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Oncogene (2014) 33, 1975–1985 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE SPROUTY2 is a b- and FOXO3a target indicative of poor prognosis in colon cancer

P Ordo´n˜ ez-Mora´n1,2, A Irmisch2,6, A Barba´chano1,6, I Chicote3, S Tenbaum3, S Landolfi4, J Tabernero5, J Huelsken2, A Mun˜oz1 and HG Pa´lmer3

SPROUTY2 (SPRY2) is an intracellular regulator of receptor tyrosine kinase signaling involved in growth, differentiation and tumorigenesis. Here, we show that SPRY2 is a target gene of the Wnt/b-catenin pathway that is abnormally activated in more than 90% of colon . In human colon cancer cells, SPRY2 expression is induced by b-catenin in co-operation with the transcription factor FOXO3a instead of lymphoid enhancer factor/T-cell factor . We found binding of b-catenin to the SPRY2 promoter at FOXO3a response elements. In vivo, cells marked by nuclear b-catenin and FOXO3a express SPRY2 in proliferative epithelial tissues, such as intestinal mucosa and . Consistently, inducible b-catenin deletion in mice reduced Spry2 expression in the small intestine. Moreover, SPRY2 expression correlated with nuclear b-catenin and FOXO3a colocalization in human colon carcinomas. Importantly, the amount of SPRY2 protein correlated with shorter overall survival of colon cancer patients. Our data reveal SPRY2 as a novel Wnt/b-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer.

Oncogene (2014) 33, 1975–1985; doi:10.1038/onc.2013.140; published online 29 April 2013 Keywords: SPROUTY2; wnt pathway; b-catenin; FOXO3a; colon cancer

INTRODUCTION induce a complete set of involved in colon cancer 8 (CRC) is the second cause of death by cancer . In this context, we have now evaluated in detail worldwide.1 Constitutive activation of the Wnt/b-catenin pathway SPROUTY2 (SPRY2) as a potential target gene of the Wnt/b-catenin by mutations is an initiating event in more than 90% of pathway by enhancing FOXO3a transcriptional activity. Indeed, human CRC.2 In normal tissues without Wnt pathway activation, transcriptome and promoter analyses of mouse cytosolic b-catenin is recruited to a destruction complex that identified Spry2 as a direct FoxO target gene that mediates 12 contains the scaffold Axin and Adenomatous polyposis coli endothelial cell morphogenesis and vascular homeostasis. proteins, casein kinase a/e and glycogen synthase kinase 3b. SPRY2 expression is downregulated in the breast, prostate, b-Catenin phosphorylation by these two kinases commits it hepatocellular and in B-cell diffuse lymphoma, 13–17 towards degradation by the proteasome. When Wnt signaling suggesting a tumor suppressive role. Contrarily, it is is activated, the destruction complex is disassembled, and upregulated in melanoma cell lines concomitantly to N-RAS and 18,19 b-catenin is not phosphorylated, translocating to the cell B-RAF activating mutations. Data regarding colon cancer are nucleus where it binds the lymphoid enhancer factor/T-cell contradictory. While it was reported that SPRY2 RNA levels are 13 factor (lymphoid enhancer factor, LEF/human T-cell factor 1, downregulated in some intestinal tumors, we and others have TCF) family of transcription factors. b-Catenin/TCF complexes reported an increased expression of SPRY2 in high-grade control the expression of many genes involved in cell proliferation undifferentiated carcinomas as compared with normal intestinal 20,21 and invasion, thus promoting tumor initiation and progression.3 mucosa in colon cancer patients. Furthermore, SPRY2 reduces In addition, b-catenin has been shown to bind other transcrip- the expression of the tumor suppressor E-cadherin responsible of tion factors enhancing the expression of alternative sets of target retaining cell-to-cell contacts and inhibiting cell motility. genes and directing cell fate in tissue homeostasis, development Contrarily, SPRY2 knockdown diminishes cell invasion in human 20,21 and cancer.4–8 One of these transcription factors is FOXO3a, that colon cancer cells. To help clarifying the role of SPRY2 in induces genes responsible for apoptosis, cell cycle arrest and cancer, we decided to study the molecular mechanisms response to oxidative stress.9 Furthermore, FOXO3a and TCF/LEF that control its expression and to analyze its potential value as factors compete for the binding of limited amounts of nuclear prognostic marker in colon cancer, both relevant aspects that b-catenin as transcriptional co activator: while nuclear have not yet been explored in detail. FOXO3a accumulation reduces b-catenin/TCF transcriptional In this study, we reveal that SPRY2 expression is regulated by activity,10 b-catenin/FOXO3a complexes regulate genes involved FOXO3a and b-catenin nuclear activity. Simultaneous accumula- in longevity and oxidative stress.11 In addition, we recently tion of both factors in the nucleus induced an increase of SPRY2 described that nuclear b-catenin and FOXO3a co-operate to RNA and protein in colon cancer cells. We detected b-catenin

1Instituto de Investigaciones Biome´dicas ’Alberto Sols’, Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´ noma de Madrid, Madrid, Spain; 2Swiss Institute for Experimental Cancer Research, E´cole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland; 3Stem Cells and Cancer Laboratory, Translational Research Program, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain; 4Department of Pathology, Vall d’Hebron University Hospital, Universitat Auto` noma de Barcelona, Barcelona, Spain and 5Medical Oncology Department, Vall d’Hebron University Hospital, Universitat Auto`noma de Barcelona, Barcelona, Spain. Correspondence: Dr HG Pa´lmer, Stem Cells and Cancer Laboratory, Translational Research Program, Vall d’Hebron Institute of Oncology (VHIO), Passeig Vall d’Hebron, 119-129, Barcelona 08035, Spain. E-mail: [email protected] 6These authors contributed equally to this work. Received 25 September 2012; revised 2 March 2013; accepted 4 March 2013; published online 29 April 2013 SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1976 recruited to the SPRY2 gene at FOXO3a response elements by located next to the transcriptional start site (DBE2 and DBE3) chromatin immunoprecipitation (ChIP) assays, confirming that it is (Figures 1d and e). The maximum b-catenin enrichment upon DOX a direct b-catenin target gene most probably through direct treatment was detected 1700 bp upstream of the SPRY2 start FOXO3a binding. Consistently, in normal mouse small intestine codon (DBE2) (Figures 1d and e). b-Catenin was also enriched and human colon tissue, SPRY2 is expressed at the bottom of the upon DOX and reduced upon 4OHT (FOXO3a) treatment at TCF crypts, the proliferative compartment where Wnt/b-catenin path- binding sites on LEF1 promoter (Supplementary Figure 1c).10 way is most active and FOXO3a is accumulated in the cell nucleus. Finally, we observed that exogenous nuclear b-catenin induced Similarly, Spry2 is expressed in cells with nuclear b-catenin the activity of SPRY2 gene promoter region, extending and FOXO3a proteins in the matrix of mouse follicles, a from À 1826 to þ 221 bp, upon DOX treatment in DLD1Fb system whose growth is well established to be stimulated by Wnt (Figures 1d and f). This region contained the FOXO3a binding ligands.5,22 In fact, Spry2 expression is reduced upon inducible site DBE2, where b-catenin was most recruited in ChIP assays intestinal deletion of b-catenin in mice, confirming that its (Figures 1d and e). expression is dependent on Wnt/b-catenin signaling activity in vivo. Furthermore, we found a direct correlation between SPRY2 protein expression and nuclear b-catenin and FOXO3a SPRY2 expression in human colon and epidermis colocalization content in human colon carcinomas. Remarkably, correlates with nuclear FOXO3a and b-catenin colocalization SPRY2 expression correlates with shorter overall survival of colon We next studied the expression of FOXO3a, b-catenin and SPRY2 cancer patients. Finally, we observed that simultaneous activation proteins in human colon by immunofluorescence analyses. of b-catenin and FOXO3a enhances SPRY2 expression and Whereas FOXO3a was equally accumulated in the nucleus of epidermal growth factor receptor/extracellular signal-regulated epithelial cells all across the human colon mucosa, b-catenin was kinase–AKT (EGFR/ERK-AKT). nuclear only in some epithelial cells at the bottom of the crypts Together, these data demonstrate that SPRY2 is a Wnt/b-catenin where the Wnt pathway is highly activated (Figures 2a–c). and FOXO3a target gene indicative of poor prognosis in colon Interestingly, SPRY2 protein was also enriched in epithelial cells cancer patients. at the bottom of colon crypts, and was found preferentially in the cytoplasm (Figures 2d and e). In this same crypt area, epithelial cells express high levels of the b-catenin/TCF target CD44, high Ki67 and low , indicating high proliferation and poor RESULTS differentiation (Supplementary Figure 2a–c). In summary, SPRY2 SPRY2 is a b-catenin and FOXO3a target gene in colon cancer cells expression was concentrated at the bottom of the colon crypts, a To explore the possible regulation of SPRY2 expression by FOXO3a region where cells present nuclear FOXO3a and b-catenin, and b-catenin, we expressed a 4-hydroxytamoxifen (4OHT)- Wnt/b-catenin pathway activation,3 high proliferation rate and inducible, AKT-insensitive FOXO3A mutant (FOXO3A-ER)23 and a low differentiation (Supplementary Figure 2d). SPRY2 is also doxycycline (DOX)-inducible, stabilized (non-degradable) expressed in stromal cells surrounding the intestinal epithelium. b-catenin mutant (b-cateninS33Y) in human DLD1 colon cancer We also analyzed FOXO3a, b-catenin and Spry2 expression in cells (DLD1Fb). DLD1 cells were chosen for these experiments as mouse hair follicles, in which growth is directly linked to Wnt they have low basal nuclear FOXO3a or b-catenin content and pathway activation.5,22 b-Catenin and FOXO3a colocalized in the low b-catenin/TCF-driven transcriptional activity.8 Both FOXO3a nucleus of epithelial cells at the matrix region of the growing and b-catenin proteins accumulated in the nucleus upon anagen hair follicles (Supplementary Figures 3a and b). These corresponding inducible treatments (Figure 1a). Exposure to epithelial cells are directly exposed to Wnt ligands secreted by the 4OHT or DOX increased SPRY2 RNA amounts, and the combinatory specialized stromal cells that populate the dermal papilla during treatment further induced its expression in DLD1Fb cells the anagen hair growth phase5,22 (Supplementary Figure 4d). (Figure 1b). The increase was also detected at protein level, and Spry2 protein was detected just in the same hair matrix region, again it was higher when b-catenin and FOXO3a were simulta- where FOXO3a and b-catenin simultaneously accumulated in the neously localized in the nucleus (Figure 1c). SPRY2 phosphory- cell nucleus (Supplementary Figures 3d and e). The expression of lation is associated with electrophoretic mobility shift. Two bands CD44 and Lef1 Wnt/b-catenin target genes was high in this matrix are detected for SPRY2 protein corresponding to, residues region, where b-catenin was nuclear and Spry2 was phosphorylated form (upper band) and non-phosphorylated positive (Supplementary Figures 3d and 4a and b). We observed form (lower band). Phosphorylation of SPRY2 likely has a role in that Spry2 expression coincided with that of Ha1, which is determining its stability and function status.24–26 Regulation was a marker of early hair shaft differentiation (Supplementary specific as the expression of Sprouty family member SPRY1 did Figure 4c). Finally, we detected an enrichment of b-catenin bound not increase following 4OHT and/or DOX addition. As expected, to the DBE present at the proximal region of the Spry2 gene DOX induced the b-catenin/TCF target lymphoid enhancer- promoter, by performing ChIP assays with extracts of mouse binding factor-1 (LEF1), whose expression was reduced that presented a majority of hair follicles at growing anagen phase by 4OHT-mediated FOXO3a induction probably owing to compe- (Supplementary Figures 3c and 4e). tition for b-catenin binding.10 Besides, the expression of the cell In summary, these data indicate that Spry2 is expressed in cells cycle regulator p21CIP1, a reported FOXO3a target, was induced by that accumulate high levels of nuclear FOXO3a and b-catenin and 4OHT treatment. The inducible expression of an exogenous in their immediate cell offspring in two highly proliferative tissues, dominant-negative DNTCF4 in LS174T colon cancer cells human intestinal colon mucosa and mouse growing hair follicles repressed TCF/LEF-dependent transcription and the expression (Figures 2g and 3g). of target genes like C-MYC or AXIN227 but did not reduce SPRY2 expression, indicating that it is not a canonical TCF/LEF target gene (Supplementary Figures 1a and b). Spry2 expression in mouse intestine requires b-catenin We next examined whether b-catenin binds to the SPRY2 gene We extended the study of Spry2 expression to the mouse promoter at putative FOXO3a binding sites (DAF-16 binding intestine. Similarly to human tissue (Supplementary Figure 2c), element, DBEs) in DLD1Fb cells by ChIP assays. Among the nine Spry2 was absent from the differentiated villi and accumulated in putative DBEs that we identified across the sequence of the the proliferative area, colocalizing with Ki67 at the bottom of the human SPRY2 coding and upstream promoter sequences, we intestinal crypts (Figure 3a). However, Spry2 was predominantly only observed enrichment in b-catenin precipitation on those sites detected at the cell membrane in mouse small intestinal tissue,

Oncogene (2014) 1975 – 1985 & 2014 Macmillan Publishers Limited SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1977 abc VEHICLE DOX4OHT DOX+4OHT 2.5 FOXO3a P = 0.0005 *** SPRY2 2.0 ** *

(r. u.) LEF1 β- 1.5 B SPRY2 1.0

CIP1 Merge mRNA 0.5 p21

0 LAMIN B DOX - + - + 4OHT --+ + d SPRY1 DBE1 DBE2 DBE3 DBE4,5,6 DBE7,8,9 dsCtrl LAMIN B

ABCD-1826 +221 EDOX - + - + 4OHT -- + + 9 P < 0.0001 *** ef8 *** 4 20x105 P = 0.0061 P < 0.0001 ** * 7 * * 6 3 15x105 5 4 2 10x105 3

5 2 1 5x10 SPRY2 protein (r. u.) 1 Fold Enrichment (r. u.) Luciferase activity (r. u.) 0 0 0 DOX --+ + --+ + --+ + --+ + DOX - + DOX --+ + 4OHT --+ + - -++ --+ + -- ++ 4OHT --+ + ABC D Figure 1. SPRY2 expression is induced by nuclear b-catenin and FOXO3a. (a) Immunofluorescence analysis of FOXO3a (green) and b-catenin (red) expression in DLDFb cells treated with DOX and/or 4OHT. Nuclei were stained with Hoechst 33342 (blue). Dashed lines mark nuclei positive for FOXO3a and/or b-catenin. Scale bar, 50 mm. (b) Normalized SPRY2 RNA levels in DLDFb cells treated with DOX and/or 4OHT were measured by real-time reverse transcription–PCR. Mean±s.d. of five independent experiments. P-value for non-parametric analysis of variance (ANOVA) analysis with Kruskal–Wallis test is indicated. Asterisk marks significant difference between groups quantified by Dunn´ s multiple comparison test. (c) Protein levels of SPRY1 and 2, LEF1 and p21CIP1 in DLDFb cells treated with DOX and/or 4OHT were estimated by western blot. Lamin B served as loading control. Black arrowhead indicates phosphorylated SPRY2 and gray arrowhead indicates non-phosphorylated SPRY2. Lower panel shows quantification of six independent experiments, mean±s.d. Black bars correspond to the quantifications of phosphorylated and gray bars to non-phosphorylated SPRY2 protein. P-value for non-parametric ANOVA analysis with Kruskal–Wallis test is indicated. Asterisk marks significant difference between groups quantified by Dunn´ s multiple comparison test. (d) Scaled diagram representing human SPRY2 gene. The nine putative FOXO3a-binding elements (DBE) are indicated. qPCR amplified regions (A, B, C and D) that cover different DBE and a downstream control region (dsCtrl, E) are shown. Exons are represented as gray bars. Yellow bar indicates the promoter region tested by luciferase assays in panel f.(e) Graph representing the quantification by qPCR of b-catenin enrichment upon ChIP assay on DBE sequences identified in the human SPRY2 gene. Values are ordered by amplified region in SPRY2 gene (A to D) and cells treatments are indicated. All values correspond to specific b-catenin enrichment versus precipitation with control immunoglobulin G and amplification of an unrelated dsCtrl. (f) Activity of SPRY2 gene promoter region tested in DLD1Fb cells treated as indicated. Bars represent the mean±s.d. of four replicates. Luciferase reporter gene was under the control of SPRY2 gene promoter region extending from À 1826 to þ 221 bp from transcription starting site. Region tested is shown in the diagram in panel (d) (yellow bar). Values of luciferase are relative to Renilla activity. P-value corresponds to non-parametric t-test. (b, c, e, f) Relative units (r. u.).

whereas it was mostly cytoplasmic in human colonic intestine SPRY2 expression correlates with nuclear FOXO3a and b-catenin (Figures 2d and e, and 3a and c). content in colon carcinomas and is indicative of shorter patients To determine whether b-catenin is essential for Spry2 expres- survival sion in vivo, we used a mouse model of 4OHT-inducible ablation of We first analyzed the correlation of SPRY2 expression with nuclear b-catenin from epithelial cells of the intestine (villin-creERT2-b- accumulation of FOXO3a and b-catenin in a panel of colon tumors cateninlox/lox).28 b-Catenin deletion upon 4OHT treatment at different stages of progression (Figure 4a). Most adenomas and provoked reduced expression of the b-catenin/TCF target a proportion of carcinomas (Patient 1, P1) presented b-catenin CD44 and concomitant downregulation of Spry2 expression in mostly localized at cell-to-cell contacts and at very low levels in the intestinal crypts (Figures 3b–d). Therefore, the activity of the nucleus. In contrast, less differentiated carcinomas (P2, P3) and b-catenin is required for the expression of proper amounts of metastases showed high accumulation of nuclear b-catenin. Spry2 protein at the proliferative mouse crypts, similarly to other Nuclear FOXO3a was detected in adenomas, a subset of Wnt targets, such as CD44. carcinomas (P3) and liver metastases. In summary, concomitant

& 2014 Macmillan Publishers Limited Oncogene (2014) 1975 – 1985 SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1978

Figure 2. SPRY2 is expressed in cells that accumulate nuclear FOXO3a and b-catenin at the bottom of the crypts in human colon epithelium. (a) Representative picture showing protein expression by double immunofluorescence and confocal microscopy of FOXO3a (green), b-catenin (red) and colocalization (yelow). Insets below show magnifications with split or merged channels corresponding to an area at the bottom of the crypt delineated by a dashed square on the main picture. Dashed lines in insets mark four nuclei with the highest double b-catenin and FOXO3a colocalization signal (yellow). (b) Image representing nuclear b-catenin and FOXO3a colocalization at the region shown in the inset of panel (a). Dashed lines mark nuclei with double staining for b-catenin and FOXO3a. (c) Color scatter plot showing the corresponding intensity correlation analysis of green (FOXO3a) and red (b-catenin) channels of the nuclei mark by dashed lines in panel (b). Yellow dots represent colocalization signal. (d, e) Representative picture showing protein expression by double immunofluorescence and confocal microscopy of (d) b-catenin (green) and SPRY2 (red) or (e) FOXO3a (green) and SPRY2 (red). Insets below show magnifications with split or merged channels corresponding to an area at the bottom of the crypt delineated by a dashed square on the main picture. SPRY2 expression is pointed by arrows in intestinal and by asterisks in stromal cells. (a, d, c). Nuclei were stained with Hoechst 33342 (blue). Scale bars, 50 mm.

nuclear FOXO3a and b-catenin accumulation was only found in FOXO3a and b-catenin enhance the EGFR/ERK-AKT pathway some advanced carcinomas and liver metastases. Interestingly, We investigated the possible role of SPRY2 as a FOXO3a/b-catenin SPRY2 expression was very high only in these tumors exhibiting target gene capable of regulating EGFR.29–31 First, we treated co-expression of FOXO3a and b-catenin in the cell nucleus DLD1Fb cells with EGF during different times (from 30 to 180 min) (Figure 4b). Furthermore, high SPRY2 expression corresponded either under control conditions or after accumulating nuclear to carcinomas with low staining indicative of poor b-catenin and FOXO3a by pretreating with DOX þ 4OHT differentiation (Supplementary Figure 5). In contrast to SPRY2, (Figures 6a and b). The induction of nuclear b-catenin high expression of the b-catenin/TCF target CD44 correlated and FOXO3a increased SPRY2 and EGFR expression and the with nuclear b-catenin accumulation even in the absence of concomitant activation of pAKT and pERK. Addition of EGF FOXO3a (Figure 4b). reduced the amount of its receptor EGFR by inducing its SPRY2 expression and nuclear b-catenin and FOXO3a degradation,29,31 but simultaneously increased pEGFR, pAKT and colocalization were quantified in 30 human Stage III colon pERK. Such activation was transient, decaying at later time points. carcinomas (Supplementary Table 1). A significant correlation When cells were pretreated with DOX þ 4OHT, EGF-induced (P ¼ 0.0045) existed between SPRY2 expression and the amount of activation of EGFR, AKT and ERK equally occurred, but signal nuclear b-catenin and FOXO3a colocalization signal (Figure 5a). decay was delayed and persisted longer time. In summary, cells Most importantly, those patients with the highest SPRY2 with nuclear FOXO3a and b-catenin presented enhanced expression presented a 14-months shorter survival (low ¼ 36 vs amplitude of response to EGF signal. high ¼ 22 months. P ¼ 0.0237. HR, 0.3892; 95% CI, 1.148–2.125) To explore the particular relevance of SPRY2 as a FOXO3a/ (Figure 5b). b-catenin target gene regulating EGFR, we knocked down its

Oncogene (2014) 1975 – 1985 & 2014 Macmillan Publishers Limited SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1979 a b β-catenin CD44 Spry2 +/lox -catenin β

* villin-creERT2-

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Spry2 * * *

Merge d +/lox lox/lox * β-cat

Spry2

β-

Figure 3. Tissue-specific deletion of b-catenin decreases Spry2 expression in mouse intestinal epithelium. (a) Double immunofluorescence staining reveals cells expressing Spry2 (green) located at the proliferative region (Ki67, red) of mouse small intestinal crypts pointed by white arrowheads. Magnifications below show that Spry2 expression is high in Ki67-positive cycling cells at the intestinal crypts pointed by white arrows. White arrows point Ki67 and Spry2 double positive cells shown in the magnifications. (b) b-Catenin deletion was induced in b-cateninlox/lox-villin-creERT2 mice by tamoxifen injection; b-catenin þ /lox-villin-creERT2 mice were used as a control. Immunohistochemical staining of murine small intestine for b-catenin and the Wnt target CD44 show a decrease in the expression of both proteins upon treatment. Dashed squares mark the crypts were Wnt activity is enhanced. (c) Double immunofluorescence analysis show the lost of b-catenin (red) and the reduction of Spry2 (green) in the crypts of the small intestine in b-cateninlox/lox-villin-creERT2 mice four days after tamoxifen treatment. White arrowhead point to intestinal crypts. (a, c) Nuclei were stained with Hoechst 33342 (blue). Asterisks indicate stromal cells expressing Spry2. Dashed lines delineate the epithelium-stroma edge. (a–c) Scale bars: 50 mm. (d) Western blots showing the expression of b-catenin and Spry2 proteins in the crypts of the small intestine in b-cateninlox/lox-villin-creERT2 and control mice 4 days after tamoxifen treatment. b-Actin was used as loading control. expression by expressing specific short hairpin RNAs in DLD1Fb DOX þ 4OHT. The limited reduction of SPRY2 expression cells, and performed the corresponded analysis (Figures 6c and d). promoted a reduction of EGFR and AKT activation upon EGF We first observed that the pool of lentivirus expressing short treatment that was not statistically significant. No effect was hairpin RNA against SPRY2 decreased its expression to 58% upon observed on ERK activation. DOX þ 4OHT treatment. Next, control (c) and knockdown (sh) cells Finally, we observed that the 4OHT-induced ablation of were treated for 180 min with EGF after pretreatment or not with b-catenin from epithelial cells of the intestine in villin-creERT2-b-

& 2014 Macmillan Publishers Limited Oncogene (2014) 1975 – 1985 SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1980

Figure 4. SPRY2, nuclear b-catenin and FOXO3a expression in human colon cancer. (a) Representative pictures of double immunofluorescence staining and confocal analysis of b-catenin (red) and FOXO3a (green) in a human colon adenoma, three colon carcinomas (P1–P3) and a liver metastasis. Dashed squares delineate areas shown as magnifications below to visualize nuclear FOXO3a and b-catenin colocalization. (b) Representative pictures of immunofluorescence staining and confocal analysis of CD44 or SPRY2 expression (green) in the same panel of patients. The asterisks correspond to SPRY2 staining in stromal cells. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 100 mm.

cateninlox/lox mice promoted the decrease of pEGFR level adapter proteins, which allow them to modulate the signaling of (Figure 6e). Therefore, the activity of b-catenin is required several growth factor tyrosine kinase receptors.32 Tyrosine kinase for the proper expression and/or activation of EGFR at the receptor signaling is central in controlling the homeostasis proliferative mouse crypts, where nuclear FOXO3a, b-catenin and of small intestine and colon epithelia.33 In particular, SPRY2 Spry2 content is high. modulates the EGFR signaling pathway.29–31 Upon EGF stimulation of its receptor, the c-Cbl E3 ligase binds and ubiquitinates EGFR, targeting it to internalization and destruction by the endocytotic DISCUSSION machinery. In this scenario, active SPRY2 competes with c-Cbl The mammalian SPRY protein family consists of four members for the binding to EGFR, thus reducing receptor endocytosis that bind to a variety of endogenous kinases, phosphatases and and increasing the amplitude and duration of downstream

Oncogene (2014) 1975 – 1985 & 2014 Macmillan Publishers Limited SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1981 a b 0.06 100 SPRY2 content HIGH n = 30 LOW P = 0.0045 0.05 80

0.04 60 0.03 40 0.02

Nuclear FOXO3a & 20 -catenin content (r. u.) 0.01 Patients surviving (%) β n = 30 P = 0.0237 0 0 0 100 200 300 400 500 600 020406080100 SPRY2content (a. u.) Time (months) Figure 5. SPRY2 expression correlates with nuclear FOXO3a and b-catenin colocalization signal. The expression of b-catenin, FOXO3a and SPRY2 proteins was analyzed by immunofluorescence and confocal microscopy in serial tissue sections of 30 colon carcinoma patients. Quantification was performed as described in Materials and methods. Relative expression values are show in Supplementary Table 1. (a) Scatter plot showing the correlation between nuclear FOXO3a and b-catenin colocalization signal and SPRY2 content for each patient. P-value corresponds to a Spearman test. (b) Kaplan–Meier survival analysis of colon cancer patients separated into two groups by the median for SPRY2 signal. Higher signal is in red (n ¼ 14) and lower signal in black (n ¼ 16). P-values by Log-rank (Mantel-Cox) test.

Ras/mitogen-activated protein kinase pathway activation.34–36 that include the analysis of conditional tissue-specific Spry2 null SPRY2 also interacts with the hepatocyte growth factor- mice should be performed to reveal its possible role in the regulated tyrosine kinase substrate (Hrs) interfering with the physiology of adult stem cells and for the homeostasis of trafficking of ligand-activated EGFR receptor from early to late epithelial tissues. endosomes, and thus increasing the signaling.37 In contrast, in Indeed, mice carrying Spry2 null alleles die as a consequence of certain cellular contexts, SPRY proteins not only fail to enhance gastrointestinal abnormalities at postnatal day 21–28.44 Although but may even inhibit tyrosine kinase receptors , indicating a these data evidence that Spry2 is crucial for intestinal complexity of SPRY function that warrants further detailed homeostasis, its exact role for intestinal stem cells physiology studies.24,26,38,39 remains to be analyzed. Other processes dependent on the Wnt/ Although such modulation of tyrosine kinase receptors b-catenin pathway like limb, lung or kidney development are signaling could have a relevant role in fundamental processes perturbed in overexpressing the SPRY2 gene.44 during development, adult tissue homeostasis and cancer Altogether, these data suggest that the tight control of the initiation or progression,33 no data about the regulation of precise amounts of SPRY2 by the Wnt/b-catenin pathway could be SPRY2 expression was available up to now. essential for an orderly execution of different biological processes In this study, we show that nuclear colocalization of FOXO3a during development or the homeostasis of various adult tissues. and b-catenin induces SPRY2 RNA and protein expression in Abnormal activation of the Wnt/b-catenin pathway by muta- colorectal carcinoma cells. We found that b-catenin interacts with tions, that promote nuclear accumulation of b-catenin, is an two regions on the SPRY2 gene promoter where FOXO binding initiating event in more than 90% of colon cancer patients. sequences are located, but which lack response elements for Contrarily, FOXO3a is considered a tumor suppressor, as it can TCF/LEF or other transcription factors that interact with b-catenin. promote cell cycle arrest and apoptosis in cancer cells. Indeed, the Besides, b-catenin enhances the activity of the proximal promoter combined knockdown of FOXO1, 3 and 4 in mice is responsible for region of SPRY2 gene. Altogether these data indicate that SPRY2 is tumor initiation.12 However, we have recently demonstrated that a FOXO3a and b-catenin target gene induced through the the accumulation of high amounts of both FOXO3a and b-catenin recruitment of both factors to specific regulatory gene regions. in the nucleus of cancer cells imposes a high motile phenotype, It remains unknown whether FOXO3a and b-catenin would form promoting metastasis in mice and correlating with metastatic part of the same regulatory protein complex on the SPRY2 gene stages and shorter overall survival of colon cancer patients.8 promoter or would act independently. Similarly, we describe here that high levels of SPRY2 also correlate In vivo, we have identified high SPRY2 expression in a with shorter survival of colon cancer patients. Furthermore, subpopulation of Wnt/b-catenin-activated epithelial cells that also supporting the idea of SPRY2 as an oncogene, we previously accumulate nuclear FOXO3a, either at the bottom of human showed its capacity to increase the expression of ZEB1, an inducer intestinal crypts or in the matrix of mouse anagen hair follicles. of epithelial-to-mesenchymal transition, which promotes cell SPRY2 expression is extended to the immediate offspring migration and invasion in colon cancer cells.20 of proliferative cells activated by FOXO3a and b-catenin. These In this scenario, SPRY2 could have a role in the malignant results together with the loss of Spry2 expression in the small phenotype of colon cancer cells as a b-catenin and FOXO3a target intestine of b-catenin null mice, confirm SPRY2 as a b-catenin gene. Its activity could also modulate EGF signaling, which in fact target gene. is central in many colon carcinomas. Indeed, the use of anti-EGFR Interestingly, the same cell populations at the bottom of the , such as Cetuximab has become one of the most intestinal crypts and the matrix of the growing hair follicles important clinical strategies to treat K-RAS wild-type patients with expressing SPRY2 are highly activated by EGF ligands.40 It is advanced metastatic disease. Therefore, SPRY2 expression could therefore possible that SPRY2, as a FOXO3a/b-catenin target gene, be used to determine the response to Cetuximab as previously could be a key factor modulating the crosstalk between Wnt/ suggested.45 b-catenin and EGF signaling pathways in vivo. The cell populations Our data provide a better understanding of the consequences located within these regions contain adult stem and progenitor of Wnt/b-catenin pathway activation by including SPRY2 as a new cells, and both Wnt and EGF signals from the corresponding target gene, which may modulate other signaling pathways like specialized niches are fundamental to sustain their distinct EGFR, relevant for tissue homeostasis and tumor progression. pluripotency and self-renewal capacities.28,41–43 Further studies In fact, we observed a decrease in pEGFR content in mouse colon

& 2014 Macmillan Publishers Limited Oncogene (2014) 1975 – 1985 SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1982 intestines knocked out for b-catenin, indicating the interplay of the expression of multiple common target genes involved in cell both pathways in vivo. signaling, proliferation and malignancy in colon cancer. The limited effects of SPRY2 knockdown on EGFR signaling Further investigations would be required to identify which are could indicate that FOXO3a and b-catenin increase the amplitude the FOXO3a/b-catenin target genes other than SPRY2 that of EGFR signaling by regulating other factors. Indeed, determine such enhanced EGFR signaling. However, as in our we previously described that b-catenin and FOXO3a regulate experiments the expression of SPRY2 was only reduced by 58%, it

Oncogene (2014) 1975 – 1985 & 2014 Macmillan Publishers Limited SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1983 is also possible that a more efficient knockdown of SPRY2 Quantitative reverse transcription–PCR expression may be necessary to detect an effect on the induction Total cellular RNA was purified using RNeasy mini kit (Qiagen, Hilden, of EGFR signaling by FOXO3a and b-catenin. Furthermore, our Germany). Values were normalized versus housekeeping gene succinate data suggest that b-catenin and FOXO3a could execute part of dehydrogenase complex subunit A. The reaction was performed in a Light- their oncogenic activity by sustaining an enhanced EGFR-RAS-ERK/ Cycler apparatus using the LightCycler-FastStart DNA MasterPLUS SYBR AKT signaling. Such mechanism could determine tumor malig- Green I Kit (Roche Diagnostics, Mannheim, Germany) or the High Capacity nancy and progression, as the EGFR signaling drives central cDNA Reverse Transcription Kit, TaqMan SPRY2 Hs00183386_m1 and TaqMan Universal PCR Master Mix, no Amperase, (Applied Biosystems, aspects of the cancer cell phenotype: hyperproliferation, Darmstadt, Germany). All experiments were performed by triplicate. enhanced survival, motility or migration. We found that SPRY2 is the only member of its family upregulated by b-catenin and FOXO3a. Such results are in line Western blotting with previous evidences showing SPRY2 as the only member Whole-cell extracts were prepared by washing the monolayers twice in upregulated in colon cancer tumors compared with adjacent phosphate-buffered saline (PBS) and cell lysis by incubation in radio- immunoprecipitation assay buffer (50 mM Hepes pH 7.4, 150 mM NaCl, normal mucosa.21 Altogether, these data highlight the relevance 1.5 mM MgCl2, 10% glycerol, 4 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% of SPRY2 in colon cancer malignancy. In summary, our results deoxycholate) plus phosphatase and protease-inhibitor mixture (25 mM reveal SPRY2 as a new b-catenin and FOXO3a target gene that is b-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 mg/ml expressed in Wnt-activated cells in normal and tumoral tissues leupeptin, 10 mg/ml aprotinin) for 15 min on ice followed by centrifugation and correlates with shorter overall survival of colon cancer at 13 000 r.p.m. for 10 min at 4 1C. Western blotting of cell lysates was patients. It should therefore be considered as a prognosis performed by electrophoresis in SDS–polyacrylamide gel electrophoresis marker and a potential target to direct therapeutic intervention and protein transfer to Immobilon P membranes (Millipore, Darmstadt, in the future. Germany). The membranes were incubated with the appropriate primary and secondary horseradish peroxidase-conjugated antibodies, and the binding was visualized using the ECL detection system (GE Healthcare, Chalfont St Giles, UK). Different exposure times of the films MATERIALS AND METHODS were used to ensure that bands were not saturated. Quantification of the Cells, culture and treatments films was performed by densitometry using ImageJ software (Bethesda, DLD1 human colon cancer cells were cultured in DMEM plus 10% fetal MD, USA). bovine serum (Invitrogen, Darmstadt, Germany). Cells were transfected with pcDNA-FOXO3A:ER46 and neomycin-selected to obtain a clone of ChIP and sequence analysis S33Y DLD1F cells. b-Catenin was introduced in DLDF (Dong Ling Di Fu) cells 47 S33Y ChIP was carried out as described previously using b-catenin (BD by with pSIN-TRE-flag-b-catenin -IRES-GFP lentivirus to Bioscience, Oxford, UK) or a species matched IgG control (Millipore) generate the DLDFb cell line. Lentiviral particles were produced by and LEF1 and SPRY2 promoter-specific primers. Previously described transfecting 293T cells with lentiviral construct and packaging vectors 8 sequences of FOXO3a response elements (DBE) in the mouse Spry2 gene (pPAX2 and pMD2.G, Addgene, Cambridge MA, USA). Cells were treated were identified in the homologous human SPRY2 gene using NTI software as follows: 2.5 mg/ml Doxycycline (DOX, Sigma-Aldrich, St Louis, MO, USA) (Life Technologies, Beverly, MA, USA) and primers were designed for 12 h before 50 nM 4-Hydroxytamoxifen (4OHT, Sigma-Aldrich) for 24 h. accordingly for the ChIP analysis. Identification of putative response In experiments using EGF, cells were treated with DOX (same elements of other transcription factors able to interact with b-catenin concentration) for 24 h before an additional 16 h incubation in serum- on the SPRY2 gene sequence, was performed by TRANSFAC software free medium in the presence of DOX and 4OHT (same concentrations), (http://www.gene-regulation.com). We used a set of primers to before the treatment with EGF (100 ng/ml) or vehicle. analyze TCF/LEF binding sites in the human LEF1 gene.48 Primer SPRY2 knockdown was performed in DLD1Fb cells by infecting sequences were as follows: Spry2 A, forward 50-GTTTCCAGTCCTTCAA simultaneously with eight different lentivirus expressing short hairpin GCAATC-30, reverse 50-AATTGGGAGTGGCTGTAACAAA-30; Spry2 B, forward RNAs specific against human SPRY2 mRNA (Open BioSystems, Pittsburgh, 0 0 0 8 5 -GGTAGACTTGAAATGGATTCTGG-3 , reverse 5 -ACTAATCTGGGCCACGC PA, USA) and following previously described protocols. DLD1Fb cells AGT-30; Spry2 C, forward 50-CTCTCTCTCTTCCGTTCTTCGT-30, reverse 50-CGC infected with lentivirus expressing control non-silencing short hairpin RNA TTGATGACTTTCTTCCTG-30; Spry2 D, forward 50-TTCTCCAGAGAGAATGTGC were use as control. TATATTT-30, reverse 50-TCACATTCTTCCGAATGTACAAC-30, Spry2 E, forward 50-CCCAAGAGATGTTTGTCTCTGAC-30, reverse 50-AGTCCAGGTGACATCAAG 0 0 0 0 Mice ACCTA-3 , Lef1 F, forward 5 -TTCTTGGCAAACTTCTCTTTCTC-3 , reverse 5 - CTCGAGGCTCATTTAATCTGCTA-30; Lef1 G, forward 50-TGGACGTTTCGTT b-Catenin ablation was induced by intraperitoneal injection of 1 mg ATCTTCTGAT-30, reverse 50-GGATGCCGACTTAGTAGTTTGTTT-30. of tamoxifen (Sigma-Aldrich) per 20 g of body weight into 10-week-old b-catenin -villin-creERT228 mice, for 4 consecutive days before analysis. Both inducible mutant (b-cateninlox/lox-villin-creERT2) and control Luciferase assays (b-catenin þ /lox-villin-creERT2) animals were injected according to the Cells were transfected using the X-tremeGENE HP DNA reagent (Roche). In same regimen. transient transfections, Firefly (Luc) and Renilla reniformis luciferase (Rluc)

Figure 6. FOXO3a and b-catenin regulate EGFR. (a) Western blots showing the expression levels of the indicated proteins in DLD1Fb cells, pretreated 24 h with DOX and an additional 16 h incubation in serum-free medium in the presence of DOX and 4OHT, prior treatment with EGF during the indicated times. (b) Plots representing the quantification of the indicated proteins measured by western blot (panel a)at different treatment time points. Values correspond to the mean±s.d. of three independent experiments. Each value was corrected by the corresponding protein expression at time 0 in cells treated or not with EGF. (c) DLD1Fb cells were infected with a pool of lentivirus expressing eight different short hairpin (shRNA) against SPRY2 mRNA (sh) or an scramble shRNA (control) (c). The expression of the indicated proteins was evaluated by western blot in both types of DLD1Fb cells (c and sh) treated 180 min with DOX þ 4OHT and or EGF. (d) Graphs representing the quantification of the indicated proteins measured by western blot (panel c). Values were corrected by the expression of each protein in DLD1Fb cells infected with sh scramble control and treated with vehicle. Reduction of SPRY2 expression is indicated as percentage of expression in sh-SPRY2 cells versus sh scramble control cells. Bars correspond to the mean±s.e.m. of two independent experiments. (b, d) Relative units (r. u.). (e) b-Catenin deletion was induced in b-cateninlox/lox-villin-creERT2 mice by tamoxifen injection; b-catenin þ /lox-villin- creERT2 mice were used as a control. Immunohistochemical staining of murine small intestine for b-catenin (green) and the pEGFR (red) show a decrease in the expression of both proteins upon treatment. Nuclei were stained with Hoechst 33342 (blue). Asterisks indicate unspecific staining in the crypt lumen. Scale bar, 100 mm.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1975 – 1985 SPRY2 is a b-catenin/FOXO3a target gene P Ordo´n˜ez-Mora´n et al 1984 activities were measured separately using the Dual Luciferase reagent kit Ethics statement 3 (Promega) and a Centro XS LB960 luminometer (Berthold, Bad Wildbad, Animal work within this article was carried out in strict accordance with the Germany). Luc activity was normalized to the Rluc activity. The SPRY2 recommendations by the European Union (ECC directive 86/609/EEC, promoter activity was studied using the pGL-hSPRY2-Luc ( À 1826/ þ 221) November 1986). All experiments were approved by the Ethical Committee 49 construct. To study b-catenin/TCF transcriptional activity, we used the for Animal Experimentation, and all efforts were made to minimize animal TOP-Flash and FOP-Flash plasmids containing multimerized wild-type suffering. The work involving human samples was conducted according to (50-CCTTTGATC-30) or mutated (50-CCTTTGGCC-30) TCF/LEF1 binding sites the principles expressed in the Declaration of Helsinki. The study was 50 upstream of a minimal c-fos promoter driving luciferase gene expression approved by the Research Ethics Board of Vall d’Hebron Institute (a gift from Dr.H Clevers, Utrecht, The Netherlands). of Oncology. All patients provided written informed consent for the collection of samples and subsequent analysis. Immunofluorescence and confocal microscopy Cultured cells were fixed in methanol at À 20 1C for 3 min and then washed four times in PBS. Cells were incubated with the primary antibodies diluted CONFLICT OF INTEREST in PBS/3% BSA O/N at 4 1C. After four washes in PBS, the cells were The authors declare no conflict of interest. incubated with secondary antibodies for 45 min at room temperature, washed three times in PBS and mounted using Prolong Gold antifade reagent (Molecular Probes-Invitrogen, Darmstadt, Germany). Conventional ACKNOWLEDGEMENTS fixed and paraffin-embedded sections of human Stage III CRC tumors from a We thank T Martı´nez for technical assistance and Hans Clevers for providing the collection available in the Pathology Service of the Vall d’Hebron Hospital LS174T-tetOn-DNTCF4 cells. Experiments were supported by grants from Fondo 22 were prepared and immunolabelled as described elsewhere. Briefly, Europeo de Desarrollo Regional-Instituto de Salud Carlos III and Spanish Cooperative antigens were retrieved by microwaving in 10 mM citrate buffer (pH 6.0) for Research Network on Cancer (RTICC) (FIS-PI081356, RD12/0036/0001, RD12/0036/ 10 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). Non- 0021 and RD12/0036/0012), Plan Nacional de Biomedicina, Ministerio de Economı´ay specific binding was blocked by incubating the sections in 10% BSA (Sigma- Competitividad (SAF-18302) and Comunidad de Madrid (S2010/BMD-2344 Colomics2). Aldrich) for 1 h. Cell imaging was performed on a Leica TCS SP5 DMI6000 HGP was supported by the Miguel Servet Program, Instituto de Salud Carlos III (ISCIII) microscope using argon ion (488 nm), HeNe (543 nm) and violet diode and PO-M by an EMBO research fellowship. (405 nm) lasers. Images were acquired sequentially by direct register using Leica Confocal Software (LAS AF). Fluorescence images were analyzed and quantified with the MBF ‘ImageJ for Microscopy’ program REFERENCES (www.macbiophotonics.ca) using the criteria described earlier8,20 (Arque´s, O. et al51. ‘Standardized Relative Quantification of Immunofluorescence 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. Tissue Staining’. Protocol Exchange. 2012). 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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