EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells.

The androgen receptor (AR) is a primary target for treating prostate cancer (PCa), forming the bedrock of its clinical management. Despite their efficacy, resistance often hampers AR-targeted therapies, necessitating new strategies against therapy-resistant PCa. These resistances involve various mechanisms, including AR splice variant overexpression and altered activities of transcription factors like the glucocorticoid receptor (GR) and FOXA1. These factors rely on common coregulators, such as EP300/CREBBP, suggesting a rationale for coregulator-targeted therapies. Our study explores EP300/CREBBP acetyltransferase inhibition's impact on steroid receptor and FOXA1 signaling in PCa cells using genome-wide techniques. Results reveal that EP300/CREBBP inhibition significantly disrupts the AR-regulated transcriptome and receptor chromatin binding by reducing the AR-gene expression. Similarly, GR's regulated transcriptome and receptor binding were hindered, not linked to reduced GR expression but to diminished FOXA1 chromatin binding, restricting GR signaling. Overall, our findings highlight how EP300/CREBBP inhibition distinctively curtails oncogenic transcription factors' signaling, suggesting the potential of coregulatory-targeted therapies in PCa.

Chromatin accessibility and pioneer factor FOXA1 restrict glucocorticoid receptor action in prostate cancer.

Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300's enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.

MCF10CA Breast Cancer Cells Utilize Hyaluronan-Coated EV-Rich Trails for Coordinated Migration.

Invasion of tumor cells through the stroma is coordinated in response to migratory cues provided by the extracellular environment. One of the most abundant molecules in the tumor microenvironment is hyaluronan, a glycosaminoglycan known to promote many hallmarks of tumor progression, including the migratory potential of tumor cells. Strikingly, hyaluronan is also often found to coat extracellular vesicles (EVs) that originate from plasma membrane tentacles of tumor cells crucial for migration, such as filopodia, and are abundant in tumor niches. Thus, it is possible that hyaluronan and hyaluronan-coated EVs have a cooperative role in promoting migration. In this work, we compared the hyaluronan synthesis, EV secretion and migratory behavior of normal and aggressive breast cell lines from MCF10 series. Single live cell confocal imaging, electron microscopy and correlative light and electron microscopy experiments revealed that migrating tumor cells form EV-rich and hyaluronan -coated trails. These trails promote the pathfinding behavior of follower cells, which is dependent on hyaluronan. Specifically, we demonstrated that plasma membrane protrusions and EVs left behind by tumor cells during migration are strongly positive for CD9. Single cell tracking demonstrated a leader-follower behavior, which was significantly decreased upon removal of pericellular hyaluronan, indicating that hyaluronan promotes the pathfinding behavior of follower cells. Chick chorioallantoic membrane assays in ovo suggest that tumor cells behave similarly in 3D conditions. This study strengthens the important role of extracellular matrix production and architecture in coordinated tumor cell movements and validates the role of EVs as important components and regulators of tumor matrix. The results suggest that tumor cells can modify the extracellular niche by forming trails, which they subsequently follow coordinatively. Future studies will clarify in more detail the orchestrated role of hyaluronan, EVs and other extracellular cues in coordinated migration and pathfinding behavior of follower cells.

M1 Macrophages Induce Protumor Inflammation in Melanoma Cells through TNFR-NF-κB Signaling.

The tumor microenvironment, with distinctive cell types and a complex extracellular matrix has a tremendous impact on cancer progression. In this study, we investigated the effects of proinflammatory (M1) and immunosuppressive (M2) macrophages on hyaluronan (HA) matrix formation and inflammatory response in melanoma cells. Proinflammatory factors secreted from M1 macrophages stimulated the formation of a thick pericellular HA matrix in melanoma cells due to upregulation of HA synthase 2 (HAS2). HAS2 silencing reversed the effect of M1 conditioned medium on pericellular HA coat formation, and interestingly, it also partly downregulated the M1 conditioned medium‒induced upregulation of inflammation-related genes (IL1ß, IL6), as did the inhibitors for TNFR and IKKγ. Gene set enrichment analysis revealed that genes related to inflammatory responses and TNF-α signaling via NF-κB are enriched in the M1 conditioned medium‒treated melanoma cells. Moreover, the expression of matrix metalloproteinase 9 and three-dimensional cell invasion were induced in these cells, whereas M2 macrophages had no effect on HA synthesis, inflammatory response, or invasion. Our results indicate that the activation of TNFR-NF-κB signaling in M1 conditioned medium‒treated cells leads to HAS2 upregulation, which associates with a protumor inflammatory and invasive phenotype of melanoma cells.

The role of FoxP3+ regulatory T cells and IDO+ immune and tumor cells in malignant melanoma - an immunohistochemical study.

BACKGROUND: FoxP3+ Regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) participate in the formation of an immunosuppressive tumor microenvironment (TME) in malignant cutaneous melanoma (CM). Recent studies have reported that IDO expression correlates with poor prognosis and greater Breslow's depth, but results concerning the role of FoxP3+ Tregs in CM have been controversial. Furthermore, the correlation between IDO and Tregs has not been substantially studied in CM, although IDO is known to be an important regulator of Tregs activity. METHODS: We investigated the associations of FoxP3+ Tregs, IDO+ tumor cells and IDO+ stromal immune cells with tumor stage, prognostic factors and survival in CM. FoxP3 and IDO were immunohistochemically stained from 29 benign and 29 dysplastic nevi, 18 in situ -melanomas, 48 superficial and 62 deep melanomas and 67 lymph node metastases (LNMs) of CM. The number of FoxP3+ Tregs and IDO+ stromal immune cells, and the coverage and intensity of IDO+ tumor cells were analysed. RESULTS: The number of FoxP3+ Tregs and IDO+ stromal immune cells were significantly higher in malignant melanomas compared with benign lesions. The increased expression of IDO in melanoma cells was associated with poor prognostic factors, such as recurrence, nodular growth pattern and increased mitotic count. Furthermore, the expression of IDO in melanoma cells was associated with reduced recurrence-free survival. We further showed that there was a positive correlation between IDO+ tumor cells and FoxP3+ Tregs. CONCLUSIONS: These results indicate that IDO is strongly involved in melanoma progression. FoxP3+ Tregs also seems to contribute to the immunosuppressive TME in CM, but their significance in melanoma progression remains unclear. The positive association of FoxP3+ Tregs with IDO+ melanoma cells, but not with IDO+ stromal immune cells, indicates a complex interaction between IDO and Tregs in CM, which demands further studies.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

In Vivo Characterization of the Ultrapotent Monoacylglycerol Lipase Inhibitor {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048).

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Increased tonic cannabinoid CB1R activity and brain region-specific desensitization of CB1R Gi/o signaling axis in mice with global genetic knockout of monoacylglycerol lipase.

In mammalian brain, monoacylglycerol lipase (MAGL) is the primary enzyme responsible for terminating signaling function of the endocannabinoid 2-arachidonoylglycerol (2-AG). Previous in vivo studies with mice indicate that both genetic and chronic pharmacological inactivation of MAGL result in 8-30-fold increase of 2-AG concentration in the brain, causing desensitization and downregulation of cannabinoid CB1 receptor (CB1R) activity, leading to functional and behavioral tolerance. However, direct evidence for reduced CB1R activity in the brain is lacking. In this study, we used functional autoradiography to assess basal and agonist-stimulated CB1R-dependent Gi/o protein activity in multiple brain regions of MAGL-KO mice in comparison to their wild-type (WT) littermates. In addition, the role of endogenous cannabinoids in basal CB1R signaling was assessed after comprehensive pharmacological blockade of 2-AG hydrolysis by determining the contents of endocannabinoids (eCBs) in WT and MAGL-KO brain tissues by LC/MS/MS technology. To show whether lack of MAGL cause compensatory alterations in the serine hydrolase activity, we compared serine hydrolase pattern of WT and MAGL-KO using activity-based protein profiling. Consistent with studies using chronic pharmacological MAGL inactivation in vivo, we observed a statistically significant decrease of CB1R-Gi/o signaling in most of the studied brain regions. In MAGL-KO brain sections, elevated 2-AG levels were mirrored to heightened basal CB1R-dependent Gi/o-activity, as well as, dampened agonist-evoked responses in several brain regions. The non-selective serine hydrolase inhibitor methylarachidonoylfluorophosphonate (MAFP) was able to significantly elevate 2-AG levels in brain sections of MAGL-KO mice, indicating that additional serine hydrolases possess 2-AG hydrolytic activity in MAGL-KO brain sections.

Optimization of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors.

At present, inhibitors of α/ß-hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the development of 1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized, and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4-morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430) potently and irreversibly inhibited hABHD6 (IC50 =44 nM) and showed ∼230-fold selectivity over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off-targets of related compounds. Additionally, activity-based protein profiling indicated that JZP-430 displays good selectivity among the serine hydrolases of the mouse brain membrane proteome. JZP-430 has been identified as a highly selective, irreversible inhibitor of hABHD6, which may provide a novel approach in the treatment of obesity and type II diabetes.

Brain regional cannabinoid CB(1) receptor signalling and alternative enzymatic pathways for 2-arachidonoylglycerol generation in brain sections of diacylglycerol lipase deficient mice.

Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The principal brain endocannabinoid, 2-arachidonoylglycerol (2-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB1 receptors in a retrograde manner. The primary pathway for 2-AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLα and DAGLß. Previous studies with DAGL-deficient mice indicated that DAGLα is the major enzyme needed for retrograde synaptic 2-AG signalling. The current study investigated whether the CB1 receptor-mediated Gi/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate 2-AG in brain cryosections. Functional autoradiography indicated that brain regional CB1 receptor-Gi/o-activity largely remained unaltered in DAGLα-knockout and DAGLß-knockout mice when compared to wild-type littermates. Following comprehensive pharmacological blockade of 2-AG hydrolysis, brain sections generated sufficient amounts of 2-AG to activate CB1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of 2-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLα or DAGLß. We conclude that in addition to the sn-1-specific DAGLs, additional 2-AG generating enzymatic pathways are active in brain sections.

Quantification of endocannabinoids in postmortem brain of schizophrenic subjects.

Numerous studies have implicated the endocannabinoid system in the pathophysiology of schizophrenia. Endocannabinoids have been measured in blood and cerebrospinal fluid in schizophrenic patients but, to the date, there are no published reports dealing with measurements of endocannabinoid levels in schizophrenics' brain tissue. In the present study, postmortem brain samples from 19 subjects diagnosed with schizophrenia (DSM-IV) and 19 matched controls were studied. In specific brain regions, levels of four endocannabinoids (2-arachidonoylglycerol (2-AG), arachidonoylethanolamine (anandamide, AEA), dihomo-γ-linolenoylethanolamine (LEA), and docosahexaenoylethanolamine (DHEA)) and two cannabimimetic compounds (palmitoyl-ethanolamine (PEA) and oleoyl-ethanolamine (OEA)) were measured using quantitative liquid chromatography with triple quadrupole mass spectrometric detection. Suffering from schizophrenia significantly affects the brain levels of 2-AG (p<0.001), AEA (p<0.0001), DHEA (p<0.0001), LEA (p<0.01) and PEA (p<0.05). In schizophrenic subjects, the three studied brain regions (cerebellum: 130±18%; p=0.16; hippocampus: 168±28%, p<0.01; prefrontal cortex: 237±45%, p<0.05) showed higher 2-AG levels when compared to matched controls. Conversely, AEA levels were lower in all brain regions of schizophrenic subjects (cerebellum: 66±7%, p<0.01; hippocampus: 66±7%, p<0.01; prefrontal cortex: 75±10%, p=0.07). Statistically significant lower levels of DHEA were also found in cerebellum (60±6%, p<0.001) and hippocampus (68±7%, p<0.05) of schizophrenic subjects. PEA (71±6%, p<0.05) and LEA (72±6%, p<0.05) levels were also found to be lower in cerebellum. No significant differences were found in OEA levels. Our results evidence specific alterations in the levels of some endocannabinoids in different brain regions of schizophrenic subjects. Furthermore, these data evidence the involvement of the endocannabinoid system in the pathophysiology of schizophrenia.

Piperazine and piperidine triazole ureas as ultrapotent and highly selective inhibitors of monoacylglycerol lipase.

Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, 2-arachidonoylglycerol (2-AG). During 2-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.

Lipid phosphate phosphatase inhibitors locally amplify lysophosphatidic acid LPA1 receptor signalling in rat brain cryosections without affecting global LPA degradation.

BACKGROUND: Lysophosphatidic acid (LPA) is a signalling phospholipid with multiple biological functions, mainly mediated through specific G protein-coupled receptors. Aberrant LPA signalling is being increasingly implicated in the pathology of common human diseases, such as arteriosclerosis and cancer. The lifetime of the signalling pool of LPA is controlled by the equilibrium between synthesizing and degradative enzymatic activity. In the current study, we have characterized these enzymatic pathways in rat brain by pharmacologically manipulating the enzymatic machinery required for LPA degradation. RESULTS: In rat brain cryosections, the lifetime of bioactive LPA was found to be controlled by Mg2+-independent, N-ethylmaleimide-insensitive phosphatase activity, attributed to lipid phosphate phosphatases (LPPs). Pharmacological inhibition of this LPP activity amplified LPA1 receptor signalling, as revealed using functional autoradiography. Although two LPP inhibitors, sodium orthovanadate and propranolol, locally amplified receptor responses, they did not affect global brain LPA phosphatase activity (also attributed to Mg2+-independent, N-ethylmaleimide-insensitive phosphatases), as confirmed by Pi determination and by LC/MS/MS. Interestingly, the phosphate analog, aluminium fluoride (AlFx-) not only irreversibly inhibited LPP activity thereby potentiating LPA1 receptor responses, but also totally prevented LPA degradation, however this latter effect was not essential in order to observe AlFx--dependent potentiation of receptor signalling. CONCLUSIONS: We conclude that vanadate- and propranolol-sensitive LPP activity locally guards the signalling pool of LPA whereas the majority of brain LPA phosphatase activity is attributed to LPP-like enzymatic activity which, like LPP activity, is sensitive to AlFx- but resistant to the LPP inhibitors, vanadate and propranolol.

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography-electrospray tandem mass spectrometry.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid-liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02-1.0muM for LPAs. The quantification limit of the assay was 54fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.

Neuroanatomical mapping of juvenile rat brain regions with prominent basal signal in [(35)S]GTPgammaS autoradiography.

[(35)S]GTPgammaS autoradiography represents a powerful functional approach to detect receptor-dependent G(i/o) protein activity in anatomically defined brain structures. Inherent to this technique, however, is the notable basal signal evident in several brain regions in the absence of receptor stimulation by exogenously added agonist. In the rat brain, much of this basal labelling derives from tonic activation of adenosine A(1) and lysophosphatidic acid LPA(1) receptors in the gray and white matter regions, respectively. Despite the elimination of the two receptor activities, prominent basal [(35)S]GTPgammaS labelling is still evident in discrete brain structures, possibly reflecting regional enrichment of G(i/o) and/or constitutive receptor activity or the presence of still unknown endogenous ligands activating their orphan receptors. Here, the anatomical distribution of the enhanced basal signal was systematically mapped in brain sections of 4-week-old male Wistar rats. Regions with prominent basal [(35)S]GTPgammaS labelling represented neuroanatomically distinct structures, in particular various thalamic and hypothalamic nuclei. For instance, the paraventricular thalamic nucleus, the bed nucleus of the stria terminalis and the subfornical organ were highly labelled, as were the periaqueductal gray and the nucleus of the solitary tract. Pre-treatment with N-ethylmaleimide (NEM), an alkylating agent preventing all known receptor-driven G protein activity in cryostat sections markedly decreased the basal binding in all examined regions. In preliminary screening, selective antagonists for various brain-enriched G(i/o)-coupled receptors failed to suppress the basal signal in any of the studied regions.