Small Molecules Revealed in a Screen Targeting Epithelial Scattering Are Inhibitors of Microtubule Polymerization

Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in the detachment of cell-cell junctions and initiation of cell migration. Instead of coordinating collective cell behavior within a tissue, cells become solitary and have few cell-cell interactions. Since epithelial scattering is recapitulated in cancer progression and since HGF signaling drives cancer metastasis in many cases, inhibitors of HGF signaling have been proposed to act as anticancer agents. We previously sought to better understand critical components required for HGF-induced epithelial scattering by performing a forward chemical genetics screen, which resulted in the identification of compounds with no previously reported biological activity that we report here. In efforts to determine the mechanism of these compounds, we find that many compounds have broad antiproliferative effects on cancer cell lines by arrest of cell division in G2/M with minimal induction of apoptosis. This effect is reminiscent of microtubule-targeting agents, and we find that several of these scaffolds directly inhibit microtubule polymerization. Compounds are assessed for their toxicity and pharmacokinetics in vivo. The identification of novel small-molecule inhibitors of microtubule polymerization highlights the role of the microtubule cytoskeleton in HGF-induced epithelial scattering.

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Voltammetric Detection of Tetrodotoxin Real-Time In Vivo of Mouse Organs using DNA-Immobilized Carbon Nanotube Sensors

Current Analytical Chemistry ◽

10.2174/1573411014666180510145320 ◽

2019 ◽

Vol 15 (5) ◽

pp. 567-574

Author(s):

Huck Jun Hong ◽

Suw Young Ly

Keyword(s):

Carbon Nanotube ◽

Real Time ◽

Anticancer Agents ◽

Cancer Metastasis ◽

Anodic Stripping Voltammetry ◽

Stripping Voltammetry ◽

Takifugu Rubripes ◽

In Vivo Detection ◽

Voltammetric Detection

Background: Tetrodotoxin (TTX) is a biosynthesized neurotoxin that exhibits powerful anticancer and analgesic abilities by inhibiting voltage-gated sodium channels that are crucial for cancer metastasis and pain delivery. However, for the toxin’s future medical applications to come true, accurate, inexpensive, and real-time in vivo detection of TTX remains as a fundamental step. Methods: In this study, highly purified TTX extracted from organs of Takifugu rubripes was injected and detected in vivo of mouse organs (liver, heart, and intestines) using Cyclic Voltammetry (CV) and Square Wave Anodic Stripping Voltammetry (SWASV) for the first time. In vivo detection of TTX was performed with auxiliary, reference, and working herring sperm DNA-immobilized carbon nanotube sensor systems. Results: DNA-immobilization and optimization of amplitude (V), stripping time (sec), increment (mV), and frequency (Hz) parameters for utilized sensors amplified detected peak currents, while highly sensitive in vivo detection limits, 3.43 µg L-1 for CV and 1.21 µg L-1 for SWASV, were attained. Developed sensors herein were confirmed to be more sensitive and selective than conventional graphite rodelectrodes modified likewise. A linear relationship was observed between injected TTX concentration and anodic spike peak height. Microscopic examination displayed coagulation and abnormalities in mouse organs, confirming the powerful neurotoxicity of extracted TTX. Conclusion: These results established the diagnostic measures for TTX detection regarding in vivo application of neurotoxin-deviated anticancer agents and analgesics, as well as TTX from food poisoning and environmental contamination.

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Cellular dynamics of EMT: lessons from live in vivo imaging of embryonic development

Cell Communication and Signaling ◽

10.1186/s12964-021-00761-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Jeffrey D. Amack

Keyword(s):

Extracellular Matrix ◽

Embryonic Development ◽

In Vivo Imaging ◽

Cytoskeletal Proteins ◽

Cell Junctions ◽

Model Organisms ◽

Cellular Dynamics ◽

Mesenchymal Transition ◽

Cell Cell

AbstractEpithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell–cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT—and the reverse mesenchymal-epithelial transition (MET)—are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell–cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos—gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos—that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer’s vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET.

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Cooperation of Cancer Stem Cell Properties and Epithelial-Mesenchymal Transition in the Establishment of Breast Cancer Metastasis

Journal of Oncology ◽

10.1155/2011/591427 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 30

Author(s):

Tetsu Hayashida ◽

Hiromitsu Jinno ◽

Yuko Kitagawa ◽

Masaki Kitajima

Keyword(s):

Breast Cancer ◽

Stem Cell ◽

Cancer Stem Cell ◽

Cancer Progression ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Metastatic Tumor ◽

Breast Cancer Metastasis ◽

Cell Junctions ◽

Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a multistep process in which cells acquire molecular alterations such as loss of cell-cell junctions and restructuring of the cytoskeleton. There is an increasing understanding that this process may promote breast cancer progression through promotion of invasive and metastatic tumor growth. Recent observations imply that there may be a cross-talk between EMT and cancer stem cell properties, leading to enhanced tumorigenicity and the capacity to generate heterogeneous tumor cell populations. Here, we review the experimental and clinical evidence for the involvement of EMT in cancer stem cell theory, focusing on the common characteristics of this phenomenon.

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lncRNA NORAD Contributes to Colorectal Cancer Progression by Inhibition of miR-202-5p

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504018x15190844870055 ◽

2018 ◽

Vol 26 (9) ◽

pp. 1411-1418 ◽

Cited By ~ 24

Author(s):

Jie Zhang ◽

Xiao-Yan Li ◽

Ping Hu ◽

Yuan-Sheng Ding

Keyword(s):

Colorectal Cancer ◽

Cancer Progression ◽

Noncoding Rna ◽

Cancer Metastasis ◽

Cellular Proliferation ◽

Inhibitory Effect ◽

Migration And Invasion ◽

Competing Endogenous Rna

Previous study indicates that long noncoding RNA NORAD could serve as a competing endogenous RNA to pancreatic cancer metastasis. However, its role in colorectal cancer (CRC) needs to be investigated. In the present study, we found that the expression of NORAD was significantly upregulated in CRC tissues. Furthermore, the expression of NORAD was positively related with CRC metastasis and patients’ poor prognosis. Knockdown of NORAD markedly inhibited CRC cell proliferation, migration, and invasion but induced cell apoptosis in vitro. In vivo experiments also indicated an inhibitory effect of NORAD on tumor growth. Mechanistically, we found that NORAD served as a competing endogenous RNA for miR-202-5p. We found that there was an inverse relationship between the expression of NORAD and miR-202-5p in CRC tissues. Moreover, overexpression of miR-202-5p in SW480 and HCT116 cells significantly inhibited cellular proliferation, migration, and invasion. Taken together, our study demonstrated that the NORAD/miR-202-5p axis plays a pivotal function on CRC progression.

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Serum ANGPTL2 Levels Reflect Clinical Features of Breast Cancer Patients: Implications for the Pathogenesis of Breast Cancer Metastasis

The International Journal of Biological Markers ◽

10.5301/jbm.5000080 ◽

2014 ◽

Vol 29 (3) ◽

pp. 239-245 ◽

Cited By ~ 22

Author(s):

Motoyoshi Endo ◽

Yutaka Yamamoto ◽

Masahiro Nakano ◽

Tetsuro Masuda ◽

Haruki Odagiri ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Clinical Features ◽

Cancer Progression ◽

Breast Cancer Cells ◽

Cancer Metastasis ◽

Ductal Carcinoma ◽

Breast Cancer Metastasis ◽

Breast Cancer Patients

Introduction Breast cancer is a leading cause of cancer-related death in women worldwide, and its metastasis is a major cause of disease mortality. Therefore, identification of the mechanisms underlying breast cancer metastasis is crucial for the development of therapeutic and diagnostic strategies. Our recent study of immunodeficient female mice transplanted with MDA-MB231 breast cancer cells demonstrated that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) accelerates metastasis through both increasing tumor cell migration in an autocrine/paracrine manner, and enhancing tumor angiogenesis. To determine whether ANGPTL2 contributes to its clinical pathogenesis, we asked whether serum ANGPTL2 levels reflect the clinical features of breast cancer progression. Methods We monitored the levels of secreted ANGPTL2 in supernatants of cultured proliferating MDA-MB231 cells. We also determined whether the circulating ANGPTL2 levels were positively correlated with cancer progression in an in vivo breast cancer xenograft model using MDA-MB231 cells. Finally, we investigated whether serum ANGPTL2 levels were associated with clinical features in breast cancer patients. Results Both in vitro and in vivo experiments showed that the levels of ANGPTL2 secreted from breast cancer cells increased with cell proliferation and cancer progression. Serum ANGPTL2 levels in patients with metastatic breast cancer were significantly higher than those in healthy subjects or in patients with ductal carcinoma in situ or non-metastatic invasive ductal carcinoma. Serum ANGPTL2 levels in patients negative for estrogen receptors and progesterone receptors, particularly triple-negative cases, reflected histological grades. Conclusions These findings suggest that serum ANGPTL2 levels in breast cancer patients could represent a potential marker of breast cancer metastasis.

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The Role of Immunoglobulin Superfamily Cell Adhesion Molecules in Cancer Metastasis

International Journal of Cell Biology ◽

10.1155/2012/340296 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 87

Author(s):

Chee Wai Wong ◽

Danielle E. Dye ◽

Deirdre R. Coombe

Keyword(s):

Cell Adhesion ◽

Adhesion Molecules ◽

Cancer Progression ◽

Cell Adhesion Molecules ◽

Cancer Metastasis ◽

Clinical Problem ◽

Metastatic Lesion ◽

Immunoglobulin Superfamily ◽

Metastatic Pathway ◽

Cell Cell

Metastasis is a major clinical problem and results in a poor prognosis for most cancers. The metastatic pathway describes the process by which cancer cells give rise to a metastatic lesion in a new tissue or organ. It consists of interconnecting steps all of which must be successfully completed to result in a metastasis. Cell-cell adhesion is a key aspect of many of these steps. Adhesion molecules belonging to the immunoglobulin superfamily (Ig-SF) commonly play a central role in cell-cell adhesion, and a number of these molecules have been associated with cancer progression and a metastatic phenotype. Surprisingly, the contribution of Ig-SF members to metastasis has not received the attention afforded other cell adhesion molecules (CAMs) such as the integrins. Here we examine the steps in the metastatic pathway focusing on how the Ig-SF members, melanoma cell adhesion molecule (MCAM), L1CAM, neural CAM (NCAM), leukocyte CAM (ALCAM), intercellular CAM-1 (ICAM-1) and platelet endothelial CAM-1 (PECAM-1) could play a role. Although much remains to be understood, this review aims to raise the profile of Ig-SF members in metastasis formation and prompt further research that could lead to useful clinical outcomes.

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A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0157 ◽

2011 ◽

Vol 22 (14) ◽

pp. 2509-2519 ◽

Cited By ~ 44

Author(s):

Jian J. Liu ◽

Rebecca A. Stockton ◽

Alexandre R. Gingras ◽

Ararat J. Ablooglu ◽

Jaewon Han ◽

...

Keyword(s):

Endothelial Cell ◽

Small Gtpases ◽

Cell Junctions ◽

Physical Interaction ◽

Dependent Manner ◽

Cardiovascular Development ◽

Cavernous Malformations ◽

Cell Cell

Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

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The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.

Blood ◽

10.1182/blood.v108.11.1795.1795 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1795-1795

Author(s):

Virginie Deleuze ◽

Elias Chalhoub ◽

Rawan El-Hajj ◽

Christiane Dohet ◽

Mikael Le Clech ◽

...

Keyword(s):

Endothelial Cells ◽

Ectopic Expression ◽

Cell Junctions ◽

Small Interfering Rnas ◽

Hek 293 ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Cell Cell

Abstract The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.

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Direct laser manipulation reveals the mechanics of cell contacts in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418732112 ◽

2015 ◽

Vol 112 (5) ◽

pp. 1416-1421 ◽

Cited By ~ 123

Author(s):

Kapil Bambardekar ◽

Raphaël Clément ◽

Olivier Blanc ◽

Claire Chardès ◽

Pierre-François Lenne

Keyword(s):

Cell Shape ◽

Constitutive Law ◽

Cell Junctions ◽

Scale Model ◽

Tissue Morphogenesis ◽

Cell Contacts ◽

Cell Shape Changes ◽

Shape Changes ◽

Cell Cell

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

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Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel

10.20944/preprints202110.0400.v1 ◽

2021 ◽

Author(s):

Samriddhi Arora ◽

Jyoti Tanwar ◽

Nutan Sharma ◽

Suman Saurav ◽

Rajender K. Motiani

Keyword(s):

Pancreatic Cancer ◽

Survival Time ◽

Cell Lines ◽

Cancer Progression ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Critical Role ◽

Mesenchymal Transition

Pancreatic cancer (PC) is one of the most lethal forms of cancers with 5-year mean survival rate of less than 10%. Most of the PC associated deaths are due to metastasis to secondary sites. Calcium (Ca2+) signaling plays a critical role in regulating hallmarks of cancer progression including cell proliferation, migration and apoptotic resistance. Store operated Ca2+ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous pathway responsible for Ca2+ influx into non-excitable cells. In this study, we performed extensive bioinformatic analysis of publicly available datasets and observed that Orai3 expression is inversely associated with the mean survival time of PC patients. Orai3 expression analysis in a battery of PC cell lines corroborated its differential expression profile. We then carried out thorough Ca2+ imaging experiments in 6 PC cell lines and found that Orai3 forms a functional SOCE in PC cells. Our in vitro functional assays show that Orai3 regulates PC cell cycle progression, apoptosis and migration. Most importantly, our in vivo xenograft studies demonstrate a critical role of Orai3 in PC tumor growth and secondary metastasis. Mechanistically, Orai3 controls G1 phase progression, matrix metalloproteinase expression and epithelial-mesenchymal transition in PC cells. Taken together, this study for the first time reports that Orai3 drives aggressive phenotypes of PC cells i.e. migration in vitro and metastasis in vivo. Considering that Orai3 expression is inversely associated with the PC patients survival time, it appears to be a highly attractive therapeutic target.

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