Pamela Austin

Toward [18F]-labeled aryltrifluoroborate radiotracers: in vivo positron emission tomography imaging of stable aryltrifluoroborate clearance in mice.

The use of a boronic ester as a captor of aqueous [(18)F]-fluoride has been previously suggested as a means of labeling biomolecules in one step for positron emission tomography (PET) imaging. For this approach to be seriously considered, the [(18)F]-labeled trifluoroborate should be humorally stable such that it neither leaches free [(18)F]-fluoride to the bone nor accumulates therein. Herein, we have synthesized a biotinylated boronic ester that is converted to the corresponding trifluoroborate salt in the presence of aqueous [(18)F]-fluoride. In keeping with its in vitro aqueous kinetic stability at pH 7.5, the trifluoroborate appears to clear in vivo quite rapidly to the bladder as the stable trifluoroborate salt with no detectable leaching of free [(18)F]-fluoride to the bone. When this labeled biotin is preincubated with avidin, the pharmacokinetic clearance of the resulting complex is visibly altered. This work validates initial claims that boronic esters are potentially useful as readily labeled precursors to [(18)F]-PET reagents.

Preventing the Activation or Cycling of the Rap1 GTPase Alters Adhesion and Cytoskeletal Dynamics and Blocks Metastatic Melanoma Cell Extravasation into the Lungs

The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin-rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM.

Chemical Synthesis and Biological Activity of the Neopetrosiamides and Their Analogues: Revision of Disulfide Bond Connectivity

Neopetrosiamides A and B (2) from the marine sponge Neopetrosia sp. are two diastereomeric tricyclic peptides that inhibit tumor cell invasion associated with metastasis. The reported structures were chemically synthesized using solid-phase peptide synthesis and sequential stepwise disulfide bond formation in solution. The disulfide bond connectivity of the originally proposed structures was revised and confirmed by chemical synthesis together with a combination of HPLC analysis, disulfide mapping, and biological activity testing. This methodology was also utilized to generate analogues containing methionine or norleucine in place of the methionine sulfoxide at position 24. Compounds 4 and 6 demonstrated potent bioactivity comparable to that of the parent peptides.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface.

The cell surface mucin podocalyxin regulates collective breast tumor budding.

BackgroundOverexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression.MethodsMCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice.ResultsForced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture.ConclusionsPodocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.

The Invasion Inhibitor Sarasinoside A1 Reverses Mesenchymal Tumor Transformation in an E-Cadherin–Independent Manner

During metastatic progression, an aberrant epithelial-to-mesenchymal transformation (EMT) that is most often driven by the loss of the cell–cell adhesion molecule E-cadherin generates noncohesive tumor cells that are highly invasive. We used mesenchymally transformed, E-cadherin–negative MDA-MB-231 breast carcinoma cells in a natural product screen and determined that the triterpenoid saponin sarasinoside A1 inhibited their invasion and the invasion of a number of other tumor cell lines. Sarasinoside A1 also caused MDA-MB-231 cells to become cohesive in a three-dimensional basement membrane and collagen gel cultures. In two-dimensional culture, sarasinoside A1 initiated a morphologic re-epithelialization of MDA-MB-231 cells wherein preexisting nonepithelial cadherins and the junction-associated proteins β-catenin and ZO-1 all relocalized to sites of cell–cell contact. In addition, the intercellular space between neighboring cells narrowed considerably, the stability of polymerized actin at cell-cell contact sites increased, and there was a recruitment and stabilization of nectin-based adhesion complexes to these sites, all of which strongly suggested that functional cell–cell junctions had formed. Importantly, sarasinoside A1 induced nascent cell–cell junction formation that did not require changes in gene expression and was not associated with an induction of E-cadherin but resulted in increased activation of Rap GTPases. Therefore, our findings with sarasinoside A1 suggest that it may be possible to re-epithelialize metastatic tumor cells with phenotypic consequence even when E-cadherin is completely absent. Mol Cancer Res; 11(5); 530–40. ©2013 AACR.

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor

ABSTRACT Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo. Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated. Summary: Tension applied through 3D matrices directs invasion of tumor cells along the tensional axis. Rap1 senses the tension and polarizes the cytoskeletal and subsequent adhesion remodeling.

Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

ABSTRACT A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C–COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain complexes required for their proliferation. Summary: Altered metabolism in tumours creates metabolic acid stress in tumour cells, which is a target for chemotherapeutics. We identify six new complexes with roles in resistance to metabolic acid stress.

The small molecule genkwanine M induces single mode, mesenchymal tumor cell motility.

Individual tumor cells utilize one of two modes of motility to invade the extracellular matrix, mesenchymal or amoeboid. We have determined that the diterpenoid genkwanine M (GENK) enhances the mesenchymal mode of cell motility that is intrinsic to HT-1080 osteosarcoma cells, stimulates a mesenchymal mode of motility in stationary MDA-MB-453 breast carcinoma cells, and induces a shift to a mesenchymal mode of cell motility in LS174T colorectal adenocarcinoma cells that normally utilize the alternate amoeboid mode of motility. The ability of GENK to stimulate or induce mesenchymal motility was preceded by a rapid cell spreading, elongation and polarization that did not require new gene expression. However, these initial morphologic changes were integrin dependent and they were associated with a reorganization of focal contacts and focal adhesions as well as an activation of the focal adhesion kinase. Therefore, GENK induces a mesenchymal mode of cell motility in a wide variety of tumor cell types that may be mediated, at least in part, by an activation of integrin-associated signaling.