Activation Of Cdc42 Research Articles

A novel HDGF-ALCAM axis promotes the metastasis of Ewing sarcoma via regulating the GTPases signaling pathway.

Ewing sarcoma (ES) is a type of highly aggressive pediatric tumor in bones and soft tissues and its metastatic spread remains the most powerful predictor of poor outcome. We previously identified that the transcription factor hepatoma-derived growth factor (HDGF) promotes ES tumorigenesis. However, the mechanisms underlying ES metastasis remain unclear. Here, we show that HDGF drives ES metastasis in vitro and in vivo, and HDGF reduces metastasis-free survival (MFS) in two independent large cohorts of human ES patients. Integrative analyses of HDGF ChIP-seq and gene expression profiling in ES cells reveal that HDGF regulates multiple metastasis-associated genes, among which activated leukocyte cell adhesion molecule (ALCAM) emerges as a major HDGF target and a novel metastasis-suppressor in ES. HDGF down-regulates ALCAM, induces expression and activation of the downstream effectors Rho-GTPase Rac1 and Cdc42, and promotes actin cytoskeleton remodeling and cell-matrix adhesion. In addition, repression of ALCAM and activation of Rac1 and Cdc42 are required for the pro-metastatic functions of HDGF in vitro. Moreover, analyses in murine models with ES tumor orthotopic implantation and experimental metastasis, as well as in human ES samples, demonstrate the associations among HDGF, ALCAM, and GTPases expression levels. Furthermore, high HDGF/low ALCAM expression define a subgroup of patients harboring the worst MFS. These findings suggest that the HDGF/ALCAM/GTPases axis represents a promising therapeutic target for limiting ES metastasis.

Cell-ECM contact-guided intracellular polarization is mediated via lamin A/C dependent nucleus-cytoskeletal connection

Cell polarization plays a crucial role in dynamic cellular events, such as cell proliferation, differentiation, and directional migration in response to diverse extracellular and intracellular signals. Although it is well known that cell polarization entails highly orchestrated intracellular molecular reorganization, the underlying mechanism of repositioning by intracellular organelles in the presence of multiple stimuli is still unclear. Here, we show that front-rear cell polarization based on the relative positions of nucleus and microtubule organizing center is precisely controlled by mechanical interactions including cellular adhesion to extracellular matrix and nucleus-cytoskeletal connections. By modulating the size and distribution of fibronectin-coated adhesive spots located in the polarized cell shape mimicking micropatterns, we monitored the alterations in cell polarity. We found that the localization of individual adhesive spots is more dominant than the cell shape itself to induce intracellular polarization. Further, the degree of cell polarization was diminished significantly by disrupting nuclear lamin A/C. We further confirm that geometrical cue-guided intracellular polarization determines directional cell migration via local activation of Cdc42. These findings provide novel insights into the role of nucleus-cytoskeletal connections in single cell polarization under a combination of physical, molecular, and genetic cues, where lamin A/C acts as a critical molecular mediator in ECM sensing and signal transduction via nucleus-cytoskeletal connection.

RhoGAP RGA-8 supports morphogenesis in C. elegans by polarizing epithelia.

ABSTRACTCDC-42 regulation of non-muscle myosin/NMY-2 is required for polarity maintenance in the one-cell embryo of Caenorhabditis elegans. CDC-42 and NMY-2 regulate polarity throughout embryogenesis, but their contribution to later events of morphogenesis are less understood. We have shown that epidermal enclosure requires the GTPase CED-10/Rac1 and WAVE/Scar complex, its effector, to promote protrusions that drive enclosure through the branch actin regulator Arp2/3. Our analysis here of RGA-8, a homolog of SH3BP1/Rich1/ARHGAP17/Nadrin, with BAR and RhoGAP motifs, suggests it regulates CDC-42, so that actin and myosin/NMY-2 promote ventral enclosure during embryonic morphogenesis. Genetic and molecular data suggest RGA-8 regulates CDC-42, and phenocopies the CDC-42 pathway regulators WASP-1/WSP-1 and the F-BAR proteins TOCA-1 and TOCA-2. Live imaging shows RGA-8 and WSP-1 enrich myosin and regulate F-actin in migrating epidermal cells during ventral enclosure. Loss of RGA-8 alters membrane recruitment of active CDC-42. We propose TOCA proteins and RGA-8 use BAR domains to localize and regenerate CDC-42 activity, thus regulating F-actin levels, through the branched actin regulator WSP-1, and myosin enrichment. RhoGAP RGA-8 thus polarizes epithelia, to promote cell migrations and cell shape changes of embryonic morphogenesis.

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn2+/Ca2+, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.

Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

P53‑p72‑Δ225‑331‑V31I identified in a cholangiocarcinoma cell line promotes migration and invasiveness via the downregulation of claudin‑1 expression and the activation of Cdc42.

TP53 is the most common gene mutated in human cancers, including in cholangiocarcinoma (CCA). The gain‑of‑function properties of p53 variants are often involved in cancer progression. The present study demonstrated that a truncated del p53 variant, del p53M213, exhibited gain‑of‑function properties and was highly expressed in the invasive liver fluke Opisthorchisviverrini‑associated CCA cell line, KKU‑M213. The del p53M213 variant lacked exons 7‑9 and contained a V31I substitution (p53‑p72‑Δ225‑331‑V31I). Stably transfected p53‑null human non‑small cell lung H1299 cells exhibited a del p53M213 localization in both the cell cytosol and nucleus. Del p53M213 lacked anti‑growth functions, and instead enhanced migration and invasiveness. In addition, this p53 variant downregulated claudin‑1 expression and promoted Cdc42 activation, consistent with the roles of claudin‑1 and Cdc42 in inhibiting cell‑cell dissociation and promoting cell migration, respectively. On the whole, although del p53M213 is an important driver of cancer cell migration and invasiveness, other properties related to its novel gain‑of‑function properties require further investigation in order to develop effective treatment strategies for cancers bearing this truncated TP53 allele.

Repolarization of HSC attenuates HSCs failure in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a bone marrow failure (BMF) syndrome associated with an increased risk of myelodysplasia and leukemia. The molecular mechanisms of SDS are not fully understood. We report that primitive hematopoietic cells from SDS patients present with a reduced activity of the small RhoGTPase Cdc42 and concomitantly a reduced frequency of HSCs polar for polarity proteins. The level of apolarity of SDS HSCs correlated with the magnitude of HSC depletion in SDS patients. Importantly, exogenously provided Wnt5a or GDF11 that elevates the activity of Cdc42 restored polarity in SDS HSCs and increased the number of HSCs in SDS patient samples in surrogate ex vivo assays. Single cell level RNA-Seq analyses of SDS HSCs and daughter cells demonstrated that SDS HSC treated with GDF11 are transcriptionally more similar to control than to SDS HSCs. Treatment with GDF11 reverted pathways in SDS HSCs associated with rRNA processing and ribosome function, but also viral infection and immune function, p53-dependent DNA damage, spindle checkpoints, and metabolism, further implying a role of these pathways in HSC failure in SDS. Our data suggest that HSC failure in SDS is driven at least in part by low Cdc42 activity in SDS HSCs. Our data thus identify novel rationale approaches to attenuate HSCs failure in SDS.

Yap1-Scribble polarization is required for hematopoietic stem cell division and fate

AbstractYap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

Murine Epsins Play an Integral Role in Podocyte Function.

Epsins, a family of evolutionarily conserved membrane proteins, play an essential role in endocytosis and signaling in podocytes. Podocyte-specific Epn1, Epn2, Epn3 triple-knockout mice were generated to examine downstream regulation of serum response factor (SRF) by cell division control protein 42 homolog (Cdc42). Podocyte-specific loss of epsins resulted in increased albuminuria and foot process effacement. Primary podocytes isolated from these knockout mice exhibited abnormalities in cell adhesion and spreading, which may be attributed to reduced activation of cell division control protein Cdc42 and SRF, resulting in diminished β1 integrin expression. In addition, podocyte-specific loss of Srf resulted in severe albuminuria and foot process effacement, and defects in cell adhesion and spreading, along with decreased β1 integrin expression. Epsins play an indispensable role in maintaining properly functioning podocytes through the regulation of Cdc42 and SRF-dependent β1 integrin expression.

HCF-1 promotes cell cycle progression by regulating the expression of CDC42

The eukaryotic cell cycle involves a highly orchestrated series of events in which the cellular genome is replicated during a synthesis (S) phase and each of the two resulting copies are segregated properly during mitosis (M). Host cell factor-1 (HCF-1) is a transcriptional co-regulator that is essential for and has been implicated in basic cellular processes, such as transcriptional regulation and cell cycle progression. Although a series of HCF-1 transcriptional targets have been identified, few functional clues have been provided, especially for chromosome segregation. Our results showed that HCF-1 activated CDC42 expression by binding to the −881 to −575 region upstream of the CDC42 transcription start site, and the regulation of CDC42 expression by HCF-1 was correlated with cell cycle progression. The overexpression of a spontaneously cycling and constitutively active CDC42 mutant (CDC42F28L) rescued G1 phase delay and multinucleate defects in mitosis upon the loss of HCF-1. Therefore, these results establish that HCF-1 ensures proper cell cycle progression by regulating the expression of CDC42, which indicates a possible mechanism of cell cycle coordination and the regulation mode of typical Rho GTPases.

DOCK8 controls survival of group 3 innate lymphoid cells in the gut through Cdc42 activation.

Innate lymphoid cells (ILCs) are a family of developmentally related leukocytes that rapidly secrete polarized sets of cytokines to combat infection and promote tissue repair at mucosal barriers. Among them, group 3 ILCs (ILC3s) play an important role in maintenance of the gut homeostasis by producing IL-22, and their development and function critically depend on the transcription factor RORγt. Although recent evidence indicates that RORγt+ ILC3s are reduced in the gut in the absence of the Cdc42 activator DOCK8 (dedicator of cytokinesis 8), the underlying mechanism remains unclear. We found that genetic deletion of Dock8 in RORγt+-lineage cells markedly reduced ILC3s in the lamina propria of the small intestine. By analyzing BrdU incorporation, it was revealed that DOCK8 deficiency did not affect the cell proliferation. Furthermore, when lineage marker-negative (Lin-) α4β7+ CD127+ RORγt- fetal liver cells were cultured with OP9 stromal cells in the presence of stem cell factor (SCF) and IL-7 in vitro, RORγt+ ILC3s normally developed irrespective of DOCK8 expression. However, DOCK8-deficient ILC3s exhibited a severe defect in survival of ILC3s under the condition with or without IL-7. Similar defects were observed when we analyzed Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Thus, DOCK8 acts in cell-autonomous manner to control survival of ILC3s in the gut through Cdc42 activation.

Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast.

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

Mechanical force-induced morphology changes in a human fungal pathogen

BackgroundThe initial step of a number of human or plant fungal infections requires active penetration of host tissue. For example, active penetration of intestinal epithelia by Candida albicans is critical for dissemination from the gut into the bloodstream. However, little is known about how this fungal pathogen copes with resistive forces upon host cell invasion.ResultsIn the present study, we have used PDMS micro-fabrication to probe the ability of filamentous C. albicans cells to penetrate and grow invasively in substrates of different stiffness. We show that there is a threshold for penetration that corresponds to a stiffness of ~ 200 kPa and that invasive growth within a stiff substrate is characterized by dramatic filament buckling, along with a stiffness-dependent decrease in extension rate. We observed a striking alteration in cell morphology, i.e., reduced cell compartment length and increased diameter during invasive growth, that is not due to depolarization of active Cdc42, but rather occurs at a substantial distance from the site of growth as a result of mechanical compression.ConclusionsOur data reveal that in response to this compression, active Cdc42 levels are increased at the apex, whereas active Rho1 becomes depolarized, similar to that observed in membrane protrusions. Our results show that cell growth and morphology are altered during invasive growth, suggesting stiffness dictates the host cells that C. albicans can penetrate.

LncRNA deleted in lymphocytic leukaemia 1 (DLEU1) promotes the migration and invasion of human embryonic trophoblast cells

SummaryTo investigate the roles of lncRNA deleted in lymphocytic leukaemia 1 (DLEU1) on migration and invasion of human trophoblast cells. Human chorionic trophoblast cell line HTR8/SVneo was cultured and transfected using lncRNA DLEU1 small interfering RNA. Real-time quantitative polymerase chain reaction was used to detect lncRNA DLEU1 expression. The activity of migration regulatory protein CDC42 was detected by western blot. The downstream miRNA targets of lncRNA and mRNAs targeted by corresponding miRNAs were respectively predicted using bioinformatics analyses. Compared with the control group, the expression of lncRNA DLEU1 in the small interfering RNA group was significantly decreased (P < 0.05). There was no significant change in cell proliferation capacity for transfected cells (lncRNA DLEU1 siRNA-1, P = 0.537; lncRNA DLEU1 siRNA-2, P = 0.384), but cell migration (lncRNA DLEU1 siRNA-1, P = 0.025; lncRNA DLEU1 siRNA-2, P = 0.019) and invasion (lncRNA DLEU1 siRNA-1, P = 0.0327; lncRNA DLEU1 siRNA-2, P = 0.021) was significantly reduced. CDC42 activity in the lncRNA DLEU1 knockdown group decreased and the phosphorylation of cofilin increased. Therefore, downregulation of lncRNA DLEU1 suppressed the migration and invasion of human trophoblast cells.

Differential regulation of rho GTPases during lung adenocarcinoma migration and invasion reveals a novel role of the tumor suppressor StarD13 in invadopodia regulation

BackgroundLung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549.MethodsWe knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation.ResultsWe observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation.ConclusionIn conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases.92FP97T_AMtXGPZ_u9jGL2Video abstract.

RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. Invivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.

Compete or Coexist? Why the Same Mechanisms of Symmetry Breaking Can Yield Distinct Outcomes.

Cellular morphogenesis is governed by the prepattern based on the symmetry-breaking emergence of dense protein clusters. Thus, a cluster of active GTPase Cdc42 marks the site of nascent bud in the baker’s yeast. An important biological question is which mechanisms control the number of pattern maxima (spots) and, thus, the number of nascent cellular structures. Distinct flavors of theoretical models seem to suggest different predictions. While the classical Turing scenario leads to an array of stably coexisting multiple structures, mass-conserved models predict formation of a single spot that emerges via the greedy competition between the pattern maxima for the common molecular resources. Both the outcome and the kinetics of this competition are of significant biological importance but remained poorly explored. Recent theoretical analyses largely addressed these questions, but their results have not yet been fully appreciated by the broad biological community. Keeping mathematical apparatus and jargon to the minimum, we review the main conclusions of these analyses with their biological implications in mind. Focusing on the specific example of pattern formation by small GTPases, we speculate on the features of the patterning mechanisms that bypass competition and favor formation of multiple coexisting structures and contrast them with those of the mechanisms that harness competition to form unique cellular structures.

P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

The P2X7 receptor is an ATP-gated cation channel with a still ambiguous role in cancer progression, proposed to be either pro- or anti-cancerous, depending on the cancer or cell type in the tumour. Its role in mammary cancer progression is not yet defined. Here, we show that P2X7 receptor is functional in highly aggressive mammary cancer cells, and induces a change in cell morphology with fast F-actin reorganization and formation of filopodia, and promotes cancer cell invasiveness through both 2- and 3-dimensional extracellular matrices in vitro. Furthermore, P2X7 receptor sustains Cdc42 activity and the acquisition of a mesenchymal phenotype. In an immunocompetent mouse mammary cancer model, we reveal that the expression of P2X7 receptor in cancer cells, but not in the host mice, promotes tumour growth and metastasis development, which were reduced by treatment with specific P2X7 antagonists. Our results demonstrate that P2X7 receptor drives mammary tumour progression and represents a pertinent target for mammary cancer treatment.

Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein).

AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.