ORIGINAL PAPER Journal of Pathology J Pathol 2011; 225: 525–534 Published online 10 June 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.2901 Zic2 synergistically enhances Hedgehog signalling through nuclear retention of Gli1 in cervical cancer cells David W Chan, 1 Vincent WS Liu, 1 Ling Yang Leung, 1 Kwok Ming Yao, 2 Karen KL Chan, 1 Annie NY Cheung 3 and Hextan YS Ngan 1 * 1 Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, University of Hong Kong, People’s Republic of China 2 Department of Biochemistry, LKS Faculty of Medicine, University of Hong Kong, People’s Republic of China 3 Department of Pathology, LKS Faculty of Medicine, University of Hong Kong, People’s Republic of China *Correspondence to: Hextan YS Ngan, Department of Obstetrics and Gynaecology, 6/F Professorial Block, Queen Mary Hospital, Pokfulam, Hong Kong SAR, People’s Republic of China. e-mail: hysngan@hkucc.hku.hk Abstract Aberrant activation of Hedgehog (Hh) signalling has been implicated in the pathogenesis of human cancers. However, the cognate molecular mechanisms contributing to this disregulated pathway are incompletely understood. In this study, we showed that Zic2 was frequently over-expressed and associated with high-grade cervical cancer (p = 0.032), high levels of Gli1 (p < 0.001) and CyclinD1 (p < 0.001) by immunohistochemical and quantitative RT–PCR analyses. Further biochemical studies using luciferase reporter, co-immunoprecipitation, subcellular fractionation and immunofluorescence analyses demonstrated that Zic2 can physically interact with Gli1 and retain it in the nucleus, which in turn increases Gli-mediated transcriptional activity. Gain- and loss-of-function analyses of Zic2 showed that Zic2 could increase Hh signalling activity, cell proliferation and anchorage-independent growth ability in cervical cancer cells. Conversely, deletion of the zinc finger domain at C-terminus of Zic2 significantly abrogated its interaction with Gli1, the retention of Gli1 in the nucleus, effects on Hh signalling activity and oncogenic properties in cervical cancer cells. Our findings suggest that Zic2 is a positive modulator increasing Gli1 transcriptional and oncogenic activity by retaining Gli1 in the nucleus of cervical cancer cells. Copyright  2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: Zic2; Gli1; Hedgehog; cervical cancer; tumourigenicity Received 3 December 2010; Revised 11 March 2011; Accepted 17 March 2011 No conflicts of interest were declared. Introduction Cervical cancer is the second most common malig- nancy in females worldwide. However, it has now become a potentially preventable and curable disease due to advances in screening, diagnosis and treatment. It is well known that human papillomavirus (HPV) is the single most significant aetiological agent in cervi- cal cancer and its viral oncoproteins, such as E6 and E7, are the main factors linking to tumour progression. However, evidence suggests that HPV infection alone is insufficient to induce malignant changes. Thus, the transformation from low-grade lesion to invasive can- cer must involve other cellular and genetic changes, which consist of aberrant up-regulation of oncogenes, deletion of tumour suppressor genes or activation of signal transduction pathways. The Hedgehog (Hh) signalling pathway has been identified in Drosophila as a key regulatory mechanism of cell fate determination during embryogenesis [1]. Aberrant activation of the Hh signalling pathway due to Gli1 over-expression, mutation or translocation of the suppressors and effectors of Hh signalling was associated with increased cell proliferation, tumour growth and metastasis in skin [2–4], prostate [5–7] and hepatocellular carcinomas [8]. This indicates that Hh signalling pathway plays a significant role in cancer development. However, the role of Hh signalling pathway in cervical cancer is rarely reported. A recent report has shown that the expressions of Gli1, Gli2 and Gli3 in Hh signalling pathway are up-regulated in uterine cervical carcinoma and its precursor lesions [9]. The expressions of these molecules are significantly elevated in cervical intraepithelial neoplasia (CIN) and carcinoma [9]. These results strongly suggest that the Hh signalling pathway is extensively activated in carcinoma and CIN of uterine cervix. Zic family genes encode zinc-finger proteins Zic1, Zic2 and Zic3, which play crucial roles in vertebrate development [10]. The zinc-finger domains are highly conserved between Zic proteins and show a remarkable homology to those of Gli family proteins. Zic and Gli have been found to cooperate with each other in neural and skeletal development [11]. Gli and Zic Copyright  2011 Pathological Society of Great Britain and Ireland. J Pathol 2011; 225: 525–534 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com