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.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer.

PURPOSE: In HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient's outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors. METHODS: We evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated. RESULTS: FoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively. CONCLUSION: In the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

Neuron-astrocyte transmitophagy is altered in Alzheimer's disease.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains. We also demonstrate that the degradation of neuronal mitochondria by astrocytes is increased in AD mice at the age of 6 months onwards. Furthermore, we demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and AD astrocytes together with significant increases in the mitophagy regulator and reactive oxygen species in aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

The prognostic and predictive role of the neutrophil-to-lymphocyte ratio and the monocyte-to-lymphocyte ratio in early breast cancer, especially in the HER2+ subtype.

PURPOSE: The aim of this study was to investigate the prognostic impact of two systemic inflammatory markers, the neutrophil-to-lymphocyte ratio (NLR) and the monocyte-to-lymphocyte ratio (MLR), and their possible predictive role regarding the efficacy of adjuvant trastuzumab, in 209 early breast cancer cases, 107 of which were HER2-positive. METHODS: Baseline NLR and MLR values were divided into two groups, high and low, according to cut-off-points determined from the ROC curve (2.2 for NLR and 0.22 for MLR). Cox's model was utilized for survival analyses. RESULTS: High NLR and MLR correlated with poor overall survival (OS) and breast cancer specific survival (BCSS) among all the patients (p ≤ 0.030). Among the HER2+ patients whose adjuvant treatment did not include trastuzumab (n = 64), the survival rates were remarkably lower in patients with a high NLR as compared to those with low; 31% vs. 71% for OS and 42% vs. 74% for BCSS (p ≤ 0.014). Similarly, high MLR correlated with poor survival among these patients (p ≤ 0.020). On the contrary, among the patients who had received adjuvant trastuzumab (n = 43), NLR or MLR did not correlate with survival. Furthermore, trastuzumab was beneficial for the HER2+ patients with high NLR/MLR, while the survival of the HER2+ patients with low NLR/MLR was good irrespective if they received adjuvant trastuzumab. CONCLUSIONS: Our results suggest that trastuzumab modulates the systemic inflammatory conditions and overcomes the poor prognostic impact of high NLR/MLR. This finding may also provide a rationale for combining trastuzumab with immuno-oncological treatments in HER2+ breast cancer.

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Advanced nanoengineered-customized point-of-care tools for prostate-specific antigen.

Change in the level of human prostate-specific antigen (PSA) is a major element in the development and progression of prostate cancer (PCa). Most of the methodologies are currently restricted to their application in routine clinical screening due to the scarcity of adequate screening tools, false reading, long assay time, and cost. Innovative techniques and the integration of knowledge from a variety of domains, such as materials science and engineering, are needed to provide sustainable solutions. The convergence of precision point-of-care (POC) diagnostic techniques, which allow patients to respond in real time to changes in PSA levels, provides promising possibilities for quantitative and quantitative detection of PSA. This solution could be interesting and relevant for use in PCa diagnosis at the POC. The approaches enable low-cost real-time detection and are simple to integrate into user-friendly sensor devices. This review focuses on the investigations, prospects, and challenges associated with integrating engineering sciences with cancer biology to develop nanotechnology-based tools for PCa diagnosis. This article intends to encourage the development of new nanomaterials to construct high-performance POC devices for PCa detection. Finally, the review concludes with closing remarks and a perspective forecast.

Tumor microenvironment and breast cancer survival: combined effects of breast fat, M2 macrophages and hyaluronan create a dismal prognosis.

PURPOSE: Tumor microenvironment, including inflammatory cells, adipocytes and extracellular matrix constituents such as hyaluronan (HA), impacts on cancer progression. Systemic metabolism also influences tumor growth e.g. obesity and type 2 diabetes (T2D) are risk factors for breast cancer. Here, in 262 breast cancer cases, we explored the combined impacts on survival of M2-like tumor associated macrophages (TAMs), the abundance of breast fat visualized as low density in mammograms, and tumor HA, and their associations with T2D. METHODS: Mammographic densities were assessed visually from the diagnostic images and dichotomized into very low density (VLD, density ≤ 10%, "fatty breast") and mixed density (MID, density > 10%). The amounts of TAMs (CD163+ and CD68+) and tumor HA were determined by immunohistochemistry. The data of T2D was collected from the patient records. Statistical differences between the parameters were calculated with Chi square or Mann-Whitney test and survival analyses with Cox's model. RESULTS: A combination of fatty breasts (VLD), abundance of M2-like TAMs (CD163+) and tumor HA associated with poor survival, as survival was 88-89% in the absence of these factors but only 40-47% when all three factors were present (p < 0.001). Also, an association between T2D and fatty breasts was found (p < 0.01). Furthermore, tumors in fatty breasts contained more frequently high levels of M2-like TAMs than tumors in MID breasts (p = 0.01). CONCLUSIONS: Our results demonstrate a dramatic effect of the tumor microenvironment on breast cancer progression. We hypothesize that T2D as well as obesity increase the fat content of the breasts, subsequently enhancing local pro-tumoral inflammation.

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.

Correlative light and electron microscopy is a powerful tool to study interactions of extracellular vesicles with recipient cells.

Extracellular vesicles (EVs) and their interactions with recipient cells constitute a rapidly growing research area. However, due to the limitations in current methodologies, the mechanisms of these interactions are still unclear. Microscopic studies of EVs are challenging, because their typical diameter is near the resolution limit of light microscopy, and electron microscopy has restricted possibilities for protein labelling. The objective of this study was to combine these two techniques to demonstrate in detail the interactions of EVs by recipient cells. Hyaluronan synthase 3 (HAS3) is an integral transmembrane protein that is enriched in EVs. In this work, GFP-HAS3 was utilized to study the interactions of EVs with the recipient cells. Surprisingly, confocal analysis correlation with scanning electron microscopy (SEM) revealed that most of the EVs were indeed lying on the recipient cell's plasma membrane, while the level of EV-derived intracellular signal was low. Immunoelectron microscopy supported this finding. Furthermore, hyaluronan oligosaccharides decreased the numbers of bound EVs, suggesting that CD44 participates in the regulation of their binding. This study indicates that correlative light and electron microscopy is a reliable method to analyze EV interactions with recipient cells. Detailed 3D confocal imaging of EV carrying a GFP-label on their plasma membrane combined with high-resolution electron microscopy provides significantly more information than either of the techniques alone. In the future studies it is crucial to utilize these techniques and their combinations to solve in detail the ambiguous fate of EV in target cells. Furthermore, live cell imaging at high resolution will be required to obtain definite answers on the detailed mechanisms of binding, fusion and endocytosis of EVs.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism.

BACKGROUND: Orotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena. METHODS: In our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography-mass spectrometry. RESULTS: Our data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles. CONCLUSIONS: The present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

Hyaluronan histochemistry-a potential new tool to assess the progress of liver disease from simple steatosis to hepatocellular carcinoma.

Nonalcoholic fatty liver disease is among the most common liver diseases worldwide and one cause of cirrhosis that can result in the development of hepatocellular carcinoma (HCC). Hyaluronan (HA) is a high-molecular-mass glycosaminoglycan with diverse functions in tissue injury and repair, for instance, in inflammation and fibrogenesis. The aim of the present study was to investigate the relationships between the HA synthesizing and degrading enzymes in a spectrum of liver pathologies. This was realized by histological staining of liver sections from controls and patients with simple steatosis, steatohepatitis, cirrhosis and HCC (n = 90). HA-positive staining intensified in connective tissue in all liver pathologies, and staining of CD44, the major HA receptor, similarly increased in steatohepatitis and cirrhosis. HA synthase 1 (HAS1)-positive staining was reduced in steatosis, steatohepatitis and HCC. Staining of HAS3, which produces HA of a lower molecular mass, promotes inflammation and is pathogenic in animal models, increased in all diagnoses. The responses in staining intensity of HAS2 and hyaluronidases 1-2 were specific for different cell types. These findings suggest that HAS1-2 are responsible for HA synthesis in healthy livers, while HAS3 increases in importance in liver diseases. It is noteworthy that the pathological changes in HA metabolism are already visible in simple steatosis and, thus, precede the histological changes of inflammation and fibrosis. It could be possible to intervene in disease progression at an early stage by influencing HA metabolism. The results could have potential clinical applications with HAS3 immunostaining supplementing the existing HCC diagnostics.

Elevated expression of hyaluronan synthase 2 associates with decreased survival in diffusely infiltrating astrocytomas.

BACKGROUND: Diffusely infiltrating astrocytomas originate from astrocytic glial cells or their precursor cells and are the most common type of brain tumors in adults. In this retrospective study, we investigated the content of hyaluronan, its cell surface receptor, CD44 and the expression of hyaluronan metabolizing enzymes, in these aggressive tumors. Hyaluronan is the main component of extracellular matrix in the brain. In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential. METHODS: Our material consisted of 163 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, hyaluronan synthases 1-3 (HAS1-3) and hyaluronidase 2 (HYAL2). The immunostaining results were compared with χ2 -test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression. RESULTS: Hyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade or any other clinicopathological parameters whereas high HAS2 staining intensity was observed in IDH1 negative tumors (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity correlated with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). In the Cox regression analysis HAS2 expression turned out to be a significant independent prognostic factor (p = 0.008). CONCLUSIONS: This study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas.

DHCR24 exerts neuroprotection upon inflammation-induced neuronal death.

BACKGROUND: DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer's disease. METHODS: Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. RESULTS: Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. CONCLUSIONS: These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo.

UDP-sugar substrates of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracellular shedding and correlate with melanoma progression.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation.

Nucleoli are not only organelles that produce ribosomal subunits. They are also overarching sensors of different stress conditions and they control specific nucleolar stress pathways leading to stabilization of p53. During DNA replication, ATR and its activator TopBP1 initiate DNA damage response upon DNA damage and replication stress. We found that a basal level of TopBP1 protein associates with ribosomal DNA repeat. When upregulated, TopBP1 concentrates at the ribosomal chromatin and initiates segregation of nucleolar components--the hallmark of nucleolar stress response. TopBP1-induced nucleolar segregation is coupled to shut-down of ribosomal RNA transcription in an ATR-dependent manner. Nucleolar segregation induced by TopBP1 leads to a moderate elevation of p53 protein levels and to localization of activated p53 to nucleolar caps containing TopBP1, UBF and RNA polymerase I. Our findings demonstrate that TopBP1 and ATR are able to inhibit the synthesis of rRNA and to activate nucleolar stress pathway; yet the p53-mediated cell cycle arrest is thwarted in cells expressing high levels of TopBP1. We suggest that inhibition of rRNA transcription by different stress regulators is a general mechanism for cells to initiate nucleolar stress pathway.

Interleukin-1ß-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats.

The proinflammatory cytokine interleukin-1ß (IL-1ß) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1ß turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1ß did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1ß caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1ß increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1ß (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1ß changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes.