Mono-ubiquitination mediated regulation of KMT5A and its role in prostate cancer

PhD ThesisAbstract: Prostate cancer (PC) is the most common cancer and the second cause of cancer related death in men. Central to this, is the role of the androgen receptor (AR) which acts as a transcription factor, regulating the expression of genes required for normal prostate growth and cancer development. Consequently, the AR remains the primary target for therapeutic intervention. However, these treatments become ineffective, resulting in castrate resistant prostate cancer (CRPC) which generally retains AR expression. The AR interacts with several co-regulatory proteins which can perturb AR-targeted therapies in CRPC. Targeting these co-regulatory proteins to indirectly target the AR signalling cascade may prove beneficial. Recently, our group identified KMT5A as a potential regulator of AR through selective siRNA library screening. KMT5A is a lysine methyltransferase that mono-methylates histone 4 lysine 20 and non-histone proteins, including p53. Using a relevant in vitro CRPC model it was shown that KMT5A acquires AR co-activator activity which is in contrast to androgen sensitive models where KMT5A co-represses AR activity. This highlights the importance of studying KMT5A regulation. KMT5A protein levels are tightly regulated by multiple E3 ligases for cell cycle-dependent poly-ubiquitination-mediated degradation. KMT5A poly-ubiquitination by E3 ligases SCFβ-TRCF, CRL4Cdt2 and APCCdh1 promotes degradation in G1, 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 direct supporting evidence is currently absent. Additionally, Skp2 is suggested to directly regulate the AR signaling pathway. It is also unknown whether KMT5A could be modified directly by ubiquitination without promoting its degradation. As such, we aimed to investigate KMT5A mono-ubiquitination and the role of Skp2 in regulating KMT5A as well as independently regulating the AR signaling cascade. Mono-ubiquitinated KMT5A was demonstrated in a panel of PC cell lines. Its existence was further confirmed by performing ubiquitination assays in COS7 cells. Furthermore, the KMT5A C-terminal SET domain was identified as the target for mono-ubiquitination. Moreover, mono-ubiquitinated KMT5A was highly enriched in S phase cells, coincident with extremely low levels of unmodified KMT5A. Mono-ubiquitinated KMT5A was exclusively cytoplasmic and its abundance was greatly enhanced by Skp2, but not associated with protein turnover. Together, this data suggests that cell cycle-dependent KMT5A mono-ubiquitination is an important mechanism to diminish nuclear, unmodified KMT5A levels to facilitate cell cycle progression. Thus, insight for the physiological significance of mono-ubiquitinated KMT5A ii may provide a novel therapeutic target to indirectly target the AR. Finally, Skp2 was not found to have a direct effect on AR signaling

The novel anti-androgen candidate galeterone targets deubiquitinating enzymes, USP12 and USP46, to control prostate cancer growth and survival

Metastatic castration resistant prostate cancer is one of the main causes of male cancer associated deaths worldwide. Development of resistance is inevitable in patients treated with anti-androgen therapies. This highlights a need for novel therapeutic strategies that would be aimed upstream of the androgen receptor (AR). Here we report that the novel small molecule anti-androgen, galeterone targets USP12 and USP46, two highly homologous deubiquitinating enzymes that control the AR-AKT-MDM2-P53 signalling pathway. Consequently, galeterone is effective in multiple models of prostate cancer including both castrate resistant and AR-negative prostate cancer. However, we have observed that USP12 and USP46 selectively regulate full length AR protein but not the AR variants. This is the first report of deubiquitinating enzyme targeting as a strategy in prostate cancer treatment which we show to be effective in multiple, currently incurable models of this disease

CDC20 is regulated by the histone methyltransferase, KMT5A, in castration resistant prostate cancer

The methyltransferase, KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis we have performed a microarray study in a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and Gene Ontology analysis revealed apoptosis and DNA damage signal- ing are up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase depend- ent manner to modulate both histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilized as a biomarker for effective therapeutic targeting

CDC20 Is Regulated by the Histone Methyltransferase, KMT5A, in Castration-Resistant Prostate Cancer

The methyltransferase KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis, we have performed a microarray study on a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of the transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and gene ontology analysis revealed that apoptosis and DNA damage signalling were up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase-dependent manner to modulate histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples, further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilised as a biomarker for effective therapeutic targeting

Molecular mechanism of the TP53-MDM2-AR-AKT signalling network regulation by USP12

The TP53-MDM2-AR-AKT signalling network plays a critical role in the development and progression of prostate cancer. However, the molecular mechanisms regulating this signalling network are not completely defined. By conducting transcriptome analysis, denaturing immunoprecipitations and immunopathology, we demonstrate that the TP53-MDM2-AR-AKT cross-talk is regulated by the deubiquitinating enzyme USP12 in prostate cancer. Our findings explain why USP12 is one of the 12 most commonly overexpressed cancer-associated genes located near an amplified super-enhancer. We find that USP12 deubiquitinates MDM2 and AR, which in turn controls the levels of the TP53 tumour suppressor and AR oncogene in prostate cancer. Consequently, USP12 levels are predictive not only of cancer development but also of patient’s therapy resistance, relapse and survival. Therefore, our findings suggest that USP12 could serve as a promising therapeutic target in currently incurable castrate-resistant prostate cancer

Antibody-induced nonapoptotic cell death in human lymphoma and leukemia cells is mediated through a novel reactive oxygen species-dependent pathway.

Monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell malignancies. Although Fc-dependent mechanisms of mAb-mediated tumor clearance have been extensively studied, the ability of mAbs to directly evoke programmed cell death (PCD) in the target cell and the underlying mechanisms involved remain under-investigated. We recently demonstrated that certain mAbs (type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD through an actin-dependent, lysosome-mediated process. Here, we reveal that the induction of PCD by these mAbs, including the type II anti-CD20 mAb GA101 (obinutuzumab), directly correlates with their ability to produce reactive oxygen species (ROS) in human B-lymphoma cell lines and primary B-cell chronic lymphocytic leukemia cells. ROS scavengers abrogated mAb-induced PCD indicating that ROS are required for the execution of cell death. ROS were generated downstream of mAb-induced actin cytoskeletal reorganization and lysosome membrane permeabilization. ROS production was independent of mitochondria and unaffected by BCL-2 overexpression. Instead, ROS generation was mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These findings provide further insights into a previously unrecognized role for NADPH oxidase-derived ROS in mediating nonapoptotic PCD evoked by mAbs in B-cell malignancies. This newly characterized cell death pathway may potentially be exploited to eliminate malignant cells, which are refractory to conventional chemotherapy and immunotherapy