Background
Colorectal cancer (CRC), a common malignant tumor of the digestive tract, is one of the leading causes of cancer death in both developed and developing nations. This disease has an estimated annual worldwide incidence of more than one million new cases, and approximately one of every three people who develop CRC dies from the disease [
1]. The current treatment for CRC patients is mainly based on comprehensive surgical treatment with chemotherapy and/or targeting therapies. Although the molecular mechanisms of CRC development and progression have been extensively researched, the prognosis of patients with CRC remains unsatisfactory, especially for patients with lymph node metastases [
2]. Therefore, a better understanding of the molecular mechanisms of CRC tumorigenesis and the development of new therapeutic targets based on these mechanisms are of great significance.
The zinc-finger transcription factor Gli1 is a key downstream effector of the Hedgehog (Hh) signaling pathway, which functions via a membrane-protein complex that consists of Patched-1 (Ptch1) and Smoothened (Smo) [
3]. Physiologically, the activation of Hh signaling is initiated by the binding of the Hh ligand to the Ptch1 receptor. As a result of this binding, Smo is activated, which consequently activates transcription factor Glis. Three Gli proteins are known, and they exert both activator and repressor functions. Specifically, Gli1 acts as a transcriptional activator, Gli2 is a composite of positive and negative regulatory domains, and Gli3 acts primarily as a transcriptional repressor [
4]. The activated Gli proteins translocate to the nucleus and transactivate many downstream target genes, such as Gli1 itself, Ptch1, Cyclin D1, p21 and Snail [
5]. The aberrant activation of Hh-Gli signaling has been implicated in the promotion of tumorigenesis in several types of carcinoma, including hepatocellular carcinoma [
6], gastric cancer [
7,
8], lung cancer [
9] and basal cell carcinomas [
10]. Similar results were also reported in other studies of CRC [
11‐
13]. Although these authors found that the Gli1 mRNA and protein expression levels were significantly increased in CRC tissues, the exact mechanism underlying this increase remained unclear. Therefore, the molecular mechanisms by which the aberrant activation of Hh signaling promotes CRC cell proliferation and tumor growth need to be further elucidated.
Forkhead box M1 (FoxM1) is a transcription factor of the forkhead family, which consists of more than 50 transcription factors that share a conserved forkhead or winged-helix DNA-binding domain [
14]. FoxM1 is expressed in embryonic tissues and dividing cells of epithelial and mesenchymal origin, but not in terminally differentiated, non-dividing cells [
15]. FoxM1 plays a critical role in cell cycle progression. Specifically, the expression of FoxM1 increases at the G1 to S phase transition and reaches a maximal level during the G2 to M phase transition, thereby promoting M phase entry [
16,
17]. FoxM1 controls the expression of a number of cell cycle regulatory proteins, including cyclin B1 [
17], and genes that are essential for faithful chromosome segregation and mitosis, such as Cdc25B, Aurora B kinase, Survivin, PLK1, centromere protein A (CENPA), and CENPB [
16,
18]. Moreover, FoxM1 has been described to be involved in a broad range of human malignancies [
19‐
22]. Recently, Zhang et al. found that the overexpression of FOXM1 contributed to the progression of CRC [
23]. Furthermore, another study indicated that FoxM1D promoted epithelial-mesenchymal transition and metastasis by interacting with ROCK2 in CRC [
24]. However, the molecular mechanisms by which FoxM1 promotes CRC cell proliferation have not been fully elucidated.
In our previous gene expression profile analysis (GSE54936 and GSE53464) [
25,
26], FoxM1 was downregulated in human glioma and ovarian cancer cells after treatment with the Hh-Gli signaling pathway inhibitor GANT61 [
27,
28]. Thus, we speculate that Gli1 promotes CRC cell proliferation by regulating FoxM1 expression. To further explore the mechanisms by which Gli1 regulates FoxM1, we constructed ChIP and luciferase reporter assays in this study and identified FoxM1 as a downstream target gene of Gli1 in CRC. Our results provide evidence that Gli1 transcriptionally activates FoxM1 expression by directly binding to the promotor of FoxM1. We also show that Gli1 promotes the proliferation of CRC cells by transactivating FoxM1 and upregulating the expression of FoxM1.
Methods
Cell culture, small molecular reagents and constructs
HEK293T and six CRC cell lines (HT-29, HCT116, LoVo, Caco-2, SW620 and SW480) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HEK293T cells were cultured in basal Dulbecco’s Modified Eagle Medium. The basal medium for the HT-29 and HCT116 cell lines was ATCC-formulated Modified McCoy’s 5α Medium; the basal media for LoVo and Caco-2 cells were ATCC-formulated F-12 K Medium and Eagle’s Minimum Essential Medium, respectively; and Leibovitz’s L-15 Medium was used for SW620 and SW480 cells. Each basal medium was supplemented with 10% fetal bovine serum (Gibco-Life Technologies, Grand Island, NY). The cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The small molecular regents were obtained from the following sources: purmorphamine (Selleck Chemicals, Houston, TX), GANT61 (Sigma-Aldrich, St. Louis, MO), thiostrepton (Sigma-Aldrich, St. Louis, MO), and cyclopamine (Sigma-Aldrich, St. Louis, MO); DMSO was used as the solvent for these regents and the vehicle control.
The human full-length FoxM1 (NM_202002) construct was subcloned into pcDNA3.1-Myc/His (Invitrogen, Carlsbad, CA). The human full-length Gli1 (NM_005269) construct was subcloned into pUB6-V5/hisB (Invitrogen, Carlsbad, CA). The miRNAi-FoxM1 expression vectors that suppress FoxM1 expression and the miRNAi-Gli1 expression vector were generated using the BLOCK-iT™Pol II miR-RNAi Expression Vector System (K4936-00, Invitrogen, Carlsbad, CA) as discribed earlier [
29,
30]. Briefly, based on an analysis of the human FoxM1 and Gli1 sequences using a program provided by Invitrogen, three regions were cloned into the pcDNA™6.2-GW/EmGFP-miR expression vectors to yield miRNAi-FoxM1 or miRNAi-Gli1. They were co-transfected with FoxM1/Gli1 expression construct into HEK293T cells to identify effective clones based on their ability to suppress FoxM1/Gli1 expression by Western blotting analysis. The following oligonucleotide sequences were used to generate the miRNAi constructs: for miRNAi-FoxM1-1692 (targeting nucleotides 1692 to 1712 of FoxM1), 5′ -CTC TTT CTT CTG CAG GAC CAG -3′, and for miRNAi-Gli1-2855 (targeting nucleotides 2855 to 2875 of Gli1, 5′-AGA GTC CCA AGT TTC TGG GGG-3′. The authenticity of all constructs was verified by DNA sequencing.
Western blotting and antibodies
Cells were harvested by trypsinization, lysed in 1× sodium dodecyl sulfate lysis buffer, and denatured for 10 min at 100 °C. After immunoblotting, the membranes were blocked with 5% nonfat dry milk in TBS/0.1% Tween-20 and then incubated with the primary antibodies in 1% nonfat dry milk in TBS/0.1% Tween-20. Subsequently, the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) and visualized with enhanced chemiluminescence (Invitrogen, Carlsbad, CA).
The immunoreagents used for Western blotting were rabbit polyclonal antibody against Gli1 (Abcam, ab92611, diluted 1:500) and rabbit polyclonal anti-FoxM1 (Abcam, ab137647, diluted 1:500). Mouse monoclonal antibody against CCNB1 was purchased from Cell Signaling Technology (CST, 4135, diluted 1:2000), and anti-β-actin antibody (Anbo, E0012, diluted 1:5000) or anti-GAPDH antibody (Millipore, MAB374, diluted 1:2000) was used as a loading control.
Immunohistochemistry
First, 3-μm-thick CRC tissue sections were deparaffinized, rehydrated, and treated with 3% H2O2 to block endogenous peroxidase activity. After the sections were pretreated for antigen retrieval by microwaving them in ethylenediamine tetraacetic acid (EDTA) (pH 9.0) for 25 min, they were rinsed in phosphate-buffered saline (PBS) and incubated with various primary antibodies overnight at 4 °C in a humidified chamber. The next morning, the slides were rinsed with PBS and then incubated for 40 min at 37 °C with the appropriate biotinylated immunoglobulins (Zhongshan Biotechnology, China) before visualizing the immunoreactivity using a Polink-2 HRP DAB Detection kit (Zhongshan Biotechnology, China) following the manufacturer’s protocol. Negative controls were performed in each case by replacing the primary antibody with normal IgG. The following primary antibodies were used: anti-Gli1 (Abcam, ab92611, diluted 1:100) and anti-FoxM1 (Santa Cruz, SC-502, diluted 1:300). An FSX100 microscope equipped with a digital camera system (Olympus, Japan) was used to obtain the immunohistochemistry images.
Real-time PCR
Total RNA was harvested from CRC cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) and evaluated by real-time PCR. Briefly, 1 μg of RNA was reverse-transcribed to cDNA using the PrimeScript
® RT reagent Kit (Takara, Japan). To quantify the mRNA levels, cDNA was amplified by real-time PCR with the SYBR Premix Ex Taq RT-PCR kit (Takara, Japan) on an ABI StepOnePlus™ Real-Time PCR System, and GAPDH was used as the internal control. The sequences of primers used for real-time PCR are shown in Additional file
1: Table S1.
Transient transfections and luciferase assays
The human FoxM1 promoter was amplified from a human genomic DNA template and inserted into the pGL4.20 basic vector (Promega, Madison, WI). A mutant Gli1 binding motif was generated using a PCR mutagenesis kit (ToYoBo, Japan) with the forward primer (mutation sites underlined) 5′- ACA CAC CCA CGC GGC GGG GAC CCC T-3′ and a reverse complement primer. Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. For the luciferase reporter assays, cells were seeded in 24-well plates and transfected with the indicated plasmids. The luciferase activities were measured 48 h after transfection using a Dual Luciferase Reporter Assay System Kit (Promega, Madison, WI).
Chromatin Immunoprecipitation (ChIP) Assay
HT29 cells were cross-linked with 1% formaldehyde, and the reaction was terminated by adding 0.125 M glycine. Chromatin was collected in 1 ml of IP buffer and sheared using a sonicator with a 4-mm tip probe using 10 3-s pulses (80 W, 90-s intervals) in an ice box. Soluble chromatin was immunoprecipitated with 4 μg of anti-Gli1 goat polyclonal antibody (Santa Cruz, sc-6152) or anti-Gli2 goat polyclonal antibody (Santa Cruz, sc-20290), and 4 μg of goat normal IgG (Santa Cruz, sc-2028) was added as a random control. DNA-protein immune complexes were eluted and reverse cross-linked by adding 0.2 M NaCl overnight at 65 °C, and DNA was extracted with phenol/chloroform and precipitated. The FoxM1 promoter domain containing the predicted Gli1 binding motifs was identified in immunoprecipitated DNA by PCR using four pairs of primers, whose sequences are shown in Additional file
2: Table S2.
Cell viability, cell cycle and colony formation assays
Cell viability was measured using a modified MTT (3-(4, 5-dimethylthiazol-z-yl)-2, 5-diphenyltetrazolium bromide) assay. Briefly, 1 × 104 cells were seeded in a 96-well plate, 0.5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO) was added to each well, and the absorbance of the resultant formazan blue crystals was detected at 490 nm using a microplate ELISA reader (Bio-Rad, Hercules, CA). Moreover, a cell cycle analysis was performed by flow cytometry. After trypsinization, the cells were fixed in 70% ethanol overnight at 4 °C and stained with propidium iodide (PI). For the colony formation assay, HCT116 and Caco2 cells were seeded at the same density in 6-well dishes (2 × 103 cells/well). After 20 h, the cells were treated with a different inhibitor or activator or were transiently transfected with myc-FoxM1, miR-FoxM1 or control vector. Transfectants were selected using blasticidin (4 μg/ml) for 2 weeks and stained with crystal violet. The total number of colonies in each well from three independent treatments was counted.
Cell proliferation assay
Cell proliferation was assessed using a BrdU assay. Briefly, CRC cells were seeded in 6-well plates (2 × 106 cells/well) and transfected with the miR-FoxM1, myc-FoxM1 or control vector plasmid for 24 to 48 h. After being labeled with BrdU (Sigma-Aldrich, St. Louis, MO) for 48 h, the cells were fixed and incubated with 0.5% Triton X-100 to permeabilize them. After antigen retrieval, the endogenous peroxidase activity was blocked with 3% H2O2, and the cells were then incubated with anti-BrdU antibody (Abcam, ab6326, diluted 1:40) at 4 °C overnight, followed by incubation with the appropriate biotinylated secondary antibody. Immunoreactivity was visualized using a Polink-2 HRP DAB Detection kit (Zhongshan Biotechnology, China) according the manufacturer’s protocol. Cells from the same population that were not labeled with BrdU were used as a negative cell-staining control. The relative proliferation rates are presented as percentages of the control.
Statistical analysis
Densitometric analyses of protein bands were conducted using the ImageJ software. All data are expressed as the mean ± SD for experiments performed at least three times. Differences between 2 groups were analyzed using a two-sided paired or unpaired Student’s t-test. A p-value less than 0.05 was considered significant.
Discussion
Previous studies have linked the Hh-Gli1 signaling pathway to numerous human cancers, including CRC [
40]. However, the molecular mechanisms underlying the Hh-Gli1 signaling-mediated maintenance of CRC remain largely unclear. Here, we identified Hh-Gli1-FoxM1 as a new signaling axis in the proliferation of CRC and clarified this signaling axis pathway as a potential target for the future development of anti-CRC therapy.
Gli1, a transcriptional factor of the Hh signaling pathway, is upregulated in most digestive tumors, including pancreatic cancer, hepatocellular carcinoma and gastric cancer [
41‐
44]. Similar results were also reported in other studies of CRC [
12,
13,
45]. Although these studies found that the Gli1 mRNA and protein expression levels were significantly increased in CRC tissues, the exact mechanism underlying this increase remained unclear. The present study reports that FoxM1 activity is required for the Gli1-mediated promotion of CRC cell proliferation. Specifically, Gli1 ectopic overexpression using the Hh signaling pathway activator purmorphamine promoted CRC cell proliferation, whereas the simultaneous inhibition of FoxM1 with the FoxM1 inhibitor thiostrepton inhibited CRC cell proliferation. A very recent study indicates that Gli1 promotes CRC metastasis in a FoxM1-dependent manner by activating EMT and PI3K-AKT signaling [
46], which is consistent with our results. However, we demonstrate that Gli1 promotes cell proliferation by directly binding to the promoter of FoxM1 and transactivating FoxM1 in CRC cells. The inhibition of Gli1 also slowed the progression from the G1 to the S phase, as evidenced by a cell cycle assay (Fig.
4e and
f). In addition to promoting the proliferation of CRC cells, Gli1 mediated multiple aspects of cellular processes, including cell survival, invasion and metastasis [
11,
45,
47,
48].
FoxM1 is a member of the forkhead box family of transcription factors and is involved in the control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Increasing evidence has shown that FoxM1 expression is upregulated in many types of tumors [
19,
21,
22,
49]. In this study, we found that FoxM1 expression was also elevated in CRC tumor tissues compared with the matched normal colorectal mucosa. Teh et al. suggested that FoxM1 is a downstream target of Gli1 in basal cell carcinomas [
50], but their study lacked direct evidence. In the present study, we demonstrated that FoxM1 is a direct target of Gli1 using ChIP and luciferase reporter assays. Specifically, we identified one potential Gli1-binding site (GCCCACCCA) in the FoxM1 promoter, and the mutation of this site significantly attenuated the Gli1-mediated transactivation of FoxM1 promoters (Fig.
2b-d). Moreover, we found that the Hh-Gli1 signaling pathway regulated the expression of FoxM1 in CRC cells and that the inhibition of FoxM1 impeded Gli1-mediated CRC cell proliferation. FoxM1 expression was also recently reported to be modulated by many other transcription factors, and Her2 reportedly upregulated FoxM1 expression in gastric cancer [
51]. FoxM1 was also shown to be transactivated by HSF1, which promoted the survival of glioma cells under heat shock stress [
52]. An increasing number of studies have reported that FoxM1 mediates drug resistance in many types of cancers, including gastric cancer [
53], breast cancer [
54,
55] and glioblastoma [
56], by regulating the expression of downstream targets. Together with these findings, our results suggest that the Hh-Gli1-FoxM1 axis can serve as a novel target for cancer therapy. Thus, further screening and validation of drugs that target Hh-Gli1-FoxM1 signaling would be interesting and significant.
Acknowledgments
Not applicable.