Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Extracts of Lindera obtusiloba induce antifibrotic effects in hepatic stellate cells via suppression of a TGF-beta-mediated profibrotic gene expression pattern

Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.

Clostridium perfringens beta-toxin binding to vascular endothelial cells in a human case of enteritis necroticans

Clostridium perfringens type C-induced enteritis necroticans is a rare but often fatal disease in humans. A consistent histopathological finding is an acute, deep necrosis of the small intestinal mucosa associated with acute vascular necrosis and massive haemorrhage in the lamina propria and submucosa. Retrospective immunohistochemical investigations of tissues from a diabetic adult who died of enteritis necroticans revealed endothelial localization of C. perfringens beta-toxin in small intestinal lesions. Our results indicate that vascular necrosis might be induced by a direct interaction between C. perfringens beta-toxin and endothelial cells and that targeted disruption of endothelial cells plays a role in the pathogenesis of enteritis necroticans.

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Expression and function of $\beta$-tubulin isotypes in Chinese hamster ovary (CHO) cells

The availability of isotype specific antisera for $\beta$-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of $\beta$-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor $\beta$-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. Their stoichiometry is approximately 1:1:0.7:0.2. All three isotypes assemble into both cytoplasmic and spindle microtubules, and are similar in their responses to cold, colcemid, and calcium induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that $\beta$-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid resistant mutants with a demonstrable alteration in $\beta$-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist. Under various conditions of the cell growth, the relative proportion of each expressed isotype does not significantly seem to change except in the early G1 phase of the cell cycle. At this time the synthesis of isotype V increases more than two fold relative to isotype I and IV, while at the same time, total $\beta$-tubulin synthesis is decreased about 60-70%. ^

Function and formation of $\beta$1,4-galactosyltransferase-specific adhesions during growth and differentiation of F9 embryonal carcinoma cells

Galactosyltransferase (GalTase) is localized in the Golgi, where it functions in oligosaccharide synthesis, as well as on the cell surface where it serves as a cell adhesion molecule. GalTase-specific adhesions are functional in a number of important biological events, including F9 embryonal carcinoma (EC) cell adhesions. GalTase-based adhesions are formed by recognition and binding to terminal N-acetylglucosamine (GlcNAc) residues on its glycoprotein counterpart on adjacent cell surfaces. The object of this work has been to investigate the formation and function of GalTase-specific adhesions during F9 cell growth and differentiation. We initially investigated GalTase synthesis during differentiation and found that the increase in GalTase activity was specific for the Golgi compartment; surface GalTase levels remained constant during differentiation. These data indicated that the increase in cell adhesions expected with increased cell-matrix interaction in differentiated F9 cells is not the consequence of increased surface GalTase expression and, more interestingly, that the two pools of GalTase are under differential regulation. Synthesis and recognition of the consociate glycoprotein component was next investigated. Surface GalTase recognized several surface glycoproteins in a pattern that changes with differentiation. Uvomorulin, lysosome-associated membrane protein-1 (LAMP-1), and laminin were recognized by surface GalTase and are, therefore, potential components in GalTase-specific adhesions. Furthermore, these interactions were aberrant in an adhesion-defective F9 cell line that results, at least in part, from abnormal oligosaccharide synthesis. The function played by surface GalTase in growth and induction of differentiation was examined. Inhibition of surface GalTase function by a panel of reagents inhibited F9 cell growth. GalTase expression at both the transcription and protein levels were differentially regulated during the cell cycle, with surface expression greatest in the G1 phase. Disruption of GalTase adhesion by exposure to anti-GalTase antibodies during this period resulted in extension of the G2 phase, a result similar to that seen with agents known to inhibit growth and induce differentiation. Finally, other studies have suggested that a subset of cell adhesion molecules have the capability to induce differentiation in EC cells systems. We have determined in F9 cells that dissociating GalTase adhesion by galactosylation of and release of the consociate glycoproteins induces differentiation, as defined by increased laminin synthesis. The ability to induce differentiation by surface galactosylation was greatest in cells grown in cultures promoting cell-cell adhesions, relative to cultures with minimal cell-cell interactions. ^

Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

A PHARMACOLOGICAL EVALUATION OF THE MECHANISM OF CYTOTOXICITY OF 9-BETA-D- XYLOFURANOSYLADENINE IN CHINESE HAMSTER OVARY CELLS

The biochemical determinants of cytotoxicity of the purine nucleoside analog, 9-(beta)-D-xylofuranosyladenine (xyl-A) were studied in wild-type Chinese hamster ovary cells and in nucleoside kinase deficient mutants. It was found that {('3)H}xyl-A was readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase deficient mutant, but not by the adenosine kinase deficient cells. Values for the apparent Km and Vmax of this uptake process were 43.9 (mu)M and 118.7 nmol/min/10('9) cells, respectively. Cloning procedures indicated that the viability of CHO cells was decreased 90 per cent by a 5-hr incubation with 10 (mu)M xyl-A. However, the toxicity of xyl-A was increased 100-fold by the addition of a nontoxic concentration (10 (mu)M) of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to the medium. High-pressure liquid chromatographic analysis indicated that after 5 hr, the concentration of 9-(beta)-D-xylofuranosyladenine 5'-triphosphate (xyl-ATP) in cells incubated with xyl-A plus EHNA was 2.0 mM, four times greater than in those cells incubated with xyl-A alone. Incubation with xyl-A plus EHNA had no significant effect on the cellular concentrations of 5-phosphoribosyl-1-pyrophosphate after 1 hr whereas, treatment with 3'-dexoyadenosine (cordycepin) decreased the concentration of this metabolite. Determinations of the cellular nucleoside triphosphates indicated that under conditions that resulted in an intracellular accumulation of 500 (mu)M xyl-ATP, the endogenous concentrations of neither the ribonucleoside triphosphates nor deoxyribonucleoside triphosphates were significantly different from those of control cells. The ID(,50) for {('3)H}thymidine incorporation into DNA, 105 (mu)M xyl-ATP, was four-fold less than the ID(,50) for {('3)H}uridine incorporation into RNA suggesting that the process of DNA synthesis is more sensitive to the presence of xyl-ATP. When removed from exogenous xyl-A, CHO cells failed to recover their ability to synthesize RNA and DNA, although the intracellular xyl-ATP concentration decreased to less than 35 (mu)M. The selective inhibition of RNA synthesis by 6-azauridine did not prevent the expression of toxicity by xyl-ATP. However, the selective inhibition of DNA synthesis by ara-C significantly spared toxicity in cells that had accumulated an otherwise lethal concentration of xyl-ATP. It is shown that in cells which had accumulated 1.27 mM {('3)H}xyl-ATP, {('3)H}xyl-A was found to terminate cellular RNA chains at a frequency of 1.42 (mu)mol of {('3)H}xyl-A 3' termini per mol of mononucleotide. These results indicate that a general mechanism for the toxicity of xyl-A to CHO cells includes the cellular accumulation of xyl-ATP, which serves as a substrate for RNA synthesizing enzymes and subsequently is incorporated into nascent RNA transcripts as a chain terminator. A specific mechanism involving the premature termination of RNA primers required for the initiation of DNA synthesis is proposed to account for the inhibitory action of xyl-ATP on DNA synthesis. ^

Hyperplasia to neoplasia sequence of duodenal and pancreatic neuroendocrine diseases and pseudohyperplasia of the PP-cells in the pancreas.

Hyperplastic changes of the neuroendocrine cell system may have the potential to evolve into neoplastic diseases. This is particularly the case in the setting of genetically determined and hereditary neuroendocrine tumor syndromes such as MEN1. The review discusses the MEN1-associated hyperplasia-neoplasia sequence in the development of gastrinomas in the duodenum and glucagon-producing tumors in the pancreas. It also presents other newly described diseases (e.g., glucagon cell adenomatosis and insulinomatosis) in which the tumors are (or most likely) also preceded by islet cell hyperplasia. Finally, the pseudohyperplasia of PP-rich islets in the pancreatic head is defined as a physiologic condition clearly differing from other hyperplastic-neoplastic neuroendocrine diseases.

Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

Increased number of non-degranulated mast cells in pancreatic ductal adenocarcinoma but not in acute pancreatitis.

Increasing evidence indicates that tumor microenvironment (TME) is crucial in tumor survival and metastases. Inflammatory cells accumulate around tumors and strangely appear to be permissive to their growth. One key stroma cell is the mast cell (MC), which can secrete numerous pro- and antitumor molecules. We investigated the presence and degranulation state of MC in pancreatic ductal adenocarcinoma (PDAC) as compared to acute ancreatitis (AP). Three different detection methods: (a) toluidine blue staining, as well as immunohistochemistry for (b) tryptase and (c) c-kit, were utilized to assess the number and extent of degranulation of MC in PDAC tissue (n=7), uninvolved pancreatic tissue derived from tumor-free margins (n=7) and tissue form AP (n=4). The number of MC detected with all three methods was significantly increased in PDAC, as compared to normal pancreatic tissue derived from tumor-free margins (p<0.05). The highest number of MC was identified by c-kit, 22.2∓7.5 per high power field (HPF) in PDAC vs 9.7∓5.1 per HPF in normal tissue. Contrary to MC in AP, where most of the detected MC were found degranulated, MC in PDAC appeared intact. In conclusion, MC are increased in number, but not degranulated in PDAC, suggesting that they may contribute to cancer growth by permitting selective release of pro-tumorogenic molecules.

Accumulation of FOXP3+T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma.

Here we explore the role of the interplay between host immune response and epithelial-mesenchymal-transition (EMT)-Type tumor-budding on the outcome of pancreatic adenocarcinoma (PDAC).CD4+, CD8+, and FOXP3+T-cells as well as iNOS+ (M1) and CD163+- macrophages (M2) were assessed on multipunch tissue-microarrays containing 120 well-characterized PDACs, precursor lesions (PanINs) and corresponding normal tissue. Counts were normalized for the percentage of tumor/spot and associated with the clinico-pathological features, including peritumoral (PTB) and intratumoral (ITB) EMT-Type tumor-budding and outcome.Increased FOXP3+T-cell-counts and CD163-macrophages and decreased CD8+T-cell-counts were observed in PDACs compared with normal tissues and PanINs (p < 0.0001). Increased peritumoral FOXP3+T-cell-counts correlated significantly with venous invasion, distant metastasis, R1-status, high-grade ITB, PTB and independently with reduced survival. Increased intratumoral FOXP3+T-cells correlated with lymphatic invasion, N1-stage, PTB and marginally with adverse outcome. High peritumoral CD163-counts correlated with venous invasion, PTB and ITB. High intratumoral CD163-counts correlated with higher T-stage and PTB.PDAC-microenvironment displays a tumor-favoring immune-cell composition especially in the immediate environment of the tumor-buds that promotes further growth and indicates a close interaction of the immune response with the EMT-process. Increased peritumoral FOXP3+T-cell density is identified as an independent adverse prognostic factor in PDAC. Patients with phenotypically aggressive PDACs may profit from targeted immunotherapy against FOXP3.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.