PKG inhibits TCF signaling in colon cancer cells by ...

Oncogene (2010) 1–12

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ORIGINAL ARTICLE

PKG inhibits TCF signaling in colon cancer cells by blocking b-catenin expression and activating FOXO4 I-K Kwon1, R Wang1, M Thangaraju1, H Shuang1, K Liu1, R Dashwood2, N Dulin3, V Ganapathy1 and DD Browning1 1 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA; 2Cancer Chemoprotection Program, Linus Pauling Institute, Oregon State University, Corvallis, OR, USA and 3Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, IL, USA

Activation of cGMP-dependent protein kinase (PKG) has anti-tumor effects in colon cancer cells but the mechanisms are not fully understood. This study has examined the regulation of b-catenin/TCF signaling, as this pathway has been highlighted as central to the anti-tumor effects of PKG. We show that PKG activation in SW620 cells results in reduced b-catenin expression and a dramatic inhibition of TCF-dependent transcription. PKG did not affect protein stability, nor did it increase phosphorylation of the amino-terminal Ser33/37/Thr41 residues that are known to target b-catenin for degradation. However, we found that PKG potently inhibited transcription from a luciferase reporter driven by the human CTNNB1 promoter, and this corresponded to reduced b-catenin mRNA levels. Although PKG was able to inhibit transcription from both the CTNNB1 and TCF reporters, the effect on protein levels was less consistent. Ectopic PKG had a marginal effect on b-catenin protein levels in SW480 and HCT116 but was able to inhibit TCF-reporter activity by over 80%. Investigation of alternative mechanisms revealed that cJun-N-terminal kinase (JNK) activation was required for the PKG-dependent regulation of TCF activity. PKG activation caused b-catenin to bind to FOXO4 in colon cancer cells, and this required JNK. Activation of PKG was also found to increase the nuclear content of FOXO4 and increase the expression of the FOXO target genes MnSOD and catalase. FOXO4 activation was required for the inhibition of TCF activity as FOXO4-specific shortinterfering RNA completely blocked the inhibitory effect of PKG. These data illustrate a dual-inhibitory effect of PKG on TCF activity in colon cancer cells that involves reduced expression of b-catenin at the transcriptional level, and also b-catenin sequestration by FOXO4 activation. Oncogene advance online publication, 29 March 2010; doi:10.1038/onc.2010.91 Keywords: PKG; cGMP; FOXO; b-catenin; TCF; colon cancer

Correspondence: Dr DD Browning, Department of Biochemistry and Molecular Biology, CB2605, Medical College of Georgia, 1459 Laney Walker Blvd, 1120, 15th Street, Augusta, GA 30912, USA. E-mail: [email protected] Received 2 October 2009; revised 28 January 2010; accepted 21 February 2010

Introduction Guanylin/uroguanylin are endogenous peptides that regulate electrolyte homeostasis and control differentiation along the crypt-villus axis in the intestine (Pitari et al., 2001; Forte, 2004). These ligands bind to receptor guanylyl-cyclase (GC-C) on the intestinal epithelium causing increased cGMP levels and can suppress tumor burden in the ApcMin/ þ mouse cancer model (Shailubhai et al., 2000; Uzzau and Fasano, 2000). The nature of the tumor suppressive properties of uroguanylin is presently not known, but as central mediators of cGMP signaling in cells, the cyclic-GMP-dependent protein kinases (PKG) are likely mediators (reviewed in Francis and Corbin, 1999; Ruth, 1999; Lincoln et al., 2001). Type 2 PKG phosphorylates ion exchangers and channels in the intestinal epithelium to carryout the natriuretic functions of uroguanylin (Forte et al., 2000). The function of type 1 PKG in epithelial cells is not known, but its expression is reduced in colon tumors and cell lines relative to matched normal tissue (Browning, 2008). Evidence for tumor suppressive functions of PKG in colon cancer cells is derived from ectopically expressed PKG, which can increase apoptosis, and reduce tumor growth and angiogenesis in xenografts (Deguchi et al., 2004; Hou et al., 2006a, b; Kwon et al., 2008). Further evidence for growth-inhibitory effects of cGMP comes from studies with exisulind, which increases cGMP levels by inhibiting phosphodiesterases (Liu et al., 2001; Deguchi et al., 2004; Zhu et al., 2005). This drug can induce apoptosis in colon cancer cells in a type 1 PKGdependent manner (Goluboff, 2001; Haanen, 2001; Liu et al., 2001). The signal transduction pathways involved in the anti-tumor effects of PKG are not fully understood, but activation of cJun-N-terminal kinase (JNK) (Soh et al., 2000), downregulation of b-catenin (Thompson et al., 2000; Liu et al., 2001), and more recently activation of SP1 (Cen et al., 2008) have all been implicated as important. The regulation of b-catenin levels by PKG is particularly significant because aberrant overexpression of this protein is a hallmark of intestinal tumorigenesis (Giles et al., 2003) and also some breast and bone marrow-derived cancers (Lin et al., 2000; Clevers, 2006; Schlange et al., 2007). Elevations in b-catenin expression

PKG inhibits TCF signaling in colon cancer cells I-K Kwon et al

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promote interaction with TCF/LEF transcription factors to activate growth-related target genes such as cMyc, cyclin D1, and c-Jun (Behrens, 2000; Lustig and Behrens, 2003). In most non-cancer cells, the b-catenin levels are minimal because it associates with the adenomatous polyposis coli (APC) complex leading to phosphorylation by glycogen synthase kinase 3b (GSK3b) and subsequent ubiquitination and degradation in proteasomes (Behrens, 1999, 2000). Many colorectal tumors have truncating mutations in APC that render it unable to bind b-catenin, or in the phosphorylation sites of b-catenin itself, which leads to excessive levels (Sparks et al., 1998; Strate and Syngal, 2005). It has been suggested that PKG has a similar role to GSK3b and directly phosphorylates b-catenin/TCF leading to proteasomal degradation (Thompson et al., 2000; Liu et al., 2001). However, in addition to the canonical pathway involving degradation of b-catenin, TCF signaling is subject to strict regulation by diverse factors that interfere with Wnt signaling or with the interaction of b-catenin with TCF in the nucleus (reviewed recently in Jin et al., 2008). Evidence is accumulating that oxidative stress can inhibit TCF signaling by activating forkhead box O (FOXO) transcription factors, which compete with TCF for b-catenin (Hoogeboom and Burgering, 2009). In cancer cells, Akt phosphorylates FOXO, which leads to interactions with 14-3-3 that sequesters them in the cytosol (Huang and Tindall, 2007; Burgering, 2008). However, in response to oxidative stress, phosphorylation by JNK causes dissociation from 14-3-3 and FOXO activation (Essers et al., 2004). This study has examined the mechanism of b-catenin/TCF downregulation by PKG in colon cancer cells and has identified two pathways. PKG was found to reduce the steady-state levels of b-catenin protein by inhibiting transcription of the CTNNB1 gene and not by stimulating protein degradation. In addition, we show that PKG can activate FOXO4 in colon cancer cells, and this inhibits TCF-dependent transcription by recruiting b-catenin to FOXO4. These results support previous work highlighting the anti-tumor properties of cGMP signaling in colon cancer cells and for the first time show activation FOXO by PKG.

Results Downregulation of b-catenin by PKG does not involve increased protein turnover The tumor suppressive properties of PKG have been attributed in part to its ability to block signaling through b-catenin/TCF and this study addresses the mechanism. Activation of PKG has been reported to reduce b-catenin levels in SW480 and HT29 cells grown in vitro (Thompson et al., 2000; Liu et al., 2001), and also in SW620 xenografts (Kwon et al., 2008). In support of these earlier studies, PKG activation could dose dependently inhibit b-catenin protein expression on western blots (Figures 1a and b). In these experiments, Oncogene

PKG overexpression by itself, or treatment with 8BrcGMP alone did not affect the b-catenin protein level, which underscores the requirement for active PKG. It has been suggested that PKG can mimic GSK3b by directly phosphorylating b-catenin, leading to increased proteasomal degradation (Thompson et al., 2000; Li et al., 2002). To test this idea we used phospho-specific antibodies against the amino-terminal Ser33/37/Thr41 residues known to target b-catenin for degradation (Figure 1c). The constitutive phosphorylation of these residues was easily observed in HEK293 cells treated with the phosphatase inhibitor calyculin, and this was blocked by pretreatment with lithium, indicating the involvement of GSK3b. However, we were unable to detect any Ser33/37/Thr41 phosphorylation in SW620 cells overexpressing PKG. In support of this result, analysis of the b-catenin sequence using a phosphorylation target database (Blom et al., 2004) revealed that the amino-terminal GSK3b target sites are likely to be poor substrates for PKG, but Ser552 and Ser675 were identified as potential PKG targets. Indeed, 8Br-cGMP could increase phosphorylation of these sites in PKGexpressing SW620 colon cancer cells (Supplementary Figure S1). To test the possibility that an unconventional route to degradation was involved, the effect of PKG on b-catenin protein levels was measured in the presence of cycloheximide (Figure 1d). These experiments revealed a nearly linear decrease in b-catenin protein levels over time, with B30% remaining after 20 h in both parental cells and those expressing PKG. Treatment with 8Br-cGMP did not affect the rate of protein degradation in the parental SW620 cells (3.1±0.7% per hour), but slightly increased stability in the PKG-expressing cells (3.2±0.8 to 2.9±0.1% per hour) although this stabilization effect was not statistically significant. PKG inhibits transcription from the CTNNB1 gene in colon cancer cells The inability of PKG to induce b-catenin degradation indicated a mechanism different from GSK3b and underscored the possibility that PKG could affect expression at the transcriptional or translational levels. As a first step, the effect of PKG activation on the steadystate levels of b-catenin mRNA was measured using semiquantitative reverse transcriptase polymerase chain reaction (RT–PCR) (Figure 2a). Activation of PKG in several inducible clones of SW620 resulted in a dramatic reduction of b-catenin message, whereas treatment of parental SW620 cells with inducer and 8Br-cGMP had no effect. To determine whether the reduced b-catenin mRNA levels were due to PKG-dependent regulation of transcription, a reporter construct previously shown to encode the essential promoter regions of the b-catenin gene (CTNNB1) was used (Li et al., 2004). PKG activation in the inducible SW620 clone inhibited basal transcription by B60%, whereas the PKG activator 8BrcGMP or PKG induction alone has little effect (Figure 2b). Suppression of CTNNB1 activity was also observed in several colon cancer cells lines following


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Figure 1 Downregulation of b-catenin expression by PKG in colon cancer cells does not involve increased degradation. (a) SW620 colon cancer cells with inducible PKG expression were treated with increasing doses of mifepristone (Mif) and 100 mM 8Br-cGMP for 48 h, or (b) with either treatment alone. Cell lysates were separated by SDS–PAGE and immunoblotted with anti-b-catenin, anti-PKG, and anti-b-actin (for protein loading). (c) Phosphorylation of b-catenin by PKG was examined by treating inducible SW620 cells with 1 nM mifepristone for 24 h and then 100 mM 8Br-cGMP for 2 h in the presence of the phosphatase inhibitor calyculin (10 nM). Treatment of HEK293T cells with calyculin was used as a positive control and lithium (1 mM) was added as indicated as a GSK3b inhibitor. Cell lysates were separated by SDS–PAGE and immunoblotted with anti-phospho Ser33/37/Thr41, anti-b-catenin C-terminus (total), antiPKG and anti-b-actin antibodies (for protein loading). (d) Parental SW620 cells (triangles) and inducible SW620 (squares; pretreated for 24 h with1 nM mifepristone) were incubated with 100 mM 8Br-cGMP (dashed lines) or without (solid lines). The translation inhibitor cycloheximide (10 mg/ml) was added to the cells and at the indicated times cell lysates were harvested and subjected to SDS–PAGE and immunoblotted with anti-b-catenin and anti-b-actin antibodies. Band intensities were quantitated using Image-Quant (Molecular Dynamics Inc.) and the b-catenin expression was normalized to b-actin at each time point. The results from at least three independent experiments are shown as a function of the basal amount, and bars show the s.e.m.

transient transfection and activation of PKG (Figure 2c). The degree of inhibition varied between the different cell types but was most dramatic in SW480 cells with an 80% reduction in CTNNB1 transcription. Notably, the

inhibitory effect of PKG was unique to the colon cancer cells and did not affect CTNNB1 activation in nontumor-derived cells such as CCD841 and HEK293 (Supplementary Figure S2). Oncogene


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Figure 2 Downregulation of b-catenin expression by PKG occurs at the transcriptional level. (a) Parental SW620 cells and three inducible clones were treated with or without 1 nM mifepristone inducer (Mif) and 100 mM 8Br-cGMP for 24 h as indicated (±). Total RNA was isolated and subjected to semiquantitative RT–PCR with primers for b-catenin, PKG, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) as an internal loading control. (b) Inducible SW620 cells were transiently transfected with a luciferase-reporter plasmid driven by the human CTNNB1 promoter, and either induced to express PKG (Mif), treated with 100 mM 8Br-cGMP (cG), or both (as indicated). Relative transcription was calculated by standardizing luciferase with b-galactosidase activity derived from cotransfected CMV-LacZ, and then subtracting similar results using the basal pGL3 vector used in parallel dishes. Lysates were subjected to immunoblotting with anti-PKG and anti-b-actin antibodies (lower panels). (c) The regulation of CTNNB1 transcription by exogenous PKG was also measured by transient transfection of SW620, SW480, HT29, and HCT116 cells. The effect of 100 mM 8Br-cGMP alone (gray bars), or together with transfected PKG (black bars), was compared with untreated controls (white bars). The reporter assay and immunoblotting were performed as described above. Experiments were reproduced at least three times and bars indicate s.e.m. Asterisks indicate statistical significance (Po0.05).

Inhibition of TCF transcription by PKG requires activation of JNK The inhibition of CTNNB1 activity by PKG explains the reduced b-catenin expression in colon cancer cells. Downregulation of b-catenin expression by PKG has been shown to block transcription of TCF-dependent target genes in SW480 cells (Thompson et al., 2000; Deguchi et al., 2004). In our inducible SW620 colon cancer cells, activation of PKG dose dependently inhibited TCF activity as measured using the TOP-flash TCF-luciferase-reporter system (Figure 3a) and reduced TCF-gene target expression as measured by RT–PCR (Figure 3b). We also examined the inhibition kinetics of Oncogene

both b-catenin protein expression and TCF activity by PKG in SW620 cells (Figure 3b). In these experiments, B60% of the maximum TCF inhibition (measured at 24 h) was observed after 6 h stimulation with 8BrcGMP, at a time when there was negligible effect on b-catenin protein. We also created doxacycline-inducible cell lines using SW480, HCT116 and HT29 cells, and all clones showed inhibition of TCF activity by PKG activity. However, the apparent disconnect between the downregulation of b-catenin protein and TCF activity was even more prominent in some of these cells, particularly SW480 and HCT116 cells, which exhibited a dramatic inhibition of TCF activity (>80%), which


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Figure 3 TCF inhibition by PKG involves more than regulation of b-catenin expression. (a) SW620 cells were cotransfected with TOP-flash reporter (or the mutant FOP-flash) and CMV-b-gal. Subsequently, the cells were induced to express increasing levels of PKG using mifepristone and treated with 100 mM 8Br-cGMP (cGMP). After 24 h, the luciferase activity was determined as described in Materials and methods. Lysates were subjected to immunoblotting with anti-b-catenin, anti-PKG, and anti-b-actin antibodies (upper panels). (b) SW620 cells were induced to express PKG and treated with 100 mM 8Br-cGMP. After 24 h, the total RNA was extracted and subjected to semiquantitative RT–PCR to detect steady-state levels of TCF target genes LEF1, LGR, and SOX9. (c) The TCF activity in PKG-expressing SW620 cells was measured (as described above) at different times following addition of 100 mM 8Br-cGMP. The relative inhibition of TCF activity by 8Br-cGMP at the different times is expressed as a percentage of the maximal inhibition measured at 24 h. Cell lysates harvested at the indicated times were immunoblotted with anti-b-catenin and anti-b-actin antibodies. (d) TCF activity was measured in inducible SW480, HCT116, and HT29 cells using the TOP-flash-reporter system as described above. The effect of PKG (induced with 2 mg/ml doxacycline and activated with 100 mM 8Br-cGMP) on TCF activity is shown as a percentage of the activity in untreated cells. Lysates from treated (cGMP þ Dox) and untreated cells were subjected to immunoblotting with antib-catenin and anti-b-actin antibodies (upper panels). Experiments were reproduced at least three times and bars indicate s.e.m. Asterisks indicate statistical significance (Po0.05).

was not paralleled by the comparatively small effect of PKG on the b-catenin protein levels. Taken together, these results indicate that mechanisms in addition to the regulation of b-catenin expression, must contribute to the inhibition of TCF signaling by PKG. The regulation of b-catenin/TCF signaling can be complex as there are families of diffusible Wnt antagonists and diverse endogenous inhibitors of the b-catenin/TCF interaction (reviewed recently in Jin et al., 2008). To better understand how PKG inhibits TCF activity, we first ruled out a role for Ser552/675 phosphorylation, which did not affect nuclear localization of b-catenin, and phospho-deficient mutants did not differ from wild type in their ability to rescue the inhibitory effect of PKG on TCF (Supplementary Figure S1). Our attention focused on the potential

involvement of FOXO transcription factors, which have been reported to inhibit TCF activity in response to oxidative stress (Essers et al., 2005; Almeida et al., 2007). FOXO regulation was particularly attractive because activation of FOXO4 by oxidative stress requires JNK (Essers et al., 2004), which has previously been shown to be activated by PKG (Soh et al., 2000). We confirmed that PKG can activate JNK in colon cancer cells using phospho-specific antibodies (Figure 4a). We then sought to verify that oxidative stress could inhibit TCF activity, and found that stimulation of transiently transfected SW480 cells with either 100 mM 8Br-cGMP or 200 mM H2O2 for 6 h resulted in 50% inhibition of the basal TCF activity (Figure 4b). Cotransfection of the cells with a dominantnegative JNK (JNK-DN) construct blocked the Oncogene


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Figure 4 Inhibition of TCF activity by PKG requires JNK. (a) SW480 and HCT116 cells were induced to express PKG and treated with 8Br-cGMP. After 24 h, the cell lysates were analyzed by immunoblotting with anti-phospho-JNK, anti-total JNK, and anti-bactin antibodies for protein loading. (b) SW480 cells were transiently cotransfected with TCF reporters, PKG (as indicated), and either empty vector (white bars) or dominant-negative JNK (JNK-DN; black bars). Cells were subsequently treated with 100 mM 8Br-cGMP or 200 mM H2O2, and after 6 h, the luciferase and b-galactosidase content was measured in cell lysates. Parallel cell lysates were subjected to western blotting to detect expression of the transfected JNK and PKG constructs (lower panels). (c) SW480 cells were transiently cotransfected with TCF reporters, and either PKG or empty vector. The cells were stimulated for 6 h with either 100 mM 8Br-cGMP or 200 mM H2O2 in the presence (black bars) or absence (white bars) of 10 mM NAC before measuring TCF activity in the cell lysates as described above. Experiments were reproduced at least three times and bars indicate s.e.m. Asterisks indicate statistical significance (Po0.05) between the bracketed groups.

TCF-inhibition by PKG and H2O2. These data indicate that PKG and H2O2 share a common mechanism involving JNK activation to inhibit TCF activity in colon cancer cells. The ability of the JNK-DN to only partially block TCF-inhibition by PKG (56% rescue) compared with H2O2 (90% rescue), likely reflects the significance of reduced b-catenin expression downstream of PKG. The inhibition of TCF activity by H2O2 but not PKG was blocked by coincubation of the cells with the antioxidant N-acetylcysteine (NAC) (Figure 4c). This indicates that JNK activation by PKG is not due to increased oxidative stress, and is consistent with an earlier report showing that JNK activation by PKG involves direct phosphorylation of MEKK (Soh et al., 2001). Oncogene

PKG activates FOXO4 in colon cancer cells The FOXO transcription factors are a recently recognized family of tumor suppressors that are gaining significant interest as targets for cancer therapy (Arden, 2006; Dansen and Burgering, 2008; Maiese et al., 2008; Weidinger et al., 2008). These proteins are generally inactive in cancer cells owing to constitutive Akt, which promotes interaction with 14-3-3 proteins and cytosolic localization (Dansen and Burgering, 2008; Fu and Tindall, 2008; Maiese et al., 2008). Our studies focused on FOXO4 because this isoform has unique JNK phosphorylation sites that are involved in H2O2-induced FOXO activation in colon cancer cells (Essers et al., 2004). Transient transfection of a flag-FOXO4 construct into colon cancer cells showed both cytosolic and


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Figure 5 Exogenous PKG activates FOXO4 in colon cancer cells. (a) SW480 cells were grown on coverslips and cotransfected with plasmids for flag-FOXO4 and either PKG or empty vector (control). PKG-expressing cells were stimulated with 8Br-cGMP for 6 h, and then all coverslips were processed for immunofluorescence with anti-flag antibodies and counter-stained with DAPI as indicated. Scale bars represent 25 mm. (b) SW480 cells were cotransfected to express flag-FOXO4 with PKG and dominant-negative JNK (JNK-DN) as indicated. Cells were stimulated with 100 mM 8Br-cGMP for 6 h before immunoprecipitating FOXO4 with anti-flag antibodies. Immunoprecipitates were separated by SDS–PAGE, western blotted to nitrocellulose, and the blots probed with anti-bcatenin and anti-flag antibodies. Cell lysates before IP were immunoblotted with anti-PKG and anti-b-catenin antibodies to show input loading. (c) PKG-expressing SW620 colon cancer cells were either untreated or stimulated with 100 mM 8Br-cGMP for 24 h. Total RNA was isolated and subjected to semiquantitative RT–PCR with primers for PKG, catalase, and manganese-dependent superoxide dismutase (MnSOD). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) as an internal loading control. (d) SW620 cells were induced to express PKG and treated for 24 h with 100 mM 8Br-cGMP. Cell lysates were analyzed by immunoblotting with anti-catalase, anti-PKG, and anti-b-actin (loading control) antibodies. All results shown are representative of at least three independent experiments.

nuclear localization of the protein by immunofluorescence (Figure 5a). However, in cells cotransfected to express PKG together with FOXO4, 8Br-cGMP caused a striking mobilization of the cytosolic fraction to the nucleus. FOXO proteins regulate gene expression not

only by activating FOXO target genes, but also by interacting with other transcription factors (van der Vos and Coffer, 2008). Several groups have shown that FOXO binds strongly to b-catenin, which increases the transcription of FOXO target genes but inhibits Oncogene


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TCF transcription by diverting b-catenin (Essers et al., 2005; Almeida et al., 2007; Hoogeboom et al., 2008). Immunoprecipitation experiments demonstrated that transient expression of flag-FOXO4 could bind endogenous b-catenin under control conditions (Figure 5b). However, activation of cotransfected PKG dramatically increased b-catenin binding to FOXO4, and this was completely blocked by cotransfection of the JNK-DN. These results strengthen the notion that PKG can activate FOXO in colon cancer cells and supports previous work by other groups that have reported that activated FOXO binds to b-catenin. FOXO can activate several classes of genes, including those involved in cell cycle arrest (p27kip), DNA repair (GADD45), and antioxidant pathways (catalase, MnSOD) (Huang and Tindall, 2007; Burgering, 2008; Calnan and Brunet, 2008; Ho et al., 2008). Activation of PKG in SW620 colon cancer cells caused increased expression of the classical FOXO target genes encoding catalase and MnSOD (Figures 5c and d), but in these studies PKG did not activate other target genes such as p27kip or GADD45 (not shown). These data are evidence that PKG can activate FOXO4 in colon cancer cells. Activation of FOXO4 is required for the inhibition of TCF by PKG To determine whether FOXO4 activation is involved in TCF inhibition by PKG, we generated short-interfering RNA (siRNA) to knockdown FOXO4 expression. Compared with a non-targeting siRNA, the FOXO4 siRNA-1 generated from position 829 of the coding region had better than 90% knockdown of FOXO4, whereas siRNA-2 (position 1267) was less efficient with close to 70% knockdown (Figure 6a). Knockdown of FOXO4 with the specific siRNAs but not the controls was able to inhibit activation of catalase expression by PKG in the SW620 colon cancer cells (Supplementary Figure S3). Moreover, silencing FOXO4 also prevented PKG from inhibiting TCF transcription (Figure 6b). The more efficient siRNA-1 completely blocked the effect of PKG and actually raised the basal TCF activity slightly higher than control. We looked at the effect of PKG on the proliferation of various colon cancer cell lines, and found small but significant inhibition in SW480, HCT116, and HT29 cells, but not SW620 cells (Figure 6c), which is consistent with earlier studies (Thompson et al., 2000; Kwon et al., 2008). As the effect of PKG was most pronounced in HT29 cells (30% inhibition), we tested the role of FOXO4 using specific siRNA (Figure 6d). We found that both FOXO4specific siRNAs were able to partially but significantly rescue the PKG-dependent inhibition of proliferation relative to non-targeting siRNA. Taken together, these results outline a model in which PKG activity can inhibit TCF activity by two mechanisms (Figure 6e). One effect is to block transcription from the CTNNB1 gene, resulting in reduced total b-catenin levels. Another mechanism is to activate FOXO4, which by binding to b-catenin, limits the pool available to interact with TCF. Oncogene

Discussion Elevated b-catenin expression is a common feature of colon cancer cells (Sparks et al., 1998), where it promotes proliferation and angiogenesis and blocks differentiation (Fodde and Brabletz, 2007; Katoh and Katoh, 2007). The importance of b-catenin to colon tumorigenesis and progression has made this protein an important therapeutic target (Luu et al., 2004; Takahashi-Yanaga and Sasaguri, 2007). There is growing evidence that type 1 PKG has anti-tumor effects in colon cancer cells, including promoting apoptosis and inhibiting growth and angiogenesis (Deguchi et al., 2004; Hou et al., 2006a, b; Kwon et al., 2008; Soh et al., 2008). Some of these effects may be due to inhibition of b-catenin/TCF signaling by PKG, which has been reported by independent laboratories (Thompson et al., 2000; Kwon et al., 2008). This study has focused on the mechanism of b-catenin/TCF inhibition by PKG to better understand the therapeutic potential of this enzyme. Earlier studies have suggested that PKG overrides APC deficiency by mimicking GSK3b and directly phosphorylating b-catenin leading to its degradation (Thompson et al., 2000; Liu et al., 2001). This study makes this model unlikely because overexpression of PKG in SW620 colon cancer cells did not increase protein turnover or cause phosphorylation of the sites known to target b-catenin for degradation. This point is strengthened by the observation that PKG is also an effective inhibitor of TCF activity in HCT116 cells, which possess mutant b-catenin that is a poor substrate for GSK3b (Sparks et al., 1998). The phosphorylation of b-catenin by PKG as reported earlier is most likely due to Ser552 and Ser675, which are also targeted by PKA (Taurin et al., 2006). Phosphorylation of these residues does not promote degradation, but instead has been found to increase b-catenin stability and enhance transcriptional activity (Hino et al., 2005; Fang et al., 2007; Xu and Kimelman, 2007). In agreement with those studies, stimulation of PKG in HEK293 cells increased b-catenin levels and TCF activity (Supplementary Figure S2). Results shown here indicate that downregulation of b-catenin protein by PKG is due to inhibition of CTNNB1 gene transcription. The mechanism underlying regulation of CTNNB1 transcription by PKG is not presently known, and warrants further study. Downregulation of b-catenin could not account for the dramatic inhibition of TCF activity resulting from short-term activation of PKG, and led to the novel finding that PKG activates FOXO4 in colon cancer cells. The important role of JNK in FOXO4 activation by PKG is consistent with earlier studies showing that PKG activates JNK in colon cancer cells (Soh et al., 2000, 2001), and that JNK activates FOXO4 (Essers et al., 2004). Activation of FOXO4 can increase resistance to oxidative stress but may slow growth by competing with TCF for b-catenin binding (Essers et al., 2005; Almeida et al., 2007; Hoogeboom et al., 2008). As type 1 PKG can be stimulated by redox stress in addition to cGMP, it is plausible that activation of FOXO4 by PKG may be a key component of the


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Figure 6 Activation of FOXO4 is necessary for the inhibition of TCF activity by PKG. (a) SW480 cells were transfected with nontargeting control (Ctrl) or two different FOXO4-specific (F4) siRNAs as indicated. After 48 h, cell lysates were immunoblotted with anti-FOXO4 and anti-b-actin (loading control) antibodies. (b) SW480 cells were treated with control or FOXO4-targeting siRNAs for 36 h and subsequently cotransfected with TCF-reporter plasmids together with either empty vector (white bars) or PKG (shaded bars) as indicated. PKG-transfected cells were treated with 100 mM 8Br-cGMP for 6 h before harvesting cells for quantitation of TCF activity as described in Materials and methods. Results are shown as TCF activity in cells with active PKG as a percentage of those without active PKG. (c) Colon cancer cell lines were induced to express PKG and treated with 100 mM 8Br-cGMP. After 48 h, the growth relative to untreated controls was measured using the MTT assay. Error bars show s.e.m. (n ¼ 3) and asterisks indicate statistical significance. (d) HT29 cells were transfected with non-targeting (Ctrl) or two FOXO4-specific (F4) siRNAs as indicated. After 24 h, the cells were treated with doxacycline and 100 mM 8Br-cGMP for an additional 48 h before quantitating cell growth using the MTT assay. Experiments were reproduced three times and bars indicate s.e.m. Asterisks in panels indicate statistical significance relative to control siRNA (Po0.05). (e) A model illustrating the role of FOXO4 in the inhibition of TCF activity is shown. Dashed line represents the nuclear envelope; double lines show promoters for FOXO (forkhead responsive element containing; FHRE) or TCF (T-cell factor) target genes.

antioxidant response (Burgoyne et al., 2007). Oxidative stress is carcinogenic in the colon (Federico et al., 2007), and we have found that 8Br-cGMP can activate catalase expression in the mucosa of colon explants (Supplementary Figure S3). The ability of FOXO4 activation in the

intestinal epithelium to protect against oxidative stress and also suppress aberrant b-catenin/TCF signaling, suggests a possible tumor prevention role for PKG. This is consistent with the ability of cGMP-elevating uroguanylin to suppress tumor burden in the ApcMin/ þ Oncogene


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mouse (Shailubhai et al., 2000), but the role for PKG/ FOXO in this phenomenon will require further study. In summary, we have examined the regulation of bcatenin/TCF signaling by PKG in colon cancer cells, and report dual-inhibitory activities. PKG can inhibit transcriptional activation of the CTNNB1 gene, which can lead to reduced b-catenin protein levels. A potentially more important finding is that PKG can activate FOXO4, which leads to a more rapid inhibition of TCF activity by sequestering b-catenin. These findings are consistent with reports of anti-tumor properties of PKG in colon cancer cells, and further suggest the possibility of a tumor preventative role.

Materials and methods Tissue culture and reagents All cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in 5% CO2 in RPMI-1640 medium containing 10% FBS, and supplemented with 200 mM L-glutamine, 10 IU/ml penicillin, 10 mg/ml streptomycin. We have previously described the creation and characterization of SW620 colon carcinoma cell lines made inducible for type 1 PKG expression in response to mifepristone (Mif) (Hou et al., 2006a, b; Kwon et al., 2008). The medium used to maintain stocks of the H3Z6 clone of inducible SW620 was supplemented with 300 mg/ml each of hygromycin and zeosin. Before experimentation, the cells were grown up for at least one passage in the absence of antibiotics. Additional clones of SW480, HCT116 and HT29 cells made inducible for PKG1b expression by doxacycline (dox) were created using the Lenti-X Tet-On Advanced Inducible Expression System (Clontech, Mountain View, CA, USA). Recombinant virus were produced in HEK-293T cells according to the manufacturer’s instructions. Cells were first infected with a virus encoding the dox-inducible PKG1b, and after puromycin selection, the surviving cells were re-infected with tet-activator virus and colonies were selected using G418. The Mif, calyculin, DAPI and 8Br-cGMP were from Calbiochem (San Diego, CA, USA). NP-40, Tween-20, Lithium, dox, and cycloheximide were from Sigma (St Louis, MO, USA), and all other chemicals were from Fisher Scientific (Pittsburgh, PA, USA). Antibodies and constructs The antibody against Catalase was from R&D systems (Minneapolis, MN, USA). The antibodies specific to JNK, phospho-JNK, FOXO4 and b-catenin (N-terminus and phosphorylated) were all from Cell Signaling (Beverly, MA, USA). The b-actin and anti-flag epitope antibodies were from Sigma. The polyclonal anti-PKG1 antibodies raised against the common C-terminus, and the PKG expression vectors have been described earlier (Browning et al., 2001). The TopFlashreporter system was from UpState (Billerica, MA, USA) and the CTNNB1 reporter has been described earlier (Li et al., 2004). The flag-JNK-DN and the flag-FOXO4 constructs were purchased from Addgene (Cambridge, MA, USA). Immunofluorescence Cells were grown on coverslips in six-well dishes until 50% confluent and then transiently transfected to express flag-FOXO4 and PKG1b. After treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde and permeablized with 0.1% Triton-X-100. After blocking 1 h at Oncogene

37 1C in PBS containing 5% goat serum, the coverslips were incubated overnight at 4 1C in blocking buffer containing 1/500 anti-Flag-M2 mAb (Sigma). Coverslips were then incubated for 2 h at room temperature with Alexa Fluor 568 goat anti-mouse secondary antibodies (Invitrogen, Carlsbad, CA, USA) and counterstained with DAPI for 5 min, before affixing to slides using Mowiol (Calbiochem, La Jolla, CA, USA). Images were captured using a Nikon TE-300 inverted epifluorescence microscope equipped with a SPOT RT3camera and SPOT Software version 4.7 (Diagnostic Instruments Inc., Sterling Heights, MI, USA). Western blotting and immunoprecipitation For PAGE analysis, cells were lysed by incubation with icecold lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate) supplemented with phosphatase and protease inhibitor cocktails (Calbiochem, La Jolla, CA, USA). Lysates were clarified by centrifugation, boiled in PAGE buffer, separated on 10% mini-gels followed by electrophoretic transfer to nitrocellulose. The blots were blocked with 5% BSA in PBS containing Tween 20 (PTS) and incubated with primary antibodies overnight at 4 1C. Following addition of 1/3000 peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA, USA), the bands were visualized using chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). The turnover of b-catenin protein in SW620 cells was determined by incubating the cells in 10 mg/ml of cycloheximide to block protein synthesis. At different times over a 24 h period, the cells were harvested and lysates were analyzed for b-catenin content by western blotting. The intensity of the b-catenin band was quantitated using Molecular Dynamics ImageQuant software (GE Healthcare, Piscataway, NJ, USA), and the levels were normalized to similarly quantitated b-actin, and expressed as a percentage of the starting amount. Results were reproduced in at least three independent experiments; the slopes from best-fit lines were determined for each replicate. Interactions between FOXO4 and b-catenin were determined by immunoprecipitation using anti-flag-M2-sepharose beads (Sigma). Cells were transiently transfected with expression vectors for flag-FOXO4 and either empty pCDNA3 or PKG1b. In these experiments, 20 ml of beads were added to 1 ml clarified lysate (10 cm dish of cells), which was rocked for 2 h at 4 1C and then washed three times in RIPA buffer before analysis of b-catenin content by western blot. Measurement of steady-state message levels Semiquantitative RT–PCR was used to determine the relative steady-state levels of mRNA for b-catenin, PKG, and both TCF and FOXO target genes. Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen), and cDNA generated using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA, USA). PCR was performed using 1 ml RT product in reactions with 0.2 U TaKAra Taq (Fisher Scientific, Pittsburgh, PA, USA), 30 cycles at 60 1C anneal temperature. Control reactions used primers specific for the hypoxanthineguanine-phosphoribosyltransferase (HPRT1). The sequences of the primers used are listed in Supplementary Table 1. Luciferase-reporter assays For measurements of transcription, the cells were cultured in 12-well plates, and triplicate wells were transfected with luciferase-reporter plasmids using Lipofectamine 2000 reagent (Invitrogen). Measurement of b-catenin transcriptional activity was carried out with TCF-reporter plasmids using the TOP flash system according to the manufacturer’s protocols


11 (UpState). In these experiments, cells were cotransfected with 0.2 mg TOP flash luciferase reporter/well (or mutant control FOP flash vector) and 0.2 mg CMV-b-galactosidase to control for cell number and transfection efficiency. After 16 h, the medium was changed and cells were stimulated with 100 mM 8Br-cGMP for an additional 6–8 h before enzyme assay. Cell extracts were prepared and analyzed for luciferase and bgalactosidase activities as described earlier (Taurin et al., 2006). The luciferase activity was standardized with regard to respective b-galactosidase activity, and the TCF-specific luciferase was expressed as the net TOP flash luciferase after subtraction of the associated FOP flash activity. Measuring the transcription of the b-catenin gene followed an almost identical procedure except that 0.2 mg CTNNB1 luciferase was used, and basal luciferase was controlled using the empty pGL3 vector. This construct encodes the promoter regions of human b-catenin and has been described earlier (Li et al., 2004). The effect of FOXO4 knockdown on TCF activity was done by creating specific siRNA using BLOCK-iT RNAi Designer (Invitrogen). The siRNAs used correspond to position 829 (CCCUGCACAGCAAGUUCAU; designated FOXO4-1) and 1268 (GCUGUUAGAUGGGCUCAAU; designated FOXO4-2) and a non-targeting control (GCUGAU GUACGGCUGUAAU). The cells were transfected with the siRNAs using Lipofectamine 2000 (Invitrogen) and after 48 h, they were transfected again with TCF reporter as described above. After an additional 24 h, the cells were harvested for luciferase determinations.

according to the manufacturer’s instructions (ATCC, Manassas, VA, USA). Cells were plated in 96-well plates and after 24 h, the cell numbers were measured (day 1). In parallel wells either normal medium or medium containing dox inducer and 8Br-cGMP was added. After an additional 48 h, the cell number was again measured (day 3), and the growth rate was calculated based upon the difference in number. Testing the role of FOXO4 on growth was determined by first transfecting the cells in the 96-well plate with siRNAs and measuring the cell number 24 h later (for day 1) and then following the procedure above. Quadruplicate wells were used for each treatment and the experiment was performed at least three times.

Cell proliferation The effect of PKG on cell proliferation was assessed by measuring viable cell number using the MTT assay kit

Supported by grants from the American Cancer Society (RSG-07-174-01-CSM to DDB and RSG-09-209-01-TBG to KL) and the National Institutes of Health (CA133085 to KL).

Statistical analysis Results are expressed as the mean±s.e.m. of at least three independent experiments and statistical comparisons used Student’s t test. A probability value of o0.05 was considered to be significant.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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