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Hypoxia promotes uveal melanoma invasion through enhanced Notch and MAPK activation.

PloS one (2014-08-29)
Laura Asnaghi, Michael H Lin, Kah Suan Lim, Kah Jing Lim, Arushi Tripathy, Murilo Wendeborn, Shannath L Merbs, James T Handa, Akrit Sodhi, Eli E Bar, Charles G Eberhart
RESUMEN

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.

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Sodium orthovanadate, ≥90% (titration)
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Sodium orthovanadate, 99.98% trace metals basis
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Rapamycin, Ready Made Solution, 2.5 mg/mL in DMSO (2.74 mM), from Streptomyces hygroscopicus
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Digoxin, United States Pharmacopeia (USP) Reference Standard
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Digoxin, analytical standard
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Rapamycin, VETRANAL®, analytical standard
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Digoxin, European Pharmacopoeia (EP) Reference Standard
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Digoxin for peak identification, European Pharmacopoeia (EP) Reference Standard
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