Abstract B48: Regulation of mutant KRAS protein stability via SMURF2:UBCH5 complex mediated degradation of beta-TrCP1

Abstract

Abstract Attempts to target mutant KRAS have been unsuccessful. Most of the current therapeutic approaches are indirect, mainly via inhibiting KRAS down-stream signaling, which have been marginally successful. Here we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2), a HECT-type ubiquitin ligase (E3) as a critical regulator of mutant KRAS protein stability. We show that the loss of SMURF2 either by si-/sh-RNA mediated gene silencing or by overexpression of a catalytically inactive SMURF2 Cys716Ala (CA) mutant, can cause lysosome-mediated KRAS degradation; whereas, overexpression of wild type SMURF2 enhances KRAS protein stability. Most importantly, we found that mutant KRAS protein is more susceptible to SMURF2 alterations in that mutant protein half-life decreased from >12h in control siRNA-treated cells to <3h with Smurf2 siRNA treatment, whereas only marginal differences were noted for wild-type KRAS protein upon similar treatments. Importantly, this loss of mutant KRAS protein could be rescued by overexpressing a siRNA-resistant (si-R) wild type SMURF2. Our data further show that SMURF2 along with an ubiquitin conjugating enzyme (E2), UBCH5, poly-ubiquitinates and degrades a Ras family E3, beta-Transducing Repeat Containing Protein 1 (beta-TrCP1). Conversely, beta-TrCP1 is accumulated upon the loss of SMURF2, leading to its increased binding and degradation of KRAS. Therefore, as expected, beta-TrCP1 knockdown following Smurf2 siRNA treatment rescues Smurf2 siRNA-mediated mutant KRAS loss. Functionally, we found a positive correlation between SMURF2 and KRAS protein levels among different cancer cell lines and demonstrated that a loss of SMURF2 reduces the clonogenic survival in vitro and prolongs tumor latency of mutant KRAS-dependent cells in vivo. Taken together, we show that SMURF2 catalytic activity is critical in maintaining mutant KRAS protein stability and propose that targeting of SMURF2 may be a unique strategy to degrade mutant KRAS and thus kill mutant KRAS-addicted cancer cells. Citation Format: Shirish Shukla, Uday Shankar Allam, Aarif Ahsan, Guoan Chen, David G. Beer, Theodore S. Lawrence, Mukesh K. Nyati, Dipankar Ray. Regulation of mutant KRAS protein stability via SMURF2:UBCH5 complex mediated degradation of beta-TrCP1. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B48. doi: 10.1158/1557-3125.RASONC14-B48