Fas-associated Factor 1 Is a Scaffold Protein That Promotes β-Transducin Repeat-containing Protein (β-TrCP)-mediated β-Catenin Ubiquitination and Degradation*

Background: Wnts activate transcriptional responses via stabilization and nuclear translocation of β-catenin. Results: FAF1 associates with SCF-β-TrCP E3 ligase complex and promotes cytosolic β-catenin polyubiquitination and degradation. Conclusion: FAF1 is a scaffold protein that promotes β-TrCP-mediated β-catenin degradation. Significance: FAF1 may have tumor suppressive activity by antagonizing Wnt/β-catenin signaling. FAS-associated factor 1 (FAF1) antagonizes Wnt signaling by stimulating β-catenin degradation. However, the molecular mechanism underlying this effect is unknown. Here, we demonstrate that the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) is required for FAF1 to suppress Wnt signaling and that FAF1 specifically associates with the SCF (Skp1-Cul1-F-box protein)-β-TrCP complex. Depletion of β-TrCP reduced FAF1-mediated β-catenin polyubiquitination and impaired FAF1 in antagonizing Wnt/β-catenin signaling. FAF1 was shown to act as a scaffold for β-catenin and β-TrCP and thereby to potentiate β-TrCP-mediated β-catenin ubiquitination and degradation. Data mining revealed that FAF1 expression is statistically down-regulated in human breast carcinoma compared with normal breast tissue. Consistent with this, FAF1 expression is higher in epithelial-like MCF7 than mesenchymal-like MDA-MB-231 human breast cancer cells. Depletion of FAF1 in MCF7 cells resulted in increased β-catenin accumulation and signaling. Importantly, FAF1 knockdown promoted a decrease in epithelial E-cadherin and an increase in mesenchymal vimentin expression, indicative for an epithelial to mesenchymal transition. Moreover, ectopic FAF1 expression reduces breast cancer cell migration in vitro and invasion/metastasis in vivo. Thus, our studies strengthen a tumor-suppressive function for FAF1.

Wnt proteins control many biological processes, including embryonic development and tumorigenesis (1)(2)(3)(4). One of the critical components of the canonical Wnt signaling pathway is ␤-catenin, the levels of which are tightly controlled via protein degradation. In the absence of Wnt ligands, the majority of ␤-catenin is associated with E-cadherin on the plasma membrane, whereas the levels of intracellular ␤-catenin are kept low in a phosphorylation-dependent manner by components of the proteasomal degradation pathway. Casein kinase 1 (CK1) and GSK3␤ sequentially catalyze ␤-catenin phosphorylation at residues Ser-45, Thr-41, Ser-37, and Ser-33, thereby acting in a destruction complex with adenomatous polyposis coli and Axin. Phosphorylated ␤-catenin is subsequently recognized by the F-box protein ␤-transducin repeat-containing protein (␤-TrCP), 4 a component of the ubiquitin ligase complex that triggers ubiquitin-dependent degradation of ␤-catenin by the proteasome (5,6). Wnt ligands induce the disruption of the cytosolic ␤-catenin degradation complex (7,8) and thereby enable ␤-catenin to enhance lymphoid enhancer-binding factor 1 (LEF)/T cell factor-mediated transcription in the nucleus (9,10).
As a member of the Fas death-inducing signaling complex, FAS-associated factor 1 (FAF1) was first identified to potentiate apoptosis in L cells and T cells (19 -22). Faf1 gene-targeted mice show embryonic lethality at the two-cell stage (23). In cellular signaling, FAF1 is linked to the regulation of NF-B activity as well as to valosin containing protein-mediated ubiquitination and proteasomal degradation (24 -27). Decreased FAF1 expression was found to be correlated with a high percentage of human gastric carcinomas (28), and genomic loss or deletion of FAF1 has been reported in uterine cervix carcinomas and mantle cell lymphoma (29,30). Therefore, FAF1 has been postulated to act as a tumor suppressor. We previously identified FAF1 as a potent negative regulator of canonical Wnt signaling and a strong repressor of Wnt/␤-catenin-induced osteoblast differentiation and bone formation (31). However, the underlying mechanism by which FAF1 promotes ␤-catenin degradation is unclear.
In this study, we screened components of the ␤-catenin destruction complex for their ability to interact with FAF1. FAF1 was shown to form a ternary complex with ␤-catenin and ␤-TrCP and thereby to promote ␤-TrCP-mediated ␤-catenin ubiquitination and degradation. Moreover, we found FAF1 to inhibit epithelial to mesenchymal transition and invasion/metastasis in breast cancer cells, enforcing the notion of its action as tumor suppressor.
Luciferase Reporter Assay-Cells were transfected and lysed as described (36, 39 -41), and luciferase activities were measured by a luminometer (Berthold Technologies). Reporter activity was normalized to ␤-gal activity, resulting from a cotransfected internal control plasmid. Experiments were performed in triplicate.
Transfection, Immunoprecipitation, and Immunoblotting-Cells were transiently transfected using calcium phosphate or Lipofectamine (Invitrogen). 40 h after transfection, cells were lysed with 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 25 mM NaF, 1% Triton X-100) plus protease inhibitors (Sigma) for 30 min at 4°C. After centrifugation at 12 ϫ 10 3 g for 15 min, the lysates were immunoprecipitated with specific antibody and protein A-Sepharose (Zymed Laboratories Inc.) for 3 h at 4°C. Thereafter, the precipitants were washed three times with washing buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), and the immune complexes were eluted with sample buffer containing 1% SDS for 5 min at 95°C and analyzed by SDS-PAGE. Immunoblotting was performed with specific antibody and secondary anti-mouse or anti-rabbit antibodies that were conju-gated to horseradish peroxidase (Amersham Biosciences). Proteins were visualized by chemiluminescence.
Lentiviral Transduction and Generation of Stable Cell Lines-Lentiviruses were produced by transfecting pLKO-1 (for the shRNA-knockdown) or pLV-bc-CMV (for cDNA expression) plasmids together with helper plasmids pCMV-VSVG, pMDLg-RRE (gag/pol), and pRSV-REV into HEK293T cells. Cell supernatants were harvested 48 h after transfection and were either used to infect cells or stored at Ϫ80°C. To obtain stable cell lines, cells were infected at low confluence (20%) for 24 h with lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 5 ng/ml Polybrene (Sigma). 48 h after infection, cells were placed under 5 g/ml puromycin selection for 1 week and then passaged before use. His-ubiquitin-expressing HEK293T cells were obtained by transfection with His-ubiquitin construct followed by selection in 1 g/ml neomycin.
Preparation of Cytosolic and Nuclear Fractions-Cytosolic and membrane fractions were prepared using the Proteo-Extract kit (Calbiochem) according to the manufacturer's standard procedures.
Embryo Preparation and Tumor Cell Implantation-Dechorionized 2-day postfertilization Casper zebrafish transgenic lines Tg(fli1:GFP) were anesthesized with 0.003% tricaine (Sigma) and positioned on a 10-cm Petri dish coated with 3% agarose. mCherry-labeled MDA-MB-231 cells were trypsinized into single cell suspensions, resuspended in DMEM, kept on ice before injection. The cell suspension was loaded into borosilicate glass capillary needles (1-mm outer diameter ϫ 0.78-mm inner diameter; Harvard Apparatus), and the injections were performed using a Pneumatic Pico pump and a manipulator (WPI) within cell trypsinization. Approximately 400 cells were injected at the duct of Cuvier site in the yolk sac. The zebrafish embryos were subsequently maintained at 33°C for 6 days after implantation. The number of embryos which displayed tumor cell invasion and micrometastasis in the posterior tail fin area were counted using fluorescent microscopy.
Microscopy and Analysis-Live zebrafish analysis and image acquisition were performed using a Leica MZ16FA stereo microscope. Fixed embryos were imaged in PBST with a Leica SP5 STED confocal microscope. Confocal stacks were processed for maximum intensity projections with Leica software FAF1 Promotes ␤-TrCP-mediated ␤-Catenin Degradation or Adobe Photoshop CS4 software. Overlays were adjusted for brightness and contrast using Adobe Photoshop CS4.
Statistical Analysis-Statistical analyses were performed with a two-tailed unpaired t test. p Ͻ 0.05 was considered statistically significant.

Effect of FAF1 on WT and Mutant ␤-Catenin-induced
Transcription-We previously identified FAF1 as a novel inhibitory factor of Wnt signaling by promoting ␤-catenin polyubiquitination and degradation (31). To investigate the underlying mechanism, we first examined the effects of FAF1 on ␤-catenin-dependent transcriptional reporters when activated by either wild-type or constitutively active ␤-catenin mutants (␤-catenin SA or ␤-catenin OEN). These mutants are resistant to degradation by the adenomatous polyposis coli-Axin-GSK3␤ destruction complex (5). Consistent with our previous observations (31), ectopic expression of human FAF1 in 293T cells dose dependently inhibited the activation of the Wnt/␤-catenintranscriptional reporter constructs TopFlash-luciferase (44) and LEF-luciferase (45) when activated by wild-type ␤-catenin. However, FAF1 failed to inhibit the transcriptional activation initiated by constitutively active ␤-catenin SA or ␤-catenin OEN (Fig. 1, A and B). We next tested the effect of FAF1 knockdown on wild type or mutant ␤-catenin-induced transcriptional responses. As previously reported (31), shRNA-mediated FAF1 depletion potentiated Wnt reporter activity stimulated by wildtype ␤-catenin. However, FAF1 knockdown showed no effect on ␤-catenin SA-induced transcriptional responses. Because constitutively active ␤-catenin is resistant to the (phosphorylation-dependent) degradation and FAF1 knockdown did not interfere with ␤-catenin phosphorylation (31), these results suggested that FAF1 acts upstream of phosphorylation-dependent polyubiquitination of ␤-catenin.

FAF1 Promotes ␤-TrCP-mediated ␤-Catenin Degradation
tide was used to elute the complex from the beads. Subsequently, the eluate was immunoprecipitated with anti-Myc or control IgG, followed by Western blotting to detect HA-␤catenin. As shown in Fig. 4C, HA-␤-catenin was present in the final Myc-FAF1 immunoprecipitation but not in the IgG control sample. This result indicates that ␤-TrCP, FAF1, and ␤-catenin are part of a common ternary complex.
FAF1 Facilitates Recognition of ␤-Catenin by ␤-TRCP-To further verify the scaffolding function of FAF1 for ␤-catenin and ␤-TrCP, we expressed ectopic FLAG-tagged ␤-TrCP and checked its association with endogenous ␤-catenin in the  absence or presence of FAF1 knockdown. In the absence of MG132, we detected a weak association between ␤-TrCP and ␤-catenin, and this interaction was increased upon short term MG132 treatment. This is not unexpected as ␤-TrCP stimulates ␤-catenin for degradation. Upon depletion of endogenous FAF1, ␤-TRCP-associated ␤-catenin was partially decreased (Fig. 6A). To further validate this result, we expressed FAF1 wild type or FAF1-dUBX together with ␤-TRCP and tested their potential to affect ␤-TrCP interaction with ␤-catenin. As shown in Fig. 6B, wild-type FAF1 but not FAF1-dUBX could increase the association between ␤-TRCP and endogenous ␤-catenin. Together, these results demonstrate that FAF1 enhances ␤-catenin degradation by ␤-TrCP by increasing ␤-catenin recognition.
FAF1 Down-regulation May Correlate with Wnt/␤-Catenin Activation during EMT of Breast Cancer Cells-Activation of Wnt/␤-catenin signaling triggers many biological events, including epithelial to mesenchymal transition (EMT) (18,49). Data mining from the oncomine data base (50) indicated that FAF1 expression is significantly down-regulated in human breast carcinoma compared with normal tissue (Fig. 7A). Moreover, we found FAF1 expression to be higher in epithelial-like MCF7 than in mesenchymal-like MDA-MB-231 human breast cancer cells (Fig. 7B). In these cells FAF1 expression inversely correlated with the levels of cytoplasmic ␤-catenin, and positively and negatively correlated with the epithelial marker E-cadherin and the mesenchymal marker vimentin (51), respectively (Fig. 7B). This suggested that FAF1 might play a negative role in Wnt-induced EMT. To verify this, we depleted endogenous FAF1 in control (MCF7-Mock) and Wnt3a-transfected MCF7 (MCF7-Wnt) cells and examined activation of the ␤-catenin-Axin2 signaling cascade. As shown in Fig. 7, C and D, FAF1 depletion led to cytosolic ␤-catenin accumulation, ␤-catenin-induced Axin2 expression, as well as increased expression of vimentin. These results suggest that loss of FAF1 expression in breast cancer cells may lead to Wnt/␤-catenininduced EMT.
Ectopic FAF1 Expression Reduces Breast Cancer Cell Migration in Vitro and Invasion/Metastasis in Vivo-Wnt/␤-catenin stimulates EMT, invasion, and metastasis of breast cancer cells (52)(53)(54). Active Wnt/␤-catenin signaling also correlates with poor prognosis in breast cancer patients (55,56). Importantly, highly metastatic MDA-MB-231 cells showed reduced cytosolic ␤-catenin level upon ectopic FAF1 expression, (Fig. 8A) as well as mitigated migratory ability (Fig. 8B). We next examined the effect of FAF1 in vivo using a zebrafish embryo xenograft invasion-metastasis model (57). Control or ectopic FAF1-expressing MDA-MB-231 cells were injected into the ducts of Cuvier 48 h postfertilization and analyzed over 6 days after injection. 53% of embryos showed invasion and metastasis in the case of control cells, whereas FAF1 expression significantly reduced this amount to 37% (Fig. 8, C and D). Taken together, these results indicate that FAF1 reduces soluble ␤-catenin and inhibits breast cancer invasion and metastasis.

DISCUSSION
Although FAF1 was initially identified as a member of the FAS death-inducing signaling complex (19), subsequent work has revealed that FAF1 functions in diverse biological processes. FAF1 has been shown to play an important role in normal development and neuronal cell survival, whereas FAF1  A, HEK293T cells stably expressing His-Ub were transfected with Myc-tagged FAF1 full-length (fl), truncation lacking UBX domain (dUBX), and Wnt3a as indicated. Cells were treated with MG132 for 4 h and then harvested for nickelbead pulldown assay followed by immunoblot (IB) analysis. B, His-Ub-expressing HEK293T cells were transfected with or without Myc-FAF1 and infected with sh␤-TrCP lentivirus as indicated. Cells were treated with MG132 for 4 h and then harvested for nickel-bead pulldown assay followed by immunoblot analysis. FAF1 Promotes ␤-TrCP-mediated ␤-Catenin Degradation down-regulation may contribute to multiple aspects of tumorigenesis. In the field of molecular cellular signaling, FAF1 was first demonstrated to inhibit NF-B activation induced by TNF-␣, interleukin-1␤ (IL-1␤), or lipopolysaccharide (LPS) through multiple mechanisms (24,25). Recent studies identified FAF1 as a suppressor of canonical Wnt signaling in osteoblast differentiation and bone formation although the detailed mechanism remained unclear (31).
To uncover the mechanism of action underlying the ability of FAF1 to promote ␤-catenin degradation, we examined multiple essential components of the Wnt signaling cascade for their ability to interact with FAF1 and thereby identified ␤-TrCP as a strong FAF1-binding factor. FAF1 was also found to interact with SKP1 and CUL1, two other components of the ␤-catenin ubiquitination apparatus. ␤-TrCP cooperated with FAF1 in antagonizing Wnt signaling, and loss of endogenous ␤-TrCP attenuated FAF1-induced ␤-catenin polyubiquitination. More-over, we could show that FAF1 forms a ternary trimeric complex with ␤-TrCP and ␤-catenin. Thus, FAF1 can function as a scaffold and thereby facilitate ␤-catenin recognition by ␤-TrCP. As a consequence, FAF1 depletion results in accumulation of cytosolic ␤-catenin, and potentiation of Wnt-induced EMT in human breast cancer cells. In mesenchymal-like highly metastatic breast cancer cells, ectopic expression of FAF1 was found to block the accumulation of soluble ␤-catenin and to antagonize tumor cell invasion and metastasis in zebrafish in vivo. It should be noted that E-cadherin, a strong binding partner of ␤-catenin in the membrane, can restrict the level of soluble ␤-catenin. However, we found ectopic FAF1 not to affect E-cadherin expression or to bind E-cadherin. 5 We do not exclude the possibility that FAF1 may regulate other signaling  FAF1 Promotes ␤-TrCP-mediated ␤-Catenin Degradation AUGUST 31, 2012 • VOLUME 287 • NUMBER 36 pathways involved in breast cancer progression, in addition to the Wnt/␤-catenin pathway.
Our results suggest that down-regulation of FAF1 may contribute to the aberrant activation of Wnt/␤-catenin signaling during breast cancer progression. In addition to breast cancer, a decreased expression of FAF1 may also occur in many other types of cancer (58), including human gastric carcinomas and uterine cervix carcinomas (28). For instance, recurrent monoallelic and homozygous deletion of FAF1 has been reported in mantle cell lymphoma (30). Furthermore, single nucleotide polymorphism testing revealed that FAF1 may be associated with a genetic locus implicated in susceptibility to Crohn disease, an inflammatory bowel disorder that conveys an increased risk for colorectal cancer (60). Considering that Wnt/␤-catenin signaling is a tumor promoting pathway, it would be worthwhile to figure out whether FAF1 loss of expression leads to aberrant Wnt/␤-catenin activation in the cases mentioned above.
Finally, Faf1 gene-targeted mice are embryonic lethal and based on the pro-apoptotic function of FAF1, it was postulated that it might act as a tumor suppressor in cancers of varying origins (30,48,58,60). Our studies on the inhibitory role of FAF1 in canonical Wnt signaling in breast cancer cells strengthen this notion.