The Investigation Of Biological Function,Clinical Significance Of CDK1 And Related Inhibitors In Pancreatic Cancer

BackgroundPancreatic cancer is one of the most malignant gastrointestinal tumors,and its overall 5-year survival rate is less than 8%.In the past few decades,our ever-growing understanding of the complex genetic,epigenetic and metabolic alteration of cancer cells has not yet resulted in a dramatic change in the overall outcome for patients with pancreatic cancer.Currently,traditional chemotherapeutics have limited efficacy in patients with pancreatic cancer,and targeted therapy for pancreatic cancer has been difficult.In 2005,the U.S.FDA approved "gemcitabine+erlotinib" for the treatment of patients with locally advanced unresectable pancreatic cancer or distant metastasis.However,the survival time of only about 10 days makes it difficult to obtain large clinical benefits.In 2019,the U.S.FDA approved the PARP inhibitor olapa for the maintenance treatment of patients with metastatic pancreatic cancer carrying BRCA gene mutations.However,the population of patients with metastatic pancreatic cancer carrying BRCA gene mutations is limited.Therefore,there is an urgent need to explore new anti-cancer targets and develop new therapeutic strategies for future treatment of pancreatic cancer.Cyclin-dependent kinases(CDKs)play a central role in the regulation of the cell cycle.At present,CDKs have been found to contain 20 subtypes in humans.Among them,CDK1?6 and CDK14?18 are directly involved in cell cycle regulation,and other CDKs mainly regulate gene transcription.Previous studies have confirmed that the abnormal activation of CDKs can lead to the imbalance of cell cycle progression,which contribute to the abnormal proliferation of tumor cells.In recent years,several small molecule CDK4/6 inhibitors have been approved by the FDA for treatment of patients with advanced or metastatic breast cancer.Although the evidence for a benefit in inhibiting CDKs for treatment of pancreatic cancer is still scarce,existing studies have found that CDKs play an important role in the pathobiology of pancreatic cancer and have the potential to become a new anti-pancreatic cancer target.It has already been found that CDK5 is overexpressed in the tissue of pancreatic cancer patient and promotes the proliferation,migration and invasion of pancreatic cancer cells.Moreover,KRAS mutation can stimulate the expression of CDK8 and promote the invasion and migration of pancreatic cancer cells through the Wnt/?-catenin signaling pathway.Among CDKs,CDK1 has a very critical role in cell cycles.Under normal physiological conditions,CDK1 forms a complex with Cyclin A or Cyclin B to regulate the cell cycle G2/M,and the abnormal activation of CDK1 is closely related to the initiation and development of tumors.Previous studies have indicated that CDK1 is overexpressed in many tumors such as colorectal cancer,liver cancer and lung cancer,and the high expression of CDK1 is related to the poor prognosis of patients.Also,CDK1 selective inhibitor RO3306 exhibited good antiproliferative activity against leukemia,breast cancer and other tumor cells.However,there are still few studies on CDK1 in pancreatic cancer.One study has found that CDC25B inhibitors can increase the phosphorylation level of CDK1 and block pancreatic cancer cells in the G2/M phase by inhibiting CDK1 activity.Another study found that CDK1 and BUB1 are highly expressed in the pancreatic cancer tissues and are related to the poor prognosis of patients.To date,there is still no research reports the effects of direct inhibition of CDK1 on the growth of pancreatic cancer cells.Therefore,the biological function and clinical significance of CDK1 in pancreatic cancer need to be further explored.In addition,although studies have found that pan-CDKs inhibitors have good anti-pancreatic cancer activities,the anti-pancreatic cancer activity and mechanism of action of RO3306,a selective inhibitor targeting CDK1,have not been reported.Therefore,it is still necessary to further study the potential of CDK1 as an anti-pancreatic cancer target and explore new strategies for treatment of pancreatic cancer.On the other hand,abnormal regulation of apoptosis is also an important reason for the development of pancreatic cancer.Previous studies have shown that the anti-apoptotic protein Mcl-1 is abnormally expressed in pancreatic cancer,and inhibiting Mcl-1 can suppress the growth of pancreatic cancer.Moreover,the regulation of Mcl-1 protein as well as endogenous apoptosis pathway is an important mechanism for CDKs inhibitors to induce tumor cell apoptosis.Therefore,the effect of CDK1 inhibitor RO3306 on Mcl-1 protein can be studied to further analyze its mechanism of action.In addition,many studies have shown that Mcl-1 small molecule inhibitors have good anti-pancreatic cancer activity.Therefore,screening of new Mcl-1 inhibitors will help to explore new anti-pancreatic cancer strategies.PurposeThe aim of this study is to explore the biological functions of CDK1 in pancreatic cancer,to analyze the expression and clinical significance of CDK1 in pancreatic cancer patients,and also to investigate the anti-proliferative activity as well as mechanism of action of known CDK1 inhibitor RO3306.Furthermore,we performed virtual screening to identify new CDK1 inhibitors and Mcl-1 inhibitors and studied the anti-pancreatic cancer activity of new Mcl-1 inhibitors.Hopefully,our study would provide a basis for future clinical treatment research of pancreatic cancer.Methods and resultsPart I.The effects of knocking down CDKl on the growth of pancreatic cancer cells in vitro and in vivo1.First,by combine use of bioinformatics analysis,gene knockdown and cell proliferation experiments,we found that knocking down of CDK1 can significantly inhibit the proliferation of pancreatic cells in vitro.(1)The expressions of CDK1-CDK10 in pancreatic cancer tissues and normal tissues were analyzed through the bioinformatics GEPIA platform and TCGA database.According to the results,CDK1,CDK2 and CDK6 were high in pancreatic cancer patients(p<0.01),and their high expressions are closely related to the poor overall survival of pancreatic cancer patient(p<0.05).(2)The effects of knocking down CDK1,CDK2 and CDK6 on the proliferation of Panc-1 cells were measured using Celigo cell counting system and results indicated that CDK1 knockdown had exhibited the most significant inhibitory effect on Panc-1 cell proliferation(p<0.005).2.We further studied the effects of CDK1 knockdown on the cell proliferation,cell cycle,cell apoptosis and cell migration in pancreatic cancer cells.The inhibitory effect of CDK1 knwodown were further confirmed in two pancreatic cancer cells(Panc-1 and Aspc-1)using MTT experiments(p<0.05).The effects of CDK1 knockdown on the cell cycle and apoptosis of pancreatic cancer cells(Panc-1 and Aspc-1)were studied using flow cytometry.Results showed that CDK1 knockdown can block pancreatic cancer cells in G2/M phase(p<0.05),however,exhibited no significant effect on the cell apoptosis of Panc-1 and Aspc-1 cells(p>0.05).Moreover,Transwell experiments showed that knocking down CDK1 inhibited the migration of Panc-1 cells(p<0.05),however,showed no significant effect on the migration of Aspc-1 cells(p>0.05).3.Using a xenograft mouse model,we studied the effect of CDK1 knockdown on the growth of pancreatic cancer in vivo.The results showed the growth of pancreatic cancer in mice was significantly inhibited after knocking downing CDK1(p<0.05).Part ?.The expression and clinical significance of CDK1 in pancreatic cancer patients1.First,the expressions of CDK1 in 90 cases of pancreatic ductal adenocarcinoma tissues and the corresponding 60 cases of adjacent tissues were detected using immunohistochemical experiments.The results showed that in 44 pairs of pancreatic tissues with adjacent tissues,CDK1 was mainly expressed in the cytoplasm,and partially expressed in the nucleus.Compared with the expression in adjacent tissues,CDK1 was significantly highly expressed in pancreatic ductal adenocarcinoma tissues(p<0.05).2.We further analyzed the relationship between CDK1 expression and clinicopathological characteristics in 73 pancreatic ductal adenocarcinoma tissue patients.The results showed that the cytoplasmic expression of CDK1 was correlated with tumor grade,p53 expression level and Ki67 expression level(p<0.05);nuclear expression of CDK1 was only correlated with p53 expression(p<0.05).3.Finally,the relationship between CDK1 expression and the prognosis of 73 pancreatic ductal adenocarcinoma patients was analyzed.Kaplan-Meier curve and univariate analysis showed that patients with high CDK1 cytoplasmic expression had a shorter overall survival(p<0.05).COX multivariate analysis showed that pathological grade and N stage were independent prognostic factors of survival(p<0.05),while the CDK1 cytoplasmic high expression group and low expression group had no significant difference with overall survival(p>0.05).There was no statistical significance between the nuclear expression of CDK1 and the overall survival of patients with pancreatic ductal adenocarcinoma(p>0.05).Part ?.Anti-pancreatic cancer activity and mechanism of action of CDK1 inhibitor RO33061.First,the anti-proliferative activity of RO3306 was investigated in five different pancreatic cancer cells.(1)The expression of CDK1 in five different pancreatic cancer cells was detected by Western-blot experiment,and it was found that the SW1990 cells possessed the highest CDK1 expression level.(2)Using SRB cell proliferation experiments,we found that RO3306 could inhibit the proliferation of five different pancreatic cancer cells.The anti-proliferative activity of RO3306 in SW1990 cells(IC50=2.9?M),is comparable with the chemotherapy drug 5-Fu(IC50=4.9?M)2.We further explored the mechanism of anti-pancreatic cancer effects of RO3306.(1)Flow cytometry was used to detect the effect of RO3306 on the cell cycle of SW1990,and it was found that RO3306 can block the cell cycle in G2/M and S phases(p<0.05).Western-blot experiment suggested that RO3306 may block the S phase of the cell cycle by activating the p5 3-p21 signaling pathway.(2)Flow cytometry was also used to detect the effect of RO3306 on cell apoptosis of SW1990 cells(p<0.005).Results indicated that RO3306 can significantly induce the apoptosis of SW1990 cells.Western-blot experiments showed that RO3306 can manipulate the Bcl-2-Bax signaling pathway and as well as regulate the phosphorylation level of Mcl-1 protein,which accounts for the ability of RO3306 in induction of cell apoptosis.3.Finally,we measured the anti-proliferative effect of RO3306 when combined with different chemotherapeutics(5-Fu,gemcitabine,paclitaxel and oxaliplatin).The results indicated that RO3306 can enhance the anti-proliferative activities of 5-Fu and paclitaxel on pancreatic cancer cell SW1990.Whereas RO3306 showed less effects on gemcitabine and oxaliplatin.Part ?.Screening of new CDK1 new inhibitors and new Mcl-1 inhibitors1.New CDK1 inhibitors and Mcl-1 inhibitors were discovered using a computer virtual screening platform,which combined the use of three-dimensional pharmacophore and molecular docking technology.We virtually screened a commercial compound library,which contains more than 210,000 molecules.The CDK1 inhibitory activity and Mcl-1 binding affinity of selected hit compounds were measured and we identified compounds with preliminary CDK1 inhibitory activity.Specially,we found new Mcl-1 inhibitors M02(Ki=5.4?M)and M08(Ki=0.53?M),which possess comparable Mcl-1 binding affinities with known Mcl-1 inhibitor Gossypol.2.We further studied the anti-pancreatic cancer activity of new Mcl-1 inhibitors(M02 and M08).Western-blot experiments showed that Mcl-1 protein was expressed in five kinds of microparticles,and the highest Mcl-1 expression was found in SW1990 cells.According to our results,M02 and M08 exhibited anti-proliferative activity(IC50?44.4-48.4?M),arrest cell cycle in G2/M phase(p<0.05)and induce cell apoptosis(p<0.005)in SW1990 cells.Further drug combination experiments showed that M08 can improve the anti-proliferative activity of gemcitabine or 5-Fu,while M02 has little effect on the anti-proliferative activity of gemcitabine or 5-Fu.Conclusions1.Knocking down of CDK1 can inhibit the growth of pancreatic cancer in vivo and in vitro,implying that CDK1 has the potential to serve as a new anti-pancreatic cancer target2.CDK1 is highly expressed in pancreatic ductal adenocarcinoma patients,and its high expression is related to tumor grade,p53 expression,Ki67 expression and other clinicopathological characteristics.High CDK1 cytoplasmic expression is related with poor overall survival of pancreatic ductal adenocarcinoma patients.3.The CDK1 selective inhibitor RO3306 can exert anti-pancreatic cancer activity by arresting the cell cycle and inducing cell apoptosis.RO3306 and can enhance the anti-proliferative activity of both 5-Fu and paclitaxel in pancreatic cancer cells.4.Identified new compounds with CDK1 inhibitory activity,as well as new Mcl-1 inhibitors M02 and M08 that are comparable to the known Mcl-1 inhibitor.Specially,M02 and M08 exhibited anti-pancreatic cancer activity and M08 can enhance the sensitivity of gemcitabine and 5-Fu to pancreatic cancer cells.