Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor in the signaling pathway that controls the hypoxic response of cancer cells. Activation of the HIF-1 pathway has been observed in many types of cancer.HIF-1 is composed of a constitutively expressed subunit HIF-1β, and an oxygen-sensitive subunit HIF-1a. HIF-1a is constantly produced but rapidly degraded by von Hippel Lindau (VHL) E3 ligase under normoxic conditions, whereas reduced oxygen concentration results in stabilization of HIF-1a. Looking for HIF-1a new regulators we found that β-TrCP interacts with HIF-1a protein. β-TrCP1 is an F-box protein and a known E3 ligase, and is part of the ubiquitin ligase SKP1–CUL1–F-box protein (SCF) complex. To study the functional consequences of this interaction we used PC-3 cancer cells and HEK 293T cells to transiently overexpressβ-TrCP1. For β-TrCP1 knockdown we used stably expressing Tet on/Tet off inducible short-hairpin RNA LNCaP and HCT-116 cells. β-TrCP1 overexpression specifically induced HIF-1a, but not HIF-2a, protein expression and HIF-1 transcriptional activity under normoxic and hypoxic conditions. Silencingb-TrCP1 significantly reduced HIF-1a protein expression and HIF-1 transcriptional activity. HIF-1α ubiquitination was decreased in the presence ofβ-TrCP1 in an oxygen independent manner. Based on these findings we hypothesized that β-TrCP1 affects HIF-1aindirectly through a mediator, which eventually downregulates HIF-1a. Analyzing different b-TrCP1 expression conditions by mass spectroscopy HSP70 was identified as a candidate player. The interaction between HSP70 and b-TrCP1 was confirmed in PC-3 cells. Moreover, HSP70/HIF-1a interaction was decreased in the presence of b-TrCP. β-TrCP1 also reduced HSP70 protein levels. These results strongly support the hypothesis that b-TrCP1 upregulates HIF-1a by preventing HSP70/HIF-1a interaction leading to enhanced transcriptional activity, independent of the well-established oxygen/VHL pathway.
