Human salivary gland adenoid cystic carcinoma (ACC), that accounts for approximately 10% of all neoplasm in the salivary glands,1,2 is a malignant tumor that is commonly encountered by oral and maxillofacial surgeons. ACC is an aggressive and often indolent tumor with a high incidence of distant metastasis to the lung which is responsible for a rather low long-term survival rate.
A20, also referred to as tumor necrosis factor α induced protein 3 (TNFaip3), is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NF-κB) activity and prevents tumor necrosis factor (TNF)-mediated programmed cell death. Recently, many researchers have found that A20 was important in the development of skin epidermis and hair follicles, but it is not detectable in normal adult skin epidermis or oral mucosa. However, high expression of A20 was observed in undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma, suggesting a role of A20 in the pathogenesis of these tumors.3 One function of A20 is to terminate the activation of NF-κB that follows stimulation by various agents, including TNFα.4,5 NF-κB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and antiapoptosis.6,7 Moreover, NF-κB has been found to be associated with the development and progression of several human malignancies, including pancreatic cancer, breast cancer and prostatic carcinoma.8-11 Several studies have implicated that the NF-κB signal pathway is associated with angiogenesis and clinico-pathological factors in ACC of salivary glands.12-14 However, the function of A20 protein in this cancer is not very clear.
In this investigation we determined if the overexpression of A20 in ACC cells could influence the behavior of ACC cells following stable transfection of full-length A20 cDNA. We found that A20 could partly inhibit NF-κB activation, either constitutive or TNFα induced, and matrigel assays demonstrated that the adenoid cystic carcinoma cell invasion was significantly reduced. These results suggest that A20 may inhibit adenoid cystic carcinoma of salivary glands via blocking NF-κB activity.
The human salivary ACC cell line ACC-2 was established by the Department of Oral and Maxillofacial Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air and routinely propagated in RPMI 1640 (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA).
A20 expression plasmid construction and validation
Complementary DNA encoding full-length wild-type of A20 was constructed by polymerase chain reaction (PCR) using specific oligonucleotide primers (5′-GCGATTTCC-ACAATGGCTGAACAAGTCCTTCCTC-3′ and 5′-CG-GGTACCTTAGCCATACATCTGCTTGAACTG-3′), and the resulting DNA fragments were cloned into the EcoRI and KpnI polylinker sites of the eukaryotic expression vector pEGGFPN3 using T4-DNA ligase (Sigma). This construct was designated as pEGGFPN3-A20. The amplified products were digested with EcoRI and KpnI, and validated by DNA sequencing and restriction endonuclease analysis.
Cell culture and stable transfection
The ACC-2 cells were grown to 95% confluence on 24-well plates in the growth medium without antibiotics. The cells were transfected with 1 μg pEGGFPN3-A20 or control vector pEGGFPN3 using Lipofectamine 2000 (Invitrogen, USA). Transfections were carried out according to the manufacturer's instructions. Six hours after transfection the medium was changed to serum-containing medium and the cells were incubated for another 48 hours at 37°C. Cells were then selected with standard medium containing G418 at 800 μg/ml. Two to three weeks later neo-resistant colonies were isolated by trypsinization and cultured in standard medium containing G418 at 200 μg/ml. The new subcultures are referred to as ACC-2-A20 and ACC-2-GFP and were verified by realtime-PCR and Western blot.
Verification of stable gene transfer
RNA was isolated from cell samples by Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. cDNA was generated from total RNA with a Reverse Transcriptase Kit (Biotec, China) using 1-2 μg of total RNA as template. The resulting cDNA pool was used for realtime-PCR. The Sequence Detection System 7300 (Applied Biosystems) was used for amplification and specific sequence detection. Forward and reverse PCR primers were used at a final concentration of 200 nmol/L, and the SYBR Premix Ex Taq Kit (TaKaRa, Japan) was used in this experiment. Expression of A20 was evaluated relative to the mRNA expression of the housekeeping gene β-actin. Cycling parameters were 10 seconds, 95°C; 40 times: 5 seconds, 95°C; 31 seconds, 59°C; 1 minute, 72°C; and finally a melting curve of 15 seconds at 95°C; 30 seconds at 60°C; 15 seconds at 95°C Every reaction was repeated three times.
Western blot analysis
For Western blotting cells were pelleted and lysed with lysis buffer containing 1% Nonidet P-40, 5% sodium deoxycholate, 1 mmol/L phenylmethyl sulfonylfluoride (PMSF), 100 mmol/L sodium orthovanadate, and 1% protease inhibitor cocktail (Sigma-Aldrich, USA). The protein concentration was assayed according to the BCA protein assay kit manufacturer's protocol (PIERCE, USA) and stored at −70°C. Before loading protein samples were boiled in a sample buffer (62.5 mmol/L Tris-HCl (pH 6.8), 10% (w/v) glycerol, 100 mmol/L DTT, 2.3% sodium dodecylsulfate (SDS), 0.002% bromphenol blue) for 10 minutes. The samples were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Arlington Heights, USA). The membranes were blotted with 5% fat-free milk overnight at 4°C and probed with the primary antibodies. The immunocomplexes were visualized with horseradish peroxidase (HRP)-coupled goat anti-mouse IgG (Promega, USA) using the ECL plus reagents (Amersham Pharmacia Biotech, Arlington Heights, USA). The primary Abs were obtained from the following sources: monoclonal antibody to human A20 (IMGENEX, USA) and anti-β-actin (Sigma).
NF-κB luciferase reporter assay
The ACC-2, ACC-2-A20 or ACC-2-GFP cells (2×105) growing in 6-well plates were transfected with the 2×NF-κB-luciferase plasmid by the Lipofectamine2000 reagents according to the manufacturer's instructions (Invitrogen Life Technoligies). Cells were cotransfected with the pRL-TK Renilla luciferase reporter for normalizing transfection efficiency. Six hours after transfection the complex medium was replaced with growth medium and the cells were incubated at 37°C for additional 48 hours. The second group of cells were treated with TNFα (50 ng/ml) for 12 hours; TNFα can stimulate the activation of NF-κB and luciferase activities were measured with a dual luciferase system (Promega).
Triplicate samples of 2×103 log phase cells were plated in 96-well tissue culture plates. Cell growth was examined daily for 9 consecutive days by adding 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma-Aldrich, USA) to each well and incubating for 4 hours at 37°C. The supernatant was then discarded and 150 μl of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) was added. Absorbance was determined by spectrophotometry (Biohit, BP800, Finland) using a wavelength of 570 nm with 630 nm as a reference.
Cell migration assays
The effect of overexpression of A20 protein on cell migration was examined using the Matrigel Invasion chamber (BD Bioscience, USA) following the manufacturer's instruction. The upper surface of the chamber contained a transwell filter (8-μm pores), coated with Matrigel, fibronectin and vitronectin. Cells (1×105) were added to the upper chamber and incubated for 24 hours in a humidified tissue culture incubator, at 37°C, 5% CO2 atmosphere. The next day a cotton-tipped swab was inserted into the chamber to remove non-invading cells by applying gentle but firm pressure while moving the tip around the membrane surface. The cells on the lower surface of the insert chambers were stained with hematoxylin for 10 minutes. The cell number was counted under a microscope at 400× magnifications and the mean number of cells per field in 10 random fields was recorded. The data were expressed as the average number (±SD) of cells from 10 fields that migrated to the lower surface of the filter from each of 3 experiments performed.
Values are presented as mean ± standard deviation (SD). Results were analyzed with the Student t test and a one-way repeated-measures analysis of variance (ANOVA) using SPSS software package (SPSS, USA). In this study a P<0.05 was considered statistically significant.
To determine the function of the A20 protein in human adenoid cystic carcinoma the A20 expression plasmid was constructed. Complementary DNA encoding full-length wild-type of A20 was generated by PCR; the full length of the A20 cDNA fragment was 2373 bp (Fig. 1A). When the amplified products were digested with EcoRI and KpnI a A20 fragment, which was 2373 bp, was released from the recombined expression plasmid pEGFPN3-A20 while the control pEGFPN3 vector did not generate this fragment (Fig. 1B). In addition, DNA sequencing was also performed to ensure that the full-length of A20 in the recombined expression plasmid was accurate. Sequencing results showed that the A20 gene, which was inserted into the pEGFPN3 plasmid, contained the complete coding region of the A20 gene sequence reported in the GeneBank and the reading frame was not shifted. The sequence was identical with the sequence reported for the A20 sequence in the NCBI and GeneBank databases (Fig. 1C).
A20 gene was stably transferred into ACC-2 cells
To confirm whether the cell line transfected with A20 expressed the transgene, we carried out realtime-PCR to compare the expression of the A20 gene versus the housekeeping gene, actin, using mRNA isolated from transfected and control cells. The ratio of the mRNA levels of ACC-2-GFP and ACC-2-A20 to ACC-2 are shown in Fig. 2A. The expression of the A20 gene was significantly up-regulated in ACC-2-A20 cells, by approximately 4.39 folds, in comparison with expression in ACC-2-GFP cells. But no difference in A20 expression was found between the ACC-2-GFP and ACC-2 cell lines. Western blot analysis further showed that A20 protein levels were also significantly enhanced in ACC-2-A20 cells (Fig. 2B). These data verified that the transgene was expressed in the stably transfected cell lines.
ACC-2-A20 cells had a lower level of NF-κB activity To provide direct evidence as to whether A20 could inhibit constitutive NF-κB activity in ACC-2 cells the 2×NF-κB-luciferase reporter and Rinilla were cotransfected into the ACC-2-A20 and control cells, ACC-2-GFP. As shown in Fig. 3, constitutive NF-κB promoter activity was significantly down-regulated in ACC-2-A20 cells which had a 30.65% decrease of NF-κB activity. When cells were treated with TNFα, the NF-κB activity of ACC-2-A20 cells could be down-regulated about 46.32% in comparison to the ACC-2-GFP cells (P<0.05).
A20 inhibited ACC-2 cell growth and invasion
To determine if A20 could effectively inhibit proliferation of adenoid cystic carcinoma the effect of ectopic expression of A20 on ACC-2 cell growth was examined. Fig. 4A shows the results of representative MTT assays. A20 potently inhibited growth of A20 transfectants ACC-2-A20 cells compared with cells from the control vector transfected groups and the ACC-2 empty control group (P<0.05). In addition, we analyzed whether A20 could reduce the cell migration which was seen in a transwell assay. To that end, 1×105 ACC-2, ACC-2-GFP and ACC-2-A20 cells were placed in the upper compartment of an invasion chamber. After 24 hours of incubation the cells that penetrated to the lower surface of the filter were stained and counted under a microscope. As shown in Fig. 4B, the ACC-2-A20 cells showed significantly reduced ability to invade through Matrigel-coated filters than ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% (P<0.05) (Fig. 4C).
Analysis of our data presented here demonstrates that stable overexpression of A20 in adenoid cystic carcinoma cells partially inhibited NF-κB activity, resulting in a significant reduction of cell growth and invasion. To the best of our knowledge this is the first demonstration that the A20 gene is associated with decreasing tumor invasion; in part through down-regulation of NF-κB expression.
Our studies demonstrate that A20 can partly inhibit the NF-κB signaling pathway in ACC cells, both constitutive and TNFα induced. A number of studies have reported that overexpression of A20 in a number of cell lines results in partial resistance to TNF-induced apoptosis, but this inhibition to apoptosis has not been observed in all cell lines analyzed.15-17 In addition, overexpression of A20 was subsequently shown to block the activation of NF-κB by TNF, IL-1, lipopolysaccharide (LPS), phorbol esters and hydrogen peroxide in different cell types.16,18-20 The underlying mechanism by which A20 down-regulates NF-κB activation in response to some pro-inflammatory cytokines, such as TNF, was shown to exert two opposing activities: sequential de-ubiquitination and ubiqutination of the TNF receptor-interacting protein (RIP), thereby targeting RIP to proteasomal degradation.21-23 In our study, the cells which were transfected with A20 showed a down-regulation of NF-κB promoter activity. Recent data showed that the expression of A20 is itself under the control of NF-κB and suggests that A20 is involved in the negative feed-back regulation of NF-κB activation.24 Our study suggests that overexpression of A20 in ACC cells may simulate the feed-back action in vivo, resulting in NF-κB inhibition.
In addition, NF-κB has been found to be associated with the development and progression of several human malignancies, including breast cancer, pancreatic cancer and head and neck cancer. Several studies have also implicated the NF-κB signaling pathway as being associated with the tumor genesis and clinical outcome of adenoid cystic carcinoma.13,14 Our data provide direct evidence that A20 could down-regulate the ACC cells' growth and invasion though inhibiting the NF-κB activity. But how A20 affects cell growth and invasion through the NF-κB signal pathway is still unclear and is under active investigation. Some researches found that A20 could bind to IKK-γ (also named NEMO) via its C-terminal zinc finger-containing domain.18 In contrast to IKK-α and IKK-β, IKK-γ lacks enzymatic activity and it is believed to transfer the upstream activator signal to IKK-α and IKK-β.25,26 There were some reports that A20 could negatively regulated NF-κB-dependent gene expression by interfering with upstream signaling pathways that were involved in the activation and regulation of NF-κB.24,27,28 In addition, some invasion and metastasis related genes are under the control of NF-κB; such as vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP), urokinase plasminogen activator (uPA) and IL-8.29-31 These studies may provide an explanation of our results that A20 finally inhibited ACC cells growth and invasion.
The increased understanding of the activation and regulation of NF-κB has opened the way for the development of new treatments of human malignancies. Currently, several synthetic and naturally occurring inhibitors of NF-κB-dependent gene expression have been described. Some investigators are encouraging an accelerated FDA approval for their use; such as PS-341 to be used in myelomas. However, some inhibitors could exert a profound immunosuppressive effect.32 Alternatively, inhibitors that target specific cytokine receptor associated molecules involved in NF-κB activation might provide NF-κB inhibition initiated by specific cytokines. In this context A20 might be an interesting candidate for gene therapy. Our study, which showed that A20 inhibited ACC cell growth and invasion, may suggest that A20 can serve as a factor to mimic the activity of endogenous cellular inhibitors of NF-κB, thus providing an additional candidate of therapeutic tools.
1. Khan AJ, DiGiovanna MP, Ross DA, Sasaki CT, Carter D, Son YH, et al. Adenoid cystic carcinoma: a retrospective clinical review. Int J Cancer 2001; 96:149-158.
2. Whatley WS, Thompson JW, Rao B. Salivary gland tumors in survivors of childhood cancer. Otolaryngol Head Neck Surg 2006; 134: 385-393.
3. Codd JD, Salisbury JR, Packham G, Nicholson LJ. A20
RNA expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma. J Pathol 1999; 187: 549-555.
4. Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP, et al. Failure to regulate TNF-induced NF-kappaB and cell death responses in A20
-deficient mice. Science 2000; 289: 2350-2354.
5. Beyaert R, Heyninck K, Van Huffel S. A20
-binding proteins as cellular inhibitors of nuclear factor-kappa B-dependent gene expression and apoptosis. Biochem Pharmacol 2000; 60: 1143-1151.
6. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell 2002; 109 Suppl: S81-96.
7. Aggarwal BB. Nuclear factor-kappaB: the enemy within. Cancer Cell 2004; 6: 203-211.
8. Helbig G, Christopherson KW 2nd, Bhat-Nakshatri P, Kumar S, Kishimoto H, Miller KD, et al. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem 2003; 278: 21631-21638.
9. McNulty SE, Tohidian NB, Meyskens FL Jr. RelA, p50 and inhibitor of kappa B alpha are elevated in human metastatic melanoma cells and respond aberrantly to ultraviolet light B. Pigment Cell Res 2001; 14: 456-465.
10. Sasaki N, Morisaki T, Hashizume K, Yao T, Tsuneyoshi M, Noshiro H, et al. Nuclear factor-kappaB p65 (RelA) transcription factor is constitutively activated in human gastric carcinoma tissue. Clin Cancer Res 2001; 7: 4136-4142.
11. Shishodia S, Aggarwal BB. Nuclear factor-kappaB activation mediates cellular transformation, proliferation, invasion
angiogenesis and metastasis of cancer. Cancer Treat Res 2004; 119: 139-173.
12. Zhang JL, Peng B, Chen XM. The relation of nuclear factor kappa B to angiogenesis and clinical outcomes in adenoid cystic carcinoma of salivary glands. Chin J Stomatol (Chin) 2005; 40: 495-499.
13. Zhang J, Peng B, Chen X. Expressions of nuclear factor kappaB, inducible nitric oxide synthase, and vascular endothelial growth factor in adenoid cystic carcinoma of salivary glands: correlations with the angiogenesis and clinical outcome. Clin Cancer Res 2005; 11: 7334-7343.
14. Fukuda M, Fukuda F, Horiuchi Y, Oku Y, Suzuki S, Kusama K, et al. Expression of CYLD, NF-kappaB and NF-kappaB-related factors in salivary gland tumors. In Vivo
2006; 20: 467-472.
15. Opipari AW Jr, Hu HM, Yabkowitz R, Dixit VM. The A20
zinc finger protein protects cells from tumor necrosis factor cytotoxicity. J Biol Chem 1992; 267: 12424-12427.
16. Jaattela M, Mouritzen H, Elling F, Bastholm L. A20
zinc finger protein inhibits TNF and IL-1 signaling. J Immunol 1996; 156: 1166-1173.
17. De Valck D, Jin DY, Heyninck K, Van de Craen M, Contreras R, Fiers W, et al. The zinc finger protein A20
interacts with a novel anti-apoptotic protein which is cleaved by specific caspases. Oncogene 1999; 18: 4182-4190.
18. Zhang SQ, Kovalenko A, Cantarella G, Wallach D. Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20
bind to NEMO (IKKgamma) upon receptor stimulation. Immunity 2000; 12: 301-311.
19. De Valck D, Heyninck K, Van Criekinge W, Vandenabeele P, Fiers W, Beyaert R. A20
inhibits NF-kappaB activation independently of binding to 14-3-3 proteins. Biochem Biophys Res Commun 1997; 238: 590-594.
20. Heyninck K, Beyaert R. The cytokine-inducible zinc finger protein A20
inhibits IL-1-induced NF-kappaB activation at the level of TRAF6. FEBS Lett 1999; 442: 147-150.
21. Heyninck K, Beyaert R. A20
inhibits NF-kappaB activation by dual ubiquitin-editing functions. Trends Biochem Sci 2005; 30: 1-4.
22. Evans PC, Ovaa H, Hamon M, Kilshaw PJ, Hamm S, Bauer S, et al. Zinc-finger protein A20
, a regulator of inflammation and cell survival, has de-ubiquitinating activity. Biochem J 2004; 378: 727-734.
23. Evans PC, Smith TS, Lai MJ, Williams MG, Burke DF, Heyninck K, et al. A novel type of deubiquitinating enzyme. J Biol Chem 2003; 278: 23180-23186.
24. Krikos A, Laherty CD, Dixit VM. Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20
, is mediated by kappa B elements. J Biol Chem 1992; 267: 17971-17976.
25. Rothwarf DM, Zandi E, Natoli G, Karin M. IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature 1998; 395: 297-300.
26. Yamaoka S, Courtois G, Bessia C, Whiteside ST, Weil R, Agou F, et al. Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation. Cell 1998; 93: 1231-1240.
27. Lee SY, Lee SY, Choi Y. TRAF-interacting protein (TRIP): a novel component of the tumor necrosis factor receptor (TNFR)- and CD30-TRAF signaling complexes that inhibits TRAF2-mediated NF-kappaB activation. J Exp Med 1997; 185: 1275-1285.
28. Rothe M, Xiong J, Shu HB, Williamson K, Goddard A, Goeddel DV. I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction. Proc Natl Acad Sci USA 1996; 93: 8241-8246.
29. Hah N, Lee ST. An absolute role of the PKC-dependent NF-kappaB activation for induction of MMP-9 in hepatocellular carcinoma cells. Biochem Biophys Res Commun 2003; 305: 428-433.
30. Bancroft CC, Chen Z, Dong G, Sunwoo JB, Yeh N, Park C, et al. Coexpression of proangiogenic factors IL-8 and VEGF by human head and neck squamous cell carcinoma involves coactivation by MEK-MAPK and IKK-NF-kappaB signal pathways. Clin Cancer Res 2001; 7: 435-442.
31. Bobrovnikova-Marjon EV, Marjon PL, Barbash O, Vander Jagt DL, Abcouwer SF. Expression of angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 is highly responsive to ambient glutamine availability: role of nuclear factor-kappaB and activating protein-1. Cancer Res 2004; 64: 4858-4869.
32. Olivier S, Robe P, Bours V. Can NF-kappaB be a target for novel and efficient anti-cancer agents? Biochem Pharmacol 2006; 72: 1054-1068.