Canonical Wnt Suppressor, Axin2, Promotes Colon Carcinoma Oncogenic Activity

Canonical Wnt suppressor, Axin2, promotes colon carcinoma oncogenic activity

Zhao-Qiu Wua, Thomas Brabletzb, Eric Fearona, Amanda L. Willisa, Casey Yuexian Hua, Xiao-Yan Lia, and Stephen J. Weissa,1

aDivision of Molecular Medicine and Genetics, Department of Internal Medicine, and the Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; and bDepartment of Visceral Surgery and Comprehensive Cancer Center Freiburg, University of Freiburg, 79106 Freiburg, Germany

Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved May 30, 2012 (received for review February 22, 2012)

Aberrant activation of canonical Wingless-type MMTV integration β-catenin, new therapeutics have been promoted that increase site family (Wnt) signaling is pathognomonic of colorectal cancers Axin2 (as well as Axin1) levels by targeting the poly-ADP ribo- (CRC) harboring functional mutations in either adenomatous sylating enzymes, tankyrase 1 and tankyrase 2 (5–7). polyposis coli or β-catenin. Coincident with Wnt cascade activa- Although Axin2 is uniformly categorized as a tumor suppres- – tion, CRCs also up-regulate the expression of Wnt pathway feed- sor in CRCs (1, 2, 5 7), its precise function in carcinomatous states is unknown, particularly with regard to the molecular back inhibitors, particularly the putative tumor suppressor, Axin2. “ ” Because Axin2 serves as a negative regulator of canonical Wnt rationale that underlies the decision of neoplastic cells to signaling in normal cells, recent attention has focused on the util- maintain high levels of a putative suppressor in vitro and in vivo. ity of increasing Axin2 levels in CRCs as a means to slow tumor Herein, we demonstrate that endogenous Axin2 promotes, rather than suppresses, a β-catenin/TCF-initiated, EMT program progression. However, rather than functioning as a tumor sup- in both β-catenin–mutant and APC-mutant CRCs. Up-regulation pressor, we demonstrate that Axin2 acts as a potent promoter of Axin2 levels in CRCs triggers marked increases in Snail1 ac- of carcinoma behavior by up-regulating the activity of the tran- tivity and induces EMT, whereas silencing endogenous Axin2 scriptional repressor, Snail1, inducing a functional epithelial- expression initiates a mesenchymal-epithelium–like switch that mesenchymal transition (EMT) program and driving metastatic ac- not only down-regulates Snail1, but also triggers widespread tivity. Silencing Axin2 expression decreases Snail1 activity, changes in the canonical Wnt signaling transcriptome. Because reverses EMT, and inhibits CRC invasive and metastatic activities Axin2 and Snail1 are further shown to be colocalized at the in- MEDICAL SCIENCES in concert with global effects on the Wnt-regulated cancer cell vasive front of CRC tissues, these studies suggest that Axin2 transcriptome. The further identiﬁcation of Axin2 and nuclear serves as an effective tumor promoter—rather than tumor sup- Snail1 proteins at the invasive front of human CRCs supports a repressor—that not only controls the induction of a Snail1-de- vised model wherein Axin2 acts as a potent tumor promoter pendent EMT program, but also exerts global control over gene in vivo. expression networks critical to invasive and metastatic behaviors.

invasion | E-cadherin | basement membrane | GSK3β | tankyrase Results Canonical Wnt Signaling Triggers a Colon Cancer Invasion Program. In olorectal cancers (CRCs) are generally characterized by ab- HCT116 or SW48 CRC cells that harbor heterozygous, stabi- Cerrant Wingless-type MMTV integration site family (Wnt) lizing mutations in β-catenin, each of the lines constitutively signaling (1–3). Neoplastic cells located at the invasive front of expresses β-catenin and Axin2 and TOPflash reporter activity CRCs frequently display increased levels of nuclear β-catenin (Fig. 1 A and B). In the presence of exogenous Wnt3a, however, and adopt a mesenchymal-like phenotype—events precipitated the cells respond with a significant increase in β-catenin protein by mutations predominantly occurring in adenomatous polypo- levels, Axin2 mRNA levels, and TOPflash reporter activity (Fig. sis coli (APC) or, more rarely, β-catenin (1–3). After nuclear 1 A and B). By contrast, in APC-defective SW620 cells, consti- translocation, β-catenin complexes with DNA-binding proteins tutive levels of β-catenin, Axin2, or TOPflash activity are maxi- of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family mally activated and unaffected by Wnt3a (Fig. 1 A and B). Both and triggers the expression of downstream genes linked to the β-catenin and APC mutant cell lines trigger canonical Wnt sig- induction of an invasion program that allows CRC cells to naling via a β-catenin/TCF-dependent pathway because Axin2 transmigrate the underlying basement membrane, gain access to mRNA levels are significantly suppressed when either HCT116 the host interstitial matrix, and infiltrate surrounding tissues (1– or SW620 cells are stably transfected with a dominant-negative, 4). However, in addition to supporting CRC invasion, the cohort FLAG-tagged TCF-4 construct (TCF-DN) (Fig. 1C) (8). Fur- of β-catenin/TCF-regulated target genes also includes feedback ther, although both HCT116 and SW620 populations are dom- inhibitors that serve to modulate the activity of the canonical inated by E-cadherin–negative or E-cadherin–low-expressing Wnt signaling cascade (1, 2). cells and assume mesenchymal cell-like phenotypes in vitro, the One of the most extensively studied canonical Wnt pathway TCF-DN stable transfectants up-regulate E-cadherin protein feedback inhibitors in CRCs is the scaffold protein, Axin2/con- levels and E-cadherin promoter activity while adopting an epi- ductin (1, 2). Under physiologic conditions, free β-catenin is thelial cell-like appearance (Fig. 1 C and D). targeted for degradation by a multiprotein complex composed of The most definitive characteristic of cancer cell EMT pro- APC, GSK3β, CK1, and either Axin1 or Axin2 (1, 2). Although grams is the ability of the transformed cell to traverse an intact, 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 effi- Author contributions: Z.-Q.W., E.F., and S.J.W. designed research; Z.-Q.W., T.B., A.L.W., ciently supports the GSK3β-dependent phosphorylation of C.Y.H., X.-Y.L., and S.J.W. performed research; T.B. contributed new reagents/analytic β-catenin in normal cells, which subsequently marks the post- tools; Z.-Q.W., T.B., E.F., X.-Y.L., and S.J.W. analyzed data; and Z.-Q.W. and S.J.W. wrote translationally modified protein for β-TrCP–dependent ubiq- the paper. uitination and proteosomal degradation (1, 2). However, the The authors declare no conflict of interest. ability of elevated levels of Axin2 to modulate β-catenin levels in This article is a PNAS Direct Submission. APC- or β-catenin–mutant CRC cells is muted (1, 2). Indeed, in 1To whom correspondence should be addressed. E-mail: [email protected]. an effort to silence the aberrant transcriptional programs acti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vated in CRCs as a function of the constitutive stabilization of 1073/pnas.1203015109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203015109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 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. (DLeft) 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.

type IV collagen-rich BM (4, 9). As such, HCT116 or SW620 tissues is known to trigger robust Axin2 expression in vivo in cells were cultured atop the chorioallantoic membrane (CAM) a presumed effort to down-regulate Wnt signaling (1, 2). Un- of live, 11-d-old chicken embryos wherein the upper epithelial expectedly, when Axin2 is overexpressed in HCT116 or SW620 cell layer is subtended by an intact BM (9). Under these con- cells not only is TOPflash activity maintained (Fig. S3), but in- ditions, both CRC cell lines rapidly degrade the underlying BM vasive activity is enhanced (see below). Because these results and invade the subjacent interstitial tissues (Fig. S1). By contrast, raise the possibility that endogenous Axin2 promotes an EMT- TCF-DN-transfectants of each cell line lose invasive potential like program in CRC cells, Axin2 levels were stably repressed by and remain confined to the upper CAM surface (Fig. S1). ∼80% in HCT116 or SW620 cells with either of two specific shRNA constructs (without affecting Axin1 levels) (Fig. 2C). β-Catenin/TCF Signaling Induces Snail-Regulated EMT and Tumor Under these conditions, both cell lines reexpress E-cadherin, Invasion in Colon Cancer Cells. During development and carcino- increase E-cadherin promoter activity by more than twofold, and genesis, activation of canonical Wnt signaling has been linked to assume an epithelial phenotype (Fig. 2 C and D and Fig. S3). In expression of the zinc-finger transcriptional repressor, Snail1 (3, tandem with the increased expression of E-cadherin and the 8, 10). To determine whether the constitutive Wnt signaling expected decrease in the pool of free β-catenin, TOPflash re- activity associated with CRC cells is sufficient to trigger Snail1 porter activity is also decreased by 25% (Fig. S3). protein expression, HCT116, SW48, or SW620 cells were cul- Recent studies indicate that canonical Wnt activation controls tured in the absence or presence of exogenous Wnt3a. Although Snail1 protein levels by regulating GSK3β activity, wherein phos- Wnt3a did not alter Snail1 mRNA expression levels in any of the phorylation of an N-terminal, serine-rich motif in Snail1 triggers cell lines tested, Snail1 protein levels increased significantly in its β-TRCP–dependent ubiquitination and proteosomal degra- Wnt3a-treated HCT116 or SW48 cells, whereas APC-mutant dation (8, 10). Consistent with an operative GSK3β-Snail1 axis in SW620, SW480, or DLD1 cells express Snail1 protein in a con- CRC cells, HCT116 or SW620 cells cultured in the presence stitutive fashion (Fig. 2A and Fig. S1). Induction of Snail1 pro- of the GSK3 inhibitor, CHIR 99021 (15), increase Snail1 protein tein expression is linked directly to the canonical Wnt pathway (but not mRNA) levels (Fig. 2E). In tandem with increased levels because HCT116 or SW620 cells that stably express a TCF-DN of Snail1 protein, levels of p-GSK3β(S9), an inactive form of the construct decrease Snail1 protein levels in the absence or pres- kinase (15–17), decrease slightly, whereas protein levels of p- ence of Wnt3a without affecting Snail1 mRNA expression (Fig. GSK3β(Y216), the catalytically active form of the kinase, fall to 2B and Fig. S2). Although the expression of Snail1 correlates background (Fig. 2E). Immunohistochemical analysis confirms with a Wnt-dependent EMT program, multiple transcription that the CHIR 99021-induced increase in Snail1 protein ex- factors have been reported to trigger similar EMT-like responses pression is confined to the nuclear compartment where an in CRCs, particularly Snail2/Slug or ZEB1 (11–13). However, expected decrease in the kinase activity of GSK3β is observed down-regulating β-catenin/TCF activity with the TCF-DN con- (Fig. S3). Likewise, Snail1 protein accumulates to higher levels struct did not affect mRNA expression levels of either of these when CRC cells are transfected with an epitope-tagged Snail1 transcription factors (Fig. S2). Further, when Snail1 expression expression vector harboring a stabilizing S→A(96) point muta- is silenced in either HCT116 or SW620 cells by either of two tion in its GSK3β phosphorylation motif relative to a wild-type shRNA constructs, E-cadherin is reexpressed, whereas TOP- Snail1 construct (Fig. 2F) (8, 10). Given that Axin family mem- Flash reporter activity is down-regulated in a manner consistent bers can potentially impact GSK3 intracellular localization and with the ability of Snail1 to modulate β-catenin/TCF activity (Fig. substrate phosphorylation (10, 18, 19), the ability of Axin2 to S2) (14). Finally, the EMT-associated invasion programs ex- modulate Snail1 protein levels was assessed. Importantly, when hibited by SW620 or HCT116 cells are suppressed by more than Axin2-silenced colon cancer cells are transfected with the wild- 80% after Snail1 knockdown in vivo (Fig. S2). Hence, the con- type epitope-tagged Snail1 construct, Snail1 protein levels reach stitutive Wnt signaling activity that distinguishes the majority of a barely detectable level relative to that observed in control all CRCs triggers a Snail1-dependent EMT program that is CRC cells (Fig. 2F). By contrast, when Axin2-silenced cells marked by both E-cadherin repression and BM invasion. are transfected with the stabilized S→A(96) mutant, Snail1 is maintained at levels comparable to those detected in the control Axin2-Dependent Augmentation of the CRC EMT Program. The population (Fig. 2F). Conversely, when endogenous Axin2 levels constitutive activation of the β-catenin/TCF pathway in CRC are increased by culturing CRCs with the tankyrase inhibitor,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203015109 Wu et al. Downloaded by guest on September 28, 2021 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 in- cubated 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- MEDICAL SCIENCES shRNA-4, or Axin2-shRNA-5. (DLeft) Stable transfectants of CRC cells were ﬁxed and stained with anti–E-cadherin antibody. Nuclei were stained with TOTO3 (blue). (Scale bar: 40 μm.) (DRight) 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.

XAV939 (5, 6), Snail1 levels increase while E-cadherin expres- with the ability of Axin2 to increase Snail1 levels, exogenous sion falls (Fig. S3). Taken together, these results indicate that Axin2 reverses the induction of E-cadherin observed in HCT116 increasing Axin2 levels—either via APC/β-catenin mutations or or SW620 cells that are transduced with TCF-DN (Fig. 1C). pharmacologic intervention—promotes CRC EMT. Indeed, Finally, confirming the important role of Y216 in regulating consistent with the ability of Axin2 to stabilize Snail1, nuclear GSK3β-mediated Snail1 phosphorylation, whereas Snail1 is ac- β-catenin, Axin2, and Snail1 are colocalized at the invasive front tively phosphorylated in vitro by recombinant wild-type GSK3β of colon carcinoma tissues (Fig. S4). (but not a catalytically inactive K85A mutant), the GSK3β Y216F mutant displays an ∼90% loss in Snail1 phosphorylating Axin2-Dependent Control of Nuclear GSK3β Activity. Axin2 has been activity (Fig. 3E). Hence, Axin2 serves as a negative regulator of reported to stabilize nuclear Snail1 protein levels in Wnt-stim- nuclear GSK3β activity that allows for the stabilization of nuclear ulated breast carcinoma cells by inducing the export of GSK3β Snail1 levels and activity. from the nuclear compartment (10). However, silencing Axin2 in SW620 or HCT116 CRCs did not alter the subcellular distribu- Axin2 and the Regulation of the CRC Transcriptome and Invasion tion of GSK3β (Fig. 3A and Fig. S5), indicating the existence of Programs. To assess the impact of Axin2 expression on global alternate schemes for regulating the Axin2/Snail1 axis. In Axin2- CRC function, mRNA was isolated from control and Axin2-si- silenced CRCs, no changes are detected in the levels of p-GSK3β lenced SW620 cells for expression profiling. Using a minimum of (S9), but nuclear levels of the active, Y216 phosphorylated form twofold change in both of the two Axin2-silenced SW620 cell of GSK3β increase significantly in parallel with enhanced nuclear populations as a cutoff, depressing endogenous Axin2 levels GSK3β kinase activity and decreased nuclear Snail1 levels (Fig. four- to fivefold alters the expression of almost 3,000 unique 3B and Fig. S5) (15–17). Axin2 silencing did not affect cytosolic transcripts (1,291 transcripts increased and 1,506 transcripts levels of p-GSK3β(Y216), cytosolic GSK3β kinase activity, or decreased; Dataset S1). Gene ontology (GO) analysis further β-catenin content (Fig. 3A and Fig. S5). Conversely, when Axin2 highlights major changes in cell cycle regulation, cell death, and levels are increased in transfected SW620 or HCT116 CRC cells, metabolic processes (Fig. S6 and Dataset S2). Consistent with nuclear Snail1 protein levels increase in tandem with a decrease the proposition that at least a subset of the affected genes reflect in nuclear p-GSK3β(Y216) content and nuclear GSK3β kinase their identity as Snail1 and/or EMT-associated target transcripts, activity (Fig. 3 C and D and Fig. S5). Furthermore, consistent Axin2 silencing decreases expression of mesenchymal cell

Wu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 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.

markers, vimentin, and fibronectin, in tandem with significant MMP, IL8, PLAUR, CD44s; refs. 9 and 21–25)—are all down- reductions in stem cell-associated markers (e.g., BMI1, CD44, regulated after Axin2 silencing in both SW620 and SW480 cells and CTGF), tumor-promoting matrix metalloproteinases (e.g., (Fig. 4A and Fig. S6), suggesting that the ability of Axin2 to drive MT1-MMP and MMP-7), and Snail1 regulatory factors (e.g., Snail1 activity and trigger EMT-like programs overrides its LOXL2) (all targets confirmed by real-time PCR; Fig. 4A and ability to act as a feedback inhibitor of β-catenin/TCF signaling Fig. S6) (18–20). In turn, Axin2 silencing led to up-regulated in CRC cells. expression of multiple epithelial-associated transcripts, including Given the apparent mesenchymal-epithelial shift in CRC be- E-cadherin, H-cadherin, and claudin 3 (Fig. 4A). Interestingly, havior after Axin2 knockdown, the ability of Axin2-silenced a series of the affected transcripts have been identified as targets cancer cells to breach the CAM basement membrane was assessed of the β-catenin/TCF transcriptional network, including L1CAM, in vivo. As shown, Axin2-silenced SW620 cells, HCT116, DLD1, IL8, CYR61, and MMP7 (Fig. 4B and Fig. S6)(www.stanford. or SW480 are no longer able to invade the CAM interstitium as edu/group/nusselab/cgi-bin/wnt). Indeed, although Axin2 silenc- assessed by H&E staining, CRC immunolocalization with anti- ing would be predicted to increase the expression of β-catenin/ human cytokeratin antibodies or immunofluorescence (Fig. 5 A TCF targets (i.e., by increasing the steady-state concentration of and B and Figs. S1 and S7). By contrast, Axin2 overexpression β-catenin/TCF complexes), the majority of canonical Wnt sig- accelerates the invasive properties of HCT116 or SW620 cells, naling targets identified in the Axin2 dataset—many of which most readily observed when the incubation time in the chicken represent Snail1 and/or EMT targets (e.g., FZD7, MET, MT1- embryo system is shortened from 4 to 2 d (Fig. 5C and Fig. S7).

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.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203015109 Wu et al. Downloaded by guest on September 28, 2021 The ability of Axin2-silenced CRCs to express invasive activity a Snail1-dependent EMT program that directs tissue-invasive can, however, be reconstituted, but only when Snail1 protein potential (Fig. S9). levels are reestablished by expressing the stabilized Snail1-S96A During CRC progression, cancer cells situated at the invasion mutant (Fig. 5D and Fig. S7). Because Snail1 has been shown to front have been reported to display an EMT-like phenotype fi play a critical role in driving metastatic events in mouse models (3, 11). Indeed, invasive CRC cells frequently display the n- in vivo (26, 27), the ability of Axin2-silenced SW620 cells to gerprint of canonical Wnt signaling in tandem with the expres- support metastatic activity after tail vein injection in nude mice sion of EMT-inducing transcription factors, including Snail1 (11, 13, 29). Given the required role for β-catenin/TCF signaling was assessed. As predicted, Axin2-suppressed CRCs display in the Snail1-dependent EMT program activated in CRCs, a profound inhibition of macrometastatic and micrometastatic coupled with the feedback inhibitor role normally assigned to activity over an 8-wk time course (Fig. S8), reinforcing the Axin2, overexpressing Axin2 would be predicted to suppress prooncogenic activity of sustained Axin2 expression. canonical Wnt signaling pathways and decrease tissue-invasive activity. However, Axin2 overexpression not only failed to inhibit Discussion β-catenin/TCF reporter activity, but also stimulated invasion. Virtually all CRCs are characterized by an inappropriate activa- More importantly, silencing endogenous Axin2 expression re- tion of the canonical Wnt pathway that triggers the induction of versed the CRC EMT program, while also blocking BM trans- the β-catenin/TCF target, Axin2 (1–3). In an effort to squelch migration and metastatic activity in vivo. Unexpectedly, Axin2 β-catenin/TCF activity in carcinomatous states, recent efforts have exerted far more complex effects on the CRC transcriptome than focused on developing pharmacologic interventions that increase anticipated. The elevated levels of Axin2 commonly found in Axin1 or Axin2 expression in CRC cells to levels capable of CRC cells increased—rather than decreased—the expression of β suppressing Wnt signaling (1–3). Although the possibility that multiple -catenin/TCF targets in tandem with the silencing of CRCs might preserve Axin2 expression for more ominous pur- a large cohort of tumor suppressor-associated transcripts, including CYLD, MeOX2, and CADM1 (Dataset S1) (30–32). poses has seldom been considered (28), we demonstrate that CRC fi cells use the elevated endogenous levels of Axin2 to promote These ndings complement a recent study describing an un- expected, 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 fi inducing EMT (34). Nevertheless, our ndings demonstrate that MEDICAL SCIENCES 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 mecha- nisms 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 sub- strates (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 Fig. 5. Axin2-dependent CRC cell invasion. (A and B) SW620 cells stably the opportunity to form heterodimers with the wild-type Axin2 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 monomers expressed by the host cell. Further studies in Axin2- – silenced CRCs will be required to map the essential domains re- H&E staining (Left), by immunohistochemical analysis with anti cytokeratin- β 18 antibody (Center), and by fluorescence microscopy (Right; labeled cells, sponsible for regulating GSK3 activity (38). Consistent with our in vitro findings, and complementary green; type IV collagen, red; cell nuclei, blue). Invasion was assessed as de- β scribed (mean ± SEM; n = 3). (Scale bar: 400 μm.) Relative invasive activity is reports documenting increased levels of nuclear -catenin, quantified in B.(C) HCT116 and SW620 cells transfected with Flag or Flag- Axin2, or Snail1 in CRCs in vivo (1–3, 29), these critical players Axin2 construct were cultured on the chick CAM for 2 d, and relative invasive were identified at the invasive front of a limited set of patient activity of the cells is quantified. (D) SW620 cells stably expressing Axin2- samples. Caution should, however, be exercised in attempting to shRNA were transfected with mock or Flag-Snail-S96A vectors and cultured on develop simple correlations between nuclear Axin2 or Snail1 the chick CAM for 4 d and relative invasive activity quantified. **, ##, P < 0.01. levels and disease status. First, the effects of nuclear Axin2 on

Wu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Snail1 activity are confined to posttranslational events impacting The ability of Axin2 to promote a protumorigenic phenotype GSK3β regulation of Snail1 protein activity rather than Snail1 in CRC challenges current dogma, but we note that aberrant transcript levels. Consequently, Axin2-dependent EMT will only Wnt/β-catenin signaling has also been reported to induce chro- be realized under conditions known to up-regulate Snail1 tran- mosomal instability in CRC via an Axin2-dependent process (28, β scription (e.g., FGF, TNF or TGF ) or translation (39). Second, 38). Taken together, these findings suggest that the long-held while the presence of nuclear Snail1 protein is potentially in- supposition that therapeutic efforts aimed at increasing Axin2 dicative of its EMT-inducing potential, it should be stressed that fi its transcriptional repressor activity is further regulated by levels in CRC patients should prove bene cial in the clinical complex phosphorylation events mediated by as yet uncharac- setting requires reevaluation as does the contention that tank- terized kinases (35). Likewise, with the realization that Snail1 yrase inhibitors exert their effects by targeting Axin family silences target genes by forming complexes with a multiplicity of members alone (5–7). Indeed, we alternatively conclude that the repressors, including HDAC1/2, Sin3a, LSD1, AJUBA/PRMT5, tumor suppressor function of Axin2 in normal cells is most likely and EZH2 (39), the terminal impact of Snail1 on cell function coopted by cancer cells to promote, rather than suppress, key will also be modulated by the coexpression of these and other aspects of the cancer progression program. accessory factors. Finally, when fully engaged, the ability of the β-catenin/TCF–Axin2–Snail1 axis to engage an EMT-like pro- Materials and Methods gram accompanied by tissue-invasive activity may not correlate Detailed protocols regarding cell culture, plasmid and shRNA construction, with effects on other tumorigenic events such as proliferation or pharmacologic treatment of cells, cell extract preparation, antibodies, immu- metastatic potential (40). For example, in CRC, tissue-invasive noprecipitation, in vitro kinase assays, luciferase reporter assay, recombinant cells migrating away from primary tumor sites have been shown protein purification, metastasis assays, transcriptional profiling, immuno- to down-regulate proliferative activity (3, 11), although we do histochemistry, Chick CAM invasion assay, and statistical analysis are de- not detect changes in CRC proliferation in vitro after Axin2 silencing (Fig. S10). Similarly, cancer cell EMT programs can scribed in SI Materials and Methods. All cell lines were obtained from ATCC. promote local invasion and intravasation, but the adoption of Animal protocols were approved by the Unit for Laboratory Animal Medi- mesenchymal cell-like properties may not necessarily prove cine at the University of Michigan. permissive for postextravasation growth at metastatic sites, although this contention also remains controversial (27, 40). ACKNOWLEDGMENTS. This work was supported by National Institutes of Health Grants CA116516 (to S.J.W. and E.F.) and R01 CA071699 (to S.J.W.), Nevertheless, the ability of Snail1 to induce EMT, trigger BM the Breast Cancer Research Foundation, and Michigan Diabetes Research invasion, exert antiapoptotic effects, and imbue expressing cells and Training Center Cell and Molecular Biology Core National Institutes of with stem cell-like properties is noteworthy (3, 25, 39). Health Grant P60 DK020572.

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