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.

A Novel Notch–YAP Circuit Drives Stemness and Tumorigenesis in Embryonal Rhabdomyosarcoma

“…All cell lines were grown in RPMI-1640 + 10% FBS in 5% CO 2 except CCA which were grown in DMEM + 20% FBS in 7.5% CO 2 . YAP shRNA (46,47), SOX2 shRNA (48), RBPJ shRNA (49,50), and ICN constructs (7) were previously described. pCW107-Notch1 ICN and pCW107-Luciferase constructs were gifts from Kris Wood (Duke University) (51).…”

Evidence for the Role of Blue Light in the Development of Uveal Melanoma

“…A central player in this process is vascular endothelial growth factor (VEGF), a signaling protein that stimulates both vasculogenesis and angiogenesis. Hypoxia is known to induce VEGF production by UM cells [ 54 , 55 ], and high levels of VEGF have been reported in both the aqueous and vitreous humors of patients with UM [ 56 , 57 ]. Furthermore, VEGF is an associated biomarker of UM metastasis [ 58 , 59 ].…”

Endothelial‐to‐hematopoietic transition: Notch‐ing vessels into blood

“…Subsequent conditional loss‐of‐function experiments in murine VE‐cadherin–expressing ECs confirmed an essential role for Hif1α in HE specification and HSC production . Interestingly, while not yet connected to Notch during blood development, Hif1α was previously linked to the Notch pathway in vascular cells and a subset of cancer cells . Molecularly, Hif1α regulated Mindbomb, an E3 ubiquitin ligase essential for Notch activity, and was suggested to suppress Notch2 .…”