In this study, we report that loss of the desmosomal cadherin Dsc2 confers a tumorigenic phenotype to transformed colonic epithelial cells. Our data demonstrate that decreased expression of Dsc2 enhances β-catenin signaling and promotes proliferation of colonic epithelial cells. Furthermore, loss of Dsc2 enables cells to grow as tumors in immunodeficient mice, a phenomenon that is not observed in parental cell lines. Importantly, our results also identify activated Akt as a key component driving β-catenin–dependent transcription in Dsc2-deficient cells. Taken together, these data provide the first mechanistic evidence that loss of Dsc2 may contribute to the malignant transformation of intestinal epithelial cells and demonstrate a novel mechanism to explain the regulation of β-catenin signaling by desmosomal cadherin family members.
Dsc2 is the only desmocollin expressed by simple epithelial tissues such as the colon. Dsc2 loss or down-regulation has been observed in sporadic and inflammation-associated colonic adenocarcinomas (Khan et al.
) and in highly tumorigenic colonic adenocarcinoma cell lines (Funakoshi et al.
). In support of these findings, we also have observed decreased Dsc2 protein expression and/or diffuse membrane localization in moderately and poorly differentiated colonic adenocarcinomas (Supplemental Figure 7). Dsc2 and Dsc3 proteins, which are both expressed in stratified epithelial tissues, are down-regulated or mislocalized in breast, skin, esophageal, and hepatocellular carcinomas (Kurzen et al.
; Oshiro et al.
; Cao et al.
; Fang et al.
). Decreased expression of Dsc3 in oral squamous cell carcinomas correlated with poor histological grade, lymph node metastasis, and altered localization of β-catenin (Wang et al.
), and Fang et al.
) recently reported similar prognostic correlations for Dsc2 loss in esophageal carcinomas. These data suggest that loss of Dsc proteins may contribute to tumor formation and/or progression; however, no study to date has examined whether Dsc has a direct role in tumorigenesis.
Here we report that down-regulation of Dsc2 in a colonic epithelial cell line increased the activation of β-catenin/TCF signaling, enhanced cell proliferation, and conferred tumorigenic capacity to SK-CO15 cells. These findings are in agreement with a large body of evidence that strongly implicates aberrant activation of β-catenin signaling in the development and progression of colorectal cancers (Munemitsu et al.
; Korinek et al.
; Morin et al.
). Importantly, increased activity of β-catenin/TCF transcription has been shown to drive cell proliferation by directly enhancing the expression of proproliferative target genes (Wong et al.
; Tetsu and McCormick, 1999
; Rimerman et al.
), and activation of this pathway also contributes to cellular transformation and the acquisition of invasive properties (Behrens et al.
; Birchmeier et al.
; Shimizu et al.
). Interestingly, Dsc2-deficient colonic epithelial cells appear to share similar properties to those described for colon cancer stem cells (hyperproliferation, activation of Akt/β-catenin, expression of putative stem cell markers such as CD44, and tumorigenic capability). It is tempting to speculate that loss or low levels of Dsc2 may play an important role in the maintenance of colon cancer stem cells (Vermeulen et al.
Germline or acquired somatic mutations in proteins of the Wnt/β-catenin signaling pathway occur in the majority of colorectal cancers and interfere with β-catenin degradation (Fodde and Brabletz, 2007
; Markowitz and Bertagnolli, 2009
). However, Wnt-independent mechanisms are also known to regulate the activity of the β-catenin/TCF transcriptional complex, including regulation by cell adhesion proteins, such as E-cadherin (Hermiston et al.
; Jeanes et al.
), as well as regulation by the serine/threonine kinase Akt (He et al.
; Fang et al.
), which has recently emerged as an important regulator of β-catenin transcriptional activity in the intestine (Vermeulen et al.
; Brown et al.
; Lee et al.
; Nava et al.
As opposed to the extensively studied classical cadherin E-cadherin, the mechanism(s) by which the desmosomal cadherins regulate β-catenin signaling remain unclear. Unlike the findings on E-cadherin, our findings reveal that Dsc2 does not associate with β-catenin in colonic epithelial cells, indicating that activation of β-catenin is not due to “release” of this protein from a Dsc/β-catenin complex, as has been proposed for E-cadherin. Furthermore, the amount of β-catenin bound to E-cadherin does not appear to be altered in Dsc2-deficient cells (unpublished data), suggesting that the effects on β-catenin signaling are not due to changes in the interaction between E-cadherin and β-catenin. In addition, we have not detected a change in E-cadherin membrane localization by immunofluorescence/confocal imaging following Dsc2 down-regulation (unpublished data). The total levels of E-cadherin, as assessed by immunoblotting, are unchanged in transient knockdown cells () and are modestly decreased (by ~20%) in stable shDsc2-expressing cells (unpublished data), which mirrors the effects we observed for total β-catenin levels. At the present time, it is not clear why there is a difference between transient and stable Dsc2 knockdown cells; however, these results suggest that decreased expression of E-cadherin in SK-CO15 cells may be a longer term consequence of loss of Dsc2.
Studies in the epidermis have suggested that Dsc proteins may regulate β-catenin signaling indirectly through effects on plakoglobin, a catenin family member that interacts with Dscs and may “compete” with and displace β-catenin from E-cadherin when plakoglobin is in excess (Zhurinsky et al.
; Miravet et al.
; Hardman et al.
). However, simultaneous knockdown experiments in which Dsc2 and β-catenin or plakoglobin were down-regulated in SK-CO15 cells demonstrated that β-catenin but not plakoglobin knockdown completely abolished the activation of the β-catenin transcriptional reporter and reversed the proliferation phenotype of Dsc2-deficient cells (; unpublished data).
Instead we observed that loss of Dsc2 induced the activation of serine/threonine kinase Akt, which has been shown to positively regulate β-catenin signaling in a number of cell types, including intestinal epithelial cells. Akt has been proposed to enhance β-catenin–dependent transcription through direct phosphorylation and activation of β-catenin (Ser-552) or through the inhibition of GSK-3β (Ser-9), a signaling component that is shared with the Wnt/β-catenin pathway. In our study, activation of Akt in Dsc2-deficient cells was associated with an increase in the number of cells staining positively for phospho-β-catenin (Ser-552), an Akt phosphorylation site on β-catenin that is associated with enhanced β-catenin transcription (Fang et al.
; He et al.
). Furthermore, we observed an enrichment of phospho-β-catenin (Ser-552) in the nucleus of mitotic cells (unpublished data), a phenomenon that has been reported previously for β-catenin (Kaplan et al.
; Zhang et al.
). We also observed an increase in phosphorylation of the inhibitory Ser-9 residue of GSK-3β, which has been proposed to enhance β-catenin signaling by promoting the accumulation of β-catenin protein levels, in a manner analogous to Wnt-mediated GSK-3β inactivation (Desbois-Mouthon et al.
; Sharma et al.
; Naito et al.
; Mulholland et al.
). Interestingly, despite the inhibition of GSK-3β, we observed a decrease in the total levels of β-catenin in stable Dsc2 knockdown cells, suggesting that Akt-mediated GSK-3β inhibition may promote the nuclear redistribution of β-catenin and enhance β-catenin–dependent transcription without inducing stabilization of total β-catenin protein levels. These findings are in agreement with a number of reports suggesting that it is the nuclear localization of β-catenin, rather than the total protein levels, that indicates enhanced transcriptional activity of β-catenin (Miller and Moon, 1997
; Gottardi and Gumbiner, 2004
; Maher et al.
) and signify that in addition to GSK-3β, other cellular components (e.g., cell–cell adhesion complexes) likely also influence the total levels of β-catenin in cells. Furthermore, our results also suggest that inhibition of GSK-3β by Akt may enhance β-catenin signaling in a more distinct manner than Wnt-mediated GSK-3β inactivation, even though this component is shared by both Wnt/β-catenin and Akt/β-catenin pathways (see ). Of note, while numerous reports have demonstrated a clear role for Akt activation in the regulation of β-catenin–dependent transcription, PI3K/Akt does not enhance the transcriptional activity of β-catenin in all cell types, suggesting that Akt/β-catenin signaling may occur in a tissue- and/or context-dependent manner (Ng et al.
FIGURE 6: Working model. Loss of Dsc2 promotes cellular transformation and proliferation through activation of the Akt/β-catenin signaling pathway. (A1) Down-regulation of Dsc2 leads to activation of the EGFR (A2) and PI3K/Akt-dependent activation of β-catenin/TCF (more ...)
Our data suggest that loss of Dsc2 may promote Akt/β-catenin signaling through the EGFR, which has been previously shown to regulate β-catenin–dependent transcription (Hu and Li, 2010
). We find that EGFR protein levels are increased following Dsc2 down-regulation () and that inhibition of EGFR diminishes the activation of β-catenin–dependent transcription and cell proliferation following Dsc2 knockdown (; Supplemental Figure 6B). Furthermore, treatment of SK-CO15 cells with EGF enhances β-catenin signaling, an effect that is diminished by treatment with EGFR, PI3K, and Akt inhibitors or by targeted depletion of Akt using siRNA (). Thus EGFR activation by EGF recapitulates the effect of Dsc2 down-regulation on β-catenin–dependent transcription and strongly supports a role for EGFR in the activation of Akt/β-catenin signaling in Dsc2-deficient cells. These findings complement a recent report linking increased desmosomal cadherin expression to the suppression of EGFR signaling in a model of epidermal differentiation (Getsios et al.
Interestingly, activation of EGFR/Akt/β-catenin signaling appears to be specific to loss of Dsc2 rather than a general response to the disruption of desmosomal adhesion, as down-regulation of the related desmosomal cadherin Dsg2 does not appear to activate the same signaling cascade in SK-CO15 cells (unpublished data).
Finally, the mechanisms by which Dsc2 may be down-regulated in colorectal cancers are not well defined. Funakoshi et al.
) reported that loss of the intestine-specific homeobox transcription factor CDX2 correlated with loss of Dsc2 expression in colon cancer cell lines and that reexpression of CDX2 restored Dsc2 expression, suggesting that transcriptional regulation may lead to its down-regulation. In contrast, Khan et al.
) noted that loss of Dsc2 protein in colonic adenocarcinomas occurred without changes in mRNA levels, indicating changes in protein stability rather than transcriptional down-regulation. Furthermore, these authors found evidence of “Dsc switching” in colonic adenocarcinoma, as loss of Dsc2 was associated with increased expression of Dsc3. In our study, we found no evidence of Dsc3 protein or mRNA induction following down-regulation of Dsc2, suggesting that loss of Dsc2 is not sufficient to induce Dsc3 expression. Last, others have proposed that enhanced proteolytic cleavage of the extracellular domain of Dsc2 may contribute to its loss in transformed cells (Mathias et al.
). While these studies provide important insight, additional work is required to better characterize the mechanisms regulating changes in Dsc expression during tumorigenesis.
Based on the data from the current study, a hypothetical model to explain how loss of Dsc2 contributes to tumor formation and growth can be proposed (). In this model, down-regulation of Dsc2 induces the activation of the EGFR, which stimulates PI3K activity and enhances PIP3
levels. Akt is recruited to the membrane via interactions with PIP3
and is activated by phosphorylation. Activated Akt phosphorylates and inhibits GSK-3β and phosphorylates β-catenin directly, increasing the nuclear localization and transcriptional activity of β-catenin. Interestingly, our data suggest that loss of Dsc2 may lead to activation of Akt/β-catenin signaling through the presence of “solitary” (Schmitt et al.
) or excess Dsg2, which has been shown to activate Akt (Brennan et al.
; our unpublished data). Although not addressed in the work presented here, in addition to promoting β-catenin signaling and cell proliferation, active Akt also phosphorylates other downstream targets, which are known to promote the survival of tumor cells (dashed arrows). These prosurvival effects of Akt may also contribute to the enhanced tumorigenicity of Dsc2-deficient SK-CO15 cells by allowing the cells to “seed” a tumor in vivo.
In summary, this work defines a mechanistic role for Dsc2 in the progression of colorectal cancer and identifies Akt as a novel link between the desmosomal cadherins and β-catenin signaling.