation can be obviously inhibited by inhibiting phosphorylation of the MSX1Ser136 site. Based on the discovery, a CDK1 inhibitor Ro3306 is selected as an MSX1Ser136 site phosphorylation inhibitor, a small-molecule targeted drug with Ro3306 and a platinum drug DDP as active components is developed in a targeted manner, it is proved that the combination of Ro3306 and DDP has a synergistic inhibition effect on gastric cancer cells, and a potential new strategy is provided for clinical gastric cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule targeted drug targeting the MSX1 phosphorylation site and an application thereof in the treatment of gastric cancer. Background Art

[0002] Malignant tumors are major diseases that endanger the health of the Chinese people. Gastric cancer is a highly prevalent malignant tumor in my country, ranking third among the causes of cancer-related deaths in the country. The number of new cases of gastric cancer in my country each year accounts for nearly half of the new cases worldwide, severely limiting the health of Chinese residents and placing a heavy burden on national health. Most newly diagnosed gastric cancers in my country are in the advanced stage. Even with metastatic disease, patients with extended radical resection face an extremely high risk of recurrence and metastasis. Postoperative chemotherapy and targeted therapy are the main strategies for controlling and treating gastric cancer recurrence. Because gastric cancer is a highly heterogeneous tumor, the effect of chemotherapy is unsatisfactory, especially for patients with advanced gastric cancer: ACTS-GC and CLASSIC studies have shown that even though adjuvant S-1 or XELOX chemotherapy can significantly improve the 5-year overall survival and recurrence-free survival of patients with advanced gastric cancer, the later the stage, the less benefit patients receive from chemotherapy and the higher the risk of postoperative recurrence and metastasis. Improving the prognosis of patients with advanced gastric cancer is a clinical challenge.

[0003] The ToGA study established the role of trastuzumab in the first-line treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer, but the clinical HER2-positivity rate in gastric cancer is less than 20%. With the approval of immune checkpoint inhibitors (ICIs) targeting programmed death receptor 1 (PD-1) antibodies for the treatment of unresectable / metastatic gastric cancer, immunotherapy has brought hope to the treatment of advanced gastric cancer. However, the microsatellite instability (MSI-H) status, which predicts a better response, accounts for less than 25% of gastric cancers, limiting the target population for ICIs. Therefore, elucidating the key molecules in the development and progression of gastric cancer and the mechanisms that mediate tumor treatment resistance has become an urgent issue to improve the treatment outcomes for gastric cancer patients.

[0004] The homeobox gene MSX is a crucial transcription factor during mammalian development. It participates in the early development of multiple tissues and organs, including muscle, bone, central nervous system, and mammary gland. Its main family members, MSX1 and MSX2, share a common characteristic during the development of different tissues and organs: they are expressed only in proliferating cells. Once differentiation is initiated, their expression levels decrease, and they are absent in terminally differentiated cells. This suggests that MSX may play a role in regulating cell proliferation and differentiation. The research team previously discovered that MSX1 promotes the proliferation of muscle and bone precursor cells. During mammalian limb development, it promotes cell proliferation by activating the fibroblast growth factor (FGF)-mitogen-activated protein kinase (MAPK) signaling pathway. Phosphorylation of the MSX1 Ser136 phosphorylation site is a key regulatory node in this process. Knockout of MSX attenuates FGF-MAPK signaling in bone precursor cells, slowing cell proliferation and ultimately leading to bone developmental defects such as decreased limb bone mass, short limbs, and radial and ulnar deficiency. These results suggest that MSX1 plays a crucial role in cell fate determination. It regulates the expression of multiple genes, influencing biological processes such as cell differentiation, proliferation, and apoptosis. During development, the expression pattern of MSX1 is crucial for cell differentiation and organ morphogenesis.

[0005] Important transcriptional regulators that determine cell fate during development are often aberrantly activated during tumor development and progression, thereby mediating tumor progression. Existing databases have shown that MSX1 is abnormally overexpressed in various tumor types, and its expression level is significantly negatively correlated with patient survival. However, relatively few reports exist on its role in tumor development and progression, and the molecular mechanisms underlying this process. Furthermore, there are no reports on the development of drugs specifically targeting the role of MSX1 in gastric cancer development and progression to improve treatment outcomes for gastric cancer patients. Summary of the Invention

[0006] To solve the above problems, the present invention provides a small molecule targeted drug targeting the MSX1 phosphorylation site, which can effectively treat gastric cancer and provide a potential new strategy for clinical gastric cancer treatment.

[0007] During a screening study for differentially expressed genes at risk-associated single nucleotide polymorphism (SNP) sites in gastric cancer, we identified MSX1, a gene with a risk-associated SNP, as a potential biomarker for gastric cancer diagnosis and treatment. Furthermore, through analysis of clinical samples and public databases, we found that MSX1 is abnormally highly expressed in gastric cancer, and its expression level is significantly negatively correlated with patient survival. Therefore, we hypothesize that MSX1 may play an important role in the development and progression of gastric cancer.

[0008] Based on this, our previous studies have found that the expression of MSX1 promotes the proliferation of gastric cancer cells. In gastric cancer cell lines, MSX1 can promote cell cycle progression and inhibit cell apoptosis, which may mediate tumor chemotherapy resistance. Proliferation experiments revealed that under the treatment of the chemotherapy drug cisplatin (DDP), the survival ability of HGC27 cells lacking MSX1 decreased and the DDP IC50 value was lower than that of wild-type cells. The MSX1 S136A point mutation (i.e., the mutation in which serine Ser at position 136 of the MSX1 protein is replaced by alanine Ala) increased the cell's chemotherapy sensitivity to DDP. Therefore, phosphorylated MSX1 plays a key role in the chemotherapy resistance of gastric cancer cells. We previously reported that CDK1 is a phosphorylation kinase at the MSX1 Ser136 site, and the CDK1 inhibitor Ro3306 effectively prevented the phosphorylation of the MSX1 Ser136 site (Nucleic Acids Res. 2020; 48(20): 11452-67.). Therefore, we hypothesized that combining the CDK1 inhibitor Ro3306 with the apoptosis-inducing agent DDP could enhance the inhibition of gastric cancer cell viability and that Ro3306 treatment might sensitize gastric cancer cells to DDP treatment. Experimental results showed that the combination of DDP and Ro3306 significantly enhanced cytotoxicity in gastric cancer cells. DDP and Ro3306 exhibited a synergistic effect in inhibiting gastric cancer cell proliferation. Even when the doses of Ro3306 and DDP were halved, the inhibitory effect of the combined drug treatment remained significant.

[0009] Based on these research contents, the present invention proposes the following technical solutions:

[0010] In a first aspect, the present invention provides use of an MSX1 phosphorylation site as a target in the preparation of a drug for treating gastric cancer, wherein the MSX1 phosphorylation site is the MSX1 Ser136 site.

[0011] In a second aspect, the present invention provides use of an inhibitor of MSX1 Ser136 phosphorylation in the preparation of a drug for treating gastric cancer.

[0012] Preferably, the MSX1 Ser136 phosphorylation inhibitor is a CDK1 inhibitor.

[0013] More preferably, the CDK1 inhibitor is Ro3306.

[0014] In a third aspect, the present invention provides the use of an MSX1 Ser136 phosphorylation inhibitor combined with an apoptosis-inducing agent in the preparation of a drug for treating gastric cancer.

[0015] Preferably, the MSX1 Ser136 site phosphorylation inhibitor is a CDK1 inhibitor, and the combined apoptosis-inducing agent is a platinum drug.

[0016] More preferably, the CDK1 inhibitor is Ro3306, and the platinum drug is DDP.

[0017] In a fourth aspect, the present invention provides a small molecule targeted drug for the MSX1 phosphorylation site for treating gastric cancer, which includes a CDK1 inhibitor Ro3306 and an apoptosis inducing agent DDP.

[0018] Furthermore, in addition to the two active ingredients, Ro3306 and DDP, the drug may also contain pharmaceutically acceptable excipients as needed. "Pharmaceutically acceptable" means that when the excipients are properly administered to animals or humans, they do not produce adverse, allergic, or other untoward reactions. The pharmaceutically acceptable excipients may be selected from one or more of diluents, fillers, surfactants, absorption enhancers, disintegrants, wetting agents, dispersants, and the like.

[0019] Furthermore, the dosage form of the drug includes but is not limited to any one of injection, emulsion, tablet, powder, granule, gel, ointment, capsule, and oral solution.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discovered the MSX1 Ser136 site as a target for gastric cancer treatment, and inhibiting its phosphorylation significantly inhibits gastric cancer cell proliferation. Based on this discovery, the present invention selected the CDK1 inhibitor Ro3306 as an inhibitor of MSX1 Ser136 phosphorylation. A targeted small molecule targeted drug containing Ro3306 and the platinum drug DDP as active ingredients was developed. The combination of Ro3306 and the platinum drug DDP demonstrated a synergistic inhibitory effect against gastric cancer cells, providing a potential new strategy for the clinical treatment of gastric cancer.

[0022] In addition, the present invention also explores the role and mechanism of the MSX1-FBXW7 molecular axis in the occurrence and progression of gastric cancer. The phosphorylation of MSX1 Ser136 plays an important role in MSX1 activation, degradation of the tumor suppressor factor FBXW7 and regulation of the stability of downstream oncoproteins, laying the foundation for in-depth study of the role of MSX1 in the occurrence and progression of gastric cancer and the development of targeted gastric cancer treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Results of experiments related to the promotion of gastric cancer cell proliferation by phosphorylation of MSX1 Ser136 in Example 1. Figures: A shows the expression levels of MSX1 in different tumors analyzed in the TCGA database; B shows the detection of MSX1 mRNA levels in various human cancer cell lines by qPCR; C shows the detection of MSX1 protein levels in various human cancer cell lines by Western blot; D shows the comparison of cell proliferation rates between MSX1-knockout HGC27 gastric cancer cell lines (MSX1 KO-1 and MSX1 KO-2) and wild-type HGC27 cell lines (WT); E shows the comparison of cell proliferation rates between HGC27 cell lines with an MSX1 S136A insertion mutation and wild-type HGC27 cell lines; F, G, and H show the effects of MSX1 knockout on the subcutaneous tumorigenicity, tumor volume, and tumor weight of HGC27 cells, respectively; I, J, and K show the effects of the MSX1 S136A mutation on the subcutaneous tumorigenicity, tumor volume, and tumor weight of HGC27 cells, respectively.

[0024] Figure 2 : Experimental results related to enhancing the sensitivity of gastric cancer cells to chemotherapy by inhibiting MSX1 phosphorylation in Example 2. In the figure: A and B are flow cytometric analysis of the cell cycle progression and cell count results at different stages after MSX1 knockout in HGC27 cell line; C and D are flow cytometric analysis of the cell cycle progression and cell count results at different stages after MSX1 S136A mutation in HGC27 cell line; E and F are flow cytometric analysis of cell apoptosis after MSX1 knockout or MSX1S136A mutation in HGC27 cell line; G is the quantitative result of E and F; H and I are the results of drug sensitivity experiments of HGC27 cells with MSX1 deletion and MSX1S136A mutation under DDP treatment conditions; J is the IC of Ro3306 in HGC27 cells and MGC803 cells determined by drug sensitivity experiments 50 (half maximal inhibitory concentration) value; K and L are the proliferation inhibitory effects of Ro3306 and DDP alone or in combination on HGC27 cells; M is the proliferation inhibitory effect of Ro3306 and DDP alone or in combination on MGC803 cells.

[0025] Figure 3: Experimental results related to MSX1-mediated FBXW7 degradation via the proteasome pathway in Example 3. In the figure: A and B are the FBXW7 protein level and FBXW7 mRNA level after stable overexpression of Flag-MSX1 in AGS gastric cancer cells with low MSX1 expression, respectively; C and D are the FBXW7 protein level and FBXW7 mRNA level after knocking out MSX1 in HGC27 cells, respectively; E and F are the FBXW7 protein level and FBXW7 mRNA level 48 hours after transfection of different amounts (0 μg, 1 μg and 4 μg) of Flag-MSX1 overexpression plasmid in MGC803 cells, respectively; G is Western blot detection of FBXW7 protein degradation in HEK293T cells overexpressing FBXW7 after overexpressing MSX1; H is the degradation curve of FBXW7 protein in HEK293T cells overexpressing FBXW7 after overexpressing MSX1; I is Western blot detection of FBXW7 protein level after reintroduction of MSX1 into MSX1-KO HGC27 cell line; J is Western blot detection of Blot analysis was used to detect the FBXW7 protein level in HEK293T cells overexpressing FBXW7 after overexpressing MSX1 and simultaneously using MG132 or CQ to inhibit the proteasome and autophagy pathways, respectively; K is a ubiquitination experiment to detect the effect of overexpression of MSX1 on the ubiquitination of FBXW7.

[0026] Figure 4 Results of experiments related to the MSX1-FBXW7 axis regulating oncoprotein levels in Example 3. Figures: A shows c-MYC and MCL-1 protein levels in AGS cells after MSX1 overexpression; B and C show c-MYC and MCL-1 protein levels in MSX1-KO and endogenous MSX1 S136A mutant HGC27 cell lines, respectively; D shows c-MYC and MCL-1 protein levels in AGS cells after MSX1 overexpression or Ser136 mutation; E and F show MSX1 mRNA levels in normal gastric tissue (normal) and tumor tissue (tumor) from 12 gastric cancer patients; G and H show MSX1, FBXW7, c-MYC, and MCL1 protein levels in normal gastric tissue (N) and tumor tissue (T) from 10 gastric cancer patients. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] Example 1: Phosphorylation of MSX1 Ser136 promotes gastric cancer cell proliferation

[0029] To elucidate the functional role of MSX1 in tumorigenesis, we first analyzed the expression pattern of MSX1 in tumors using the UALCAN online tool (http: / / ualcan.path.uab.edu / ), which integrates the TCGA and GTEx databases. The analysis showed that the expression level of MSX1 was significantly higher than that of normal adjacent tissues in a variety of tumor samples, including colorectal adenocarcinoma (COAD), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), cutaneous melanoma (SKCM), and gastric adenocarcinoma (STAD). Figure 1 A). We then examined the expression levels of MSX1 in widely used human cancer cell lines, including gastric, colorectal, liver, prostate, and breast cancer cell lines. We found that MSX1 expression levels were high in some cell lines ( Figure 1 B and 1C), suggesting that MSX1 may be associated with the occurrence of various tumors. Notably, consistent with the elevated expression of MSX1 observed in human gastric cancer (STAD) and colorectal cancer (COAD) samples, we found high expression of MSX1 in gastric cancer cell lines (HGC27 and MGC803) and colorectal cancer cell line (HCT116) ( Figure 1 To further analyze the mechanism of MSX1 in cancer, we selected three gastric cancer cell lines, HGC27, MGC803, and AGS, whose MSX1 expression levels were high, medium, and low, respectively.

[0030] We used the MSX1-overexpressing HGC27 cell line to construct an MSX1-knockout HGC27 cell line. The results showed that the proliferation rate of MSX1-knockout HGC27 cells was decreased compared with wild-type HGC27 cells ( Figure 1 D). Previous studies by the research team have shown that phosphorylation of the MSX1 Ser136 site plays a key role in promoting cell proliferation (Nucleic Acids Res. 2020; 48(20): 11452-67.). Therefore, we used the CRISPR / Cas9 system to construct an endogenous MSX1S136A (Ser136 to Ala) insertion mutation in the HGC27 cell line to prevent phosphorylation of the MSX1 Ser136 site. Proliferation was detected and it was found that the proliferation rate of MSX1 S136A cells was significantly reduced compared to wild-type HGC27 cells ( Figure 1 E) Therefore, similar to its function in embryonic development, MSX1 may also play a role in promoting cell proliferation in gastric cancer cell lines, and its phosphorylation at Ser136 plays a key role.

[0031] Next, we used a nude mouse subcutaneous tumorigenesis assay to examine the effects of MSX1 knockout or S136A mutation on HGC27 cell tumorigenesis in vivo. The results showed that compared with wild-type HGC27 cells, the tumorigenic ability of MSX1 knockout cells was significantly impaired, and the tumor volume and weight were significantly reduced ( Figure 1 FH). Similarly, the subcutaneous tumorigenicity of MSX1S136A point mutation cells was significantly weakened compared with the control group, and the tumor volume and weight were reduced ( Figure 1 These findings suggest that MSX1 promotes tumorigenesis and progression in vivo, and phosphorylation of MSX1 at Ser136 is crucial for this process.

[0032] Example 2: Inhibition of MSX1 phosphorylation enhances the sensitivity of gastric cancer cells to chemotherapy

[0033] Based on the results of Example 1, we further studied the role of MSX1 in promoting cell cycle and inhibiting apoptosis. First, flow cytometry analysis showed that knocking out MSX1 in the HGC27 cell line led to a delay in the DNA synthesis stage and subsequent cell cycle progression ( Figure 2 Similarly, the endogenous MSX1 S136A mutation in HGC27 cells also leads to delayed cell cycle progression ( Figure 2 C and 2D). On the other hand, flow cytometry analysis of apoptosis showed that knockout of MSX1 or endogenous MSX1 S136A mutation promoted apoptosis in HGC27 cells ( Figure 2 E and 2F), and significantly increased the proportion of apoptotic cells ( Figure 2 G). These findings confirm that MSX1 plays a dual role in promoting cell cycle progression and inhibiting apoptosis, with phosphorylation of MSX1 Ser136 being key. These results are consistent with the finding that knockout of MSX1 or endogenous MSX1 S136A mutation leads to decreased survival of gastric cancer cells ( Figure 1 D and 1E). Thus, in gastric cancer cell lines, MSX1 enhances cell survival by promoting cell cycle progression and inhibiting apoptosis, and its underlying mechanism depends on the phosphorylation of MSX1 Ser136.

[0034] DDP is a DNA-damaging metallo-anticancer drug widely used clinically, which has the effect of inducing apoptosis in cancer cells. Given that our results show the role of MSX1 in inhibiting apoptosis in gastric cancer cells, we speculate that MSX1 may be involved in the resistance of gastric cancer cells to the chemotherapy drug DDP. Proliferation experiments revealed that under DDP treatment, MSX1-deficient HGC27 cells had decreased viability and lower DDP IC50 values ​​compared to wild-type cells ( Figure 2H and 2I). In wild-type HGC27 cells, the IC50 was 26.00 μM, while that in MSX1 knockout HGC27 cells was 16.89 μM ( Figure 2 H). Consistently, the IC50 of MSX1 S136A point mutation in HGC27 cells was 18.13 μM ( Figure 2 H and 2I), indicating that the MSX1S136A point mutation enhances chemosensitivity to DDP compared with wild-type cells. Therefore, phosphorylated MSX1 plays a key role in chemoresistance in gastric cancer cells.

[0035] These results suggest that reducing dephosphorylation of MSX1 or Ser136 may be a potential therapeutic strategy for gastric cancer. We previously reported that CDK1 is a phosphorylation kinase at the MSX1 Ser136 site, and the CDK1 inhibitor Ro3306 effectively prevented the phosphorylation of the MSX1 Ser136 site (Nucleic Acids Res. 2020; 48(20): 11452-67.). Therefore, we speculate that the CDK1 inhibitor Ro3306 combined with the apoptosis-inducing agent DDP can enhance the inhibition of gastric cancer cell viability, and Ro3306 treatment may sensitize gastric cancer cells to DDP treatment.

[0036] We then evaluated the synergistic effect of Ro3306 and DDP on gastric cancer. The experimental methods and results are as follows:

[0037] Ro3306 was dissolved in DMSO to prepare solutions at concentrations of 2.5 μM and 5 μM; DDP was dissolved in DMF to prepare solutions at concentrations of 5 μM, 10 μM, and 20 μM. HGC27 cells were treated with the following groups: DMSO, Ro3306 (5 μM), DDP (20 μM), a combination of Ro3306 (5 μM) and DDP (20 μM), and a combination of Ro3306 (2.5 μM) and DDP (10 μM). MGC803 cells were treated with the following groups: DMSO, Ro3306 (5 μM), DDP (10 μM), a combination of Ro3306 (5 μM) and DDP (10 μM), and a combination of Ro3306 (2.5 μM) and DDP (5 μM). The cells were seeded into 24-well plates and incubated for 2 weeks. The cells were then treated with Ro3306 and / or DDP for 24 hours, fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet, and then eluted with 33% acetic acid. The absorbance was measured at 570 nm using a multimode microplate reader to determine the relative cell viability.

[0038] The results of the half-maximal inhibitory concentration experiment showed that the IC50 of Ro3306 in HGC27 cells was 6.935μM ( Figure 2J). The results of the clone formation experiment showed that the combined application of Ro3306 (5μM) and DDP (20μM) could effectively inhibit the survival of HGC27 cells ( Figure 2 K and 2L), significantly enhancing the cytotoxicity of DDP. Using Jin's formula (q = Ea + b / (Ea + Eb - Ea × Eb)), the q value was 1.21, which is greater than 1.15, indicating that DDP and Ro3306 have a synergistic effect in inhibiting tumor proliferation, and the combination of the two enhances the anti-tumor response of gastric cancer. It is worth noting that even if the dose of Ro3306 and DDP is halved, the inhibitory effect of the combined drug treatment is still significant ( Figure 2 K and 2L). Similarly, after calculating the IC50 of Ro3306 and DDP against MGC803 cells ( Figure 2 J), we also observed a significant synergistic inhibitory effect of DDP and Ro3306 on the viability of MGC803 cells ( Figure 2 These results indicate that the combination of CDK1 inhibitors and the platinum drug DDP has a synergistic inhibitory effect on the survival of gastric cancer cells, providing a potential new strategy for the clinical treatment of gastric cancer.

[0039] Example 3: Study on the mechanism of involvement of MSX1 protein Ser136 phosphorylation in tumorigenesis and development

[0040] 1. MSX1 mediates FBXW7 degradation through the proteasome pathway

[0041] In our previous studies, we identified multiple phosphorylation sites on MSX1, including Ser148, Ser152, and Ser160, in addition to Ser136, but we did not identify their roles. We hypothesized that these phosphorylation sites may play a role in MSX1 stability and degradation. Our investigations into the regulation of MSX1 stability and degradation revealed that, despite possessing a CPD motif specifically recognized by FBXW7, MSX1 is not a substrate of FBXW7.

[0042] However, we unexpectedly found that MSX1 negatively regulates the expression or stability of FBXW7. After stably overexpressing Flag-MSX1 in AGS gastric cancer cells with low MSX1 expression, we found that FBXW7 protein levels decreased ( Figure 3 A), FBXW7 mRNA levels did not change significantly ( Figure 3 B); After knocking out MSX1 in HGC27 cells, FBXW7 protein levels increased ( Figure 3 C), and the FBXW7 mRNA level did not change significantly ( Figure 3D). Next, we transfected different amounts (0 μg, 1 μg, and 4 μg) of Flag-MSX1 overexpression plasmid into MGC803 cells, which express moderate levels of MSX1. The results showed that as the level of Flag-MSX1 gradually increased, the level of FBXW7 protein decreased in a dose-dependent manner ( Figure 3 E); however, FBXW7 mRNA levels remained unchanged ( Figure 3 F). In addition, overexpression of MSX1 in 293T cells resulted in a shortened half-life of FBXW7 protein ( Figure 3 G and 3H), reintroduction of MSX1 into the MSX1-KO HGC27 cell line suppressed the upregulation of FBXW7 caused by MSX1 knockout ( Figure 3 I). Therefore, our results indicate that MSX1 negatively regulates FBXW7 expression at the protein level.

[0043] To explore the regulatory mechanism of MSX1 on FBXW7 protein stability, we used MG132 and CQ to inhibit the proteasome and autophagy pathways, respectively. The results showed that inhibition of the proteasome pathway effectively inhibited the degradation of FBXW7 induced by MSX1 ( Figure 3 J), whereas inhibition of the autophagy pathway had little effect on MSX1-mediated FBXW7 degradation ( Figure 3 J). Consistently, ubiquitination experiments demonstrated that overexpression of MSX1 promoted the ubiquitination of FBXW7 ( Figure 3 K). Therefore, we concluded that MSX1 mediates the degradation of FBXW7 through the proteasome pathway.

[0044] 2. The MSX1-FBXW7 axis regulates oncoprotein levels

[0045] FBXW7 is widely considered a tumor suppressor due to its targeting of several oncogenes, including c-MYC, c-JUN, and MCL-1. Given the negative regulation of FBXW7 protein by MSX1, we investigated whether MSX1 regulates the protein levels of these oncogenes. To this end, we measured c-MYC and MCL-1 protein levels in gastric cancer cell lines and clinical samples.

[0046] By overexpressing MSX1 in AGS cells, we found that c-MYC and MCL-1 protein levels were significantly upregulated ( Figure 4 A). Conversely, in HGC27 cell lines with MSX1-KO or endogenous MSX1 S136A mutation, the protein levels of c-MYC and MCL-1 were reduced compared with those in wild-type cell lines ( Figure 4B and 4C). In addition, overexpression of the MSX1 S136A mutant in AGS cells prevented MSX1 from upregulating the expression of c-MYC and MCL-1, whereas the MSX1 S136D mutant retained the ability to upregulate the expression of c-MYC and MCL-1 ( Figure 4 D) Therefore, in gastric cancer cell lines, FBXW7 oncoprotein substrates are positively regulated by MSX1 expression, and phosphorylation of MSX1 at Ser136 is crucial for this process.

[0047] Next, we investigated the association between MSX1 and these oncogenes in gastric cancer tissue samples from 12 gastric cancer patients. By qPCR analysis, we found that the expression of MSX1 in gastric cancer tissues was significantly higher than that in the corresponding normal tissues in 8 patient samples ( Figure 4 E and 4F), which is consistent with the results of analyzing gastric cancer samples from the TCGA (the Cancer Genome Atlas) database and the ACRG (the Asian Cancer Research Group) dataset. In addition, the levels of c-MYC and MCL1 proteins were increased in the eight patients with high MSX1 expression, while the level of FBXW7 protein was significantly decreased ( Figure 4 G and 4H). Collectively, these findings provide clinical evidence that high MSX1 expression in gastric cancer may increase the expression of oncogene proteins such as c-MYC and MCL1, possibly through MSX1 degradation of FBXW7 protein. The MSX1-FBXW7 molecular axis has been confirmed in clinical patient samples.

[0048] Oncoproteins such as c-MYC and MCL1 exhibit oncogenic activity by promoting cell cycle progression or inhibiting apoptosis. We have found that MSX1 positively regulates c-MYC and MCL1 by promoting the degradation of FBXW7; therefore, we infer that the MSX1-FBXW7 axis plays a key role in both promoting cell cycle progression and inhibiting apoptosis.

[0049] In summary, we have preliminarily explored the role and mechanism of the MSX1-FBXW7 axis in the development and progression of gastric cancer. Phosphorylation of MSX1 at Ser136 plays a crucial role in MSX1 activation, FBXW7 degradation, and regulation of the stability of downstream target proteins. The MSX1 / FBXW7 signaling pathway plays a crucial role in regulating the malignant progression of gastric cancer, laying the foundation for further investigation of the role of MSX1 in the development and progression of gastric cancer and the development of targeted therapeutic strategies for this disease.

[0050] This specific implementation is merely an explanation of the present invention and is not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Application of the MSX1 Ser136 site as a target in the preparation of drugs for the treatment of gastric cancer.

2. Application of MSX1 Ser136 site phosphorylation inhibitors in the preparation of drugs for the treatment of gastric cancer.

3. The use according to claim 2, characterized in that The MSX1 Ser136 site phosphorylation inhibitor is a CDK1 inhibitor.

4. The use according to claim 3, characterized in that The CDK1 inhibitor is Ro3306.

5. Application of MSX1 Ser136 site phosphorylation inhibitor combined with apoptosis-inducing reagent in the preparation of drugs for the treatment of gastric cancer.

6. The use according to claim 5, characterized in that The MSX1 Ser136 site phosphorylation inhibitor is a CDK1 inhibitor, and the combined apoptosis inducing agent is a platinum drug.

7. The use according to claim 6, characterized in that The CDK1 inhibitor is Ro3306, and the platinum drug is DDP.

8. A small molecule targeted drug for the MSX1 phosphorylation site for treating gastric cancer, characterized in that: Including CDK1 inhibitor Ro3306 and apoptosis inducing reagent DDP.

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