Study On The Alteration Of PI3K/AKT/mTOR Signal Pathway And Its Mechanisms In Diffuse Large B Cell Lymphoma

Objective:The aims of this study are to investigate the activation of PI3K/AKT/mTOR signal pathway in diffuse large B cell lymphoma(DLBCL) and its association with clinicopathologic characteristics,to elucidate the pathogenesis and development of DLBCL from the viewpoint of signal transduction,with the purpose of providing new markers for the diagnosis and prognosis of DLBCL;and further investigate the mechanisms of this pathway's activation from the level of subunits of PI3K.In addition,we developed a mice model of DLBCL,to investigate the involvement of AKT pathway and its effects in DLBCL in vivo,which not only provided an animal model for the in vivo study of DLBCL,but also provided new therapeutic targets for the treatment of DLBCL.Methods:Part 1:(1) The frozen tissue samples and corresponding formalin-fixed paraffin-embedded samples of 75 DLBCL cases and 10 cases of reactive hyperplasia (RH) acquired from 2002-2007 were obtained from the Tumor Tissue Bank in the Department of Pathology,Cancer Hospital of Fudan University.Among the 75 DLBCL cases,follow-up data were available for 54 cases.(2) Immunohistochemistry(IHC) staining on frozen section was used to detect the expression of pAKT and pmTOR in the 75 DLBCL and 10 RH cases.IHC was performed on formalin-fixed paraffin-embedded sections to investigate the expression of Bcl6,CD10 and MUM1,according to which the DLBCL cases were divided into two subtypes,germinal center-B cell like(GCB) and non-GCB. Real-time quantitative reverse transcription polymerase chain reaction(Real-time RT-PCR) was performed to detect the mRNA expression of Bcl6 in the above cases. (3) The expression of pAKT and pmTOR in DLBCL and RH was compared and the association of these two molecules expression with Bcl6 expression,DLBCL subclassification,clinical biomarkers and the overall survival were analyzed.Part 2:(1) Real-time PCR was performed to investigate the mRNA expression of the 4 catalytic subunits of PI3K on 54 DLBCL cases from part 1 and 9 RH cases. Then the expression level of the 4 subunits was compared.The difference of their expression in DLBCL and RH and the association with pAKT expression were analyzed.(2) Genomic DNA of 76 DLBCL cases was extracted using phenol-chloroform and ethanol precipitation.Polymerase chain reaction(PCR) was performed to amplify the fragment with reported hotspots of mutation,and amplification products were then purified and directly sequenced.Part 3:(1) DLBCL-bearing mice model was first developed using human DLBCL cell line LY8.The hlstologic characteristics and the immunophenotype were examined.PCR amplification was performed to investigate the IgH cloned rearrangement and three microsatellite loci of xenografted tumor samples and LY8 cell line.(2) Tumor-beating mice were divided randomly into 3 groups when the tumor mass reached 200-400mm³:(â…°) LY294002(0.1mg/kg),(â…±) LY294002 (0.5mg/kg),(â…²) vehicle alone(control).Each group consisted of 10 mice and treated each day continually for 10 days.Tumor size was measured 3 times per week.At the end of the experiments,the mice were humanly sacrificed and the tumors were excised and bisected.Western blot was used to detect the activation of AKT,and histological appearance was examined using hematoxylin-eosin(HE) staining and Ki67 staining was detected using IHC test and then proliferation index was analyzed accordingly.The weight inhibitory rate and the volume inhibitory rate were calculated.(3) DLBCL-bearing mice were divided randomly into 4 groups when the tumor mass reached 600-800mm³.Each group included 4 mice,which were administered intraperitoneally 0.02mg,0.1mg,and 1.0mg LY294002 and 20% DMSO,respectively.The 4 groups were sacrificed after treated for 6h,18h,24h,and 48h,respectively.Tumor masses were excised and cell cycle and apoptosis were detected using flow cytometry(FCM).(4) DLBCL-bearing mice were divided randomly into the following 4 groups when the tumor mass reached 200-400mm³:(â…°) LY294002(0.5mg/kg,each day for 10 days),(â…±) DOX(5mg/kg,the 1^st day and the 3^rd day),(â…²) DOX(5mg/kg,the 1^st day and the 3^rd day)+LY294002(0.5mg/kg,each day for 10 days)(â…³) vehicle alone(20%DMSO).Each group consisted of 10 mice. LY294002 and vehicle were administered intraperitoneally and DOX was given intravenously.Tumor size measurement,histological appearance examination, proliferation index analysis,and weight inhibitory rate and the volume inhibitory rate calculation were performed according to(2).Results:Part 1:(1) The expression of pAKT and pmTOR was 73.3%(55/75) and 74.7% (56/75),respectively,and the expression of the two proteins was in consistent with each other.(2) The expression of pAKT and pmTOR in non-GCB group was 82.5% (47/57) and 84.2%(48/57),respectively,which were significantly higher than that in GCB group,44.4%(8/18) and 44.4%(8/18),respectively(both p＜0.01).(3) The expression of Bcl6 protein and mRNA in pAKT and pmTOR high-expression group was significantly lower than that in low-expression group(both p＜0.01).(4) The percentage of patients with abnormal LDH in pAKT and pmTOR positive groups was higher than that in negative groups,although it only reached borderline significance(both 10=0.055).There was no relationship between the expression of pAKT and pmTOR and age,sex,stage,KPS and B symptoms(p＞0.05).The overall survival of patients with negative pAKT was better than that with positive pAKT, although it did not reach statistical significance(p＞0.05).Part 2:(1) Quantitative analysis using real-time PCR showed that PIK3CD expression was the most predominant among the 4 isoforms,with PIK3CB closely following it,while PIK3CG expression was the lowest.PIK3CD expression was significantly higher than PIK3CA and PIK3CG(p＜0.05).(2) The PIK3CB expression was also significantly higher than PIK3CA and PIK3CG(p＜0.05) and its expression in DLBCL was significantly higher than that in RH(p＜0.05).(3) Both PIK3CB and PIK3CD expression level were significantly associated with the pAKT expression,with high mRNA level of PIK3CB and PIK3CD in cases with positive pAKT expression(both p＜0.05).(4) Only 1 out of 76 primary DLBCLs(1.32%) displayed mutations.(5) Three mutation points were found.One was a missense mutation(c.1634A＞C) which occurred in one of the reported hotspots.The other mutations,c.1658G＞C and c.1659delT,had not been hitherto reported in hematopoietic or solid tumors.Part 3:(1) We successfully developed DLBCL-bearing mice using human DLBCL cell line LY8,and it grew with high stability.By the time we analyzed,the mice had been steadily transferred to the 9^th generation;the total number of xenografted mice was 124,among which 114 mice were successfully transplanted with DLBCL,with the tumor formation rate remaining 91.1%.The grow characteristics of each generation were similar.Generally,the tumor mass appeared in about 2 weeks after inoculation,and then grew fast,and the tumor volume reached 1000mm³ in diameter in about 3 weeks and 3000mm³ in about 4 weeks.The histologic appearance,IHC detection,gene rearrangement detection,and Microsatellite DNA amplification of the xenotransplanted tumors and LY8 cell line showed that the histologic and genetic features,and Microsatellite DNA of xenotransplanted tumors were identical with that of the DLBCL cell line LY8.(2) Compared with the control group,high dose LY294002 group showed significant anti-tumor effect in a time-depended manner, and both the tumor volume and tumor weight in high dose group were dramatically reduced(p＜0.01).The low dose group did not show noticeable anti-tumor effect (p＞0.05).pAKT expression in low and high dose groups were significantly reduced compared with the control,with the high dose group being more dramatic.(3) Both the proliferation activity of low dose group and high dose group were dramatically lower than that of control group(both p＜0.05).(4) The ratio of cells in S phase in each dose of 6h and 18h group was slightly reduced compared with the control,but the effect was not significant(p＞0.05).The ratio of cells in S phase was significantly reduced in high dose of 24h group and the low and high dose of 48h group(p＜0.05). In our study,48h is the best time period for LY294002 in cell cycle arrest.In addition,we showed that LY294002 did not induce cell apoptosis in this in vivo study(p＞0.05).(5) The mice of LY294002 combined with DOX group showed significant inhibition of tumor growth much earlier than that of the LY294002 alone, DOX alone and control group.In addition,we showed that the tumor cell proliferation activity of the combined group was dramatically lower than that of control and LY294002 alone and DOX alone group(p＜0.01).Conclusions:Part 1:PI3K/AKT pathway might play an important role in the development of DLBCL,and it was closely related to the low-or non-expression of Bcl6 and non-GCB subgroup.This pathway might serve as new target in the treatment of certain group of DLBCL.We have not found any related reports in China and only one abroad,which did not investigate the correlation of this pathway with DLBCL subtype and Bcl6 expression.Part 2:To the best of our knowledge,there was little is known about the mechanisms of PI3K/AKT pathway activation and the expression of the 4 PI3K catalytic isoforms in DLBCL.In the current research,we suggested that both PIK3CD and PIK3CB high expression might play important roles in the activation of PI3K/AKT pathway in DLBCL.PIK3CA mutations are infrequent in DLBCL, indicating that this mutation might not be a common route of frequent activation of the PI3K pathway in DLBCL.Part 3:We successfully developed a mice model of human DLBCL,providing a nice model for further investigation of DLBCL in vivo.We showed that LY294002 might inhibit the activation of AKT and the tumor growth of DLBCL-bearing mice, probably through the regulation of cell cycle instead of cell apoptosis.LY294002 also inhibited the proliferative activity of tumor cells.We also demonstrated that LY294002 might enhance the anti-tumor effect of DOX in vivo.