Wolfgang Kemmner

Vascular Endothelial Growth Factor-D and Its Receptor VEGFR-3: Two Novel Independent Prognostic Markers in Gastric Adenocarcinoma

PURPOSE Vascular endothelial growth factor (VEGF)-D and its homolog VEGF-C influence lymphangiogenesis through activation of VEGF receptor 3 (VEGFR-3), and have been implicated in lymphatic tumor spread. Nodal dissemination of gastric adenocarcinomas critically determines clinical outcome and therapeutic options of affected patients. Therefore, we analyzed expression and prognostic significance of VEGF-D along with VEGF-C, and VEGFR-3 in gastric adenocarcinomas. MATERIALS AND METHODS VEGF-C, VEGF-D, and VEGFR-3 were analyzed in 91 R(0)-resected primary gastric adenocarcinomas, corresponding noncancerous gastric mucosa, and lymph node metastases employing immunohistochemistry and/or in situ hybridization. Blood and lymph vessel densities were assessed after staining with CD31 and LYVE-1-specific antibodies. RESULTS VEGF-D and VEGF-C were detected in 67.0% and 50.5% of gastric cancers, respectively. Healthy gastric mucosa was negative for VEGF-C and in 12.5% positive for VEGF-D. Presence of VEGF-D (P = .005) or VEGF-C (P = .006) was correlated with lymphatic metastases and decreased survival (VEGF-D, P < .05; VEGF-C, P < .05). VEGFR-3 was correlated with reduced carcinoma-specific survival (P < .05), and Cox multivariate regression analysis qualified VEGF-D and VEGFR-3, but not VEGF-C, as independent prognostic parameters. In lymph node-positive gastric cancers, presence of VEGF-D/VEGFR-3 was associated with poor survival, whereas absence of VEGF-D/VEGFR-3 defined a subgroup of patients with clearly favorable prognosis. CONCLUSION VEGF-D and VEGFR-3 are novel independent prognostic marker molecules aiding to identify patients with poor prognosis after curative resection of gastric adenocarcinomas. Combined analysis of the VEGF-C/VEGF-D/VEGFR-3 system can be useful to identify patients with unfavorable clinical outcome and thereby may help to refine therapeutic decisions in gastric cancer.

Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases.

BACKGROUND/AIMS Biosynthesis of carbohydrate structures is tissue specific and developmentally regulated by glycosyltransferases such as fucosyltransferases, sialyltransferases, and N-acetylgluco- saminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumour subpopulations with different adhesion properties. Therefore alterations in glycosyltransferase mRNA expression in colorectal carcinomas were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). METHODS Colorectal carcinoma specimens were classified and characterised according to the WHO/UICC system. Expression of fucosyltransferases FT-I, FT-III, FT-IV, FT-V, FT-VI, and FT-VII, sialyltransferases ST3Gal-I, ST3Gal-III, ST3Gal-IV, and ST6Gal-I, β1,4-galacto- syltransferase, and β1,6-Nacetylgluco- saminyltransferase V (GNT-V) was screened simultaneously in extracts of 22 homogenised tumour specimens by RT-PCR and compared with corresponding mucosa from each patient. Also 12 adenomas and 17 liver metastases of colorectal carcinomas were examined. RESULTS GNT-V expression was enhanced in colorectal adenomas (p = 0.039), carcinomas (p<0.001), and liver metastases of colorectal carcinomas (p<0.001). Also, expression of fucosyltransferase FT-IV was increased in colorectal adenomas (p = 0.039) and carcinomas (p<0.001). In addition, fucosyltransferase FT-I (p<0.001) and sialyltransferases ST6Gal-I (p = 0.004) and ST3Gal-III (p = 0.001) showed increased expression in carcinoma specimens. On the other hand, fucosyltransferase FT-III was less abundantly expressed in carcinomas exhibiting distant metastases (p = 0.046) and in highly invasive tumours (p = 0.041). CONCLUSIONS Glycosyltransferase mRNA expression is significantly altered in colorectal adenomas and carcinomas isolated from surgical specimens. RT-PCR determination of specific glycosyltransferases may be helpful for earlier detection of carcinomas and for tumour prognosis.

Identification of early molecular markers for breast cancer

BackgroundThe ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma. Since DCIS is a curable disease, it would be highly desirable to identify molecular markers that allow early detection. Mice transgenic for the WAP-SV40 early genome region were used as a model for DCIS development. Gene expression profiling was carried out on DCIS-bearing mice and control animals. Additionally, a set of human DCIS and invasive mammary tumors were analyzed in a similar fashion. Enhanced expression of these marker genes in human and murine samples was validated by quantitative RT-PCR. Besides, marker gene expression was also validated by immunohistochemistry of human samples. Furthermore in silico analyses using an online microarray database were performed.ResultsIn DCIS-mice seven genes were identified that were significantly up-regulated in DCIS: DEPDC1, NUSAP1, EXO1, RRM2, FOXM1, MUC1 and SPP1. A similar up-regulation of homologues of the murine genes was observed in human DCIS samples. Enhanced expression of these genes in DCIS and IDC (invasive ductal carcinoma) was validated by quantitative RT-PCR and immunohistochemistry.ConclusionsBy comparing murine markers for the ductal carcinoma in situ (DCIS) of the mammary gland with genes up-regulated in human DCIS-samples we were able to identify a set of genes which might allow early detection of DCIS and invasive carcinomas in the future. The similarities between gene expression in DCIS and invasive carcinomas in our data suggest that the early detection and treatment of DCIS is of utmost relevance for the survival of patients who are at high risk of developing breast carcinomas.

Amperometric Immunosensor for Carcinoembryonic Antigen in Colon Cancer Samples Based on Monolayers of Dendritic Bipodal Scaffolds

Detection of proteins that signal the presence or recurrence of cancer is a powerful therapeutic tool for effective early diagnosis and treatment. Carcinoembryonic antigen (CEA) has been extensively studied as a tumor marker in clinical diagnosis. We report on the development of an amperometric biosensor for the detection of CEA based on the immobilization of anti-CEA monoclonal antibody on a novel class of bipodal thiolated self-assembled monolayers containing reactive N-hydroxysuccinimide (NHS) ester end groups. The current variations showed a linear relationship with the concentration of CEA over the range of 0-200 ng/mL with a sensitivity of 3.8 nA x mL x ng(-1) and a detection limit of 0.2 ng/mL, which is well below the commonly accepted concentration threshold (5 ng/mL) used in clinical diagnosis. Real time and accelerated stability studies of the reporter antibody under various storage conditions demonstrated that the enzymatic activity and antibody affinity of the conjugate is retained for long periods of time in commercial stabilizing buffers such as StabilGuard Biomolecule Stabilizer, and a prediction of the stability trends was carried out using the kinetic and thermodynamic parameters obtained from the Arrhenius equation. The developed immunosensor as well as a commercially available enzyme-linked immunosorbent assay (ELISA) kit were successfully applied to the detection of CEA in serum samples obtained from colon cancer patients, and an excellent correlation of the levels of CEA measured was obtained. Ongoing work is looking at the incorporation of the developed biosensor into a platform for multiplexed simultaneous detection of several breast cancer related biomarkers.

Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer

Hemes and heme proteins are vital components of essentially every cell of virtually every eukaryote organism. Previously, we demonstrated accumulation of the heme precursor protoporphyrin‐IX (PplX) in gastrointestinal tumor tissues. To elucidate the mechanisms of PplX accumulation by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), we studied expression of the relevant enzymes of the heme synthetic pathway. Here, we describe a significant down‐regulation of ferrochelatase (FECH) mRNA expression in gastric, colonic, and rectal carcinomas. Accordingly, in an in vitro model of several carcinoma cell lines, ferrochelatase down‐regulation and loss of enzymatic activity corresponded with an enhanced PpIX‐dependent fluorescence. Direct detection of PpIX in minute amounts was achieved by a specifically developed pulsed solid‐state laser dual delay fluorimetry setup. Silencing of FECH using small interfering RNA (siRNA) technology led to a maximum 50‐fold increased PplX accumulation, imageable by a specifically adapted two‐photon microscopy unit. Our results show that in malignant tissue a transcriptional down‐regulation of FECH occurs, which causes endogenous PpIX accumulation. Furthermore, accumulation of intracellular PplX because of FECH siRNA silencing provides a small‐molecule‐based approach to molecular imaging and molecular therap.—Kemmner, W., Wan, K., Rüttinger, S., Ebert, B., Macdonald, R., Klamm, U., Moesta, K. T. Silencing of human ferrochelatase causes abundant protoporphyrin‐IX accumulation in colon cancer. FASEB J. 22, 500–509 (2008)

Altered mRNA expression of glycosyltransferases in human gastric carcinomas.

Biosynthesis of carbohydrate structures is tissue-specific and developmentally regulated by glycosyltransferases like fucosyl-, sialyl- and N-acetylglucosaminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumor subpopulations with different adhesion properties. The aim of this contribution was to directly compare mRNA expression of several glycosyltransferases in surgical specimens of gastric carcinomas. Carcinoma specimens were classified and characterized according to the WHO/UICC system. In each case, the expression of 12 glycosyltransferase enzymes was studied simultaneously by RT-PCR. For semi-quantitative analysis, amplification of the sample sequence was compared with that of beta-actin, co-amplified within the same tube. Expression of N-acetylglucosaminyltransferase V in gastric carcinomas was significantly enhanced compared to normal tissue. Also, expression of sialyltransferase ST3Gal-IV and fucosyltransferase FT-IV was significantly enhanced in carcinoma tissue. No significant differences in glycosyltransferase expression were found in samples positive for Helicobacter pylori or between the different gastric regions. Thus, carcinogenesis is characterized by specific alterations in mRNA expression of several glycosyltransferases. Future studies will show whether RT-PCR detection of the expression of these enzymes could be helpful for prognostic purposes.

Helicobacter pylori stimulates host vascular endothelial growth factor-A (vegf-A) gene expression via MEK/ERK-dependent activation of Sp1 and Sp3

VEGF‐A is a key regulator of inflammatory and tumor‐associated angiogenesis. H. pylori plays a critical role in the pathogenesis of benign and malignant gastric diseases. It has been suggested that H. pylori infection is associated with activation of host angiogenesis, however, underlying mechanisms as well as angiogenic growth factors activated by the bacterium have not yet been identified. Therefore, we investigated the influence of the bacterium on VEGF‐A as a candidate host target gene in vivo and in vitro. We show that H. pylori potently up‐regulates production and release of VEGF‐A protein as well as vegf‐A mRNA levels, and we provide strong evidence that enhanced recruitment of Sp1 and Sp3 transcription factors to two proximal GC‐rich vegf‐A promoter elements mediates H. pylori‐triggered vegf‐A gene expression. In addition, H. pylori infection increased the transactivating capacity of both Sp1 and Sp3, which suggests additional mechanism(s) of vegf‐A gene regulation by the bacterium. Signaling studies identified the MEK>ERK1/‐2 kinase cascade as principal host signaling pathway mediating H. pylori‐stimulated vegf‐A transcription. By identifying H. pylori as potent activator of vegf‐A gene expression and characterization of underlying molecular mechanisms, our results provide novel insights into pathways linking the bacterium to host angiogenesis and may help to develop strategies to influence vegf‐A gene expression in the setting of H. pylori infection.

Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway dependent gene signatures in colorectal cancer cells.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

Colon carcinoma glycoproteins carrying α 2,6-linked sialic acid reactive with Sambucus nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-TN antigen

In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O‐acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor‐associated sialylated antigens may be constitutively expressed in O‐acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma‐associated expression of α 2,6‐linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de‐acetylation of normal and transitional colonic mucosa, in contrast to sialyl‐Tn antigen, does not result in SNA binding. Further, the α 2,6‐linked sialic acid recognized by SNA is distinct from that of sialyl‐Tn antigen. This is confirmed by Northern blotting detecting transcripts for α 2,6 sialyltransferase of N‐glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub‐lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins.Int. J. Cancer 70:575–581.

Sialic acid dependent cell adhesion to collagen IV correlates with in vivo tumorigenicity of the human colon carcinoma sublines HCT116, HCT116a and HCT116b

Cell surface sialylation of three metastasizing sublines HCT116, HCT116a and HCT116b of a human colon carcinoma was shown to correlate with their in vivo tumorigenicity. Lectin binding studies revealed further differences in cell surface glycosylation between HCT116a and HCT116 sublines. Binding to collagen IV correlated with the in vivo aggressiveness of the cells, whereas binding to fibronectin did not. On a laminin substrate the most tumorigenic line adhered best, but binding of the other lines was similar. Sialidase treatment of the cells had no effect on cell binding to laminin and fibronectin, but resulted in a decrease of cell binding to collagen IV.