Background
Esophageal cancer (EC) is a common malignancy worldwide, with Asia being one of the high-prevalence areas [
1]. Although the incidence rates for esophageal adenocarcinoma have been increasing in several Western countries, esophageal squamous cell carcinoma (ESCC) is the most common histological type in Eastern countries, such as China, where it accounts for more than 90 % of EC cases [
2]. Despite improvements in surgical techniques, perioperative management, and surgery combined with chemotherapy and/or radiotherapy, the prognosis for ESCC remains poor, particularly in advanced stages [
3]. Therefore, the development of new therapy modalities, particularly targeted therapies based on knowledge of the biology and genetics of the disease, may offer the potential for improving treatment response and quality of life for ESCC patients.
In the past decade, great interest has been directed toward the use of agents targeting cell surface receptors that are responsible for the development and progression of various cancers. Epidermal growth factor receptor (EGFR) is one of the most commonly altered receptors in human malignancies. This receptor is mainly involved in regulating cellular processes including cell apoptosis, proliferation, angiogenesis, migration, and adhesion through activation of PI3K-Akt, STAT3, and Ras-Raf-MAPK signaling pathways [
4]. In a variety of human cancers, increased expression of EGFR has been identified and shown to be associated with advanced disease, development of metastases, and poor clinical prognosis in a subset of these cancers [
5]. However, study results on the prognostic effect of EGFR in ESCC remain conflicting [
6‐
10]. To investigate the role of EGFR-related pathway activities in ESCC progression, we used tissue microarray (TMA) technology and immunohistochemical (IHC) analysis to evaluate the activities of EGFR and its downstream effectors AKT, ERK, and STAT3 in ESCC; we also analyzed the relationships of these markers and their association with prognosis in ESCC patients.
Discussion
In this study, we tested the protein expression and activities of EGFR as well as several key nodes on its downstream pathways for ESCC patients and found that expression of p-AKT1, p-AKT2, p-ERK1/2, and p-STAT3 was significantly related to the expression of p-EGFR. We also found that activation of AKT1 independently influenced patients’ survival, with higher expression of p-AKT1 being linked to poorer OS; neither EGFR nor p-EGFR expression, however, had a prognostic effect in ESCC patients in this cohort.
Currently, the role of EGFR in tumor development in ESCC is not clear, although elevated expression of EGFR has been reported in 50–90 % of patients with ESCC [
16‐
20]. Several studies have shown that EGFR overexpression was associated with poor OS and poor disease-free survival in ESCC patients [
6‐
8]; other studies, however, did not find a prognostic effect for EGFR overexpression in ESCC [
9,
10]. The inconsistent conclusions drawn from the various studies might be due to differences in patient selection, treatments, and the methods used for detecting and scoring EGFR expression.
In the present study, the prognostic value of EGFR was not found. Several researchers [
9,
21] have reported that EGFR expression has predictive value for the therapeutic effect of chemotherapy and radiotherapy in ESCC; specifically, patients with elevated EGFR expression had better treatment outcomes after chemoradiotherapy. To rule out the influence of adjuvant chemotherapy and/or radiotherapy in the prognosis, we performed Cox regression analyses for the 106 patients who did not receive adjuvant therapy in our group and still found no association between EGFR expression and OS (data not shown). Our future work will explore whether alterations of EGFR, including gene mutation and amplification, have prognostic values in ESCC.
EGFR is a tyrosine kinase receptor, and the phosphorylated tyrosine residue serves as a docking site to exert its biological roles. After being phosphorylated, EGFR is activated and then in turn activates multiple downstream intracellular signaling pathways, mainly PI3K-Akt, STAT3, and Ras-Raf-MAPK pathways. Our finding that p-EGFR was highly correlated to the phosphorylation of AKT1, AKT2, ERK1/2, and STAT3 indicated that p-EGFR possibly contributed to the activation of these downstream pathways in ESCC, suggesting that the EGFR pathways might be active in some patients with ESCC. However, we also observed that p-AKT1 could predict the prognosis of ESCC, while EGFR and p-EGFR could not be, suggesting that the activation of AKT1 resulted from other factors in some patients. Besides EGFR stimulation, several other ways of activating AKT1 have been reported, including other growth factor receptors such as VEGF and PDGF, mutations of PI3K or RAS, inactivation of tumor suppressor gene PTEN, and AKT1
E17K somatic mutations [
22‐
26]. The exact mechanisms of this phenomenon in ESCC are unclear and need further investigation.
AKT, a serine/threonine protein kinase, is the central mediator of the canonical PI3K pathway, which can mediate various cellular functions including cell metabolism, growth, proliferation, survival, apoptosis, and angiogenesis [
27]. A number of studies have demonstrated the overactivation of AKT in many human solid tumors and hematological malignancies [
28]. AKT has three isoforms: AKT1, AKT2, and AKT3. Although these AKT family members share a similar domain structure, they have distinct substrates and different physiological behaviors [
29]. These AKT isoforms seem to mediate different functions in cancer pathophysiology; for example, AKT1 appears to promote mammary tumor induction, whereas AKT2 promotes metastasis in previous reports [
29,
30]. This may explain the difference in the prognostic effects between p-AKT1 and p-AKT2 in ESCC patients in our study.
The prognostic values of p-AKT1 have been studied for several malignancies. Interestingly, many studies have shown that activation of AKT1 was associated with poor prognosis [
31‐
33], whereas other studies have shown AKT1 activation to be a favorable prognostic indicator [
34‐
36]. To the best of our knowledge, few studies have examined the association between AKT1 activation and clinical outcome in ESCC. Yoshioka et al. [
37] used IHC analysis to examine p-AKT expression in 235 ESCC patients who underwent surgery with or without preoperative chemotherapy and found that p-AKT expression was associated with poor prognosis in those who had received chemotherapy but did not correlate with survival in those who had not received chemotherapy. However, that study did not specify the isoform of AKT1. Nowadays, the PI3K/AKT pathway has been recognized as an important pathway in the development of cancers [
38]. Our study suggested the potential of AKT1 as a target for anticancer therapeutics in ESCC.
Acknowledgments
We thank the Department of Scientific Publication at The University of Texas MD Anderson Cancer Center for editorial review of this paper. This work was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and Foundation of Shanghai Municipal Commission of Health and Family Planning, and partially supported by Shanghai Committee of Science and Technology, Natural Science Foundation of Shanghai, China (NO.11ZR1407300).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KLZ, XLF, ZZF and WWY conceived and designed the study. ZZF, WWY, MHS, QW and DLL performed the experiments. ZZF, WWY and DLL analyzed the data. HCC, JXX, HCL, YWZ and WXZ contributed reagents/materials/analysis tools. ZZ and WY wrote the paper. ZZF, WWY, XLF, MHS, QW, DLL, HQC, JXX, HCL, YWZ, WXZ and KLZ read and revised the manuscript, accepted the final version. All authors read and approved the final manuscript.