The histone demethylase, JMJD2B, as a potential therapeutic target in prostate cancer


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Kelly Armstrong1, Lynsey Rogerson1, Dhuha AlKaraif1, Jacqueline Stockley1, Luke Gaughan1, Craig Robson1
1Newcastle University, Newcastle upon Tyne, United Kingdom

Background

The androgen receptor (AR) is a key molecule in prostate cancer (CaP) development and progression. Proteins which regulate AR activity could prove useful drug targets. Recently, histone methyltransferase (HMT) and demethylase (HDM) enzymes have been described as potential AR regulators. In this study HDM enzymes were tested for their ability to regulate the AR.

Method

To determine which HDMs are involved in AR signalling, siRNA screening with a PSA ELISA endpoint was used on LNCaP cells stimulated with the ligand dihydrotestosterone (DHT). Data validation was achieved using QPCR for PSA mRNA levels and knockdown confirmation. Reporter assays were used to confirm effects on AR transcriptional activity whilst Western blotting was used to identify whether knockdown of JMJD2B results in altered AR protein levels. WST-1 assays were used to assess knockdown effects on proliferation. Histone marks on androgen responsive promoters were assessed by chromatin immunoprecipitation in response to DHT.

Results

JMJD2B was identified as a potential AR co-activator by siRNA screening and QPCR. Further characterisation of JMJD2B revealed that its enzymatic activity is essential for its co-activator function. Upon JMJD2B knockdown in multiple androgen-responsive CaP cell lines, AR protein levels were greatly reduced and proliferation was decreased. JMJD2B protein was found at both the PSA enhancer and promoter where tri-methylation of histone H3 at lysine 9 (H3K9Me3) is reduced in response to DHT. Knockdown of JMJD2B reduced AR recruitment, enhanced H3K9Me3 levels and reduced acetylation of H3K9 at these regulatory sites suggesting JMJD2B is a key molecule in androgen regulated transcription.

Conclusion

JMJD2B enzymatic activity appears to be important in regulating the transcriptional activity and protein levels of the AR. Thus, targeting this enzyme with a drug may help to overcome mis-regulation of the AR in CaP.