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Allenstown Elementary School, NH, United States

DeCastro A.J.,Program in Experimental and Molecular Medicine | Dunphy K.A.,University of Massachusetts Amherst | Hutchinson J.,Program in Experimental and Molecular Medicine | Balboni A.L.,Program in Experimental and Molecular Medicine | And 2 more authors.
Cell Death and Disease | Year: 2013

During reproductive life, the mammary epithelium undergoes consecutive cycles of proliferation, differentiation and apoptosis. Doing so relies on the retained proliferative capacity, prolonged lifespan and developmental potency of mammary stem cells (MaSCs). ΔNp63α, the predominant TP63 isoform in mammary epithelia, is robustly expressed in MaSCs and is required for preservation of self-renewing capacity in diverse epithelial structures. However, the mechanism(s) underlying subversion of this activity during forfeiture of self-renewing capacity are poorly understood. MicroRNAs (miRNAs) govern critical cellular functions including stem cell maintenance, development, cell cycle regulation and differentiation by disrupting translation of target mRNAs. Data presented here indicate that expression of miR203, a miRNA that targets ΔNp63α and DNp63b is activated during luminal epithelial differentiation and that this pattern is observed in the murine mammary hierarchy. In addition, we present evidence that the transcription factor Zeb1 represses miR203 expression, thus enhancing ΔNp63α protein levels. Furthermore, ectopic miR203 suppresses ΔNp63α expression, proliferation and colony formation. The anti-clonogenic effects mediated by miR203 require suppression of ΔNp63α. In addition, ectopic miR203 promotes mesenchymal-to-epithelial transition and disrupts activities associated with epithelial stem cells. These studies support a model in which induction of miR203 mediates forfeiture of self-renewing capacity via suppression of ΔNp63α and may also have anti-tumorigenic activity through its reduction of EMT and cancer stem cell populations. © 2013 Macmillan Publishers Limited All rights reserved.

Stan R.,Heart and Vascular Research Center | Stan R.,Norris Cotton Cancer Center | Smits N.,Heart and Vascular Research Center | Buitendijk M.,Program in Experimental and Molecular Medicine | And 8 more authors.
Developmental Cell | Year: 2012

Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding. Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition. Plasmalema vesicle-associated protein (PV1) is critical for the formation of diaphragms in endothelial caveolae, fenestrae, and transendothelial channels. Using mice with loss and gain of PV1 function, Stan et al. show that the diaphragms of fenestrae are critical for the control of basal permeability, blood composition, and survival. © 2012 Elsevier Inc.

Osipovitch D.C.,Program in Experimental and Molecular Medicine | Parker A.S.,Dartmouth | Makokha C.D.,Trinity Partners LLC | Desrosiers J.,University of Rhode Island | And 2 more authors.
Protein Engineering, Design and Selection | Year: 2012

The unparalleled specificity and activity of therapeutic proteins has reshaped many aspects of modern clinical practice, and aggressive development of new protein drugs promises a continued revolution in disease therapy. As a result of their biological origins, however, therapeutic proteins present unique design challenges for the biomolecular engineer. For example, protein drugs are subject to immune surveillance within the patient's body; this anti-drug immune response can compromise therapeutic efficacy and even threaten patient safety. Thus, there is a growing demand for broadly applicable protein deimmunization strategies. We have recently developed optimization algorithms that integrate computational prediction of T-cell epitopes and bioinformatics-based assessment of the structural and functional consequences of epitope-deleting mutations. Here, we describe the first experimental validation of our deimmunization algorithms using Enterobacter cloacae P99 β-lactamase, a component of antibody-directed enzyme prodrug cancer therapies. Compared with wild-type or a previously deimmunized variant, our computationally optimized sequences exhibited significantly less in vitro binding to human type II major histocompatibility complex immune molecules. At the same time, our globally optimal design exhibited wild-type catalytic proficiency. We conclude that our deimmunization algorithms guide the protein engineer towards promising immunoevasive candidates and thereby have the potential to streamline biotherapeutic development. © 2012 The Author.

Balboni A.L.,Program in Experimental and Molecular Medicine | Hutchinson J.A.,Program in Experimental and Molecular Medicine | DeCastro A.J.,Program in Experimental and Molecular Medicine | Liby K.,Norris Cotton Cancer Center | And 9 more authors.
Cancer Research | Year: 2013

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of regenerative stasis within diverse epithelial tissues. In squamous carcinomas, TP63 is commonly amplified, and ΔNp63a confers a potent survival advantage. Genome-wide occupancy studies show that ΔNp63 promotes bidirectional target gene regulation by binding more than 5,000 sites throughout the genome; however, the subset of targets mediating discreet activities of TP63 remains unclear. We report that ΔNp63a activates bone morphogenic proteins (BMP) signaling by inducing the expression of BMP7. Immunohistochemical analysis indicates that hyperactivation of BMP signaling is common in human breast cancers, most notably in the basal molecular subtype, as well as in several mouse models of breast cancer. Suppression of BMP signaling in vitro with LDN193189, a small-molecule inhibitor of BMP type I receptor kinases, represses clonogenicity and diminishes the cancer stem cell-enriched ALDH1+ population. Importantly, LDN193189 blocks reconstitution of mixed ALDH1+/ALDH1- cultures indicating that BMP signaling may govern aspects of cellular plasticity within tumor hierarchies. These results show that BMP signaling enables reversion of committed populations to a stem-like state, potentially supporting progression and maintenance of tumorigenesis. Treatment of a mouse model of breast cancer with LDN193189 caused reduced expression of markers associated with epithelial-to-mesenchymal transition (EMT). Furthermore, in vivo limiting dilution analysis assays revealed that LDN193189 treatment suppressed tumor-initiating capacity and increased tumor latency. These studies support a model in which ΔNp63a-mediated activation of BMP signaling governs epithelial cell plasticity, EMT, and tumorigenicity during breast cancer initiation and progression. © 2012 AACR.

Balboni A.L.,Program in Experimental and Molecular Medicine | DeCastro A.J.,Program in Experimental and Molecular Medicine | Cheng C.,Norris Cotton Cancer Center | DiRenzo J.,Norris Cotton Cancer Center
Molecular Cancer Research | Year: 2015

The TGFβ superfamily regulates a broad range of cellular processes, including proliferation, cell-fate specification, differentiation, and migration. Molecular mechanisms underlying this high degree of pleiotropy and cell-type specificity are not well understood. The TGFβ family is composed of two branches: (i) TGFβs, activins, and nodals, which signal through SMAD2/3, and (ii) bone morphogenetic proteins (BMP), which signal through SMAD1/5/8. SMADs have weak DNA-binding affinity and rely on coactivators and corepressors to specify their transcriptional outputs. This report reveals that p53 and ΔNp63α act as transcriptional partners for SMAD proteins and thereby influence cellular responses to TGFβ and BMPs. Suppression of p53 or overexpression of ΔNp63α synergistically enhance BMP-induced transcription. Mechanistically, p53 and ΔNp63α physically interact with SMAD1/5/8 proteins and co-occupy the promoter region of inhibitor of differentiation (ID2), a prosurvival BMP target gene. Demonstrating further convergence of these pathways, TGFβinduced canonical BMP regulated transcription in a ΔNp63αand p53-dependent manner. Furthermore, bioinformatic analyses revealed that SMAD2/3 and ΔNp63α coregulate a significant number of transcripts involved in the regulation of epithelial-tomesenchymal transition. Thus, p53 and ΔNp63α are transcriptional partners for a subset of TGFβ-and BMP-regulated SMAD target genes in the mammary epithelium. Collectively, these results establish an integrated gene network of SMADs, p53, and ΔNp63α that contribute to EMT and metastasis. Implications: This study identifies aberrant BMP activation as a result of p53 mutation or ΔNp63α expression. Mol Cancer Res; 13(4); 732-42. © 2015 AACR.

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