The Pro402/Pro564 hydroxylated Hif-1α protein has increased affinity for the E3 ubiquitin-protein ligase complex composed of the von Hippel–Lindau tumor suppressor protein VHL, elongin B and C and cullin 2. Under normoxia, Hif-1α is hydroxylated at two conserved proline residues (Pro402 and Pro564) by prolyl-hydroxylases (PHDs) ( Epstein et al., 2001 Jaakkola et al., 2001). In the Hif-1 complex, the Hif-1α subunit is constitutively present within the cell, whereas Hif-1β is stabilized under hypoxic conditions ( Semenza, 1999). Hif-1 consists of two subunits, Hif-1α and Hif-1β. The key transcription factor for the hypoxic response pathways is hypoxia-inducible factor 1 (Hif-1) ( Iyer et al., 1998 Semenza, 2000 Schofield and Ratcliffe, 2004). The main cellular response to hypoxic stress is the upregulation of hypoxia-responsive genes, including VEGF, Glut-1, and CA9 ( Semenza, 1999). Solid tumor growth is associated with areas of poor oxygen supply within the tumor mass. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies. In addition, levels of Hif-1α and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1α protein levels and Hif-1-mediated gene expression under hypoxia. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. The hydroxylation of Hif-1α by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation.
Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1α (Hif-1α), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis.
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