Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells

Bulut, Güllay; Hong, Saahoon; Chen, K.; Beauchamp, Em M.; Rahim, Said; Kosturko, Gw W.; Glasgow, Eric; Dakshanamurthy, Sivanesan; Lee, H.-S.; Daar, Ira; Toretsky, Ja A.; Khanna, Chand; Üren, Aykut

doi:10.1038/onc.2011.245

Cited by 149 publications

(159 citation statements)

References 40 publications

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“…A critical threonine residue is present in the C terminus of each of the ERM family members that regulates the interaction with f-actin, and the N-terminal FERM (Band4.1, Ezrin, Radixin, Moesin) domain regulates an interaction with MTs (22). A quinolone-based compound (NSC668394) was previously developed to bind this threonine and prevent its phosphorylation, disrupting f-actin binding (23). Addition of NSC668394 at 50 μM to the dynamic protocol (during the no chemoattractant phase) resulted in a reduction of directional memory from 90% to 60% (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Although moesin exhibited the long polarization time scale indicative of a directional memory (exhibiting faster decay and delay in depolarization), inhibition via the small quinolone molecule did not change moesin dynamics but did interrupt memory. The action of NSC668394 is to block T558 phosphorylation on moesin and decouple moesin from f-actin (23). This decoupling may accelerate the depolarization of the actual memory element.…”

Section: Discussionmentioning

confidence: 99%

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Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Prentice-Mott

Meroz

Carlson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a longlived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstream of RhoA (Ras homolog gene family, member A), stabilized moesin and directional memory while depolymerization of microtubules (MTs) disoriented moesin deposition and also reduced directional memory. Our study reveals that long-lived polarized cytoskeletal structures, specifically moesin, actomyosin, and MTs, provide a directional memory in neutrophil-like cells even as they respond on short time scales to external chemical cues. . In particular, chemical cues activate signaling at the cell surface and are integrated within the cell cytoplasm to give rise to a polarization in the direction of the local gradient. Although these processes have been the subject of detailed experimental study and theoretical and computational modeling, the mechanisms by which this orientation is achieved and maintained in the face of environmental noise remains incomplete.Although migration might be thought of as being very sensitive to the variations in the external environment, instead, we see robust migration in a variety of fluctuating environments. These observations all point to the existence of a directional memory in chemotactic cells-a biochemical pathway that stores information about cellular orientation and prevents the loss of orientation in the face of fluctuations, transient loss of polarization, or saturation of the receptors. For example, recent work has demonstrated memory-based behavior in Dictyostelium amoebae under fluctuating waves of chemoattractant (6, 7), although the authors do not identify potential molecular elements that store this information.Here, we use microchannel-based microfluidic devices to observe cell polarization and movement in confined mammalian neutrophil-like cells. Cells in this environment exhibit a strong bias to repolarize in the previous direction of motion after a period of depolarization. This memory is time-dependent and decays when the cell is unstimulated. To describe these results, we construct a minimal phenomenological model coupling membrane and cytoskeletal polarization lifetimes and show that thi...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Prentice-Mott

Meroz

Carlson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…These results support the important role of ROCKmediated phosphorylation of ezrin on Threonine567 in the biological activities of ezrin as also reported in other types of cancer cells. 18,19 As the induction of CRC metastasis to the liver by L1 is mediated by ezrin and NF-kB signaling, 9 we wished to determine the target genes involved in this process. We used global gene expression analyses to identify genes that are induced by L1 and whose levels are suppressed in L1-transfected cells by shRNA to ezrin.…”

Section: Discussionmentioning

confidence: 99%

Global analysis of L1-transcriptomes identified IGFBP-2 as a target of ezrin and NF-κB signaling that promotes colon cancer progression

et al. 2012

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1L1, a neuronal cell adhesion receptor of the immunoglobulin-like protein family is expressed in invading colorectal cancer (CRC) cells as a target gene of Wnt/b-catenin signaling. Overexpression of L1 in CRC cells enhances cell motility and proliferation, and confers liver metastasis. We recently identified ezrin and the IkB-NF-kB pathway as essential for the biological properties conferred by L1 in CRC cells. Here, we studied the underlying molecular mechanisms and found that L1 enhances ezrin phosphorylation, via Rho-associated protein kinase (ROCK), and is required for L1-ezrin co-localization at the juxtamembrane domain and for enhancing cell motility. Global transcriptomes from L1-expressing CRC cells were compared with transcriptomes from the same cells expressing small hairpin RNA (shRNA) to ezrin. Among the genes whose expression was elevated by L1 and ezrin we identified insulin-like growth factor-binding protein 2 (IGFBP-2) and showed that its increased expression is mediated by an NF-kB-mediated transactivation of the IGFBP-2 gene promoter. Expression of a constitutively activated mutant ezrin (Ezrin567D) could also increase IGFBP-2 levels in CRC cells. Overexpression of IGFBP-2 in CRC cells lacking L1-enhanced cell proliferation (in the absence of serum), cell motility, tumorigenesis and induced liver metastasis, similar to L1 overexpression. Suppression of endogenous IGFBP-2 in L1-transfected cells inhibited these properties conferred by L1. We detected IGFBP-2 in a unique organization at the bottom of human colonic crypts in normal mucosa and at increased levels throughout human CRC tissue samples co-localizing with the phosphorylated p65 subunit of NF-kB. Finally, we found that IGFBP-2 and L1 can form a molecular complex suggesting that L1-mediated signaling by the L1-ezrin-NF-kB pathway, that induces IGFBP-2 expression, has an important role in CRC progression.Oncogene ( In recent studies, we identified members of the immunoglobulin-like cell adhesion receptors (Nr-CAM and L1), mostly known for their function in nerve cells, 3,4 but also in many cancer cell types, 5 as target genes of b-catenin-TCF signaling in CRC cells. 6,7 We detected L1 in a subpopulation of cells at the invasive front of CRC tissue displaying nuclear b-catenin localization. 7 We further found that the expression of L1 in human CRC cells, lacking endogenous L1, confers enhanced motility and metastasis to the liver in a mouse metastasis model.

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“…Because ERM phosphorylation is drastically reduced upon LOK cleavage by caspases, we speculated that this dephosphorylation could play an active role during cell death. A selective inhibitor of ezrin, able to directly bind its target (21), induced a dose-dependent decrease of ERM phosphorylation. Interestingly, ezrin dephosphorylation is associated with induction of T-cell line apoptosis, suggesting that dephosphorylation of ERM could play a direct role in the regulation of apoptotic cell death.…”

Section: Discussionmentioning

confidence: 98%

“…To assess the involvement of ERM phosphorylation during T cell apoptosis, we took advantage of a cell-permeable ezrin inhibitor developed recently that is able to bind directly to ezrin and to inhibit its phosphorylation at Thr-567 (21). We first demonstrated that the inhibitor of ezrin NSC668394 inhibited, in a dose-dependent manner, the ERM phosphorylation in Jurkat cells, as analyzed by flow cytometry (Fig.…”

Section: The P50 N-lok Fragment Generated During Apoptosis Is Anmentioning

confidence: 99%

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis

Leroy

Belkina

Long

et al. 2016

Journal of Biological Chemistry

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The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK ؊/؊ and LOK ؉/؊ lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments.

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Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells

Cited by 149 publications

References 40 publications

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Global analysis of L1-transcriptomes identified IGFBP-2 as a target of ezrin and NF-κB signaling that promotes colon cancer progression

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis

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