Filopodia Formation Research Articles

Endoglin and MMP14 Contribute to Ewing Sarcoma Spreading by Modulation of Cell-Matrix Interactions.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell–matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.

P1249: INHIBITION OF THE PENTOSE-PHOSPHATE-PATHWAY (PPP) MEDIATES MACROPHAGE REPROGRAMMING FROM SUPPORTIVE BYSTANDER FUNCTION TOWARDS ANTIBODY-DEPENDENT LYMPHOMA CELL CLEARANCE AND LYMPHOMA SUPPRESSION

Background: Treatment response in lymphoma depends on the interaction and functional status of macrophages as important effector cells for tumor cell clearance by antibody-dependent cellular phagocytosis (ADCP). In the tumor microenvironment (TME) there is a limited supply of nutrients which implicates metabolic reprograming of immune cells such as changed phagocytic capacity of macrophages. Aims: We aimed to identify metabolic pathways regulating macrophages in the TME to improve macrophage effector cell function in lymphoma therapy. Methods: We addressed macrophage lymphoma cell co-cultures using macrophage cell lines, primary murine macrophages and human monocyte-derived macrophages. For pathway inhibition specific compounds as well as shRNA-mediated knockdown were used for functional read-out of ADCP and viability in lymphoma co-culture. Macrophage reprogramming was assessed by microscopy, FACS-immunophenotyping, ELISA, (phospho)-proteomic and metabolomic assessment. C57BL/6 mice and the humanized aggressive lymphoma model hMB were used for in vivo treatment with the pentose phosphate pathway (PPP) inhibitor S3 and analysis of macrophage programming and overall survival. Results: Inhibition of AMP kinase, glycolysis or mitochondrial ATP-production did not increase the phagocytosis rate in ADCP co-cultures. Inhibiting the PPP induced an increased lymphoma cell phagocytosis by macrophages. Under PPP-inhibition, the oxygen consumption and the glycolysis of macrophages was increased and the cells developed a more activated morphology with formation of filopodia, which are needed for phagocytosis initiation. Moreover, a down-regulation of immune-inhibitory surface proteins PD-L1 and SIRP1α was seen and cytokine secretion was shifted from anti-inflammatory IL10 to pro-inflammatory IL6 secretion. PPP-inhibition of macrophages significantly reduced CLL-cell support in vitro abrogating the “nurse-like” effect of macrophages. The increased lymphoma cell clearance and phenotypic alterations of macrophages were also observed in PPP-enzyme knockdown of transketolase (TKT) and 6-phosphogluconatedehydrogenase (6PGD) in macrophages and by inhibiting the PPP in primary murine and human macrophages. In a multiomics assessment of PPP-inhibition on proteome, phosphoproteome and metabolome level, we found protein expression alterations regulating metabolism and immunity. A connection between PPP inhibition and changed immune profile by modulation of the Stat1-Irg1-itaconate axis was identified and validated. In a humanized lymphoma mouse model the addition of PPP-inhibitor S3 led to significant prolonged survival of lymphoma bearing mice and an increased maturation and pro-inflammatory polarization of macrophages. Summary/Conclusion: An increased maturation, activation and repolarization of macrophages towards a pro-inflammatory and phagocytic active phenotype was observed by inhibiting the pentose-phosphate-pathway (PPP). This led to a significant survival benefit in in vivo lymphoma mouse model. We were able to point out the underlying Stat1-Irg1-itaconate signalling axis, connecting metabolic regulation and immune-phenotype of macrophages. We hypothesize the PPP as a key regulator of macrophage function determining the support of malignant B cell growth versus the capacity of lymphoma cell clearance and thereby therapy outcome. By that, we have identified the PPP as a targetable modulator of macrophage polarization to improve efficacy of B cell lymphoma and CLL therapy.

Abstract 143: Novel HSP40 or J domain protein inhibitors that deplete misfolded mutant p53

Abstract Background: Accumulation of oncogenic mutant p53 (mutp53) greatly contributes to cancer progression. Heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), has been implicated in stabilization of misfolded mutp53. Specifically, knockdown of DNAJA1, a member of HSP40/JDPs, results in degradation of mainly misfolded mutp53, leading to inhibition of tumor progression. Since no DNAJA1 inhibitors are currently available in clinics, these findings prompted us to identify potential DNAJA1 inhibitors which can induce mutp53 degradation. Methods/Design: An in-silico docking study was performed for binding of the DNAJA1 J-domain to compounds in zinc data base, which identified compound Number7-3. Further analysis suggests three critical amino acids in DNAJA1 for the binding, including Y7, K44, and Q47. Results: Eighteen commercially available analogs of Number7-3 were further examined. Of these, a compound, namely A11, had the strongest activity to deplete both DNAJA1 and misfolded mutp53. A11 reduced protein levels of misfolded mutp53 in concentration- and time-dependent manners without affecting mRNA levels of DNAJA1 and mutp53. Importantly, A11 showed minimal effects on the levels of wild-type p53 and DNA contact mutp53. Treatment of cells expressing misfolded mutp53 with A11 resulted in reduced filopodia formation and cell migration, which was not observed in cells lacking DNAJA1. Moreover, protein levels of a triple mutant DNAJA1 (Y7A/K44A/Q47A) were not altered by A11. Intriguingly, A11 reduced protein levels of several other HSP40/JDPs. Conclusions: Our study, for the first time, has identified a small compound that depletes DNAJA1, leading to reduced levels of misfolded mutp53 and inhibited malignant properties of cancer cells. Citation Format: Tomoo Iwakuma, Shigeto Nishikawa, Atsushi Kaida, Atul Ranjan, Alejandro Parrales, Mohamed A. Alalem, David K. Johnson. Novel HSP40 or J domain protein inhibitors that deplete misfolded mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 143.

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti‐GPIbβ antibody derivative

BackgroundGlycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb‐IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R‐IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb‐IX‐mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild‐type mice. Previously, an anti‐GPIbβ antibody RAM.1 was developed. RAM.1 inhibits GPIbα‐dependent platelet signaling and activation. Further, RAM.1 also inhibits anti‐GPIbα antibody‐mediated filopodia formation. ObjectiveTo investigate whether RAM.1 can ameliorate trigger sequence exposure‐mediated platelet clearance. MethodsSpontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488‐labeled anti‐mouse GPIX antibody. ResultTransfected Chinese hamster ovary cells stably expressing the same chimeric IL4R‐Ibα protein complex as in IL4R‐IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R‐IbαTg platelets. ConclusionThese results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb‐IX‐mediated thrombocytopenia.

The Process of Filopodia Induction during HPV Infection.

Human Papillomavirus 16 (HPV16) infects mucosal and epithelial cells and has been identified as a high-risk HPV type that is an etiologic agent of human cancers. The initial infectious process, i.e., the binding of the virus particle and its entry into the host cell, has been studied extensively, although it is not fully understood. There is still a gap in understanding the steps by which the virus is able to cross the plasma membrane after receptor binding. In this study, we demonstrate that after HPV16 comes into contact with a plasma membrane receptor, there are cytoskeletal changes resulting in an increase of filopodia numbers. This increase in filopodia numbers was transient and was maintained during the first two hours after virus addition. Our data show that there is a statistically significant increase in infection when filopodia numbers are increased by the addition of drug and virus simultaneously, and a decrease in virus infection when filopodia formation is inhibited. We describe that HPV16 binding results in the activation of Cdc42 GTPase that in turn results in an increase in filopodia. siRNA directed at Cdc42 GTPase resulted in a statistically significant reduction of infection and a corresponding lack of filopodia induction.

The RAL Enigma: Distinct Roles of RALA and RALB in Cancer.

RALA and RALB are highly homologous small G proteins belonging to the RAS superfamily. Like other small GTPases, the RALs are molecular switches that can be toggled between inactive GDP-bound and active GTP-bound states to regulate diverse and critical cellular functions such as vesicle trafficking, filopodia formation, mitochondrial fission, and cytokinesis. The RAL paralogs are activated and inactivated by a shared set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) and utilize similar sets of downstream effectors. In addition to their important roles in normal cell biology, the RALs are known to be critical mediators of cancer cell survival, invasion, migration, and metastasis. However, despite their substantial similarities, the RALs often display striking functional disparities in cancer. RALA and RALB can have redundant, unique, or even antagonistic functions depending on cancer type. The molecular basis for these discrepancies remains an important unanswered question in the field of cancer biology. In this review we examine the functions of the RAL paralogs in normal cellular physiology and cancer biology with special consideration provided to situations where the roles of RALA and RALB are non-redundant.

Intrinsic epigenetic control of angiogenesis in induced pluripotent stem cell-derived endothelium regulates vascular regeneration

Human-induced pluripotent stem cell-derived endothelial cells (iECs) provide opportunities to study vascular development and regeneration, develop cardiovascular therapeutics, and engineer model systems for drug screening. The differentiation and characterization of iECs are well established; however, the mechanisms governing their angiogenic phenotype remain unknown. Here, we aimed to determine the angiogenic phenotype of iECs and the regulatory mechanism controlling their regenerative capacity. In a comparative study with HUVECs, we show that iECs increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) mediates their highly angiogenic phenotype via regulation of glycolysis enzymes, filopodia formation, VEGF mediated migration, and robust sprouting. We find that the elevated expression of VEGFR2 is epigenetically regulated via intrinsic acetylation of histone 3 at lysine 27 by histone acetyltransferase P300. Utilizing a zebrafish xenograft model, we demonstrate that the ability of iECs to promote the regeneration of the amputated fin can be modulated by P300 activity. These findings demonstrate how the innate epigenetic status of iECs regulates their phenotype with implications for their therapeutic potential.

TIP60 acts as a regulator of genes involved in filopodia formation and cell migration during wound healing

Wound healing is a complex phenomenon that requires coordination of numerous molecular and cellular changes to facilitate timely and efficient repair of the damaged tissue. Although many of these molecular pathways have been detailed, others remain to be elucidated. In the present work, we show for the first time, roles for the acetyltransferase TIP60 and nuclear receptor transcription factor PXR in this process, participating in wound healing by altering actin dynamics and cellular motility. We found that in response to wound-injury, TIP60 induces rapid formation of filopodia at the wounded cell front, leading to enhanced cell migration and faster closure of the wound. Further, qPCR analysis revealed heightened expression of Cdc42 and ROCK1 genes, key regulators involved in filopodia formation and actin reorganization, exclusively in TIP60-PXR-expressing cells upon wound-induction. We also performed ChIP assays to confirm the context-specific binding of TIP60 on the ROCK1 promoter and demonstrated that the TIP60 chromodomain is essential for loading of the TIP60–PXR complex onto the chromatin. Results from immunoprecipitation assays revealed that during the wounded condition, TIP60 alters the chromatin microenvironment by specifically acetylating histones H2B and H4, thereby modulating the expression of target genes. Overall, findings of this study show that TIP60 is a novel regulator of the wound healing process by regulating the expression of wound repair-related genes.

Investigation of co‐electrospun gelatin:TiO2/polycaprolactone:silk fibroin scaffolds for wound healing applications

AbstractElectrospinning is a facile technique to create porous nanofiber scaffolds with a high capability of natural environment mimicking. Here, co‐electrospinning of gelatin:TiO2, polycaprolactone:silk fibroin (G:T/P:F) was investigated with the aim of wound healing. Polycaprolactone improved the mechanical properties and gelatin:TiO2enhanced cellular interactions. Silk fibroin was extracted from theBombyx morisilkworm and increased the cellular and mechanical properties of the designed scaffolds. The morphology of the scaffolds was observed with field emission scanning electron microscopy (FESEM) and confirmed bead free porous nanofibrous structure. The fiber diameter rose after silk fibroin and TiO2addition but it was in the favorable fiber range. The contact angle, swelling, and mechanical properties of the G:T/P:F scaffolds supplied the requirements for skin tissue engineering. The designed scaffold showed over 80% cell viability with filopodia formation after 48 h according to cell adhesion observations and MTT assay results. The DAPI and LIVE/DEAD staining results confirmed high cell viability and the scratch assay observations proved the high wound healing potential of the G:T/P:F scaffolds. Furthermore, antibacterial properties were shown. It seems the designed scaffold can be useful in skin tissue regeneration. Further research may address the suggested skin scaffold.

Evaluation of the Anti‐Migratory Activity of Small‐molecule Gankyrin Inhibitors in A549 and MDA‐MB‐231 Cells

IntroductionGankyrin is a 25 kDa oncoprotein that plays a crucial role in cellular growth, proliferation, and migration. It is highly conserved and has 7 ankyrin repeats which bind to the S6 ATPase subunit of the 26S proteasome. It has been shown that gankyrin exerts its oncogenic activities by interacting with numerous tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein (Rb), which in turn promotes their proteasomal degradation. While Rb plays an important role in cell adhesion, p53 inhibits Rac/CDC42 which is responsible for lamellipodia and filopodia formation. Gankyrin is also reported to increase the levels of interleukin‐6 (IL‐6) which activates the JAK/STAT3 pathway. The phosphorylation of STAT3 to p‐STAT3 and the subsequent nuclear translocation of p‐STAT3 results in transcription of migratory factors. The inhibition of these gankyrin‐based protein‐protein interactions (PPI) by a small molecule inhibitor, should result in an anti‐migratory effect. Cjoc42 is the first known binder of gankyrin, and since its discovery we have developed two derivatives with an enhanced ability to bind gankyrin, – AFM‐2 and JA‐38 In order to determine if next‐generation gankyrin inhibitors could affect cancer cell migration, we evaluated both compounds in gankyrin overexpressing cell lines A549 and MDA‐MB‐231. Their ability to disrupt multiple gankyrin‐based PPIs is hypothesized to be due to their ability to induce a conformational change in gankyrin upon binding. Various assays such as the scratch assay, Boyden chamber assay, western blotting, co‐immunoprecipitation (co‐IP) assay and fluorescence‐based assays were used to evaluate our hypotheses.MethodsWe evaluated the anti‐migratory potential of the molecules by scratch and Boyden chamber assay. The molecules’ ability to disrupt gankyrin’s interactions with p53, Rb and the S6 proteasome was evaluated by co‐IP assay. Using western blotting, we evaluated the changes in the levels of various TSPs and migratory proteins upon treatment with the molecules. To test our hypothesis of induction of conformational change in gankyrin upon treatment with the molecules, we utilized florescence‐based experiments such as intrinsic fluorescence evaluation and thermal shift.DiscussionThe cell‐based scratch and Boyden chamber assay showed significant decreases in cellular migration in both cell lines upon treatment with the molecules. Co‐IP experiments demonstrated that both molecules disrupt various gankyrin‐based PPIs. Disruption of gankyrin’s interactions with the TSPs would prevent their subsequent proteasomal degradation and subsequently affect the levels of downstream proteins such as IL‐6 and Rac/CDC42. Significant reduction in the migratory proteins and a significant increase in the TSPs was confirmed by western blotting. Lastly, using fluorescence‐based experiments, we believe that the binding of the two molecules caused a conformation change in gankyrin, which could be why gankyrin’s multiple PPIs are disrupted.ConclusionGankyrin plays a key role controlling cellular migratory pathways through its various PPIs. By inducing a conformational change in gankyrin, our molecules disrupt these PPI and exhibit significant anti‐migratory activity which could prevent cancer metastasis.

HIF1\u03b1-SP1 interaction disrupts the circ-0001875/miR-31-5p/SP1 regulatory loop under a hypoxic microenvironment and promotes non-small cell lung cancer progression

BackgroundCircular RNAs (circRNAs) play an important role in the progression of non-small cell lung cancer (NSCLC), especially under tumor hypoxia. However, the precise functions and underlying mechanisms of dysregulated circRNAs in NSCLC are largely unknown.MethodsHigh-throughput RNA sequencing was performed to identify significantly expressed circRNAs in NSCLC tissues. The functions of circ-0001875 in NSCLC cells were investigated in vitro and in vivo. The regulatory relationships of circ-0001875, miR-31-5p and SP1 were examined by dual luciferase reporter assays and rescue experiments. The signal pathway of epithelial-to-mesenchymal transition and the formation of filopodia were analyzed by western blot and immunofluorescence staining. The binding of SP1 to Alu elements was evaluated by RNA immunoprecipitation, and the HIF1α and SP1 interaction was detected by co-immunoprecipitation.ResultsWe identified the novel Has_circ_0001875 as a significantly upregulated circRNA in NSCLC tissues and cell lines. circ-0001875 promoted the proliferation and metastasis of NSCLC both in vitro and in vivo, and induced NSCLC cells to extend filopodia. Mechanistically, circ-0001875 sponged miR-31-5p to regulate SP1, influencing epithelial-to-mesenchymal transition via the TGFβ/Smad2 signal pathway. SP1 negatively regulated circ-0001875 formation through an AluSq-dependent feedback loop, which was disrupted by competitive binding of HIF1α to SP1 under hypoxia condition. The circ-0001875/miR-31-5p/SP1 axis was associated with the clinical features and prognosis of NSCLC patients.ConclusionsOur results revealed that the circ-0001875/miR-31-5p/SP1 axis and the complex regulatory loops influence NSCLC progression. These findings provide new insights into the regulation of circRNA formation under tumor hypoxia.

Induction of Filopodia During Cytomegalovirus Entry Into Human Iris Stromal Cells.

Many viruses exploit thin projections of filopodia for cell entry and cell-to-cell spread. Using primary cultures of human iris stromal (HIS) cells derived from human eye donors, we report a significant increase in filopodia formation during human cytomegalovirus (HCMV) infection. Using confocal microscopy, we observed a large number of virions being frequently associated along the filopodia prior to cell infection. Depolymerization of actin filaments resulted in a significant inhibition of HCMV entry into HIS cell. Our results further revealed that the transient expression of HCMV envelope glycoprotein B (gB) triggers the induction of the filopodial system. Since gB is known to bind the diverse chains of heparan sulfate (HS), a comparative study was performed to evaluate the gB-mediated filopodial induction in cells expressing either wild-type HS and/or 3-O sulfated HS (3-OS HS). We found that cells co-expressing HCMV gB together with the 3-O sulfotranseferase-3 (3-OST-3) enzyme had a much higher and robust filopodia induction compared to cells co-expressing gB with wild-type HS. The above results were further verified by pre-treating HIS cells with anti-3-OS HS (G2) peptide and/or heparinase-I before challenging with HCMV infection, which resulted in a significant loss in the filopodial counts as well as decreased viral infectivity. Taken together, our findings highlight that HCMV entry into HIS cells actively modulates the actin cytoskeleton via coordinated actions possibly between gB and the 3-OS HS receptor to influence viral infectivity.

Dynamics of Endothelial Engagement and Filopodia Formation in Complex 3D Microscaffolds

The understanding of endothelium–extracellular matrix interactions during the initiation of new blood vessels is of great medical importance; however, the mechanobiological principles governing endothelial protrusive behaviours in 3D microtopographies remain imperfectly understood. In blood capillaries submitted to angiogenic factors (such as vascular endothelial growth factor, VEGF), endothelial cells can transiently transdifferentiate in filopodia-rich cells, named tip cells, from which angiogenesis processes are locally initiated. This protrusive state based on filopodia dynamics contrasts with the lamellipodia-based endothelial cell migration on 2D substrates. Using two-photon polymerization, we generated 3D microstructures triggering endothelial phenotypes evocative of tip cell behaviour. Hexagonal lattices on pillars (“open”), but not “closed” hexagonal lattices, induced engagement from the endothelial monolayer with the generation of numerous filopodia. The development of image analysis tools for filopodia tracking allowed to probe the influence of the microtopography (pore size, regular vs. elongated structures, role of the pillars) on orientations, engagement and filopodia dynamics, and to identify MLCK (myosin light-chain kinase) as a key player for filopodia-based protrusive mode. Importantly, these events occurred independently of VEGF treatment, suggesting that the observed phenotype was induced through microtopography. These microstructures are proposed as a model research tool for understanding endothelial cell behaviour in 3D fibrillary networks.

SARM1 Suppresses Axon Branching Through Attenuation of Axonal Cytoskeletal Dynamics.

Axon branching is a fundamental aspect of neuronal morphogenesis, neuronal circuit formation, and response of the nervous system to injury. Sterile alpha and TIR motif containing 1 (SARM1) was initially identified as promoting Wallerian degeneration of axons. We now report a novel function of SARM1 in postnatal sensory neurons; the suppression of axon branching. Axon collateral branches develop from axonal filopodia precursors through the coordination of the actin and microtubule cytoskeleton. In vitro analysis revealed that cultured P0-2 dorsal root ganglion sensory neurons from a SARM1 knockout (KO) mouse exhibit increased numbers of collateral branches and axonal filopodia relative to wild-type neurons. In SARM1 KO mice, cutaneous sensory endings exhibit increased branching in the skin in vivo with normal density of innervation. Transient axonal actin patches serve as cytoskeletal platforms from which axonal filopodia emerge. Live imaging analysis of axonal actin dynamics showed that SARM1 KO neurons exhibit increased rates of axonal actin patch formation and increased probability that individual patches will give rise to a filopodium before dissipating. SARM1 KO axons contain elevated levels of drebrin and cortactin, two actin regulatory proteins that are positive regulators of actin patches, filopodia formation, and branching. Live imaging of microtubule plus tip dynamics revealed an increase in the rate of formation and velocity of polymerizing tips along the axons of SARM1 KO neurons. Stationary mitochondria define sites along the axon where branches may arise, and the axons of SARM1 KO sensory neurons exhibit an increase in stationary mitochondria. These data reveal SARM1 to be a negative regulator of axonal cytoskeletal dynamics and collateral branching.

Platelet Membrane: An Outstanding Factor in Cancer Metastasis.

In addition to being biological barriers where the internalization or release of biomolecules is decided, cell membranes are contact structures between the interior and exterior of the cell. Here, the processes of cell signaling mediated by receptors, ions, hormones, cytokines, enzymes, growth factors, extracellular matrix (ECM), and vesicles begin. They triggering several responses from the cell membrane that include rearranging its components according to the immediate needs of the cell, for example, in the membrane of platelets, the formation of filopodia and lamellipodia as a tissue repair response. In cancer, the cancer cells must adapt to the new tumor microenvironment (TME) and acquire capacities in the cell membrane to transform their shape, such as in the case of epithelial−mesenchymal transition (EMT) in the metastatic process. The cancer cells must also attract allies in this challenging process, such as platelets, fibroblasts associated with cancer (CAF), stromal cells, adipocytes, and the extracellular matrix itself, which limits tumor growth. The platelets are enucleated cells with fairly interesting growth factors, proangiogenic factors, cytokines, mRNA, and proteins, which support the development of a tumor microenvironment and support the metastatic process. This review will discuss the different actions that platelet membranes and cancer cell membranes carry out during their relationship in the tumor microenvironment and metastasis.

SREBP1 promotes invasive phenotypes by upregulating CYR61/CTGF via the Hippo-YAP pathway.

Aberrant lipid metabolism provides bioenergetic, biosynthetic, and redox supplies to cancer cells. Previous studies have reported differential lipid profiling in thyroid malignancies. Sterol regulatory element-binding protein 1 (SREBP1), encoded by the SREBF1 gene, is a master regulator of cellular lipid homeostasis. The clinical and functional significance of SREBP1 in thyroid cancer is not well understood. Here, we showed that SREBP1 expression is significantly upregulated in invasive thyroid cancer than in normal thyroid tissue or benign thyroid nodules. High tumoral SREBP1 expression was associated with extrathyroidal extension, advanced disease stage, and shorter disease-specific survival in patients with differentiated thyroid cancer. SREBP1 overexpression significantly increased the oxygen consumption rate, filopodia formation, and migratory and invasive capacities of thyroid cancer cells. Knockdown of SREBF1 or treatment with an SREBP1 activation inhibitor fatostatin had the opposite effect. RNA-Seq analysis showed that modulation of SREBP1 expression was accompanied by corresponding changes in the expression of epithelial-mesenchymal transition markers and CYR61/CTGF. SREBP1-facilitated cell invasion could be abrogated by treatment with a YAP inhibitor such as verteporfin or genetic silencing of CYR61 or CTGF. In summary, SREBP1 upregulation can be used as a prognostic indicator for thyroid cancer and SREBP1 overexpression is involved in cancer invasiveness, at least partly, through upregulation of CYR61/CTGF via the Hippo-YAP pathway.

The "Biological Weapons" of Ehrlichia chaffeensis: Novel Molecules and Mechanisms to Subjugate Host Cells.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging, potentially fatal tick-borne infectious disease. The bacterium enters human cells via the binding of its unique outer-membrane invasin EtpE to the cognate receptor DNase X on the host-cell plasma membrane; this triggers actin polymerization and filopodia formation at the site of E. chaffeensis binding, and blocks activation of phagocyte NADPH oxidase that catalyzes the generation of microbicidal reactive oxygen species. Subsequently, the bacterium replicates by hijacking/dysregulating host-cell functions using Type IV secretion effectors. For example, the Ehrlichia translocated factor (Etf)-1 enters mitochondria and inhibits mitochondria-mediated apoptosis of host cells. Etf-1 also induces autophagy mediated by the small GTPase RAB5, the result being the liberation of catabolites for proliferation inside host cells. Moreover, Etf-2 competes with the RAB5 GTPase-activating protein, for binding to RAB5-GTP on the surface of E. chaffeensis inclusions, which blocks GTP hydrolysis and consequently prevents the fusion of inclusions with host-cell lysosomes. Etf-3 binds ferritin light chain to induce ferritinophagy to obtain intracellular iron. To enable E. chaffeensis to rapidly adapt to the host environment and proliferate, the bacterium must acquire host membrane cholesterol and glycerophospholipids for the purpose of producing large amounts of its own membrane. Future studies on the arsenal of unique Ehrlichia molecules and their interplay with host-cell components will undoubtedly advance our understanding of the molecular mechanisms of obligatory intracellular infection and may identify hitherto unrecognized signaling pathways of human hosts. Such data could be exploited for development of treatment and control measures for ehrlichiosis as well as other ailments that potentially could involve the same host-cell signaling pathways that are appropriated by E. chaffeensis.

MiR-520d-3p/MIG-7 axis regulates vasculogenic mimicry formation and metastasis in osteosarcoma.

Vasculogenic mimicry (VM) refers to a novel mode of tumor microcirculation, which provides an escape route for tumor metastasis, and thereby correlates with a poor prognosis. We previously reported MIG-7 plays a pivotal role in osteosarcoma (OS) VM. However, the precise mechanism of MIG-7 in regulating OS VM remains to be elucidated. The expression levels of miR-520d-3p and MIG-7 were measured in OS cell lines. The effects of the miR-520d-3p/MIG-7 axis were investigated by in vitro functional assays. An orthotopic xenograft model was established to assess the role of the miR-520d-3p/MIG-7 axis in OS cells in vivo. Phalloidin staining, western blot, immunohistochemistry, ELISA assays were carried out to explore the molecular events that were involved in the miR-520d-3p/MIG-7 axis-mediated VM formation. The miR-520d-3p expression level was inversely correlated with MIG-7 in these cell lines. miR-520d-3p overexpression suppressed the proliferation, migration, invasion, VM, and promotes the adhesion of OS cells in vitro. miR-520d-3p could directly bind to the 3'-UTR of MIG-7 and regulated MIG-7 expression, which led to impaired lamellipodia and filopodia formation and inactivation of the PI3K/MMPs/Ln-5γ2 signaling pathway. The anti-metastatic and anti-VM effects of miR-520d-3p were confirmed in vivo. Our findings suggest miR-520d-3p acts as a tumor suppressor by inhibiting VM formation in OS via targeting MIG-7.

Transcriptomic and Genetic Analyses Identify the Krüppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development.

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. We provide for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, we identified the Krüppel-like factor Dar1 as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. Our findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors.

Sleep promotes the formation of dendritic filopodia and spines near learning-inactive existing spines

Changes in synaptic connections are believed to underlie long-term memory storage. Previous studies have suggested that sleep is important for synapse formation after learning, but how sleep is involved in the process of synapse formation remains unclear. To address this question, we used transcranial two-photon microscopy to investigate the effect of postlearning sleep on the location of newly formed dendritic filopodia and spines of layer 5 pyramidal neurons in the primary motor cortex of adolescent mice. We found that newly formed filopodia and spines were partially clustered with existing spines along individual dendritic segments 24 h after motor training. Notably, posttraining sleep was critical for promoting the formation of dendritic filopodia and spines clustered with existing spines within 8 h. A fraction of these filopodia was converted into new spines and contributed to clustered spine formation 24 h after motor training. This sleep-dependent spine formation via filopodia was different from retraining-induced new spine formation, which emerged from dendritic shafts without prior presence of filopodia. Furthermore, sleep-dependent new filopodia and spines tended to be formed away from existing spines that were active at the time of motor training. Taken together, these findings reveal a role of postlearning sleep in regulating the number and location of new synapses via promoting filopodial formation.