Epigenetic silencing of tumor suppressor genes controls various aspects of carcinogenesis, including proliferation, differentiation, and apoptosis (Momparler, 2003
). This widespread mechanism has been implicated in regulating critical signaling cascades, including Notch, sonic hedgehog, and Wnt (Jones and Baylin, 2002
). Aberrant silencing of tumor suppressor genes has been associated with methylation of their promoter regions in medulloblastoma (Lindsey et al., 2005
). Little is known, however, about how epigenetic histone modifications may alter gene expression in medulloblastoma. Using D283 cells, a well-characterized medulloblastoma cell line, we examined global epigenetic changes in medulloblastoma and identified genes belonging to multiple pathways important in tumorigenesis. Similar approaches in tumor cell lines by us and others have yielded several promising candidate tumor suppressor genes (Foltz et al., 2006; Suzuki et al., 2002; Takai et al., 2005; Yamashita et al., 2002
). In the present screen, we identified DKK1
, a Wnt signaling antagonist, and confirmed its silencing in medulloblastoma cell lines, primary tumor cells, and medulloblastoma patient tissue.
The Wnt signaling pathway regulates multiple processes in development, tissue homeostasis, and stem cell maintenance (Nusse, 2005
). Genetic mutations that disrupt Wnt signaling can cause tumors, the best-studied case being colon adenocarcinoma (Suzuki et al., 2004
). Although mutations in Wnt signaling components, APC, GSK3β, and β-catenin have all been linked to colon cancer progression, mutations in these molecules occur only in a small subset of medulloblastoma patients (Koch et al., 2001
), with most being the APC mutations in Turcot’s syndrome (Marino, 2005
). Our work demonstrates that Wnt signaling is also disrupted in medulloblastoma pathogenesis via the epigenetic silencing of DKK1
We demonstrated that restoring DKK1
expression in medulloblastoma cells induced apoptosis and suppressed colony formation. Consistent with our data, others showed that expressing DKK1
in HeLa cells also suppressed transformation (Lee et al., 2004; Mikheev et al., 2004
), and similar to our results, DKK1
inhibited growth by inducing apoptosis, not cell cycle arrest (Lee et al., 2004
). In gliomas as well as models of ischemic neuronal apoptosis, DKK1
was also shown to be a pro-apoptotic factor (Cappuccio et al., 2005; Shou et al., 2002
). Thus, DKK1
’s tumor-suppressing activity is likely important in regulating proliferation in many cell types.
Our data raise two important questions with regard to DKK1
activity in medulloblastoma. The first is how DKK1
induces apoptosis in medulloblastoma. One possibility is that DKK1
suppresses the canonical Wnt signaling pathway, thus down-regulating prosurvival molecules such as Bcl-2. Alternatively, DKK1
might stimulate pro-apoptotic pathways via noncanonical signaling mechanisms. Clues to DKK1
function in medulloblastoma might be provided by its role during vertebrate limb development where DKK1
inhibits proproliferative activities of canonical Wnt signaling and independently regulates apoptosis (Mukhopadhyay et al., 2001
). Although the molecular mechanisms that allow DKK1
to regulate apoptosis are not well understood, some data suggest that it regulates the JNK pathway. In mesothelioma, DKK1
antagonizes Wnt signaling in the absence of β-catenin by inducing JNK-mediated apoptosis.
A second question is whether DKK1
is required for medulloblastoma tumor initiation or is associated with tumor progression. Recent evidence from colon cancer supports its role in tumor progression (Aguilera et al., 2006
). Investigating DKK1
gene knockdown in mouse models of medulloblastoma will provide insight into its biological role in medulloblastoma tumorigenesis.
In this study, we demonstrated the feasibility and robustness of a systematic approach to determine the role of epigenetically silenced genes in medulloblastoma. Our preliminary data suggest that DKK1 gene is a potent tumor suppressor and that Wnt signaling is important in medulloblastoma pathogenesis, a factor not previously appreciated. We are now investigating the mechanistic basis of DKK1 activity in medulloblastoma. Recent studies indicate that Wnt signaling is negatively regulated by secreted Wnt antagonists such as secreted frizzled–related proteins and Dickkopf proteins. We found Wif1 and sFRP1 also to be silenced in medulloblastoma cell lines and up-regulated on HDAC inhibition by TSA (data not shown). A systematic approach aimed to elucidate molecular mechanisms that various Wnt antagonists use to induce apoptosis in medulloblastoma may indicate new, more effective therapeutic targets. Similarly, studies with other epigenetically silenced genes will delineate their roles in malignant transformation and identify pathways involved in tumorigenesis.