Uwe Karsten

Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors

ABSTRACT Activation of Wnt signaling through β-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of β-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3β. Here we show that conductin is upregulated in human tumors that are induced by β-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of β-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity.

Summary Report on the ISOBM TD-4 Workshop: Analysis of 56 Monoclonal Antibodies against the MUC1 Mucin

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.

Patterns of cytokeratin and vimentin expression in the human eye

SummaryWe studied the expression of the various cytokeralin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaiclike staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.

Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study.

A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens. TF (Galβ1-3GalNAcα1-R), Tn (TF precursor, GalNAcα1-R), sialosyl-Tn (NeuAcα2-6GalNAcα1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF. These antigens or, more correctly, glycotopcs, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry. For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence. Staining with mAbs appeared to be more restricted than with the lectins used. Distribution patterns among normal epithelia were different for all four antigens. These antigens were also detected in some non-epithelial tissues. They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF> >sialosyl-Tn>Tn>TF. Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas. The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase fromVibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF [NeuAcα2-3Galβ1-3GalNAcα1-R and Galβ1-3 (NeuAcα2-6)GalNAcα1-R] in normal human tissues. From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.

Expression of Thomsen-Friedenreich-related antigens in primary and metastatic colorectal carcinomas : a reevaluation

Background. Expression of the pancarcinoma Thomsen‐Friedenreich (TF) carbohydrate antigen or, more correctly, hapten, in colorectal carcinomas is not generally agreed on. Furthermore, its suggested role in liver metastasis so far has not been substantiated by direct immunohistochemical evidence.

Immunodetection of epithelial mucin (MUC1, MUC3) and mucin-associated glycotopes (TF, Tn, and sialosyl-Tn) in benign and malignant lesions of colonic epithelium: apolar localization corresponds to malignant transformation

Abstract Epithelial mucins are present at the apical membranes of gastrointestinal epithelial cells or in their secretions. In this study, we examined the occurrence of peptide epitopes of the mucins MUC1 and MUC3 and of three mucin-associated glycotopes (TF, Tn, and s-Tn) in a series of colorectal tissue samples (normal colon, adenomas with different grades of dysplasia, carcinoma in situ, and invasive carcinomas). A new monoclonal antibody to a conformation-dependent peptide epitope of MUC1 was employed, which does not react with the fully glycosylated mucin as found in normal gastrointestinal mucosa. We found that adenomas acquired the ability to expose Tn, s-Tn, TF and MUC1 epitopes, and this correlated with increasing malignant potential. The secretory mucin, MUC3, revealed a different pattern: it was detectable in all sections, with maximum expression in adenomas and decrease in carcinomas. Most importantly, normal mucosa and benign lesions showed supranuclear and/or apical distribution of these antigens, but malignant lesions and lesions with a very high risk of malignancy revealed diffuse cytoplasmic and basolateral membrane localization. The immunohistological response to a combination of MUC1-related antibodies may assist in assessing the malignant potential and status of lesions of the colon.

Mucins (MUC1 and MUC3) of Gastrointestinal and Breast Epithelia Reveal Different and Heterogeneous Tumor-associated Aberrations in Glycosylation

In a comprehensive study, we examined the expression of the membrane and secretory mucins MUC1 and MUC3, respectively, in normal and neoplastic gastrointestinal and breast epithelia before and after specific alterations of their glycan structures by neuraminidase, α-fucosidase, or carbohydrate-specific periodate oxidation. MUC1 mRNA was also identified in normal colorectal tissues by in situ hybridization. The data revealed that normal colorectal epithelia express both MUC1 mRNA and protein, which were detectable after periodate oxidation with all tested MUC1-specific antibodies. During tumorigenesis in the colon, MUC1 became recognizable without periodate treatment concomitantly with highly dysplastic lesions and the malignant state. In the breast, in which MUC1 is detectable with most antibodies in normal epithelium as well as in carcinomas, staining could be enhanced by pretreatment with periodate and casually by enzyme treatments. MUC3 was detectable in normal and neoplastic colorectal tissues and was more intensely stained after periodate oxidation. It was absent in normal breast even after pretreatment but was expressed in seven of 20 breast carcinomas. Therefore, incomplete glycosylation, abnormal distribution, and ectopic expression of mucins are characteristics of malignancy. Periodate oxidation may be widely applicable to immunohistochemistry for examining changes in glycosylation and for detecting antigens masked by glycans. (J Histochem Cytochem 45:1547–1557, 1997)

PankoMab: a potent new generation anti-tumour MUC1 antibody

Recently, we described a new carbohydrate-induced conformational tumour-epitope on mucin-1 (MUC1) with the potential for improvement of immunotherapies [29, 30]. PankoMab is a novel antibody, which binds specifically to this epitope and was designed to show the highest glycosylation dependency and the strongest additive binding effect when compared to other MUC1 antibodies. This enables PankoMab to differentiate between tumour MUC1 and non-tumour MUC1 epitopes. It has a high-affinity towards tumour cells (e.g. KD [M] of 0.9 and 3×10−9 towards NM-D4 and ZR75-1, respectively) and detects a very large number of binding sites (e.g. 1.0 and 2.4×106 for NM-D4 and ZR75-1, respectively). PankoMab is rapidly internalised, and after toxin coupling is able to induce very effectively toxin-mediated antigen-specific tumour cell killing. PankoMab reveals a potent tumour-specific antibody-dependent cell cytotoxicity (ADCC). PankoMab is, therefore, distinguished by a combination of advantages compared to other MUC1 antibodies in clinical development, including higher tumour specificity, higher affinity, a higher number of binding sites, largely reduced binding to shed MUC1 from colon and pancreatic carcinoma patients, no binding to mononucleated cells from peripheral blood (except ~7% of activated T cells), stronger ADCC activity and rapid internalisation as required for toxin-mediated cell killing. This renders it a superior antibody for in vivo diagnostics and various immunotherapeutic approaches.

What Makes MUC1 a Tumor Antigen

The epithelial mucin 1 (MUC1) is an accepted serum tumor marker and cellular tumor antigen. We discuss recent views on the difference(s) between normal and tumor MUC1, and its implication for the development of cancer vaccines and antibody therapies, with special emphasis on the role of glycosylation.

Expression of MUC1, Thomsen-Friedenreich antigen, Tn, sialosyl-Tn, and α2,6-linked sialic acid in hepatocellular carcinomas and preneoplastic hepatocellular lesions

Abstract The expression of epithelial mucins and Thomsen-Friedenreich-related antigens in preneoplastic and neoplastic hepatocellular lesions was systematically investigated using an in situ immunohistochemical staining approach. MUC1, MUC2, TF, sialosyl-TF, Tn, sialosyl-Tn, α2,3-linked sialic acid, and α2,6-linked sialic acid were examined in normal and cirrhotic human liver and in human hepatocellular carcinomas (HCCs) and cholangiocarcinomas. Normal hepatocytes and preneoplastic foci of altered hepatocytes did not express MUC1, MUC2, TF, Tn, s-Tn, or α2,6-linked sialic acid. In contrast, HCCs showed positive reactions for MUC1, TF, Tn, s-Tn, and α2,6-linked sialic acid. MUC2 was absent in normal biliary epithelial cells, but present in cholangiocarcinomas. The staining of MUC1, or s-Tn and α2,6-linked sialic acid in human normal liver tissues and various liver diseases did not change after specific treatments such as periodate oxidation or saponification, indicating that their expression in HCC does not result from incomplete glycosylation or low O-acetylation, respectively. MUC1, TF, Tn, s-Tn, and α2,6-linked sialic acid may be useful as indicators of progression of HCC in tissue sections, and perhaps also as targets for diagnostic and therapeutic approaches in vivo.