Increased Nanog Expression Promotes Tumor Development and Cisplatin Resistance in Human Esophageal Cancer Cells

Background/Aims: Nanog plays a key role in stem cell self-renewal and pluripotency differentiation in embryonic stem cells ( ESCs). Recently, some studies reported that abnormal expression of Nanog could be detected in several tumors, indicating that Nanog might be related to tumor development. However, studies on the correlation between Nanog expression and esophageal cancer are sparse. Methods: In this study, we established two esophageal cancer cell lines 9706-Nanog and 9706-shNanog which stably expressed Nanog and Nanog-short-hairpin RNA (shRNA) genes. Results: We found that Nanog expression could promote the proliferation and invasiveness of the cancer cells, and inhibit the apoptosis. We also treated 9706-Nanog, EC9706 and 9706-shNanog cell lines with cisplatin and evaluated the drug sensitivity of the three cell lines. We found that the sensitivity of cisplatin was decreased with increased expression of Nanog. The expression of MDR-1 was also increased in 9706Nanog cells. Conclusions: Nanog may play an important role in human esophageal cancer development, and could be used as a therapeutic target in esophageal cancer treatment.


Introduction
Esophageal cancer is one of the most common malignant tumors with high mortality rates in Hebei, Henan and east coast of China [1]. The treatments for esophageal cancer include surgery, chemotherapy and radiotherapy but the ef�icacy of these therapies is unsatisfactory [2,3]. Thus it's important to develop new therapeutic strategies for esophageal cancer. Previous studies found that a core regulatory network including three stemness factors Sox2, Oct3/4, and Nanog coordinately determines embryonic stem cells (ESCs) selfrenewal and differentiation, and further studies have demonstrated that Nanog is expressed at high levels in ESCs and that the expression levels decrease after differentiation of ESCs [4][5][6][7]. There were many similar biological properties between cancer cells and embryonic stem (ES) cells, such as the characteristics of continuous proliferation and uncontrollable differentiation, which indicating these ESC self-renewal molecules may conceptually also contribute to tumorigenesis and development. Some studies have suggested that these factors could regulate self-renewal and pluripotency differentiation of cancer stem cell [8] and may play a role in human malignancy [9]. Previous studies have found that Sox2 might promote cell proliferation and tumorigenesis of breast cancer [10], and Oct3/4 might associated with the early stage of pancreatic cancer carcinogenesis [11,12], and even correlated with lymph node metastasis in colorectal cancer cells [13]. High expression of OCT-4 has been observed in esophageal cancer [14], suggesting association between OCT-4 and proliferation of esophageal cancer.
Nanog is a member of ANTP class NK family genes and plays a key role in stem cell self-renewal and pluripotency differentiation [4,5]. In addition to self-renewal regulation of embryonic development, the abnormal expression of Nanog gene is found in malignant germ cell tumors, such as embryonic carcinoma and seminoma [8]. The abnormal expression of Nanog is also detected in solid tumors, such as pulmonary [15], breast [16], cervix [17], oral cavity [18], ovary [19], gastrointestinal [20] and kidney [21] cancer. Jeter CR et al. [9] evaluated the expression, origin, and functions of NANOG in different tumor cells, and found that multiple tumor cells in vitro and in vivo express NANOG (NANOG mRNA is derived from a Transcribed pseudogene, NANOGP8), and down regulation of NANOG inhibits tumor cells development associated with an inhibition of cell proliferation, clonal expansion, and clonogenic growth of tumor cells. This systematical investigation demonstrated that NANOG expression in human cancer cells is biologically functional in regulating tumor development. In addition, researchers also found that Nanog overexpression may induce chemo-resistance in oral squamous cell carcinoma [22] and prostate cancer [23], promote the tumor recurrence to resist cisplatin. Comprehensive and systematic studies of NANOG expression in human tumor cells have been proceeded, but research of the correlation between Nanog expression and esophageal cancer cells development is lacking.
In our pilot study, the supression effect of shRNA target Nanog gene was demonstrated in vitro. The suppression effect was compared with the off-target effect of control shRNA, and eukaryotic expression vectors pcDNA3.1-Nanog and pSUPER-EGFP-shNanog, which could respectively express and knock-down Nanog gene in human esophageal cancer cell line EC9706, were also constructed. In this study, we detected the expression of Nanog in EC9706 cells, and used pcDNA3.1-Nanog and pSUPER-EGFP-shNanog to transfect into EC9706 cells, and established two esophageal cancer cell lines 9706-Nanog and 9706-shNanog, which could express Nanog and Nanog-shRNA gene stably. Using these cell lines, the impact of Nanog expression on the tumor development including proliferation, apoptosis and invasion behavior of esophageal cancer was evaluated. In addition, drug resistance of cisplatin, a widely used chemotherapeutic agent for esophageal cancer treatment, was also investigated in these cell lines.
Establishment of esophageal cancer cell lines EC9706 cells were transfected with pcDNA3.1-Nanog and pSUPER-EGFP-shNanog respectively using liposome 2000, and screened by G418 reagent, and the cells that have stably incorporated pcDNA3.1-Nanog and pSUPER-EGFP-Nanog-shRNA could not be killed by G418. Initially these cells were treated with 400 µg/mL of G418 then the dose was increased to 800µg/mL after 2 weeks. EC9706 cells transfected with normal medium were regarded as control. Cells were cultured until cell clusters derived from replication of cell clones were observed. Then, one of the cell clusters was picked by trypsin �ilter paper and expanded for further experiments. Finally, Real-time PCR and western blot was carried out for identify the Nanog expression of cell lines.

Real-time PCR and Western blot
Real-time PCR and Western blot analysis were used to evaluate mRNA and protein expression of target gene. Total RNA and protein was isolated from esophageal cancer cells. cDNA was synthesized by random priming and real-time PCR was performed used SYBR green mixture. The sequences of the primers used for real-time PCR are as follows: Nanog-F(5'-ACCTGGTGCACCCAATCCTGG -3'); Nanog-R(5'-CCCCAGCAGCTTCCAAGGCAG -3'). MDR1-F(5'-CCGAGCACACCTGGGCATCG -3'); MDR-1-R(5'-GGCCTCCTTTGCTGCCCTCAC -3'); β-actin-R (5'-GTCGTCGACAACGGCTCCGG -3'); β-actin-F(5'-TGGGCCTCGTCGCCCACATA -3'). Following ampli�ication, compare CT values of samples (normalized to β-actin) in order to assess fold differences in mRNA levels of the target genes. 2-Delta Delta CT Method was used in relative gene expression data analysis. Total protein was subjected to SDS-PAGE, and transferred to PVDF membrane, probed with the antibodies as indicated. Primary antibodies were applied and incubated overnight at 4°C as follows : anti-Nanog and anti-MDR-1 (Santa Cruz Biotechnology) at 1:200; β-actin (Santa Cruz Biotechnology) at 1:500. Immunoreactive bands were visualized by ECL Chemiluminescence method and quanti�ied with Gelpro32 image processing and analyzing program.

Colony formation assay
Colony formation was analyzed by plating clone assay. First, 1000 cells/well in logarithmic growth were seeded onto 6-well, and incubated for 10 days. After incubation cells were �ixed with 4% paraformaldehyde for 20 min and stained with haematoxylin for 5 min. Finally, staining solution was washed away and colony formation was recorded the microphotographs. The numbers of colony were counted by viewing multiple �ields under a microscope.

FACS analysis
EC9706 cells were collected and washed twice using cold PBS, centrifuged at 1000 rpm for 5 min and re-suspend in 500 µL PBS. The re-suspension was added with 5µL Annexin V-FITC and 2.5 µL PI and cultured for 15 min in darkroom before studied by �low cytometry. The test was described as below: intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). Rate of apoptotic cells was calculated. Transwell �ilter invasion assay Transwell �ilter invasion assay is one of the most frequently used methods to analyze cell migration in vitro assays. This assay involved a two-compartment system where cells may be induced to migrate from an upper compartment through a porous membrane into a lower compartment. Invasion assays were performed in a 24-well Transwell chamber (Corning, Lowell, MA, USA). Six Transwell chambers were set up for each group, and each chamber was coated with 100 µg Matrigel, and 1 × 10 5 cells were seeded to each chamber. The lower compartments were �illed with 500 µL of RPMI 1640 medium containing 10% FBS, and were incubated in a 5% CO 2 humidi�ied incubator at 37°C for 48 h. To determine the extent to which cells have migrated, the �ilter inserts are removed from culture wells, the cells that have not migrated are cleared from the top surface of the �ilter, and the remaining cells that have migrated to the underside of the �ilter were �ixed with 95% alcohol for 10 min and stained with hematoxylin-eosin for 10 min. Finally, the invasion behavior of the cells were quantitated by viewing multiple �ields under a microscope, an area corresponding to 20% of the �ilter are counted.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
MTT assay and trypan bluetrypan blue staining were used to determine the cisplatin sensitivity of EC9706 cell lines. Brie�ly, cells were harvested and seeded in a 96-well. After 24 h, fresh RPMI 1640 medium containing cisplatin (3µg/mL) was added. After incubation for different time intervals (12,24,36,48, 60, 72 h), 10 µL of MTT (5 mg/mL) was added to each well and the cells were further incubated at 37°C for 4 h. The supernatant was then removed and 100 µL DMSO was added into each well. The absorbance at 450 nm was measured using a microplate reader. Six replicate wells were used for each group.

Statistical analysis
Statistical analysis was performed with one-way ANOVA and student t test. Statistical signi�icance was set at P <0.05.

Esophageal cancer cell lines expressing Nanog and shNanog
After 30 days of culture by G418 screening, stable clones of these cells were selected and expanded, and two esophageal cancer cells lines were established and named as 9706-Nanog and 9706-shNanog (Fig. 1). Observation of morphology showed that the growth of 9706-Nanog was faster than 9706-shNanog. Real-time PCR and Western blot were performed to identify the expression of Nanog in these cell lines. The results showed that expression of Nanog in 9706-Nanog was up-regulated and higher than normal EC9706 cells at both mRNA and protein levels (P <0.05). The expression of Nanog in 9706-shNanog was lower than normal EC9706 cells because of the down-regulation of Nanog by shRNA (P <0.05, Fig. 2).

Nanog promote clonogenicity and proliferation of esophageal cancer cells
To determine the proliferation and replication abilities of 9706-Nanog and 9706-shNanog cell lines, colony formation assay was performed. The results showed that clonogenicity of 9706-Nanog cell was enhanced according to the size and number of cell colonies. Obviously, colonies of 9706-Nanog were bigger than EC9706 and 9706-shNanog, meanwhile the number was also greater than other groups. To the contrary, cell colonies of 9706-shNanog were smaller and less. In addition, colony formation rate of 9706-Nanog, EC9706 and 9706-shNanog was 34.55±4.66%, 21.11±3.56% and 12.8±3.33% respectively. The clonogenicity and proliferation abilities of 9706-Nanog cells were stronger than in the EC9706 and 9706-shNanog cells (P <0.05, Fig. 3).

Apoptosis of esophageal cancer cells
Flow cytometry was applied to assess the apoptosis of 9706-Nanog, EC9706 and 9706-shNanog cell lines. There was little apoptosis being observed in 9706-Nanog and EC9706 cells, and the apoptotic rate of 9706-Nanog and EC9706 cells was 1.63±1.08% and 1.54±0.98%, respectively (P >0.05, Fig. 4). The apoptotic rate of 9706-shNanog cells was 19.97±2.08%, which was higher than in the 9706-Nanog and EC9706 cells (P <0.05, Fig. 4).    method. The results showed that cisplatin inhibited proliferation of EC9706 cells, and the inhibition rate increased with time dependence. Compared to EC9706, cells of 9706-Nanog exhibited a weaker proliferation inhibition, most of 9706-Nanog cells could remain a good growth condition even 48 h after treatment with cisplatin (Fig. 6). But the growth of 9706-shNanog cells was extremely inhibited and most of cells were dead 48 h after treatment with cisplatin. Compared to EC9706, the growth inhibition rate of cells by cisplatin was increased in 9706-shNanog and decreased in 9706-Nanog (P <0.05), and these data indicated that Nanog could in�luence the cisplatin sensitivity of EC9706.
The changes in apoptosis upon treatment with cisplatin were also evaluated by Flow cytometry. There was an obvious apoptosis of EC9706 cells after treatment with cisplatin (16.44±2.48%), and the apoptotic cells of 9706-Nanog was less than EC9706 (7.9±1.52%), while more apoptotic cells in 9706-shNanog cells (23.59±1.78%) was detected (P <0.05, Fig. 7).  To more precisely determine the effect of Nanog on cisplatin sensitivity of EC9706, we also detected the expression of MDR-1 which was regarded as an important factor on drug resistance and sensitivity of chemotherapy to esophageal cancer. The real-time and Western blot assay showed that MDR-1 expression was related closely with Nanog expression. Compared to EC9706, the Expression of MDR-1 was increased in 9706-Nanog cells and decreased in 9706-shNanog cells at both mRNA and protein levels with signi�icant difference (P <0.05) (Fig. 8).

Discussion
Nanog serves as a novel transcription factor which maintains self-renewal and pluripotency of stem cells [4,5]. As a key self-renewal molecule, Nanog is detected not only in embryonic stem cells (ESCs), but also in germ cell tumors [8]. Ezeh et al. [16] showed that the Nanog protein was expressed in tissues of breast cancer. Zhang et al. [24] reported that several tumor cell lines express NANOGP8, a processed pseudogene of Nanog. Jeter CR et al. [9] previously �inished a systematical investigation on the expression, origin, and functions of NANOG in different tumor cells, and found that multiple tumor cells in vitro and in vivo express NANOG and demonstrated that NANOG expression in human cancer cells might be related to tumor development. The results suggest that Nanog may play an important role in tumor development.
In the present study, we used two Eukaryotic expression vectors, pcDNA3.1-Nanog and pSUPER-EGFP-shNanog, which were transfected into human esophageal cancer cell line EC9706, and established two cell lines 9706-Nanog and 9706-shNanog by screening cells with G418 reagent. In addition, Real-time PCR and Western blot assay were carried out to identify the expression of Nanog in 9706-Nanog and 9706-shNanog cell lines. Based on these cell lines, we evaluated the relations between Nonog and the biological characteristics of human esophageal cancer cells, including clonogenicity and proliferation, invasion and apoptosis of cell lines with Nanog expression and loss-of-function. We showed that the colony formation rate of 9706-Nanog cells was higher than EC9706 and 9706-shNanog cell lines, and there were more invasion cells in 9706-Nanog than in other cell lines. These results indicate that expression of Nanog could promote clonogenicity, proliferation and invasion abilities of human esophageal cancer cell line EC9706. Our data also indicate that Nanog inhibits apoptosis of esophageal cancer cells, as increased apoptotic cells were observed by FACS in 9706-shNanog cells. Taken together, not only Nanog is expressed in human esophageal cancer cell line EC9706, it is also closely related to the malignant characteristics which usually gave rise to tumorigenesis and progression of esophageal cancer. Furthermore, inhibition of Nanog expression may be an effective treatment for patients with esophageal cancer. Multidrug resistance (MDR) enables cancer cells to resist anticancer drugs of a wide variety of structure and function [25,26], and inducing reduced drug sensitivity in cancer cells [27,28]. Cisplatin is a commonly used chemotherapeutic drug for human esophageal cancer, but drug resistance has become a major issue. Expression of Nanog was increased when cancer cells turned into MDR condition [22], suggesting a possible relationship between Nanog and MDR in cancer cells [18,29]. In our study, we treated 9706-Nanog, EC9706 and 9706-shNanog cell lines with cisplatin and evaluated the drug sensitivity of these three cell lines. We found that the expression of MDR-1 was increased in 9706-Nanog cells but it was inhibited in 9706-shNanog cells. The sensitivity to cisplatin was decreased as Nanog expression levels were increased. These data indicate Nanog was related to the expression of MDR-1 gene and further changed the drug sensitivity of human esophageal cancer to cisplatin. Therefore, Nanog may be used as a novel target to study drug resistance of esophageal cancer.
In this study, we were unable to discriminate between Nanog and NanogP8 gene, so we could not demonstrate the expression level of these two genes and biological differences among Nanog and its pseudogenes in esophageal cancer. Future studies are required to assay for Nanog and NanogP8 mRNA, and to perform the sequence analysis of 3'UTR region to discriminate among the Nanog alleles and NanogP8.
In conclusion, this study showed that Nanog was expressed in human esophageal cancer cell line EC9706 and promotes tumor cells development. Overexpression or loss of function of Nanog was associated with ability of clonogenicity, proliferation and invasion, and apoptosis in EC9706 cells. In addition, expression of Nanog was related to cisplatin resistance of EC9706. However, the biologically function of NANOG expressed in human esophageal cancer cells remains unclear, and more importantly, the function of multiple pseudogenes of Nanog genes in regulating tumorigenesis and tumor development is still controversial.