WNT7B mediates autocrine Wnt/ß-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma.

Developmental and cancer models show Wnt/ß-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/ß-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/ß-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression-based assays revealed wide heterogeneity in Wnt/ß-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally generated, pancreas-specific gene expression signature of Wnt/ß-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early-stage PDAC tumors (N=41). In this cohort, higher Wnt/ß-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease-specific survival (median survival time 20.3 versus 43.9 months, log-rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/ß-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small-molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/ß-catenin activity. Gene-knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B-mediated cell autonomous Wnt/ß-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/ß-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/ß-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.

The chemokine receptor CXCR4 is expressed in pancreatic intraepithelial neoplasia.

OBJECTIVE: The chemokine CXCL12, together with its specific receptor, CXCR4, have been shown to mediate invasiveness and metastatic behaviour in pancreatic cancer cells. The expression of CXC12/CXCR4 has not been previously examined in pancreatic intraepithelial neoplasias (PanIN), the accepted precursor lesions to pancreatic duct cancer. DESIGN: In this study we sought to characterise the expression of CXCL12 and CXCR4 during the progression of PanIN using both a murine model and human tissues. RESULTS: These studies reveal that both CXCL12 and CXCR4 are expressed in PanIN and that the frequency increases during PanIN progression (0% CXCR4 expression in normal mouse and human ducts vs 100% in mouse PanIN 3 and 77% in human PanIN 3). Next we demonstrate a dose-dependent increase in the proliferation of murine PanIN cells when exposed to CXCL12. Finally, we show that expression of CXCR4 in murine PanIN cells is partially dependent on mitogen-activated protein kinase (MAPK) signalling and that the effect of CXCL12 on PanIN proliferation can be abrogated by an MAPK inhibitor. CONCLUSIONS: Together these results demonstrate that CXCL12/CXCR4 expression begins in the pre-invasive stages of pancreatic neoplasia, and suggest that the presence of an autocrine loop that is at least partially regulated by MAPK signalling. Further studies that define the role of CXCR4 signalling in PanIN progression will determine if CXCR4 could serve as a novel target for chemoprevention and early stage therapy in pancreatic cancer.

Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas.

Most human lymphomas originate from transformed germinal center (GC) B lymphocytes. While activating mutations and translocations of MYC, BCL2 and BCL6 promote specific GC lymphoma subtypes, other genetic and epigenetic modifications that contribute to malignant progression in the GC remain poorly defined. Recently, aberrant expression of the TCL1 proto-oncogene was identified in major GC lymphoma subtypes. TCL1 transgenic mice offer unique models of both aggressive GC and marginal zone B-cell lymphomas, further supporting a role for TCL1 in B-cell transformation. Here, restriction landmark genomic scanning was employed to discover tumor-associated epigenetic alterations in malignant GC and marginal zone B-cells in TCL1 transgenic mice. Multiple genes were identified that underwent DNA hypermethylation and decreased expression in TCL1 transgenic tumors. Further, we identified a secreted isoform of EPHA7, a member of the Eph family of receptor tyrosine kinases that are able to influence tumor invasiveness, metastasis and neovascularization. EPHA7 was hypermethylated and repressed in both mouse and human GC B-cell non-Hodgkin lymphomas, with the potential to influence tumor progression and spread. These data provide the first set of hypermethylated genes with the potential to complement TCL1-mediated GC B-cell transformation and spread.

An enzyme immunoassay for intrinsic factor in urine.

An enzyme immunoassay for intrinsic factor has been used on urine. The assay can measure intrinsic factor in native urine from healthy people and from patients with pernicious anaemia with no antibodies. The urinary intrinsic factor concentration in healthy individuals ranged from 40 to 54 pmol/l. Intrinsic factor antibodies, demonstrated by testing the recovery of added intrinsic factor, interfered with the assay. Cobalamin at high concentrations also affected the assay result. A low intrinsic factor concentration or the presence of antibodies to intrinsic factor was found in the urine of individuals with pernicious anaemia.

Pigment epithelium-derived factor: a potent inhibitor of angiogenesis.

In the absence of disease, the vasculature of the mammalian eye is quiescent, in part because of the action of angiogenic inhibitors that prevent vessels from invading the cornea and vitreous. Here, an inhibitor responsible for the avascularity of these ocular compartments is identified as pigment epithelium-derived factor (PEDF), a protein previously shown to have neurotrophic activity. The amount of inhibitory PEDF produced by retinal cells was positively correlated with oxygen concentrations, suggesting that its loss plays a permissive role in ischemia-driven retinal neovascularization. These results suggest that PEDF may be of therapeutic use, especially in retinopathies where pathological neovascularization compromises vision and leads to blindness.

Three distinct D-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat.

Mal II, a 19-residue peptide derived from the second type 1 properdin-like repeat of the antiangiogenic protein thrombospondin-1 (TSP-1), was inactive in angiogenesis assays. Yet the substitution of any one of three L-amino acids by their D-enantiomers conferred on this peptide a potent antiangiogenic activity approaching that of the intact 450-kDa TSP-1. Substituted peptides inhibited the migration of capillary endothelial cells with an ED50 of 8.5 nM for the D-Ile-15 substitution, 10 nM for the D-Ser-4 substitution, and 0.75 nM for the D-Ser-5 substitution. A peptide with D-Ile at position 15 could be shortened to its last seven amino acids with little loss in activity. Like whole TSP-1, the Mal II D-Ile derivative inhibited a broad range of angiogenic inducers, was selective for endothelial cells, and required CD36 receptor binding for activity. A variety of end modifications further improved peptide potency. An ethylamide-capped heptapeptide was also active systemically in that when injected i.p. it rendered mice unable to mount a corneal angiogenic response, suggesting the potential usefulness of such peptides as antiangiogenic therapeutics.

CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells.

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.

Malnutrition: folate and cobalamin deficiency.

Malnutrition of folate and cobalamin occurs on a world-wide scale. Millions of individuals, for a variety of cultural, religious and socio-economic reasons, ingest less than the daily amounts required to maintain body stores. Assessment of intake depends on the population under study, method of food preparation and assay technique. Up to 90% of folate may be destroyed by cooking and, although less, significant amounts of cobalamin can also be lost in this way. Estimates of the proportion of both vitamins absorbed from a mixed diet vary, but may be as little as 50%. The need for supplementation is more common with folate than cobalamin. However, recent advances have highlighted subtle sub-clinical metabolic changes in some groups, particularly the elderly. Further investigation into their requirements is indicated. New assays for metabolites of cobalamin and folate are highly sensitive but lack specificity and are not readily available.

High incidence of type II autoantibodies in pernicious anaemia.

AIMS: To investigate the incidence of type II autoantibodies to intrinsic factor in pernicious anaemia. METHODS: Three hundred and forty four serum samples submitted for intrinsic factor antibody (IFAB) analysis on clinical or laboratory grounds were tested by an established radioassay and a new enzyme linked immunosorbent assay (ELISA) method for type I and total IFAB, respectively. Sixty of these were found to be positive by ELISA; this method was used to test further, 40 samples of adequate volume for types I and II antibodies. RESULTS: Type II antibodies were detected in 39 of the 40 sera tested. A comparative analysis indicated that seven samples contained pure type II antibody, being positive for total and type II by ELISA, but negative for type I by both the ELISA and radioassay technique. CONCLUSIONS: The occurrence of type II antibody, both alone and in combination with type I, seems to be more common than has previously been recognised, and emphasises the advantage of using a technique which will detect both types of antibody.

The accuracy and clinical interpretation of serum ferritin assays.

The accuracy of methods used to assay serum ferritin was determined in two ways. In one a serum, to which the UK ferritin standard had been added, was issued to the participants in an inter-regional quality assurance scheme. The overall recovery was close to that expected. For the second assessment a series of sera from individuals of known iron status were issued. Differences between assay results related to the method of assay though the immunoradiometric and ELISA methods gave results which were close to each other. The variation in clinical interpretation applied to assay results when ferritin concentrations around the upper and lower limits of normal were assayed was more than could be accounted for by method imprecision and indicates that inappropriate reference ranges are in common use. 'Indeterminate', a proper clinical interpretation when the imprecision of a technique should prevent a definite response, was inadequately used.

Dietary deficiency of vitamin B12 is associated with low serum cobalamin levels in non-vegetarians.

A prospective study of 106 patients with low serum cobalamin (vitamin B12) levels showed that, in 37, it was unexplained. The dietary intake of the vitamin was assessed in these patients by questionnaire and was found to be low in 10 (37%). None of these patients was vegetarian and they were of varying age and social circumstance. Dietary deficiency may be the sole cause of a low serum cobalamin in a significant proportion of non-vegetarians. An assessment of dietary intake should be part of the investigation of cobalamin deficiency.

New enzyme immunoassay for detecting total, type I, and type II intrinsic factor antibodies.

A method for the detection of total, type I, and type II intrinsic factor antibodies was devised. The technique comprises a two-site solid phase enzyme linked immunosorbent assay (ELISA), with human intrinsic factor conjugated with horseradish peroxidase as label and attached to polystyrene tubes as solid phase. One conjugation provides sufficient material to assay more than 10,000 patient samples. The label proved stable during the course of this evaluation and was still in use more than 12 months after preparation. When applied to 45 serum samples from cases of pernicious anaemia, intrinsic factor antibodies were shown in 30 (67%). Simplicity, high capacity, low cost and label stability, combined with relatively high clinical sensitivity make the method suitable for cost effective screening of large numbers of samples. Simple modifications to the basic assay reagents permitted type I and type II intrinsic factor antibodies to be differentiated.

Intrinsic factor antibody tests.

The sensitivity of methods to detect antibodies to intrinsic factor was assessed. Five sera of known antibody content were tested in 31 laboratories and 30 sera from patients with pernicious anaemia were tested in one laboratory. Five non-commercial methods and two kits for type I antibodies and one non-commercial method for types I and II antibodies are in current use. Differences in sensitivity of the non-commercial methods for type I antibodies related more to the antigen: antibody ratio in the test system than to the method itself. A radioimmune assay for types I and II antibodies showed the best sensitivity but that of an enzyme linked immunosorbent assay (ELISA) method was poor.

Experiences with dual protein bound aqueous vitamin B12 absorption test in subjects with low serum vitamin B12 concentrations.

A dual isotope vitamin B12 absorption test in which vitamin B12 is given both in aqueous solution and bound to protein (chicken serum), was evaluated in 26 controls and 68 patients with subnormal serum vitamin B12 concentrations (19 with pernicious anaemia, 13 with iron deficiency, seven after partial gastrectomy, seven with malabsorptive states, five with folate deficiency, four with chronic alcoholism and 13 in whom no cause was apparent). In control patients protein bound absorption decreased with age; isotope excretion was 1.0% or over in those aged under 60 and 0.5% or over in those aged 60 and above. Malabsorption of protein bound vitamin B12 with normal aqueous absorption occurred in five patients with iron deficiency, three with alcoholism, two after partial gastrectomy, two with folate deficiency and in one with a malabsorptive state. In alcoholics abstinence produced an improvement in protein bound absorption. All patients in the group for whom no cause could be found for the subnormal serum vitamin B12 concentration had normal aqueous absorption but four had malabsorption of protein bound vitamin. Although the dual isotope test gave reproducible results and was consistent with the standard Schilling test some anomalies were detected; nine patients had reduced aqueous absorption with normal protein bound absorption. Despite this the dual test may prove useful in determining the importance of a subnormal vitamin B12 concentration where the cause is not clinically apparent. Further development is needed before it can be considered for routine use.

Laboratory diagnosis of megaloblastic anaemia: current methods assessed by external quality assurance trials.

The results of an Interregional quality assurance scheme for tests in the diagnosis of megaloblastic anaemia were reviewed to assess the methods used. Serum folate assays showed great variation between methods, partly due to limitations in assessment by external quality assurance. Red cell folate assays yielded widely different results and much imprecision due both to the differences in preparation of the haemolysate and to the problems inherent in radioassay of a mixture of folate compounds. Intrinsic factor antibody tests showed appreciable variation in sensitivity. There was considerable inconsistency in the detection of polymorph nuclear hypersegmentation.

Plasma cobalamin-binding and serum cobalamin in patients with folate deficiency.

Plasma unsaturated R-binder and transcobalamin 2 (TC 2) levels were measured in 62 patients with folate deficiency and compared with 80 control subjects and 52 patients with pernicious anaemia. An increase in unsaturated R-binder concentration was found in the majority of patients with folate deficiency and with PA. In folate deficiency, however, the unsaturated R-binder was often elevated whether the serum cobalamin (Cbl) was low or normal, more frequently when the serum Cbl was normal. Results of a separate in vivo study of plasma retention of injected 57Co cyanocobalamin were consistent with these findings. An elevated TC2 was found in a small number of patients with folate deficiency and with PA. The serum Cbl appears to the maintained at a normal level in some patients with folate deficiency by an increase in R-binder, which is caused by folate deficiency itself.

Malabsorption of protein bound vitamin B12.

Patients with subnormal serum vitamin B12 concentrations were tested for absorption of protein bound vitamin B12 and compared with controls. Absorption of the protein bound vitamin appeared to decrease with increasing age in healthy subjects. Differences between the result of this test and the result of the Schilling test in patients who had undergone gastric surgery were confirmed; such differences were also seen in some patients who had iron deficiency anaemia, an excessive alcohol intake, or folate deficiency. Defective absorption was also found in six patients with an adequate dietary intake of vitamin B12, normal Schilling test results, low serum vitamin concentrations, and tissue changes responding to treatment with vitamin B12. Malabsorption of the vitamin from protein bound sources, which is not detected by the Schilling test, may produce vitamin B12 deficiency of clinical importance.