15-Lipoxygenase 1 Promotes Hypoxia-Inducible Factor 1α Degradation Dependent of Essential Enzymatic Function
H Zhong, R Wang, U Kelavkar, M Ohh, JW Simons. New York University School of Medicine, New York; Emory University School of Medicine, Atlanta; University of Pittsburgh School of Medicine, Pittsburgh; University of Toronto, Toronto, Canada
Background: Hypoxia inducible factor-1α (HIF-1α) is the major subunit of HIF-1, critical in transcriptional regulation of genes whose products mediate angiogenesis and hypoxic adaptation. HIF-1α is overexpressed in human cancers, predominately through defects in degradation pathway. Mammalian lipoxygenases (LOXs) are key mediators of arachidonic acid metabolism relevant to carcinogenesis. 15-LOX-1 has recently been demonstrated to be down-regulated in several common malignant tumors, and the degree of its down-regulation is reversely correlated to advanced tumor stages. Our prior studies have shown that inhibiting 15-LOX-1 increases HIF-1α level and HIF-1 transcriptional activity (2002 AACR) while mechanistic details remain elusive.
Design: Prostate cancer and colon cancer cell lines and human tissue microarray were employed. 15-LOX-1 expression vector was subcloned so as to manipulate its enzymatic activity. Stable or transient transfection plus luciferase assays, immunoprecipitation, HIF-1α ubiquitination assay, and immunohistochemistry were utilized in the study.
Results: Here, we report that 15-LOX-1 promotes HIF-1α ubiquitination and degradation, depending on the classic Pro564/hydroxylation/26S proteasome system under normoxia. Both enzymatic activity and intracellular membrane binding function of 15-LOX-1 are required to degrade HIF-1α. The enhanced HIF-1α degradation by 15-LOX-1 overexpression is not abolished even if proteasomal degradation is inhibited by hypoxia or CoCl2. No physical association between immunoprecipitated HIF-1α and 15-LOX-1 occurs, which further supports that enzymatic products of 15-LOX-1 likely mediate the decrease in HIF-1α. In addition, induction of endogenous 15-LOX-1 simultaneously reduces hypoxia-induced HIF-1α accumulation in a dose dependent pattern. Finally, 15-LOX-1 down-regulation was demonstrated by immunohistochemical studies in breast or other common malignancies compared to their benign counterparts.
Conclusions: Our findings provide a novel mechanism for the HIF-1α regulation that combines oxygen dependent and oxygen insensitive actions. This is the first evidence demonstrating that a lipoxygenase involves HIF-1α regulation. Lowered 15-LOX-1 is probably one of the many reasons of HIF-1α overexpression in human cancers, which contributes to diseases progression.
Monday, March 22, 2010 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 227, Monday Morning