Notch-1 knockdown suppresses proliferation, migration and metastasis of salivary adenoid cystic carcinoma cells

Notch-1 promotes invasion and metastasis of cancer cells but its role in salivary adenoid cystic carcinoma (SACC) remains unelucidated. Here, we sought to investigate the effect of Notch-1 knockdown on the invasion and metastasis of SACC cells. Stable ACC-M cells whose Notch-1 was silenced by lentiviral vectors were established. Cellular proliferation was evaluated by the MTT assays and clonogenic assays, apoptosis by flow cytometry and the migration of ACC-M cells by Transwell assays. Metastasis was evaluated by examining the number of lung nodules in Balb⁄c nu⁄nu nude mice bearing subcutaneous SACC xenografts. Our MTT assay revealed that Notch-1 knockdown significantly suppressed the proliferation of ACC-M cells compared with non-infected or scrambled control cells. Clonogenic assays further showed that Notch-1 knockdown significantly suppressed the clonogenic growth of ACC-M cells (p < 0.01 vs. controls). Our flow cytometry demonstrated that Notch-1 knockdown was associated with a significantly higher proportion of late apoptotic and necrotic cells (p < 0.01 vs. controls). Transwell assays revealed that Notch-1 knockdown markedly reduced the migratory capacity of ACC-M cells (p < 0.01 vs. controls) and xenograft studies showed that the number of metastatic nodules in the lung surface was significantly lower in nude mice bearing xenografts with Notch-1 knockdown compared to those bearing control xenografts (p < 0.01 vs. controls). Notch-1 knockdown suppresses the growth and migration of SACC cells in vitro and the metastasis of SACC cells in vivo. Notch-1 may be a new candidate target in SACC.


Background
Salivary adenoid cystic carcinoma (SACC) accounts for approximately 7.5 % of all salivary gland epithelial malignancies and 4 % of all salivary gland neoplasms [1]. It is characterized by heterogeneous phenotypic features and persistently progressive biological behavior. SACC patients have a dismal long-term outcome: in a cohort of 160 SACC patients, the overall survival stood at 89 % at 5 years, but declined to 40 % at 15 years [2]. Major contributors to the poor long-term prognosis of SACC patients are local recurrence associated with perineural invasion (PNI) and distant hematogenous metastasis, particularly to the lungs [3,4].
The Notch signaling pathway is a highly evolutionarily conserved signaling pathway that regulates various physiological processes, including stem cell maintenance, differentiation, proliferation and apoptosis [5,6]. Recently, accumulating evidence has implicated aberrant Notch signaling in the oncogenesis of many human malignancies. However, little is known about the differential role of various components of the Notch pathway in SACC. Notch-1, one of the key receptors in the Notch signaling pathway, encodes an important member of the Notch family proteins [7]. Whole genome sequencing of adenoid cystic carcinoma cells has identified mutations in Notch-1 though the functional significance of the mutations is unknown [8]. Additionally, Notch-1 expression was found to be elevated in SACC tissues [9]. Notch-1 has also been shown to promote the proliferation of SACC and head and neck cancer cells [9,10]. We have demonstrated by microarray analysis that Notch-1 and Notch-4 are implicated in the pathobiology of SACC by influencing perineural invasion [11]. Furthermore, Notch-1 cross-talk has also been reported with other major cell growth and apoptotic regulatory pathways through modulating the activity of transcription factors such as nuclear factor-κB (NF-κB) and Wnt/β-catenin signaling [12,13].
Notch-1 was shown to promote the invasion and metastasis of esophageal carcinoma cells by inducing epithelialmesenchymal transition (EMT) [14]. Activation of Notch-1 signaling was demonstrated in gefitinib-resistant lung cancer cells undergoing EMT [15]. However, a role of Notch-1 has not been suspected in the invasion and metastasis of SACC cells. We hypothesized that Notch-1 may be involved in the pathogenesis of SACC by promoting the invasion and metastasis of SACC cells. In the current study, we sought to elucidate the role of Notch-1 in invasion and metastasis of SACC cells by examining the proliferation and migration of SACC cells whose Notch-1 expression was knocked down by lentiviral vector-mediated RNA interference (RNAi), and by evaluating the presence of metastatic nodules in nude mice bearing SACC xenografts.

Materials and methods
Cell lines and cell culture condition ACC-M and 293 T cells were purchased from the Cell Bank for Type Culture Collection, Chinese Academy of Sciences (Beijing, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen. Carlsbad, CA, USA) supplemented with 10 % of fetal bovine serum (FBS) (Gibco, Invitrogen), 2.05 mM L-glutamine, 100 μg/mL of penicillin and 100 μg/mL of streptomycin at 37°C with 5 % CO 2 .

Generation of stable Notch-1-silencing cells
Lentiviral vectors (pLenR-GPH, pMDLg/pRRE, pRSV-Rev, and pMD2.G) were purchased from Innovation Biotechnology (Shanghai, China). The sequences of siRNAs are shown in Table 1. SiRNAs were designed according to the cDNA sequence of the Notch-1 gene (NM_017617.3) in GenBank and were inserted into the BamHI and EcoRI site of linearized pLenR-GPH according to the manufacturer's instructions. The recombinant vectors were confirmed by endonuclease restriction analysis with KpnI and EcoRI and sequencing and were designated pLenR-Notch-1-shRNA1, 2, and 3 ( Fig. 1).
ACC-M cells were seeded at a density of 2 × 10 5 ⁄ well in 6-well plates. After 24 h incubation, the cells were transfected with appropriate lentivirus vectors at a multiplicity of infection of 10 as instructed by the manufacturer (Innovation Biotechnology). Stable ACC-M cells containing the lentivirus vectors were established after selections with appropriate antibiotics. Fluorescent microscopy revealed a transfection rate of 90 % (Fig. 2).

Western blotting assays
Cells were washed twice with cold phosphate-buffered saline and lysed on ice in buffer containing protease inhibitors. Equal amounts of protein (20 μg/lane) from the cell lysates were electrophoresed on 10 % acrylamide gels. After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 2 h in PBS plus 0.1 % Tween 20 and 5 % non-fat skim milk to block non-specific binding. Subsequently, the membrane was incubated for 2 h with an antibody against Notch-1 (Abcam, Cambridge, UK) and an anti-GAPDH antibody. The protein bands were visualized using an ECL detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Notch-1 expression was normalized against GAPDH. The experiment was repeated three times.

Cell proliferation and clonogenic assays
For cell proliferation studies, cells were seeded at a density of 1 × 10 3 per well on 96-well plates. After 8 days, cell viability was assessed by the tetrazolium-based semiautomated colorimetric 3-[4, 5-dimethylthiazol-2-yl] -2, 5diphenyltetrazolium bromide (MTT) reduction assays. Spectrometric absorbance was read at 490 nm using a microplate reader. For clonogenic survival assays, cells were plated at 400 cells/well in 6-well culture dishes 96 h post lentiviral infection. After 1 week, cell colonies were fixed with 4 % paraformaldehyde and stained with crystal violet (Sigma). An aggregate of at least 50 cells was considered a colony and was counted. Each group was repeated three times.

Flow cytometry
Stable Notch-1 knockdown ACC-M cells were washed once with PBS, trypsinized and washed again in PBS with 2 % FBS and fixed in 70 % ice-cold ethanol for 2 h. Cells were washed, and stained with propidium iodide

Xenograft studies
Six-to 8-week-old Balb⁄ c nu⁄ nu nude mice were inoculated with 2 × 10 5 ACC-M cells in 200 μL culture medium via the tail vein. Six weeks after inoculation, all animals were euthanized and the lungs were removed. The number of metastatic nodules on the surface of the lungs was counted. Then, the metastatic nodules were paraffin-embedded for immunohistochemical staining.
The study protocol was approved by the local animal ethics committee of Jinling Hospital (Project NO.2014GJJ-003) and was carried out in accordance with the local and state regulations regarding the use of animals for experimental purposes.

Immunohistochemistry
An S-P Kit (Gene Tech, Shanghai) was used for immunohistochemical analysis. In brief, paraffin sections (4 μm thick) were deparaffinized in xylene and rehydrated in gradient alcohol. Antigen retrieval was performed by microwave treatment in 0.01 mol/L sodium citrate buffer (pH = 6.0) for 15 min, cooled to room temperature for 1 h, and then washed with phosphate-buffered saline (PBS). Endogenous peroxidase activity was inactivated with 0.3 % hydrogen peroxide for 30 min and non-specific blocked with avidin/biotin blocking solutions and 10 % normal goat serum. The sections were then incubated overnight with primary antibody against Notch-1 (1:150, Abcam, Cambridge, UK). Biotinylated goat anti-rabbit IgG were used as secondary antibody. The immunoreactions were detected by staining with 3,3′diaminobenzidine. Negative controls were performed by omitting the primary antibody. The slides were

Statistical analysis
Data were expressed as mean ± standard deviation (SD) and analyzed using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). Data were tested for statistical significance using analysis of variance (ANOVA) or twotailed unpaired Student's t-test. Normally distributed, continuous variables were compared using one-way ANOVA. When ANOVA produced a significant difference between groups, multiple comparisons of group means were performed using the Bonferroni procedure with a type I error adjustment. All p values were two-sided, and significance was defined as p < 0.05.

Efficiency of Notch-1 knockdown by lentiviral vectors
We infected ACC-M cells with a panel of lentiviral vectors bearing siRNA targeting Notch-1 and quantitative RT-PCR revealed that all three lentiviral vectors caused significant reduction in the mRNA transcript levels of Notch-1 (p < 0.01 vs. non-infected ACC-M cells (negative control) or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (positive control, ACC-M Mock)) (Fig. 3a). Furthermore, pLenR-Notch-1-shRNA1 caused the most significant inhibition in Notch-1 mRNA transcript levels. Consistently, immunoblotting assays showed marked reduction in the protein level of Notch-1 by the lentiviral vectors, most noticeably pLenR-Notch-1-shRNA1 (Fig. 3b), which was used for subsequent experiments.

Notch-1 knockdown suppresses clonogenic growth of SACC cells in vitro
To examine whether lentiviral vector-mediated knockdown of Notch-1 impacted on the proliferation of cancer cells, we examined the growth of lentivirally infected ACC-M cells by MTT assays. We found that siRNA cells (p < 0.01) (Fig. 4a). Clonogenic assays further showed that Notch-1 knockdown significantly suppressed the clonogenic growth of ACC-M cells (116 ± 7) compared with non-infected ACC-M cells (176 ± 13) or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (164 ± 12) (p < 0.01) (Fig. 4b, c). These findings demonstrated that Notch-1 knockdown significantly suppressed the clonogenic growth of salivary adenoid cystic carcinoma cells in vitro.

Notch-1 knockdown induced late apoptotic and necrotic death of SACC cells in vitro
We first investigated whether the growth-inhibitory effect of Notch-1 knockdown on ACC-M cells was due to reduced DNA synthesis. Our flow cytometric analysis revealed no statistically significant difference in the proliferation index of Notch-1 knocked down cells, non-infected ACC-M cells or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (p > 0.05) ( Table 2, Fig. 5), indicating that Notch-1 downregulation did not impact on the cell cycle distribution of ACC-M cells. We next investigated whether the growth-inhibitory effect of Notch-1 knockdown was associated with changes with apoptotic or necrotic death of lentivirally infected ACC-M cells. Our flow cytometry showed that Notch-1 knockdown was associated with a significantly higher proportion of late apoptotic cells (63.7 ± 3.8 %) compared to non-infected ACC-M cells (45.4 ± 2.6 %) or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (46.3 ± 2.7 %) (p < 0.01) ( Fig. 6a and b). Furthermore, Notch-1 knockdown was also associated with a significantly higher proportion of necrotic cells (17.2 ± 1.8 %) compared to non-infected ACC-M cells (3.7 ± 0.5 %) or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (2.4 ± 0.7 %) (p < 0.01) (Fig. 6c). We also noticed that lentiviral vectors bearing scrambled siRNA caused a noticeable increase in the proportion of early apoptotic cells (31.2 ± 1.4 %) vs. Notch-1 knockdown (9.8 ± 0.8 %; p < 0.01). These results suggest that knockdown of Notch-1 caused a significant increase in the proportion of late apoptotic and necrotic ACC-M cells.

Notch-1 knockdown inhibits the migratory capacity of SACC cells
Notch-1 has been implicated in tumor metastasis in a variety of cancer types [14]. We investigated whether Notch-1 downregulation influenced the migratory capacity of salivary adenoid cystic carcinoma cells. Our Transwell assays showed that ACC-M cells infected with pLenR-Notch-1-shRNA1 exhibited a markedly reduced migratory capacity compared with non-infected ACC-M cells or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (p < 0.01) (Fig. 7). We then inoculated nude mice with lentivirally infected ACC-M cells and evaluated the presence of metastatic nodes in the lungs after 6 weeks. Immunohistochemical staining of tumor xenografts showed a marked reduction in the expression of Notch-1 in ACC-M cells whose Notch-1 was knocked down by pLenR-Notch-1-shRNA1 compared to non-infected ACC-M cells or ACC-M cells infected with lentiviral vectors bearing scrambled siRNA (immunoreactivity score: Notch-1 knockdown, 0.0 vs. scrambled siRNA, 5.0; p < 0.01) (Fig. 8c). Furthermore, the number of metastatic nodules in the lung surface was significantly lower in nude mice bearing xenografts with Notch-1 knockdown compared to those bearing control xenografts ( Fig. 8a and b). The results indicated that Notch-1 was also implicated in the metastasis of salivary adenoid cystic carcinoma cells in vivo.

Discussion
Notch signaling regulates cell fate, stem cell maintenance and the initiation of differentiation in many tissues and aberrant Notch signaling is intimately implicated in oncogenesis and development. Notch-1 expression was found to be elevated in SACC tissues [9], but its role in the growth and metastasis of SACC remains largely undefined. In the current study, we demonstrated that Notch-1 downregulation significantly suppresses the clonogenic growth and the migratory capacity of SACC cells in vitro. More importantly, Notch-1 knockdown markedly inhibited the formation of metastatic lung nodules in a mouse model bearing SACC xenograft. Our study has provided the piece of experimental evidence that Notch-1 is implicated in the growth and invasion of SACC in vitro and in vivo, paving the way for further elucidation of the role of Notch-1 in the oncogenesis and development of SACC.
We further demonstrated that the growth inhibitory effect of Notch-1 knockdown was at least partially associated with significantly enhanced late apoptosis and necrotic death of SACC cells. This is consistent with the findings by Ye et al. who demonstrated that Notch-1 knockdown promoted docetaxel induced growth inhibition and apoptosis of prostate cancer cells in vitro [16]. Li et al. also found that Notch-1 activation inhibited apoptosis of MDA-MB-231 breast cancer cells [17]. We further demonstrated that the growth inhibitory effect of Notch-1 knockdown was not due to altered cell cycle distributions of SACC cells. Our findings and those by other investigators suggest that Notch-1 regulates cancer cell growth at least partially by modulating apoptosis of these cancer cells.
Our previous microarray study revealed that Notch-1 and Notch-4 were notably overexpressed in the perineural invasion group, providing the hint of the involvement of Notch-1 in the invasion and metastasis of SACC [11,18]. Notch-1 was actively involved in promoting the metastasis of esophageal carcinoma cells by inducing EMT [14] and its activation was also implicated in potentiating EMT of gefitinib-resistant lung cancer cells [15]. The current study demonstrated that Notch-1 knockdown significantly inhibits the migratory capacity of salivary adenoid cystic carcinoma cells and reduces the number of metastatic nodules in the lung surface of mice bearing SACC xenografts, indicating that Notch-1 is implicated in the metastasis of salivary adenoid cystic carcinoma cells in vivo. Tumor progression is the process by which tumor cells acquire malignant properties, such as aggressive growth, perineural invasion, and metastasis. By targeting critical signaling molecules in the acquisition of malignant phenotypes of SACC, we may slow down the progression of SACC. Our results indicate that Notch-1 is implicated in the invasion and metastasis of SACC cells, and may be manipulated therapeutically to delay the migration and metastasis of SACC cells. Our findings of Notch-1 as a critical signaling molecule in SACC would lead to additional studies to delineate the role of Notch-1 in the oncogenesis and development of SACC and the underlying mechanism.

Conclusions
In conclusion, we demonstrate that Notch-1 knockdown suppresses the growth and migration of SACC cells in vitro and the metastasis of SACC cells in vivo. As a key regulator of growth and metastasis of SACC cells, Notch-1 may be a new candidate target for the treatment of SACC.

Competing interest
The authors declare that they have no competing interests.
Authors' contributions WC conceived of and designed the study, carried out the Notch-1-silencing experiments and drafted the manuscript. GC carried out the quantitative RT-PCR and helped to draft the manuscript. XRY carried out the flow cytometry and helped to draft the manuscript. XZ carried out the western blotting. QQZ carried out the cell proliferation and clonogenic assays. YLZ participated in the cell migration assays. ZD participated in the xenograft studies. SLZ conceived of and designed the study, carried out the immunohistochemistry and helped to draft the manuscript. All authors read and approved the final manuscript.