Mitochondrial Dysfunction Inhibits Hypoxia-Induced HIF-1α Stabilization and Expression of Its Downstream Targets.

mtDNA variations often result in bioenergetic dysfunction inducing a metabolic switch toward glycolysis resulting in an unbalanced pH homeostasis. In hypoxic cells, expression of the tumor-associated carbonic anhydrase IX (CAIX) is enhanced to maintain cellular pH homeostasis. We hypothesized that cells with a dysfunctional oxidative phosphorylation machinery display elevated CAIX expression levels. Increased glycolysis was observed for cytoplasmic 143B mutant hybrid (m.3243A>G, >94.5%) cells (p < 0.05) and 143B mitochondrial DNA (mtDNA) depleted cells (p < 0.05). Upon hypoxia (0.2%, 16 h), genetic or pharmacological oxidative phosphorylation (OXPHOS) inhibition resulted in decreased CAIX (p < 0.05), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-alpha (HIF-1α) expression levels. Reactive oxygen species (ROS) and prolyl-hydroxylase 2 (PHD2) levels could not explain these observations. In vivo, tumor take (>500 mm3) took longer for mutant hybrid xenografts, but growth rates were comparable with control tumors upon establishment. Previously, it has been shown that HIF-1α is responsible for tumor establishment. In agreement, we found that HIF-1α expression levels and the pimonidazole-positive hypoxic fraction were reduced for the mutant hybrid xenografts. Our results demonstrate that OXPHOS dysfunction leads to a decreased HIF-1α stabilization and subsequently to a reduced expression of its downstream targets and hypoxic fraction in vivo. In contrast, hypoxia-inducible factor 2-alpha (HIF-2α) expression levels in these xenografts were enhanced. Inhibition of mitochondrial function is therefore an interesting approach to increase therapeutic efficacy in hypoxic tumors.

Distinct radiation responses after in vitro mtDNA depletion are potentially related to oxidative stress.

Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ0). Overall survival after irradiation was increased (p<0.001) for BEAS-2B ρ0 cells, while decreased for both tumor ρ0 lines (p<0.05). In agreement, increased residual DNA damage was observed after mtDNA depletion for A549 and 143B cells. Intrinsic radiosensitivity (surviving fraction at 2Gy) was not influenced. We investigated whether ROS levels, oxidative stress and/or antioxidant responses were responsible for altered radiation responses. Baseline ROS formation was similar between BEAS-2B parental and ρ0 cells, while reduced in A549 and 143B ρ0 cells, compared to their parental counterparts. After irradiation, ROS levels significantly increased for all parental cell lines, while levels for ρ0 cells remained unchanged. In order to investigate the presence of oxidative stress upon irradiation reduced glutathione: oxidized glutathione (GSH:GSSG) ratios were determined. Irradiation reduced GSH:GSSG ratios for BEAS-2B parental and 143B ρ0, while for A549 this ratio remained equal. Additionally, changes in antioxidant responses were observed. Our results indicate that mtDNA depletion results in varying radiation responses potentially involving variations in cellular ROS and antioxidant defence mechanisms. We therefore suggest when mitotoxic drugs are combined with radiation, in particular at high dose per fraction, the effect of these drugs on mtDNA copy number should be explored.

Mitochondrial DNA copy number in colorectal cancer: between tissue comparisons, clinicopathological characteristics and survival.

Low mitochondrial DNA (mtDNA) copy number in tumors has been associated with worse prognosis in colorectal cancer (CRC). This study further deciphers the role of mtDNA copy number in CRC by comparing mtDNA copy number between healthy, adenoma and carcinoma tissue, by investigating its association according to several clinicopathological characteristics in CRC, and by relating it to CRC-specific survival in CRC patients. A hospital-based series of samples including cancer, adenoma and adjacent histologically normal tissue from primary CRC patients (n = 56) and recurrent CRC (n = 16) was studied as well as colon mucosa samples from healthy subjects (n = 76). Furthermore, mtDNA copy number was assessed in carcinomas of 693 CRC cases identified from the population-based Netherlands Cohort Study (NLCS). MtDNA copy number was significantly lower in carcinoma tissue (P = 0.011) and adjacent tissue (P < 0.001) compared to earlier resected adenoma tissue and in primary CRC tissue compared to recurrent CRC tissue (P = 0.011). Within both study populations, mtDNA copy number was significantly lower in mutated BRAF (P = 0.027 and P = 0.006) and in microsatellite unstable (MSI) tumors (P = 0.033 and P < 0.001) and higher in KRAS mutated tumors (P = 0.004). Furthermore, the association between mtDNA and survival seemed to follow an inverse U-shape with the highest HR observed in the second quintile of mtDNA copy number (HR = 1.70, 95% CI = 1.18, 2.44) compared to the first quintile. These results might reflect an association of mtDNA copy number with various malignant processes in cancer cells and warrants further research on tumor energy metabolism in CRC prognosis.

No association between TGF-ß1 polymorphisms and radiation-induced lung toxicity in a European cohort of lung cancer patients.

This study aimed at validating the previously published association between TGF-ß1 single nucleotide polymorphisms and a reduced risk for radiation-induced lung toxicity. We were not able to confirm the reported association, neither using maximum dyspnea score nor after correction for baseline dyspnea score.