Identification of oral squamous cell carcinoma markers MUC2 and SPRR1B downstream of TANGO.

PURPOSE: Transport and Golgi organization protein 1 (TANGO) promotes angiogenesis and lymphangiogenesis in oral squamous cell carcinoma (OSCC). To elucidate the underlying mechanisms, this study aims to identify and characterize elements downstream of TANGO that mediate its involvement in OSCC. METHODS: In this study, microarray analysis compared gene expression between control and TANGO-repressed HSC3 cells. Protein expression in 213 OSCC tissue samples was analyzed immunohistochemically. RESULTS: TANGO repression decreased or increased expression of Mucin 20 (MUC20) and small proline-rich protein 1B (SPRR1B), respectively. MUC20 increased the growth and invasiveness of OSCC cells via altered matrix metalloproteinase (MMP)-2 and E-cadherin expression and c-met phosphorylation. MUC20 induced angiogenesis and lymphangiogenesis by activating vascular endothelial growth factors A and C. In well-differentiated OSCC, SPRR1B expression was high (P = 0.0091) and correlated with keratinization markers and promoted proliferation by inducing mitogen-activated protein kinase p38 phosphorylation. MUC20 expression correlated significantly with clinical stage (P = 0.0024), lymph node metastasis (P = 0.0036), and number of blood and lymph vessels (P < 0.0001). MUC20-expressing cases had a significantly worse prognosis than non-expressing cases (P < 0.0001). CONCLUSION: MUC20 and SPRR1B located downstream of TANGO may be useful molecular markers for OSCC.

Modulation of the gut microbiota with antibiotic treatment suppresses whole body urea production in neonatal pigs.

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.

Lamellar structures of MUC2-rich mucin: a potential role in governing the barrier and lubricating functions of intestinal mucus.

Mucus is a ubiquitous feature of mammalian wet epithelial surfaces, where it lubricates and forms a selective barrier that excludes a range of particulates, including pathogens, while hosting a diverse commensal microflora. The major polymeric component of mucus is mucin, a large glycoprotein formed by several MUC gene products, with MUC2 expression dominating intestinal mucus. A satisfactory answer to the question of how these molecules build a dynamic structure capable of playing such a complex role has yet to be found, as recent reports of distinct layers of chemically identical mucin in the colon and anomalously rapid transport of nanoparticles through mucus have emphasized. Here we use atomic force microscopy (AFM) to image a MUC2-rich mucus fraction isolated from pig jejunum. In the freshly isolated mucin fraction, we find direct evidence for trigonally linked structures, and their assembly into lamellar networks with a distribution of pore sizes from 20 to 200 nm. The networks are two-dimensional, with little interaction between lamellae. The existence of persistent cross-links between individual mucin polypeptides is consistent with a non-self-interacting lamellar model for intestinal mucus structure, rather than a physically entangled polymer network. We only observe collapsed entangled structures in purified mucin that has been stored in nonphysiological conditions.

Chronic anti-inflammatory drug therapy inhibits gel-forming mucin production in a murine xenograft model of human pseudomyxoma peritonei.

BACKGROUND: Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP. METHODS: The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays. RESULTS: Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count. CONCLUSIONS: Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

Proteomic study of the mucin granulae in an intestinal goblet cell model.

Goblet cells specialize in producing and secreting mucus with its main component, mucins. An inducible goblet-like cell line was used for the purification of the mucus vesicles stored in these cells by density gradient ultracentrifugation, and their proteome was analyzed by nanoLC-MS and MS/MS. Although the density of these vesicles coincides with others, it was possible to reveal a number of proteins that after immunolocalization on colon tissue and functional analyses were likely to be linked to the MUC2 vesicles. Most of the proteins were associated with the vesicle membrane or their outer surface. The ATP6AP2, previously suggested to be associated with vesicular proton pumps, was colocalized with MUC2 without other V-ATPase proteins and, thus, probably has roles in mucin vesicle function yet to be discovered. FAM62B, known to be a calcium-sensitive protein involved in vesicle fusion, also colocalized with the MUC2 vesicles and is probably involved in unknown ways in the later events of the MUC2 vesicles and their secretion.