Focal Adhesion Assembly Research Articles

Initiation of focal adhesion assembly by talin and kindlin: A dynamic view.

Focal adhesions (FAs) are integrin-containing protein complexes regulated by a network of hundreds of protein-protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion-dependent physiological and pathological responses.

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line.

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.

Integrin-mediated adhesion and mechanosensing in the mammary gland

The mammary gland is dynamically remodelled during its postnatal development and the reproductive cycles. This inherent plasticity has been suggested to increase the susceptibility of the organ to carcinogenesis. Morphological changes in the mammary epithelium involve cell proliferation, differentiation, apoptosis, and migration which, in turn, are affected by cell adhesion to the extracellular matrix (ECM). Integrin adhesion receptors function in the sensing of the biochemical composition, patterning and mechanical properties of the ECM surrounding the cells, and strongly influence cell fate. This review aims to summarize the existing literature on how different aspects of integrin-mediated adhesion and mechanosensing, including ECM composition; stiffness and topography; integrin expression patterns; focal adhesion assembly; dynamic regulation of the actin cytoskeleton; and nuclear mechanotransduction affect mammary gland development, function and homeostasis. As the mechanical properties of a complex tissue environment are challenging to replicate in vitro, emphasis has been placed on studies conducted in vivo or using organoid models. Outright, these studies indicate that mechanosensing also contributes to the regulation of mammary gland morphogenesis in multiple ways.

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFβ1 (transforming growth factor β1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

Surface Co-presentation of BMP-2 and integrin selective ligands at the nanoscale favors α5β1 integrin-mediated adhesion

Here we present the use of surface nanopatterning of covalently immobilized BMP-2 and integrin selective ligands to determine the specificity of their interactions in regulating cell adhesion and focal adhesion assembly. Gold nanoparticle arrays carrying single BMP-2 dimers are prepared by block-copolymer micellar nanolithography and azide-functionalized integrin ligands (cyclic-RGD peptides or α5β1 integrin peptidomimetics) are immobilized on the surrounding polyethylene glycol alkyne by click chemistry. Compared to BMP-2 added to the media, surface immobilized BMP-2 (iBMP-2) favors the spatial segregation of adhesion clusters and enhances focal adhesion (FA) size in cells adhering to α5β1 integrin selective ligands. Moreover, iBMP-2 copresented with α5β1 integrin ligands induces the recruitment of αvβ3 integrins in FAs. When copresented with RGD, iBMP-2 induces the assembly of a higher number of FAs, which are not affected by α5β1 integrin blocking. Our dual-functionalized platforms offer the possibility to study the crosstalk between integrins and BMP receptors, and more in general they could be used to address the spatial regulation of growth factors and adhesion receptors crosstalk on biomimetic surfaces.

Syndecan-4 Modulates Cell Polarity and Migration by Influencing Centrosome Positioning and Intracellular Calcium Distribution.

Efficient cell migration requires cellular polarization, which is characterized by the formation of leading and trailing edges, appropriate positioning of the nucleus and reorientation of the Golgi apparatus and centrosomes toward the leading edge. Migration also requires the development of an asymmetrical front-to-rear calcium (Ca2+) gradient to regulate focal adhesion assembly and actomyosin contractility. Here we demonstrate that silencing of syndecan-4, a transmembrane heparan sulfate proteoglycan, interferes with the correct polarization of migrating mammalian myoblasts (i.e., activated satellite stem cells). In particular, syndecan-4 knockdown completely abolished the intracellular Ca2+ gradient, abrogated centrosome reorientation and thus decreased cell motility, demonstrating the role of syndecan-4 in cell polarity. Additionally, syndecan-4 exhibited a polarized distribution during migration. Syndecan-4 knockdown cells exhibited decreases in the total movement distance during directional migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Super-resolution direct stochastic optical reconstruction microscopy images of syndecan-4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of the migrating cells. Given the crucial importance of myoblast migration during embryonic development and postnatal muscle regeneration, we conclude that our results could facilitate an understanding of these processes and the general role of syndecan-4 during cell migration.

LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 12 Syndecan-4 Modulates Cell Polarity and Migration by Influencing Centrosome Positioning and Intracellular Calcium Distribution

Efficient cell migration requires cellular polarization, which is characterized by the formation of leading and trailing edges, appropriate positioning of the nucleus and reorientation of the Golgi apparatus and centrosomes towards the leading edge. Migration also requires the development of an asymmetrical front-to-rear calcium gradient to regulate focal adhesion assembly and actomyosin contractility. Here we demonstrate that silencing of syndecan-4, a transmembrane heparan sulfate proteoglycan, interferes with the correct polarization of migrating mammalian myoblasts (i.e., activated satellite stem cells). In particular, syndecan-4 knockdown completely abolished the intracellular calcium gradient, abrogated centrosome reorientation and thus decreased cell motility, demonstrating the role of syndecan-4 in cell polarity. Additionally, syndecan-4 exhibited a polarized distribution during migration. Syndecan-4 knockdown cells exhibited decreases in the total movement distance during directional migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Super-resolution direct stochastic optical reconstruction microscopy (dSTORM) images of syndecan-4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of migrating cells. Given the crucial importance of myoblast migration during embryonic development and postnatal muscle regeneration, we conclude that our results could facilitate an understanding of these processes and the general role of syndecan-4 during cell migration.

Abstract 4183: Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator

Abstract Background and objectives: Despite the outstanding antitumor activity of halichondrins in mice, the limited supply from the natural sources has prevented drug development using intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) under good manufacturing practice (GMP) conditions. E7130 is not only a novel microtubule dynamics inhibitor, but also a novel tumor-microenvironment ameliorator. E7130 can increase intratumoral CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts (CAFs) at pharmacologically relevant compound concentrations. Here, we analyzed the molecular mechanisms of the α-SMA-positive CAF reduction with E7130. Methods: The expression levels of α-SMA, ECM proteins, ER-TR7 (pan-fibroblast marker), and Ki67 were examined using E7130-treated tumors. In vitro co-culture experiments, normal human fibroblasts were co-cultured with human cancer cells. To examine the effects of E7130 on the TGF-β-induced CAF activation, normal human fibroblasts were treated with TGF-β (final concentration of 1 ng/mL) and E7130 at the several concentrations, and gene expression analysis and immunocytochemical analysis were conducted. Results: E7130 reduced α-SMA-positive CAF and malignant ECM proteins without changing total fibroblast number in tumors. The in vitro co-culture system revealed that TGF-β from cancer cells activate normal human fibroblast and increased α-SMA expression. In addition, treatment with E7130 interfered with α-SMA induction by TGF-β in fibroblasts without growth inhibitory activity. We further found that E7130 inhibited TGF-β-induced PI3K/AKT/mTOR pathway, which resulted in the reduction of α-SMA expression in fibroblasts. Moreover, TGF-β treatment enhanced β-tubulin expression and focal adhesions formation, which were diminished by co-treatment with E7130 in fibroblasts. Many signaling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway. Conclusions: Drug discovery using halichondrins was based on their outstanding in vivo antitumor (inhibitory) activity in mice and bone metastasis in mouse melanoma model first reported in 1996. From these results, we hypothesized that halichondrins are not simple microtubule-targeted compounds in tumor cell, and have developed E7130, which harbors unique tumor microenvironment ameliorating effects. Here we proved that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process without killing the fibroblast by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4183.

Abstract B32: ASAP1 regulates differentiation in myoblasts and PAX-FOXO1 fusion-negative rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS), the most frequently diagnosed soft-tissue sarcoma in children, is caused by a differentiation defect in skeletal muscle precursor cells. Despite aggressive, multimodal therapies, the prognosis for recurrent PAX-FOXO1 fusion-negative RMS (FN-RMS) remains poor. Inducing differentiation in diseases defined by defective differentiation programs has led to curative therapies in cancers such as acute promyelocytic leukemia. A recent study showed that inducing differentiation slowed tumor growth and extended survival in a xenograft model of FN-RMS, identifying differentiation as a promising therapeutic target in FN-RMS. However, genes associated with differentiation are frequently found to be hijacked by tumor cells and repurposed for proinvasive programs. Therefore, better understanding of differentiation signaling and its relation to invasion could reveal novel therapeutic opportunities for patients with advanced FN-RMS. We hypothesize that ASAP1, an Arf GTPase-activating protein implicated in differentiation in normal cells and invasion in carcinoma, promotes progression by controlling proinvasive elements of differentiation signaling through focal adhesion assembly. ASAP1, an Arf GTPase-activating protein (Arf GAP), regulates integrin adhesion complexes, critical regulators of biologic processes such as proliferation, migration, and differentiation that are commonly dysregulated in cancer. ASAP1 is overexpressed in several cancers and correlates with increased metastasis and poor patient prognosis but has also been shown to promote differentiation. The mechanisms by which ASAP1 affects cancer progression and differentiation and the relationship between these effects are not yet understood. We found that ASAP1 is overexpressed in FN-RMS. ASAP1 overexpression inhibits proliferation in myoblasts, but not in FN-RMS. Knockdown of ASAP1 inhibits differentiation in both myoblast and FN-RMS cell lines, while overexpression enhances differentiation. Moreover, gene set enrichment analysis shows that myoblast differentiation-associated genes fail to become enriched upon knockdown of ASAP1. Finally, knockdown of Arf1 and Arf5, established binding partners of ASAP1, also blocks differentiation of FN-RMS cell lines, indicating that ASAP1 may regulate differentiation through its interaction with Arf GTPases. These data support our hypothesis that ASAP1 regulates the continuum of differentiation and invasion in FN-RMS. As a continuing test of the hypothesis, future studies will investigate focal adhesion assembly, dynamics, and signaling, which are processes known to be affected by ASAP1, as a mechanism for ASAP1-mediated regulation of myoblast differentiation. Citation Format: Katie E. Hebron, Olivia Feehan-Nelson, Xiaoying Jian, Sofia A. Girald, Paul A. Randazzo, Marielle E. Yohe. ASAP1 regulates differentiation in myoblasts and PAX-FOXO1 fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B32.

Circulating extracellular vesicles from patients with breast cancer enhance migration and invasion via a Src‑dependent pathway in MDA‑MB‑231 breast cancer cells.

Triple negative breast cancer (TNBC) is a breast cancer subtype associated with high rates of metastasis, heterogeneity, drug resistance and a poor prognosis. Extracellular vesicles (EVs) are vesicles of endosomal and plasma membrane origin, and are secreted by healthy and cancer cells. In cancer, EVs contribute to tumor progression by mediating escape from the immune system surveillance, and are involved in extracellular matrix degradation, invasion, angiogenesis, migration and metastasis. Furthermore, EVs have been identified in several human fluids. However, the role of EVs from patients with breast cancer in the migration and invasion of human breast cancer cells is not fully understood. The present study investigated whether EVs isolated from Mexican patients with breast cancer can induce cellular processes related to invasion in breast cancer. Moreover, plasma fractions enriched in EVs and deprived of platelet-derived EVs obtained from blood samples of 32 Mexican patients with biopsy-diagnosed breast cancer at different clinical stages who had not received treatment were analyzed. Furthermore, one control group was included, which consisted of 20 Mexican healthy females. The present results demonstrated that EVs from women with breast cancer promote migration and invasion, and increase matrix metalloproteinase (MMP)-2 and MMP-9 secretion in TNBC MDA-MB-231 cells. In addition, it was found that EVs from patients with breast cancer induced Src and focal adhesion kinase activation, and focal adhesions assembly with an increase in focal adhesions number, while the migration and invasion was dependent on Src activity. Collectively, EVs from Mexican patients with breast cancer induce migration and invasion via a Src-dependent pathway in TNBC MDA-MB-231 cells.

Ppp2r2a Knockout Mice Reveal That Protein Phosphatase 2A Regulatory Subunit, PP2A-B55α, Is an Essential Regulator of Neuronal and Epidermal Embryonic Development.

The serine/threonine protein phosphatase 2A (PP2A) is a master regulator of the complex cellular signaling that occurs during all stages of mammalian development. PP2A is composed of a catalytic, a structural, and regulatory subunit, for which there are multiple isoforms. The association of specific regulatory subunits determines substrate specificity and localization of phosphatase activity, however, the precise role of each regulatory subunit in development is not known. Here we report the generation of the first knockout mouse for the Ppp2r2a gene, encoding the PP2A-B55α regulatory subunit, using CRISPR/Cas9. Heterozygous animals developed and grew as normal, however, homozygous knockout mice were not viable. Analysis of embryos at different developmental stages found a normal Mendelian ratio of Ppp2r2a–/– embryos at embryonic day (E) 10.5 (25%), but reduced Ppp2r2a–/– embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live Ppp2r2a–/– pups were observed at birth. Ppp2r2a–/– embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. Ppp2r2a–/– embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in Ppp2r2a–/– embryos were highly disorganized, with reduced immunolabeling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the Ppp2r2a–/– embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Thus PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis.

Abstract A29: The gastric cancer-associated mutations R5W and Y42C in the RAS homologous RHOA protein cause distinct biochemical alterations, exhibit gain-of-function signaling and oncogenic activities

Abstract Missense mutations of the RAS homologous RHOA gene have recently been identified in ~25% of diffuse gastric cancer (DGC). Unexpectedly, in contrast to the gain-of-function hotspot mutations found in RAS in cancer, DGC-associated RHOA mutations (e.g., R5W, Y42C) are localized at different hotspots that instead suggest loss-of-function alterations. First, we utilized bacterially expressed recombinant proteins and determined the biochemical consequences of these mutations on RHOA function. We found that RHOA WT and Y42C, but not R5W nucleotide exchange, was catalyzed by the ECT2 RhoGEF. Similarly, WT and Y42C, but not R5W GTP hydrolysis activity, was stimulated by the p190RhoGAP. Surprisingly, Y42C and to a lesser degree R5W exhibited impaired intrinsic GTP hydrolysis activity. Interestingly, Y42C showed both loss- and gain-of-function interaction with effectors. Second, we ectopically expressed WT and mutant RHOA proteins in NIH/3T3 fibroblast, and surprisingly, found that Y42C and R5W, similar to the lab-generated constitutively GTP-bound Q63L mutant (analogous to RAS Q61L), stimulated actin stress fiber formation and focal adhesion assembly. Finally, when ectopically expressed in beta-catenin-deficient (another mutation found in gastric cancer) mouse gastric organoids, Y42C disrupted the 3D architecture and induced a DGC-like phenotype in vitro and in vivo. In summary, our studies suggest mutation-specific biochemical defects that act as gain-of-function oncogenic RHOA mutations. Citation Format: Antje Schaefer, Richard G. Hodge, Devon R. Blake, Haisheng Zhang, Adam J. Bass, Channing J. Der. The gastric cancer-associated mutations R5W and Y42C in the RAS homologous RHOA protein cause distinct biochemical alterations, exhibit gain-of-function signaling and oncogenic activities [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A29.

Bisphenol A induces focal adhesions assembly and activation of FAK, Src and ERK2 via GPER in MDA-MB-231 breast cancer cells

Bisphenol A (BPA) is an industrial synthetic chemical used in the production of polycarbonate plastics and epoxy resins. Human exposition to BPA is primarily through eating food, and drinking liquids, because BPA can leach from polycarbonate plastic containers, beverage cans and epoxy resins. BPA induces proliferation and migration in human breast cancer cells. The G protein-coupled estrogen receptor (GPER) is a G protein-coupled receptor coupled with Gs proteins that is activated by estrogen and estrogenic compounds and it is the receptor for BPA. However, the signal transduction pathways that mediate migration via BPA/GPER in triple negative breast cancer (TNBC) cells has not been studied in detail. Here, we demonstrate that BPA induces an increase of GPER expression and activation of FAK, Src and ERK2, and an increase of focal adhesion assembly via GPER in TNBC MDA-MB-231 cells. Moreover, BPA induces FAK and ERK2 activation, focal adhesion assembly and migration via epidermal growth factor receptor (EGFR) transactivation. Collectively our data showed that BPA via GPER and/or EGFR transactivation induces activation of signal transduction pathways that mediate migration in TNBC MDA-MB-231 cells.

C-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly.

ABSTRACTEndothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions or focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we have identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on the role of c-Src in the maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate that c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue- and context-dependent manner.

OPN binds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature.

Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.

Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex.

Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.

Attenuation of fibrogenesis by PDE4 inhibitor via decreased cytoskeleton remodeling, activation and contractility of hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix (ECM) proteins that occurs due to chronic liver injury. Activated hepatic stellate cells (HSCs) are main source of ECM in the liver. Rho‐associated kinase (Rho‐kinase/ROCK) promotes the assembly of focal adhesions and actin stress fibers by regulating the organization of the cytoskeleton in HSCs. Exchange protein directly regulated by cAMP 1 (EPAC1) attenuated Rho kinase signaling in HSCs and alleviated the development of liver fibrosis. We previously showed that cAMP degrading phosphodiesterase 4 (PDE4) enzymes play a critical role in the pathogenesis of liver fibrosis suggesting that hepatic cAMP signaling is compromised during fibrogenesis. Based on these observations, we hypothesize that preserving cAMP/EPAC1 signaling by inhibiting PDE4 will attenuate HSC activation and fibrotic signaling.C57Bl/6 mice were subjected to repeated carbon tetrachloride (CCl4) injection for 4 weeks to estabilesh liver fibrosis. One group of mice received PDE4 inhibitor, Rolipram (targeted to the liver) twice a week. To examine the direct effect of EPAC1/PDE4 pathway on HSCs, in vitro studies using human HSC cell line (LX2) were also performed. Activation of LX2 HSCs was achieved by using recombinant TGFβ1. Liver tissue proteomic analysis was performed. Pathway enrichment analysis was done using Clarivate analytics software, MetaCore. Sirius Red staining and hydroxyproline (HYP) assay was performed to assess collagen deposition. Real time qPCR and Western blot were performed to validate the findings from proteomic analysis. Statistical analysis was performed using ANOVA.CCl4 administration resulted in a significant accumulation of collagen confirmed by Sirius red staining and hydroxyproline (HYP) content in mice. Importantly, Rolipram prevented HYP accumulation and the expression of genes involved in collagen synthesis (Serpinh1) and crosslinking (Loxl1 and Lox). Additionally, Smad3 (central modulator of canonical TGFβ1 signaling) activation and mRNA levels of Mmp2 and Timp2 were markedly decreased by Rolipram. Pathway enrichment analysis of proteomic data identified several fibrotic pathways impacted by Rolipram, including HSC activation, integrin signaling and cytoskeleton remodeling regulated by Rho‐kinase. Western blot analyses confirmed that Rolipram significantly decreased phosphorylation of myosin light chain (MLC), target of Rho‐associated protein kinase. The effect of PDE4 inhibition on TGFβ1 inducible Smad3 and MLC phosphorylation was recapitulated in LX2 cells by using Rolipram and EPAC1 specific agonist. Moreover, Rolipram decreased Endothelin 1 (ET1) levels both in vivo and in vitro LX2 cells indicating a reduced contractility of HSCs. In LX2 cells TGFβ1 inducible nuclear translocation of Yes‐associated protein (YAP) was also inhibited by Rolipram suggesting that PDE4 inhibition promotes the crosstalk between TGFβ1 and YAP pathway to repress TGFβ1 signaling.These results demonstrate that PDE4 inhibition attenuates liver fibrosis by affecting multiple pathways involved in HSC activation and perpetuation of fibrosis.Support or Funding InformationNIH P20GM113226

Enhancing Stem Cell‐based Toxicity Assays by Engineering the Culture Niche in a High‐throughput, Assay‐agnostic Manner

Human induced pluripotent stem cell (hiPSC)‐based assays hold great promise for ameliorating human disease and improving the translation of new therapies, but they have shortcomings that hamper their ability to accurately model human physiology. For example, hiPSC‐Cardiomyocytes (CMs) mis‐classify several compounds of known cardiotoxicity. One likely source of this is the maturity of the cells; most culture techniques result in hiPSC‐CMs that express fetal phenotypes, leading to inaccurate or difficult‐to‐translate results. While engineering the extracellular matrix (ECM) has demonstrated improvements to both structural and functional phenotypes, it is often hampered by challenges with integration into industry‐standard assays or instrumentation. Further, the ability to engineer increasingly intricate and multi‐faceted artificial culture environments often drives decisions that trade off biological complexity with experimental throughput. Here, we developed bioengineering techniques to create biomimetic patterns that mimic the shape and structure of the ECM on a transparent polymer‐coated glass layer. The resultant structure directs focal adhesion assembly mechanisms in individual cells to drive higher‐ordered structuring of 2D tissues. This forms 2D anisotropic syncytia from iPSC‐CMs in industry‐standard microplate formats that are compatible with transmitted light microscopy. The method is extended to micro‐electrode arrays (MEA) in a manner that maintains both the quality of MEA recordings and baseline electrophysiological properties. iPSC‐CMs grown this way exhibit several structural and biochemical phenotypes including adult protein isoform expression, myofibril alignment, and sarcomere spacing, length, and width. We show that biomimetic cues enhance the electrophysiological response of cardiomyocytes to various drugs of known effect. Since ECM structuring recapitulates the polarized nature of cells and their concomitant gap junctions, we used the gap junction blocker carbenoxolone for validation studies. Polarized tissues enable differential conduction block measurements between longitudinal and transverse directions. Our MEA data show that patterned tissues exhibit increased sensitivity to carbenoxolone over unpatterned constructs (IC50’s of 14.71 nM versus 398.1 nM, respectively) comparing favorably to clinically relevant unbound concentrations reported in the low nM range. Our biomimetic engineering approach is instrument‐ and assay agnostic, effective on a variety of cell types and lines (including neuronal cells; examples to be presented), and compatible with high‐throughput approaches.Support or Funding InformationNational Institutes of Health Small Business Innovation Research Grant.(A) decellularized ex‐vivo cardiac tissue slices demonstrate a polarized meshwork of extracellular matrix (ECM) fibers; (B) our biomimetic patterning technique recapitulates the nanoscale structure and orientation of the ECM; (C) the technique can be expanded to the surface of high‐throughput MEAs without degradation to electrical signal detection while maintaining the nanoscale pattern (inset); (D) FCDI iCell2 Cardiomyocytes cultured on traditional plastic surfaces show unstructured morphology; (E) iCell2 Cardiomyocytes cultured on our biomimetic surfaces recapitulate the anisotropic structure of adult heart tissue.Figure 1(A) sarcomere lengths (B) and widths of cells on biomimetic nanosurfaces (green) compared to conventional surfaces (gray) are increased; (C) adult isoforms of structural proteins are enhanced with biomimetic nanopatterning; (D) patterned cells exhibit higher oxygen consumption rate; (E) patch clamp data demonstrate increased action potential duration on patterned tissues; (F) asymmetric conduction on patterned tissues enhances the sensitivity of MEA assays to conduction blockers;Figure 2

Chemoresistant ovarian cancer enhances its migration abilities by increasing store-operated Ca2+ entry-mediated turnover of focal adhesions

BackgroundAmong gynecological cancers, ovarian carcinoma has the highest mortality rate, and chemoresistance is highly prevalent in this cancer. Therefore, novel strategies are required to improve its poor prognosis. Formation and disassembly of focal adhesions are regulated dynamically during cell migration, which plays an essential role in cancer metastasis. Metastasis is intricately linked with resistance to chemotherapy, but the molecular basis for this link is unknown.MethodsTranswell migration and wound healing migration assays were used to analyze the migration ability of ovarian cancer cells. Real-time recordings by total internal reflection fluorescence microscope (TIRFM) were performed to assess the turnover of focal adhesions with fluorescence protein-tagged focal adhesion molecules. SOCE inhibitors were used to verify the effects of SOCE on focal adhesion dynamics, cell migration, and chemoresistance in chemoresistant cells.ResultsWe found that mesenchymal-like chemoresistant IGROV1 ovarian cancer cells have higher migration properties because of their rapid regulation of focal adhesion dynamics through FAK, paxillin, vinculin, and talin. Focal adhesions in chemoresistant cells, they were smaller and exhibited strong adhesive force, which caused the cells to migrate rapidly. Store-operated Ca2+ entry (SOCE) regulates focal adhesion turnover, and cell polarization and migration. Herein, we compared SOCE upregulation in chemoresistant ovarian cancer cells to its parental cells. SOCE inhibitors attenuated the assembly and disassembly of focal adhesions significantly. Results of wound healing and transwell assays revealed that SOCE inhibitors decreased chemoresistant cell migration. Additionally, SOCE inhibitors combined with chemotherapeutic drugs could reverse ovarian cancer drug resistance.ConclusionOur findings describe the role of SOCE in chemoresistance-mediated focal adhesion turnover, cell migration, and viability. Consequently, SOCE might be a promising therapeutic target in epithelial ovarian cancer.Graphical abstract

Cell Adhesiveness Serves as a Biophysical Marker for Metastatic Potential

Tumors are heterogeneous and composed of cells with different dissemination abilities. Despite significant effort, there is no universal biological marker that serves as a metric for metastatic potential of solid tumors. Common to disseminating cells from such tumors, however, is the need to modulate their adhesion as they detach from the tumor and migrate through stroma to intravasate. Adhesion strength is heterogeneous even among cancer cells within a given population, and using a parallel plate flow chamber, we separated and sorted these populations into weakly and strongly adherent groups; when cultured under stromal conditions, this adhesion phenotype was stable over multiple days, sorting cycles, and common across all epithelial tumor lines investigated. Weakly adherent cells displayed increased migration in both two-dimensional and three-dimensional migration assays; this was maintained for several days in culture. Subpopulations did not show differences in expression of proteins involved in the focal adhesion complex but did exhibit intrinsic focal adhesion assembly as well as contractile differences that resulted from differential expression of genes involved in microtubules, cytoskeleton linkages, and motor activity. In human breast tumors, expression of genes associated with the weakly adherent population resulted in worse progression-free and disease-free intervals. These data suggest that adhesion strength could potentially serve as a stable marker for migration and metastatic potential within a given tumor population and that the fraction of weakly adherent cells present within a tumor could act as a physical marker for metastatic potential. SIGNIFICANCE: Cancer cells exhibit heterogeneity in adhesivity, which can be used to predict metastatic potential.