The histone demethylase enzymes KDM3A and KDM4B co-operatively regulate chromatin transactions of the estrogen receptor in breast cancer

Many estrogen receptor (ER)-positive breast cancers develop resistance to endocrine therapy but retain canonical receptor signalling in the presence of selective ER antagonists. Numerous co-regulatory proteins, including enzymes that modulate the chromatin environment, control the transcriptional activity of the ER. Targeting ER co-regulators has therefore been proposed as a novel therapeutic approach. By assessing DNA-binding dynamics in ER-positive breast cancer cells, we have identified that the histone H3 lysine 9 demethylase enzymes, KDM3A and KDM4B, co-operate to regulate ER activity via an auto-regulatory loop that facilitates the recruitment of each co-activating enzyme to chromatin. We also provide evidence that suggests that KDM3A primes chromatin for deposition of the ER pioneer factor FOXA1 and recruitment of the ER-transcriptional complex, all prior to ER recruitment, therefore establishing an important mechanism of chromatin regulation involving histone demethylases and pioneer factors, which controls ER functionality. Importantly, we show via global gene-expression analysis that a KDM3A/KDM4B/FOXA1 co-regulated gene signature is enriched for pro-proliferative and ER-target gene sets, suggesting that abrogation of this network could be an efficacious therapeutic strategy. Finally, we show that depletion of both KDM3A and KDM4B has a greater inhibitory effect on ER activity and cell growth than knockdown of each individual enzyme, suggesting that targeting both enzymes represents a potentially efficacious therapeutic option for ER-driven breast cancer

Survival outcome and EMT suppression mediated by a lectin domain interaction of Endo180 and CD147

Epithelial cell-cell contacts maintain normal glandular tissue homeostasis, and their breakage can trigger epithelial-to-mesenchymal transition (EMT), a fundamental step in the development of metastatic cancer. Despite the ability of C-type lectin domains (CTLD) to modulate cell-cell adhesion, it is not known if they modulate epithelial adhesion in EMT and tumor progression. Here, the multi-CTLD mannose receptor, Endo180 (MRC2/uPARAP), was shown using the Kaplan-Meier analysis to be predictive of survival outcome in men with early prostate cancer. A proteomic screen of novel interaction partners with the fourth CTLD (CTLD4) in Endo180 revealed that its complex with CD147 is indispensable for the stability of three-dimensional acini formed by nontransformed prostate epithelial cells (PEC). Mechanistic study using knockdown of Endo180 or CD147, and treatment with an Endo180 mAb targeting CTLD4 (clone 39.10), or a dominant-negative GST-CTLD4 chimeric protein, induced scattering of PECs associated with internalization of Endo180 into endosomes, loss of E-cadherin (CDH1/ECAD), and unzipping of cell-cell junctions. These findings are the first to demonstrate that a CTLD acts as a suppressor and regulatory switch for EMT; thus, positing that stabilization of Endo180-CD147 complex is a viable therapeutic strategy to improve rates of prostate cancer survival

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

AGE-modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell–cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180ΔEx2–6/ΔEx2–6 mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors

Deubiquitinating enzyme Usp12 regulates the interaction between the androgen receptor and the Akt pathway

The androgen receptor (AR) is a transcription factor involved in prostate cell growth, homeostasis and transformation regulated by post-translational modifications, including ubiquitination. We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity. In this study we have investigated the relationship between Usp12, PHLPP and PHLPPL tumour suppressors in the regulation of AR transcriptional activity in prostate cancer (PC). PHLPP and PHLPPL are pro-apoptotic phosphatases that dephosphorylate and subsequently deactivate Akt. Phosphorylated Akt is reported to deactivate AR in PC by phosphorylation at Ser213 and Ser791 leading to ligand dissociation and AR degradation. In contrast, PHLPP- and PHLPPL-mediated dephosphorylation and inactivation of Akt elevates the levels of active AR. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt. Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity. Additionally, we observe that depleting Usp12 sensitises PC cells to therapies aimed at Akt inhibition irrespectively of their sensitivity to androgen ablation therapy. We propose that Usp12 inhibition could offer a therapeutic alternative for castration resistant prostate cancer

Development and exploitation of a novel mutant androgen receptor modelling strategy to identify new targets for advanced prostate cancer therapy

The persistence of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the unmet clinical need for the development of more effective AR targeting therapies. A key mechanism of therapy-resistance is by selection of AR mutations that convert anti-androgens to agonists enabling the retention of androgenic signalling in CRPC. To improve our understanding of these receptors in advanced disease we developed a physiologically-relevant model to analyse the global functionality of AR mutants in CRPC. Using the bicalutamide-activated ARW741L/C mutation as proof of concept, we demonstrate that this mutant confers an androgenic-like signalling programme and growth promoting phenotype in the presence of bicalutamide. Transcriptomic profiling of ARW741L highlighted key genes markedly up-regulated by the mutant receptor, including TIPARP, RASD1 and SGK1. Importantly, SGK1 expression was found to be highly expressed in the KUCaP xenograft model and a CRPC patient biopsy sample both of which express the bicalutamide-activated receptor mutant. Using an SGK1 inhibitor, ARW741L transcriptional and growth promoting activity was reduced indicating that exploiting functional distinctions between receptor isoforms in our model may provide new and effective therapies for CRPC patients

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

Targeting the DNA Double Strand Break Repair Machinery in Prostate Cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways

Regulation of the androgen receptor by SET9-mediated methylation

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment