Abstract 5634: Characterization of molecular mechanisms of cytoplasmic trafficking and nuclear translocation of AR splice variants ARv7
Authors: Eiman Mukhtar, Seaho Kim, Paraskevi Giannakakou
Journal: Cancer Research (2017)

Abstract Androgen receptor (AR) signaling is critical to not only hormone-sensitive but also advanced castration-resistant prostate cancer. AR inhibitors (abiraterone and enzalutamide), the next generation of androgen deprivation therapy (ADT) have been used for metastatic castration-resistant PC (mCRPC) treatment, however, the majority of patients progress due to the development of drug resistance. AR variants has emerged as one of the mechanisms of resistance to these drugs. ARv7 and Arv567 splice variants that found lacking the ligand -binding domain are constitutively active in the nucleus and thus restore AR function despite AR inhibitors treatment. We have reported that microtubules and dynein motor protein is required as transportation system for AR for its nuclear translocation and activity and that taxanes inhibit AR signaling downstream of microtubule inhibitors. In addition, we identified that the AR hinge region mediates binding to microtubules is present in ARv567 but missing from ARv7. ARv7 is expressed in about 60% of CRPC patients and has been shown to confer clinical resistance to next generation AR signaling inhibitors. Currently, there is no therapeutic modality targeting specifically ARv7 expression or function. Using fluorescent recovery after photobleaching experiments (FRAP) we have shown that ARv7 translocation to the nucleus occurs much faster than AR-FL (t1/2 11s and 23 s respectively) and that its nuclear import is independent of the importin a/b pathway utilized by AR-FL and proteins with canonical NLS. Furthermore, transient expression of the Q69L RanGTP mutant protein, which acts as dominant negative by occluding the nucleoporins, had minimal effect on ARv7 nuclear import, while it completely blocked AR-FL and AR-v567. Collectively, these data suggest that the nuclear import pathway for ARv7 is distinct from that utilized by AR-FL. We believe that ARv7 undoubtedly uses facilitated transport because of its size (75 kDa), therefore we are tackling three possibilities of nuclear translocation of ARv7. First, ARv7 could interact directly with FG repeat nucleoporins (without receptor) as reported for beta catenin. We are investigating this by the use of importin β-(71-876) mutant cell line for in vitro studies which blocks all members of the importin family. In addition, we are investigating the role of karyopherin transport receptors by individual knockdown experiments coupled with live cell imaging and kinetic quantitation of ARv7 nucleocytoplasmic trafficking. Further, ARv7could translocate to nucleus by utilizes a piggyback on another protein with a classical nuclear localization signals. Overexpression of glutathione S-transferase- importin β binding domain fragment which will block all importin β pathways will exclude this mechanism. Elucidation of this mechanism will be vital to facilitate design an alternative therapeutic modality to halt AR signaling for CRPC treatment. Citation Format: Eiman Mukhtar, Seaho Kim, Paraskevi Giannakakou. Characterization of molecular mechanisms of cytoplasmic trafficking and nuclear translocation of AR splice variants ARv7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5634. doi:10.1158/1538-7445.AM2017-5634