| Background and PurposeGastric cancer ranked the fourth in morbidity and fifth in mortality among 36 cancer types in 185 countries around the world according to the data released by the International Agency for Research on Cancer(I ARC)of World Health Organization(WHO)in 2020.Gastric cancer remains a major global health burden.The main risk factors for gastric cancer include Helicobacter pylori(H.p)infection,Epstein-Barr virus(EBV)infection,family history,alcohol consumption,smoking,and obesity.About 80%of gastric cancer is related to H.p infection,and persistent chronic H.p infection can cause atrophic gastritis,which could develop into gastric cancer when combined with other risk factors.The successful eradication of H.p can reduce the risk of gastric cancer by 30%,but H.p is difficult to eradicate and prone to repeated infection.Currently,there is no effective drug for the treatment of atrophic gastritis in clinical practice,which increases the challenge in the prevention and treatment of gastric cancer.The highest incidence of gastric cancer in the world is in Asia and is concentrated in East Asia.China and Japan are the countries with the higher incidence.In recent years,the incidence and mortality of gastric cancer in Japan have gradually decreased,while the survival time has gradually increased,which is attributed to the early diagnosis of gastric cancer and the eradication policy of H.p in Japan.However,the incidence and mortality of gastric cancer in China remain high,and most of gastric cancer patients are in advanced stage when they are diagnosed.However,there is a lack of effective treatment options for advanced gastric cancer,and there is a lack of therapeutic targets and effective inhibitors for gastric cancer treatment in clinic.Data mining based on gastric cancer in TCGA database showed that Cyclin-dependent kinase 12(CDK12)and Glutamate/proline-tRNA ligase(EPRS/GluRS)were highly expressed in gastric tumor tissues,and CDK12 was correlated with EPRS.CDK12 can regulate cell cycle,transcription,and translation.Previous studies have shown that CDK12 plays an important role in human cancers,both as a biomarker and as a therapeutic target.CDK12 can participate in PI3K/AKT signaling pathway,non-classical NF-κB signaling pathway,DNA damage response,and regulate MYC expression.CDK12 has different functions in different cancer types.CDK12 plays a tumor suppressive role in prostate cancer and a tumor promoter role in liver cancer.However,the role of CDK12 in gastric cancer has not been elucidated.EPRS/GluRS is a member of the amino-acetyl-tRNA synthases(AARSs)superfamily,involved in protein synthesis.At present,there are few reports on the role of EPRS in cancer.Existing studies have shown that EPRS is highly expressed in breast cancer,and the highly expressed EPRS predicts poor overall survival,suggesting that EPRS may play a key role in cancer.However,the role of EPRS in gastric cancer has not been reported.Studies on specific inhibitors of CDK12 and EPRS are still in progress and have not been used in clinic.Computer docking modeling has been well employed in drug discovery,using this technology in combination with the FDA approved drugs database can be beneficial to the development of CDK12 and EPRS inhibitors.Therefore,it is of great significance to study the function,molecular mechanism,and inhibitor of CDK12 and EPRS in gastric cancer for elucidating new therapeutic target and treatment strategies of gastric cancer.In this study,we conducted an in-depth study on the function of CDK12 and EPRS in gastric cancer,clarified the promoter role of CDK12 and EPRS in gastric cancer,elucidated the molecular mechanism of CDK12 and EPRS in the progression of gastric cancer.and confirmed the rationality,feasibility,and significance of CDK12 and EPRS act as potential therapeutic targets for gastric cancer.In addition,the computer docking modeling was used to screen CDK12 and EPRS inhibitors.The pull-down assay,kinase assay,and surface plasmon resonance assay were applied to verify the procaterol hydrochloride can bind with CDK12 and restrain its kinase activity,xanthoangelol(XA)and 4-hydroxyderricin(4-HD)can bind with EPRS and inhibit EPRS mediated signaling pathways.Finally,the safety and efficacy of procaterol hydrochloride,XA and 4-HD were verified in cells,patient-derived xenograft mouse models(PDXs),window phase clinical follow-up trial,and H.p combined with alcohol induced gastric tumorigenesis mouse model.In summary,it was revealed that CDK12 and EPRS may be new therapeutic targets for gastric cancer,and procaterol hydrochloride,XA and 4-HD are expected to be used in the clinical prevention and treatment of atrophic gastritis and gastric cancer.Methods(1)Explore the role of CDK12 in gastric cancerFirstly,the expression of CDK12 in gastric tumor tissues was searched on the TCGA and GEPIA databases,then the expression of CDK12 in gastric tumor tissues was detected by immunohistochemistry(IHC)using a tissue array.The expression of CDK12 in gastric cancer cells was detected by western blotting(WB).The lentivirus-mediated knockdown or overexpression of CDK12 was detected by WB.MTT and soft agar assay were used to check the effects of down regulation or up regulation of CDK12 on cell proliferation and colony formation.The flow cytometry assay was used to check the cell cycle and cell apoptosis.Cell-derived xenograft NU/NU mouse models(CDXs)and patient-derived xenograft NOD/SCID mouse models(PDXs)were used to verify the effects of CDK12 down regulation on tumor growth.(2)Elucidate the molecular mechanism of CDK12 in gastric cancer developmentThe proteins interacting with CDK12 were identified by immunoprecipitation(IP)and mass spectrometry(MS),and the co-localization of CDK12 with their partners in cells was obtained by immunofluorescence(IF).PAK2 expression vectors with different transcripts were constructed,and IP was used to identify the binding region between CDK12 and PAK2.The regulation of related signaling pathways after CDK12 knockdown or overexpression were detected by WB,and verified in tumor tissues by IHC.The phosphorylation status of CDK12 and PAK2 were analyzed and predicted by Net Phos K.2.0 software,and verified by kinase assay.Phospho-MS was used to identify the phosphorylation sites of PAK2,and PAK2 with phosphorylation site mutations was constructed,expressed,purified,and finally verified by kinase assay.The effects of PAK2 with phosphorylation site mutation on cell proliferation and tumor growth were detected by MTT assay,crystal violet foci formation assay,and CDX model.(3)Screen CDK12 inhibitorPotential inhibitors of CDK12 were explored by computer docking modeling,and were verified by pull-down assay and kinase assay.The screened potential inhibitors of CDK 12 were used to treat the cancer cells and the effects on cell proliferation and related signaling pathways were detected by MTT assay,soft agar assay,WB,and IHC.The effects and safety of the selected inhibitors on tumor growth were verified in the PDX models.IHC was used to detect the effects of procaterol hydrochloride oral solution on MAPK signaling pathway related molecules in tumor tissues of patients with gastric cancer in the window clinical follow-up trial.(4)Study the functional role of EPRS in gastric cancerThe expression of EPRS in gastric tumor tissues was analyzed and detected by databases and IHC,and the expression of EPRS in gastric cancer cells was detected by WB.The effects of EPRS down-regulation on cell proliferation were detected by MTT,crystal violet foci formation,and soft agar assay,as well as verified in CDX and PDX models.(5)Identify the mechanism of EPRS in accelerating gastric cancer progressionThe binding of EPRS with SCYL2 and their co-localization in cells were detected by IP and IF.The expression of SCYL2 in gastric cancer tissues and its correlation with EPRS were detected by IHC.The effect of SCYL2 down-regulation on cell growth was detected by MTT,crystal violet foci formation and soft agar colony formation assay.WB and IHC were used to detect the changes of WNT/GSK-3β/β-catenin signaling pathway related molecules in cancer cells and tumor tissues after down-regulation or up-regulation of EPRS,and IF was used to detect the localization of β-catenin in cells.(6)Screen the inhibitors of EPRSThe inhibitors of EPRS were screened by computer docking model and verified by mass spectrometry,pull-down assay,and surface plasma resonance assay.The effects of EPRS,SCYL2,and EPRS inhibitors on the serine phosphorylation and localization of β-catenin in cells were detected by kinase assay and IF.The effects of EPRS inhibitors on cell proliferation was detected by soft agar assay,and finally identified in PDX model and H.p combined with alcohol induced gastric tumorigenesis mouse model.Results(1)CDK12 played an oncogenic role in gastric cancer developmentThe expression of CDK12 was significantly higher in gastric tumor tissues than that in adjacent tissues,and was positively correlated with the age of the patient.The expression of CDK12 was obviously higher in patients older than 60 years compared to patients younger than 60 years.CDK12 down regulation could dramatically inhibit the proliferation of gastric cancer cells,arrest the cell cycle at G2 phase,and inhibit the tumor growth of CDX and PDX models,while overexpression of CDK12 could significantly promote the colony formation of gastric cancer cells.(2)CDK12 could bind and phosphorylate PAK2 to activate MAPK signaling pathwayCDK12 could bind to PAK2 and co-localize in the cytoplasm and nucleus.The high expression of PAK2 in gastric tumor tissues was positively correlated with the expression of CDK12 in the same patient.PAK2 knockdown significantly inhibited gastric cancer cell proliferation and tumor growth.CDK12 could bind to PAK2 and phosphorylate PAK2 at threonine 134/169 to activate MAPK signaling pathway.The mutation of the phosphorylation sites of threonine 134/169 in PAK2 significantly inhibited proliferation and tumor growth of gastric cancer.(3)Procaterol hydrochloride is an effective CDK12 inhibitorProcaterol hydrochloride could bind to CDK12 and inhibit the kinase activity of CDK12.Procaterol hydrochloride could induce cell cycle arrest at G2 phase and induce cell apoptosis.Procaterol hydrochloride oral solution could significantly suppress gastric cancer cell proliferation and tumor growth without obvious toxicity.Procaterol hydrochloride oral solution could inhibit the expression of MAPK signaling pathway related molecules in gastric tumor tissues of patients in window-phase clinical trial.(4)EPRS accelerated gastric cancer progressionEPRS was dramatically overexpressed in gastric tumor tissues,and the highly expressed EPRS was associated with poor prognosis of gastric cancer patients.The expression of EPRS was negatively correlated with P53 and E-cadherin,and the expression of EPRS in mutant P53 tumor tissues was higher than that in wild-type P53 tumor tissues.Down regulation of EPRS could significantly inhibit the cancer cell proliferation and tumor growth.EPRS plays an oncogenic role in gastric cancer development.(5)EPRS could bind to SCYL2 to enhance WNT/GSK-3β/β-catenin signaling pathway and accumulation of β-catenin in the nuclear.EPRS could bind to SCYL2 and co-localize in the cytoplasm.The expression of SCYL2 was significantly higher in gastric tumor tissues compared to adjacent tissues,and was positively correlated with EPRS.SCYL2 knockdown could dramatically inhibit the growth of gastric cancer cells.SCYL2 plays an important role in the development of gastric cancer.EPRS-SCYL2 promoted the transduction of WNT/GSK-3β/β-catenin signaling pathway and accumulation of β-catenin in the nuclear.(6)XA and 4-HD are EPRS inhibitorsXA and 4-HD could bind to EPRS and inhibit the WNT/GSK-3β/β-catenin cascade mediated by EPRS-SCYL2 and the accumulation of β-catenin in nuclear.XA and 4-HD dramatically inhibited cancer cell proliferation and tumor growth.XA and 4-HD could restrain the formation of atrophic gastritis and gastric tumorigenesis in mice induced by H.p combined with alcohol.ConclusionCDK12 binds to PAK2 and phosphorylates PAK2 at threonine 134/169 to activate the MAPK signaling pathway,promoting cell proliferation and tumor growth.Procaterol hydrochloride is an effective CDK12 inhibitor without obvious toxicity.These results indicate that CDK12 may be a new target for gastric cancer treatment,and procaterol hydrochloride is expected to be used in clinical prevention and treatment of gastric cancer.EPRS can bind with SCYL2 to enhance WNT/GSK-3β/β-catenin signal transduction and the accumulation of β-catenin in the nuclear,thus increasing cell proliferation and tumor growth.XA and 4-HD act as EPRS inhibitors significantly inhibit tumor growth and the formation of atrophic gastritis and gastric tumorigenesis induced by H.p and alcohol.These results suggest that EPRS may be a potential therapeutic target for gastric cancer,meanwhile,XA and 4-HD are expected to be used as effective reagents in clinic for the treatment and prevention of atrophic gastritis and gastric cancer. |
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