Abstract
Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis.
Highlights
Chromosome region maintenance 1, CRM1, was originally identified in yeast as a gene required for maintaining chromosome structure [1]
HCT116 cells were treated with CBS9106, and the levels of CRM1 protein were analyzed by Western blotting using an anti-CRM1 antibody
We investigated how CBS9106 promotes the degradation of CRM1 protein
Summary
Chromosome region maintenance 1, CRM1, was originally identified in yeast as a gene required for maintaining chromosome structure [1]. CRM1 protein is known to be a nuclear export receptor belonging to the karyopherin b family of transport receptors [2]. CRM1 binds to different cargo proteins bearing leucine-rich nuclear export sequences leading to nuclear export of cargo proteins and mRNAs [3, 4]. Exported cargo proteins include tumor suppressors, such as p53, BRCA1, survivin, nucleophosmin, and adenomatous polyposis coli that shuttle between the nucleus and cytoplasm [5]. CRM1 is overexpressed in various malignancies, such as breast, cervical, ovarian, and pancreatic cancers, glioma, and osteosarcoma [6].
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