Oncogene (2014), 1–9 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc
ORIGINAL ARTICLE
Myocyte enhancer factor 2C regulation of hepatocellular carcinoma via vascular endothelial growth factor and Wnt/β-catenin signaling XL Bai1,2,7, Q Zhang1,2,7, LY Ye1,2, F Liang3, X Sun1, Y Chen4, QD Hu1, QH Fu1, W Su1, Z Chen5, ZP Zhuang6 and TB Liang1,2 Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide. Myocyte enhancer factor 2C (MEF2C) was traditionally regarded as a development-associated factor and was recently reported to be an oncogene candidate. We have previously reported overexpression of MEF2C in HCC; however, the roles of MEF2C in HCC remain to be clarified. In this study, HCC cell lines and a xenograft mouse model were used to determine the functions of MEF2C in vitro and in vivo, respectively. Specific plasmids and small interfering RNA were used to upregulate and downregulate MEF2C expression, respectively. Functional assays were performed to assess the influence of MEF2C on cell proliferation, and VEGF-induced vasculogenic mimicry, migration/invasion as well as angiogenesis. Co-immunoprecipitation was conducted to identify the interaction of MEF2C and β-catenin. Human HCC tissue microarrays were used to investigate correlations among MEF2C, β-catenin and involved biomarkers. MEF2C was found to mediate VEGF-induced vasculogenic mimicry, angiogenesis and migration/invasion, with involvement of the p38 MAPK and PKC signaling pathways. However, MEF2C itself inhibited tumor growth in vitro and in vivo. MEF2C was upregulated by and directly interacted with β-catenin. The nuclear translocation of β-catenin blocked by MEF2C was responsible for MEF2C-mediated growth inhibition. The nuclear translocation of MEF2C was associated with intracellular calcium signaling induced by β-catenin. HCC microarrays showed correlations of nuclear MEF2C with the angiogenesis-associated biomarker, CD31, and cytosolic MEF2C with the proliferation-associated biomarker, Ki-67. MEF2C showed double-edged activities in HCC, namely mediating VEGF-induced malignancy enhancement while inhibiting cancer proliferation via blockade of Wnt/β-catenin signaling. The overall effect of MEF2C in HCC progression regulation was dictated by its subcellular distribution. This should be determined prior to any MEF2C-associated intervention in HCC. Oncogene advance online publication, 20 October 2014; doi:10.1038/onc.2014.337
INTRODUCTION Hepatocellular carcinoma (HCC) is the third leading cause of human cancers for which there is currently no satisfactory treatment.1 Although the mechanisms of initiation and progression of HCC have been widely studied for decades, the details of these processes remain to be elucidated. Myocyte enhancer factor 2C (MEF2C) is a member of the MEF2 family, which is known to be a central regulator of cell differentiation and organogenesis.2 MEF2C is a co-transcription factor, and was previously thought to be predominantly involved in the differentiation of myocytes, endothelial cells, neurons, lymphocytes, chondrocytes and neural crest cells.2 Recently, however, the oncogenic role of MEF2C has been revealed. For instance, previous studies implicated MEF2C as a potential oncogene in leukemia, colon cancer and pancreatic cancer.3–5 Our previous work showed that MEF2C was upregulated in human HCC tissues compared with the levels in peritumoral tissues, suggesting a possible role in HCC;6 however, the mechanism is awaiting investigation.
Vascular endothelial growth factor (VEGF) is a classic secreted inducer of angiogenesis under both physiological and pathological conditions.7 The close relationship of VEGF and MEF2C in vascular endothelial cells has been comprehensively demonstrated.8 Both VEGF and MEF2C are responsible for vascular development.9 Furthermore, in human umbilical vein endothelial cells, MEF2C was shown to mediate the transcriptional activation of VEGF on phosphatase of regenerating liver 3 (PRL-3), which is frequently overexpressed in vascular endothelial cells in many cancers.10 Intriguingly, MEF2C-null mice showed reduced expression of VEGF in cardiac cells during development,11 and MEF2C was reported to control the transcriptional level of VEGF expression in angiogenesis.12 These observations suggested that the transcriptional factor MEF2C probably binds to the DNA domain that promotes vegf transcription. However, the association between VEGF and MEF2C in HCC is less well characterized but may contribute to the role of MEF2C in HCC. Dysregulation of Wnt/β-catenin signaling is involved in tumor proliferation, invasion and epithelial–mesenchymal transition,
1 Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 2Key Laboratory of Cancer Prevention and Intervention, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 3Department of Neurosurgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 4Department of General Surgery, The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, China; 5Zhejiang Key Laboratory of Gastro-Intestinal Pathophysiology, Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou, China and 6National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA. Correspondence: Professor TB Liang, Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou 310009, China. E-mail: [email protected] 7 These authors contributed equally to this work. Received 16 January 2014; revised 15 August 2014; accepted 29 August 2014
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leading to enhanced HCC malignancy.13,14 To date, direct mutual regulation and interaction between MEF2C and β-catenin have not been reported, with the exception of some secondary relationships mediated by Mastermind-like 1 and glycogen synthase kinase 3β, or NKX2-5 in non-cancer cells.15,16 In this study, we explored the roles of MEF2C in HCC cells, showing complex regulation of HCC progression mediated by MEF2C, with the critical involvement of VEGF and β-catenin. Previous reports have shown distinct roles of MEF2C in the cytoplasm and nucleus as well as its regulation mechanism.15,17 Here we show preliminary evidence of double-edged characteristics of MEF2C in HCC progression that are associated with the subcellular location of MEF2C. RESULTS Activation of MEF2C by VEGF and corresponding signaling Considering the potent pro-tumor effects of VEGF and the validity of VEGF-targeted therapy in cancer,18 we first investigated whether MEF2C-mediated VEGF signaling in HCC cells as it does in endothelial cells.8,10 In Huh-7 and Sk-hep-1 cell lines, we found an obvious concentration- and time-dependent upregulation of MEF2C and phosphorylated MEF2C (p-MEF2C; Figures 1a and b). In Huh-7 cells treated with 25 ng/ml VEGF for 6 h, the transcriptional activity of MEF2C was found to be significantly enhanced in luciferase report assays (Figure 1c). To explore the signaling pathway involved in VEGF-mediated regulation of MEF2C, we inhibited PKC, JNK, PI3K, ERK1/2 and p38 MAPK signaling individually by use of specific pharmacological inhibitors. Compared with non-inhibited controls, the upregulation of p-MEF2C induced by VEGF was attenuated in both cell lines by the PKC and p38 MAPK inhibitors, whereas inhibition of JNK and ERK1/2 signaling affected only Sk-hep-1 cells and inhibition of PI3K showed no influence on both cell lines (Figure 1d). To confirm these findings, Huh-7 cells was further treated with VEGF in the presence or absence of PKC/p38 MAPK inhibitors. The VEGFinduced overexpression of MEF2C was completely blocked in presence of either inhibitor (Supplementary Figure S1A and B). Considering decreased VEGF expression in MEF2C-null mice, we hypothesize a possible positive circuit regulation as VEGF-MEF2CVEGF. Changes of MEF2C expression positively correlated with VEGF in Huh-7 cells (Supplementary Figure S1C). Moreover, VEGF transient pre-treatment induced subsequent VEGF expression and secretion, which was abrogated by MEF2C knockdown (Figures 1e and f). VEGF-induced vasculogenic mimicry, invasion and angiogenesis were mediated by MEF2C VEGF has been reported to influence cancer cells directly by autocrine or paracrine pathways, including promoting vasculogenic mimicry,19,20 migration and invasion.21,22 Based on the VEGF-induced change in MEF2C expression, we investigated the role of MEF2C in mediating these pro-tumor effects of VEGF in HCC. Huh-7 and Skhep-1 cells exhibited vasculogenic mimicry in the presence of VEGF, and this effect was diminished by MEF2C interference using siRNA (Figure 2a and Supplementary Figure S2A). In vitro transwell and wound-healing assays indicated that VEGF-enhanced cancer cell migration, which was again blocked by MEF2C siRNA (Figure 2b, Supplementary Figure S2B and C). Additionally, VEGF-MEF2C signaling was also responsible for Huh-7 cell invasion, with significantly increased passage of cells through the Matrigel matrix, an effect that was dramatically inhibited by MEF2C knockdown (Figure 2c). The enhanced invasive ability of cancer cells suggested increased expression of matrix degradation-associated factors, such as matrix metallopeptidase 9 and membrane type 1-matrix metalloproteinase in HCC cells.23 In Huh-7 cells, which showed epithelial traits, matrix metallopeptidase 9 mRNA levels were significantly elevated by VEGF and reduced by MEF2C siRNA Oncogene (2014), 1 – 9
(Figure 2d and Supplementary Figure S2D). The mRNA level of tissue inhibitor of metalloproteinase 2, a negative regulator of matrix degradation-associated factor balance, showed a similar changing pattern to that of matrix metallopeptidase 9; however, ratio of MMP 9 and tissue inhibitor of metalloproteinase 2 mRNAs revealed advantages with regard to matrix degradation induced by VEGF and inhibited by MEF2C interference (Figure 2e). No such alterations in any of these factors were detected in mesenchymal-like Sk-hep-1 cells or the membrane type 1-matrix metalloproteinase in Huh-7 cells (Figure 2d and Supplementary Figure S2D). Moreover, we assessed the influence of MEF2C on VEGF-induced angiogenesis in vivo. MEF2C overexpression increased blood perfusion on the surfaces of tumors in mice (Figure 2f), and MEF2C overexpressing xenografts showed greater microvessel density as indicated by immunohistochemistry (IHC) staining of CD31 (Supplementary Figure S2E). MEF2C-inhibited cell proliferation by interfering with Wnt/βcatenin signaling To investigate the role of MEF2C in tumor growth, we used siRNA and plasmid transfection to decrease and increase MEF2C expression, respectively. In Cell Counting Kit-8 (CCK-8) assays in vitro, we found that MEF2C inhibited cell proliferation (Figure 3a); this effect was further confirmed in xenograft models. MEF2C overexpressing xenografts exhibited reduced volume and weight compared with controls (Figure 3b and Supplementary Figure S3A). In addition, fewer Ki-67 positive cells were detected by IHC in MEF2C overexpressing xenografts (Supplementary Figure S3B). Wnt/β-catenin is one of the most important signaling pathways involved in the regulation of cancer cell proliferation; therefore, we investigated the effects of MEF2C on Wnt/β-catenin signaling. Downregulation of MEF2C slightly enhanced expression of βcatenin, as well as that of its target-gene transcription products, c-myc and cyclin D1 (Figure 3c, left panel). Upregulation of MEF2C, in turn, inhibited expression of these proteins (Figure 3c, right panel). In accordance with this, β-catenin transcription activity, assessed in a TOP/FOPFlash plasmid and luciferase reporter system, correlated inversely with changes in MEF2C protein levels (Figure 3d). As the activation of canonical Wnt signaling is marked by nuclear location of β-catenin, we further assessed the subcellular distribution of β-catenin. Unexpectedly, MEF2C overexpression reduced the nuclear location of β-catenin, and vice versa (Figure 3e). Co-expression and interaction between MEF2C and β-catenin Given the mutual regulation between MEF2C and β-catenin, we investigated the potential relationship between these two molecules in four HCC cell lines (Huh-7, HepG2, Hep3B and Skhep-1) and an additional normal liver cell line HL-7702. Generally MEF2C and β-catenin were overexpressed in HCC cell lines compared with HL-7702, with a positive correlation observed in the expression of both proteins (Supplementary Figure S4A). In human HCC tissues derived from surgical resection samples, five out of seven patients showed overexpression of MEF2C and βcatenin in tumor tissues compared to the corresponding peritumor controls (Figure 4a). Relative levels of β-catenin protein were found to correlate with total MEF2C and its phosphorylated forms (Figure 4b). Real-time PCR showed similar changes at the mRNA level with those of the protein after altering Wnt/β-catenin signaling (Supplementary Figure S4B), suggesting that MEF2C expression is positively regulated by β-catenin. In accordance with these results, MEF2C transcription activity was enhanced in the presence of Wnt3a, but was inhibited by siRNA-mediated knockdown of β-catenin (Figure 4c). In Huh-7 cells, MEF2C and β-catenin were found to be colocated to some extent (Figure 4d). Furthermore, MEF2C formed complexes with β-catenin in both HL-7702 and Huh-7 cells (Supplementary Figure S4C). The ratio of β-catenin interacting © 2014 Macmillan Publishers Limited
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Figure 1. MEF2C was activated by VEGF through PKC and p38 MAPK signaling. (a) MEF2C and p-MEF2C were upregulated in Huh-7 and Skhep-1 cells by VEGF in a concentration-dependent manner. Cells were exposed to different concentrations of VEGF for 6 h. (b) MEF2C and p-MEF2C expression was upregulated by VEGF (25 ng/ml) at different time-points in both cell lines. (c) VEGF (25 ng/ml, 6 h) enhanced MEF2C transcription activity in Huh-7 cells. (d) Involvement of PKC and p38 MAPK in VEGF-induced MEF2C overexpression in both cell lines. Cells were treated with specific concentrations of five signaling pathway inhibitors (25 μM for PI3Ki, 5 μM for p38 MAPKi, 10 μM for JNKi, PKCi and ERK1/2i) for 18 h and then treated for 6 h with VEGF (25 ng/ml) or vehicle control. (e) Huh-7 cells were pretreated with VEGF (25 ng/ml) or vehicle after transfected with or without siRNA. VEGF was removed completely 6 h later. Cells were lysed after another 24 h and immunoblotting was conducted. (f) Huh-7 cells were treated as in (E) and relative concentration of VEGF in medium was determined by ELISA after 48 h of siRNA transfection.
with MEF2C and total β-catenin suggested only partial complexation of β-catenin with MEF2C. The interaction between MEF2C and β-catenin was then further confirmed in Huh-7 cells by using different co-immunoprecipitation strategies (Figure 4e). Next, to study the influence of VEGF signaling on this complex formation and protein distribution, separate lysates were obtained from the nucleus and cytosol of cells treated with or without VEGF. The MEF2C/β-catenin complex formed in the nucleus and cytosol; however, VEGF diminished cytosolic MEF2C and MEF2C/β-catenin complexation (Figure 4f), which was consistent with the observation that VEGF promoted nuclear translocation of MEF2C (Supplementary Figure S5A). Subcellular distribution of MEF2C and its influences in human HCC tissues Although MEF2C is a cofactor for transcription factors and predominantly acts in the nucleus, its location in the cytoplasm has also been observed under certain conditions.24 Furthermore, © 2014 Macmillan Publishers Limited
the nuclear translocation of MEF2C is attributed to its nuclear location signal.25 We detected cytosolic MEF2C in HCC cell lines as well as in human HCC tissues. Therefore, we assessed the subcellular distribution of MEF2C under the conditions of our study. VEGF not only induced MEF2C overexpression but also promoted translocation of MEF2C from the cytoplasm to the nucleus in Huh-7 and Sk-hep-1 cells (Supplementary Figure S5A). Moreover, siRNA-mediated knockdown of β-catenin resulted in significant accumulation of MEF2C in the nucleus, while upregulation of β-catenin by Wnt3a led to MEF2C overexpression in the cytoplasm (Figure 5a). We further confirmed that β-cateninregulated nuclear location of MEF2C was mediated by intracellular calcium signaling (Supplementary Figure S6-S10). We identified different patterns of MEF2C expression in human HCC tissues; namely non-expression, nuclear, cytosolic and pancellular expression (data not shown). More than one-third of patients had nuclear expression of MEF2C, while cytosolic expression was even more frequent. Intriguingly, only nuclear expression of MEF2C correlated with CD31 expression (Figure 5b Oncogene (2014), 1 – 9
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Figure 2. VEGF-induced malignant characteristics in cancer cell was mediated by MEF2C. (a) Treatment of VEGF (25 ng/ml, 6 h) induced vasculogenic mimicry in Huh-7 cells. Knockdown of MEF2C by siRNA significantly decreased the quantity of vessel-like structures (VLS). (b and c) VEGF (25 ng/ml, 6 h) increased the (c) migration and (d) invasion capacity of Huh-7 cells; this effect was blocked by MEF2C siRNA transfection. Cell number was calculated as the average of three independent chambers, for which five random fields of vision were countered. (d and e) After the indicated treatments, the mRNA levels of matrix metallopeptidase 9, tissue inhibitor of metalloproteinase 2 and membrane type 1-matrix metalloproteinase were assessed by quantitative reverse transcription-PCR. Fold changes were calculated and were calibrated to β-actin. VEGF increased transcription of (d) matrix metallopeptidase 9, tissue inhibitor of metalloproteinase 2 as well as (e) the ratio of matrix metallopeptidase 9 and tissue inhibitor of metalloproteinase 2; these effects were attenuated by MEF2C siRNA in Huh-7 cells. (f) In a Huh-7 xenograft model, MEF2C overexpressing xenografts showed much higher blood perfusion on the surface of tumors compared with that of controls. **Po 0.01, ***Po 0.001.
and c and Supplementary Figure S5B), suggesting that MEF2Cassociated angiogenesis was related to changes in MEF2C gene transcription. In contrast, cytosolic location of MEF2C correlated negatively with the expression of Ki-67 and β-catenin (Figure 5d and e and Supplementary Figure S5C), which was consistent with the role of MEF2C in blocking β-catenin translocation to the nucleus. In another HCC microarray from Shanghai Biochip National Engineering Research Center (Shanghai, China), expression of cytosolic MEF2C was proven to correlate with maximum Oncogene (2014), 1 – 9
diameter of tumor (Supplementary Figure S5D). Together, these data distinguished two independent roles of MEF2C in cancer cell regulation, which were relevant to the subcellular distribution of MEF2C (Figure 5f). DISCUSSION MEF2C has recently been identified as a novel candidate oncogene, although evidence is limited to very few types of © 2014 Macmillan Publishers Limited
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Figure 3. MEF2C-inhibited cancer cell proliferation with concomitant inhibition of Wnt/β-catenin signaling. (a) Knockdown of MEF2C by siRNA increased cell viability, while ectopic overexpression of MEF2C by plasmid transfection reduced cell viability. (b) Representatives of MEF2C overexpressing xenografts and vehicle controls. (c) Knockdown of MEF2C increased expression of β-catenin and its target genes, and vice versa. (d) MEF2C interference upregulated β-catenin transcription activity, which was enhanced by overexpression of MEF2C. (e) Huh-7 cells overexpressing MEF2C showed predominant cytosolic location of β-catenin, while MEF2C knockdown resulted in predominantly nuclear location of β-catenin. **P o0.01, ***P o0.001.
malignancies.3–5 Whether MEF2C is a universal or tissue-specific oncogene remains to be clarified. We previously demonstrated overexpression of MEF2C in human HCC tissues.6 Based on this observation, we studied the roles of MEF2C and the underlying mechanisms in HCC progression. In contrast to its pure oncogenic role in acute T-cell leukemia, our study showed a double-edged role for MEF2C in HCC. On one hand, MEF2C-mediated VEGF induction of vasculogenic mimicry, migration and invasion as well as angiogenesis of cancers; on the other hand, MEF2C inhibited tumor growth via crosstalk with Wnt/β-catenin signaling. In addition, these two roles were mediated by nuclear and cytosolic MEF2C, respectively. Thus, the overall effects of MEF2C overexpression in HCC depend on its predominant subcellular © 2014 Macmillan Publishers Limited
distribution, as well as the concomitant expression of β-catenin and the amount of VEGF in the microenvironment. Patients were clinically identified with multi-modal expression of MEF2C. In types of HCC with pan-cellular expression, both functions of MEF2C may be performed simultaneously, which suggests a subtype of HCC that is small in size but with abundant angiogenesis as well as the potential for early metastasis. Although proliferation and invasion are both characteristics of malignant cancers and together contribute to subsequent metastasis, these are two independent processes with distinct mechanisms that are not necessarily coupled in cells.26,27 In our study, MEF2C-mediated enhancement of VEGF-induced cell invasion and inhibition of cell proliferation by reducing canonical Oncogene (2014), 1 – 9
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Figure 4. Concurrent expression and mutual regulation of MEF2C and β-catenin. (a) Concurrent expression of MEF2C and β-catenin was shown in tissues from five representative patients. (b) Wnt3a treatment (200 ng/ml, 12 h) upregulated MEF2C and p-MEF2C expression; this effect was inhibited by β-catenin knockdown. (c) Wnt3a treatments enhanced MEF2C transcription activity was enhanced by Wnt3a treatment and reduced by β-catenin siRNA treatment. (d) MEF2C was located in the cytoplasm and nucleus, whereas β-catenin showed pan-cellular distribution. Of note, co-location of these proteins was observed in the cytoplasm and nucleus. (e) Increased β-catenin expression and MEF2C/ β-catenin complexes were detected in Huh-7 cells compared with those in HL-7702 cells. (f) Lysates from the cytoplasm and nucleus were extracted from Huh-7 cells treated with or without VEGF (25 ng/ml, 6 h). MEF2C/β-catenin complexes were detected in both the cytoplasm and nucleus. *P o0.05, **P o0.01, ***P o0.001.
Wnt/β-catenin signaling. Similar to the functions of transforming growth factor-β,28 these double-edged characteristics highlight the complicated roles of MEF2C in HCC. Unfortunately, we were unable to investigate the influence of MEF2C on circulating cancer cells and metastatic foci in vivo. Inhibition of cancer proliferation has been reported to be concomitant with epithelialmesenchymal transition and to facilitate metastasis.26 In addition, MEF2C is included as a critical factor in a scoring system for predicting cancer recurrence.29 Taking these reports into consideration, we speculated that overexpression of MEF2C may benefit cancer metastasis in HCC. Thus, subcellular location of MEF2C, as well as VEGF and β-catenin status, should be investigated carefully before adopting MEF2C-associated strategies in cancer treatment. Tumor initiation is a multistep process and tumors undergo complicated alterations. During this period, different changes occurs sequentially.30–32 Considering the high frequency of concomitant overexpression of MEF2C and β-catenin and the fact that β-catenin upregulated MEF2C, while MEF2C inhibited βcatenin, we speculate that the event of β-catenin overexpression precedes that of MEF2C. This supposition suggested manifold functions of β-catenin in different steps of cancer development. In the early stages of HCC development, β-catenin was upregulated by some mechanism and induced MEF2C, which in turn inhibited β-catenin. However, MEF2C can be induced and activated by the presence of abundant VEGF in the microenvironment in a βcatenin-independent manner. Furthermore, MEF2C mutations may also exist as they do in other diseases,33,34 although they have not been verified to date. Consistent with our results, reduced MEF2C has been reported previously to be associated with nuclear accumulation of βcatenin in breast cancer cells.35 However, the direct regulation of β-catenin by MEF2C has not been studied comprehensively. As a co-transcription factor, MEF2C might recruit histone deacetylase 5 and deacetylate the β-catenin promoter, leading to inhibition of Oncogene (2014), 1 – 9
CTNNB1 gene transcription.36 Mechanically, histone deacetylase 5 has been shown to deacetylate histone 3.37,38 Our study also showed dramatically decreased levels of acetylated histone 3 after VEGF treatment, which was then inhibited by MEF2C knockdown in Huh-7 cells (data not shown). According to our results, deacetylation of VEGF could be indirectly realized by VEGFinduced nuclear translocation of MEF2C, which then recruits histone deacetylase 5, resulting in deacetylation of histone 3. The possible interaction of MEF2C and β-catenin has been indicated by bioinformatic analysis.4 Although attempts to identify direct binding of MEF2 and β-catenin have been unsuccessful in myoblasts,17 in our study, the MEF2C/β-catenin complex was detected. This discrepancy may due to differences in the cell types investigated, with the possibility of mutations in cancer cells, differences in the affinity of immunoprecipitating and antibodies and distinct experimental conditions. It is a limitation of this study that an investigation of the interacting domains was not conducted. Complex formation is not necessary to suppress nuclear translocation, and MEF2C has a nuclear location sequence.25 Consequently, binding may affect the nuclear location sequence of MEF2C; however, this requires further studies. The mechanisms responsible for MEF2C translocation under the context of aberrant β-catenin expression were still full of questions, though our data showed β-catenin-limited intracellular calcium release could be a reasonable mechanism (Supplementary Figure S6-S10). Hepatitis B virus infection (Supplementary Table S2) might also be the possible correlation; however, these need further investigation. Additionally, our results showed reduced expression of βcatenin when MEF2C was overexpressed. MEF2C has been reported to locate in the β-catenin promoter and to inhibit gene transcription by recruiting histone deacetylase 5.36 Thus, MEF2C overexpression could result in higher frequency of deacetylation in the β-catenin promoter and consequent downregulation of β-catenin expression. © 2014 Macmillan Publishers Limited
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Figure 5. Subcellular distribution of MEF2C and the underlying distinct functions. (a) β-Catenin siRNA increased nuclear location of MEF2C in Huh-7 cells; this effect was partially reversed in the presence of Wnt3a (200 ng/ml, 12 h). (b) Representatives of manifold patterns of MEF2C and CD31 expression in human HCC tissues. Only nuclear MEF2C expression was associated with high CD31 expression. (c) In human HCC tissue microarrays, nuclear expression of MEF2C strongly and positively correlated with CD31 expression (n = 344). Dash lines show 95% confidence interval. (d) Cytosolic expression of MEF2C significantly and negatively correlated with Ki-67 expression (n = 338). (e) Cytosolic expression of MEF2C negatively correlated with nuclear expression of β-catenin. (f) Scheme of MEF2C function and its regulation of HCC.
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Oncogene (2014), 1 – 9
Myocyte enhancer factor 2C regulation of hepatocellular carcinoma XL Bai et al
8 In summary, we provide evidence of MEF2C overexpression and its double-edged characteristics in HCC by means of in vitro, in vivo analyses of HCC cell lines, xenograft mice models as well as clinical human tissues. MEF2C-mediated VEGF-induced vasculogenic mimicry, angiogenesis and invasion, as well as inhibition of βcatenin-induced tumor growth. Our study provides the first identification of MEF2C in HCC and a novel understanding of its 'oncogenic' function. MATERIALS AND METHODS Cell culture and reagents The HL-7702 and HCC cell lines, Huh-7, Sk-hep-1 and Hep3B were obtained from the Shanghai Institute for Biological Science (Shanghai, China); HepG2 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC cells were cultured in appropriate medium with 10% FBS and 1% penicillin/streptomycin, and were maintained in standard conditions. Hl-7702 cells were cultured in RPMI-1640 media with 20% FBS. The plasmid used for ectopic overexpression of MEF2C was a gift from Professor Xu Qiang from State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, China. All types of small interfering RNA (siRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant VEGF and signalingpathway inhibitors (chelerythrine chloride for PKC inhibition, PD98059 for ERK1/2 inhibition, SB203580 for p38 MAPK inhibition, SP600125 for JNK inhibition, and LY-294002 hydrochloride for PI3K inhibition) and ionomycin were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant Wnt3a was purchased from R&D Systems (Minneapolis, MN, USA). Fluo-4AM and BAPTA-AM was purchased from Life Technologies (Carlsbad, CA, USA). TOPFlash, FOPFlash and renilla luciferase pRL-TK reporter plasmids were purchased from Millipore (Billerica, MA, USA) and 3 × MEF2C reporter plasmid was a gift from Prof. Ron Prywes from Department of Biological Science, Columbia University (Addgene plasmid 32967).
Cell proliferation assays Cancer cell proliferation was evaluated with a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Following treatments, cells were incubated with CCK-8 solution for 3 h, and absorbance was measured at 450 nm using a Varioskan Flash Multimode Reader (Thermo Scientific, CA, USA). Relative proliferation capacity was expressed as a percentage of the indicated controls.
(Olympus, Tokyo, Japan). Staining without primary antibody was used as negative controls. For immunohistochemical analyses, paraffin-embedded tumor tissue samples from nude mice or patients were cut into 5 μm-thick serial sections. The slides were incubated with CD31 (1:50), Ki-67 (1:100), MEF2C (1:50), or β-catenin (1:100) antibodies. The slides were then incubated with horseradish peroxidase-conjugated antibodies against rabbit immunoglobulin using Histostain-Plus Kit (ZSGB-BIO, Beijing, China) and counterstained with hematoxylin. Staining without primary antibody was used as negative controls. The pathological score was evaluated as previously described.14 The scoring system is illustrated using representative samples of each score in Supplementary Figure S0. For co-immunoprecipitation, ~ 300 μg of protein extract was incubated with 1 μg of protein-A/G sepharose (Santa Cruz) at 4 °C for 4 h. The samples were then centrifuged, rinsed and were mixed with SDS loading buffer for immunoblotting.
Quantitative reverse transcription-PCR As previously described and briefly, total RNA was extracted and reverse transcribed. The primers used were showed in Supplementary Table S1. Real-time PCR was performed on the ABI 7900 Prism HT (Applied Biosystems, Shanghai, China). Fold change in gene expression change was calculated using delta delta CT method, followed by normalization to β-actin. Each treatment was tested in triplicate in three independent experiments.
Transwell assays and wound healing assays Transwell chambers (Corning, NY, USA) containing 6.5 mm diameter polycarbonate filters (8 μm pore) with or without gel matrix (Matrigel; BD, Franklin Lakes, NJ, USA) were used for evaluation of cell invasion or migration, respectively. Cells were seeded at a density of 5 × 104 per chamber. FBS-free medium was added to the upper compartments, and medium with 10% FBS was added to the lower compartments of the chambers. Cells on the upper surface of the filters were removed after 48 h, and cells attached to the lower surface were fixed using 4% polyphosphate formaldehyde (Beyotime, Shanghai, China). Stained cells were counted in five randomly selected fields of vision for each chamber. Wound-healing assays were performed in 6-well plates by scratching monolayers of cells at 90% confluence. The wound area was quantified after 24 and 48 h using Image-Pro Plus 6.0 software (IPP; Media Cybernetics, Acton, MA, USA). All experiments were repeated three times.
Animals, xenograft model and tumor blood perfusion detection Transient transfection and luciferase reporter assays Transfections of siRNA (50 nM) and plasmid were performed using X-tremeGENE siRNA Transfection Reagent and X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland), respectively. For luciferase reporter assays, cells were seeded in 6-well plates and TOPFlash, FOPFlash or 3 × MEF2C reporter plasmids were co-transfected with pRL-TK reporter plasmid. Reporter activity was assayed using the dual luciferase reporter system (Promega, Madison, WI, USA).
Immunoblotting, immunofluorescence staining, immunohistochemistry and co-immunoprecipitation These experiments were performed as described previously.14 Briefly, cells were treated as indicated for certain periods, collected, lysed and proteins were extracted and quantified. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). Specific primary antibodies and secondary antibodies were sequentially incubated, and membranes were visualized. Anti-GAPDH antibody was purchased from Kangchen Biotechnology (Shanghai, China) and was diluted at 1:5000. Other primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and were used at a 1:1000 dilution. For immunofluorescence staining, cells were treated as indicated, and were treated with 4% polyphosphate formaldehyde and subsequent 0.1% Triton X-100 (Sigma). Cells were incubated with anti-MEF2C or anti-βcatenin (1 : 100) at 4 °C overnight, followed by incubation with fluorescein isothiocyanate or cyanine 3-conjugated secondary antibodies (1:1000, Invitrogen, Grand Island, NY, USA). Counterstaining with DAPI (1:10,000, Sigma) was carried out before inspection under a fluorescence microscope Oncogene (2014), 1 – 9
Male Balb/c nude mice (aged 4–6 weeks) were obtained from the Shanghai Laboratory Animal Center, and maintained under standard pathogen-free conditions. Animal experiments were authorized by the Animal Care Committee of Zhejiang University School of Medicine. For xenograft models, Huh-7 cells were transfected with the MEF2C overexpression plasmid or the pcDNA3.1 vehicle plasmid. One million cells of each group were suspended in 200 μl PBS and injected subcutaneously into either flank of mice. Blood perfusion detection was performed at 4 weeks post-inoculation. Mice were anesthetized with 4% chloral hydrate (Sigma) and tumor blood flow was measured at three sites on the surface of each tumor using a Laser Doppler Perfusion and Temperature Monitor (moorVMS-LDF1; Moor Instruments, Devon, UK). All mice were then sacrificed and xenografts were obtained, weighed, and fixed with 10% formaldehyde.
Statistical analysis Statistical calculations were performed using Prism 5 software (GraphPad, San Diego, CA, USA). Statistical analyses were performed using one-way analysis of variance or F test following two-tailed unpaired Student’s t-tests, as appropriate, unless otherwise specified. Data of in vivo tumor blood perfusion are presented as mean ± standard deviation (s.d.) of six mice. Other data are presented as means ± s.d. of three independent experiments. Correlation analysis of tissue microarrays were performed using the Spearman method. Best-fit values and 95% confidence intervals were calculated using linear regression. For all tests, Po0.05 was considered to be statistically significant.
CONFLICT OF INTEREST The authors declare no conflict of interest.
© 2014 Macmillan Publishers Limited
Myocyte enhancer factor 2C regulation of hepatocellular carcinoma XL Bai et al
ACKNOWLEDGEMENTS We thank Professor Xu Qiang (State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, China) for the generous gift of MEF2C overexpression plasmid. We appreciate Mr Xie Shangzhi, Mr Hu Liqiang, and Miss Chen Conglin (The Second Affiliated Hospital, Zhejiang University School of Medicine, China) for their great help in certain experiments. This study was financially supported by the National Natural Science Foundation of China (No. 81171884), the National Key Basic Research Program of China (No. 2014CB542101), the MinistryProvince Co-supportive Project of China, and Innovation and High-Level Talent Training Program of Department of Health of Zhejiang.
AUTHOR CONTRIBUTIONS XLB, QZ, ZZP and TBL designed the experiments. XLB, QZ, LYY, FL, XS, YC, QDH, QHF, ZC and WS performed the experiments and analyzed the data. TBL supervised the project. XLB, QZ and TBL wrote the report.
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9 17 De Angelis L, Borghi S, Melchionna R, Berghella L, Baccarani-Contri M, Parise F et al. Inhibition of myogenesis by transforming growth factor beta is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm. Proc Natl Acad Sci USA 1998; 95: 12358–12363. 18 Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer 2008; 8: 579–591. 19 Seftor RE, Hess AR, Seftor EA, Kirschmann DA, Hardy KM, Margaryan NV et al. Tumor cell vasculogenic mimicry: from controversy to therapeutic promise. Am J Pathol 2012; 181: 1115–1125. 20 Kirschmann DA, Seftor EA, Hardy KM, Seftor RE, Hendrix MJ. Molecular pathways: vasculogenic mimicry in tumor cells: diagnostic and therapeutic implications. Clin Cancer Res 2012; 18: 2726–2732. 21 Goel HL, Mercurio AM. VEGF targets the tumor cells. Nat Rev Cancer 2013; 13: 871–882. 22 Zhou L, Wang DS, Li QJ, Sun W, Zhang Y, Dou KF. Downregulation of the Notch signaling pathway inhibits hepatocellular carcinoma cell invasion by inactivation of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor. Oncol Rep 2012; 28: 874–882. 23 Maatta M, Soini Y, Liakka A, Autio-Harmainen H. Differential expression of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP in hepatocellular and pancreatic adenocarcinoma: implications for tumor progression and clinical prognosis. Clin Cancer Res 2000; 6: 2726–2734. 24 Chen SL, Wang SC, Hosking B, Muscat GE. Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells. Mol Endocrinol 2001; 15: 783–796. 25 Borghi S, Molinari S, Razzini G, Parise F, Battini R, Ferrari S. The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4. J Cell Sci 2001; 114: 4477–4483. 26 Evdokimova V, Tognon C, Ng T, Sorensen PH. Reduced proliferation and enhanced migration: two sides of the same coin? Molecular mechanisms of metastatic progression by YB-1. Cell Cycle 2009; 8: 2901–2906. 27 Hugo HJ, Pereira L, Suryadinata R, Drabsch Y, Gonda TJ, Gunasinghe NP et al. Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells. Breast Cancer Res 2013; 15: R113. 28 Akhurst RJ, Derynck R. TGF-beta signaling in cancer--a double-edged sword. Trends Cell Biol 2001; 11: S44–S51. 29 Oka M, Hamamoto Y, Okabe N. Evaluating system for predicting cancer return, Patent Number: CN100398663(C), granted on 02 July 2008. 30 Liu Y, Sanchez-Tillo E, Lu X, Huang L, Clem B, Telang S et al. Sequential inductions of the ZEB1 transcription factor caused by mutation of Rb and then Ras proteins are required for tumor initiation and progression. J Biol Chem 2013; 288: 11572–11580. 31 Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990; 61: 759–767. 32 Jen KY, Song IY, Banta KL, Wu D, Mao JH, Balmain A. Sequential mutations in Notch1, Fbxw7, and Tp53 in radiation-induced mouse thymic lymphomas. Blood 2012; 119: 805–809. 33 Bienvenu T, Diebold B, Chelly J, Isidor B. Refining the phenotype associated with MEF2C point mutations. Neurogenetics 2013; 14: 71–75. 34 Novara F, Rizzo A, Bedini G, Girgenti V, Esposito S, Pantaleoni C et al. MEF2C deletions and mutations versus duplications: a clinical comparison. Eur J Med Genet 2013; 56: 260–265. 35 Yang M, Chen J, Su F, Yu B, Lin L, Liu Y et al. Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells. Mol Cancer 2011; 10: 117. 36 Zhao JX, Yue WF, Zhu MJ, Du M. AMP-activated protein kinase regulates beta-catenin transcription via histone deacetylase 5. J Biol Chem 2011; 286: 16426–16434. 37 McGee SL, van Denderen BJ, Howlett KF, Mollica J, Schertzer JD, Kemp BE et al. AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5. Diabetes 2008; 57: 860–867. 38 Tate CR, Rhodes LV, Segar HC, Driver JL, Pounder FN, Burow ME et al. Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat. Breast Cancer Res 2012; 14: R79.
Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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ORIGINAL ARTICLE
Myocyte enhancer factor 2C regulation of hepatocellular carcinoma via vascular endothelial growth factor and Wnt/β-catenin signaling XL Bai1,2,7, Q Zhang1,2,7, LY Ye1,2, F Liang3, X Sun1, Y Chen4, QD Hu1, QH Fu1, W Su1, Z Chen5, ZP Zhuang6 and TB Liang1,2 Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide. Myocyte enhancer factor 2C (MEF2C) was traditionally regarded as a development-associated factor and was recently reported to be an oncogene candidate. We have previously reported overexpression of MEF2C in HCC; however, the roles of MEF2C in HCC remain to be clarified. In this study, HCC cell lines and a xenograft mouse model were used to determine the functions of MEF2C in vitro and in vivo, respectively. Specific plasmids and small interfering RNA were used to upregulate and downregulate MEF2C expression, respectively. Functional assays were performed to assess the influence of MEF2C on cell proliferation, and VEGF-induced vasculogenic mimicry, migration/invasion as well as angiogenesis. Co-immunoprecipitation was conducted to identify the interaction of MEF2C and β-catenin. Human HCC tissue microarrays were used to investigate correlations among MEF2C, β-catenin and involved biomarkers. MEF2C was found to mediate VEGF-induced vasculogenic mimicry, angiogenesis and migration/invasion, with involvement of the p38 MAPK and PKC signaling pathways. However, MEF2C itself inhibited tumor growth in vitro and in vivo. MEF2C was upregulated by and directly interacted with β-catenin. The nuclear translocation of β-catenin blocked by MEF2C was responsible for MEF2C-mediated growth inhibition. The nuclear translocation of MEF2C was associated with intracellular calcium signaling induced by β-catenin. HCC microarrays showed correlations of nuclear MEF2C with the angiogenesis-associated biomarker, CD31, and cytosolic MEF2C with the proliferation-associated biomarker, Ki-67. MEF2C showed double-edged activities in HCC, namely mediating VEGF-induced malignancy enhancement while inhibiting cancer proliferation via blockade of Wnt/β-catenin signaling. The overall effect of MEF2C in HCC progression regulation was dictated by its subcellular distribution. This should be determined prior to any MEF2C-associated intervention in HCC. Oncogene advance online publication, 20 October 2014; doi:10.1038/onc.2014.337
INTRODUCTION Hepatocellular carcinoma (HCC) is the third leading cause of human cancers for which there is currently no satisfactory treatment.1 Although the mechanisms of initiation and progression of HCC have been widely studied for decades, the details of these processes remain to be elucidated. Myocyte enhancer factor 2C (MEF2C) is a member of the MEF2 family, which is known to be a central regulator of cell differentiation and organogenesis.2 MEF2C is a co-transcription factor, and was previously thought to be predominantly involved in the differentiation of myocytes, endothelial cells, neurons, lymphocytes, chondrocytes and neural crest cells.2 Recently, however, the oncogenic role of MEF2C has been revealed. For instance, previous studies implicated MEF2C as a potential oncogene in leukemia, colon cancer and pancreatic cancer.3–5 Our previous work showed that MEF2C was upregulated in human HCC tissues compared with the levels in peritumoral tissues, suggesting a possible role in HCC;6 however, the mechanism is awaiting investigation.
Vascular endothelial growth factor (VEGF) is a classic secreted inducer of angiogenesis under both physiological and pathological conditions.7 The close relationship of VEGF and MEF2C in vascular endothelial cells has been comprehensively demonstrated.8 Both VEGF and MEF2C are responsible for vascular development.9 Furthermore, in human umbilical vein endothelial cells, MEF2C was shown to mediate the transcriptional activation of VEGF on phosphatase of regenerating liver 3 (PRL-3), which is frequently overexpressed in vascular endothelial cells in many cancers.10 Intriguingly, MEF2C-null mice showed reduced expression of VEGF in cardiac cells during development,11 and MEF2C was reported to control the transcriptional level of VEGF expression in angiogenesis.12 These observations suggested that the transcriptional factor MEF2C probably binds to the DNA domain that promotes vegf transcription. However, the association between VEGF and MEF2C in HCC is less well characterized but may contribute to the role of MEF2C in HCC. Dysregulation of Wnt/β-catenin signaling is involved in tumor proliferation, invasion and epithelial–mesenchymal transition,
1 Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 2Key Laboratory of Cancer Prevention and Intervention, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 3Department of Neurosurgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 4Department of General Surgery, The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, China; 5Zhejiang Key Laboratory of Gastro-Intestinal Pathophysiology, Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou, China and 6National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA. Correspondence: Professor TB Liang, Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou 310009, China. E-mail: [email protected] 7 These authors contributed equally to this work. Received 16 January 2014; revised 15 August 2014; accepted 29 August 2014
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leading to enhanced HCC malignancy.13,14 To date, direct mutual regulation and interaction between MEF2C and β-catenin have not been reported, with the exception of some secondary relationships mediated by Mastermind-like 1 and glycogen synthase kinase 3β, or NKX2-5 in non-cancer cells.15,16 In this study, we explored the roles of MEF2C in HCC cells, showing complex regulation of HCC progression mediated by MEF2C, with the critical involvement of VEGF and β-catenin. Previous reports have shown distinct roles of MEF2C in the cytoplasm and nucleus as well as its regulation mechanism.15,17 Here we show preliminary evidence of double-edged characteristics of MEF2C in HCC progression that are associated with the subcellular location of MEF2C. RESULTS Activation of MEF2C by VEGF and corresponding signaling Considering the potent pro-tumor effects of VEGF and the validity of VEGF-targeted therapy in cancer,18 we first investigated whether MEF2C-mediated VEGF signaling in HCC cells as it does in endothelial cells.8,10 In Huh-7 and Sk-hep-1 cell lines, we found an obvious concentration- and time-dependent upregulation of MEF2C and phosphorylated MEF2C (p-MEF2C; Figures 1a and b). In Huh-7 cells treated with 25 ng/ml VEGF for 6 h, the transcriptional activity of MEF2C was found to be significantly enhanced in luciferase report assays (Figure 1c). To explore the signaling pathway involved in VEGF-mediated regulation of MEF2C, we inhibited PKC, JNK, PI3K, ERK1/2 and p38 MAPK signaling individually by use of specific pharmacological inhibitors. Compared with non-inhibited controls, the upregulation of p-MEF2C induced by VEGF was attenuated in both cell lines by the PKC and p38 MAPK inhibitors, whereas inhibition of JNK and ERK1/2 signaling affected only Sk-hep-1 cells and inhibition of PI3K showed no influence on both cell lines (Figure 1d). To confirm these findings, Huh-7 cells was further treated with VEGF in the presence or absence of PKC/p38 MAPK inhibitors. The VEGFinduced overexpression of MEF2C was completely blocked in presence of either inhibitor (Supplementary Figure S1A and B). Considering decreased VEGF expression in MEF2C-null mice, we hypothesize a possible positive circuit regulation as VEGF-MEF2CVEGF. Changes of MEF2C expression positively correlated with VEGF in Huh-7 cells (Supplementary Figure S1C). Moreover, VEGF transient pre-treatment induced subsequent VEGF expression and secretion, which was abrogated by MEF2C knockdown (Figures 1e and f). VEGF-induced vasculogenic mimicry, invasion and angiogenesis were mediated by MEF2C VEGF has been reported to influence cancer cells directly by autocrine or paracrine pathways, including promoting vasculogenic mimicry,19,20 migration and invasion.21,22 Based on the VEGF-induced change in MEF2C expression, we investigated the role of MEF2C in mediating these pro-tumor effects of VEGF in HCC. Huh-7 and Skhep-1 cells exhibited vasculogenic mimicry in the presence of VEGF, and this effect was diminished by MEF2C interference using siRNA (Figure 2a and Supplementary Figure S2A). In vitro transwell and wound-healing assays indicated that VEGF-enhanced cancer cell migration, which was again blocked by MEF2C siRNA (Figure 2b, Supplementary Figure S2B and C). Additionally, VEGF-MEF2C signaling was also responsible for Huh-7 cell invasion, with significantly increased passage of cells through the Matrigel matrix, an effect that was dramatically inhibited by MEF2C knockdown (Figure 2c). The enhanced invasive ability of cancer cells suggested increased expression of matrix degradation-associated factors, such as matrix metallopeptidase 9 and membrane type 1-matrix metalloproteinase in HCC cells.23 In Huh-7 cells, which showed epithelial traits, matrix metallopeptidase 9 mRNA levels were significantly elevated by VEGF and reduced by MEF2C siRNA Oncogene (2014), 1 – 9
(Figure 2d and Supplementary Figure S2D). The mRNA level of tissue inhibitor of metalloproteinase 2, a negative regulator of matrix degradation-associated factor balance, showed a similar changing pattern to that of matrix metallopeptidase 9; however, ratio of MMP 9 and tissue inhibitor of metalloproteinase 2 mRNAs revealed advantages with regard to matrix degradation induced by VEGF and inhibited by MEF2C interference (Figure 2e). No such alterations in any of these factors were detected in mesenchymal-like Sk-hep-1 cells or the membrane type 1-matrix metalloproteinase in Huh-7 cells (Figure 2d and Supplementary Figure S2D). Moreover, we assessed the influence of MEF2C on VEGF-induced angiogenesis in vivo. MEF2C overexpression increased blood perfusion on the surfaces of tumors in mice (Figure 2f), and MEF2C overexpressing xenografts showed greater microvessel density as indicated by immunohistochemistry (IHC) staining of CD31 (Supplementary Figure S2E). MEF2C-inhibited cell proliferation by interfering with Wnt/βcatenin signaling To investigate the role of MEF2C in tumor growth, we used siRNA and plasmid transfection to decrease and increase MEF2C expression, respectively. In Cell Counting Kit-8 (CCK-8) assays in vitro, we found that MEF2C inhibited cell proliferation (Figure 3a); this effect was further confirmed in xenograft models. MEF2C overexpressing xenografts exhibited reduced volume and weight compared with controls (Figure 3b and Supplementary Figure S3A). In addition, fewer Ki-67 positive cells were detected by IHC in MEF2C overexpressing xenografts (Supplementary Figure S3B). Wnt/β-catenin is one of the most important signaling pathways involved in the regulation of cancer cell proliferation; therefore, we investigated the effects of MEF2C on Wnt/β-catenin signaling. Downregulation of MEF2C slightly enhanced expression of βcatenin, as well as that of its target-gene transcription products, c-myc and cyclin D1 (Figure 3c, left panel). Upregulation of MEF2C, in turn, inhibited expression of these proteins (Figure 3c, right panel). In accordance with this, β-catenin transcription activity, assessed in a TOP/FOPFlash plasmid and luciferase reporter system, correlated inversely with changes in MEF2C protein levels (Figure 3d). As the activation of canonical Wnt signaling is marked by nuclear location of β-catenin, we further assessed the subcellular distribution of β-catenin. Unexpectedly, MEF2C overexpression reduced the nuclear location of β-catenin, and vice versa (Figure 3e). Co-expression and interaction between MEF2C and β-catenin Given the mutual regulation between MEF2C and β-catenin, we investigated the potential relationship between these two molecules in four HCC cell lines (Huh-7, HepG2, Hep3B and Skhep-1) and an additional normal liver cell line HL-7702. Generally MEF2C and β-catenin were overexpressed in HCC cell lines compared with HL-7702, with a positive correlation observed in the expression of both proteins (Supplementary Figure S4A). In human HCC tissues derived from surgical resection samples, five out of seven patients showed overexpression of MEF2C and βcatenin in tumor tissues compared to the corresponding peritumor controls (Figure 4a). Relative levels of β-catenin protein were found to correlate with total MEF2C and its phosphorylated forms (Figure 4b). Real-time PCR showed similar changes at the mRNA level with those of the protein after altering Wnt/β-catenin signaling (Supplementary Figure S4B), suggesting that MEF2C expression is positively regulated by β-catenin. In accordance with these results, MEF2C transcription activity was enhanced in the presence of Wnt3a, but was inhibited by siRNA-mediated knockdown of β-catenin (Figure 4c). In Huh-7 cells, MEF2C and β-catenin were found to be colocated to some extent (Figure 4d). Furthermore, MEF2C formed complexes with β-catenin in both HL-7702 and Huh-7 cells (Supplementary Figure S4C). The ratio of β-catenin interacting © 2014 Macmillan Publishers Limited
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Figure 1. MEF2C was activated by VEGF through PKC and p38 MAPK signaling. (a) MEF2C and p-MEF2C were upregulated in Huh-7 and Skhep-1 cells by VEGF in a concentration-dependent manner. Cells were exposed to different concentrations of VEGF for 6 h. (b) MEF2C and p-MEF2C expression was upregulated by VEGF (25 ng/ml) at different time-points in both cell lines. (c) VEGF (25 ng/ml, 6 h) enhanced MEF2C transcription activity in Huh-7 cells. (d) Involvement of PKC and p38 MAPK in VEGF-induced MEF2C overexpression in both cell lines. Cells were treated with specific concentrations of five signaling pathway inhibitors (25 μM for PI3Ki, 5 μM for p38 MAPKi, 10 μM for JNKi, PKCi and ERK1/2i) for 18 h and then treated for 6 h with VEGF (25 ng/ml) or vehicle control. (e) Huh-7 cells were pretreated with VEGF (25 ng/ml) or vehicle after transfected with or without siRNA. VEGF was removed completely 6 h later. Cells were lysed after another 24 h and immunoblotting was conducted. (f) Huh-7 cells were treated as in (E) and relative concentration of VEGF in medium was determined by ELISA after 48 h of siRNA transfection.
with MEF2C and total β-catenin suggested only partial complexation of β-catenin with MEF2C. The interaction between MEF2C and β-catenin was then further confirmed in Huh-7 cells by using different co-immunoprecipitation strategies (Figure 4e). Next, to study the influence of VEGF signaling on this complex formation and protein distribution, separate lysates were obtained from the nucleus and cytosol of cells treated with or without VEGF. The MEF2C/β-catenin complex formed in the nucleus and cytosol; however, VEGF diminished cytosolic MEF2C and MEF2C/β-catenin complexation (Figure 4f), which was consistent with the observation that VEGF promoted nuclear translocation of MEF2C (Supplementary Figure S5A). Subcellular distribution of MEF2C and its influences in human HCC tissues Although MEF2C is a cofactor for transcription factors and predominantly acts in the nucleus, its location in the cytoplasm has also been observed under certain conditions.24 Furthermore, © 2014 Macmillan Publishers Limited
the nuclear translocation of MEF2C is attributed to its nuclear location signal.25 We detected cytosolic MEF2C in HCC cell lines as well as in human HCC tissues. Therefore, we assessed the subcellular distribution of MEF2C under the conditions of our study. VEGF not only induced MEF2C overexpression but also promoted translocation of MEF2C from the cytoplasm to the nucleus in Huh-7 and Sk-hep-1 cells (Supplementary Figure S5A). Moreover, siRNA-mediated knockdown of β-catenin resulted in significant accumulation of MEF2C in the nucleus, while upregulation of β-catenin by Wnt3a led to MEF2C overexpression in the cytoplasm (Figure 5a). We further confirmed that β-cateninregulated nuclear location of MEF2C was mediated by intracellular calcium signaling (Supplementary Figure S6-S10). We identified different patterns of MEF2C expression in human HCC tissues; namely non-expression, nuclear, cytosolic and pancellular expression (data not shown). More than one-third of patients had nuclear expression of MEF2C, while cytosolic expression was even more frequent. Intriguingly, only nuclear expression of MEF2C correlated with CD31 expression (Figure 5b Oncogene (2014), 1 – 9
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Figure 2. VEGF-induced malignant characteristics in cancer cell was mediated by MEF2C. (a) Treatment of VEGF (25 ng/ml, 6 h) induced vasculogenic mimicry in Huh-7 cells. Knockdown of MEF2C by siRNA significantly decreased the quantity of vessel-like structures (VLS). (b and c) VEGF (25 ng/ml, 6 h) increased the (c) migration and (d) invasion capacity of Huh-7 cells; this effect was blocked by MEF2C siRNA transfection. Cell number was calculated as the average of three independent chambers, for which five random fields of vision were countered. (d and e) After the indicated treatments, the mRNA levels of matrix metallopeptidase 9, tissue inhibitor of metalloproteinase 2 and membrane type 1-matrix metalloproteinase were assessed by quantitative reverse transcription-PCR. Fold changes were calculated and were calibrated to β-actin. VEGF increased transcription of (d) matrix metallopeptidase 9, tissue inhibitor of metalloproteinase 2 as well as (e) the ratio of matrix metallopeptidase 9 and tissue inhibitor of metalloproteinase 2; these effects were attenuated by MEF2C siRNA in Huh-7 cells. (f) In a Huh-7 xenograft model, MEF2C overexpressing xenografts showed much higher blood perfusion on the surface of tumors compared with that of controls. **Po 0.01, ***Po 0.001.
and c and Supplementary Figure S5B), suggesting that MEF2Cassociated angiogenesis was related to changes in MEF2C gene transcription. In contrast, cytosolic location of MEF2C correlated negatively with the expression of Ki-67 and β-catenin (Figure 5d and e and Supplementary Figure S5C), which was consistent with the role of MEF2C in blocking β-catenin translocation to the nucleus. In another HCC microarray from Shanghai Biochip National Engineering Research Center (Shanghai, China), expression of cytosolic MEF2C was proven to correlate with maximum Oncogene (2014), 1 – 9
diameter of tumor (Supplementary Figure S5D). Together, these data distinguished two independent roles of MEF2C in cancer cell regulation, which were relevant to the subcellular distribution of MEF2C (Figure 5f). DISCUSSION MEF2C has recently been identified as a novel candidate oncogene, although evidence is limited to very few types of © 2014 Macmillan Publishers Limited
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Figure 3. MEF2C-inhibited cancer cell proliferation with concomitant inhibition of Wnt/β-catenin signaling. (a) Knockdown of MEF2C by siRNA increased cell viability, while ectopic overexpression of MEF2C by plasmid transfection reduced cell viability. (b) Representatives of MEF2C overexpressing xenografts and vehicle controls. (c) Knockdown of MEF2C increased expression of β-catenin and its target genes, and vice versa. (d) MEF2C interference upregulated β-catenin transcription activity, which was enhanced by overexpression of MEF2C. (e) Huh-7 cells overexpressing MEF2C showed predominant cytosolic location of β-catenin, while MEF2C knockdown resulted in predominantly nuclear location of β-catenin. **P o0.01, ***P o0.001.
malignancies.3–5 Whether MEF2C is a universal or tissue-specific oncogene remains to be clarified. We previously demonstrated overexpression of MEF2C in human HCC tissues.6 Based on this observation, we studied the roles of MEF2C and the underlying mechanisms in HCC progression. In contrast to its pure oncogenic role in acute T-cell leukemia, our study showed a double-edged role for MEF2C in HCC. On one hand, MEF2C-mediated VEGF induction of vasculogenic mimicry, migration and invasion as well as angiogenesis of cancers; on the other hand, MEF2C inhibited tumor growth via crosstalk with Wnt/β-catenin signaling. In addition, these two roles were mediated by nuclear and cytosolic MEF2C, respectively. Thus, the overall effects of MEF2C overexpression in HCC depend on its predominant subcellular © 2014 Macmillan Publishers Limited
distribution, as well as the concomitant expression of β-catenin and the amount of VEGF in the microenvironment. Patients were clinically identified with multi-modal expression of MEF2C. In types of HCC with pan-cellular expression, both functions of MEF2C may be performed simultaneously, which suggests a subtype of HCC that is small in size but with abundant angiogenesis as well as the potential for early metastasis. Although proliferation and invasion are both characteristics of malignant cancers and together contribute to subsequent metastasis, these are two independent processes with distinct mechanisms that are not necessarily coupled in cells.26,27 In our study, MEF2C-mediated enhancement of VEGF-induced cell invasion and inhibition of cell proliferation by reducing canonical Oncogene (2014), 1 – 9
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Figure 4. Concurrent expression and mutual regulation of MEF2C and β-catenin. (a) Concurrent expression of MEF2C and β-catenin was shown in tissues from five representative patients. (b) Wnt3a treatment (200 ng/ml, 12 h) upregulated MEF2C and p-MEF2C expression; this effect was inhibited by β-catenin knockdown. (c) Wnt3a treatments enhanced MEF2C transcription activity was enhanced by Wnt3a treatment and reduced by β-catenin siRNA treatment. (d) MEF2C was located in the cytoplasm and nucleus, whereas β-catenin showed pan-cellular distribution. Of note, co-location of these proteins was observed in the cytoplasm and nucleus. (e) Increased β-catenin expression and MEF2C/ β-catenin complexes were detected in Huh-7 cells compared with those in HL-7702 cells. (f) Lysates from the cytoplasm and nucleus were extracted from Huh-7 cells treated with or without VEGF (25 ng/ml, 6 h). MEF2C/β-catenin complexes were detected in both the cytoplasm and nucleus. *P o0.05, **P o0.01, ***P o0.001.
Wnt/β-catenin signaling. Similar to the functions of transforming growth factor-β,28 these double-edged characteristics highlight the complicated roles of MEF2C in HCC. Unfortunately, we were unable to investigate the influence of MEF2C on circulating cancer cells and metastatic foci in vivo. Inhibition of cancer proliferation has been reported to be concomitant with epithelialmesenchymal transition and to facilitate metastasis.26 In addition, MEF2C is included as a critical factor in a scoring system for predicting cancer recurrence.29 Taking these reports into consideration, we speculated that overexpression of MEF2C may benefit cancer metastasis in HCC. Thus, subcellular location of MEF2C, as well as VEGF and β-catenin status, should be investigated carefully before adopting MEF2C-associated strategies in cancer treatment. Tumor initiation is a multistep process and tumors undergo complicated alterations. During this period, different changes occurs sequentially.30–32 Considering the high frequency of concomitant overexpression of MEF2C and β-catenin and the fact that β-catenin upregulated MEF2C, while MEF2C inhibited βcatenin, we speculate that the event of β-catenin overexpression precedes that of MEF2C. This supposition suggested manifold functions of β-catenin in different steps of cancer development. In the early stages of HCC development, β-catenin was upregulated by some mechanism and induced MEF2C, which in turn inhibited β-catenin. However, MEF2C can be induced and activated by the presence of abundant VEGF in the microenvironment in a βcatenin-independent manner. Furthermore, MEF2C mutations may also exist as they do in other diseases,33,34 although they have not been verified to date. Consistent with our results, reduced MEF2C has been reported previously to be associated with nuclear accumulation of βcatenin in breast cancer cells.35 However, the direct regulation of β-catenin by MEF2C has not been studied comprehensively. As a co-transcription factor, MEF2C might recruit histone deacetylase 5 and deacetylate the β-catenin promoter, leading to inhibition of Oncogene (2014), 1 – 9
CTNNB1 gene transcription.36 Mechanically, histone deacetylase 5 has been shown to deacetylate histone 3.37,38 Our study also showed dramatically decreased levels of acetylated histone 3 after VEGF treatment, which was then inhibited by MEF2C knockdown in Huh-7 cells (data not shown). According to our results, deacetylation of VEGF could be indirectly realized by VEGFinduced nuclear translocation of MEF2C, which then recruits histone deacetylase 5, resulting in deacetylation of histone 3. The possible interaction of MEF2C and β-catenin has been indicated by bioinformatic analysis.4 Although attempts to identify direct binding of MEF2 and β-catenin have been unsuccessful in myoblasts,17 in our study, the MEF2C/β-catenin complex was detected. This discrepancy may due to differences in the cell types investigated, with the possibility of mutations in cancer cells, differences in the affinity of immunoprecipitating and antibodies and distinct experimental conditions. It is a limitation of this study that an investigation of the interacting domains was not conducted. Complex formation is not necessary to suppress nuclear translocation, and MEF2C has a nuclear location sequence.25 Consequently, binding may affect the nuclear location sequence of MEF2C; however, this requires further studies. The mechanisms responsible for MEF2C translocation under the context of aberrant β-catenin expression were still full of questions, though our data showed β-catenin-limited intracellular calcium release could be a reasonable mechanism (Supplementary Figure S6-S10). Hepatitis B virus infection (Supplementary Table S2) might also be the possible correlation; however, these need further investigation. Additionally, our results showed reduced expression of βcatenin when MEF2C was overexpressed. MEF2C has been reported to locate in the β-catenin promoter and to inhibit gene transcription by recruiting histone deacetylase 5.36 Thus, MEF2C overexpression could result in higher frequency of deacetylation in the β-catenin promoter and consequent downregulation of β-catenin expression. © 2014 Macmillan Publishers Limited
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Figure 5. Subcellular distribution of MEF2C and the underlying distinct functions. (a) β-Catenin siRNA increased nuclear location of MEF2C in Huh-7 cells; this effect was partially reversed in the presence of Wnt3a (200 ng/ml, 12 h). (b) Representatives of manifold patterns of MEF2C and CD31 expression in human HCC tissues. Only nuclear MEF2C expression was associated with high CD31 expression. (c) In human HCC tissue microarrays, nuclear expression of MEF2C strongly and positively correlated with CD31 expression (n = 344). Dash lines show 95% confidence interval. (d) Cytosolic expression of MEF2C significantly and negatively correlated with Ki-67 expression (n = 338). (e) Cytosolic expression of MEF2C negatively correlated with nuclear expression of β-catenin. (f) Scheme of MEF2C function and its regulation of HCC.
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Myocyte enhancer factor 2C regulation of hepatocellular carcinoma XL Bai et al
8 In summary, we provide evidence of MEF2C overexpression and its double-edged characteristics in HCC by means of in vitro, in vivo analyses of HCC cell lines, xenograft mice models as well as clinical human tissues. MEF2C-mediated VEGF-induced vasculogenic mimicry, angiogenesis and invasion, as well as inhibition of βcatenin-induced tumor growth. Our study provides the first identification of MEF2C in HCC and a novel understanding of its 'oncogenic' function. MATERIALS AND METHODS Cell culture and reagents The HL-7702 and HCC cell lines, Huh-7, Sk-hep-1 and Hep3B were obtained from the Shanghai Institute for Biological Science (Shanghai, China); HepG2 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC cells were cultured in appropriate medium with 10% FBS and 1% penicillin/streptomycin, and were maintained in standard conditions. Hl-7702 cells were cultured in RPMI-1640 media with 20% FBS. The plasmid used for ectopic overexpression of MEF2C was a gift from Professor Xu Qiang from State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, China. All types of small interfering RNA (siRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant VEGF and signalingpathway inhibitors (chelerythrine chloride for PKC inhibition, PD98059 for ERK1/2 inhibition, SB203580 for p38 MAPK inhibition, SP600125 for JNK inhibition, and LY-294002 hydrochloride for PI3K inhibition) and ionomycin were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant Wnt3a was purchased from R&D Systems (Minneapolis, MN, USA). Fluo-4AM and BAPTA-AM was purchased from Life Technologies (Carlsbad, CA, USA). TOPFlash, FOPFlash and renilla luciferase pRL-TK reporter plasmids were purchased from Millipore (Billerica, MA, USA) and 3 × MEF2C reporter plasmid was a gift from Prof. Ron Prywes from Department of Biological Science, Columbia University (Addgene plasmid 32967).
Cell proliferation assays Cancer cell proliferation was evaluated with a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Following treatments, cells were incubated with CCK-8 solution for 3 h, and absorbance was measured at 450 nm using a Varioskan Flash Multimode Reader (Thermo Scientific, CA, USA). Relative proliferation capacity was expressed as a percentage of the indicated controls.
(Olympus, Tokyo, Japan). Staining without primary antibody was used as negative controls. For immunohistochemical analyses, paraffin-embedded tumor tissue samples from nude mice or patients were cut into 5 μm-thick serial sections. The slides were incubated with CD31 (1:50), Ki-67 (1:100), MEF2C (1:50), or β-catenin (1:100) antibodies. The slides were then incubated with horseradish peroxidase-conjugated antibodies against rabbit immunoglobulin using Histostain-Plus Kit (ZSGB-BIO, Beijing, China) and counterstained with hematoxylin. Staining without primary antibody was used as negative controls. The pathological score was evaluated as previously described.14 The scoring system is illustrated using representative samples of each score in Supplementary Figure S0. For co-immunoprecipitation, ~ 300 μg of protein extract was incubated with 1 μg of protein-A/G sepharose (Santa Cruz) at 4 °C for 4 h. The samples were then centrifuged, rinsed and were mixed with SDS loading buffer for immunoblotting.
Quantitative reverse transcription-PCR As previously described and briefly, total RNA was extracted and reverse transcribed. The primers used were showed in Supplementary Table S1. Real-time PCR was performed on the ABI 7900 Prism HT (Applied Biosystems, Shanghai, China). Fold change in gene expression change was calculated using delta delta CT method, followed by normalization to β-actin. Each treatment was tested in triplicate in three independent experiments.
Transwell assays and wound healing assays Transwell chambers (Corning, NY, USA) containing 6.5 mm diameter polycarbonate filters (8 μm pore) with or without gel matrix (Matrigel; BD, Franklin Lakes, NJ, USA) were used for evaluation of cell invasion or migration, respectively. Cells were seeded at a density of 5 × 104 per chamber. FBS-free medium was added to the upper compartments, and medium with 10% FBS was added to the lower compartments of the chambers. Cells on the upper surface of the filters were removed after 48 h, and cells attached to the lower surface were fixed using 4% polyphosphate formaldehyde (Beyotime, Shanghai, China). Stained cells were counted in five randomly selected fields of vision for each chamber. Wound-healing assays were performed in 6-well plates by scratching monolayers of cells at 90% confluence. The wound area was quantified after 24 and 48 h using Image-Pro Plus 6.0 software (IPP; Media Cybernetics, Acton, MA, USA). All experiments were repeated three times.
Animals, xenograft model and tumor blood perfusion detection Transient transfection and luciferase reporter assays Transfections of siRNA (50 nM) and plasmid were performed using X-tremeGENE siRNA Transfection Reagent and X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland), respectively. For luciferase reporter assays, cells were seeded in 6-well plates and TOPFlash, FOPFlash or 3 × MEF2C reporter plasmids were co-transfected with pRL-TK reporter plasmid. Reporter activity was assayed using the dual luciferase reporter system (Promega, Madison, WI, USA).
Immunoblotting, immunofluorescence staining, immunohistochemistry and co-immunoprecipitation These experiments were performed as described previously.14 Briefly, cells were treated as indicated for certain periods, collected, lysed and proteins were extracted and quantified. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). Specific primary antibodies and secondary antibodies were sequentially incubated, and membranes were visualized. Anti-GAPDH antibody was purchased from Kangchen Biotechnology (Shanghai, China) and was diluted at 1:5000. Other primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and were used at a 1:1000 dilution. For immunofluorescence staining, cells were treated as indicated, and were treated with 4% polyphosphate formaldehyde and subsequent 0.1% Triton X-100 (Sigma). Cells were incubated with anti-MEF2C or anti-βcatenin (1 : 100) at 4 °C overnight, followed by incubation with fluorescein isothiocyanate or cyanine 3-conjugated secondary antibodies (1:1000, Invitrogen, Grand Island, NY, USA). Counterstaining with DAPI (1:10,000, Sigma) was carried out before inspection under a fluorescence microscope Oncogene (2014), 1 – 9
Male Balb/c nude mice (aged 4–6 weeks) were obtained from the Shanghai Laboratory Animal Center, and maintained under standard pathogen-free conditions. Animal experiments were authorized by the Animal Care Committee of Zhejiang University School of Medicine. For xenograft models, Huh-7 cells were transfected with the MEF2C overexpression plasmid or the pcDNA3.1 vehicle plasmid. One million cells of each group were suspended in 200 μl PBS and injected subcutaneously into either flank of mice. Blood perfusion detection was performed at 4 weeks post-inoculation. Mice were anesthetized with 4% chloral hydrate (Sigma) and tumor blood flow was measured at three sites on the surface of each tumor using a Laser Doppler Perfusion and Temperature Monitor (moorVMS-LDF1; Moor Instruments, Devon, UK). All mice were then sacrificed and xenografts were obtained, weighed, and fixed with 10% formaldehyde.
Statistical analysis Statistical calculations were performed using Prism 5 software (GraphPad, San Diego, CA, USA). Statistical analyses were performed using one-way analysis of variance or F test following two-tailed unpaired Student’s t-tests, as appropriate, unless otherwise specified. Data of in vivo tumor blood perfusion are presented as mean ± standard deviation (s.d.) of six mice. Other data are presented as means ± s.d. of three independent experiments. Correlation analysis of tissue microarrays were performed using the Spearman method. Best-fit values and 95% confidence intervals were calculated using linear regression. For all tests, Po0.05 was considered to be statistically significant.
CONFLICT OF INTEREST The authors declare no conflict of interest.
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Myocyte enhancer factor 2C regulation of hepatocellular carcinoma XL Bai et al
ACKNOWLEDGEMENTS We thank Professor Xu Qiang (State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, China) for the generous gift of MEF2C overexpression plasmid. We appreciate Mr Xie Shangzhi, Mr Hu Liqiang, and Miss Chen Conglin (The Second Affiliated Hospital, Zhejiang University School of Medicine, China) for their great help in certain experiments. This study was financially supported by the National Natural Science Foundation of China (No. 81171884), the National Key Basic Research Program of China (No. 2014CB542101), the MinistryProvince Co-supportive Project of China, and Innovation and High-Level Talent Training Program of Department of Health of Zhejiang.
AUTHOR CONTRIBUTIONS XLB, QZ, ZZP and TBL designed the experiments. XLB, QZ, LYY, FL, XS, YC, QDH, QHF, ZC and WS performed the experiments and analyzed the data. TBL supervised the project. XLB, QZ and TBL wrote the report.
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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Oncogene (2014), 1 – 9
