Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth

Triple-negative breast cancer (TNBC) comprises ∼20% of all breast cancers and is the most aggressive mammary cancer subtype. Devoid of the estrogen and progesterone receptors, along with the receptor tyrosine kinase ERB2 (HER2), that define most mammary cancers, there are no targeted therapies for patients with TNBC. This, combined with a high metastatic rate and a lower 5-year survival rate than for other breast cancer phenotypes, means there is significant unmet need for new therapeutic strategies. Herein, the anti-neoplastic effects of the electrophilic fatty acid nitroalkene derivative, 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA), were investigated in multiple preclinical models of TNBC. NO2-OA reduced TNBC cell growth and viability in vitro, attenuated TNFα-induced TNBC cell migration and invasion, and inhibited the tumor growth of MDA-MB-231 TNBC cell xenografts in the mammary fat pads of female nude mice. The up-regulation of these aggressive tumor cell growth, migration, and invasion phenotypes is mediated in part by the constitutive activation of pro-inflammatory nuclear factor κB (NF-κB) signaling in TNBC. NO2-OA inhibited TNFα-induced NF-κB transcriptional activity in human TNBC cells and suppressed downstream NF-κB target gene expression, including the metastasis-related proteins intercellular adhesion molecule-1 and urokinase-type plasminogen activator. The mechanisms accounting for NF-κB signaling inhibition by NO2-OA in TNBC cells were multifaceted, as NO2-OA (a) inhibited the inhibitor of NF-κB subunit kinase β phosphorylation and downstream inhibitor of NF-κB degradation, (b) alkylated the NF-κB RelA protein to prevent DNA binding, and (c) promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO2-OA more selectively inhibited TNBC function. This was attributed to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO2-OA–glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.

to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/ disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO 2 -OA-glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.
Triple-negative breast cancer (TNBC) 4 is characterized by an absence of estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor-2 expression (2,3). TNBC accounts for up to 20% of breast cancer incidence and is the subtype with the worst prognosis (4). The majority of TNBC tumors are "basal-like", with 5-year survival rates lower than for all other breast cancer phenotypes (ϳ77% versus ϳ93%, respectively) (3). TNBC patients are also at greater risk for relapse during the first 5 years post-chemotherapy. The recurrent tumors are more aggressive and invasive (5,6), resulting in a life expectancy of 3-22 months after reappearance (7,8). Consequently, there is an urgent unmet need for new therapeutic strategies for TNBC, beyond the limited options of standard chemotherapy, ionizing radiation, and surgery.
Activation of nuclear factor-B (NF-B) is strongly linked with TNBC development and progression (9 -11), with NF-B signaling constitutively activated in ER-negative breast cancer cell lines and primary tumors (10 -13). The inhibition of NF-B activation, induced by overexpression of the non-degradable inhibitor of NF-B (IB␣) superrepressor (Ser-32/36 mutations of IB␣), significantly inhibits the growth of several TNBC cell lines (13). The pro-inflammatory cytokine TNF␣ also contributes significantly to this complex inflammatory microenvironment that promotes tumor progression. TNF␣ activates tumor metastasis and invasion through NF-Bmediated up-regulation of extracellular matrix degradation enzymes and adhesion molecule expression (14). Notably, a meta-analysis revealed that TNBC patients with elevated TNF␣ expression have an increased risk of tumor metastasis to distant organs (15). Thus, NF-B activation and the downstream signaling actions of its pro-inflammatory mediators play a critical role in TNBC malignancy. This motivates the development of novel NF-B inhibition strategies as a chemotherapeutic approach for countering metastatic TNBC.
Electrophilic fatty acid nitroalkene derivatives (NO 2 -FA) are endogenously formed by the acidic conditions of digestion and the complex redox milieu that is up-regulated during inflammation. These environments facilitate the reaction of the nitric oxide ( ⅐ NO) and nitrite (NO 2 Ϫ )-derived nitrating species nitrogen dioxide ( ⅐ NO 2 ) (16) with biological targets, such as unsaturated fatty acids. Basal plasma and urinary NO 2 -FA concentrations in healthy humans range from 2 to 20 nM, with additional pools of NO 2 -FA present as (a) Michael addition products with the abundant biological nucleophiles present in tissues and fluids and (b) esterified species in complex neutral and polar lipids (17,18). Tissue NO 2 -FA levels are affected by both dietary lipid and nitrogen oxide concentrations and during metabolic stress can rise to concentrations as high as 1 M (19,20).
The unique electrophilic character of fatty acid nitroalkene substituents promotes kinetically rapid and reversible Michael addition with nucleophilic Cys and, to a lesser extent, His residues of proteins (21,22). This reversible protein adduction by fatty acid nitroalkenes decreases the potential for toxicity stemming from the accumulation of Schiff's base and Michael addition products characteristic of other lipid electrophiles, such as ␣,␤-unsaturated oxo (or keto) and cyclopentanone derivatives (21,23,24). Through transient post-translational modification (PTM) reactions with hyperreactive protein thiols, NO 2 -FA modulate signaling pathways involved in cell proliferation and inflammatory responses. This occurs as a result of the alkylation of functionally significant Cys residues in transcriptional regulatory proteins, including the Kelch-like ECH-associated protein-1 (Keap1) regulator of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling, the nuclear lipid receptor peroxisome proliferator-activated receptor ␥ (PPAR␥), and NF-B (25)(26)(27). Of relevance to the present study, NO 2 -FA inhibit NF-B-mediated signaling in diverse cell and murine models of metabolic and inflammatory stress to cardiovascular, pulmonary, and renal systems (27)(28)(29). NO 2 -FA specifically alkylate Cys-38 of the RelA subunit of NF-B, a functionally significant, lipid electrophile-reactive thiol located in the DNA-binding domain of RelA. Redox-dependent PTMs of RelA Cys-38 inhibit DNA binding and downstream pro-inflammatory mediator gene expression (27). Current data indicate that other electrophilic species, such as the isothiocyanate derivative sulforaphane, mediate therapeutic actions in preclinical models of breast cancer (30, 31), thus motivating the present studies. Moreover, the pleiotropic signaling actions of NO 2 -FA include the activation of angiogenesis via up-regulation of HIF-1␣ signaling during hypoxia (32). Because these effects may potentially modulate cancer cell and tumor properties, it was important to test the impact of an electrophilic NO 2 -FA in both in vitro and in vivo models of an aggressive cancer phenotype, TNBC.
This study reports the inhibition of TNBC (MDA-MB-231 and MDA-MB468) cell proliferation, invasion, and metastasis by a synthetic homolog of an endogenous electrophilic NO 2 -FA found in species ranging from plants to humans (10-nitro-octadec-9-enoic acid, termed nitro-oleic acid and NO 2 -OA). NO 2 -OA displayed lower cytotoxic and anti-proliferative effects on non-tumorigenic breast ductal epithelium (MCF-10A and MCF7) versus triple-negative human breast ductal epithelial cells, due to the more stable mechanisms for maintaining redox homeostasis in MCF-10A and MCF7 cells. NO 2 -OA also attenuated TNF␣-induced TNBC cell migration and invasion via inhibition of NF-B signaling. Two newly discovered mechanisms also accounted for NO 2 -OA inhibition of TNBC NF-B transcriptional activity. First, NO 2 -OA alkylated the inhibitor of NF-B subunit kinase ␤ (IKK␤), leading to inhibition of its kinase activity and downstream IB␣ phosphorylation. Second, NO 2 -OA alkylated NF-B RelA protein, a reaction that not only inhibited DNA binding, but also promoted proteasomal RelA degradation. As a consequence, NO 2 -OA inhibited the expression of two NF-B-regulated, TNF␣-induced genes that are central to tumor metastasis, intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator (uPA). Finally, in a nude mouse xenograft model, NO 2 -OA reduced the growth of established MDA-MB-231 tumors. In aggregate, these findings reveal that electrophilic NO 2 -FA can mediate chemotherapeutic actions in treating TNBC and possibly other inflammation-related cancers.

NO 2 -OA inhibits TNBC cell growth and viability
The endogenously occurring lipid electrophile NO 2 -OA and its non-electrophilic control fatty acids (NO 2 -SA and OA) were evaluated for their impact on normal and cancerous breast ductal epithelial cell growth and signaling responses (Fig. 1A). To examine whether NO 2 -OA preferentially inhibited TNBC cell growth, Hoechst 33258 was used for counting non-tumorigenic breast epithelial cells (MCF-10A), an ER ϩ breast cancer cell line (MCF7), and two TNBC cell lines (MDA-MB-231 and MDA-MB-468). Each cell line was treated with a range of NO 2 -OA concentrations (0 -15 M) for 48 h. NO 2 -OA significantly inhibited the growth of both TNBC cell lines but not ER ϩ or MCF-10A cells (Fig. 1, B, C, and D). The IC 50 for NO 2 -OA was significantly greater for non-cancerous MCF-10A cells (7.7 Ϯ 1.93 M) and MCF7 (11.61 Ϯ 3.59 M), as opposed to TNBC MDA-MB-231 (2.7 Ϯ 0.11 M) and MDA-MB-468 (1.6 Ϯ 0.11 M) cells (Fig. 1E). In addition to preferential TNBC cell growth inhibition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) detection of intact cell electron transfer mechanisms revealed that NO 2 -OA also significantly reduced the viability of both MDA-MB-231 and MDA-MB-468 cells, but not MCF7 or MCF-10A cells (Fig. S1, A-C). No cytotoxicity was detectable in any cell line for up to 24 h at the 5 M NO 2 -OA concentrations typically used for subsequent cell signaling and functional studies that had durations ranging from 1 to 8 h. Non-electrophilic NO 2 -SA, structurally NO 2 -OA inhibits breast cancer cell function related to NO 2 -OA (Fig. 1A), did not affect TNBC cell growth (Fig. S2), affirming that NO 2 -OA-mediated TNBC cell growth inhibition is attributable to the electrophilic nitroalkene moiety.

NO 2 -OA reduces MDA-MB-231 xenograft tumor growth
Given that TNBC cell growth and viability are inhibited by NO 2 -OA, the efficacy of NO 2 -OA on tumor growth was examined in a murine xenograft model of TNBC. MDA-MB-231 cells were injected into the fourth inguinal mammary fat pad of 6-week-old female athymic nude mice. Oral gavage with NO 2 -OA (7.5 mg/kg/day), NO 2 -SA (7.5 mg/kg/day), or sesame oil (vehicle control) was initiated and continued for 4 weeks after the average tumor sizes reached between 50 and 100 mm 3 . There was significantly reduced tumor growth in the mice treated with NO 2 -OA versus vehicle controls and NO 2 -SA-treated mice at 27 days post-treatment (Fig. 1F). During the course of treatment, there was no weight loss in NO 2 -OAtreated or control mice (Fig. S3). These results indicate that NO 2 -OA mediates in vivo growth suppression of MDA-MB-231 cells with no overt toxic effects.

NO 2 -OA induces cell cycle arrest and apoptotic cell death in TNBC cells
To determine whether the decreased cell numbers were due to NO 2 -OA-induced cell cycle alterations, FACS analysis was performed. NO 2 -OA significantly increased the percentage of cells at G 2 /M phase and decreased the percentage of cells in G 0 /G 1 upon 24-h treatment in MDA-MB-231 and MDA-MB-468 cells (Fig. 2, A and B). Notably, all cell cycle phase populations (G 0 /G 1 , S, and G 2 /M) of MCF-10A cells were not affected by NO 2 -OA (Fig. 2C). The cell cycle inhibition by NO 2 -OA was Also, it is possible that the increase in p21 blocks cell cycle entry into the S phase, resulting in the increase in sub-G 1 cells. To further investigate apoptotic signaling responses to NO 2 -OA in TNBC cells, the activation of initiator caspases (caspase-8 for the extrinsic pathway and caspase-9 for the intrinsic pathway) was analyzed using antibodies that detect both the pro-caspase and activated (cleaved) forms of these initiator caspases.  2F). In aggregate, these results confirm that NO 2 -OA selectively modulates cell cycle arrest and apoptosis in TNBC cells versus MCF-10A cells.

Extracellular NO 2 -OA-glutathione adduct efflux is linked with multidrug resistance protein-1 (MRP1) expression
In the intracellular compartment, GSH and its reactive Cys moiety is more abundant than protein thiols; thus, GSH and other low-molecular weight thiols are the primary targets for oxidation and alkylation by free radicals, oxidants, and electrophiles (33). In the case of NO 2 -OA, which readily diffuses and gains access to the intracellular compartment and subcellular organelle protein targets (26,34), GSH conjugates (NO 2 -OA-SG) are formed that can be actively transported from cells by the GSH-conjugate efflux pump MRP1 (1). This

NO 2 -OA inhibits breast cancer cell function
non-cancerous cell lines. Western blot analysis detected MRP1 protein expression in MCF-10A cells, but MRP1 was undetectable in both TNBC cell lines (Fig. 3B). MRP4 mRNA was detected at low levels in all three cell types, but protein expression was not evident by Western blotting (not shown).

MRP1 influences NO 2 -OA bioactivity in MCF-10A cells
Two strategies, use of the organic anion transport inhibitor probenecid, often used as an MRP inhibitor, and siRNA knockdown of MRP1, facilitated investigation of the role of MRP1 in

GSH and GSSG responses to NO 2 -OA in MCF-10A cells versus TNBC cells
LC-MS quantitation of GSH and GSSG from 0 to 12 h after treatment with 5 M NO 2 -OA revealed that basal GSH levels in MCF-10A cells (19.3 Ϯ 1.9 nmol/10 6 cells) were Ͼ2-fold that of MDA-MB-231 (8.3 Ϯ 0.8 nmol/10 6 cells) and ϳ1.5-fold greater than MDA-MB-468 cells (12.9 Ϯ 0.5 nmol/10 6 cells) (Fig. 4A). GSSG levels ( indicate that there will be a more extensive reaction expected between NO 2 -OA and cellular protein targets in TNBC cells because of the more favorable pharmacokinetics (greater intracellular concentration and longer t 0.5 ) lent by the lower GSH concentrations and the suppression of NO 2 -OA-SG export by the MRP1-deficient TNBC cell phenotype. In MCF-10A cells, NO 2 -OA will be more readily glutathionylated and exported, thus limiting reactions with signaling pathway proteins.

NO 2 -OA inhibits TNF␣-induced TNBC cell migration and invasion
Inflammatory stimuli, such as TNF␣, induce responses in the tumor microenvironment that promote TNBC tumor metastasis and invasion (14). Because electrophilic NO 2 -FAs mediate anti-inflammatory signaling actions (27,28), the impact of

NO 2 -OA inhibits TNF␣-induced NF-B transcriptional activity in TNBC cells
The inhibition of MDA-MB-468 cell invasion by JSH-23 ( Fig.  5D) suggests that NO 2 -OA may also inhibit TNF␣-induced breast cancer cell mobility due to a capacity to inhibit NF-B signaling. To test this concept, the effect of NO 2 -OA on TNF␣activated NF-B transcriptional activity in TNBC cells was examined. MDA-MB-231 and MDA-MB-468 cells were transiently transfected with an NF-B luciferase reporter plasmid and treated with 5 M NO 2 -OA for 2 h, followed by activation with 20 ng/ml TNF␣ for 4 h. In addition to NO 2 -OA, the nonelectrophilic lipid controls NO 2 -SA (5 M) and OA (5 M) were also examined. NO 2 -OA significantly inhibited NF-B-dependent transcription of luciferase in both TNBC cell lines, compared with TNF␣ alone, whereas NO 2 -SA and OA had no effect. Moreover, the extent of inhibition of NF-Bdependent luciferase expression by NO 2 -OA was similar to that induced by the NF-B inhibitor JSH-23 (20 M; Fig. 6, A and B). These data indicate that the electrophilic reactivity of NO 2 -OA accounts for the inhibition of TNF␣-induced NF-B transcriptional activity in TNBC cells.

NO 2 -OA inhibits breast cancer cell function NO 2 -OA inhibits NF-B-regulated gene expression linked with TNBC tumor metastasis
Inhibition of NF-B transcriptional activity by NO 2 -OA suggested that the expression of metastasis-related downstream target genes may be decreased. To investigate this, key NF-B target genes were evaluated via RT 2 profiler PCR array analysis of MDA-MB-468 cells treated with NO 2 -OA (5 M) for 24 h. The expression levels of NF-B target genes that were regulated by NO 2 -OA were compared with untreated MDA-MB-468 cells as a control. Data revealed that treatment with NO 2 -OA decreased the mRNA expression of multiple NF-B target genes, including ICAM-1 and uPA, two critical mediators of tumor progression and metastasis (Fig. 6C). TNF␣ induces the expression of both ICAM-1 and uPA in MDA-MB-231 cells (37,38). To more directly examine whether NO 2 -OA suppressed TNF␣-induced expression of ICAM-1 and uPA in TNBC cells, MDA-MD-231 or MDA-MD-468 cells were treated with 5 M NO 2 -OA and 20 ng/ml TNF␣. Simultaneous treatment with either NO 2 -OA or RelA siRNA led to suppression of TNF␣-induced expression of ICAM-1 and uPA genes in TNBC cells (Fig. 6, D, E, G, and H). The impact of NO 2 -OA and RelA siRNA on RelA-dependent target gene expression was further evaluated by real-time qPCR (Fig. 6, F and I). RelA mRNA levels were suppressed by RelA siRNA treatment, but not NO 2 -OA. Both NO 2 -OA and RelA siRNA inhibited gene expression of TNF␣-induced ICAM-1 and uPA gene expression via NF-B-dependent mechanisms. To determine whether NO 2 -OA suppressed TNF␣-induced pro-metastatic ICAM-1 and uPA gene expression during cell migration, transcript levels of ICAM-1 and uPA genes were evaluated in MDA-MB-468 cells being studied in Boyden chamber migration assays (Fig. 5C). Under these conditions, NO 2 -OA significantly inhibited TNF␣-induced expression of ICAM-1 and uPA in migrating tumor cells (Fig. S6, A and B), again indicating that

NO 2 -OA suppresses TNF␣-induced IKK␤/IB␣ signaling in TNBC
To better define mechanisms accounting for NO 2 Fig. 7C). This indicates that NO 2 -OA suppresses TNF␣-induced IKK␤ phosphorylation and IB␣ degradation, with these actions in turn inhibiting downstream NF-B signaling in TNBC cells.

NO 2 -OA alkylates IKK␤ and RelA proteins
Cys-179, located in the activation loop of IKK␤, is a target for oxidation and electrophile alkylation reactions (39,40). Because NO 2 -OA suppresses TNF␣-induced phosphorylation of IKK␤ and IB␣ in TNBC cells (Fig. 7, A and C), the potential for NO 2 -OA to alkylate IKK␤ was investigated. Biotinylated  and then all alkylated proteins were pulled down from whole-cell lysates using streptavidinconjugated beads. Western blotting revealed that IKK␤ was pulled down by Bt-NO 2 -OA, but not by non-electrophilic control fatty acids (Fig. 7D). Similarly, Bt-NO 2 -OA (but not control fatty acids) promoted the pull-down NF-B RelA (Fig. 8A).    whereas NO 2 -SA and OA had no effect (Fig. 8B). In contrast, RelA protein levels in MCF-10A cells were not altered by NO 2 -OA (Fig. 8B). In all three cell lines, RelA mRNA levels were

NO 2 -OA inhibits breast cancer cell function
not altered by NO 2 -OA (Fig. S9). These data indicate that NO 2 -OA impacts RelA protein stability via alkylation of RelA in TNBC cells. RelA is regulated by ubiquitin-and proteasomedependent degradation signals that govern NF-B activation (42)(43)(44). To determine whether RelA modification by NO 2 -OA induced ubiquitination of endogenous RelA in TNBC cells, MDA-MB-231 or MDA-MB-468 cells were treated with 5 M NO 2 -OA or NO 2 -SA for 5 h. RelA protein was immunoprecipitated, and its polyubiquitination was detected by anti-ubiquitin. NO 2 -OA, but not NO 2 -SA, promoted polyubiquitination of RelA in both TNBC cell lines (Fig. 8C). This indicates that NO 2 -OA interacts with RelA and destabilizes RelA protein by promoting ubiquitination and proteasomal degradation in TNBC cells.

Discussion
Compared with other breast cancer phenotypes, TNBC is an aggressive subtype with a poor prognosis (3). Patients are 4 times more likely to show visceral metastases to the lung, liver, and brain within 5 years after diagnosis (45). Because TNBC does not respond to endocrine therapy or other more targeted chemotherapeutic agents, DNA damage-inducing strategies, such as ionizing radiation, cisplatin, and doxorubicin, remain mainstay treatments. Adverse systemic responses to DNA-directed chemotherapeutic agents, including cardiac and renal toxicity, limit chemotherapy options because of cytotoxic effects on non-cancerous cells (46 -48). Herein, NO 2 -OA inhibited cultured TNBC cell viability, motility, and tumor cell proliferation-related signaling reactions to an extent where in vivo tumor growth in MDA-MB-231 xenografted mice was attenuated by oral administration of NO 2 -OA. This initial observation motivates more detailed dose-timing, dose-response, and structure-function studies of nitroalkene-based drug candidates, with respect to effects on tumor growth and metastasis of multiple breast cancer phenotypes, both in vitro and in preclinical animal models.
At lower concentrations, there was selective cytotoxicity of NO 2 -OA toward TNBC cells, compared with non-tumorigenic MCF-10A breast ductal epithelial cells. One significant explanation for this selectivity of action stemmed from the analysis of both basal GSH levels and the formation and fate of NO 2 -OA-SG adducts in control and TNBC cells. Because of the abundance and reactivity of the GSH thiolate, GSH is a primary intracellular reaction target of endogenously generated and exogenously administered oxidants and electrophilic species (49). The rate of MRP1-mediated efflux of GSH-adducted electrophiles from cells is important, as it contributes to defining the net intracellular concentration, half-life, alternative reactions with target proteins, and thus the net cellular and tissue responses to lipid electrophiles (1,50,51).  (Fig. 3A). This more extensive export of NO 2 -OA-SG by MCF-10A cells, relative to MDA-MB-231 and MDA-MB-468 cells, was also notable because basal GSH concentrations and the GSH/GSSG ratio of MCF-10A cells were more stable after treatment with NO 2 -OA. In contrast, the GSH concentrations and GSH/GSSG ratio in MDA-MB-231 and MDA-MB-468 cells quickly decreased after treatment with NO 2 -OA (Fig. 4). These results indicate that MRP1 export of NO 2 -OA-SG and the more sufficient antioxidant capacity of the MCF10A cell line, as opposed to TNBC cells (52), plays a role in defining the vulnerability of TNBC cells to NO 2 -OA signaling actions. Another electrophile, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), displays antitumor activity by inducing apoptosis in a variety of cancers. CDDO rapidly decreases mitochondrial GSH and induces increased generation of reactive species in pancreatic cancer cells (53,54). In contrast, NO 2 -OA did not significantly impact cellular rates of H 2 O 2 production after both short-term and extended (6-h) treatment of TNBC cells, indicating that NO 2 -OA inhibition of TNBC cell growth and viability are not due to induction of oxidative stress (Fig. S10).
When the MRP1 transport activity of MCF-10A cells was inhibited by the organic acid probenecid (55), a more TNBClike phenotype was conferred in the context of sensitivity to NO 2 -OA. For example, the impact of NO 2 -OA on cell growth arrest and killing (Fig. 3, C and D), cell cycle arrest (cyclin D1, p21), and apoptosis-regulating mediators (PARP-1, caspase-3) all supported the concept that NO 2 -OA signaling actions are enhanced in MRP1-depleted cells because of more favorable pharmacokinetics in the intracellular compartment. This affirms that the cellular concentrations of GSH, the reaction of GSH with NO 2 -OA, and the subsequent MRP1 export of NO 2 -OA-SG all influence downstream responses to NO 2 -OA. It is possible that other mechanisms, yet to be described, are also responsible for this differentiation of breast epithelial cell responses.
Anti-proliferative actions of NO 2 -OA on macrophages, vascular smooth muscle cells, and fibroblasts are observed in models of chronic vascular and pulmonary disease (56 -61), but the impact of fatty acid nitroalkenes on cancer cell proliferation had not been considered. This motivated experimental consideration, because there are a limited number of reports suggesting that the up-regulation of Nrf2 signaling may result in intrinsic or acquired chemoresistance (62). In contrast, we observed the in vitro and in vivo inhibition of TNBC growth by NO 2 -OA (Fig. 1, B-E). This growth inhibition of TNBC cells was the result of alterations in signaling responses that were specific to TNBC cells and not non-transformed MCF-10A cells. Increased p21 and decreased cyclin D1 expression (Fig. 2D) were observed, along with an increase in the sub-G 1 population of TNBC cells (Fig. 2, A-C). Two distinct pathways of apoptotic signaling were engaged by NO 2 -OA in TNBC cells, initiated by both mitochondria-regulated (caspase-9 activation) and death receptor-regulated (caspase-8 activation; Fig. 2F) mechanisms. In aggregate, these data reveal that NO 2 -OA displays pleiotropic anti-cancer properties via the inhibition of cell proliferation and induction of apoptosis in TNBC. At this point, more detailed mechanisms of NO 2 -OA-induced apoptotic cell death remain to be defined; however, the electrophilic thiocyanate

NO 2 -OA inhibits breast cancer cell function
sulforaphane also decreases Bcl-2 expression, activates cytochrome c release from the mitochondria, and increases FasL expression in TNBC cells (30). These actions imply that electrophilic fatty acid nitroalkene derivatives might mediate similar actions in the regulation of apoptosis.
The inhibition of NF-B signaling by NO 2 -OA also limits TNBC cell migration and invasion. Pro-inflammatory cytokines, such as TNF␣, enhance the metastatic potential of TNBC, with the up-regulation of TNF␣ expression and activity in TNBC patients strongly linked with tumor metastasis phenotype (63). TNF␣ stimulates the expression of the epithelialmesenchymal transition and chemokine genes via the activation of AP-1 and NF-B signaling in TNBC cells (14). Herein, NO 2 -OA significantly inhibited TNF␣-induced TNBC cell migration and invasion (Fig. 5). Decreased expression of the pro-metastasis genes uPA and ICAM-1, via a decrease in NF-B transcriptional activity, was also induced by NO 2 -OA (Fig. 6 (D  and E) and Fig. S6 (A and B)). Consistent with this, electrophilic 15-deoxy-⌬ 12,14 -prostaglandin J 2 , dithiolethione, and dimethyl fumarate also inhibit breast cancer cell migration (38,64,65). NO 2 -OA also limited the migration of MDA-MB-231 cells in the absence of TNF␣ induction (Fig. 5B). It is likely that NO 2 -OA inhibits cell mobility upon reaction with molecular targets in addition to NF-B, because the electrophilic cyclopentenone 15-deoxy-⌬ 12,14 -prostaglandin J 2 also interferes with mammary cancer cell migration via inhibition of F-actin reorganization and focal adhesion disassembly (64). Additional studies are in progress to identify other metastasis-related protein targets and signaling pathways that could be impacted by NO 2 -OA-mediated alkylation reactions.
The proteolytic degradation of NF-B subunits contribute to the termination of NF-B activation. RelA protein is regulated by ubiquitin-and proteasome-dependent degradation signals that terminate NF-B activation (42)(43)(44)66). Thiol-alkylating and S-nitrosating agents also promote the degradation of the NF-B subunit p50 via post-translational modification of Cys-62 in HT29 and HCT116 tumor cell lines (41). Thus, the NO 2 -OA alkylation of NF-B RelA induces functional responses similar to other alkylating agents (41). Notably, the alkylation of RelA by NO 2 -OA induced an increase in RelA ubiquitination in TNBC cells, an effect not observed for nonelectrophilic NO 2 -SA (Fig. 7D). PPAR␥ acts as an E3 ubiquitin ligase, inducing RelA protein ubiquitination and degradation via physically interacting with RelA protein. The PPAR␥ ligands troglitazone and pioglitazone increase PPAR␥ E3 ligase activity by promoting its interaction with RelA protein, in turn, decreasing RelA half-life (67). Because NO 2 -OA is a partial agonist of PPAR␥ (26), one can speculate that NO 2 -OA also activates PPAR␥ E3 ligase activity, thus further destabilizing RelA protein in TNBC.
The inhibition of NF-B signaling represents a viable anticancer strategy, especially because the aberrant activation of NF-B is closely linked with the development of diverse human cancers (68,69). The immunomodulatory electrophile dimethyl fumarate, approved by the Food and Drug Administration as an oral drug for treating multiple sclerosis, also inhibits NF-B activity in breast cancer cells and inhibits TNBC cell proliferation (65). The present results, in which NO 2 -OA inhib-ited multiple TNBC cell functions (proliferation, survival, mobility, and invasion), imply that electrophilic lipid nitroalkene species may display promising utility as pleiotropic chemotherapeutic agents.
In summary, we report that the lipid electrophile NO 2 -OA impacts NF-B signaling in TNBC at multiple levels, including the suppression of IKK␤ phosphorylation, inhibition of IB␣ degradation, and enhanced ubiquitination and proteasomal degradation of RelA. These actions in turn contribute to the inhibition of TNBC cell migration and invasion in vitro. TNBC cells are in part more sensitive to NO 2 -OA due to lower GSH concentrations and suppression of NO 2 -OA export as the NO 2 -OA-SG adduct, a consequence of lower MRP1 expression. This GSH insufficiency-induced redox vulnerability of TNBC cells (70) in turn promotes more extensive protein thiol alkylation and oxidation reactions and instigates chemotherapeutic signaling responses at lower electrophile concentrations. The concentrations of endogenous free NO 2 -FAs, which are not protein-adducted or esterified to complex lipids, in healthy human plasma and urine are typically 1-5 nM (16,18,19). The oral administration of NO 2 -OA increased murine tumor NO 2 -OA levels to an extent sufficient to induce pharmacological responses, as evidenced by inhibition of MDA-MB-231 xenograft tumor growth. These results motivate more detailed future investigation of dose-response relationships and the impact of other lipid electrophiles on tumor growth and metastasis. At present, NO 2 -OA has cleared preclinical toxicology and pharmacokinetics testing in human Phase 1 safety trials of both oral and IV formulations (IV IND 122583; oral IND 124524) and is entering Phase 2 trials for treating chronic renal and pulmonary diseases. This present preclinical study provides the biochemical foundations for evaluating whether electrophilic NO 2 -FAs represent a useful new therapeutic candidate for treating breast cancer patients and possibly providing selectivity for treating TNBC, a cancer that currently lacks effective treatment options.

NO 2 -FA synthesis and use
OA was purchased from Nu-Chek Prep (Elysian, MN). Nitrostearic acid (NO 2 -SA; 10-nitro-octadecanoic acid) was obtained by the reduction of 10-nitro-oleic acid. Specifically, NO 2 -OA was dissolved in tetrahydrofuran/methanol and cooled, and then sodium borohydride was added. The flask was stirred, and aliquots were monitored by UV analysis until there was full loss of the nitroalkene, and then the reactions were quenched with acetic acid. NO 2 -SA was purified by first adducting any remaining NO 2 -OA with added cysteine, and then NO 2 -SA was chromatographically fractionated on silica gel, using an ethyl acetate/hexane gradient. OA, NO 2 -OA, and NO 2 -SA were dissolved in absolute methanol and diluted in culture medium immediately before use in all experiments, at a maximum methanol concentration of 0.1% (v/v). Biotinylated

Cell growth assay
Cells were plated at a cell density of 5000 cells/well in 96-well plates. After attachment overnight, the medium was replaced, and cells were treated with 0 -15 M NO 2 -OA, NO 2 -SA, or 0.1% methanol (vehicle) for 48 h. In an MRP inhibition study, MCF-10A cells were pretreated with 0.25 mM probenecid for 1 h, followed by 0 -25 M NO 2 -OA for 48 h. Cells were counted using the FluoReporter dsDNA quantitation kit (Molecular Probes) according to the manufacturer's instructions. Fluorescence was measured using a SpectraMax M2 plate reader (Molecular Devices). The half-maximal inhibitory concentration (IC 50 ) of NO 2 -OA was determined by using CalcuSyn software from Biosoft. Three individual experiments were done (n ϭ 5/each), and statistical comparison between two cell lines across doses was determined by two-way analysis of variance followed by Tukey post-test.

FACS
MCF-10A, MDA-MB-231, and MDA-MB-468 cells were plated at a cell density of 2.5 ϫ 10 5 cells in 6-well plates for 24 h before treatment with 0.1% methanol (vehicle), 5 M NO 2 -OA, NO 2 -SA, or OA for 24 h. Adherent and nonadherent cells were collected, centrifuged at 2000 rpm for 10 min, washed with ice-cold phosphate-buffered saline, fixed with cold 70% ethanol at 4°C for 30 min, and stained with 50 g/ml propidium iodide (Sigma-Aldrich). FACS analysis was performed at the University of Pittsburgh Department of Immunology Unified Flow Core Facility. Three individual experiments were done, and statistical comparisons among phases (G 0 /G 1 , S, and G 2 /M) were determined by one-way analysis of variance followed by Tukey post-test.

Cell migration analysis
MDA-MB-231 and MDA-MB-468 cells were subjected to cell migration analysis in Boyden chambers. The bottom of a 12-well membrane filter (BD Biosciences) was coated with 10 g/ml fibronectin for 12 h before each experiment. Cells were pretreated with 5 M NO 2 -OA or NO 2 -SA for 1 h and then in the absence or presence of TNF␣ (20 ng/ml) for an additional 2 h in culture medium containing 1% FBS. Cells were trypsinized and washed with migration medium (DMEM containing 0.1% fatty acid-free BSA) to remove serum. Cells at a density of 10 5 /well were then placed in the upper chamber with migration medium containing the same pretreatment conditions. The cells were allowed to migrate toward the 5% FBS chemoattractant for 5 h. Non-migrated cells from the top surface were removed with cotton swabs. Migrated cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and then stained with 0.5% crystal violet (Sigma-Aldrich) for 15 min. Migrated cell density on the filters was observed by microscopy. The crystal violet on migrated cells was destained with 10% acetic acid, and the absorbance in individual filters was determined at A 573 nm . Images are representative of three individual experiments, and statistical comparison among treatments was determined by one-way analysis of variance followed by Tukey post-test.

Cell invasion assay
MDA-MB-468 cells were pretreated with NO 2 -OA (5 M), NO 2 -SA (5 M), or NF-B inhibitor JSH-23 (10 M) for 1 h and then in the absence or presence of TNF␣ (20 ng/ml) for an additional 2 h in culture medium containing 1% FBS. Cells were then suspended in migration medium and placed in the top well of invasion chambers (EMD Millipore). Chemoattractant (5% FBS) was placed in the lower chamber for 24 h at 37°C to attract invasive cells. Cells were then harvested, and invasion rates were determined according to the manufacturer's protocol. Three individual experiments were done, and statistical comparison among treatments was determined by one-way analysis of variance followed by Tukey post-test.

Luciferase analysis of NF-B activity
Luciferase chemiluminescence-based analysis of NF-B transcriptional activity was performed as described previously NO 2 -OA inhibits breast cancer cell function (27) with minor modifications. MDA-MB-231 and MDA-MB-468 cells (ϳ70% confluence) in 12-well plates were transiently transfected with a NF-B-luciferase reporter plasmid (Stratagene, La Jolla, CA) with Lipofectamine 3000. After transfection (24 h), cells were pretreated with NO 2 -OA (5 M), NO 2 -SA (5 M), OA (5 M), or JSH-23 (20 M) for 2 h, followed by 20 ng/ml TNF␣ for an additional 4 h. Each transfection was performed in triplicate. Luciferase activity was measured using the Dual-Luciferase assay kit (Promega). Relative light units (RLU) were measured using a 96-well plate luminometer, according to the manufacturer's instructions (Victor II, PerkinElmer Life Sciences). Protein concentration was determined using the BCA assay (Thermo Fisher Scientific). Data represent the ratio of treated samples to controls in the context of mean RLU/ protein content Ϯ S.D. Three individual experiments were done, and statistical significance was determined by Kruskal-Wallis test followed by Dunn's post-test with Bonferroni corrections for multiple comparisons.

NO 2 -FA protein alkylation reactions
To determine whether NO 2 -FAs bind to RelA (p65) or IKK␤ in TNBC cells, MDA-MB-231 or MDA-MB-468 cells were treated with 5 M Bt-NO 2 -OA, Bt-NO 2 -SA, or Bt-OA in DMEM containing 5% FBS. After 2 h, cells were harvested in lysis buffer containing 1% Triton X, 10% glycerol, 150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA and supplemented with a mixture of protease and phosphatase inhibitors (Roche Applied Science) (26). Total cell lysates (0.5-1 mg) were mixed and incubated with streptavidin-agarose beads (Sigma-Aldrich) at 4°C overnight. Beads were washed three times using lysis buffer. After SDS-PAGE, immunoblotting was performed using anti-RelA mouse monoclonal antibody (Santa Cruz Biotechnology) or anti-IKK␤ rabbit polyclonal antibody (Cell Signaling). Proteomics analysis for the alkylation of RelA by NO 2 -OA was also conducted using recombinant RelA protein and LC-MS/MS analysis. See supporting Methods for more detail.

Immunoprecipitation and NO 2 -OA-induced RelA protein polyubiquitination
To determine the induction level of RelA protein polyubiquitination by NO 2 -FA, MDA-MB-231 and MDA-MB-468 cells were treated with 0.1% methanol (vehicle), NO 2 -OA (5 M), or NO 2 -SA (5 M) for 6 h, and then cell lysates were harvested in lysis buffer supplemented with a mixture of protease and phosphatase inhibitors. Lysates were clarified by centrifugation at 14,000 ϫ g for 10 min. Protein lysates (1 mg) were incubated with anti-RelA antibody and Protein G/A-conjugated agarose beads (EMD Millipore, Bedford, MA) at 4°C overnight. Immunoprecipitation fractions were obtained by centrifugation at 14,000 ϫ g for 1 min at room temperature and washed with lysis buffer three times. The immunoprecipitated RelA was resolved by an 8% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad) for immunoblotting probed with an anti-ubiquitin antibody (Santa Cruz Biotechnology). The blot was then stripped and probed with an anti-RelA antibody to assess amounts of RelA protein pulldown.

RNA extraction, quantitative PCR, and RT 2 profiler PCR array
To determine the effect of NO 2 -OA on expression of NF-B target genes in TNF␣-induced MDA-MB-231 and MDA-MB-468 cells, cells were pretreated with NO 2 -OA (5 M) for 2 h and then stimulated with TNF␣ (20 ng/ml) for 6 h. Total RNA samples of tissues or cells were extracted using TRIzol reagents according to the manufacturer's instructions (Invitrogen). Total RNA (1 g) was reverse transcribed using the iScript cDNA kit (Bio-Rad) according to the manufacturer's instructions. cDNA (25 ng) was used for each subsequent real-time qPCR. All real-time qPCR was performed on the StepOne PLUS PCR system (Thermo Fisher Scientific) using TaqMan gene expression assays. -Fold change was calculated using the ⌬⌬Ct method with 18S ribosomal RNA or human ␤-actin RNA serving as the internal control. Three individual experiments were done, and statistical significance was determined by oneway analysis of variance followed by Tukey post-test. For the RT 2 profiler PCR array, MDA-MB-468 cells were treated or untreated with NO 2 -OA (5 M) for 24 h. The expression of 84 human NF-B target genes was analyzed with a 96-well plate format as instructed in the manufacturer's handbook (Qiagen). PCR amplification was conducted by the StepOne PLUS PCR system, and -fold change of gene expression was calculated according to the manufacturer's instructions.

Analysis of NO 2 -OA-SG and NO 2 -OA in cell medium
MCF-10A, MDA-MB-231, or MDA-MB-468 cells were cultured in 6-well plates (1 ϫ 10 6 cells/well) for 24 h. Before treatments, cell medium was replaced with DMEM containing 5% FBS. NO 2 -OA (5 M) was added to the medium, and cells were incubated at 37°C for 60 min before the cell culture medium was collected. For MRP1 inhibition studies, MCF-10A cells were pretreated with 1 mM probenecid for 1 h and then cotreated with 5 M NO 2 -OA for an additional 1 h. For MRP1 siRNA knockdown studies, MCF-10A cells were transiently transfected with non-target siRNA (scrambled) or MRP1 siRNA for 48 h before treatment with 5 M NO 2 -OA for 1 h. Cells were washed with PBS and then gently scraped off of the plate in 1 ml of PBS. 100 l of cell suspensions was lysed by sonication and used for protein concentration measurements via a BCA protein assay. The remaining 0.9 ml of cell suspen-

NO 2 -OA inhibits breast cancer cell function
sion was used to determine the amount of intracellular NO 2 -OA-SG. NO 2 -OA-SG and free NO 2 -OA were extracted using a modified Bligh-Dyer method with NO 2 -OA-SG partitioning into the polar phase and NO 2 -OA into the organic. The cell culture medium was spiked with 15 NO 2 -d 4 -OA (5 nM) as an internal standard for free NO 2 -OA before extraction. Samples were centrifuged at 2800 rpm at room temperature for 5 min. The bottom (organic) layer was transferred to a clean vial, dried, and reconstituted in methanol before MS analysis. The upper (aqueous) layer containing NO 2 -OA-SG was desalted and concentrated using 3 ml of C18 SPE columns (Thermo Fisher Scientific). Columns were preconditioned with 1 column volume of 100% methanol, followed by 2 column volumes of 5% methanol before sample addition. Samples were vortexed and equilibrated at 4°C for 5 min before extraction. Samples were washed with 2 column volumes of 5% methanol, and the column was dried under vacuum for 30 min before elution with 3 ml of 100% methanol. Solvent was then evaporated under N 2 , and the samples were reconstituted in methanol for further analysis. 15 N 2 ]GSSG, and 25 mM NEM) was added to each well and incubated for 15 min at room temperature. Next, 50 l of 10% (w/v) sulfosalicylic acid solution was immediately added to each well to stabilize GSH and GSSG. Supernatant was collected by centrifugation at 15,000 rpm for 10 min at 4°C. Samples were diluted 1:5 in 5% sulfosalicylic acid, and 20 l was injected for HPLC-MS/MS analysis. Cell numbers at time 0 were quantitated by a Hoechst 33258 DNA stain assay and used to normalize GSH or GSSG levels expressed as nmol/cells (ϫ 10 6 ).

LC-MS/MS
NO 2 -OA, NO 2 -OA-SG, GSH, and GSSG were analyzed by high-performance LC-MS/MS using a Shimadzu/CTC PAL HPLC coupled to a Sciex 5000 triple quadrupole mass spectrometer (Sciex, San Jose, CA). NO 2 -OA, NO 2 -OA-SG gradient solvent systems consisted of water ϩ 0.1% acetic acid (solvent A) and acetonitrile ϩ 0.1% acetic acid (solvent B). NO 2 -OA and its metabolites were resolved using a Luna C18 reversed phase column (2 mm ϫ 100 mm, Phenomenex, Torrence, CA) at a flow rate of 0.65 ml/min. Samples were applied to the column at 30% B and eluted with a linear increase in solvent B (30 -100% in 9.7 min). The column was washed at 100% B for 3 min before returning to initial conditions for equilibration (2 min). NO 2 -OA-SG conjugates were resolved using a Luna C18 reversed phase column (2 mm ϫ 150 mm; Phenomenex) at a 0.25 ml/min flow rate. Samples were applied to the column at 20% B, held for 5 min, and eluted with a linear increase in solvent B (20 -98% solvent B in 20 min), followed by a wash step at 98% B for 4.5 min, and switched back to initial conditions for 4 min. MS analyses for NO 2 -FAs used electrospray ionization in the negative-ion mode with the collision gas set at 5 units, curtain gas at 40 units, ion source gas number 1 at 55 units and number 2 at 60 units, ion spray voltage at Ϫ4500 V, and temperature at 600°C. The declustering potential was Ϫ80 eV, entrance potential Ϫ5, collision energy Ϫ35, and the collision exit potential Ϫ3. Multiple-reaction monitoring (MRM) was used for the analysis of lipids showing loss of a nitro group (m/z 46) upon collision-induced dissociation (MRM: 326.2/46 and 331/47 for NO 2 -OA and 15 NO 2 -d 4 -OA, respectively) in negative-ion mode. The following parameters for the mass spectrometers were used for NO 2 -OA-SG conjugates in positive-ion mode: gas number 1, 50 units; gas number 2, 55 units; ion spray voltage, 5000 V; source temperature, 550°C; declustering potential, 70 eV; entrance potential, 5; collision energy, 17; and collision exit potential, 5. The following MRM transitions were used: 635.2/506.2 and 640.2/511.2 for NO 2 -OA-SG and 15 NO 2 -d 4 -OA-SG (Fig. S7), respectively.
The method for simultaneous determination of GSH and GSSG involved sample (20 l) separation on a Phenomenex C18 (2.1 ϫ 150 mm; 3.5-m pore size) column. The solvent system employed aqueous 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B) with a net flow rate of 0.6 ml/min. A linear gradient of 2% B to 75% B from 0.1 to 6.2 min, followed by wash with 100% B for 2 min and re-equilibration with 2% B for 6 min, was employed for separation. Unlabeled and 13 C 4 15 N 2labeled GSSG eluted at 2 min, whereas unlabeled and 13 C 2 15 Nlabeled GS-NEM eluted at ϳ2.7 min. The Sciex 5000 mass spectrometer settings were as follows: CAD, 4 units; curtain gas, 40 units; GS1, 45 units; GS2, 50 units; ion spray voltage, 5500 V; source temperature, 550°C; EP, 5 V; and CXP, 10 V. Multiplereaction monitoring was performed in positive-ion mode. Transitions for respective species were as follows: GSH (Q1 308.3 3 Q3 179.1; declustering potential (DP) 60 V, collision energy (CE) 18.5 V). 13  . Calibration curves were generated using known GSH and GSSG standards and isotopic internal standards and showed linearity over 5 orders of magnitude, and the limit of quantification (71) for both GS-NEM and GSSG was 1 nM. Sample [GSH] and [GSSG] were determined from analyte/internal standard area ratios, and intracellular GSH and GSSG were normalized to cell number (10 6 ), with results expressed as nmol of GSH or GSSG per 10 6 cells.

Statistical analysis
Data analyses were conducted using Prism version 6 software (GraphPad Software). Results are presented as mean Ϯ S.D. tumor volumes except in Fig. 1E, where results are presented as mean Ϯ S.E. Statistical analysis was performed using Student's t test, one-way or two-way analysis of variance as appropriate. Statistical significance was achieved with p Ͻ 0.05.