DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells

  • Authors:
    • Xiangling Feng
    • Cui Li
    • Weidong Liu
    • Huan Chen
    • Wen Zhou
    • Lei Wang
    • Bin Zhu
    • Kaitai Yao
    • Xingjun Jiang
    • Caiping Ren
  • View Affiliations

  • Published online on: April 8, 2013     https://doi.org/10.3892/ijo.2013.1885
  • Pages: 1973-1984
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Abstract

In our previous study we demonstrated the downregulation or loss of deleted in liver cancer‑1 (DLC-1) gene expression in nasopharyngeal carcinoma (NPC). In this study, we report the effects of the DLC-1 gene on NPC cells and its mechanisms of action. DLC-1 expression was restored in the 5-8F NPC cell line, which lacks DLC-1 expression, and the biological characteristics of 5-8F-DLC‑1 cells were analyzed by MTT assay, colony formation assay, flow cytometry (FCM), tumorigenesis analysis in nude mice, as well as invasion and migration assay. Differentially expressed genes in response to DLC-1 expression were screened using microarray analysis and identified by RT-PCR. The re-expression of DLC-1 in the NPC cells attenuated the proliferation and colony formation ability of the cells in vitro, blocked NPC cells at the G0/G1 phase, reduced tumorigenicity potential in vivo, inhibited the invasion and migration ability of NPC cells and resulted in the reorganization of the actin cytoskeleton. DLC-1 altered the gene expression profile in 5-8F cells. Some tumor suppressor genes (TSGs) were upregulated and some oncogenes were downregulated. These results demonstrate that DLC-1 gene can partially reverse the malignant phenotype of NPC cells by changing the tumor-related gene expression profile, and may be a candidate tumor suppressor gene and a promising diagnostic and therapeutic target in NPC.

Introduction

Nasopharyngeal carcinoma (NPC), an epithelial malignancy with high incidence in Southeast Asia and Southern China, is associated with genetic alteration and environmental factors (14). Although much progress has been made by studies on NPC, the exact molecular mechanisms underlying nasopharyngeal carcinogenesis remain unclear, and no significant breakthrough on the early detection and treatment of NPC has been achieved. It is widely considered that Epstein-Barr virus (EBV), chemical carcinogens and genetic susceptibility are three main factors involved in NPC pathogenesis. Previous studies have revealed numerous NPC-related genes, most of which are potential tumor suppressor genes (TSGs). However, these genes are only a small proportion of NPC-related genes and they are not sufficient to fully elucidate the molecular carcinoge nesis of NPC (5).

Deleted in liver cancer-1 (DLC-1) gene, which was originally cloned by Yuan et al from liver tissue using representational difference analysis (RDA), is located at chromosome 8p22, and its full-length cDNA covers 6,044 bp (GeneBank ID: NM006094) (6). It encodes one of the Rho GTPase-activating proteins (GAPs), a negative regulator of the Rho family proteins by stimulating their intrinsic GTPase activity. DLC-1 is extensively expressed in normal tissues, but is downregulated or deleted in liver, gastric, lung and prostate cancer, as well as in multiple myeloma. Furthermore, it can significantly suppress proliferation and metastasis of many kinds of tumors (7,8). Loss of heterozygosity (LOH) and promoter hypermethylation are two of the main causes associated with the transcriptional silencing of DLC-1 in these tumors. Previously we and others have shown that DLC-1 is normally expressed in chronic nasopharyngitis tissues and normal nasopharyngeal epithelium, but is significantly downregulated or absent in NPC cell lines and tissues, and so it may play a critical role in carcinogenesis and the progression of NPC (9,10). Promoter hypermethylation plays a key role in inactivating DLC-1 in NPC, although LOH is also involved in the aberrant expression of DLC-1. In this study, we constructed a recombinant eukaryotic vector of DLC-1 and established a 5-8F cell line stably expressing high level of DLC-1. Furthermore, we investigated the function of DLC-1, as well as the effects of DLC-1 re-expression on the malignant phenotype and gene expression profile of NPC cells.

Materials and methods

Ethics statement

All animal experiments were performed according to the institutional guidelines approved by the Animal Care and Use Committee of Central South University, Changsha, China. We also received ethics approval from the Institutional Review Board of Central South University.

Cell culture

The NPC cell line, 5-8F, a subclone of SUNE-1 which was isolated from poorly differentiated squamous cell carcinoma tissue and exhibited high metastatic and tumorigenic ability, was preserved by our institute. It was cultured in RPMI-1640 supplemented with 10% newborn calf serum (NCS) at 37°C in a humidified 5% CO2 atmosphere.

Plasmid construction, transfection and stable colony selection

RNA from human normal spleen tissue was reverse transcribed into cDNA with M-MLV reverse transcriptase (Promega, Madison, WI, USA). The entire open reading frame (ORF) fragment (approximately 3.3 kb) of DLC-1 gene was amplified by RT-PCR with the cDNA as the template. The DLC-1 ORF forward and reverse amplification primers were 5′-GCCTGCCGTGCTTGATGTGC-3′ and 5′-TGGTGGAA GCGGTTGCGTTG-3′, respectively. The PCR product was TA-cloned into the pBS-T vector (Tiangen, Beijing, China) and sequenced. The entire DLC-1 ORF sequence was removed from pBS-T/DLC-1 following BamHI and KpnI digestion and was subcloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA). The recombinant vector (pcDNA3.1/DLC-1) was transformed into DH5α Escherichia coli and the plasmid DNA was isolated using the Miniprep kit (Qiagen, Hilden, Germany) for transfection.

The 5-8F cells were seeded into six-well plates with RPMI-1640 medium at a density of 3×105 cells/well. When reached 70–80% confluence, the cells were transfected with 2 μg plasmid DNA (pcDNA3.1(+) or pcDNA3.1/DLC-1) using 8 μl FuGENE 6 as described by the manufacturer (Roche, Basel, Switzerland). After 48 h, the medium was replaced with fresh RPMI-1640 medium with 10% NCS and Geneticin (G418) in order to select clones stably harboring DLC-1 or the empty vector. The selected clones were named 5-8F-DLC-1 or 5-8F-vector, respectively.

Western blot analysis and immunocytochemistry (ICC)

Immunoblotting experiments and ICC analysis were performed according to the procedure outlined in our previous study (11). For each cell line, 2×106 cells were harvested and cell lysates were prepared using commercial cell lysis buffer (Pierce Biotechnology, Inc., Rockford, IL, USA) in western blot analysis. Equal amounts of protein (30 μg) from whole cell lysates were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBST buffer (1× Tris-buffered saline and 0.1% Tween-20) for 2 h, and then incubated overnight at 4°C with mouse monoclonal anti-human DLC-1 antibody (1:300, BD Biosciences, Franklin Lakes, NJ, USA) and β-actin antibody (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) which was used as a loading control. After washing with TBST buffer three times, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:2,000, Santa Cruz Biotechnology). Finally, the ECL western blotting detection system (Pierce Biotechnology, Inc.) and X-ray films were used to develop the blot images.

A 1:150 dilution of mouse monoclonal anti-human DLC-1 antibody was used in immunocytochemistry analysis. Briefly, cells seeded on slides were washed in PBS, fixed with 4% paraformaldehyde in PBS for 20 min, and treated with 0.5% Triton X-100 in PBS (PBST) for 30 min. Subsequently, endogenous peroxidase was inactivated by incubation in 3% H2O2 in dH2O for 15 min at room temperature. After being rinsed with PBST, the cells were treated with 5% bovine serum albumin (BSA) in PBST (2 h at room temperature). DLC-1 antibody, the Histostain™-Plus kit (Zymed Laboratories Inc., South San Francisco, CA, USA) and the diaminobenzidine (DAB) substrate kit were then used to detect DLC-1 protein. Finally, the slides were lightly counterstained with hematoxylin, dehydrated, mounted with neutral balsam (Shanghai Specimen and Model Factory, Shanghai, China) and photographed under a microscope. For the negative control, the primary DLC-1 antibody was replaced with PBS.

MTT assay

MTT assay was performed as previously described (12). The 5-8F-DLC-1 and 5-8F-vector cells were seeded in 96-well plates at a density of 2×103 cells/well in 200 μl culture medium (RPMI-1640 with serum). Three wells had no cells and were used as the control for the minimum absorbance. Cells were propagated at 37°C in an incubator with humidified 5% CO2 atmosphere for one to seven days. The medium was discarded and the cells were incubated with 20 μl/well MTT solution (5 mg/ml) for 4 h at 37°C followed by the addition of 150 μl/well DMSO. Ten minutes later, the absorbance value was measured with an ELISA plate reader (ELx800, BioTek Instruments, Inc., Winooski, VT, USA) at a test wavelength of 490 nm. The growth curves were drawn by EXCEL software.

Colony formation assay

The 5-8F-vector and 5-8F-DLC-1 cells were seeded in six-well plates at 1×103 cells/well and each cell line was seeded in triplicate. Cells were cultured in RPMI-1640 supplemented with 10% NCS at 37°C in a humidified 5% CO2 atmosphere for eight days. After discarding the culture medium and washing with D-Hank’s solution (three times), the cells were fixed with methanol for 15 min and stained with 0.4% crystal violet for 10–30 min. After washing with water and drying in the air, clones containing >50 cells were counted under an inverse microscope (TE2000U; Nikon, Osaka, Japan).

Flow cytometry (FCM)

As has been described in our previous study (11), the 5-8F-vector and 5-8F-DLC-1 cells (1×106 cells per sample) were collected and washed with PBS, fixed in 70% ethanol and stored at 4°C. All samples were resuspended in PBS containing RNase A (100 U/ml, Sigma, St. Louis, MO, USA) and incubated at 37°C for 30 min, stained with propidium iodide (PI, 50 μg/ml, Sigma), and analyzed by FCM (BD FACSCalibur™; BD Biosciences). The cell cycle distribution was calculated from the resultant DNA histogram using Mod Fit LT software.

In vivo tumorigenicity assay

A total of 12 (six male and six female) four to six-week-old BALB/c-nu/nu nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The 5-8F-DLC-1 and 5-8F-vector cells (5×106 cells/each type) were injected subcutaneously into the proximal dorsal midline of three four to six-week-old male nude mice and three four to six-week-old female nude mice. Tumor size was subsequently measured in two dimensions twice a week. All the mice were sacrificed 45 days after injection and were examined for the presence of tumors. Tumors were removed from the nude mice, measured and weighed for tumorigenicity analysis.

Wound healing assay

The 5-8F-vector and 5-8F-DLC-1 cells were seeded into six-well plates at a density of 1×106 cells/well. The following day, the confluent cell monolayer was wounded with a sterile 200 μl tip and plates were returned to cell culture incubator. Images were captured at the beginning and after 24 h of cultivation, and the migration ability of the cells was evaluated by measuring the width of the wounds.

Cell migration and invasion assays

Transwell inserts were loaded into 24-well plates, and 200 μl migration buffer (0.1% BSA in RPMI-1640 medium) were added to the top chamber at 37°C for 1 h. A total of 5×105 cells in 200 μl migration buffer were seeded into the top chamber pre-treated with migration buffer and incubated for 18 h. The lower chamber was supplemented with 600 μl complete medium with 15% fetal calf serum (FCS). After an 18-h incubation, the cells on the upper side of the membranes were removed using a cotton swab and cells on the lower side were fixed in methanol and stained with 0.1% crystal violet and counted in five independent microscopic fields at ×200 magnification.

The protocol for invasion assay was similar to that for cell migration assay except that Transwell chambers were coated with Matrigel.

Detection of cytoskeletal proteins

Climbing slices of 5-8F-DLC-1 and 5-8F-vector cells were prepared and washed with ice-cold PBS buffer on the following day. The cells were then fixed with ice-cold 4% paraformaldhyde for 20 min, incubated in 0.2% (v/v) Triton X-100 for 20 min and stained with 1 μg/ml phalloidin-TRITC for 40 min at room temperature. The slices were washed with PBS three times (10 min per time) after each of the above steps. The cytoskeletal distribution was observed under a ZEISS LSM 5 confocal laser microscope (Carl Zeiss Inc., Oberkochen, Germany).

Microarray analysis

Total RNA from the 5-8F-vector and 5-8FDLC-1 cells was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. Microarray analysis was performed by CapitalBio Corp. (Beijing, P.R. China) using Human Genome U133 Plus2.0 GeneChip Arrays (Affymetrix, Sunnyvale, CA, USA) which covers >54,000 probe sets representing 47,000 transcripts and variants and including 38,500 human genes as previously described (13). The raw data were normalized with Robust Multichip Analysis (RMA). The differential expression of genes was detected with Significance Analysis of Microarrays (SAM). A two-fold or greater change in intensity was used as the criterion for inclusion in our filtered data set and the P-value was set ≤0.05.

RT-PCR analysis

To detect the expression of DLC-1 in 5-8F cells transfected with DLC-1 or the pcDNA3.1(+) vector and to confirm the microarray results, RT-PCR analysis was performed as previously described (11). Total cellular RNA was extracted using TRIzol (Invitrogen) and reverse transcribed using a Reverse Transcription kit (Fermentas, Hanover, MD, USA) according to the manufacturer’s instructions. The cDNAs obtained were used as the template for amplification with Taq enzyme mixture (Fermentas). The primer sets, the length of PCR products, annealing temperature and cycles for amplification are listed in Table I. DLC-1 primers were taken from a previous study (10).

Table I

Primers for RT-PCR analysis.

Table I

Primers for RT-PCR analysis.

Gene symbolPrimers (5′→3′)Product (bp)Annealing temperature (°C)Cycles (N)
GAPDHF: GAGATCCCTCCAAAATCAAGTG2825830
R: GAGTCCTTCCACGATACCAAAG
DLC-1F: GGACACCATGATCCTAACAC2625535
R: CTCATCCTCGTCTGAATCGT
WNT5AF: ATTCTTGGTGGTCGCTAGGTA1595228
R: CGCCTTCTCCGATGTACTGC
TNS1F: GCGGGCTAAAGTGAAGTT3355831
R: GGATGATGGAGTGCTGGTA
FHL1F: GACATCTGCTCTCGGTTATT2076031
R: TAGTGGCAGTCAAACTTCTC
S100A2F: AAGAGGGCGACAAGTTCA2446231
R: AGAGTTCTGCTTCAGGGTC
RECKF: GTCTTGTATTGTTGGAGGAA2605834
R: ACTGATGGTCTTGGAGGC
DUSP2F: CTGTGGAGATCTTGCCCTA2215832
R: CACCCAGTCAATGAAGCCTA
CASP9F: TGCGTGGTGGTCATTC1946030
R: TGGTCTTTCTGCTCCC
IGFBP7F: AACAAGGTAAAAAGGGGTCA2736230
R: TGGCTGTGAGATTTATTGTGTA
EGFRF: GAAGGCTGTCCAACGAAT2936030
R: CAGAGTCCCTTATACACCGT
CDCP1F: TGACTTGAGTAATGAGCGAG2156030
R: CTGTGCAGCTTATGGTTTTT
KRASF: GCAAAGACAAGACAGGGTG2646030
R: GGTAAAAGCTAACAGTCTGC
TGFβ2F: CCATCCCGCCCACTTTCTAC1956030
R: AGCTCAATCCGTTGTTCAGGC
AKT3F: AGAGAATCCAAACCCTAAAGCTGA1856030
R: ATCCTATGAATGAGCCATCTGT
MMP7F: GAACAGGCTCAGGACTATCT2836032
R: GATCCACTGTAATATGCGGT
MUC4F: CGTTCTGGGACGATGCTGAC2306032
R: GATGGCTTGGTAGGTGTTGCT
BCL10F: GTGAAGAAGGACGCCTTAGAAA2066032
R: TCAACAAGGGTGTCCAGACCT
PTK6F: TGCCCCATTGGGATGACTG2216032
R: GTACAGCGCCAGGATGTGTTT
Statistical analysis

MTT assay, colony formation assay, FCM, and cell migration and invasion assay were carried out in triplicate. The data are presented as the means ± SD and processed with SPSS for Windows 11.5 by using an independent-sample t-test. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

Construction of eukaryotic expression vector pcDNA3.1(+)/DLC-1 and establishment of NPC cell line stably expressing DLC-1

The recombinant vector was sequenced and the results showed that the coding sequence was correct except for a single nucleotide polymorphism (SNP) site at +1380 (G→A, V354M) (Fig. 1A). This vector was designated as pcDLC-1. Plasmid, either pcDLC-1 or pcDNA3.1(+) was transfected into 5-8F cells using FuGENE 6 and the cell lines stably expressing pcDLC-1 or pcDNA3.1(+) plasmids were named 5-8F-DLC-1 or 5-8F-vector, respectively. DLC-1 was highly expressed in the 5-8F-DLC-1 cells while it was absent in the 5-8F-vector cells when detected by RT-PCR (Fig. 1B) and western blot analysis (Fig. 1C). Immunocytochemical detection showed that the DLC-1 protein was mainly present in the cytoplasm of the 5-8F-DLC-1 cells (Fig. 1D).

Effects of DLC-1 on the proliferation of NPC cells

We analyzed changes in the biological characteristics of 5-8F-DLC-1 cells. Compared with the 5-8F-vector cells, the 5-8F-DLC-1 cells showed significant growth inhibition (P<0.05, Fig. 2A) and a reduction in cloning efficiency as measured by MTT analysis and colony formation assay (23.1 vs. 52.5%, respectively) (Fig. 2C). FCM showed that the 5-8F-DLC-1 cells were arrested at the G0/G1 phase (Fig. 2B). Compared to the 5-8F-vector cells, the ratio of 5-8F-DLC-1 cells at the G0/G1 phase increased (67.25 vs. 45.39%), while the ratio of cells at the S phase (21.24 vs. 31.26%) and the G2/M phase (11.51 vs. 23.35%) decreased significantly. In vivo tumorigenicity analysis confirmed that the size of the tumors formed by 5-8F-DLC-1 cells in nude mice was much smaller than that of the tumors formed by 5-8F-vector cells (P<0.05, Fig. 2D). These results indicated that the stable expression of DLC-1 blocked 5-8F cells at the G0/G1 phase and resulted in attenuated proliferation and colony formation ability in vitro and a lower tumorigenicity potential in vivo.

Effects of DLC-1 on cell migration and invasion of NPC cells

To determine whether DLC-1 affects the motility of NPC cells, in vitro wound healing assay, migration assay and invasion assay were performed. Compared with the 5-8F-vector cells, the 5-8F-DLC-1 cells closed the wound more slowly (Fig. 3A) and the number of cells penetrating the Transwell membrane was significantly lower (18±4.0 vs. 42±5.6; P<0.00) (Fig. 3B), indicating that the motility of DLC-1-expressing cells was significantly impaired. When observed after a 24 h-cultivation, the number of 5-8F-DLC-1 and 5-8F-vector cells penetrating the Transwell membrane coated with Matrigel was 191.40±16.6 and 278±20.7, respectively (n=5, P<0.05) (Fig. 3C), indicating a significant reduction in the invasive ability of 5-8F-DLC-1 cells.

Effect of DLC-1 on cytoskeletal formation

Phalloidin is a virulent alkaloid extracted from a toxic mushroom and can solidly bind with the cell membrane and fibrous actin (F-actin). Fluorescently-labeled phalloidin was used to specifically mark cell surface morphology and changes in F-actin distribution. In the 5-8F-vector cells, abundant F-actin was observed at the cell periphery, a prominent site of the cytoplasm, and throughout the cytoplasm (Fig. 4); while in the 5-8F-DLC-1 cells, microfilaments exhibited polar and circular distribution, focused along the cell periphery and were significantly decreased in the cytoplasm as compared with those in the 5-8F-vector cells (Fig. 4). This is an indication that DLC-1 plays a crucial role in cytoskeletal formation, which may be one of the mechanisms behind its effect on NPC cell motility.

DLC-1 expression induces differential expression of certain tumor-related genes in NPC cells

To investigate the molecular mechanism behind the inhibitory effect of DLC-1 expression on the biological characteristics of tumor cells, microarray analysis was performed to compare gene expression profiles in 5-8F-vector and 5-8F-DLC-1 cell groups. The gene expression levels were compared between the two groups. The expression of 840 genes and 151 expressed sequence tags (ESTs) was significantly altered in the 5-8F-DLC-1 group as compared to that in the 5-8F-vector group. Among these genes, 454 were upregulated and 386 were downregulated. The most differentially expressed or altered critical tumor-related genes are listed in Tables II and III. As shown in Table II, genes that act as tumor suppressors, such as IGFBP7, TNS1, TP53 and TP63, were significantly upregulated by the DLC-1 gene. Those acting as oncogenes, such as EGFR, KRAS and TGFβ2, were significantly downregulated (Table III).

Table II

Representative upregulated genes induced by DLC-1 gene expression in the 5-8F cell line.

Table II

Representative upregulated genes induced by DLC-1 gene expression in the 5-8F cell line.

Gene symbolDescriptionRefSeqFold change (DLC-1 vs. vector)
CASP9Caspase 9, apoptosis-related cysteine peptidaseNM_0329965.00
DUSP2Dual specificity phosphatase 2NM_00441824.97
FHL1Four and a half LIM domains 1NM_0014492.65
GAS1Growth arrest-specific 1NM_0020486.15
HDAC4Histone deacetylase 4NM_0060377.59
IGFBP7Insulin-like growth factor binding protein 7NM_0015533.09
PDCD4Programmed cell death 4 (neoplastic transformation inhibitor)NM_0144562.93
RASSF6Ras association (RalGDS/AF-6) domain family member 6NM_1775325.09
RECK Reversion-inducing-cysteine-rich protein with kazal motifsNM_0211112.67
S100A2S100 calcium binding protein A2NM_0059783.00
TANKTRAF family member-associated NF-κB activatorNM_0041802.03
TLN2Talin 2NM_0150593.31
TNS1Tensin 1NM_0226482.89
TP53TG1TP53 target 1 (non-protein coding)NR_0153813.60
TP63Tumor protein p63NM_0011149802.98
VWA5AVon Willebrand factor A domain containing 5ANM_1983157.60
WNT5AWingless-type MMTV integration site family, member 5ANM_0033924.99
WWOXWW domain containing oxidoreductaseNM_0163733.40

[i] RefSeq, reference sequence.

Table III

Representative downregulated genes induced by DLC-1 gene expression in the 5-8F cell line.

Table III

Representative downregulated genes induced by DLC-1 gene expression in the 5-8F cell line.

Gene symbolDescriptionRefSeqFold change (DLC-1 vs. vector)
AKT3V-akt murine thymoma viral oncogene homolog 3 (protein kinase B, γ)NM_0054650.52
BCL10B-cell CLL/lymphoma 10NM_0039210.44
CD44CD44 molecule (Indian blood group)NM_0006100.23
CDCP1CUB domain containing protein 1NM_0228420.37
EGFREpidermal growth factor receptor [erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian]NM_0052280.50
ETS1v-ets erythroblastosis virus E26 oncogene homolog 1 (avian)NM_0052380.45
FGF2Fibroblast growth factor 2 (basic)NM_0020060.46
FOSL1FOS-like antigen 1NM_0054380.35
KRASv-Ki-ras2 Kirsten rat sarcoma viral oncogene homologNM_0333600.23
LAMC2Laminin, γ2NM_1446520.14
MMP7Matrix metallopeptidase 7 (matrilysin, uterine)NM_0024230.15
MUC4Mucin 4, cell surface associatedNM_0184060.33
NEXNNexilin (F-actin binding protein)NM_1445730.22
PCSK6Proprotein convertase subtilisin/kexin type 6NM_0025700.37
PTK6PTK6 protein tyrosine kinase 6NM_0059750.48
STYK1 Serine/threonine/tyrosine kinase 1NM_0184230.48
ST14Suppression of tumorigenicity 14 (colon carcinoma)NM_0219780.38
TGFβ2Transforming growth factor, β2NM_0011355990.36
WIPF1WAS/WASL interacting protein family, member 1NM_0033870.37

[i] RefSeq, reference sequence.

To validate our array expression findings, 17 of the differentially expressed genes, such as WNT5A, TNS1, FHL1, S100A2, RECK, DUSP2, CASP9, IGFBP7, EGFR, CDCP1, KRAS, TGFβ2, AKT3, MMP7, MUC4, BCL10 and PTK6 were selected for further verification with RT-PCR analysis. As shown in Fig. 5, eight of these genes, namely WNT5A, TNS1, FHL1, S100A2, RECK, DUSP2, CASP9 and IGFBP7 were found to be upregulated, while nine genes, including EGFR, CDCP1, KRAS, TGFβ2, AKT3, MMP7, MUC4, BCL10 and PTK6 were downregulated in the 5-8F-DLC-1 cells as compared to the control 5-8F-vector cells. The trends for either the up- or downregulation of mRNA expression obtained by RT-PCR were consistent with the microarray results.

Furthermore, using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) software, we analyzed the microarray dataset to identify whether specific biological pathways were differentially affected by the DLC-1 gene. As shown in Fig. 6, proteins encoded by these genes were mainly observed in the cytoplasm and nucleus. Their molecular functions include protein binding, metal ion binding and nucleotide binding and they participate in some important biological process mainly associated with cell adhesion, negative regulation of cell proliferation, cell cycle arrest and the inhibition of apoptosis. Pathway analysis indicated that the altered genes were associated with a number of essential biological processes, such as tumor-related pathways including focal adhesion, MAPK signaling, VEGF and TGFβ signaling and apoptotic pathways along with several specific cancer-related and metabolism-related pathways (Fig. 7).

Discussion

The DLC-1 gene is considered an important TSG candidate (5). It is located at chromosome 8p22, a region that exhibits high loss frequency in NPC tissues and is closely associated with NPC (10). In order to further investigate the biological function of DLC-1 in NPC, we transfected the constructed pcDLC-1 plasmid, a DLC-1 eukaryotic expression vector, into 5-8F NPC cells and established a cell line (5-8F-DLC-1) stably expressing DLC-1. 5-8F is a highly tumorigenic, highly metastatic NPC cell line and a good model for studying the invasive and metastatic mechanisms of NPC (14). Although there was a missense SNP site in the obtained ORF sequence, bioinformatics analysis and literature investigation indicated that this site was not in any of the known important domains. Furthermore, secondary structure prediction did not reveal any obvious changes and there was no report about any effects of this SNP on protein function. Therefore, we continued to use the 5-8F-DLC-1 cell line to investigate changes in the biological characteristics of NPC cells induced by the re-expression of DLC-1.

NPC is a malignancy with predisposition to early metastasis. It is often at the middle or advanced stage at clinical examination. Studies have shown that recovering the expression of DLC-1 in hepatic carcinoma (15,16), breast cancer (17), non-small cell lung cancer (NSCLC) (18) and multiple myeloma cells (19) results in the reduction of cytoskeletal content, alteration of cell morphology and the inhibition of tumor growth and metastasis. Seng et al reported that DLC-1 suppresses the colony formation of CNE2 NPC cells (10) and Loyo et al showed that DLC-1 decreases the growth and invasion capacity in a human nasal epithelial (HNE) cell line (20). Our results indicated that DLC-1 not only suppressed cell growth and proliferation, but also inhibited the motility, migration and invasion ability of 5-8F cells. Compared with those in 5-8F-vector cells, the microfilaments were reduced and exhibited circular/polar distribution around the cell membrane in the 5-8F-DLC-1 cells, as observed in multiple myeloma cells (19). Therefore, our results further suggest that DLC-1 plays an important role in the tumorigenesis and metastasis of NPC.

DLC-1 is the earliest cloned member of the DLC gene family, which consists of the RhoGAP, START and SAM domains and unstructured middle regions, such as the serine-rich region, tensin-binding motif (SIYDNV) and LD motif (LDDILYHV) (7,21,22). DLC-1 is a GTPase-activating protein (GAP) and inhibits tumor growth and progression through the activation of intrinsic GTPase of Rho family proteins by the RhoGAP domain, which subsequently results in the inactivation of Rho protein. However, some studies have reported that DLC-1 participates in suppressing tumor growth and metastasis by binding with the SH2 domain of tensin through 440–445 amino acid residues (SIYDNV) and the inhibition of Rho protein activity through RhoGAP domain was not required (23,24). In addition, DLC-1 has been reported to bind talin protein through its LD motif and perform its function. Thus far, there is no report on the specific mechanisms underlying the tumor suppressive function of DLC-1 in NPC. Whether it functions through the inhibition of certain Rho protein activity, binding with tensins or other mechanisms remains to be elucidated.

Using Affymetrix Human Genome U133 Plus2.0 GeneChip Array, we found that DLC-1 affected the expression of a series of genes. Most of the upregulated genes exhibited tumor suppressor characteristics. For example, tensin 1 (TNS1) is a protein localized at focal adhesions acting as a scaffold for the signaling pathway. Furthermore, the SH2 domain of TNS1 is associated with DLC-1. Human breast, prostate and head and neck squamous cell carcinoma, as well as melanoma exhibit low expression levels of TNS1, suggesting that it functions as a tumor suppressor. The re-expression of TNS1 has been shown to promote the formation of focal adhesions and to decrease the migration and invasion of MDA-MB-231 human breast cancer cells (25). Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) is a cysteine-rich, extracellular protein with protease inhibitor-like domains whose expression is significantly suppressed in several tumors (26), including NPC (27). RECK has been reported to inhibit tumor angiogenesis, invasion and metastasis by negatively regulating matrix metalloproteinases (MMPs) (28). Dual specificity phosphatase 2 (DUSP2), originally named phosphatase of activated cells-1 (PAC-1), is a member of the dual specificity protein phosphatase subfamily and predominantly inactivates ERK, but also inactivates p38 MAPK, although to a lesser degree (29,30). DUSP2 mRNA and protein are markedly reduced or completely absent in many types of cancer. The re-expression of DUSP2 in xenograft mouse models of cancer has been reported to increase drug sensitivity and induce tumor regression (31).

By contrast, some of the downregulated genes exhibit oncogenic characteristics. Epidermal growth factor receptor (EGFR) is one of the most widely known receptor tyrosine kinases. The overexpression of EGFR is common in NPC and most NPC cell lines and approximately 85% of Chinese NPC patients have a moderate to strong expression of EGFR. Moreover, the overexpression of EGFR in primary tumors is associated with tumor metastasis, recurrence and a low survival rate in patients with NPC, suggesting that EGFR plays a crucial role in the development and progression of NPC (32). The role of TGFβ in tumorigenesis is complex. In different cell types and stages, TGFβ has either positive or negative effects. High levels of TGFβ2 have been observed in many tumor cell lines, which promotes the survival of tumor cells by activating nuclear factor-κB (NF-κB) (33). Additionally, MMP7, a member of the MMP family, is overexpressed in a variety of epithelial and mesenchymal tumors and its expression correlates with tumor progression, metastasis and unfavorable prognosis in human esophageal, colon and gastric carcinoma (3436). CUB domain containing protein 1 (CDCP1), a type I transmembrane protein containing three extracellular CUB domains, has been found to be overexpressed in many types of cancer and its overexpression is significantly associated with poor prognosis in relation to disease-free and overall survival (37,38). The downregulation of CDCP1 by RNA interference in lung and gastric carcinoma cells has been shown to result in suppressed invasion and experimental metastasis (38,39). Our array results suggested that DLC-1 suppressed NPC cell growth and metastasis through the upregulation of tumor suppressors and the downregulation of oncogenes or metastasis-related genes.

A variety of aberrantly altered signals is an important factor mediating the biological behavior of NPC including carcinogenesis and development. Such signaling pathways are critical for cell survival, growth and metastasis. A number of studies have shown that Wnt, PI3K-Akt, MAPK, focal adhesion, apoptosis and EGRF signaling pathways contribute to the tumorigenesis of NPC (40,41). Using KEGG software, we also found that focal adhesion, MAPK, VEGF and apoptosis signaling pathways, etc., were altered in 5-8F-DLC-1 cells, suggesting that DLC-1 performed its biological function including suppressing cell growth and metastasis in NPC by regulating such tumor-related pathways. In addition, many altered metabolism associated pathways were be found in this study, suggesting that metabolic regulation play an important role in inhibitory effects of DLC-1 on NPC cells. An ever-increasing amount of evidence demonstrates that metabolism is another important factor contributing to carcinogenesis and tumor development (42). The role of DLC-1 as a tumor suppressor and its effects on metabolism in NPC remain to be further elucidated.

In conclusion, our results demonstrate that DLC-1 partially alters the malignant phenotypes of NPC cells, which includes the inhibition of cell proliferation and the suppression of invasion and metastasis etc., through the regulation of the expression of tumor suppressors, oncogenes or metastasis-related genes and affecting cancer-related pathways. This study provides a theoretical and experimental basis for comprehensively understanding the biological function of the DLC-1 gene in NPC and further elucidating the molecular mechanisms of NPC carcinogenesis. Our data open up a new avenue for the more effective diagnosis and treatment of NPC.

Acknowledgements

This study was supported by grants from the National Basic Research Program of China (2010CB833605), the Program for New Century Excellent Talents in University (NCET-10-0790), the National Natural Science Foundation of China (30801322, 81172206, 81272972), the Provincial Natural Science Foundation of Hunan (10JJ4025), Science and Technology Project of Hunan Province (2010FJ3005), the Incubation Program for National Natural Science Funds for Distinguished Young Scholar of Central South University (2010QYZD006), and the Open-End Fund for the Valuable and Precision Instruments of Central South University (CSUZC2012009, CSUZC2012010).

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June 2013
Volume 42 Issue 6

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Spandidos Publications style
Feng X, Li C, Liu W, Chen H, Zhou W, Wang L, Zhu B, Yao K, Jiang X, Ren C, Ren C, et al: DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells. Int J Oncol 42: 1973-1984, 2013
APA
Feng, X., Li, C., Liu, W., Chen, H., Zhou, W., Wang, L. ... Ren, C. (2013). DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells. International Journal of Oncology, 42, 1973-1984. https://doi.org/10.3892/ijo.2013.1885
MLA
Feng, X., Li, C., Liu, W., Chen, H., Zhou, W., Wang, L., Zhu, B., Yao, K., Jiang, X., Ren, C."DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells". International Journal of Oncology 42.6 (2013): 1973-1984.
Chicago
Feng, X., Li, C., Liu, W., Chen, H., Zhou, W., Wang, L., Zhu, B., Yao, K., Jiang, X., Ren, C."DLC-1, a candidate tumor suppressor gene, inhibits the proliferation, migration and tumorigenicity of human nasopharyngeal carcinoma cells". International Journal of Oncology 42, no. 6 (2013): 1973-1984. https://doi.org/10.3892/ijo.2013.1885