Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis.
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-kappaB 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-1alpha 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.
Shah
Meera
M
Signal Transduction Program, Burnham Institute for Medical Research, La Jolla, CA, USA.
Stebbins
John L
JL
Dewing
Antimone
A
Qi
Jianfei
J
Pellecchia
Maurizio
M
Ronai
Ze'ev A
ZA
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins; D051379:Mice; D008819:Mice, Nude; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009368:Neoplasm Transplantation; D014183:Transplantation, Heterologous; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D024483:Vitamin K 3 / Q000737:chemistry / Q000378:metabolism*; D014815:Vitamins / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C485561:SPRY2 protein, human; D014815:Vitamins; D024483:Vitamin K 3; C533102:Siah2 protein, mouse; D044767:Ubiquitin-Protein Ligases; D020928:Mitogen-Activated Protein Kinases