Mitochondrial Bioenergetics of Metastatic Breast Cancer Cells in Response to Dynamic Changes in Oxygen Tension: Effects of HIF-1α

<div><p>Solid tumors are characterized by regions of low oxygen tension (OT), which play a central role in tumor progression and resistance to therapy. Low OT affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low OT to maintain the cellular bioenergetics. In this study, a novel experimental approach was developed to examine the real-time bioenergetic changes in breast cancer cells (BCCs) during adaptation to OT (from 20% to <1% oxygen) using sensitive extracellular flux technology. Oxygen was gradually removed from the medium, and the bioenergetics of metastatic BCCs (MDA-MB-231 and MCF10CA clones) was compared with non-tumorigenic (MCF10A) cells. BCCs, but not MCF10A, rapidly responded to low OT by stabilizing HIF-1α and increasing HIF-1α responsive gene expression and glucose uptake. BCCs also increased extracellular acidification rate (ECAR), which was markedly lower in MCF10A. Interestingly, BCCs exhibited a biphasic response in basal respiration as the OT was reduced from 20% to <1%. The initial stimulation of oxygen consumption is found to be due to increased mitochondrial respiration. This effect was HIF-1α-dependent, as silencing HIF-1α abolished the biphasic response. During hypoxia and reoxygenation, BCCs also maintained oxygen consumption rates at specific OT; however, HIF-1α silenced BCC were less responsive to changes in OT. Our results suggest that HIF-1α provides a high degree of bioenergetic flexibility under different OT which may confer an adaptive advantage for BCC survival in the tumor microenvironment and during invasion and metastasis. This study thus provides direct evidence for the cross-talk between HIF-1α and mitochondria during adaptation to low OT by BCCs and may be useful in identifying novel therapeutic agents that target the bioenergetics of BCCs in response to low OT.</p></div

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model

Determination of the bioenergetic flexibility of MB231 and MB231^shHIF-1α cells during de-oxygenation and re-oxygenation MB231 (circles) and MB231^shHIF-1α (square) cells were equilibrated to atmospheric OT and is de-oxygenated to 4% and then to 1%.

<p>The chamber is re-oxygenated step-wise to 4% and then back to atmospheric OT. Several OCR measurements were made at each step and were plotted over time. A representative O₂ trace (dotted line) during the course of the experiment is shown for reference.</p

Effect of different OTs in MB231 and MB231^shHIF-1α cells on OCR and ECAR.

<p>MB231 cells (circles) or MB231 cells deficient in HIF-1α (triangles) were exposed to reducing OT as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068348#pone-0068348-g001" target="_blank">Figure 1</a>. OCR (<b>A</b>) and ECAR (<b>B</b>) were simultaneously measured over time.</p

Validation of experimental setup to measure OCR and ECAR of breast epithelial and cancer cells under different OTs.

<p>A schematic representation of exposure to different OTs in the Seahorse XF24 Analyzer is shown (<b>A</b>). Representative O₂ traces in media were obtained using XF24 Analyzers equilibrated within the sealed chamber at ∼1% O₂) (<b>B</b>). MCF10A and MB231 cells were exposed to ∼1% O₂ for 0–5 h, and nuclear HIF-1α and cytoplasmic LDHA protein levels were assessed using Western blot analysis (representative image shown). Lamin A/C and β-actin were used as protein loading controls for nuclear and cytoplasmic fractions, respectively (<b>C</b>). Cells were exposed to atmospheric OT (closed bars) or ∼1% O₂ (open bars) for 5 h, and the rate of 2-NBDG (0.3 mM) uptake, a fluorescence glucose analog, was measured. 2-NBDG fluorescence was normalized to protein levels (<b>D</b>). Values represent means ± SEM, n = 12. * p≤0.05 compared MCF10A. # p≤0.05 compared to cultures under normoxic conditions.</p

Effect of reducing OT on oxygen consumption rate (OCR) in breast epithelial and cancer cells.

<p>OCR was determined in non-tumorigenic (MCF10A) and MB231 over time during reducing OT as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068348#pone-0068348-g001" target="_blank">Figure 1</a>. A representative O₂ trace (dotted line) during the course of the experiment is shown for reference. OCR was measured over time in MCF10A (circles), and MB231 (triangles) (<b>A upper panel</b>)<b>.</b> OCR measured over time equilibrated at atmospheric air (<b>A lower panel</b>). OCR traces of MB231(circles) and MCF10CA d1.α at stable 4% OT are shown (<b>B</b>). The bioenergetic state of MCF10A (circles) and MB231 (triangles) cells was determined from data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068348#pone-0068348-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068348#pone-0068348-g003" target="_blank">3</a> at atmospheric air OT (closed) or at low OT (open) at 100 (<b>C upper panel</b>) and 300 (<b>C lower panel</b>) min by constructing a 2D plot of OCR versus ECAR.</p

A Novel Class of Mitochondria-Targeted Soft Electrophiles Modifies Mitochondrial Proteins and Inhibits Mitochondrial Metabolism in Breast Cancer Cells through Redox Mechanisms

<div><p>Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs) has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration <i>in vitro</i>. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins.</p></div