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


RESULTS
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.


CONCLUSIONS
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.
Mills
Caroline N
CN
Department of Biochemistry and Microbiology, Joan C, Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
Joshi
Sandeep S
SS
Niles
Richard M
RM
D002448:Cell Adhesion / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D050505:Mutant Proteins / Q000378:metabolism; D009362:Neoplasm Metastasis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D048049:Extracellular Signal-Regulated MAP Kinases