The Function And Regualtional Mechanism Of LASP-1 In Pancreatic Cancer

BackgroundLIM and SH3 1（LASP-1）is the first protein to connect LIM and SH3 domains,and there are two repeatedly nebulin,mainly focused on the focal adhesion of cells,the front foot and dense shaped pseudo filopodia,enable cell adhesion,migration and cellular communication and other important functions.The LASP-1 protein is overexpressed in many solid tumors.More importantly,LASP-1 is associated with the function of malignant tumors,such as proliferation,invasion and metastasis.And now,LASP-1 is recognized as a metastasis-related marker.Our data showed that LASP-1 was upregulated in tissues and cell lines of pancreatic ductal adenocarcinoma（PDAC）compared with normal tissues and cell lines.We designed the experiment to further clarify（1）the LASP-1 expression pattern in pancreatic cancer tissues and cells;（2）the function and regulational mechanism of LASP-1 at clinical,cellular and animal levels.Method1.Analysis the expression of LASP-1 in fresh and paraffin-fixed PDAC samples by using the immunohistochemistry staining,Western blot and RT-PCR experimental methods.2.Measurement the expression of LASP-1 in PDAC cell lines by using the Western blot and RT-PCR methods.Furthermore,assessment the function of LASP-1 on invasion and metastasis by using wound-healing assays,Transwell and immunofluorescence methods.3.The Hypoxia inducible factor-1α（HIF-1α）-overexpressing plasmid and HIF-1α-siRNA was constructed.Western blot and RT-PCR were used to verify the correlation between HIF-1αand LASP-1 expression by transfecting the plasmid and siRNA.4.We have found four hypoxia reactive elements（HREs:A/GCGTG）by using genetic information analysis technology.Next,we explore weather HIF-1αdirectly binds to the promoter of LASP-1 by Chromatin immunoprecipitation assay（ChIP）and dual-luciferase experiment.5.Xenograft mouse model was constructed.We inhibited the expression HIF-1αby digoxin and then measureed the proliferation of the tumors.Ananylsis the correlation between HIF-1αand LASP-1 in samples of mouse and human by immunohistochemistry staining and Western blot methods.6.The ASLP-1-overexpressing was constructed.Lentiviral stable transfection system was used to establish stably overexpressing LASP-1 cell line.Orthotopic pancreatic tumor model in nude nu/nu mice was used to validate the role of LASP-1in promoting pancreatic cancer cell metastasis in vivo.7.The expression pattern of LASP-1 was analyzed is SPSS software.The clinical data including the follow-up information was collected.The relationship between the expression levels of LASP-1 and clinicopathological parameters was analyzed.The results1.Through the Western blot and RT-PCR experimental methods,we veried that LASP-1 protein and mRNA were over-expressed in fresh samles of PDAC;By immunohistochemical staining in the 91 cases of pancreatic tissue we found that LASP-1 protein was upregulated,while the corresponding normal pancreatic tissues and benign or low-grade tumors do not express or weakly expressed LASP-1.2.Firstly,we measuered the baseline of LASP-1 in PDAC cell lines and found that CFPAC-1 and MIA PaCa-2 cell lines were relative upregulate expression of LASP-1,but BxPC-3 and PANC-1 cell lines were low expression.To investigate the role ofLASP-1 in the aggressive phenotypes of PDAC cellsin vitro,we used siRNA transfection to knock down LASP-1 expression in two PDAC cell lines with high endogenous LASP-1 levels（CFPAC-1 and MIA-PaCa-2）.Cell migration and invasion analysis using Transwell assay suggested that LASP-1 depletion in CFPAC-1 and MIA-PaCa-2 cells evidently reduced cell migration and invasion.In the wound-healing assays,the migratory activity of CFPAC-1 and MIA-PaCa-2 cells was also inhibited by LASP-1 silencing.Immunofluorescence analysis confirmed regional colocalization between LASP-1 and F-actin.Interestingly,compared with CFPAC-1/siNC and MIA-PaCa-2/siNC cells,the morphology of CFPAC-1/siLASP-1and MIA-PaCa-2/siLASP-1 cells lacked thin and long pseudopods.3.To determine whether HIF1 regulates transcription of LASP-1 inpancreatic cancer cells,we used specific siRNAs targeting HIF-1αto effectively reduce HIF1αexpression.Knockdown of HIF-1αexpression decreased LASP-1 mRNA and protein expression（p<0.05）.Moreover,when HIF-1αwas overexpressed in Panc-1 cells,LASP-1 mRNA and protein expression markedly increased.Four PDAC cell lines were cultured under normoxia（21%O₂）and hypoxia（1.5%O₂）for 12 hours and the LASP-1 expression levels were determined by Western blot analysis.The data showed that LASP-1 expression increased about 3.85-fold after hypoxic treatment when compared with normoxia cultured cells.These results suggest that HIF-1αplays a critical role in LASP-1expression.4.We surveyed the promoter region of human LASP-1 gene and identified four HREs.In chromatin fractions pulled down by an anti-HIF-1αantibody,only the HRE of LASP-1 promoter located at-1,005 to-1,001 was detected.The fragment immunoprecipitated by anti-HIF-1αantibody significantly increased under hypoxia,suggesting that hypoxia promoted the binding of HIF-1αto LASP-1 promoter.Luciferase analysis showed that HIF-1αoverexpression（pcDNA-HIF-1α）significantly increased LASP-1 promoter activity in Panc-1 cells.5.We injected Panc-1 cells subcutaneously into the rightflank of nude nu/nu mice.When the tumors reached 100 mm³,the mice were intraperitoneally injected with saline or digoxin（2 mg/kg）on a daily basis to inhibit HIF activity.Compared with the saline control group,the average tumor volume in the digoxin group was reduced obviously.Next,we evaluated the association between HIF-1αand LASP-1by Western blot analysis and immunohistochemistry and the results suggest that expression of LASP-1 was decreased as a result of the HIF-1αlevel reduction by digoxin.Importantly,HIF-1αexpression in PDAC specimens significantly correlated with the levels of LASP-16.To understand the role of LASP-1 in HIF-1α-mediated migration,we ectopically expressed LASP-1 in HIF-1αknockdown Panc-1 and CFPAC-1 cells.As shown in Figures,LASP-1 overexpression at least partially rescued the inhibitory effect of HIF1aknockdown on PDAC cell migration（p<0.05）,suggesting that LASP-1 was involved in HIF1amediated migration.The results of orthotopic pancreatic cancer mouse model indictaed that overexpression of LASP-1 developed a significantly larger primary pancreatic tumor and higher number of metastatic lesions in liver,gut,and mesentery and is critical for HIF-1α-mediated PDAC metastasis.7.LASP-1 signal measurement byimmunohistochemistry was detected in most（87.9%）of PDAC tissues.LASP-1 expression was correlated with the pathologic tumor node metastasis stage（p<0.05）and lymph node metastasis（p<0.01）of PDAC samples.Importantly,patients with PDAC with high or medium LASP-1 protein expression had significantly worse overall survival than those with negative or low LASP-1 expression（p=0.008;median time,8 and 16months）.Conclusions1.LASP-1 was over-expressed in clinical samples and cell lines of PDAC and associated with invasion and metastasis of PDAC.2.HIF-1αdirectly binds to the promoter of LASP-1 at-1,005 to-1,001 and significantly increased LASP-1 promoter activity.3.LASP-1 was correlated with the pathologic tumor node metastasis stage and lymph node metastasis of PDAC samples.Importmantly,LASP-1 influences the overall survival of patients with PDAC.