Mitochondrial respiration supports autophagy to provide stress resistance during quiescence.

Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.

MiR-301a-3p Suppresses Estrogen Signaling by Directly Inhibiting ESR1 in ERα Positive Breast Cancer.

BACKGROUND/AIMS: MiRNA-301a-3p is an oncogenic miRNA whose expression is associated with tumor development, metastases and overall poor prognosis. Estrogen receptor α (ERα) is one of the estrogen hormone-activated transcription factors, which regulates a large number of genes and is involved in the mammary gland development. Expression of ERα is considered to be a good indicator for endocrine therapy and breast cancer survival. Loss of ERα in breast cancer patients indicates invasiveness and poor prognosis. In this study, we focus on the regulation of ERα by miR-301a and its role in transition from estrogen-dependent to estrogen-independent breast cancer. METHODS: Expression of miR-301a-3p was measured by qRT-PCR in tumor tissue samples from 111 patients with primary breast carcinoma and in mammospheres representing in vitro model of cancer stem-like cells. Dual reporter luciferase assay and complementary experiments were performed to validate ESR1 as a direct target of miR-301a-3p. The effect of miR-301a-3p on estrogen signaling was evaluated on the level of gene and protein expression and growth response to estrogens. Finally, the effect of miR-301a-3p expression on tumor growth was studied in nude mice. RESULTS: We identified ESR1 as a direct target of miR-301a-3p. Ectopic miR-301a-3p causes a decrease in ESR1 mRNA and protein level and modulates the expression of ERα target genes in ERα positive breast cancer cells. Consistently, miR-301a-3p causes a decrease in sensitivity of MCF7 cells to 17ß-estradiol and inhibits the growth of estrogen dependent tumor in nude mice. Yet, the mice tumors have significantly increased expression of genes related to cancer stem-like cells and epithelial to mesenchymal transition suggesting enrichment of the population of cells with more invasive properties, in line with our observation that miR-301a-3p expression is highly increased in mammospheres which show a decrease in estrogenic signaling. Importantly, miR-301a-3P level is also increased in primary breast cancer samples exhibiting an ER/PR negative phenotype. CONCLUSION: Our results confirm ESR1 as a direct target of miR-301a-3p and suggest that miR-301a-3p likely contributes to development of estrogen independence, which leads to a more invasive phenotype of breast cancer.

Antioxidant defense in quiescent cells determines selectivity of electron transport chain inhibition-induced cell death.

Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 - thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death.

Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2high Breast Cancer.

AIMS: Expression of the HER2 oncogene in breast cancer is associated with resistance to treatment, and Her2 may regulate bioenergetics. Therefore, we investigated whether disruption of the electron transport chain (ETC) is a viable strategy to eliminate Her2high disease. RESULTS: We demonstrate that Her2high cells and tumors have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam, a novel mitochondrial-targeted derivative of tamoxifen. Unlike tamoxifen, MitoTam efficiently suppresses experimental Her2high tumors without systemic toxicity. Mechanistically, MitoTam inhibits complex I-driven respiration and disrupts respiratory SCs in Her2high background in vitro and in vivo, leading to elevated reactive oxygen species production and cell death. Intriguingly, higher sensitivity of Her2high cells to MitoTam is dependent on the mitochondrial fraction of Her2. INNOVATION: Oncogenes such as HER2 can restructure ETC, creating a previously unrecognized therapeutic vulnerability exploitable by SC-disrupting agents such as MitoTam. CONCLUSION: We propose that the ETC is a suitable therapeutic target in Her2high disease. Antioxid. Redox Signal. 26, 84-103.