Kelly Coffey

Regulation of the androgen receptor by post-translational modifications

The androgen receptor (AR) is a key molecule in prostate cancer and Kennedys disease. Understanding the regulatory mechanisms of this steroid receptor is important in the development of potential therapies for these diseases. One layer of AR regulation is provided by post-translational modifications including phosphorylation, acetylation, sumoylation, ubiquitination and methylation. While these modifications have mostly been studied as individual events, it is becoming clear that these modifications can functionally interact with each other in a signalling pathway. In this review, the effects of all modifications are described with a focus on interplay between them and the functional consequences for the AR.

Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer

Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an in vitro HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC50 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8–27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.

The lysine demethylase, KDM4B, is a key molecule in androgen receptor signalling and turnover

The androgen receptor (AR) is a key molecule involved in prostate cancer (PC) development and progression. Post-translational modification of the AR by co-regulator proteins can modulate its transcriptional activity. To identify which demethylases might be involved in AR regulation, an siRNA screen was performed to reveal that the demethylase, KDM4B, may be an important co-regulator protein. KDM4B enzymatic activity is required to enhance AR transcriptional activity; however, independently of this activity, KDM4B can enhance AR protein stability via inhibition of AR ubiquitination. Importantly, knockdown of KDM4B in multiple cell lines results in almost complete depletion of AR protein levels. For the first time, we have identified KDM4B to be an androgen-regulated demethylase enzyme, which can influence AR transcriptional activity not only via demethylation activity but also via modulation of ubiquitination. Together, these findings demonstrate the close functional relationship between AR and KDM4B, which work together to amplify the androgen response. Furthermore, KDM4B expression in clinical PC specimens positively correlates with increasing cancer grade (P < 0.001). Consequently, KDM4B is a viable therapeutic target in PC.

Deubiquitinating Enzyme Usp12 Is a Novel Co-activator of the Androgen Receptor

Background: Androgen receptor (AR) is the principle therapeutic target in prostate cancer. Result: We have established that Usp12 deubiquitinates and stabilizes the AR resulting in increased transcriptional and pro-proliferative activity. Conclusion: We have identified Usp12 to be a novel positive regulator of AR. Significance: Usp12 presents a therapeutic target upstream of AR that could enable bypassing the limitations of therapeutics aimed specifically at AR. The androgen receptor (AR), a member of the nuclear receptor family, is a transcription factor involved in prostate cell growth, homeostasis, and transformation. AR is a key protein in growth and development of both normal and malignant prostate, making it a common therapeutic target in prostate cancer. AR is regulated by an interplay of multiple post-translational modifications including ubiquitination. We and others have shown that the AR is ubiquitinated by a number of E3 ubiquitin ligases, including MDM2, CHIP, and NEDD4, which can result in its proteosomal degradation or enhanced transcriptional activity. As ubiquitination of AR causes a change in AR activity or stability and impacts both survival and growth of prostate cancer cells, deubiquitination of these sites has an equally important role. Hence, deubiquitinating enzymes could offer novel therapeutic targets. We performed an siRNA screen to identify deubiquitinating enzymes that regulate AR; in that screen ubiquitin-specific protease 12 (Usp12) was identified as a novel positive regulator of AR. Usp12 is a poorly characterized protein with few known functions and requires the interaction with two cofactors, Uaf-1 and WDR20, for its enzymatic activity. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity. Our data show that Usp12 acts in a pro-proliferative manner by stabilizing AR and enhancing its cellular function.

The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer

Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved in breast cancer progression. To date, a small number of enzymes that control methylation status of histones have been identified as co-regulators of ER signalling. We have identified the histone H3 lysine 9 mono- and di-methyl demethylase enzyme KDM3A as a positive regulator of ER activity. Here, we demonstrate that depletion of KDM3A by RNAi abrogates the recruitment of the ER to cis-regulatory elements within target gene promoters, thereby inhibiting estrogen-induced gene expression changes. Global gene expression analysis of KDM3A-depleted cells identified gene clusters associated with cell growth. Consistent with this, we show that knockdown of KDM3A reduces ER-positive cell proliferation and demonstrate that KDM3A is required for growth in a model of endocrine therapy-resistant disease. Crucially, we show that KDM3A catalytic activity is required for both ER-target gene expression and cell growth, demonstrating that developing compounds which target demethylase enzymatic activity may be efficacious in treating both ER-positive and endocrine therapy-resistant disease.

KDM4B is a Master Regulator of the Estrogen Receptor Signalling Cascade

The importance of the estrogen receptor (ER) in breast cancer (BCa) development makes it a prominent target for therapy. Current treatments, however, have limited effectiveness, and hence the definition of new therapeutic targets is vital. The ER is a member of the nuclear hormone receptor superfamily of transcription factors that requires co-regulator proteins for complete regulation. Emerging evidence has implicated a small number of histone methyltransferase (HMT) and histone demethylase (HDM) enzymes as regulators of ER signalling, including the histone H3 lysine 9 tri-/di-methyl HDM enzyme KDM4B. Two recent independent reports have demonstrated that KDM4B is required for ER-mediated transcription and depletion of the enzyme attenuates BCa growth in vitro and in vivo. Here we show that KDM4B has an overarching regulatory role in the ER signalling cascade by controlling expression of the ER and FOXA1 genes, two critical components for maintenance of the estrogen-dependent phenotype. KDM4B interacts with the transcription factor GATA-3 in BCa cell lines and directly co-activates GATA-3 activity in reporter-based experiments. Moreover, we reveal that KDM4B recruitment and demethylation of repressive H3K9me3 marks within upstream regulatory regions of the ER gene permits binding of GATA-3 to drive receptor expression. Ultimately, our findings confirm the importance of KDM4B within the ER signalling cascade and as a potential therapeutic target for BCa treatment.

Human α2β1HI CD133+VE Epithelial Prostate Stem Cells Express Low Levels of Active Androgen Receptor

Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α2β1 HI CD133+VE), transiently amplifying (α2β1 HI CD133–VE) and terminally differentiated (α2β1 LOW CD133–VE) cells. AR transcript and protein expression was confirmed in α2β1 HI CD133+VE and CD133–VE progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α2β1 HI CD133+VE stem cells and 68% (±12%) in α2β1 HI CD133–VE transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α2β1 HI CD133+VE stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133+VE cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis.

Gene Silencing by RNAi in Mammalian Cells

This unit provides information how to use short interfering RNA (siRNA) for sequence‐specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented.

Ubiquitin-specific protease 12 interacting partners Uaf-1 and WDR20 are potential therapeutic targets in prostate cancer

The androgen receptor (AR) is a key transcription factor in the initiation and progression of prostate cancer (PC) and is a major therapeutic target for the treatment of advanced disease. Unfortunately, current therapies are not curative for castration resistant PC and a better understanding of AR regulation could identify novel therapeutic targets and biomarkers to aid treatment of this disease. The AR is known to be regulated by a number of post-translational modifications and we have recently identified the deubiquitinating enzyme Usp12 as a positive regulator of AR. We determined that Usp12 deubiquitinates the AR resulting in elevated receptor stability and activity. Furthermore, Usp12 silencing was shown to reduce proliferation of PC cells. Usp12 is known to require the co-factors Uaf-1 and WDR20 for catalytic activity. In this report we focus further on the role of Uaf-1 and WDR20 in Usp12 regulation and investigate if these co-factors are also required for controlling AR activity. Firstly, we confirm the presence of the Usp12/Uaf-1/WDR20 complex in PC cells and demonstrate the importance of Uaf-1 and WDR20 for Usp12 stabilisation. Consequently, we show that individual silencing of either Uaf-1 or WDR20 is sufficient to abrogate the activity of the Usp12 complex and down-regulate AR-mediated transcription via receptor destabilisation resulting in increased apoptosis and decreased colony forming ability of PC cells. Moreover, expression of both Uaf-1 and WDR20 is higher in PC tissue compared to benign controls. Overall these results highlight the potential importance of the Usp12/Uaf-1/WDR20 complex in AR regulation and PC progression. Highlights: Androgen receptor is a key transcriptional regulator in prostate cancer Usp12/Uaf-1/WDR20 complex plays a crucial role in androgen receptor stability and activity Destabilising an individual Usp12/Uaf-1/WDR20 complex member reduces the protein levels of the whole complex and diminishes androgen receptor activity Protein levels of all members of the Usp12/Uaf-1/WDR20 complex are significantly increased in PC

Abstract 4464: The role of KMT5A in prostate cancer

Castrate resistant prostate cancer (CRPC) remains a significant clinical problem with no currently available cure. The Androgen receptor (AR) continues to function in CRPC through a plethora of mechanisms and the AR remains the primary target for therapeutic intervention in CRPC. Targeting AR co-regulating proteins to indirectly target AR signaling may prove beneficial. Recently, our group identified KMT5A as a potential regulator of AR from selective siRNA library screening. KMT5A is a methyltransferase which monomethylates histone 4 on lysine 20. The establishment of this epigenetic mark directly impacts chromatin structure, genome stability and cell cycle progression, all hallmarks of cancer. In addition, it monomethylates non-histone proteins such as p53. It is therefore important that KMT5A activity and its cellular localization are under tight regulation. Using a relevant in vitro CRPC model we have shown that KMT5A acts as an AR co-activator while in androgen sensitive models KMT5A restricts AR activity. This highlights the importance of studying how KMT5A itself is regulated. KMT5A is primarily regulated through post-translational modifications in a cell cycle dependent manner. KMT5A polyubiquitination by E3 ligases CUL4-Cdt2 and APC-Cdh1 causes its proteasomal mediated degradation in S and late mitosis cell cycle phases, respectively. Moreover, the Skp2 E3 ligase has been suggested to play a role in KMT5A ubiquitination and degradation; but there is no direct evidence to show this to date. Therefore, the role of Skp2 in regulating KMT5A is understudied and remains elusive. It is also not known whether KMT5A could be modified directly by ubiquitination without leading to its degradation. As such, we aimed to investigate KMT5A monoubiquitination and the role of Skp2 in regulating KMT5A. We demonstrated monoubiquitinated KMT5A is present in a panel of PC cell lines. This was further confirmed by performing ubiquitination assays in COS7 cells. We determined that the KMT5A C-terminal SET domain is targeted for monoubiquitination and identification of the site(s) is ongoing. Furthermore, monoubiquintinated KMT5A was found to be exclusively cytoplasmic. In addition, Skp2 markedly enhances KMT5A monoubiquitination in a concentration dependent manner. We were also able to show that KMT5A and Skp2 interact. Skp2 mediated monoubiquitination of KMT5A however is not associated with protein turn over as addition of MG132 (proteasome inhibitor) does not further stabilize KMT5A. Interestingly, Skp2 mediated monoubiquitination of KMT5A downregulates MG132 mediated stabilization of KMT5A, possibly indicating this monoubiquitination impacts the accessibility of KMT5A for other ligases which promote KMT5A polyubiquitination and degradation. The newly characterized modification of KMT5A is a prevalent form of total KMT5A which can affect its function. Thus, insight into its physiological significance may provide a novel therapeutic target to indirectly target the AR. Citation Format: Mahsa Azizyan, Kelly Coffey, Craig N. Robson. The role of KMT5A in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4464.