Gabriel Fenteany

Lactacystin and clasto-Lactacystin β-Lactone Modify Multiple Proteasome β-Subunits and Inhibit Intracellular Protein Degradation and Major Histocompatibility Complex Class I Antigen Presentation

The antibiotic lactacystin was reported to covalently modify β-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [3H]lactacystin treatment modifies all the proteasome’s catalytic β-subunits. Lactacystin and its more potent derivative β-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short- and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the β-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-γ-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all β-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition.

Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets

BACKGROUND Sheets of cells move together as a unit during wound healing and embryonic tissue movements, such as those occurring during gastrulation and neurulation. We have used epithelial wound closure as a model system for such movements and examined the mechanisms of closure and the importance of the Rho family of Ras-related small GTPases in this process. RESULTS Wounds induced in Madin-Darby canine kidney (MDCK) epithelial cell monolayers close by Rac- and phosphoinositide-dependent cell crawling, with formation of lamellipodia at the wound margin, and not by contraction of a perimarginal actomyosin purse-string. Although Rho-dependent actin bundles usually form at the margin, neither Rho activity nor formation of these structures is required for wound closure to occur at a normal rate. Cdc42 activity is also not required for closure. Inhibition of Rho or Cdc42 results, however, in statistically significant decreases in the regularity of wound closure, as determined by the ratio of wound margin perimeter over the remaining denuded area at different times. The Rac-dependent force generation for closure is distributed over several rows of cells from the wound margin, as inhibition of motility in the first row of cells alone does not inhibit closure and can be compensated for by generation of motile force in cells behind the margin. Furthermore, we observed high levels of Rac-dependent actin assembly in the first few rows of cells from the wound margin. CONCLUSIONS Wounds in MDCK cell sheets do not close by purse-string contraction but by a crawling behavior involving Rac, phosphoinositides and active movement of multiple rows of cells. This finding suggests a new distributed mode of signaling and movement that, nevertheless, resembles individual cell motility. Although Rho and Cdc42 activities are not required for closure, they have a role in determining the regularity of closure.

Polycomb Protein EZH2 Regulates Tumor Invasion via the Transcriptional Repression of the Metastasis Suppressor RKIP in Breast and Prostate Cancer

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Cell Surface Actin Remodeling

Actin filament remodeling at cell surfaces is a fundamental aspect of cellular life. Except for minor sequence variations, actin proteins are structurally identical. They have similar self-association properties and ATPase activities that accommodate their assembly into polarized semi-flexible

Raf kinase inhibitor protein suppresses nuclear factor-κB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression

Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor-κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIPs function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.

Diversity through a branched reaction pathway: generation of multicyclic scaffolds and identification of antimigratory agents.

A library of 91 heterocyclic compounds composed of 16 distinct scaffolds has been synthesized through a sequence of phosphine-catalyzed ring-forming reactions, Tebbe reactions, Diels-Alder reactions, and, in some cases, hydrolysis. This effort in diversity-oriented synthesis produced a collection of compounds that exhibited high levels of structural variation both in terms of stereochemistry and the range of scaffolds represented. A simple but powerful sequence of reactions thus led to a high-diversity library of relatively modest size with which to explore biologically relevant regions of chemical space. From this library, several molecules were identified that inhibit the migration and invasion of breast cancer cells and may serve as leads for the development of antimetastatic agents.

A Non-Antibacterial Oxazolidinone Derivative that Inhibits Epithelial Cell Sheet Migration

We have developed a high‐throughput assay for screening chemical libraries for compounds that affect cell sheet migration during wound closure in epithelial cell monolayers. By using this assay, we have discovered a new inhibitor of cell sheet migration. This compound (UIC‐1005) is a 3,4‐disubstituted oxazolidinone that bears an electrophilic α,β‐unsaturated N‐acyl group required for activity. UIC‐1005 also inhibits growth in an epithelial cell proliferation assay. The molecule does not display general toxicity at concentrations at which it potently inhibits cell sheet migration and growth. Unlike certain 3,5‐disubstituted oxazolidinones, it exhibits no antibacterial activity. UIC‐1005 therefore represents a new class of bioactive oxazolidinone derivative that may prove useful as a probe for signaling pathways leading to cell motility.

Selective deprotection of either alkyl or aryl silyl ethers from aryl, alkyl bis-silyl ethers

Abstract A pair of complementary methods was developed using CeCl 3 ·7H 2 O/CH 3 CN and LiOH/DMF to selectively deprotect alkyl and aryl silyl ethers, respectively, from the corresponding bis-silyl ethers in excellent yields.

Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics.

Background Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton. Methodology/Principal Findings We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I. Conclusions/Significance Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.

Raf kinase inhibitor protein positively regulates cell–substratum adhesion while negatively regulating cell–cell adhesion

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin‐Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E‐cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO‐1, while expression of the cell–substratum adhesion protein β1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three‐dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP‐overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference‐based silencing of RKIP expression results in decreased cell–substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell–substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch‐wound assay, although not as pronounced as that previously reported for RKIP‐knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell–substratum adhesion while negatively controlling cell–cell adhesion, and underscore the complex functions of RKIP in cell physiology. J. Cell. Biochem. 103: 972–985, 2008.