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FEBS Letters 587 (2013) 2105–2111

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FOXO6 promotes gastric cancer cell tumorigenicity via upregulation of C- ⇑ Li Qinyu a,b, , Cui Long a,b, Du Zhen-dong c, Shi Min-min a, Wu Wei-ze a,b, Yang Wei-ping a,b, ⇑ Peng Cheng-hong a,b,

a Shanghai Institute of Digestive Surgery, Shanghai Jiaotong University School of Medicine, Shanghai, China b Department of Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China c Department of Clinical Laboratory, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

article info abstract

Article history: The aberrant regulation of many related is involved in the development and progression of Received 26 February 2013 gastric carcinoma. In the present study, we show that mRNA and levels of FOXO6 are upreg- Revised 12 May 2013 ulated in gastric cancer tissues. Forced overexpression of FOXO6 promotes gastric cancer cell prolif- Accepted 13 May 2013 eration, while knockdown of FOXO6 expression inhibits proliferation. We show that ectopic FOXO6 Available online 25 May 2013 expression induces the expression of C-myc. Furthermore, we found that FOXO6 physically interacts with the hepatic nuclear factor 4 (HNF4) in gastric cancer cells. FOXO6 induces Edited by Angel Nebreda C-myc expression by associating to HNF4 and mediating histone acetylation, and the dissociation of HDAC3 from the promoter of the C-myc . Therefore, our results suggest a previously unknown Keywords: FOXO6/HNF4/C-myc molecular network controlling gastric cancer development. Gastric cancer Cell proliferation Structured summary of protein interactions: FOXO6 FOXO6 physically interacts with HNF4 by anti bait coimmunoprecipitation (View Interaction: 1, 2) C-myc HNF4 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction down-regulation of the cell-cycle regulator Cyclin D1 [10–12]. Forkhead box protein O 6 (FOXO6) is also a member of the FOXO Gastric cancer has become the fourth most common malig- family, however, its biological functions were very limited. Previ- nancy and the second cause of death [1,2]. Numerous studies indi- ous studies have shown that FOXO6 plays an important role in cate that the development and progression of gastric cancer arises the regulation of hepatic glucose homeostasis and synaptic func- via miss-regulation of many related genes such as C-myc, and tion in mice [13,14]. Interestingly, the clinical relevance of FOXO6 PTEN [3–5]. However, the regulatory mechanisms remain poorly in cancer biology was not fully understood. In the present study, understood. Therefore, discovery of critical carcinogenic pathways we found that FOXO6 expression was up-regulated in gastric can- may be beneficial for the identification of new therapeutic targets cer tissues. Overexpression of FOXO6 promoted proliferation of for gastric cancer. gastric cancer cells. In addition, we discovered that FOXO6 played The FOXO family of Forkhead transcription factors has attracted a role in gastric cancer development through regulation of C-myc a lot of interest because of its conserved role in the regulation of expression. Identification the roles of FOXO6 might help to further many cellular processes, including proliferation, apoptosis, differ- understand the pathological mechanisms of gastric cancer cell ini- entiation and metabolic pathways [6,7]. Mammals have four FOXO tiation and proliferation. isoforms (FOXO 1, FOXO3, FOXO4, and FOXO6). Growing evidence suggested that FOXO1 could inhibit cell proliferation in many types 2. Materials and methods of tumor cells, including gastric cancer cells [8,9]. For example, FOXO1 can lead to cell-cycle arrest through up-regulation of the 2.1. Tissue samples cyclin-dependent kinase (CDK) inhibitors, p27and p21, and 25 paired primary gastric cancer tissues and normal gastric tis- sues were collected from routine therapeutic surgery at our ⇑ Corresponding authors at: Shanghai Institute of Digestive Surgery, Shanghai Jiaotong University School of Medicine, Shanghai, China. department. All samples were obtained with informed consent E-mail addresses: [email protected] (L. Qinyu), [email protected] (P. Cheng- and approved by Rui-Jin Hospital, Shanghai Jiao Tong University hong). School of Medicine.

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.05.027 2106 L. Qinyu et al. / FEBS Letters 587 (2013) 2105–2111

2.2. Cell culture inghamshire, U.K.). After blocking with 10% non-fat milk in PBS, membranes were immunoblotted with antibodies as indicated, fol- Gastric cancer cell lines (SNU-16 and NCI-N87) used were pur- lowed by HRP-linked secondary antibodies (Cell Signaling). The chased from The Cell Bank of Type Culture Collection of Chinese signals were detected by SuperSignal West Pico Chemiluminescent Academy of Sciences (CAS, Shanghai), and cultured in Dulbecco Substrate kit (Pierce, Rockford, IL, USA) according to manufac- modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) sup- turer’s instructions. Anti-b-actin antibody was purchased from plemented with 10% fetal calf serum, 100 IU/ml penicillin and (USA). Anti-FOXO6, anti-FOXO1, anti-C-myc, anti-Cyclin D1, anti- 100 mg/ml streptomycin. Cultures were maintained at 37 °Cina Cyclin D2, anti-Cyclin E, anti-Cdc25A and anti-HNF4 antibodies humidified atmosphere with 5% CO2. were from Abcam (USA).

2.3. Expression plasmids and retro-viruses 2.7. ChIP assays

Wild-type FOXO6 and FOXO6 with DNA binding domain dele- Chromatin immunoprecipitation (ChIP) assay kits were used tion (FOXO6-DBD del) plasmids were purchased from Genechem (Upstate, USA). In short, Cells or tissues were fixed with 1% formal- (Shanghai, China). HNF4 expression plasmids were purchased from dehyde to cross-link the and DNA, followed by sonication Bioyare Technology (Shanghai, China). Viral supernatants were in an ultrasound bath on ice. DNA was sheared to fragments at produced in HEK293T cells co-transfected with the pBabe-puro 200–1000 bp using sonication. The chromatin was then incubated constructs and packaging vectors GAGPOL and VSV-G (Clontech). and precipitated with the indicated antibodies. The immunopre- Viral supernatants were collected 48 h after transfection, filter- cipitated DNA fragments were detected using Real-time PCR anal- sterilized, and stored at 80 °C. ysis. The primer used in ChIP assays is listed as following: Forward (50-TGAGTCTCCTCCCCACCT-30) and Reverse (50-GTCCAGACCCT 2.4. Promoter construction and Luciferase assays CGCATTA-30).

Human C-myc promoter was cloned from the human genomic 2.8. Statistical analysis DNA and inserted into pGL4.15 vector (Promega). Mutant HNF4 binding motif was generated using a PCR mutagenesis kit (Toyobo) All the cellular experiments were performed three or four times. with a primer (mutation sites underlined): 50-TTATCAGTCCCAGC- Data are presented as mean ± SEM. Statistical differences were TAGCAG-30. For luciferase reporter assays, cells were seeded into determined by a two-tailed t test. Statistical significance is dis- 24-well plates and transfected with 200 ng promoter plasmids by played as ⁄(P < 0.05), ⁄⁄(P < 0.01) or ⁄⁄⁄(P < 0.001). Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. pRL-SV40 (Promega) expressing renilla luciferase was 3. Results used to normalize the luciferase activities, which were measured using the Dual Luciferase Reporter Assay System (Promega, USA). 3.1. Up-regulation of FOXO6 in gastric cancer tissues

2.5. siRNA, RNA extraction and Real-time analysis At the first, FOXO6 mRNA and protein levels were compared in 25 paired gastric cancer tissues and normal gastric tissues. We found Cells were seeded into 6-well plates and then transfected with that FOXO6 mRNA expression was significantly up-regulated in can- 10 nM siGENOME non-targeting siRNA, human FOXO6 or HNF4 cer tissues (Fig. 1A). Besides, Western blot also showed the increased (Dharmacon, USA). Total RNAs were isolated from tissues or cells protein contents of FOXO6 in four pairs of cancer samples (Fig. 1B). by TRIzol reagent (Invitrogen, USA), and reverse transcriptions were performed by Takara RNA PCR kit (Takara, China), following 3.2. FOXO6 regulates cell proliferation in gastric cancer cells the manufacturer’s instructions. In order to determine the tran- scripts of the interest genes, Real-time PCR was performed using To further determine the potential effects of FOXO6 in gastric a SYBR Green Premix Ex Taq (Roche, Switzerland) on Light Cycler cancer cells, we introduced SNU-16 cells with retro-viruses con- 480 (Roche). b-Actin gene was used as an internal control. The taining empty vector (Ctrl) or FOXO6 (Fig. 2A). As shown in primers used were as following: FOXO6 (Forward: 50- Fig. 2B, FOXO6 overexpression led to a significant increase in cell GGCCGCGCTCGTGTACC-30; Reverse: 50-TACACGAGCGCGGCCG-30); number of SNU-16 cells (Fig. 2B). Moreover, bromodeoxyuridine C-myc (Forward: 50-GGCTCCTGGCAAAAGGTCA-30; Reverse: 50-CT (BrdU) analysis also suggested that forced FOXO6 overexpression GCGTAGTTGTGCTGATGT-30); CylinD1 (Forward: 50-TGGAGCCCGTG promoted cell proliferation (Fig. 2C). Indeed, FOXO6 overexpress- AAAAAGAGC-30; Reverse: 50-TCTCCTTCATCTTAGAGGCCAC-30); Cy- ing SNU-16 cells had a significantly lower percentage of cells in linD2 (Forward: 50-ACCTTCCGCAGTGCTCCTA-30; Reverse: 50-CCCA the G1/G0 phase and increased percentage of cells in the S phase, GCCAAGAAACGGTCC-30); CylinE (Forward: 50-ACTCAACGTGCA compared to empty vector-transfected cells (Fig. 2D). Next, SNU- AGCCTCG-30; Reverse: 50-GCTCAAGAAAGTGCTGATCCC-30); Cdc25A 16 cells were transfected with small interfering RNA (siRNA) tar- (Forward: 50-GTGAAGGCGCTATTTGGCG-30; Reverse: 50-TGGTTG geting FOXO6 to knockdown endogenous FOXO6 expression CTCATAATCACTGCC-30); HNF4 (Forward: 50-CGAAGGTCAAGCTAT- (Fig. 2E and F). As a result, down-regulation of FOXO6 led to a GAGGACA-30; Reverse: 50-ATCTGCGATGCTGGCAATCT-30); b-actin marked decrease in cell number and proliferation in SNU-16 cells (Forward: 50-CATGTACGTTGCTATCCAGGC-30; Reverse: 50-CTCCT (Fig. 2G and H). Additionally, silencing of FOXO6 significantly in- TAATGTCACGCACGAT-30) creased the percentage of cells in the G0/G1 phase and decreased the percentage of cells in the S phase (Fig. 2I). 2.6. Western blot

Cells or tissues were harvested and lysed with ice-cold lysis buf- 3.3. FOXO6 overexpression induces C-myc transcription in gastric fer (50 mM Tris–HCl, pH 6.8, 100 mM 2-ME, 2% w/v SDS, 10% glyc- cancer cells erol). After centrifugation at 20000g for 10 min at 4 °C, proteins in the supernatants were quantified and separated by 10% SDS– As described above, FOXO6 plays a critical role in the prolifera- PAGE, transferred to NC membrane (Amersham Bioscience, Buck- tion of gastric cancer cells. In agreement, we further found that a L. Qinyu et al. / FEBS Letters 587 (2013) 2105–2111 2107

Fig. 1. Up-regulation of FOXO6 in lung cancer tissues and cell lines. (A and B) mRNA and protein levels of FOXO6 were analyzed by Real-time PCR (A) and Western blot (B) in normal tissues (normal) or gastric cancer tissues. ⁄⁄⁄P < 0.001, compared with normal tissues.

Fig. 2. FOXO6 promotes cell proliferation in SNU-16 cells. (A) FOXO6expression was determined by Western blot in SNU-16 cells transfected with retro-viruses expressing empty vector (Ctrl) or FOXO6. (B) The growth curve of SNU-16 cells transfected with empty vector (Ctrl) or FOXO6. ⁄P < 0.05 compared with empty vector (Ctrl). (C) The cell proliferative potential (BrdU) was determined in SNU-16 cells transfected with empty vector (Ctrl) or FOXO6. A450 absorption was assayed after transfection for 24 h. ⁄⁄P < 0.01 compared with empty vector (Ctrl). (D)The cell cycle phase of SNU-16 cells transfected with empty vector (Ctrl) or FOXO6 were analyzed by flow cytometry. Cells were labeled for 15 min with PI and immediately analyzed by flow cytometry. Histograms represent the percentage of cells in each phase of the cell cycle (G0/G1, S and G2/ M). (E and F) mRNA and protein levels of FOXO6 expression was determined by Real-time PCR (E) and Western blot (F) in SNU-16 cells transfected with siRNA oligos targeting FOXO6 (FOXO6-1, FOXO6-2) or scramble siRNA (Ctrl). Cells were seeded into 6-well plates and transfected with siRNA for 24 or 48 h to harvest RNA or proteins, respectively. ⁄⁄P < 0.01 compared with scramble siRNA oligos (Ctrl). (G) The growth curve of SNU-16 cells transfected with transfected with siRNA oligos targeting FOXO6 (FOXO6-1, FOXO6-2) or scramble siRNA (Ctrl). ⁄⁄P < 0.01 compared with control siRNA oligos (Ctrl). (H) The cell proliferative potential (BrdU) was determined in SNU-16 cells as indicated. ⁄P < 0.05, ⁄⁄P < 0.01 compared with control siRNA oligos (Ctrl). (I) The cell cycle phase of SNU-16 cells transfected with siRNA oligos targeting FOXO6 (FOXO6-1, FOXO6-2) or scramble siRNA (Ctrl) were analyzed by flow cytometry. series of genes relevant to the cell cycle entry were up-regulated in (Fig. 3C and D). Previous studies have focused on C-myc’s effect SNU-16 cells overexpressing FOXO6, including Cyclin D1 and D2, on these regulatory proteins of the transition of the cell cycle Cyclin E and Cdc25A (Fig. 3A and B). Consistently, FOXO6 inhibition [15,16], which prompted us to investigate whether C-myc could by siRNA oligos led to a decreased expression of these genes be a target gene of FOXO6. As shown in Fig. 3E and F, overexpres- 2108 L. Qinyu et al. / FEBS Letters 587 (2013) 2105–2111

Fig. 3. FOXO6 up-regulates c-myc expression in SNU-16 cells. (A and B) mRNA (A) and protein (B) levels of cell-cycle regulatory genes were analyzed by Real-time PCR or Western blot in SNU-16 cells transfected with empty vector (Ctrl) or FOXO6 for 24 or 48 h, respectively. ⁄P < 0.05, ⁄⁄P < 0.01 compared with empty vector (Ctrl). (C and D) mRNA (C) and protein (D) levels of cell-cycle regulatory genes were analyzed by Real-time PCR or Western blot in SNU-16 cells transfected with siRNA oligos targeting FOXO6 (FOXO6-1) or scramble siRNA (Ctrl) for 24 or 48 h, respectively. ⁄P < 0.05, ⁄⁄P < 0.01 compared with scramble siRNA (Ctrl). (E and F) mRNA (E) and protein (F) levels of C-myc were determined in SNU-16 cells administrated with retro-viruses expressing empty vector (Ctrl) or FOXO6 for 24 or 48 h, respectively. ⁄⁄⁄P < 0.001 compared with empty vector (Ctrl). (G and H) mRNA (G) and protein (H) levels of c-myc in SNU-16 cells transfected with siRNA oligos targeting FOXO6 (FOXO6-1) or scramble siRNA (Ctrl) for 24 or 48 h, respectively. ⁄⁄P < 0.01 compared with scramble siRNA (Ctrl). sion of FOXO6 increased C-myc mRNA and protein levels. In con- expression of FOXO6 was abrogated by knockdown of HNF4 trast, FOXO6 depletion dramatically led to a reduction of C-myc (Fig. 4H and I). contents (Fig. 3G and H). Moreover, this regulation was also ob- served in another gastric cancer cell, NCI-N87 cells (Supplementary 3.5. FOXO6 cooperates HNF4 to trans-activate C-myc expression Fig. 1A–D), suggesting that FOXO6 was an up-stream regulator of C-myc transcription. Next, we used coimmunoprecipitation to assess whether the ability of FOXO6 to enhance HNF4-dependent C-myc expression 3.4. FOXO6 up-regulates C-myc promoter activity through an HNF4 is linked to a physical interaction between FOXO6 and HNF4. When binding site a nuclear fraction isolated from gastric cancer cells was immuno- precipitated with an antibody against FOXO6, HNF4 was detected To delineate the regulatory pathway from FOXO6 to C-myc in the immunoprecipitate (Fig. 5A). In the reciprocal experiment, expression, we generated luciferase reporters fused to the pro- endogenous HNF4 was also able to co-precipitate FOXO6 moter region of C-myc (positions 1200 to +1). We then tested (Fig. 5B). Besides, we found that the AF-2 domain of HNF4 was re- whether FOXO6 induced activity of C-myc promoter fused lucifer- quired for this interaction. ase reporter. When FOXO6 was overexpressed, the activity of C- To further explore the molecular mechanism by which FOXO6 myc was markedly higher (Fig. 4A). Unexpectedly, FOXO6 with induces C-myc expression, we measured the amount of FOXO6 deletion of DNA binding domain (FOXO6-DBD- del) could also and HNF4 associated with the C-myc promoter by ChIP assays. up-regulate C-myc promoter activity, to the similar extent with Our results revealed that FOXO6 was associated with the C-myc wild-type FOXO6 (Fig. 4A). Indeed, overexpression of FOXO6- promoter, and this association was reduced when HNF4 was DBD-del also increased C-myc mRNA and protein levels in SNU- knocked down (Fig. 5C). Notably, the association of HNF4 with 16 cells (Fig. 4B and C), suggesting that FOXO6 may regulate C- the C-myc promoter was substantially increased when FOXO6 myc transcription through a transactivation mechanism. Consis- was overexpressed but markedly reduced in FOXO6 deficiency tently, FOXO6-DBD-del overexpression also promoted cell prolifer- cells (Fig. 5D and E). Sequential ChIP experiments indicated that ation in SNU-16 cells using BrdU assays (data not shown). FOXO6 and the HNF4 complex reside together on the C-myc pro- Next, through serial deletion of C-myc promoter, we defined a moter (Supplementary Fig. 3). Because HNF4 belongs to a family minimal FOXO6-responsive region (254–246 bp upstream of the of nuclear receptors, which mediated activation of downstream transcriptional start site) that contained a consensus binding sites target genes involves dissociation of the HDACs-containing tran- for HNF4 (Fig. 4D). Indeed, overexpression of HNF4 increased lucif- scription corepressor complex from the promoter [17,18].We erase activity of wild-type C-myc promoter, but not a mutant C- examined the association of HDACs with the C-myc promoter in myc promoter carrying an alteration in the HNF4 binding site the presence and absence of FOXO6. The association of HDAC3 (Fig. 4E). Besides, the binding of HNF4 to this region was also con- (Fig. 5F and G), but not HDAC1 or HDAC2 (data not shown), with firmed by chromatin immunoprecipitation (ChIP) assays (Supple- the C-myc promoter was dramatically reduced in cells over- mentary Fig. 2A). Moreover, stimulation by FOXO6 was also not expressing FOXO6, whereas the knockdown of FOXO6 increased observed in this mutant promoter (Fig. 4F) or when HNF4 was the association of HDAC3 with the C-myc promoter. Consistently, knocked down (Supplementary Figs. 2B and C and 4G). Consis- the overexpression of FOXO6 resulted in markedly more acetyla- tently, the induction of endogenous C-myc expression by the over- tion of histone 3 at Lys4 and Lys9 (Ac-H3K4 and Ac-H3K9), two L. Qinyu et al. / FEBS Letters 587 (2013) 2105–2111 2109

Fig. 4. FOXO6 trans-activates C-myc expression independent of its DNA binding domain. (A) SNU-16 cells were co-transfected with the wild-type FOXO6 or FOXO6 with DNA binding domain deficiency (FOXO6-DBD del) together with human C-myc luciferase promoter (1200 bp +1 bp) and the luciferase activity was measured after co- transfection for 36 h. The transcription start site was considered as +1 bp. ⁄⁄P < 0.001 compared with empty vector (Ctrl). (B and C) mRNA (B) and protein (C) levels of C-myc were analyzed by Real-time PCR or Western blot in SNU-16 cells transfected with empty vector (Ctrl), FOXO6 or FOXO6-DBD del for 24 or 48 h, respectively. ⁄⁄⁄P < 0.001 compared with empty vector (Ctrl). (D) SNU-16 cells were co-transfected with series of C-myc promoter as indicated. ⁄⁄⁄P < 0.001 compared with empty vector (Ctrl). (E) SNU- 16 cells were co-transfected with the wild-type C-myc promoter (WT) or HNF4 binding site mutant promoter (Mut) together with empty vector (Ctrl) or HNF4. The luciferase activity was measured after co-transfection for 36 h. ⁄⁄⁄P < 0.001 compared with mutant promoter. (F) SNU-16 cells were co-transfected with the wild-type C-myc promoter (WT) or HNF4 binding site mutant promoter (Mut) together with empty vector (Ctrl), HNF4, FOXO6 or FOXO6 plus HNF4. The luciferase activity was measured after co- transfection for 36 h. ⁄⁄⁄P < 0.001 compared with mutant groups. (G) SNU-16 cells were co-transfected with the wild-type C-myc promoter together with empty vector (Ctrl) or FOXO6. Cells were pre-transfected with siRNA oligos targeting HNF4 or scramble siRNA (Ctrl siRNA) for 24 h. ⁄⁄⁄P < 0.001 compared with other two groups. (H and I) mRNA (H) and protein (I) levels of C-myc were analyzed by Real-time PCR or Western blot in SNU-16 cells transfected with empty vector (Ctrl) or FOXO6 for 24 or 48 h, respectively. Cells were pre-transfected with siRNA oligos targeting HNF4 or scramble siRNA (Ctrl siRNA) for 24 h. ⁄⁄⁄P < 0.001 compared with other two groups. markers of active chromatin [19,20], at the C-myc promoter roles to promoter tumor formation in vivo and in vitro [21,22].It (Fig. 5H). Moreover, although the mRNA and protein levels of is now well established that the dys-regulated expression of c- HNF4 were not changed between normal and gastric cancer tissues myc plays a significant role in human cancer development [23]. (Supplementary Fig. 4A and B), the association of HNF4 with the C- Evidently, given that aberrant up-regulation of C-myc is present myc promoter was significantly increased in tumor tissues (Fig. 5I). in most, if not all, human cancers and is associated with a poor Taken together, these data indicate that by interacting with HNF4, prognosis [24]. Therefore, down-regulation of C-myc expression FOXO6 strengthens the association of HNF4 with the C-myc pro- or activity has been considered as a useful strategy for the control moter and increases histone acetylation to trans-activate C-myc of human cancers, including gastric cancers. For example, it was expression (Fig. 5J). Consistently, we found that mRNA levels of shown that S-adenosylmethionine (SAM) can effectively inhibit C-myc were elevated in gastric cancer tissues (Supplementary the growth of cancer cells by reversing the hypomethylation on Fig. 5A). Moreover, correlation analysis revealed a significant posi- promoters of C-myc, thus down-regulating its expression in human tive correlation between mRNA levels of FOXO6 and C-myc, further gastric cancer [25]. Besides, RAD001, an orally bioavailable deriva- confirming the roles of FOXO6 on the regulation of C-myc (Supple- tive of rapamycin, markedly inhibited proliferation of human gas- mentary Fig. 5B). tric carcinoma cells by down-regulating expression of C-myc [26]. Although many down-stream target genes of C-myc have been 4. Discussion identified, the molecular networks controlling C-myc expression remain largely unexplored. The c-myc oncoprotein, as a transcription factor, is a master In the present study, our data characterize FOXO6 as a novel regulator of genes involved in diverse cellular processes [21]. Situ- transcription factor that plays an important role in the develop- ated upstream of signalling pathways regulating cellular replica- ment of gastric carcinoma. FOXO6 expression, at the first, was tion/growth as well as apoptosis/growth arrest, C-myc may help shown to be up-regulated in gastric cancer tissues, although the integrate processes determining cell numbers and tissue size in mechanisms leading to its overexpression will be investigated in physiology and disease [21]. In cancer, c-myc acts as oncogenic our further studies. Being classified to the FOXO family, FOXO6 is 2110 L. Qinyu et al. / FEBS Letters 587 (2013) 2105–2111

Fig. 5. FOXO6 cooperates HNF4 to transactivate C-myc expression. (A and B) Immunoblot (IB) showing coprecipitation of HNF4 (A) or FOXO6 (B) upon immunoprecipitation (IP) of FOXO6 (A) or HNF4 (B) in nuclear extracts from SNU-16 cells. Anti-IgG was used as a negative control. (C) ChIP assays showing the binding of FOXO6 on the C-myc promoter. SNU-16 cells were transfected with siRNA oligos targeting HNF4 or scramble siRNA (Ctrl) for 24 h. Then cells were fixed with 1% formaldehyde and immunoprecipitated with antibodies to IgG or FOXO6. ⁄⁄⁄P < 0.001 compared with scramble siRNA (Ctrl). (D) ChIP assays showing the binding of HNF4 on the C-myc promoter in SNU-16 cells after transfection with empty vector or FOXO6 for 24 h. ⁄⁄⁄P < 0.001 compared with empty vector (Ctrl). (E) ChIP assays showing the binding of HNF4 on the C- myc promoter in SNU-16 cells after transfection with siRNA oligos targeting HNF4 or scramble siRNA (Ctrl) for 24 h. ⁄⁄⁄P < 0.001 compared with scramble siRNA (Ctrl). (F) ChIP assays showing the binding of HDAC3 on the C-myc promoter in SNU-16 cells after transfection with empty vector (Ctrl) or FOXO6 for 24 h. ⁄⁄⁄P < 0.001 compared with empty vector (Ctrl). (G) ChIP assays showing the binding of HDAC3 on the C-myc promoter in SNU-16 cells after transfection with siRNA oligos targeting HNF4 or scramble siRNA (Ctrl) for 24 h. ⁄⁄⁄P < 0.001 compared with scramble siRNA (Ctrl). (H) ChIP assays showing the binding of acetylated histone 3 lysine 4 and lysine 9 (Ac-H3K4 and Ac-H3K9) on the C-myc promoter in SNU-16 cells after transfection with empty vector (Ctrl) or FOXO6 for 24 h. ⁄⁄⁄P < 0.001 compared with empty vector (Ctrl). (I) ChIP analysis of HNF4 binding to the C-myc promoter in normal or gastric cancer tissues. (n = 8). ⁄⁄⁄P < 0.001 compared with normal tissues. (J) Working model: In gastric cancer tissues, FOXO6 interacts with HNF4, promotes its association with the C-myc promoter and increases histone acetylation to trans-activate C-myc expression.

evolutionarily diverged from other FOXO members. FOXO6 shares histone acetylation and increases mRNA transcription. Interest- the least homology (30%) in amino acid sequence with FOXO1, con- ingly, a recent study also indicates that FOXO1 could also regulate tains only two Akt/PKB phosphorylation sites (Thr26 and Ser184), cell proliferation independent of its transcriptional activity [27]. and lacks the nuclear export signal, a structural motif that is con- Therefore, together with this report, our study may broad the served in the carboxyl domain of other members of the FOXO fam- knowledge of transcriptional regulatory roles of FOXO family. ily. In addition, while FOXO1 acts as a tumor suppressor in many However, further studies are still required to investigate more di- tumor tissues [10–12], our data suggest that FOXO6 might be an rect and indirect target genes of FOXO6 in gastric cancer tissues. oncogene, which positively regulates cell proliferation. Indeed, as Moreover, HNF4, classified as an orphan , is en- shown in the Supplementary Fig. 6A–D, overexpression of FOXO1 riched in liver tissues [28]. HNF4 is essential for the development inhibited proliferation of gastric cancer cells, while knockdown of and maintenance of the hepatic epithelium and also has been asso- FOXO1 promoted it. Although the reason for this inconsistency be- ciated to several human diseases, including diabetes, colitis, and tween FOXO6 and other FOXO members remains unclear now, the cancer [29,30]. Interestingly, recent studies suggest that HNF4 roles of FOXO6 in other tumors (hepatocyte carcinoma, colon can- exerts both tumor-promoting and tumor-suppressing roles in cer, etc.) remain to be explored in the future. hepatocyte carcinoma, while promotes intestinal cancer in mice At the molecular level, our results demonstrated that FOXO6 [31–33]. Therefore, its precise roles in cancer cell proliferation could trans-activate C-myc expression in its DNA binding do- and tumor formation remain to be defined in other tumors. main-independent manner. FOXO6 interacted with HNF4 and en- Until now, little is known about the role of FOXO6 in gastric hanced its association with C-myc promoter, which facilitates the cancer. Our results will provide another potential pathogenic L. Qinyu et al. / FEBS Letters 587 (2013) 2105–2111 2111 mechanism of gastric cancer, which may be useful for the develop- [15] Cascon, A. and Robledo, M. (2012) MAX and MYC: a heritable breakup. Cancer ment of anti-gastric cancer therapeutics. Res. 72, 3119–3124. [16] Secombe, J., Pierce, S.B. and Eisenman, R.N. (2004) Myc: a weapon of mass destruction. Cell 117, 153–156. Appendix A. Supplementary data [17] Lonard, D.M., Lanz, R.B. and O’Malley, B.W. (2007) Nuclear receptor coregulators and human disease. Endocr. Rev. 28, 575–587. [18] Xie, Y.B., Nedumaran, B. and Choi, H.S. (2009) Molecular characterization of Supplementary data associated with this article can be found, in SMILE as a novel corepressor of nuclear receptors. Nucleic Acids Res. 37, 4100– the online version, at http://dx.doi.org/10.1016/j.febslet.2013. 4115. 05.027. [19] Guillemette, B. et al. (2011) H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation. PLoS Genet. 7, e1001354. Reference [20] Krejci, J., Uhlirova, R., Galiova, G., Kozubek, S., Smigova, J. and Bartova, E. (2009) Genome-wide reduction in H3K9 acetylation during human embryonic stem cell differentiation. J. Cell. Physiol. 219, 677–687. [1] Parkin, D.M., Bray, F., Ferlay, J. and Pisani, P. (2005) Global cancer statistics, [21] Zimonjic, D.B. and Popescu, N.C. (2012) Role of DLC1 tumor suppressor gene 2002. CA Cancer J. Clin. 55, 74–108. and MYC oncogene in pathogenesis of human hepatocellular carcinoma: [2] Cervantes, A., Roda, D., Tarazona, N., Rosello, S. and Perez-Fidalgo, J.A. (2013) potential prospects for combined targeted therapeutics (review). Int. J. Oncol. Current questions for the treatment of advanced gastric cancer. Cancer Treat. 41, 393–406. Rev. 39, 60–67. [22] Dang, C.V. (2012) MYC on the path to cancer. Cell 149, 22–35. [3] Tamura, G. (2006) Alterations of tumor suppressor and tumor-related genes in [23] Cairo, S., Armengol, C. and Buendia, M.A. (2012) Activation of Wnt and Myc the development and progression of gastric cancer. World J. Gastroenterol. 12, signaling in hepatoblastoma. Front. Biosci. (Elite Ed) 4, 480–486. 192–198. [24] Liu, X., Cai, H., Huang, H., Long, Z., Shi, Y. and Wang, Y. (2011) The prognostic [4] Asaoka, Y., Ikenoue, T. and Koike, K. (2011) New targeted therapies for gastric significance of apoptosis-related biological markers in Chinese gastric cancer cancer. Expert Opin. Investig. Drugs 20, 595–604. patients. PLoS One 6, e29670. [5] Almhanna, K., Strosberg, J. and Malafa, M. (2011) Targeting AKT protein kinase [25] Luo, J., Li, Y.N., Wang, F., Zhang, W.M. and Geng, X. (2010) S- in gastric cancer. Anticancer Res. 31, 4387–4392. adenosylmethionine inhibits the growth of cancer cells by reversing the [6] Weigel, D., Jurgens, G., Kuttner, F., Seifert, E. and Jackle, H. (1989) The homeotic hypomethylation status of c-myc and H-ras in human gastric cancer and colon gene fork head encodes a nuclear protein and is expressed in the terminal cancer. Int. J. Biol. Sci. 6, 784–795. regions of the Drosophila embryo. Cell 57, 645–658. [26] Xu, D.Z. et al. (2010) Activated mammalian target of rapamycin is a potential [7] Greer, E.L. and Brunet, A. (2005) FOXO transcription factors at the interface therapeutic target in gastric cancer. BMC Cancer 10, 536. between longevity and tumor suppression. Oncogene 24, 7410–7425. [27] Zhao, Y. et al. (2010) Cytosolic FoxO1 is essential for the induction of [8] Kloet, D.E. and Burgering, B.M. (2011) The PKB/FOXO switch in aging and autophagy and tumour suppressor activity. Nat. Cell Biol. 12, 665–675. cancer. Biochim. Biophys. Acta 1813, 1926–1937. [28] Zhong, W., Sladek, F.M. and Darnell Jr., J.E. (1993) The expression pattern of a [9] Kim, J.H. et al. (2007) Constitutive phosphorylation of the FOXO1A Drosophila homolog to the mouse transcription factor HNF-4 suggests a transcription factor as a prognostic variable in gastric cancer. Mod. Pathol. determinative role in gut formation. EMBO J. 12, 537–544. 20, 835–842. [29] Parviz, F. et al. (2003) Hepatocyte nuclear factor 4alpha controls the [10] Medema, R.H., Kops, G.J., Bos, J.L. and Burgering, B.M. (2000) AFX-like development of a hepatic epithelium and liver morphogenesis. Nat. Genet. Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB 34, 292–296. through p27kip1. Nature 404, 782–787. [30] Gupta, R.K. and Kaestner, K.H. (2004) HNF-4alpha: from MODY to late-onset [11] Schmidt, M., Fernandez de Mattos, S., van der Horst, A., Klompmaker, R., Kops, type 2 diabetes. Trends Mol. Med. 10, 521–524. G.J., Lam, E.W., Burgering, B.M. and Medema, R.H. (2002) Cell cycle inhibition [31] Xu, L. et al. (2001) Expression profiling suggested a regulatory role of liver- by FoxO forkhead transcription factors involves downregulation of cyclin D. enriched transcription factors in human hepatocellular carcinoma. Cancer Res. Mol. Cell. Biol. 22, 7842–7852. 61, 3176–3181. [12] Kops, G.J., Medema, R.H., Glassford, J., Essers, M.A., Dijkers, P.F., Coffer, P.J., [32] Yin, C. et al. (2008) Differentiation therapy of hepatocellular carcinoma in Lam, E.W. and Burgering, B.M. (2002) Control of cell cycle exit and entry by mice with recombinant adenovirus carrying hepatocyte nuclear factor-4alpha protein kinase B-regulated forkhead transcription factors. Mol. Cell. Biol. 22, gene. Hepatology 48, 1528–1539. 2025–2036. [33] Darsigny, M., Babeu, J.P., Seidman, E.G., Gendron, F.P., Levy, E., Carrier, J., [13] Kim, D.H. et al. (2011) FoxO6 integrates signaling with Perreault, N. and Boudreau, F. (2010) Hepatocyte nuclear factor-4alpha gluconeogenesis in the liver. Diabetes 60, 2763–2774. promotes gut neoplasia in mice and protects against the production of [14] Salih, D.A. et al. (2012) FoxO6 regulates memory consolidation and synaptic reactive oxygen species. Cancer Res. 70, 9423–9433. function. Genes Dev. 26, 2780–2801.