Identification of Novel Cellular Targets in Biliary Tract Cancers Using Global Gene Expression Technology

Abstract

Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage diagnosis, lack of serum markers, and limited information regarding biliary tumor pathogenesis. RNAbased global analyses of gene expression have led to the identification of a large number of up-regulated genes in several cancer types. We have used the recently developed Affymetrix U133A gene expression microarrays containing nearly 22,000 unique transcripts to obtain global gene expression profiles from normal biliary epithelial scrapings (n ‫؍‬ 5), surgically resected biliary carcinomas (n ‫؍‬ 11), and biliary cancer cell lines (n ‫؍‬ 9). Microarray hybridization data were normalized using dCHIP (http://www.dCHIP.org) to identify differentially up-regulated genes in primary biliary cancers and biliary cancer cell lines and their expression profiles was compared to that of normal epithelial scrapings using the dCHIP software as well as Significance Analysis of Microarrays or SAM (http:// www-stat.stanford.edu/ϳtibs/SAM/). Comparison of the dCHIP and SAM datasets revealed an overlapping list of 282 genes expressed at greater than threefold levels in the cancers compared to normal epithelium (t-test P <0.1 in dCHIP, and median false discovery rate <10 in SAM). Several pathways integral to tumorigenesis were up-regulated in the biliary cancers, including proliferation and cell cycle antigens (eg, cyclins D2 and E2, cdc2/p34, and geminin), transcription factors (eg, homeobox B7 and islet-1), growth factors and growth factor receptors (eg, hepatocyte growth factor, amphiregulin, and insulin-like growth factor 1 receptor), and enzymes modulating sensitivity to chemotherapeutic agents (eg, cystathionine ␤ synthase, dCMP deaminase, and CTP synthase). In addition, we identified several "pathway" genes that are rapidly emerging as novel therapeutic targets in cancer (eg, cytosolic phospholipase A2, an upstream target of the cyclooxygenase pathway, and ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E, two important downstream mediators of the mitogenic Akt/mTOR signaling pathway). Overexpression of selected up-regulated genes was confirmed in tissue microarrays of biliary cancers by immunohistochemical analysis (n ‫؍‬ 4) or in situ hybridization (n ‫؍‬ 1), and in biliary cancer cell lines by reverse transcriptase PCR (n ‫؍‬ 2). The majority of genes identified in the present study has not been previously reported in biliary cancers, and represent novel potential screening and therapeutic targets of this cancer type. Biliary tract carcinomas, which include cancers of the gallbladder and intra-and extrahepatic biliary tree, affect 7500 individuals in the United States each year, and nearly 3500 patients die as a direct consequence of this lethal disease.1 Once established, biliary tract cancers are notoriously challenging to diagnose and treat. At present, only surgical excision of detectable malignancy is associated with improvement in ...

Gene Expression Profiling of Intrahepatic Cholangiocarcinoma

“…The current study of 15 Thai ICC patients (8 periductal infiltrating and 7 intraductal growing tissues) using Affymetrix GeneChip ® Human Exon 1.0ST identified 2,821 up-regulated and 1,361 down-regulated genes at the same fold change cut off (≥2). Using unsupervised hierarchical clustering analysis, with fold change cut-off >20, 42 genes were up-regulated in tumor tissues and 204 down-regulated with only 3 up-regulated genes (BUB1B, HOXB7 and TOP2A) in agreement with the previous report (Hansel et al, 2003;Obama et al, 2005;Jinawath et al, 2006). BUB1B [budding uninhibited by benzimidazones 1 homolog beta (yeast)] encodes a kinase involved in spindle checkpoint function and plays a role in the inhibition of anaphase-promoting complex/cyclosome, delaying the onset of anaphase and ensuring proper chromosome segregation (Davenport et al, 1999), over expression of BUB1B is significantly correlation with a less advanced pathologic stage in oral squamous cell carcinoma (Rizzardi et al, 2011).…”

“…Global gene expression profiling of billiary tract cancer has been applied in 7 cases of gallbladder carcinoma, 2 ICC, 2 distal bile duct carcinoma and 9 biliary cancer cell lines, revealing 282 up-regulated and 513 downregulated genes (Hansel et al, 2003). Gene expression profile of 25 Japanese ICC patients (10 mass-forming, 2 periductal infiltrating, 11 mixed subtypes (mass-forming and periductal infiltrating) and 2 unknown subtype), using an in-house cDNA microarray demonstrated 52 up-regulated and 421 down-regulated genes (Obama et al, 2005).…”

“…Although various genetic markers in different types cancers and their sub-classification have been widely exploited using this technique (Chee et al, 1996;Golub et al, 1999;Kim et al, 2004), there are only two publications using in-house cDNA microarrays to produce a comprehensive analysis of gene expression profiles in ICC mass forming subtype (Obama et al, 2005;Jinawath ,et al, 2006) and another for gene expression profile in billiary tract cancer (gallbladder carcinoma, ICC and distal bile duct carcinoma) using Affymetrix GeneChip U133A expression array (Hansel et al, 2003).…”

The Replicative Regulator Protein Geminin on Chromatin in the HeLa Cell Cycle

“…Similarly, replacing the wild type geminin allele with a mutant allele expressing a non-degradable version of geminin causes a reduction in the tumor-promoting potential of HCT116 cancer cells, and this is explained by the fact that in this case the cell cycle is somewhat slower than normal but not altogether abolished (21). Although geminin acts to slow down cellular proliferation in vitro, it is highly expressed in numerous malignancies (22,23) in contrast to other negative cell cycle regulators like cyclin-dependent kinase inhibitors, which are clearly down-regulated in many human tumors.…”

Prediction of carcinogenic potential by a toxicogenomic approach using rat hepatoma cells

“…The long-term rodent bioassay, the standard method to predict carcinogenic hazard of chemicals to humans, is costly in terms of time and material resources, therefore interest has concentrated on shortor medium-term bioassays. (1)(2)(3)(4)(5) One approach is to study change in gene expression in biological models in response to chemical exposure. Signatures of genetic alteration cannot be defined using classical methods for investigation of individual genes but with the advent of toxicogenomics, and the use of cDNA microarrays, the potential roles of multiple genes in concert can now be readily investigated in high throughput assays that are more sensitive and predictive than, for example, cell death assays.…”