Molecular Mechanism Of Type Two Diabetes Promote Cell Centrosome Amplification In Colon Cancer

Type 2 diabetes is a common chronic disease whichcauses enormous economic and health burdens.Epidemiological studies show that diabetes is positively associated with the cancer initiation and development,but the underline molecular mechanisms remainunknown.Centrosome amplification is a common phenomenon in tumor cells,which can initiate tumorigenesis and promote cancer invasion.In this thesis,in search for the biological link between type 2 diabetes and cancer as well as the molecular basis,I investigated whether and how type 2 diabetes can promote centrosome amplification.The whole thesis consists of three parts:Part One: Type 2 diabetes promotes cell centrosome amplificationI recruited 12 normal subjects and 32 diabetic patients and compared their levels of centrosome amplification in peripheral blood mono nuclear cells(PBMC).Clinical data showed that t The diabetic patients had increased fasting blood glucose,glycosylated hemoglobin A1 c and body mass index than the healthy controls.PBMC were separated using lymphocyte separation fluid and the centrosome number was visalized by immunofluorescentstaining.The results showed that the centrosome amplification rate of the diabetic group was 2.6 times higher than that in the healthy group(8.7%±2.54% vs 3.4%±1.49%,p<0.05);When the diabetic patients were divided into two groups according to their fasting blood glucose level less or greater than 10 m M,there was a step-wise increase in centrosome amplification;For all the volunteers and in the diabetic patients,the extent of the cell centrosome amplification was positively correlated with the level of Hb A1c(R2=0.6448,p<0.01 and R2=0.2381,p<0.01 respectively);Moreover,I compared the levels of centrosome amplification in the colon cancer and adjacent non-cancerous tissues to examine whether diabetes promotes cell centrosome amplification.I found that in the colon cancer tissues from the patients with type 2 diabetes had a higher level of centrosome amplification compared with the those from the patients without diabetes 16.7%±1.7% v.s.8.9%±1.7%,p<0.01).Centrosome amplification in cancer adjacent tissues was too low to analyze.In conclusion,the results show that type 2 diabetes promotes cell centrosome amplification,at least in PBMC and colon cancer.Part Two: The molecular basis of the diabetes-associated centrosome amplificationType 2 diabetes presents pathophysiological features of hyperglycemia,hyperinsulinnemia and increased free fatty acids.Palmitic acid is the most common saturated free fatty acid,which is commonly used for the investigations of the inverse effects of free fatty acids.I treated colon cancer HCT116 cells with high glucose(Glu),insulin(Ins)and palmitic acid(Pal),which was used as my experimental model.Treatment with Glu,Ins and Pal,alone or in combinations,increased the level of centrosome amplification,in the order of three factors > two factors > single factor;thus the treatment with three factors was performed for all the subsequent experiments.The treatment increased the intracellular level of the reactive oxygen species(ROS),with was inhibitred by ROS inhibitor N-acetylcysteine(NAC)which was able to inhibit the centrosome amplification.AKT was activated,which was inhibited by its chemical inhibitor,si RNA and NAC.High-throughput transcriptomic profiling,bioinformatics analysis were performed in search for the molecular signals,which showed that the gene expression levels of 14-3-3? and ROCK1 were increased,which were confirmed by Western blot,which could be inhibited using their specific si RNA.The treatmentincreased centrosome amplification was attenuated by AKT inhibitor or si RNA species of AKT,14-3-3? and ROCK1.Subsequently,the upstream or downstream relationship amongst AKT,ROCK1,14-3-3? in signal transduction was studied.Immunofluorescent staining revealed that the treatment increased the centrosome localization of ROCK1 and14-3-3?,and ROCK1 bound to and mediated the itrs centrosomal localization.The final signal transduction pathway underlying the centrosome amplification was ROS-AKT-ROCK1/14-3-3? complex.Aiming at the central role of ROCK1,I further examined the ROCK1 binding partners for the centrosome amplification.Proteins interacting with ROCK1 were enriched by immunoprecipitation and identified by LC-MS/MS technique.Functional clustering,protein interaction network,subcellular localization,TCGA-based tumor m RNA level expression and prognosis analyses were performed to interpret the identified proteins.The results show that both KIF2 A and DCTN2 were in the same protein complex located in the centrosome(possibly mother centrosome).Blocking the ROCK1 kinase structure with ROCK1 inhibitor GSK269962 A did not inhibited the centrosome amplification and caused significantly decrease in KIF2 A protein levels.Protein coimmunoprecipitation showed that ROCK1,KIF2 A,and DCTN2 were present in the same complex after treatment but the inhibitor GSK269962 A could not block the interaction between ROCK1 and KIF2A-DCTN2.Subsequently,using Discovery Studio version 3.5 to molecularly dock ROCK1 with KIF2 A and ROCK1 inhibitor GSK269962 A,the results showed that ROCK1 and KIF2 A docked well,DOCK value=20.32,inhibitor GSK269962 A and ROCK1 docked well,CDOCKER_ENERGY scored 18.85,inhibitor blocking site of ROCK1 does not have a repeating amino acid at the ROCK1-KIF2 A binding site.In conclusion,my results show that the pathophysiological factors of high glucose,palmitic acid,insulin could cause centrosome amplification via The ROS-AKT-ROCK/14-3-3? complex signaling pathway,and suggest that ROCK1 localizes to the centrosome,which binds to and recruits 14-3-3?to centrosome;ROCK1 binds to the KIF2A-DCTN2 complex and to promote centrosome amplificationPart Three: Advanced glycation end products(AGEs)-triggered centrosome amplification and the molecular mechanisms.Since increased level of AGEs is another typical clinical feature in type 2 diabetes,I further investigated whether and how AGEs could promote centrosome amplification.BSA-AGEs products were prepared using incubation of bovine serum albumin(BSA)with glucose.The basic characteristics of the prepared AGEs such as molecular mass,fluorescent spectrum aned their cell binding as well as proliferation promoting effects were examined to confirm the quality of the products.The results showed that the prepared AGEs could trigger centrosome amplification of HCT116 cells in a time-and dose-dependent manner.After AGEs treatment,the protein levels of PLK4,Aurora A and MDM2 were significantly up-regulated,while Rb andp53 were decreased,which were all inhibited by antibody or si RNA for,AGEs' cell surface receptor RAGE.Subsequent studies showed that the colonic tumor-associated transcription factor KLF5 was increased in the nuclear location after treatment.si RNA knockdown KLF5 couldsignificantly inhibit the centrosome amplification by the AGEs;KLF5 knockdown could inhibit the expression of MDM2,and inhibition of MDM2 led to inhibition of the expansion of centrosomes.Chromatin immunoprecipitation(CHIP)showed that KLF5 directly bound to the transcriptional regulatory region of MDM2 after 12 hours of AGEs treatment,directly regulating the transcription of MDM2.For information for the in vivo setting,in the,I constructed the mouse model of type 1 diabetes using streptozocin.Protein levels of KLF5 and MDM2 were significantly increased in the colon of diabetic mice.The CHIP experiment was performed using the colon tissue,and results showed that KLF5 could bind to the transcriptional regulatory region of mouse MDM2 in the diabetic state.(Finally,AGEs were used to treat the noncancerous intestinal cell IEC6 for a long time.After long-term treatment,the cell volume increased and the boundary was more obvious,multilayer growth could be observed in the plate culture.When cell were cultured in soft agar plate,chronic AGEs treatment could form clone in soft agar medium with or without the stimulation of AGEs.It is indicated that after chronic treatment of AGEs cells could overcoming contact inhibition.In conclusion,AGEs in vitro could promote centrosome amplification via KLF5-MDM2 pathway,in which directly regulated the transcription of MDM2,which was also evidenced under the in vivo setting.In summary,my results suggest that type 2 diabetes promotes cell centrosome amplification via multiple signal transduction pathways which include AKT-ROSROCK1/ 14-3-3? complex,ROCK-DCTN2-KIF2 A and KLF5-MDM2.Centrosome amplification is a candidate biological link between type 2 diabetes and cancer.