Die Bedeutung der Expression von Myoglobin in Mammakarzinomzellen für die Anpassung an Hypoxie
Anna Katharina Heymann, Joachim Fandrey, Timm Schreiber
Institute of Physiology, University Duisburg-Essen, Germany
Myoglobin is well known as an oxygen-binding protein in cardiac and skeletal myocytes. It is believed to function as a transport and temporary storage protein for O2. However, myoglobin has recently also been identified as a hallmark in luminal breast cancer and was shown to be robustly induced under hypoxia. In general, cellular responses to hypoxia are regulated by the transcription factor hypoxia-inducible factor (HIF). Here, we hypothesize that myoglobin in breast cancer cells has a function under normoxic (21% O2) and hypoxic (1% O2) conditions with respect to HIF activation.
For exploring the function of myoglobin in breast cancer, we employed the human cell line MDA-MB-468, grown in a 3D multicellular spheroid model that mimics the in vivo avascular tumor architecture and physiology. Myoglobin expression was knocked down by siRNA (kindly provided by Dr. Thomas Gorr, University of Zurich) and the ability of adaptation to hypoxia was investigated. Myoglobin knockdown was successful both in monolayer cultures and in the tumor spheroids with efficiencies over 90%.
Due to their three dimensional architecture, spheroids have a heterogeneous cell population of proliferating cells in the rim, hypoxic cells in the middle region and non-cycling or necrotic cells in the core region. In the core region hypoxia to anoxia results in cell death, called central necrosis. This central necrosis was increased after myoglobin knockdown. Another potential function of myoglobin is scavenging of reactive oxygen species (ROS) that can initiate cell death. We found that myoglobin knockdown lead to higher ROS levels in the cells after treatment with H2O2. In addition, we showed that hypoxia inducible factors (HIFs) accumulated under conditions of hypoxia, indicating that MDA-MB-468 cells can adapt to hypoxic conditions. Moreover, myoglobin knockdown causes higher levels of HIF-1α
after treatment with NO.
Our data indicate that myoglobin plays an important role in HIF activation and in survival of breast cancer cells due to its ROS and NO scavenging properties.