Expression, Inactivation And Function Of Tumor Suppressor DLC-1 Gene In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC),a kind of epithelial malignancy with high incidence in Southeast Asia and Southern China,shows a distinct ethnic aggregation and geographic distribution.Although lots of progresses have been achieved in the studies on nasopharyngeal carcinogenesis,early diagnosis and treatment of NPC,the exact molecular mechanism underlying nasopharyngeal carcinogenesis still remains unclear and no great breakthrough on early detection and therapy of NPC has been obtained.It has been considered widely that Epstein-Barr virus(EBV), chemical carcinogens and genetic susceptibility are three main factors involved in NPC pathogenesis nowadays.Recent studies have revealed numerous NPC-related genes,most of which are potential tumor suppressor genes(TSGs).However,these genes are just a small proportion of NPC-related genes and they cannot make the molecular carcinogenesis of NPC thorough understanding.DLC-1(deleted in liver cancer-1) was originally isolated from a primary hepatocellular carcinoma by representational difference analysis (RDA) as a candidate TSG.It is one of Rho GTPase-activating proteins (GAPs),negative regulators of the Rho family by stimulating the intrinsic GTPase activity.DLC-1 mRNA is widely expressed in adult human tissues,while it is down-regulated or absent in a number of common human cancers.LOH(loss of heterozygous) and promoter hypermethylation are associated with transcriptional silencing of DLC-1 in these tumors.Moreover,restoration of DLC-1 in DLC1-deficient cell lines from hepatocellular carcinoma,breast cancer,non-small cell lung carcinoma,prostate carcinoma and multiple myeloma has led to suppress tumor cell proliferation,migration and invasion and induce apoptosis in vitro.A recent study shows that DLC-1 inactivation cooperated with c-Myc over-expression can induce hepatocullular carcinoma in mouse confirmed that DLC-1 is a promising TSG.Although Peng et al and Seng et al have performed some studies on DLC-1 expression and its inactivation in NPC,some discrepancies have been found in methods and results between them.Moreover,the actual function of DLC-1 has not been reported in NPC till now.Therefore,we examined the expression and localization of DLC-1 in NPC,and further made most specimens of same batch to analyze the possible mechanisms leading to its aberrant expression in NPC.In addition,we further investigated the biological function of DLC-1 in NPC by gene transfection method.【The expression status of DLC-1 in NPC】RT-PCR and real-time RT-PCR were performed to analyze the expression of DLC-1 at the transcription level in primary cultural embryonic nasopharyngeal cells and a normal nasopharyngeal cell line,7 NPC cell lines,18 chronic nasopharyngitis biopsies and 32 NPC biopsies. The results showed that DLC-1 expression was stable in the normal nasopharyngeal cells and 88.9%(16/18) of the chronic nasopharyngitis tissues,while aberrant expression(loss or down-regulation) of DLC-1 was detected in 100%(7/7) of NPC cell lines and 68.75%(22/32) of NPC biopsies.Methods of in situ hybridization(ISH) and immuno-histochemistry (IHC) were also used to analyze the expression and location of DLC-1 at the mRNA and protein levels in various cell types of the nasopharynx. The results showed that varying degree of the mRNA and protein expression level was found in pseudo-stratified ciliated columnar epithelial,stratified squamous epithelial,glandular epithelial, inflammatory and tumor cells.The positive signal was mainly located in the cytoplasm.Additionally,74.3%(26/35) of the NPC specimens showed negative or very low expression level of DLC-1 protein.The negative expression ratio differed within different clinical stages(P<0.05). The ratio in TNMⅠ～Ⅱand TNMⅢ～Ⅳis 50%(6/12) and 87%(20/23) respectively.All the above results indicated that DLC-1 protein might be negatively associated with NPC progression.It provided experimental evidence for further studying the molecular mechanisms underlying the DLC-1 inactivation in NPC. 【Exploration of the possible molecular mechanisms causing DLC-1 aberrant expression in NPC】Genetic and epigenetic alterations are two aspects of molecular mechanisms associated with TSG inactivation.First,PCR-SSCP and subsequent sequencing were utilized to detect DLC-1 mutation spanning 5' UTR,open reading frame(ORF) and exon-intron junctions in 30 primary NPC biopsies as well as their matched control peripheral blood samples.One missense mutation(A→G transition,Glu→Gly) was found at codon +2398 of exon 8 in 3.3%(1/30) of primary NPC tissues. In addition,five single nucleotide polymorphism(SNP) sites were identified including one SNP in DLC-1 promoter,three exonic SNPs and one intronic SNP.The low mutation frequency of DLC-1 implied that mutation was unlikely to play an important role in the inactivation of DLC-1 in NPC.To evaluate the correlation between SNP -29A/T in the promoter region of DLC-1 and risk of NPC,a total of 521 samples from Chinese population including 320 healthy individuals and 201 NPC patients were collected for SNP analysis by PCR-SSCP and sequencing. The differences in allelic and genotypic frequency between NPC patients and controls were tested using logistic regression statistical method.As a result,no significant differences were found in allelic or genotypic frequency between NPC patients and controls or between NPC patients from Guangdong and Hunan or among different NPC clinical stages. Hence,our data indicated that the SNP -29A/T of DLC-1 was not associated with NPC susceptibility.Secondly,microsatellite markers located at gene locus or flanking the gene(upstream and downstream) usually were used to indirectly reflect deletion.Using PCR amplification,followed by denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining,four microsatellite markers,i.e.D8S1827,D8S552,D8S1754 and D8S1790 were used for LOH analysis in 30 primary NPC biopsies as well as their matched control peripheral blood samples.The results showed that total loss frequency of DLC-1 was detected in at least 23.3%(7/30) of NPC tissues.It indicated that LOH played a certain role in DLC-1 inactivation in NPC. Thirdly,aberrant promoter methylation is one of the most common reasons for epigenetic alteration of TSGs.A CpG island spanning from position -592 to +355 was found,when a promoter region of DLC-1 spanning from -2005bp to +355bp relative to the transcription start site was examined by CpG Island Searcher software.Besides,only CpG dinucleotides scattered in the region from -2005bp to -592bp,but no CpG island was revealed.With genomic DNA prepared from human normal peripheral blood samples as templates,four fragments spanning from position -1983 to -377,-396 to +16,-3 to +339 and -396 to +339 were amplified by PCR,respectively.Luciferase reporter assay showed that two fragments spanning from position -396 to +16 and -396 to +339, not the other two fragments(-1983 to -377 and -396 to +339),exhibited a strong promoter activity.Specific primers detecting the methylated or unmethylated alleles of DLC-1 promoter were designed to aim directly at the region spanning -396 to +16.The methylation status of DLC-1 promoter was determined by methylation-specific PCR(MSPCR) followed by bisulfite DNA sequencing in 18 chronic nasopharyngitis tissues,39 primary NPC tissues and 7 NPC cell lines.MSPCR analysis revealed DLC-1 promoter hypermethylation in 7 NPC cell lines,32 of 39(82.1%) primary NPC and 2 of 18(11.1%) chronic nasopharyngitis tissues.Statistical analysis indicated that there was a significant difference in methylation frequency of DLC-1 between chronic nasopharyngitis samples and primary NPC tissues(χ~2 test,P=0.00).Restoration of DLC-1 expression along with decrease in methylated allele of DLC-1 could be achieved in 5-8F and 6-10B cell lines after the treatment with 5-aza-2'-deoxycytidine(Aza),a DNA methyltransferase inhibitor.It suggested that promoter hypermethylation should be the key mechanism responsible for inactivation of DLC-1 in NPC.Comparative analyses of DLC-1 expression,mutation,LOH and methylation status in the same batch of NPC specimens showed that in 22 NPC biopsies with down-regulation or loss of DLC-1 expression, only 4.5%(1/22) of NPC biopsies showed both mutation and methylation,only 4.5%(1/22) of NPC biopsies showed LOH,22.7% (5/22) of NPC biopsies showed both LOH and methylation alterations, while 68.2%(15/22) of NPC biopsies showed only promoter methylation,further strongly indicated that promoter hypermethylation played a key role while LOH played a certain role in DLC-1 inactivation in NPC.To investigate the role of mammalian DNA methyltransferases (DNMTs) in regulating the DLC-1 promoter activity,three sets of siRNAs,specifically interfering DNMT1,DNMT3a and DNMT3b,were transiently transfected into 5-8F cell line,then DLC-1 expression was examined.The results showed differential effects on DNA demethylation and gene reactivation in the treated cells.The DNMT1 siRNA treatment led to a partial removal of DNA methylation from DLC-1 inactive promoter CpG islands and restored the expression of DLC-1.The epigenetic alterations appeared less effective in cells transfected with DNMT3a or DNMT3b siRNA.Thus,our data suggested that DNMT1 might play a relatively important role in methylation maintenance of DLC-1 in NPC.Moreover,we found that DLC-1 promoter activity of the region spanning from position -396 to +16 and -396 to +339 was repressed,when patch methylation of this region in vitro followed by luciferase reporter assay was performed in 5-8F and 6-10B cells.These results further indicated that hypermethylation of DLC-1 promoter region spanning from position -396 to +16 was mediated mostly by DNMT1 and this might contribute to down-regulation or absent expression of DLC-1 in NPC.【Investigation of DLC-1 functions in NPC cells】In order to investigate the functions of DLC-1 in NPC,the entire ORF sequence(3.3kb) of DLC-1 was amplified by RT-PCR,using normal embryonic spleen RNA as a template.Sequencing results showed that the amplified cDNA sequence contained a missense SNP (+1380A→G,V354M),but this had no influence on DLC-1 function according to bioinformatic analysis.Subsequently,the DLC-10RF was cloned into pcDNA3.1(+) vector and the eukaryotic expression vector of DLC-1(named pcDLC1) was constructed.Then,pcDLC1 vector was transferred into 5-8F cells by Lipofectamine 2000.After G418 selection, several G418-resistant cell clones were obtained.It was demonstrated that both mRNA and protein of DLC-1 expressed stably in the G418-resistant cell clones assessed by RT-PCR and Western Blotting, respectively,showing that the 5-8F cells stably expressing DLC-1 (named as 5-SF-DLC1 cells) was successfully established. Immunocytochemical(ICC) experiments revealed that DLC-1 protein was localized in the cytoplasm of 5-8F-DLC1 cells.Then,we analyzed the changes in biological characteristics 5-8F-DLC1 cells.Proliferation and clonogenic ability of 5-SF-DLC1 were measured by MTT analysis and colony formation assay, respectively.Compared with empty vector-transfected 5-8F cells(named as 5-8F-vector cells) or untransfected 5-8F cells,5-8F-DLC1 cells showed significant growth inhibition(P<0.05) and reduction in the cloning efficiency(23.1%vs 52.5%/49.4%).Flow cytometry(FCM) analysis showed that 5-8F-DLC1 cells could be arrested in G0/G1 phase, as the G0/G1 phase cells increased(67.25%vs 45.39%/46.53%) while S phase(21.24%vs 31.26%/31.55%) and G2/M phase cells reduced (11.51%vs 23.35%/21.92%).In vivo tumorigenicity experiment confirmed that the size of the tumor formed by 5-8F-DLC-1 cells in nude mice was much smaller than that formed by 5-8F-vector cells(P <0.05).These results indicated that DLC-1 stable expression blocked 5-8F cells at G0/G1 phase resulting in attenuated proliferation and colony forming ability in vitro and lower tumorigenicity potential in vivo.In addition,in vitro wound healing assay,migration assay and invasion assay were carried out to demonstrate that restoration of DLC-1 expression could also significantly decrease the mobility and invasion ability of 5-8F cells.To examine the effect of DLC-1 expression on cytoskeletal organization in 5-8F cells,the 5-8F-DLC1 cells were stained with phalloidin and observed under a confocal laser-scanning microscope.As a result,5-8F-DLC1 showed that remarkably fewer muscle actin filaments which distributed mainly in the perimeter region displaying an obvious structural polarity in contrast to 5-8F-vector cells or untransfected 5-8F cells.This suggested that DLC-1 gene could inhibit migration and invasion of NPC cells by influencing the cytoskeleton formation.In summary,based on previous work of our laboratory,more systematic and comprehensive studies on expression and distribution of the tumor suppressor DLC-1 in normal nasopharyngeal cells,NPC cell lines,chronic nasopharyngitis tissues and N-PC biopsies have been done. Meanwhile,potential mechanisms of expression causing DLC-1 inactivation in NPC and the functions of DLC-1 in NPC cells have been investigated.Our results suggest that DLC-1 expresses normally in chronic nasopharyngitis tissues and normal nasopharyngeal epithelia, but is significantly down-regulated or absent in NPC cell lines and tissues.Promoter hypermethylation plays a key role in inactivating DLC-1 in NPC,while LOH also plays a certain role in aberrant expression of DLC-1.Re-expression of DLC-1 in NPC cells can confer partial reversion of the malignant phenotype of NPC cells,for example, it can inhibit the ability of cell proliferation,migration and invasion of NPC cells.These results will help us comprehensively understand the role of DLC-1 in molecular carcinogenesis of NPC,elucidate the molecular mechanisms of NPC carcinogenesis,and provide the theoretical and experimental evidence for its potential clinical applications.