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, NO-OA), were investigated in multiple preclinical models of TNBC. NO-OA reduced TNBC cell growth and viability , 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. NO-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 NO-OA in TNBC cells were multifaceted, as NO-OA () inhibited the inhibitor of NF-κB subunit kinase β phosphorylation and downstream inhibitor of NF-κB degradation, () alkylated the NF-κB RelA protein to prevent DNA binding, and () promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO-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 NO-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.
Pulmonary hypertension (PH) and its severe subtype pulmonary arterial hypertension (PAH) encompass a set of multifactorial diseases defined by sustained elevation of pulmonary arterial pressure and pulmonary vascular resistance leading to right ventricular failure and subsequent death. Pulmonary hypertension is characterized by vascular remodeling in association with smooth muscle cell proliferation of the arterioles, medial thickening, and plexiform lesion formation. Despite our recent advances in understanding its pathogenesis and related therapeutic discoveries, PH still remains a progressive disease without a cure. Nevertheless, development of drugs that specifically target molecular pathways involved in disease pathogenesis has led to improvement in life quality and clinical outcomes in patients with PAH. There are presently more than 12 Food and Drug Administration-approved vasodilator drugs in the United States for the treatment of PAH; however, mortality with contemporary therapies remains high. More recently, there have been exuberant efforts to develop new pharmacologic therapies that target the fundamental origins of PH and thus could represent disease-modifying opportunities. This review aims to summarize recent developments on key signaling pathways and molecular targets that drive PH disease progression, with emphasis on new therapeutic options under development.
Pulmonary arterial hypertension (PAH) refers to a set of heterogeneous vascular diseases defined by elevation of pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR), leading to right ventricular (RV) remodeling and often death. Early increases in pulmonary artery stiffness in PAH drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling. Dysregulation of microRNAs can drive PAEC dysfunction. However, the role of vascular stiffness in regulating pathogenic microRNAs in PAH is incompletely understood. Here, we demonstrated that extracellular matrix (ECM) stiffening downregulated miR-7 levels in PAECs. The RNA binding protein Quaking (QKI) has been implicated in the biogenesis of miR-7. Correspondingly, we found that ECM stiffness up-regulated QKI, and QKI knockdown led to increased miR-7. Downstream of the QKI-miR-7 axis, the serine and arginine rich splicing factor 1 (SRSF1) was identified as a direct target of miR-7. Correspondingly, SRSF1 was reciprocally up-regulated in PAECs exposed to stiff ECM and was negatively correlated with miR-7. Decreased miR-7 and increased QKI and SRSF1 were observed in lungs from PAH patients and PAH rats exposed to SU5416/hypoxia. Lastly, miR-7 upregulation inhibited human PAEC migration, while forced SRSF1 expression reversed this phenotype, proving that miR-7 depended upon SRSF1 to control migration. In aggregate, these results define the QKI-miR-7-SRSF1 axis as a mechanosensitive mechanism linking pulmonary arterial vascular stiffness to pathogenic endothelial function. These findings emphasize implications relevant to PAH and suggest the potential benefit of developing therapies that target this miRNA-dependent axis in PAH.
Homologous recombination (HR)-directed DNA doublestrand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO 2 ) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and ␥-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO 2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO 2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO 2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO 2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO 2 , octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO 2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO 2 -mediated Cys-319 alkylation on ABL1 binding and found that OA-NO 2 inhibits RAD51-ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51-ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO 2 and suggests further investigation of lipid electrophile-based combinational therapies for TNBC.
Rationale: Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined. Objective: We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH. Methods and Results: Using miR-210 replete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210–positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210–positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT; Cx3cr1 knockout mice (deficient in myeloid recruitment) or miR-210 WT; Rag1 knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210. Conclusions: Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.
Triple negative breast cancer (TNBC) is an aggressive and therapy‐resistant metastatic cancer. Patients with metastatic TNBC are difficult to treat due to the absence of effective target therapies, thus leading to poor clinical outcome. Constitutive activation of the nuclear factor kB (NF‐kB) pathway is a main driver to promote TNBC tumor aggressive phenotypes including cell survival, migration, and invasion. This study investigates the anticancer effects of electrophilic anti‐inflammatory mediators nitroalkene fatty acids in TNBC in vitro and in vivo. Decreased inflammatory metastatic potential of TNBC cells by an exemplary electrophilic nitrolipid, nitro‐oleic acid (NO2‐OA), was shown through inhibition of cell motility using transwell cell migration and invasion assays. NO2‐OA treatment inhibited NF‐kB transcriptional activity and expression of two TNFa‐induced NF‐kB‐regulated genes involved in tumor metastasis, intercellular adhesion molecule‐1 (ICAM‐1) and urokinase‐type plasminogen activator (uPA) in TNBC cells. The mechanism of NF‐kB signaling inhibition by NO2‐OA was due to suppression of IKKb phosphorylation and IkBa degradation. Notably, NO2‐OA also caused polyubiquitination and degradation of NF‐kB RelA protein via protein nitroalkylation in TNBC cells. In an MDA‐MB‐231 xenograft model, NO2‐OA inhibited tumor burden resulted in reduced tumor growth and incidence of metastasis in mice. Taken together, electrophilic NO2‐OA shows potential as a therapeutic agent that may display high selectivity for inhibition of TNBC progression and metastasis.
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