Abstract 4595: RPL18A as putative target of rigosertib

Abstract

Abstract Rigosertib (ON 01910.Na) is clinical stage anti-mitotic cancer inhibitor that causes spindle abnormalities in neoplastic cells. Rigosertib inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously we reported rigosertib led to hyperphosphorylation of RanGAP1•SUMO1, regulator of RanGTP/RanGDP cycle, and hypothesized phosphatase inhibition [Cancer Res 2011; 71; 4968-76]. Using biotin-conjugated rigosertib and avidin-conjugated agarose beads, we sought rigosertib molecular target from HeLa cell lysates by pull-down assay. Long-linker 1910-biotin showed an IC50 for cytotoxicity similar to unmodified rigosertib and efficient avidin binding. Asynchronously grown HeLa cell lysates were incubated with designated ligands and avidin beads. Bound lysate proteins were recovered from beads, resolved on SDS-PAGE and stained. Specific protein band of ∼20 kDa was detected only with long-linker 1910-biotin. Identification of proteins from the gel was performed by LC-ESI-MS/MS. Four ribosomal proteins possessed Mascot score above cut-off level with RPL18A, score of 367, and other proteins scored 153 or less. Specific dose-dependent binding of long-linker 1910-biotin to RPL18A was confirmed by Western blot analysis in replicate pull-down experiments using HeLa or MOLT-3 cell lysates. Recombinant RPL18A possessed specific reactivity to 1910-biotin in the pull-down assay. Knock-down of RPL18A with siRNA causes apoptosis in cancer cell lines [Sudo et al, Genomics 2010; 95; 210-6; and our data]. We propose that nucleocytoplasmic transport of RPL18A is impaired when hyperphosphorylated RanGAP1•SUMO1 fails to provide RanGTP/GDP gradient for nuclear pore complex. Importin-9 (IPO-9) is a major nucleocytoplasmic transporter for RPL18A. Among other cargos of IPO-9 are HSP27 (important anti-apoptotic player), scaffold subunit A of PP2A (major mitotic phosphatase), core histones and ribosomal proteins. Considering the essential role of nucleocytoplasmic transport in general and probable consequences of impeded transport of HSP27, PP2A and/or core histones, that actually match those observed with rigosertib, we hypothesize that binding with RPL18A in the presence of hyperphosphorylated RanGAP1•SUMO1 interferes with normal function of IPO-9, resulting in a variety of malfunctions and apoptosis observed in rigosertib treated cancer cells. Citation Format: Irina Oussenko, Saikrishna Divakar, M. V. Ramana Reddy, James F. Holland, E. Premkumar Reddy, Takao Ohnuma. RPL18A as putative target of rigosertib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2014-4595