C89
Identification of Androgen receptor gene expression signatures in prostate cancer with high through-put validation
Charlie Massie1, Ladan Fazli2, Andy Lynch1, Rory Stark1, Elena Grigorenko3, Naomi Sharma1, Helen Scott1, David Neal1, Ian Mills1
1CRUK Cambridge Research Institute, Cambridge, UK, 2The Prostate Centre at Vancouver General Hospital, Vancouver, Canada, 3BioTrove, Woburn, USA
The AR signalling axis is central to Prostate Cancer (PrCa) biology, shown by its use in both screening (PSA) and treatment of the disease (androgen deprivation therapy). Identifying direct target genes of the AR will provide a better understanding the key signalling pathways controlled by the AR in PrCa and could lead to improved biomarkers and future therapeutic targets for the disease.
To this end we have performed fine mapping of androgen responsive gene expression in prostate cancer cell lines using Illumina bead-arrays, coupled with AR chromatin-immunoprecipitation and Solexa sequencing technology (ChIP-seq). This approach generated a long list of candidate AR target genes, of which only a small percentage could be validated by conventional quantitative Realtime PCR. Therefore we have undertaken large scale, cross-platform validation using BioTrove OpenArray Realtime PCR panels which allow the measurement of >600 transcripts simultaneously.
Using this combination of approaches we have identified and validated several hundred AR regulated genes. Androgen regulated protein kinases are of particular interest given that these enzymes often show altered activity in cancer and can be targeted by small molecule inhibitors. Using the BioTrove qPCR Kinome panel we were able to identify 82 androgen regulated kinases, of which 22 were detected on the Illumina expression arrays. Calmodulin kinase kinase 2 (CAMKK2) was detected as an androgen regulated gene on both platforms, contained a promoter AR binding site by ChIP-seq and showed increased expression in clinical PrCa samples. A small molecule inhibitor of CAMKK2 (STO-609) inhibited the growth of a panel of PrCa cell lines. Interestingly, the levels of CAMKK2 were reduced in PrCas following androgen deprivation therapy and were raised in Castrate Resistant disease, showing that CAMKK2 is AR regulated and remains a potential therapeutic target in anti-androgen resistant disease.
In summary, these data show that combining expression analysis, ChIP-seq and high through-put validation allows the identification pathway signatures and can help to identify important signalling molecules.
