Hypoxia enhances migration and invasion in glioblastoma by promoting a mesenchymal shift mediated by the HIF1α-ZEB1 axis.

Glioblastoma (GBM) is the most common brain tumor in adults and the mesenchymal GBM subtype was reported to be the most malignant, presenting severe hypoxia and necrosis. Here, we investigated the possible role of a hypoxic microenvironment for inducing a mesenchymal and invasive phenotype. The exposure of non-mesenchymal SNB75 and U87 cells to hypoxia induced a strong change in cell morphology that was accompanied by enhanced invasive capacity and the acquisition of mesenchymal marker expression. Further analyses showed the induction of HIF1α and HIF2α by hypoxia and exposure to digoxin, a cardiac glycoside known to inhibit HIF1/2 expression, was able to prevent hypoxia-induced mesenchymal transition. ShRNA-mediated knockdown of HIF1α, and not HIF2α, prevented this transition, as well as the knockdown of the EMT transcription factor ZEB1. We provide further evidence for a hypoxia-induced mesenchymal shift in GBM primary material by showing co-localization of GLUT1, ZEB1 and the mesenchymal marker YKL40 in hypoxic regions of the tumor. Collectively, our results identify a HIF1α-ZEB1 signaling axis that promotes hypoxia induced mesenchymal shift and invasion in GBM in a cell line dependent fashion.

[1]  Jun O. Liu,et al.  Digoxin and other cardiac glycosides inhibit HIF-1α synthesis and block tumor growth , 2008, Proceedings of the National Academy of Sciences.

[2]  Brian Keith,et al.  Hypoxia-Inducible Factors, Stem Cells, and Cancer , 2007, Cell.

[3]  S. Gabriel,et al.  Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. , 2010, Cancer cell.

[4]  Yiting Cao,et al.  Tumor angiogenic and hypoxic profiles predict radiographic response and survival in malignant astrocytoma patients treated with bevacizumab and irinotecan. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[5]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[6]  Rakesh K. Jain,et al.  Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases , 2011, Nature Reviews Drug Discovery.

[7]  Robert A. Weinberg,et al.  Poised Chromatin at the ZEB1 Promoter Enables Breast Cancer Cell Plasticity and Enhances Tumorigenicity , 2013, Cell.

[8]  M. Westphal,et al.  Cost of migration: invasion of malignant gliomas and implications for treatment. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[9]  Daniel J Brat,et al.  Pseudopalisades in Glioblastoma Are Hypoxic, Express Extracellular Matrix Proteases, and Are Formed by an Actively Migrating Cell Population , 2004, Cancer Research.

[10]  Baocun Sun,et al.  Hypoxia promotes vasculogenic mimicry formation by inducing epithelial-mesenchymal transition in ovarian carcinoma. , 2014, Gynecologic oncology.

[11]  John B. Shoven,et al.  I , Edinburgh Medical and Surgical Journal.

[12]  Michael E. Berens,et al.  Molecular Mechanisms of Glioma Cell Migration and Invasion , 2004, Journal of Neuro-Oncology.

[13]  Guofei Zhou,et al.  cAMP-dependent protein kinase is essential for hypoxia-mediated epithelial-mesenchymal transition, migration, and invasion in lung cancer cells. , 2012, Cellular signalling.

[14]  G. Nikkhah,et al.  Epithelial-to-mesenchymal(-like) transition as a relevant molecular event in malignant gliomas. , 2013, Cancer letters.

[15]  C. Hsieh,et al.  NADPH Oxidase Subunit 4-Mediated Reactive Oxygen Species Contribute to Cycling Hypoxia-Promoted Tumor Progression in Glioblastoma Multiforme , 2011, PloS one.

[16]  E. Vellenga,et al.  Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation. , 2010, Blood.

[17]  Adrian L. Harris,et al.  Hypoxia — a key regulatory factor in tumour growth , 2002, Nature Reviews Cancer.

[18]  Jianping Zhang,et al.  HIFs enhance the migratory and neoplastic capacities of hepatocellular carcinoma cells by promoting EMT , 2014, Tumor Biology.

[19]  Hong Liao,et al.  The HIF-2alpha dependent induction of PAP and adenosine synthesis regulates glioblastoma stem cell function through the A2B adenosine receptor. , 2014, The international journal of biochemistry & cell biology.

[20]  G. Semenza.  Intratumoral hypoxia, radiation resistance, and HIF-1. , 2004, Cancer cell.

[21]  B. Teicher.  Hypoxia and drug resistance , 1994, Cancer and Metastasis Reviews.

[22]  Daniel J Brat,et al.  'Pseudopalisading' Necrosis in Glioblastoma: A Familiar Morphologic Feature That Links Vascular Pathology, Hypoxia, and Angiogenesis , 2006, Journal of neuropathology and experimental neurology.

[23]  B. Liang.  Effects of hypoxia on drug resistance phenotype and genotype in human glioma cell lines , 1996, Journal of Neuro-Oncology.

[24]  S. White,et al.  Dynamic, site-specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein. , 2001, Cancer research.

[25]  M. Wolter,et al.  A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. , 2010, Brain : a journal of neurology.

[26]  Peter T. Nelson,et al.  Hypoxia Is Important in the Biology and Aggression of Human Glial Brain Tumors , 2004, Clinical Cancer Research.

[27]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[28]  R. Davuluri,et al.  miR-218 opposes a critical RTK-HIF pathway in mesenchymal glioblastoma , 2013, Proceedings of the National Academy of Sciences.

[29]  Trevor Hastie,et al.  Gene Expression Programs in Response to Hypoxia: Cell Type Specificity and Prognostic Significance in Human Cancers , 2006, PLoS medicine.

[30]  C. Brennan,et al.  Molecular subclassification of diffuse gliomas: Seeing order in the chaos , 2011, Glia.

[31]  Å. Borg,et al.  Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype. , 2006, Cancer cell.

[32]  R. Jensen,et al.  Brain tumor hypoxia: tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic target , 2009, Journal of Neuro-Oncology.

[33]  Santosh Kesari,et al.  Malignant gliomas in adults. , 2008, The New England journal of medicine.

[34]  K. Varani,et al.  Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells. , 2006, Biochemical pharmacology.

[35]  K. Bhat,et al.  TGF-β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion , 2014, Cell Death and Disease.

[36]  Joan Pérez Roca,et al.  Hypoxia-inducible factors : , 2015 .

[37]  J. Uhm,et al.  The transcriptional network for mesenchymal transformation of brain tumours , 2010 .

[38]  P. Vaupel,et al.  Hypoxia in cancer: significance and impact on clinical outcome , 2007, Cancer and Metastasis Reviews.

[39]  I. Herr,et al.  Triptolide reverses hypoxia-induced epithelial–mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation , 2013, International journal of cancer.

[40]  Thomas D. Wu,et al.  Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. , 2006, Cancer cell.

[41]  S. Vegter,et al.  TGF-β is an inducer of ZEB 1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion , 2014 .

[42]  Ganesh Rao,et al.  The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma. , 2011, Genes & development.

[43]  Eric C. Holland,et al.  Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma , 2010, Nature Reviews Cancer.

[44]  Hui Wang,et al.  Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. , 2009, Cancer cell.

[45]  Se Hoon Kim,et al.  Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma. , 2013, Cancer cell.

[46]  D. Steindler,et al.  The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance , 2013, EMBO molecular medicine.

[47]  Robert A. Weinberg,et al.  Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. , 2008, Developmental cell.

[48]  Tahsin Kurc,et al.  The tumor microenvironment strongly impacts master transcriptional regulators and gene expression class of glioblastoma. , 2012, The American journal of pathology.

[49]  L. Poellinger,et al.  Mechanism of regulation of the hypoxia‐inducible factor‐1α by the von Hippel‐Lindau tumor suppressor protein , 2000, The EMBO journal.

[50]  p53 regulates epithelial-mesenchymal transition (EMT) and stem cell properties through modulating miRNAs , 2010, Nature Cell Biology.