EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 2, 102-115, February 2009 Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells Hee-Jung Yoo1, Bo-Ra Yun1, tumor suppressor genes (p53 and FHIT) and the ex- Jung-Hee Kwon2, Hyuk-Soo Ahn1, pression of Fas/FasL gene, well known CC-related re- Min-A Seol1, Mi-Jin Lee,1 ceptor and its ligand, in these four CC cell lines. The de- Goung-Ran Yu1, Hee-Chul Yu3, letion mutation of p53 was found in the sarcomatoid SCK cells. These cells expressed much less Fas/FasL BeeHak Hong2, KwanYong Choi2 1,4 mRNAs than did the other ordinary CC cells. We further and Dae-Ghon Kim characterize the gene expression pattern that is in- 1 volved in the sarcomatous progression of CC, using Division of Gastroenterology and Hepatology cDNA microarrays that contained 18,688 genes. Com- Department of Internal Medicine parison of the expression patterns between the sarco- Institute for Medical Sciences matoid SCK cells and the differentiated Choi-CK cells Chonbuk National University Medical School and Hospital enabled us to identify 260 genes and 247 genes that Jeonju 561-712, Korea were significantly over-expressed and under-ex- 2Department of Life Sciences pressed, respectively. Northern blotting of the 14 ran- Pohang University of Science and Technology domly selected genes verified the microarray data, in- Pohang 790-784, Korea 3 cluding the differential expressions of the LGALS1, Department of General Surgery Chonbuk National University Medical School and Hospital TGFBI, CES1, LDHB, UCHL1, ASPH, VDAC1, VIL2, Jeonju 561-712, Korea CCND2, S100P, CALB1, MAL2, GPX1, and ANXA8 4Corresponding author: Tel, 82-63-250-1681; mRNAs. Immunohistochemistry also revealed in part Fax, 82-63-254-1609; E-mail, [email protected] the differential expressions of these gene proteins. DOI 10.3858/emm.2009.41.2.013 These results suggest that those genetic and gene ex- pression alterations may be relevant to the sarcoma- Accepted 3 November 2008 tous change/EMT in CC cells. Abbreviations: CC, cholangiocarcinoma; Dox, doxorubicin; EMT, Keywords: cholangiocarcinoma; gene expression epithelial mesenchymal transition; FDR, false discovery rate; FHIT, profiling; oligonucleotide array sequence analysis; fragile histidine triad; KRTs, keratins; SAM, significance analysis sarcoma of microarray Introduction Abstract Cholangiocarcinomas (CC) are primary malignan- Cholangiocarcinoma (CC) is an intrahepatic bile duct cies of the biliary tract, and patients with these carcinoma with a high mortality rate and a poor prog- tumors have a poor prognosis because it is difficult nosis. Sarcomatous change/epithelial mesenchymal to make an early diagnosis and the standard transition (EMT) of CC frequently leads to aggressive therapy is not very effective (Ahrendt et al., 2001). intrahepatic spread and metastasis. The aim of this Chronic biliary tract inflammation resulting from study was to identify the genetic alterations and gene bacterial infection or parasitic infestation or from a chronic inflammatory disease such as primary expression pattern that might be associated with the sclerosing cholangitis predisposes a patient to the sarcomatous change in CC. Previously, we estab- development of CC (Chang et al., 1983). Most lished 4 human CC cell lines (SCK, JCK1, Cho-CK, and hepatic tumors with sarcomatous features are Choi-CK). In the present study, we characterized a typi- sarcomatoid carcinomas, whereas true hepatic cal sarcomatoid phenotype of SCK, and classified the sarcomas are extremely rare tumors (Kakizoe et other cell lines according to tumor cell differentiation al., 1987). These sarcomatoid cells are the result (a poorly differentiated JCK, a moderately differ- of a reversal of differentiation of ordinary hepa- entiated Cho-CK, and a well differentiated Choi-CK tocellular carcinoma cells or adenocarcinomatous cells), both morphologically and immunocytologi- CC cells (Oda et al., 1994). Such tumors are cally. We further analyzed the genetic alterations of two predominantly composed of spindle-cell, giant cells Gene expression alterations in sarcomatoid CC cells 103 and bizarre cells that express cytokeratins (KRTs) generate a cell-autonomous EMT-like process via as an epithelial marker and vimentin as a mesen- oncogenic activation is still unclear. One approach chymal marker. It has been suggested that this to understanding how genetic and molecular tumor has an epithelial origin and it is then trans- changes can lead to EMT is to compare the gene formed to the sarcomatous type. The incidence of expression between tumor cells with EMT and the sarcomatous change in CC is approximately 5%. paired tumor cells without EMT. cDNA microarray However, patients with these tumors show aggre- is a well established technique to perform large- ssive intrahepatic spread of tumor and widespread scale surveys of the expression of genes and to metastasis, and they have a poorer prognosis than identify candidate target genes. We previously esta- those patients with ordinary CC. Epithelial tumor blished four distinct CC cell lines (SCK, JCK1, cells lose their restriction during the last steps of Cho-CK, and Choi-CK) and we analyzed their tumorigenesis, concomitant with the loss of epithe- chromosomal aberrations by cross-species color lial characteristics and the acquisition of motile banding (Kim et al., 2001). One of them (SCK) was behavior. These changes contribute to a transition derived from the sarcomatoid CC. In this study, we from an epithelial morphology toward a more me- verified the grade of differentiation in the four cell senchymal fibroblastic (sarcomatoid) phenotype, lines using a xenotransplanted nude mice model and this is referred to as epithelial to mesenchymal and examined a typical sarcomatoid phenotype of transition (EMT) (Boyer et al., 1997). SCK cells both morphologically and immunocyto- The molecular mechanism whereby cancer cells logically. We further analyzed the status of tumor Figure 1. The morphological and immunohistochemical characteristics of the 4 CC cell lines. (A) Phase contrast images of the 4 CC cell lines that were o monolayer-cultured in DMEM supplemented with 10% FBS and 1% antibiotics-antimycotics in a 5% CO2-air conditioned 37 C incubator. (B) Histochemical findings of the xenotransplanted tumors derived from the 4 CC cell lines (SCK, JCK1, Cho-CK or Choi-CK). The anaplastic spindle cells with a fascicular arrangement in a xenotransplanted tumor derived from SCK cells. The poorly differentiated adenocarcinoma derived from the JCK1 cells. The moderately differentiated and the well differentiated adenocarcinomas derived from the Cho-CK and Choi-CK cells, respectively (H&E, ×100; upper panels). Immunohistochemistry (IHC) with an anti-KRT7 antibody stained for the xenotransplanted tumors derived from the 4 CC cell lines. SCK cell-derived tumor; most of the spindle cells were barely stained. IHC with anti-KRT7 antibody in the xenotransplanted tumor tissues, according to the tumor differentiation (lower panels). (C) Immunohistochemical staining of the original tumor tissues. Vimentin and KRT19 expressions were examined in the sarcomatoid (SCK) and the differentiated (Choi-CK) CC tissues, respectively. 104 Exp. Mol. Med. Vol. 41(2), 102-115, 2009 suppressor genes, including the p53 gene and the Table 1. The expression patterns of marker proteins by immuno- fragile histidine triad (FHIT) gene, and the expre- histochemical staining. ssion of a well-known CC-related receptor and its ligand Fas/Fas-ligand (FasL) in the sarcomatous Antigen SCK JCK1 Cho-CK Choi-CK CC cell line as well as in other carcinomatous CC KRT7 - +/- + ++ cell lines. In addition, we used cDNA microarrays KRT19 +/- + + +/- to investigate the genes that are associated with KRT20 - - - - EMT in CC. We found up- or down-regulated CEA +/- +/- + ++ genes in the sarcomatous SCK cells, as compared EMA - (+)* + + + VIM ++ - - - with the differentiated Choi-CK cells. The genes AFP - - - - identified or characterized through this approach Albumin - - - - are potential candidates for those genes involved Fibrinogen - - - - in the cancer metastasis and invasiveness of CC. -, ＞ 95% of the cells could not be determined as positive; +/-, the cells are focally positive between 5% and 24%; +, the percentage of cells de- Results termined as positive is from 25% up to 75%; ++, ＞ 75% of the cells are positive with clearly distinguishable strength. *The immunoreactivity for KRT7 or EMA was positive in the primary tumors, but it was negative in The histology and immunohistochemistry of the xenotransplanted tumors (KRT, keratin; CEA, carcinoembryonic anti- the xenoplanted tumors gen; EMA, epithelial membrane antigen; VIM, vimentin; AFP, alpha-feto- The SCK, JCK and Cho-CK cells were originally protein). derived from a peripheral type of intrahepatic CC. The Choi-CK cells were cultured from a hilar type of infiltrative CC. The SCK cells showed the glandular luminal borders. Immunoreactivity for spindle- shaped (fibroblastoid) morphology and EMA was positive in the primary SCK tumors, but loose cell- cell adhesion. In contrast, the Cho-CK this was negative in the xenoplanted tumors from and Choi- CK cells showed the polygonal and the SCK cells. All the other markers of hepa- epitheloid morphology, and tight cell-cell adhesion. tocellular carcinoma that were tested were nega- The JCK1 cells showed the intermediate type of tive for all 4 types of tumors. morphology between the SCK and Cho-CK cells (Figure 1A). All four