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.

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.

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.

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.

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.

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.

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.

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.

Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

Decreased hyaluronidase 1 expression is associated with early disease recurrence in human endometrial cancer.

OBJECTIVE: Hyaluronidases (HYAL1 and HYAL2) are key enzymes in the degradation of hyaluronan, and their expression has been altered in various cancer types. We previously showed that hyaluronan accumulation in endometrial carcinomas was correlated with decreased mRNA expression of the HYAL genes. In this study, we analyzed HYAL1 and HYAL2 protein expressions in normal and precancerous endometrial tissues and in endometrial carcinomas. We also investigated whether the protein levels were associated with clinicopathological factors, invasion, and disease recurrence. METHODS: A total of 343 tissue specimens from normal, atrophic, hypertrophic, and neoplastic endometria were analyzed immunohistochemically for HYAL1 and HYAL2 expressions. The results were correlated with clinicopathological factors, the expression of the epithelial-mesenchymal transition marker, E-cadherin, and disease recurrence. RESULTS: Reduced HYAL1 expression was associated with the progression of endometrial carcinomas towards higher grades and also with large tumor sizes, lymph node metastasis, and lymphovascular invasion. Reduced expression of both HYAL1 and HYAL2 was associated with deep myometrial invasion. HYAL2 expression was primarily constant in neoplastic tissues, but its expression was altered in different phases of the endometrial cycle. In addition, a reduction in HYAL1 expression was associated with the depletion of E-cadherin. In a multivariate analysis, reduced HYAL1 expression was an independent prognostic factor for early disease recurrence (HR 5.13, 95% CI: 1.131-23.270, p=0.034). CONCLUSIONS: This study showed that reduced HYAL1 expression was associated with endometrial carcinoma aggressiveness, which further supported the role of hyaluronan degradation in cancer progression.

High numbers of macrophages, especially M2-like (CD163-positive), correlate with hyaluronan accumulation and poor outcome in breast cancer.

AIMS: High amounts of tumour-associated macrophages (TAMs) and hyaluronan (HA) correlate with tumour aggressiveness in breast cancer, but the relationship between these parameters is unclear. The aim of this study was to assay the numbers of TAMs in 278 human breast cancer cases, and their correlations with HA-related factors, clinical variables, and outcome. METHODS AND RESULTS: The immunoreactivities for CD163 and CD68 were considered as indicators for M2-like and all TAMs, respectively. The numbers of TAMs were counted in at least four hot spots, and averaged to represent the numbers of TAMs in each section. In the statistical analyses, the numbers were graded as either low or high according to the median. High numbers of TAMs correlated with a high tumour HA content, HA synthases, CD44 positivity, and poor outcome. The number of CD163-positive cells represented a strong independent prognostic factor. There was also a significant correlation between obesity and a high number of CD163-positive cells. CONCLUSIONS: Concurrent increases in TAMs and HA in breast cancer indicate that the accumulation of HA facilitates macrophage infiltration and inflammatory responses during human breast cancer progression.

Evidence of endoplasmic reticulum stress and liver inflammation in the American mink Neovison vison with benign hepatic steatosis.

We investigated the presence of inflammatory signs in the progression of fatty liver disease induced by fasting. Sixty standard black American mink (Neovison vison) were fasted for 0, 1, 3, 5, or 7 days and one group for 7 days followed by re-feeding for 28 days. Liver sections were evaluated histologically and liver mRNA levels indicating endoplasmic reticulum (ER) stress, adipogenic transformation, and inflammation were assessed by quantitative real-time PCR. After 3 days of fasting, the mink had developed moderate liver steatosis. Increased hyaluronan reactivity in lymphocytic foci but no Mallory-Denk bodies were seen in livers of the mink fasted for 5-7 days. Up-regulation of glucose-regulated protein, 78 kDa was observed on day 7 indicating ER stress, especially in the females. Liver lipoprotein lipase and monocyte chemoattractant protein 1 mRNA levels increased in response to 5-7 days of food deprivation, while tumor necrosis factor α (TNF-α) was the highest in the mink fasted for 5 days. The expression of the genes of interest, except for TNF-α, correlated with each other and with the liver fat content. The mRNA levels were found to change more rapidly below n-3/n-6 polyunsaturated fatty acid ratio threshold of 0.15. Following re-feeding, hepatocyte morphology and mRNA abundance returned to pre-fasting levels. Within the studied timeframe, evidence for ER stress, adipogenic transformation, and liver inflammation suggested incipient transition from steatosis to steatohepatitis with potential for development of more severe liver disease. This may present a possibility to influence disease progression before histologically observable steatohepatitis.

Hyaluronan-coated extracellular vesicles--a novel link between hyaluronan and cancer.

The synthesis of hyaluronan (HA) on the plasma membrane is a unique and still partly mysterious way of macromolecular biosynthesis. HA forms pericellular coats around many cell types and accumulates in the extracellular matrix (ECM) of growing and renewing tissues. It is secreted to high concentrations in body fluids with antifriction properties like pleural, peritoneal, and synovial fluids, but is also detectable in plasma, saliva, and urine. In pathological states, like cancer and inflammation, the amount of HA is increased around cells, in the ECM, and in the body fluids. HA is an indicator of poor prognosis for cancer patients and creates a favorable environment for cellular growth and motility. The recent finding that HA-coated extracellular vesicles act both as a product of HA synthase activity and as special vehicles for HA, and perhaps carry signals important for malignant growth, provides a novel link between HA and cancer. HA could be carried on the surface of these vesicles in tissues and body fluids, creating beneficial environments by itself, or by associated molecules, for the invasion and metastasis of cancer cells. The HA-coated plasma membrane protrusions and vesicles shed from them are potential biomarkers in cancer and other HA-associated disease states.

Isothiocyanate analogs targeting CD44 receptor as an effective strategy against colon cancer.

Inflammatory pathway plays an important role in tumor cell progression of colorectal cancers. Although colon cancer is considered as one of the leading causes of death worldwide, very few drugs are available for its effective treatment. Many studies have examined the effects of specific COX-2 and 5-LOX inhibitors on human colorectal cancer, but the role of isothiocyanates (ITSCs) as COX-LOX dual inhibitors engaged in hyaluronan-CD44 interaction has not been studied. In the present work, we report series of ITSC analogs incorporating bioisosteric thiosemicarbazone moiety. These inhibitors are effective against panel of human colon cancer cell lines including COX-2 positive HCA-7, HT-29 cells lines, and hyaluronan synthase-2 (Has2) enzyme over-expressing transformed intestinal epithelial Apc10.1Has2 cells. Specifically, our findings indicate that HA-CD44v6-mediated COX-2/5-LOX signaling mediate survivin production, which in turn, supports anti-apoptosis and chemo-resistance leading to colon cancer cell survival. The over-expression of CD44v6shRNA as well as ITSC treatment significantly decreases the survival of colon cancer cells. The present results thus offer an opportunity to evolve potent inhibitors of HA synthesis and CD44v6 pathway and thus underscoring the importance of the ITSC analogs as chemopreventive agents for targeting HA/CD44v6 pathway.