Publications by authors named "Amanda G Ammer"

6 Publications

  • Page 1 of 1

Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia.

Free Radic Biol Med 2021 Sep 5;175:226-235. Epub 2021 Sep 5.

Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine, Morgantown, WV, USA; West Virginia University Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA. Electronic address:

B-cell acute lymphoblastic leukemia (ALL) affects both pediatric and adult patients. Chemotherapy resistant tumor cells that contribute to minimal residual disease (MRD) underlie relapse and poor clinical outcomes in a sub-set of patients. Targeting mitochondrial oxidative phosphorylation (OXPHOS) in the treatment of refractory leukemic cells is a potential novel approach to sensitizing tumor cells to existing standard of care therapeutic agents. In the current study, we have expanded our previous investigation of the mitoNEET ligand NL-1 in the treatment of ALL to interrogate the functional role of the mitochondrial outer membrane protein mitoNEET in B-cell ALL. Knockout (KO) of mitoNEET (gene: CISD1) in REH leukemic cells led to changes in mitochondrial ultra-structure and function. REH cells have significantly reduced OXPHOS capacity in the KO cells coincident with reduction in electron flow and increased reactive oxygen species. In addition, we found a decrease in lipid content in KO cells, as compared to the vector control cells was observed. Lastly, the KO of mitoNEET was associated with decreased proliferation as compared to control cells when exposed to the standard of care agent cytarabine (Ara-C). Taken together, these observations suggest that mitoNEET is essential for optimal function of mitochondria in B-cell ALL and may represent a novel anti-leukemic drug target for treatment of minimal residual disease.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.09.003DOI Listing
September 2021

Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection.

Immun Inflamm Dis 2019 12 5;7(4):326-341. Epub 2019 Nov 5.

Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia.

Introduction: Stroke-associated pneumonia (SAP) is a major cause of mortality in patients who have suffered from severe ischemic stroke. Although multifactorial in nature, stroke-induced immunosuppression plays a key role in the development of SAP. Previous studies using a murine model of transient middle cerebral artery occlusion (tMCAO) have shown that focal ischemic stroke induction results in functional defects of lymphocytes in the spleen, thymus, and peripheral blood, leading to spontaneous bacterial infection in the lungs without inoculation. However, how ischemic stroke alters immune cell niche and the expression of cytokines and chemokines in the lungs has not been fully characterized.

Methods: Ischemic stroke was induced in mice by tMCAO. Immune cell profiles in the brain and the lungs at 24- and 72-hour time points were compared by flow cytometric analysis. Cytokine and chemokine expression in the lungs were determined by multiplex bead arrays. Tissue damage and bacterial burden in the lungs following tMCAO were evaluated.

Results: Ischemic stroke increases the percentage of alveolar macrophages, neutrophils, and CD11b dendritic cells, but reduces the percentage of CD4 T cells, CD8 T cells, B cells, natural killer cells, and eosinophils in the lungs. The alteration of immune cell niche in the lungs coincides with a significant reduction in the levels of multiple chemokines in the lungs, including CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, CXCL5, CXCL9, and CXCL10. Spontaneous bacterial infection and tissue damage following tMCAO, however, were not observed.

Conclusion: This is the first report to demonstrate a significant reduction of lymphocytes and multiple proinflammatory chemokines in the lungs following ischemic stroke in mice. These findings suggest that ischemic stroke directly impacts pulmonary immunity.
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http://dx.doi.org/10.1002/iid3.277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842816PMC
December 2019

The NAC family transcription factor GmNAC42-1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean.

BMC Genomics 2019 Feb 20;20(1):149. Epub 2019 Feb 20.

Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia, 26506, USA.

Background: Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis.

Results: Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42-1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42-1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42-1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42-1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42-1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system.

Conclusions: Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42-1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42-1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.
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http://dx.doi.org/10.1186/s12864-019-5524-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381636PMC
February 2019

Cortactin Phosphorylation by Casein Kinase 2 Regulates Actin-Related Protein 2/3 Complex Activity, Invadopodia Function, and Tumor Cell Invasion.

Mol Cancer Res 2019 04 4;17(4):987-1001. Epub 2019 Jan 4.

Program in Cancer Cell Biology, Department of Biochemistry, West Virginia University, Morgantown, West Virginia.

Malregulation of the actin cytoskeleton enhances tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin-related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in head and neck squamous cell carcinoma (HNSCC) and other cancers, corresponding with elevated tumor progression and poor patient outcome. Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. Although cortactin-mediated activation of Arp2/3 complex and invadopodia regulation has been well established, signaling pathways responsible for governing cortactin binding to Arp2/3 are unknown and potentially present a new avenue for anti-invasive therapeutic targeting. Here we identify casein kinase (CK) 2α phosphorylation of cortactin as a negative regulator of Arp2/3 binding. CK2α directly phosphorylates cortactin at a conserved threonine (T24) adjacent to the canonical Arp2/3 binding motif. Phosphorylation of cortactin T24 by CK2α impairs the ability of cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2α phosphorylation-null cortactin mutants, shRNA-mediated CK2α knockdown, and with the CK2α inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2α-mediated cortactin phosphorylation at T24 is critical in regulating cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. IMPLICATIONS: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion. http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445698PMC
April 2019

Permeability across a novel microfluidic blood-tumor barrier model.

Fluids Barriers CNS 2017 Jan 23;14(1). Epub 2017 Jan 23.

Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University HSC, 1 Medical Center Dr., Morgantown, WV, 26506, USA.

Background: The lack of translatable in vitro blood-tumor barrier (BTB) models creates challenges in the development of drugs to treat tumors of the CNS and our understanding of how the vascular changes at the BBB in the presence of a tumor.

Methods: In this study, we characterize a novel microfluidic model of the BTB (and BBB model as a reference) that incorporates flow and induces shear stress on endothelial cells. Cell lines utilized include human umbilical vein endothelial cells co-cultured with CTX-TNA2 rat astrocytes (BBB) or Met-1 metastatic murine breast cancer cells (BTB). Cells were capable of communicating across microfluidic compartments via a porous interface. We characterized the device by comparing permeability of three passive permeability markers and one marker subject to efflux.

Results: The permeability of Sulforhodamine 101 was significantly (p < 0.05) higher in the BTB model (13.1 ± 1.3 × 10, n = 4) than the BBB model (2.5 ± 0.3 × 10, n = 6). Similar permeability increases were observed in the BTB model for molecules ranging from 600 Da to 60 kDa. The function of P-gp was intact in both models and consistent with recent published in vivo data. Specifically, the rate of permeability of Rhodamine 123 across the BBB model (0.6 ± 0.1 × 10, n = 4), increased 14-fold in the presence of the P-gp inhibitor verapamil (14.7 ± 7.5 × 10, n = 3) and eightfold with the addition of Cyclosporine A (8.8 ± 1.8 × 10, n = 3). Similar values were noted in the BTB model.

Conclusions: The dynamic microfluidic in vitro BTB model is a novel commercially available model that incorporates shear stress, and has permeability and efflux properties that are similar to in vivo data.
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http://dx.doi.org/10.1186/s12987-017-0050-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5260004PMC
January 2017

Src binds cortactin through an SH2 domain cystine-mediated linkage.

J Cell Sci 2012 Dec 24;125(Pt 24):6185-97. Epub 2012 Oct 24.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA.

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.
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http://dx.doi.org/10.1242/jcs.121046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585525PMC
December 2012
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