Elsevier

Human Pathology

Volume 43, Issue 7, July 2012, Pages 1068-1076
Human Pathology

Original contribution
Reduced nuclear and ectopic cytoplasmic expression of lysyl oxidase–like 2 is associated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma

https://doi.org/10.1016/j.humpath.2011.07.027Get rights and content

Summary

Lysyl oxidase family members have various roles in cancer progression. The aim of this study was to investigate their expression and clinical significance in esophageal squamous cell carcinoma. We examined messenger RNA expression of lysyl oxidase family members including lysyl oxidase and lysyl oxidase–like proteins (lysyl oxidase L) in 10 esophageal squamous cell carcinoma cell lines and 83 pairs of tumor samples by quantitative real-time polymerase chain reaction. All except lysyl oxidase L3 were expressed at high levels in esophageal squamous cell carcinoma, but only lysyl oxidase L2 was associated with lymph node metastasis (P = .034). We examined lysyl oxidase L2 protein further by immunohistochemistry staining in 178 surgically resected esophageal squamous cell carcinoma tissue samples. The protein manifested decreased nuclear expression and increased cytoplasmic expression. Moreover, these 2 events both had significant correlation with the presence of lymph node metastasis (P = .001 and P < .001). Overall survival rates of the patients with esophageal squamous cell carcinoma with decreased nuclear expression or increased cytoplasmic expression of lysyl oxidase L2 were significantly lower than those of the patients with esophageal squamous cell carcinoma with the reverse expression pattern (P = .040 or P = .022). Multivariate analyses revealed that nuclear expression of lysyl oxidase L2 was an independent prognostic factor for esophageal squamous cell carcinoma. These results suggest that lysyl oxidase L2 exerts a critical effect on esophageal squamous cell carcinoma progression and can be a predictive marker of lymph node metastasis and outcome.

Introduction

Esophageal squamous cell carcinoma (ESCC), the most common histopathologic form of esophageal cancer, is one of the most prevalent cancers worldwide and is the fourth leading cause of cancer deaths in China [1], [2]. It has a poor survival rate because ESCC frequently exhibits local invasion and metastasis when discovered [3]. Thus, it is important to establish biological markers for determining treatment.

Lysyl oxidase (LOX) was described initially as a cuproenzyme that catalyzes oxidative deamination of lysine residues in collagen and elastin and makes extracellular matrix stable and strong [4]. Besides LOX, 4 human isoenzymes of LOX-like protein (LOXL) have been described. The LOX family is present in a variety of human tissues including the placenta, heart, lung, kidney, and pancreas [5], [6], [7], [8], [9]. It is involved in growth and development, senescence, chemotaxis, and cell mobility [10]. Many studies focused mainly on their function in tumor formation and progression. In this regard, LOX, perhaps, had a contradictory effect. Both the catalytic activity of LOX and its messenger RNA (mRNA) expression were dramatically down-regulated in most cancer cells. However, many studies demonstrated that the amount of LOX mRNA is elevated in highly invasive cancer cells [11]. High expression of LOXL2 was detected in many tumor cell lines and found to correlate closely with tumor invasion and metastasis [12], [13], [14], [15]. Similarly, we found it to be increased in cholangiocarcinoma, where it could promote lymph node and distant metastasis [16], [17]. Also, LOXL4 was up-regulated in some tumor cells such as bladder cancer and relative-to-normal epithelial cells. It could promote tumor cell colony formation and invasion [18], [19]. Nevertheless, the function of LOXL1 and LOXL3 in cancer was largely unknown. Some experiments indicated that all of them were expressed in highly invasive/metastatic breast cancer cells but not in poorly invasive/metastatic ones [20].

The functions of the LOX family in ESCC were not known, so we examined each member's expression in ESCC and analyzed its clinical relevance. The aim was to evaluate the effect of LOX family proteins on ESCC and identify risk factors.

Section snippets

Cell lines and culture

Human kidney 293T cells and human ESCC cell lines (SHEEC, EC18, EC109, EC171, EC8712, KYSE70, KYSE140, KYSE150, KYSE180, KYSE510, COLO-680N) were used. The KYSE and COLO-680N lines were cultured in RPMI 1640 medium (HYCLONE, Beijing, China). The EC cell lines were cultured in 199 medium (HYCLONE) and SHEEC in 45% Dulbecco modified Eagle medium/45% Ham Nutrient Mixture F12 (GIBCO, Grand Island, NY). All media were supplemented with 10% fetal bovine serum and antibiotics (penicillin 100 U/mL and

Expression of LOX family in ESCC cell lines

The expression of LOX family mRNA was investigated in 10 ESCC cell lines by quantitative RT-PCR. Compared with 293T cells, only LOXL2 mRNA had higher expression in half of all ESCC cell lines (5/10), whereas other members showed mainly low expression (Fig. 1).

Messenger RNA levels of LOX family in ESCC tissues

Each of the LOX family members' expression at the mRNA level was investigated in 83 pairs of ESCC tissues by quantitative RT-PCR. Greater mRNA expression compared with normal esophageal tissues was documented in 56 ESCC tissues (67%) for

Discussion

The LOX family members are extracellular enzymes that catalyze oxidative deamination of lysine residues. More and more reports have shown that LOX family proteins also can locate in the intracellular domain to exert many complicated biological functions. Nevertheless, the roles of LOX family members in cancer are not clear, and the function in ESCC, if any, is largely unknown. In our study, the LOX mRNA concentrations in ESCC tissues were significantly higher than in normal tissues except for

Supplementary data

The following is the Supplementary data to this article.

. Clinicopathological features of patients with ESCC for quantitative real-time RT-PCR analysis.

. Clinicopathological features of patients with ESCC for LOXL2 immunohistochemical analysis.

. Relationship between LOX mRNA expression and clinicopathological features.

. Relationship between LOXL1 mRNA expression and clinicopathological features.

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    This work was supported by grants from the National Nature Science Foundation of China (No. 81000900; Beijing, China) and the National Natural Science Foundation of China-Guangdong Joint Fund (No. U0932001; Beijing, China).

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