Zhao S, El-Deiry WS

The major adaptive response to hypoxia involves hypoxia-inducible factor HIF-1? which is regulated by von Hippel Lindau (VHL) E3 ligase. We previously observed a stabilization of HIF-1? by cyclin-dependent kinases CDK1 and CDK4/6 that is independent of VHL, hypoxia or p53, and found that CDK4/6 inhibitors destabilize HIF-1? under normoxia ...

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and hypoxia. To further investigate the molecular mechanism of HIF-1? destabilization by CDK1 or CDK4/6 inhibitors, we performed a proteomic screen on immunoprecipitated HIF-1? from hypoxic colorectal cancer cells that were either untreated or treated with CDK1 inhibitor Ro3306 and CDK4/6 inhibitor palbociclib. Our proteomics screen identified a number of candidates that were enriched in palbociclib-treated hypoxic cells including SMAD specific E3 ubiquitin protein ligase 2 (Smurf2). We also identified a HIF-1? peptide that appeared to be differentially phosphorylated after palbociclib treatment. Gene knockdown of SMURF2 increased basal expression of HIF-1? even in the presence of Ro3306 or two different CDK4/6 inhibitors, palbociclib and abemaciclib. Overexpression of Smurf2 inhibited expression of HIF-1? and enhanced HIF-1? ubiquitination in normoxia. Proteasome inhibitor MG-132 partially rescued HIF-1? expression when Smurf2 was overexpressed. Smurf2 overexpression also inhibited HIF-1? expression level in two other cell lines, SW480 and VHL-deficient RCC4. Overexpression of SMURF2 mRNA is correlated with improved disease-free survival and overall survival in clear cell renal cell cancer. Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1? destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1? regulation.

Date: Sep. 28, 2021

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