Background
Gastrointestinal stromal tumour (GIST) is the most common soft tissue sarcoma and often localizes to the gastrointestinal tract [
1,
2]. Currently, the majority of studies indicate that GISTs originate from the mesenchymal pacemaker cells of the gastrointestinal tract known as the interstitial cells of Cajal (ICCs) that harbour multi-oncogenic mutations, such as KIT and PDGFRA [
3,
4]. Increasing evidence has demonstrated that those oncogenes play a critical role in GIST tumourigenesis, proliferation, and metastasis. Given the important role of oncogenes in GIST progression, molecular targeted drugs (imatinib) have been employed to cure GISTs harbouring mutant KIT or PDGFRA [
5]. Although targeted drugs have revolutionized the treatment of GIST, a significant number of GIST patients experience recurrence within two years due to resistance [
6,
7]. In addition, there is no promising treatment for wild-type KIT/PDGFRA GISTs [
8]. Thus, to develop novel therapeutic strategies, further understanding of the molecular mechanisms of GISTs is crucial.
Recently, numerous studies have implied that epigenetic alterations play critical roles in a wide range of tumours [
9,
10]. Previous studies have also demonstrated that epigenetic alterations are responsible for GIST development [
11]. Both DNA hypomethylation and DNA hypermethylation are reported to be closely related to GIST progression. Igarashi S. reported that LINE-1 methylation was associated with malignant GIST profiles and poor prognosis. In addition, more genes are methylated in advanced GIST compared with benign GIST [
12]. More important, DNA methylation is associated with aggressive clinical characteristics, strongly indicating that DNA methylation is involved in GIST progression and may act as a novel treatment approach for GIST patients [
13]. In addition to DNA methylation, histone methylation is another major epigenetic modification that is a reversible process. Previous studies have implied that changes in histone methylation could lead to gene activation or repression and effect tumour progression [
14,
15]. In GIST, histone H2AX is a direct mediator of gastrointestinal stromal tumour cell apoptosis upon treatment with imatinib mesylate [
16]. Histones can be modified by methylation and demethylation. Numerous demethylases are involved in diverse tumour development [
17]. For example, KDM4 family members demethylate different sites of histones to activate or suppress gene expression [
18‐
20]. However, the potential role of demethylases in GIST remains largely unknown. Importantly, the molecular mechanisms by which demethylases regulate GIST progression remain unclear.
Herein, we demonstrate that KDM4D mRNA and protein levels are upregulated in GIST compared with the matched normal tissues. Further, KDM4D overexpression strongly promotes GIST cell proliferation, migration, invasion and tumour angiogenesis. In contrast, silencing KDM4D reduced cell proliferation, migration, invasion and tumour angiogenesis. More importantly, these biologic effects mediated by KDM4D might be dependent on the Hif1β/VEGFA signalling pathway. These findings highlight novel evidence for a functional link between demethylase and GIST development. We believe that the KDM4D/Hif1β/VEGFA signalling pathway may act as a potential therapeutic target for GIST patients.
Methods
Cell lines and regents
GIST882 cells, a kind gift of Jonathan Fletcher (Dana-Farber Cancer Institute, Boston, MA), were derived from a GIST patient with a homozygous missense mutation in KIT exon 13 (K642E). GIST-T1 cells obtained from Biowit Technologies (Shenzhen, China) were derived from a GIST patient with an in-frame deletion of 57 nucleotides in KIT exon 11 (V560Y579del). Both cell lines used in this study were cultured at 37 °C with 5% CO2 in DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. Antibodies to KDM4D (ab93694) and VEGFA (ab1316) were purchased from Abcam (Cambridge, MA, USA). HIF1a (#36169), H3K36me3 (#4909) and H3K9me3 (#13969) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies to GAPDH (sc-47,724), HIF1β (sc-17,811), Histone3 (sc-517,576) and mouse IgG (sc-69,786) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The human VEGFA ELISA Kit (EK0539) was obtained from Boster Biological Technology (Wuhan, China).
Immunohistochemistry
Human GISTs samples were obtained from Tongji Hospital. For immunohistochemistry, formalin-fixed paraffin-embedded tissues were cut into 4-mm sections. Then, slides were chosen to stain with antibodies against KDM4D, VEGFA and CD31, separately. Two experienced pathologists examined immunostaining results. The intensity stained scores were evaluated according to the intensity of the nucleic or cytoplasmic staining (no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3). Finally, the IHC score was generated by combining the intensity scores with the percentage of positively stained cells.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol Reagent (Invitrogen) and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR was performed using the ABI 7300 real-time PCR system (Applied Biosystems) with KDM4D and GAPDH specific primers. The fold-change in KDM4D expression was calculated using the 2-△△CT method, and GAPDH mRNA levels served as the control. The sense primer for KDM4D was 5’-GGGCAGGGGTGTTTACTCAAT-3′, the antisense primer for KDM4D was 5’-TGTTTGCCAAATGGCGATACT-3′. The sense primer for GAPDH was 5’-ACCACAGTCCATGCCATCAC-3′, the antisense primer for GAPDH was 5’-TCCACCACCCTGTTGCTG TA-3′.
Western blot
Total cell protein was isolated using NP40 lysates with protease and phosphatase inhibitors. Protein samples were separated by SDS-PAGE and then transferred to PVDF membranes. Blots were incubated with primary antibodies overnight at 4 °C. Then, blots were incubated with the corresponding HRP-conjugated secondary antibodies. Immunoreactive bands were examined with ECL regents (Thermo Scientific).
Cell viability assay
Cells were seeded into 96-well plates. Overnight, the medium was exchanged with 100 μl of medium supplemented with 10 μl CCK8. The plates were incubated for 2 h. Afterwards, absorbance at 450 nm was measured to obtain OD value. Similarly, other time points (24, 48, 72 and 96 h) were also assessed according to the procedures.
Briefly, 1% noble agar was added to the bottom of 6-well plates. After the layer of agar solidified, 500 cells were seeded into the 6-well plates. The time required for adequate colony formation varies for each cell line and is typically approximately two weeks. The medium was replaced every three days. After approximately two weeks, photographs of colonies were obtained using a microscope.
ChIP assay
Chromatin immunoprecipitation assays were performed as described previously [
20]. The primers used for CHIP assays are provided below: HIF1β upstream sense, 5’-CGCTCTTGTTGCCCAGACTGG-3′; HIF1β downstream sense, 5’-TCTGTAATCCCAGCACTTTGGG-3′.
Luciferase reporter assay
Cells were co-transfected with 0.2 μg of HIF1β promoter-luciferase reporter plasmid (PGL3-HIF1β promoter) and 10 ng Renilla luciferase control vector. Firefly luciferase activity was detected using dual-luciferase Reporter Assays (Promega) 48 h after transfection and normalized to Renilla luciferase activity. PGL3-HIF1β promoter was generated using the following primers. The sense primer for the HIF1β promoter was 5’-CTTTGTGATCCGCCCTCCTTG-3′, and the anti-sense primer for the HIF1β promoter was 5’-AGTAGGCGGAGTCAACACAC-3′.
BrdU/PI assay
BrdU/PI assays were performed as described previously [
21]. Briefly, cells were cultured in a 6-well plate overnight followed by a 20-min incubation with 10 μg/ml BrdU. Then, cells were washed twice with PBS and fixed with 70% ethanol at − 20 °C overnight.
Next, cells were denatured in 2 N HCL for 45 min and stained with FITC secondary antibody for 1 h at room temperature. Cells were further incubated with 40 g/ml RNase A and 200 g/ml PI for 30 min and finally analysed by flow cytometry.
GIST cells were treated as indicated overnight in DMEM supplemented without serum. Then, 20,000 HUVECs cells per well incubated with GIST cell-conditioned media were seeded into the Matrigel in 96-well plates. Microscopic images of tube formation were obtained, and the number of branches in the formed HUVEC tubes was assessed.
Enzyme-linked immunosorbent assay (ELISA)
GISTs cells were treated as indicated overnight in DMEM supplemented without serum. Overnight, cell-conditioned medium was collected for VEGFA-ELISA analysis according to the manufacturer’s instructions. The OD values were obtained based on absorbance at 470 nm.
Wound healing and Transwell assays
For the wound-healing assay, GISTs cells were seeded on six-well plates. When 95% confluence was achieved, the cell monolayer was gently scratched using a 200-μm sterile plastic pipette tip. Then, the wound was photographed. After 24 h, the healing wound was photographed. For Transwell migration or invasion assays, 4 × 104 cells suspended in medium without serum were seeded in the upper chamber membranes coated without/with Matrigel (BD Biosciences). Then, 600 μl medium with 10% FBS was added to the lower chamber. After 24 h, the underside of the membrane was fixed for 30 min and stained with 0.1% crystal violet. The inner side of the membrane was wiped with a cotton swab. Then, cells were quantified under a microscope.
Mouse xenograft tumour assay
All mice experiments were approved by the Animal Care and Use Committee of Tongji Hospital. Four-week Balb/c nude mice were obtained from Beijing Huafukang Bioscience, and 12 mice were randomly divided into 2 groups. We resuspended GIST882 cells stably expressing vector or KDM4D in PBS, mixed the cells with Matrigel in a 1:1 ratio and injected the suspension into the subcutaneous right groin tissue of Balb/c nude mice. After these mice developed a visible tumour mass, tumour volumes were calculated as (length×width×width)/2. Thirty days after subcutaneous injection, mice were sacrificed. Of note, for the mouse xenograft tumor model from ShNC cells and ShKDM4D cells, mice were sacrificed after subcutaneous injection 25d.
Statistical analysis
Statistical analysis was performed using Prism 5.0 (GraphPad Software). Statistical differences between two groups of data were determined using the unpaired two-tailed Student t-test. Data were considered significant when p < 0.05.
Discussion
Most GISTs harbouring mutant KIT or PDGFRA are treated with targeted therapies, such as imatinib. Although limited success is noted at the beginning of treatment, the majority of patients ultimately develop drug resistance [
24]. More importantly, wild type GISTs do not respond to molecular targeted therapy [
25‐
29]. Thus, further understanding of the molecular mechanism regulating GIST progression is urgent. Novel treatment strategies should be identified to treat patients with GIST, especially those with unresectable and advanced GIST.
In this study, we found that KDM4D is overexpressed in GISTs compared with corresponding normal tissues. This finding suggests that epigenetic post-translational modifications may be involved in GIST progression. Consistent with our findings, previous data also demonstrated that epigenetic alterations play important roles in GIST development and even have predictive value in GISTs. For example, CD133 methylation percentage is inversely correlated with CD133 protein expression, and hypermethylation in small (< 2 cm) GIST reflects tumour size. Niimura et al. demonstrated that multiple genes in the HOXC cluster are marked by active histone H3K4me3 in malignant GISTs and revealed that HOTAIR might be a potentially useful biomarker in malignant GIST [
30]. These data suggested that histone-modifying enzymes could act as novel therapeutic targets in GISTs. KDM4D expression in GISTs has not been previously assessed. Herein, we first evaluated the roles of KDM4D in GIST cell proliferation, migration, and invasion. Surprisingly, KDM4D overexpression strongly increased GIST cell proliferation, migration, and invasion. In contrast, KDM4D depletion significantly attenuated GIST cell proliferation, migration, and invasion, indicating that KDM4D regulated GIST progression. Targeting KDM4D might offer an effective method to control the development of GISTs.
Angiogenesis is a complicated process that is crucial for cancer proliferation and metastasis [
31]. Evidence indicates that the Hif/VEGFA signalling pathway is a potent driver of angiogenesis [
32]. In the present study, we evaluated the role of KDM4D in tumour angiogenesis. We found that KDM4D overexpression strongly accelerates HUVEC tube formation both in vitro and in vivo. In addition, increased VEGFA secretion was observed in KDM4D cells compared with control vector cells. Consistent with the overexpression experiments, we further found that silencing KDM4D significantly abolished tube formation and decreased VEGFA secretion, indicating that KDM4D regulates GIST development in an angiogenesis-dependent manner. The Hif family plays a critical role in angiogenesis, especially HIF1a, which is mainly regulated by hypoxia. Other Hif members also cooperate with HIF1a to play an important role in tumour progression, including tumour angiogenesis. HIF1β also regulates angiogenesis in a HIF1a-independent manner [
33]. To explain the underlying molecular mechanisms of KDM4D-induced angiogenesis, we examined HIF1a and HIF1β protein expression. Interestingly, KDM4D markedly upregulated HIF1β expression but not HIF1a expression.
KDM4D regulates gene expression by regulating the methylation levels of both H3K36me3 and H3K9me3 [
34,
35]. Consistent with previous reports, KDM4D overexpression significantly increased H3K36me3 and H3K9me3 methylation levels compared with vector cells. Importantly, CHIP assays revealed that KDM4D interacts with the HIF1β promoter. In addition, changes in KDM4D strongly affect the interaction between the HIF1β promoter and both H3K36me3 and H3K9me3. These findings indicated that KDM4D induces tumour angiogenesis via HIF1β expression. In addition, compared with the control vector group in Balb/c nude mice, the growth of KDM4D-overexpressing cells was increased along with increased VEGFA expression as assessed by immunohistochemistry. These results indicate the pro-tumour effect of KDM4D via tumour angiogenesis.
In summary, our study presents the following lines of evidence supporting the notion that KDM4D plays a vital role in GIST progression. First, KDM4D expression is upregulated in GIST samples. Second, we demonstrated that KDM4D plays a central role in GIST proliferation, migration and invasion both in vitro and in vivo. Third, we demonstrated that KDM4D is required for tumour angiogenesis via regulating VEGFA secretion. Finally, we further revealed that KDM4D directly binds to and activates the HIF1β gene promoter.