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The proinvasive activity of Wnt-2 is mediated through a noncanonical Wnt pathway coupled to GSK-3ß and c-Jun/AP-1 signaling

Nathalie Le Floch, Christine Rivat, Olivier De Wever^*, Erik Bruyneel^*, Marc Mareel^*, Trevor Dale and Christian Gespach¹

INSERM U482, Signal transduction and Cellular Functions in Diabetes and Digestive Cancers, Hôpital Saint-Antoine, Paris Cedex, France;
^* Laboratory of Experimental Cancerology, University Hospital, Gent, Belgium; and
Cardiff School of Biosciences, Cardiff, UK

¹Correspondence: INSERM Unité U482, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: gespach{at}st-antoine.inserm.fr

SPECIFIC AIMS

Inappropriate activation of the Wnt/APC/ß-catenin signaling pathways plays a critical role in human cancer. We reported that Wnt-2 is overexpressed during the neoplastic progression of human colonic tumors. However, the role of Wnt-2 and the relative contribution of the Wnt pathways in tumor cell invasion are still hypothetical. We therefore examined the impact of Wnt-2 and classical regulators of the Wnt pathways on the invasive potential of human colorectal and kidney cancer cells in collagen type I gels.

PRINCIPAL FINDINGS

1. The proinvasive activity of Wnt-2 is determined by heterotrimeric G-proteins coupled to a noncanonical Wnt transcription pathway
First, we observed that activation or inhibition of the canonical Wnt transcription pathway through signaling intermediates targeting Dvl-2 or the multimolecular complex comprising Axin, GSK-3ß, and ß-catenin did not alter cancer cell invasiveness. Moreover, the Wnt pathway inhibitor wt-Axin was unable to abrogate the invasive phenotype determined by ß-catenin regulators src, HGF/Met, and trefoil peptides. Dvl-2 and Ax501-560 exerted a permissive cross-talk with low and ineffective concentrations of HGF and ITF at the threshold invasion response (2 U/mL HGF and 30 nM ITF). Upstream activation of the Wnt pathways by forced expression of Wnt-2 or addition of the conditioned medium prepared from Wnt-2-transformed cells (Wnt-2 CM) induced invasion through several oncogenic pathways, including PI3-kinase, the Rho/Rho-kinase (ROCK) cascade, phospholipase C, p42/p44, p38 MAPK/SAPK (Fig. 1 A), and the EGFR tyrosine kinase (Iressa; not shown). Accordingly, invasion induced by Wnt-2 was prevented by pertussis toxin (PTx), which inactivates members of the Go/i subfamily of heterotrimeric G-proteins, Gß subunits sequestrant ct-ßARK (Fig. 1A, B ), and the soluble Wnt inhibitors sFRP-3 (Fig. 1C ) and sFRP-1. As expected, Wnt-2 CM activity was alleviated by the dominant negative form of the small GTPase RhoA (T19N-RhoA), whereas the corresponding Rac1 and Cdc42 (T17N) mutants as well as wt-Axin were ineffective (Fig. 1C ). Immunodepletion of Wnt-2 in the Wnt-2 CM impaired the proinvasive potential of this CM, suggesting that Wnt-2 is the direct proinvasive factor secreted by Wnt-2 transformed cells (Fig. 1D ). Purified Wnt-3a peptide also stimulated collagen invasion by HEK-293T and HCT8/S11 cells.

View larger version (31K): [in this window] [in a new window]   Figure 1. The proinvasive activity of Wnt-2 is determined by a noncanonical Wnt pathway in human colorectal cancer cells HCT8/S11. A) Ectopic expression of Wnt-2 induces invasion. Wnt-2 overexpression was analyzed by Western blot with an anti-HA antibody. Data presented were obtained with clone 3; pool 1 and clone 8 gave similar results. PTx and the PKA inhibitor KT5720 were added for 24 h at concentrations of 200 ng/mL and 1 µM, respectively. B) Sequestration of Gß subunits by ct-ßARK abolished Wnt-2 proinvasive activity. HCT8/S11-Wnt-2 (clone 3) was stably transfected with an expression vector encoding the C-terminal domain of the ß-adrenergic receptor kinase. Positive clones (C11 and C12) were selected for invasion assays. C) Wnt-2 CM induces invasion independently of the canonical Wnt pathway. Parental and HCT8/S11 cell lines stably expressing wt-Axin or dominant negative mutants of Rho (T19N-RhoA) and Rac (T17N-Rac) were incubated for 24 h in the absence or presence of conditioned media harvested from HEK-293T-pcDNA3 (pcDNA3 CM) or HEK-293T-Wnt-2 kidney cancer cells (Wnt-2 CM). D) Wnt-2 is responsible for the proinvasive activity of Wnt-2 CM. Left: HEK-293T cells were pretreated for 1 h with sFRP-3 before incubation with Wnt-2 CM or pS2. Right: Wnt-2 CM was immunodepleted by an antibody directed against the HA epitope of ectopic Wnt-2. Number of immunoprecipitation rounds performed is indicated beneath the graph. Residual levels of Wnt-2 were detected by immunoblot. All data are means ± SE of 3 or 4 experiments.

2. Inhibition of GSK-3ß promotes cell invasiveness
Invasiveness promoted by the GSK-3ß inhibitors lithium and SB216763 required PI3-kinase, the Rho/ROCK axis, and was insensitive to PTx, as expected (Fig. 2 B, C). Upstream activation of the Wnt pathways by the Wnt-2 CM was indeed abrogated by the toxin (Fig. 2C ). GSK-3ß inhibitors lithium (30 mM) and SB216763 (10–50 µM) both induced the stabilization and accumulation of ß-catenin in our model (Fig. 2A ). Accordingly, expression of a kinase-dead mutant (GSK-3ß-K85R) or a GSK-3ß mutant unable to bind primed substrates (R96E) confers a constitutive invasive phenotype via PI-3K and Rho/ROCK-dependent and Go/i-Gß-independent pathways (Fig. 2D, E ).

View larger version (21K): [in this window] [in a new window]   Figure 2. Inhibition of GSK-3ß promotes cell invasiveness. A) Stabilization of ß-catenin by the GSK-3ß inhibitors SB216763 and LiCl. HEK-293T cells were incubated for 6 h with SB216763 (10–50 µM) or LiCl (30 mM). Total ß-catenin levels by Western blot analysis; ß-actin as a loading control. B) GSK-3ß inhibitors induce the invasive phenotype in HEK-293T or HCT8/S11 cells in the 24 h invasion assay. C) Effects of pharmacological inhibitors with selectivity for PI3K, Rho/ROCK, and Go/i proteins against invasion induced by the Wnt-2 CM or GSK-3ß inhibitors. D, E) Stable expression of the dominant negative mutants targeting GSK-3ß promotes cell invasion in HEK-293T cells. Positive individual clones (C2-C6 or C8) and pools of clones (P1-P2) were identified by Western blot (antibody against the HA tag of the constructs). Data are means ± SE of 3 experiments.

3. Requirement of Jun/AP-1 activity for Wnt-2-induced matrix metalloprotease matrilysin (MMP-7) expression and cellular invasion
Finally, we demonstrated that Wnt-2 activated expression of the matrilysin gene and increased accumulation of the cleavage-activated form of the matrilysin protease (18 kDa) through reciprocal implication of the AP-1 and LEF/Tcf binding sites at the MMP-7 promoter (Mp). In agreement, the proinvasive activity of Wnt-2 was abrogated by the c-jun dominant negative mutant TAM-67. Axin partly reversed Wnt-2-induced Mp activation, suggesting that the invasive phenotype determined by Wnt-2 might be monitored through signaling components using the canonical Wnt pathway and matrilysin as matrix protease.

CONCLUSIONS AND SIGNIFICANCE

We have identified a new function of Wnt-2 as a proinvasive agent acting through a noncanonical Wnt transcription pathway coupled to GSK-3ß and AP-1 signaling at the matrilysin promoter (Fig. 3 ). The reciprocal interdependence of the AP-1/Jun and LEF/Tcf elements in the matrilysin promoter activated by Wnt-2 signals provides an important new insight into the mechanisms involved in the control of cellular invasion and expression of the matrix metalloprotease MMP-7. This molecular interdependence is closely related to the cross-talk between canonical Wnt activators (Dvl-2 and Ax501-560) and other signaling pathways activated by HGF and ITF, as shown here. Matrilysin and Dvl-2 overexpression as well as loss-of-functions of Axin are reported in human cancers, including several human solid tumors of breast, prostate, lung, digestive tract, and 90% of adenomas from familial adenomatous polyposis patients. The AP-1 response element is present in the promoter region of several oncogenic elements as well as serine proteases and matrix metalloprotease genes implicated in cellular invasion, including urokinase plasminogen activators MMP-1, -3, -9, and -13. Thus, GSK-3ß exerts a key role in the determination of the invasive potential of Wnt-2 and other invasion promoters inhibiting GSK-3ß including EGF-R, insulin, LPA, integrin-linked kinase, PI3-K/Akt, and the cAMP-PKA cascade. Accordingly, selective depletion of the GSK-3ß protein by RNA interference impaired the proinvasive activity of Wnt-2 in our study (Fig. 3) . CD44 has been shown to mediate the interaction of MMP-7 with pro HB-EGF and EGFR. Within this complex, pro HB-EGF is processed by MMP-7 and the resulting mature HB-EGF can activate EGFR. After EGFR stimulation, several proinvasive pathways are activated, including PI-3K/Akt and MAPK pathways, both targeting GSK-3ß inhibition.

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating a suggested mechanism of Wnt-2 proinvasive pathways. Proinvasive activity of Wnt-2 is mediated through a noncanonical Wnt pathway using GSK-3ß and the c-jun oncogene in human kidney and colorectal cancer cells. Internal Wnt regulators such as Dvl-2 and Axin respectively exert permissive and negative controls on cellular invasion and matrilysin promoter activity, suggesting that the canonical Wnt pathway can modulate the activity of other proinvasive agents in matrix substrates and corresponding proteases. Thus, inhibition of GSK-3ß and activation of MMP-7 by Wnt-2 and G-protein coupled Fz receptors initiate and amplify a constitutive proinvasive loop using HB-EGF shedding, EGFR and PI3K/Akt activation, and, in turn, GSK-3ß inhibition. Fz: frizzled receptors; sFRP-3: soluble inhibitor preventing the Wnt/Fz interaction; ct-ßARK: carboxyl-terminal end of the ßARK kinase; PTx: pertussis toxin; PI3K/Akt: phosphatidyl inositol 3' kinase activating the PKB/Akt kinase; lithium and SB216763: GSK-3ß inhibitors; DN-GSK-3ß: dominant negative forms of GSK-3ß; pro HB-EGF: precursor form of the heparin binding epidermal growth factor; CD44: cell surface proteoglycan; EGFR: epidermal growth factor receptor; Iressa: pharmacological inhibitor of the EGFR tyrosine kinase.

Our data provide a potential clue to understanding the cross-talk between Wnt activators and other proinvasive pathways in relation to matrix substrates and effective proteases within the tumors. Indeed, Wnt factors and GSK-3ß are connected to a large array of signaling pathways and cellular functions linked to normal development (embryonic stem cells, body pattern, progenitor self-renewal), inflammatory and metabolic diseases (brain, blood and peripheral system), and the neoplastic progression in human solid tumors. It is thus of great interest to better understand the mechanisms underlying the activity of Wnt-2 and other proinvasive factors acting through GSK-3ß inhibition and Wnt pathways, as it will probably allow the design of new therapeutic interventions to circumvent the progression of colorectal cancer toward metastasis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2373fje;

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