During the past decade, mounting evidence has well demonstrated that accumulation and nuclear localization is a hallmark of Wnt/β-catenin pathway activation. In addition to controlling cell-cell adhesion through its binding with cadherin at the membrane, β-catenin acts as a transcriptional activator in the nucleus where it interacts with LEF1 and TCF transcription factors and regulates transcription of target genes responsible for cell proliferation and differentiation. Thus, subcellular localization of β-catenin from cell membrane to the nucleus determines its two distinct functions. In agreement with these findings, cytoplasmic and nuclear β-catenin expression has been reported to be associated with a poorer prognosis in patients with cancers of breast, liver, and colon [21–23].
The prognostic roles of β-catenin expression in NSCLC have been extensively studied in the past years [24, 25]. In a very recent study reported by Chiu et al. on 370 cases of NSCLC, β-catenin expression was found to be negative in 28% of tumors and positive in 72% of tumors. The lack of β-catenin expression was significantly associated with poor survival and retained independent prognostic significance . Similar results were also observed in a number of early studies. In the present study, we evaluated the expression of β-catenin in 309 tissue specimens of NSCLC. In order to more precisely analyze IHC results in relation to patient prognosis and survival, we have classified the IHC staining results into three categories according to subcellular localization of β-catenin. We showed that patients with negative expression of membranous β-catenin had a trend of shorter survival (p = 0.067) than those with positive expression. This result is in agreement with the findings reported by others previously.
In addition to the similarity, there are some striking differences between our results and those of earlier studies. We found that increased expression of nuclear β-catenin was strongly associated with poor prognosis of patients with NSCLCs. In addition, multivariate analysis revealed that the expression of nuclear β-catenin was an independent prognosticator for OS. These data are consistent with recent observations that increased Wnt/β-catenin pathway activity is associated with metastasis and relapse in primary lung adenocarcinoma . Importantly, even though we revealed that cytoplasmic β-catenin was a prognosticator, these observations do not exclude the possibility that nuclear β-catenin acts primarily on the prognosis, since a majority of cases over expressing cytoplasmic β-catenin also contain nuclear β-catenin expression. We further demonstrated by immunofluorescent staining and/or western blot analysis that independent to Wnt, nuclear accumulation of β-catenin can be induced by EGFR pathway that is critically involved in tumorigenesis of NSCLC. Our in vitro results provide not only supportive evidence to our IHC finding that nuclear β-catenin is significantly associated with Nanog expression in primary NSCLC specimens, but also an additional mechanism by which β-catenin activation is regulated by growth factor signaling in a Wnt-independent manner in NSCLC.
In contrast to our investigation, some earlier studies evaluated only the overall intensity of IHC staining of β-catenin in tumor cells without stratification of the data based on subcellular localization of the protein. Thus the distinct impact of β-catenin at different cellular compartments on patient prognosis and survival was ignored. Some of the studies [28, 29] evaluated cytoplasmic or nuclear β-catenin specifically, but found no correlation between cytoplasmic or nuclear staining and clinicopathological parameters or survival rates, probably due to low numbers of the positive cases identified in those studies. One of the early study  showed that increased expression of cytoplasmic and nuclear β-catenin can predict favorable prognosis of NSCLCs patients. However, β-catenin immunoactivity was found to be associated with increased proliferation as suggested by high Ki-67 expression in the same set of specimens.
Nanog protein is elevated in many human tumors including NSCLC and might positively regulate tumor metastasis through enhancing EMT in lung adenocarcinoma . We found that Nanog is expressed in significant portion of specimens and Nanog immunoactivity was observed in the nucleus. Expression of Nanog is significantly correlated with nuclear, but not membranous nor cytoplasmic, β-catenin. Furthermore, our IHC staining demonstrated that increased expression of Nanog and nuclear translocation of β-catenin occurred concomitantly in response to EGFR signaling in A549 and H23 lung adenocarcinoma cells. This correlation is in agreement with a previous report demonstrating that β-catenin up-regulates Nanog expression in embryonic stem cells . We proposed that Nanog contributes to tumorigenesis and represents an important prognostic marker of poor prognosis in patients with NSCLC.
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 IHC staining with established lung cancer cell lines revealed that increased expression of Nanog and nuclear translocation of β-catenin occurred concomitantly in response to EGFR signaling. In conclusion, we propose that evaluation of subcellular localization of β-catenin and Nanog expression is of clinical significance for patients with NSCLC.