WNT6 Is a Novel Target Gene of Caveolin-1 Promoting Chemoresistance to Epirubicin in Human Gastric Cancer Cells

ORIGINAL ARTICLE WNT6 is a novel target gene of caveolin-1 promoting chemoresistance to epirubicin in human gastric cancer cells

G Yuan1,2, I Regel1, F Lian2, T Friedrich1, I Hitkova3, RD Hofheinz4, P Stro¨ bel5, R Langer6, G Keller6,CRo¨ cken7, W Zimmermann8, RM Schmid1, MPA Ebert3 and E Burgermeister3

Resistance to chemotherapy is a major obstacle for curative treatment of human gastric cancer (GC). However, the underlying molecular mechanisms are largely unknown. Wingless-type MMTV integration site family members (WNTs) are secreted glycoproteins involved in embryogenesis and, on inappropriate expression in the adult, in cancer. Here, we show expression of WNT6 in GC patient specimens, human GC cell lines and in a mouse model of GC. In human GC cells, WNT6 expression was enhanced by caveolin-1 (Cav1), a scaffold protein of plasma membrane caveolae. WNT6 knock-down and overexpression experiments demonstrated that WNT6 increased the resistance to apoptotic cell death induced by the anthracycline chemotherapeutics epirubicin (Epi) and doxorubicin (Dox). Epi increased the activity of the human WNT6 promoter through Cav1-dependent binding of b-catenin to the proximal WNT6 promoter. Epi increased both WNT6/Wnt6 and Cav1 expression in human GC cells and within the tumor area of a murine model of GC (CEA424-SV40 TAg). In GC patients, WNT6 expression was positively associated with the tumor stage and the nodal status, and inversely correlated with the response to ECF (Epi, cisplatin, 5-ﬂuorouracil) chemotherapy. These results showed that WNT6 and Cav1 are upregulated by chemotherapeutics and enhance the resistance of GC cells to anthracycline drugs. Understanding the molecular mechanisms driving WNT6/Cav1- induced drug resistance will provide beneﬁts in developing new therapies for GC.

Oncogene (2013) 32, 375--387; doi:10.1038/onc.2012.40; published online 27 February 2012 Keywords: WNT; caveolin; stomach; gastric cancer; epirubicin

INTRODUCTION activity of the enzyme glycogen synthase kinase-3b, augmenting Despite a recent decrease, gastric cancer (GC) remains one of the b-catenin translocation to the nucleus and the transcription of most common types of human malignancy and the second target genes. WNT signaling prevents b-catenin degradation and leading cause of cancer death in the world.1,2 Surgery combined results in cytoplasmic/nuclear accumulation of b-catenin.12 WNT with chemotherapy is the current treatment of choice.3 However, signaling has been described to promote chemoresistance in the outcome among patients with advanced GC is poor. multiple myeloma,13 neuroblastoma14 and hepatocellular carcino- Identification of biological markers that have a role in determining ma cells.15 WNT6 is an evolutionary conserved morphogenic factor the sensitivity or resistance of GC cells to chemotherapeutic promoting differentiation, survival and epithelial-to-mesenchymal treatment is necessary to improve prognosis. The combination of transitions during embryonal organogenesis.16,17 WNT6 has been epirubicin (Epi), cisplatin and 5-fluorouracil (5-FU) (ECF) comprises shown to be overexpressed in different human cancer cell lines.18 one of the current standard chemotherapy regimens for the Its function in the embryo predisposes this factor, on inappropri- palliative as well as for the perioperative treatment of GC.4,5 ate expression in the adult, for a role in tumorigenesis, metastasis However, the underlying mechanisms of chemoresistance in GC and chemoresistance. remain largely unknown. Caveolin (Cav) is present in lipid rafts, which are microdomains Wingless-type MMTV integration site family members (WNTs) of plasma membranes enriched in cholesterol and sphingoli- constitute a family of cysteine-rich secreted ligands that are pids.19,20 Cav1 is their essential structural protein. Cav1 is involved essential for a wide array of developmental and physiological in lipid transport, membrane trafficking, gene regulation and processes.6,7 WNTs have diverse functions in governing cell fate, signal transduction.21,22 Our previous studies demonstrated that proliferation, migration, polarity and death. Deregulation of WNT Cav1 expression is reduced in primary human GC compared with signaling in the adult, due to mutations in WNT pathway normal stomach tissue, but increased in metastatic human GC cell components such as adenomatous polyposis coli (APC) and lines.23 Cav1 is also upregulated in numerous human drug- b-catenin, is a hallmark of many gastrointestinal cancers.8--11 WNT resistant tumor cells, such as multidrug-resistant colon adenocar- activates at least three intracellular signaling pathways including cinoma cells, doxorubicin (Dox)-resistant breast adenocarcinoma the b-catenin (canonical WNT pathway), planar cell polarity and cells and multidrug-resistant lung cancer cells.24 --27 Cav1 is Ca2 þ pathways. The canonical WNT signaling cascade inhibits the positively correlated with the tumor grade and its progression

1Department of Medicine II, Klinikum rechts der Isar, Technische Universita¨tMu¨nchen, Mu¨nchen, Germany; 2Department of Gastroenterology, The First Afﬁliated Hospital of Sun Yat-sen University, Guangzhou, PR China; 3Department of Medicine II, Universita¨tsklinikum Mannheim, Universita¨t Heidelberg, Baden-Wu¨rttemberg, Germany; 4Department of Medicine III, Universita¨tsklinikum Mannheim, Universita¨t Heidelberg, Baden-Wu¨rttemberg, Germany; 5Institute of Pathology, Universita¨tsklinikum Mannheim, Universita¨t Heidelberg, Baden-Wu¨rttemberg, Germany; 6Institute of Pathology, Klinikum rechts der Isar, Technische Universita¨tMu¨nchen, Mu¨nchen, Germany; 7Institute of Pathology, Christian-Albrechts Universita¨t Kiel, Kiel, Germany and 8Tumor Immunology Laboratory, LIFE Center, Universita¨tMu¨nchen, Mu¨nchen, Germany. Correspondence: Dr E Burgermeister, Department of Medicine II, Universita¨tsklinikum Mannheim, Universita¨t Heidelberg, Theodor-Kutzer Ufer 1-3, Mannheim, Baden-Wu¨ rttemberg D-68167, Germany. E-mail: [email protected] Received 9 May 2011; revised 11 January 2012; accepted 15 January 2012; published online 27 February 2012 WNT6 in gastric cancer G Yuan et al 376 stage, and in some cases, the expression of Cav1 was shown to be an independent predictor of poor prognosis, as reviewed in Shatz and Liscovitch.28 Cav1 upregulation has been associated with poor therapeutic response in patients with lung, kidney, meningioma, prostate and nasopharyngeal cancers.27,29 --32 Here, we investigated the expression and function of WNT6 in GC, and its relationship to Cav1 expression and chemoresistance to anthracycline chemotherapeutics, Epi and Dox. We observed that Cav1 promotes the resistance of human GC cells to apoptosis induced by anthracycline drugs via augmenting WNT6 expression. This phenomenon was associated with b-catenin-dependent chromatin modiﬁcations and transcription. Our data reveal a novel role for Cav1 in promoting WNT6 survival signaling, which facilitates cellular chemoresistance. These results support the notion that targeting Cav1 and/or WNT6 may be a valuable alternative for developing new therapeutic strategies to improve the treatment outcome for GC patients.

RESULTS WNT6 is expressed in GC cell lines and patient specimens To determine WNT6 expression, we studied a series of seven well- characterized human GC cell lines. Western blot (WB) analysis using the monoclonal WNT6 antibody revealed the presence of a band in the range of the predicted molecular weight for WNT6 (46 kDa) (Figure 1a). Consistent with this finding, real-time quantitative PCR (RT--qPCR) confirmed expression of WNT6 mRNA in these cell lines. WNT6 expression was low in AGS and SNU1 cells, originated from a primary gastric tumor, but high in cells derived from a distant metastasis such as MKN45, MKN7 and NCI- N87. Subcellular fractionation confirmed WNT6 protein expression in the membrane, the cytoplasm and in the nucleus (Figure 1b). To assess whether WNT6 is secreted from GC cells, conditioned medium was collected from MKN45 cells, which express high levels of WNT6 and Cav1. WBs detecting acid-precipitated protein from these media confirmed secretion of WNT6 into the medium as compared with mouse esophagus tissue lysate and HEK293 Figure 1. Expression of WNT6 in human GC cell lines and tissues. (two positive controls) (Figure 1c). WNT6 protein was also (a) Top panel: RT--qPCR detection of WNT6 mRNA. CT-values were detectable in tissue lysates from frozen specimens of four normalized against b2-microglobulin (b2M) and presented as randomly selected GC patients, both in normal and tumor tissue means±s.e. (n ¼ 3); *Po0.05 versus AGS. Bottom panel: WB (Figure 1d). Interestingly, WNT6 mRNA expression was down- detecting WNT6 protein in whole-cell lysates (TCL) of human GC cell regulated in 19/26 (73%) of human GC specimens as compared lines using the monoclonal Ab. (b) WB detecting cell-associated and with matched normal gastric tissue, but upregulated in 7/26 (27%) secreted WNT6. Membrane, cytosolic and nuclear fractions of human samples (Figure 1e). This differential WNT6 expression was also GC cell lines are shown. (c) WB detecting secreted WNT6 in conditioned medium (CM) of MKN45 cells as compared with whole evident in immunohistochemical analyses of tissue sections from tissue lysate from mouse forestomach/esophagus (positive control) 53 GC patient tumor specimens (Table 1). WNT6 was expressed in and whole cell lysate of HEK293 cells (positive control). (d)WB the normal gastric foveolar epithelium in the apical gland region detecting WNT6 protein expression in whole-tissue lysates from frozen and in intestinal metaplasia (Figure 2), both in the cytoplasm and GC specimens of four randomly selected patients. Matched tumor (TU) in the nuclei. Plasma cells and macrophages in the lamina propria and adjacent non-neoplastic (NT) tissue is shown. (e)RelativeWNT6 were positive for WNT6 as well. Wnt6 expression was further mRNA expression in human GC (n ¼ 26 patients) compared with confirmed in the mouse stomach tissue predominantly at the base matched normal gastric tissue. Results from RT--qPCRs are calculated of antral glands. As positive controls, the basal layer of the mouse and presented as in (a). Matched normal tissue was set to 1. esophagus and intestinal crypts are shown.

Cav1 promotes WNT6 expression in human GC cells that a gene cluster for Dox resistance (DOXO_GASTRIC_UP) was We have shown previously that Cav1 augments stress resistance signiﬁcantly enriched in AGS/Cav1 cells (Figure 3a). Within this of human GC cell lines.23 AGS cells, which are naturally devoid of gene cluster the heparin-binding growth factor midkine was Cav1, were stably transfected with Cav1 (AGS/Cav1) or empty identiﬁed as the gene consistently upregulated in all drug- vector (AGS/EV), respectively. Vice versa, stable knockdown of resistant GC cell lines tested for the three drugs Dox, cisplatin and endogenous Cav1 in MKN45 cells was achieved using Cav1- 5-FU.34 The second member of the midkine family pleiotrophin directed (MKN45/RNA interference (RNAi)) or control RNAi was also 26-fold upregulated in our array, indicative of a drug- (MKN45/Cav1). To explore the underlying molecular mechanisms resistant phenotype of the AGS/Cav1 cells. Interestingly, WNT6 of Cav1 action, DNA microarrays were hybridized to total RNAs was upregulated 15-fold in AGS/Cav1 versus AGS/EV cells together from the AGS/EV and AGS/Cav1 clonal cell lines.33 AGS/Cav1 cells with other gastroprotective factors such as annexin (15-fold) and displayed a 17-fold increase in Cav1 mRNA together with a panel serpin A1, a serine protease inhibitor (45-fold). These data of mRNAs involved in gastric cell protection and survival indicated that Cav1 overexpression induces gene signatures (Supplementary Table S1). Gene set enrichment analysis revealed associated with chemoresistance.

Oncogene (2013) 375 --387 & 2013 Macmillan Publishers Limited WNT6 in gastric cancer G Yuan et al 377 Table 1. Patient clinicopathologic characteristics and correlation with Wnt6 expression in GC

WNT6 + WNT6 À WNT6 + (%) WNT6 À (%)

Gender (n ¼ 53) Female 11/15 4/15 73 27 Male 23/38 13/38 61 34

NS Fisher exact test Age (n ¼ 53) 44--59 12/16 4/16 75 25 60--70 10/15 5/15 67 33 71--91 12/22 8/22 55 36 NS Fisher exact test Localization (n ¼ 53) Cardia 4/8 3/8 50 38 Corpus 15/26 10/26 58 38 Antrum 9/13 4/13 69 31 NS Fisher exact test Lauren type (n ¼ 30)a Diffuse 8/11 3/11 73 27 Intestinal 10/16 5/16 63 31 NS Fisher exact test Matched normal (n ¼ 53) TU 34/53 17/53 64 32 NT 39/53 6/53 74 11 P ¼ 0.031 Fisher exact test (TU/NT) G grade (n ¼ 53) G2 13/18 5/18 72 28 G3 19/32 11/32 59 34 NS Fisher exact test (G2/G3) T stage (n ¼ 53) T0, T1 6/11 5/11 55 45 T2 16/28 11/28 57 39 T3, T4 12/14 1/14 86 7 P ¼ 0.038 Fisher exact test (T0, 1, 2/T3, 4) N status (n ¼ 53) N0 13/24 11/24 54 46 N1 13/16 3/16 81 19 N2 8/13 3/13 62 23 P ¼ 0.086 Fisher exact test; P ¼ 0.038 Unpaired t-test (N0/N1, 2) ECF response (n ¼ 23)b NR (G3)c 10/11 1/11 91 9 R (G1, 2) 4/12 8/12 33 67 P ¼ 0.01 Fisher exact test (NR/R) Abbreviations: ECF, epirubicin/cisplatin/5-ﬂuorouracil; GC, gastric cancer; NR, non-responder; NS, nonsigniﬁcant; NT, normal tissue; TU, tumor tissue; WNT, wingless-type MMTV integration site family member. aLauren histology data were available from untreated GC patients only (n ¼ 30). Matched TU and NT gastric tissue was not present in all specimens. bClinical response data were analysed from ECF-treated GC patients (n ¼ 23). cBecker regression grade (Becker et al., 2003).

To further explore the relationship between Cav1 and WNT6, we the ECF regimen, Epi (Dox), cisplatin and 5-FU, by MTT assay. AGS analyzed WNT6 expression in the GC clonal cell lines. As shown for and MKN45 parental cell lines and derived clones with modiﬁed the AGS/EV cells, MKN45/RNAi cells with low Cav1 levels also had Cav1 status were treated with Epi and Dox for up to 48 h low expression of WNT6 mRNA compared with AGS/Cav1 cells (Figure 4). Determinations of the IC50 values for the various cell and MKN45/Cav1 cells (Figure 3b). We then measured WNT6 in lines tested showed that GC cells with high Cav1 expression levels parental AGS and HEK293 cells that had been transiently were more resistant to Epi- and Dox-induced cell death than cells transfected with pcDNA3-Cav1 plasmid. Results from qRT--PCRs expressing low Cav1 levels (Supplementary Table S2). Concentra- (Figure 3c) showed that AGS and HEK cells transfected with Cav1 tion response curves in MTT assays showed a signiﬁcant shift in expressed higher levels of WNT6 mRNA than those transfected the IC50 values for the anthracycline drugs, but not for 5-FU or with EV. Similar results were obtained for WNT6 protein in WBs. cisplatin (Supplementary Figure S1). Parental AGS cells, which are These results supported the notion that Cav1 promotes the naturally devoid of Cav1, were more sensitive to anthracyclines expression of WNT6 in GC cells, and raised the hypothesis that than parental MKN45 or NCI-N87 cells with endogenous Cav1 WNT6 acts as a downstream mediator of Cav1 in the response of (Figure 4a). AGS/Cav1 and MKN45/Cav1 clones were more GC cells to chemotherapeutic agents. resistant than AGS/EV and MKN45/RNAi cells (Figure 4b). Anthra- cyclines are topoisomerase inhibitors and intercalate into the DNA WNT6 promotes resistance to anthracycline drugs in human to promote apoptotic cell death. WB analysis showed that Epi GC cells triggered an enhanced apoptotic response in MKN45/RNAi cells as To further examine the functional role of Cav1-mediated WNT6 compared with MKN45/Cav1, as demonstrated by the parallel upregulation, cells were subjected to genotoxic stress. We increase in the cleavage of both caspase-3 and 8 (Figure 4c). These measured the cytotoxicity evoked by the three components of results provided evidence that Cav1 is an important determinant

& 2013 Macmillan Publishers Limited Oncogene (2013) 375 --387 WNT6 in gastric cancer G Yuan et al 378

Figure 2. Immunohistochemical detection of WNT6 in human GC tissue and Wnt6 in gastrointestinal organs from wild-type C57BL/6 mice. Parafﬁn sections were stained with polyclonal WNT6 antibody. WNT6 staining was visible both in the cytoplasm and nuclei. Human: normal stomach tissue and intestinal metaplasia with goblet cells; positive controls: mouse tissues. Magniﬁcation Â 100.

for the resistance of human GC cells toward induction of apoptosis To test whether ectopic overexpression of WNT6 desensitizes by Epi and Dox. cells to chemotherapy, we cloned the full-length mouse To conclusively establish the involvement of WNT6 in Cav1- Wnt6 complementary DNA (Wnt6) from colon tissue of APCmin/ þ mediated drug resistance, we investigated whether the inhibition mice by RT--PCR and inserted it into pTarget expression plasmid. of WNT6 activity could increase the drug sensitivity of cells. Mouse Wnt6 and human WNT6 share 97% homology in their Endogenous WNT6 was knocked down by transient transfection of amino-acid sequence. Mouse Wnt6 was overexpressed by small interfering RNA (siRNA) oligonucleotides in parental MKN45 transient transfection in parental AGS and MKN45 cells, resulting and HEK293 cells, resulting in a marked decrease in the level of in a strong increase in the level of Wnt6 mRNA and protein WNT6 mRNA and protein (Figure 5a). Cells with WNT6-knockdown (Figure 6a).35 Transient transfection of the Wnt6 expression were then exposed to Dox and Epi for up to 48 h. Results from plasmid into AGS/Cav1 and MKN45/Cav1 cells signiﬁcantly MTT assays showed that, relative to cells transfected with control increased cell survival in presence of Epi as compared with Cy5-RNAi, HEK293 and MKN45 cells with silenced WNT6 expres- mock-transfected cells (Figure 6b). In contrast, AGS/EV and sion displayed a signiﬁcant increase in Dox- and Epi-induced cell MKN45/RNAi cells, which both lack endogenous Cav1 did not death (Figure 5b). These data underlined the involvement of show a change in survival. These results showed that Wnt6 WNT6 in the drug response mechanism and proposed WNT6 as an positively cooperates with Cav1 in human GC cells to enhance important mediator of the pro-survival function exerted by Cav1 in their resistance to the anthracycline chemotherapeutics Epi human GC cells. and Dox.

Oncogene (2013) 375 --387 & 2013 Macmillan Publishers Limited WNT6 in gastric cancer G Yuan et al 379 WNT6 protein was increased on drug exposure. To elucidate the molecular mechanisms of this upregulation, the proximal WNT6 promoter (À500/ATG) was cloned from genomic DNA of normal human stomach tissue and inserted into pGL3 luciferase reporter plasmid (hWNT6p-pGL3). AGS and MKN45 cells were transiently transfected with hWNT6p-pGL3 and then treated with Epi for 48 h. Retinoic acid served as a positive control.17 Epi and retinoic acid evoked a dose-dependent increase inWNT6 reporter gene luciferase activity (Figure 7b). b-Catenin is a key regulator in the WNT signal transduction cascade.7 To determine whether the effect of WNT6 on drug resistance was mediated by a b-catenin-dependent pathway, b-catenin luciferase reporter activity was measured after drug treatment. b-catenin activity was measured with the commonly used TOPﬂash assay, which utilizes multimeric transcription factor 4 (TCF)-binding sites to drive luciferase activity. Treatment of MKN45 cells with Epi evoked a concentration-dependent increase in b-catenin reporter gene activity as shown before for the WNT6 promoter (Figure 7c). These results showed that WNT6 upregulation occurs at the transcriptional level and is dependent on active b-catenin. To gain further insight into the involvement of Cav1 in the effect of Epi on b-catenin activity, the mouse Wnt6 expression plasmid was co-transfected with the b-catenin reporter into MKN45/RNAi and MKN45/Cav1 clones (Figure 7d). Notably, Epi increased b-catenin reporter gene activity to a higher extent in Wnt6-overexpressing MKN45/Cav1 cells as compared with MKN45/RNAi cells. Collectively, these data corroborated that Cav1 and Wnt6 functionally cooperate to promote b-catenin activity.

Anthracyclines promote b-catenin binding to a conserved TCF site in the WNT6 promoter Using transcription element search system, we identified a putative DNA-binding site for TCF in the proximal WNT6/Wnt6 promoters in both the mouse and human DNA loci (Supplemen- tary Table S3). To analyze b-catenin/TCF binding to this region, chromatin immunoprecipitation primers were designed flanking this predicted site. The amplicons comprised the distal (À500/ À250) and proximal (À250/ þ 1) promoter regions (Figure 7e). To investigate the effect of Epi on b-catenin binding to this promoter region, we performed chromatin immunoprecipitation on the human genomic WNT6 locus in parental MKN45 cells. MKN45 cells were treated for 24 h with 0.5 mM Epi and were subjected to chromatin immunoprecipitation with a polyclonal b-catenin anti- Figure 3. Cav1 promotes expression of WNT6 mRNA and protein. body followed by genomic qPCR. Specific amplification products (a) Gene set enrichment analysis (GSEA) of signatures from AGS cells were visualized together with the quantification of normalized stably transfected with Cav1 (AGS/Cav1) as compared with empty cycle threshold values. Compared with vehicle-treated cells, Epi vector (AGS/EV), respectively. Left panel: Dox resistance gene cluster led to an increased binding of b-catenin to the distal (À500/À250) (DOXO_GASTRIC_UP) was enriched in AGS/Cav1 cells. Right panel: Heatmap of resistance genes; red: upregulated, blue: downregulated. WNT6 promoter region. Collectively, these data corroborated (b) Cav1 increases WNT6 mRNA in AGS and MKN45 clones. RT--qPCR WNT6 as a positive regulator of b-catenin transcriptional activity results were normalized against b2-microglobulin (b2M) and are and chemoresistance (Figure 7f). shown as means±s.e. (n ¼ 3); *Po0.05 AGS/Cav1 versus AGS/EV; MKN45/Cav1 versus MKN45/RNAi. (c) Transient expression of ectopic Epi induces expression of Cav1 and Wnt6 in a mouse model of GC Cav1 in AGS and HEK293 cells increases WNT6 mRNA and protein. Top panel: results from RT--qPCRs normalized against b2M are shown We next explored whether the functional interaction between as means±s.e. (n ¼ 3); *Po0.05 Cav1 versus EV. Bottom panel: Cav1 and Wnt6, observed to have a role in determining the drug representative WB are shown. response of human GC cells in vitro, may be also involved in the drug response in vivo. To test this, we evaluated the expression of Cav1 and Wnt6 in CEA424-SV40 TAg transgenic mice. CEA424- SV40 TAg is a transgenic C57BL/6 mouse strain harboring the SV40 T-antigen gene under the control of the human À424/À8 bp CEA Anthracyclines activate the WNT6 promoter via a b-catenin- gene promoter, which leads to the development of a highly dependent mechanism proliferative carcinoma in the pyloric part of the stomach.36,37 By To assess whether the anthracycline drugs themselves induce day 50, the whole pyloric mucosa has been replaced by carcinoma expression of WNT6, AGS cells that naturally have low WNT6 cells, and mice are moribund after 3 month. Eight weeks old expression were treated with Epi and Dox for 48 and 72 h. qRT-- CEA424-SV40 TAg mice were treated with Epi (3 mg/kg, intraper- PCRs revealed that drug treatment increases WNT6 expression itoneally once a week) for 3 weeks, and tissue samples were in a time-dependent manner (Figure 7a). We also found that extracted from the pylori. Immunohistochemical and RT--qPCR

& 2013 Macmillan Publishers Limited Oncogene (2013) 375 --387 WNT6 in gastric cancer G Yuan et al 380

Figure 4. Cav1 promotes survival of human GC cell lines in presence of the anthracyline chemotherapeutics Epi and Dox. Cells were treated with increasing concentrations of Epi and Dox for 48 h. Cell viability was assessed by MTT assay. Optical density (OD) values are calculated as % survival±s.e. (n ¼ 3) of vehicle-treated controls. (a) Parental cell lines: AGS (Cav1-negative); MKN45 and N87 (Cav1-positive). (b) Derived cell clones: AGS/Cav1 versus AGS/EV; MKN45/Cav1 versus MKN45/RNAi. (c) Cav1 inhibits Epi-induced apoptosis. MKN45/Cav1 and MKN45/RNAi clones were treated with 6 mM Epi for the times indicated. Representative WBs of cleaved caspases are shown.

results showed an increased expression of Wnt6 and Cav1 mRNA chemotherapy, a retrospective analysis of 23 GC patients who had levels (Figure 8a) and a more intense and frequent nuclear staining been treated with preoperative chemotherapy using a combina- of Wnt6 in the tumor regions (Figure 8b). These results suggested tion of Epi, cisplatin and 5-FU (ECF regimen) was performed. The that the functional link between Cav1 and Wnt6 in promoting entire paraffin-embedded tumor beds of patient specimens were chemoresistance in GC cells may be also existent in the mouse stained for WNT6 and evaluated histologically (Figure 9). The gastric tumor. Immunohistochemical detection of the proliferation histopathologic regression was divided into three grades accord- marker Ki-67 demonstrated that the area of residual tumor nests ing to Becker: grade 1, complete or subtotal tumor regression; but not the intratumoral proliferation rate was significantly grade 2, partial tumor regression; and grade 3, minimal or no reduced in presence of Epi (Regel et al., unpublished observation). tumor regression.38 There was no correlation of WNT6 positivity to These data indicated that, by upregulation of Cav1/Wnt6 expres- patient survival, presumably because of limited case numbers. sion, Epi may promote chemoresistance instead of anti-tumor However, 10 out of 11 (91%) non-responding patients (Becker efficacy in vivo. Collectively, these in vivo findings support a regression grade 3) had WNT6 staining in the tumor area, whereas possible clinical significance of the in vitro data for novel 8 out of 12 (67%) responding patients (Becker regression grades 1 therapeutic strategies against chemoresistant human GC. and 2) were negative for WNT6 in the tumor area (Table 1). These findings indicated an inverse relationship between WNT6 expres- Inverse relationship of WNT6 expression and response to ECF sion in GC and clinical response to ECF therapy. treatment in GC patients To assess whether WNT6 expression correlates with other To determine whether WNT6 expression in human GC clinicopathologic characteristics, a tissue microarray with paraffin- correlates with a patient’s histomorphological response to ECF embedded biopsies from 30 GC patients, who had not yet

Oncogene (2013) 375 --387 & 2013 Macmillan Publishers Limited WNT6 in gastric cancer G Yuan et al 381

Figure 5. Knock-down of WNT6 expression decreases GC cell survival in presence of anthracyclines. (a) Functional validation of WNT6 siRNA. Parental MKN45 and HEK293 cells were transiently transfected with WNT6 siRNA and Cy5 control RNAi, respectively, and were treated with the indicated concentrations of Epi for 48 h. Results from RT--qPCRs were normalized to b2-microglobulin (b2M) and are shown as means±s.e. (n ¼ 3). Representative WBs detecting WNT6 and HSP90 as control in whole cell lysates are shown below. (b) Decreased cell survival on WNT6 knock-down in parental MKN45 and HEK293 cells in presence of Epi and Dox. Cells were transfected and treated as in (a). Cell Viability was measured by MTT assay. OD values were calculated as % survival±s.e. (n ¼ 3) of vehicle-treated cells; *Po0.05 WNT6 siRNA versus Cy5 control siRNA. received any chemotherapy, was stained for WNT6. The combined the adult, which facilitates the development of resistance in GC statistical analysis of the two patient cohorts (n ¼ 53) revealed that cells to chemotherapeutic drugs. Our results further indicate that there was no association of WNT6 positivity with gender, age, tumor the pro-survival function of Cav1 was mediated by the increased localization (cardia, corpus, antrum) or histological subtype (diffuse expression and/or secretion of the WNT6 glycoprotein. These or intestinal according to Lauren) (Table 1). Nonetheless, WNT6 was events may ultimately render GC cells resistant to apoptosis, thus positively correlated with the T (tumor) stage and the N (nodal) favoring tumor cell survival and further progression of their status, both major prognostic factors for poor survival in patients malignant phenotype. with GC. Future studies have to evaluate the potential of WNT6 as a We hypothesize, that the high number of membrane-bound putative novel response or negative predictive marker in prospec- Cav1 molecules in drug-resistant cancer cells may facilitate the tivestudiesinlargerseriesofpatientswithchemorefractoryGC. secretion of active glycosylated WNT6 molecules into the medium. Alternatively, Cav1 itself may support canonical WNT/b-catenin signaling in an autocrine or paracrine mode, where drug-resistant DISCUSSION cells secrete WNT6 into the medium to activate neighbor cells to In this study, we describe to our knowledge for the first time, increase their survival potential. On WNT ligand binding, WNT6 expression in human GC patients, human GC cell lines and a transmembranous frizzled receptors trigger an intracellular mouse model of GC. We demonstrated that Cav1 and WNT6 signaling cascade leading to the translocation of active b-catenin positively cooperate in promoting chemoresistance of GC cells to to the nucleus and activation of the TCF/lymphoid enhancer- DNA-damaging anthracycline drugs, such as Epi and Dox, through binding factor 1 transcription complex to promote target gene activation of the canonical WNT/b-catenin pathway. Information expression.6,7 It has been shown that canonical WNTs are first on the function of WNT6 in general and on the underlying secreted to the cell surface through the endoplasmatic reticulum/ molecular mechanisms of drug response in GC is very limited. Our Golgi system, then compartmentalized into endosomes and data represent the first description of a role for WNT6 as an recycled to the cell surface.39 Cav1 is one of the major drivers of example of a tumor-related re-expression of an embryonic gene in clathrin-independent endocytosis.40 Reports claim a dual role of

& 2013 Macmillan Publishers Limited Oncogene (2013) 375 --387 WNT6 in gastric cancer G Yuan et al 382

Figure 6. Overexpression of ectopic Wnt6 promotes resistance of human GC cells to anthracyclines. (a) Validation of Wnt6 mRNA and protein overexpression. Parental AGS and MKN45 cells (and HEK293 as control) were transiently transfected with mouse Wnt6 expression plasmid (Wnt6) or empty vector (EV), respectively. Data from RT--qPCR were normalized to b2-microglobulin (b2M) and are shown for AGS (left panel) and HEK293 (right panel) as---fold change±s.e. (n ¼ 3) of empty vector (EV) transfected cells; *Po0.05 Wnt6 versus EV. Representative WBs detecting Wnt6 and HSP90 in AGS (left panel) and MKN45 (right panel) cells are shown. (b) AGS and MKN45 clones were transiently transfected as in (a) and treated with the concentrations indicated of Epi for 48 h. Cell viability was measured by MTT assay. OD values were calculated as % survival±s.e. (n ¼ 3) of vehicle-treated cells transfected with EV; *Po0.05 Wnt6 versus EV.

Figure 7. Epi upregulates human WNT6 promoter activity through a b-catenin-dependent pathway. (a) Epi increases expression of WNT6 mRNA and protein. Parental AGS cells were treated with 0.1 mM Epi for the times indicated. Results from RT--qPCRs normalized to b2-microglobulin (b2M) are presented as means±s.e. (n ¼ 3) of untreated cells; *Po0.05 Epi versus DMSO. Representative WBs from total cell lysates are shown. (b) Epi induces activation of the proximal human WNT6 promoter. Parental MKN45 cells were transiently co-transfected with pGL3 firefly luciferase reporter plasmid driven by the proximal human WNT6 promoter and with renilla luciferase reporter plasmid, and were stimulated for 48 h with Epi (0.2, 0.3 and 0.6 mM). Firefly luciferase activity was normalized to renilla and presented as % ±s.e. (n ¼ 3) of vehicle-treated controls; *Po0.05 Epi versus DMSO. As positive control parental AGS and MKN45 cells were transfected as above and stimulated for 48 h with retinoic acid (RA, 10 mM). Data are presented as above; *Po0.05 RA versus DMSO. (c) Epi increases b-catenin transcriptional activity. Parental MKN45 cells were transiently co-transfected with b-catenin-driven firefly and renilla luciferase reporter plasmids, and were stimulated for 48 h with Epi (0.2 , 0.3 and 0.6 mM). Data are presented as in (b); *Po0.05 Epi versus DMSO. (d) WNT6 augments Epi-induced b-catenin activity in the presence of Cav1. MKN45/RNAi and MKN45/Cav1 clones were transiently co-transfected with b-catenin firefly and renilla luciferase reporter plasmids in presence or absence of Wnt6 expression plasmid or empty vector (EV), respectively. Cells were stimulated for 48 h with Epi (0.6 mM). Data are presented as in (b); *Po0.05 Epi versus DMSO. (e) Epi promotes b-catenin binding to the proximal human WNT6 promoter. Top panel: Scheme of the proximal (À500/ þ 1) human WNT6 genomic locus. Primers sites for chromatin immunoprecipitation (ChIP) and the putative conserved TCF/lymphoid enhancer binding factor site are indicated. Lower panels: ChIP. Parental MKN45 cells were treated for 24 h with vehicle or 0.5 mM Epi, respectively. ChIP was performed with rabbit polyclonal b-catenin antiserum or control immunoglobulin G (IgG). Genomic qPCR reactions were visualized by gel electrophoresis. CT-values from qPCRs of immunoprecipitated DNA were normalized to the CT-values of input DNA and are expressed as means±s.e. (n ¼ 3) increase in binding by Epi compared with vehicle controls; *Po0.05 Epi versus DMSO. (f) Proposed scheme of Epi-induced drug resistance. Anthracyline drugs introduce double-strand breaks (DSB) into the DNA, which trigger an Ataxia telangiectasia mutated (ATM)-mediated DNA-damage response that upregulates Cav1 and WNT-pathway genes via activation of p53. Cav1 and WNT6 then promote WNT/b-catenin-signaling by feeding an auto- or paracrine amplification loop toward chemoresistance.

Oncogene (2013) 375 --387 & 2013 Macmillan Publishers Limited WNT6 in gastric cancer G Yuan et al 383 Cav1 in (i) activation of WNT signaling, for example, by In fact, it has been reported that the induction of apoptosis in endocytosis of ligated WNT receptors (frizzled, LRP6)41,42 and in GC cells by Dox relates to b-catenin.44 However, as Epi and Dox are (ii) sequestration of b-catenin to membranes via E-cadherin, agents that directly damage DNA, it is possible that double-strand thereby reducing the availability of nuclear b-catenin for the breaks introduced by these drugs trigger a specific DNA-damage formation of a persistent, transcriptionally active complex response involving ataxia telangiectasia mutated kinase and between b-catenin and TCF/lymphoid enhancer-binding factor.43 p53.45,46 The p53 protein is a known regulator of the Cav1 gene47 For example, siRNA-knockdown of Cav1 causes a reduction of and several WNT-related genes.48,49 Thus, an anthracycline b-catenin stabilization upon WNT signaling, suggesting that damage-specific mechanism may activate WNT-dependent survi- caveolae-mediated endocytosis contributes positively to WNT val signaling by co-upregulation of Cav1 and WNT6. These two signaling.41 Our result support this positive role of Cav1 in proteins then positively cooperate in an auto- or paracrine activation of WNT signaling by promoting expression and/or amplification loop to foster b-catenin-dependent activation of secretion of WNT6, and this effect was dependent on b-catenin. their own synthesis and other genes involved in proliferation and Thus, further experimentation is required to ascertain the cell survival (Figure 7f). mechanisms, which act downstream of WNT6 to determine the Our in vivo data corroborated that anthracyclines function response of GC cells to chemotherapeutic treatment. as chemoresistance promoters also in the context of murine GC.

& 2013 Macmillan Publishers Limited Oncogene (2013) 375 --387 WNT6 in gastric cancer G Yuan et al 384 stage of GC, under stress conditions, for example, during metastasis or chemotherapy, WNT6 as a driver of cell survival may be re-upregulated to protect GC cells against apoptosis. In sum, the data from our study evinced Cav1 as an important determinant of the resistance of GC cells to anthracycline-induced apoptosis, and suggest that WNT6 is a critical mediator of the prosurvival functions of Cav1. Although a larger series of human GC specimens needs to be evaluated, the results presented here support the notion that the development of new Cav1- and/or WNT6-targeting strategies may enhance the efﬁcacy of current GC therapies and improve the treatment outcome for patients with GC, particularly those presenting with advanced, metastatic or recurrent disease.

MATERIALS AND METHODS Ethics statement The patient study was approved by the Ethics Committee of the Technische Universita¨tMu¨nchen and the Universita¨t Heidelberg. Animal studies were conducted in agreement with ethical guidelines of the Technische Universita¨tMu¨nchen and approved by the appropriate government authorities.

Subjects Human GC (TU) and non-tumorous (NT) tissues were collected and stored as described previously.23 Tissue microarrays from parafﬁn-embedded specimens from GC patients (n ¼ 30) receiving no previous chemotherapy Figure 8. Epi upregulates Cav1 and Wnt6 expression in vivo. were provided by GK (Klinikum rechts der Isar, Technische Universita¨t (a) Transgenic CEA424-SV40 TAg mice with GC (n ¼ 5 per group) Mu¨nchen). Specimens from GC patients (n ¼ 23) resected on neoadjuvant were treated with Epi (3 mg/kg intraperitoneally (i.p.) once a week) ECF therapy were provided by RH (Universita¨tsklinikum Mannheim, or NaCl (control group) for 3 weeks. Macroscopic tumor lesions Universita¨t Heidelberg). Clinicopathological data were provided by GK were resected for RNA analysis. Results from RT--qPCR normalized and RH, respectively. to b2-microglobulin (b2M) are shown as means±s.e. (n ¼ 5 per group); *Po0.05 Epi versus NaCl. (b) Epi increases Wnt6 protein expression in murine GC. Immunohistochemical detection Animals of Wnt6 in nuclei of tumor areas in stomach pylori of Epi-treated Transgenic CEA424-SV40 T-antigen (CEA424-SV40 TAg) mice were CEA424-SV40 TAg mice. Representative images are shown; described previously36 and maintained on a pure C57BL/6 background. Tu, tumor; Gl, gland; magniﬁcation Â 100. Mice (female, 8 weeks old) were randomized (n ¼ 5 per group) and were treated with Epi for 3 weeks (3 mg/kg, intraperitoneally once a week).

Cell culture Epi per se upregulated Cav1 and Wnt6 expression in the tumor area. This event was manifest independently of a substantial drug- Human embryonic kidney HEK-293 cells and human GC cell lines AGS, NCI- mediated anti-tumor efficacy, as measured by tumor area and N87, KATO-III, SNU-1, SNU-5 (all from LGC Standards GmbH, Wesel, Ki-67 proliferation index (Regel et al., unpublished observation). Germany), MKN-7 and MKN-45 (both from Japanese Collection of Research Bioresources (JCRB)-Cell Bank, National Institute of Biomedical Innovation, This finding implicated that chemotherapy itself may promote 23 chemoresistance by increasing the expression of secreted Osaka, Japan) and stable clones were cultivated as described. For resistance/survival proteins in addition to classical multidrug collection of conditioned medium, MKN45 cells were grown to subcon- resistance transporters. Despite other reports, as reviewed in fluency in a 10 cm dish. Conditioned medium was replaced with RPMI Goetz et al.,50 we did not detect a substantial effect of Cav1 on containing 0% (v/v) fetal calf serum, 48 h before collection of conditioned expression levels of a series of 11 multidrug resistance proteins medium under sterile conditions. Conditioned mediums were stored at 1 (data not shown). Thus, specific resistance mechanisms may work À20 C. For acid precipitation, medium was thawed and mixed 1:2 independently of simple drug extrusion, and thus may constitute with 21% (v/v) trichloric acid, and the mixture was incubated on ice for putative novel selective targets for anticancer therapy. 16 h. The precipitate was collected by centrifugation at 4000 r.p.m. for 1 In support of this concept, the patient data confirm a positive 30 min at 4 C. The supernatant was discarded, and the pellet was dried correlation of WNT6 with GC progression and reduced clinical and resuspended in 3 M Tris-HCl, pH 8.8 for WB analysis. response to ECF therapy (Table 1). The fact that overall WNT6 mRNA and protein was reduced in GC compared with matched DNA constructs normal gastric tissue, indicates that elevated WNT expression in The full-length mouse Wnt6 complementary DNA (NM_009526) was non-responder patients may be either maintained throughout amplified from colon tissue of APCmin/ þ mice by RT--PCR and inserted tumorigenesis or re-acquired after initial loss. WNT acts both as a into the EcoRI site of the pTarget expression plasmid (Promega factor of differentiation7 or tumor aggressiveness,51,52 depending GmbH, Mannheim, Germany). The proximal human WNT6 promoter on the age of the organism or the progression stage of the cancer. (À500/ATG) (NC_000002.11) was amplified from genomic DNA of normal Thus, in the embryo or the adult normal tissue, differentiation will human stomach tissue and inserted into the XhoI/HindIII-digested prevail, for example, in stem cell niches of the gastrointestinal pGL3 firefly luciferase reporter plasmid (Promega). Cells were transiently tract. In primary cancer sites, WNT6 may impede rapid prolifera- transfected with TurboFect (Thermofisher Scientific, Schwerte, tion at early stages of tumorigenesis and its expression can be Germany) according to the manufacturer’s instructions. Firefly luciferase silenced, for example, by methylation.53 In contrast, in advanced reporter activity was measured 48 h post transfection and normalized

Oncogene (2013) 375 --387 & 2013 Macmillan Publishers Limited WNT6 in gastric cancer G Yuan et al 385

Figure 9. Inverse correlation of WNT6 expression and the clinical response to ECF therapy in GC patients (see also Table 1). Immunohistochemical detection of nuclear and cytoplasmic WNT6 expression. Top panels: normal foveolar glands (left) and mucus pit regions (right, zoomed-in) were positive for WNT6 in non-neoplastic gastric tissue (NT) adjacent to GC tissue (TU) in the same patient; Middle panels: positive WNT6 staining in differentiated (left) and undifferentiated (right) gastric tumors of two different ECF non-responder (NR) patients; Bottom panels: two different ECF responder (R) patients with absence of WNT6 staining in differentiated (left) and undifferentiated (right) gastric tumors are shown. Magniﬁcation Â 100, Â 200.

to renilla luciferase activity using the Dual Luciferase Assay system 48 h. The siGENOME SMART pool siRNA WNT6 was purchased from (Promega). Dharmacon/Thermoﬁsher, Schwerte, Germany.

MTT cell viability assay Western blotting Cells were plated and grown in 96-well plate in 0.1 ml Dulbecco’s WB was performed as previously described.23,56 The following antibodies modified Eagle’s medium or RPMI containing 5% (v/v) fetal calf serum at were used: polyclonal sheep anti-human WNT6 (#AF4109, R&D, Wiesbaden, 37 1C for 24 h. Thereafter, the medium was changed and 0.1 ml fresh Germany), polyclonal rabbit anti-WNT6 (#ab50030, Abcam, Cambridge, medium containing drug was added and the cells were incubated for UK), cleaved caspase-3 (Asp175), cleaved caspase-8 (Asp391) (Cell additional 48 h. The number of viable cells was determined by using the Signaling Technology, Beverly, MA, USA), Hsp90 (Santa Cruz Biotechnology, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay Santa Cruz, CA, USA). as described.54,55 Immunohistochemistry Reverse transcription PCR (RT-PCR) and qPCR The method was performed as described previously.57 Anti-WNT6 (rabbit Total RNAs were extracted from cell lines and tissues using RNeasy Mini kit polyclonal, 1:100; Abcam) was used as primary antibody. Images were (Qiagen GmbH, Hilden, Germany). Total RNA was reverse transcribed using collected using a Zeiss Axio Imager A1 microscope, AxioCam MRC camera Verso cDNA kit (Thermofisher Scientific), and RT--PCR was performed and Axiovision Rel 4.8 acquisition software (Zeiss, Luton, UK). using the SYBR Green Mix (Roche, Nutley, NJ, USA) on LightCycler480 system (Roche). Each reaction was performed in duplicate. Results were normalized to b2-microglobulin mRNA. Primer sequences are listed in Chromatin immunoprecipitation Supplementary Table S4. Cells were treated for 24 h with Epi before cross-linking, lysis and sonication, as described previously.58 Chromatin fragments were immunoprecipitated with b-catenin antibody (C-18, Santa Cruz Biotechnology). WNT6 knock-down by siRNA To ablate endogenous WNT6 expression, 2 Â 105 cells per well in a six-well plate were transfected with 50 nM WNT6 siRNA or a control Cy 5-labelled Statistical analysis siRNA oligonucleotide (Silencer, Ambion/Applied Biosystems, Darmstadt, The software (GraphPad Software, Inc., La Jolla, CA, USA) was used to Germany) using the Interferin siRNA transfection reagent as recommended analyze the data. The *P-value o0.05 was considered statistically by the manufacturer (Peqlab, Erlangen, Germany). Cells were incubated significant. All values are given as means ±s.e. from at least five animals with siRNA for 12 h followed by treatment with Epi and Dox for additional per group or at least three independent cell experiments.

& 2013 Macmillan Publishers Limited Oncogene (2013) 375 --387 WNT6 in gastric cancer G Yuan et al 386 ABBREVIATIONS 21 Anderson RG, Jacobson K. A role for lipid shells in targeting proteins to caveolae, WNT, wingless-type MMTV integration site family member; GC, gastric rafts, and other lipid domains. Science 2002; 296: 1821 --1825. cancer; Cav1, caveolin-1; TCF, transcription factor 4; Dox, doxorubicin; Epi, 22 Williams TM, Lisanti MP. Caveolin-1 in oncogenic transformation, cancer, and epirubicin; 5-FU, 5-fluorouracil; ECF, epirubicin/cisplatin/5-fluorouracil. metastasis. Am J Physiol Cell Physiol 2005; 288: C494 --C506. 23 Burgermeister E, Xing X, Ro¨cken C, Juhasz M, Chen J, Hiber M et al. Differential expression and function of caveolin-1 in human gastric cancer progression. Cancer Res 2007; 67: 8519 --8526. CONFLICT OF INTEREST 24 Lavie Y, Fiucci G, Liscovitch M. Up-regulation of caveolae and caveolar The authors declare no conflict of interest. constituents in multidrug-resistant cancer cells. J Biol Chem 1998; 273: 32380 --32383. 25 Yang CP, Galbiati F, Volonte D, Horwitz SB, Lisanti MP. Upregulation of caveolin-1 ACKNOWLEDGEMENTS and caveolae organelles in Taxol-resistant A549 cells. FEBS Lett 1998; 439: 368 --372. We are grateful to Minhu Chen, Xiao Chen, Jie Chen for support on collaborations. 26 Be´langer MM, Gaudreau M, Roussel E, Couet J. Role of caveolin-1 in etoposide M Ebert is supported by grants from the Deutsche Krebshilfe (107885, 108096), the DFG resistance development in A549 lung cancer cells. Cancer Biol Ther 2004; 3: (SFB 824, TP B1), the Else Kro¨ner Stiftung (Nr P14/07//A104/06), the BMBF (Mobimed 954 --959. 01EZ0802 and KMU-innovativ Initiative of the BMBF No 0315116B). E Burgermeister 27 Ho CC, Kuo SH, Huang PH, Huang HY, Yang CH, Yang PC. Caveolin-1 expression is receives funds from the Deutsche Krebshilfe (108287) and DFG (BU-2285). significantly associated with drug resistance and poor prognosis in advanced non-small cell lung cancer patients treated with gemcitabine-based chemotherapy. Lung Cancer 2008; 59: 105 --110. REFERENCES 28 Shatz M, Liscovitch M. Caveolin-1 and cancer multidrug resistance: coordinate 1 Kelley JR, Duggan JM. Gastric cancer epidemiology and risk factors. J Clin regulation of pro-survival proteins? Leuk Res 2004; 28: 907 --908. Epidemiol 2003; 56:1--9. 29 Joo HJ, Oh DK, Kim YS, Lee KB, Kim SJ. Increased expression of caveolin-1 and 2 Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence microvessel density correlates with metastasis and poor prognosis in clear cell and mortality rates and trends. Cancer Epidemiol Biomarkers Prev 2010; 19: 1893 -- renal cell carcinoma. BJU Int 2004; 93: 291 --296. 1907. 30 Barresi V, Cerasoli S, Paioli G, Vitarelli E, Giuffre` G, Guiducci G et al. Caveolin-1 in 3 Becker K, Langer R, Reim D, Novotny A, Meyer Zum Buschenfelde C, Engel J et al. meningiomas: expression and clinico-pathological correlations. Acta Neuropathol Significance of histopathological tumor regression after neoadjuvant chemother- 2006; 112: 617 --626. apy in gastric adenocarcinomas: a summary of 480 cases. Ann Surg 2011; 253: 31 Karam JA, Lotan Y, Roehrborn CG, Ashfaq R, Karakiewicz PI, Shariat SF. Caveolin-1 934 --939. overexpression is associated with aggressive prostate cancer recurrence. Prostate 4 Cunningham D, Allum WH, Stenning SP, Thompson JN, Van de Velde CJ, Nicolson 2007; 67: 614 --622. M et al. Perioperative chemotherapy versus surgery alone for resectable 32 Du ZM, Hu CF, Shao Q, Huang MY, Kou CW, Zhu XF et al. Upregulation of caveolin- gastroesophageal cancer. N Engl J Med 2006; 355:11--20. 1 and CD147 expression in nasopharyngeal carcinoma enhanced tumor cell 5 Cunningham D, Starling N, Rao S, Iveson T, Nicolson M, Coxon F et al. migration and correlated with poor prognosis of the patients. Int J Cancer 2009; Capecitabine and oxaliplatin for advanced esophagogastric cancer. N Engl J Med 125: 1832 --1841. 2008; 358: 36 --46. 33 Burgermeister E, Friedrich T, Hitkova I, Gruner-Regel I, Einwa¨chter H, Zimmermann 6 Logan CY, Nusse R. The WNT signaling pathway in development and disease. W et al. The ras inhibitors caveolin-1 and docking protein-1 activate PPARgamma Annu Rev Cell Dev Biol 2004; 20: 781 --810. through spatial relocalization at helix-7 of its ligand-binding domain. Mol Cell Biol 7 Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006; 127: 2011; 31: 3497 --3510. 469 --480. 34 Kang HC, Kim IJ, Park JH, Shin Y, Ku JL, Jung MS et al. Identification of genes with 8 Wei Y, Fabre M, Branchereau S, Gauthier F, Perilongo G, Buendia MA. Activation of differential expression in acquired drug-resistant gastric cancer cells using high- beta-catenin in epithelial and mesenchymal hepatoblastomas. Oncogene 2000; density oligonucleotide microarrays. Clin Cancer Res 2004; 10: 272 --284. 19: 498 --504. 35 Burrus LM, McMahon AP. Biochemical analysis of murine WNT proteins reveals 9 Taniguchi K, Roberts LR, Aderca IN, Dong X, Qian C, Murphy LM et al. Mutational both shared and distinct properties. Exp Cell Res 1995; 220: 363 --373. spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and 36 Thompson J, Epting T, Schwarzkopf G, Singhofen A, Eades-Perner AM, van Der hepatoblastomas. Oncogene 2002; 21: 4863 --4871. Putten H et al. A transgenic mouse line that develops early-onset invasive gastric 10 Hadjihannas MV, Bru¨ckner M, Jerchow B, Birchmeier W, Dietmaier W, Behrens J. gastric carcinoma provides a model for carcinoembryonic antigen-targeted tumor Aberrant WNT/beta-catenin signaling can induce chromosomal instability in therapy. Int J Cancer 2000; 86: 863 --869. colon cancer. Proc Natl Acad Sci USA 2006; 103: 10747 --10752. 37 Bourquin C, von der Borch P, Zoglmeier C, Anz D, Sandholzer N, Suhartha N et al. 11 Asciutti S, Akiri G, Grumolato L, Vijayakumar S, Aaronson SA. Diverse mechanisms Efficient eradication of subcutaneous but not of autochthonous gastric tumors by of WNT activation and effects of pathway inhibition on proliferation of human adoptive T cell transfer in an SV40 T antigen mouse model. J Immunol 2010; 185: gastric carcinoma cells. Oncogene 2011; 30: 956 --966. 2580 --2588. 12 Kikuchi A, Yamamoto H, Sato A. Selective activation mechanisms of Wnt signaling 38 Becker K, Muller JD, Schulmacher C, Ott K, Fink U, Busch R et al. Histomorphology pathways. Trends Cell Biol 2009; 19: 119 --129. and grading of regression in gastric carcinoma treated with neoadjuvant 13 Kobune M, Chiba H, Kato J, Kato K, Nakamura K, Kawano Y et al. Wnt3/RhoA/ROCK chemotherapy. Cancer 2003; 98: 1521 --1530. signaling pathway is involved in adhesion-mediated drug resistance of multiple 39 Hausmann G, Ba¨nziger C, Basler K. Helping Wingless take flight: how WNT myeloma in an autocrine mechanism. Mol Cancer Ther 2007; 6: 1774 --1784. proteins are secreted. Nat Rev Mol Cell Biol 2007; 8: 331 --336. 14 Flahaut M, Meier R, Coulon A, Nardou KA, Niggli FK, Martinet D et al. The Wnt 40 Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003; 422: receptor FZD1 mediates chemoresistance in neuroblastoma through activation of 37 --44. the Wnt/beta-catenin pathway. Oncogene 2009; 28: 2245 --2256. 41 Yamamoto H, Komekado H, Kikuchi A. Caveolin is necessary for WNT-3a- 15 Noda T, Nagano H, Takemasa I, Yoshioka S, Murakami M, Wada H et al. Activation dependent internalization of LRP6 and accumulation of beta-catenin. Dev Cell of Wnt/beta-catenin signalling pathway induces chemoresistance to interferon- 2006; 11: 213 --223. alpha/5-fluorouracil combination therapy for hepatocellular carcinoma. Br J 42 Bilic J, Huang YL, Davidson G, Zimmermann T, Cruciat CM, Bienz M et al. WNT Cancer 2009; 100: 1647 --1658. induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phos- 16 Nyeng P, Norgaard GA, Kobberup S, Jensen J. FGF10 signaling controls stomach phorylation. Science 2007; 316: 1619 --1622. morphogenesis. Dev Biol 2007; 303: 295 --310. 43 Galbiati F, Volonte D, Brown AM, Weinstein DE, Ben-Ze’ev A, Pestell RG et al. 17 Krawetz R, Kelly GM. WNT6 induces the specification and epithelialization of F9 Caveolin-1 expression inhibits WNT/beta-catenin/Lef-1 signaling by recruiting embryonal carcinoma cells to primitive endoderm. Cell Signal 2008; 20: 506 --517. beta-catenin to caveolae membrane domains. J Biol Chem 2000; 275: 23368 -- 18 Kirikoshi H, Sekihara H, Katoh M. WNT10A and WNT6, clustered in human 23377. chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in 44 Dvory-Sobol H, Sagiv E, Liberman E, Kazanov D, Arber N. Suppression of gastric SW480 cells. Biochem Biophys Res Commun 2001; 283: 798 --805. cancer cell growth by targeting the beta-catenin/T-cell factor pathway. Cancer 19 Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal 2007; 109: 188 --197. physiology. Pharmacol Rev 2002; 54: 431 --467. 45 Huang J, Yang J, Maity B, Mayuzumi D, Fisher RA. Regulator of G protein signaling 20 Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol 2007; 8: 6 (RGS6) mediates doxorubicin-induced ATM and p53 activation by a reactive 185 --194. oxygen species (ROS)-dependent mechanism. Cancer Res 2011; 71: 6310 --6319.

Oncogene (2013) 375 --387 & 2013 Macmillan Publishers Limited WNT6 in gastric cancer G Yuan et al 387 46 Millour J, de Olano N, Horimoto Y, Monteiro LJ, Langer JK, Aligue R et al. ATM and 53 Ordway JM, Bedell JA, Citek RW, Nunberg A, Garrido A, Kendall R et al. p53 regulate FOXM1 expression via E2F in breast cancer epirubicin treatment and Comprehensive DNA methylation profiling in a human cancer genome identifies resistance. Mol Cancer Ther 2011; 10: 1046 --1058. novel epigenetic targets. Carcinogenesis 2006; 27: 2409 --2423. 47 Bist A, Fielding CJ, Fielding PE. p53 regulates caveolin gene transcription, 54 Fiucci G, Ravid D, Reich R, Liscovitch M. Caveolin-1 inhibits anchorage- cell cholesterol, and growth by a novel mechanism. Biochemistry 2000; 39: independent growth, anoikis and invasiveness in MCF-7 human breast cancer 1966 --1972. cells. Oncogene 2002; 21: 2365 --2375. 48 Li Q, Lin S, Wang X, Lian G, Lu Z, Guo H et al. Axin determines cell fate by 55 Ravid D, Maor S, Werner H, Liscovitch M. Caveolin-1 inhibits cell detachment- controlling the p53 activation threshold after DNA damage. Nat Cell Biol 2009; 11: induced p53 activation and anoikis by upregulation of insulin-like growth factor-1 1128 --1134. receptors and signaling. Oncogene 2005; 24: 1338 --1347. 49 Lee KH, Li M, Michalowski AM, Zhang X, Liao H, Chen L et al. A genomewide study 56 Chen J, Ro¨ cken C, Hoffmann J, Kru¨ger S, Lendeckel U, Rocco A et al. Expression identifies the Wnt signaling pathway as a major target of p53 in murine of carbonic anhydrase 9 at the invasion front of gastric cancers. Gut 2005; 54: embryonic stem cells. Proc Natl Acad Sci USA 2010; 107:69--74. 920 --927. 50 Goetz JG, Lajoie P, Wiseman SM, Nabi IR. Caveolin-1 in tumor progression: the 57 Ebert MP, Fei G, Kahmann S, Mu¨ ller O, Yu J, Sung JJ et al. Increased beta-catenin good, the bad and the ugly. Cancer Metastasis Rev 2008; 27: 715 --735. mRNA levels and mutational alterations of the APC and beta-catenin gene are 51 Kurayoshi M, Oue N, Yamamoto H, Kishida M, Inoue A, Asahara T et al. Expression present in intestinal-type gastric cancer. Carcinogenesis 2002; 23: 87 --91. of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell 58 Burgermeister E, Schnoebelen A, Flament A, Benz J, Stihle M, Gsell B et al. migration and invasion. Cancer Res 2006; 66: 10439 --10448. A novel partial agonist of peroxisome proliferator-activated receptor-gamma 52 Yamamoto H, Kitadai Y, Yamamoto H, Oue N, Ohdan H, Yasui W et al. Laminin (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride gamma2 mediates Wnt5a-induced invasion of gastric cancer cells. Gastroenter- accumulation, and potentiates insulin signaling in vitro. Mol Endocrinol 2006; ology 2009; 137: 242 --252. 20: 809 --830.

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

& 2013 Macmillan Publishers Limited Oncogene (2013) 375 --387