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.

Combination of Wnt/ß-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer.

Metastasis is directly linked to colorectal cancer (CRC) patient survival. Wnt signaling through ß-catenin plays a key role. Metastasis-inducing S100A4 is a Wnt/ß-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 ß-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.

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.

C/EBPß 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/EBPß is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBPß knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPß. Cross-species comparison of gene expression profiles between C/EBPß-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPß 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/EBPß and that cross-regulation between C/EBPß-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis. KEY MESSAGE: C/EBPß controls proliferation and differentiation balance in the stomach. Homeostatic differentiation/proliferation balance is altered in gastric cancer. RUNX1t1 is a C/EBPß-associated tumor suppressor. RUNX1t1 negatively regulates C/EBPß pro-oncogenic functions.

High MACC1 expression in combination with mutated KRAS G13 indicates poor survival of colorectal cancer patients.

BACKGROUND: The metastasis-associated in colon cancer 1 (MACC1) gene has been identified as prognostic biomarker for colorectal cancer (CRC). Here, we aimed at the refinement of risk assessment by separate and combined survival analyses of MACC1 expression with any of the markers KRAS mutated in codon 12 (KRAS G12) or codon 13 (KRAS G13), BRAF V600 mutation and MSI status in a retrospective study of 99 CRC patients with tumors UICC staged I, II and III. FINDINGS: We showed that only high MACC1 expression (HR: 6.09, 95% CI: 2.50-14.85, P < 0.001) and KRAS G13 mutation (HR: 5.19, 95% CI: 1.06-25.45, P = 0.042) were independent prognostic markers for shorter metastasis-free survival (MFS). Accordingly, Cox regression analysis revealed that patients with high MACC1 expression and KRAS G13 mutation exhibited the worst prognosis (HR: 14.48, 95% CI: 3.37-62.18, P < 0.001). Patients were classified based on their molecular characteristics into four clusters with significant differences in MFS (P = 0.003) by using the SPSS 2-step cluster function and Kaplan-Meier survival analysis. CONCLUSION: According to our results, patients with high MACC1 expression and mutated KRAS G13 exhibited the highest risk for metachronous metastases formation. Moreover, we demonstrated that the "Traditional pathway" with an intermediate risk for metastasis formation can be further subdivided by assessing MACC1 expression into a low and high risk group with regard to MFS prognosis. This is the first report showing that identification of CRC patients at high risk for metastasis is possible by assessing MACC1 expression in combination with KRAS G13 mutation.

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.

Wdr66 is a novel marker for risk stratification and involved in epithelial-mesenchymal transition of esophageal squamous cell carcinoma.

BACKGROUND: We attempted to identify novel biomarkers and therapeutic targets for esophageal squamous cell carcinoma by gene expression profiling of frozen esophageal squamous carcinoma specimens and examined the functional relevance of a newly discovered marker gene, WDR66. METHODS: Laser capture microdissection technique was applied to collect the cells from well-defined tumor areas in collaboration with an experienced pathologist. Whole human gene expression profiling of frozen esophageal squamous carcinoma specimens (n = 10) and normal esophageal squamous tissue (n = 18) was performed using microarray technology. A gene encoding WDR66, WD repeat-containing protein 66 was significantly highly expressed in esophageal squamous carcinoma specimens. Microarray results were validated by quantitative real-time polymerase chain reaction (qRT-PCR) in a second and independent cohort (n = 71) consisting of esophageal squamous cell carcinoma (n = 25), normal esophagus (n = 11), esophageal adenocarcinoma (n = 13), gastric adenocarcinoma (n = 15) and colorectal cancers (n = 7). In order to understand WDR66's functional relevance siRNA-mediated knockdown was performed in a human esophageal squamous cell carcinoma cell line, KYSE520 and the effects of this treatment were then checked by another microarray analysis. RESULTS: High WDR66 expression was significantly associated with poor overall survival (P = 0.031) of patients suffering from esophageal squamous carcinomas. Multivariate Cox regression analysis revealed that WDR66 expression remained an independent prognostic factor (P = 0.042). WDR66 knockdown by RNA interference resulted particularly in changes of the expression of membrane components. Expression of vimentin was down regulated in WDR66 knockdown cells while that of the tight junction protein occludin was markedly up regulated. Furthermore, siRNA-mediated knockdown of WDR66 resulted in suppression of cell growth and reduced cell motility. CONCLUSIONS: WDR66 might be a useful biomarker for risk stratification of esophageal squamous carcinomas. WDR66 expression is likely to play an important role in esophageal squamous cell carcinoma growth and invasion as a positive modulator of epithelial-mesenchymal transition. Furthermore, due to its high expression and possible functional relevance, WDR66 might be a novel drug target for the treatment of squamous carcinoma.

Microarray meta-analysis defines global angiogenesis-related gene expression signatures in human carcinomas.

Angiogenesis is a prerequisite for progression of cancers. The number of genes linked to angiogenesis suggests the existence of complex gene-networks, which remain to be elucidated. To identify angiogenesis genes deregulated in carcinomas, we performed a meta-profiling analysis of published gene expression microarray studies. Own microarray and quantitative RT-PCR data were obtained from a colorectal carcinoma cohort. Applying highly stringent inclusion criteria, 15 cancer array studies were suitable for our analysis. These studies provided 789 tumor specimens and 190 samples of healthy tissues yielding a total of approx. 1,000,000 gene expression measurements. Meta-analysis on the expression of 480 angiogenesis-related genes in 10 cancer types identified a characteristic, entity-independent "global" cancer expression signature of 25 angiogenesis-related genes showing high frequency down-regulation when compared to corresponding healthy tissues. Furthermore, we characterized 25 genes displaying frequent up-regulation, yet less often than the 25 down-regulated genes. Comparative inter-study cross-validation revealed that both signatures discriminate cancers from healthy tissues with high accuracy in independent test sets. Moreover, own microarray data of colorectal carcinomas confirmed the specific and sensitive discriminating potential of both signatures. These results were validated by quantitative RT-PCR for eight genes displaying the highest differences in the microarray analysis. Our study for the first time defines global gene expression signatures linked to angiogenesis in carcinomas. Our findings suggest that gene down-regulation may represent a central aspect of tumor angiogenesis.

Cardiovascular medications in angiogenesis--how to avoid the sting in the tail.

Cancer and cardiovascular disease are the leading causes of death worldwide. Cardiovascular medications have recently been found to have favorable effects also for the treatment of noncardiovascular diseases, including cancer. In this review, we use a reverse bedside-to-bench approach to investigate the effects of common cardiovascular medications on tumor angiogenesis and vascular angiogenesis. Aspirin seems to reduce the risk of developing cancer, particularly colon cancer. However, whether the protective influence of aspirin is due to antiangiogenesis effect is still unclear. ß-Blockers, which are normally used to reduce heart rate and prolong diastole, trigger an increase in stretch-associated release of proangiogenic growth factors thereby inducing angiogenesis. However, according to other studies ß-blockers are able to inhibit angiogenesis via multiplicate mechanisms. Similarly, angiotensin converting enzyme inhibitor and angiotensin II type 1 receptor blocker have controversial effects for the regulation of cell proliferation and angiogenesis. Statins can augment collateral vascular growth in ischemic tissues and restrict the development of cancer. So this topical anti-inflammatory drug seems to be of high value for further therapy. Finally, suggestions on how this pilot experience may guide the conduct of future preclinical investigations, and clinical trials are discussed.

In vivo tumor imaging using a novel RNAi-based detection mechanism.

A new concept of tumor imaging is introduced using a siRNA-based probe that is capable of amplifying a specific endogenous fluorescence emission in cancerous tissue. In previous studies, we demonstrated a significant downregulation of Ferrochelatase (FECH) mRNA-expression in colorectal carcinomas leading to the accumulation of protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis. In this article, we report on first in vivo experiments in xenografted nude mice using folate-coupled liposomes or dendritic polyglycerolamine nanoparticles carrying ferrochelatase-siRNA to enhance PpIX-derived fluorescence in the tumor tissue. Tiny tumor foci could be monitored by the emission of PpIX fluorescence in vivo. Due to the omnipresence of the heme synthesis pathway, targeted application of ferrochelatase-siRNA may provide a general means for molecular imaging. FROM THE CLINICAL EDITOR: A new concept of tumor imaging is presented in this paper using a siRNA-based probe detecting protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis previously demonstrated to accumulate in cancer tissue.

Loss of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) induces apoptotic processes in pancreatic carcinoma cells.

Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) was silenced, or p16 was overexpressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4-ATF3-CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up-regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE-dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.

THBS4, a novel stromal molecule of diffuse-type gastric adenocarcinomas, identified by transcriptome-wide expression profiling.

Gastric adenocarcinomas can be divided into two major histological types, the diffuse and intestinal type (Laurén classification). Since they diverge in many clinical and molecular characteristics, it is widely accepted that they represent distinct disease entities that may benefit from different therapeutic approaches. Gene expression profiling studies have identified numerous genes that are differentially expressed between them. However, none of these studies covered the whole transcriptome and the published gene lists reveal little overlap, raising the need for further, more comprehensive analyses. Here, we present the first transcriptome-wide expression profiling study comparing the two types (diffuse n=19, intestinal n=24), which identified >1000 genes that are differentially expressed. Among them, thrombospondin 4 (THBS4) showed the strongest correlation to histological type, with vast overexpression in the diffuse type. Quantitative real-time PCR validated this strong overexpression and revealed that intestinal tumors generally lack THBS4 expression. Immunohistochemistry demonstrated THBS4 overexpression on the protein level (n=10) and localized THBS4 to the stromal aspect. Its expression was primarily observed within the extracellular matrix surrounding the tumor cells, with the highest intensities found in regions of high tumor cell density and invasion. Intestinal tumors and matched non-neoplastic gastric epithelium and stroma did not feature any relevant THBS4 expression in a preliminary selection of analyzed cases (n=5). Immunohistochemical colocalization and in vitro studies revealed that THBS4 is expressed and secreted by cancer-associated fibroblasts. Furthermore, we show that THBS4 transcription in fibroblasts is stimulated by tumor cells. This study is the first to identify THBS4 as a powerful marker for diffuse-type gastric adenocarcinomas and to provide an initial characterization of its expression in the course of this disease.

Latent transforming growth factor binding protein 4 (LTBP4) is downregulated in mouse and human DCIS and mammary carcinomas.

BACKGROUND: Transforming growth factor beta (TGF-ß) is able to inhibit the proliferation of epithelial cells and is involved in the carcinogenesis of mammary tumors. Three latent transforming growth factor-ß binding proteins (LTBPs) are known to modulate TGF-ß functions. METHODS: The current study analyses the expression profiles of LTBP4, its isoforms LTBP1 and LTBP3, and TGF-ß1, TGF-ß2, TGF-ß3, and SMAD2, SMAD3 and SMAD4 in human and murine (WAP-TNP8) DCIS compared to invasive mammary tumors. Additionally mammary malignant (MCF7, Hs578T, MDA-MB361) and non malignant cell lines (Hs578BsT) were analysed. Microarray, q-PCR, immunoblot, immunohistochemistry and immunofluorescence were used. RESULTS: In comparison to non-malignant tissues (n = 5), LTBP4 was downregulated in all human and mouse DCIS (n = 9) and invasive mammary adenocarcinomas (n = 5) that were investigated. We also found decreased expression of bone morphogenic protein 4 (BMP4) and increased expression of its inhibitor gremlin (GREM1). Treatment of the mammary tumor cell line (Hs578T) with recombinant TGF-ß1 rescued BMP4 and GREM1 expression. CONCLUSION: We conclude that the lack of LTBP4-mediated targeting in malignant mammary tumor tissues may lead to a possible modification of TGF-ß1 and BMP bioavailability and function.

Identification of early molecular markers for breast cancer.

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: By 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.

Integrated microfluidic platform for the electrochemical detection of breast cancer markers in patient serum samples.

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.

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.

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.

REG1A expression is a prognostic marker in colorectal cancer and associated with peritoneal carcinomatosis.

By expression profiling of early staged colon carcinomas, we found regenerating islet-derived 1 alpha (REG1A) to be upregulated in patients with an unfavorable clinical outcome. For validation, REG1A expression was quantified in another colorectal cancer (CRC) patient cohort by Taqman PCR. Aside from tumor and normal tissue from 63 nonpretreated CRC patients, 31 mucosa biopsies from healthy individuals as well as 22 adenomas were included in the investigation. REG1A was significantly upregulated in tumor specimens (p < 0.001) and adenoma (p < 0.01) as compared to normal colorectal tissue. REG1A expression in normal peritumoral tissue in turn proved to be significantly elevated compared to mucosa from healthy individuals (p < 0.01). Determination of REG1A expression might be useful for early tumor diagnosis with a sensitivity of 90.6%, and a specificity of 77.9%. REG1A expression was significantly increased in tumors with peritoneal carcinomatosis (p < 0.01). Moreover, REG1A turned out to be a significant predictor of disease-free survival (p < 0.05). In conclusion, we present evidence that REG1A is a molecular marker of prognostic value and is associated with peritoneal carcinomatosis in CRC. REG1A turned out to be already significantly raised in peritumoral normal tissue compared to mucosa from healthy individuals, suggesting a molecular field effect of secreted REG1A.

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 (PpIX) in gastrointestinal tumor tissues. To elucidate the mechanisms of PpIX 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 PpIX 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 PpIX because of FECH siRNA silencing provides a small-molecule-based approach to molecular imaging and molecular therapy.

Tumor suppressor p16INK4a--modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells.

Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta1,4-galactosyltransferases (down-regulation of beta1,4-galactosyltransferases-I/V and up-regulation of beta1,4-galactosyltransferase-IV) as well as decreased alpha2,3-sialylation of O-glycans and alpha2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.