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. RNA-based 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/ approximately tibs/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 beta 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.

Figure 1.

Cluster analysis of biliary cancers. A: Cluster analysis of 9 biliary cancer cell lines (BCL), 11 primary biliary cancers (BT), and 5 normal biliary epithelial samples (NBE). The colored bars under the tumor samples indicate their location in the biliary tree: purple, gallbladder; green, extrahepatic bile duct; orange, intrahepatic bile duct. B: Representative panel demonstrating results of principal component analysis (PCA) of biliary cancers and normal biliary epithelium. Red indicated relative overexpression, green indicates relative underexpression (TREEVIEW software).

Figure 2.

Immunohistochemical validation of genes overexpressed in biliary cancers using a biliary cancer tissue microarray. Bone morphogenetic protein receptor type 1A (BMPR1A) expression in normal biliary epithelium (A) and in adenocarcinoma of the biliary tract (B). BMPR1A is absent in normal biliary epithelium, but is diffusely expressed in a cytoplasmic and membranous distribution in the invasive adenocarcinoma. Geminin expression is absent in normal biliary epithelium (C) while biliary tract adenocarcinoma is diffusely positive (D). Cdc2 (p34) expression is absent in normal biliary epithelium (E), while it is diffusely expressed in biliary tract adenocarcinoma (F). Topoisomerase II α expression is absent in normal biliary epithelium (G) but diffuse nuclear labeling is seen in adenocarcinoma of the biliary tract (H).

Figure 3.

Non-radioisotopic in situ hybridization for validation of Islet-1 expression in biliary cancer using a biliary cancer tissue microarray. Islet-1 transcripts are detected in a biliary adenocarcinoma using the ISL-1 antisense riboprobe (arrows) (A), but transcripts are not detectable in normal biliary epithelium using an antisense riboprobe (B), or in the serial section of the cancer using the ISL-1 sense (control) riboprobe (C).

Figure 4.

RT-PCR validation of overexpressed genes in biliary cancers. Two genes overexpressed in the Affymetrix analysis, homeobox B7 (HoxB7) and dickkopf-1 homolog (Dkk1), were selected for RT-PCR in five biliary cancer cell lines (GBH3, SNU1079, SNU308, SNU245, and HuCCT1), and three samples of normal biliary epithelium. HoxB7 expression is absent in the normal epithelium, but is present in 4 of 5 biliary cancer cell lines; water control is appropriately negative (top panel). Dkk-1 expression is present in normal epithelium, but its expression is up-regulated in 5 of 5 cell lines; water control is appropriately negative (middle panel). GAPDH is used as an external control (bottom panel).