Mitochondrial-targeted nitroxides disrupt mitochondrial architecture and inhibit expression of peroxiredoxin 3 and FOXM1 in malignant mesothelioma cells.

Mitochondria are dynamic organelles, constantly adapting their structure and function in response to environmental cues and intracellular signals (Mitra et al., 2009; Hamanaka and Chandel, 2010; Antico Arciuch et al., 2012). Beyond their role as the primary source of ATP in the cell, mitochondria have emerged as signaling hubs that regulate normal and pathological cellular processes through redox-responsive signaling cascades, as reviewed in (Hamanaka and Chandel, 2010; Tait and Green, 2010). It has long been appreciated that cancer cells harbor mitochondria with altered energy production and structural aberrations (de Oliveira et al., 2012). The “Warburg effect” first described altered metabolism in malignant tissues that is characterized by increases in aerobic glycolysis, lactic acid production, and loss of oxidative phosphorylation (Diaz-Ruiz et al., 2011). Along with altered energy metabolism, the mitochondria of tumor cells produce increased amounts of oxidants (Fried and Arbiser, 2008; Klaunig et al., 2011), mainly through electron leakage to molecular oxygen in the electron transport chain (ETC) located in the inner mitochondrial membrane. Leakage of electrons from the ETC to molecular oxygen leads to the formation of superoxide radical which is spontaneously and enzymatically dismutated to hydrogen peroxide, the primary oxidant capable of freely crossing membranes (Jones, 2006; Rhee, 2006; Janssen-Heininger et al., 2008; Murphy, 2009). Through oxidation of reactive cysteine residues in signaling factors, hydrogen peroxide has been implicated in the modulation of regulatory pathways that control proliferation, apoptosis, metabolism, migration, and survival (Droge, 2002; Jones, 2010). It is important to note that the balance between oxidant production and metabolism, as well as the array of susceptible targets expressed in the cell, is critical in determining phenotypic responses. Moreover, redox-signaling by endogenous hydrogen peroxide involves significant spatial and temporal regulation, as either too little or too much hydrogen peroxide impairs cell cycle progression and viability (Burhans and Heintz, 2009). Activation of certain oncogenes, such as Ras, leads to increased production of cellular oxidants, a metabolic response that in most normal cells induces senescence (Lee et al., 1999). Tumor cells evade senescence and tolerate constitutive increases in the production of cellular oxidants, either through loss of checkpoint function or adaptive responses, including the up-regulation of anti-oxidant enzymes. Indeed, some tumor types appear to rely on enhanced production of oxidants for viability and other properties of malignancy (Fried and Arbiser, 2008; Gupta et al., 2012). FOXM1, a redox-responsive transcription factor that regulates genes involved in S phase and the G2/M transition, functions at the interface between oxidative stress, aging, and cancer (Laoukili et al., 2007; Myatt and Lam, 2007; Park et al., 2009). Because FOXM1 is up-regulated in all carcinomas examined to date, and is expressed only in proliferating cells (Laoukili et al., 2007), FOXM1 has emerged as a promising therapeutic target in cancer treatment (Wang et al., 2010). FOXM1 has also been shown to respond to changes in cellular redox status, with its expression increasing in response to exposure to low levels of exogenous hydrogen peroxide and decreasing following overnight treatment of cells with the free radical scavenger TEMPOL (Park et al., 2009). Through up-regulation of anti-oxidant enzymes that include mitochondrial superoxide dismutase (SOD2) and peroxiredoxin 3 (PRX3), FOXM1 permits cells to escape senescence induced by activated Ras (Park et al., 2009). Previously, we showed that malignant mesothelioma (MM) cells in culture constitutively produce twofold to threefold more mitochondrial superoxide than non-transformed mesothelial cells, and that compounds that inactivate the major mitochondrial anti-oxidant network of thioredoxin reductase 2 (TR2)—thioredoxin 2 (TRX2)—PRX3 increase mitochondrial oxidative stress and block FOXM1 expression (Newick et al., 2012), albeit through an unknown pathway. Other small molecules that perturb mitochondrial redox status may therefore prove useful for inhibiting FOXM1 expression and the clinical management of MM. Due to its high negative membrane potential, compounds can be selectively targeted to mitochondria through the conjugation of a triphenylphosphonium (TPP) moiety, which provides a large, dispersed positive charge to the test agent (Murphy, 1997). The selective accumulation of TPP-containing compounds in mitochondria has allowed for targeted delivery of a large number of test agents, with levels that can be 100- to 500-fold higher than the bulk concentration (Murphy, 1997; Dhanasekaran et al., 2005). The higher negative membrane potential of tumor mitochondria also facilitates increased accumulation in tumor cell mitochondria versus normal cell mitochondria (Modica-Napolitano and Aprille, 2001; Millard et al., 2010). In this study we evaluated the activity of two TPP conjugated mitochondrial-targeted nitroxides, Mito-carboxy proxyl (MCP) (Dhanasekaran et al., 2005) and Mito-TEMPOL (MT) (Trnka et al., 2008) on MM tumor cell proliferation and survival (Supplementary Fig. S1). Our results indicate that MCP and MT inhibit FOXM1 expression by inducing marked mitochondrial fragmentation and increased production of mitochondrial oxidants, a phenotypic response that appears distinct from mitochondrial fission. In contrast, the parent compounds, carboxy proxyl (CP), TEMPOL, and TPP alone had no effect on FOXM1 expression, mitochondrial architecture, or cell viability at equivalent concentrations. These observations demonstrate that altered mitochondrial energy and oxidant metabolism in tumor cells is linked to FOXM1 expression, thereby providing a rationale for exploiting this relationship for cancer therapy.