In Drosophila, SINA has been shown to co-operate with Phyllopod (PHYL), Ebi and UBCD1 to facilitate the ubiquitylation and degradation of the transcriptional co-repressor tramtrack 88 (TTK88) ( Boulton et al., 2000 Li et al., 1997 Tang et al., 1997). Drosophila Seven in Absentia (SINA) and mammalian Seven in Absentia Homologue (Siah) are RING-containing proteins that function in protein degradation as ubiquitin ligases. Specificity is achieved by ubiquitin ligases recognising specific degradation signals, or “degrons” ( Laney & Hochstrasser, 1999). The specificity of the process is defined by the final step in which a ubiquitin ligase, either alone or as part of a complex, transfers ubiquitin to the substrate protein ( Glickman & Ciechanover, 2002). Protein ubiquitylation is a three-step, enzymatic process ( Hochstrasser, 2000). Recently we and others have shown that, under hypoxic conditions, members of the Siah ubiquitin ligase family can poly-ubiquitylate and target PHD and FIH for proteasomal degradation ( Fukuba et al., 2007 Khurana et al., 2006 Nakayama et al., 2004 Nakayama et al., 2007), thus stabilizing Hif-1alpha.
This hydroxylation prevents the interaction between Hif-1alpha and transcriptional co-activators CBP/p300 ( Lando et al., 2002). Furthermore Hif-1alpha is hydroxylated at Asn803 by the asparaginyl hydroxylase factor inhibiting Hif-1 (FIH) ( Lando et al., 2002). After being poly-ubiquitylated, Hif-1alpha is degraded by the 26S proteasome ( Huang et al., 1998 Maxwell et al., 1999 Salceda & Caro, 1997). The Pro402/Pro564 hydroxylated Hif-1alpha protein has increased affinity for the E3 ubiquitin-protein ligase complex composed of the von Hippel-Lindau tumor suppressor protein VHL, elongin B & C and cullin 2. Under normoxia, Hif-1a 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-1beta subunit is constitutively present within the cell whereas Hif-1alpha is stabilized under hypoxic conditions ( Semenza, 1999). Hif-1 consists of two subunits, Hif-1alpha and Hif-1beta. The key transcription factor for the hypoxic response pathways is hypoxia-inducible factor 1 (Hif-1) ( Iyer et al., 1998 Schofield & Ratcliffe, 2004 Semenza, 2000). The main cellular response to hypoxic stress is the up-regulation 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 demonstrate, 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 anti-tumor therapies. In addition, levels of Hif-1alpha and its target Glut-1 are reduced in the inhibitor expressing tumors. In a syngeneic mouse model of breast cancer, the inhibitor reduced tumor growth and neoangiogenesis and prolonged survival of the mice. Furthermore, cells stably expressing the inhibitor display reduced up-regulation of Hif-1alpha 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 peptide derived from a Drosophila protein interferes with the PHD degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves poly-ubiquitylated and degraded by Siah ubiqiuitin ligases.
The hydroxylation of Hif-1alpha by PHD proteins during normoxia serves as a recognition motif for its proteasomal degradation. Interference with the Hif-1 pathway and neoangiogenesis is an attractive anti-tumor target. Tumor hypoxia induces the up-regulation of Hif-1alpha 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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