Stromal hyaluronan accumulation is associated with low immune response and poor prognosis in pancreatic cancer.

Hyaluronan (HA) accumulation has been associated with poor survival in various cancers, but the mechanisms for this phenomenon are still unclear. The aim of this study was to investigate the prognostic significance of stromal HA accumulation and its association with host immune response in pancreatic ductal adenocarcinoma (PDAC). The study material consisted of 101 radically treated patients for PDAC from a single geographical area. HA staining was evaluated using a HA-specific probe, and the patterns of CD3, CD8, CD73 and PD-L1 expression were evaluated using immunohistochemistry. HA staining intensity of tumour stromal areas was assessed digitally using QuPath. CD3- and CD8-based immune cell score (ICS) was determined. High-level stromal HA expression was significantly associated with poor disease-specific survival (p = 0.037) and overall survival (p = 0.013) In multivariate analysis, high-level stromal HA expression was an independent negative prognostic factor together with histopathological grade, TNM stage, CD73 positivity in tumour cells and low ICS. Moreover, high-level stromal HA expression was associated with low ICS (p = 0.017). In conclusion, stromal HA accumulation is associated with poor survival and low immune response in PDAC.

Cellular hyaluronan is associated with a poor prognosis in renal cell carcinoma.

PURPOSE: Hyaluronan, a major glycosaminoglycan of the extracellular matrix, can act as an oncogenic component of the tumor microenvironment in many human malignancies. We characterized the hyaluronan content of renal cell carcinomas (RCCs) and investigated its correlations with clinicopathological parameters and patient survival. PATIENTS AND METHODS: This retrospective study included data from 316 patients that had undergone surgery for RCC in Kuopio University Hospital in 2000 to 2013. The hyaluronan content of surgical tumor samples were histochemically stained with a biotinylated hyaluronan-specific affinity probe. The amount of tumor infiltrating lymphocytes was evaluated in each tumor. Kaplan-Meier and univariate and multivariate Cox-regression analyses were performed to estimate the impact of hyaluronan content on overall survival, disease-specific survival, and metastasis-free survival. RESULTS: Detectable cellular hyaluronan was associated with higher tumor grades and the presence of tumor infiltrating lymphocytes. Cellular hyaluronan identified a prognostically unfavourable subgroup among low-grade carcinomas. Multivariate analyses showed that measurable cellular hyaluronan was an independent negative prognostic factor for overall survival (hazard ratio [HR] 1.4; 95% confidence interval [CI]: 1.02-2.0; P = 0.039), Disease-specific survival (HR 2.07; 95% CI: 1.2-3.3; P = 0.002), and metastasis-free survival (HR 2.45; 95% CI: 1.37-4.4; P = 0.003). CONCLUSIONS: Cellular hyaluronan was significantly associated with unfavourable features and a poor prognosis in RCC. Further studies are needed to investigate the biological mechanism underlying hyaluronan accumulation in RCC.

Peritumoral ADC values in breast cancer: region of interest selection, associations with hyaluronan intensity, and prognostic significance.

OBJECTIVES: We aimed to evaluate the differences in peritumoral apparent diffusion coefficient (ADC) values by four different ROI selection methods and to validate the optimal method. Furthermore, we aimed to evaluate if the peritumor-tumor ADC ratios are correlated with axillary lymph node positivity and hyaluronan accumulation. METHODS: Altogether, 22 breast cancer patients underwent 3.0-T breast MRI, histopathological evaluation, and hyaluronan assay. Paired t and Friedman tests were used to compare minimum, mean, and maximum values of tumoral and peritumoral ADC by four methods: (M1) band ROI, (M2) whole tumor surrounding ROI, (M3) clockwise multiple ROI, and (M4) visual assessment of ROI selection. Subsequently, peritumor/tumor ADC ratios were compared with hyaluronan levels and axillary lymph node status by the Mann-Whitney U test. RESULTS: No statistically significant differences were found among the four ROI selection methods regarding minimum, mean, or maximum values of tumoral and peritumoral ADC. Visual assessment ROI measurements represented the less time-consuming evaluation method for the peritumoral area, and with sufficient accuracy. Peritumor/tumor ADC ratios obtained by all methods except the clockwise ROI (M3) showed a positive correlation with hyaluronan content (M1, p = 0.004; M2, p = 0.012; M3, p = 0.20; M4, p = 0.025) and lymph node metastasis (M1, p = 0.001; M2, p = 0.007; M3, p = 0.22; M4, p = 0.015), which are established factors for unfavorable prognosis. CONCLUSIONS: Our results suggest that the peritumor/tumor ADC ratio could be a readily applicable imaging index associated with axillary lymph node metastasis and extensive hyaluronan accumulation. It could be related to the biological aggressiveness of breast cancer and therefore might serve as an additional prognostic factor. KEY POINTS: • Out of four different ROI selection methods for peritumoral ADC evaluation, measurements based on visual assessment provided sufficient accuracy and were the less time-consuming method. • The peritumor/tumor ADC ratio can provide an easily applicable supplementary imaging index for breast cancer assessment. • A higher peritumor/tumor ADC ratio was associated with axillary lymph node metastasis and extensive hyaluronan accumulation and might serve as an additional prognostic factor.

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.

Hyaluronan synthesis supports glutamate transporter activity.

Hyaluronan is synthesized, secreted, and anchored by hyaluronan synthases (HAS) at the plasma membrane and comprises the backbone of perineuronal nets around neuronal soma and dendrites. However, the molecular targets of hyaluronan to regulate synaptic transmission in the central nervous system have not been fully identified. Here, we report that hyaluronan is a negative regulator of excitatory signals. At excitatory synapses, glutamate is removed by glutamate transporters to turn off the signal and prevent excitotoxicity. Hyaluronan synthesized by HAS supports the activity of glial glutamate transporter 1 (GLT1). GLT1 also retracted from cellular processes of cultured astrocytes after hyaluronidase treatment and hyaluronan synthesis inhibition. A serial knockout study showed that all three HAS subtypes recruit GLT1 to cellular processes. Furthermore, hyaluronidase treatment activated neurons in a dissociated rat hippocampal culture and caused neuronal damage due to excitotoxicity. Our findings reveal that hyaluronan helps to turn off excitatory signals by supporting glutamate clearance. Cover Image for this issue: doi: 10.1111/jnc.14516.

Melanocyte Hyaluronan Coat Fragmentation Enhances the UVB-Induced TLR-4 Receptor Signaling and Expression of Proinflammatory Mediators IL6, IL8, CXCL1, and CXCL10 via NF-κB Activation.

Skin is constantly exposed to UVR, the most critical risk factor for melanoma development. Hyaluronan is abundant in the epidermal extracellular matrix and may undergo degradation by UVR. It is hypothesized that an intact hyaluronan coat around the cells protects against various agents including UVR, whereas hyaluronan fragments promote inflammation and tumorigenesis. We investigated whether hyaluronan contributes to the UVB-induced inflammatory responses in primary melanocytes. A single dose of UVB suppressed hyaluronan secretion and the expression of hyaluronan synthases HAS2 and HAS3, the hyaluronan receptor CD44, and the hyaluronidase HYAL2, as well as induced the expression of inflammatory mediators IL6, IL8, CXCL1, and CXCL10. Silencing HAS2 and CD44 partly inhibited the inflammatory response, suggesting that hyaluronan coat is involved in the process. UVB alone caused little changes in the coat, but its removal with hyaluronidase during the recovery from UVB exposure dramatically enhanced the surge of these inflammatory mediators via TLR4, p38, and NF-κB. Interestingly, exogenous hyaluronan fragments did not reproduce the inflammatory effects of hyaluronidase. We hypothesize that the hyaluronan coat on melanocytes is a sensor of tissue injury. Combined with UVB exposure, repeated injuries to the hyaluronan coat could maintain a sustained inflammatory state associated with melanomagenesis.

Analysis of UDP-Sugars from Cultured Cells and Small Tissue Samples.

UDP-sugars are important substrates for the synthesis of various cellular glycans and glycoconjugates, many of which play essential roles in the pathobiology of diseases associated with deranged glucose metabolism, such as cancer and type 2 diabetes. Hence, their analysis from cultured cells and especially from tissue samples can give valuable information. This chapter describes a method for UDP-sugar isolation from various sources utilizing ion-pair solid-phase extraction with graphitized carbon cartridges, and their analysis using anion-exchange high-performance liquid chromatography.

Activated hyaluronan metabolism in the tumor matrix - Causes and consequences.

Hyaluronan accumulates in the stroma of several solid tumors and promotes their progression. Both enhanced synthesis and fragmentation of hyaluronan are required as a part of this inflammatory process resembling wound healing. Increased expression of the genes of hyaluronan synthases (HAS1-3) are infrequent in human tumors, while posttranslational modifications that activate the HAS enzymes, and glucose shunted to the UDP-sugar substrates HASs, can have crucial contributions to tumor hyaluronan synthesis. The pericellular hyaluronan influences virtually all cell-cell and cell-matrix interactions, controlling migration, proliferation, apoptosis, epithelial to mesenchymal transition, and stem cell functions. The catabolism by hyaluronidases and free radicals appears to be as important as synthesis for the inflammation that promotes tumor growth, since the receptors mediating the signals create specific responses to hyaluronan fragments. Targeting hyaluronan metabolism shows therapeutic efficiency in animal experiments and early clinical trials.

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins.

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

Extracellular ATP activates hyaluronan synthase 2 (HAS2) in epidermal keratinocytes via P2Y2, Ca2+ signaling, and MAPK pathways.

Extracellular nucleotides are used as signaling molecules by several cell types. In epidermis, their release is triggered by insults such as ultraviolet radiation, barrier disruption, and tissue wounding, and by specific nerve terminals firing. Increased synthesis of hyaluronan, a ubiquitous extracellular matrix glycosaminoglycan, also occurs in response to stress, leading to the attractive hypothesis that nucleotide signaling and hyaluronan synthesis could also be linked. In HaCaT keratinocytes, ATP caused a rapid and strong but transient activation of hyaluronan synthase 2 (HAS2) expression via protein kinase C-, Ca2+/calmodulin-dependent protein kinase II-, mitogen-activated protein kinase-, and calcium response element-binding protein-dependent pathways by activating the purinergic P2Y2 receptor. Smaller but more persistent up-regulation of HAS3 and CD44, and delayed up-regulation of HAS1 were also observed. Accumulation of peri- and extracellular hyaluronan followed 4-6 h after stimulation, an effect further enhanced by the hyaluronan precursor glucosamine. AMP and adenosine, the degradation products of ATP, markedly inhibited HAS2 expression and, despite concomitant up-regulation of HAS1 and HAS3, inhibited hyaluronan synthesis. Functionally, ATP moderately increased cell migration, whereas AMP and adenosine had no effect. Our data highlight the strong influence of adenosinergic signaling on hyaluronan metabolism in human keratinocytes. Epidermal insults are associated with extracellular ATP release, as well as rapid up-regulation of HAS2/3, CD44, and hyaluronan synthesis, and we show here that the two phenomena are linked. Furthermore, as ATP is rapidly degraded, the opposite effects of its less phosphorylated derivatives facilitate a rapid shut-off of the hyaluronan response, providing a feedback mechanism to prevent excessive reactions when more persistent signals are absent.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

UDP-sugar accumulation drives hyaluronan synthesis in breast cancer.

Increased uptake of glucose, a general hallmark of malignant tumors, leads to an accumulation of intermediate metabolites of glycolysis. We investigated whether the high supply of these intermediates promotes their flow into UDP-sugars, and consequently into hyaluronan, a tumor-promoting matrix molecule. We quantified UDP-N-Acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA) in human breast cancer biopsies, the levels of enzymes contributing to their synthesis, and their association with the hyaluronan accumulation in the tumor. The content of UDP-GlcUA was 4 times, and that of UDP-GlcNAc 12 times higher in the tumors as compared to normal glandular tissue obtained from breast reductions. The surge of UDP-GlcNAc correlated with an elevated mRNA expression of glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), one of the key enzymes in the biosynthesis of UDP-GlcNAc, and the expression of GFAT1 was also elevated. The contents of both UDP-sugars strongly correlated with tumor hyaluronan levels. Interestingly, hyaluronan content did not correlate with the mRNA levels of the hyaluronan synthases (HAS1-3), thus emphasizing the role of the UDP-sugar substrates of these enzymes. The UDP-sugars showed a trend to higher levels in ductal vs. lobular cancer subtypes. The results reveal for the first time a dramatic increase of UDP-sugars in breast cancer, and suggest that their high supply drives the accumulation of hyaluronan, a known promoter of breast cancer and other malignancies. In general, the study shows how the disturbed glucose metabolism typical for malignant tumors can influence cancer microenvironment through UDP-sugars and hyaluronan.

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 µm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

High extent of O-GlcNAcylation in breast cancer cells correlates with the levels of HAS enzymes, accumulation of hyaluronan, and poor outcome.

PURPOSE: Obesity and oversupply of glucose, e.g., due to nutritional factors may shape the tumor microenvironment favorable for tumor progression. O-GlcNAcylation, a reversible modification of intracellular proteins, influences on several cellular functions and is connected to many diseases including cancer. Glycosaminoglycan hyaluronan (HA) enhances tumor progression and in breast cancer HA accumulation associates strongly with poor outcome. In vitro studies have suggested that O-GlcNAcylation may enhance HA synthesis. The aim of this study was to investigate the correlations between O-GlcNAcylation, HA-related parameters, and disease outcome in a clinical breast cancer material consisting of 278 breast cancer cases. METHODS: In microscopic analyses, O-GlcNAc staining of the breast carcinoma cells was evaluated in several randomly picked high-power fields of each section. The extent of cytoplasmic O-GlcNAc staining was graded as either low or high according to the intensity of the staining and the percentage of stained cells. The extent of nuclear O-GlcNAc staining was categorized as either low or high according to the percentage of stained nuclei. RESULTS: A high extent of both cytoplasmic and nuclear O-GlcNAcylation correlated with an increased relapse rate, development of distant metastases, and poor outcome. A high extent of cytoplasmic O-GlcNAcylation correlated also with the accumulation of all hyaluronan synthase (HAS1-3) proteins and with a large amount of HA in the tumor stroma. In addition, a high extent of nuclear O-GlcNAcylation associated with obesity. CONCLUSIONS: The results suggest a mechanistic association between increased O-GlcNAcylation and HA synthesis, leading to a HA-rich microenvironment favorable for breast cancer progression.

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.

Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.

Hyaluronan-positive plasma membrane protrusions exist on mesothelial cells in vivo.

Previous observations of our research group showed that HAS2 and HAS3 overexpression in cultured cells induces the formation of long and numerous microvillus-like cell protrusions, which are present also in cultured cell types with naturally high hyaluronan secretion and the cell protrusions resemble those found in mesothelial cells. The aim of this study was to investigate whether these hyaluronan secreting, actin-dependent protrusions exist also in vivo. It was found that rat mesothelium in vivo is positive for hyaluronan and Has1-3. Also microvilli in rat mesothelium and live primary cultures of mesothelial cells were found to be hyaluronan positive, and the cells expressed all Has isoforms. Furthermore, ultrastructure of the cell protrusions in rat mesothelium was similar to that induced by overexpression of HAS2 and HAS3, and the number and orientation of actin filaments supporting the cell protrusions was identical. The results of this study show that HA-positive protrusions exist in vivo and support the idea that hyaluronan secretion from plasma membrane protrusions is a general process. This mechanism is potentially crucial for the normal function and maintenance of tissues and body fluids and may be utilized in many therapeutic applications.

The reciprocal association between mammographic breast density, hyaluronan synthesis and patient outcome.

Low mammographic breast density (MBD) and increased hyaluronan (HA) synthesis have been shown to have adverse effects on breast cancer prognosis. We aimed at elucidating the background of risk associated with mammographic characteristics, MBD and HA and its synthesizing isoforms in an attempt to uncover potential underlying biological mechanisms. MBD and mammographic characteristics of 270 patients were classified according to percentile density (very low density VLD, ≤25 %; mixed density MID, >25 %) and the BI-RADS 5th edition lexicon. Breast density and mammographic features were correlated with the localization and expression of HA, CD44, and HAS1-3 isoforms, and their combined effect on patients' survivals was explored. VLD showed an increased level of HA-positive carcinoma cells and stromal HA, HAS2, and HAS3. Tumors presenting as masses had more HA-positive carcinoma cells and more stromal HAS2 and HAS3. Indistinct margin tumors showed more stromal HA and HAS3. Patients who combined both VLD breasts with either high HA in carcinoma cells or stroma showed a worse prognosis compared to low levels (carcinoma cells 58.0 vs. 80.5 %, p = 0.001; stroma 64.2 vs. 79.6 %, p = 0.017), while no similar HA-related effect was observed in MID breasts. Our findings suggest a strong reciprocal relationship between low MBD and HA expression and synthesis. The expression of both factors simultaneously leads to an especially adverse prognostic effect which might have an impact on treatment decision in the future. Moreover, HA around cancer cells may inhibit chemotherapy agents and antibody treatments from reaching cancer cells.

The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation.

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion.

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.