Expression and function of hypoxia inducible factor-1 alpha in human melanoma under non-hypoxic conditions

Hypoxia inducible factor-1 alpha (HIF-1alpha) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1alpha protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1alpha RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas.

HIF-1alpha mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1alpha mRNA splice variant that lacks part of the oxygen-dependent regulation domain in WM1366 and WM9 melanoma cells. Over-expression of HIF-1alpha and its splice variant in the RGP cell line SbCl2 resulted in a small increase in soft agar colony formation and a large increase in matrigel invasion relative to control transfected cells. Knockdown of HIF-1alpha expression by siRNA in the MET WM9 melanoma cell line resulted in a large decrease in both soft agar colony formation and matrigel invasion relative to cells treated with non-specific siRNA. There is a high level of ERK1/2 phosphorylation in WM9 cells, indicating an activated Ras-Raf-MEK-ERK1/2 MAPK pathway. Treatment of WM9 cells with 30 microM U0126 MEK inhibitor, decreased ERK1/2 phosphorylation and resulted in a decrease in HIF-1alpha expression. However, a 24 h treatment with 10 microM U0126 totally eliminated Erk1/2 phosphorylation, but did not change HIF-1alpha levels. Furthermore, siRNA knockdown of MEK siRNA did not change HIF-1alpha levels.

We speculate that metabolic products of U0126 decrease HIF-1alpha expression through "off target" effects. Overall our data suggest that increased HIF-1alpha expression under normoxic conditions contributes to some of the malignant phenotypes exhibited by human melanoma cells. The expanded role of HIF-1alpha in melanoma biology increases its importance as a therapeutic target.