Prostate Cancer Metastases Are Strongly Inhibited by Agonistic Epha2 Ligands in an Orthotopic Mouse Model.

The EphA2 tyrosine kinase receptor is highly expressed in several types of solid tumors. In our recent studies, we targeted EphA2 in pancreatic cancer with agonistic agents and demonstrated that suppression of EphA2 significantly reduced cancer-cell migration in cell-based assays. In the present study, we focused on targeting EphA2 in prostate cancer.

Stability and Cell Permeability of Sulfonyl Fluorides in the Design of Lys-Covalent Antagonists of Protein-Protein Interactions.

Recently we reported on aryl-fluorosulfates as possible stable and effective electrophiles for the design of lysine covalent, cell permeable antagonists of protein-protein interactions (PPIs). Here we revisit the use of aryl-sulfonyl fluorides as Lys-targeting moieties, incorporating these electrophiles in XIAP (X-linked inhibitor of apoptosis protein) targeting agents. We evaluated stability in buffer and reactivity with Lys311 of XIAP of various aryl-sulfonyl fluorides using biochemical and biophysical approaches, including displacement assays, mass spectrometry, SDS gel electrophoresis, and denaturation thermal shift measurements.

The added value of CT virtual angioscopy to MDCT angiography in the evaluation of aortic diseases.

Purpose: Incorporation of virtual angioscopy (VA) in the diagnostic work-up of aortic diseases could improve the clinical value and efficiency of multidetector computed tomography angiography (MDCTA). We aim to evaluate the clinical usefulness of virtual aortic navigation by CT angiography in various aortic diseases as a complement to standard MDCTA.

Materials And Methods: We retrospectively selected 211 patients who performed MDCTA for suspected or operated aortic diseases.

Therapeutic Targeting of Pancreatic Cancer via EphA2 Dimeric Agonistic Agents.

Recently, we reported on potent EphA2 targeting compounds and demonstrated that dimeric versions of such agents can exhibit remarkably increased agonistic activity in cellular assays compared to the monomers. Here we further characterize the activity of dimeric compounds at the structural, biochemical, and cellular level. In particular, we propose a structural model for the mechanism of receptor activation by dimeric agents and characterize the effect of most potent compounds in inducing EphA2 activation and degradation in a pancreatic cancer cell line.

N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity.

Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy.

We have recently investigated the reactivity of aryl-fluorosulfates as warheads to form covalent adducts with Lys, Tyr, and His residues. However, the rate of reaction of aryl-fluorosulfates seemed relatively slow, putting into question their effectiveness to form covalent adducts in cell. Unlike the previously reported agents that targeted a relatively remote Lys residue with respect to the target's binding site, the current agents were designed to more directly juxtapose an aryl-fluorosulfate with a Lys residue that is located within the binding pocket of the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP).

Covalent Inhibitors of Protein-Protein Interactions Targeting Lysine, Tyrosine, or Histidine Residues.

We have recently reported a series of Lys-covalent agents targeting the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) using a benzamide-sulfonyl fluoride warhead. Using XIAP as a model system, we further investigated a variety of additional warheads that can be easily incorporated into binding peptides and analyzed their ability to form covalent adducts with lysine and other amino acids, including tyrosine, histidine, serine, and threonine, using biochemical and biophysical assays. Moreover, we tested aqueous, plasma stability, cell permeability, and cellular efficacy of the most effective agents.

Structure-Based Design of Novel EphA2 Agonistic Agents with Nanomolar Affinity in Vitro and in Cell.

EphA2 overexpression is invariably associated with poor prognosis and development of aggressive metastatic cancers in pancreatic, prostate, lung, ovarian, and breast cancers and melanoma. Recent efforts from our laboratories identified a number of agonistic peptides targeting the ligand-binding domain of the EphA2 receptor. The individual agents, however, were still relatively weak in affinities (micromolar range) that precluded detailed structural studies on the mode of action.

Design of Potent pan-IAP and Lys-Covalent XIAP Selective Inhibitors Using a Thermodynamics Driven Approach.

Recently we reported that rapid determination of enthalpy of binding can be achieved for a large number of congeneric agents or in combinatorial libraries fairly efficiently. We show that using a thermodynamic Craig plot can be very useful in dissecting the enthalpy and entropy contribution of different substituents on a common scaffold, in order to design potent, selective, or pan-active compounds. In our implementation, the approach identified a critical Lys residue in the BIR3 domain of XIAP.

Reduction of Circulating Cancer Cells and Metastases in Breast-Cancer Models by a Potent EphA2-Agonistic Peptide-Drug Conjugate.

EphA2 overexpression has been associated with metastasis in multiple cancer types, including melanomas and ovarian, prostate, lung, and breast cancers. We have recently proposed the development of peptide-drug conjugates (PDCs) using agonistic EphA2-targeting agents, such as the YSA peptide or its optimized version, 123B9. Although our studies indicated that YSA- and 123B9-drug conjugates can selectively deliver cytotoxic drugs to cancer cells in vivo, the relatively low cellular agonistic activities (i.

Potent and Selective EphA4 Agonists for the Treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Recent studies identified the receptor tyrosine kinase EphA4 as a disease-modifying gene that is critical for the progression of motor neuron degeneration. We report on the design and characterization of a family of EphA4 targeting agents that bind to its ligand binding domain with nanomolar affinity.

hBfl-1/hNOXA Interaction Studies Provide New Insights on the Role of Bfl-1 in Cancer Cell Resistance and for the Design of Novel Anticancer Agents.

Upregulation of antiapoptotic Bcl-2 proteins in certain tumors confers cancer cell resistance to chemotherapy or radiations. Members of the antiapoptotic Bcl-2 proteins, including Bcl-2, Mcl-1, Bcl-xL, Bcl-w, and Bfl-1, inhibit apoptosis by selectively binding to conserved α-helical regions, named BH3 domains, of pro-apoptotic proteins such as Bim, tBid, Bad, or NOXA. Five antiapoptotic proteins have been identified that interact with various selectivity with BH3 containing pro-apoptotic counterparts.

Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay.

Small-molecule inhibitors of DNA repair pathways are being intensively investigated as primary and adjuvant chemotherapies. We report the discovery that cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, are potent inhibitors of DNA double-strand break (DSB) repair. Our data suggest that cardiac glycosides interact with phosphorylated mediator of DNA damage checkpoint protein 1 (phospho-MDC1) or E3 ubiquitin-protein ligase ring finger protein 8 (RNF8), two factors involved in DSB repair, and inhibit the retention of p53 binding protein 1 (53BP1) at the site of DSBs.

WNT/β-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues.

Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing.

Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multiple sub-pathways, some of which still remain poorly understood.

Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.

Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA-damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radiosensitization.

Cigarette smoke metabolically promotes cancer, via autophagy and premature aging in the host stromal microenvironment.

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored.

Creating a tumor-resistant microenvironment: cell-mediated delivery of TNFα completely prevents breast cancer tumor formation in vivo.

Here, we provide the necessary proof of concept, that it is possible to metabolically create a non-permissive or "hostile" stromal microenvironment, which actively prevents tumor engraftment in vivo. We developed a novel genetically engineered fibroblast cell line that completely prevents tumor formation in mice, with a 100% protection rate. No host side effects were apparent.

Mitochondria "fuel" breast cancer metabolism: fifteen markers of mitochondrial biogenesis label epithelial cancer cells, but are excluded from adjacent stromal cells.

Here, we present new genetic and morphological evidence that human tumors consist of two distinct metabolic compartments. First, re-analysis of genome-wide transcriptional profiling data revealed that > 95 gene transcripts associated with mitochondrial biogenesis and/or mitochondrial translation were significantly elevated in human breast cancer cells, as compared with adjacent stromal tissue. Remarkably, nearly 40 of these upregulated gene transcripts were mitochondrial ribosomal proteins (MRPs), functionally associated with mitochondrial translation of protein components of the OXPHOS complex.

Mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth and confers autophagy resistance.

Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would "fuel" enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) overexpressing PGC-1α and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC-1α and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy resistance under conditions of starvation and increased tumor growth by up to ~3-fold.

Downregulation of stromal BRCA1 drives breast cancer tumor growth via upregulation of HIF-1α, autophagy and ketone body production.

Our recent studies have mechanistically demonstrated that cancer-associated fibroblasts (CAFs) produce energy-rich metabolites that functionally support the growth of cancer cells. Also, several authors have demonstrated that DNA instability in the tumor stroma greatly contributes to carcinogenesis. To further test this hypothesis, we stably knocked-down BRCA1 expression in human hTERT-immortalized fibroblasts (shBRCA1) using an shRNA lentiviral approach.

Two-compartment tumor metabolism: autophagy in the tumor microenvironment and oxidative mitochondrial metabolism (OXPHOS) in cancer cells.

Previously, we proposed a new paradigm to explain the compartment-specific role of autophagy in tumor metabolism. In this model, autophagy and mitochondrial dysfunction in the tumor stroma promotes cellular catabolism, which results in the production of recycled nutrients. These chemical building blocks and high-energy "fuels" would then drive the anabolic growth of tumors, via autophagy resistance and oxidative mitochondrial metabolism in cancer cells.

Caveolin-1 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition.

Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we over-expressed Cav-1 in a pancreatic cancer cell line, Panc 10.

Involvement of osteopontin in the matrix-degrading and proangiogenic changes mediated by nicotine in pancreatic cancer cells.

Background: Substantial evidence indicates that exposure to cigarette smoke is associated with an elevated risk of pancreatic ductal adenocarcinoma (PDA). However, the mechanisms underlying the effects of nicotine on the development or progression of PDA remain to be investigated. Previously, we showed that nicotine promotes the expression of osteopontin c (OPNc), an isoform of OPN protein that confers on cancer cells a migratory phenotype.

Induction of monocyte chemoattractant protein-1 by nicotine in pancreatic ductal adenocarcinoma cells: role of osteopontin.

Background: Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers.