Abstract
We previously reported a gradual increase of relative mitochondrial DNA (mtDNA) copy number during the progression of esophageal squamous cell carcinoma (ESCC). Because mitochondria are the intracellular organelles responsible for ATP production, we investigated the associations among mtDNA copy number, mitochondrial bioenergetic function, tumor invasion and the expression levels of epithelial mesenchymal transition (EMT) markers in a series of seven ESCC cell lines, including 48T, 81T, 146T, TE1, TE2, TE6 and TE9. Among them, TE1 had the highest relative mtDNA copy number of 240.7%. The mRNA of mtDNA-encoded ND1 gene (2.80), succinate-supported oxygen consumption rate (11.21 nmol/min/106 cells), ATP content (10.7 fmol/cell), and the protein level of mitochondrial transcription factor A (TFAM) were the highest and the lactate concentration in the culture medium (3.34 mM) was the lowest in TE1. These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria. Furthermore, TE1 showed the highest trans-well migration activity of 223.0 cells/field, the highest vimentin but the lowest E-cadherin protein expression levels, which suggest that TE1 had the highest invasion capability. We then conducted a knockdown study using pLKO.1-based lentiviral particles to infect TE1 cells to suppress the expression of TFAM. Molecular analyses of the parental TE1, control TE1-NT and TFAM knockdown TE1-sh-TFAM(97) cells were performed. Interestingly, as compared to the control TE1-NT, TE1-sh-TFAM(97) exhibited lower levels of the relative mtDNA copy number (p = 0.001), mRNA of mtDNA-encoded ND1 gene (p = 0.050), succinate-supported oxygen consumption rate (p = 0.065), and ATP content (p = 0.007), but had a higher lactate concentration in the culture medium (p = 0.010) and higher protein level of lactate dehydrogenase. A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97). Significantly, compared to the control TE1-NT, TE1-sh-TFAM(97) had a lower trans-well migration activity (p < 0.001), a higher E-cadherin level but a lower vimentin protein level, which indicates a decrease of invasiveness. Taken together, we suggest that high relative mtDNA copy number and bioenergetic function of mitochondria may confer an advantage for tumor invasion of ESCC.
Highlights
Esophageal squamous cell carcinoma (ESCC) is an aggressive neoplasm in Asia, especially in Southern China, Hong Kong and Taiwan [1,2,3,4,5]
In a previous study of 72 pairs of ESCC clinical samples, we found that the relative copy number mitochondrial DNA was increased gradually from the non-cancerous esophageal mucosa to cancerous ESCC nest and the metastatic lymph node [6]
In light of these findings, and the fact that mitochondria are the intracellular organelles responsible for ATP production to meet the energy demand of human cells [7], we investigated whether mitochondrial DNA (mtDNA) copy number is related to energy production and plays an important role in the progression of ESCC
Summary
Esophageal squamous cell carcinoma (ESCC) is an aggressive neoplasm in Asia, especially in Southern China, Hong Kong and Taiwan [1,2,3,4,5]. In a previous study of 72 pairs of ESCC clinical samples, we found that the relative copy number mitochondrial DNA (mtDNA) was increased gradually from the non-cancerous esophageal mucosa to cancerous ESCC nest and the metastatic lymph node [6] In light of these findings, and the fact that mitochondria are the intracellular organelles responsible for ATP production to meet the energy demand of human cells [7], we investigated whether mtDNA copy number is related to energy production and plays an important role in the progression of ESCC. In contrast to normal human tissues, human cancers can display an avid glucose uptake with profound lactate production to generate ATP, even if the surrounding oxygen supply is sufficient This special phenomenon is termed Warburg effect, which was first observed by Dr Otto Warburg eight decades ago. One of these 7 cell lines, subsequently underwent TFAM knockdown for a further study of the relationships among the relative mtDNA copy number, bioenergetic function and the invasive phenotype of ESCC
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