Metastasis by cancer cells relies upon the acquisition of the ability to evade anoikis, a cell death process elicited by detachment from extracellular matrix (ECM). The molecular mechanisms that ECM-detached cancer cells use to survive are not understood. Striking increases in reactive oxygen species (ROS) occur in ECM-detached mammary epithelial cells, threatening cell viability by inhibiting ATP production, suggesting that ROS must be neutralized if cells are to survive ECM-detachment. Here, we report the discovery of a prominent role for antioxidant enzymes, including catalase and superoxide dismutase, in facilitating the survival of breast cancer cells after ECM-detachment. Enhanced expression of antioxidant enzymes in nonmalignant mammary epithelial cells detached from ECM resulted in ATP elevation and survival in the luminal space of mammary acini. Conversely, silencing antioxidant enzyme expression in multiple breast cancer cell lines caused ATP reduction and compromised anchorage-independent growth. Notably, antioxidant enzyme-deficient cancer cells were compromised in their ability to form tumors in mice. In aggregate, our results reveal a vital role for antioxidant enzyme activity in maintaining metabolic activity and anchorage-independent growth in breast cancer cells. Furthermore, these findings imply that eliminating antioxidant enzyme activity may be an effective strategy to enhance susceptibility to cell death in cancer cells that may otherwise survive ECM-detachment. Cancer Res; 73(12); 3704-15. Ó2013 AACR.

For decades, it has been recognized that cancer cells display a unique metabolism; specifically, cancer cells have been shown to preferentially utilize glycolysis instead of mitochondrial respiration. This phenomenon is commonly known as the "Warburg effect" after Otto Warburg who first made this observation in 1927. The discovery of the Warburg effect has lead to new methods of detection and differentiation of cancerous tissue and normal tissue. More recently, alterations in cancer metabolism have been researched as a possible target for chemotherapeutic intervention in a number of cancers. The push to understand the metabolism of cancer cells has been particularly acute in breast cancer cells, where multiple novel metabolic mechanisms have recently been described and characterized. However, despite this recent progress, the completion of additional studies on the cellular metabolism of breast cancer cells is necessary before drugs that target cancer cell metabolism could be available to disease-afflicted women. Here, we review recent discoveries in breast cancer cell metabolism as well as current logical drug targets that could be used to alter cell metabolism to promote the selective elimination of breast cancer cells.

Tumor heterogeneity is recognized as a major issue within clinical oncology, and the concept of personalized molecular medicine is emerging as a means to mitigate this problem. Given the vast number of cancer types and subtypes, robust pre-clinical models of cancer must be studied to interrogate the molecular mechanisms involved in each scenario. In particular, mouse models of tumor metastasis are of critical importance for pre-clinical cancer research at the cancer cell molecular level. In many of these experimental systems, tumor cells are injected intravenously, and the distribution and proliferation of these cells are subsequently analyzed via ex vivo methods. These techniques require large numbers of animals coupled with time-consuming histological preparation and analysis. Herein, we demonstrate the use of two facile and noninvasive imaging techniques to enhance the study of a pre-clinical model of breast cancer metastasis in the lung. Breast cancer cells were labeled with a near-infrared fluorophore that enables their visualization. Upon injection into a living mouse, the distribution of the cells in the body was detected and measured using whole animal fluorescence imaging. X-ray computed tomography (CT) was subsequently used to provide a quantitative measure of longitudinal tumor cell accumulation in the lungs over six weeks. A nuclear probe for lung perfusion, 99mTc-MAA, was also imaged and tested during the time course using single photon emission computed tomography (SPECT). Our results demonstrate that optical fluorescence methods are useful to visualize cancer cell distribution patterns that occur immediately after injection. Longitudinal imaging with X-ray CT provides a convenient and quantitative avenue to measure tumor growth within the lung space over several weeks. Results with nuclear imaging did not show a correlation between lung perfusion (SPECT) and segmented lung volume (CT). Nevertheless, the combination of animal models and noninvasive optical and CT imaging methods provides better research tools to study cancer cell differences at the molecular level. Ultimately, the knowledge gleaned from these improved studies will aid researchers in uncovering the mechanisms mediating breast cancer metastasis, and eventually improve the treatments of patients in the clinic.

<p>Catalase deficiency in T47D cells does not impact cell proliferation, Catalase deficiency in T47D cells does not impact cell viability, Diminished SOD2 expression in T47D cells has no on glucose uptake, SOD2 deficiency in T47D cells does not impact cell proliferation.</p>

<p>SOD2 deficiency in T47D cells has a negligible impact on cell viability, SOD1 deficiency in T47D cells diminishes ATP levels in ECM-detached cells, Nrf2 deficiency in T47D cells diminishes ATP levels in ECM-detached cells, SOD2 deficiency in ErbB2 positive SKBR3 cells leads to a decrease in ATP levels during ECM-detachment.</p>

<p>Catalase elimination in MDA-MB-231 cells does not alter glucose uptake activity, Catalase elimination in MDA-MB-231cells does not alter cell proliferation, Catalase deficiency in MDA-MB-231 cells has no bearing on cell viability, Catalase deficiency in T47D cells does not affect glucose uptake.</p>

<p>Antioxidant compound treatment does not affect glucose uptake in detached MCF-10A cells, Antioxidant compound treatment does not alter cell viability in attached E7/Bcl2 cells, Antioxidant compound treatment can be reversible, Catalase expression has no impact on glucose uptake in ECM-detached MCF-10A cells.</p>

<p>The expression of antioxidant enzymes is unchanged during ECM-detachment and is comparable between untransformed and transformed cells, The expression of catalase leads to viability changes in the absence of apoptosis, Catalase re-expression rescues ATP levels in catalase-deficient MDA-MB-231 cells.</p>