The Molecular Mechanism Of LASP1 Regulating Colorectal Cancer Invasion And Metastasis By Targeting 14-3-3? In Synergism With COPS5

Background and Objection:Colorectal cancer is one of the most common digestive malignant tumors in the modern time,the morbidity and cancer-related mortality rang the upper third of all tumors.In recent years,the incidence of CRC has increased rapidly in our country,and age at onset is becoming younger and younger.About 90%Grade I patients can be cured by operation,while approximately 40%?50%of colorectal cancer patients have been found tumor micro metastasis when they are diagnosed clinically.Local transfer of colorectal cancer occurs much earlier than distant metastasis of colorectal cancer,approximately 60?80%of the patients could be found tumor metastasis locally,metastasis is one of the most important cause of mortality in CRC patients.Colorectal cancer has severely impact patients' quality of lives and physical health,and result in heavy financial burdens to patients and society.Although lots of research and investigation on colorectal cancer have been carried out at present,but the molecular basis and mechanisms of development and metastasis of colorectal cancer is not fully understood.Carcinogenesis of CRC is a complex process with variety of tumorigenic factors involved.The intestinal epithelial cells loss the normal regulation of growth,result in cell cycle disorder,followed by immortalized growth,and eventually tumorigenesis.Metastasis of colorectal cancer is a multistep,multistage,multiple gene mutations,accompanied by oncogene activation,tumor suppressor genes inactivation,apoptosis-regulating genes and DNA repair gene.Cancer cells in the primary tumor site escape into the surrounding matrix,subsequently the circulation system and lymphatic system.After adhering endothelia cell wall and migrate extravascular,the cells invade in distant,eventually develop new metastases.There are more than hundreds of genes have been reported to take part in metastasis of colorectal cancer.For the sake of early warning,diagnosis,intervention and outcoming judgment of colorectal cancer,it is very significant to in-depth understand the molecular mechanisms of colorectal cancer,and to seek effective,sensitive and specific molecular markers of colorectal cancer in large amounts of pathogenic factors.LASP1(LIM and SH3 proteinl)was previously identified in a cDNA library of breast cancer metastasis.Chromosomal mapping showed that the LASP1 gene is localized on the q12-q21 region of the long arm of chromosome 7.LASP1 cDNA encodes a putative protein of 261 residues,named LASP1(LIM and SH3 protein)since it contains a LIM motif and a domain of Src homology region 3(SH3)at the amino-and the C-terminal parts of the protein,respectively.Thus,LASP1 defines a new LIM protein subfamily.LASP1 is an important cytoskeleton protein,as an actin-binding protein and zyxin scaffold protein,LASP1 accumulates in many key locations of actin assembly like focal adhesion,lamellipodium,membrane fold,pseudopodium.To date,more and more investigations have proved that LASPI is significantly overexpressed in numerous different cancer entities and affects cancer aggressiveness.In previous studies,we found that LASP1 significant overexpression contributes to colorectal cancer especially aggressive type.In previous studies,we illustrated that miR-1 and miR-133a could inhibit LASP1 expression by directly binding with its 3'UTR region in CRC cells.Epigenetic silencing of miR-1 and miR-133a by promote hypermethylation resulted in over-expression of LASP1 in CRC tissues.An over-expression of LASP1 was required for TGF?-mediated epithelial-mesenchymal transition(EMT)and aggressive phenotypes of cancer cells,thereby promoting cancer progression.Clinically,the expression of this protein was closely correlated with lymph node status,thereby improving the overall survival rates of patients with CRC.These results indicated that LASP1 might be a promising molecule that could be used in developing treatments for patients with advanced CRC.For the sake of elucidating the molecular mechanism of LASP1 in CRC metastasis,we use two-dimensional difference gel electrophoresis and yeast two hybrid system to screening proteins that interact with LASP1.Of all the candidate proteins,we select 14-3-3? and COPS5 for further research because of their close relation with tumor on the basis of proven evidence.In this study,we identified 14-3-3? and COPS5 as LASP1 interact proteins using proteomic strategy.Furthermore,we investigate the expression of 14-3-3? and COPS5 in CRC metastasis,and analyzed the relation between the up-regulate expression and clinicopathological parameter.Vivo and vitro experiments were taken for insight into the function and underlying mechanism of both proteins in colorectal cancer.We want to deepen our understanding of CRC metastasis and provide the experimental basis for targeted treatment of patients with advanced CRC.MethodsPart one.The study of 14-3-3?,one of the LASP1 interacting proteins,which was selected by the two-dimensional difference gel electrophoresis1 Identification of 14-3-3? as a LASP1 interaction partner1.1 The detection of the expression of 14-3-3? and the mutual manipulation between LASP1 and 14-3-3? in colorectal cancer cell lines.1.1.1 Using western blot to detect endogenous expression of 14-3-3? in all seven colorectal cancer cell lines.1.1.2 Using western blot to detect the mutual manipulation between LASP1 and 14-3-3? in colorectal cancer cell lines.1.1.3 Transiently transferring the LASP1 overexpression vector and LASP1 siRNA fragments into the colorectal cancer cell lines sw480 and HCT116 cell,extracting the RNA and protein of these LASP1 transient overexpression and LASP1 transient silencing cells,followed by using QPCR and western blotting to detect the expression changes of LASP1 and 14-3-3 ?.1.2 Using CO-IP to detect the LASP1 physical interaction with 14-3-3?.1.3 Using IF(immunofluorescence)to testify the co-location of LASP1 and 14-3-3? inside colorectal cancer cell lines.2 The biological function of 14-3-3? in colorectal cancer2.1 14-3-3? suppresses cell migration via inhibiting phosphorylation of AKT2.1.1 The 14-3-3? overexpression vector was constructed with pCDNA3 as a negative control vector followed by being transiently transferred into colorectal cancer cell lines SW480 and HCT116.The siRNA-14-3-3? by synthesis was transiently transferred into colorectal cancer cell lines RKO and SW620,and treatment of inhibitor LY294002 at the same time.Using western blotting to detect the phosphorylation of AKT in CRC cells,using transwell assay to detect the ability of cell migration.2.1.2 Using CO-IP to detect the AKT physical interaction with 14-3-3?.Using IF to testify the co-location of AKT and 14-3-3? inside colorectal cancer cell lines.2.2 Loss of 14-3-3? is essential for LASP1-mediated cell aggressiveness2.2.1 Using transwell assay to test the migratory captivity of CRC cells in which LASP1 was overexpressed and knockdown or LASP1 and 14-3-3? were co-overexpress or co-knockdown.2.2.2 Using western blot to detect the phosphorylation of AKT in CRC cells in which LASP1 was overexpressed and knockdown or LASP1 and 14-3-3? were co-overexpress or co-knockdown.3 The expression of 14-3-3? in colorectal cancer and the Clinical significance analysis.3.1 Western blot technique was used to detect LASP1 and 14-3-3? expression in 24 cases of fresh CRC tissues.3.2 To evaluate expression and subcellular localization of 14-3-3? protein,we performed immunohistochemical assay in 52 and 116 paraffin-embedded,archival normal colorectal mucosa and CRC tissues,respectively.To evaluate the clinical relevance of 14-3-3? expression,we analyzed its relationship with pathological features.Part 2 The study of COPS5,one of the LASP1 interacting proteins,which was selected by the yeast two-hybrid system1 Identification of COPS5 as a LASP1 interaction partner;1.1 Using 2-D difference gel electrophoresis(2-D DIGE)-based proteomic strategy,one of the candidates LASP1-modulated proteins was identified as 14-3-3?.1.2 The protein interaction was also validated by co-Immunoprecipitation(Co-IP)assay in protein extraction of SW480 and HCT116 cells.1.3 we observed clearly co-localization between LASP1 and COPS5 in the cytoplasm of colorectal cancer SW480?HCT116?SW620?RKO?LS174T?H29?LOVO?SW480/M5 cell lines by CO-IP.1.4 COPS5 truncates COPS5(1?195a),COPS5(44?195a),COPS5(190?334a)and LASP1 truncates LASP1(1?61a),LASP1(1?134a)?LASP1(60?198a)?LASP1(132?198a)?LASP1(132?261a)were successfully constructed into GST fused prokaryotic expression vector.GST-pull down assay result show that the COPS5(1?195a)?COPS5(44?195a)binds with LASP1(132?261a),which indicate the SH3 domain of LASP1 binds directly with the MPN domain of COPS5.2 The biological function of COPS5 in colorectal cancer2.1 We detected the endogenous expression of COPS5 protein in all eight CRC cell lines,the expression difference was statistically significant(p<0.01).COPS5 expression lever is much higher in RKO?SW620?H29?LS174T?M5 when compared with HCT116?LOVO?SW480.2.2 After transiently transferring 2 COPS5 siRNA fragments which were purchased as commercial goods into colorectal cancer cell lines RKO and SW620 and transiently transferring COPS5 overexpression vector pCDNA3-COPS5 into SW480 and HCT116,western blot was performed to detect the COPS5 expression changes among groups.The result indicate COPS5 expression was overexpress or knockdown with statistically significant.2.3 The COPS5 gene was packaged into virus,the virus supernatant was used to infect colorectal cancer cell line HCT116.Western blotting was carried out to detect the COPS5 exogenous expression.2.4 Using CCK8(cell counting kit 8),transwell assay,cell colonies formation assays,cell cycle detection assays to detect the influence overexpression and silencing of COPS5 have on colorectal cancer cells biology behaviors.2.5 Observing the influence of COPS5 overexpression has on in vain tumor migration ability in SPF class BALB/c nude mice models.3 The mechanism analysis of COPS5-LASP1 interaction regulation on colorectal cancer invasion and metastasis through 14-3-3?3.1 Using CO-IP to detect the physical interaction of LASP1,COPS5,14-3-3? in colorectal cells RKO and SW480.3.2Western blot indicate that LASP1-COPS5 interaction synergistic effects on down-regulation of 14-3-3? expression and phosphorylation of AKT.3.3 Western blot technique was used to detect the MAPK?PI3K/Akt signaling pathway proteins after COPS5 transient overexpression and transient silencing.Results:Part one.The study of 14-3-3?,one of the LASP1 interacting proteins,which was selected by the two-dimensional difference gel electrophoresis1 LASP1 negatively regulates 14-3-3? expression by protein interaction.1.1 Using 2-D difference gel electrophoresis(2-D DIGE)-based proteomic strategy,one of the candidates LASP1-modulated proteins was identified as 14-3-3?,which was negatively correlated with LASP1 expression.Western blot analysis showed that decreased expression of 14-3-3? was observed in LASP1-overexpressing SW480 and HCT116 cells,meanwhile increased expression of 14-3-3a was detected in LASP1-silenced SW620 and HCT116 cells,which are consistent with proteomic results.Using western blot to detect the mutual manipulation between LASP1 and 14-3-3? in colorectal cancer cell lines.1.2 The LASP1 physical interaction with 14-3-3? was validate by CO-IP.1.3 The co-location of LASP1 and 14-3-3? inside colorectal cancer cell lines was testified by IF.1.4 Transiently transferring the LASP1 overexpression vector and LASP1 siRNA fragments into the colorectal cancer cell lines sw480 and HCT116 cell,extracting the RNA and protein of these LASP1 transient overexpression and LASP1 transient silencing cells,followed by using QPCR and western blotting to detect the expression changes of LASP1 and 14-3-3?,the results showed that LASP1 did not affect the expression of 14-3-3? mRNA.2 The biological function of 14-3-3? in colorectal cancer2.1 14-3-3? suppresses cell migration via inhibiting phosphorylation of AKT2.1.1 We detected the endogenous expression of 14-3-3? protein in all seven CRC cell lines.Interestingly,a significantly decreased expression of 14-3-3a was found in SW620 cells derived from lymph node metastasis,compared with SW480 cells derived from the primary lesion in the same patient.2.1.2 In vitro loss-and gain-of-function assays were carried out to investigate the effect of 14-3-3? on CRC cellular biological behaviors.Transwell assay showed that exogenous introduction of 14-3-3? resulted in decreased ability of cell migration in SW620 cells with a relatively low 14-3-3? expression level.siRNA-mediated depletion of endogenous 14-3-3? expression enhanced migratory ability in SW480 and HCT116 cells.2.1.3 To uncover the mechanism underlying tumor suppressor induced by 14-3-3?,we performed western blot analysis to detect the phosphorylation level of AKT.The results showed that 14-3-3? suppressed the phosphorylation of AKT in CRC cells.Meanwhile,treatment of PI3K inhibitor LY294002 markedly prevented phosphorylation of AKT and subsequently counteract aggressive phenotype mediated by siRNA of 14-3-3?.2.1.4 we observed clearly co-localization between AKT and 14-3-3? in SW480 and HCT116 cells.The protein interaction was also validated by co-IP assay in protein extraction of SW480 and HCT116 cells.2.2 Loss of 14-3-3? is essential for LASP1-mediated cell aggressiveness2.2.1 To address the pivotal role of 14-3-3? in LASP1-mediated cell aggressive phenotype,we performed the rescued experiment to detect activation of PI3K/AKT signaling pathway and aggressive capacity of CRC cells.To consistent with our previous study,LASP1 could promote migratory captivity of CRC cells via activating PI3K/AKT signaling pathway.Restoring expression of 14-3-3? weakened cell migration induced by LASP1 in CRC cells,whereas depletion of 14-3-3? recovered aggressive capacity of CRC cells suppressed by siRNA of LASP1.2.2.2 Western blot analysis confirmed that loss of 14-3-3? was required for PI3K/AKT signaling pathway activated by LASP1.3 The expression of 14-3-3? in colorectal cancer and the Clinical significance analysis.3.1 14-3-3? is frequently down-regulated in CRC tissues.3.1.1 To evaluate expression and subcellular localization of 14-3-3? protein,we performed immunohistochemical assay in 52 and 116 paraffin-embedded,archival normal colorectal mucosa and CRC tissues,respectively.Positive signals of 14-3-3?were predominantly detected in the cytoplasm of benign and malignant epithelial cells,decreased expression of 14-3-3? was frequently found in CRC tissues compared with adjacent non-tumorous tissues tested.Interestingly,deletion of 14-3-3?expression was also observed in carcinogenetic lesions surrounding normal mucosa,especially in malignant cells derived from benign gland.3.1.2 Immunohistochemistry clearly allowed to localize 14-3-3? expression in 92.3%(48 of 52)of adjacent non-tumorous tissues tested.As compared to these non-tumorous tissues,we observed 14-3-3? protein expression in 87.1%(101 of 116)of all CRC samples(P=0.433).According to reclassification as described above,14-3-3? was evaluated as high expression in 50.0%(58 of 116)of tumor samples,compared with 71.2%(37 of 52)of adjacent non-tumorous samples(P=0.011).3.2 Down-regulation of 14-3-3? is associated with tumor progression and poor prognosis of patients with CRC.To evaluate the clinical relevance of 14-3-3? expression,we analyzed its relationship with pathological features.Expression of 14-3-3? was negatively associated with N classification(lymph node metastasis)of patients with CRC(P=0.001).Kaplan-Meier survival curves revealed a significant trend towards poorer survival for patients whose primary tumors showed low 14-3-3? expression,compared with those patients whose primary tumors showed high 14-3-3? expression(Log Rank=16.351,P=0.000).Furthermore,multivariate analysis confirmed low expression of 14-3-3?,including gender,N classification and M classification,as independent prognostic factors for CRC.3.3 14-3-3? expression is inversely correlated with LASP1 expression in CRC tissues.3.3.1 Western blot technique was used to detect LASP1 and 14-3-3? expression in 24 cases of fresh CRC tissues.The results suggested that 14-3-3? expression was mainly lower in CRC tissues than in paired non-cancerous colorectal tissues(P=0.0031)and had a negative correlation with LASP1 expression(R=-0.505,P=0.012).3.3.2 Immunohistochemical results from a larger population-based cohort study showed that relatively low expression of 14-3-3? was frequently observed in CRC samples with LASP1 overexpression.There was also a negative correlation between 14-3-3? and LASP1 expression(R=-6.458,P<0.001).3.3.3 Based on the above data,we then tested a combination of 14-3-3? and LASP1 and studied its predictive value for overall survival.Intriguingly,patients with high LASP1 and low 14-3-3? had significantly worse outcome,and patients with low LASP1 and high 14-3-3? had better outcome,indicating the opposing effects of LASP1 and 14-3-3? on CRC patient survival.Part TWO.The study of COPS5,one of the LASP1 interacting proteins,which was screened by the yeast two-hybrid system.1 Identification of COPS5 as a LASP1 interaction partner1.1 Using CLONTECH MATCHMAKER Two-Hybrid system to screening COPS5 as one of LASP1 interaction partner.1.2 Using CO-IP to detect the LASP1 physical interaction with COPS5.Using IF to testify the co-location of LASP1 and COPS5 inside colorectal cancer cell lines and frozen sections.1.3 GST-COPS5 and GST-LASP1 truncates prokaryotic expression vector were constructed and GST-pull down assay were performed to detect the interaction region between the genes.2 The biological function of COPS5 in colorectal cancer2.1 We detected the endogenous expression of COPS5 protein in all eight CRC cell lines.2.2 After transiently transferring siRNA-COPS5-1,siRNA-COPS5-2 fragments into colorectal cancer cell lines RKO and SW620 and transiently transferring COPS5 overexpression vector pCDNA3-COPS5 into SW480 and HCT116,western blot was performed to detect the COPS5 expression changes among groups.The result indicate the COPS5 overexpression in SW480/COPS5 and HCT116/COPS5,the siRNA-COPS5-land siRNA-COPS5-2 can transient silence COPS5 expression in RKO and SW620 efficiently about 50?60%.2.3 The COPS5 gene was packaged into virus,the virus supernatant was used to infect colorectal cancer cell line HCT116.The COPS5 exogenous expression was detected by Western blot.We can observe the green fluorescence expressed in infected cells.2.4 Using CCK8(cell counting kit 8)assay to detect the change of proliferation ability in vitro in SW620/NC and SW620/siCOPS5,RKO/NC and RKO/siCOPS5,HCT116/V-CON and HCT116/V-COPS5,SW480/V-CON and SW480/V-COPS5,followed by drawing the growth curve.Factorial analysis results showed that there was significant difference on the level of growth time between COPS5 overexpression and negative control cell lines(p<0.001),however the ability of cell proliferation had significantly differences between cell groups(p<0.001);time and group interaction effect had significant difference(p<0.001).There was significant difference on the level of growth time in COPS5 transient silencing cell lines(p<0.001),the ability of cell proliferation had significantly differences between cell groups(p<0.001),and time and group interaction effect had no significant difference(p<0.001).The results indicate that the proliferation ability in COPS5 transient silencing groups were down-regulated compared to the NC control group.2.5 Cell clone formation experiments show that the cell proliferation speed in RKO/siCOPS5 group is slowdown compared to RKO/NC group.Independent-sample T test statistics showed that cell clone formation ability existed significant differences between the two groups in RKO/siCOPS5 and RKO/NC cells.2.6The cell cycle assay by Flow Cytometer was using in the RKO/NC and RKO/siCOPS5.The cell number in G2(second gap)of RKO/siCOPS5 is much less than that of RKO/NC.The result suggest that COPS5 can regulate the colorectal cancer cell cycle.2.7 Transwell assay show that much more cells transported out of the chamber in SW480/COPS5 and HCT116/COPS5 cell groups compared to that of vector cell group(p<0.05).Furthmore,there were much less cells transported out of the chamber membrane in RKO/siCOPS5 and SW620/siCOPS5 compared to that of NC cells(p<0.05).The results indicate COPS5 promote the invasion ability of colorectal cancer cells.2.8 Observing the influence of COPS5 overexpression has on in vain tumor migration ability in SPF class BALB/c nude mice models.The influence that COPS5 has on colorectal cancer in vivo biologic behaviors.we construct the stably COPS5 overexpressing HCT116 cell lines by the virus infection as well as the control cell lines.Nude mice tail intravenous injection transfer experiment in vivo results shows that there were visionable transferred colorectal cancer mass in mice lungs,and the lung metastasis rates in the two groups are 6/6,however the number of lung metastasis tumors in HCT116/V-COPS5 group is increased significantly(p<0.05).The result suggests that COPS5 promotes the tumor metastasis in nude mices.3 The mechanism analysis of COPS5-LASP1 interaction regulation on colorectal cancer invasion and metastasis through 14-3-3?.3.1 The physical interactions of LASP1,COPS5 and 14-3-3? in colorectal cells RKO and SW480 were testify by CO-IP.3.2Western blot technique was used to detect the influence of LASP1-COPS5 interaction on 14-3-3? expression and phosphorylation of AKT.3.3 Western blot technique was used to detect the MAPK?PI3K/Akt signaling pathway proteins after COPS5 transient overexpression and transient silencing.Conclusion1 The study of 14-3-3?,one of the LASP1 interacting proteins,which was selected by the two-dimensional difference gel electrophoresis1)LASP1 negatively regulates 14-3-3? expression by protein interaction;2)14-3-3? suppresses cell migration via inhibiting phosphorylation of AKT;3)Loss of 14-3-3? is essential for LASP1-mediated cell aggressiveness;4)14-3-3? is frequently down-regulated in CRC tissues;5)Down-regulation of 14-3-3? is associated with tumor progression and poor prognosis of patients with CRC;6)14-3-3? expression is inversely correlated with LASP1 expression in CRC tissues.7)The study of COPS5,one of the LASP1 interacting proteins,which was screened by the yeast two-hybrid system.8)COPS5 is one of the LASP1 interaction partners which co-localized with LASP1 in cytoplasm of colorectal cancer cells.The SH3 domain of LASP1 binds directly with the MPN domain of COPS5.9)COPS5 was up-regulated in colorectal cancer tissues and promote the proliferation,invasion and migration ability of colorectal cancer cells.10)COPS5 participates in the LASP1 negatively regulated 14-3-3? expression by PI3K/Akt signaling pathway and promote the colorectal cancer invasion and metastasis.