EFNA3 long noncoding RNAs induced by hypoxia promote metastatic dissemination

The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.

[1]  Kotb Abdelmohsen,et al.  Posttranscriptional gene regulation by long noncoding RNA. , 2013, Journal of molecular biology.

[2]  A. Dopazo,et al.  Cooperativity of Stress-Responsive Transcription Factors in Core Hypoxia-Inducible Factor Binding Regions , 2012, PloS one.

[3]  Yan Wang,et al.  Long non-coding RNAs: versatile master regulators of gene expression and crucial players in cancer. , 2012, American journal of translational research.

[4]  M. Celeste Simon,et al.  The impact of O2 availability on human cancer , 2008, Nature Reviews Cancer.

[5]  Elena B Pasquale,et al.  Eph-Ephrin Bidirectional Signaling in Physiology and Disease , 2008, Cell.

[6]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[7]  L. Holmberg,et al.  Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts , 2005, Breast Cancer Research.

[8]  Jin Chen.  Regulation of tumor initiation and metastatic progression by Eph receptor tyrosine kinases. , 2012, Advances in cancer research.

[9]  G. Semenza,et al.  HIF-1-dependent Expression of Angiopoietin-like 4 and L1CAM Mediates Vascular Metastasis of Hypoxic Breast Cancer Cells to the Lungs , 2011, Oncogene.

[10]  Borivoj Vojnovic,et al.  MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU , 2010, PloS one.

[11]  Elena B. Pasquale,et al.  Eph receptors and ephrins in cancer: bidirectional signalling and beyond , 2010, Nature Reviews Cancer.

[12]  Joseph Loscalzo,et al.  MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. , 2009, Cell metabolism.

[13]  Christopher J Schofield,et al.  Hypoxia-inducible Factor (HIF) Asparagine Hydroxylase Is Identical to Factor Inhibiting HIF (FIH) and Is Related to the Cupin Structural Family* , 2002, The Journal of Biological Chemistry.

[14]  J. Gómez-Skarmeta,et al.  A role for insulator elements in the regulation of gene expression response to hypoxia , 2011, Nucleic acids research.

[15]  Y. Fujii‐Kuriyama,et al.  Hypoxia-inducible Transcription Factor-2α in Endothelial Cells Regulates Tumor Neovascularization through Activation of Ephrin A1* , 2008, Journal of Biological Chemistry.

[16]  W. Kaelin,et al.  Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. , 2008, Molecular cell.

[17]  L. del Peso,et al.  Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene. , 2005, The Biochemical journal.

[18]  E. Liu,et al.  Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation , 2005, Nature Methods.

[19]  Van,et al.  A gene-expression signature as a predictor of survival in breast cancer. , 2002, The New England journal of medicine.

[20]  J. Casal,et al.  In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion, Migration, and Invasion* , 2013, Molecular & Cellular Proteomics.

[21]  J. Loscalzo,et al.  Hypoxia: a master regulator of microRNA biogenesis and activity. , 2013, Free radical biology & medicine.

[22]  J. Aragonés,et al.  Acute Vhl Gene Inactivation Induces Cardiac HIF-Dependent Erythropoietin Gene Expression , 2011, PloS one.

[23]  Robert M. Miura,et al.  Prediction of mRNA polyadenylation sites by support vector machine , 2006, Bioinform..

[24]  Michael I. Wilson,et al.  Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O2-Regulated Prolyl Hydroxylation , 2001, Science.

[25]  G. Semenza.  Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. , 2012, Trends in pharmacological sciences.

[26]  C. Wykoff,et al.  The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis , 1999, Nature.

[27]  Ravi Salgia,et al.  The role of ephrins and Eph receptors in cancer. , 2004, Cytokine & growth factor reviews.

[28]  D. Peet,et al.  Asparagine Hydroxylation of the HIF Transactivation Domain: A Hypoxic Switch , 2002, Science.

[29]  Gianluca Bontempi,et al.  Predicting prognosis using molecular profiling in estrogen receptor-positive breast cancer treated with tamoxifen , 2008, BMC Genomics.

[30]  A. Pause,et al.  An oxygen-regulated switch in the protein synthesis machinery , 2012, Nature.

[31]  Michael I. Wilson,et al.  C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation , 2001, Cell.

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

[33]  Piero Carninci,et al.  Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat , 2012, Nature.

[34]  E. Pasquale,et al.  The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization , 2000, Oncogene.

[35]  Ling Zhou,et al.  WSS25 inhibits Dicer, downregulating microRNA-210, which targets Ephrin-A3, to suppress human microvascular endothelial cell (HMEC-1) tube formation. , 2013, Glycobiology.

[36]  Fabio Martelli,et al.  MicroRNA-210 Modulates Endothelial Cell Response to Hypoxia and Inhibits the Receptor Tyrosine Kinase Ligand Ephrin-A3* , 2008, Journal of Biological Chemistry.

[37]  J. Steffensen,et al.  Hypoxia-induced metastasis model in embryonic zebrafish , 2010, Nature Protocols.

[38]  G. Semenza,et al.  Transactivation and Inhibitory Domains of Hypoxia-inducible Factor 1α , 1997, The Journal of Biological Chemistry.

[39]  Borisas Bursteinas,et al.  ROCK: a breast cancer functional genomics resource , 2010, Breast Cancer Research and Treatment.

[40]  J. Caro,et al.  Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. , 1997, The Journal of biological chemistry.

[41]  Takashi Fukaya,et al.  MicroRNAs mediate gene silencing via multiple different pathways in drosophila. , 2012, Molecular cell.

[42]  George A. Calin,et al.  A MicroRNA Signature of Hypoxia , 2006, Molecular and Cellular Biology.

[43]  Diego Villar,et al.  Genome-wide identification of hypoxia-inducible factor binding sites and target genes by a probabilistic model integrating transcription-profiling data and in silico binding site prediction , 2010, Nucleic acids research.

[44]  F. Frey,et al.  Hypoxia up‐regulates expression of Eph receptors and ephrins in mouse skin , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[45]  Yihai Cao,et al.  Pathological angiogenesis facilitates tumor cell dissemination and metastasis , 2010, Cell cycle.

[46]  Xin Huang,et al.  MiR-210--micromanager of the hypoxia pathway. , 2010, Trends in molecular medicine.

[47]  E. Nievergall,et al.  Eph-dependent cell-cell adhesion and segregation in development and cancer , 2011, Cellular and Molecular Life Sciences.

[48]  M. Ivan,et al.  HIFα Targeted for VHL-Mediated Destruction by Proline Hydroxylation: Implications for O2 Sensing , 2001, Science.

[49]  Xin Lu,et al.  Hypoxia and Hypoxia-Inducible Factors: Master Regulators of Metastasis , 2010, Clinical Cancer Research.

[50]  Ricky T. Tong,et al.  Hypoxia-inducible mir-210 regulates normoxic gene expression involved in tumor initiation. , 2009, Molecular cell.

[51]  G. Semenza.  Targeting HIF-1 for cancer therapy , 2003, Nature Reviews Cancer.

[52]  J. Ragoussis,et al.  Extensive regulation of the non-coding transcriptome by hypoxia: role of HIF in releasing paused RNApol2 , 2013, EMBO reports.

[53]  G. Semenza,et al.  Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. , 1995, Proceedings of the National Academy of Sciences of the United States of America.