Prognostic implications of ezrin and phosphorylated ezrin expression in non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death worldwide, with over one million cases diagnosed each year [1]. Approximately 85% of lung cancers are non-small cell lung cancer (NSCLC) [2]. Molecular target therapy is one of the promising field of NSCLC treatment, and its target includes EGFR (epidermal growth factor receptor), EML4-ALK (echinoderm microtubule associated protein like4-anaplastic lymphoma kinase). EGFR TKI (gefitinib and erlotinib) and EML4/ALK inhibitor (Crizotinib) have achieved better results in the clinical therapy of advanced NSCLC [3, 4]. Despite progress in the multimodality treatment of lung cancer, prognosis is still poor, with 10–15% 5-year survival rates. More than 90% of deaths from NSCLC are attributable to metastases [5].

The cytoskeletal organizer ezrin was first identified as an important metastatic regulator in rhabdomyosarcoma and osteosarcoma [6, 7]. Ezrin is a member of the ezrin-radixin-moesin (ERM) family and acts as a cross-linker between the plasma membrane and the actin cytoskeleton [8]. In its inactive form, ezrin is located in the cytoplasm and its C-terminal domain, an F-actin-binding site, is masked by the N-terminal domain of ezrin or other ERM family member proteins. Once ezrin is activated by threonine and tyrosine phosphorylation, it assumes an active form, in which its N-terminal domain binds the cell membrane and its C-terminal domain binds to F-actin [8, 9]. Ezrin plays important roles not only in cell motility, cell adhesion, and apoptosis, but also in various cell signaling pathways. Ezrin is synthesized in a dormant form in which the N-terminal domain in bound to the C-terminal domain, thus mutually blocking their capacity to bind other molecules [10].

Phosphatidylinositol 4, 5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (Thr-567) and tyrosine 353 (Tyr-353) in ezrin are involved in the switching of ezrin from the dormant form to its active state. Once phosphorylated, ezrin binds more tightly to the membrane. Phosphorylated ezrin (p-ezrin) is often associated with the stimulation of cellular functions. Phosphorylation of Tyr-353 in ezrin is regulated through the PI3-kinase/Akt pathway, and phosphorylation of Thr-567 depends on the p38 MAP-kinase activity, and these phosphorylation events promote tumor amplification, metastasis and invasion. Phosphorylation of Thr-567 and Tyr-353 are key events that regulate the transition from the dormant state of ezrin to its active form [11]. These findings suggest that phosphorylated ezrin might be a potential molecular target for cancer therapy.

A few studies to date have reported an association between ezrin/p-ezrin expression and clinicopathological parameters, as well as its prognostic role in lung cancer. Thus, we analyzed the expression and localization of ezrin/p-ezrin in NSCLC compared with the normal counterparts, determined its relationship with clinicopathological parameters, and investigated its prognostic value for NSCLC patients based on tumor stage and survival data. We found that ezrin and p-ezrin (ezrin^Thr-567 and ezrin^Tyr-353) are frequently upregulated in NSCLC compared with the normal counterparts, and are related with the poor differentiation and late clinical stage of NSCLC. However, only ezrin^Thr-567 was related with the presence of lymph node metastasis and the overall survival time of NSCLC patients, indicating that ezrin, especially ezrin^Thr-567, may prove to be useful as a novel prognostic biomarker of NSCLC.

The human ezrin gene maps to chromosome 6q25.2-q26 and the total length of the mRNA is 3166 bp, encoding 585 amino acids [6]. Ezrin is known to be a component of cell surface structures that are involved in cell adhesion to the extracellular matrix, as well as in cell-cell interactions, receptor tyrosine kinase signaling, signal transduction through Rho GTPase and interactions with Akt-mediated cellular apoptotic machinery [14, 15]. It is present in the cytoplasm in an inactive form but after threonine and tyrosine phosphorylation, ezrin assumes an active form, moving to the cell membrane and tethering F-actin to the cell membrane [8]. Recent studies showed that ezrin likely regulates adhesion molecules and participates in cell signal transduction and other channels in these processes [6, 16].

In previous studies, ezrin was found to have important roles in the tumorigenesis and metastasis of several malignancies. Khanna et al. [6] showed that high ezrin expression was associated with early development of metastasis and poor outcome in osteosarcoma. Xie et al. [17] reported that ezrin affected the growth and invasiveness of esophageal SCC cells through the MAPK and transforming growth factor β pathway. However, Moilanen et al. found that serous ovarian cancer with low expression of ezrin protein had poor prognosis [18]. Karmakar et al. [19] also reported that the invasion of choriocarcinoma cells actually strengthened when ezrin protein expression decreased. Here we also found that ezrin was significantly upregulated in NSCLC compared with the adjacent non-tumor tissues and normal lung tissue counterparts (P < 0.01). Ezrin overexpression was significantly correlated with the advanced clinical stage and poor differentiation of NSCLC (P < 0.01), but no correlation was found with lymph node metastasis and invasion of NSCLC in our data. However, Zhang et al. [20] reported that ezrin expression was significantly associated with increased tumor stage and LN metastasis of NSCLC. These studies suggested that there might be cell- and tissue-specific functions for ezrin in tumor progression.

To date, two important phosphorylation sites in ezrin have been identified: ezrin^Thr567 and ezrin^Tyr353. Ezrin directly interacts with signaling enzymes such as phosphoinositide 3-kinase (PI3-kinase), and the phosphorylated tyrosine 353 (Tyr-353) residue of ezrin is regulated through the PI3-kinase/Akt pathway [14]. Ezrin is also preferentially degraded and resynthesized after the phosphorylation at threonine 567 (Thr-567) depends on the p38 MAP-kinase activity [21]. Phosphorylation at Thr-567 has received a great deal of attention, as this phosphorylation event is believed to relieve the N- to C-terminal binding of ezrin, transforming ezrin into an active state with domains accessible for binding to membrane and F-actin [22]. This suggests that ezrin^Thr-567 alters ezrin molecule plasticity, and it is associated with numerous biological behaviors. Chen et al. [23] revealed that ezrin^Thr-567 has an important role in liver cancer metastasis. Krishnan et al. [24] found that transfection of the ezrin-Thr-567A mutant blocked the ezrin^Thr-567-inhibited metastases in Ewing’s sarcoma, suggesting that ezrin^Thr-567 is closely related to malignancy metastasis. Additionally, ezrin^Tyr-353 has been known to be related to subunit p85, which activates the PI3K/Akt pathway and plays an important role in modulating tumor cell survival, invasion, and metastasis. Cui et al. [25] reported that expression of ezrin^Tyr-353 correlated with less tumor differentiation and the presence of lymph node metastasis in pancreatic cancer. Lan et al. [26] found that induction of p-ezrin via the p38 MAP kinase signaling pathway was involved in the formation of microvilli during development of epithelial cell polarization. However, few reports to date have identified the favorable roles and prognostic value of p-ezrin in NSCLC. Zhang et al. [20] revealed that p-ezrin expression was significantly higher in NSCLC than in normal lung tissues, and was closely correlated with clinical stage and LN metastasis in NSCLC. In the present study, we found that p-ezrin exhibited cytosolic and membranous staining patterns in NSCLC, and the percentages of ezrin^Thr-567 and ezrin^Tyr-353 expression were significantly higher in NSCLC than in adjacent non-tumor tissues and normal tissue counterparts (P < 0.01). Interestingly, we found that total ezrin staining was lower than p-ezrin staining. Similarly, Oda et al. revealed that the expression rate of the ezrin was 42.5%, but 55.0% for ezrin^Tyr353 and 42.5% for ezrin^Tyr354 in intraductal papillary mucinous neoplasms of pancreas. Moreover, ezrin expression rate was 32.4%, but higher for ezrin^Tyr353 (41.2%) in intestinal-type pancreatic neoplasms [27]. Cui et al. and Di Cristofano et al. also reported the similar conclusions in pancreatic cancer and osteosarcoma, respectively [25, 28]. The cause might be related with that some cases mainly expressed p-ezrin, but showed lower ezrin level or not, suggesting that although total ezrin is sum of p-ezrin and non-phosphorylated ezrin in cell level, it is not suitable in the case study. It needs the further study to verify the detailed mechanism.

Among the clinicopathological features, both ezrin^Thr-567 and ezrin^Tyr-353 protein overexpression were significantly correlated with the poor differentiation and late clinical stage of NSCLC (P < 0.01). However, only ezrin^Thr-567 overexpression was significantly correlated with the presence of lymph node metastasis, suggesting that ezrin^Thr-567 was important for the invasion and metastasis processes in NSCLC. Interestingly, Orr et al. [29] recently found that EGF can induce ezrin phosphorylation (Thr567) via activation of the SK/S1P pathway, and Antelmi et al. [30] revealed that p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-locolized in a functional complex with EGFR and β1-integrin in metastatic breast cancer cell line MDA-MB-231, suggesting that ezrin might be related with EGF and EGFR in cancer progression. These results impelled us to study the detailed mechanism of the correlation between EGFR and ezrin, and whether EGFR-TKI can suppress the metastasis of NSCLC via ezrin phosphorylation in future.

In regard to survival, Zhang et al. [20] showed that ezrin-positive expression independently predicted inferior overall survival and disease-free survival. Additionally, ezrin overexpression was helpful to predict the poor survival of patients with early stage of NSCLC. Cui et al. [25] also found that overall survival of patients with pancreatic cancer was significantly associated with ezrin^Tyr-353, but not with total ezrin or ezrin^Thr-567. However, Di Cristofano et al. [28] reported no statistical significance regarding the relationship between p-ezrin expression and survival time in osteosarcoma. Here we demonstrated that ezrin^Tyr-353 has no correlation with the survival of patients with NSCLC. However, ezrin^Thr-567 expression was significantly correlated with adverse outcomes with respect to overall survival time, and both ezrin and ezrin^Thr-567 overexpressions were correlated with shorter survival time in patients with early stage NSCLC. The high proportion and prognostic value of ezrin and ezrin^Thr-567 expression in NSCLC suggested that ezrin, especially ezrin^Thr-567, could be a potential biomarker for NSCLC. However, more extensive investigations are needed to clarify the exact roles of ezrin and ezrin^Thr-567 in the development and progression of NSCLC.

Ezrin, ezrin^Thr-567 and ezrin^Tyr-353 were all significantly upregulated in NSCLC compared with normal tissues, and all correlated with the poor differentiation and late clinical stage of NSCLC. However, only ezrin^Thr-567 overexpression correlated with the presence of lymph node metastasis of NSCLC. Additionally, ezrin^Thr-567 correlated with the overall survival time of patients with NSCLC, and both ezrin and ezrin^Thr-567 overexpression were correlated with shorter survival time in patients with early stage NSCLC. In this regard, ezrin, especially ezrin^Thr-567, may prove to be useful as a novel prognostic biomarker of NSCLC.