Abstract 1424: F-box protein fbxl5 nuclear retention by specific inhibitors of nuclear export induces snail ubiquitination leading to reversal of EMT
Authors: Muqbil Irfana , Aboukameel Amro , Landesman Yosef , Kauffman Michael , Shacham Sharon , Mohammad Ramzi M. , Azmi Asfar S. 
Published: August 2015

Abstract
Background: FBXL5 belonging to SKP1-cullin 1-F-box protein E3 ligase complexes (F-BOX) been shown to promote snail nuclear ubiquitination thereby regulating snail induced epithelial-to-mesenchymal transition (EMT) processes. However, cancer associated enhancement in the nuclear exporter Exportin1 (Xpo1) expression results in nuclear expulsion of FBXL5 causing snail stability and EMT. Here we demonstrate that Xpo1 inhibition by specific inhibitors of nuclear export (SINEs) results in nuclear retention FBXL5, causing nuclear degradation of SNAIL leading to reversal of mesenchymal phenotype to epithelial in HMLE-SNAIL models.
Methods: Molecular assays (MTT, Annexin V FITC, Histone DNA ELISA, Spheroid formation, Western Blotting, Confocal microscopy, Co-immunoprecipitation, Xpo1 site directed mutagenesis) and computational techniques (gene expression microarray, pathway analysis) were used. In vivo activity of Selinexor was evaluated in xenografts developed from HMLE-SNAIL cells in ICR-SCID mice.
Results: SINEs [Selinexor (KPT-330) and +ve controls KPT-185 and Leptomycin B (LMB)) not KPT-301 (-ve control)] reverse mesenchymal morphology, induce growth inhibition and apoptosis in HMLE-SNAIL and Kras-HMLE-SNAIL cells and prevent spheroid formation (IC50s ∼150 nM). Immunofluorescence analysis demonstrated that SINE treatment resulted in nuclear retention of FBXL5 that was concurrent with nuclear degradation of snail. Co-immunoprecipitation experiments showed nuclear ubiquitination of snail by SINEs. Western blotting analysis verified nuclear enhancement of FBXL5 that was consistent with down-regulation of EMT markers (Vimentin, snail, EpCAM) and enhancement of E-Cadherin. SiRNA against FBXL5 or transfecting cells with cys528 mut-Xpo1 that lacks SINE binding site markedly abrogated SINE activity thereby verifying the Xpo1 and FBXL5 mediated mechanisms of action. Pathways analysis of quadruplet microarray expression arrays from SINE treated HMLE-SNAIL cells demonstrated differential expression of F-Box family proteins [FBXO2, FBXL17, FBXO33, FBXO37, FBXW7 (p&lt;0.001)] and suppression of snail network. Most significantly, oral administration of SINE (Selinexor at 15 mg/kg three times a week for three weeks) resulted in complete cure of HMLE-SNAIL tumors (tumor free at 120 days). SINE exposed animals showed normal spleen size and morphology in comparison to control animals that showed spleen enlargement (p&lt;0.001). Quantitative sandwich ELISA of spleen tissue extracts showed suppression of snail expression in SINE treatment animals.
Conclusion: This is the first proof of concept study demonstrating that targeted inhibition of Xpo1 can inhibit EMT through nuclear retention of FBOX protein, particularly FBXL5 and consequent snail ubiquitination and degradation. Our findings open a unique possibility to block EMT at the nuclear pore.
Citation Format: Irfana Muqbil, Amro Aboukameel, Yosef Landesman, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad, Asfar S. Azmi. F-box protein fbxl5 nuclear retention by specific inhibitors of nuclear export induces snail ubiquitination leading to reversal of EMT. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2015-1424