Dominant-negative Construct Research Articles

Human trafficking in nairaland digital community: A corpus-assisted critical discourse study

Human trafficking in Nigeria as a topical issue has enjoyed more sociological interrogation with very scant attention in linguistics and discourse. This paper applies a corpus-assisted critical discourse study to examine representative posts on human trafficking in Nigeria (2019 to 2022) retrieved from the Nairaland digital community. Using the Sketch Engine corpus tool and social actors representation model, this paper investigates how different constructions were deployed by participants to represent human trafficking, human traffickers and traffick victims in Nigeria. Findings suggest four constructions oriented to negativity: prostitution/commodification of sex, abuse of underage for sexual satisfaction, maltreatment of others for huge labour, dismembering of humans for occultism and health-assurance. Participants deployed role allocation, nominalisation, and others to negatively evaluate human traffickers, especially as economic usurpers, exploiters, and fraudsters, while the traffick-victims were represented as naïve, non-violent, armless, defenceless and (in)active recipients of the activities of the human traffickers. The dominant negative constructs manifested implicitly and explicitly through tagging, negative comparison, appeal to sentimentalities, and expression of detest, while the positive constructs of victims manifested through pity and appeal to humanity. Online participants attack the political class, and declare their ideological stances on human traffickers in Nigeria, making efforts to project suppressed stances.

PI3K couples long-term synaptic potentiation with cofilin recruitment and actin polymerization in dendritic spines via its regulatory subunit p85α

Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.

The Ubiquitin Ligase Adaptor NDFIP1 Interacts with TRESK and Negatively Regulates the Background K+ Current.

The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.

Abstract 5829: Elucidating the significance of amphiregulin and its implications for EGFR inhibition strategies in LKB1-mutant NSCLC

Abstract LKB1-mutant non-small cell lung cancer (NSCLC) is aggressive and treatment-refractory, underscoring the need for new therapeutic strategies. Our comparative study involving mouse lung tumors driven by oncogenic KrasG12D and LKB1 loss as well as tumors driven solely by KrasG12D, revealed that LKB1 loss upregulated amphiregulin (Areg), a pro-growth EGFR ligand. Therefore, this study investigates how the inactivation of LKB1 enhances AREG expression, the significance of elevated AREG in sustaining lung cancer growth, and the responses of AREG-high lung cancer to EGFR inhibitors. We first evaluated the potential clinical significance of AREG using The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset and the Cancer Cell Line Encyclopedia (CCLE) database. We observed that LKB1-null tumors with high AREG levels were associated with shorter patient survival and exhibited transcriptional signatures similar to EGFR-mutant tumors. High AREG expression also correlated positively in NSCLC cell lines with LKB1 mutations and Western blot analysis confirmed that elevated AREG levels in lung cancer cells were associated with constitutive EGFR signaling. We next explored the impact of LKB1 loss on AREG expression in lung cancer. Our prior RNA-seq and ChIP-seq investigations demonstrated CRTC2 and CREB enrichment on the AREG promoter in LKB1-null lung cancer cells. Upon LKB1 reintroduction, the enrichment diminished, indicating that CRTC-CREB transcriptionally induces AREG in LKB1-null lung cancer. To corroborate, reintroducing LKB1 or expressing a dominant-negative construct (dnCRTC), disrupting CREB/CRTC interaction, decreased AREG levels, as confirmed by RT-qPCR in LKB1-null lung cancer cells. This supports that AREG is a direct target of CRTC-CREB activation induced by LKB1 loss. To assess the impact of elevated AREG in LKB1-mutant lung cancer growth, we used RNA interference and antibodies to inhibit AREG. Both interventions decreased cell growth and colony formation, underscoring AREG is critical for LKB1-mutant lung cancer growth. Moreover, we investigated the potential correlation between AREG and the response to the EGFR tyrosine-kinase inhibitor Erlotinib in NSCLC cell lines, utilizing AREG expression data from CCLE and Erlotinib response data from the Genomics of Drug Sensitivity in Cancer (CDSC) databases. Stratifying NSCLC cell lines into sensitive, intermediate, and resistant categories revealed a potential positive association between AREG expression and Erlotinib sensitivity. Notably, LKB1-null lung cell lines with high AREG expression demonstrated heightened sensitivity compared to those with low AREG expression. Our study revealed that LKB1 loss increases AREG, triggering aberrant EGFR signaling critical for lung cancer growth, and AREG expression might impact the Erlotinib response in LKB1-inactive cells. Citation Format: Xzaviar Kaymar Solone, Xin Zhou, Mu Yu, Jennifer W. Li, Finn Rimay, Frederic Kaye, Lizi Wu. Elucidating the significance of amphiregulin and its implications for EGFR inhibition strategies in LKB1-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5829.

Etv5a suppresses neural progenitor cell proliferation by inhibiting sox2 transcription.

Neural progenitor cells are self-renewable, proliferative, and multipotent cell populations that generate diverse types of neurons and glia to build the nervous system. Transcription factors play critical roles in regulating various cellular processes; however, the transcription factors that regulate the development of neural progenitors are yet to be identified. In the present study, we demonstrated that zebrafish etv5a is expressed in the neural progenitor cells of the neuroectoderm. Downregulation of endogenous Etv5a function by etv5a morpholino or an etv5a dominant-negative variant increased the proliferation of sox2-positive neural progenitor cells, accompanied by inhibition of neurogenesis and gliogenesis. These phenotypes in Etv5a-depleted embryos could be rescued by co-injection with etv5a cRNA. Etv5a overexpression reduced sox2 expression. Direct binding of Etv5a to the regulatory elements of sox2 was affirmed by chromatin immunoprecipitation. These data revealed that Etv5a directly suppressed sox2 expression to reduce the proliferation of neural progenitor cells. In addition, the expression of foxm1, a putative target gene of Etv5a and a direct upstream transcription factor of sox2, was upregulated in Etv5a-deficient embryos. Moreover, the suppression of Foxm1 function by the foxm1 dominant-negative construct nullified the phenotype of upregulated sox2 expression caused by Etv5a deficiency. Overall, our results indicated that Etv5a regulates the expression of sox2 via direct binding to the sox2 promoter and indirect regulation by inhibiting foxm1 expression. Hence, we revealed the role of Etv5a in the transcriptional hierarchy that regulates the proliferation of neural progenitor cells.

‘A threat to national unity, an emancipator’: discourse construction of the Yoruba nation secessionist agitation in selected Nigerian digital communities

ABSTRACT The recently resurged Yoruba Nation (YN, henceforth) agitation joins some socio-political movements, social protests, and resistance group discourse in Nigeria that continue to gain traction in (critical) discourse studies. Guided by the theoretical paradigms of van Leeuwen’s representational strategies and Martin and White’s appraisal framework, 24 representative posts out of a thousand posts culled from Nairaland, Gistmania, and Naijaloaded generated between October 2020 and October 2021 were purposively selected and subjected to discourse analysis. Two levels of construction were realised: YN agitators and YN agitation. YN agitators were associated with four constructions: cowards, violence mongers, terrorists, and organised strategists. YN agitation was associated with three constructions: a threat to sovereignty and national unity, and emancipation from oppression. Negative labels manifested more than positives. The dominant negative constructs of the YN agitation manifested implicitly and explicitly through labelling, negative comparison, appeal to sentiments, expression of detest, and flaming, while the positive constructs manifested through encomium and appeal to ethnic benefit. Online participants do not only project and spread their ideological stances on the YN agitation; they also make efforts to suppress antithetical stances.

Inverse agonists of retinoic acid receptor/retinoid X receptor signaling as lineage-specific antitumor agents against human adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a lethal malignancy of exocrine glands, characterized by the coexistence within tumor tissues of 2 distinct populations of cancer cells, phenotypically similar to the myoepithelial and ductal lineages of normal salivary epithelia. The developmental relationship linking these 2 cell types, and their differential vulnerability to antitumor treatments, remains unknown. Using single-cell RNA sequencing, we identified cell-surface markers (CD49f, KIT) that enabled the differential purification of myoepithelial-like (CD49fhigh/KITneg) and ductal-like (CD49flow/KIT+) cells from patient-derived xenografts (PDXs) of human ACCs. Using prospective xenotransplantation experiments, we compared the tumor-initiating capacity of the 2 cell types and tested whether one could differentiate into the other. Finally, we searched for signaling pathways with differential activation between the 2 cell types and tested their role as lineage-specific therapeutic targets. Myoepithelial-like cells displayed higher tumorigenicity than ductal-like cells and acted as their progenitors. Myoepithelial-like and ductal-like cells displayed differential expression of genes encoding for suppressors and activators of retinoic acid signaling, respectively. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) signaling (all-trans retinoic acid, bexarotene) promoted myoepithelial-to-ductal differentiation, whereas suppression of RAR/RXR signaling with a dominant-negative RAR construct abrogated it. Inverse agonists of RAR/RXR signaling (BMS493, AGN193109) displayed selective toxicity against ductal-like cells and in vivo antitumor activity against PDX models of human ACC. In human ACCs, myoepithelial-like cells act as progenitors of ductal-like cells, and myoepithelial-to-ductal differentiation is promoted by RAR/RXR signaling. Suppression of RAR/RXR signaling is lethal to ductal-like cells and represents a new therapeutic approach against human ACCs.

Abstract 1435: A GABP dominant negative approach to targeting tumor cell immortality

Abstract Immortality is a hallmark of human cancer cells and therapeutic reversal is of great interest. Telomerase Reverse Transcriptase promoter (TERTp) mutations reactivate TERT expression, the rate limiting step in telomerase activity and cellular immortality. While prior telomerase blocking therapies lack tumor selectivity and were poorly tolerated, TERTp mutations and their regulation offer a unique opportunity for tumor specific reversal of cellular immortality. TERTp mutations are the most common non-coding mutation across all cancers, including glioblastoma (GBM), melanoma, urothelial carcinoma, and many others. The mutations generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression. While GABP can form both a dimeric and tetrameric complex, we found that the tetrameric complex is responsible for reactivation of the mutant TERTp, suggesting selectively targeting the tetramer may induce telomere shortening and tumor cell death. However, through knocking out the tetramer, we found that the GABP dimer is sufficiently upregulated to maintain TERT expression. In the absence of the dimer and the tetramer isoform, many tumor cells senesce or undergo cell death. However, some escape this fate via upregulation of a second tetramer forming GABP paralogue that maintains TERT expression. Therefore, we devised a transactivation domain null GABP subunit that we hypothesized would act in a dominant negative manner to block the GABP dimer and both GABP tetramers. Introduction of the dominant negative construct into TERTp mutant GBM, meningioma, medulloblastoma, oligodendroglioma, and urothelial carcinoma cells significantly reduced TERT expression while not altering expression in TERTp wildtype GBM cells. Furthermore, the dominant negative is able to shorten telomere length in TERTp mutant GBM cells. While therapeutic targeting of the three GABP complexes seems particularly challenging, our initial dominant negative appears to do so. Ultimately the dominant negative could be delivered directly into tumors via clinically approved viral delivery systems such as retroviral replicating vectors. Citation Format: Nicholas O. Stevers, Sara A. Collins, Samuel H. Wu, Noriyuki Kasahara, Joseph F. Costello. A GABP dominant negative approach to targeting tumor cell immortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1435.

Expression of DNAJB9 and some other genes is more sensitive to SWCNTs in normal human astrocytes than glioblastoma cells.

Objective. Single-walled carbon nanotubes (SWCNTs) are considered to be one of the nanomaterials attractive for biomedical applications, particularly in the health sciences as imaging probes and drug carriers, especially in the field of cancer therapy. The increasing exploitation of nanotubes necessitates a comprehensive evaluation of the potential impact of these nanomaterials, which purposefully accumulate in the cell nucleus, on the human health and the function of the genome in the normal and tumor tissues. The aim of this study was to investigate the sensitivity of the expression of DNAJB9 and some other genes associated with the endoplasmic reticulum (ER) stress and cell proliferation to low doses of SWCNTs in normal human astrocytes (NHA/TS) and glioblastoma cells (U87MG) with and without an inhibition of ERN1 signaling pathway of the ER stress. Methods. Normal human astrocytes, line NHA/TS and U87 glioblastoma cells stable transfected by empty vector or dnERN1 (dominant-negative construct of ERN1) were exposed to low doses of SWCNTs (2 and 8 ng/ml) for 24 h. RNA was extracted from the cells and used for cDNA synthesis. The expression levels of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 mRNAs were measured by a quantitative polymerase chain reaction and normalized to ACTB mRNA. Results. It was found that the low doses of SWCNTs up-regulated the expression of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 genes in normal human astrocytes in dose-dependent (2 and 8 ng/ml) and gene-specific manner. These nanotubes also increased the expression of most studied genes in control (transfected by empty vector) U87 glioblastoma cells, but with much lesser extent than in NHA/TS. However, the expression of CLU gene in control U87 glioblastoma cells treated with SWCNTs was down-regulated in a dose-dependent manner. Furthermore, the expression of TOB1 and P4HA2 genes did not significantly change in these glioblastoma cells treated by lower dose of SWCNTs only. At the same time, inhibition of ERN1 signaling pathway of ER stress in U87 glioblastoma cells led mainly to a stronger resistance of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, and P4HA2 gene expression to both doses of SWCNTs. Conclusion. The data obtained demonstrate that the low doses of SWCNTs disturbed the genome functions by changing the levels of key regulatory gene expressions in gene-specific and dose-dependent manner, but their impact was much stronger in the normal human astrocytes in comparison with the tumor cells. It is possible that ER stress, which is constantly present in tumor cells and responsible for multiple resistances, also created a partial resistance to the SWCNTs action. Low doses of SWCNTs induced more pronounced changes in the expression of diverse genes in the normal human astrocytes compared to glioblastoma cells indicating for a possible both genotoxic and neurotoxic effects with a greater extent in the normal cells.

Members of Venezuelan Equine Encephalitis complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner

Pixuna virus (PIXV) and Río Negro virus (RNV) are mosquito-borne alphaviruses belonging to the Venezuelan Equine Encephalitis (VEE) complex, which includes pathogenic epizootic and enzootic subtypes responsible for life-threatening diseases in equines. Considering that the first steps in viral infection are crucial for the efficient production of new progeny, the aim of this study was to elucidate the early events of the replication cycle of these two viruses. To this end, we used chemical inhibitors and the expression of dominant-negative constructs to study the dependence of clathrin and endosomal pH on PIXV and RNV internalization mechanisms. We demonstrated that both viruses are internalized primarily via clathrin-mediated endocytosis, where the low pH in endosomes is crucial for viral replication. Contributing knowledge regarding the entry route of VEE complex members is important to understand the pathogenesis of these viruses and also to develop new antiviral strategies.

A NAC Transcription Factor from 'Sea Rice 86' Enhances Salt Tolerance by Promoting Hydrogen Sulfide Production in Rice Seedlings.

Soil salinity severely threatens plant growth and crop performance. Hydrogen sulfide (H2S), a plant signal molecule, has been implicated in the regulation of plant responses to salinity stress. However, it is unclear how the transcriptional network regulates H2S biosynthesis during salt stress response. In this study, we identify a rice NAC (NAM, ATAF and CUC) transcription factor, OsNAC35-like (OsNACL35), from a salt-tolerant cultivar ‘Sea Rice 86′ (SR86) and further show that it may have improved salt tolerance via enhanced H2S production. The expression of OsNACL35 was significantly upregulated by high salinity and hydrogen peroxide (H2O2). The OsNACL35 protein was localized predominantly in the nucleus and was found to have transactivation activity in yeast. The overexpression of OsNACL35 (OsNACL35-OE) in japonica cultivar Nipponbare ramatically increased resistance to salinity stress, whereas its dominant-negative constructs (SUPERMAN repression domain, SRDX) conferred hypersensitivity to salt stress in the transgenic lines at the vegetative stage. Moreover, the quantitative real-time PCR analysis showed that many stress-associated genes were differentially expressed in the OsNACL35-OE and OsNACL35-SRDX lines. Interestingly, the ectopic expression of OsNACL35 triggered a sharp increase in H2S content by upregulating the expression of a H2S biosynthetic gene, OsDCD1, upon salinity stress. Furthermore, the dual luciferase and yeast one-hybrid assays indicated that OsNACL35 directly upregulated the expression of OsDCD1 by binding to the promoter sequence of OsDCD1. Taken together, our observations illustrate that OsNACL35 acts as a positive regulator that links H2S production to salt stress tolerance, which may hold promising utility in breeding salt-tolerant rice cultivar.

FGF/MAPK/Ets signaling in Xenopus ectoderm contributes to neural induction and patterning in an autonomous and paracrine manner, respectively.

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue.

The involvement of oxysterol-binding protein related protein (ORP) 6 in the counter-transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.

MAPKAP1 (Sin1), a Key Component of the MTORC2 Complex, Confers Resistance to Sorafenib and Correlates with Adverse Clinical Outcomes in Acute Myeloid Leukemia

Introduction: The mammalian target of rapamycin (mTOR) functions as a critical catalytic core in two distinct intracellular complexes: the rapamycin-sensitive mTORC1 and the rapamycin-insensitive mTORC2. The latter is capable of activating Akt through phosphorylation at the Ser473 residue. Constitutive activation of Akt is identified in up to 70% of acute myeloid leukemia (AML) patients and has been associated with an adverse impact on survival. Akt activation also mediates, at least in part, the leukemogenic effects of activating FLT3 internal tandem duplication (ITD) mutations, which in turn lead to activation of mTORC1. Mitogen-activated protein kinase-associated protein 1 (MAPKAP1 or Sin1) is required for mTORC2 integrity and activation and is also required for phosphorylation and activation of Akt. In addition, the pleckstrin homology (PH) domain of MAPKAP1 directly binds the PI3K product PtdIns P3 to promote mTORC2 kinase activation and membrane localization, thereby revealing a mechanistic link between PI3K and mTORC2. However, the role of MAPKAP1 in AML pathogenesis and its impact on clinical outcomes are as of yet unknown.Methods: The in vitro system included 5 AML cell lines (2 FLT3 -mutated and 3 FLT3 -unmutated) and 6 BA/F3 (murine pro-B cells) stable clones bearing specific FLT3 mutations. Experiments were performed using transient transfections with siRNA or plasmids, pharmacologic studies, Western blot analysis, q-RT-PCR, flow cytometry, immunofluorescence, confocal microscopy and proteomics. The patient cohort included 224 adult AML patients diagnosed and treated at The University of Texas MD Anderson Cancer Center (Houston, TX) and the National University Hospital (Singapore) with available cytogenetics and mutation status data. Potential associations of MAPKAP1 with clinical outcomes were evaluated using mRNA expression data from the The Cancer Genome Atlas (TCGA) AML dataset and protein expression data assessed by immunohistochemistry on bone marrow biopsy specimens from patients in our study group using a monoclonal anti-MAPKAP1 antibody (Cell Signaling). Overall survival (OS) was defined as time from AML diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from AML diagnosis to relapse, death, or last follow-up. Survival analysis was based on the Kaplan-Meier method using the Log-rank test.Results: MAPKAP1 is frequently expressed in AML cells and regulated by STAT3, which is predominantly phosphorylated at Ser727 residue in AML cell lines and patient samples. Inhibition of STAT3 activity by pharmacologic agents, dominant negative constructs, or STAT3 gene silencing leads to decreased MAPKAP1 levels and MTORC2 activity associated with Akt dephosphorylation. In silico analysis reveals 4 potential STAT3 binding sites on the MAPKAP1 gene promoter suggesting transcriptional regulation. Knocking down the MAPKAP1 gene in AML cell lines results in Akt deactivation associated with decreased cell proliferation, and these effects are mediated by cell cycle regulators. MAPKAP1 also confers resistance to sorafenib in vitro, as MAPKAP1 is shown to be upregulated and more active in BA/F3 clones bearing sorafenib-resistant FLT3 mutations. Conversely, STAT3 or MAPKAP1 inhibition sensitizes resistant BA/F3 clones to sorafenib treatment. Using the TCGA dataset, high MAPKAP1 mRNA levels are significantly associated with poorer EFS and OS (Figure 1); these findings were independent of AML risk group or the presence of FLT3 or NPM1 mutations. Similarly, high MAPKAP1 protein levels assessed by immunohistochemistry on bone marrow specimens prior to treatment correlate with adverse clinical outcomes in a cohort of AML patients treated with equivalent regimens.Conclusions: MAPKAP1 is involved in AML pathogenesis, likely through a STAT3-MTORC2-Akt crosstalk mechanism, and may represent a novel target of investigational therapy. MAPKAP1 mRNA and protein levels are significantly associated with clinical outcomes in AML._ [Display omitted] DisclosuresKhoury:Pfizer: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromics: Research Funding. Kantarjian:Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

Novel PITX2 Homeodomain-Contained Mutations from ATRIAL Fibrillation Patients Deteriorate Calcium Homeostasis

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10% in 80+ years patients. Pituitary Homeobox 2, Paired Like Homeodomain 2 (PITX2c) loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factor plays a pivotal role in atrial electrophysiology and calcium homeostasis and point to PITX2 as a candidate gene for AF. To address this issue, we recruited 31 AF patients for genetic analyses of both the known risk alleles and PITX2c open reading frame (ORF) re-sequencing. We found two-point mutations in the homedomain of PITX2 and three other variants in the 5’untranslated region. A 65 years old male patient without 4q25 risk variants but with recurrent AF displayed two distinct HD-mutations, NM_000325.5:c.309G>C (Gln103His) and NM_000325.5:c.370G>A (Glu124Lys), which both resulted in a change within a highly conserved amino acid position. To address the functional impact of the PITX2 HD mutations, we generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2c MD4 and PITX2c MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, similar to the PITX2c DN (50%), while Shox2 promoter repression was also impaired. Co-transactivation with other cardiac-enriched co-factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2c function. Furthermore, when expressed in HL1 cardiomyocyte cultures, the PITX2 mutants impaired endogenous expression of calcium regulatory proteins and induced alterations in sarcoplasmic reticulum (SR) calcium accumulation. This favored alternating and irregular calcium transient amplitudes, causing deterioration of the beat-to-beat stability upon elevation of the stimulation frequency. Overall this data demonstrate that these novel PITX2c HD-mutations might be causative of atrial fibrillation in the carrier.

RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11.

The small GTPase Rab11 (herein referring to the Rab11A and Rab11B isoforms) plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain from one of its effectors, for example, the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab-binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, for example, to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 allowed visualization of endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking - tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

Leptin activates Akt in oesophageal cancer cells via multiple atorvastatin-sensitive small GTPases

Obesity is a risk factor for Barrett’s oesophagus and oesophageal adenocarcinoma. Adipose tissue secretes the hormone leptin. Leptin is a growth factor for several cell types, including Barrett’s cells and oesophageal adenocarcinoma cells. Statins are associated with reduced rates of Barrett’s oesophagus and oesophageal cancer and exhibit anti-cancer effects in vitro. The mechanisms of these effects are not fully established. We have examined the effects of leptin and the lipid-soluble statin, atorvastatin, on signalling via monomeric GTP-binding proteins and Akt. Proliferation and apoptosis were assessed in OE33 cells. Akt activity was quantified by cell-based ELISA and in vitro kinase assay. Specific small-molecule inhibitors and a dominant-negative construct were used to reduce Akt activity. Small GTPases were inhibited using transfection of dominant-negative plasmids, prenylation inhibitors and pretreatment with atorvastatin. Leptin stimulated Akt activity and cell proliferation and inhibited camptothecin-induced apoptosis in an Akt-sensitive manner. Leptin induced phosphorylation of Bad and FOXO1 in an Akt-sensitive manner. Leptin activated Ras, Rac, RhoA and cdc42. Transfection of dominant-negative plasmids confirmed that leptin-induced Akt activation required Ras, RhoA cdc42 but not Rac. Atorvastatin inhibited leptin-induced activation of Ras, RhoA, cdc42 and Akt. Co-treatment with mevalonate prevented these effects of atorvastatin. The protein kinase Akt is essential to the growth-promoting and anti-apoptotic effects of leptin in oesophageal adenocarcinoma cells. Akt is activated via Ras-, Rho- and cdc42-dependant pathways. Atorvastatin reduces leptin-induced Akt activation by inhibiting prenylation of small GTPases. This may explain the reduced incidence of oesophageal adenocarcinoma in statin-users.

Knockdown of slit signaling during limb development leads to a reduction in humerus length.

Slits (1-3) and their Robo (1-3) receptors play multiple non-neuronal roles in development, including in development of muscle, heart and mammary gland. Previous work has demonstrated expression of Slit and Robo family members during limb development, where their functions are unclear. In situ hybridisation confirmed strong expression of Slit2, Slit3, Robo1, and Robo2 throughout mouse limb and joint development. No expression of Slit1 or Robo3 was detected. Analysis of Slit1/2 or Slit3 knockout mice revealed normal limb development. In contrast, locally blocking Slit signaling though grafting of cells expressing a dominant-negative Robo2 construct in the proximo-central region of developing chicken limb buds caused significant shortening of the humerus. These findings demonstrate an essential role for Slit/Robo signaling in regulating bone length during chicken limb development.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

Activation of ATM/Akt/CREB/eNOS Signaling Axis by Aphidicolin Increases NO Production and Vessel Relaxation in Endothelial Cells and Rat Aortas.

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5’-serially deleted eNOS promoters revealed that Tax-responsive element site, located at −962 to −873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser133 level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser1981 and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473, p-CREB-Ser133, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser1981, p-Akt-Ser473, p-CREB-Ser133, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.