Epigenetic Silencing Of DUSP9 Induces The Proliferation Of Human Gastric Cancer By Activating JNK Signaling

BackgroundA total of 989,600 new gastric cancer (GC) cases and 738,000 mortalities are estimated to have occurred in 2008, accounting for 8% of the total cases and 10% of total deaths worldwide. Over 70% of new cases and deaths occur in developing countries and GC is a leading cause of cancer-associated mortality in China. Dysregulation of normal signaling pathways is a critical process in the pathogenesis of GC. Mitogen-activated protein kinase (MAPK) signaling activities play important roles in many of the processes involved in the initiation and genesis of cancer, and MAPK signaling pathway abnormalities have been shown to be involved in various human malignancies, including GC.MAPK pathways constitute a highly conserved family of kinase modules that it serve to relay information from extracellular signals to the effectors which control various cell processes such as proliferation, differentiation, migration and apoptosis. MAPKs are activated by phosphorylation on the threonine and tyrosine residues of a conserved signature T-X-Y motif within the activation loop of the kinase. There are MAPK phosphatases (MKPs) that act as negative regulators of MAPK activity in mammalian. The MKPs constitute a distinct subgroup of 10 catalytically active enzymes within the larger family of cysteine-dependent dual-specificity protein phosphatases (DUSPs). The dual-specificity phosphatase 9 (DUSP9) gene, also known as mitogen-activated protein kinase phosphatase 4 (MKP4), was first described in 1997 by Muda et al. This gene is a member of the large family of protein tyrosine phosphatases (PTPs). It consists of 4 exons and has a size of 8,884 bp and is localized on chromosome Xq28, which codes for a functional protein of 41.9 kDa.DNA methylation of promoter-associated CpG islands may function as an alternate mechanism of silencing tumor suppressor genes in numerous neoplasias, including GC. De novo methylation of genes appears to be an early and frequent event in most neoplasias. Hypermethylation occurs at different stages in the development of cancer and in different cell networks. In the genesis of many types of cancer, hypermethylation of the CpG islands in the promoter regions of tumor suppressor genes is a major event, and is able to affect genes involved in the cell cycle, DNA repair, and the metabolism of carcinogens, cell-to-cell interaction, apoptosis, and angiogenesis.DUSP9 has been shown to be downregulated in colorectal cancer, renal and hepatocellular carcinomas, and squamous cell carcinoma. In a mouse model, the tumor-suppressor capacity of DUSP9 was confirmed on squamous cell carcinoma and NSCLC tissues. However, to the best of our knowledge, no studies have been performed regarding DUSP9 expression in human GC tissues. Additionally, no studies have been performed with the regard to the mechanism of DUSP9 inactivation in GC. The present study was performed using 30 matched GC and normal gastric mucosa tissues. The analysis was conducted using bisulfite sequencing PCR (BSP) after bisulfite treatment of DNA. The lentiviral transfection of these cells with an inducible DUSP9 transfect led to a re-expression of DUSP9 was resulting in a repression of proliferation in GC cell lines.Materials and Methods1. Cell culture. GC cell lines MKN-1, TMK-1, MKN45, BGC823, NCI-N87, NUGC3, AGS and MKN-7 were maintained in RPMI-1640 supplemented with 10% FCS at 37℃ in a 5% CO2 incubator.2. RNA isolation, reverse transcription, and RT-qPCR. Total RNA was extracted from frozen tumor samples using TRIzol reagent. cDNA was synthesized from 500 ng total RNA using the PrimeScript RT Master Mix. Each sample was analyzed in triplicate. The sequences of the primers used for PCR were designed by Primer Bank (http://pga.mgh.harvard.edu/primerbank/). The relative expressions of genes were calculated by the 2-△△Ct method. Data are presented as the relative quantity of target mRNA normalized to expression of GAPDH mRNA and relative to a calibrator sample. Each assay was performed three times.3. Bisulfite sequencing. Clinical samples were homogenized and digested overnight with proteinase K. Clinical sample DNA was modified by the bisulfite reaction using an EpiTect Bisulfite kit (Qiagen, Germany). PCR products were examined after gel electrophoresis in 1.5% agarose in order to confirm that a single band was obtained. Forward and reverse primer sequences of DUSP9 for bisulfite sequencing were designed by MethPrimer, and together they amplified a 138 bp product containing 11 CpG sites in the promoter. Sequence homologies were identified using the BLAST program of the National Center for Biotechnology Information available at http://www.ncbi.nlm.nih.gov/BLAST/.4. Western blot analysis. Cell lysate was prepared using RIPA buffer with protease inhibitors and quantified using the BCA protein assay. Protein was loaded onto a 10% SDS-PAGE gel that was then transferred onto PVDF membrane and incubated with rabbit anti-DUSP9, rabbit anti-p21, rabbit anti-CDK4, rabbit anti-CDK6, rabbit anti-CCNDl, rabbit anti-c-Jun at 4℃ overnight in blocker followed by incubation with HRP-conjugated secondary anti mouse. The bands were visualized by eECL Western Blot Kit. The images were captured with ChemiDocTM CRS+Molecular Imager.5. Tumor growth assay. Animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of CUHK. Following fluorescence-activated cell sorting, the green fluorescence protein positive cells were isolated and a total of 5x105 infected cells were injected subcutaneously into the dorsal flank of nude mice. Each group contained 7 mice and the experiment was repeated 3 times.6. Plasmid construction and transfection. Eukaryotic expression vectors of the wild type (pEGFP-DUSP9) was constructed by Guangzhou Sagene. MKN-1 cells were transfected with empty vector (pEGFP-Cl), the wild-type vector (pEGFP-DUSP9) by Lipofectamine 2000. Culture medium containing G418 was used to select stable transfectants.7. Establishment of lentivirus-delivered LV-sh-DUSP9 and LV-DUSP9 in GC cells. The preparation of lentivirus expressing human DUSP9 short hairpin RNA was performed using the pLVTHM-GFP lentiviral RNAi expression system. The lentiviral particles were used to infect GC cell lines MKN-1. Lentivirus particles carrying DUSP9 and its control were purchased from GeneChem, Shanghai, China. The lentiviral transduction of MKN-1 cells was carried out according to the manufactures’ protocol. The resulting cells were seeded onto 96-well plates and cultured for 3 weeks to produce a stable DUSP9-overexpressing MKN-1 cells and MKN-1 control cells. The high expression of DUSP9 was validated by quantitative RT-qPCR and Western blot.8. Transient transfection with siRNAs for DUSP9. siRNAs were transfected at a working concentration of 100 nmol/L using Lipofectamine 2000 reagent. The siRNA for DUSP9, a nonspecific control were all purchased from Guangzhou RiboBio. The sequence of each gene and their controls are shown in Table 3. Twenty-four hours before transfection, MKN-1 cells were plated onto a 96-well plate or a 6-well plate at a 30-50% confluence. They were then transfected into cells using TurboFect siRNA Transfection Reagent according to the manufacturer’s protocol. Cells were collected after 48-72 h for the further experiments.9. Colony formation assay. After 72 hours of infection, the cells were plated in 6-well plates at 200 per well and grown for 2 weeks. After 2 weeks, the cells were washed twice with PBS, fixed with methanol/acetic acid (3:1, v/v), and stained with 0.5% crystal violet. The number of colonies was counted under the microscope.10. Cell proliferation and cell cycle analyses. Cell proliferation was analyzed using MTT assay. Briefly, MKN-1 cells (5x103) were plated onto 96-well plates respectively in 100 μL of growth medium and allowed to adhere overnight. The cells were then transfected with DUSP or Si-DUSP9 and control respectively. At different time points (24 h,48 h,72 h and 96 h), the culture medium was removed and replaced with culture medium containing 10 μL of sterile MTT dye. After incubation at 37℃ for 4 hours, the MTT solution was removed, and 150μL of DMSO was added to dissolve the formazan crystals. Spectrometric absorbance at 490 nm was measured by BioTek ELx800 microplate photometer. For cell cycle analysis, LV-DUSP9-infected MKN-1 cells, LV-sh-DUSP9-infected MKN-1 cells and negative control cells were fixed in 70% ice-cold ethanol for 48 hours at 4℃, stained by incubation with PBS containing 10 μg/mL propidium iodide and 0.5 mg/mL RNase A for 15 min at 37℃, and analyzed for the DNA content of labeled cells by Cytometry. Each experiment was done in triplicate.Results1. DUSP9 expression is reduced in GC cell lines.A panel of human GC cell lines was first analyzed to quantify the expression level of DUSP9. The results showed that the expression level of DUSP9 was decreased in the seven GC cell lines examined, compared with the normal gastric mucosa tissues. DUSP9 transcript levels were significantly decreased in all the stages of GC compared with the normal gastric mucosa tissues. DUSP9 transcript levels were also reduced in premalignant lesions (adenomas) compared with those in normal gastric mucosa tissues, indicating that DUSP9 loss occurs early in the progression to tumorigenesis. These data supported that the DUSP9 transcript level was downregulated in GC.2. DUSP9 is silenced via hypermethylation in malignancy.The DUSP9 promoter contains a large CpG island from-1297 to+1104 from the transcription start site. We performed BSP in 30 matched GC and normal gastric mucosa tissues. BSP of the DUSP9 promoter included 11 CpG sites. In normal gastric mucosa tissues, the DUSP9 promoter demonstrated low methylation levels (average methylation level was 20.3%). By contrast, DUSP9 promoter methylation was significantly increased at each individual CpG site examined and reached an average of 74.5% in the cancerous tissues.To confirm the role of DNA methylation in the transcriptional regulation of DUSP9, we treated MKN-1 cells with 2 or 8 μM 5-aza-2’-deoxycytidine for 72 h and examined DUSP9 promoter methylation and mRNA expression changes. DUSP9 gene expression was restored following 5-aza-2’-deoxycytidine treatment. This re-expression was accompanied by a decrease in promoter DNA methylation from 94 to 72%. These results indicated that promoter hypermethylation is one mechanism mediating transcriptional silencing of DUSP9 in GC.3. DUSP9 induces growth inhibition in GC cells in vitro and in vivo.To examine the effect of DUSP9 on cell growth, MKN-1 cells were transiently transfected with DUSP9 vector or negative control vector, respectively. The results of MTT assay showed that DUSP9 inhibited cell growth in MKN-1 cells by 52%, whereas Si-DUSP9 promoted cell growth in MKN-1 cells by 74%. By contrast, the DUSP9-negative control or Si-DUSP9 control had no effect on cell growth, indicating that the effect caused by DUSP9 was highly specific. As demonstrated by the colony formation assay, DUSP9-infected MKN-1 cells exhibited much fewer and smaller colonies compared with LV-coninfected cells.We used lentiviral vectors to stably restore the expression of DUSP9 in MKN-1 cells and examined cell-cycle distribution. Compared with the negative control, LV-DUSP9-infected MKN-1 cells showed an increased percentage of cells in the Gl phase and fewer cells in the S phase, while the cell-cycle distribution had a significant difference between LV-sh-DUSP9 control and LV-sh-DUSP9-transfected cells. These results suggested that the growth-suppressive effect of DUSP9 was partly due to a G0/G1 phase arrest.MKN-1 cells were infected with LV-sh-DUSP9 and then injected subcutaneously into the dorsal flank of nude mice. As early as 2 weeks post-implantation, the growth of transplanted tumors between two groups became statistically significant. At 25 days after implantation, those mice injected with LV-sh-DUSP9 carried larger burdens. As compared with the LV-sh-DUSP9-treated group, the average tumor volume of the control group was markedly reduced by>60%.4. DUSP9 inhibits progression of the cell cycle at S-G2/M phase by regulating cell cycle-related molecules.To investigate the mechanisms leading to loss of cell proliferati 生存 on by DUSP9, we assessed whether the observed inhibitory effects of DUSP9 on cell proliferation were due to induction of cell-cycle arrest. As previously described, infection with LV-sh-DUSP9 increased the percentage of cells in the S and G2/M phase. DUSP-mediated cycle-related molecules downregulation was observed in the transient expression system in MKN-1 cells. RT-qPCR showed that, the mRNA expression level of the CCND1 gene was shown to be decreased 0.81-fold at 24 h and 0.35-fold at 48 h, and this level was sustained up to 72 h after the transfection of DUSP9 into MKN-1 cells. The mRNA expression levels of CDK4 and CDK6 in DUSP9 gene-transfected MKN-1 cells were at the same level as that of CCND1 mRNA expression throughout the time course. By contrast, Si-DUSP9-mediated cycle-related molecules upregulation was observed in the transient expression system in MKN-1 cells.We also examined the effect of DUSP9 on cell cycle-regulatory molecules, including c-Jun, CCND1, CDK4, CDK6, and p21. The infection of MKN-1 cells with LV-DUSP9 resulted in downregulation in the levels of c-Jun protein as well as the levels of CCND1. Downregulation of CDK4 and CDK6 was also observed in MKN-1 cells. The level of p21 protein increased markedly following the infection of MKN-1 cells with LV-DUSP9. By contrast, the infection of MKN-1 cells with LV-sh-DUSP9 resulted in upregulation in the levels of c-Jun protein as well as the levels of CCND1. Upregulation of CDK4 and CDK6 was also observed in MKN-1 cells. In addition, the expression of p21 increased in LV-sh-DUSP9-infected cells in comparison with the controls. However, the infection of MKN-1 cells with LV-sh-DUSP9/LV-DUSP9 resulting in regulation of the levels of cycle-related molecules was inhibited when the JNK inhibitor SP600125 was added, suggesting that the drug inhibits the DUSP9-mediated JNK activation of c-Jun, CCND1, CDK4, CDK6 and inactivation of p21. Thus, the results of the present study confirm that DUSP9 induces regulation in the levels of cycle-related molecules via JNK signaling.ConclusionsIn conclusion, DUSP9 was frequently methylated in human GC and the expression of DUSP9 was silenced by the promoter region hypermethylation. DUSP9 suppresses GC proliferation by inhibiting JNK signaling pathways. As was the case in the present study, overexpression of DUSP9 correlated with reduced JNK activity. However, DUSP9-induced JNK kinase inactivation can be specifically blocked by the inhibitor SP600125. This results suggest that therapeutic intervention to increase the expression or activity of DUSP9 may enable the activation of the anti-proliferative signals in malignant cells.