Abstract LB-185: Novel activity of selective inhibitors of nuclear export in epithelial-to-mesenchymal transition models
Authors: Azmi Asfar S. , Shacham Sharon , Kauffman Michael , McCauley Dilara , Aboukameel Amro , Mohammad Ramzi M. 
Published: May 2015

Epithelial-to-mesenchymal transition (EMT) that confers tumors an aggressive phenotype is a fine tuned process regulated by a number of molecules that are strategically distributed in the nuclear and cytosolic compartments of cancer cells. Major EMT regulating proteins and transcription factors (TFs), such as wnt/βcatenin, notch, TGF-β, Twist and Snail are recognized to undergo nuclear-cytosolic shuttling using specialized transporters; karyopherins. The export of most of the EMT regulating TFs is mediated exclusively by Exportin1/XPO1/CRM1 through nuclear exclusion sequence (NES) recognition. These observations indicate that nuclear transport may play a critical role in the development of EMT. However, to date there have been no studies evaluating the impact of nuclear export inhibition on EMT. We have earlier developed Specific Inhibitors of Nuclear Export (SINEs) that block XPO1 leading to nuclear retention of various tumor suppressor proteins (TSPs) and TFs. This is the first report showing that XPO1 inhibition by SINEs induce global re-organization of proteins leading to reversal of EMT in Robert Weinberg's snail transduced immortalized human mammary epithelial (HMLE-Snail) cells. Snail is a NES carrying transcription factor that has been documented to regulate EMT from either the nucleus or cytosol. Exposure of HMLE-Snail cells to SINEs to clinically relevant concentration of 0-200 nM [i.e. KPT-185, its inactive analog KPT-TRANS, and clinical agent Selinexor (KPT-330)] resulted in reversal of cellular morphology from mesenchymal to epithelial (MET) that was concurrent with growth inhibition and apoptosis. KPT-185 treatment at 150 nM (IC50) concentration suppressed spheroid forming ability of HMLE-snail cells. Molecular analysis of KPT-185 treated HMLE-snail cells and corresponding spheroids showed suppression of key EMT markers including snail, Twist, vimentin and EpCAM. Co-immunoprecipitation studies verified the disruption of the pre-dominantly cytosolic snail-XPO1 interaction upon SINE treatment. In order to understand the mechanism of EMT reversal, RNA from quadruplet SINE treated HMLE-Snail cells were subjected to mRNA microarrays and systems biology analysis. KPT-185 treatment resulted in differential expression of 720 genes (fold change p&lt;0.001). The statistically significant differentially expressed genes were subjected to pathway analysis (upstream and downstream regulator analysis and similarity analysis to known EMT gene networks), which showed downregulation in Snail, HDAC1 and Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) networks. Conversely, the activated networks included apoptosis and p73 signaling. The network genes were validated at the expression level using, RT-PCR and western blotting. The anti-tumor activity of SINEs against HMLE-snail derived spheroids grown subcutaneously in mice is currently being evaluated. In conclusion, this is the first study demonstrating the role of XPO1 in EMT. Selinexor is currently being evaluated in 3 phase 1 studies in patients with advanced solid tumor and hematological malignancies. Therefore, in view of our results, XPO1 inhibition through SINEs can be a new form of therapy for highly aggressive and metastatic tumors that harbor EMT.

Citation Format: Asfar S. Azmi, Sharon Shacham, Michael Kauffman, Dilara McCauley, Amro Aboukameel, Ramzi M. Mohammad. Novel activity of selective inhibitors of nuclear export in epithelial-to-mesenchymal transition models. [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 LB-185. doi:10.1158/1538-7445.AM2014-LB-185