Cell Motility In Response Research Articles

Galectin-3 Increases Gastric Cancer Cell Motility by Up-regulating Fascin-1 Expression

Galectin-3 is a beta-galactoside-binding protein that increases gastric cancer cell motility in response to integrin signaling and is highly expressed in gastric tumor cells. Galectin-3 induces cytoskeletal remodeling to increase cell motility, but the mechanisms of this process are not understood. We investigated the effects of galectin-3 on fascin-1, an actin-bundling protein. We collected malignant and normal tissues from gastric cancer patients and examined the expression levels of galectin-3 and fascin-1. We silenced galectin-3 expression in human gastric cancer cell lines using small interfering RNA and lenti-viral constructs and determined the effects on fascin-1 expression, cell motility, and invasion. Malignant gastric tissues expressed high levels of galectin-3 and fascin-1, compared with normal gastric tissues. Silencing of galectin-3 resulted in altered cancer cell morphology, reduced fascin-1 expression, decreased cell motility, and reduced malignant cell invasion. Galectin-3 overexpression reversed these effects. Silencing of fascin-1 also reduced cell motility and caused changes in cell shape, as did silencing of galectin-3. Furthermore, galectin-3 silencing inhibited the interaction between glycogen synthase kinase (GSK)-3beta, beta-catenin, and T-cell factor (TCF) 4, and the binding of beta-catenin/TCF-4 to the fascin-1 promoter. Nuclear localization of GSK-3beta and beta-catenin were not detected when galectin-3 was silenced. Overexpression of mutated galectin-3 (with mutations in the GSK-3beta binding and phosphorylation motifs) did not increase fascin-1 levels, in contrast to overexpression of wild-type galectin-3. Galectin-3 increases cell motility by up-regulating fascin-1 expression. Galectin-3 might be a potential therapeutic target for the prevention and treatment of gastric cancer progression.

Interplay of anionic charge, poly(ethylene glycol), and iodinated tyrosine incorporation within tyrosine‐derived polycarbonates: Effects on vascular smooth muscle cell adhesion, proliferation, and motility

Regulation of smooth muscle cell adhesion, proliferation, and motility on biomaterials is critical to the performance of blood-contacting implants and vascular tissue engineering scaffolds. The goal of this study was to examine the underlying substrate-smooth muscle cell response relations, using a selection of polymers representative of an expansive library of multifunctional, tyrosine-derived polycarbonates. Three chemical components within the polymer structure were selectively varied through copolymerization: (1) the content of iodinated tyrosine to achieve X-ray visibility; (2) the content of poly(ethylene glycol) (PEG) to decrease protein adsorption and cell adhesivity; and (3) the content of desaminotyrosyl-tyrosine (DT), which regulates the rate of polymer degradation. Using quartz crystal microbalance with dissipation, we quantified differential serum protein adsorption behavior because of the chemical components DT, iodinated tyrosine, and PEG: increased PEG content within the polymer structure progressively decreased protein adsorption but the simultaneous presence of both DT and iodinated tyrosine reversed the effects of PEG. The complex interplay of these components was next tested on the adhesion, proliferation, and motility behavior cultured human aortic smooth muscle cells. The incorporation of PEG into the polymer reduced cell attachment, which was reversed in the presence of iodinated tyrosine. Further, we found that as little as 10% DT content was sufficient to negate the PEG effect in polymers containing iodinated tyrosine, whereas in non-iodinated polymers, the PEG effect on cell attachment was reversed. Cross-functional analysis of motility and proliferation revealed divergent substrate chemistry related cell response regimes. For instance, within the series of polymers containing both iodinated tyrosine and 10% of DT, increasing PEG levels lowered smooth muscle cell motility without a change in the rate of cell proliferation. In contrast, for non-iodinated tyrosine and 10% of DT, increasing PEG levels increased cell proliferation significantly while reducing cell motility. Clearly, the polycarbonate polymer library offers a sensitive platform to modulate cell adhesion, proliferation, and motility responses, which, in turn, may have implications for controlling vascular remodeling in vivo. Additionally, our data suggests unique biorelevant properties following the incorporation of iodinated subunits in a polymeric biomaterial as a potential platform for X-ray visible devices.

Zyxin Mediates Actin Fiber Reorganization in Epithelial–Mesenchymal Transition and Contributes to Endocardial Morphogenesis

Epithelial-mesenchymal transition (EMT) confers destabilization of cell-cell adhesion and cell motility required for morphogenesis or cancer metastasis. Here we report that zyxin, a focal adhesion-associated LIM protein, is essential for actin reorganization for cell migration in TGF-beta1-induced EMT in normal murine mammary gland (NMuMG) cells. TGF-beta1 induced the relocation of zyxin from focal adhesions to actin fibers. In addition, TGF-beta1 up-regulated zyxin via a transcription factor, Twist1. Depletion of either zyxin or Twist1 abrogated the TGF-beta1-dependent EMT, including enhanced cell motility and actin reorganization, indicating the TGF-beta1-Twist1-zyxin signal for EMT. Both zyxin and Twist1 were predominantly expressed in the cardiac atrioventricular canal (AVC) that undergoes EMT during heart development. We further performed ex vivo AVC explant assay and revealed that zyxin was required for the reorganization of actin fibers and migration of the endocardial cells. Thus, zyxin reorganizes actin fibers and enhances cell motility in response to TGF-beta1, thereby regulating EMT.

Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling

Switching between attractive and repulsive migration in cell movement in response to extracellular guidance cues has been found in various cell types and is an important cellular function for translocation during cellular and developmental processes. Here we show that the preferential direction of migration during electrotaxis in Dictyostelium cells can be reversed by genetically modulating both guanylyl cyclases (GCases) and the cyclic guanosine monophosphate (cGMP)-binding protein C (GbpC) in combination with the inhibition of phosphatidylinositol-3-OH kinases (PI3Ks). The PI3K-dependent pathway is involved in cathode-directed migration under a direct-current electric field. The catalytic domains of soluble GCase (sGC) and GbpC also mediate cathode-directed signaling via cGMP, whereas the N-terminal domain of sGC mediates anode-directed signaling in conjunction with both the inhibition of PI3Ks and cGMP production. These observations provide an identification of the genes required for directional switching in electrotaxis and suggest that a parallel processing of electric signals, in which multiple-signaling pathways act to bias cell movement toward the cathode or anode, is used to determine the direction of migration.

Modeling Skeletal Muscle Angiogenesis from the Molecular to the Tissue Level

We present a multiscale model of angiogenesis that integrates individual modules representing blood flow, oxygen transport, growth factor distribution and signaling, cell sensing, cell movement and cell proliferation. This integration will be used to better understand underlying biological mechanisms from the molecular level up through the organ systems level; examine the effects of pathological conditions on skeletal muscle capillary growth; and test new therapeutic strategies. Model methodology includes partial differential equations, stochastic models, complex logical rules, and agent‐based architectures. Spatial and time scales range from ligand‐receptor interactions and intracellular signaling occurring on the order of seconds and minutes, to cell‐level movement and cell‐matrix interactions over minutes to hours, to vessel formation and tissue level responses over hours to days.We introduce the individual modules and describe our methodology for integration, which involves wrapping models (written in different programming languages) in a Java‐based dynamic link library format that allows for a central controller to readily pass parameters between the models. We present results from the coordination of modules: the effects of hypoxia in skeletal muscle from the molecular‐detailed changes in HIF1‐initiated VEGF secretion and VEGF‐VEGFR signaling; to endothelial cell activation, cell movement in response to VEGF gradients, and capillary branching as a function of the Notch ligand DLL4; to the resulting new capillary network and altered pattern of blood flow. This angiogenesis multiscale modeling and integration will be a tool in rational drug development and therapeutic regime design, and encourage related angiogenesis experiments.

Live Cell Fluorescence Fluctuation Analysis Of Cdc42 Polarity Maintenance In Live Yeast

Biological processes such as development, cell motility, and immune response depend on the maintenance of robust cell polarization. Cdc42, a conserved Rho-GTPase and master regulator of cell polarization is recycled by two complementary mechanisms: actin-mediated transport/endocytosis and cytosolic recycling through the Rho-GDP dissociation inhibitor (GDI) Rdi1p. However, the molecular interactions controlling these recycling mechanisms are not well understood. Here, fluorescence correlation spectroscopy (FCS), and cross correlation spectroscopy (FCCS), along with fluorescence recovery after photobleaching (FRAP) are implemented to study recycling of Cdc42 at the polarized site and dynamics and interactions of cytosolic Cdc42p in living yeast cells.

Paxillin nuclear-cytoplasmic localization is regulated by phosphorylation of the LD4 motif: evidence that nuclear paxillin promotes cell proliferation

Paxillin, a major focal-adhesion complex component belongs to the subfamily of LIM domain proteins and participates in cell adhesion-mediated signal transduction. It is implicated in cell-motility responses upon activation of cell-surface receptors and can recruit, among others, the GIT1 [GRK (G-protein-coupled-receptor kinase)-interacting ARF (ADP-ribosylation factor) GAP (GTPase-activating protein)]-PIX [PAK (p21-activated kinase)-interacting exchange factor]-PAK1 complex. Several adhesion proteins including zyxin, Hic5 and Trip6 are also nuclear and can exert transcriptional effects. In the present study we show that endogenous paxillin shuttles between the cytoplasm and nucleus, and we have used a variety of tagged paxillin constructs to map the nuclear export signal. This region overlaps an important LD(4) motif that binds GIT1 and FAK1 (focal-adhesion kinase 1). We provide evidence that phosphorylation of Ser(272) within LD(4) blocks nuclear export, and we show that this modification also reduces GIT1, but not FAK1, binding; however, Ser(272) phosphorylation does not appear to be mediated by PAK1 as previously suggested. Expression of nuclear-localized paxillin LIM domains stimulate DNA synthesis and cell proliferation. By real-time PCR analysis we have established that overexpression of either full-length paxillin or a truncated nuclear form suppresses expression of the parental imprinted gene H19, and modulation of this locus probably affects the rate of NIH-3T3 cell proliferation.

C‐Fos accelerates hepatocyte conversion to a fibroblastoid phenotype through ERK‐mediated upregulation of paxillin‐Serine178 phosphorylation

Transforming growth factor beta (TGF-beta) exerts an important role in the late steps of carcinogenesis by cooperating with Ras to induce cell motility and tumor invasion. The transcription complex AP-1 has been implicated in the regulation of genes involved in motility and invasion, by mechanisms not yet delineated. We utilized a model of immortalized human hepatocytes (IHH) overexpressing c-Fos (IHH-Fos) or not (IHH-C) to investigate the role of c-Fos on cell motility in response to a prolonged treatment with TGF-beta, EGF or a combination of both. Cotreatment with EGF and TGF-beta, but neither cytokine alone, induced the conversion of hepatocytes to a fibroblastoid phenotype and increased their motility in Boyden chambers. EGF/TGF-beta cotreatment induced a higher effect on ERK phosphorylation compared to TGF-beta treatment alone. It also induced an increase in total and phosphorylated Ser(178) paxillin, a protein previously implicated in cell motility. This response was inhibited by two specific MEK inhibitors, indicating the involvement of the ERK pathway in paxillin activation. Overexpression of c-Fos correlated with increased cell scattering and motility, higher levels of ERK activation and phospho Ser(178) paxillin, increased levels of EGF receptor (EGF-R) mRNA and higher EGF-R phosphorylation levels following EGF/TGF-beta cotreatment. Conversely, siRNA-mediated invalidation of c-Fos delayed the appearance of fibroblastoid cells, decreased EGF-R mRNA and downregulated ERK and Ser(178) paxillin phosphorylations, indicating that c-Fos activates hepatocyte motility through an EGF-R/ERK/paxillin pathway. Since c-Fos is frequently overexpressed in hepatocarcinomas, this newly identified mechanism might be involved in the progression of hepatic tumors in vivo.

CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules

The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor κB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

A radiation damage repair model for normal tissues

A cellular Monte Carlo model describing radiation damage and repair in normal epithelial tissues is presented. The deliberately simplified model includes cell cycling, cell motility and radiation damage response (cell cycle arrest and cell death) only. Results demonstrate that the model produces a stable equilibrium system for mean cell cycle times in the range 24–96 h. Simulated irradiation of these stable equilibrium systems produced a range of responses that are shown to be consistent with experimental and clinical observation, including (i) re-epithelialization of radiation-induced lesions by a mixture of cell migration into the wound and repopulation at the periphery; (ii) observed radiosensitivity that is quantitatively consistent with both rate of induction of irreparable DNA lesions and, independently, with the observed acute oral and pharyngeal mucosal reactions to radiotherapy; (iii) an observed time between irradiation and maximum toxicity that is consistent with experimental data for skin; (iv) quantitatively accurate predictions of low-dose hyper-radiosensitivity; (v) Gomperzian repopulation for very small lesions (∼2000 cells) and (vi) a linear rate of re-epithelialization of 5–10 µm h−1 for large lesions (>15 000 cells).

Endothelial Function of von Hippel-Lindau Tumor Suppressor Gene: Control of Fibroblast Growth Factor Receptor Signaling

von Hippel-Lindau (VHL) disease results from germline and somatic mutations in the VHL tumor suppressor gene and is characterized by highly vascularized tumors. VHL mutations lead to stabilization of hypoxia-inducible factor (HIF), which up-regulates proangiogenic factors such as vascular endothelial growth factor (VEGF). This pathway is therefore believed to underlie the hypervascular phenotypes of the VHL tumors. However, recent studies have identified novel VHL functions that are independent of the HIF-VEGF pathway. In addition, a potential role of VHL in the tumor microenvironment, which carries heterozygous VHL mutations in VHL patients, has been overlooked. Here, we report a novel HIF-independent VHL function in the endothelium. VHL knockdown in primary human microvascular endothelial cells caused defective turnover of surface fibroblast growth factor (FGF) receptor, increased extracellular signal-regulated kinase signaling, and ETS1 activation, leading to increased cell motility in response to FGF and three-dimensional cord formation in vitro. HIF-alpha knockdown in VHL loss-of-function endothelial cells does not impede their elevated in vitro angiogenic activity. Importantly, the elevated angiogenic response to FGF is recapitulated in Vhl-heterozygous mice. Thus, partial loss of function of VHL in endothelium may be a contributing factor in tumor angiogenesis through a HIF-VEGF-independent mechanism.

Development and Characterization of T cell Specific Insulin Receptor Transgenic Mouse Model To Study Type‐1 Diabetes

In the nonobese diabetic (NOD) mouse, type1 diabetes (T1D) is an autoimmune disease characterized by insulitis and T cell‐mediated destruction of pancreatic islet â cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined since it was shown that IR is a chemotactic receptor capable of directing cell movement in response to insulin. Our published data shows that IR High/+ T cells aggressively transfer insulitis and diabetes while IR Low/− T cells are capable of neither. Therefore, IR may function to deliver T cells to the islet. Recently, it has been shown in NOD mice that benign insulitis can occur in the absence of T cell‐MHC/Ag interactions. The object of this work is to test whether T cells overexpressing IR, can move into the pancreas based on insulin chemotaxis alone, without being targeted to the pancreas by antigenic specificity. To that end a transgenic mouse is made wherein FLAG tagged IR expression is targeted to all T cells. If IR expression is a mechanism to move T cells into the islet then down regulation of IR expression or blocking of chemotactic IR activity specifically in T cells, will provide a new therapeutic target to block cell movement into the pancreas, thus preventing diabetes.

Analysis of cytoskeletal features in dried and living cells and slow dynamic experiments using atomic force microscopy

The ability to interact with live cells in vitro in combination with either sequential image acquisition or continuous force sensing provide the means for tracking the dynamics of intra- or inter-cell processes. The former operational mode is relatively slow and is most suitable for events where the temporal evolution takes place on a time scale of several minutes to hours. For instance, aspects of cytoskeletal dynamics, cell motility or conformational response to external stimuli fall into this category.In this study we demonstrate cell responses from exposure to cytochalasin, glutaraldehyde and a tetrazolium salt. Significant changes in mechanical properties and structural features are observed on time scales up to 3 hrs. In addition, comparative studies of cytoskeletal components of dried and living cells are undertaken.

Positive Feedback between Vascular Endothelial Growth Factor-A and Autotaxin in Ovarian Cancer Cells

Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and motogen lysophosphatidic acid (LPA). In the current study, we show a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA(4), modulates VEGF responsiveness by inducing VEGF receptor (VEGFR)-2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and lysophosphatidylcholine. These effects are accompanied by decreased LPA(4) and VEGFR2 expression as well as by increased release of soluble VEGFR1. Because LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.

Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.

Binding of Rac1, Rnd1, and RhoD to a Novel Rho GTPase Interaction Motif Destabilizes Dimerization of the Plexin-B1 Effector Domain

Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, beta-strands 3 and 4 and a short alpha-helical segment of the plexin domain. The 2.0 A resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel beta-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.

The Mechanisms Underlying Primitive Streak Formation in the Chick Embryo

Formation of the primitive streak is one of the key events in the early development of amniote embryos. The streak is the site where during gastrulation the mesendoderm cells ingress to take up their correct topographical positions in the embryo. The process of streak formation can be conveniently observed in the chick embryo, where the streak forms as an accumulation of cells in the epiblast in the posterior pole of the embryo and extends subsequently in anterior direction until it covers 80% of the epiblast. A prerequisite for streak formation is the differentiation of mesoderm, which is induced in the epiblast at the interface between the posterior Area Opaca and Area Pellucida in a sickle shaped domain overlying Koller's sickle. Current views on the molecular mechanisms of mesoderm induction by inducing signals from the Area Opaca and inhibitory signals from the hypoblast are briefly discussed. During streak formation the sickle of mesoderm cells transforms into an elongated structure in the central midline of the embryo. We discuss possible cellular mechanisms underlying this process, such as oriented cell division, cell-cell intercalation, chemotactic cell movement in response to attractive and repulsive signals and a combination of chemotaxis and contact following. We review current experimental evidence in favor and against these different hypotheses and outline some the outstanding questions. Since many of the interactions between cells signaling and moving are dynamic and nonlinear in nature they will require detailed modeling and computer simulations to be understood in detail.

P-Rex1 Links Mammalian Target of Rapamycin Signaling to Rac Activation and Cell Migration

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.

FLT3 D835/I836 mutations predict worse disease-free survival (DFS) in younger adults with cytogenetically normal acute myeloid leukemia (CN AML) without FLT3 internal tandem duplications (ITD): A Cancer and Leukemia Group B (CALGB) study

10532 Background: The adverse prognostic impact of FLT3 ITD is well-recognized in younger adults with CN AML, whereas the clinical relevance of D835/I836 mutations in the second tyrosine kinase domain of FLT3 (FLT3 TKD), that occur in 5%-14% of patients (pts), remains controversial. We evaluated the prognostic impact of FLT3 TKD, alone and with other prognostic markers, in 139 de novo CN AML adults, aged <60 years, without FLT3 ITD, and treated on similar CALGB protocols (9621 and 19808). Methods: FLT3 TKD was detected by RT-PCR/EcoRV digestion. Affymetrix U133 plus 2.0 gene expression profiles derived from FLT3 TKD+ and FLT3 wild-type (FLT3 WT) pts with available RNA were compared. Results: Relative to FLT3 WT pts, FLT3 TKD+ pts [n=16 (11.5%)] presented with higher white blood cell counts (WBC) (median: 57.2 v 14.6 × 109/L; P<.001), % bone marrow (BM) blasts (median: 75.5 v 63; P=.001), and % blood blasts (median: 62.5 v 46; P=.03). All 16 FLT3 TKD+ pts achieved complete remission versus 85% of FLT3 WT pts (P=.13). With a median follow-up of 4.6 years, FLT3 TKD+ pts had worse DFS (P=.01; 3-year DFS rates: 31% v 60%) and a trend for worse overall survival (OS) (P=.17, 3-year OS rates: 40% v 65%) compared to FLT3 WT pts. Other variables moderately associated with a worse DFS (P<.10) included higher % BM blasts and NPM1 WT. Adjusting for these two variables, FLT3 TKD remained significantly associated with adverse DFS (P=.02; hazard ratio=2.3). A gene expression signature consisting of 333 probe sets (P<.001) differentiated FLT3 TKD+ (n=9) from FLT3 WT (n=50) pts. Genes represented in this signature include those involved in cell motility (e.g., C3AR1, VNN1, VNN2, ADAM8; all over-expressed in FLT3 TKD+), development (e.g., HOXB5, HOXB6; both over-expressed in FLT3 TKD+) and stress response (e.g., MPO; under-expressed in FLT3 TKD+). Conclusions: FLT3 TKD appears to be an independent predictor for DFS among younger CN AML adult pts without FLT3 ITD. Gene expression profiling provides additional insight into the biology of FLT3 TKD+ AML and may lead to identification of additional targets for improved therapy. No significant financial relationships to disclose.

Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases

Development, angiogenesis, wound healing, and metastasis all involve the movement of cells in response to changes in the extracellular environment. To determine whether caveolin-1 plays a role in cell migration, we have used fibroblasts from knockout mice. Caveolin-1–deficient cells lose normal cell polarity, exhibit impaired wound healing, and have decreased Rho and increased Rac and Cdc42 GTPase activities. Directional persistency of migration is lost, and the cells show an impaired response to external directional stimuli. Both Src inactivation and p190RhoGAP knockdown restore the wild-type phenotype to caveolin-1–deficient cells, suggesting that caveolin-1 stimulates normal Rho GTP loading through inactivation of the Src–p190RhoGAP pathway. These findings highlight the importance of caveolin-1 in the establishment of cell polarity during directional migration through coordination of the signaling of Src kinase and Rho GTPases.