Oncogene (2012) 31, 4233–4244 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Dickkopf 4 (DKK4) acts on Wnt/b-catenin pathway by influencing b-catenin in hepatocellular carcinoma S Fatima1,7, NP Lee1,7, FH Tsang1, FT Kolligs2, IOL Ng3, RTP Poon1, ST Fan1 and JM Luk1,4,5,6 1Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong; 2Department of Medicine II, Klinikum Grosshadern, University of Munich, Marchioninistrasse, Munich, Germany; 3Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong; 4Department of Pharmacology and Department of Surgery, National University of Singapore, Singapore; 5Cancer Science Institute and Institute of Molecular and Cell Biology, Singapore and 6Department of Oncology, pRED China, Roche R&D Center, Shanghai, China Deregulation of Wnt/b-catenin pathway is a hallmark of Introduction major gastrointestinal cancers including hepatocellular carcinoma (HCC). The oncogenic role of b-catenin is well Liver cancer ranks as the second and sixth leading cause defined but reasons for its accumulation in HCC remain of cancer death worldwide in men and women, unclear. Dickkopf 4 (DKK4) acts as a negative regulator respectively, and hepatocellular carcinoma (HCC) of Wnt/b-catenin pathway but its functional role in liver accounts for 70–80% of total liver cancers (Jemal carcinogenesis has not been studied. We investigated the et al., 2011). HCC associates a dismal prognosis because role of DKK4 in b-catenin regulation in HCC. Reduced of late presentation of the disease and high risk of expression of DKK4 was found in 47% (38/81) of HCC, postoperative tumour recurrence (Hao et al., 2009). as measured by quantitative real time PCR. Ectopic Although the risk factors for HCC are well known expression of DKK4 in two HCC cell lines, PLC/PRF/5 (Kemp et al., 2005), the cellular and molecular mechan- (PLC) and MHCC97L (97L), attenuated b-catenin isms contributing to hepatocarcinogenesis are poorly responsive luciferase activity, and decreased both understood. The Wnt/b-catenin pathway is critical for b-catenin and cyclin D1 protein levels. To study the effect embryonic development, cell proliferation, survival, self- of DKK4 on cell growth and tumourigenicity, two stable renewal and regeneration (Gonzalez, 2006; Nejak-Bowen HCC cell lines were established from PLC and 97L cells. and Monga, 2011). However, its deregulation is a Functional assays demonstrated that overexpression of hallmark of several cancers including HCC (Inagawa DKK4 hampered cell proliferation, reduced colony et al., 2002). Surplus b-catenin in the cytoplasm is formation and retarded cell migration. When DKK4- phosphorylated by a multiprotein destruction complex expressing 97L stable cells were used to induce tumour consisting of glycogen synthase kinase 3b (GSK3b), Axin xenografts in nude mice (n ¼ 8), reduction in tumour sizes and adenomatous polyposis coli (APC). The phosphory- was observed (P ¼ 0.027). Furthermore, immunohisto- lated b-catenin is then ubiquitinated and degraded by the chemical studies showed that decreased expression of proteasome (Orford et al., 1997; MacDonald et al., DKK4 was associated with b-catenin accumulation in 2009). Upon binding of Wnt to one of the members of HCC tissues. Additionally, inhibition of the proteasome the frizzled family receptors and to the low-density using specific inhibitor in DKK4-expressing 97L stable lipoprotein-related protein-5 (LRP5) or LRP6 co-recep- cells masked the effect of b-catenin. Our findings suggest tors, the phosphorylation and degradation of b-catenin is a potential tumour suppressive role of DKK4 as well as inhibited, and it accumulates in the cytoplasm and that of an important regulator of HCC. translocates to the nucleus (Zeng et al., 2005). Inside the Oncogene (2012) 31, 4233–4244; doi:10.1038/onc.2011.580; nucleus, b-catenin forms a complex with T-cell factor published online 16 January 2012 (TCF)/lymphoid enhancer factor and upregulates target genes that are implicated in cancer development for Keywords: hepatocellular carcinoma; Wnt/b-catenin example, c-Myc and cyclin D1 (Logan and Nusse, 2004). signalling; DKK4; tumourigenesis Although Wnt/b-catenin signalling is frequently activated in HCC, reasons for its activation are not well understood. Somatic mutations of b-catenin have only been observed in 12–26% of HCC (de La Coste et al., 1998; Wong et al., 2001), but cytoplasmic and nuclear accumulation of b-catenin have been reported Correspondence. Current address: Professor JM Luk, Department of more commonly in 40–70% of HCC (Huang et al., 1999; Oncology, Roche R&D Center, pRED China, 720 Cai Lun Road, Wong et al., 2001). Mutations of other Wnt/b-catenin Shanghai 201203, China. signalling components like Axin1 and Axin2 have only E-mail: [email protected] been observed in 10% and 3% of HCC cases, 7These authors contributed equally to this work. Received 18 July 2011; revised 17 October 2011; accepted 9 November respectively (Taniguchi et al., 2002). Furthermore, 2011; published online 16 January 2012 inactivating mutations of APC are unlikely to contribute Action of DKK4 on Wnt/b-catenin pathway S Fatima et al 4234 significantly as they are very rare (Chen et al., 1998), frequent in HCC tissues compared with their matched implying that other undefined mechanisms may con- non-cancerous cirrhotic tissues, implying that methyla- tribute to Wnt/b-catenin activation in HCC. tion of DKK2 and DKK3 may not be an early event in The dickkopf (DKK) family is important for verte- HCC but may function in the progression of HCC brate development and comprises four secreted glyco- (Yang et al., 2010). Additionally, methylation of DKK3 proteins (DKK1, DKK2, DKK3 and DKK4) of has been significantly associated with short progression- 255–350 amino acids (Niehrs, 2006). They are classified free survival in HCC patients (Ding et al., 2009; Yang as inhibitors of Wnt/b-catenin signalling but members of et al., 2010). Although reduced expression of DKK4 has the DKK family exert different effects on Wnt/b-catenin only been reported in HCC cell lines (Maehata et al., signalling, and their mechanism of action has not been 2008), its function in hepatocarcinogenesis with respect fully delineated (Fatima et al., 2011). With the exception to the Wnt/b-catenin pathway has not been explored. of DKK3, whose receptor has not been identified, Therefore, in this study we investigated the functional DKK1, DKK2 and DKK4 compete with frizzled family role of DKK4 in HCC. receptors binding to the extracellular domain of LRP5/ LRP6 receptor complex in response to attenuation of Wnt activity (Krupnik et al., 1999; Semenov et al., 2001; Results Niehrs, 2006). DKK1, DKK2 and DKK4 also bind to Kremens, another class of transmembrane receptors Reduced expression of DKK4 in HCC cell lines (Mao et al., 2002; Mao and Niehrs, 2003; Nakamura and tissues and Matsumoto, 2008), although the exact mechanism Eight HCC cell lines were evaluated for DKK4 mRNA of how Krm-DKKs-LRP6 complex inhibit Wnt activity expression using quantitative PCR (qPCR) and com- has not been defined. DKK1 is an antagonist of the pared with immortalized normal hepatocytes MIHA. Wnt/b-catenin pathway and also a downstream target of Marked reduction of DKK4 was found in all HCC cell Wnt/b-catenin signalling (Niida et al., 2004; Chamorro lines (Figure 1a). To test whether the reduced expression et al., 2005; Gonzalez-Sancho et al., 2005). However, in of DKK4 in HCC cells is due to DNA methylation, HCC its negative feedback mechanism is abrogated and representative HCC cell lines, such as Hep3B, HuH-7, overexpression of DKK1 in HCC has been associated PLC/PRF/5 (PLC) and MHCC97L (97L), were treated with cytoplasmic and nuclear b-catenin accumulation with increasing concentrations of 5-azacytidine, a (Yu et al., 2009). DKK2 can serve as both an inhibitor pharmacological inhibitor of DNA methyltransferase and activator depending on cellular context (Mao et al., to rule out the effects of DNA methylation. Figure 1b 2001), and the role of DKK3 remains unclear. Previous illustrates unaltered DKK4 expression following treat- study suggested DKK3 to have no effect on Wnt/b- ment with 5-azacytidine, suggesting other reasons for its catenin signalling (Krupnik et al., 1999), whereas others reduced expression in HCC. We next checked the have demonstrated nuclear accumulation of b-catenin as expression of DKK4 in 81 matched pairs of HCC a result of reduced DKK3 expression in lung cancer tumours and their corresponding adjacent non-tumour (Yue et al., 2008). Furthermore, DKK2 and DKK3 are liver tissues of the same patient. DKK4 was reduced in both methylated in HCC and their methylation is more 47% (38/81) of HCC tumour tissues (Figure 1c). Figure 1 Reduced expression of DKK4 mRNA in HCC cell lines and tissues. qPCR was used to determine the mRNA expression of DKK4 in HCC cells and tissues. (a) Reduced expression of DKK4 mRNA was found in HCC cells (HepG2, Hep3B, HuH-7, PLC, H2P, H2M, 97L and 97H) when compared with immortalized normal hepatocytes MIHA. (b) This reduction observed in HCC cells was not associated with DNA methylation when representative HCC cells (Hep3B, HuH-7, PLC and 97L) were treated with increasing concentrations of 5-azacytidine and no effects on DKK4 mRNA expression were observed. (c) Reduced mRNA expression of DKK4 was found in tumour (T) tissues when compared with their adjacent non-tumour liver (NT) tissues in 81 patients with primary HCC. DKK4 expression of each sample is presented by the fold ratio against all NT tissues. *Po 0.05. Oncogene Action of DKK4 on Wnt/b-catenin pathway S Fatima et al 4235 Whereas DKK4-negative was defined as having a DKK4 suppresses in vivo tumourigenicity tumour/non-tumour expression ratio less than twofold, To further explore the in vivo tumour suppressive ability DKK4-positive had this ratio greater than twofold.