Over-expression of the special AT rich sequence binding protein 1 (SATB1) promotes the progression of nasopharyngeal carcinoma: association with EBV LMP-1 expression

Background Special AT rich sequence binding protein 1 (SATB1) plays a crucial role in the biology of various types of human cancer. However, the role of SATB1 in human nasopharyngeal carcinoma (NPC) remains unknown. In the present study, we sought to investigate the contribution of aberrant SATB1 expression in the progression of NPC and its association with the Epstein Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1). Methods Immunohistochemical analysis was performed to detect SATB1 and LMP-1 protein in clinical samples, and the association of SATB1 protein expression with patient clinicopathological characteristics and LMP-1 expression were analyzed. SATB1 expression profiles were evaluated in well-differentiated NPC cell line CNE1, poorly-differentiated CNE2Z, undifferentiated C666-1 and immortalized nasopharyngeal epithelia NP-69 cells using quantitative RT-PCR, western blotting and fluorescent staining. After inhibition the SATB1 expression by using SATB1 specific small interfering RNA in these cell lines, the change of cell proliferation was investigated by western blotting analysis of PCNA (proliferating cell nuclear antigen) expression and CCK-8 assay, and the cell migration was assessed by Transwell migration assay. Finally, the expressions of SATB1 and PCNA were examined in CNE1 cells that forced LMP-1 expression by fluorescent staining and RT-PCR. Results Immunohistochemical analysis revealed that SATB1 protein expression was elevated in NPC tissues compared to benign nasopharyngeal tissues (P = 0.005). Moreover, high levels of SATB1 protein expression were positively correlated with clinical stage (P = 0.025), the status of lymph node metastasis (N classification) (P = 0.018), distant metastasis (M classification) (P = 0.041) and LMP-1 expression status (r = 2.35, P < 0.01) in NPC patients. In vitro experiments demonstrated that an inverse relationship between SATB1 expression and NPC differentiation status, with SATB1 weakly expressed in NP-69 cells and CNE1 cells, and significant increasingly expressed in CNE-2Z and C666-1 cells. Targeted knockdown of SATB1 expression obviously attenuated the proliferation and migration of highly SATB1-expressing CNE2Z and C666-1 cells, but not NP-69 and CNE1 cells. Interestingly, forced LMP-1 expression in CNE1 cells led to a surprisingly increasing SATB1 expression and nuclear location, companying with an up-regulated PCNA expression. Conclusions Our results reveal that EBV LMP-1-mediated over-expression of SATB1 is associated with NPC progression, suggesting SATB1 may represent a promising biomarker and therapeutic target for NPC.


Background
The nuclear factor special AT-rich sequence binding protein 1 (SATB1), which was cloned in 1992, is considered to be a global chromatin organizer [1]. By providing a nuclear architectural platform, SATB1 regulates gene expression by promoting chromatin remodeling and regulating the levels of histone acetylation and methylation [2,3]. Under normal physiological conditions, SATB1 is highly expressed in the thymus, with low levels also present in the testes, fetal brain and osteoblasts; however, expression is virtually undetectable in other tissues [4,5]. SATB1-null mice display abnormal expression of numerous genes involved in T-cell development [5], implying a critical role for SATB1 in the maintenance of the immune system.
Recently, accumulating data suggests that over-expression of SATB1 contributes to tumor progression [6]. Studies by Han et al. revealed that SATB1 is over-expressed in aggressive breast cancer cells and the level of SATB1 expression had high prognostic significance. In this study, targeted silencing of SATB1 led to altered expression of >1000 genes, predominantly controlling cell adhesion, signaling, extracellular matrix formation and cell cycle [7]. This report opened a new window regarding the role of SATB1 in tumor biology, despite controversial studies [8,9]. To date, high expression of SATB1 has been linked to the progression of human gastric cancer [10][11][12], ovarian carcinoma [13], liver cancer [14][15][16], rectal cancer [17,18], laryngeal squamous cell carcinoma [19], cutaneous malignant melanoma [20], and prostate cancer [21,22]. In contrast to these reports, however, Selinger et al. reported that loss of SATB1 is associated with poor prognosis in lung squamous cell carcinoma [23]. Thus, the role of SATB1 in tumorigenesis remains unclear and requires further investigation.
Nasopharyngeal carcinoma (NPC) is a malignant cancer derived from the nasopharyngeal epithelium. Over the past decades, epidemiological studies have shown that NPC appeared the unique prevalence features, including regional, racial familial aggregation and Epstein-Barr virus (EBV) infection status. NPC is one of the most common malignant diseases in Chinese and persons of south-east Asian descent, and the provinces of Guangdong, Guangxi, Hainan, Hunan and Fujian are the five most endemic areas in China [24]. Histologically, non-keratinizing carcinoma accounts for >97% of NPC cases in endemic areas [25]. Clinically, most NPC patients present with cervical lymphadenopathy at primary diagnosis [26], which represents a more advanced stage. Therefore, current approaches aimed at improving the rate of early detection, diagnosis and treatment in NPC patients, deserves significant attention.
NPC is an EBV associated carcinoma, and EBV-encoded oncogene latent membrane protein 1 (LMP-1) contributes to the carcinogenesis of NPC. Previous studies in our department focused on the role of the LMP-1 in the pathogenesis of NPC [27][28][29][30][31]. In the present study, we aimed to clarify the role of aberrant SATB1 expression in NPC progression. We investigated the correlation between SATB1 protein expressions with NPC patient clinicopathologic parameters. Furthermore, we analyzed the expression of SATB1 in a panel of NPC cell lines characterized by varied differentiation and LMP-1 expression status. Using RNA interference (RNAi), we investigated the effect of targeted knockdown of SATB1 on NPC cell proliferation and migration. Finally, the expression of LMP-1 and its association with SATB1 expression in NPC were investigated.

Patients and specimens
Clinical, paraffin-embedded samples were obtained from 119 patients from the Affiliated Hospital of the Guangdong Medical College (Zhanjiang, Guangdong, China) and the People's Hospital of Zhongshan City (Zhongshan, Guangdong, China). All patients did not receive preoperative radiotherapy or chemotherapy. The use of the human tissue in this study was approved by the Ethics Council of the Affiliated Hospital of the Guangdong Medical College and the People's Hospital of Zhongshan City for Approval of Research Involving Human Subjects. Subjects comprised NPCs (n = 95; 71 men, 24 women) and nasopharyngeal epithelial hyperplasia (NEH, n = 24; 18 men, 6 women). Clinical data of patients was reviewed based on the pathology tumor-node-metastasis (pTNM) system (AJCC ⁄ UICC 2002). Among 119 cases, the clinicopathologic variables of 115 patients were shown as described previously [27], and the remained 4 cases were newly involved. NPC patients were diagnosed for the first time at an average age of 42.9 years (range, 23-72 years). There were 5 samples in stage I, 10 samples in stage II, 36 samples in stage III, and 44 samples in stage IV. All NPC patients were diagnosed with non-keratinizing carcinoma following histological examination.

In vitro cell proliferation assay
Cell proliferation was assessed 24 hours and 48 hours post-transfection with siRNAs using the Cell Counting Kit-8 (CCK-8) assay, in accordance with the manufacturer's instructions (Biyuntian, Jiangsu, China). Briefly, transfected cells (4 × 10 3 per well) were seeded in 96-well plates and cultured for either 24 hours or 48 hours, after washed with 1 × PBS, 10 μl CCK-8 reagent plus 100 μl basal DMEM medium was added per well and incubated at 37°C for 2 hours. The optical density (OD) was subsequently measured using a microplate reader (Multiskan MKS, Thermo Scientific, Waltham, MA, USA) by dual wavelength mode (450/630 nm). Data represent the mean ± SD of three independent experiments.

Transwell migration assay
In vitro cell migration assays were performed as previously described [30] using Transwell chambers (8-μm polycarbonate membranes, BD Biosciences). In brief, 48 hours following transfection with si-SATB1, si-Control or no transfection, cells were harvested in DMEM supplemented with 0.5% FBS. 2 × 10 4 cells were seeded in the upper chamber which was placed over the lower chamber containing DMEM medium supplemented with 10% FBS. Migration was allowed to proceed for 12 hours at 37°C, and insert membranes were fixed for 20 minutes with 70% ethanol, and stained with 0.5% Eosin. Membranes were washed with PBS and cut from the inserts. Cells on the upper surface of the membrane were removed with a cotton swab and membranes were mounted with glycerol.
The number of migrated cells on the lower surfaces of the membranes was determined by counting fifteen representative fields from triplicate inserts.

Statistical analyses
Statistical analyses were performed using PRISM Software of differences between two groups, Student's t-test was performed. For multiple groups, ANOVA was performed followed by the Student-Newman-Keuls test. P values <0.05 were considered statistically significant.

Results
Over-expression of SATB1 protein is correlated with clinicopathological features of human NPC  Figure 1), suggesting a crucial role for SATB1 over-expression in NPC pathogenesis. We next analyzed the association of SATB1 expression with the clinical parameters of NPC patients. While no correlation was observed between SATB1 high expression and patient gender, age or smoking condition, a positive correlation with clinical classification (I-II vs III-IV, P = 0.025), N classification (N0 vs N1-N3, P = 0.018) and M classification (M0 vs M1, P = 0.041) was observed (Table 1). We also observed an association between SATB1 expression and T classification (T1-T2 vs T3-T4, P = 0.077), although this was not statistically significant. Together these results strongly indicate that high expression of SATB1 protein contributes to the clinical progression of human NPC.

Expression features of SATB1 in NPC and NP-69 cell lines
To further examine the relationship between SATB1 expression and NPC pathogenesis, we analyzed the expression of SATB1 in three cell lines representing various differentiation stages of NPC. As shown in Figures 2 and 3, the expression of SATB1 mRNA and protein varied between the different NPC cell lines. In general, we observed a very low level of SATB1 expression in immortalized NP-69 cells, which was predominantly localized in the cytoplasm. In contrast, SATB1 expression was increasingly observed in well-differentiated CNE1 cells, poorly-differentiated CNE2Z cells and undifferentiated C666-1 cells. SATB1 mainly located in cytoplasm in CNE1 and CNE2Z cells, but increasing nucleus location in C666-1 cells was observed ( Figure 3). These results suggest that SATB1 expression is inversely associated with the cellular differentiation status of NPC.

Inhibition of SATB1 attenuates NPC cell proliferation
To investigate the effect of SATB1 on NPC cell proliferation, we performed knockdown of SATB1 in C666-1 cells using three independent siRNAs targeting the SATB1 coding region. Initial testing of these siRNAs revealed that the cocktail of si-SATB1-1 and si-SATB1-2 displayed the significant degree of SATB1 knockdown in C666-1 cells (inhibitory rate >85%, data not shown), and this cocktail of two pair of siRNA oligos was therefore utilized in subsequent experiments. And the results of western blotting confirmed that transfection this siRNA cocktails to NPC cells led to a significant decrease of SATB1 protein expression, besides, attenuated PCNA protein expressions were also observed ( Figure 4). Then the cell proliferation was examined by CCK-8 assays post transfection these 4 cell line with SATB1 specific siRNA cocktails. As shown in Figure 5, transfection of the NPC cell lines with this siRNA cocktails led to significantly damaged cell viability in CNE2Z and C666-1 cells, but not the CNE1 and NP-69 cells. Taken together, these results demonstrate that over-expression of SATB1 plays a crucial role in NPC cell proliferation.

Inhibition of SATB1 represses NPC cell migration
Cervical lymph node metastasis is one of the most common clinical symptoms of NPC patients at primary diagnosis [26], therefore blocking the migration of NPC cells may represent a promising treatment strategy. Indeed, treatment of highly SATB1-expressing CNE2Z and C666-1 cells with SATB1-siRNAs led to a significant decrease in cell migration, while migration was unaffected in CNE1 cells and immortalized NP-69 cells ( Figure 6). These results suggest that SATB1 over-expression in NPC cells promotes migration. samples were the same SATB1 high expression (IRS ≥6).
A positive correlation of LMP-1 to SATB1 expression in clinical NPC samples was appeared (r = 2.35, P < 0.01, Figure 7). Further, we performed immunflurescent staining, western blotting and RT-PCR analysis to assess LMP-1 expression in CNE1, CNE2Z and C666-1 cells lines. As results, there were no obviously LMP-1 protein expressions in three NPC cell lines, which were verified by results of immunflurescent staining and western blotting, however, the transcripts of LMP-1 could be notably detected in C666-1 cells; interestingly, LMP-1 transcripts expression levels in NPC cell lines were positively associated with SATB1 mRNA expression (Figure 7). To further investigate the relationship between LMP-1 and SATB1 in NPC cells, a LMP-1 over-expression plasmid (PAT-LMP-1-GFP) was transfected into CEN1 cells to generate LMP-1 stably expressed NPC cell line CNE1-GL [31], then we found that forced LMP-1 expression in CNE1 cells led to a surprisingly increased SATB1 expression and nuclear location, companying with the up-regulation of PCNA ( Figure 8).
Together these results referred that LMP-1 may mediate SATB1 expression in NPC cells.
Discussion SATB1 promotes tumor metastasis by providing a nuclear architectural platform regulating the expression of >1000 genes, many of which are related to cell growth and translocation [7]. In the present study, we detected over-expression of SATB1 protein (IRS ≥6) in 65.3% of NPCs (62/95), which was significantly higher than that observed in benign NEH (8/24), suggesting that SATB1 may play a role in NPC pathogenesis. We then further assessed the correlation between SATB1 protein overexpression and NPC patient clinical parameters. We observed that SATB1 protein over-expression was positively reports showing that high expression of SATB1 promotes cancer progression in multiple human carcinomas [7,[11][12][13][14]17,19,32]. We next examined SATB1 expression in NPC cell lines at various stages of differentiation. We found that when compare with a basal levels of SATB1 expression in the immortalized NP-69 cells, the well-differentiated CNE1 cells displayed a low levels of SATB1 expression, while the highly proliferative and poorly-differentiated CEN2Z cells, and undifferentiated C666-1 cells exhibited increasing SATB1 expression and a increase of nuclear localization (Figure 2 and 3). These results indicate that the expression of SATB1 is inversely correlated to NPC cell differentiation. Our findings support the very recent report of Barboro et al., demonstrating that SATB1, a DNA matrix attachment regions (MARs) binding protein, is involved in the differentiation of prostate cancer cells [33]. In the present study, SATB1 expression co-localized with PCNA, a marker of cell proliferation, in NPC cells (Figure 3). This is in accordance with a study by Chu et al. [34], which showed that SATB1 expression was positively correlated with Ki67 expression in gliomas.
In human epithelium-derived carcinoma cells such as breast cancer cells, inhibition of SATB1 expression by siRNA or Decoy-DNA led to reversal of cell malignancy [7,32]. In addition, knockdown of SATB1 in human nonepithelium-derived cancers such as U251 glioma cells also led to inhibition of cell growth, invasion, metastasis and angiogenesis [34]. These results highlight the possibility of molecularly targeting SATB1 in cancer treatment. In this study, we found that treatment of SATB1-expressing NPC cells with SATB1-specific siRNAs, attenuated cell proliferation and motility which verified by the changes of expression of PCNA (Figure 4), cell viability ( Figure 5) and cell migration ( Figure 6). Our results support the rationale that targeting SATB1 may represent an alternative therapeutic strategy in the treatment of NPC.
NPC is an EBV associated malignant carcinoma, especially in endemic area such as Southern China. Previous studies in our department focused on the role of the EBVencoded LMP-1 in the pathogenesis of NPC [31,[35][36][37][38], and the results of our lab and others have previously indicated that the LMP-1 could promote NPC progression. In the present study, our preliminary data revealed that LMP1 expression was positively associated with SATB1 expression in clinical NPC specimens (r = 2.35, P < 0.01); in addition, the expression level of SATB1 was correlation with LMP-1 expression status in NPC cell lines ( Figure 7); much interestingly, ectopic expression of LMP-1 strongly promoted SATB1 expression and its nuclear location, as well as an up-regulation of PCNA, in well-differentiated CNE1 cells ( Figure 8). Combining results about the SATB1 expression styles in CNE1, CNE2Z, C666-1 and CNE-GL cells and its correlation to LMP-1 expression in clinical NPC samples, it seemed that LMP-1 stimulates cell proliferation partly via up-regulating of SATB1 expression in NPC cells.
The process of epithelial-to-mesnchymal transition (EMT) is one of several key factors involved in tumor metastasis. Previous reports have shown that NPC cells have undergone the process of EMT [39][40][41], in particular the neoplastic spindle cells in NPC [40]. Our present results showed that knockdown of STAB1 expression by SATB1 specific siRNA led to an obvious up-regulation of E-cadherin and a down-regulation of Vinmentin, the marker for EMT (Additional file 2: Figure S1). These results therefore support previous reports regarding the role of EBV infection in EMT in NPC [41][42][43]. Very recently, a report of Endo K and colleagues had revealed that EBV LMP1 could induce SATB1 expression in human NPC [44]. Thus, we hypothesized that EBV may promote NPC progression partly by modulation the process of EMT via LMP1-mediated SATB1 expression.

Conclusions
Taken together, our study provides the evidence that EBV LMP-1 expression may associate with SATB1 expression in NPC; over-expression of SATB1 is closely related to the clinical progression of NPC, SATB1 was universally expressed in all NPC cell lines examined, and its expression levels were inversely related with cell differentiation; knockdown of SATB1 by specific siRNA led to attenuated cell proliferation and migration. Our results highlight the possibility of SATB1 as a promising biomarker and therapeutic target for NPC. Future studies will focus on i) factors such as LMP-1 controlling SATB1 expression in the NPC cells, ii) the combination of factors working in concert with SATB1 in NPC cells, and iii) investigating how SATB1 mediates the process of EMT in NPC cells.

Additional files
Additional file 1: Primer pairs used for PCR.
Additional file 2: Figure S1. Knockdown of SATB1 in NPC cells lead to changes of EMT marker genes.