Genome-wide analysis of barrett's adenocarcinoma. a first step towards identifying patients at risk and developing therapeutic paths

BACKGROUND: Barrett's esophagus metaplasia is the key precursor lesion of esophageal adenocarcinoma. The aim of this study was to find a subset of markers that may allow the identification of patients at risk for esophageal adenocarcinoma, and to determine genes differentially expressed in esophageal squamous cell carcinoma. METHODS:Laser capture microdissection technique was applied to procure cells from defined regions. Genome-wide RNA profiling was performed on esophageal adenocarcinoma (n = 21), Barrett's esophagus (n = 20), esophageal squamous carcinoma (n = 9) and healthy esophageal biopsies (n = 18) using the Affymetrix Human Genome U133plus 2.0 array. Microarray results were validated by quantitative real-time polymerase chain reaction in a second and independent cohort and by immunohistochemistry of two putative markers in a third independent cohort. RESULTS:Through unsupervised hierarchical clustering and principal component analysis, samples were separated into four distinct groups that match perfectly with histology. Many genes down-regulated in esophageal cancers belong to the epidermal differentiation complex or the related GO-group "cornified envelope" of terminally differentiated keratinocytes. Similarly, retinol metabolism was strongly down-regulated. Genes showing strong overexpression in esophageal carcinomas belong to the GO groups extracellular region /matrix such as MMP1, CTHRC1, and INHBA. According to an analysis of genes strongly up-regulated in both esophageal adenocarcinoma and Barrett's esophagus, REG4 might be of particular interest as an early marker for esophageal adenocarcinoma. CONCLUSIONS:Our study provides high quality data, which could serve for identification of potential biomarkers of Barrett's esophagus at risk of esophageal adenocarcinoma progression

Alectinib treatment improves photodynamic therapy in cancer cell lines of different origin

BACKGROUND: Photodynamic therapy with a photosensitizer such as protoporphyrin-IX, a light sensitive metabolite of heme synthesis, is a highly selective treatment for various carcinomas. In previous studies, we found a significant down regulation of the relevant enzyme ferrochelatase in gastrointestinal carcinomas leading to an accumulation of protoporphyrin-IX within the tumor cells. Recent studies showed that a novel anti-cancer drug, Alectinib, an orally available, highly selective, potent second-generation inhibitor of anaplastic lymphoma tyrosinkinase binds to ferrochelatase. Therefore, we were interested to see whether Alectinib treatment might lead to an accumulation of protoporphyrin IX. METHODS: Tumor cells of different origin were cultured, treated with LED-light and Alectinib. Results were gained by flow cytometry, immunohistochemistry and western blotting. Apoptosis was determined by flow cytometric analysis of Annexin V-FITC stained cells. In addition, cells were counterstained with propidium iodide to distinguish early apoptotic cells and late apoptotic/necrotic cells. RESULTS: Here, we report that photodynamic treatment of tumor cell lines of different origin in combination with Alectinib increased protoporphyrin-IX specific fluorescence and concomitantly cell death. CONCLUSIONS: The usage of Alectinib could be another step for enhancing the effectiveness of photodynamic therapy. Further experiments will show whether photodynamic therapy in combination with Alectinib could be a new strategy for the treatment of e.g. peritoneal disseminated carcinomas

C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

Cancer of the stomach is among the leading causes of death from cancer worldwide. The transcription factor C/EBPβ is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBP{beta} knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPβ. Cross-species comparison of gene expression profiles between C/EBPβ-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPβ signature. Within this signature, the RUNX1t1 tumor suppressor transcript was down-regulated in 38% of gastric tumor samples. The RUNX1t1 promoter was frequently hypermethylated and ectopic expression of RUNX1t1 in gastric cancer cells inhibited proliferation and enhanced TFF1 expression. These data suggest that the tumor suppressor activity of both RUNX1t1 and TFF1 are mechanistically connected to C/EBPβ and that cross-regulation between C/EBPβ-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis

Combination of Wnt/β-catenin targets S100A4 and DKK1 improves prognosis of human colorectal cancer

Metastasis is directly linked to colorectal cancer (CRC) patient survival. Wnt signaling through β-catenin plays a key role. Metastasis-inducing S100A4 is a Wnt/β-catenin target gene and a prognostic biomarker for CRC and other cancer types. We aimed to identify S100A4-dependent expression alterations to better understand CRC progression and metastasis for improved patient survival. S100A4-induced transcriptome arrays, confirmatory studies in isogenic CRC cell lines with defined β-catenin genotypes, and functional metastasis studies were performed. S100A4-regulated transcriptome examination revealed the transcriptional cross-regulation of metastasis-inducing S100A4 with Wnt pathway antagonist Dickkopf-1 (DKK1). S100A4 overexpression down-regulated DKK1, S100A4 knock-down increased DKK1. Recombinant DKK1 reduced S100A4 expression and S100A4-mediated cell migration. In xenografted mice, systemic S100A4-shRNA application increased intratumoral DKK1. The inverse correlation of S100A4 and DKK1 was confirmed in five independent publicly available CRC expression datasets. Combinatorial analysis of S100A4 and DKK1 in two additional independent CRC patient cohorts improved prognosis of overall and metastasis-free survival. The newly discovered transcriptional cross-regulation of Wnt target S100A4 and Wnt antagonist DKK1 is predominated by an S100A4-induced Wnt signaling feedback loop, increasing cell motility and metastasis risk. S100A4 and DKK1 combination improves the identification of CRC patients at high risk

Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression

Loss of the coxsackie and adenovirus receptor (CAR) has previously been observed in gastric cancer. The role of CAR in gastric cancer pathobiology, however, is unclear. We therefore analysed CAR in 196 R0-resected gastric adenocarcinomas and non-cancerous gastric mucosa samples using immunohistochemistry and immunofluorescence. Coxsackie and adenovirus receptor was found at the surface and foveolar epithelium of all non-neoplastic gastric mucosa samples (n=175), whereas only 56% of gastric cancer specimens showed CAR positivity (P<0.0001). Loss of CAR correlated significantly with decreased differentiation, increased infiltrative depths, presence of distant metastases, and was also associated with reduced carcinoma-specific survival. To clarify whether CAR impacts the tumorbiologic properties of gastric cancer, we subsequently determined the role of CAR in proliferation, migration, and invasion of gastric cancer cell lines by application of specific CAR siRNA or ectopic expression of a human full-length CAR cDNA. These experiments showed that RNAi-mediated CAR knock down resulted in increased proliferation, migration, and invasion of gastric cancer cell lines, whereas enforced ectopic CAR expression led to opposite effects. We conclude that the association of reduced presence of CAR in more severe disease states, together with our findings in gastric cancer cell lines, suggests that CAR functionally contributes to gastric cancer pathogenesis, showing features of a tumour suppressor

FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas

Colorectal carcinoma cells have recently been shown to express Fas ligand (FasL). This ligand could allow the tumour cells to evade activated tumour-infiltrating lymphocytes (TILs) by inducing their apoptosis and would thus promote tumour survival and possibly metastasis formation. To test this hypothesis in vivo we analysed the expression of FasL mRNA and protein in paired tissue samples of normal colonic mucosa (N), primary colorectal carcinomas (T) and their metastases (M) from a total of 21 patients by four different methods. Additionally, the presence and activation status of infiltrating lymphocytes, which might contribute to the total amount of FasL in the tissue, was determined by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) in the same samples. The frequency of FasL detection was 30–40% in T and was 60–100% in M, depending on the sensitivity of the method. Simultaneously, the amount of CD25 mRNA, used as a measure of the number of activated TILs, was in 90% of patients lower in M than in T. The increased frequency of FasL detection in liver metastases was therefore not due to the presence of activated TILs. We conclude that metastasizing subpopulations of colorectal tumour cells express FasL more frequently than the primary carcinomas and may be able to eliminate activated TILs in vivo via Fas/FasL-induced apoptosis or other hitherto unknown mechanisms. © 1999 Cancer Research Campaig

HIF-1α determines the metastatic potential of gastric cancer cells

Gastric adenocarcinoma is characterised by rapid emergence of systemic metastases, resulting in poor prognosis due to vanished curative treatment options. Better understanding of the molecular basis of gastric cancer spread is needed to design innovative treatments. The transcription factor HIF-1α (hypoxia-inducible factor 1α) is frequently overexpressed in human gastric cancer, and inhibition of HIF-1α has proven antitumour efficacy in rodent models, whereas the relevance of HIF-1α for the metastatic phenotype of gastric adenocarcinoma remains elusive. Therefore, we have conducted a comprehensive analysis of the role of HIF-1α for pivotal metastasis-associated processes of human gastric cancer. Immunhistochemistry for HIF-1α showed specific staining at the invading tumour edge in 90% of human gastric cancer samples, whereas normal gastric tissue was negative and only a minority of early gastric cancers (T1 tumours) showed specific staining. Hypoxia-inducible factor 1α-deficient cells showed a significant reduction of migratory, invasive and adhesive properties in vitro. Furthermore, the HIF-1α-inhibitor 2-methoxy-estradiol significantly reduced metastatic properties of gastric cancer cells. The accentuated expression at the invading edge together with the in vitro requirement of HIF-1α for migration, invasion and adherence argues for a pivotal role of HIF-1α in local invasion and, ultimately, systemic tumour spread. These results warrant the exploration of HIF-1α-inhibiting substances in clinical treatment studies of advanced gastric cancer

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

Rationale and study design of the prospective, longitudinal, observational cohort study “rISk strAtification in end-stage renal disease” (ISAR) study

Background: The ISAR study is a prospective, longitudinal, observational cohort study to improve the cardiovascular risk stratification in endstage renal disease (ESRD). The major goal is to characterize the cardiovascular phenotype of the study subjects, namely alterations in micro-and macrocirculation and to determine autonomic function. Methods/design: We intend to recruit 500 prevalent dialysis patients in 17 centers in Munich and the surrounding area. Baseline examinations include: (1) biochemistry, (2) 24-h Holter Electrocardiography (ECG) recordings, (3) 24-h ambulatory blood pressure measurement (ABPM), (4) 24 h pulse wave analysis (PWA) and pulse wave velocity (PWV), (5) retinal vessel analysis (RVA) and (6) neurocognitive testing. After 24 months biochemistry and determination of single PWA, single PWV and neurocognitive testing are repeated. Patients will be followed up to 6 years for (1) hospitalizations, (2) cardiovascular and (3) non-cardiovascular events and (4) cardiovascular and (5) all-cause mortality. Discussion/conclusion: We aim to create a complex dataset to answer questions about the insufficiently understood pathophysiology leading to excessively high cardiovascular and non-cardiovascular mortality in dialysis patients. Finally we hope to improve cardiovascular risk stratification in comparison to the use of classical and non-classical (dialysis-associated) risk factors and other models of risk stratification in ESRD patients by building a multivariable Cox-Regression model using a combination of the parameters measured in the study

Concerted down-regulation of immune-system related genes predicts metastasis in colorectal carcinoma

BACKGROUND: This study aimed at the identification of prognostic gene expression markers in early primary colorectal carcinomas without metastasis at the time point of surgery by analyzing genome-wide gene expression profiles using oligonucleotide microarrays. METHODS: Cryo-conserved tumor specimens from 45 patients with early colorectal cancers were examined, with the majority of them being UICC stage II or earlier and with a follow-up time of 41-115 months. Gene expression profiling was performed using Whole Human Genome 4x44K Oligonucleotide Microarrays. Validation of microarray data was performed on five of the genes in a smaller cohort. RESULTS: Using a novel algorithm based on the recursive application of support vector machines (SVMs), we selected a signature of 44 probes that discriminated between patients developing later metastasis and patients with a good prognosis. Interestingly, almost half of the genes was related to the patients' immune response and showed reduced expression in the metastatic cases. CONCLUSIONS: Whereas up to now gene signatures containing genes with various biological functions have been described for prediction of metastasis in CRC, in this study metastasis could be well predicted by a set of gene expression markers consisting exclusively of genes related to the MHC class II complex involved in immune response. Thus, our data emphasize that the proper function of a comprehensive network of immune response genes is of vital importance for the survival of colorectal cancer patients