Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin

Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1‐dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluro...

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Published in:	The Journal of pathology Vol. 234; no. 3; pp. 351 - 364
Main Authors:	Meier, Claudia, Spitschak, Alf, Abshagen, Kerstin, Gupta, Shailendra, Mor, Joel M., Wolkenhauer, Olaf, Haier, Jörg, Vollmar, Brigitte, Alla, Vijay, Pützer, Brigitte M.
Format:	Journal Article
Language:	English
Published:	Chichester, UK John Wiley & Sons, Ltd 01-11-2014 Wiley Subscription Services, Inc
Subjects:	Animals Blotting, Western Cell Line, Tumor Chromatin Immunoprecipitation docking analyses E2F1 transcription factor E2F1 Transcription Factor - metabolism Enzyme-Linked Immunosorbent Assay Extracellular Matrix Proteins - metabolism extravasation feed-forward loop fibronectin Fibronectins - biosynthesis Fluorescent Antibody Technique Gene Expression Regulation, Neoplastic - physiology Heterografts human tissues Humans Hyaluronan Receptors - metabolism Immunoprecipitation Mice Neoplasm Invasiveness - physiopathology Neoplastic Cells, Circulating - metabolism Reverse Transcriptase Polymerase Chain Reaction RHAMM Transcription, Genetic Up-Regulation feed-forward loop extravasation E2F1 transcription factor docking analyses fibronectin human tissues RHAMM
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Summary:	Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1‐dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluronan‐mediated motility receptor RHAMM. E2F1 directly up‐regulates RHAMM, which in turn acts as a co‐activator of E2F1 to stimulate expression of the extracellular matrix protein fibronectin. Enhanced fibronectin secretion links E2F1/RHAMM transcriptional activity to integrin‐β1–FAK signalling associated with cytoskeletal remodelling and enhanced tumour cell motility. RHAMM depletion abolishes fibronectin expression and cell transmigration across the endothelial layer in E2F1‐activated cells. In a xenograft model, knock‐down of E2F1 or RHAMM in metastatic cells protects the liver parenchyma of mice against extravasation of CTCs, whereas the number of transmigrated cells increases in response to E2F1 induction. Expression data from clinical tissue samples reveals high E2F1 and RHAMM levels that closely correlate with malignant progression. These findings suggest a requirement for RHAMM in late‐stage metastasis by a mechanism involving cooperative stimulation of fibronectin, with a resultant tumourigenic microenvironment important for enhanced extravasation and distant organ colonization. Therefore, stimulation of the E2F1–RHAMM axis in aggressive cancer cells is of high clinical significance. Targeting RHAMM may represent a promising approach to avoid E2F1‐mediated metastatic dissemination. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Bibliography:	ArticleID:PATH4400 Figure S1 Immunofluorescence microscopy of RHAMM: green, RHAMM; blue, DAPI; scale bar = 10 µmFigure S2 Functional comparability analysis of different shRNAs against E2F1 and RHAMM in SK-Mel-147 cells. (A) TRITC-phalloidin staining of the actin cytoskeleton in cells depleted for E2F1 (shE2F#2, #3; left) and RHAMM (shRHAMM#2, #3; right); scale bar = 10 µm. (B) Effect of #2 and #3 shRNAs on cell migration; bar graphs represent mean ± SD; ***p < 0.001. (C) Immunoblotting of FN expression after silencing of E2F1 (left) or RHAMM (right); RHAMM, E2F1 and actin levels are indicatedFigure S3 In silico protein interaction analyses. (A) 3D model of E2F1 and RHAMM; proteins are shown as solid ribbon model, which is coloured on the basis of position of amino acid residues from N-terminus (blue) to C-terminus (red). (B) Interaction pose between E2F1 (surface-coloured based on atom charges) and RHAMM (green); amino acid residues of E2F1 involved in DNA binding domain are marked as red ball and stickFigure S4 Luciferase activities in H1299 cells measured 24 h after co-transfection of TP73 promoter construct with RHAMM, E2F1 or both; bar graphs show results from three independent experiments as relative software units; data represent mean ± SDFigure S5 Dependency of integrin-β1 activation on E2F1 and RHAMM. (A) Western blot of serum-starved SK-Mel-147 cells treated with 500 µm MnCl2 for 5 min served as positive control for active integrin-β1. (B) Immunofluorescence staining of total and activated integrin-β1 in E2F1- or RHAMM-depleted SK-Mel-147; scale bar = 10 µmFigure S6 Exogenous FN rescues the polarity phenotypes of E2F1- and RHAMM-depleted SK-Mel-147 cells, as examined by confocal microscopy; scale bar = 10 µmTable S1 Detail of intermolecular H-bonds in the best pose of interaction between E2F1 and RHAMMTable S2 Primer sequences and antibodiesTable S3 Putative E2F1 binding sites identified within the proximal RHAMM promoter by the open-access JASPAR database and primer sequences for cloning of the RHAMM and FN promoter constructs Deutsche Krebshilfe istex:A549AF63230B8A1FAF35E8575FB0EDEA10B3E627 German Federal Ministry of Education and Research ark:/67375/WNG-NDN639FN-X FORUN programme of Rostock University Medical Centre ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0022-3417 1096-9896
DOI:	10.1002/path.4400