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The Prognostic Significance and Therapeutic Potential of Hedgehog Signaling in Intrahepatic Cholangiocellular Carcinoma

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The Prognostic Significance and Therapeutic Potential of Hedgehog Signaling in Intrahepatic Cholangiocellular Carcinoma Published OnlineFirst March 14, 2013; DOI: 10.1158/1078-0432.CCR-12-0349 Clinical Cancer Human Cancer Biology Research The Prognostic Significance and Therapeutic Potential of Hedgehog Signaling in Intrahepatic Cholangiocellular Carcinoma Liang Tang1, Ye-Xiong Tan1, Bei-Ge Jiang2, Yu-Fei Pan1, Shuang-Xi Li1, Guang-Zhen Yang1, Min Wang1, Qing Wang1, Jian Zhang1, Wei-Ping Zhou2, Li-Wei Dong1, and Hong-Yang Wang1,3 Abstract Purpose: The correlation of the hedgehog signaling pathway with the progression, prognosis, and therapeu- tics of intrahepatic cholangiocellular carcinoma (ICC) has not been well documented. The study aimed to investigate the expression, prognostic significance, and therapeutic value of hedgehog components in ICC. Experimental Design: Two independent cohorts of 200 patients with ICC were enrolled. By real-time PCR and immunohistochemistry assay, hedgehog components expression was evaluated. The prognostic values of hedgehog proteins were identified and verified. Cyclopamine or siRNA-targeting Gli was used to block the hedgehog signaling. Cell proliferation and apoptosis were observed by CCK8, cell cycle, and annexin V staining assays. In vivo murine tumor model was used to evaluate the role of hedgehog in ICC. Results: In ICC tissues, the Gli1 nuclear immune-intensity was associated with intrahepatic metastasis and the expression of Gli2 was associated with intrahepatic metastasis, venous invasion, and Unio Internationale Contra Cancrum (UICC) pT characteristics. In survival analysis, high Gli1 or Gli2 expressers had an unfavorable overall survival (OS) prognosis and a shorter disease-free survival (DFS) than those with low expression. In multivariate analysis, Gli1 expression was found to be an independent prognostic factor of OS, which was validated by another independent cohort. Furthermore, blocking the hedgehog signaling by cyclopamine or siRNA-targeting Gli1 resulted in apoptosis and growth inhibition in ICC cells. Conclusions: This study shows, for the first time, activation of hedgehog pathway associated with the progression and metastasis in ICC, which may provide prognostic and therapeutic values for this tumor. Clin Cancer Res; 19(8); 1–11. Ó2013 AACR. Introduction malignant liver tumor compared with hepatocellular carci- Intrahepatic cholangiocellular carcinoma (ICC) is a noma (HCC). Unfortunately, the vast majority of patients malignant neoplasm originating from epithelium of the with cholangiocarcinoma do not have optimal situation for biliary tree with high mortality (1) and a rare primary curative surgery when diagnosis is confirmed. Patients with resection generally have the higher recurrence rate (2, 3). The conventional chemotherapy and radiotherapy to date Authors' Affiliations: 1International Cooperation Laboratory on Signal have proved to play limited effect in improving long-term Transduction, Eastern Hepatobiliary Surgery Institute; 2Department of Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical survival and it still cannot be satisfied (4). So, the mortality University; and 3State Key Laboratory of Oncogenes and Related Genes, from ICC is very high, with the 5-year survival rates being Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University less than 15% to 20% in most series (5, 6). Although several School of Medicine, Shanghai, P.R. China molecules have been reported to be associated with prog- Note: Supplementary data for this article are available at Clinical Cancer nosis and metastasis of ICC (7), more valuable biomarkers Research Online (http://clincancerres.aacrjournals.org/). are needed to predict the clinical outcome or provide L. Tang, Y.-X. Tan, and B.-G. Jiang contributed equally to this work. therapeutic values of patients with ICC. Hedgehog was first identified in a Drosophila screen for Current address for L. Tang: Department of Hepatobiliary Surgery, Jinan Military General Hospital, 25 Shifan Road, 250031, Jinan, Shandong genes important in early embryonic development (8). Sonic Province, P.R. China. hedgehog (Shh), Indian hedgehog (Ihh), and desert hedge- Corresponding Authors: Hong-Yang Wang, International Cooperation hog (Dhh) are 3 mammalian hedgehog genes that have been Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, identified (9). Two transmembrane proteins function to Second Military Medical University, 225 Changhai Road, 200438, Shang- hai, P.R. China. Phone: 86-21-81875361; Fax: 86-21-6556-6851; E-mail: transduce the hedgehog signal: patched (Ptch) and smooth- [email protected]; Li-wei Dong. E-mail: [email protected]; ened (Smo). Smo can transduce signals intracellularly, and and Wei-ping Zhou. E-mail: [email protected] this results in the nuclear localization of the transcription doi: 10.1158/1078-0432.CCR-12-0349 factor Glioma-associated oncogene homolog (Gli). Three Ó2013 American Association for Cancer Research. Gli proteins, Gli1, Gli2, and Gli3 are known to be present in www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 14, 2013; DOI: 10.1158/1078-0432.CCR-12-0349 Tang et al. 2004). The study population consisted of patients with ICC Translational Relevance as confirmed by pathologic analysis. Tumor differentiation This study shows that hedgehog signaling is activated was defined according to the Edmondson grading system. in intrahepatic cholangiocellular carcinoma (ICC) com- Tumor staging was determined according to the sixth edi- pared with normal cholangiocytes. Higher Gli1 and Gli2 tion of the tumor–node–metastasis (TNM) classification of protein levels in ICC tissues are significantly associated the International Union Against Cancer. The selection of with unfavorable overall survival (OS) prognosis and this specific material was conducted to include patients who shorter disease-free survival (DFS). Gli1 is an indepen- underwent surgery alone without chemotherapy or radio- dent prognostic factor for clinical outcome in patients therapy at a time when these adjunctive therapies were not with ICC. Blocking the hedgehog signaling by chemical the standard of treatment. In this way, the analysis of data in inhibitor or siRNA-targeting Gli1 resulted in apoptosis this series will reflect the actual impact of the tumor biology and growth detention in cultured cells and xenograft on the clinical outcome. The patients included in this series tumors. These findings imply that the intratumoral Gli1 also had available paraffin-embedded tumor tissue and the levels of ICC can be used to identify subgroups of tumor cell subtype proven by immunohistochemistry. patients with a favorable or poor OS prognosis and Gli1 Patients were excluded from this cohort with the following may have a potential clinical application as a biomarker exclusion criteria: uncontrolled infection; previously and a therapeutic target for ICC. Regulation of hedgehog received any anticancer therapy; pregnancy and lactation; signaling by agents may influence proliferation and prior malignancy; impaired heart, lung, liver, or kidney apoptosis of ICC cells during tumor progression that function; and previous malignant disease. The follow-up provides an innovative strategy for future clinical trials. period was defined as the interval from the date of operation to the date of death or the last follow-up. Deaths from other causes were treated as censored cases. All patients were observed until December 2010, ranged from 2 to 82 months vertebrates. The 3 Gli proteins act in a combinatorial fash- (median, 17 months). ion, with Gli1 and Gli2 being the major positive Fresh-frozen ICC tissues from 41 patients and normal intermediaries of hedgehog signaling. Hedgehog interact- intrahepatic bile duct epithelia from 5 patients who under- ing protein (Hhip) was identified as a protein that can bind went primary hepatectomy between 2001 and 2007 were to Shh and inhibit hedgehog signaling (10). Recent evi- used for RNAextraction. Overall survival (OS) was defined as dence has shown that hedgehog plays an important role in the interval between the dates of surgery and death. Disease- multiple tumor types, for example, basal cell carcinoma (11, free survival (DFS) was defined as the interval between the 12), small cell lung cancer (13), prostate cancer (14), gastric dates of surgery and recurrence; if recurrence was not diag- cancer (15), esophageal cancer (16), pancreatic cancer nosed, patients were censored on the date of death or the last (15, 17), and HCC (18, 19). The reports have been shown follow-up. Informed consent was obtained from all patients that dysregulation of hedgehog signaling may contribute to before subsequent use of their resected tissues. The present tumorigenesis. study was conducted in accordance with the ethical stan- Although limited studies have reported the role of hedge- dards of the Helsinki Declaration in 1975, after approval of hog in cholangiocellular carcinoma, the correlation of the the Ethics Committee of Human Experimentation of Second hedgehog signaling pathway with the prognosis and ther- Military Medical University (Shanghai, P.R. China). apeutics of ICC has not been well documented. In the current study, expression of the hedgehog signaling path- Tissue microarray and immunohistochemistry way in ICC was detected and the relationship between Tissue microarray slides were prescreened with hematox- hedgehog proteins and clinicopathologic characteristics ylin and eosin staining (Shanghai Biochip Company,
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