Increase In Steady-state Levels Research Articles

Abstract P2056: The Transcription Factor TFEB Protects Against Cardiac Hypertrophy By Increasing Lysosomal Capacity And Activating AMPKα

Introduction: Pathological cardiac remodeling in response to elevated afterload can eventually progress to heart failure. Lysosome-assisted processes such as autophagy play an important role in this remodeling. The lysosome is also a nexus for some of the same signaling pathways that are involved in hypertrophic growth. The transcription factor EB (TFEB) has emerged as a key regulator of lysosomal genes in multiple tissues, especially in response to external stress. Methods and Results: Here, we report that TFEB is rapidly activated and translocates to the nucleus in cardiomyocytes under hypertrophic stress conditions, activating a lysosomal gene program independent of autophagy gene changes. We engineered a cardiomyocyte-specific TFEB knockout mouse (CTKO). At baseline, contractile function measured by echocardiography appears normal in these mice compared with their Cre-negative littermates. However, in the setting of pressure-overload stress induced by TAC (thoracic aortic constriction), the CTKO mice manifest an exacerbated hypertrophic response leading quickly to heart failure. Unlike the WT hearts, CTKO hearts fail to increase lysosomal capacity after TAC and manifest an increase in steady-state levels of autophagosome-associated proteins such as LC3II and p62, as well as accumulation of ubiquitinated proteins, suggesting a decrease in protein turnover. Surprisingly, AMPK signaling, while normal at baseline is significantly decreased in the CTKO hearts after TAC. This reliance on TFEB for PE-induced AMPK signaling is also observed in vitro , as are the anti-hypertrophic effects of TFEB activation, supporting a direct role of TFEB in this process. Finally, we show that exogenous activation of AMPK in the absence of TFEB can completely rescue the exacerbated hypertrophic response both in vitro and in vivo , independent of lysosomal function. Surprisingly, blunting of the hypertrophic response had no impact on the decreased cardiac function observed in the TAC-treated CTKO mice. Conclusion: Overall, our findings suggest that TFEB antagonizes pathological hypertrophic cardiac remodeling through up-regulation of lysosomal capacity and AMPK signaling.

USP5 enhances SGTA mediated protein quality control.

Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

Sirt6 activation blocks LPS induced expression of inflammatory cytokines in cardiac cells

Sepsis induced myocardial dysfunction is a common complication that has been linked to an increased mortality. The activation of NFkB pathway and excessive production of pro‐inflammatory cytokines is considered to be one of the most important signaling pathway involved in the pathogenesis of sepsis. Earlier studies have shown that Sirt6, a member of class III of histone deacetylases, attenuates hyper‐activation of NFkB signaling that contributes to inflammation and the aging process. In the present study we tested the hypothesis whether pharmacological activation of Sirt6 can prevent LPS induced myocardial inflammation. Primary cultures of adult rat cardiac fibroblasts were treated with UBCS039, an allosteric activator of Sirt6, for 48 hours. The results showed a dose‐dependent increase in steady‐state levels of Sirt6 as well as deacetylation of its target H3K9 by Western analysis. A similar effect of UBCS039 treatment was observed in H9C2 cardiac myocytes. We also tested effects of UBCS039 on LPS induced NFkB activation and cytokine production in these cells. Our data showed that pretreatment of cardiac fibroblasts with UBCS039 for 48 hrs significantly reduced LPS (100ng/ml)‐induced phosphorylated levels of NFkB (pNFkB). By real‐time PCR analysis we found that LPS led to increased expression (4‐9 fold) of mRNA of IL‐6, IL‐1b, TLR4 and CD‐38 in cardiac fibroblasts within 6h of treatment. This effect of LPS in cardiac fibroblasts was completely blocked when cells were pretreated with UBCS039. These data thus indicated that pharmacological activation of Sirt6 is capable of blocking LPS induced inflammatory response in cardiac cells, and has the potential to alleviate sepsis induced myocardial dysfunction and related health consequences.

Triphenylphosphonium derivatives disrupt metabolism and inhibit melanoma growth in vivo when delivered via a thermosensitive hydrogel.

Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5–16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.

Characterization of glycan substrates accumulating in GM1 Gangliosidosis

IntroductionGM1 gangliosidosis is a rare autosomal recessive genetic disorder caused by the disruption of the GLB1 gene that encodes β-galactosidase, a lysosomal hydrolase that removes β-linked galactose from the non-reducing end of glycans. Deficiency of this catabolic enzyme leads to the lysosomal accumulation of GM1 and its asialo derivative GA1 in β-galactosidase deficient patients and animal models. In addition to GM1 and GA1, there are other glycoconjugates that contain β-linked galactose whose metabolites are substrates for β-galactosidase. For example, a number of N-linked glycan structures that have galactose at their non-reducing end have been shown to accumulate in GM1 gangliosidosis patient tissues and biological fluids. ObjectiveIn this study, we attempt to fully characterize the broad array of GLB1 substrates that require GLB1 for their lysosomal turnover. ResultsUsing tandem mass spectrometry and glycan reductive isotope labeling with data-dependent mass spectrometry, we have confirmed the accumulation of glycolipids (GM1 and GA1) and N-linked glycans with terminal beta-linked galactose. We have also discovered a novel set of core 1 and 2 O-linked glycan metabolites, many of which are part of structurally-related isobaric series that accumulate in disease. In the brain of GLB1 null mice, the levels of these glycan metabolites increased along with those of both GM1 and GA1 as a function of age. In addition to brain tissue, we found elevated levels of both N-linked and O-linked glycan metabolites in a number of peripheral tissues and in urine. Both brain and urine samples from human GM1 gangliosidosis patients exhibited large increases in steady state levels for the same glycan metabolites, demonstrating their correlation with this disease in humans as well. ConclusionsOur studies illustrate that GLB1 deficiency is not purely a ganglioside accumulation disorder, but instead a broad oligosaccharidosis that include representatives of many β-linked galactose containing glycans and glycoconjugates including glycolipids, N-linked glycans, and various O-linked glycans. Accounting for all β-galactosidase substrates that accumulate when this enzyme is deficient increases our understanding of this severe disorder by identifying metabolites that may drive certain aspects of the disease and may also serve as informative disease biomarkers to fully evaluate the efficacy of future therapies.

Abstract 3527: Inhibition of mitochondrial pyruvate transport selectively sensitizes cancer cells to metabolic oxidative stress

Abstract To determine if mitochondrial pyruvate transport could represent a therapeutic target for sensitizing cancer cells to oxidative stress, lung and breast cancer cells were treated with 5 µM UK5099 to inhibit the mitochondrial pyruvate carrier (MPC). Treatment with UK5099 selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione using 1 mM buthionine sulfoximine (BSO; a glutathione synthesis inhibitor) for 48 h and 72 h, relative to normal lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant, N-acetylcysteine (NAC; 20 mM), independent of GSH levels, indicating a mechanism of toxicity involving reduced thiols and metabolic oxidative stress. In addition, treatment with UK5099 alone for 48 h also decreased levels of total glutathione in cancer cells that could be reversed by NAC. Using oxidation-sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment of lung and breast cancer cells for 24 h and 48 h with UK5099 induced increases in steady state levels of pro-oxidants (presumably hydroperoxides and mitochondrial superoxide), which were further increased with BSO. Finally, treatment of breast cancer cells with UK5099 for 24 and 48 hours significantly sensitized breast cancer cells to clonogenic cell killing mediated by paclitaxel. These data support the hypothesis that inhibition of the MPC selectively causes an impairment of antioxidant capability in cancer cells that is enhanced by depletion of glutathione. Furthermore, these results also support the hypothesis that inhibition of the MPC represents a significant target for sensitizing human breast cancer cells to chemotherapy agents thought to induce oxidative stress. Supported by R01CA182804. Citation Format: Shane R. Solst, Samuel N. Rodman, Melissa A. Fath, Eric B. Taylor, Douglas R. Spitz. Inhibition of mitochondrial pyruvate transport selectively sensitizes cancer cells to metabolic oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3527.

Cdc48/p97 segregase is modulated by cyclin‐dependent kinase to determine cyclin fate during G1 progression

Cells sense myriad signals during G1, and a rapid response to prevent cell cycle entry is of crucial importance for proper development and adaptation. Cln3, the most upstream G1 cyclin in budding yeast, is an extremely short-lived protein subject to ubiquitination and proteasomal degradation. On the other hand, nuclear accumulation of Cln3 depends on chaperones that are also important for its degradation. However, how these processes are intertwined to control G1-cyclin fate is not well understood. Here, we show that Cln3 undergoes a challenging ubiquitination step required for both degradation and full activation. Segregase Cdc48/p97 prevents degradation of ubiquitinated Cln3, and concurrently stimulates its ER release and nuclear accumulation to trigger Start. Cdc48/p97 phosphorylation at conserved Cdk-target sites is important for recruitment of specific cofactors and, in both yeast and mammalian cells, to attain proper G1-cyclin levels and activity. Cdk-dependent modulation of Cdc48 would subjugate G1 cyclins to fast and reversible state switching, thus arresting cells promptly in G1 at developmental or environmental checkpoints, but also resuming G1 progression immediately after proliferative signals reappear.

Transmission of microRNA antimiRs to mouse offspring via the maternal-placental-fetal unit.

The emergence of microRNA as regulators of organogenesis and tissue differentiation has stimulated interest in the ablation of microRNA expression and function during discrete periods of development. To this end, inducible, conditional modulation of microRNA expression with doxycycline-based tetracycline-controlled transactivator and tamoxifen-based estrogen receptor systems has found widespread use. However, the induction agents and components of genome recombination systems negatively impact pregnancy, parturition, and postnatal development; thereby limiting the use of these technologies between late gestation and the early postnatal period. MicroRNA inhibitor (antimiR) administration also represents a means of neutralizing microRNA function in vitro and in vivo. To date, these studies have used direct (parenteral) administration of antimiRs to experimental animals. As an extension of this approach, an alternative means of regulating microRNA expression and function is described here: the maternal–placental–fetal transmission of antimiRs. When administered to pregnant dams, antimiRs were detected in offspring and resulted in a pronounced and persistent reduction in detectable steady-state free microRNA levels in the heart, kidney, liver, lungs, and brain. This effect was comparable to direct injection of newborn mouse pups with antimiRs, although maternal delivery resulted in fewer off-target effects. Furthermore, depletion of steady-state microRNA levels via the maternal route resulted in concomitant increases in steady-state levels of selected microRNA targets. This novel methodology permits the temporal regulation of microRNA function during late gestation and in neonates, without recourse to conventional approaches that rely on doxycycline and tamoxifen, which may confound studies on developmental processes.

141 - Inhibition of Mitochondrial Pyruvate Transport Selectively Sensitizes Cancer Cells to Metabolic Oxidative Stress

To determine if mitochondrial pyruvate transport could represent a therapeutic target for sensitizing cancer cells to oxidative stress, lung and breast cancer cells were treated with 5 µM UK5099 to inhibit the mitochondrial pyruvate carrier (MPC). Treatment with UK5099 selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione using 1 mM buthionine sulfoximine (BSO; a glutathione synthesis inhibitor) for 48 h and 72 h, relative to normal lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant, N-acetylcysteine (NAC; 20 mM), independent of GSH levels indicating a mechanism of toxicity involving reduced thiols and metabolic oxidative stress. In addition, treatment with UK5099 alone for 48 h also decreased levels of total glutathione in cancer cells that could be reversed by NAC. Using oxidation sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment of lung and breast cancer cells for 24 h and 48 h with UK5099 induced increases in steady state levels of pro-oxidants (presumably hydroperoxides and mitochondrial superoxide) which were further increased with BSO. Finally, treatment of breast cancer cells with UK5099 for 24 and 48 hours significantly sensitized breast cancer cells to clonogenic cell killing mediated by paclitaxel. These data support the hypothesis that inhibition of the MPC selectively causes an impairment of antioxidant capability in cancer cells that is enhanced by depletion of glutathione. Furthermore, these results also support the hypothesis that inhibition of the MPC represents a significant target for sensitizing human breast cancer cells to chemotherapy agents thought to induce oxidative stress.

Intertwined Role of Reactive Oxygen, Nitrogen and Carbon in Diabetic State

Diabetes is a metabolic disorder of multiple etiologies, characterized by chronic hyperglycemia caused by defects in the secretion of insulin or the action of insulin, or both. Production of reactive oxygen species (ROS) and lipid peroxidation are increased in diabetic patients, especially in those with poor glycemic control. An increase in steady-state level of reactive oxygen species (ROS) or reactive carbonyl species (RCS) may result in so-called “oxidative stress” or “carbonyl stress”, respectively. Reactive nitrogen species (RNS) is also a subset of ROS. ROS and RNS include hydroxyl radical ( • OH), hydrogen peroxide (H2O2), superoxide (O2 •), nitric oxide (NO•), peroxynitrite (ONOO−). In physiological conditions, mitochondria are the major site of intracellular ROS production, due to electron leakage along the respiratory chain. Protein kinase C also becomes activated in retinal endothelial cells under hyperglycemic conditions and can lead to activation of NADH oxidase. Metal-catalyzed auto oxidation of reducing carbohydrates could be involved in the formation of AGEs and ROS. Hexosamine biosynthesis pathway is an additional pathway of glucose metabolism that may mediate some of the toxic effects of glucose Oxidized lipoproteins activate inflammatory signaling, which in turn aggravates the inflammation. Therefore, clearance of oxidative and nitrosative stress and inhibition of inflammation have been considered good strategies for the treatment of diabetic complications.

Escape of X-linked miRNA genes from meiotic sex chromosome inactivation

Past studies have indicated that transcription of all X-linked genes is repressed by meiotic sex chromosome inactivation (MSCI) during the meiotic phase of spermatogenesis in mammals. However, more recent studies have shown an increase in steady-state levels of certain X-linked miRNAs in pachytene spermatocytes, suggesting that either synthesis of these miRNAs increases or that degradation of these miRNAs decreases dramatically in these cells. To distinguish between these possibilities, we performed RNA-FISH to detect nascent transcripts from multiple miRNA genes in various spermatogenic cell types. Our results show definitively that Type I X-linked miRNA genes are subject to MSCI, as are all or most X-linked mRNA genes, whereas Type II and III X-linked miRNA genes escape MSCI by continuing ongoing, active transcription in primary spermatocytes. We corroborated these results by co-localization of RNA-FISH signals with both a corresponding DNA-FISH signal and an immunofluorescence signal for RNA polymerase II. We also found that X-linked miRNA genes that escape MSCI locate non-randomly to the periphery of the XY body, whereas genes that are subject to MSCI remain located within the XY body in pachytene spermatocytes, suggesting that the mechanism of escape of X-linked miRNA genes from MSCI involves their relocation to a position outside of the repressive chromatin domain associated with the XY body. The fact that Type II and III X-linked miRNA genes escape MSCI suggests an immediacy of function of the encoded miRNAs specifically required during the meiotic stages of spermatogenesis.

Inflammation: gone with translation.

Inflammation: gone with translation.

Evaluating the effects of CELF1 deficiency in a mouse model of RNA toxicity

Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by an expanded (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The toxic RNA transcripts produced from the mutant allele alter the function of RNA-binding proteins leading to the functional depletion of muscleblind-like (MBNL) proteins and an increase in steady state levels of CUG-BP1 (CUGBP-ETR-3 like factor 1, CELF1). The role of increased CELF1 in DM1 pathogenesis is well studied using genetically engineered mouse models. Also, as a potential therapeutic strategy, the benefits of increasing MBNL1 expression have recently been reported. However, the effect of reduction of CELF1 is not yet clear. In this study, we generated CELF1 knockout mice, which also carry an inducible toxic RNA transgene to test the effects of CELF1 reduction in RNA toxicity. We found that the absence of CELF1 did not correct splicing defects. It did however mitigate the increase in translational targets of CELF1 (MEF2A and C/EBPβ). Notably, we found that loss of CELF1 prevented deterioration of muscle function by the toxic RNA, and resulted in better muscle histopathology. These data suggest that while reduction of CELF1 may be of limited benefit with respect to DM1-associated spliceopathy, it may be beneficial to the muscular dystrophy associated with RNA toxicity.

Common chromosomal fragile site FRA16D tumor suppressor WWOX gene expression and metabolic reprograming in cells

The WWOX gene spans the FRA16D common chromosomal fragile site and is able to suppress tumor growth. FRA16D is a frequent site of DNA instability in cancer resulting in reduced levels of WWOX expression. Altered levels of WWOX have been shown to affect metabolism. Whereas metabolic reprograming of cells from oxidative phosphorylation to aerobic glycolysis is a major hallmark of tumors, the relationship between common chromosomal fragile site genes and altered metabolism has been unclear. Here we report that altering metabolism from glycolysis to oxidative phosphorylation causes stable increase in steady-state levels of transcripts of the WWOX gene. Consistent with this, exposure to hypoxic conditions, in which cells rely on glycolysis, causes a downregulation of WWOX mRNA. The function of WWOX is therefore intimately integrated with metabolism, as WWOX not only contributes to the metabolic state of cells, its transcript levels are also linked to intracellular metabolic state.

Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG(1-3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase-mediated telomere repeat addition is negatively modulated by the levels of telomere-bound Rap1-Rif1-Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere-binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady-state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C-terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere-bound Est2 is increased, while telomere-bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.

Abstract 1778: C-MYC plays a novel role in driving the error-prone double-strand break repair in triple negative breast cancers.

Abstract Approximately 10-20% of breast cancers lack estrogen, progesterone or HER2 receptors, the so-called triple negative breast cancer cells (TNBCs). These women have a poor prognosis and treatment options are limited. Therefore, the development of novel therapies are urgently required. It is well established that repair of DNA double-strand breaks (DSBs), one of the most lethal forms of DNA damage, is abnormal in breast cancers and can lead to genomic instability. We have recently reported that an alternative and highly error-prone form of NHEJ (ALT NHEJ) is found to be upregulated in TNBCs, as evidenced by increased protein levels of ALT NHEJ components, DNA ligase III (LIG3) and poly ADP ribose polymerase 1 (PARP1) and increased ALT NHEJ activity. Notably, these cells are highly sensitive to a combination of DNA ligase and PARP inhibitors in colony survival assays. This suggests that ALT NHEJ may not only contribute to the genomic instability, but it is also important for survival. The aim of this study is to determine the mechanisms underlying ALT-NHEJ upregulation in TNBCs that will likely identify potential novel targets for therapy. Herein, we demonstrate that c-MYC plays a novel role in driving error-prone repair by ALT NHEJ. A significant decrease in PARP1 and LIG3 is observed with siRNA-mediated knockdown (KD) of c-MYC in several TNBC cell lines (N=4). Furthermore, overexpression of c-MYC in wild-type breast epithelial cells (WT) with c-MYC-pcDNA3 construct leads to &amp;gt;2-fold increase in steady-state levels of PARP1 and LIG3. To determine whether c-MYC expression affects the functional outcome of DSB repair via ALT NHEJ, we performed in vivo and in vitro plasmid-based end-joining assays following knockdown or overexpression of c-MYC. Notably, c-MYC KD led to an increase in fidelity of NHEJ repair in TNBCs. In contrast, c-MYC overexpression leads to an increase in DSB repair errors, characterized by larger DNA deletions. Given the pro-proliferative role of c-MYC we determined whether error-prone ALT NHEJ was S-phase specific. In vivo NHEJ assays on cells released from synchronization at S- and G2/M phases of cell cycle reveal that DSB repair errors in the plasmid are introduced mainly in the S-phase. Putative c-MYC binding sites are present on the promoters of both PARP1 and LIG3. To investigate whether c-MYC is involved in transcriptional regulation of ALT NHEJ proteins, we cloned the PARP1 promoter into a luciferase vector and transfected this construct into WT and TNBCs (N=4). PARP1 promoter activity decreases upon c-MYC knockdown in TNBCs and increases upon c-MYC overexpression in WT cells, suggesting that c-MYC is likely involved in direct regulation of PARP1. Our findings demonstrate a novel role of c-MYC in driving error-prone DSB repair by ALT NHEJ pathway, which could have important implications in genomic instability, disease progression and the development of novel therapies in TNBCs. Citation Format: Pratik K. Nagaria, Khadiza Chowdhury, Angela Brodie, Feyruz Rassool. C-MYC plays a novel role in driving the error-prone double-strand break repair in triple negative breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2013-1778

Judge, Jury, and Executioner: DXO Functions as a Decapping Enzyme and Exoribonuclease in Pre-mRNA Quality Control

In this issue of Molecular Cell, Jiao et al. (2013) describe the mammalian enzyme DXO, which has pyrophosphohydrolase, decapping, and 5'-3' exoribonuclease activity and functions as an important checkpoint in cotranscriptional capping of RNA polymerase II (Pol II) pre-mRNA transcripts.

The Bcl-2/Bcl-xL inhibitor BH3I-2′ affects the dynamics and subcellular localization of sumoylated proteins

Sumoylation modulates many proteins implicated in apoptosis such as Fas, TNFR1, Daxx, p53 and its regulator MDM2. Some of these proteins, such as DRP-1, are involved in the intrinsic apoptosis pathway. The intrinsic pathway is regulated at the mitochondrial level by the Bcl-2 family of proteins. The small-molecule inhibitor BH3I-2′ binds to the hydrophobic groove of the BH3 domain of anti-apoptotic proteins Bcl-xL and Bcl-2. Following treatment with this inhibitor in various experimental conditions, we observed decreased levels of detergent-soluble SUMO-1, an increase in the relative levels of detergent-insoluble sumoylated proteins, or both. Accordingly, immunofluorescence microscopy revealed that the relative numbers and intensities of endogenously or exogenously expressed SUMO-1 foci in the nucleus were increased following BH3I-2′ treatment. MG132 caused a large increase in steady-state levels of SUMO-1 and of sumoylated proteins, and this was especially true for detergent-insoluble proteins. The conjugation-incompetent GG-to-AA SUMO-1 mutant, which did not form nuclear foci, was only present in the detergent-soluble lysate fraction and was insensitive to BH3I-2′, implying that BH3I-2′ specifically affects SUMO in its conjugated form. Finally, BH3I-2′ had similar effects on SUMO-2 and SUMO-3 as it had on SUMO-1. In conclusion, BH3I-2′ causes an intracellular redistribution of sumoylated proteins, more specifically their targeting to PML and non-PML nuclear bodies in which they may be degraded by the proteasome. Interestingly, knocking down Bcl-2 also altered levels of sumoylated proteins and their presence in detergent-insoluble compartments, confirming the role of Bcl-2 as a modulator of the sumoylation pathway.

Maintenance of higher H2O2 levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A

We assessed the catalase bioactivity and hydrogen peroxide (H2O2) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H2O2 steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H2O2 which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O2-) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O2- produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H2O2 levels and led to apoptosis of these cells. The H2O2 produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O2- leads to an increase in steady-state levels of H2O2 in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.

Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4β2 nAChR binding site density and β2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4β2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and β2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (∼1.2 fold), suggesting that the increase in α4 and β2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.