Metabolic Reprogramming in Response to Alterations of Mitochondrial DNA and Mitochondrial Dysfunction in Gastric Adenocarcinoma

Abstract

We used gastric cancer cell line AGS and clinical samples to investigate the roles of mitochondrial DNA (mtDNA) alterations and mitochondrial respiratory dysfunction in gastric adenocarcinoma (GAC). A total of 131 clinical samples, including 17 normal gastric mucosa (N-GM) from overweight patients who had received sleeve gastrectomy and 57 paired non-cancerous gastric mucosae (NC-GM) and GAC from GAC patients who had undergone partial/subtotal/total gastrectomy, were recruited to examine the copy number and D310 sequences of mtDNA. The gastric cancer cell line AGS was used with knockdown (KD) mitochondrial transcription factor A (TFAM) to achieve mitochondrial dysfunction through a decrease of mtDNA copy number. Parental (PT), null-target (NT), and TFAM-KD-(A/B/C) represented the parental, control, and TFAM knocked-down AGS cells, respectively. These cells were used to compare the parameters reflecting mitochondrial biogenesis, glycolysis, and cell migration activity. The median mtDNA copy numbers of 17 N-GM, 57 NC-GM, and 57 GAC were 0.058, 0.055, and 0.045, respectively. The trend of decrease was significant (p = 0.030). In addition, GAC had a lower mean mtDNA copy number of 0.055 as compared with the paired NC-GM of 0.078 (p < 0.001). The mean mtDNA copy number ratio (mtDNA copy number of GAC/mtDNA copy number of paired NC-GM) was 0.891. A total of 35 (61.4%) GAC samples had an mtDNA copy number ratio ≤0.804 (p = 0.017) and 27 (47.4%) harbored a D310 mutation (p = 0.047), and these patients had shorter survival time and poorer prognosis. After effective knockdown of TFAM, TFAM-KD-B/C cells expressed higher levels of hexokinase II (HK-II) and v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT, but lower levels of phosphorylated pyruvate dehydrogenase (p-PDH) than did the NT/PT AGS cells. Except for a higher level of p-PDH, the expression levels of these proteins remained unchanged in TFAM-KD-A, which had a mild knockdown of TFAM. Compared to those of NT, TFAM-KD-C had not only a lower mtDNA copy number (p = 0.050), but also lower oxygen consumption rates (OCR), including basal respiration (OCR_BR), ATP-coupled respiration (OCR_ATP), reserve capacity (OCR_RC), and proton leak (OCR_PL)(all with p = 0.050). In contrast, TFAM-KD-C expressed a higher extracellular acidification rate (ECAR)/OCR_BR ratio (p = 0.050) and a faster wound healing migration at 6, 12, and 18 h, respectively (all with p = 0.050). Beyond a threshold, the decrease in mtDNA copy number, the mtDNA D310 mutation, and mitochondrial dysfunction were involved in the carcinogenesis and progression of GACs. Activation of PDH might be considered as compensation for the mitochondrial dysfunction in response to glucose metabolic reprogramming or to adjust mitochondrial plasticity in GAC.

Keywords: D310 mutation; copy number; gastric adenocarcinoma (GAC); metabolic reprogramming; mitochondrial DNA (mtDNA); mitochondrial transcription factor A (TFAM); prognosis.

Figure 1

Kaplan-Meier survival curves, p-values (Log-rank test), and their HRs (including 95% CI, Cox proportional-hazards regression, univariate) about the prognostic roles of (A) mtDNA copy number ratio, high vs. low and (B) D310 mutation, no vs. yes in GAC patients are illustrated. GAC patients harboring a low mtDNA copy number ratio (p = 0.017) or D310 mutation (p = 0.047) had poorer prognosis. CI: confidence interval; GAC: gastric adenocarcinoma; HR: hazard ratio; High: mtDNA copy number ratio, >0.804; Low: mtDNA copy number ratio, ≤0.804; No: without the D310 mutation; Yes: with the D310 mutation.

Figure 2

(A) In the left two lanes, the Western blot shows that clones NT and PT had similar TFAM/PDH-E1α/p-PDH-E1α(S293)/HK-II/LDH/AKT expression levels. The α-tubulin is used as the internal control. In the first row, clones TFAM-KD-B/C expressed obviously lower levels and clone TFAM-KD-A expressed a mildly lower level of TFAM than did clones NT/PT. In the second row, clones TFAM-KD-B/C expressed lower levels of p-PDH-E1α(S293), but clone TFAM-KD-A expressed a higher level of p-PDH-E1α(S293) than did clones NT/PT. In the third row, clones TFAM-KD-A/B/C expressed similar levels of PDH-E1α to those of clones NT/PT. In the fourth row, clones TFAM-KD-B/C expressed higher levels of HK-II, and clone TFAM-KD-A expressed a similar level of HK-II to those of clones NT/PT. In the fifth row, clones TFAM-KD-A/B/C expressed similar levels of LDH to those of clones NT/PT. In the sixth row, clones TFAM-KD-B/C expressed higher levels of AKT, and clone TFAM-KD-A expressed a similar level of AKT to those of clones NT/PT. (B) The clone TFAM-KD-C had a lower mtDNA copy number (0.610 ± 0.154 vs. 0.958 ± 0.168, p = 0.050) than did the clone NT. (C) The clone TFAM-KD-C had lower OCRs (pmol/min/10⁶ cells), including those for basal respiration (OCR_BR)(945.4 ± 67.5 vs. 2618.8 ± 410.9, p = 0.050), ATP-coupled (OCR_ATP)(534.0 ± 139.3 vs. 1257.1 ± 243.8, p = 0.050), reserve capacity (OCR_RC)(586.5 ± 309.8 vs. 2205.7 ± 397.9, p = 0.050), and proton leak (OCR_PL)(411.4 ± 134.5 vs. 1361.8 ± 374.8, p = 0.050), than did the clone NT. (D) The clone TFAM-KD-C had a higher ECAR/OCR_BR ratio (1.248 ± 0.071 vs. 0.730 ± 0.098, p = 0.050) than did the clone NT. (E) The clone TFAM-KD-C had a shorter wound width (μm) at 6 hr (333.8 ± 19.7 vs. 459.0 ± 13.6, p = 0.050), 12 hr (251.9 ± 2.0 vs. 361.7 ± 3.1, p = 0.050), and 18 hr (115.7 ± 11.1 vs. 330.0 ± 22.0, p = 0.050), respectively, than did the clone NT. NT: Null target; PT: Parental; TAFM: mitochondrial transcription factor A; PDH-E1α: pyruvate dehydrogenase E1α subunit; p-PDH-E1α (293): phosphorylated pyruvate dehydrogenase E1α subunit; HK-II: hexokinase II; LDH: lactate dehydrogenase; AKT: v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT; mtDNA: mitochondrial DNA; OCR: oxygen consumption rate; ECAR: extracellular acidification rate; hr: hour.