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.

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.

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.

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.

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.

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.