14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation.

Ten-eleven translocation-2 (TET2) is a member of the methylcytosine dioxygenase family of enzymes and has been implicated in cancer and aging because of its role as a global epigenetic modifier. TET2 has a large N-terminal domain and a catalytic C-terminal region. Previous reports have demonstrated that the TET2 catalytic domain remains active independently of the N-terminal domain. As such, the function of the N terminus of this large protein remains poorly characterized. Here, using yeast two-hybrid screening, co-immunoprecipitation, and several biochemical assays, we found that several isoforms of the 14-3-3 family of proteins bind TET2. 14-3-3 proteins bound TET2 when it was phosphorylated at Ser-99. In particular, we observed that AMP-activated protein kinase-mediated phosphorylation at Ser-99 promotes TET2 stability and increases global DNA 5-hydroxymethylcytosine levels. The interaction of 14-3-3 proteins with TET2 protected the Ser-99 phosphorylation, and disruption of this interaction both reduced TET2 phosphorylation and decreased TET2 stability. Furthermore, we noted that protein phosphatase 2A can interact with TET2 and dephosphorylate Ser-99. Collectively, these results provide detailed insights into the role of the TET2 N-terminal domain in TET2 regulation. Moreover, they reveal the dynamic nature of TET2 protein regulation that could have therapeutic implications for disease states resulting from reduced TET2 levels or activity.

Trichomonas vaginalis Virus Among Women With Trichomoniasis and Associations With Demographics, Clinical Outcomes, and Metronidazole Resistance.

BACKGROUND: Trichomonas vaginalis virus (TVV) is a non-segmented, 4.5-5.5 kilo-base pair (kbp), double-stranded RNA virus infecting T. vaginalis. The objectives of this study were to examine the TVV prevalence in US Trichomonas vaginalis isolates and TVV's associations with patient demographics, clinical outcomes, and metronidazole resistance. METHODS: Archived T. vaginalis isolates from the enrollment visits of 355 women participating in a T. vaginalis treatment trial in Birmingham, Alabama, were thawed and grown in culture. Their total RNA was extracted using a Trizol reagent. Contaminating, single-stranded RNA was precipitated using 4.0 M Lithium Chloride and centrifugation. The samples were analyzed by gel electrophoresis to visualize a 4.5 kbp band representative of TVV. In vitro testing for metronidazole resistance was also performed on 25/47 isolates obtained from the women's test of cure visits. RESULTS: TVV was detected in 142/355 (40%) isolates at the enrollment visit. Women with TVV-positive (TVV+) isolates were significantly older (P = .01), more likely to smoke (P = .04), and less likely to report a history of gonorrhea (P = .04). There was no association between the presence of clinical symptoms or repeat T. vaginalis infections with TVV+ isolates (P = .14 and P = .44, respectively). Of 25 test of cure isolates tested for metronidazole resistance, 0/10 TVV+ isolates demonstrated resistance, while 2/15 TVV-negative isolates demonstrated mild to moderate resistance (P = .23). CONCLUSIONS: Of 355 T. vaginalis isolates tested for TVV, T. vaginalis isolates tested for TVV, the prevalence was 40%. However, there was no association of TVV+ isolates with clinical symptoms, repeat infections, or metronidazole resistance. These results suggest that TVV may be commensal to T. vaginalis.

In Vitro Study of the Susceptibility of Clinical Isolates of Trichomonas vaginalis to Metronidazole and Secnidazole.

Nitroimidazoles (metronidazole [MTZ] and tinidazole [TNZ]) are the only drugs recommended for treatment of Trichomonas vaginalis infections. MTZ resistance occurs in 4% to 10% of cases of vaginal trichomoniasis (R. D. Kirkcaldy et al., Emerg Infect Dis 18:939-943, 2012; J. R. Schwebke and F. J. Barrientes, Antimicrob Agents Chemother 50:4209-4210, 2006) and TNZ resistance in 1% of cases (J. R. Schwebke and F. J. Barrientes, Antimicrob Agents Chemother 50:4209-4210, 2006). Emerging nitroimidazole-resistant trichomoniasis is concerning, because few alternatives to standard therapy exist. We assessed the prevalence of in vitro aerobic MTZ and secnidazole resistance among T. vaginalis isolates collected in 2015 to 2016 from 100 women in Birmingham, Alabama, with positive cultures. Archived specimens were treated with secnidazole or MTZ (0.2 to 400 µg/ml) for 48 h, according to U.S. Centers for Disease Control and Prevention protocols. Ninety-six (96%) of the 100 clinical Trichomonas isolates tested demonstrated lower minimum lethal concentrations for secnidazole than for MTZ, suggesting that secnidazole has better in vitro activity than MTZ.

Kinomic profiling identifies focal adhesion kinase 1 as a therapeutic target in advanced clear cell renal cell carcinoma.

The introduction of targeted therapies has caused a paradigm shift in the treatment of metastatic clear cell (cc)-renal cell carcinoma (RCC). We hypothesized that determining differential kinase activity between primary and metastatic tumor sites may identify critical drivers of progression and relevant therapeutic targets in metastatic disease. Kinomic profiling was performed on primary tumor and metastatic tumor deposits utilizing a peptide substrate microarray to detect relative tyrosine phosphorylation activity. Pharmacologic and genetic loss of function experiments were used to assess the biologic significance of the top scoring kinase on in vitro and in vivo tumor phenotypes. Kinomics identified 7 peptides with increased tyrosine phosphorylation in metastases that were significantly altered (p<0.005). Based on these peptides, bioinformatics analyses identified several candidate kinases activated in metastases compared to primary tumors. The highest ranked upstream kinase was Focal Adhesion Kinase 1 (FAK1). RCC lines demonstrate evidence of elevated FAK1 activation relative to non-transformed renal epithelial cells. Pharmacologic inhibition of FAK1 with GSK2256098 suppresses in vitro tumor phenotypes. In turn, FAK1 knockdown in RCC cells suppresses both in vitro phenotypes and in vivo tumor growth. Collectively, these data demonstrate functional activation of FAK1 in metastases and provide preclinical rationale for targeting this kinase in the setting of advanced ccRCC.

Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

L-2-Hydroxyglutarate: an epigenetic modifier and putative oncometabolite in renal cancer.

UNLABELLED: Through unbiased metabolomics, we identified elevations of the metabolite 2-hydroxyglutarate (2HG) in renal cell carcinoma (RCC). 2HG can inhibit 2-oxoglutaratre (2-OG)-dependent dioxygenases that mediate epigenetic events, including DNA and histone demethylation. 2HG accumulation, specifically the d enantiomer, can result from gain-of-function mutations of isocitrate dehydrogenase (IDH1, IDH2) found in several different tumors. In contrast, kidney tumors demonstrate elevations of the l enantiomer of 2HG (l-2HG). High-2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC), consistent with 2HG-mediated inhibition of ten-eleven translocation (TET) enzymes, which convert 5-methylcytosine (5mC) to 5hmC. l-2HG elevation is mediated in part by reduced expression of l-2HG dehydrogenase (L2HGDH). L2HGDH reconstitution in RCC cells lowers l-2HG and promotes 5hmC accumulation. In addition, L2HGDH expression in RCC cells reduces histone methylation and suppresses in vitro tumor phenotypes. Our report identifies l-2HG as an epigenetic modifier and putative oncometabolite in kidney cancer. SIGNIFICANCE: Here, we report elevations of the putative oncometabolite l-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer.

CHOP potentially co-operates with FOXO3a in neuronal cells to regulate PUMA and BIM expression in response to ER stress.

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in various neurodegenerative diseases including Parkinson Disease, Alzheimer Disease and Huntington Disease. PUMA (p53 upregulated modulator of apoptosis) and BIM (BCL2 interacting mediator of cell death), pro-apoptotic BH3 domain-only, BCL2 family members, have previously been shown to regulate ER stress-induced cell death, but the upstream signaling pathways that regulate this response in neuronal cells are incompletely defined. Consistent with previous studies, we show that both PUMA and BIM are induced in response to ER stress in neuronal cells and that transcriptional induction of PUMA regulates ER stress-induced cell death, independent of p53. CHOP (C/EBP homologous protein also known as GADD153; gene name Ddit3), a critical initiator of ER stress-induced apoptosis, was found to regulate both PUMA and BIM expression in response to ER stress. We further show that CHOP knockdown prevents perturbations in the AKT (protein kinase B)/FOXO3a (forkhead box, class O, 3a) pathway in response to ER stress. CHOP co-immunoprecipitated with FOXO3a in tunicamycin treated cells, suggesting that CHOP may also regulate other pro-apoptotic signaling cascades culminating in PUMA and BIM activation and cell death. In summary, CHOP regulates the expression of multiple pro-apoptotic BH3-only molecules through multiple mechanisms, making CHOP an important therapeutic target relevant to a number of neurodegenerative conditions.

Deficiency of pro-apoptotic Hrk attenuates programmed cell death in the developing murine nervous system but does not affect Bcl-x deficiency-induced neuron apoptosis.

The BCL-2 family includes both pro- and anti-apoptotic proteins, which regulate programmed cell death during development and in response to various apoptotic stimuli. The BH3-only subgroup of pro-apoptotic BCL-2 family members is critical for the induction of apoptotic signaling, by binding to and neutralizing anti-apoptotic BCL-2 family members. During embryonic development, the anti-apoptotic protein BCL-X(L) plays a critical role in the survival of neuronal populations by regulating the multi-BH domain protein BAX. In this study, the authors investigated the role of Harakiri (HRK), a relatively recently characterized BH3-only molecule in disrupting the BAX-BCL-X(L) interaction during nervous system development. Results indicate that HRK deficiency significantly reduces programmed cell death in the nervous system. However, HRK deficiency does not significantly attenuate the widespread apoptosis seen in the Bcl-x (-/-) embryonic nervous system, indicating that other BH3-only molecules, alone or in combination, may regulate BAX activation in immature neurons.

Lysosome dysfunction triggers Atg7-dependent neural apoptosis.

Macroautophagy (autophagy) is a process wherein bulk cytosolic proteins and damaged organelles are sequestered and degraded via the lysosome. Alterations in autophagy-associated proteins have been shown to cause neural tube closure defects, neurodegeneration, and tumor formation. Normal lysosome function is critical for autophagy completion and when altered may lead to an accumulation of autophagic vacuoles (AVs) and caspase activation. The tumor suppressor p53 is highly expressed in neural precursor cells (NPCs) and has an important role in the regulation of both autophagy and apoptosis. We hypothesized that altered lysosome function would lead to NPC death via an interaction between autophagy- and apoptosis-associated proteins. To test our hypothesis, we utilized FGF2-expanded NPCs and the neural stem cell line, C17.2, in combination with the lysosomotropic agent chloroquine (CQ) and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1). Both CQ and Baf A1 caused concentration- and time-dependent AV accumulation, p53 phosphorylation, increased damage regulator autophagy modulator levels, caspase-3 activation, and cell death. Short hairpin RNA knockdown of Atg7, but not Beclin1, expression significantly inhibited CQ- and Baf A1-induced cell death, indicating that Atg7 is an upstream mediator of lysosome dysfunction-induced cell death. Cell death and/or caspase-3 activation was also attenuated by protein synthesis inhibition, p53 deficiency, or Bax deficiency, indicating involvement of the intrinsic apoptotic death pathway. In contrast to lysosome dysfunction, starvation-induced AV accumulation was inhibited by either Atg7 or Beclin1 knockdown, and Atg7 knockdown had no effect on starvation-induced death. These findings indicate that Atg7- and Beclin1-induced autophagy plays a cytoprotective role during starvation but that Atg7 has a unique pro-apoptotic function in response to lysosome dysfunction.

Cytoplasmic p53 and activated Bax regulate p53-dependent, transcription-independent neural precursor cell apoptosis.

The prodeath effects of p53 are typically mediated via its transcriptional upregulation of proapoptotic Bcl-2 family members, including PUMA, Noxa, and/or Bax. We previously reported that staurosporine (STS), a broad-spectrum kinase inhibitor and prototypical apoptosis-inducing agent, produced p53-dependent, Bax-dependent, neural precursor cell (NPC) apoptosis, but that this effect occurred independently of new gene transcription and PUMA expression. To further characterize the mechanism by which p53 regulates NPC death, we used primary cerebellar NPCs derived from wild-type, p53-deficient, and Bax-deficient neonatal mice and the mouse cerebellar neural stem cell line, C17.2. We found that STS rapidly increased p53 cytoplasmic immunoreactivity in neuritic-like processes in C17.2 cells, which preceded Bax activation and caspase-3 cleavage. Confocal microscopy analysis of STS-treated cells revealed partial colocalization of p53 with the mitochondrial marker pyruvate dehydrogenase as well as with conformationally altered "activated" Bax, suggesting an interaction between these proapoptotic molecules in triggering apoptotic death. Nucleophosmin (NPM), a CRM1-dependent nuclear chaperone, also exhibited partial colocalization with both activated Bax and p53 following STS treatment. These observations suggest that cytoplasmic p53 can trigger transcription-independent NPC apoptosis through its potential interaction with NPM and activated Bax.

bcl-2/Adenovirus E1B 19-kd interacting protein 3 (BNIP3) regulates hypoxia-induced neural precursor cell death.

Perinatal hypoxia-ischemia may result in long-term neurological deficits. In addition to producing neuron death, HI causes death of neural precursor cells (NPCs) in the developing brain. To characterize the molecular pathways that regulate hypoxia-induced death of NPCs, we treated a mouse neural stem cell line (C17.2 cells) and fibroblastic growth factor II-expanded primary NPCs derived from wild-type or gene-disrupted mice, with oxygen glucose deprivation or the hypoxia mimetics desferrioxamine or cobalt chloride. Neural precursor cells undergoing hypoxia exhibited time- and concentration-dependent caspase-3 activation and cell death, which was significantly reduced by treatment with a broad caspase inhibitor or protein synthesis inhibition. Bax/Bak-deficient NPCs were protected from desferrioxamine-induced death and exhibited minimal caspase-3 activation. Oxygen glucose deprivation or hypoxia-mimetic exposure also resulted in increased hypoxia-inducible factor alpha and bcl-2/adenovirus E1B 19-kd interacting protein 3 (BNIP3) expression. BNIP3 shRNA treatment failed to affect hypoxia-induced caspase-3 activation but inhibited cell death and nuclear translocation of apoptosis-inducing factor, indicating that BNIP3 is an important regulator of caspase-independent NPC death after hypoxia. These studies demonstrate that hypoxia activates both caspase-dependent and -independent NPC death pathways that are critically regulated by multiple Bcl-2 family members.

The proapoptotic BH3-only, Bcl-2 family member, Puma is critical for acute ethanol-induced neuronal apoptosis.

Synaptogenesis in humans occurs in the last trimester of gestation and in the first few years of life, whereas it occurs in the postnatal period in rodents. A single exposure of neonatal rodents to ethanol during this period evokes extensive neuronal apoptosis. Previous studies indicate that ethanol triggers the intrinsic apoptotic pathway in neurons, and that this requires the multi-BH domain, proapoptotic Bcl-2 family member Bax. To define the upstream regulators of this apoptotic pathway, we examined the possible roles of p53 and a subclass of proapoptotic Bcl-2 family members (i.e. the BH3 domain-only proteins) in neonatal wild-type and gene-targeted mice that lack these cell death inducers. Acute ethanol exposure produced greater caspase-3 activation and neuronal apoptosis in wild-type mice than in saline-treated littermate controls. Loss of p53-upregulated mediator of apoptosis (Puma) resulted in marked protection from ethanol-induced caspase-3 activation and apoptosis. Although Puma expression has been reported to be regulated by p53, p53-deficient mice exhibited a similar extent of ethanol-induced caspase-3 activation and neuronal apoptosis as wild-type mice. Mice deficient in other proapoptotic BH3-only proteins, including Noxa, Bim, or Hrk, showed no significant protection from ethanol-induced neuronal apoptosis. Collectively, these studies indicate a p53-independent, Bax- and Puma-dependent mechanism of neuronal apoptosis and identify Puma as a possible molecular target for inhibiting the effects of intrauterine ethanol exposure in humans.