Hyaluronic Acid Synthesis Inhibitor Research Articles

4-Methylumbelliferone Targets Revealed by Public Data Analysis and Liver Transcriptome Sequencing.

4-methylumbelliferone (4MU) is a well-known hyaluronic acid synthesis inhibitor and an approved drug for the treatment of cholestasis. In animal models, 4MU decreases inflammation, reduces fibrosis, and lowers body weight, serum cholesterol, and insulin resistance. It also inhibits tumor progression and metastasis. The broad spectrum of effects suggests multiple and yet unknown targets of 4MU. Aiming at 4MU target deconvolution, we have analyzed publicly available data bases, including: 1. Small molecule library Bio Assay screening (PubChemBioAssay); 2. GO pathway databases screening; 3. Protein Atlas Database. We also performed comparative liver transcriptome analysis of mice on normal diet and mice fed with 4MU for two weeks. Potential targets of 4MU public data base analysis fall into two big groups, enzymes and transcription factors (TFs), including 13 members of the nuclear receptor superfamily regulating lipid and carbohydrate metabolism. Transcriptome analysis revealed changes in the expression of genes involved in bile acid metabolism, gluconeogenesis, and immune response. It was found that 4MU feeding decreased the accumulation of the glycogen granules in the liver. Thus, 4MU has multiple targets and can regulate cell metabolism by modulating signaling via nuclear receptors.

Effects of pharmacological inhibition of hyaluronic acid synthesis on experimental endometriosis.

Dysregulated hyaluronic acid (HA) metabolism has been shown to be implicated in several pathologies including endometriosis. 4-Methylumbelliferone (4MU) is an HA synthesis inhibitor with proven antitumour activity. In this study, we aim to evaluate the effect of 4MU on endometriosis development both in vivo and in vitro. Endometriosis was surgically induced by uterine tissue auto-transplantation in 32 two-month-old BALB/c mice. Animals were designated into the early or late starting treatment group, which initiated on day 2 or day 15 after surgery, respectively. Within each group, 4MU 200 mg/kg/day or vehicle (Control) were administered by oesophageal gavage for 28 days. After sacrifice, the percentage of developed lesions, lesion size, cell proliferation, vascularization and HA deposition within the endometriotic-like lesions were evaluated. Cell viability was assessed in endometrial epithelial cells (ECC-1) and in endometrial stromal cells (t-HESC); and migration was evaluated in t-HESC. There was a significant reduction in the percentage of developed lesions in mice that started the 4MU treatment on day 2 compared with its respective control group, and compared with those that started treatment on day 15. However, no significant changes were found when analysing endometriotic-like lesion's cell proliferation, vascularization and HA deposition. In vitro, both cell viability and migration were inhibited by 4MU treatment. The inhibition of HA synthesis could be a beneficial and alternative option to treat endometriosis at the early stage of the disease. Further research is necessary to elucidate 4MU's mechanism of action and better strategies for delivering this promising drug.

Cell-Type-Specific Expression of Hyaluronan Synthases HAS2 and HAS3 Promotes Goblet Cell Hyperplasia in Allergic Airway Inflammation.

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.

Hymecromone: a clinical prescription hyaluronan inhibitor for efficiently blocking COVID-19 progression

Currently, there is no effective drugs for treating clinically COVID-19 except dexamethasone. We previously revealed that human identical sequences of SARS-CoV-2 promote the COVID-19 progression by upregulating hyaluronic acid (HA). As the inhibitor of HA synthesis, hymecromone is an approved prescription drug used for treating biliary spasm. Here, we aimed to investigate the relation between HA and COVID-19, and evaluate the therapeutic effects of hymecromone on COVID-19. Firstly, HA was closely relevant to clinical parameters, including lymphocytes (n = 158; r = −0.50; P < 0.0001), C-reactive protein (n = 156; r = 0.55; P < 0.0001), D-dimer (n = 154; r = 0.38; P < 0.0001), and fibrinogen (n = 152; r = 0.37; P < 0.0001), as well as the mass (n = 78; r = 0.43; P < 0.0001) and volume (n = 78; r = 0.41; P = 0.0002) of ground-glass opacity, the mass (n = 78; r = 0.48; P < 0.0001) and volume (n = 78; r = 0.47; P < 0.0001) of consolidation in patient with low level of hyaluronan (HA < 48.43 ng/mL). Furthermore, hyaluronan could directly cause mouse pulmonary lesions. Besides, hymecromone remarkably reduced HA via downregulating HAS2/HAS3 expression. Moreover, 89% patients with hymecromone treatment had pulmonary lesion absorption while only 42% patients in control group had pulmonary lesion absorption (P < 0.0001). In addition, lymphocytes recovered more quickly in hymecromone-treated patients (n = 8) than control group (n = 5) (P < 0.05). These findings suggest that hymecromone is a promising drug for COVID-19 and deserves our further efforts to determine its effect in a larger cohort.

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone Suppresses the Secretory Processes That Ensure the Invasion of Neutrophils into Tissues and Induce Inflammation.

Integrin-dependent adhesion of neutrophils to tissue, accompanied by the development of neutrophil-induced inflammation, occurs both in the focus of infection and in the absence of infection in metabolic disorders such as reperfusion after ischemia, diabetes mellitus, or the development of pneumonia in patients with cystic fibrosis or viral diseases. Hyaluronic acid (HA) plays an important role in the recruitment of neutrophils to tissues. 4-methylumbilliferon (4-MU), an inhibitor of HA synthesis, is used to treat inflammation, but its mechanism of action is unknown. We studied the effect of 4-MU on neutrophil adhesion and concomitant secretion using adhesion to fibronectin as a model for integrin-dependent adhesion. 4-MU reduced the spreading of neutrophils on the substrate and the concomitant secretion of granule proteins, including pro-inflammatory components. 4-MU also selectively blocked adhesion-induced release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, which can influence cell invasion by modifying the extracellular matrix. Finally, 4-MU inhibited the formation of cytonemes, the extracellular membrane secretory structures containing the pro-inflammatory bactericides of the primary granules. The anti-inflammatory effect of 4-MU may be associated with the suppression of secretory processes that ensure the neutrophil invasion and initiate inflammation. We suggest that HA, due to the peculiarities of its synthesis, can promote the release of secretory carriers from the cell and 4-MU can block this process.

PD49-12 BIOMARKER-TARGETED NOVEL COMBINATION FOR RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (PD49)1 Sep 2021PD49-12 BIOMARKER-TARGETED NOVEL COMBINATION FOR RENAL CELL CARCINOMA Eric Thomas, Soum Lokeshwar, Jiaojiao Wang, Andre Jordan, Zachary Klaassen, Martha Terris, and Vinata Lokeshwar Eric ThomasEric Thomas More articles by this author , Soum LokeshwarSoum Lokeshwar More articles by this author , Jiaojiao WangJiaojiao Wang More articles by this author , Andre JordanAndre Jordan More articles by this author , Zachary KlaassenZachary Klaassen More articles by this author , Martha TerrisMartha Terris More articles by this author , and Vinata LokeshwarVinata Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002071.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The survival of patients with metastatic renal cell carcinoma (mRCC) is < 10% at 5-years, despite treatment. Hyaluronic acid (HA) promotes tumor growth and metastasis through its receptors CD44 and RHAMM. 4-methylumbelliferone (MU) is a non−toxic small molecule HA synthesis inhibitor and down regulates CD44 and RHAMM mRNA. In this study we examined if MU in combination with an FDA-approved treatments could abrogate mRCC in preclinical models and can the target be validated in a clinical cohort and TCGA dataset. METHODS: Response of Vector-only (EV), HAS3-overexpressing (HAS3) or HAS3-knockdown (HAS3-KO) transfectants of 786-O and Caki-1 cells, primary RCC spheroids and endothelial cells co-cultured with these transfectants to MU combined with Sorafenib (SF), Sunitinib or Everolimus was examined in proliferation, apoptosis, motility and invasion assays. Immunoblotting and RT-qPCR were performed for downstream signaling studies. HAS3 promoter was cloned and effect of SF and HC on HAS3 promoter activity was determined. Treatment efficacy was evaluated in the Caki−1 s.c. and orthotopic xenograft models. HAS3 levels were analyzed in a clinical-cohort (51 normal; 83 tumor), RCC TCGA-dataset (TCGA-cohort; n=542). RESULTS: MU synergized with SF to inhibit RCC cell and primary tumor spheroid proliferation > 90%, motility/invasion (>80%) and induced apoptosis (5-fold). Combination of MU (0.1, 0.2 mM) with SF (5 µM) downregulated (>80%) HAS3 expression, HAS3 promoter activity and HA synthesis in RCC cells. While HAS3 transfectants were resistant to SF+MU treatment, HAS3-KO transfectants were sensitive to SF alone. Endothelial cells co-cultured with HAS3 transfectants were resistant to the combination. SF+MU up-regulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in HAS3 transfectants. MU (100 or 200 mg/kg) and SF (30 mg/kg) oral combination abrogated Caki-1 tumor growth (>80%), without serum/tissue toxicity. MU+SF treatment decreased HAS3/HA levels, Ki67 index and microvessel density. HAS-overexpressing tumors were resistant to treatment. HAS3 levels were 4-fold elevated in mRCC patients’ tumors and significantly predicted metastasis (p=0.0086). HAS3 levels also significantly correlated with overall survival in TCGA dataset (p=0.029). CONCLUSIONS: This is the first study to demonstrate that HAS3 significantly predicts metastasis. By downregulating HAS3 expression, MU+SF combination effectively abrogates RCC growth and metastasis. HAS3 is a potential biomarker to predict clinical outcome in RCC patients. Source of Funding: This is the first study to demonstrate that HAS3 significantly predicts metastasis. By downregulating HAS3 expression, MU+SF combination effectively abrogates RCC growth and metastasis. HAS3 is a potential biomarker to predict clinical outcome in RCC patients © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e849-e850 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eric Thomas More articles by this author Soum Lokeshwar More articles by this author Jiaojiao Wang More articles by this author Andre Jordan More articles by this author Zachary Klaassen More articles by this author Martha Terris More articles by this author Vinata Lokeshwar More articles by this author Expand All Advertisement Loading ...

Inhibition of hyaluronan secretion by novel coumarin compounds and chitin synthesis inhibitors.

Elevated plasma levels of hyaluronic acid (HA) is a disease marker in liver pathology and other inflammatory disorders. Inhibition of HA synthesis with coumarin 4-methylumbelliferone (4MU) has a beneficial effect in animal models of fibrosis, inflammation, cancer and metabolic syndrome. 4MU is an active compound of approved choleretic drug hymecromone with low bioavailability and a broad spectrum of action. New, more specific and efficient inhibitors of hyaluronan synthases (HAS) are required. We have tested several newly synthesized coumarin compounds and commercial chitin synthesis inhibitors to inhibit HA production in cell culture assay. Coumarin derivative compound VII (10′-methyl-6′-phenyl-3′H-spiro[piperidine-4,2′-pyrano[3,2-g]chromene]-4′,8′-dione) demonstrated inhibition of HA secretion by NIH3T3 cells with the half-maximal inhibitory concentration (IC50) = 1.69 ± 0.75 μΜ superior to 4MU (IC50 = 8.68 ± 1.6 μΜ). Inhibitors of chitin synthesis, etoxazole, buprofezin, triflumuron, reduced HA deposition with IC50 of 4.21 ± 3.82 μΜ, 1.24 ± 0.87 μΜ and 1.48 ± 1.44 μΜ, respectively. Etoxazole reduced HA production and prevented collagen fibre formation in the CCl4 liver fibrosis model in mice similar to 4MU. Bioinformatics analysis revealed homology between chitin synthases and HAS enzymes, particularly in the pore-forming domain, containing the proposed site for etoxazole binding.

4-Methylumbelliferone promotes the migration and odontogenetic differentiation of human dental pulp stem cells exposed to lipopolysaccharide in vitro.

Hyaluronic acid (HA), a major component of the extracellular matrix, is essential to inflammatory regulation. 4-Methylumbelliferone (4-mu), as the specific inhibitor of HA synthesis, is an anti-inflammatory in multiple systems. However, there have been no studies, to our knowledge, regarding 4-mu treatment in pulp inflammation. Therefore, the purpose of this study was to investigate the effects of 4-mu on biological behaviors in human dental pulp stem cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro. hDPSCs were exposed to LPS to construct the inflammation model in vitro. Immunocytochemistry, quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, scratch/Transwell assay, and alizarin red staining/alkaline phosphatase staining were selected to explore the effect of 4-mu on the expression of inflammatory factors, cell proliferation, cell migration, and the odontogenic differentiation ability of hDPSCs. LPS stimulated hDPSCs to highly express the related inflammatory factors and CD44 (the major HA receptor), which were all inhibited by 0.1 mM of 4-mu. In addition, the cell proliferation ability of hDPSCs was suppressed by 4-mu, while cell migration and odontogenic differentiation abilities were significantly improved under inflammation. In conclusion, 4-mu suppressed inflammatory cytokines in inflamed hDPSCs and had a positive effect on the migration and odontogenic differentiation of hDPSCs.

4-Methylumbelliferone Attenuates Macrophage Invasion and Myocardial Remodeling in Pressure-Overloaded Mouse Hearts.

[Figure: see text].

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone as an Anti-Inflammatory Modulator of LPS-Mediated Astrocyte Responses.

Astrocytes are glial cells that play an important role in neuroinflammation. Astrocytes respond to many pro-inflammatory stimuli, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4). Regulatory specificities of inflammatory signaling pathways are still largely unknown due to the ectodermal origin of astrocytes. Recently, we have shown that hyaluronic acid (HA) may form part of astrocyte inflammatory responses. Therefore, we tested 4-methylumbelliferone (4-MU), a specific inhibitor of HA synthesis, as a possible regulator of LPS-mediated responses. Rat primary astrocytes were treated with LPS with and without 4-MU and gene expression levels of inflammatory (interleukins 1β, (IL-1β), 6, (IL-6), tumor necrosis factor alpha TNFα,) and resolution interleukin 10 (IL-10) markers were evaluated via real-time PCR and western blot. The release of cytokines and HA was determined by ELISA. Oxylipin profiles were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Our data show that 4-MU (i) has anti-inflammatory effects in the course of TLR4 activation, decreasing the cytokines level TNFα, IL-6 and IL-1β and increasing IL-10, (ii) downregulates prostaglandin synthesis but not via cyclooxygenases COX-1 and COX-2 pathways, (iii) modulates HA synthesis and decreases LPS-induced HA synthase mRNA expression (HAS-1, HAS-2) but does not have an influence on HAS-3, HYAL1 and HYAL2 mRNAs; (iv) the effects of 4-MU are predominantly revealed via JNK but not p38, ERK mitogen-activated protein kinases (MAPKs) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. For the first time, it is shown that 4-MU possesses the useful potential to regulate an inflammatory astrocyte response.

Inhibition of HAS2 and hyaluronic acid production by 1,25-Dihydroxyvitamin D3 in breast cancer.

1,25-Dihydroxyvitamin D3 (1,25D3) induces growth arrest and apoptosis in breast cancer cells in vivo and in vitro, however the exact mechanisms are unclear. Although the vitamin D receptor (VDR), a ligand dependent transcription factor, is required for growth regulation by vitamin D, the specific target genes that trigger these effects are unknown. Genomic profiling of murine mammary tumor cells with differential VDR expression identified 35 transcripts that were altered by the 1,25D3-VDR complex including Hyaluronan Synthase-2 (Has2). Here we confirmed that 1,25D3 reduces both HAS2 gene expression and hyaluronic acid (HA) synthesis in multiple models of breast cancer. Furthermore, we show that the growth inhibitory effects of 1,25D3 are partially reversed in the presence of high molecular weight HA. HAS2 expression and HA production are elevated in immortalized human mammary epithelial cells induced to undergo epithelial-mesenchymal transition (EMT) through stable expression of TGFβ, SNAIL or TWIST and in those expressing oncogenic H-RASV12, indicating that deregulation of HA production may be an early and frequent event in breast tumorigenesis. 1,25D3 also reduces HA secretion and acts additively with an HA synthesis inhibitor to slow growth of cells expressing TGFβ, SNAIL and TWIST. Analysis of mammary gland and tumors from Vdr knockout mice suggest that loss of VDR is associated with enhanced HAS2 expression and HA production in vivo. These data define a novel role for 1,25D3 and the VDR in control of HA synthesis in epithelial tissues that likely contributes to its anti-cancer actions.

MP08-17 BIOMARKER-TARGETED NOVEL COMBINATION FOR RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP08)1 Apr 2020MP08-17 BIOMARKER-TARGETED NOVEL COMBINATION FOR RENAL CELL CARCINOMA Eric Webb*, Soum Lokeshwar, Rashid Sayyid, Andre Jordan, Zachary Klaassen, Martha Terris, and Vinata Lokeshwar Eric Webb*Eric Webb* More articles by this author , Soum LokeshwarSoum Lokeshwar More articles by this author , Rashid SayyidRashid Sayyid More articles by this author , Andre JordanAndre Jordan More articles by this author , Zachary KlaassenZachary Klaassen More articles by this author , Martha TerrisMartha Terris More articles by this author , and Vinata LokeshwarVinata Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000828.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The survival of patients with metastatic renal cell carcinoma (mRCC) is < 10% at 5-years, despite treatment. Hyaluronic acid (HA) promotes tumor growth and metastasis through its receptors CD44 and RHAMM. 4-methylumbelliferone (MU) is a non−toxic small molecule HA synthesis inhibitor and down regulates CD44 and RHAMM mRNA. In this study we examined if MU in combination with an FDA-approved treatment could abrogate mRCC in preclinical models and whether the target can be validated in a clinical cohort and TCGA dataset. METHODS: Response of Vector-only (EV), HAS3-overexpressing (HAS3) or HAS3-knockdown (HAS3-KO) transfectants of 786-O and Caki-1 cells, primary RCC spheroids and endothelial cells co-cultured with these transfectants to MU combined with Sorafenib (SF), Sunitinib or Everolimus was examined in proliferation, apoptosis, motility and invasion assays. Immunoblotting and RT-qPCR were performed for downstream signaling studies. HAS3 promoter was cloned and effect of SF and HC on HAS3 promoter activity was determined. Treatment efficacy was evaluated in the Caki−1 s.c. and orthotopic xenograft models. HAS3 levels were analyzed in a clinical-cohort (51 normal; 83 tumor), RCC TCGA-dataset (TCGA-cohort; n=542). RESULTS: MU synergized with SF to inhibit RCC cell and primary tumor spheroid proliferation > 90%, motility/invasion (>80%) and induced apoptosis (5-fold). Combination of MU (0.1, 0.2 mM) with SF (5 µM) downregulated (>80%) HAS3 expression, HAS3 promoter activity and HA synthesis in RCC cells. While HAS3 transfectants were resistant to SF+MU treatment, HAS3-KO transfectants were sensitive to SF alone. Endothelial cells co-cultured with HAS3 transfectants were resistant to the combination. SF+MU up-regulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in HAS3 transfectants. MU (100 or 200 mg/kg) and SF (30 mg/kg) oral combination abrogated Caki-1 tumor growth (>80%), without serum/tissue toxicity. MU+SF treatment decreased HAS3/HA levels, Ki67 index and microvessel density. HAS-overexpressing tumors were resistant to treatment. HAS3 levels were 4-fold elevated in mRCC patients’ tumors and significantly predicted metastasis (P=0.0086). HAS3 levels also significantly correlated with overall survival in TCGA dataset (P=0.029). CONCLUSIONS: This is the first study to demonstrate that HAS3 significantly predicts metastasis. By downregulating HAS3 expression, MU+SF combination effectively abrogates RCC growth and metastasis. HAS3 is a potential biomarker to predict clinical outcome in RCC patients. Source of Funding: 5R01CA176691-05 (VBL); 1F31 CA210612-01 to ARJ © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e112-e112 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eric Webb* More articles by this author Soum Lokeshwar More articles by this author Rashid Sayyid More articles by this author Andre Jordan More articles by this author Zachary Klaassen More articles by this author Martha Terris More articles by this author Vinata Lokeshwar More articles by this author Expand All Advertisement PDF downloadLoading ...

Inhibition of Hyaluronic Acid Synthesis Decreases Endometrial Cell Attachment, Migration, and Invasion.

To characterize the effects of 4-methylumbelliferone (4-MU) on expression of the hyaluronic acid (HA) system and on attachment, migration, and invasion of endometrial epithelial (EECs) and stroma cells (ESCs) to peritoneal mesothelial cells (PMCs), this in vitro study was performed in an Academic Center. De-identified endometrial tissue samples used were from reproductive-aged women. EECs and ESCs isolated from menstrual endometrial biopsies were treated with 4-MU or vehicle. Real-time polymerase chain reaction and western blot were used to assess expression of HA synthases (HAS), hyaluronidase, and standard CD44. Established in vitro assays were used to assess attachment, migration, and invasion with and without treatment with 4-MU. Chi square and Student's t-test were used to analyze the results as appropriate. The addition of 4-MU decreased mRNA and protein expression of HAS 2, HAS 3, and CD44 in EECs and ESCs compared to control. Treatment with 4-MU also decreased attachment, migration, and invasion of EECs and ESCs to PMCs compared to control. 4-MU decreases endometrial cell adhesion, migration, and invasion to PMCs. This effect appears to be mediated by a decrease in HAS 2, HAS 3, and CD44. 4-MU is a potential treatment for endometriosis. Future in vivo studies are needed to evaluate 4-MU as a therapeutic agent for endometriosis.

CD44 splice variant (CD44v3) promotes progression of urothelial carcinoma of bladder through Akt/ERK/STAT3 pathways: novel therapeutic approach.

The incidence of Urothelial carcinoma of bladder (UBC) is gradually increasing by changing lifestyle and environment. The development of a tumor has been noted to be accompanied by modifications in the extracellular matrix (ECM) consisting of CD44, hyaluronic acid (HA) and its family members. The importance of CD44 splice variants and HA family members has been studied in UBC. The cohort of study included 50 UBC patients undergoing radical cystectomy and 50 healthy subjects. The molecular expression of CD44 and HA family members was determined. Effect of CD44 variant-specific silencing on downstream signaling in HT1376 cells was investigated. Combinatorial treatment of 4-MU (4-methylumbelliferone) with cisplatin or doxorubicin on chemosensitivity was also explored. Higher expression of HA, HAS2, and CD44 was observed in Indian UBC patients which also showed the trend with severity of disease. Splice variant assessment of CD44 demonstrated the distinct role of CD44v3 and CD44v6 in bladder cancer progression. shRNA-mediated downregulation of CD44v3 showed an increase effect on cell cycle, apoptosis and multiple downstream signaling cascade including pAkt, pERK and pSTAT3. Furthermore, 4-MU, an HA synthesis inhibitor, observed to complement the effect of Cisplatin or Doxorubicin by enhancing the chemosensitivity of bladder cancer cells. Our findings exhibit involvement of CD44 splice variants and HA family members in UBC and significance of 4-MU in enhancing chemosensitivity suggesting their novel therapeutic importance in disease therapeutics.

Hyaluronic Acid Synthesis Contributes to Tissue Damage in Systemic Lupus Erythematosus.

Hyaluronic acid (HA), a component of the extracellular matrix, is the ligand for CD44 and has been implicated in the pathogenesis of kidney inflammation in patients with systemic lupus erythematosus (SLE), but its direct role and mechanism of action have not been studied. Here we show that administration of hymecromone (4-Methylumbelliferone, 4-MU), an HA synthesis inhibitor, to lupus-prone mice suppressed dramatically lupus-related pathology. Interestingly, 4-MU stopped the appearance of disease when administered prior to its onset and inhibited the progression of disease when administered after its appearance. Inhibition of HA synthesis in vivo reduced tissue damage and the number of intrarenal lymphoid cell infiltrates including double negative CD3+CD4–CD8– T cells which are known to be involved in the pathogenesis of SLE. Exposure of human peripheral blood mononuclear cells to HA in vitro increased the generation of CD3+CD4–CD8– T cells through a mechanism involving Rho-associated kinase. Our results signify the importance of the HA-rich tissue microenvironment in the activation of lymphocytes to cause tissue damage in SLE and suggest the consideration of inhibition of HA synthesis to treat patients.

Abstract 1521: Tumor-produced hyaluronan contributes to the formation tolerogenic immunosuppressive microenvironment

Abstract Immunosuppressive ligand PD-L1 considered as a major player in mechanism of negative regulation of anti-tumor immune response and its expression is highly up-regulated in human and experimental tumors, including bladder cancer. Bladder cancer is characterized by aberrant hyaluronan metabolism resulting in increased HA production in tumor tissue. Hyaluronan or hyaluronic acid (HA) is a prominent component of tumor stroma/microenvironment. Membrane-bound or free extracellular HA favors tumor progression by inducing tumor cell motility, invasive properties, proliferation, production of growth factors and epithelial-mesenchymal transition. Here, we provide evidence that tumor-produced hyaluronan, one of the key tumor stroma components, contributes to the formation of tolerogenic/immunosuppressive tumor microenvironment and stimulated PD-L1 expression. We found that tumor-derived HA specifically binds to CD44 receptor expressed by myeloid precursors, such as myeloid-derived suppressor cells (MDSCs) and promotes its expansion with differentiation toward PD-L1-expressing F4/80+ macrophages. Inhibition of HA synthesis in tumor cells with pharmacologic inhibitor 4-MU, or blockade of CD44 signaling in myeloid cells with antagonistic anti-CD44 antibody, prevented the macrophage differentiation and tumor-induced up-regulation of PD-L1 expression. We also found that tumor-derived HA stimulated production of immunosuppressive and inflammatory factors IL-6, IL-10, TNF-alpha, IL-1beta and PGE2 by myeloid cells in CD44-dependent manner. Moreover, tumor-produced HA stimulated development of PD-L1-expressing macrophages in both murine and human myeloid cells. Our study reveals that tumors may evade the immune system by creating a protective tolerogenic “shield” in the form of tumor-produced HA, which binds to the CD44-expressing tumor-recruited MDSCs, stimulating production of immunosuppressive factors and promoting development of the PD-L1+ macrophages. Inhibiting of hyaluronan synthesis by tumors or targeting HA-CD44 signaling could provide an attractive approach to break tumor-induced immune tolerance and unleash the anti-tumor immune response. Citation Format: Paul R. Dominguez-Gutierrez, Paul Crispen, Sergei A. Kusmartsev. Tumor-produced hyaluronan contributes to the formation tolerogenic immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1521.

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

Endometrial cell invasion is decreased with inhibition of hyaluronic acid synthesis

Hyaluronic Acid (HA) plays a role in the early development of the endometriotic lesion.1 4-Methylumbelliferone (4-MU), a coumarin, has been shown to inhibit endometriotic lesion formation in mice.2 Here, we assessed the effects of 4-MU treatment of endometrial epithelial (EEC) and stromal cell (ESC) on: (1) expression of hyaluronic acid synthases (HAS), and (2) invasion of EECs and ESCs. In vitro studies. EECs and ESCs were isolated from menstrual endometrial biopsies. Cell viability after treatment was previously confirmed. EEC and ESC mRNA expression of HAS 2 and HAS 3 were quantified by RT-PCR with and without 4-MU (2mM). An established in vitro assay using peritoneal mesothelial cell (PMC) monolayers on a Matrigel matrix was used to assess EEC and ESC invasion of 4-MU treated and untreated cells. Results were analyzed with unpaired t-tests. RT-PCR confirmed a significant decrease in HAS 2 and HAS 3 mRNA expression in 4-MU treated EECs and ESCs (p <0.01). EECs invasion to PMCs decreased with the addition of 4-MU compared to control (668 ± 37 vs 1217 ± 51; p<0.0001). ESC invasion to PMCs with addition of 4-MU also decreased compared to control (765 ± 47 vs 1187 ± 64; p=0.04). 4-MU decreases HAS 2 and HAS 3 expression and invasiveness of EECs and ESCs. These findings further suggest that the CD44/HA system is involved in the development of the early endometriotic lesion and may serve as a therapeutic target to prevent the disease.

Abstract 4037: Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells

Abstract Evidence suggests that growth of cancer stem cells (CSCs) drives intratumor heterogeneity, a major clinical problem that results in drug resistance, recurrence of tumors, and metastatic progression of breast cancer. Therapeutic strategies to eliminate CSCs require identification of signaling pathways that facilitate the emergence and/or expansion of CSCs. These signaling pathways are implicated in altering cell behavior, as well as promoting the expression of genes involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Previous work demonstrated that a cooperative STAT3/SMAD3 gene transcription program increased the expression of Hyaluronan Synthase 2 (HAS2), an enzyme that produces Hyaluronic Acid (HA) which serves as a CD44 ligand and facilitates EMT. We hypothesize that STAT3 is a critical regulator of HA synthesis and signaling which enhances EMT and stemness of human mammary epithelial cells, driving tumor progression. We tested this hypothesis in a TGFβ triggered model of EMT. Immortalized human mammary epithelial cells (HMLE) expressing either a control vector (pWZL-GFP) or a vector containing TGFβ1 (pWZL-TGFβ1-GFP) were characterized with respect to STAT3 signaling, HA synthesis, and mammosphere formation. Our data revealed that HMLE-pWZL-TGFβ1-GFP cells have significantly higher expression of active STAT3 (Y705) compared to control cells, but total STAT3 gene and protein expression were similar in the two cell lines. HMLE-pWZL-TGFβ1-GFP cells exhibited a significant increase in expression of HAS2 and HAS2-AS1, a long non-coding RNA that enhances the transcription of HAS2. Preliminary results show that overexpression of TGFβ1 increased HA secretion in media compared to control cells. HMLE-pWZL-TGFβ1-GFP cells demonstrated significantly higher mammosphere forming ability compared to control cells, suggesting that TGFβ increases the percentage of stem cells in the population. Initial results demonstrated changes in mammosphere size and morphology of HMLE-pWZL-TGFβ1-GFP cells treated with the HA synthesis inhibitor 4-Methylumbelliferone (4MU). Together, these results suggest STAT3 and TGFβ1 co-regulate HAS2 expression and HA production in a model of EMT supporting the use of the HMLE-pWZL cell series as a promising model to study STAT3 regulation of the HA-CD44 signaling pathway. Citation Format: Brenda G. Trevizo-Barresi, JoEllen Welsh. Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4037.

Role of Hyaluronic Acid on Apolipoprotein A1 and HDL-Mediated Cholesterol Efflux in Macrophages

Diabetic macroangiopathy are characterized by increased arterial stiffness and hyaluronic acid (HA) deposition in the large elastic arteries. It is noteworthy that systemic inhibition of HA synthesis by 4-methylumbelliferone interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, inflammation, and progression of atherosclerosis. Apolipoprotein A1 (ApoA1) and High-density lipoprotein (HDL) mediates cholesterol efflux, which is the initial and rate-limiting step of reverse cholesterol transport preventing from atherosclerosis. The aim of the present study was to investigate the role of HA on ApoA1 and HDL-mediated cholesterol efflux in macrophages. Murine J774.1 macrophages and human THP-1 macrophages were labeled with 3H-cholesterol and incubated with ApoA1 (10 μg/ml) for 24 hours or HDL (50 μg/ml) for 4 hours. The percentage cholesterol efflux was calculated by dividing the media-derived radioactivity by the sum of the radioactivity in the media and the cells. The effect of HA on the cholesterol efflux was studied, adding that into the medium before the analysis of the cholesterol efflux. Under the existence of medium molecular weight HA (75-350 kDa), the HDL-mediated cholesterol efflux had significantly increased by 10.9% (n=4, p&amp;lt;0.01) in J774.1 macrophages. We also researched the relationship between HA and ATP-binding cassette transporter G1 (ABCG1), because expression of ABCG1 enhances cholesterol efflux to HDL. Predictably, HA significantly increased ABCG1 mRNA expression in macrophage (n=4, p&amp;lt;0.01), determined by Real-time Quantitative RT-PCR. These data suggest that HA increases HDL-mediated cholesterol efflux via ABCG1 in macrophages and the difference of molecular weight on HA may influence ApoA1 and HDL-mediated cholesterol efflux. The relationship between HA and cholesterol efflux might be one of the important mechanisms to prevent progression of atherosclerosis and diabetic macroangiopathy. Disclosure K. Matsuki: None. M. Ogura: None. M. Harada-Shiba: Advisory Panel; Self; Aegerion Pharmaceuticals. Speaker's Bureau; Self; Amgen Inc., Astellas Pharma US, Inc., Sanofi, Boehringer Ingelheim Pharmaceuticals, Inc., AstraZeneca, Daiichi Sankyo Company, Limited, MSD K.K.. Research Support; Self; Sanofi, Aegerion Pharmaceuticals, Kowa Pharmaceuticals America, Inc..