Pharmacological suppression of the OTUD4/CD73 proteolytic axis revives antitumor immunity against immune-suppressive breast cancers.

Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-ß signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.

New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy.

Despite the development of cancer therapies, the success of most treatments has been impeded by drug resistance. The crucial role of tumor cell plasticity has emerged recently in cancer progression, cancer stemness and eventually drug resistance. Cell plasticity drives tumor cells to reversibly convert their cell identity, analogous to differentiation and dedifferentiation, to adapt to drug treatment. This phenotypical switch is driven by alteration of the transcriptome. Several pluripotent factors from the KLF and SOX families are closely associated with cancer pathogenesis and have been revealed to regulate tumor cell plasticity. In this review, we particularly summarize recent studies about KLF4, KLF5 and SOX factors in cancer development and evolution, focusing on their roles in cancer initiation, invasion, tumor hierarchy and heterogeneity, and lineage plasticity. In addition, we discuss the various regulation of these transcription factors and related cutting-edge drug development approaches that could be used to drug "undruggable" transcription factors, such as PROTAC and PPI targeting, for targeted cancer therapy. Advanced knowledge could pave the way for the development of novel drugs that target transcriptional regulation and could improve the outcome of cancer therapy.

Proteolytic regulation of CD73 by TRIM21 orchestrates tumor immunogenicity.

Despite the rapid utilization of immunotherapy, emerging challenges to the current immune checkpoint blockade need to be resolved. Here, we report that elevation of CD73 levels due to its aberrant turnover is correlated with poor prognosis in immune-cold triple-negative breast cancers (TNBCs). We have identified TRIM21 as an E3 ligase that governs CD73 destruction. Disruption of TRIM21 stabilizes CD73 that in turn enhances CD73-catalyzed production of adenosine, resulting in the suppression of CD8+ T cell function. Replacement of lysine 133, 208, 262, and 321 residues by arginine on CD73 attenuated CD73 ubiquitylation and degradation. Diminishing of CD73 ubiquitylation remarkably promotes tumor growth and impedes antitumor immunity. In addition, a TRIM21high/CD73low signature in a subgroup of human breast malignancies was associated with a favorable immune profile. Collectively, our findings uncover a mechanism that governs CD73 proteolysis and point to a new therapeutic strategy by modulating CD73 ubiquitylation.

Active dry yeast supplementation improves the growth performance, rumen fermentation, and immune response of weaned beef calves.

The objective of this experiment was to investigate the potential benefits of active dry yeast (ADY) on the growth performance, rumen fermentation, nutrient digestibility, and serum parameters of weaned beef calves. Thirty Simmental crossbred male calves (body weight = 86.47 ± 4.41 kg and 70 ± 4 d of age) were randomly divided into 2 groups: control (CON) (fed basal ration) and ADY (fed basal ration and 5 g/d ADY per calf). The dietary concentrate-to-roughage ratio was 35:65. All the calves were regularly provided rations 3 times a day at 07:00, 13:00, and 19:00 and had free access to water. The experiment lasted for 60 d. The average daily gain of ADY group was higher (P = 0.007) than that of the CON group, and the ratio of feed intake to average daily gain in the ADY group was reduced (P = 0.022) as compared to the CON group. The concentration of ruminal ammonia-N was higher (P = 0.023) in the CON group than that in the ADY group, but an opposite trend of microbial protein was found between the 2 groups. Also, the ruminal concentrations of propionate and butyrate were higher (P < 0.05) in the ADY group than those in the CON group. Calves fed ADY exhibited higher (P < 0.05) crude protein and neutral detergent fiber digestibility. Supplementation of ADY increased (P < 0.05) the contents of glucose, glutathione peroxidase, superoxide dismutase, immunoglobulin A, immunoglobulin M, and interleukin 10 in the serum of calves, but an opposite trend was observed in malondialdehyde, interleukin 1 beta, and tumor necrosis factor alpha contents between the 2 groups. In conclusion, dietary supplementation with ADY could improve the growth performance, rumen fermentation, nutrient digestibility, antioxidant ability, and immune response of weaned beef calves.

Supercritical Fluid CO2 Extraction and Microcapsule Preparation of Lycium barbarum Residue Oil Rich in Zeaxanthin Dipalmitate.

The scope of this investigation aimed at obtaining and stabilizing bioactive products derived from Lycium barbarum seeds and peels, which were the byproducts in the processing of fruit juice. Zeaxanthin dipalmitate is a major carotenoid, comprising approximately 80% of the total carotenoid content in the seeds and peels. The method of obtainment was supercritical fluid CO2 extraction, studying different parameters that affect the oil yield and content of zeaxanthin dipalmitate. The optimized protocol to enact successful supercritical fluid CO2 extraction included optimum extraction pressure of 250 bar, temperature at 60 °C over a time span of 2.0 h, and a CO2 flow of 30 g/min, together with the use of a cosolvent (2% ethanol). The yields of oil and zeaxanthin dipalmitate under these optimal conditions were 17 g/100 g and 0.08 g/100 g, respectively. The unsaturated fatty acids were primarily linoleic acid (C18:2), oleic acid (C18:1), and γ-linolenic acid (C18:3), with their contents being as high as 91.85 ± 0.27% of the total fatty acids. The extract was a red-colored oil that was consequently microencapsulated through spray-drying with octenylsuccinate starch, gum arabic, and maltodextrin (13.5:7.5:3, w/w) as wall materials to circumvent lipid disintegration during storage and add to fruit juice in a dissolved form. The mass ratio of core material and wall material was 4:1. These materials exhibited the highest microencapsulation efficiency (92.83 ± 0.13%), with a moisture content of 1.98 ± 0.05% and solubility of 66.22 ± 0.24%. The peroxide content level within the microencapsulated zeaxanthin dipalmitate-rich oil remained at one part per eight in comparison to the unencapsulated oil, following fast-tracked oxidation at 60 °C for 6 weeks. This indicated the potential oxidation stability properties of microcapsule powders. Consequently, this microencapsulated powder has good prospects for development, and can be utilized for a vast spectrum of consumer health and beauty products.

MnSOD Lysine 68 acetylation leads to cisplatin and doxorubicin resistance due to aberrant mitochondrial metabolism.

Manganese superoxide dismutase (MnSOD) acetylation (Ac) has been shown to be a key post-translational modification important in the regulation of detoxification activity in various disease models. We have previously demonstrated that MnSOD lysine-68 (K68) acetylation (K68-Ac) leads to a change in function from a superoxide-scavenging homotetramer to a peroxidase-directed monomer. Here, we found that estrogen receptor positive (ER+) breast cancer cell lines (MCF7 and T47D), selected for continuous growth in cisplatin (CDDP) and doxorubicin (DXR), exhibited an increase in MnSOD-K68-Ac. In addition, MnSOD-K68-Ac, as modeled by the expression of a validated acetylation mimic mutant gene (MnSODK68Q ), also led to therapy resistance to CDDP and DXR, altered mitochondrial structure and morphology, and aberrant cellular metabolism. MnSODK68Q expression in mouse embryo fibroblasts (MEFs) induced an in vitro transformation permissive phenotype. Computerized molecular protein dynamics analysis of both MnSOD-K68-Ac and MnSOD-K68Q exhibited a significant change in charge distribution along the α1 and α2 helices, directly adjacent to the Mn2+ binding site, implying that this decrease in surface charge destabilizes tetrameric MnSOD, leading to an enrichment of the monomer. Finally, monomeric MnSOD, as modeled by amber codon substitution to generate MnSOD-K68-Ac or MnSOD-K68Q expression in mammalian cells, appeared to incorporate Fe to maximally induce its peroxidase activity. In summary, these findings may explain the mechanism behind the observed structural and functional change of MnSOD-K68-Ac.

Systemic application of honokiol prevents cisplatin ototoxicity without compromising its antitumor effect.

Cisplatin is a potent drug used in about 40% of cancer treatment but also leads to severe deafness in 60-80% of the cases. Although the mechanism is known to be related to the accumulation of reactive oxygen species (ROS), no drug or FDA approved treatment is currently available to prevent cisplatin ototoxicity. With this study, we show for the first time that honokiol (HNK), a pleiotropic poly-phenol prevents cisplatin-induced hearing loss. HNK also improves the wellbeing of the mice during the treatment, determined by the increase in the number of surviving animals. In a transgenic tumor mouse model, HNK does not hinder cisplatin's antitumor effect. The mechanism is related to the activation of sirtuin 3, a deacetylase in mitochondria essential for ROS detoxification. We expect a paradigm shift in cisplatin chemotherapy based on the current study and future clinical trials, where honokiol is applied to reduce side effects including hearing loss.

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter.

Manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, once tumorigenesis occurs, clinical data suggest MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in the regulation of metabolism. Here we show, using in vitro transformation and xenograft growth assays that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) functions as a tumor promoter. Interestingly, in various breast cancer and primary cell types the expression of MnSODK68Q is accompanied with a change of MnSOD's stoichiometry from a known homotetramer complex to a monomeric form. Biochemical experiments using the MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as a peroxidase, distinct from the established MnSOD superoxide dismutase activity. MnSODK68Q expressing cells exhibit resistance to tamoxifen (Tam) and cells selected for Tam resistance exhibited increased K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, carcinogenesis and tumor progression.

Using hyperspectral analysis as a potential high throughput phenotyping tool in GWAS for protein content of rice quality.

BACKGROUND: The advances of hyperspectral technology provide a new analytic means to decrease the gap of phenomics and genomics caused by the fast development of plant genomics with the next generation sequencing technology. Through hyperspectral technology, it is possible to phenotype the biochemical attributes of rice seeds and use the data for GWAS. RESULTS: The results of correlation analysis indicated that Normalized Difference Spectral Index (NDSI) had high correlation with protein content (PC) with RNDSI 2 = 0.68. Based on GWAS analysis using all the traits, NDSI was able to identify the same SNP loci as rice protein content that was measured by traditional methods. In total, hyperspectral trait NDSI identified all the 43 genes that were identified by biochemical trait PC. NDSI identified 1 extra SNP marker on chromosome 1, which annotated extra 22 genes that were not identified by PC. Kegg annotation results showed that traits NDSI annotated 3 pathways that are exactly the same as PC. The cysteine and methionine metabolic pathway identified by both NDSI and PC was reported important for biosynthesis and metabolism of some of amino acids/protein in rice seeds. CONCLUSION: This study combined hyperspectral technology and GWAS analysis to dissect PC of rice seeds, which was high throughput and proven to be able to apply to GWAS as a new phenotyping tool. It provided a new means to phenotype one of the important biochemical traits for the determination of rice quality that could be used for genetic studies.

Homeostatic Roles of STING in Cell Proliferation and Chromosomal Instability.

In this issue of Cancer Research, Ranoa and colleagues report on the role of STING (stimulator of IFN genes, TMEM173) in regulating critical tumor cell-intrinsic functions including cell-cycle progression, chromosomal stability, and cellular response to therapeutic ionizing radiation. The authors used multiple methods including RNA expression profiling, molecular and biochemical techniques, cell biology, and reagents from genetically modified murine models to test their hypothesis that downregulating the STING pathway in cancer cells promotes cellular transformation through accumulation of chromosomal instability and premature progression of the cell cycle. Their findings demonstrate that STING is a tumor suppressor that inhibits cell proliferation by restricting entry to mitosis as well as protecting cells against aneuploidy. These findings significantly advance our understanding of the role of STING as a tumor gate keeper.See related article by Ranoa et al., p. 1465.

The development of low-calorie sugar and functional jujube food using biological transformation and fermentation coupling technology.

Jujube juice has been used as ingredient in a range of foods and dietary supplements. In this study, an enzyme transformation and fermentation coupling technology was applied to increase the nutritional value of concentrated/extracted Jinsi jujube juice. Two enzymes, D-glucose isomerase (GI) and D-allulose 3-epimerase (DAE), were employed to convert the glucose and fructose to a low-calorie sweeter D-allulose with a concentration of 110 g/L in jujube juice. Furthermore, the mixed cultures of Pediococcus pentosaceus PC-5 and Lactobacillus plantarum M were employed to increase the content of nutrition components related to bioactivities and flavor volatiles in jujube juice. Accordingly, this fermentation accumulated 100 mg/L gamma-aminobutyric acid (GABA), which has neurotransmission, hypotension, diuretic, and tranquilizer effects, and increased the content of branched-chain amino acids (BCAAs) and many free amino acids (Asp, Glu, Gly, and Ala) at different level. The fermentation not only maintained the concentration of native functional components such as cyclic adenosine monophosphate (cAMP) and minerals, but also increased the content of iron (Fe2+) and zinc (Zn2+), which have blood and eyesight tonic function. The value-added jujube juice might serve as a low-calorie and probiotic functional beverage and show high application potential in food industry.

Biosynthesis of dendroketose from different carbon sources using in vitro and in vivo metabolic engineering strategies.

BACKGROUND: Asymmetric aldol-type C-C bond formation with ketones used as electrophilic receptor remains a challenging reaction for aldolases as biocatalysts. To date, only one kind of dihydroxyacetone phosphate (DHAP)-dependent aldolases has been discovered and applied to synthesize branched-chain sugars directly using DHAP and dihydroxyacetone (DHA) as substrate. However, the unstable and high-cost properties of DHAP limit large-scale application. Therefore, biosynthesis of branched-chain sugar from low-cost and abundant carbon sources is essential. RESULTS: The detailed catalytic property of l-rhamnulose-1-phosphate aldolase (RhaD) and l-fuculose-1-phosphate aldolase (FucA) from Escherichia coli in catalyzing the aldol reactions with DHA as electrophilic receptors was characterized. Furthermore, we calculated the Bürgi-Dunitz trajectory using molecular dynamics simulations, thereby revealing the original sources of the catalytic efficiency of RhaD and FucA. A multi-enzyme reaction system composed of formolase, DHA kinase, RhaD, fructose-1-phosphatase, and polyphosphate kinase was constructed to in vitro produce dendroketose, a branched-chain sugar, from one-carbon formaldehyde. The conversion rate reached 86% through employing a one-pot, two-stage reaction process. Moreover, we constructed two artificial pathways in Corynebacterium glutamicum to obtain this product in vivo starting from glucose or glycerol. Fermentation with glycerol as feedstock produced 6.4 g/L dendroketose with a yield of 0.45 mol/mol glycerol, representing 90% of the maximum theoretical value. Additionally, the dendroketose production reached 36.3 g/L with a yield of 0.46 mol/mol glucose when glucose served as the sole carbon resource. CONCLUSIONS: The detailed enzyme kinetics data of the two DHAP-dependent aldolases with DHA as electrophilic receptors were presented in this study. In addition, insights into this catalytic property were given via in silico simulations. Moreover, the cost-effective synthesis of dendroketose starting from one-, three-, and six-carbon resources was achieved through in vivo and in vitro metabolic engineering strategies. This rare branched-chain ketohexose may serve as precursor to prepare 4-hydroxymethylfurfural and branched-chain alkanes using chemical method.

Emerging evidence for targeting mitochondrial metabolic dysfunction in cancer therapy.

Mammalian cells use a complex network of redox-dependent processes necessary to maintain cellular integrity during oxidative metabolism, as well as to protect against and/or adapt to stress. The disruption of these redox-dependent processes, including those in the mitochondria, creates a cellular environment permissive for progression to a malignant phenotype and the development of resistance to commonly used anticancer agents. An extension of this paradigm is that when these mitochondrial functions are altered by the events leading to transformation and ensuing downstream metabolic processes, they can be used as molecular biomarkers or targets in the development of new therapeutic interventions to selectively kill and/or sensitize cancer versus normal cells. In this Review we propose that mitochondrial oxidative metabolism is altered in tumor cells, and the central theme of this dysregulation is electron transport chain activity, folate metabolism, NADH/NADPH metabolism, thiol-mediated detoxification pathways, and redox-active metal ion metabolism. It is proposed that specific subgroups of human malignancies display distinct mitochondrial transformative and/or tumor signatures that may benefit from agents that target these pathways.

Glycogen Phosphorylase: A Novel Biomarker in Doxorubicin-Induced Cardiac Injury.

Extracellular vesicles containing glycogen phosphorylase, brain/heart (PYGB) have been demonstrated as a sensitive biomarker for normal cardiac injuries for patients after chemotherapy. Oxidative stress was suggested to be the mechanism behind the chemotherapy-induced tissue damage and augmented with mitochondrial antioxidant could be an effective means of early intervention. Clin Cancer Res; 24(7); 1516-7. ©2018 AACRSee related article by Yarana et al., p. 1644.

Manganese superoxide dismutase (SOD2): is there a center in the universe of mitochondrial redox signaling?

It is becoming increasingly clear that mitochondria drive cellular functions and in vivo phenotypes by directing the production rate and abundance of metabolites that are proposed to function as signaling molecules (Chandel 2015; Selak et al. 2005; Etchegaray and Mostoslavsky 2016). Many of these metabolites are intermediates that make up cellular metabolism, part of which occur in mitochondria (i.e. the TCA and urea cycles), while others are produced "on demand" mainly in response to alterations in the microenvironment in order to participate in the activation of acute adaptive responses (Mills et al. 2016; Go et al. 2010). Reactive oxygen species (ROS) are well suited for the purpose of executing rapid and transient signaling due to their short lived nature (Bae et al. 2011). Hydrogen peroxide (H2O2), in particular, possesses important characteristics including diffusibility and faster reactivity with specific residues such as methionine, cysteine and selenocysteine (Bonini et al. 2014). Therefore, it is reasonable to propose that H2O2 functions as a relatively specific redox signaling molecule. Even though it is now established that mtH2O2 is indispensable, at least for hypoxic adaptation and energetic and/or metabolic homeostasis (Hamanaka et al. 2016; Guzy et al. 2005), the question of how H2O2 is produced and regulated in the mitochondria is only partially answered. In this review, some roles of this indispensable signaling molecule in driving cellular metabolism will be discussed. In addition, we will discuss how H2O2 formation in mitochondria depends on and is controlled by MnSOD. Finally, we will conclude this manuscript by highlighting why a better understanding of redox hubs in the mitochondria will likely lead to new and improved therapeutics of a number of diseases, including cancer.

SIRT3-Mediated Dimerization of IDH2 Directs Cancer Cell Metabolism and Tumor Growth.

The isocitrate dehydrogenase IDH2 produces α-ketoglutarate by oxidizing isocitrate, linking glucose metabolism to oxidative phosphorylation. In this study, we report that loss of SIRT3 increases acetylation of IDH2 at lysine 413 (IDH2-K413-Ac), thereby decreasing its enzymatic activity by reducing IDH2 dimer formation. Expressing a genetic acetylation mimetic IDH2 mutant (IDH2K413Q) in cancer cells decreased IDH2 dimerization and enzymatic activity and increased cellular reactive oxygen species and glycolysis, suggesting a shift in mitochondrial metabolism. Concurrently, overexpression of IDH2K413Q promoted cell transformation and tumorigenesis in nude mice, resulting in a tumor-permissive phenotype. IHC staining showed that IDH2 acetylation was elevated in high-risk luminal B patients relative to low-risk luminal A patients. Overall, these results suggest a potential relationship between SIRT3 enzymatic activity, IDH2-K413 acetylation-determined dimerization, and a cancer-permissive phenotype. Cancer Res; 77(15); 3990-9. ©2017 AACR.

Mitochondrial protein Fus1/Tusc2 in premature aging and age-related pathologies: critical roles of calcium and energy homeostasis.

Decreased energy production and increased oxidative stress are considered to be major contributors to aging and aging-associated pathologies. The role of mitochondrial calcium homeostasis has also been highlighted as an important factor affecting different pathological conditions. Here, we present evidence that loss of a small mitochondrial protein Fus1 that maintains mitochondrial homeostasis results in premature aging, aging-associated pathologies, and decreased survival. We showed that Fus1KO mice develop multiple early aging signs including lordokyphosis, lack of vigor, inability to accumulate fat, reduced ability to tolerate stress, and premature death. Other prominent pathological changes included low sperm counts, compromised ability of adult stem cells to repopulate tissues, and chronic inflammation. At the molecular level, we demonstrated that mitochondria of Fus1 KO cells have low reserve respiratory capacity (the ability to produce extra energy during sudden energy demanding situations), and show significantly altered dynamics of cellular calcium response.Our recent studies on early hearing and memory loss in Fus1 KO mice combined with the new data presented here suggest that calcium and energy homeostasis controlled by Fus1 may be at the core of its aging-regulating activities. Thus, Fus1 protein and Fus1-dependent pathways and processes may represent new tools and targets for anti-aging strategies.

Loss of NAD-Dependent Protein Deacetylase Sirtuin-2 Alters Mitochondrial Protein Acetylation and Dysregulates Mitophagy.

AIMS: Sirtuins connect energy generation and metabolic stress to the cellular acetylome. Currently, only the mitochondrial sirtuins (SIRT3-5) and SIRT1 have been shown to direct mitochondrial function; however, Aims: NAD-dependent protein deacetylase sirtuin-2 (SIRT2), the primary cytoplasmic sirtuin, is not yet reported to associate with mitochondria. RESULTS: This study revealed a novel physiological function of SIRT2: the regulation of mitochondrial function. First, the acetylation of several metabolic mitochondrial proteins was found to be altered in Sirt2-deficient mice, which was, subsequently, validated by immunoprecipitation experiments in which the acetylated mitochondrial proteins directly interacted with SIRT2. Moreover, immuno-gold electron microscopic images of mouse brains showed that SIRT2 associates with the inner mitochondrial membrane in central nervous system cells. The loss of Sirt2 increased oxidative stress, decreased adenosine triphosphate levels, and altered mitochondrial morphology at the cellular and tissue (i.e., brain) level. Furthermore, the autophagic/mitophagic processes were dysregulated in Sirt2-deficient neurons and mouse embryonic fibroblasts. INNOVATION: For the first time it is shown that SIRT2 directs mitochondrial metabolism. CONCLUSION: Together, these findings support that SIRT2 functions as a mitochondrial sirtuin, as well as a regulator of autophagy/mitophagy to maintain mitochondrial biology, thus facilitating cell survival. Antioxid. Redox Signal. 26, 849-863.

Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism.

The goal of this study was to determine if depletion of glutathione (GSH) and inhibition of thioredoxin (Trx) reductase (TrxR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol-dependent oxidative stress. The following were used to inhibit GSH and Trx metabolism: buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc- cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2-AAPA, a GSH-reductase inhibitor. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24-ESA+ or ALDH1) and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO and radiation also significantly decreased breast cancer cell migration and invasion in a thiol-dependent manner that could be inhibited by NAC. In addition, pretreating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24-ESA+ or ALDH1. Combined administration of Au and BSO, given prior to irradiation, significantly increased the survival of mice with human breast cancer xenografts, and decreased the number of ALDH1+ cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and in vivo.