Altered mitochondrial function and energy metabolism is associated with a radioresistant phenotype in oesophageal adenocarcinoma

Abstract

Neoadjuvant chemoradiation therapy (CRT) is increasingly the standard of care for locally advanced oesophageal cancer. A complete pathological response to CRT is associated with a favourable outcome. Radiation therapy is important for local tumour control, however, radioresistance remains a substantial clinical problem. We hypothesise that alterations in mitochondrial function and energy metabolism are involved in the radioresistance of oesophageal adenocarcinoma (OAC). To investigate this, we used an established isogenic cell line model of radioresistant OAC. Radioresistant cells (OE33 R) demonstrated significantly increased levels of random mitochondrial mutations, which were coupled with alterations in mitochondrial function, size, morphology and gene expression, supporting a role for mitochondrial dysfunction in the radioresistance of this model. OE33 R cells also demonstrated altered bioenergetics, demonstrating significantly increased intracellular ATP levels, which was attributed to enhanced mitochondrial respiration. Radioresistant cells also demonstrated metabolic plasticity, efficiently switching between the glycolysis and oxidative phosphorylation energy metabolism pathways, which were accompanied by enhanced clonogenic survival. This data was supported in vivo, in pre-treatment OAC tumour tissue. Tumour ATP5B expression, a marker of oxidative phosphorylation, was significantly increased in patients who subsequently had a poor pathological response to neoadjuvant CRT. This suggests for the first time, a role for specific mitochondrial alterations and metabolic remodelling in the radioresistance of OAC.

Figure 1. Radioresistant OE33 R cells have increased mitochondrial mutagenesis and altered mitochondrial function.

(A) OE33 R cells demonstrate significantly elevated basal levels of random mitochondrial mutations, when compared to OE33 P. (B) OE33 R cells have significantly increased basal ROS levels, when compared to OE33 P. ROS levels are significantly increased in OE33 P cells at 24 h post irradiation with 2 Gy. (C) Mitochondrial mass is significantly increased in OE33 P cells at 24 h post irradiation with 2 Gy, this effect is not seen in OE33 R. Data are presented as mean±SEM from 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s t-test, *P<0.05.

Figure 2. OE33 R cells have increased basal numbers of mitochondria and display altered morphology.

The ultrastructure of OE33 P (A, C and E) and OE33 R cells (B, D and F) was assessed basally (A, B, E, F) and at 24 h post 2 Gy (C and D) by TEM. Representative images are shown. Black arrows point to mitochondria.

Figure 3. OE33 R cells display alterations in mitochondrial-associated gene expression, total cellular energy and energy metabolism pathways.

(A) Basal expression of 15 mitochondrial function and energy metabolism-associated genes were altered 1.5-fold in OE33 R cells, when compared to OE33 P. Data are presented as mean ± SEM from 2 independent experiments. (B) OE33 R cells have significantly increased basal intracellular ATP levels, when compared to OE33 P. ATP levels significantly decrease in OE33 P cells at 24 h post irradiation with 2 Gy, when compared to basal levels. Data are presented as mean ± SEM from 1 independent experiment. (C) Basal oxygen consumption rates (OCR) are significantly increased in OE33 R cells, when compared to OE33 P. Data are presented as mean ± SEM from 4 independent experiments. (D) OE33 R and OE33 P cells demonstrate similar extracellular acidification rates (ECAR). Data are presented as mean ± SEM from 4 independent experiments. Statistical analysis was performed by 2-tailed Student’s t-test, *P<0.05, **P<0.01.

Figure 4. OE33 R cells have higher ATP turnover and reduced proton leak.

(A) OE33 P and OE33 R cells demonstrate similar sensitivity to antimycin (2.6 µM). (B) OE33 R cells were significantly more sensitive to the effects of oligomycin (2 µg/mL) on OCR, when compared to OE33 P cells. At 24 h post 2 Gy, OE33 P cells were significantly more sensitive to the effects of oligomycin on OCR, when compared to unirradiated cells. (C) OE33 R cells demonstrate significantly higher percentage of mitochondrial respiration coupled to ATP synthesis, when compared to OE33 P. (D) OE33 R cells demonstrate significantly lower proton leak, when compared to OE33 P. Data are presented as mean± SEM from at least 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s t-test, *P<0.05. The contribution of mitochondrial respiration to ATP synthesis in OE33 P (E), OE33 R (F) at basal level and at 24 h post irradiation with 2 Gy (G and H).

Figure 5. OE33 R cells are more metabolically robust.

(A) OE33 P and OE33 R cells display similar sensitivities to the glycolytic inhibitor 2-deoxyglucose (55 µg/mL). (B) ECAR is significantly increased in OE33 R cells following oligomycin treatment (2 µg/mL), when compared to OE33 P. (C) OE33 R cells have significantly enhanced clonogenic survival following treatment with oligomycin (2 µg/mL) for 1.5 h, when compared to OE33 P cells. Data are presented as mean ± SEM from at least 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s t-test, *P<0.05.

Figure 6. ATP5B expression is increased in the tumour epithelium of poor responders.

ATP5B expression was assessed by immunohistochemistry in pre-treatment tumour biopsies from OAC patients who subsequently received neoadjuvant CRT (n = 23). Representative images of ATP5B staining in tumours from good (A) and poor (B) responders, magnification 10×. (C) ATP5B positivity in the epithelium is significantly increased in tumours of patients with a poor response to neoadjuvant CRT (TRG 3–5), when compared to good responders (TRG 1 and 2). (D) ATP5B positivity in the epithelium is significantly increased in tumours of patients with no evidence of regression (TRG 5), when compared to patients achieving a full (TRG 1) or major response (TRG 2). Statistical analysis was performed by unpaired 2-tailed Student’s t-test, *P<0.05.