Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively active COOH-terminally truncated AR variants lacking the AR ligand-binding domain has emerged as an important mechanism of ADT resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35-kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8579-bp deletion of AR exons 5, 6 and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted resequencing of the AR gene in CWR-R1 cells led to the discovery of a 48-kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.
Bibliographical noteFunding Information:
We are grateful to the Minnesota Supercomputing Institute and the Masonic Cancer Center Biostatistics and Bioinformatics Core for providing computing, bioinformatics, statistical, software and data storage support for this project. Cytogenetic analyses were performed in the Cytogenetics Shared Resource Laboratory at the University of Minnesota, with support from the comprehensive Masonic Cancer Center NIH Grant P30 CA077598. We thank Amanda Hemmingsen Jaeger for assistance with developing MLPA for AR gene structure analysis. SMD is a Masonic Scholar of the Masonic Cancer Center, University of Minnesota. This work was supported by a Young Investigator Award from the Prostate Cancer Foundation (SMD), DOD New Investigator Award PC094384 (SMD) NCI Grant CA141011 (SMD), and a seed grant from the Institute of Human Genetics, University of Minnesota.
- AR alternative splicing
- Androgen receptor variants
- Intragenic rearrangement
- Prostate cancer