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Abstract

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis (p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression (p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression (p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor.

Keywords

Endothelin-converting enzyme-1 survival esophageal squamous cell carcinoma

Introduction

Esophageal cancer, the eighth most common cancer causing more than 400,000 deaths per year in the world, is especially popular in Africa and East Asia.¹ In Taiwan, esophageal cancer, of which esophageal squamous cell carcinoma (ESCC) is the major subtype, is the fifth leading cause of cancer-induced deaths in males.² At the time of diagnosis, most patients have locally advanced, unresectable, or metastatic disease status (stage II–IV). Overall, 60% of patients could receive only palliative therapy.³ Even though some patients could receive curative surgery, their 5-year survival rates are usually less than 40%⁴ due to high recurrence rate (around 40%–60%).^5,6 Although neoadjuvant chemoradiotherapy (CRT) plus surgery provides better patient survival than surgery alone in patients with locally advanced esophageal cancer, these patients suffer from more postoperative mortalities.⁷ In order to provide adjuvant therapy besides surgery to patients with poor prognosis or high recurrence, we need biomarkers to identify this high-risk group. However, no such biomarkers are available now.

The endothelin (ET) family includes three isoforms of 21 amino acid peptides, ET-1, ET-2, and ET-3.⁸ All ETs are synthesized as inactive forms and converted to active ETs through serial processing. ET-converting enzyme-1 (ECE-1), which has four isoforms, catalyzes the conversion of pro-ETs into active ETs,⁹ which then bind to G protein–coupled receptors ET-A receptor (ET_AR) and ET-B receptor (ET_BR) with variable affinities.^10,11 Activation of the ET axis has great influence in many cancers.¹² Mounting evidence has shown that ET-1/ET_AR interaction induces phosphorylation of endothelial growth factor receptor,¹³ inhibits apoptosis,¹⁴ and promotes angiogenesis¹⁵ and proliferation¹⁶ of tumor cells. Moreover, preclinical data have supported the therapeutic potential of ET_AR antagonists in gynecological, urological, and breast cancers, which further proves the importance of ET axis in cancer progression.¹⁷

The role of ET axis in human ESCC has just been elucidated. Human ESCC, which has ET_AR and ET_BR on their surface,^18,19 expressed more ET-1 compared to adjacent normal tissues.²⁰ Blockade of ET signaling pathway by ET_AR/ET_BR antagonist could inhibit ESCC growth¹⁸ and reduce invasive ability of ESCC cells through downregulating cathepsin B.²⁰ Moreover, higher preoperative circulating pro-ET-1 level or tumor ET_BR expression in ESCC patients not only correlated with an advanced disease staging but also predicted a faster tumor recurrence and a worse patient survival after curative surgical resection.^19,21 These studies suggest that activation of ET axis plays a crucial role in growth and invasion of human ESCC.

ECE-1, which is involved in the ET axis, has not yet been studied in human ESCC. In this study, we investigated ECE-1 expression of human ESCC by immunohistochemical staining. We found that high tumor ECE-1 expression was associated with high risk of lymph node metastasis. Moreover, tumor ECE-1 expression could independently predict the survival in all ESCC patients and even in patients with locally advanced and potentially resectable ESCC.

Materials and methods

Patients and study design

This study consecutively enrolled 99 adults with histology-confirmed ESCC. Every patient had signed the inform consent to provide the pretreated cancer tissues for the ECE-1 expression validated by immunohistochemistry. The hospital ethics committees approved the study protocol (EMRP-20101N and BR-100-087). The clinical staging was evaluated by computed tomography (CT) and endoscopic ultrasound (EUS), and the tumor–node–metastasis (TNM) classification system by the American Joint Committee on Cancer²² was followed. Every patient was treated after a multi-disciplinary evaluation following the National Comprehensive Cancer Network (NCCN) guideline. For patients who did not have endoscopic resection or curative surgery, the initial treatment for the ESCC was definitive CRT using platinum-based regimens. All the patients received regular follow-up, and the posttreatment survival was evaluated by regular clinic visits, medical records, or telephone contact.

Immunohistochemistry staining to determine the ECE-1 expression

The esophageal tumor tissue was fixed in 10% buffered formalin, embedded in paraffin, and serially sectioned at 4 μm thickness. To stop endogenous peroxidase activity, the specimen was immersed for 20 min in 3% hydrogen peroxide and then pretreated with Dako Cytomation Target Retrieval Solution (Dako, Carpinteria, CA, USA) for antigen retrieval. The tissue section was treated with primary monoclonal antibody against ECE-1 (Catalog sc-376018; 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then the section was incubated for overnight in a humidified chamber at 4°C. The Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA) was used for blocking, linkage, and labeling for staining according to the manufacturer’s instructions. The Dako Cytomation Liquid DAB Substrate Chromogen system was used as chromogen. The section was then counterstained with hematoxylin. In total, 20 tumor-adjacent normal esophageal tissues obtained from endoscopic resection or surgery were also evaluated for ECE-1 expression and served as controls. The pathologist blinded to patients’ background scored the staining of ECE-1 expression. The expression levels of ECE-1 in the cytoplasm of tumor cells were quantified as ECE-1 score = (the intensity of staining (grade 1–4)) × (the percentage of positive-stained cells (%)) and then further dichotomized into either low ECE-1 expression (score ≤50) or high ECE-1 expression (score >50) (Figure 1).

Figure 1.

Representative pictures of tumor ECE-1 immunohistochemistry. (a) The immunohistochemistry staining showed high ECE-1 expression in the tumor part but negative ECE-1 expression over adjacent normal esophageal mucosa (original magnification: 40X). (b) A representative case of high ECE-1 level ESCC, demonstrating cytoplasmic staining of ECE-1 protein. (c) A representative case of low ECE-1 level ESCC, showing negative immunoreactivity of ECE-1 protein (original magnification: 200X).

Statistical analysis

The statistical analyses were performed using SPSS software (SPSS for Windows, version 18.0; SPSS Inc., Chicago, IL, USA). The comparisons between ECE-1 expression level in ESCCs and the clinical characteristics were carried out by χ² test, Fisher’s exact test, or Student’s t test. The cumulative survival of different groups was shown by Kaplan–Meier curves, and their differences were assessed by the two-tailed log-rank test. Based on the Cox proportional hazard model, the value of tumor ECE-1 expression in predicting patient survival was evaluated. Statistical significance was defined as p < 0.05.

Results

Correlations between tumor ECE-1 expression and clinicopathological findings of ESCC patients

The expression of ECE-1 on 99 ESCC tissues was evaluated by immunohistochemistry. Overall, 54 (54.5%) cases showed high ECE-1 expression. The adjacent normal esophageal tissues all revealed negative staining for ECE-1. To investigate the clinical significance of tumor ECE-1 expression, clinicopathological factors were compared between patients with low and high tumor ECE-1 expression (Table 1). There was no difference in the mean age; percentage of patients consuming alcohol, betel nut, or cigarettes; tumor differentiation; length or invasion depth; as well as ratio of patients with distant metastasis between the patients with low and high ECE-1 ESCC (p > 0.05). As compared to the ESCC with low ECE-1 expression, the ESCC with high ECE-1 expression had higher possibility of lymph node involvement (p < 0.05) and tended to be at more advanced stages (stage III and IV, p = 0.07).

Table 1.

Correlation of ECE-1 expression to the clinicopathological findings of patients with ESCC.

Characteristics n (%)	ECE-1 (n)	High (n = 54)	Low (n = 45)	p value
Age (years), mean ± SD (range)		52.5 ± 8.3 (30–74)	52.7 ± 9.1 (39–72)	0.91
Gender
Female	2	1 (50%)	1 (50%)	0.99
Male	97	53 (55%)	44 (45%)
Alcohol drinking
No	4	2 (50%)	2 (50%)	0.99
Yes	95	52 (55%)	43 (45%)
Betel nut chewing
No	22	10 (45%)	12 (55%)	0.35
Yes	77	44 (57%)	33 (43%)
Cigarette smoking
No	4	4 (100%)	0 (0%)	0.12
Yes	95	50 (53%)	45 (47%)
Tumor differentiation
Well or moderate	92	50 (54%)	42 (46%)	0.99
Poor	7	4 (57%)	3 (43%)
Tumor length (cm)
≤5	60	34 (57%)	26 (43%)	0.68
>5	39	20 (51%)	19 (49%)
Clinical stage
I or II	25	10 (40%)	15 (60%)	0.07
III or IV	74	44 (59%)	30 (41%)
Invasion depth
T1–2	24	12 (50%)	12 (50%)	0.64
T3–4	75	42 (56%)	33 (44%)
Lymph node
Negative	20	7 (35%)	13 (65%)	0.04
Positive	79	47 (59%)	32 (41%)
Distant metastasis
Negative	65	32 (49%)	33 (51%)	0.10
Positive	34	22 (65%)	12 (35%)

ECE-1: endothelin-converting enzyme-1, ESCC: esophageal squamous cell carcinoma; SD: standard deviation.

Associations between ECE-1 expression and survival of patients with ESCC

The Cox regression analyses confirmed the age, tumor size and the tumor differentiation were not related with the survival of ESCC. In contrast, both high ECE-1 expression levels in tumors (hazard ratio (HR) = 1.76, p = 0.030) and a clinical advanced stage (HR = 2.94, p = 0.002) were confirmed as independent factors of poor survival of ESCC after adjusted by tumor length (Table 2). Furthermore, as shown by the Kaplan–Meier curves in Figure 2(a), patients with high ECE-1 expressed ESCC had poorer 5-year overall survival than those with low ECE-1 expressed ESCC (p = 0.016).

Table 2.

Factors related to poor survival of patients with ESCC by Cox hazard regression.

Factors	Variable	Case number	95% CI	HR	p value
Univariate
Age (years)	>50 vs ≤50	56/43	0.79–2.12	1.30	0.299
Tumor length (cm)	>5 vs ≤5	39/60	0.90–2.37	1.46	0.129
Differentiation	Poor vs well or moderate	7/92	0.66–3.54	1.53	0.324
ESCC stage	III, IV vs I, II	74/25	1.64–6.03	3.14	0.001
ECE-1 level	High vs. low	54/45	1.11–3.01	1.83	0.018
Multivariate
Tumor length (cm)	>5 vs ≤5	39/60	0.64–1.82	1.08	0.784
ESCC stage	III, IV vs I, II	74/25	1.46–5.91	2.94	0.002
ECE-1 level	High vs low	54/45	1.06–2.93	1.76	0.030

ESCC: esophageal squamous cell carcinoma; CI: confidence interval; HR: hazard ratio; ECE-1: endothelin-converting enzyme-1.

Figure 2.

High tumor endothelin-converting enzyme-1 (ECE-1) expression was associated with worse survival in patients with (a) all stages of ESCC (log-rank test, p = 0.016) or (b) locally advanced and potentially resectable ESCC (stage II or III; log-rank test, p = 0.003).

Significance of tumor ECE-1 expression in patients with stage II and III ESCC

Among 99 patients, 61 patients belonged to ESCC stage II or III, in which tumors were locally advanced and potentially resectable. In this specific population, patients received either definitive CRT or surgery based regimens. As shown in Figure 2(b), patients in this population still had worse survival if their tumor ECE-1 expression was higher (p = 0.003). By univariate Cox regression analysis, definitive CRT (p = 0.025) and high tumor ECE-1 expression (p = 0.004) were predictive factors for worse survival of patients with stage II or III ESCC. Moreover, after adjustment by treatment strategy and tumor staging, high tumor ECE-1 expression (p = 0.012) still significantly predicted worse survival of patients in this population (Table 3).

Table 3.

Independent factors to predict poor survival of patients with locally advanced and potentially resectable ESCC by Cox hazard regression.

Factors	Variable	Case number	95% CI	HR	p value
Univariate
Age (years)	>50 vs ≤50	30/31	0.60–2.14	1.13	0.702
Tumor length (cm)	>5 vs ≤5	27/34	0.57–2.07	1.08	0.805
Differentiation	Poor vs well or moderate	5/56	0.53–4.22	1.49	0.449
ESCC stage	III vs II	49/12	0.70–3.64	1.60	0.265
Treatment	Definitive CRT vs OP-based	27/34	1.09–3.95	2.08	0.025
ECE-1 level	High vs low	34/27	1.40–5.61	2.80	0.004
Multivariate
ESCC stage	III vs II	49/12	0.56–3.02	1.30	0.548
Treatment	Definitive CRT vs OP-based	27/34	0.85–3.21	1.65	0.141
ECE-1 level	High vs low	34/27	1.22–5.05	2.48	0.012

ESCC: esophageal squamous cell carcinoma; CI: confidence interval; HR: hazard ratio; CRT: chemoradiotherapy; OP: operation; ECE-1: endothelin-converting enzyme-1.

Discussion

Since currently patients with ESCCs have poor prognosis even after various types of treatment, it is necessary to find a biomarker to predict the prognosis of patients. This study has provided the first evidence of ECE-1 as a potential biomarker to predict patient prognosis. This study proved that in patients with ESCC, high tumor ECE-1 expression was associated with tumor invasion to regional lymph nodes and poor survival of patients. Moreover, tumor ECE-1 expression, instead of clinical staging, could also predict survival of in-patients with locally advanced and potentially resectable ESCC.

ECE-1 has been shown to be upregulated in some types of human cancers other than ESCC. However, the role of ECE-1 in these cancers is still poorly understood. In patients with breast cancer, tumor ECE-1 overexpression was associated with higher disease recurrence.²³ Although ECE-1 was abundantly produced in endometrial adenocarcinoma,^24,25 prostate cancer,²⁶ and lung cancer cells,²⁷ its clinical value was not yet demonstrated by epidemiological research. Only some in vitro studies have shown that ECE-1 could promote cell proliferation of oral squamous carcinoma cells²⁸ as well as maintain invasion of prostate²⁹ and ovarian cancer cells.³⁰ This study demonstrates that tumor ECE-1 overexpression can predict poor survival of patients with ESCC, which strengthens the clinical value of ECE-1 in cancer situation.

In this study, high tumor ECE-1 expression was associated with regional lymph node invasion, not with tumor length, invasion depth, or differentiation. In addition, previous studies have shown that knockdown of ECE-1 not only decreases prostate cancer cell invasion and migration²⁹ but also inhibits invasive phenotype of ovarian carcinoma cells.³⁰ It indicates that tumor ECE-1 is involved in tumor invasion rather than tumor cell proliferation and transformation. This hypothesis can further explain why tumor ECE-1 level is a stronger predictive factor of survival in patients with locally advanced and potentially resectable ESCCs than in general ESCC patients. In this subpopulation, micrometastasis, which can influence tumor staging and patient prognosis, is hardly detected and evaluated precisely. High tumor ECE-1 expression might indicate high tumor invasive ability, which causes more preexisting or incoming micrometastases, resulting in worse survival in the subpopulation. Thus, tumor ECE-1 expression can be a potentially useful marker to predict survival especially in patients with locally advanced and potentially resectable ESCC.

Not only utilized as a biomarker, ECE-1 can also be therapeutically targeted. Different ECE-1 inhibitors have been synthesized recently. RO67-7447, one of the ECE-1 inhibitors, could reduce breast cancer cell invasion²³ and inhibit growth of human glioblastoma cells³¹ both in vitro. In vivo studies using these ECE-1 inhibitors are currently being conducted. ESCC patients with high tumor ECE-1 expression could be good candidates for ECE-1 inhibitor treatment.

Taken together, the ECE-1 expression level in ESCC can serve as a good biomarker to identify patients with poor survival. ESCC with high ECE-1 expression may not only implicate higher tumor invasion possibility but also become a therapeutic target in the future.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a grant from E-Da Hospital (EDAHP105046) and the Ministry of Science and Technology, Taiwan (MOST-103-2314-B-650-006).

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