Nuclear β-catenin Accumulation Is Associated With Increased Expression Of Nanog Protein And Predicts Poor Prognosis Of Non-small Cell Lung Cancer

Background:Although β-catenin and Nanog are related to the properties of the cancer stem cell, the relationship between β-catenin and Nanog in lung cancer have not been reported currently. Advances in surgical, radiotherapeutic, and chemotherapeutic approaches have been made, but the long-term survival rate remains low. The aggressive and heterogeneous nature of lung cancer has thwarted efforts to reduce mortality from the NSCLC. Thus, there is an urgent need for the determination of useful prognostic molecular markers for clinical management of patients with NSCLC.β-catenin is a key component of the canonical Wnt pathway that plays pivotal roles in pattern formation during embryogenesis and in malignant transformation. At the plasma membrane, β-catenin is associated with the cadherin class of cell-adhesion proteins and functions in regulating cell adhesion. In the absence of Wnt signal, cytoplasmic P-catenin interacts with glycogen synthase kinase-3p (GSK-3β) in a large complex known as destruction complex which also contains the adenomatous polyposis coli (APC) and the axis inhibition protein (Axin).The hypophosphorylated β-catenin is stabilized and translocated to the nucleus, where it binds T-cell factor/lymphoid enhancer factor (TCF/Lef) and activates its target gene expression.Nanog is a core transcription factor required for maintenance of the pluripotency and self-renewal of embryonic stem cells. Recent studies have revealed that Nanog is also involved in self-renewal and tumorigenicity of cancer stem cells in a variety of human cancers. Nanog is critically involved in regulation of cancer stem cells in several types of tumors and has been reported to be target gene of β-catenin, β-catenin inhibits differentiation by increasing the expression of Nanog. we also demonstrated Nanog expression could by influenced by β-catenin in nasopharyngeal carcinoma in our earlier study.Objective In this study, we have evaluated the expression levels and subcellular localization of β-catenin and Nanog proteins by immunohistochemical (IHC) staining in tissue specimens from309patients with Non-small cell lung cancer (NSCLC), and explored their association with clinicopathological features and patient outcome.Methods:1. Primary tumor specimens from309NSCLC patients who underwent curative surgical resection were evaluated in this study. None of these patients had undergone chemotherapy or radiotherapy prior to the surgery. Of these patients,203were collected from2001to2005at Hunan Hospital and the rest of96were from2002to2006at the Guilin University. Patients in the study were examined and treated according to provincial guidelines.2. Cores from formalin-fixed paraffin embedded tumor tissues of NSCLC were arrayed in triplicate onto a tissue microarray (TMA) and each TMA block consisted of up to40tissue cores, before the area of cores were chosen, the preliminary experiment of β-catenin were made to ensure the typical cores.3. Immunohistochemistry (IHC) staining, serial tumor sections and IHC staining were used to evaluate both β-catenin and Nanog proteins. The TMA blocks containing tissue sections (4μm) were subject to immunohistochemistry staining using the primary antibodies. Immunofluorescent staining.4. For immunofluorescent staining, A549or H23cells grown on the surface of cover slides were serum-starved overnight followed by stimulation with50ng/ml EGF for24hours. Cells were incubated with mouse anti-β-catenin and rabbit anti-Nanog primary antibodies. The nuclei were stained with DAPI. Slides were examined with a fluorescent confocal microscope.5. Cytoplasmic and nuclear proteins of A549and H23cells were extracted by Nuclear and Cytoplasmic Protein Extraction Kit, then equal amount of protein were subjected to electrophoresis on a SDS-PAGE gel. The separated proteins were transferred to PVDF membranes and probed with appropriate primary antibodies. Protein bands were detected by enhanced chemiluminescence reagents.6. Both pLKO.1lentiviral shRNA vector and control shRNA targeting against GFP were from Aldrich-Sigma. The sense and antisense oligonucleotides were annealed and ligated into pLKO.1lentiviral vector.Results:1. Tumor specimen from309NSCLC patients consisting of134males and175female were included in this study. The median age was52years (range:37-82years). Of these patients,167(32.9%) were diagnosed as adenocarcinoma and142(33.1%) diagnosed as squamous carcinoma. Clinical stages, histological grades, and studied proteins were correlated as shown in Table1. The tumor tissues was classified in three histological grades:grade1(126patients), grade2(112patients) and grade3(71patients). The expression of membranous and nuclear, but not cytoplasmic, β-catenin expression were correlated with the histological grade (p<0.05), as shown in Table1. Nanog expression that was localized in the nucleus was also correlated with the histological grade (p<0.01, Table2). 2. Positive membranous, cytoplasmic, and nuclear expression of β-catenin was detected in67.0%(207/309),43.0%(133/309), and45.0%(139/309) of the NSCLC tissues, respectively. Thus we further examined Nanog expression in NSCLC specimen. We found that30.4%(94/309) tumor tissues displayed Nanog immunoactivity that was located in the nucleus in most cases.3. The last follow-up date is Sep.29th,2009, with a median follow-up time52months (range7-69.5months). The1-,3-and5-year overall survival (OS) rates were82.4%,52.5%,30.6%, respectively. The survival rates of309NSCLC patients according to status of β-catenin and Nanog were shown in Table2. The survival rate of patients with nuclear β-catenin expression was significantly lower than that of patients without nuclear β-catenin expression (p<0.01). Likewise, the survival rate of patients with cytoplasmic β-catenin expression was significantly lower than that of patients without cytoplasmic β-catenin expression (p<0.01). However, there was no statistical difference in the survival rate between patients with or without membranous P-catenin (p=0.064), but with a trend toward reduced survival in patients with loss of β-catenin expression. Patients without Nanog protein expression had a significantly better prognosis and longer survival. The overall survival rate was12.3%(Nanog-positive) and38.3%(Nanog-negative) for this group (p＜0.01).4. Univariate analyses showed no significant association between OS and age (>52yr vs.<52yr), sex (female vs. male), histological subtype (adenocarcinoma vs. squamous carcinoma), T stage (T3-T4vs. T1-T2), clinical stage (stage Ⅲ vs. Ⅰ-Ⅱ). The expression levels of cytoplasmic and nuclear β-catenin and Nanog were an independent prognosticator for OS. In a multivariate analysis incorporating all clinicopathologic variables reported as shown in Table3, and the covariates included, presence of sex, age, tumor grade, tumor T stage, tumor C stage, expression levels of Nanog protein (low vs. high), and intracellular β-catenin protein expression at membrane, in cytoplasm and in nucleus (low vs. high) were performed to identify independent prognostic factors.5. We further analyzed correlation between expression status of Nanog and P-catenin. As mentioned above, positive Nanog protein staining was almost exclusively observed in nucleus and β-catenin expression was stratified according to its subcellular locations. Seventy two of the139(51.8%) tumor tissues that stained positive for nuclear P-catenin also displayed Nanog immunoactivity. Among the139specimen with nuclear β-catenin expression,148(72.7%) showed Nanog immunoreactions (β=0.036). On the contrary, although, membranous β-catenin expression is correlated with Nanog protein, among the207specimen with membranous β-catenin expression, only55(26.6%) showed Nanog immunoreactions (β<0.001). Cytoplasmic β-catenin expression is not correlated with Nanog protein even though the cytoplasmic P-catenin is associated with poor prognosis (β=0.166, Table4). Cytoplasmic β-catenin expression is correlated with nuclear β-catenin protein, among the133specimen with cytoplasmic β-catenin expression,105(78.9%) showed nuclear p-catenin immunoreactions (p<0.001).6. In the absence of EGF, P-catenin was located predominantly at the plasma membrane, with faint staining distributed in the cytoplasm. When cells were treated with EGF, β-catenin staining translocated to the nucleus in both A549and H23cells. These results suggested that nuclear accumulation of P-catenin can be induced not only by Wnt signaling, but also by EGF stimulation in NSCLC. In addition, nuclear Nanog expression was dramatically enhanced in response to EGFR activation.7. We investigated the requirement of P-catenin in the regulation of Nanog in A549and H23. To this end, β-catenin was knocked down with lentivirus-mediated shRNAs. Compared with cells infected with the control shRNA lentivirus, cells infected with β-catenin shRNA lentivirus exhibited reduced Nanog expression. Moreover, Nanog expression in the NSCLC cells with knockdown of P-catenin can not be obviously enhanced by adding EGF, but on the other hand, when the expression of β-catenin is increased by adding EGF, Nanog thereupon increased expression upon P-catenin. It is important that nuclear β-catenin rather than cytoplasm β-catenin expression in the NSCLC cells can be enhanced by adding EGF.8. P-catenin was knocked down with lentivirus-mediated shRNAs. Compared with cells infected with the control shRNA lentivirus, cells knockdown of β-catenin markedly reduced the percentage of SP.9. β-catenin was overexpressed by an lentivirus-mediated system. After expression of the P-catenin was confirmed, the SP cells were evaluated by FACS sorting. Overexpression of β-catenin resulted in increased proportion of SP.10. Nanog was silenced with lentivirus-mediated shRNAs. Compared with cells infected with the control shGFP lentivirus, cells silenced of Nanog obviously reduced the percentage of SP.11. Nanog was overexpressed by an lentivirus-mediated system. After expression of the Nanog was confirmed, the SP cells were evaluated by FACS sorting. Overexpression of Nanog resulted in increased proportion of SP.Conclusion:Taken together, we showed that negative expression of membranous β-catenin correlated with a shorter survival time than the normal expression level of the protein. However, high expression of nuclear β-catenin was strongly associated with poor prognosis and was an independent prognosticator for OS. We further found that NSCLC cells frequently exhibited nuclear Nanog protein abundance that is significantly correlated with nuclear β-catenin expression and poor prognosis. Furthermore, results from ICC staining with established lung cancer cell lines revealed that increased expression of Nanog and nuclear translocation of P-catenin occurred concomitantly in response to EGFR signaling. In conclusion, we propose that evaluation of subcellular localization of P-catenin and Nanog expression is of clinical significance for patients with NSCLC.