FOXC2 Promotes Colorectal Cancer Metastasis by Directly Targeting MET

ORIGINAL ARTICLE FOXC2 promotes colorectal cancer metastasis by directly targeting MET

Y-M Cui1,2,3,6, H-L Jiao1,2,3,6, Y-P Ye1,2,3, C-M Chen1,2,3, J-X Wang1,2,3, N Tang1,2,3, T-T Li1,2,3, J Lin1,2,3,LQi1,2,3,PWu1,2,3, S-Y Wang1,2,3, M-R He4, L Liang1,2,3, X-W Bian5, W-T Liao1,2,3 and Y-Q Ding1,2,3

Metastasis is the major cause of death in colorectal cancer (CRC). Although multiple genes have been identified to be responsible for the development of CRC, the molecular changes that enable CRC cells to undergo early local invasion and to form distant metastatic colonies still remain largely unknown. Herein, we investigated the role of Forkhead box protein C2 (FOXC2) and explored the underlying mechanisms in invasion and metastasis of CRC. We show that both high FOXC2 expression and nuclear localization of FOXC2 are significantly correlated with advanced TNM (T = primary tumor; N = regional lymph nodes; M = distant metastasis) stages. FOXC2 enhanced the invasive abilities of CRC cells in vitro and promoted local invasion and distant metastasis in an orthotopic mouse metastatic model of CRC. Microarray analysis revealed that overexpression of FOXC2 increased the proto-oncogene MET tyrosine kinase expression and activated the hepatocyte growth factor (HGF)-MET signaling pathway. Furthermore, luciferase reporter assays and chromatin immunoprecipitation assays revealed that FOXC2 directly associated with MET promoter to increase the transcriptional activity of MET. Inhibition of MET attenuates the invasive phenotype and metastatic potential of FOXC2-overexpressing CRC cells, indicating that MET is a major mediator of FOXC2-promoted metastasis. In addition, FOXC2 expression was positively correlated with MET expression in CRC tissue samples. Our findings suggest that FOXC2 has a crucial role in CRC metastasis by regulating HGF-MET signaling via inducing MET expression, highlighting FOXC2 as a potential therapeutic target for preventing or reducing metastasis in CRC.

Oncogene (2015) 34, 4379–4390; doi:10.1038/onc.2014.368; published online 10 November 2014

INTRODUCTION Forkhead box protein C2 (FOXC2; also known as mesenchyme Colorectal cancer (CRC) is one of the most common types of forkhead 1) has been recently reported to be involved in cancer and a leading cause of cancer-related deaths worldwide.1 cancer progression and metastasis. It has been documented that Metastasis and recurrence are the major causes of death in CRC. FOXC2 directly induces the transcription of CXCR4 and integrin-b3 It is estimated that over 50% of patients diagnosed with CRC will by activating their promoters, and is associated with angio- 5–7 die due to complications related to metastasis.2 Unfortunately, genesis and lymphangiogenesis. FOXC2 induces epithelial- traditional methods, such as the assessment of clinical and mesenchymal transition (EMT) and also serves as a link between 8,9 histopathologic features, and current molecular markers, such as EMT and stem cell properties in breast cancer. High expression carcinoembryonic antigen, are unsatisfactory for the early of FOXC2 is an independent prognostic factor in esophageal – diagnosis of patients with CRC who are at high risk of cancer, gastric cancer and non-small-cell lung cancer.10 12 It is also metastasis.3 reported that downregulation of FOXC2 was critical for the Although multiple genes have been identified to be responsible suppression of lung cancer metastasis by resveratrol.13 These for the development of CRC, such as mutational inactivation findings emphasize that FOXC2 may have a crucial role in of the adenomatous polyposis coli (APC) gene or activation of the endowing tumor cell malignant behavior. However, the mechan- β-catenin, Kirsten rat sarcoma viral oncogene homolog (KRAS), ism by which FOXC2 confers an increased metastatic ability to tumor protein p53 (TP53), SMAD family member 4 c (SMAD4)or cancer cells needs further investigation. phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), the In this study, we report that overexpression of FOXC2 activates molecular changes that enable CRC cells to undergo early local hepatocyte growth factor (HGF)-MET signaling in CRC cells. FOXC2 invasion and to form distant colonies still remain largely regulates MET expression and promotes invasion and metastasis unknown.4 Therefore, efforts to elucidate the molecular mechan- of CRC cells by direct activation of MET promoter. Our results isms underlying these processes are imperative in order to enable highlight a new molecular mechanism underlying the metastatic early recognition of the molecular changes involved in CRC behavior of human cancer cells and suggest that FOXC2 could metastasis, and to develop novel predictive methods and design represent a potential target for anti-metastatic therapeutic efficient treatment strategies for metastasis in CRC. strategies in CRC.

1Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, China; 2Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China; 3Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Guangzhou, China; 4Guangdong Provincial Key Laboratory of Gastroenterology and Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China and 5Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China. Correspondence: Dr W-T Liao or Professor Y-Q Ding, Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 24 February 2014; revised 24 September 2014; accepted 30 September 2014; published online 10 November 2014 FOXC2 regulates MET signaling Y-M Cui et al 4380 RESULTS and livers of mice implanted with FOXC2-overexpressing cells High expression of FOXC2 is associated with CRC metastasis (Figures 2f and g). No obvious macrometastases were detected in We examined FOXC2 mRNA expression in 40 primary colorectal the mice of control group. tumors and their paired adjacent normal tissues. FOXC2 was expressed more highly in CRC tissues than in normal tissues, Downregulation of FOXC2 represses invasiveness and reduces the especially in subjects with CRC who later developed distant metastatic potential of CRC cells metastasis (Figure 1a). To further validate the role of FOXC2 in invasiveness and Immunohistochemistry was performed to examine the protein metastasis in CRC cells, we silenced endogenous FOXC2 expres- expression level and subcellular localization of FOXC2 in 108 cases sion in CRC cells using two specific short hairpin RNAs (shRNAs). of paraffin-embedded CRC tissues. The results revealed that Ablation of FOXC2 induced a reverse change from mesenchymal FOXC2 displayed either cytoplasm or nucleic/cytoplasm expres- morphology into epithelial morphology in HCT15 and SW620 cells sion in the tumor cells (Figure 1b). Mann–Whitney U-test revealed (Supplementary Figure S1b). In addition, silencing of FOXC2 led to that FOXC2 expression level was strongly correlated with T stage enhanced expression of E-cadherin, P120-catenin and α-catenin, (P = 0.023), N stage (P = 0.042) and distant metastasis (P = 0.004). In and concomitantly, decreased expression of Vimentin and addition, tumors with nucleic/cytoplasm localization of FOXC2 N-cadherin (Figure 3a and Supplementary Figure S2). Moreover, were more aggressive that those with only cytoplasm expression knockdown of FOXC2 significantly compromised the migratory of FOXC2. Nucleic/cytoplasm localization of FOXC2 was positively and invasive ability of CRC cells (Figures 3b–d). Next, we correlated with T stage (Po0.001), N stage (P = 0.001) and distant o orthotopically transplanted SW620 cells expressing shRNAs metastasis (P 0.001) (Supplementary Table S1). against FOXC2 or a control shRNA (six mice were included in each group). SW620 cells expressing the FOXC2 shRNAs showed Overexpression of FOXC2 promotes the invasion and metastasis of reduced mesenteric metastases and distant metastasis in liver and CRC cells both in vitro and in vivo lungs, compared with the vector control group (Figures 3e and f). To investigate the role of FOXC2 in invasion and metastasis of CRC, we established FOXC2-overexpressing stable cell lines in SW480. FOXC2 regulates MET signaling pathway Stable expression of FOXC2 induced a spindle-like, fibroblastic To identify the signaling pathways activated in CRC cells in morphology (Supplementary Figure S1a), which suggested that response to FOXC2 overexpression, we performed whole-genome FOXC2-overexpressed SW480 cells may have undergone EMT. expression microarray and Ingenuity Pathway Analysis in SW480/ Moreover, overexpression of FOXC2 led to marked downregula- fi tion of E-cadherin and P120-catenin, but upregulation of vimentin vector and SW480/FOXC2 cells. Upregulation of FOXC2 signi - and N-cadherin (Figure 2a). Furthermore, overexpression of FOXC2 cantly correlated with the receptor protein tyrosine kinase strongly enhanced the migratory and invasive ability of CRC cells signaling, mitogen-activated protein kinase (MAPK), phosphatidy- β in vitro (Figures 2b–d). linositol 3 kinase/Akt and Wnt/ -catenin pathways (Figure 4a). In To determine the in vivo effect of FOXC2 on promoting CRC addition, the expression of proto-oncogene MET, a receptor fi invasion and metastasis, SW480/pBabe control cells or SW480/ tyrosine kinase, was signi cantly upregulated in SW480/FOXC2 FOXC2 cells were orthotopically implanted into the cecum of nude cells compared with the vector control cells (Figure 4b). More mice (n = 6 in each group). As shown in Figure 2e, large primary interestingly, MET had a central role in regulation of its interaction tumors in the cecum and widespread distribution of tumor foci in proteins including CTNNB1 (β-catenin), which were also upregu- the abdomina were observed in the FOXC2-overexpressing group, lated by FOXC2 (Figure 4c). Moreover, stable expression of FOXC2 whereas the primary tumors in the vector control group were markedly increased, but downregulation of FOXC2 significantly remarkably smaller (Figure 2e, upper). More importantly, the attenuated the expression of MET, phosphorylated MET, K-Ras, vector control cells formed noninvasive, oval-shaped tumors. In Raf1, p-ERK, p-AKT, p-GSK-3β and Snail (Figures 4d and e and contrast, SW480/FOXC2 cells developed diffusely distributed and Supplementary Figure S3). In addition, FOXC2 could significantly highly invasive tumors, which displayed interspersed fibroblast- increase Wnt activity (Supplementary Figure S4) and upregulate like structures (Figure 2e, lower). Notably, the tumor cells formed the both total and active β-catenin levels, as well as the target by FOXC2-overexpressing cells invaded into the vascular vessels gene LEF1 and DKK1 (Figure 4d). Furthermore, MET expression (Figure 2e, lower), and metastatic loci were observed in the lungs was increased in a dose-dependent manner at both the

Figure 1. FOXC2 is highly expressed in CRC tissues. (a) FOXC2 mRNA expression in 40 cases of normal intestinal mucosa, primary CRC with or without metastasis, as detected by quantitative real-time RT-PCR. **Po0.001. (b) FOXC2 protein expression in normal intestinal mucosa and primary CRC tissues that originated from patients with or without metastasis, as determined by immunohistochemistry.

Figure 2. Overexpression of FOXC2 promotes the invasive and metastatic abilities of CRC cells in vitro and in vivo.(a) Expression of the epithelial markers and the mesenchymal markers in SW480/pBabe or SW480/FOXC2 were assessed by western blot. α-Tubulin was used as a loading control. (b) Representative images of wound-healing assay. Original magnification, × 100. Histograms represent the average number of cells migrated into the box at the indicated times. Error bars represent mean ± s.d. from three independent experiments. (c) The migratory and invasive properties of SW480/pBabe or SW480/FOXC2 cells were analyzed using a Matrigel-coated Boyden chamber. Original magnification, × 400. Error bars represent mean ± s.d. from three independent experiments. (d) Three-dimensional morphologies assay. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Original magnification, × 400. Error bars represent mean ± s.d. (e) Images (upper) of primary tumors and scattered tumors in the excised intestines of mice orthotopically implanted with the indicated cells. Histograms represent the size of the primary tumors in the FOXC2 group (n = 6) and pBabe group (n = 6). Representative hematoxylin and eosin (H&E) staining (lower) of tumors formed by the indicated cells. Arrows (lower middle) indicate the invasive front of tumors. Representative image of the vascular invasion by SW480/FOXC2 cells was shown (lower right). Original magnification, × 200. (f) Representative images of gross specimens and H&E staining of liver and lung metastatic lesions formed in mice orthotopically implanted with SW480/pBabe (top) or SW480/FOXC2 tumors (bottom). (g) The numbers of liver and lung micrometastases and macrometastases determined using a dissection microscope were shown. *Po0.01. translational and transcriptional levels by transiently transfecting protein degradation (Supplementary Figures S5a and c). Moreover, SW480 cells with a FOXC2 expression vector (Figure 4f). silencing Snail could rescue E-cadherin expression and repress the However, FOXC2 or MET did not affect the mRNA level of Snail. migratory and invasive abilities of SW480/FOXC2, whereas ectopic Further experiments showed that FOXC2 could inhibit Snail expression of Snail could inhibit the upregulated effect of

© 2015 Macmillan Publishers Limited Oncogene (2015) 4379 – 4390 FOXC2 regulates MET signaling Y-M Cui et al 4382 shFOXC2 on E-cadherin and regain the migratory and invasive (Supplementary Figures S5d and e). In addition, overexpression abilities of SW620/shFOXC2 cells, indicating that Snail has an of MET in SW620/shFOXC2 cell could markedly restore the important role in FOXC2 upregulation-mediated metastasis expression of Snail expression and result in decrease of E-cadherin

Oncogene (2015) 4379 – 4390 © 2015 Macmillan Publishers Limited FOXC2 regulates MET signaling Y-M Cui et al 4383 expression, and rescue the migratory and invasive abilities of formation of metastatic foci by HCT15 or HCT15/FOXC2 cells SW620/shFOXC2 cell, indicating that MET is required in FOXC2- (Figure 6d). mediated Snail stabilization (Supplementary Figure S5f). Taken together, these data indicate that E-cadherin downregulation FOXC2 expression positively correlates with MET expression in mediated by FOXC2-MET is dependent of Snail. CRC We further analyzed the expression of FOXC2 and MET in eight FOXC2 associates with MET promoter freshly collected CRC biopsies. Western blot analyses indicated We performed a luciferase reporter assay to investigate whether that both FOXC2 and MET were significantly upregulated in the FOXC2 could increase MET promoter activity as a transcription eight tumor samples examined, compared with the paired factor. A 2.7-kb fragment of the full-length MET promoter region adjacent noncancerous tissues from the same patients was subcloned into a luciferase vector. The MET promoter activity (Figure 7a). Comparative real-time PCR analysis demonstrated was increased by co-transfection with a FOXC2 expression vector that the mean tumor/normal ratios of both FOXC2 and MET mRNA in SW480 cells but decreased in HCT15 and SW620 cells expression were 42-fold for all samples (Figure 7b). In addition, expressing FOXC2 shRNA in a dose-dependent manner, compared FOXC2 expression was positively correlated with MET expression with empty vectors (Figures 5a and b). To determine the effective at both protein (P = 0.003, r = 0.694) and mRNA levels (Po0.001, regions of MET promoter that FOXC2 may affect, we transfected r = 0.976) as analyzed in the eight samples (Figures 7a and b). MET promoter truncations into HCT15 cells expressing either Moreover, FOXC2 mRNA levels were significantly correlated with FOXC2 shRNA or an empty vector. As shown in Figure 5c, the MET mRNA levels in the 40 CRC biopsies described in Figure 1a luciferase activity was increased in cells carrying full-length MET (Figure 7c; Po0.001, r = 0.582). Furthermore, immunohistochem- promoter and truncations − 600, − 1200, − 1800-bp upstream of istry showed that there was a significant correlation between MET the transcription start site as well as − 1800 to − 1201 bp, but not expression and either FOXC2 expression levels (P = 0.007) or in cells carrying truncations − 2637 to − 1801bp or − 1200 to FOXC2 nuclear localization (Po0.001) in the 108 CRC samples − 601 bp. Knockdown of FOXC2 expression by co-transfection (Supplementary Table S1). Samples that had lower level of FOXC2 FOXC2 shRNA significantly decreased MET promoter activity expression also had a lower MET expression, whereas samples that (Figure 5c). Furthermore, we performed chromatin immunopreci- had higher level of FOXC2 expression especially mainly located in pitation (ChIP) assays and identified that the − 1322 to − 1635 bp the nucleus had a higher MET expression (Figure 7d). and − 693 to +49-bp regions of the MET promoter were FOXC2 protein binding sites; p120 and glyceraldehyde 3-phosphate DISCUSSION dehydrogenase (GAPDH) promoter fragments were used as positive and negative controls, respectively (Figure 5d).14 In Tumor progression toward metastasis is a complex, multistage fi process, which is classically simplified as local invasion, intravasa- addition, FOXC2 bound to the MET promoter much less ef ciently 15,16 in FOXC2-knocking down cells (Figure 5e), indicating that the tion, survival in the circulation, extravasation and colonization. association was specific. Taken together, this data identifies MET as Metastasis initiation genes are a group of metastasis-associated a direct transcriptional target of FOXC2. genes that determine the activities that allow cancer cells to invade the surrounding tissue, attract a supportive tumor microenvironment and facilitate the dispersion of cancer cells, Inhibition of MET attenuates the invasive phenotype and and probably continue to exert these actions when the cancer metastatic potential of FOXC2-overexpressing CRC cells cells infiltrate distant tissues.15 In addition, metastasis initiation To analyze the functional relationship between FOXC2 and MET, genes may promote aggressive behaviors in cancer cells, such as we knocked down MET using two MET shRNAs or suppressed MET increased cell motility, EMT, extracellular matrix degradation, bone activation using a MET inhibitor (EMD 1214063). Silencing or marrow cell mobilization, survival in the circulation and induction inhibition of MET in SW480/FOXC2, HCT15 or HCT15/FOXC2 cells of angiogenesis.15 The overexpression of metastasis initiation significantly decreased the expression of phosphorylated MET, genes and their targets often predicts a poor prognosis in various Akt, ERK and GSK-3β as well as Snail (Figures 6a and b). Moreover, types of cancer.15 FOXC2 is a highly conserved transcription factor, knocking down MET partially decreased the expression levels of which has crucial roles in angiogenesis and lymphangiogenesis, as Vimentin and N-cadherin and increased the level of E-cadherin in well as induction of the EMT.5,8,17 Functional analyses revealed these cells, indicating that FOXC2-induced EMT is partially that FOXC2 can confer a number of properties that may contribute mediated by MET activation (Figures 6a and b). to metastasis. For instance, FOXC2 can increase cell proliferation, Furthermore, the invasive and migratory abilities of SW480/ tumor angiogenesis, motility and invasion and induce cancer stem FOXC2, HCT15 or HCT15/FOXC2 cells were partially diminished by cell properties,8,9,17 yet the underlying mechanism by which inhibition of MET (Supplementary Figure S6). Orthotopic implanta- FOXC2 exerts these effects is poorly understood. In this study, we tion assay showed that FOXC2 significantly promoted lung and provided a series of in vitro and in vivo evidence which indicates liver metastases. Inversely, inhibition of MET significantly attenu- that FOXC2 may function as a metastasis initiation gene in the ated the formation of metastatic foci by SW480/FOXC2 cells invasion-metastasis cascade, similarly to previously identified (Figure 6c). Furthermore, inhibition of MET also attenuated the metastasis initiation genes such as TWIST1, SNAI1, SLUG and

Figure 3. Silencing of endogenous FOXC2 expression in CRC cells represses the invasive ability in vitro and reduces metastatic ability in vivo. (a) Expression of the indicated epithelial and mesenchymal markers assessed by western blot. (b) The invasive and migratory abilities of CRC cells evaluated using the Matrigel-coated Boyden chamber invasion assay. Each bar represents the mean ± s.d. of three independent experiments. (c) Representative images of wound-healing assay. Original magnification, × 100. Histograms represent the average number of cells migrated into the box at the indicated times. Error bars represent mean ± s.d. **Po0.001. (d) Three-dimensional morphologies of the indicated CRC cells were analyzed under culture in Matrigel. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Original magnification, × 400. Error bars represent mean ± s.d. (e) Mice were orthotopically transplanted with SW620/vector (n = 6) or SW620/shFOXC2 (n = 6) cells. Representative gross images of the intestines, livers and lungs were shown. Sections of the liver and lung were stained with hematoxylin and eosin (H&E). Arrows show the primary tumors in the intestines, and macroscopic metastatic foci in the liver. (f) The numbers of liver and lung micrometastases and macrometastases were determined using a dissection microscope. **Po0.001.

© 2015 Macmillan Publishers Limited Oncogene (2015) 4379 – 4390 FOXC2 regulates MET signaling Y-M Cui et al 4384 MET.15 Alteration of FOXC2 expression signiﬁcantly induced EMT steps of invasion and distant metastasis by CRC cells in an and affected the motility and migratory ability of CRC cells in vitro. orthotopic mouse model of CRC metastasis. More importantly, we Furthermore, FOXC2 strongly promoted both the early and late also revealed a novel cellular function of FOXC2 and identiﬁed

Figure 4. FOXC2 activates MET signaling pathway in CRC cells. (a) RNA from three separate experiments was extracted and analyzed using a whole-genome expression microarray. Gene expression was analyzed using Ingenuity Pathway Analysis. The signaling pathways activated by FOXC2-overexpressing in SW480 cells were shown. (b) Heatmap analysis showing part of the genes regulated by FOXC2 as identiﬁed by microarray proﬁling. (c) Protein–protein interaction network showing MET is in the central role of regulating the proteins upregulated by FOXC2. (d) Western blot analysis of the expression levels of indicated genes in the indicated CRC cell lines. (e) Real-time RT-PCR analyses of MET expression in the indicated cells. (f) SW480 cells were transiently transfected with the indicated amounts of pBabe/FOXC2. After 48 h, the expression of FOXC2, MET and p-MET was determined by western blotting (left), and the expression of MET mRNA was analyzed by real-time RT-PCR (right).

Figure 5. FOXC2 regulates MET transcriptional activity by binding to the MET promoter. (a, b) MET promoter activity was measured using the dual-luciferase reporter assay system by co-transfection with the indicated amounts of plasmids in the indicated cell lines. (c) The luciferase activity was measured by transfection full-length MET promoter or its truncations in HCT15/vector or HCT15/FOXC2 shRNA#1 cells. (d) Schematic illustration of the MET promoter; the regions with binding affinity for FOXC2 are indicated by +. ChIP was performed using an anti-FOXC2 antibody or a control immunoglobulin G to identify FOXC2 binding sites in the MET promoter in HCT15 cells. The p120 and GAPDH promoters were used as positive and negative controls, respectively. (e) ChIP analysis of the binding efficiency of FOXC2 toward the MET promoter in HCT15 cells expressing the vector control shRNA or FOXC2 shRNA#1. Enriched chromatin was analyzed using Bio-Profile. Error bars represent mean ± s.d. from three independent experiments.

MET as one of its transcriptional targets. FOXC2-induced invasion have demonstrated that FOXC2 induces the transcription of and metastasis were blocked by MET inhibitor or shRNAs targeting CXCR4, integrin β3 and PDGFR-β, by directly activating their FOXC2 or MET. promoters.5 In addition, FOXC2 can suppress the expression of As a transcription factor, FOXC2 may mediate the EMT and E-cadherin and upregulate the expression of matrix metallopro- angiogenesis, and promote metastasis via the transcriptional teinases (MMPs) to promote metastasis in breast cancer.8,9 Other regulation of downstream genes. However, only limited down- research showed that FOXC2 could downregulate expression of stream targets of FOXC2 have been characterized. Recent studies E-cadherin, mainly via regulation of p120-catenin by binding

Figure 6. Inhibition of MET reduces the invasive and metastatic ability of FOXC2-overexpressing cells. (a, b) Western blotting analyzed the expression of the indicated proteins in SW480 (a) or HCT15 (b) cells with indicated treatments. (c, d) Mice were orthotopically transplanted with indicated cells (n = 6 in each group). Representative gross images of the intestines, livers and lungs were shown. Sections of the liver and lung were stained with hematoxylin and eosin (H&E). Arrows show the primary tumors in the intestines, and macrometastases or micrometastases in the liver and lungs. The numbers of liver and lung macrometastases or micrometastases were determined using a dissection microscope. Error bars represent mean ± s.d. **Po0.001.

Figure 7. FOXC2 expression positively correlated with MET expression in human CRC. (a) Western blot examination of FOXC2 and MET protein expression in eight freshly collected CRC biopsies. The relative protein expression levels were quantified by comparing the gray level of each band using Quantity One Software (Life Science Research, Hercules, CA, USA) (left). Spearman’s correlation analysis was used to determine the relationship between FOXC2 and MET protein expression (right). (b) Average tumor/adjacent normal intestine epithelium (N/T) ratios of FOXC2 and MET expression were quantified by real-time RT-PCR and normalized against GAPDH (n = 8). Error bars represent the s.d. calculated from three parallel experiments. The left graph represent the relationship between FOXC2 and MET mRNA expression in the eight tumor samples analyzed by Spearman’s correlation. (c) Real-time RT-PCR analysis of the relative expression levels of MET mRNA in the 40 CRC biopsies used in Figure 1a. Spearman’s correlation analysis was used to determine the relationship between FOXC2 and MET mRNA expression in the 40 tumor samples. (d) Immunohistochemical staining of FOXC2 and MET in CRC tissues showed that FOXC2 expression was positively correlated with MET expression. directly to the p120-catenin promoter region.14 In this study, we the HGF ligand, and transmits intracellular signaling cascades via found that FOXC2 could induce EMT and promote invasion and the RAS-mitogen-activated protein kinases (MAPK) and phospha- metastasis by decreasing E-cadherin expression but increasing tidylinositol 3 kinase/Akt pathways.18–20 In CRC, MET is over- Snail expression. More interestingly, we identified that FOXC2 expressed in the vast majority of invasive carcinomas and could transcriptionally regulate the expression of MET, which is metastases, and MET has been suggested to be mainly essential for EMT phonotype and metastatic potential of CRC cells, responsible for the metastatic behavior of CRC cells.25–28 In a subsequently activating its downstream signaling cascades RAS- large majority of CRC cases, MET is activated by overexpression or mitogen-activated protein kinase (MAPK) and phosphatidylinositol gene amplification.19,29,30 However, MET gene amplification is a 3 kinase/Akt pathways.18,19 These findings highlight a new relatively rare event, and the mechanisms regulating MET protein regulatory mechanism that FOXC2 induces EMT and promotes overexpression in CRC remain largely obscure.19,30 Transcriptional metastasis by regulating the HGF-MET signaling pathway. regulation may be one major mechanism, which modulates MET The HGF-MET signaling pathway has been implicated in expression. MET has been identified to be a target of hypoxia- different processes related to malignant progression, including inducible factor-1, β-catenin and metastasis-associated in colon cell survival, growth, angiogenesis, cell motility, EMT, invasiveness cancer-1.31–35 Here, we demonstrate that FOXC2 is a novel and metastasis.18–23 Hyperactivation of the HGF-MET signaling transcriptional regulator of MET. We provided several lines of cascade leads to metastasis in various human cancers, including evidence to support the conclusion that MET is a direct target of CRC.24,25 The tyrosine kinase MET is activated following binding of FOXC2. First, MET expression was induced after FOXC2

© 2015 Macmillan Publishers Limited Oncogene (2015) 4379 – 4390 FOXC2 regulates MET signaling Y-M Cui et al 4388 transfection, and MET expression was knockdown after FOXC2 the proportion of positively stained tumor cells and the intensity of shRNA treatment. Second, MET promoter activity was significantly staining. increased after FOXC2 transfection but decreased after knockdown of FOXC2 by a specific shRNA, both in a dose-dependent Plasmids manner. Most importantly, FOXC2 was able to bind directly to the The FOXC2 construct was generated by cloning PCR-amplified full-length MET promoter, and the binding efficiency was greatly reduced by human FOXC2 cDNA into pBabe (Addgene, Inc., Cambridge, MA, USA). The knockdown of FOXC2 using a specific shRNA. In addition, we human shRNA sequences specifically targeting FOXC2 or MET (FOXC2 identified two binding fragments in MET promoter, which contain shRNA#1: 5′-CCACACGTTTGCAACCCAA-3′; FOXC2 shRNA#2: 5′-CCTCCTGG a previously reported − 223 to − 68-bp region harboring TATCTGAACCA-3′;8,39 MET shRNA#1: 5′-GAACAGAATCACTGACATA-3′; MET consensus motifs for specificity protein-1, Kruppel-like factor 4, shRNA#2: 5′-GTCATAGGAAGAGGGCATT-3′) were cloned into pSuper-retro- activator protein-2 and metastasis-associated in colon cancer-1, neo (Oligo-Engine, Seattle, WA, USA). The pBabe-puro-c-met and pBabe- described as MET regulators.33–36 Thus, we hypothesize that on puro-Snail plasmids were purchased from Addgene, Inc. one hand, MET has an essential part in FOXC2-induced metastasis, and on another hand, the abundance of FOXC2 may fundamen- Cell culture tally affect the MET-dependent aggressive behavior and meta- The SW480, HCT15 cell lines were cultured in RPMI 1640 (Invitrogen, static ability of CRC cells. These results also reveal a novel Carlsbad, CA, USA) and SW620 cells were cultured in DMEM (Invitrogen). All regulatory factor for the HGF-MET signaling pathway. medium was supplemented with 10% fetal bovine serum (PAA Labora- tories, Pasching, Austria) and 1% penicillin/streptomycin (Invitrogen). MET has been reported to be a transcription target of beta- 40 catenin and there may be a positive feedback loop between MET The stable cell lines were established as previously described. and beta-catenin to sustain CRC cell invasive growth.31,32 Our data Retroviruses were generated by transient transfection in 293FT cells. The FOXC2 gene was introduced into SW480 cells by infecting cells with a indicated that FOXC2 could also activate Wnt-β-catenin signaling β retroviral vector pBabe-FOXC2. Control cells were infected with the empty in CRC cells. Thus, we infer that FOXC2, MET and -catenin may retroviral vector pBabe. Retrovirus-infected cells were selected and form a positive feedback regulatory loop to synergisticly promote maintained in 0.5 μg/ml puromycin for 3–5 days. Western blot analysis metastatic behavior of CRC cells. However, the possible mechan- was done to confirm the expression of FOXC2 in the SW480 cells using the isms and detailed interplay fashions among them need further specific antibody against FOXC2. Cell lines with stable silencing of FOXC2 investigation. were established by a similar method, using the retroviral vector pSuper- In summary, our data suggest that FOXC2 has an essential role retro-neo. in CRC metastasis by regulating the proto-oncogene MET. As targeting the HGF-MET signaling pathway using specific anti- RNA isolation, reverse transcription (RT) and real-time PCR fi bodies and small molecules to neutralize HGF has been identi ed Total RNA from human tissues and cultured cell lines was isolated using as a valuable treatment strategy in clinical practice, blockade of the Trizol reagent (Invitrogen) according to the manufacturer's instruction. 37 the MET receptor or inhibition of its receptor kinase function, or Primers for real-time RT-PCR were designed using Primer Express v 2.0 interfering with FOXC2 may represent a alternative therapeutic software (Applied BioSystems, Foster, CA, USA). Sequences of the primers target to prevent or reduce metastasis in CRC. are summarized in Supplementary Table S2. RT was carried out with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's protocol. Real-time RT-PCR was carried out using MATERIALS AND METHODS SYBR Green I (Applied BioSystems). The data were normalized to the − ΔΔCT Patients and specimens geometric mean of housekeeping gene GAPDH and calculated as 2 method. A total of 108 cases of formalin-fixed paraffin-embedded CRC samples were collected between 2012 and 2014 at the Department of Pathology, Nanfang Hospital Southern Medical University. The 40 freshly collected Luciferase reporter assay CRC biopsies and their matched adjacent noncancerous mucosa tissues Cells at 60% confluence in 24-well plate were transfected using were collected at the operation room, Nanfang Hospital. The fresh biopsies lipofectamin2000. Forty-eight hours after transfection, luciferase activity were frozen and stored in liquid nitrogen until further use. The formalin- was measured using the dual-luciferase reporter assay system (Promega fixed paraffin-embedded specimens of these samples were obtained in the corp., Madison, WI, USA) and normalized to Renilla luciferase gene Department of Pathology, Nanfang Hospital, Southern Medical University. expression. All the experiments were performed in triplicate. The medical records of the patients were reviewed to collect the information about TNM (T = primary tumor; N = regional lymph nodes; M = distant metastasis) stages. For the use of clinical materials for research Western blotting 41 purposes, prior approval was obtained from the Southern Medical We performed western blotting according to the previous study, using University Institutional Board (Guangzhou, China). anti-FOXC2 (Bethyl Laboratories, Inc., Montgomery, TX, USA), anti-MET, anti-p-MET, anti-Akt, anti-GSK-3β, anti-ERK, anti-p-Akt, anti-p-GSK-3β, anti- p-ERK, anti-Snail, anti-Lef1, anti-non-phospho-β-catenin (Ser33/37/Thr41) Immunohistochemistry antibodies (Cell Signaling), anti-DKK1 (Abcam, Cambridge, MA, USA), anti- Immunohistochemistry was performed as previously described.38 Paraffin- E-cadherin, anti-P120-catenin, anti-β-catenin (BD Biosciences, San Diego, embedded specimens were cut into 4-μm sections and baked at 65 °C for CA, USA), anti-k-Ras, anti-Raf1, anti-α-catenin, anti-vimentin and anti-N- 30 min. The sections were deparaffinized with xylenes and rehydrated. cadherin (Bioworld Technology, Inc., Louis Park, MN, USA). Anti-α-Tubulin After treatment with 3% hydrogen peroxide in methanol to quench the monoclonal antibody (Sigma, St Louis, MO, USA) served as a loading endogenous peroxidase activity, the sections were submerged into citrate control. buffer and high-pressure boiled for antigenic retrieval, followed by incubation with 1% bovine serum albumin to block the nonspecific binding. Rabbit anti-FOXC2 (1:200; Millipore Corp., Temecula, CA, USA) or ChIP assays rabbit anti-MET (1:200; Cell Signaling, Danvers, MA, USA) was incubated ChIP assays were carried out using a kit according to the manufacture’s with the sections overnight at 4 °C. For negative controls, the rabbit anti- instruction (Millipore Corp., catalog # 17-371). Briefly, approximately 1 × 107 FOXC2 or anti-MET antibody was replaced with normal goat serum. After cells were treated with 1% formaldehyde for 10 min in a 100 mm culture washing, the tissue sections were treated with biotinylated anti-rabbit dish to crosslink chromatin-associated proteins to DNA, and then added secondary antibody (Zymed, San Francisco, CA, USA), followed by further glycine to quench unreached formaldehyde. Cells were collected in 1 ml incubation with streptavidin-horseradish peroxidase complex (Zymed). The sodium dodecyl sulfate lysis buffer supplemented with 5 ul of protease tissue sections were incubated with 3,3-diaminobenzidin (DAB) and inhibitor cocktail. Cell lysates were sonicated for 9–10 sets of 10-s pulses at counterstained with hematoxylin, dehydrated and mounted. The sections 40% output to shear the DNA to 200–1000-bp fragments. Then the lysates were reviewed and scored independently by two observers, based on both were cleared by centrifugation at 14 000 r.p.m. for 10 min at 4 °C. In all,

Oncogene (2015) 4379 – 4390 © 2015 Macmillan Publishers Limited FOXC2 regulates MET signaling Y-M Cui et al 4389 100 ul of lysates was first mixed with 900 ul of dilution buffer, and then between FOXC2 and MET expression. Student’s t-tests were used to added 60 ul of protein G agarose incubating for 1 h at 4 °C to preclear the compare the real-time RT-PCR values between subgroups. Po0.05 was chromatin. The precleared lysates were incubated with FOXC2 antibody or considered significant. normal mouse immunoglobulin G as a negative control overnight at 4 °C with rotation. Immunoprecipitation of the DNA–protein complexes was performed with 60 μl of protein G agarose for 1 h at 4 °C, followed by CONFLICT OF INTEREST fi isolation of the DNA. The human MET promoter was ampli ed by PCR. All The authors declare no conflict of interest. ChIP assays were performed three times and the sequences of the PCR primers were listed in Supplementary Table S3. ACKNOWLEDGEMENTS Migration assay This work was supported by the National Basic Research Program of China (973 A Boyden chamber with 8 -μm-pore filter membrane was used for the program, nos. 2015CB554002 and 2010CB529403), the National Natural Science in vitro migration and invasion assay. Briefly, cells (5 × 04) in culture Foundation of China (NSFC)-Guangdong Joint Fund (U1201226), the Major Projects of medium containing 1% fetal bovine serum were seeded in the upper the National Natural Science Foundation of China (no. 81090422), the National chamber, and culture medium with 10% fetal bovine serum was added in Natural Science Foundation of China (nos. 30901791, 81172055, 81472313, 81472710 the lower chamber as a chemoattractant. The upper side of the filter was and 81071735), Guangdong Provincial Natural Science Foundation of China (no. first coated with 0.2% Matrigel (BD Biosciences, San Jose, CA, USA). After S2012010009643), Zhu Jiang Science & Technology New Star Foundation in incubation for 24 h, cells on the upper side of the filter were removed with Guangzhou city (2012J2200052 and 2012J2200044), the Science and Technology cotton swabs. Cells that migrated to the lower surface of the filter were Innovation Foundation of Guangdong Higher Education (CXZD1016), the Key fixed in 4% paraformaldehyde and stained with Giemsa. The migratory Program of the National Natural Science Foundation of Guangdong, China cells were counted (10 random 200 × fields per well). Three independent (2010B031500012), and Guangzhou Science & Technology Plan Project experiments were performed and the data were presented as the (201300000056). mean ± s.e.m. REFERENCES Wound-healing assay 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)