Abstract 3251: Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma.

Abstract

WP1066 was shown to exert its potent inhibitory activity against several types of human cancer including melanoma in vitro and in vivo via suppression of JAK2/STAT3/STAT5 activation. However, if there is any other additional molecular mechanism(s) by which WP1066 generates its anti-tumor effects remain largely unknown. Here, we report a novel function for WP1066 in inducing the degradation of both HIF-1α and HIF-1β subunits in human melanoma cells. We found constitutive activation of HIF-1α and an increased expression of hexokinase II, a down target of HIF-1α, in normoxia in 6/6 of melanoma cell lines, but not in other tumor types. Both hypoxia and cobalt chloride, a transition metal that mimics hypoxia, further increased HIF-1α protein accumulation in a time-dependent manner in WM35 and SKMEL-1 melanoma cells. WP1066 selectively decreased the constitutive and hypoxia and cobalt chloride induced increase in the level of HIF-1α in a dose-dependent manner with a half-life (T1/2) of ∼13, ∼10, and ∼10 min in WM35 cells. Moreover, WP1066 decreased the T1/2 of HIF-1α following cycloheximide treatment from ∼13 to ∼7 min suggesting that WP1066 induced HIF-1α instability is not mediated via inhibition of its translation. Further, inhibition of HIF-1α protein accumulation by WP1066 was not abolished in the presence of MG132 and proteasome inhibitor I, a potent inhibitor of the 26S proteasome, suggesting that the 26S proteasome-system is not responsible for WP1066-induced HIF-1α instability. Immunoprecipitation, confocal microscopy, and cellular fractionation analyses revealed that WP1066 induced ubiquitinylation, aggregation and degradation of HIF-1α in WM35 cells. Finally, WP1066 directly inhibits activity of rhUSP5 and rUCH-L1. Altogether, our study identified a novel function for WP1066 in inducing HIF-1 instability that may also contribute to the anti-cancer effects of WP1066 in human melanoma. Citation Format: Arumugam Jayakumar, Jana Rauvolfova, Hanying Bao, Izabela Fokt, Stanislaw Skora, Amy Heimberger, Waldemar Priebe. Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2013-3251

2-Deoxy-d-Glucose and Its Analogs: From Diagnostic to Therapeutic Agents

“…Thus, these highly hypoxic and/or glycolytic cancer cells require higher concentrations of 2-DG to compete with glucose and effectively block glycolysis [74]. In such cases, combined therapy with HIF-1α inhibitors, such as WP1066, could restore cell sensitivity to 2-DG glycolysis inhibition [75].…”

Analogs of Cinnamic Acid Benzyl Amide As Nonclassical Inhibitors of Activated JAK2 Kinase

“…In view of their well-recognized biological activity, systematic assessment of all potential mechanisms of action was important. CABA analogs' ability to block the JAK2/STAT3 and JAK2/STAT5 pathways is clearly a result of multifactorial effects rather than exclusively direct inhibition of JAK2, STAT3, or STAT5 [22][23][24][25][26]. In addition, certain CABA derivatives may interact with targets outside the JAK/STAT pathways as exemplified by the CABA derivative WP1609, which inhibits the activity of the atypical protein kinase Rio1 [27].…”