Publications by authors named "Ilkka Paatero"

24 Publications

  • Page 1 of 1

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling.

Curr Biol 2021 Sep 29. Epub 2021 Sep 29.

Turku Bioscience Centre, University of Turku and Åbo Akademi University, Tykistökatu 6, Turku 20520, Finland; Department of Life Technologies, University of Turku, Tykistökatu 6, Turku 20520, Finland. Electronic address:

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.
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http://dx.doi.org/10.1016/j.cub.2021.09.022DOI Listing
September 2021

Combined genetic and chemical screens indicate protective potential for EGFR inhibition to cardiomyocytes under hypoxia.

Sci Rep 2021 08 17;11(1):16661. Epub 2021 Aug 17.

Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20014, Turku, Finland.

The return of blood flow to ischemic heart after myocardial infarction causes ischemia-reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia-reperfusion injury. Here we screened for targets for the treatment of ischemia-reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein-protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction.
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http://dx.doi.org/10.1038/s41598-021-96033-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371130PMC
August 2021

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

Development 2021 08 12;148(15). Epub 2021 Aug 12.

Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.
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http://dx.doi.org/10.1242/dev.197509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380458PMC
August 2021

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells.

Cell Mol Life Sci 2021 Aug 21;78(15):5827-5846. Epub 2021 Jun 21.

Minerva Foundation Institute for Medical Research, Biomedicum Helsinki 2U, Tukholmankatu 8, 00290, Helsinki, Finland.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.
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http://dx.doi.org/10.1007/s00018-021-03880-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316191PMC
August 2021

A feed-forward loop between SorLA and HER3 determines heregulin response and neratinib resistance.

Oncogene 2021 02 8;40(7):1300-1317. Epub 2021 Jan 8.

Turku Bioscience Centre, University of Turku and Åbo Akademi University, FI-20520, Turku, Finland.

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.
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http://dx.doi.org/10.1038/s41388-020-01604-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892347PMC
February 2021

Genetic and functional implications of an exonic TRIM55 variant in heart failure.

J Mol Cell Cardiol 2020 01 19;138:222-233. Epub 2019 Dec 19.

Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Biomedicum 2U, Tukholmankatu 8, FI-00290 Helsinki, Finland; Department of Mathematics and Statistics, University of Turku, Vesilinnantie 5, FI-20014 Turku, Finland. Electronic address:

Background: To tackle the missing heritability of sporadic heart failure, we screened for novel heart failure-associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo.

Methods And Results: Heart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/- cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wildtype genotype or overexpression of WT TRIM55, respectively.

Conclusions: A previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.
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http://dx.doi.org/10.1016/j.yjmcc.2019.12.008DOI Listing
January 2020

SORLA regulates endosomal trafficking and oncogenic fitness of HER2.

Nat Commun 2019 05 28;10(1):2340. Epub 2019 May 28.

Turku Bioscience Centre, University of Turku and Åbo Akademi University, FI-20520, Turku, Finland.

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.
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http://dx.doi.org/10.1038/s41467-019-10275-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538630PMC
May 2019

GGA2 and RAB13 promote activity-dependent β1-integrin recycling.

J Cell Sci 2019 06 7;132(11). Epub 2019 Jun 7.

Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku FIN-20520, Finland

β1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes, including cancer cell invasion. Here, we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with β1-integrin, and its role in recycling of active but not inactive β1-integrin receptors. Silencing of GGA2 limits active β1-integrin levels in focal adhesions and decreases cancer cell migration and invasion, which is in agreement with its ability to regulate the dynamics of active integrins. By using the proximity-dependent biotin identification (BioID) method, we identified two RAB family small GTPases, i.e. RAB13 and RAB10, as novel interactors of GGA2. Functionally, RAB13 silencing triggers the intracellular accumulation of active β1-integrin, and reduces integrin activity in focal adhesions and cell migration similarly to GGA2 depletion, indicating that both facilitate active β1-integrin recycling to the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.
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http://dx.doi.org/10.1242/jcs.233387DOI Listing
June 2019

An unbiased screen for activating epidermal growth factor receptor mutations.

J Biol Chem 2019 06 5;294(24):9377-9389. Epub 2019 Apr 5.

From the Institute of Biomedicine and Medicity Research Laboratories and

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites, yet only a few of these mutations have been functionally tested. Here, we describe an unbiased platform for the functional characterization of thousands of variants of a single receptor tyrosine kinase (RTK) gene in a single assay. Our een for ctivating utations (iSCREAM) platform enabled rapid analysis of mutations conferring gain-of-function RTK activity promoting clonal growth. The screening strategy included a somatic model of cancer evolution and utilized a library of 7,216 randomly mutated epidermal growth factor receptor () single-nucleotide variants that were tested in murine lymphoid Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for growth, but this dependence can be compensated by ectopic EGFR overexpression, enabling selection for gain-of-function mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel activating variant that structurally stabilized the EGFR kinase dimer interface and conferred sensitivity to kinase inhibition by afatinib. As proof of concept for our approach, we recapitulated clinical observations and identified the EGFR L858R as the major enriched EGFR variant. Altogether, iSCREAM enabled robust enrichment of 21 variants from a total of 7,216 mutations. These findings indicate the power of this screening platform for unbiased identification of activating RTK variants that are enriched under selection pressure in a model of cancer heterogeneity and evolution.
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http://dx.doi.org/10.1074/jbc.RA118.006336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579474PMC
June 2019

ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos.

Mol Biol Cell 2019 01 21;30(2):209-218. Epub 2018 Nov 21.

Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland.

Tyrosine kinase inhibitors are widely used in the clinic, but limited information is available about their toxicity in developing organisms. Here, we tested the effect of tyrosine kinase inhibitors targeting the ErbB receptors for their effects on developing zebrafish ( Danio rerio) embryos. Embryos treated with wide-spectrum pan-ErbB inhibitors or erbb4a-targeting antisense oligonucleotides demonstrated reduced locomotion, reduced diameter of skeletal muscle fibers, and reduced expression of muscle-specific genes, as well as reduced motoneuron length. The phenotypes in the skeletal muscle, as well as the defect in motility, were rescued both by microinjection of human ERBB4 mRNA and by transposon-mediated muscle-specific ERBB4 overexpression. The role of ErbB4 in regulating motility was further controlled by targeted mutation of the endogenous erbb4a locus in the zebrafish genome by CRISPR/Cas9. These observations demonstrate a potential for the ErbB tyrosine kinase inhibitors to induce neuromuscular toxicity in a developing organism via a mechanism involving inhibition of ErbB4 function.
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http://dx.doi.org/10.1091/mbc.E18-07-0460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589560PMC
January 2019

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target.

BMC Cardiovasc Disord 2018 10 20;18(1):196. Epub 2018 Oct 20.

Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520, Turku, Finland.

Background: Receptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.

Methods: RTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass, was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.

Results: In addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.

Conclusions: Several novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.
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http://dx.doi.org/10.1186/s12872-018-0933-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196006PMC
October 2018

Zebrafish Embryo Xenograft and Metastasis Assay.

Bio Protoc 2018 Sep 20;8(18):e3027. Epub 2018 Sep 20.

Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland.

Xenograft models, and in particular the mouse xenograft model, where human cancer cells are transplanted into immunocompromised mice, have been used extensively in cancer studies. Although these models have contributed enormously to our understanding of cancer biology, the zebrafish xenograft model offers several advantages over the mouse model. Zebrafish embryos can be easily cultured in large quantities, are small and easy to handle, making it possible to use a high number of embryos for each experimental condition. Young embryos lack an efficient immune system. Therefore the injected cancer cells are not rejected, and the formation of primary tumors and micrometastases is rapid. Transparency of the embryos enables imaging of primary tumors and metastases in an intact and living embryo. Here we describe a method where GFP expressing tumor cells are injected into pericardial space of zebrafish embryos. At four days post-injection, the embryos are imaged and the formation of primary tumor and distant micrometastases are analyzed.
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http://dx.doi.org/10.21769/BioProtoc.3027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328589PMC
September 2018

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction.

Nat Commun 2018 08 31;9(1):3545. Epub 2018 Aug 31.

Department of Cell Biology, Biozentrum, University of Basel, Basel, 4056, Switzerland.

Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation-similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements.
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http://dx.doi.org/10.1038/s41467-018-05851-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119192PMC
August 2018

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation.

Elife 2018 05 1;7. Epub 2018 May 1.

Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland.

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and β1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident β1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype.
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http://dx.doi.org/10.7554/eLife.32490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929907PMC
May 2018

Analyses in zebrafish embryos reveal that nanotoxicity profiles are dependent on surface-functionalization controlled penetrance of biological membranes.

Sci Rep 2017 08 21;7(1):8423. Epub 2017 Aug 21.

Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, FI-20520, Turku, Finland.

Mesoporous silica nanoparticles (MSNs) are extensively explored as drug delivery systems, but in depth understanding of design-toxicity relationships is still scarce. We used zebrafish (Danio rerio) embryos to study toxicity profiles of differently surface functionalized MSNs. Embryos with the chorion membrane intact, or dechoroniated embryos, were incubated or microinjected with amino (NH-MSNs), polyethyleneimine (PEI-MSNs), succinic acid (SUCC-MSNs) or polyethyleneglycol (PEG-MSNs) functionalized MSNs. Toxicity was assessed by viability and cardiovascular function. NH-MSNs, SUCC-MSNs and PEG-MSNs were well tolerated, 50 µg/ml PEI-MSNs induced 100% lethality 48 hours post fertilization (hpf). Dechoroniated embryos were more sensitive and 10 µg/ml PEI-MSNs reduced viability to 5% at 96hpf. Sensitivity to PEG- and SUCC-, but not NH-MSNs, was also enhanced. Typically cardiovascular toxicity was evident prior to lethality. Confocal microscopy revealed that PEI-MSNs penetrated into the embryos whereas PEG-, NH2- and SUCC-MSNs remained aggregated on the skin surface. Direct exposure of inner organs by microinjecting NH-MSNs and PEI-MSNs demonstrated that the particles displayed similar toxicity indicating that functionalization affects the toxicity profile by influencing penetrance through biological barriers. The data emphasize the need for careful analyses of toxicity mechanisms in relevant models and constitute an important knowledge step towards the development of safer and sustainable nanotherapies.
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http://dx.doi.org/10.1038/s41598-017-09312-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566213PMC
August 2017

FiloQuant reveals increased filopodia density during breast cancer progression.

J Cell Biol 2017 10 1;216(10):3387-3403. Epub 2017 Aug 1.

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland

Defective filopodia formation is linked to pathologies such as cancer, wherein actively protruding filopodia, at the invasive front, accompany cancer cell dissemination. Despite wide biological significance, delineating filopodia function in complex systems remains challenging and is particularly hindered by lack of compatible methods to quantify filopodia properties. Here, we present FiloQuant, a freely available ImageJ plugin, to detect filopodia-like protrusions in both fixed- and live-cell microscopy data. We demonstrate that FiloQuant can extract quantifiable information, including protrusion dynamics, density, and length, from multiple cell types and in a range of microenvironments. In cellular models of breast ductal carcinoma in situ, we reveal a link between filopodia formation at the cell-matrix interface, in collectively invading cells and 3D tumor spheroids, and the in vitro invasive capacity of the carcinoma. Finally, using intravital microscopy, we observe that tumor spheroids display filopodia in vivo, supporting a potential role for these protrusions during tumorigenesis.
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http://dx.doi.org/10.1083/jcb.201704045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626550PMC
October 2017

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

PLoS One 2015 29;10(12):e0145684. Epub 2015 Dec 29.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Background: Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.

Methodology/principal Findings: We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.

Conclusions/significance: Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145684PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694698PMC
July 2016

Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts.

PLoS One 2015 15;10(6):e0130340. Epub 2015 Jun 15.

Section of Genetics and Physiology, Department of Biology, University of Turku, 20500 Turku, Finland.

Background And Methods: Pim family proteins are oncogenic kinases implicated in several types of cancer and involved in regulation of cell proliferation, survival as well as motility. Here we have investigated the ability of Pim kinases to promote metastatic growth of prostate cancer cells in two xenograft models for human prostate cancer. We have also evaluated the efficacy of Pim-selective inhibitors to antagonize these effects.

Results: We show here that tumorigenic growth of both subcutaneously and orthotopically inoculated prostate cancer xenografts is enhanced by stable overexpression of either Pim-1 or Pim-3. Moreover, Pim-overexpressing orthotopic prostate tumors are highly invasive and able to migrate not only to the nearby prostate-draining lymph nodes, but also into the lungs to form metastases. When the xenografted mice are daily treated with the Pim-selective inhibitor DHPCC-9, both the volumes as well as the metastatic capacity of the tumors are drastically decreased. Interestingly, the Pim-promoted metastatic growth of the orthotopic xenografts is associated with enhanced angiogenesis and lymphangiogenesis. Furthermore, forced Pim expression also increases phosphorylation of the CXCR4 chemokine receptor, which may enable the tumor cells to migrate towards tissues such as the lungs that express the CXCL12 chemokine ligand.

Conclusions: Our results indicate that Pim overexpression enhances the invasive properties of prostate cancer cells in vivo. These effects can be reduced by the Pim-selective inhibitor DHPCC-9, which can reach tumor tissues without serious side effects. Thus, Pim-targeting therapies with DHPCC-9-like compounds may help to prevent progression of local prostate carcinomas to fatally metastatic malignancies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130340PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467846PMC
May 2016

Hypoxia-inducible factor-1α induces ErbB4 signaling in the differentiating mammary gland.

J Biol Chem 2014 Aug 25;289(32):22459-69. Epub 2014 Jun 25.

From the Department of Medical Biochemistry and Genetics, and MediCity Research Laboratory, University of Turku, 20520 Turku, Finland, the Department of Oncology, Turku University Hospital, 20520 Turku, Finland

Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.
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http://dx.doi.org/10.1074/jbc.M113.533497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139252PMC
August 2014

CYT-1 isoform of ErbB4 is an independent prognostic factor in serous ovarian cancer and selectively promotes ovarian cancer cell growth in vitro.

Gynecol Oncol 2013 Apr 9;129(1):179-87. Epub 2013 Jan 9.

Department of Medical Biochemistry and Genetics, and MediCity Research Laboratory, University of Turku, Turku, Finland.

Objective: ErbB4 is a member of the ErbB subfamily of receptor tyrosine kinases with a poorly understood biological role in ovarian cancer. Here, we have addressed the expression, subcellular localization, and prognostic relevance of ErbB4 and its alternatively spliced isoforms in serous ovarian adenocarcinoma.

Methods: A tissue microarray including 482 samples was analyzed by immunohistochemistry, and a series of 198 samples by isoform-specific real-time RT-PCR. The data were statistically analyzed for associations with clinicopathological markers and survival. The functional effect of expressing the relevant ErbB4 isoforms in ovarian cancer cells was addressed by measuring colony formation in soft agar.

Results: While ErbB4 immunoreactivity was present in 90% of the samples, total ErbB4 protein expression was not significantly associated with prognostic markers. However, real-time RT-PCR analysis of serous ovarian cancer samples indicated the presence of two alternatively spliced cytoplasmic isoforms of ERBB4, CYT-1 and CYT-2, previously demonstrated to mediate significantly different cellular activities. Expression of CYT-1, but not of CYT-2, was significantly associated with tumor grade (P=0.014) and poor overall survival (P=0.0028). CYT-1 expression was also an independent prognostic factor (P=0.021) in multivariate analysis of survival. Consistent with a biological effect specific for the one isoform, overexpression of ErbB4 CYT-1, but not of ErbB4 CYT-2, increased anchorage-independent growth of ovarian adenocarcinoma cells in vitro.

Conclusions: These results suggest that expression of a specific ErbB4 isoform, CYT-1, is associated with poor survival and enhanced growth in serous ovarian cancer.
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http://dx.doi.org/10.1016/j.ygyno.2012.12.044DOI Listing
April 2013

Interaction with ErbB4 promotes hypoxia-inducible factor-1α signaling.

J Biol Chem 2012 Mar 3;287(13):9659-9671. Epub 2012 Feb 3.

Department of Medical Biochemistry and Genetics, and MediCity Research Laboratory, University of Turku, FI-20520 Turku, Finland,; Department of Oncology, Turku University Hospital, FI-20520 Turku, Finland. Electronic address:

The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.
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http://dx.doi.org/10.1074/jbc.M111.299537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322979PMC
March 2012

ErbB4 and its isoforms: patentable drug targets?

Recent Pat DNA Gene Seq 2008 ;2(1):27-33

Department of Medical Biochemistry and Molecular Biology, and Medicity Research Laboratory, University of Turku, Finland.

Research on ErbB receptors has spearheaded the rational cancer drug design, and ErbB1 (also known as EGFR) and ErbB2 are among the first clinically validated targeted therapies. Despite the fact that applicability of ErbB4 as a drug target is still uncertain, several patents involving utilization of ErbB4 have recently been issued. Manipulation of functions of ErbB4 may be therapeutically beneficial in cancer but also in psychiatric and cardiovascular disorders. In addition, analyzing expression or mutations of ErbB4 may provide prognostic or predictive value. Contents of ErbB4-related patents, as well as biology of ErbB4 and its alternatively spliced isoforms, will be reviewed in this article.
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http://dx.doi.org/10.2174/187221508783406602DOI Listing
May 2009

Isoform-specific monoubiquitination, endocytosis, and degradation of alternatively spliced ErbB4 isoforms.

Proc Natl Acad Sci U S A 2008 Mar 11;105(11):4162-7. Epub 2008 Mar 11.

MediCity Research Laboratory and Department of Medical Biochemistry and Molecular Biology, University of Turku, FIN-20520 Turku, Finland.

Endocytosis and subsequent lysosomal degradation serve as a well characterized mechanism to fine-tune and down-regulate EGFR signaling. However, other members of the EGFR/ErbB receptor family have been reported to be endocytosis-impaired. Here we demonstrate that endocytosis of ErbB4 is regulated in an isoform-specific manner: CYT-1 isoforms were efficiently endocytosed whereas CYT-2 isoforms were endocytosis-impaired. CYT-1 isoforms in endocytic vesicles colocalized with Rab5 and Rab7 indicating trafficking via early endosomes to late endosomal/lysosomal structures. A PPXY motif within the CYT-1-specific sequence that lacks from CYT-2 was necessary both for ubiquitination and endocytosis of CYT-1 isoforms and provided a binding site for a WW domain-containing ubiquitin ligase Itch. Itch catalyzed ubiquitination of ErbB4 CYT-1, promoted its localization into intracellular vesicles, and stimulated degradation of ErbB4 CYT-1. Dominant negative Itch suppressed ErbB4 CYT-1 endocytosis and degradation. These data indicate that ErbB4 isoforms differ in endocytosis and degradation by a mechanism mediated by CYT-1-specific PPXY motif interacting with a WW domain-containing E3 ubiquitin ligase.
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http://dx.doi.org/10.1073/pnas.0708333105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2393780PMC
March 2008

Intra- and extracellular signaling by endothelial neuregulin-1.

Exp Cell Res 2007 Aug 18;313(13):2896-909. Epub 2007 Apr 18.

Department of Medical Biochemistry and Molecular Biology, and Medicity Research Laboratories, University of Turku, Turku, Finland.

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.
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http://dx.doi.org/10.1016/j.yexcr.2007.03.042DOI Listing
August 2007
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