title: Hypoxia promotes uveal melanoma invasion through enhanced Notch and MAPK activation.
abstract: The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.
authors.affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Asnaghi
authors.affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Lin
authors.affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Kah Suan
authors.initials: KS
authors.lastname: Lim
authors.affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Kah Jing
authors.initials: KJ
authors.lastname: Lim
authors.affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Arushi
authors.initials: A
authors.lastname: Tripathy
authors.affiliation: Department of Ophthalmology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Murilo
authors.initials: M
authors.lastname: Wendeborn
authors.affiliation: Department of Ophthalmology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Shannath L
authors.initials: SL
authors.lastname: Merbs
authors.affiliation: Department of Ophthalmology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: James T
authors.initials: JT
authors.lastname: Handa
authors.affiliation: Department of Ophthalmology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Akrit
authors.initials: A
authors.lastname: Sodhi
authors.affiliation: Department of Neurological Surgery, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Eli E
authors.initials: EE
authors.lastname: Bar
authors.affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Ophthalmology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Charles G
authors.initials: CG
authors.lastname: Eberhart
mesh_terms: D045744:Cell Line, Tumor; D004077:Digoxin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009361:Neoplasm Invasiveness / Q000473:pathology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014604:Uveal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*
chemical_list: D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051880:Receptors, Notch; D004077:Digoxin; D051057:Proto-Oncogene Proteins c-akt