Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis

Abstract

Summary The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-κB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.

Vascular Normalization by Loss of Siah2 Results in Increased Chemotherapeutic Efficacy

“…Because of its peptide nature, it is not suitable for assessing the systemic effect of Siah blockade on tumor progression. Recently, vitamin K3 has been reported as an inhibitor of Siah ubiquitin ligases, although with reasonably low affinity (54). Improving this Siah inhibitor further might provide a novel antiangiogenic treatment option for solid cancers in the future.…”

Siah: A Promising Anticancer Target

“…1). Mice treated with menadione had reduced tumor growth rates associated with decreased Hif-1a stabilization (37). Although the mechanism of Siah2 inhibition of menadione has not been entirely elucidated, this study provides evidence for the screening and functional validation of Siah inhibitors to prevent cancer progression.…”

“…The only drug targeting Siah described so far, vitamin K3 (menadione), was identified as an inhibitor of Siah2 ubiquitin ligase activity in a screen of U.S. Food and Drug Administration-approved therapeutic drugs (37). Menadione inhibits both arms of the Siah2 downstream signaling network, the Ras/MAPK pathway and the hypoxic response pathway (37), suggesting that it might act through the SBD and RING domain (Fig.…”

“…Menadione inhibits both arms of the Siah2 downstream signaling network, the Ras/MAPK pathway and the hypoxic response pathway (37), suggesting that it might act through the SBD and RING domain (Fig. 1).…”

Novel Function of E3 Ubiquitin Ligase Siah2 to Regulate ROS Metabolism

“…Inhibition of the binding of Siah with the substrates through PHYL also reduced tumor growth, angiogenesis, and metastatic spread by the inactivated hypoxic response pathway [34] . Furthermore, in vivo, a chemical inhibitor of Siah2, menadione, abolished the growth of xenograft tumors in nude mice [35] . The genetic knockout of Siah proteins is valuable in establishing the significance of biochemical and cell biological studies.…”