Publications by authors named "Jose Javier Bravo-Cordero"

29 Publications

  • Page 1 of 1

Actin dynamics during tumor cell dissemination.

Int Rev Cell Mol Biol 2021 24;360:65-98. Epub 2020 Nov 24.

Department of Medicine, Division of Hematology and Oncology, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States. Electronic address:

The actin cytoskeleton is a dynamic network that regulates cellular behavior from development to disease. By rearranging the actin cytoskeleton, cells are capable of migrating and invading during developmental processes; however, many of these cellular properties are hijacked by cancer cells to escape primary tumors and disseminate to distant organs in the body. In this review article, we highlight recent work describing how cancer cells regulate the actin cytoskeleton to achieve efficient invasion and metastatic colonization. We also review new imaging technologies that are capable of revealing the complex architecture and regulation of the actin cytoskeleton during motility and invasion of tumor cells.
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http://dx.doi.org/10.1016/bs.ircmb.2020.09.004DOI Listing
November 2020

Pleckstrin-2 is essential for erythropoiesis in β-thalassemic mice, reducing apoptosis and enhancing enucleation.

Commun Biol 2021 May 3;4(1):517. Epub 2021 May 3.

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.
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http://dx.doi.org/10.1038/s42003-021-02046-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093212PMC
May 2021

Effects of electret coating technology on coronary stent thrombogenicity.

Platelets 2021 Apr 15:1-8. Epub 2021 Apr 15.

Department of Medicine, AtheroThrombosis Research Unit (ATRU), Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, USA.

Stent thrombosis (ST) is a catastrophic event and efforts to reduce its incidence by altering blood-stent interactions are longstanding. A new electret coating technology that produces long-lasting negative charge on stent surface could make them intrinsically resistant to thrombosis. We assessed the thrombogenicity of stents using an annular perfusion model with confocal microscopy, and determined the efficacy of electret coating technology to confer thrombo-resistant properties to standard stents. Using an annular perfusion chamber, Bare Metal Stent (BMS), standard uncoated DES (DES), and Electret-coated DES (e-DES) were exposed to human blood under arterial flow conditions. Deposits of fibrinogen and platelets on the stent surface were analyzed using immunofluorescence staining and confocal microscopy. Surface coverage by fibrinogen and platelets and the deposit/aggregate size were quantified using computerized morphometric analysis. The experimental methodology produced consistent, quantifiable results. Area of stent surface covered by fibrinogen and platelets and the average size of the deposits/aggregates were lowest for e-DES and highest on BMS, with DES in the middle. The size of fibrinogen-deposits showed no differences between the stents. The testing methodology used in our study successfully demonstrated that electret coating confers significant antithrombotic property to DES stents. These findings warrant confirmation in a larger study.
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http://dx.doi.org/10.1080/09537104.2021.1912313DOI Listing
April 2021

Visualizing Cancer.

Cancer Cell 2020 12;38(6):753-756

Imaging has had a profound impact on our ability to understand and treat cancer. We invited some experts to discuss imaging approaches that can be used in various aspects of cancer research, from investigating the complexity and diversity of cancer cells and their environments to guiding clinical decision-making.
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http://dx.doi.org/10.1016/j.ccell.2020.11.014DOI Listing
December 2020

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Cell 2020 10;183(3):786-801.e19

Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.
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http://dx.doi.org/10.1016/j.cell.2020.09.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074872PMC
October 2020

Textures of the tumour microenvironment.

Essays Biochem 2019 10;63(5):619-629

Department of Medicine, Division of Hematology and Oncology, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, U.S.A.

In this review, we present recent findings on the dynamic nature of the tumour microenvironment (TME) and how intravital microscopy studies have defined TME components in a spatiotemporal manner. Intravital microscopy has shed light into the nature of the TME, revealing structural details of both tumour cells and other TME co-habitants in vivo, how these cells communicate with each other, and how they are organized in three-dimensional space to orchestrate tumour growth, invasion, dissemination and metastasis. We will review different imaging tools, imaging reporters and fate-mapping strategies that have begun to uncover the complexity of the TME in vivo.
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http://dx.doi.org/10.1042/EBC20190019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839695PMC
October 2019

Adipocyte-Derived Lipids Mediate Melanoma Progression via FATP Proteins.

Cancer Discov 2018 08 14;8(8):1006-1025. Epub 2018 Jun 14.

Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York.

Advanced, metastatic melanomas frequently grow in subcutaneous tissues and portend a poor prognosis. Though subcutaneous tissues are largely composed of adipocytes, the mechanisms by which adipocytes influence melanoma are poorly understood. Using and models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. Additionally, adipocytes directly transfer lipids to melanoma cells, which alters tumor cell metabolism. Adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAF in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. Pharmacologic blockade of FATPs with the small-molecule inhibitor Lipofermata abrogates lipid transport into melanoma cells and reduces melanoma growth and invasion. These data demonstrate that stromal adipocytes can drive melanoma progression through FATP lipid transporters and represent a new target aimed at interrupting adipocyte-melanoma cross-talk. We demonstrate that stromal adipocytes are donors of lipids that mediate melanoma progression. Adipocyte-derived lipids are taken up by FATP proteins that are aberrantly expressed in melanoma. Inhibition of FATPs decreases melanoma lipid uptake, invasion, and growth. We provide a mechanism for how stromal adipocytes drive tumor progression and demonstrate a novel microenvironmental therapeutic target. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-1371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192670PMC
August 2018

Distant Insulin Signaling Regulates Vertebrate Pigmentation through the Sheddase Bace2.

Dev Cell 2018 06 24;45(5):580-594.e7. Epub 2018 May 24.

Memorial Sloan Kettering Cancer Center, Department of Cancer Biology & Genetics, New York, NY 10065, USA. Electronic address:

Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.
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http://dx.doi.org/10.1016/j.devcel.2018.04.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991976PMC
June 2018

Macrophage-Dependent Cytoplasmic Transfer during Melanoma Invasion In Vivo.

Dev Cell 2017 12;43(5):549-562.e6

Fred Hutchinson Cancer Research Center, Basic Sciences Division, Seattle, WA 98109, USA.

Interactions between tumor cells and tumor-associated macrophages play critical roles in the initiation of tumor cell motility. To capture the cellular interactions of the tumor microenvironment with high-resolution imaging, we directly visualized tumor cells and their interactions with macrophages in zebrafish. Live imaging in zebrafish revealed that macrophages are dynamic, yet maintain sustained contact with tumor cells. In addition, the recruitment of macrophages to tumor cells promotes tumor cell dissemination. Using a Cre/LoxP strategy, we found that macrophages transfer cytoplasm to tumor cells in zebrafish and mouse models. Remarkably, macrophage cytoplasmic transfer correlated with melanoma cell dissemination. We further found that macrophages transfer cytoplasm to tumor cells upon cell contact in vitro. Thus, we present a model in which macrophage/tumor cell contact allows for the transfer of cytoplasmic molecules from macrophages to tumor cells corresponding to increased tumor cell motility and dissemination.
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http://dx.doi.org/10.1016/j.devcel.2017.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728704PMC
December 2017

Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation.

J Cell Biol 2017 12 23;216(12):4331-4349. Epub 2017 Oct 23.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and βPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.
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http://dx.doi.org/10.1083/jcb.201704048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716284PMC
December 2017

2D and 3D Matrices to Study Linear Invadosome Formation and Activity.

J Vis Exp 2017 06 2(124). Epub 2017 Jun 2.

INSERM U1053;

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.
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http://dx.doi.org/10.3791/54911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608231PMC
June 2017

Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments.

Nat Cell Biol 2017 02 23;19(2):120-132. Epub 2017 Jan 23.

Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029, USA.

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1/DEC2/p27/TGFβ2 and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1 expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER breast cancer cells, post-hypoxic ER breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.
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http://dx.doi.org/10.1038/ncb3465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342902PMC
February 2017

Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena-initiated invadopodium formation.

Sci Rep 2016 11 30;6:37874. Epub 2016 Nov 30.

Department of Anatomy and Structural Biology Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, United States.

The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/Mena signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell - macrophage interaction results in the upregulation of Mena through the activation of MENA transcription. Notch1 and Mena expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of Mena expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.
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http://dx.doi.org/10.1038/srep37874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129016PMC
November 2016

Rho GTPase isoforms in cell motility: Don't fret, we have FRET.

Cell Adh Migr 2014 ;8(6):526-34

a Department of Anatomy and Structural Biology ; Albert Einstein College of Medicine of Yeshiva University ; Bronx , NY USA.

The Rho-family of p21 small GTPases are directly linked to the regulation of actin-based motile machinery and play a key role in the control of cell migration. Aside from the original and most well-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerous isoforms of these key proteins have been identified and shown to have specific roles in regulating various cellular motility processes. The major difficulty in addressing these isoform-specific effects is that isoforms typically contain highly similar primary amino acid sequences and thus are able to interact with the same upstream regulators and the downstream effector targets. Here, we will introduce the major members of each GTPase subfamily and discuss recent advances in the design and application of fluorescent resonance energy transfer-based probes, which are at the forefront of the technologies available to directly probe the differential, spatiotemporal activation dynamics of these proteins in live single cells. Currently, it is possible to specifically detect the activation status of RhoA vs. RhoC isoforms, as well as Cdc42 vs. TC-10 isoforms in living cells. Clearly, additional efforts are still required to produce biosensor systems capable of detecting other isoforms of Rho GTPases including RhoB, Rac2/3, RhoG, etc. Through such efforts, we will uncover the isoform-specific roles of these near-identical proteins in living cells, clearly an important area of the Rho GTPase biology that is not yet fully appreciated.
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http://dx.doi.org/10.4161/cam.29712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594258PMC
July 2015

Talin regulates moesin-NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis.

J Cell Biol 2014 Jun 2;205(5):737-51. Epub 2014 Jun 2.

Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin-NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.
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http://dx.doi.org/10.1083/jcb.201312046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050723PMC
June 2014

A Trio-Rac1-Pak1 signalling axis drives invadopodia disassembly.

Nat Cell Biol 2014 Jun 25;16(6):574-86. Epub 2014 May 25.

Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.

Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, we developed a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. We describe here an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.
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http://dx.doi.org/10.1038/ncb2972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083618PMC
June 2014

Spatial regulation of tumor cell protrusions by RhoC.

Cell Adh Migr 2014 ;8(3):263-7

Department of Anatomy and Structural Biology; Albert Einstein College of Medicine of Yeshiva University; Bronx, NY USA; Gruss Lipper Biophotonics Center; Albert Einstein College of Medicine of Yeshiva University; Bronx, NY USA.

Systemic metastasis is the dissemination of cancer cells from the primary tumor to distant organs and is the primary cause of death in cancer patients. How do cancer cells leave the primary tumor mass? The ability of the tumor cells to form different types of actin-rich protrusions including invasive protrusions (invadopodia) and locomotory protrusions (lamellipodia [2D] or pseudopodia [3D]), facilitate the invasion and dissemination of the tumor cells. Rho-family of p21 small GTPases plays a direct role in regulating the actin dynamics in these intracellular compartments. Recent studies have shown that the signaling molecules including RhoC/p190RhoGEF/p190RhoGAP acts as a "molecular compass" in order to direct the spatial and temporal dynamics of the formation of these invasive and locomotory protrusions leading to efficient invasion.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198350PMC
http://dx.doi.org/10.4161/cam.28405DOI Listing
July 2015

Quantitative ratiometric imaging of FRET-biosensors in living cells.

Methods Cell Biol 2013 ;114:593-609

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, USA.

Biosensors based on FRET have been useful in deciphering the dynamics of protein activation events in living cells at subcellular resolutions and in time scales of seconds. These new systems allow observations of dynamic processes which were not possible previously using more traditional biochemical and cell biological approaches. The image data sets obtained from these sensors require careful processing in order to represent the actual protein activation events. Here, we will cover the basic approaches useful for processing the raw image data sets into relativistic ratiometric measurements, capable of depicting relative differences in the protein activation states within a single cell. We will discuss in detail the approaches for genetically encoded, single-chain biosensor systems based on FRET, as well as those that are based on intermolecular, dual-chain design. Additionally, the same analysis can be utilized for biosensor systems using solvatochromic dyes (Nalbant, Hodgson, Kraynov, Toutchkine, & Hahn, 2004), useful for detection of endogenous protein activation states.
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http://dx.doi.org/10.1016/B978-0-12-407761-4.00025-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789067PMC
February 2014

Live cell imaging of RhoGTPase biosensors in tumor cells.

Methods Mol Biol 2013 ;1046:359-70

Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA.

Tumor cell motility and invasion rely on actin cytoskeleton rearrangements mediated by the activation of RhoGTPase signaling pathways. Invadopodia are membrane-degrading protrusions that mediate extracellular matrix degradation. Here, we provide procedures for imaging RhoGTPase biosensors in tumor cells during the formation of invadopodia and matrix degradation.
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http://dx.doi.org/10.1007/978-1-62703-538-5_22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784404PMC
February 2014

Functions of cofilin in cell locomotion and invasion.

Nat Rev Mol Cell Biol 2013 Jul 19;14(7):405-15. Epub 2013 Jun 19.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

Recently, a consensus has emerged that cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization without changing the rate of G-actin association and dissociation at either filament end. The structural basis of actin filament severing by cofilin is now better understood. These results have been integrated with recently discovered mechanisms for cofilin activation in migrating cells, which led to new models for cofilin function that provide insights into how cofilin regulation determines the temporal and spatial control of cell behaviour.
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http://dx.doi.org/10.1038/nrm3609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878614PMC
July 2013

Spatial regulation of RhoC activity defines protrusion formation in migrating cells.

J Cell Sci 2013 Aug 23;126(Pt 15):3356-69. Epub 2013 May 23.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.

Protrusion formation is the first step that precedes cell movement of motile cells. Spatial control of actin polymerization is necessary to achieve directional protrusion during cell migration. Here we show that the spatial coordinators p190RhoGEF and p190RhoGAP regulate actin polymerization during leading edge protrusions by regulating the actin barbed end distribution and amplitude. The distribution of RhoC activity and proper balance of cofilin activation achieved by p190RhoGEF and p190RhoGAP determines the direction of final protrusive activity. These findings provide a new insight into the dynamic plasticity in the amplitude and distribution of barbed ends, which can be modulated by fine-tuning RhoC activity by upstream GEFs and GAPs for directed cell motility.
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http://dx.doi.org/10.1242/jcs.123547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730245PMC
August 2013

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors.

Intravital 2013 Apr;2(2):e25294

Department of Anatomy and Structural Biology; Albert Einstein College of Medicine; Bronx, NY USA ; Gruss Lipper Biophotonics Center; Albert Einstein College of Medicine; Bronx, NY USA.

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.
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http://dx.doi.org/10.4161/intv.25294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908591PMC
April 2013

Directed cell invasion and migration during metastasis.

Curr Opin Cell Biol 2012 Apr 30;24(2):277-83. Epub 2011 Dec 30.

Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, United States.

Metastasis requires tumor cell dissemination to different organs from the primary tumor. Dissemination is a complex cell motility phenomenon that requires the molecular coordination of the protrusion, chemotaxis, invasion and contractility activities of tumor cells to achieve directed cell migration. Recent studies of the spatial and temporal activities of the small GTPases have begun to elucidate how this coordination is achieved. The direct visualization of the pathways involved in actin polymerization, invasion and directed migration in dissemination competent tumor cells will help identify the molecular basis of dissemination and allow the design and testing of more specific and selective drugs to block metastasis.
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http://dx.doi.org/10.1016/j.ceb.2011.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320684PMC
April 2012

Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway.

J Cell Biol 2011 Nov 21;195(5):903-20. Epub 2011 Nov 21.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.

Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin-cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium-hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion.
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http://dx.doi.org/10.1083/jcb.201103045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257566PMC
November 2011

A novel spatiotemporal RhoC activation pathway locally regulates cofilin activity at invadopodia.

Curr Biol 2011 Apr 7;21(8):635-44. Epub 2011 Apr 7.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.

Background: RhoGTPases have been implicated in the regulation of cancer metastasis. Invasive carcinoma cells form invadopodia, F-actin-rich matrix-degrading protrusions that are thought to be important for tumor cell invasion and intravasation. Regulation of actin dynamics at invadopodial protrusions is crucial to drive invasion. This process requires the severing activity of cofilin to generate actin-free barbed ends. Previous work demonstrates that cofilin's severing activity is tightly regulated through multiple mechanisms, including regulation of cofilin serine phosphorylation by Rho GTPases. However, it is not known which Rho GTPase is involved in regulating cofilin's phosphorylation status at invadopodia.

Results: We show here, for the first time, how RhoC activation is controlled at invadopodia and how this activation regulates cofilin phosphorylation to control cofilin's generation of actin-free barbed ends. Live-cell imaging of fluorescent RhoC biosensor reveals that RhoC activity is spatially confined to areas surrounding invadopodia. This spatiotemporal restriction of RhoC activity is controlled by "spatially distinct regulatory elements" that confine RhoC activation within this compartment. p190RhoGEF localizes around invadopodia to activate RhoC, whereas p190RhoGAP localizes inside invadopodia to deactivate the GTPase within the structure. RhoC activation enhances cofilin phosphorylation outside invadopodia.

Conclusion: These results show how RhoC activity is spatially regulated at invadopodia by p190RhoGEF and p190RhoGAP. RhoC activation in areas surrounding invadopodia restricts cofilin activity to within the invadopodium core, resulting in a focused invadopodial protrusion. This mechanism likely enhances tumor cell invasion during metastasis.
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http://dx.doi.org/10.1016/j.cub.2011.03.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081966PMC
April 2011

An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion.

Cancer Res 2011 Mar 21;71(5):1730-41. Epub 2011 Jan 21.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.

Invasive carcinoma cells use specialized actin polymerization-driven protrusions called invadopodia to degrade and possibly invade through the extracellular matrix (ECM) during metastasis. Phosphorylation of the invadopodium protein cortactin is a master switch that activates invadopodium maturation and function. Cortactin was originally identified as a hyperphosphorylated protein in v-Src-transformed cells, but the kinase or kinases that are directly responsible for cortactin phosphorylation in invadopodia remain unknown. In this study, we provide evidence that the Abl-related nonreceptor tyrosine kinase Arg mediates epidermal growth factor (EGF)-induced cortactin phosphorylation, triggering actin polymerization in invadopodia, ECM degradation, and matrix proteolysis-dependent tumor cell invasion. Both Src and Arg localize to invadopodia and are required for EGF-induced actin polymerization. Notably, Arg overexpression in Src knockdown cells can partially rescue actin polymerization in invadopodia while Src overexpression cannot compensate for loss of Arg, arguing that Src indirectly regulates invadopodium maturation through Arg activation. Our findings suggest a novel mechanism by which an EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Furthermore, they identify Arg as a novel mediator of invadopodia function and a candidate therapeutic target to inhibit tumor invasion in vivo.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-1432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057139PMC
March 2011

Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia.

J Cell Sci 2010 Nov;123(Pt 21):3662-73

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells enriched in proteins that regulate actin polymerization. The on-off regulatory switch that initiates actin polymerization in invadopodia requires phosphorylation of tyrosine residues 421, 466, and 482 on cortactin. However, it is unknown which of these cortactin tyrosine phosphorylation sites control actin polymerization. We investigated the contribution of individual tyrosine phosphorylation sites (421, 466, and 482) on cortactin to the regulation of actin polymerization in invadopodia. We provide evidence that the phosphorylation of tyrosines 421 and 466, but not 482, is required for the generation of free actin barbed ends in invadopodia. In addition, these same phosphotyrosines are important for Nck1 recruitment to invadopodia via its SH2 domain, for the direct binding of Nck1 to cortactin in vitro, and for the FRET interaction between Nck1 and cortactin in invadopodia. Furthermore, matrix proteolysis-dependent tumor cell invasion is dramatically inhibited in cells expressing a mutation in phosphotyrosine 421 or 466. Together, these results identify phosphorylation of tyrosines 421 and 466 on cortactin as the crucial residues that regulate Nck1-dependent actin polymerization in invadopodia and tumor cell invasion, and suggest that specifically blocking either tyrosine 421 or 466 phosphorylation might be effective at inhibiting tumor cell invasion in vivo.
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http://dx.doi.org/10.1242/jcs.068163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037016PMC
November 2010

Novel lambda FRET spectral confocal microscopy imaging method.

Microsc Res Tech 2009 Jan;72(1):1-11

Biotechnology Programme, Confocal Microscopy and Cytometry Unit, Centro Nacional de Investigaciones Oncológicas (CNIO), C/ Melchor Fernández Almagro 3, Madrid E-28029, Spain.

We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (lambdaFRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. LambdaFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. LambdaFRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, lambdaFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The lambdaFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies.
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http://dx.doi.org/10.1002/jemt.20633DOI Listing
January 2009