Johannes Gebert

Transforming growth factor beta receptor 2 (TGFBR2) changes sialylation in the microsatellite unstable (MSI) Colorectal cancer cell line HCT116.

Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.

De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach

Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome.

Three molecular pathways model colorectal carcinogenesis in Lynch syndrome

Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes. MMR deficiency has long been regarded as a secondary event in the pathogenesis of Lynch syndrome colorectal cancers. Recently, this concept has been challenged by the discovery of MMR‐deficient crypt foci in the normal mucosa. We aimed to reconstruct colorectal carcinogenesis in Lynch syndrome by collecting molecular and histology evidence from Lynch syndrome adenomas and carcinomas. We determined the frequency of MMR deficiency in adenomas from Lynch syndrome mutation carriers by immunohistochemistry and by systematic literature analysis. To trace back the pathways of pathogenesis, histological growth patterns and mutational signatures were analyzed in Lynch syndrome colorectal cancers. Literature and immunohistochemistry analysis demonstrated MMR deficiency in 491 (76.7%) out of 640 adenomas (95% CI: 73.3% to 79.8%) from Lynch syndrome mutation carriers. Histologically normal MMR‐deficient crypts were found directly adjacent to dysplastic adenoma tissue, proving their role as tumor precursors in Lynch syndrome. Accordingly, mutation signature analysis in Lynch colorectal cancers revealed that KRAS and APC mutations commonly occur after the onset of MMR deficiency. Tumors lacking evidence of polypous growth frequently presented with CTNNB1 and TP53 mutations. Our findings demonstrate that Lynch syndrome colorectal cancers can develop through three pathways, with MMR deficiency commonly representing an early and possibly initiating event. This underlines that targeting MMR‐deficient cells by chemoprevention or vaccines against MMR deficiency‐induced frameshift peptide neoantigens holds promise for tumor prevention in Lynch syndrome.

Reconstitution of TGFBR2 in HCT116 colorectal cancer cells causes increased LFNG expression and enhanced N-acetyl-d-glucosamine incorporation into Notch1.

Transforming growth factor-β (TGF-β) signaling plays a key role in regulating normal cell growth and differentiation, and mutations affecting members of this pathway contribute to cancer development and metastasis. In DNA mismatch repair (MMR)-deficient colorectal cancers that exhibit the microsatellite instability (MSI) phenotype, biallelic frameshift mutations in the transforming growth factor β receptor type 2 (TGFBR2) gene occur at high frequency that lead to altered signal transduction and downstream target gene expression. Although recent evidence suggests that altered TGF-β signaling can modulate protein glycosylation patterns in MSI-high colorectal tumor cells, affected genes have not been identified. Here, we investigated in a more systematic approach, expression changes of TGFBR2-regulated genes, involved in glycosylation using a TGFBR2-reconstituted colorectal cancer cell line (HCT116-TGFBR2) and Glyco-Gene Chip analysis. Based on this oligonucleotide array of about 1000 human glycosylation-related genes, several candidates including HES1, PDGFB, JUNB and LFNG were found to be upregulated in a TGFBR2-dependent manner and subsequently validated by real-time RT-PCR analyses. Focusing on the glycosyltransferase LFNG and its target signaling protein Notch1, dual labeling with [3H]-N-acetyl-d-glucosamine ([3H]-GlcNAc) and [35S]-l-methionine revealed a significant increase in N-acetyl-d-glucosamine incorporation into immunoprecipitated Notch1 receptor upon TGFBR2 expression whereas the protein level remained unaffected. These data suggest that TGFBR2 signaling can affect Notch1 glycosylation via regulation of glycosyltransferase LFNG expression and provide a first mechanistic example for altered glycosylation in MSI colorectal tumor cells.

A new method for detection of tumor driver‐dependent changes of protein sialylation in a colon cancer cell line reveals nectin‐3 as TGFBR2 target

Protein‐linked glycans play key roles in cell differentiation, cell–cell interactions, cell growth, adhesion and immune response. Aberrant glycosylation is a characteristic feature of tumor cells and is involved in tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. It can serve as cancer biomarker and treatment target. To enable comprehensive screening for the impact of tumor driving mutations in colorectal cancer cells we present a method for specific analysis of tumor driver‐induced glycome changes. The strategy is based on a combination of three technologies, that is recombinase‐mediated cassette exchange (RMCE), Click‐It chemistry and mass spectrometry. The new method is exemplified by the analysis of the impact of inactivating mutations of the TGF‐ß‐receptor type II (TGFBR2) on sialic acid incorporation into protein‐linked glycans of the colon cancer cell line HCT116. Overall, 70 proteins were found to show de novo sialic acid incorporation exclusively upon TGFBR2 expression whereas 7 proteins lost sialylation upon TGFBR2 reconstitution. Validation of detected candidate glycoproteins is demonstrated with the cell surface glycoprotein nectin‐3 known to be involved in metastasis, invasion and prognosis of various cancers. Altogether, our new approach can help to systematically puzzle out the influence of tumor‐specific mutations in a major signaling pathway, as exemplified by the TGFBR2 tumor suppressor, on the tumor glycome. It facilitates the identification of glycan‐based tumor markers that could be used for diagnostic and therapeutic applications. In principle the outlined strategy can be adapted to any cancer cell line, tumor driver mutation and several glycan‐building blocks.

Dose-dependent effect of 2-deoxy-D-glucose on glycoprotein mannosylation in cancer cells.

High glucose consumption due to Warburg effect is one of the metabolic hallmarks of cancer. Consequently, glucose antimetabolites, such as 2‐deoxy‐glucose (2DG), can induce substantial growth inhibition of cancer cells. However, the inhibition of metabolic pathways is not the sole effect of 2DG on cancer cells. As mannose‐mimetic molecule, 2DG is believed to interfere with normal glycosylation of proteins in cells. Here, we address how 2DG influences protein glycosylation in cancer cells and discuss possible implications of the consequences of this influence. In detail, six colorectal cancer cell lines were examined for alterations of protein glycosylation by measuring monosaccharide incorporation into cellular glycoproteins and cell surface glycosylation by lectin FACS. A significant increase in mannose incorporation was observed on treatment with 2DG (1 mM for 48 h), which was also reflected by an increased binding of the mannose‐binding lectin Concanavalin A in FACS analysis. This phenomenon, which could be reversed by external addition of mannose, was not caused by 2DG‐mediated mannosidase inhibition, as shown by pulse‐chase experiments, arguing in favor of the hypothesis that 2DG directly influenced the incorporation of mannose. Increased mannose content was generally observed in cellular glycoproteins, including glycoproteins isolated from the plasma membrane fraction. Our results indicate that 2DG at low doses, which have only a limited metabolism‐related effect on glycosylation, induces a strong increase in mannose incorporation into cellular glycoproteins. On the other hand, higher 2DG concentrations (10 and 20 mM) led to a significant decrease of absolute mannose incorporation accompanied by a dramatically reduced protein synthesis rate. 2DG‐induced alterations of glycosylation may represent a novel mechanism potentially explaining the varied effects of 2DG on cancer cells. Moreover, 2DG treatment may open a path toward novel diagnostic and cancer therapeutic approaches, which specifically target altered glycoantigen structures induced by 2DG.

Reconstitution of TGFBR2-Mediated Signaling Causes Upregulation of GDF-15 in HCT116 Colorectal Cancer Cells

Although inactivating frameshift mutations in the Transforming growth factor beta receptor type 2 (TGFBR2) gene are considered as drivers of microsatellite unstable (MSI) colorectal tumorigenesis, consequential alterations of the downstream target proteome are not resolved completely. Applying a click-it chemistry protein labeling approach combined with mass spectrometry in a MSI colorectal cancer model cell line, we identified 21 de novo synthesized proteins differentially expressed upon reconstituted TGFBR2 expression. One candidate gene, the TGF-ß family member Growth differentiation factor-15 (GDF-15), exhibited TGFBR2-dependent transcriptional upregulation causing increased intracellular and extracellular protein levels. As a new TGFBR2 target gene it may provide a link between the TGF-ß branch and the BMP/GDF branch of SMAD-mediated signaling.

Hereditäre Tumoren des Gastrointestinaltrakts (FAP/HNPCC)

The identification of the disease-causing genes has enabled molecular diagnostics for the two clinically well characterized forms of hereditary colorectal cancer, familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). FAP patients carry germ line mutations in the APC gene, predominantly leading to the synthesis of truncated APC proteins and allowing a protein-based mutation screening assay. Known correlations between APC mutation site and severity of the polyposis phenotype or extracolonic manifestations facilitate mutation analysis and probably might lead to surgical decision taking in the near future. The molecular cause of HNPCC are germ line mutations in one of five human DNA mismatch repair genes. Complete loss of this DNA repair system in HNPCC tumors results in genetic instability of highly repetitive DNA sequences (microsatellite instability, MSI). Application of clinical selection criteria (Bethesda Criteria) for identification of potential HNPCC patients together with MSI classification and immunohistochemical analysis of DNA mismatch repair protein expression should be performed before subsequent germ line mutation analysis is initiated. Thus, molecular diagnostics enables identification of mutation carriers and noncarriers within affected FAP and HNPCC families. Mutation carriers can be subjected to close clinical surveillance, whereas noncarriers may then be excluded from further clinical examinations. For APC muation carriers, prophylactic restorative proctocolectomy is the therapy of choice, whereas incomplete penetrance of disease among DNA mismatch repair gene mutation carriers currently does not justify similar procedures for HNPCC patients.

Analyzing epigenetic control of galectin expression indicates silencing of galectin‐12 by promoter methylation in colorectal cancer

Galectins, a class of lectins with specificity for ß‐galactoside containing glycoconjugates, modulate several cellular processes that are involved in the control of normal cell growth, differentiation, cell‐cell, and cell matrix interactions. Pathological alterations of the galectin expression pattern have been implicated in the development and progression of cancer. We therefore analyzed epigenetic mechanisms for control of galectin expression in 9 colorectal cancer (CRC) cell lines. Our data demonstrate that expression of galectins‐1, −2, −7, −8, and −9 can be regulated by histone acetylation in CRC cell lines. In addition, the same set of galectins was also found to be modulated by DNA methylation. Of particular note, galectin‐12 is silenced in all tested CRC cell lines but known to be re‐expressed upon butyrate‐induced differentiation and present in normal colonic mucosa. Loss of galectin‐12 expression in undifferentiated CRC cells is associated with promoter hypermethylation and for the first time we provide detailed methylation analysis of the promoter region. In CRC tumor tissue, galectin‐12 expression was downregulated in 66% of CRC tissue specimens as compared to adjacent normal tissue hinting to a possible tumor‐suppressing function in CRC.

Mutationslokalisation als Wegweiser zur operativen Taktik bei FAP

Restorative proctocolectomy and ileal pouch-anal anastomosis (IPAA) is considered the operative therapy of choice for the prophylactic treatment of FAP. Recently, Vasen and coworkers [5] after correlating the incidence of metachronous rectal cancer with the site of the causative APC mutation suggested subtotal colectomy and IRA to be the primary treatment in patients with mutations proximal to codon 1250, whereas IPAA should be performed in those with mutations beyond this codon. Mutation analysis in our patients after IRA, however, shows the majority of APC mutations to be located proximal to codon 1250 even in those patients with severe rectal polyposis and metachronous rectal cancer, thus not supporting the therapeutic recommendations of Vasen and coworkers.