Abstract Number: 5430
Presentation Title: eEF1A2 is a New Target for Anticancer Therapy .
Presentation Time: Wednesday, Apr 22, 2015, 8:00 AM -12:00 PM
Author Block: Alejandro Losada1, Maria José Muñoz1, Carolina Garcia2, Juan F. Martínez-Leal1, Federico Gago3, Carmen Cuevas1, Luis F. Garcia-Fernández1, Juan M. Dominguez1, Pilar Lillo2, Carlos M. Galmarini1. 1PharmaMar, S.A., Madrid, Spain; 2Instituto Quimica Fisica Rocasolano (CSIC), Madrid, Spain; 3Universidad de Alcalá (UAH), Madrid, Spain
Abstract Body: Eukaryotic Elongation Factor 1A2 (eEF1A2) is an isoform of the alpha subunit of eEF1 complex. Differently from the A1 isoform, the expression of eEF1A2 is restricted to brain, heart and skeletal muscle. eEF1A2 is overexpressed in tumors, including multiple myeloma (MM) (Li et al, 2012, PLOS One 5, e10755), prostate (Sun et al, 2014, Biochem Biophys Res Commun 450:1-6), pancreas (Xu et al, 2013, Clin Exp Metastasis 30:933-44) and ovarian (Anand et al, 2002, Nat Genet 31:301-5), and has also an oncogenic behavior favoring tumor cell proliferation while inhibiting apoptosis (Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93). Thus, eEF1A2 is an interesting target for cancer treatment.
Aplidin (plitidepsin) is an antitumor agent, originally isolated from the marine tunicate Aplidium albicans, which is being tested in MM patients in a phase III pivotal trial in combination with dexamethasone and a phase I trial in combination with bortezomib and dexamethasone.
Herein we reveal the interaction of Aplidin with eEF1A2 using different methods; i) a DARTS assay showed that Aplidin protected eEF1A2 from digestion by the protease subtilisin (EC 3.4.21.62); ii) a saturation binding experiment using [14C]-Aplidin and eEF1A2 purified from rabbit muscle determined a Kd for the interaction of approximately 80 nM; iii) a fluorescence anisotropy test through two photon microscopy showed that Aplidin preferentially binds the GTP-bound form of eEF1A2.
Furthermore, we performed a [14C]-Aplidin binding-guided fractionation of K562 cell extracts through differential centrifugation and several chromatographic steps, including ion-exchange and size-exclusion chromatography, demonstrating that eEF1A2 is the unique cellular protein that can be retrieved through specific binding to Aplidin.
In addition, HeLa-APL-R cells, a HeLa subclone made extremely resistant to Aplidin, more than 1000 fold, (Losada et al., 2004, Br J Cancer 91:1405-13), are shown to express 8 fold less eEF1A2 than the parental cells both at the mRNA (determined with an Affimetryx HG-U133A Array) and protein (determined by iTRAQ) levels. When normal eEF1A2 levels were restored in HeLa-APL-R cells through ectopic expression of an eEF1A2-GFP construct, these cells were rendered partially sensitive to Aplidin.
Interestingly, the transfected cells recovered most of the signaling events which are typically induced by the drug in HeLa wt cells. Altogether, our results demonstrate that eEF1A2, the oncogenic isoform of the alpha subunit of eEF1, is the primary target of Aplidin and a new suitable and druggable target for anticancer therapy.