Canonical Wnt suppressor, Axin2, promotes colon carcinoma oncogenic activity

Abstract

Aberrant activation of canonical Wingless-type MMTV integration site family (Wnt) signaling is pathognomonic of colorectal cancers (CRC) harboring functional mutations in either adenomatous polyposis coli or β-catenin. Coincident with Wnt cascade activation, CRCs also up-regulate the expression of Wnt pathway feedback inhibitors, particularly the putative tumor suppressor, Axin2. Because Axin2 serves as a negative regulator of canonical Wnt signaling in normal cells, recent attention has focused on the utility of increasing Axin2 levels in CRCs as a means to slow tumor progression. However, rather than functioning as a tumor suppressor, we demonstrate that Axin2 acts as a potent promoter of carcinoma behavior by up-regulating the activity of the transcriptional repressor, Snail1, inducing a functional epithelial-mesenchymal transition (EMT) program and driving metastatic activity. Silencing Axin2 expression decreases Snail1 activity, reverses EMT, and inhibits CRC invasive and metastatic activities in concert with global effects on the Wnt-regulated cancer cell transcriptome. The further identification of Axin2 and nuclear Snail1 proteins at the invasive front of human CRCs supports a revised model wherein Axin2 acts as a potent tumor promoter in vivo.

Colorectal cancers (CRCs) are generally characterized by aberrant Wingless-type MMTV integration site family (Wnt) signaling (1–3). Neoplastic cells located at the invasive front of CRCs frequently display increased levels of nuclear β-catenin and adopt a mesenchymal-like phenotype—events precipitated by mutations predominantly occurring in adenomatous polyposis coli (APC) or, more rarely, β-catenin (1–3). After nuclear translocation, β-catenin complexes with DNA-binding proteins of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family and triggers the expression of downstream genes linked to the induction of an invasion program that allows CRC cells to transmigrate the underlying basement membrane, gain access to the host interstitial matrix, and infiltrate surrounding tissues (1–4). However, in addition to supporting CRC invasion, the cohort of β-catenin/TCF-regulated target genes also includes feedback inhibitors that serve to modulate the activity of the canonical Wnt signaling cascade (1, 2).

One of the most extensively studied canonical Wnt pathway feedback inhibitors in CRCs is the scaffold protein, Axin2/conductin (1, 2). Under physiologic conditions, free β-catenin is targeted for degradation by a multiprotein complex composed of APC, GSK3β, CK1, and either Axin1 or Axin2 (1, 2). Although low levels of Axin2 are found in the normal colon epithelium, constitutive activation of Axin2 expression by the β-catenin/TCF transcriptional complex is observed in CRCs (1–3). Axin2 efficiently supports the GSK3β-dependent phosphorylation of β-catenin in normal cells, which subsequently marks the posttranslationally modified protein for β-TrCP–dependent ubiquitination and proteosomal degradation (1, 2). However, the ability of elevated levels of Axin2 to modulate β-catenin levels in APC- or β-catenin–mutant CRC cells is muted (1, 2). Indeed, in an effort to silence the aberrant transcriptional programs activated in CRCs as a function of the constitutive stabilization of β-catenin, new therapeutics have been promoted that increase Axin2 (as well as Axin1) levels by targeting the poly-ADP ribosylating enzymes, tankyrase 1 and tankyrase 2 (5–7).

Although Axin2 is uniformly categorized as a tumor suppressor in CRCs (1, 2, 5–7), its precise function in carcinomatous states is unknown, particularly with regard to the molecular “rationale” that underlies the decision of neoplastic cells to maintain high levels of a putative suppressor in vitro and in vivo. Herein, we demonstrate that endogenous Axin2 promotes, rather than suppresses, a β-catenin/TCF-initiated, EMT program in both β-catenin–mutant and APC-mutant CRCs. Up-regulation of Axin2 levels in CRCs triggers marked increases in Snail1 activity and induces EMT, whereas silencing endogenous Axin2 expression initiates a mesenchymal-epithelium–like switch that not only down-regulates Snail1, but also triggers widespread changes in the canonical Wnt signaling transcriptome. Because Axin2 and Snail1 are further shown to be colocalized at the invasive front of CRC tissues, these studies suggest that Axin2 serves as an effective tumor promoter—rather than tumor suppressor—that not only controls the induction of a Snail1-dependent EMT program, but also exerts global control over gene expression networks critical to invasive and metastatic behaviors.

Results

Canonical Wnt Signaling Triggers a Colon Cancer Invasion Program.

In HCT116 or SW48 CRC cells that harbor heterozygous, stabilizing mutations in β-catenin, each of the lines constitutively expresses β-catenin and Axin2 and TOPflash reporter activity (Fig. 1 A and B). In the presence of exogenous Wnt3a, however, the cells respond with a significant increase in β-catenin protein levels, Axin2 mRNA levels, and TOPflash reporter activity (Fig. 1 A and B). By contrast, in APC-defective SW620 cells, constitutive levels of β-catenin, Axin2, or TOPflash activity are maximally activated and unaffected by Wnt3a (Fig. 1 A and B). Both β-catenin and APC mutant cell lines trigger canonical Wnt signaling via a β-catenin/TCF-dependent pathway because Axin2 mRNA levels are significantly suppressed when either HCT116 or SW620 cells are stably transfected with a dominant-negative, FLAG-tagged TCF-4 construct (TCF-DN) (Fig. 1C) (8). Further, although both HCT116 and SW620 populations are dominated by E-cadherin–negative or E-cadherin–low-expressing cells and assume mesenchymal cell-like phenotypes in vitro, the TCF-DN stable transfectants up-regulate E-cadherin protein levels and E-cadherin promoter activity while adopting an epithelial cell-like appearance (Fig. 1 C and D).

Fig. 1.

Canonical Wnt signaling pathway triggers a CRC cell invasion program. (A) CRC cells were treated with vehicle or Wnt3a (100 ng/mL) for 24 h. β-catenin protein and Axin2 mRNA levels were assessed by Western blotting (Upper) and qRT-PCR (Lower), respectively. (B) Cells cotransfected with TOPflash reporter and pRL-TK constructs were treated with Wnt3a, and the cell lysates were analyzed for luciferase reporter activity (mean ± SEM; n = 3). (C) Cells infected with mock or TCF-DN–expressing retrovirus were selected with G418, recovered, and reinfected with mock or an IRES-GFP-Axin2–expressing lentivirus. GFP-positive cells were sorted by FACS, cultured, and subjected to Western blotting (Upper) and qRT-PCR (Lower) analyses, respectively. (D Left) Mock or TCF-DN stably expressing CRC cells were fixed and stained with anti–E-cadherin antibody. Nuclei were stained with TOTO3 (blue). (Scale bar: 40 μm.) (D Right) CRC were cotransfected with an E-cadherin luciferase reporter and pRL-TK constructs, and luciferase reporter activity was determined (mean ± SEM; n = 3). **P < 0.01.

The most definitive characteristic of cancer cell EMT programs is the ability of the transformed cell to traverse an intact, type IV collagen-rich BM (4, 9). As such, HCT116 or SW620 cells were cultured atop the chorioallantoic membrane (CAM) of live, 11-d-old chicken embryos wherein the upper epithelial cell layer is subtended by an intact BM (9). Under these conditions, both CRC cell lines rapidly degrade the underlying BM and invade the subjacent interstitial tissues (Fig. S1). By contrast, TCF-DN-transfectants of each cell line lose invasive potential and remain confined to the upper CAM surface (Fig. S1).

β-Catenin/TCF Signaling Induces Snail-Regulated EMT and Tumor Invasion in Colon Cancer Cells.

During development and carcinogenesis, activation of canonical Wnt signaling has been linked to expression of the zinc-finger transcriptional repressor, Snail1 (3, 8, 10). To determine whether the constitutive Wnt signaling activity associated with CRC cells is sufficient to trigger Snail1 protein expression, HCT116, SW48, or SW620 cells were cultured in the absence or presence of exogenous Wnt3a. Although Wnt3a did not alter Snail1 mRNA expression levels in any of the cell lines tested, Snail1 protein levels increased significantly in Wnt3a-treated HCT116 or SW48 cells, whereas APC-mutant SW620, SW480, or DLD1 cells express Snail1 protein in a constitutive fashion (Fig. 2A and Fig. S1). Induction of Snail1 protein expression is linked directly to the canonical Wnt pathway because HCT116 or SW620 cells that stably express a TCF-DN construct decrease Snail1 protein levels in the absence or presence of Wnt3a without affecting Snail1 mRNA expression (Fig. 2B and Fig. S2). Although the expression of Snail1 correlates with a Wnt-dependent EMT program, multiple transcription factors have been reported to trigger similar EMT-like responses in CRCs, particularly Snail2/Slug or ZEB1 (11–13). However, down-regulating β-catenin/TCF activity with the TCF-DN construct did not affect mRNA expression levels of either of these transcription factors (Fig. S2). Further, when Snail1 expression is silenced in either HCT116 or SW620 cells by either of two shRNA constructs, E-cadherin is reexpressed, whereas TOPFlash reporter activity is down-regulated in a manner consistent with the ability of Snail1 to modulate β-catenin/TCF activity (Fig. S2) (14). Finally, the EMT-associated invasion programs exhibited by SW620 or HCT116 cells are suppressed by more than 80% after Snail1 knockdown in vivo (Fig. S2). Hence, the constitutive Wnt signaling activity that distinguishes the majority of all CRCs triggers a Snail1-dependent EMT program that is marked by both E-cadherin repression and BM invasion.

Fig. 2.

Axin2-dependent EMT program in CRC cells. (A) CRC cells were treated with vehicle or Wnt3a (100 ng/mL) for 24 h. Snail1 protein and mRNA levels were assessed by Western blotting (Upper) and qRT-PCR (Lower), respectively. (B) Mock or TCF-DN stably expressing cells were incubated in the presence or absence of Wnt3a, and Snail1 protein level was determined by Western blot analysis. (C) Axin2, E-cadherin, and vimentin levels were determined in cell lysates recovered from CRC cells stably expressing Scr-shRNA, Axin2-shRNA-4, or Axin2-shRNA-5. (D Left) Stable transfectants of CRC cells were fixed and stained with anti–E-cadherin antibody. Nuclei were stained with TOTO3 (blue). (Scale bar: 40 μm.) (D Right) Cells were stained with anti–E-cadherin and subjected to FACS analysis. (E) CRC cells were treated with CHIR99021 (100 ng/mL) for 24 h, and protein and mRNA levels were assessed by Western blotting and qRT-PCR, respectively. (F) CRC cells stably expressing Scr-shRNA or Axin2-shRNA were transfected with a mock, Flag-Snail1-WT, or Flag-Snail1-S96A construct, and Snail1 and β-actin levels in cell lysates were determined by Western blotting. *P < 0.05, **P < 0.01.

Axin2-Dependent Augmentation of the CRC EMT Program.

The constitutive activation of the β-catenin/TCF pathway in CRC tissues is known to trigger robust Axin2 expression in vivo in a presumed effort to down-regulate Wnt signaling (1, 2). Unexpectedly, when Axin2 is overexpressed in HCT116 or SW620 cells not only is TOPflash activity maintained (Fig. S3), but invasive activity is enhanced (see below). Because these results raise the possibility that endogenous Axin2 promotes an EMT-like program in CRC cells, Axin2 levels were stably repressed by ∼80% in HCT116 or SW620 cells with either of two specific shRNA constructs (without affecting Axin1 levels) (Fig. 2C). Under these conditions, both cell lines reexpress E-cadherin, increase E-cadherin promoter activity by more than twofold, and assume an epithelial phenotype (Fig. 2 C and D and Fig. S3). In tandem with the increased expression of E-cadherin and the expected decrease in the pool of free β-catenin, TOPflash reporter activity is also decreased by 25% (Fig. S3).

Recent studies indicate that canonical Wnt activation controls Snail1 protein levels by regulating GSK3β activity, wherein phosphorylation of an N-terminal, serine-rich motif in Snail1 triggers its β-TRCP–dependent ubiquitination and proteosomal degradation (8, 10). Consistent with an operative GSK3β-Snail1 axis in CRC cells, HCT116 or SW620 cells cultured in the presence of the GSK3 inhibitor, CHIR 99021 (15), increase Snail1 protein (but not mRNA) levels (Fig. 2E). In tandem with increased levels of Snail1 protein, levels of p-GSK3β(S9), an inactive form of the kinase (15–17), decrease slightly, whereas protein levels of p-GSK3β(Y216), the catalytically active form of the kinase, fall to background (Fig. 2E). Immunohistochemical analysis confirms that the CHIR 99021-induced increase in Snail1 protein expression is confined to the nuclear compartment where an expected decrease in the kinase activity of GSK3β is observed (Fig. S3). Likewise, Snail1 protein accumulates to higher levels when CRC cells are transfected with an epitope-tagged Snail1 expression vector harboring a stabilizing S→A(96) point mutation in its GSK3β phosphorylation motif relative to a wild-type Snail1 construct (Fig. 2F) (8, 10). Given that Axin family members can potentially impact GSK3 intracellular localization and substrate phosphorylation (10, 18, 19), the ability of Axin2 to modulate Snail1 protein levels was assessed. Importantly, when Axin2-silenced colon cancer cells are transfected with the wild-type epitope-tagged Snail1 construct, Snail1 protein levels reach a barely detectable level relative to that observed in control CRC cells (Fig. 2F). By contrast, when Axin2-silenced cells are transfected with the stabilized S→A(96) mutant, Snail1 is maintained at levels comparable to those detected in the control population (Fig. 2F). Conversely, when endogenous Axin2 levels are increased by culturing CRCs with the tankyrase inhibitor, XAV939 (5, 6), Snail1 levels increase while E-cadherin expression falls (Fig. S3). Taken together, these results indicate that increasing Axin2 levels—either via APC/β-catenin mutations or pharmacologic intervention—promotes CRC EMT. Indeed, consistent with the ability of Axin2 to stabilize Snail1, nuclear β-catenin, Axin2, and Snail1 are colocalized at the invasive front of colon carcinoma tissues (Fig. S4).

Axin2-Dependent Control of Nuclear GSK3β Activity.

Axin2 has been reported to stabilize nuclear Snail1 protein levels in Wnt-stimulated breast carcinoma cells by inducing the export of GSK3β from the nuclear compartment (10). However, silencing Axin2 in SW620 or HCT116 CRCs did not alter the subcellular distribution of GSK3β (Fig. 3A and Fig. S5), indicating the existence of alternate schemes for regulating the Axin2/Snail1 axis. In Axin2-silenced CRCs, no changes are detected in the levels of p-GSK3β(S9), but nuclear levels of the active, Y216 phosphorylated form of GSK3β increase significantly in parallel with enhanced nuclear GSK3β kinase activity and decreased nuclear Snail1 levels (Fig. 3B and Fig. S5) (15–17). Axin2 silencing did not affect cytosolic levels of p-GSK3β(Y216), cytosolic GSK3β kinase activity, or β-catenin content (Fig. 3A and Fig. S5). Conversely, when Axin2 levels are increased in transfected SW620 or HCT116 CRC cells, nuclear Snail1 protein levels increase in tandem with a decrease in nuclear p-GSK3β(Y216) content and nuclear GSK3β kinase activity (Fig. 3 C and D and Fig. S5). Furthermore, consistent with the ability of Axin2 to increase Snail1 levels, exogenous Axin2 reverses the induction of E-cadherin observed in HCT116 or SW620 cells that are transduced with TCF-DN (Fig. 1C). Finally, confirming the important role of Y216 in regulating GSK3β-mediated Snail1 phosphorylation, whereas Snail1 is actively phosphorylated in vitro by recombinant wild-type GSK3β (but not a catalytically inactive K85A mutant), the GSK3β Y216F mutant displays an ∼90% loss in Snail1 phosphorylating activity (Fig. 3E). Hence, Axin2 serves as a negative regulator of nuclear GSK3β activity that allows for the stabilization of nuclear Snail1 levels and activity.

Fig. 3.

Axin2-dependent control of nuclear GSK3β activity in CRC cells. (A) CRC cells stably expressing Scr-shRNA or Axin2-shRNA were fractioned into cytoplasmic and nuclear pools and subjected to Western blot analysis to determine Axin2, Snail1, GSK3β, p-GSK3β (Tyr216 and Ser9), or β-catenin levels. HDAC1 and β-tubulin are used as markers for nuclear and cytoplasmic fractions, respectively. (B) Cytoplasmic and nuclear fractions from the above cell lines were subjected to anti-GSK3β antibody immunoprecipitation, followed by GSK3β in vitro kinase assay with phosphoglycogen synthase peptide as a substrate. Inset displays the equal quantities of immunoprecipitated GSK3β in the nuclear fractions (mean ± SEM; n = 3). (C and D) CRC cells were transfected with Flag or Flag-Axin2 vectors, and cytoplasmic or nuclear fractions were subjected to Western blot analysis (C) or to anti-GSK3β antibody immunoprecipitation, followed by GSK3β in vitro kinase assay (D). (E) HA-tagged GSK3β protein was immunoprecipitated from HCT116 cells that were transfected with HA-GSK3β-WT, -K85A or -Y216F constructs, and the immunoprecipitated complex were subjected to GSK3β in vitro kinase assay. **P < 0.01.

Axin2 and the Regulation of the CRC Transcriptome and Invasion Programs.

To assess the impact of Axin2 expression on global CRC function, mRNA was isolated from control and Axin2-silenced SW620 cells for expression profiling. Using a minimum of twofold change in both of the two Axin2-silenced SW620 cell populations as a cutoff, depressing endogenous Axin2 levels four- to fivefold alters the expression of almost 3,000 unique transcripts (1,291 transcripts increased and 1,506 transcripts decreased; Dataset S1). Gene ontology (GO) analysis further highlights major changes in cell cycle regulation, cell death, and metabolic processes (Fig. S6 and Dataset S2). Consistent with the proposition that at least a subset of the affected genes reflect their identity as Snail1 and/or EMT-associated target transcripts, Axin2 silencing decreases expression of mesenchymal cell markers, vimentin, and fibronectin, in tandem with significant reductions in stem cell-associated markers (e.g., BMI1, CD44, and CTGF), tumor-promoting matrix metalloproteinases (e.g., MT1-MMP and MMP-7), and Snail1 regulatory factors (e.g., LOXL2) (all targets confirmed by real-time PCR; Fig. 4A and Fig. S6) (18–20). In turn, Axin2 silencing led to up-regulated expression of multiple epithelial-associated transcripts, including E-cadherin, H-cadherin, and claudin 3 (Fig. 4A). Interestingly, a series of the affected transcripts have been identified as targets of the β-catenin/TCF transcriptional network, including L1CAM, IL8, CYR61, and MMP7 (Fig. 4B and Fig. S6) (www.stanford.edu/group/nusselab/cgi-bin/wnt). Indeed, although Axin2 silencing would be predicted to increase the expression of β-catenin/TCF targets (i.e., by increasing the steady-state concentration of β-catenin/TCF complexes), the majority of canonical Wnt signaling targets identified in the Axin2 dataset—many of which represent Snail1 and/or EMT targets (e.g., FZD7, MET, MT1-MMP, IL8, PLAUR, CD44s; refs. 9 and 21–25)—are all down-regulated after Axin2 silencing in both SW620 and SW480 cells (Fig. 4A and Fig. S6), suggesting that the ability of Axin2 to drive Snail1 activity and trigger EMT-like programs overrides its ability to act as a feedback inhibitor of β-catenin/TCF signaling in CRC cells.

Fig. 4.

Axin2 is a master regulator of Wnt/β-catenin/TCF signaling gene expression programs. (A) GO terms identifying cellular processes that are differentially expressed in polyclonal populations of SW620 cells that stably express scramble- or Axin2-shRNAs. GO terms generated from up-regulated and down-regulated genes are colored red and green, respectively (P ≤ 0.05; Dataset S2). (B) Quantitative RT-PCR analysis for the indicated transcripts. (Mean ± SEM; n = 3; **P < 0.01.) (A) Heat map of predicted Wnt/β-catenin/TCF pathway targets relative to Axin2-silenced SW620 cells using either of two distinct shRNA constructs.

Given the apparent mesenchymal-epithelial shift in CRC behavior after Axin2 knockdown, the ability of Axin2-silenced cancer cells to breach the CAM basement membrane was assessed in vivo. As shown, Axin2-silenced SW620 cells, HCT116, DLD1, or SW480 are no longer able to invade the CAM interstitium as assessed by H&E staining, CRC immunolocalization with anti-human cytokeratin antibodies or immunofluorescence (Fig. 5 A and B and Figs. S1 and S7). By contrast, Axin2 overexpression accelerates the invasive properties of HCT116 or SW620 cells, most readily observed when the incubation time in the chicken embryo system is shortened from 4 to 2 d (Fig. 5C and Fig. S7). The ability of Axin2-silenced CRCs to express invasive activity can, however, be reconstituted, but only when Snail1 protein levels are reestablished by expressing the stabilized Snail1-S96A mutant (Fig. 5D and Fig. S7). Because Snail1 has been shown to play a critical role in driving metastatic events in mouse models in vivo (26, 27), the ability of Axin2-silenced SW620 cells to support metastatic activity after tail vein injection in nude mice was assessed. As predicted, Axin2-suppressed CRCs display a profound inhibition of macrometastatic and micrometastatic activity over an 8-wk time course (Fig. S8), reinforcing the prooncogenic activity of sustained Axin2 expression.

Fig. 5.

Axin2-dependent CRC cell invasion. (A and B) SW620 cells stably expressing Scr-shRNA, Axin2-shRNA-4, or Axin2-shRNA-5 were cultured on the chick CAM for 4 d. CAMs were sectioned and assessed for invasion by H&E staining (Left), by immunohistochemical analysis with anti–cytokeratin-18 antibody (Center), and by fluorescence microscopy (Right; labeled cells, green; type IV collagen, red; cell nuclei, blue). Invasion was assessed as described (mean ± SEM; n = 3). (Scale bar: 400 μm.) Relative invasive activity is quantified in B. (C) HCT116 and SW620 cells transfected with Flag or Flag-Axin2 construct were cultured on the chick CAM for 2 d, and relative invasive activity of the cells is quantified. (D) SW620 cells stably expressing Axin2-shRNA were transfected with mock or Flag-Snail-S96A vectors and cultured on the chick CAM for 4 d and relative invasive activity quantified. **, ##, P < 0.01.

Discussion

Virtually all CRCs are characterized by an inappropriate activation of the canonical Wnt pathway that triggers the induction of the β-catenin/TCF target, Axin2 (1–3). In an effort to squelch β-catenin/TCF activity in carcinomatous states, recent efforts have focused on developing pharmacologic interventions that increase Axin1 or Axin2 expression in CRC cells to levels capable of suppressing Wnt signaling (1–3). Although the possibility that CRCs might preserve Axin2 expression for more ominous purposes has seldom been considered (28), we demonstrate that CRC cells use the elevated endogenous levels of Axin2 to promote a Snail1-dependent EMT program that directs tissue-invasive potential (Fig. S9).

During CRC progression, cancer cells situated at the invasion front have been reported to display an EMT-like phenotype (3, 11). Indeed, invasive CRC cells frequently display the fingerprint of canonical Wnt signaling in tandem with the expression of EMT-inducing transcription factors, including Snail1 (11, 13, 29). Given the required role for β-catenin/TCF signaling in the Snail1-dependent EMT program activated in CRCs, coupled with the feedback inhibitor role normally assigned to Axin2, overexpressing Axin2 would be predicted to suppress canonical Wnt signaling pathways and decrease tissue-invasive activity. However, Axin2 overexpression not only failed to inhibit β-catenin/TCF reporter activity, but also stimulated invasion. More importantly, silencing endogenous Axin2 expression reversed the CRC EMT program, while also blocking BM transmigration and metastatic activity in vivo. Unexpectedly, Axin2 exerted far more complex effects on the CRC transcriptome than anticipated. The elevated levels of Axin2 commonly found in CRC cells increased—rather than decreased—the expression of multiple β-catenin/TCF targets in tandem with the silencing of a large cohort of tumor suppressor-associated transcripts, including CYLD, MeOX2, and CADM1 (Dataset S1) (30–32). These findings complement a recent study describing an unexpected, positive regulatory function for Axin1 and Axin2 in Wnt signaling (33). Nevertheless, it is unlikely that all Axin2-induced changes can be attributed to complementary changes in Snail1 activity or EMT, e.g., silencing L1CAM (a β-catenin/TCF target) has been reported to decrease CRC metastasis without inducing EMT (34). Nevertheless, our findings demonstrate that Snail1 remains a key Axin2 target as silencing Snail1 expression abrogated invasive and metastatic activities.

The functional impact of Axin2 on Snail1 activity was linked directly to its ability to regulate nuclear GSK3β activity. Because the transcriptional repressor activity of Snail1 localizes to the nuclear compartment (35), we considered the possibility that Axin2 might alter GSK3β nuclear localization in a process similar to that described for Wnt-stimulated breast cancer cells (10). Instead, nuclear Axin2 directly inhibited nuclear GSK3β activity without affecting its cellular distribution. Currently, the mechanisms by which Axin2 alters GSK3β activity and phosphorylation remain the subject of conjecture, but recent studies have linked changes in GSK3β Y216 phosphorylation and/or activity to a variety of factors, including PDGF-DD signaling or p38 MAPK activity (17, 36). Independent of changes in nuclear GSK3β activity, Axin2–GSK3β binding interactions can potentially alter the ability of kinase to recognize and phosphorylate target substrates (37). Whereas GSK3β catalyzes β-catenin phosphorylation when both components are incorporated into an APC/Axin docking complex, alternate targets, such as Snail1, are phosphorylated directly by GSK3β (10, 37). In this regard, when the ability of GSK3β to phosphorylate Snail1 was assessed under cell-free conditions in either the absence or presence of recombinant Axin2, GSK3β-dependent phosphorylation of Snail1 is inhibited as the Axin2:GSK3β ratio is increased (Fig. S10). Hence, Axin2 not only decreases nuclear GSK3β kinase activity directly, but also competitively inhibits the active kinase from targeting Snail1. Preliminary efforts designed to map Axin2 regions critical to Snail1 regulation failed to identify sites required for controlling β-catenin/TCF reporter activity or Snail1 protein levels (Fig. S10), but exogenously introduced mutants would have the opportunity to form heterodimers with the wild-type Axin2 monomers expressed by the host cell. Further studies in Axin2-silenced CRCs will be required to map the essential domains responsible for regulating GSK3β activity (38).

Consistent with our in vitro findings, and complementary reports documenting increased levels of nuclear β-catenin, Axin2, or Snail1 in CRCs in vivo (1–3, 29), these critical players were identified at the invasive front of a limited set of patient samples. Caution should, however, be exercised in attempting to develop simple correlations between nuclear Axin2 or Snail1 levels and disease status. First, the effects of nuclear Axin2 on Snail1 activity are confined to posttranslational events impacting GSK3β regulation of Snail1 protein activity rather than Snail1 transcript levels. Consequently, Axin2-dependent EMT will only be realized under conditions known to up-regulate Snail1 transcription (e.g., FGF, TNF or TGFβ) or translation (39). Second, while the presence of nuclear Snail1 protein is potentially indicative of its EMT-inducing potential, it should be stressed that its transcriptional repressor activity is further regulated by complex phosphorylation events mediated by as yet uncharacterized kinases (35). Likewise, with the realization that Snail1 silences target genes by forming complexes with a multiplicity of repressors, including HDAC1/2, Sin3a, LSD1, AJUBA/PRMT5, and EZH2 (39), the terminal impact of Snail1 on cell function will also be modulated by the coexpression of these and other accessory factors. Finally, when fully engaged, the ability of the β-catenin/TCF–Axin2–Snail1 axis to engage an EMT-like program accompanied by tissue-invasive activity may not correlate with effects on other tumorigenic events such as proliferation or metastatic potential (40). For example, in CRC, tissue-invasive cells migrating away from primary tumor sites have been shown to down-regulate proliferative activity (3, 11), although we do not detect changes in CRC proliferation in vitro after Axin2 silencing (Fig. S10). Similarly, cancer cell EMT programs can promote local invasion and intravasation, but the adoption of mesenchymal cell-like properties may not necessarily prove permissive for postextravasation growth at metastatic sites, although this contention also remains controversial (27, 40). Nevertheless, the ability of Snail1 to induce EMT, trigger BM invasion, exert antiapoptotic effects, and imbue expressing cells with stem cell-like properties is noteworthy (3, 25, 39).

The ability of Axin2 to promote a protumorigenic phenotype in CRC challenges current dogma, but we note that aberrant Wnt/β-catenin signaling has also been reported to induce chromosomal instability in CRC via an Axin2-dependent process (28, 38). Taken together, these findings suggest that the long-held supposition that therapeutic efforts aimed at increasing Axin2 levels in CRC patients should prove beneficial in the clinical setting requires reevaluation as does the contention that tankyrase inhibitors exert their effects by targeting Axin family members alone (5–7). Indeed, we alternatively conclude that the tumor suppressor function of Axin2 in normal cells is most likely coopted by cancer cells to promote, rather than suppress, key aspects of the cancer progression program.

Materials and Methods

Detailed protocols regarding cell culture, plasmid and shRNA construction, pharmacologic treatment of cells, cell extract preparation, antibodies, immunoprecipitation, in vitro kinase assays, luciferase reporter assay, recombinant protein purification, metastasis assays, transcriptional profiling, immunohistochemistry, Chick CAM invasion assay, and statistical analysis are described in SI Materials and Methods. All cell lines were obtained from ATCC. Animal protocols were approved by the Unit for Laboratory Animal Medicine at the University of Michigan.