Felix H. Brembeck

A human protein-protein interaction network : A resource for annotating the proteome

Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

A Colorectal Cancer Expression Profile That Includes Transforming Growth Factor β Inhibitor BAMBI Predicts Metastatic Potential

BACKGROUND & AIMS Much is known about the genes and mutations that cause colorectal cancer (CRC), yet only a few have been associated with CRC metastasis. We performed expression-profiling experiments to identify genetic markers of risk and to elucidate the molecular mechanisms of CRC metastasis. METHODS We compared gene expression patterns between metastatic and nonmetastatic stage-matched human colorectal carcinomas by microarray analysis. Correlations between BAMBI and metastasis-free survival were examined by quantitative real-time polymerase chain reaction (PCR) using an independent set of human colon carcinomas. Human colon cancer cell lines were analyzed for BAMBI regulation, cell motility, and experimental metastasis. RESULTS We established a signature of 115 genes that differentiated metastatic from nonmetastatic primary tumors. Among these, the transforming growth factor (TGF) beta inhibitor BAMBI was highly expressed in approximately half of metastatic primary tumors and metastases but not in nonmetastatic tumors. BAMBI is a target of canonical Wnt signaling that involves the beta-catenin coactivator BCL9-2. We observed an inverse correlation between level of BAMBI expression and metastasis-free survival time of patients. BAMBI inhibits TGF-beta signaling and increases migration in colon cancer cells. In mice, overexpression of BAMBI caused colon cancer cells to form tumors that metastasized more frequently to liver and lymph nodes than control cancer cells. CONCLUSIONS BAMBI regulates CRC metastasis by connecting the Wnt/beta-catenin and TGF-beta-signaling pathways. The metastatic expression signature we describe, along with BAMBI levels, can be used in prognosis. Developmental signaling pathways appear to act in hierarchies and cooperate in tumor cell migration, invasion, and metastasis.

Retinoids: Effects on growth, differentiation, and nuclear receptor expression in human pancreatic carcinoma cell lines

BACKGROUND & AIMS Advanced pancreatic carcinoma has a dismal prognosis despite extensive chemotherapeutic trials. The aim of this study was to evaluate the role of retinoids as an experimental therapeutic approach for pancreatic cancer. METHODS Four ductal and one acinar pancreatic tumor cell lines were investigated. Growth was determined by cell number and a human tumor clonogenic assay. In vivo growth was assessed by xenografts transplanted into nude mice. Differentiation was characterized by immunofluorescence microscopy and carbonic anhydrase II gene expression. Retinoid receptors were characterized by Northern blotting and reverse-transcriptase polymerase chain reaction. RESULTS Retinoid treatment results in a time- and dose-dependent growth inhibition in vitro and in vivo of ductal but not acinar pancreatic tumor cells. Retinoid treatment induces a more differentiated phenotype in ductal tumor cells as shown by morphological criteria and increased expression of carbonic anhydrase II. All pancreatic tumor cell lines expressed a broad panel of cellular retinoid binding proteins and nuclear retinoid receptors. Retinoic acid receptor gamma and cellular retinoic acid binding protein II were found in all retinoid-sensitive ductal tumor cell lines but not in the retinoid-resistant acinar cell lines. CONCLUSIONS Detailed knowledge of nuclear retinoid receptor expression may provide rational strategies for retinoid treatment of pancreatic cancer.

Dual function of the epithelial specific ets transcription factor, ELF3, in modulating differentiation.

The ets family of transcription factors comprises many members which contribute to diverse cellular functions that vary depending upon the cell- and tissue-type context. Recently, different groups have identified a novel member of the ets family that is epithelial-specific. Variably called ESE-1, ERT, jen, ESX, this gene is designated currently as ELF3. In order to understand transcriptional regulatory mechanisms mediated by ELF3, we investigated its effect on the human keratin 4 gene promoter based upon the role of keratin 4 in early differentiation of the esophageal squamous epithelium. Interestingly, ELF3 suppressed basal keratin 4 promoter activity in both esophageal and cervical epithelial cancer cell lines, a novel result, while simultaneously activating the late-differentiation linked SPRR2A promoter. Furthermore, serial deletion constructs of the keratin 4 promoter continued to be suppressed by ELF3, a phenomenon that was only partially rescued by ELF3 ets domain mutants, but completely abrogated by deletion of the ELF3 pointed domain. These results suggest that ELF3 may have dual functions in the transcriptional regulation of genes involved in squamous epithelial differentiation. One of these functions may not be exclusively mediated through DNA binding in the context of transcriptional suppression of the keratin 4 promoter.

BCL9-2 Promotes Early Stages of Intestinal Tumor Progression

BACKGROUND & AIMS The roles of the 2 BCL9 and 2 Pygopus genes in Wnt to β-catenin signaling are not clear in vertebrates. We examined their expression and function in normal and tumor intestinal epithelia in mice and humans. METHODS Specific antibodies were generated to characterize the BCL9 and Pygopus proteins in normal intestine and in colon tumors. Targets of BCL9 and Pygopus in colon cancer cells were analyzed using small interfering RNA analysis. Transgenic mice were created that overexpressed BCL9-2 in intestine; these were crossed with APCMin/+ mice to create BCL9-2;APCMin/+ mice. RESULTS BCL9 and Pygopus2 were expressed in all normal intestinal and colon cancer cells. BCL9-2 was detectable only in the villi, not in the crypts of normal intestine. BCL9-2 was up-regulated in adenomas and in almost all colon tumors, with a concomitant increase of Pygopus2, whereas levels of BCL9 were similar between normal and cancer cells. Transgenic overexpression of BCL9-2 in the intestine of BCL9-2; APCMin/+ mice increased formation of adenomas that progressed to invasive tumors, resulting in reduced survival time. Using small interfering RNA analysis, we found that BCL9s and Pygopus are not targets of Wnt in colon cancer cells, but Wnt signaling correlated with levels of BCL9-2. BCL9-2 regulated expression of β-catenin-dependent and -independent target genes that have been associated with early stages of intestinal tumorigenesis. CONCLUSIONS BCL9-2 promotes early phases of intestinal tumor progression in humans and in transgenic mice. BCL9-2 increases the expression of a subset of canonical Wnt target genes but also regulates genes that are required for early stages of tumor progression.

Tumor Stroma–Derived Wnt5a Induces Differentiation of Basal Cell Carcinoma of Ptch-Mutant Mice via CaMKII

Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptch(flox/flox)ERT2(+/-) knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca(2+) signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling.

The role of Pygo2 for Wnt/ß-catenin signaling activity during intestinal tumor initiation and progression

Pygo2 acts as a co-activator of Wnt signaling in a nuclear complex with ß-catenin/BCL9/BCL9-2 to increase target gene transcription. Previous studies showed that Pygo2 is upregulated in murine intestinal tumors and human colon cancer, but is apparently dispensable for normal intestinal homeostasis. Here, we have evaluated the in vivo role of Pygo2 during intestinal tumorigenesis using Pygo2 deficient mice. We analyzed chemically induced colon tumor development and conditional intestine specific mouse models harboring either Apc loss-of-function (LOF) or Ctnnb1 gain-of-function (ß-catenin GOF). Remarkably, the number and size of chemically induced tumors was significantly reduced in Pygo2 deficient mice, suggesting that Pygo2 has a tumor promoting function. Furthermore, loss of Pygo2 rescued early tumorigenesis of Ctnnb1 GOF mutants. In contrast, Pygo2 ablation was not sufficient to prevent tumor development of Apc LOF mice. The effect on tumor formation by Pygo2 knockout was linked to the repression of specific deregulated Wnt target genes, in particular of c-Myc. Moreover, the role of Pygo2 appears to be associated with the signaling output of deregulated Wnt signaling in the different tumor models. Thus, targeting Pygo2 might provide a novel strategy to suppress tumor formation in a context dependent manner.