Oncogene (2015), 1–13 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

Wnt/β-catenin signaling regulated SATB1 promotes colorectal cancer tumorigenesis and progression R Mir1, SJ Pradhan1, P Patil2, R Mulherkar3 and S Galande1,4 The chromatin organizer SATB1 has been implicated in the development and progression of multiple cancers including breast and colorectal cancers. However, the regulation and role of SATB1 in colorectal cancers is poorly understood. Here, we demonstrate that expression of SATB1 is induced upon hyperactivation of Wnt/β-catenin signaling and repressed upon depletion of TCF7L2 (TCF4) and β-catenin. Using several colorectal cancer cell line models and the APC min mutant zebrafish in vivo model, we established that SATB1 is a novel target of Wnt/β-catenin signaling. We show that direct binding of TCF7L2/β-catenin complex on Satb1 promoter is required for the regulation of SATB1. Moreover, SATB1 is sufficient to regulate the expression of β-catenin, members of TCF family, multiple downstream effectors and mediators of Wnt pathway. SATB1 potentiates the cellular changes and expression of key cancer-associated genes in non-aggressive colorectal cells, promotes their aggressive phenotype and tumorigenesis in vivo. Conversely, depletion of SATB1 from aggressive cells reprograms the expression of cancer-associated genes, reverses their cancer phenotype and reduces the potential of these cells to develop tumors in vivo. We also show that SATB1 and β-catenin bind to the promoters of TCF7L2 and the downstream targets of Wnt signaling and regulate their expression. Our findings suggest that SATB1 shares a feedback regulatory network with TCF7L2/β-catenin signaling and is required for Wnt signaling-dependent regulation of β-catenin. Collectively, these results provide unequivocal evidence to establish that SATB1 reprograms the expression of tumor growth- and metastasis-associated genes to promote tumorigenesis and functionally overlaps with Wnt signaling critical for colorectal cancer tumorigenesis. Oncogene advance online publication, 13 July 2015; doi:10.1038/onc.2015.232

INTRODUCTION Wnt signaling has an essential role in various diseases1 and is considered as a hallmark of colorectal tumorigenesis.2–4 The primary event during Wnt signaling is β-catenin stabilization. In absence of Wnt ligand, the multiprotein complex consisting of AXIN1/AXIN2, APC, CK1 and GSK3β regulates β-catenin levels.1 This multiprotein complex engages β-catenin and mediates sequential phosphorylation by CK1 and GSK3β. Phosphorylated β-catenin is recognized by β-TrCP followed by proteosomal degradation.1 In presence of Wnt ligand, β-catenin is disengaged from the multiprotein complex and is not phosphorylated by GSK3β, hence it escapes recognition by β-TrCP. Dephosphorylated β-catenin translocates into the nucleus and interacts with T cell factor/lymphoid enhancer factor (LEF/TCF) family of transcription factors to induce target gene expression. SATB1 orchestrates chromatin architecture thereby regulating global gene expression.5–7 Aberrant SATB1 expression potentiates structural and molecular changes that are critical for breast cancer tumorigenesis and metastasis.8 The role of SATB1 in development of multiple cancers has been extensively studied.7 Aberrant SATB1 expression has been shown to be associated with multiple cancers including liver cancer,9 ovarian cancer,10 Glioma,11 gastric cancer.12,13 Recently, potential role of SATB1 in colorectal cancer development has been studied.14–18 Increased SATB1 expression has been shown to be associated with poor prognosis; however, the precise molecular mechanism of how SATB1 promotes colorectal tumorigenesis is poorly understood. Meng et al.18 hypothesized that SATB1 could be involved in the development

and progression of colorectal cancer in a Wnt/β-catenin signalingdependent manner. However, since then no study had established such connection directly. Studies so far have shown that SATB1 hyperexpression is essential to induce molecular changes that potentiate tumorigenic transition. Hence, regulation of SATB1 expression could have a key role during this transition from normal to cancer phenotype. Recently Mcinnes et al.19 showed that FOXP3 and FOXP3 regulated microRNAs regulate SATB1 expression. However, this study did not reveal the regulatory network and molecular events responsible for induction of SATB1 expression. Here, we studied the regulation of SATB1 during colorectal tumorigenesis and found that Wnt signaling induces and regulates SATB1 expression. Furthermore, using APC mutant zebrafish as in vivo model system, we established that SATB1 hyperexpression is driven by activation of Wnt signaling. Importantly, various biochemical assays demonstrated that Wnt signaling-driven expression of SATB1 is also β-catenin-dependent. We found that SATB1 promotes tumorigenesis in vivo. We demonstrate that SATB1 regulates critical players of Wnt signaling and is required for Wnt-dependent stimulation of β-catenin. RESULTS Expression of SATB1 correlates with aggressive phenotype of colorectal cancer To elucidate the role of SATB1 in colorectal cancer, we analyzed expression of SATB1 in 12 colorectal cancer cell lines and in a primary colorectal cell line (CRL1790). These cell lines were chosen

1 Indian Institute of Science Education and Research, Pashan, Pune, India; 2Tata Memorial Hospital, Parel, Mumbai, India; 3Advanced Centre for Treatment Research and Education in Cancer, Kharghar, Navi Mumbai, India and 4National Centre for Cell Science, Pune, India. Correspondence: Professor S Galande, Centre of Excellence in Epigenetics, Indian Institute of Science Education and Research, Dr Homi Bhabha Road, Pashan, Pune 411008, India. E-mail: [email protected] Received 30 November 2014; revised 21 April 2015; accepted 1 May 2015

Colorectal cancer tumorigenesis by SATB1 regulation R Mir et al

2 such that they constitute a panel of cells representing various stages of colorectal tumors. The expression of SATB1 at protein level (Figure 1a, Supplementary Figure S1A) and at transcript level (Supplementary Figure S1B) was higher in potentially metastatic cell lines (type C, confined to lymph nodes, represents stage III; and type D, metastasis stage, represents stage IV) relative to primary colorectal cell line (CRL1790), type A (SW1116, confined to mucosa, represents stage I) and type B cell line (SW480, represents stage II). This data establishes direct correlation of SATB1 expression level with the aggressive phenotype of colorectal cancer cells as per the Duke’s classification scheme.20 TCF7L2/β-catenin signaling regulates SATB1 expression in colorectal cancer SATB1 has been shown to be hyperexpressed during progression of multiple types of cancers.7 However, the molecular events that lead to the induction of SATB1 expression are not clear. In this study, we show that SATB1 is upregulated in colorectal cancer cell lines exhibiting an aberrant or hyperactivated Wnt signaling, suggesting that TCF7L2/β-catenin signaling could be involved in SATB1 regulation. To test this hypothesis, we activated Wnt signaling in primary colorectal cell line (CRL1790) using the GSK3β inhibitor 6-(2-(4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethyl-amino)-nicotinonitrile, CHIR99021 (CHIR) and by knocking down GSK3β using siRNA. Both CHIR treatment and GSK3β depletion resulted in robust increase in SATB1 and β-catenin expression (Figure 1b). Quantitative PCR with reverse transcription analysis revealed that CHIR treatment and GSK3β knockdown resulted in 15–20-fold increase in SATB1 expression at transcript level as well as that of known Wnt responsive genes AXIN2 and c-Myc indicating that Wnt signaling is involved in transcriptional regulation of SATB1 (Figure 1c). Further activation of Wnt signaling in a dose-dependent manner by treatment with Wnt3A and overexpression of degradation resistant form of β-catenin (S37A-β-catenin) in HeLa cells induced SATB1 expression (Figure 1d). Furthermore, time-dependent activation also resulted in upregulation of SATB1 but not that of SATB2 thereby indicating Wnt signaling specifically induces SATB1 expression (Figure 1e). Similarly, activation of Wnt signaling by 6-bromoindirubin-30 oxime treatment resulted in robust increase in SATB1 expression at transcript level (Supplementary Figure S1C). Further, depletion of TCF7L2 and β-catenin in HCT116 cells with constitutively active β-catenin and APC null HCT-15 cells resulted in downregulation of SATB1 as well as known Wnt responsive genes at protein and transcript levels (Figures 1f and g, Supplementary Figures S1D–F). Furthermore, siRNA-mediated β-catenin depletion or induction of its degradation by treatment with CK1 activator pyrvinium pamoate (PP)21 in SW620 and COLO201 colorectal cancer cells decreased SATB1 expression (Supplementary Figure S1G). Similar decrease in SATB1 expression was observed upon β-catenin depletion in aggressive breast cancer cell line MDA-MB-231 (Figure 1h, Supplementary Figure S1H showing DKK1-mediated downregulation of SATB1). Next, we evaluated that increased SATB1 expression through hyperactivation of Wnt signaling is β-catenin-dependent, such that SATB1 expression induced upon CHIR treatment in CRL1790 was abrogated by β-catenin knockdown (Figure 1i). Further, hyperactivation of Wnt signaling in vivo as seen in APC mutant zebrafish resulted in elevated SATB1 expression at protein (Figure 1j) and transcript level (Figure 1k). These results provide compelling evidence to demonstrate that TCF7L2/β-catenin signaling cascade regulates SATB1 expression. TCF7L2/β-catenin complex binds to Satb1 promoter and directly regulates SATB1 To delineate whether TCF7L2/β-catenin complex directly induces SATB1 expression by binding to its promoter, we delineated Satb1 promoter region by in silico analysis using transcription regulatory Oncogene (2015), 1 – 13

database (https://cb.utdallas.edu/cgi-bin/TRED/tred.cgi?process=home) and transcription start site database (http://dbtss.hgc.jp/index. html?nmid=DBTSS:NM_002971) and retrieved Satb1 promoter sequence.22 We then scanned Satb1 promoter and found multiple TCF7L2 consensus sequence motifs (A/T A/T CAAAG, CTTTGNN)23–26 (Figure 2a). Next, chromatin immunoprecipitation (ChIP) assay revealed that TCF7L2 is enriched on Satb1 promoter along with increased enrichment of gene activation mark H3K4(me)3 but not gene repression mark H3K27(me)3 (Figure 2b). To gain further insight into SATB1 regulation we analyzed the occupancy of TCF7L2 and β-catenin on Satb1 promoter upon activation of Wnt signaling by CHIR treatment in CRL1790. The hyperactivation of Wnt signaling induced β-catenin expression and occupancy of TCF7L2/β-catenin complex on Satb1 promoter along with Histone mark H3K4(me)3 (Figure 2c) and thereby presumably inducing SATB1 expression at protein and transcript level (Figure 2d). Next we cloned 381 bp of human Satb1 promoter in Luciferase reporter vector and analyzed activity of Satb1 promoter upon β-catenin knockdown in HCT116 cells. Depletion of β-catenin resulted in about fivefold reduction in Satb1 promoter activity (Figure 2e, Supplementary Figure S2A). Furthermore, we also analyzed the occupancy of β-catenin on Satb1 promoter upon β-catenin depletion. Knockdown of β-catenin resulted in decreased enrichment of β-catenin on Satb1 promoter and also the known Wnt target c-Myc promoter (Figure 2f, Supplementary Figure S2B). The decreased occupancy of β-catenin on Satb1 promoter was also reflected in reduced SATB1 expression (Figure 2g). These results provide further evidence in favor of the argument that TCF7L2/βcatenin signaling positively regulates SATB1 expression. SATB1 depletion reverses the tumorigenic growth in vivo To test whether SATB1 expression correlates with cancer phenotype we examined the expression of SATB1 in colorectal tissue samples. SATB1 expression was significantly higher in at least 10 out of 11 tumor samples in comparison with their matched normal adjacent tissues (Supplementary Figure S3A) (tissue details listed in Supplementary Table S1). Furthermore, many tumors exhibited increased expression of TCF7L2, the major transcription factor driving expression of Wnt responsive genes, in correlation with SATB1 expression (Supplementary Figure S3A). To further characterize the potential role of SATB1 in aggressiveness of colorectal cancer cells, we depleted SATB1 (Supplementary figure S3B) in aggressive cell line HCT-15. SATB1 knockdown reduced cell growth, proliferative potential and migratory potential and reduced their ability to form colonies in soft agar thereby restoring their anchorage-dependent growth relative to control cells (Supplementary Figures S3C–F). Next, we investigated the potential role of SATB1 in colorectal tumorigenesis in vivo. We injected HCT-15 cells stably transfected with vector alone (control) or shSATB1 subcutaneously in six-weeks-old severe combined immunodeficient mice (SCID) and monitored the tumor growth over 4 weeks. Immunoblot and quantitative PCR with reverse transcription confirmed decreased SATB1 expression in shSATB1 stably transfected cells (Figure 3a). Depletion of SATB1 reduced the potential of HCT-15 cells to develop tumors in vivo (Figure 3b). Further SATB1 knockdown resulted in tumor regression and significantly reduced tumor volume and tumor weight in SATB1 knockdown cells relative to control cells (Figures 3c and d). For in vivo imaging we subcutaneously injected mCherry expressing control and shSATB1 cells in mice. In vivo imaging analysis revealed increased tumor growth within 2 weeks after injection with control HCT-15 cells and significant tumor regression in mice injected with HCT-15:shSATB1 cells (Figure 3e). Collectively, these results confirm that SATB1 depletion reverses the tumorigenic potential of aggressive colorectal cancer cells. © 2015 Macmillan Publishers Limited

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Figure 1. TCF7L2/β-catenin signaling regulates SATB1 expression in colorectal cancers. (a) Immunoblot for expression of SATB1 in primary colorectal cell line (CRL1790), type A (SW1116, represents stage I), type B (SW480 represents stage II), type C (HCT-15, HT-29, DLD1, COLO320, represents stage III) and type D and metastatic cell lines (COLO201, COLO205, COLO741 and T84 metastasis stage represents stage IV). Expression of SATB1 correlates with Duke’s classification and aggressive phenotype of colorectal cancers. (b) CRL1790 cells were treated with CHIR (3μM) for 48 h and/or transfected with siGSK3-β to induce Wnt signaling. Immunoblot showing expression of β-catenin, SATB1 and GSK3β upon CHIR treatment and GSK3β knockdown. Actin was used as loading control. (c) Relative mRNA levels of SATB1, c-Myc and AXIN2 under CHIR treatment and GSK3-β knockdown in CRL1790 primary cells as determined by quantitative PCR. GAPDH2 was used as endogenous control (error bar represents standard deviation from triplicates). (d) Immunoblot for expression of β-catenin and SATB1 under Wnt3A treatment for 6 h and upon overexpression of mutant S37A β-catenin in dose-dependent manner in HeLa cells. (e) Immunoblot showing expression of SATB1, β-catenin and SATB2 in time-dependent manner under CHIR treatment in HeLa cells. (f) Immunoblot showing expression of SATB1, AXIN2, TCF7 and TCF7L2 in siGFP control HCT116 in comparison with siTCF7L2 HCT116 cells. Actin was used as loading control. (g) Immunoblot for expression of SATB1, TCF7 and β-catenin in siGFP control HCT116 in comparison with siβ-catenin HCT116 cells. (h) Immunoblot for expression of SATB1 and β-catenin upon β-catenin knockdown in MDA-MB-231 cells. (i) Immunoblot for expression of SATB1 and β-catenin upon CHIR treatment in CRL1790 cells and reduction in expression of SATB1 upon depletion of β-catenin in CHIR-treated cells (compare lanes 1 and 2, lanes 2 and 3). (j) Immunoblot for expression of SATB1 (k) relative mRNA levels of SATB1 in heterozygous and APC min mutant zebrafish (48 h post fertilization). Actin was used as endogenous control for Immunoblot and for quantitative PCR.

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Figure 2. TCF7L2/β-catenin signaling directly regulates SATB1. (a) In silico analysis of Satb1 promoter sequence showing multiple TCF7L2 consensus sites upstream to TSS. Promoter sequence was retrieved by using bioinformatics tools: TRED and DBTSS. The TCF7L2-binding sequences are boxed. (b) ChIP assay showing occupancy of TCF7L2 and histone H3 lysine activation and repressor marks on Satb1 promoter in HCT116 cells. ChIP was performed using antibodies to TCF7L2, Histone H3K4(me)3 and H3K27(me)3 followed by ChIP-quantitative PCR using primers flanking the TCF7L2-binding site on Satb1 promoter (a). ChIP using rabbit IgG served as control. (c) ChIP showing the increase in the occupancy of β-catenin and TCF7L2 along with the Histone H3 activation mark on Satb1 promoter upon CHIR treatment. CRL1790 primary cells were treated with CHIR (3 μM) for 48 h followed by ChIP using antibodies to TCF7L2, β-catenin, Histone H3K4(me)3. CHIR treatment induced the expression of β-catenin, so did the occupancy of TCF7L2 and β-catenin along with enrichment of H3K4 (me)3 on Satb1 promoter. (d) Immunoblot (left panel) and quantitative PCR (right panel) showing increased expression of SATB1 and β-catenin upon CHIR treatment in CRL1790 cells used for ChIP assay in c. (e) Satb1 promoter driven Luciferase reporter assay in control cells and in β-catenin knockdown cells. The HCT116 cells were transfected with control siRNA and siβ-catenin. After 24 h, cells were transfected with pGL3 basic vector and pGL3-Satb1 promoter in both control cells and β-catenin knockdown cells. The experiment was performed in triplicates and error bars represents s.d. (f) ChIP-PCR for occupancy of β-catenin on Satb1 promoter and c-Myc promoter upon knockdown of β-catenin in HCT116 cells (g) Immunoblot showing decrease in SATB1 expression upon β-catenin knockdown in HCT116 cells used in the ChIP assay.

SATB1 induces tumorigenesis in vivo Next we investigated whether SATB1 is sufficient to induce tumorigenesis in vivo. Immunoblot analysis confirmed the overexpression of FLAG-tagged SATB1 in SW480 cells (Figure 4a). Injection of control SW480 cells subcutaneously in SCID mice did not result in significant tumor formation in vivo, whereas injection of SATB1 overexpressing SW480 cells induced tumors in all mice (Figure 4b). The tumor volume and tumor weight showed significant increase upon SATB1 overexpression (Figures 4c and d). Oncogene (2015), 1 – 13

Thus, SATB1 is sufficient to transform the non-aggressive colorectal cells to induce their aggressive phenotype and promote tumorigenesis in vivo. SATB1 modulates Wnt signaling and is required for maintenance of mesenchymal phenotype in colorectal cancer cells Since SATB1 is involved in promoting the aggressive phenotype of colorectal cancer cells and in vivo tumorigenesis, we thought to © 2015 Macmillan Publishers Limited

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Figure 3. SATB1 knockdown reverses the tumorigenic growth in vivo. (a) Immunoblot (upper panel) and Taqman quantitative PCR (lower panel) showing expression of SATB1 in knockdown cells in comparison with control cells. (b) Six-weeks-old SCID mice were injected subcutaneously with HCT-15 control cells and shSATB1 cells and tumors were monitored for 4 weeks. Knockdown of SATB1 reduced the tumor growth in shSATB1 mice in comparison with control mice. One of the control mice died within 2 weeks due to tumor overburden. (c) Graphical representation of tumor volume monitored for 4 weeks. Error bars represent s.e.m. (d) Tumor weight in control and shSATB1 mice. Error bars represent s.e.m. (e) In vivo imaging reveals tumor burden in control mice (panel on left side) in comparison with shSATB1 mice (panel on the right). Six mice were used for each experiment. Student’s paired t-test was performed to calculate significance. *Po 0.05, **Po0.01.

investigate whether SATB1 potentially modulates Wnt signaling— a major driving force for colorectal tumorigenesis.27 SATB1 depletion decreased the expression of β-catenin, c-Myc, DVL2 and DVL3 as compared with control (Figure 5a). Interestingly, SATB1 depletion also resulted in decrease of expression of TCF7L2 and Wnt responsive genes AXIN2 and TCF7 at protein level while induced the expression of SATB2 (Figure 5b). To further validate and assess whether SATB1 is sufficient to modulate Wnt signaling, we stably knocked down SATB1 in © 2015 Macmillan Publishers Limited

HCT116 cells and overexpressed SATB1 transiently in the primary cell line CRL1790. Depletion of SATB1 resulted in marked reduction in the levels of β-catenin, DVL2, DVL3, Wnt3A and exhibited drastic effect on expression of TCF7L2 and TCF7L1 (TCF3) (Figure 5c, Supplementary Figure S4A). Furthermore, knockdown using different shRNA for SATB1 and knockout of SATB1 using transcription activator like effector nucleases targeted against SATB1 in HCT116 resulted in drastic downregulation of TCF7L2 and Wnt responsive genes Oncogene (2015), 1 – 13

Colorectal cancer tumorigenesis by SATB1 regulation R Mir et al

6 (Supplementary Figure S4B). Expression analysis of Wnt signalingdriven colorectal cancer cells revealed that tumor-derived cells expressing higher SATB1 also exhibited higher TCF7L2 expression (Supplementary Figure S5C). The overexpression of SATB1 in CRL1790 induced the expression of β-catenin, DVL2, DVL3 and Wnt responsive genes c-Myc and AXIN2 (Figure 5d). Increased SATB1 expression in primary colorectal cell line CRL1790 abolished

the expression of DKK1 (Figure 5d). Further, SATB1 depletion reduced the expression of Wnt responsive genes and overexpression induced their expression at transcript level (Figures 5e and f). Next, we analyzed whether SATB1 regulates the molecular changes essential for epithelial and mesenchymal phenotype. SATB1 knockdown decreased the expression of markers essential for mesenchymal phenotype such as LEF1 and

Figure 4. SATB1 promotes tumorigenesis in vivo. (a) Immunoblot showing expression of SATB1 in FLAG SW480 cells and FLAG SATB1 SW480 cells and Taqman-quantitative PCR showing relative levels of SATB1 at transcript level in control cells and in SATB1 overexpressing cells. Actin was used as endogenous control for immunoblot and quantitative PCR. (b) Six-week-old SCID mice were subcutaneously injected with FLAG SW480 cells and FLAG-SATB1 SW480 cells and tumor growth was monitored for 8 weeks. Overexpression of SATB1 in SW480 cells induced the tumorigenesis in comparison with control. (c) Tumor volume and (d) Tumor weight measured after 8 weeks in control mice injected subcutaneously with FLAG SW480 cells and mice injected with FLAG-SATB1 SW480 cells. Error bars represent s.e.m.

Figure 5. SATB1 modulates Wnt signaling. (a) Immunoblot showing decreased expression of SATB1 in HCT-15 sh1 and sh2 SATB1 knockdown stable cells in comparison with control HCT-15 cells. Knockdown of SATB1 reduced the expression of DVL2, DVL3, β-catenin and c-Myc in comparison with control cells. γ-tubulin used as loading control (b) Immunoblot for expression of AXIN2, TCF7, TCF7L2 and SATB2 under SATB1 knockdown. Actin used as loading control. (c) Immunoblot for expression of SATB1 in control and shSATB1 HCT116 cells. Depletion of SATB1 reduced the expression of β-catenin, DVL2, DVL3, Wnt3A and TCF7L2. (d) Immunoblot for expression of SATB1 in CRL1790 transfected with FLAG and FLAG SATB1. Expression of β-catenin, DVL2, DVL3, c-Myc and AXIN2 was induced upon SATB1 overexpression. Expression of DKK1 was reduced upon SATB1 overexpression. Actin was used as loading control. (e) Relative expression of Wnt signaling downstream targets upon SATB1 knockdown in HCT116 determined by quantitative PCR. (f) Relative expression of Wnt signaling downstream targets under SATB1 overexpression in CRL1790. Data was normalized using GAPDH2 as endogenous control (error bar represents s.d. from triplicates). (g) Left panel: immunoblot for monitoring the expression of SATB1, Vimentin, LEF1 and E-cadherin in control HCT-15 cells in comparison with shSATB1 HCT-15 cells. Right panel: immunoblot for expression of SATB1, Vimentin, MMP2 and N-cadherin in control HCT116 cells in comparison with shSATB1 HCT116 cells. Oncogene (2015), 1 – 13

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7 Vimentin, while induced the expression of E-cadherin essential for epithelial phenotype (Figure 5g, left panel). Similarly, SATB1 knockdown in HCT116 resulted in decreased expression of MMP2,

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Vimentin and N-cadherin (Figure 5g, right panel). Next, we delineated the role of SATB1 in regulating expression of various cancer-associated genes and Wnt signaling mediators using

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Colorectal cancer tumorigenesis by SATB1 regulation R Mir et al

8 customized Taqman low-density gene arrays (TLDA). SATB1 depletion reduced the expression of epidermal growth signaling genes such as ERBB2, ERBB3, matrix metalloproteases such as MMP2, MMP11 involved in tumor invasion, whereas SATB1 overexpression induced the expression of these genes (Supplementary Figure S5A). Collectively, these results indicate that SATB1 is essential for regulating key Wnt signaling events during colorectal cancer development. SATB1 hyperexpression presumably leads to accumulation of molecular changes including epithelial to mesenschymal transition that are essential to promote the tumorigenic phenotype of colorectal cells. SATB1 regulates β-catenin/TCF7L2 mediated transcription We then investigated whether SATB1 directly binds to the promoters of cancer-associated genes and Wnt responsive genes and regulates their expression. We examined the occupancy of SATB1 and β-catenin on promoters of Wnt responsive genes, Mmp2 and Tcf7l2 in control and SATB1 silenced cells by performing ChIP analysis. Such analysis revealed that SATB1 and β-catenin bind to the promoters of c-Myc, Mmp2, Tcf7, Axin2 and Tcf7l2, whereas the occupancy of SATB1 and β-catenin was abrogated upon SATB1 depletion (Figures 6a–d). The reduced occupancy was reflected in the decreased expression of these Wnt responsive genes (Figure 6e, Supplementary figure S5B). Thus, these results suggest that SATB1 is an important player in promoting colorectal tumorigenesis by modulating Wnt signaling through regulation of TCF7L2 and β-catenin. Additionally, SATB1 directly binds to the promoters of downstream targets of Wnt signaling and therefore governs the outcome of Wnt signaling in this newly discovered regulatory loop. SATB1 and TCF7L2/β-catenin signaling share a positive feedback regulatory network The results described above provided multiple evidences that SATB1 and β-catenin share a positive feedback regulatory network. To prove this unequivocally, we induced Wnt signaling in CRL1790 by CHIR treatment and depleted SATB1 in CHIR treated cells. CHIR treatment induced robust expression of β-catenin and SATB1, whereas SATB1 depletion abrogated β-catenin expression in CHIR treated cells (Figure 7a). Similarly, in HeLa cells also the Wnt driven expression of β-catenin was reduced upon SATB1 depletion (Figure 7b and c). These results therefore indicate that SATB1 is regulated by Wnt signaling and is required for Wnt signaling-dependent regulation of β-catenin. We further wished to investigate whether SATB1 is sufficient to rescue the effect of β-catenin depletion on downstream targets of Wnt signaling. Depletion of β-catenin decreased SATB1 expression and that of known downstream targets. However, SATB1 re-expression was not sufficient to rescue the effect of β-catenin depletion on expression of Wnt responsive genes (Figure 7d). These results are consistent with the previous study demonstrating that β-catenin drives SATB1-dependent gene expression.28 Next, we monitored whether TCF7L2 re-expression can rescue the expression of Wnt responsive genes. SATB1 depletion decreased the expression of TCF7L2 and Wnt responsive genes AXIN2 and TCF7. However, TCF7L2 re-expression was not sufficient to re-induce the expression of downstream targets (Figure 7e). To further gain insight into the functional crosstalk of SATB1 with Wnt signaling, we ectopically expressed the N-terminal 204 amino acid region (SATB1-DN) of SATB1 in HCT116 cells that acts as dominant negative regulator for SATB1 function.29 Ectopic expression of SATB1-DN29 resulted in a dramatic decrease in TCF7L2 and known Wnt responsive genes AXIN2, TCF7 and also the novel target SATB1. However, TCF7L2 re-expession in SATB1DN HCT116 cells was not sufficient to re-induce the expression of AXIN2 and TCF7 (Figure 7f, Supplementary Figure S5C). Further to understand whether SATB1 is sufficient to induce the expression Oncogene (2015), 1 – 13

of Wnt responsive genes independent of TCF7L2, we overexpressed SATB1 in control and TCF7L2 depleted cells. SATB1 overexpression induced the expression of AXIN2 and DVL2. However, TCF7L2 depletion did not affect the expression of AXIN2 and DVL2 (Figure 7g). Thus, these results strongly argue that SATB1 regulates TCF7L2 expression and both are essential for regulation of Wnt responsive genes. Importantly, SATB1 can induce expression of Wnt responsive genes in absence of TCF7L2 but TCF7L2 is not sufficient to re-induce expression of Wnt responsive genes in the absence of SATB1. These data suggest that SATB1 regulates multiple events in the Wnt signaling cascade and regulation of SATB1 expression is an important determinant of colorectal tumorigenic transition. DISCUSSION In this study we report a regulatory network of SATB1 expression and Wnt signaling in colorectal cancer. Our results demonstrate for the first time that SATB1 expression is induced by Wnt signaling which in turn is required for Wnt-dependent regulation of downstream targets. This study sheds light on the role of Wnt signaling toward induction and regulation of SATB1. The molecular events or signaling pathways that induce SATB1 expression had not been elucidated until now. The expression of SATB1 is virtually undetectable in primary colorectal cell line CRL1790 and increases in aggressive and Wnt hyperactivated colorectal cancer cells. Thus, we reasoned that hyperactivated Wnt signaling could be responsible for induction and regulation of SATB1 expression. We analyzed SATB1 expression upon hyperactivation of Wnt signaling and established that Wnt signaling induces SATB1 expression at protein and at transcript level and we also establish that Wnt signaling-dependent SATB1 expression requires β-catenin. Further, using other cellular model systems and in vivo model of APC mutant zebrafish, we observed robust increase in SATB1 expression upon hyperactivation of Wnt signaling. Our data suggests that Wnt signaling could be the primary event leading to upregulation of SATB1 expression during tumorigenic transition in β-catenin-dependent manner. To further characterize that elevated expression of SATB1 is because of the direct binding of TCF7L2/β-catenin complex to Satb1 promoter, we analyzed the Satb1 promoter and found multiple TCF7L2 binding motifs (CTTTGNN).1,25 ChIP analysis determined that TCF7L2 binds to Satb1 promoter, promotes histone modifications such as H3K4 trimethylation and thereby regulates SATB1 expression. We further established that hyperactivation of Wnt signaling induces occupancy of TCF7L2/βcatenin complex on Satb1 promoter resulting in induction of SATB1 expression. Further, silencing of TCF7L2 and β-catenin and subsequent loss of occupancy on Satb1 promoter downregulated SATB1 as well as known downstream targets of Wnt signaling, corroborating that SATB1 expression is regulated by TCF7L2/βcatenin signaling. Most importantly, SATB1 depletion from aggressive type C cell line HCT-15 resulted in reduction of proliferation, migratory potential, restored anchorage dependence and resulted in reduction of tumorigenic potential of these cells, thereby regression of tumors in vivo. Conversely, the ectopic expression in non-aggressive cell line SW480 induced their potential to promote tumorigenesis. SATB1 expression is higher in tumor tissues in comparison with adjacent non-tumor tissues. These results firmly establish the role of SATB1 in colorectal cancer tumorigenesis in agreement with earlier reports.16,30,31 Further we observed that significant fraction of tumors overexpressing SATB1 also exhibited higher TCF7L2 expression. In contrast, few tumors with higher expression of SATB1 exhibited lower TCF7L2 expression. This heterogeneity of tumor samples could be of particular interest in future to delineate how differential © 2015 Macmillan Publishers Limited

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Figure 6. SATB1 regulates β-catenin/TCF7L2 mediated transcription. (a) ChIP-PCR demonstrating occupancy of SATB1 and β-catenin on promoters of Mmp2 and c-Myc (left panel) and ChIP-quantitative PCR for relative enrichment (right panel). (b and c) ChIP-PCR reveals occupancy of SATB1 and β-catenin on promoters of Axin2 and Tcf7 (left panel) and ChIP-quantitative PCR for relative enrichment (right panel). ChIP was performed in control HCT116 cells and shSATB1 HCT116 cells using antibodies against β-catenin and SATB1 followed by ChIP-PCR and ChIP-quantitative PCR using primers corresponding to the promoters of Mmp2, c-Myc, Axin2 and Tcf7. (d) ChIP-PCR reveals occupancy of SATB1 and β-catenin on promoter of Tcf7l2 (left panel) and ChIP-quantitative PCR depicting relative enrichment (right panel). (e) Immunoblot showing expression of AXIN2, TCF7, TCF7L2 and c-Myc under SATB1 knockdown in HCT116 cells. Actin was used as the loading control.

expression and coexpression of SATB1 and TCF7L2 influences tumorigenic outcome. Similar positive correlation was observed in tumor-derived cells—those exhibiting higher SATB1 levels also expressed higher levels of TCF7L2. Our study established that SATB1 regulates the expression of several important players of Wnt signaling and cancer associated genes to induce colorectal tumorigenic transition. We also report novel role of © 2015 Macmillan Publishers Limited

SATB1 in regulating TCF7L2 expression by binding to its promoter. Taken together, these findings suggest that SATB1 expression is a critical determinant of the molecular changes associated with colorectal tumorigenesis. Recent studies have shown that DKK132,33 and SATB234 are downregulated during colorectal tumorigenesis and are essential for good prognosis. Data presented here shows for the first time that SATB1 negatively Oncogene (2015), 1 – 13

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Figure 7. SATB1 and TCF7L2/β-catenin signaling share a positive feedback regulatory network. (a) Immunoblot showing increase in expression of SATB1 and β-catenin upon CHIR treatment in CRL1790 cells and reduction in expression of β-catenin upon knockdown of SATB1 in CHIR treated cells (compare lanes 1 and 2; lanes 2 and 3). (b) Immunoblot showing increase in expression of SATB1 and β-catenin upon Wnt3A treatment in HeLa cells and reduction in β-catenin upon SATB1 knockdown (using two different shSATB1 constructs) in WNT3A treated cells (compare lanes 1 and 2; lanes 2–4). (c) Increase in expression of SATB1 at transcript level upon Wnt3A treatment and decrease upon SATB1 knockdown in Wnt3A-treated HeLa cells. (d) Immunoblot showing expression of SATB1, AXIN2, TCF7 and β-catenin under β-catenin knockdown and under overexpression of SATB1 in β-catenin-depleted HCT116 cells. HCT116 cells were transfected with siβ-catenin in combination of GFP and GFP SATB1. (e) Immunoblot showing expression of SATB1, TCF7L2, AXIN2 and TCF7 in SATB1 knockdown HCT116 cells transfected with FLAG and FLAG-TCF7L2. Actin was used as endogenous control and FLAG antibody to show overexpression of TCF7L2. (f) Immunoblot showing expression of TCF7L2, AXIN2, TCF7 and SATB1 in HCT116 cells transfected with SATB1-DN (1–204) (dominant negative) in combination with FLAG vector and FLAG-TCF7L2. (g) Immunoblot depicting expression of SATB1, TCF7L2, AXIN2, DVL2 on SATB1 overexpression and knockdown of TCF7L2 in SATB1 overexpressing SW480 cells. SW480 cells were transfected with siTCF7L2 in combination of FLAG and FLAG-SATB1. Actin was used as an endogenous control.

regulates SATB2 and the Wnt antagonistic DKK1. Thus, our data provides unequivocal evidences that SATB1 differentially regulates the positive and negative regulators of Wnt signaling and modulates the changes in their expression profiles critical for tumorigenic phenotype. SATB1 has emerged as a key factor linking higher order chromatin organization with regulation of genes.35,36 In this study we established that SATB1 directly binds to promoters of Mmp2, Axin2, Tcf7, Tcf7l2 and c-Myc along with the occupancy of β-catenin. Based on these findings, we propose that SATB1 Oncogene (2015), 1 – 13

exerts its effect on the outcome of Wnt signaling in two ways. First, it directly regulates the expression of TCF7L2, TCF7L1, β-catenin and DVLs and secondly, it directly binds to the promoters of Wnt target genes and regulates their expression. Using multiple cellular models we demonstrated that increased β-catenin expression upon hyperactivation of Wnt signaling was reduced upon SATB1 depletion, thus suggesting that SATB1 is required for Wnt signaling-dependent regulation of β-catenin. We observed that SATB1 knockdown led to concomitant decrease in TCF7L2 and Wnt responsive genes and investigated whether © 2015 Macmillan Publishers Limited

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Figure 8. Model depicting the proposed molecular mechanism for regulation of SATB1 and its functional consequences. In Wnt-OFF state (left), the levels of β-catenin are low and therefore the expression of Wnt responsive genes and SATB1 is reduced. In Wnt-ON state (right), β-catenin levels increase. Subsequent to nuclear accumulation of β-catenin, the TCF7L2/β-catenin complex binds to Satb1 promoter thereby inducing its expression. SATB1/β-catenin complex binds to Tcf7l2 promoter to maintain its expression. The TCF7L2/β-catenin and SATB1/βcatenin complexes subsequently bind to Wnt responsive genes to induce their expression.

SATB1 regulates Wnt target genes via TCF7L2 regulation. Surprisingly, we found that TCF7L2 re-expression in SATB1 depleted cells did not re-induce the expression of Wnt target genes, thus corroborating the notion that SATB1 is a critical player essential for regulation of TCF7L2 and Wnt target genes. This is further strengthened by ChIP analysis which suggests that SATB1 directly binds to the promoters of Wnt target genes and hence regulates their expression. The N-terminal domain (1–204 amino acids) is essential for functional switching of SATB1 and acts as dominant negative for its function (SATB1-DN).29 SATB1 interacts with β-catenin during differentiation of T-helper type 2 cells.28 In the present study we found that overexpression of SATB1-DN dramatically reduced the expression of TCF7L2 and its target genes, thus establishing that crosstalk of SATB1 with its cofactors is essential determinant towards the outcome of Wnt signaling. Re-expression of TCF7L2 is not sufficient to re-induce the expression of its target genes. Furthermore, SATB1 was sufficient to induce the expression of Wnt responsive genes in TCF7L2 depleted cells, suggesting that SATB1 independently regulates expression of TCF7L2 and its target genes. We also propose that the complexes of SATB1/βcatenin and TCF7L2/β-catenin might independently occupy the promoters of Wnt target genes and regulate their expression (see model in Figure 8). In conclusion, our data suggests that SATB1 is a novel target of Wnt signaling and its increased expression regulates TCF7L2. Both SATB1 and TCF7L2 are essential for coordinated regulation of Wnt responsive genes. Elucidation of the mechanistic role of SATB1 in colorectal tumorigenesis and its regulation by Wnt signaling provides new therapeutic possibilities for cancers driven by hyperexpression of SATB1 and Wnt signaling. © 2015 Macmillan Publishers Limited

MATERIALS AND METHODS Antibodies and reagents Details of antibodies and reagents are provided in Supplementary Table S4.

Colorectal tumor specimens The study was approved by the Institutional Review Board of the Tata Memorial Center, and the scientific research committee and human ethics committee of the Tata Memorial Hospital, Parel, Mumbai, India. Paired samples from the colorectal cancer and adjacent normal colon tissues were obtained from patients during a routine diagnostic colonoscopy at Tata Memorial Hospital. Informed consent was provided by all patients before the procedure. None of the patients had received any prior systemic treatment for colorectal cancer. Tissues were snap frozen in liquid nitrogen and processed for protein extraction using RIPA buffer (20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% Sodium deoxycholate, protease inhibitor cocktail).

Plasmids TCF7L2 was cloned in pCMV9-3XFLAG. FLAG SATB1 was used as described previously.28 For GFP fusion, SATB1 was subcloned from pCMV10-3XFLAGSATB1. The N-terminal SATB1-DN (1–204) was used as described.29 SATB1 promoter sequence having multiple TCF7L2 binding was cloned in pGL3 basic vector (Promega, Madison, WI, USA). All shRNAs were cloned in pSUPER Puro vector (Oligoengine, Madison, WI, USA). Transcription activator like effector nucleases constructs for knocking out SATB1 were custom designed and procured from Applied Biosystems—Life Technologies (Waltham, MA, USA).

Cell culture, transfections and western blotting SW480, SW1116, SW620, T84 and HCT116 cell lines were grown in DMEM (Gibco, Waltham, MA, USA) with 10% FCS. COLO320, COLO205, COLO201, COLO741, HCT-15, HT-29, DLD1 cell lines were grown in RPMI (Gibco, Grand Island, NY, USA) with 10% FCS. CRL1790 in MEM with 10% FCS. Oncogene (2015), 1 – 13

Colorectal cancer tumorigenesis by SATB1 regulation R Mir et al

12 CRL1790, SW480, COLO201, COLO205, COLO320, SW620, HT-29 DLD1, HeLa and MDA-MB231 were obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). SW1116, T84, HCT116, HCT-15 were obtained from European Collection of Cell Cultures (ECACC) SIGMA (St Louis, MO, USA). SW480 FLAG, SW80 FLAG SATB1 and HCT-15 sh control and HCT-15 shSATB1 stable cell lines were established as described.8 Expression of SATB1 across different colorectal cancer cells, knockdown and overexpression of SATB1 were detected by immunoblotting and quantitative PCR. For immunoblotting 25 μg of lysate was used. The sequences of shRNAs and siRNAs used are listed in Supplementary Table S2.

width. Mice were killed and tumor weight was calculated. For in vivo imaging, briefly 1 × 105 HCT-15 pSUPER puro-mCherry or pSUPER puro-shSATB1-mCherry cells were injected subcutaneously into flanks of male NOD-SCID mice. In vivo fluorescence imaging was performed using cryogenically cooled IVIS system (Xenogen, Alameda, CA, USA) through living image acquisition and analysis software. Images were acquired and analyzed qualitatively. All mice experiments were done according to guidelines of the animal ethics committee of animal facility at the National Centre for Cell Science, Pune.

Expression analysis in zebrafish Biochemical assays To activate Wnt signaling CRL1790 cells were treated with CHIR (3 μM) and 6-bromoindirubin-30 -oxime (1 μM) for 48 h. Alternatively, cells were transfected with siGSK3β and harvested for protein and RNA extractions. HeLa cells were treated with 3 μM CHIR 99021 (GSK3β kinase inhibitor) or with varying concentrations of Wnt3A. Similarly, pyrvinium pamoate was used at concentration of 100 nM and harvested after 48 h. For SATB1 and β-catenin under CHIR and Wnt3A (in HeLa cells), cells were first transfected with sh constructs and after 12 h treated with CHIR for 48 h in case of CRL1790 cells, whereas HeLa cells were treated with CHIR and Wnt3A for 6 h after 42 h of transfection.

Reporter assays Luciferase assay was performed in HCT116 cells essentially as described37 using Satb1 promoter and control constructs. All reporter assays were performed in triplicates.

Heterozygous and APC min mutant zebrafish embryos were harvested 48 h post fertilization for RNA extraction and protein extraction. The guidelines recommended by Committee for the Purpose of Control and Supervision of Experiments nn Animals, Government of India, were followed.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank the staff of the experimental animal facility and in vivo imaging facility of NCCS. Work was supported by grants from the Centre of Excellence in Epigenetics program of the Department of Biotechnology and the Swarnajayanti Fellowship from the Department of Science and Technology, Government of India to SG. RM is supported by fellowship from the University Grants Commission, India. We thank Mahendra Sonawane for providing RNA and whole cell lysates from APC min mutant Zebrafish.

RNA isolation and PCR with reverse transcriptions RNA was isolated using Trizol reagent (Invitrogen, Waltham, MA, USA). Two micrograms of RNA was used for first strand cDNA synthesis using Superscript III (Invitrogen). The cDNA was then used for quantitative PCR as described.38 The sequences of primers used for quantitative PCR are listed in Supplementary Table S3.

ChIP assay ChIP assay was performed as described.39 ChIP-PCR primer sequences are listed in Supplementary Table S3.

Proliferation assay Proliferation assay was performed for control and SATB1 knockdown stable cell lines (Promega) as described.40

Wound healing assay Control HCT-15 and SATB1 knockdown stable cell lines were generated as essentially described (Liang et al.41). Images were acquired using Nikon Eclipse Ti microscope (Tokyo, Japan) at 10 × magnification. NIS elements BR imaging software (Nikon, Minato-ku, Tokyo, Japan) was used for measurement of distance.

Colony formation assay The colony formation assay was performed essentially as described.42 Images of plates with colonies were acquired using digital camera.

Soft agar assay Soft agar assay was performed essentially as described.38 Cells were cultured for 14 days. Images were acquired at 10 × magnification using inverted microscope (AMG Evos, Mill Creek, WI, USA).

In vivo tumor growth assay SW480 cells (1 × 106) expressing FLAG and FLAG-SATB1 were injected subcutaneously with Matrigel at 5 mg/ml in phosphate-buffered saline in a volume of 200 μl in six 8-week-old NOD-SCID mice for each group as described.8 Similarly 1 × 105 shControl HCT-15 cells and shSATB1 HCT-15 cells were injected. Tumor size was monitored for 8 weeks in case of FLAGSATB1 stable cells and for 4 weeks in case of shSATB1 HCT-15 stable cells. Tumor growth was measured using Vernier caliper and tumor volume was calculated by using formula 0.5 × L × W2, where L is length and W is Oncogene (2015), 1 – 13

AUTHOR CONTRIBUTIONS R Mir and SG conceived project and designed experiments. Experiments are performed by R Mir and SP. Tissue resources are provided by PP and RM. Manuscript is written by R Mir and SG.

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

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