Abstract A37: Blockade of ARV7:HIF1α; heterodimers after toptoecan reverses enzalutamide resistance in 22Rv1 cells
Authors: Fruehauf John P. , Farrokh Nathan , Sarkissian Sarmen , Kim Jai-Hyun 
Published: May 2017

Background: Although enzalutamide (ENZ) is initially effective for the treatment of castration-resistant prostate cancer (CRPC), development of ENZ resistance is inevitable. One mechanism for resistance is the emergence of ARV7, an alternative splicing variant of the full length androgen receptor (FL AR) which lacks the androgen binding and hinge region domains. Nuclear translocation of FL AR utilizes the hinge region for coupling to tubulin or importin. ARV7 protein appears to gain nuclear entry independent of the canonical pathway. FL AR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, facilitating its nuclear localization and transcriptional activity. We previously found that TPT blocks HIF1α translation by targeting topoisomerase I resident on HIF1α mRNA. We therefor determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ.

Methods: 22Rv1 ENZ resistant prostate cancer cells express comparable levels of full length AR and truncated ARV7 protein. 22Rv1 cells were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the Luminescent cell viability assay.

Results: Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots. Compared with control cells, TPT treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α accompanied by decreased ARV7 nuclear localization. Topo-I immunoprecipitation confirmed its linkage to HIF-1α mRNA. Surprisingly, we found that Topo-I RNA-IP pulled down AR mRNA as well. Dose-response curves for the TPT / ENZ combination demonstrated synergistic activity, with combination index values of 0.6. Under hypoxic conditions, TPT had an IC50 of 120 nM. pretreatment of 22Rv1 cells with a minimally toxic dose of 15 nM TPT reduced the ENZ IC50 from 93 nM to 60 nM.

Conclusions: Our data indicate that Topo I residing on HIF-1α mRNA provided a druggable target for TPT-mediated inhibition of HIF1α mRNA translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support further exploration of TPT modulation of ENZ resistance in the clinic.

Citation Format: John P. Fruehauf, Nathan Farrokh, Sarmen Sarkissian, Jai-Hyun Kim. Blockade of ARV7:HIF1α; heterodimers after toptoecan reverses enzalutamide resistance in 22Rv1 cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A37.