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BackgroundThe forkhead box transcription factor FOXQ1 has been shown to be upregulated in colorectal cancer (CRC) and metastatic breast cancer and involved in tumor development, epithelial-mesenchymal transition and chemoresistance. Yet, its transcriptional regulation is still unknown.MethodsFOXQ1 mRNA and protein expression were analysed in a panel of CRC cell lines, and laser micro-dissected human biopsy samples by qRT-PCR, microarray GeneChip® U133 Plus 2.0 and western blots. FOXQ1 regulation was assayed by chromatin immunoprecipitation and luciferase reporter assays.ResultsFOXQ1 was robustly induced in CRC compared to other tumors, but had no predictive value with regards to grade, metastasis and survival in CRC. Prototype-based gene coexpression and gene set enrichment analysis showed a significant association between FOXQ1 and the Wnt pathway in tumors and cancer cell lines from different tissues. In vitro experiments confirmed, on a molecular level, FOXQ1 as a direct Wnt target. Analysis of known Wnt targets identified FOXQ1 as the most suitable marker for canonical Wnt activation across a wide panel of cell lines derived from different tissues.ConclusionsOur data show that FOXQ1 is one of the most over-expressed genes in CRC and a direct target of the canonical Wnt pathway. It is a potential new marker for detection of early CRC and Wnt activation in tumors of different origins.

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Background: Environmental factors are thought to play an important role in the development of Crohn's disease (CD). Immune responses against auto-antigens or food antigens may be a reason for the perpetuation of inflammation. Methods: In a pilot study, 79 CD patients and 20 healthy controls were examined for food immunoglobulin G (IgG). Thereafter, the clinical relevance of these food IgG antibodies was assessed in a double-blind cross-over study with 40 patients. Based on the IgG antibodies, a nutritional intervention was planned. The interferon (IFN) secretion of T cells was measured. Eosinophilderived neurotoxin was quantified in stool. Results: The pilot study resulted in a significant difference of IgG antibodies in serum between CD patients and healthy controls. In 84 and 83% of the patients, respectively, IgG antibodies against processed cheese and yeast were detected. The daily stool frequency significantly decreased by 11% during a specific diet compared with a sham diet. Abdominal pain reduced and general well-being improved. IFN secretion of T cells increased. No difference for eosinophilderived neurotoxin in stool was detected. Conclusion: A nutritional intervention based on circulating IgG antibodies against food antigens showed effects with respect to stool frequency. The mechanisms by which IgG antibodies might contribute to disease activity remain to be elucidated. clusion: A nutritional intervention based on circulating IgG antibodies against food antigens showed effects with respect to stool frequency. The mechanisms by which IgG antibodies might contribute to disease activity remain to be elucidated.

BackgroundThe criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies.ResultsWe made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics.ConclusionsThis study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

Background and Aims: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. Methods: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. Results: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1 protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. Conclusions: TKTL1 expression correlated with HIF-1 protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances. © 2013 S. Karger AG, Basel.

Background: To meet the urgent need to predict individual drug responses of patients and thus support drug development, better preclinical models of solid tumors are inevitable. Here, a newly developed precision cut cancer tissue slice culture is presented and its use in drug testing was evaluated. Methods: Efficacy of therapeutic compounds from different classes, i.e. Staurosporine, IRESSATM, and Herceptin® was tested within the drug testing platform using fresh precision cut cancer slices from human colon, lung, or breast tumor tissues. The stability and significance of the model were evaluated on the level of gene expression, by antibody diffusion assays, and drug responses were detected by immunohistochemical staining, Meso Scale Discovery (Akt, pAkt) analysis, Western blotting (pAkt, pMAPK), viability (ATP) and apoptosis assays (Caspase-3/7). Results: We obtained sufficient numbers of tissue slices from cancer specimens to be able to perform a wide range of experiments for each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly due to therapeutic treatment in a dose-dependent manner. Gene expression varied remarkably in primary cultivated cells and HT-29 cells in comparison to cultivated tumor slices, which closely represent freshly isolated tumor tissue. An Alexa Fluor 488-labelled antibody showed diffusion in deeper cell layers and the ability of the system to evaluate effects of antibody therapy. Sustained viability of the precision cut cancer tissue slices over 72h enabled to test different drugs. Staurosporine, Iressa TM , and Herceptin® showed a dose-dependent reduction of viability and downstream signaling pathways like Akt and MAPK kinase phosphorylation in EGFR-or Her-2-positive Caco-2 or BT-474 cells or tissue slices, respectively. No effect was seen in EGFR-or Her-2-negative cells and tissues. Conclusions: We showed that this preclinical model is applicable to examine the effects of various anti-cancer compounds like cytotoxic chemotherapeutic drugs as well as targeted therapeutics. It may therefore have significant impact on drug development and patient selection for initial clinical trials.

Transketolase-like (TKTL) 1 indirectly replenishes NADPH preventing damage induced by reactive oxygen species (ROS) formed upon intestinal inflammation. We investigated the function of TKTL1 during murine colitis and ROS detoxification for prevention of tissue damage. Mucosal damage in TKTL1(-/-) and wild-type (WT) mice was assessed by miniendoscopy and histology during dextran sodium sulfate (DSS) colitis. mRNA levels of interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF), transketolase (TKT), and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress nitrosylation, carbonylation and antioxidative enzymes catalase (Cat), superoxide dismutase 1 and 2, as well as glutathione (GSH) were determined. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. TKTL1 knockout or DSS treatment did not influence TKT and TKTL2 mRNA or protein expression. Mucosal damage was significantly increased in TKTL1(-/-) mice indicated by miniendoscopy as well as a significantly shorter colon and more severe histological scores compared with WT mice during DSS colitis. This was associated with higher mRNA levels of IFN-γ, iNOS, IL-6, and TNF. In addition, iNOS protein expression was significantly enhanced in TKTL1(-/-) mice as well as MPO activity. Protein modification by nitric oxide (nitrotyrosine) was induced in TKTL1(-/-) mice. However, introduction of carbonyl groups by ROS was not induced in these mice. The expression of SOD1, SOD2, Cat, as well as GSH content was not significantly changed in TKTL1(-/-) mice. We conclude that induced colitis in TKTL1(-/-) mice was more severe compared with WT. This indicates a role of TKTL1 during mucosal repair and restoration.