Low Expression of the FoxO 4 Gene may Contribute to the Phenomenon of EMT in Non-small Cell Lung Cancer

Lung cancer, one of the most common malignant tumors in the world with high morbidity and mortality (Jemal et al., 2008). In a recent survey across China, the mortality of lung cancer rose by 465% in the past 30 years, which became the main cause of urban residents’ death (WenDehnel 2011). NSCLC accounts for approximately 80% of lung cancer, which is mainly classified as squamous carcinoma and adenocarcinoma. Comparing to other cancers, such as breast or colorectal cancers, the 5-year survival rate of lung cancer is the lowest (15%). As one of the ten hallmarks of cancer, metastasis is the main lethal cause of lung cancer patients. However, the molecular mechanisms of metastasis is still unclear. Hence, identification of new metastasis genes and the molecular mechanisms underlying the metastatic progression still need more efforts. Epithelial-mesenchymal transition (EMT), which participates in multiple physiological and pathological processes of human body, was first proposed in 1982. EMT pathway was found to play important roles in regulating the transcription of genes involved in embryonic


Introduction
Lung cancer, one of the most common malignant tumors in the world with high morbidity and mortality (Jemal et al., 2008).In a recent survey across China, the mortality of lung cancer rose by 465% in the past 30 years, which became the main cause of urban residents' death (WenDehnel 2011).NSCLC accounts for approximately 80% of lung cancer, which is mainly classified as squamous carcinoma and adenocarcinoma.Comparing to other cancers, such as breast or colorectal cancers, the 5-year survival rate of lung cancer is the lowest (15%).As one of the ten hallmarks of cancer, metastasis is the main lethal cause of lung cancer patients.However, the molecular mechanisms of metastasis is still unclear.Hence, identification of new metastasis genes and the molecular mechanisms underlying the metastatic progression still need more efforts.
Epithelial-mesenchymal transition (EMT), which participates in multiple physiological and pathological processes of human body, was first proposed in 1982.EMT pathway was found to play important roles in regulating the transcription of genes involved in embryonic RESEARCH ARTICLE

Low Expression of the FoxO4 Gene may Contribute to the Phenomenon of EMT in Non-small Cell Lung Cancer
Ming-Ming Xu 1& , Guo-Xin Mao 2& , Jian Liu 2 , Jian-Chao Li 1 , Hua Huang 3 , Yi-Fei Liu 3 *, Jun-Hua Liu 1 * development (Thiery et al., 2009), inflammatory response (Foroni et al., 2012), tissue regeneration (KalluriWeinberg 2009), organ fibrosis (Lopez-NovoaNieto 2009;Liu 2010), tumor invasion and tumor metastasis (Lopez-NovoaNieto 2009).Particularly, the activation of EMT pathway often precedes cancer metastasis for tumors of the epithelial origin (Wells et al., 2008).Epithelial cells lead to loosen and even loss of cell-cell adhesion throughout EMT.Besides, at the molecular level, EMT is characterized by loss or downregulation of E-cadherin and cytokeratins, with the exception of mesenchymal proteins like Vimentin, fibronectin and N-cadherin, whose expression are often upregulated (Wells et al., 2008;Ivaska 2011;SatelliLi 2011;Vaid et al., 2011).It has been shown that EGF and resultant EGFR activation could promote EMT in HCC cell lines by altered the expression and morphology of EMT-associated markers (E-cadherin/β-catenin complex) (Fan et al., 2014).Recently, Lee et al suggested that in cholangiocarcinoma cells, EGF-mediated phosphorylated FoxO4 could negatively regulate the transcription of ANXA8, leading to the morphologic changes similar to epithelial-mesenchymal transition (EMT) (Lee et al., 2009).Based on current knowledge, growth factor or insulin inhibits the transcription activity of FoxO4 by phosphorylated via phosphatidylinositol-3-kinase (PI3K)/ AKT signaling (Kops et al., 1999).It implied that FoxO4 might be an inhibitor for EMT in cholangiocarcinoma.However, no data of FoxO4 are available for NSCLC until now.
FoxO (Forkhead box O) transcription factors consists of FoxO1, FoxO3, FoxO4 and FoxO6.They are becoming an important and major target in inhibiting tumorigenesis (Katoh 2004;Burgering 2008;CalnanBrunet 2008).The functional role of FoxO4 gene is largely determined by its DNA-binding domain (DBD) (Silhan et al., 2009).DBD is characterized by three α helices, three stranded β sheet and two wing-like loops, and is thought to play important roles interacting with other proteins (Boura et al., 2007).The interaction with 14-3-3 proteins affected FoxO4 binding to the target DNA and prevented FoxO4 from entering the nucleus by interfering with the function of nuclear localization sequence (NLS) (Obsilova et al., 2005;Boura et al., 2007).Human FoxO4 gene is located on chromosome Xq13.1 and encodes a protein of 65KD.FoxO4 participates in many cellular processes, including regulation of cell cycle, proliferation, differentiation, apoptosis, ageing, metabolism, response to stress and tumorigenesis (Tran et al., 2002).Another Fox family member-FoxK1, promoted cell proliferation by inhibiting the transcription activity of FoxO4, and the repression of FoxO4 resulted in down-regulation of P21 expression in myogenic progenitor cell (MPC) (Shi et al., 2012).In the study of diabetic nephropathy, an advanced glycation endproducts (AGE)-RAGE (a receptor for AGEs) interaction promoted podocyte apoptosis by activating FoxO4 transcription factor (Chuang et al., 2007).In addition, FoxO4 was shown to play a critical role in lifespan extension, and the function was correlated with FoxO4's role in reactive oxygen species (ROS) scavenging (Dansen et al., 2009).As a transcription repressor, FoxO4 plays a particularly important role in tumorigenesis.The cell cycle dependent kinase inhibitor P27, activated by FoxO4, affects cell cycle dependent kinase (CDK), which blocks tumors' G1 cell cycle progression (Yang et al., 2005).Earlier study showed that FoxO4 inhibited tumor growth by reducing tumor size in HER-2 overexpression tumor cells (Yang et al., 2005).Recently, Chen L found that miR-421 promoted tumor proliferation and antiapoptosis by down-regulating FoxO4 downstream molecules such as cyclin dependent kinase inhibitors P21, P27, Bim (BCL-2 family) and Fasl (Fas ligand) in nasopharyngeal carcinoma (Chen et al., 2013).In addition, chemotherapy experiments in colorectal cancer and liver cancer, it showed that FoxO4 sensitized colorectal and liver cancer cells to doxorubicin-medicated cytotoxicity (Lupertz et al., 2008).In tumor hypoxic environment, FoxO4 reduces the level of hypoxia-inducible factor 1α (HIF-1α) protein, thereby inhibiting responses to hypoxia, such as the expression of glucose transporter type 1 (GLUT-1), erythropoietin (EPO) and vascular epithelial growth factor (VEGF).These processes are involved in the procession of glucose metabolism, erythropoiesis and angiogenesis, and are essential for tumor development (TangLasky 2003).Down regulation of FoxO4 also significantly associates with low-grade, lymph node metastases and TNM Ⅲ,Ⅳ tumors in colorectal cancer (Xiang-qiang et al., 2011).In colorectal SW480 cancer cells, miR-499-5p which was involved in metastasis could specifically bind to the 3'-UTR of FoxO4 and decreased the expression level of FoxO4 mRNA and protein (Liu et al., 2011).
However, the correlation between the expression of FoxO4 and EMT in NSCLC is still unclear.In this study, we investigated the expression of FoxO4 in 8-paired NSCLC cases.Immunohistochemistry was used to characterize the expression of FoxO4 and EMT indicators.In addition, we analyzed the relationship between the expression of FoxO4 and clinicopathologic parameters, EMT indicators and patients' survival.

Materials
Surgically resected 150 NSCLC samples between 2005 and 2007 from the Affiliated Hospital of Nantong University were formalin-fixed and paraffin-embedded for histopathologic diagnosis.The histological type, differentiated degree, lymph node metastasis, tumor size and TNM stage (according to the 7 th Edition) (Groome et al., 2007) were showed in the Table1.Another 10 cases of normal lung tissues were set as control.All specimens were fixed with 10% formaldehyde, dehydration, conventional paraffin embedding, made into tissue microarray (TMA) sections.In addition, we took the 8 freshly frozen NSCLC tissues and matched adjacent tissues for Western blot analysis.This study was approved by Ethics Committee of the Affiliated Hospital of Nantong University.

Immunohistochemistry
We adopted conventional immunohistochemical SP method in this study.Tissue microarray (TMA) sections of 4μm were cut, deparaffinized and rehydrated.Endogenous peroxidase was blocked for 15 min by 3% hydrogen peroxide.Antigen retrieval was conducted by microwave for 15 min within citrate buffer solution (PH=6.0).The sections were blocked with 2% normal goat serum for 30 min.The slides were then incubated with the diluted following antibodies: rabbit monoclonal anti-FoxO4 (Abcam, USA), mouse monoclonal anti-E-cadherin (Bioworld, USA) and mouse monoclonal anti-Vimentin (Bioworld, USA) overnight at 4℃.Subsequently, HRP-labeled goat anti-rabbit immunoglobulins, the secondary antibody was used for 30 min at 37℃.The 3, 3'-diaminobenzidine (DAB, Bioworld) solution was used as a chromogen.The slides were washed by water, and then counterstained with hematoxylin.Positive and negative controls were conducted throughout.

Western blot
Tissue proteins were resolved by 10% SDS-PAGE and transferred on to a polyvinylidene fluoride (PVDF) membrane.The membranes were blocked by 5% nonfat milk for 1h and incubated with primary antibody (rabbit monoclonal anti-FoxO4) for 1h at 37℃ and washed with PBST.At last, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1h at room temperature.The signals were visualized with ECL detection reagent.The density on the film were measured using ImageQuant image analysis system.GAPDH was detected using mouse anti-GAPDH antibody as a loading control.The experiment was repeated for three times.

Statistical methods
The expression level of FoxO4 protein in freshly frozen NSCLC tissues and matched adjacent tissues were normalized to GAPDH and analyzed.The Chi-square was used to analyze the statistical significance of the relationship respectively between FoxO4, E-cadherin, Vimentin and clinical pathological features.The survival curve of FoxO4's expression was computed by the method of Kaplan-Meier.The prognostic influence of factors on survival was analyzed by using a multivariate Cox regression model.A P value of less than 0.05 was considered as statistically significant.Spss 17.0 software was used for statistical analysis.

Expression of FoxO4, E-cadherin and Vimentin in NSCLC
Immunohistochemistry (IHC) was used to confirm the expression of FoxO4, E-cadherin and Vimentin in NSCLC tissues.Positive staining FoxO4 was mainly localized in NSCLC cells in nucleus and /or cytoplasm at different levels.Low expression of FoxO4 was detected in 42.67% of NSCLC tissues and was in 90.00% of matched adjacent normal tissues.Positive staining of E-cadherin and Vimentin were respectively localized in cell membrane and cytoplasma.E-cadherin immunoreactivity was identified in 58 cases (38.67%) of NSCLC tissues, while positive staining of Vimentin was detected in 83 cases (55.34%).
To investigate the difference expression of FoxO4 in paired NSCLC tissues, eight pairs of representative cases were tested by western blot.The results showed that FoxO4 protein was overexpressed in normal lung tissues compared with NSCLC tissues.

Relationship between the expression of FoxO4 protein and clinicopathological factors in NSCLC
By analyzing the NSCLC clinicopathological features, we found that there was statistically significant difference in expression of FoxO4 protein between different TNM stages (P<0.001),histological differentiation (P=0.004) and lymph node metastasis (P<0.001).In contrast, no significant association was seen in patient gender, age, histological type, tumor diameter.The association between expression of FoxO4 protein and clinicopathological features was shown in Table 1.

Relationship between FoxO4 protein and EMT representative proteins
The relationships between them were shown in Table 1.We found that positive staining of epithelial protein E-cadherin was detected in 58 cases (38.67%).However, the mesenchymal protein expression was 55.34% in the same samples.We noted that the low expression of FoxO4 correlated with a loss of E-cadherin expression (P<0.001)High expression of FoxO4 correlated with better overall survival rate Survival analysis was restricted to 150 patients with follow-up data until death.After all variable were compared separately with survival status, we found FoxO4 (P=0.046),E-cadherin (P=0.002),Vimentin (P=0.008) and clinical stage (P<0.001)significantly influenced survival (Table 2).Kaplan-Meier survival curves showed that high expression of FoxO4 correlated with better survival with statistical significance (P<0.001; Figure 3).In conclusion, multivariate analysis using the Cox's proportional hazards model proved that FoxO4 (P=0.046;Table 2) was a prognostic factor for patients' overall survival.

Discussion
In this study, we used a TMA to identify that FoxO4 played an important role in the progression of EMT.In addition, through measuring the expression of FoxO4 in NSCLC tissues, we revealed strong association between the expression of FoxO4 and several clinicopathologic parameters such as histological differentiation, TNM stage and EMT indicators.Lastly, multivariate analysis showed that FoxO4 might be used as a prognostic factor for NSCLC.
Unfortunately, at the first diagnosis, almost 56% lung cancer patients are diagnosed at advanced stage.Metastasis predicts a poor prognosis and is the main cause of NSCLC patients' death.Through metastasis, NSCLC cells are easily found in the bone, adrenal glands, liver, brain and regional lymph nodes.The progression of metastasis, namely metastatic cascade contains many steps including invasion, EMT, angiogenesis, vessels transportation, MET and secondary tumors growth (GeigerPeeper 2009).The important bases of molecular and cellular mechanisms are the diminished capacity of tumor cells adhesion while increasing cells' motility (GeigerPeeper 2009).Therefore, EMT becomes the essential role in the process of tumor invasion and metastasis, and is often considered as a prelude to tumor metastasis (Wells et al., 2008).Importantly, advanced tumor cells show an obvious down-regulation of epithelial markers like E-cadherin, cytokeratin, resulting in the reduced cell adhesion.However, over-expression of the mesenchymal proteins like Vimentin and N-cadherin are responsible for increasing cells' motility (SatelliLi 2011).E-cadherin was significantly related to the degree of EMT and tumor malignancy, for example in lung cancer (Bremnes et al., 2002), gastric cancer (JooRewChoi et al., 2002), skin cancer (McGary et al., 2002), breast cancer (BerxVan Roy 2001), pancreatic cancer (JooRewPark et al., 2002) and nasopharyhgeal cancer (Tsao et al., 2003).Recently, studies have demonstrated that the positive expression rate of E-cadherin in NSCLC tissue without lymphatic metastasis was significantly higher than that with lymphatic metastasis (47.2% vs. 21.6%)(Su et al., 2014).Earlier studies showed that expression of E-cadherin was associated with the patients' survival in NSCLC (Wang et al., 2011).For many years, we just know Vimetin is a marker for EMT, however, its function of taking part in the process of EMT is still unclear (Ivaska 2011).Some researches showed that over-expression of Vimentin

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Expression Profiles of FoxO4 in NSCLC and Non-tumorous Adjacent Tissues.Western blot was performed to study the levels of FoxO4 in 8 representative paired samples of NSCLC tissue (T) and non-tumorous adjacent tissues (N).GAPDH was used as a loading control.The same experiment was repeated at least 3 times