Date of Award

2010

Degree Name

Doctor of Philosophy

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Richard Niles

Second Advisor

Gary Rankin

Third Advisor

Beverly Delidow

Fourth Advisor

Kelley Kiningham

Fifth Advisor

Vincent Sollars

Abstract

Hypoxia inducible factor-1α (HIF-1α) protein, a key regulator of oxygen homeostasis, is stabilized under hypoxia and degraded under normal oxygen tension. Here, the human melanoma cells were found to express an elevated amount of HIF-1α mRNA and protein relative to normal human melanocytes under normoxic conditions. The amount of HIF-1α expressed is roughly correlated with the stage of melanoma from which the cell line was established. In addition, a splice variant mRNA of HIF-1α785 is expressed at higher levels than full-length HIF-1α mRNA in the more aggressive melanoma cells. This splice variant lacks part of the oxygen regulation domain. Ectopic expression of HIF-1α in a radial growth phase melanoma cell line increased their ability to form colonies in soft agar and their ability to invade and migrate through Matrigel. Conversely, knockdown of HIF-1α expression in metastatic melanoma cell line decreased their ability to form colonies in soft agar as well as their ability to invade and migrate through Matrigel. Further, it has been shown that the ERK1/2 MAPK pathway is probably not involved in the expression of HIF-1α under normoxic conditions. In addition, the normoxic expression of HIF-1α directly regulates the micropthalamia-associated transcription factor (MITF) mRNA and protein expression in human metastatic melanoma cell line. There was clear shift in MITF expression from MITF-M, a specific isoform in melanocytes, to the predominantly MITF-A in metastatic melanoma. The MITF-A promoter contains two hypoxia response elements while none were found in the MITF-M promoter region. Reporter gene and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds and transactivates the MITF-A promoter in metastatic melanoma cells. Knockdown of MITF expression in metastatic melanoma cell line decreased their viability through induction of apoptotic pathways. Overall data suggest that increased normoxic expression of HIF-1α contributes to some of the malignant properties of melanoma and increases its importance as a therapeutic target. Further, HIF-1α activates the expression of MITF-A whose expression contributes to the survival of metastatic melanoma cells.

Subject(s)

Hypoxia.

Cancer - Research.

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