The Molecular Mechnanism Of GGTase-I Or FTase On The Pathogenesis Of K-Ras-Induced Lung Cancer

PartⅠstudy in vivoBackgroundRecent studies have demonstrated that lung cancer is the leading morbility and fatality rate among malignance tumor in the worldwide.Lung cancer is a gene disease; its genesis is a multistep process involving the cooperation of oncogenic mutations and antioncogenic inactivation.Therefore,elucidating the molecular mechanism and blockage the correlated link of lung cancer is very significance in clinical study.K-ras and P53 gene are the most common oncogenes and anti-oncogene.For example,K-ras mutations occur with high frequency in human colon,pancreas and lung cancer,and the presence of this strongly correlates with disease progression to malignancy. Moreover,there is recent evidence that activation of Ras to promote cancer progression in mouse models for lung.Recently,the study of the role of K-ras gene in the genesis of lung cancer has been the hot study.More than 100 intracellular proteins contain a CAAX motif that directs isoprenylation at a carboxyterminal cysteine(the "C" of the CAAX motif).Some CAAX proteins,such as RHOA,cell division cycle 42(CDC42),and RAP1,are geranylgeranylated by protein geranylgeranyltransferase typeⅠ(GGTase-I).Others, such as K-RAS and N-RAS,are farnesylated by protein farnesyltransferase(FTase). If the "X" of the CAAX motif is a leucine,the protein is generally geranylgeranylated; otherwise,it is farnesylated.Isoprenylation renders the carboxyl terminus of the CAAX proteins more hydrophobic,enhancing their ability to bind to membranes within cells,and also regulates protein-protein interactions.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.Theirβsubunits are called Pggtlb or Fntbs,respectively.In some eukaryotic cells,GGTase-I is an essential enzyme.Null mutations in theβsubunit of GGTase-I are lethal in both Drosophila melanogaster and Saccharomyces cerevisiae.The lethality of GGTase-I deficiency in eukaryotic cells was likely due to the failure to geranylgeranylate Rho1p and Cdc42p,as the lethality could be overcome by expressing mutant Rho1p and Cdc42p proteins engineered to undergo farnesylation by FTase.The realization that the RAS proteins are farnesylated has fueled interest in protein isoprenylation.Farnesylation is important for the proper membrane targeting of RAS proteins and for their transforming ability. In mouse models,farnesyltransferase inhibitors(FTIs) have significant antitumor activity and minimal toxicity.In human clinical trials,however,FTIs have been disappointing,at least for the treatment of solid tumors,likely because K-ras and N-ras -the Ras isoforms most often implicated in human cancer-can be geranylgeranylated in the presence of an FTI.At the same time,inhibition of GGTase-I ameliorated disease phenotypes in a mouse model of multiple sclerosis, inhibited hepatitis C viral replication in hepatoma cell.According to the inhibitory role of GGTase-I or FTase gene in K-ras mutation induced lung cancer and the results of fundament study.The present study was designed to test the hypothesis that the role of inactivation of Pggtlb or Fntb gene in K-ras induced lung cancer due to its anti-proliferation in a mouse model of lung cancer created mice with a conditional knockout allele.The result of this study will provide science evidence for gene therapy.Objective(1) To establish an animal model of lung cancer that are mimic to human pathological changes and convenient for study;(2) To investigate the inhibitory role of knockout GGTase-I or FTase gene on K-ras induced lung cancer transformation;(3) To investigate the molecular mechanisms of GGTase-I or FTase gene on the genesis of K-ras induced lung cancer in vivo.Methods1.Animal model:we created mice with a conditional knockout or knockin allele for GGTase-I(Pggtlb^fl/fl),FTase(Fntb^fl/fl) and K-ras(K^LSL).And then K^LSL mice were bred with Pggtlb^fl/fl mice or Fntb^fl/flmice,generating K^LSLPggtlb^fl/fl mice and K^LSL Fntb^fl/flmice.2.According the genotype of mice,they were divided into three groups:K^LSL(A group),K^LSLPggtlb^fl/fl(B group) and K^LSLFntb^fl/fl(C group),then they were divided into two subgroups according to different adenovirus:cre-adenovirus groups(Study groups:A1 group,B1 group and C1 group) and gal-adenovirus groups(Control groups:A2 group,B2 group and C2 group).3.Adenovirus infection of animals:On the day of 14,all animals were infected with Cre-adenovirus orβ-gal-adenovirus by aerosol rebreathing method.4.Genotype:The K^LSL,Fntb^fl and Pggtlb^fl allele was genotyped by PCR amplification of genomic DNA from tail biopsies.5.Body weight:after infection with adenovirus,the weight of all mousse was measured every week.6.Survival for all animals:the life span of all animals was detected from the treatment.7.Histopathological analysis:The lung,spleen and liver were processed and examined by hematoxylin and eosin staining.8.Immunohistochemical staining was performed and the expressions of CD11b,SP-C,CC10 and Ki-67 were detected.9.Real-time PCR:The mRNA expressions of Pggtlb^fl/fl,Fntb^fl/fl and K^LSL in the lung,liver,cell lines and spleen tissue were analyzed using real-time PCR technique.10.Western blot:The protein expressions of nonprenylated RAP1,total RAP1,RHOA,phosphorylated-ERK1/2,phosphorylated-AKT,total AKT and total ERK1/2 in lung,liver,cell lines and spleen tissue were analyzed using western blot technique.11.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival rates of animal were compared with Kaplan-Meier curve test and other indexes were compared with One-Way ANOVA comparison test.P＜0.05 was considered statistically significant.Results1.General state of the experimental animals:60 mice were infected with adenovirus in all groups.19 mice in group A,19 mouse in group B and 20 mouse in group C.2.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I or FTased was silencing in the tumor tissue.3.Body weight:body weights in the control groups were essentially equal.Since two weeks after infection,body weight in group B1 and C1 were significantly higher than that in group A1(P all＜0.01).Compared with the control group,body weight in the group A1,group B1,and group C1 were significantly decreased(P all＜0.01).4.The rate of lung or spleen weight on body weightCompared A1 group,the lung weight relative to total body weight(LW/BW) of group B1 and group C1 were significantly decreased(P all＜0.01),while the spleen weight relative to total body(SW/BW) of group B1 and group C1 were significantly decreased(P all＜0.01).Compared the group B1,the LW/BW of group C1 is significantly increased(P＜0.01),and the SW/BW of group C1 is significantly increased(P＜0.01).5.Pathologic staining:Most mouse in the trial groups developed tumor cells with large and trachychromatic nuclear,diffuse hyperplasia and leukocyte infiltration were seen in the lung tumor tissue.Compared with group A1,there are little tumor cells and leukocyte infiltration in the group B1 and C1.In the group A1,the diffuse adenocarcinoma has obliterated the majority of alveolar spaces in lung.6.Immunohistochemical staining:The lung tumors of K^LSL,K^LSLPggtlb^fl/fl and K^LSLFntb^fl/fl mice were negative for the monocyte/macrophage marker,the Clara cell marker,and the T cell markers but were positive for the typeⅡpneumocyte marker SP-C as judged by immunohistochemistry.Compared with group A1,the expression of SP-C in lung tumor tissue was significantly decreased in group B1 and C1,while Ki-67 was significantly enhanced.7.Real-time PCR:The mRNA expressions of Pggtlb^fl/fl,Fntb^fl/fl and K^LSLmRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2(P＜0.01 or 0.05).Comparison the group A2,the group A1 had higher K^LSL mRNA expressions(P＜0.01).8.Western blot:The protein expression of P-AKT in the tissues of trial group were increased,and P-ERK1/2 were decreased.The nonprenylated RAP1 was only detected in the group B1 and C1.No difference was found about the total AKT and total ERK1/2.9.Survival rate:Kaplan-Meier curve showing the survival of mouse in the group B1 and group C1 was significantly improved than that in group A1,while there is no significant difference between the group B1 and group C1.Compared with the trial groups,the survival the control group was significantly improved(P＜0.001).Conclusions (1) The method of conditional knockout allele technique is an efficient and time-saving way to establish lung cancer model induced by K-ras;(2) The special gene can be expressed in lung by inhale adenovirus.(3) Inactivation of Pggtlb or Fntb by conditional knockout technique can reduce the tumor formation and improves survival in mice with K-ras-induced lung cancer;(4) GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras;(5) The molecular mechanisms of Pggtlb or Fntb inactivation to treat the lung cancer induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein. PartⅡin vitro studyBackgroundGene mutation is the main cause of the genesis of cancer and there is gene mutation in many solid tumor.Non-small cell lung cancer(NSCLC) may be occupy more than 70%of all the pulmonary carcinoma.It is the leading morbility and fatality rate among malignance tumor in the worldwide and shorten the life span.K-ras gene is the most common oncogene in NSCLC.Therefore,the study of the role of K-ras in the genesis of lung cancer has been the hot study.Recent studies have demonstrated that RAS protein belong to the intracellular proteins contain a CAAX motif.Parts of these proteins can be geranylgeranylated by protein geranylgeranyltransferase typeⅠ(GGTase-I) and the others can farnesylated by protein farnesyltransferase(FTase) alternatively.This is the prenylation process by post-transcriptional modification.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.In mouse study,We have proved that inactivation of GGTase-I or FTase can inhibit the development of lung cancer induced by K-ras.But we do not know the effects of inactivation of GGTase-I or FTase on the mouse embryo fibroblast(MEF) with K-ras mutation.To investigate the effects of the inactivation of GGTase-I or FTase on K-ras mutation MEF,The present study was designed to test the role of the inactivation GGTase-I or FTase on K-ras induced MEF through a conditional knockout technique.Objective(1) To establish the cell model with the K-ras and GGTase-I or FTase knockout; (2) To observe the effects of K-ras on the bionomics in vitro,such as curve growth, clone formation in soft agar and migration;(3) To investigate the inhibitory role of the inactivation of GGTase-I or FTase on K-ras induced MEF;(4) To investigate the molecular mechanisms of the inactivation of GGTase-I or FTase on the K-ras induced MEF.Methods1.Isolation of embryonic fibroblasts:The mouse embryonic fibroblasts(MEF) were isolated from K^LSL,K^LSLPggtlb^fl/fl and K^LSLFntb^fl/fl embryos on day 15.5 post coitum.2.cell lines groups:According the genetype of cell lines,they were divided into three groups:K^LSL(A group),K^LSLPggtlb^fl/fl(B group) and K^LSLFntb^fl/fl(C group), then they were divided into two subgroups according to different adenovirus:creadenovirus groups(Trial groups,A1 group,B1 group and C1 group) andβ-gal-adenovirus groups(Control groups,A2 group,B2 group and C2 group).3.Adenovirus infection of cell lines:To inactivate Pggtlb,Fntb^fl and activate K-RAS^G12D expression,all cells were seeded and infected with Cre-adenovirus orβ-gal-adenovirus for two times.4.Genotype:The K^LSL,Fntb^fl and Pggtlb^fl allele was genotyped by PCR amplification of genomic DNA from tail biopsies.5.Real-time PCR:The mRNA expressions of GGTase-I,FTase and K^LSL in the cell lines were analyzed using real-time PCR technique.6.Bionomics of cell lines:The bionomics of cell lines was detected,such as cell growth curve,clone formation in soft agar,migration and apoptosis.7.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival curve of cell lines were compared with One-Way ANOVA comparison test and other indexes were compared with T test.P＜0.05 was considered statistically significant.Results1.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I and FTased was inactived.2.Real-time PCR:The mRNA expressions of GGTase-I,FTase and K^LSL mRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2(P＜0.01 or 0.05).Comparison of the group A1 and group A2 revealed that group A1 had higher K^LSL mRNA expressions than group A2(P＜0.01).2.Genetype of the MEF linesThe genetype of the cell lines are the same as that in the animal.3.The growth of MEF linesIn the trial group,compared with group B1 and group C1,the group A1 grows significantly quickly.No significant difference was found between the group B1 and group C1.No significant difference was found between the trial group and the control group.There is no significant difference in the control group.4.Migration of the MEF linesCompared with the control group,the ability of migration in the trial group was increased significantly.In the trial group,the number of migration cells in the group A1 is much more than that in the group B1 and C1.There is no significant difference in the control group.5.Clone formation of MEF linesThe clone formation was only seen in the group A1,and no clone in the group B1 and C1.There is also no clone in the control group.6.Apoptosis of MEF linesThe rate of apoptosis in the group A1 is 3.31±1.34%,the group B1 is 13. 91%±1.88%,and the group C1 is 9.57%±2.45%.Compared with the group A1,the rates of apoptosis in the group B1 and C1 are increased significantly,and the group B1 is significant higher than that in the group C1.There is no significant difference in the control group.7.Western blotCompared with the group A2,the expression of Caspase-3 in the group A1 was significantly decreased.The expression of Caspase-3 in the group B1 and C1 was significantly lower than that in the group B2 and C2.The expression of phos-P70 in all the cell lines was no significant difference.The expression of PARP in the group B1 and C1 was significantly higher than that in the group B2 and C2,and no significant difference in the group A1 and A2.Conclusions(1) The method of conditional knockout allele technique is an efficient and time-saving way to establish MEF model induced by K-ras;(2) Activation of K-ras can promote the ability of cancer cell growth,migration,clone formation,and inhibit the apoptosis;(3) Inactivation of GGTase-I or FTase by conditional knockout technique can reduce the K^LSL cell viability,proliferation and enhanced the apoptosis; (4) GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras; (5) The molecular mechanisms of the inactivation of GGTase-I or FTase to affect the MEF induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein,enhance the expression of apoptosis gene,promote the apoptosis,and suppressor cell division.