Esophageal squamous cell carcinoma with low mitochondrial copy number has mesenchymal and stem-like characteristics, and contributes to poor prognosis.

Yasunori Masuike,Kiyokazu Nakajima,Tsuyoshi Takahashi,Yasuhiro Miyazaki,Tomoki Makino,Masaki Mori,Yukinori Kurokawa,Koji Tanaka,Yuichiro Doki,Makoto Yamasaki,Hyun-Sung Lee

doi:10.1371/journal.pone.0193159

Abstract

Alterations in mitochondrial DNA (mtDNA) copy numbers in various human cancers have been studied, but any such changes in esophageal squamous cell carcinoma (ESCC) are not established. In the present study, we investigated the correlation of mtDNA copy number with clinicopathologic features, prognosis, and malignant potential of ESCC. MtDNA copy numbers of resected specimens from 80 patients treated with radical esophagectomy were measured by quantitative real-time PCR analyses. Human ESCC cells, TE8 and TE11, were cultured, and depletion of mtDNA content was induced by knockdown of mitochondrial transcription factor A expression or treatment with ethidium bromide. The mRNA and protein expression, proliferation, invasion, and cell cycle were investigated. The results showed that the mtDNA copy number of cancerous portions was 56.0 (37.4–234.5) percent that of non-cancerous parts and significantly lower (p<0.01). Low mtDNA copy number in resected cancerous tissues was significantly correlated with pathological depth of tumor invasion (p = 0.045) and pathological stage (p = 0.025). Patients with lower mtDNA copy number had significantly poorer 5-year overall survival compared to patients with higher levels (p<0.01). The mtDNA-depleted TE8 and TE11 cells had morphological changes and proliferated more slowly than control cells under normoxia but proliferated at almost the same rate under hypoxic conditions. In mtDNA-depleted cells, E-cadherin mRNA expression was decreased, and N-cadherin, vimentin, zeb-1, and cd44 mRNA expression was increased. Immunoblotting and flow cytometry analysis also showed downregulated E-cadherin and upregulated N-cadherin and CD44 protein in mtDNA-depleted cells. Moreover, mtDNA-depleted cells had enhanced invasion, migration, and sphere formation abilities, and the cell cycle arrest at G0/G1 phase was induced in these cells. These results suggested that mtDNA-depleted ESCC cells had mesenchymal characteristics, cancer stemness, and tolerance to hypoxia, which played important role in cancer progression. In conclusion, a low copy number of mtDNA is associated with tumor progression in ESCC.

Highlights

Esophageal cancer is the eighth most common cancer worldwide, with an estimated 450,000 new cases annually, and the sixth most common cause of death from cancer, with an estimated 400,000 deaths each year
We found that mitochondrial DNA (mtDNA) copy number in esophageal squamous cell carcinoma (ESCC) was significantly lower than that of non-cancerous tissues and that low mtDNA copy number was associated with tumor progression and poor prognosis
The present study showed that low mtDNA copy number is correlated with tumor invasion, epithelial-to-mesenchymal transition, cancer stemness, and poor prognosis in ESCC

Summary

Introduction

Esophageal cancer is the eighth most common cancer worldwide, with an estimated 450,000 new cases annually, and the sixth most common cause of death from cancer, with an estimated 400,000 deaths each year. Mitochondria are eukaryotic intracellular organelles that produce the majority of cellular ATP through the process of oxidative phosphorylation, and play an important role in reactive oxygen species production and integrating apoptosis pathways [11,12,13]. They contain their own DNA (mtDNA), which consists of a circular double-stranded structure with 16,569 base pair and encodes 13 polypeptides that are essential for the assembly of respiratory enzyme complexes [12, 14,15,16]. The aim of this study was to clarify the correlation of mtDNA copy number with clinicopathologic features, prognosis, and cell characteristics of ESCC

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