Our studies have identified ATDC as a protein highly expressed in the majority of human pancreatic adenocarcinomas and pancreatic cancer precursor lesions. We also demonstrated that expression of ATDC in pancreatic cancer cells promoted cellular proliferation and enhanced tumor growth and metastasis. Additionally, we provided evidence that elevated levels of ATDC expression correlated with elevated β-catenin levels in pancreatic cancer cell lines and primary pancreatic cancers, and that silencing of ATDC via shRNA approaches antagonized β-catenin/TCF-mediated reporter activation and activation of TCF target genes. β-catenin was implicated in the oncogenic effects of ATDC in vitro and in vivo, and the ability of ATDC to increase β-catenin levels appeared to be attributable to ATDC’s effects on disheveled-2 protein expression. In summary, our findings implicate ATDC as an important positive regulator of β-catenin-dependent signaling in pancreatic cancer.
ATDC has been reported to be up-regulated in a number of different cancer types, including lung, bladder, colorectal, ovarian and endometrial cancers and multiple myeloma (Dyrskjot et al., 2004
; Glebov et al., 2006; Hawthorn et al., 2006; Mutter et al., 2001
; Ohmachi et al., 2006; Santin et al., 2004; Zhan et al., 2002
). A recent report correlated ATDC expression in gastric cancer and poor histological grade, large tumor size, extent of tumor invasion, and lymph node metastasis (Kosaka et al., 2007
). ATDC has also been reported to be down-regulated in some cancer types (Smith et al., 2005, Nacht et al., 1999; Zhang et al., 2006; Ernst et al., 2002) suggesting the function of ATDC may be depend on cellular context. In none of these reports was the role of ATDC in tumorigenesis examined in functional studies.
We found that ATDC upregulated β-catenin levels in pancreatic cancer cell lines and primary pancreatic cancers. A large body of data supports the contribution of activation of the canonical (β-catenin-dependent) Wnt signaling pathway in the development of colorectal cancer. Sustained β-catenin pathway activation independent of APC, Axin1 or β-catenin mutations has been demonstrated in a subset of breast and ovarian cancer (Bafico et al., 2004
). Mutations in APC or β-catenin appear to be rare in pancreatic adenocarcinoma (Zeng et al., 2006). While robust activation of the pathway due to signature mutations in components of the β-catenin signaling cascade that are commonly observed in other gastrointestinal cancers are not present in pancreatic adenocarcinoma, immunohistochemical analysis of β-catenin suggests a possible contribution of β-catenin signaling during PanIN progression and the development of invasive pancreatic cancer. Increased levels of both cytoplasmic and nuclear β–catenin, indicative of β-catenin signaling activity, have been reported in a substantial group of pancreatic adenocarcinomas and PanIN s (Zeng et al., 2006; Pasca di Magliano et al., 2007
). Pasca di Magliano and colleagues reported that 65% of pancreatic cancers have an increase in either cytoplasmic and/or nuclear β-catenin. Similar results were obtained in Pdx-Cre;KrasG12D
transgenic mice that developed PanIN lesions and subsequent invasive pancreatic cancers that were phenotypically indistinguishable from human pancreatic adenocarcinomas. Moreover, the authors showed that cancer cell survival and proliferation, depended on β-catenin signaling activity in multiple human pancreatic cancer cell lines.
The mechanisms by which ATDC levels are upregulated in pancreatic adenocarcinomas remain unclear. Interestingly, Pasca di Magliano and colleagues demonstrated that increased hedgehog signaling, one of the earliest changes in PanIN lesions, activated β-catenin signaling in transgenic mice and untransformed pancreatic duct cells, suggesting that hedgehog may play a role in upregulating β-catenin activity in some pancreatic adenocarcinomas. A possible connection between hedgehog upregulation and ATDC expression in human pancreatic cancer cell lines or primary tumors remains to be explored.
The levels of the free cytoplasmic pool of β-catenin are known to be regulated by Wnt ligands. In the absence of an activating Wnt signal, mediated via Wnt binding to the frizzled-low density lipoprotein-related (LRP) 5/6 co-receptor complex, cytoplasmic β-catenin is destabilized by a multiprotein complex containing axin, GSK3β, and APC. Axin acts as the scaffold of this complex and interacts with the other components- β catenin, APC, and GSK3β. Interaction of GSK3β with Axin in the complex facilitates efficient phosphorylation of β-catenin by GSK3β. Phosphorylated β-catenin is then ubiquinated, leading to its rapid proteosomal degradation. We found that ATDC bound to Axin and GSK-3β in pancreatic cancer cells, suggesting that ATDC interacted with the destruction complex to prevent phosphorylation and subsequent ubiquination of β-catenin.
When Wnt binds to the frizzled/LRP co-receptors at the cell surface, a cytoplasmic protein, Dvl, antagonizes GSK-3β dependent phosphorylation of β-catenin. Although it is not known if Dvl binds directly to the frizzled/LRP co-receptor or whether intermediary proteins are involved in the signal transduction between frizzled and Dvl, Dvl appears to bind to axin and inhibit GSK-3β-dependent phosphorylation of β-catenin, APC and axin. Once the phosphorylation of β-catenin is reduced, β-catenin dissociates from the axin complex, resulting in its accumulation in the cytoplasm. Once stabilized, a fraction of the β-catenin is translocated to the nucleus, where it binds to transcription factors such as the TCF/lymphoid enhancer binding factor and thereby stimulates the transcription of β-catenin target genes. We noted that ATDC expression in HEK 293 cells induced expression of Dvl-2, and demonstrated that ATDC formed a complex with Dvl-2 in pancreatic cancer cells. We demonstrated that levels of ATDC in primary pancreatic cancers correlated well with Dvl-2 levels, suggesting that ATDC upregulates Dvl-2 levels in primary pancreatic cancers, and increases β-catenin levels by this mechanism. We further show that knockdown of Dvl-2 in ATDC expressing cells abrogates enhanced TCF activity and cell proliferation induced by ATDC, directly implicating Dvl-2 as an intermediary in this process.
The regulation of the disheveled protein is still poorly understood, but recent data suggest that disheveled, like β-catenin, may be controlled by ubiquination and degradation by the proteosome (Hershko et al., 1998; Simons et al., 2005; Miyazaki et al., 2004). Dvl-1 has been reported to interact with the neuronal Homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase NEDL1 (Miyazaki et al., 2004). The proteins inversin and the interactions between PP2A phosphatase and the Wnt-induced antagonist naked cuticle have been shown to modulate the stability of Dvl-1 (Simons et al., 2005; Creyghton et al., 2005). And, in a recent manuscript published by Angers and colleagues, KLHL12-Cullin-3 ubiquitin ligase was shown to negatively regulate the Wnt-β-catenin pathway by targeting disheveled for degradation (Angers et al., 2006
). We found that ATDC did not increase Dvl-2 levels by changes in Dvl-2 gene expression but rather by enhancing the stability of the Dvl-2 protein, supporting changes in Dvl stability serve as a important mechanism in regulating the Wnt/β-catenin signaling pathway.
The data in the present study support a model for the mechanism by which ATDC functions to promote the oncogenesis of pancreatic cancer cells (). In unstimulated, normal pancreatic cells lacking ATDC, Dvl-2 is in the cytoplasm and is not bound to the Axin/Gsk-3β/APC destruction complex. This allows the destruction complex to phosphorylate β-catenin and target it for ubiquitin-mediated degradation. In pancreatic cancer cells expressing high levels of ATDC, ATDC binds to and stabilizes Dvl-2, resulting in the release of β-catenin from the destruction complex, increased β-catenin levels and subsequent activation of downstream β-catenin/TCF-regulated target genes These studies define for the first time a functional role of ATDC in human tumorigenesis and besides highlighting ATDC as a potential therapeutic target in pancreatic cancer, have defined a novel mechanism for activating Wnt/β-catenin signaling in cancer.
Model of how ATDC mediates activation of β-catenin signaling in pancreatic cancer cells