Background
Gastric carcinoma (GC), with an estimated number of one million new cases every year, is the fourth most common malignant tumor worldwide and the second most common cause of cancer-related deaths [
1],[
2]. Although extensive research on the molecular mechanisms of GC progression has been carried out, the prognosis of patients with GC is still poor. Therefore, further understanding of the molecular mechanisms of GC progression and the development of new therapeutic targets based on these mechanisms are anticipated.
The Hedgehog (Hh) signaling pathway is crucial for the growth and patterning of various tissues during embryonic development [
3]. Aberrant Hh pathway activation, driven by either mutation or ligand overexpression, is often associated with human cancers [
4],[
5]. This pathway is a highly coordinated network of Hh (Sonic Hh, Indian Hh and Desert Hh) proteins, seven-transmembrane protein Smoothened (Smo), 12-transmembrane receptors (Patched1 [Ptch1] and Patched2 [Ptch2]), and transcription factor Gli family (Gli1, Gli2, Gli3) [
6]-[
8]. Hh exerts its biological function through a signaling cascade that culminates in an alteration of the balance between activator and repressor forms of the Gli transcription factors. The vertebrate Gli repressor function largely depends on Gli3, while the primary Gli activator function largely depends on Gli2. Gli1 acts as a transcriptional activator and thus a reliable indicator of Hh pathway activity [
9],[
10]. In Drosophila, the Hh signal is mediated by the Cubitus Interruptus (Ci) transcription factor. Yet the Ci function is positively regulated by Fused (Fu) serine/threonine kinase, but suppressed by Suppressor of Fused (SuFu) and the kinesin-like molecule Costal2 (Cos2) [
11]. SuFu is antagonized by HIB (Hh-induced MATH and BTB domain protein), of which speckle-type POZ protein, SPOP, is the vertebrate homolog [
12].
SPOP is demonstrated as an E3 ubiquitin ligase adaptor. It has 374 residues and contains three domains: an N-terminal MATH domain (residues 28–166) that recruits substrates, an internal BTB domain (residues 190–297) that binds Cul3, and a C-terminal nuclear localization sequence (residues 365–374) [
13]. SPOP is mostly studied in ubiquitin-dependent proteolysis, however, its role in tumorigenesis is largely unknown [
13],[
14]. Li C., et al. demonstrated that SPOP is responsible for full length Gli2 and Gli3 ubiquitination and proteolysis [
15]. Recently, Geng C., et al. revealed that wild type SPOP functions as a critical tumor suppressor in prostate cancer cells by promoting the turnover of SRC-3 (p160 steroid receptor coactivator-3) protein and, thus suppressing androgen receptor transcription activity [
16]. Wang C., et al. showed that SPOP promotes full-length Gli2 and Gli3 degradation, which may indicate a potential tumor suppressor role of SPOP [
17]. The limited studies indicate the importance of SPOP in tumorigenesis, which prompts us to find out more evidence of it.
In this study, we evaluated SPOP expression in gastric cancer tissues and adjacent gastric tissues. We then exhibited possible correlations between SPOP expression and the clinical pathologic factors. Based on the results of clinical findings, we performed in vitro experiments and studied the effects of up-regulated or down-regulated SPOP expression on the proliferation, migration and apoptosis of GC cell lines. Our results indicate that SPOP reduces gastric cancer cell invasion and proliferation by regulating Hh/Gli2 signaling pathway.
Methods
Constructs, antibodies and reagents
Human SPOP cDNA was amplified by PCR and then inserted into KpnI and NotI sites of Pub6/V5-HisB vector. SPOP - miRNA (miR-SPOP) expressed vectors were generated using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit (K4936-00, Invitrogen, Carlsbad, CA). Clones were confirmed by DNA sequencing of both strands (Generay, Shanghai, China). The oligonucleotide sequences for miRNA constructs were as follows: for miR-SPOP-607, 5′-AAT GAC TTC ACC CAT TTC CTC-3′; for miR-SPOP-917, 5′-TAA GCT TGT CAT CAG GGA GAA-3′; and for miR-SPOP-1430, 5′-AGT TGA TGA AAT CCA CTG CCT-3′. The numbers in the miRNA constructs indicate the start of targeted nucleotide sequence of the gene. All transfectants were selected with 7 μg/ml Blasticidin for 3 weeks and selected clones were identified by Western blotting. Antibodies were purchased from Abcam (Gli2, ab26056; p16, ab108349; PTEN, ab31392), Cell Signaling (Gli1, L42B10; Caspase-3, 9665S; cleaved Caspase-3, 9664; Ki-67, 9449; Cyclin B1, 4135S; p21, 2947; p27, 3686; Daxx, 4533; p-ERK, 4376S), Novus (SPOP, H00008405-B01P), Protein-Tech (SPOP, 16750-1-AP), Santa Cruz (PCNA, sc-9857; PARP, sc-7150; Actin, sc-1616), Abgent (DUSP7, AP8450a), Millipore (GAPDH, MAB374), Thermo (goat anti-rabbit IgG, 31460). Enhanced chemiluminescence (ECL) Western blot detection reagents were from Thermo Fisher Scientific (Rockford, IL). MG-132 (Z-Leu-Leu-Leu-CHO, I-130) was from BostonBiochem. Protease inhibitor cocktail, Lubrol-PX, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO).
Cell culture and tissue specimens
Human gastric epithelial cell line GES-1, human gastric cancer cell lines AGS, MKN28, SGC-7901, MKN45 and human embryonic kidney 293 T (HEK293T) cell lines were obtained from American Type Culture Collection (ATCC, Manassas). Cells were maintained in DMEM (Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, CA) and 100 μg/ml streptomycin in humidified incubator at 37°C with 5% CO2.
Gastric cancer samples were obtained from 88 patients diagnosed gastric carcinoma from March 2007 to April 2010 in the Department of Pathology, the First Affiliated Hospital of Nanchang University. All the patients had not received chemotherapy or radiotherapy before surgery and diagnosed by clinic pathologic staging based on the 2010 American Joint Committee on Cancer (AJCC) criteria. Detailed clinic pathologic information of all patients and their correlation with SPOP expression were summarized in Table
1. This study was approved by the institutional review board of the First Affiliated Hospital of Nanchang University. All patients had given written informed consent.
Table 1
Correlation between SPOP expression and clinic pathological information
Gender | Male | 54 | 10 | | |
Female | 22 | 2 | −0.095 | 0.380 |
Age | ≤50 | 18 | 1 | | |
>50 | 62 | 7 | 0.070 | 0.518 |
Lymp node | Metastasis | 54 | 4 | | |
No metastasis | 23 | 7 | 0.236 | 0.027* |
Histopathologic grading | Poorly differentiated | 34 | 0 | | |
Moderately differentiated | 36 | 10 | | |
Well differentiated | 8 | 0 | −0.232 | 0.029* |
TNM staging | I | 13 | 1 | | |
II | 33 | 7 | | |
III + IV | 30 | 4 | −0.375 | 0.0003* |
Immunohistochemical staining
Paraffin sections (5 μm) of formalin-fixed gastric cancer tissue and adjacent tissue arrays were de-waxed, rehydrated and incubated in 3% H
2O
2 for 10 min to block endogenous peroxidase. Then the sections were autoclaved in 10 mM sodium citrate buffer (pH 6.0) for 15 min. Normal goat serum (10%) was used to block non-specific staining and then the tissue sections were exposed to indicated antibodies. All of the stainings were observed by at least 2 independent investigators blinded to the histopathologic features and patient data of the samples [
18]. The widely accepted German semi-quantitative scoring system was used for assessing the staining intensity and area extent [
19]. Each specimen was assigned a score according to the intensity of the nucleic, cytoplasmic, and/or membrane staining (no staining, not detected = 0; weak staining, light yellow = 1; moderate staining, yellowish brown = 2; strong staining, brown =3) and the extent of stained cells (no staining = 0, 1-24% = 1, 25-49% = 2, 50-74% = 3, 75-100% = 4). The final immunoreactive score was determined by multiplying the intensity score with the extent score of stained cells, ranging from 0 (the minimum) to 12 (the maximum).
Western blotting
Cells were solubilized in the extraction buffer (0.5% Lubrol-PX, 50 mM KCl, 2 mM CaCl2, 20% glycerol, 50 mM Tris–HCl, and inhibitors of proteases and phosphatases, pH 7.4) at 4°C for 30 min and centrifuged (12,000 rpm, 15 min at 4°C) to maintain the supernatant. Protein concentrations were determined by Bradford assay (Bio-Rad, CA). Proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad), and then probed with the specified primary antibody followed by the appropriate secondary antibody. The immunostaining was visualized by using Kodak X-ray films, and quantified by scanning films containing nonsaturated signals with an Epson 1680 scanner before analyzed with ImageJ software [
20].
Immunoprecipitation
Supernatant of the cell lysates was incubated with indicated antibodies and protein-G agarose beads at 4°C overnight. The beads were washed with the buffer (0.1% Lubrol-PX, 50 mM KCl, 2 mM CaCl2, 20% glycerol, 50 mM Tris–HCl, and inhibitors of proteases and phosphatases, pH 7.4), then subjected to SDS-PAGE and subsequent immunoblotting with antibodies.
Cell viability analysis
Cell viability assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, Sigma) reduction assay [
21]. Briefly, cells were seeded at a density of 300 cells per well in a 96-well plate and cultured for indicated days. Culture medium was replaced with fresh media every day. MTT (20 μl) was added for 4 h to allow formation of the colored formazan product. Then, the supernatant was replaced with 150 μl dimethyl sulfoxide (DMSO). Plates were incubated for a further 2 h and then optical density was measured using a microplate ELISA reader (Bio-Rad) at 490 nm. The relative survival rates were presented as the percentage of control. Triplicate wells were used for each cell line. Each experiment was repeated three times.
Scratching assay
Cell migration was measured using a scratch assay [
22]. AGS cells were plated in 6-well plates to create a confluent monolayer. At a confluence of 90-100%, a 200 μl pipette tip was used to make a “scratch” in the cell monolayer. Then the cells were transfected with SPOP plasmid or empty plasmid as control for 24 h. After removing debris and adding fresh media containing 2% FBS, cells were photographed using phase contrast microscopy (Olympus, IX71). The migration distance was assessed using ImageJ software. A relative migration rate was calculated by cell relative migration rate for each treatment.
One or two hundred cells were plated into 6 cm dishes and incubated in DMEM with 10% FBS at 37°C. Two weeks later, the cells were fixed and stained with 0.1% crystal violet. The number of colonies, defined as >50 cells/colony, were counted. The experiments were performed in triplicate.
Real-time PCR
Total RNA (1 μg) was employed to prepare cDNA via reverse transcription using cDNA synthesis kit (Invitrogen) according to manufacturer’s instructions and analyzed using an Applied Biosystems 7500 PCR Detection System (Applied Biosystems Inc.). Target gene mRNA expressions were normalized to the expression of GAPDH and quantified using the comparative Ct method. The primers used for real-time PCR are as follows: SPOP (forward, 5′-CAA GGC AAA GAC TGG G-3′; Reverse, 5′-AAC ACT CAC CTC GCA GA-3′); Gli1 (forward, 5′-TCC TAC CAG AGT CCC AAG TT-3′; reverse, 5′- CCC TAT GTG AAG CCC TAT TT-3′); Gli2 (forward, 5′-CCT GGC ATG ACT ACC ACT ATG AG-3′; reverse, 5′-GGC TTG GCT GGC ATG TTG-3′); β-actin (forward, 5′-CGG GAA ATC GTG CGT GAC-3′; reverse, 5′-ATG CCC AGG AAG GAA GGC T-3′).
Dual luciferase reporter assay
We inserted Gli binding sites (8 × GBS) into plasmid PGL4.2 in HEK293T cells and screened for stable cell lines [
23]. Myc-SPOP or empty vector were co-transfected into the cells for 48 h. Luciferase activity was assayed using dual luciferase reporter assay system with procedure described by the manufacturer (Promega, Madison, WI). Change of luciferase activity in treated cells was expressed as percentage of the controls.
Immunofluorescence analysis
MKN45 cells were transfected with Myc-SPOP, miR-SPOP or empty vector as control. Approximately 48 h after transfection, cells were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS), permeated with 0.1% Triton/PBS, blocked in 3% BSA/PBS for 30 min, and then incubated with indicated antibodies (1: 200) in 2% BSA/PBS at 4°C overnight. Double-labelled immunostaining was done with appropriate fluorochrome-conjugated secondary antibodies. Protein localization was visualized using a Zeiss-700 (Jena, Germany) confocal microscope.
Statistical analysis
Values were indicated as mean ± SD. The differences between the groups were evaluated by Student’s t test or one-way analysis of variance (ANOVA). For the relationship between SPOP expression and clinical pathologic factors, the Chi-square (χ2) test, Fisher’s exact test and Spearman’s correlation were used. All analyses were carried out by the use of SPSS17.0 software (SPSS Inc., Chicago, IL). Differences were considered significant if P < 0.05.
Discussion
SPOP is an E3 ubiquitin ligase adaptor and its role in tumorigenesis is largely unknown. In this study, we described the relationship between SPOP expression and clinical pathology parameters of 88 GC cases by immunohistochemical analysis. Interestingly, immunostaining of SPOP is differentially detected in GC tissues and adjacent gastric tissues. Dramatically decreased SPOP expression in GC tissues indicates a role of it in gastric cancer processing. Further correlation analysis revealed that SPOP expression was negatively related to lymph node metastasis, poor histopathologic differentiation and advanced TNM stages. In vitro experiments provide evidence that SPOP functions as a tumor suppressor in different gastric cancer cell lines and contributes to post-transcriptional modification of Gli2. Intracellular studies found that SPOP inhibited Gli2 expression inside the cytoplasm. In addition, SPOP promotes tumor cell apoptosis through regulating the expression of Hh/Gli related apoptotic proteins. Together, our study elucidates the tumor protective function of SPOP through inhibiting Hh/Gli2 pathway, and the possible molecular mechanism of Gli2 stability regulated by SPOP.
Aberrant activation of Hh pathway has been reported in the proliferation of a variety of human cancers, such as BCCs, gastric cancers, prostate cancers, breast cancers, pancreatic cancers [
27]-[
34]. As the end effectors of canonical Hh signaling pathway, Gli family play roles of transcription factors, and the abnormal regulation of Gli proteins leads to tumorigenesis [
35],[
36]. Their activity is tightly regulated through various posttranslational modifications, such as ubiquitin-mediated degradation, proteolytic processing, and nuclear-cytoplasmic shuttling. In the present study, Gli mRNA and protein were differentially affected by SPOP in human gastric epidermal cell lines, suggesting a post-transcription modification of Gli by SPOP. As an E3 ubiquitin ligase adaptor, SPOP is supposed to process in degradation of Gli depending on ubiquitin/proteasome system. In fact, a recent study confirmed our speculation [
17]. Wang C., et al. found that treatment with proteasome inhibitor MG-132 inhibited SPOP-mediated full-length Gli2 and Gli3 degradation. Co-expression of SPOP and Myc-tagged ubiquitin with either Gli2 or Gli3 resulted in increased levels of ubiquitinated forms of both Gli2 and Gli3 proteins, although the extent of ubiquitination between the two proteins was different. In our study, we demonstrated that increasing exogenous SPOP expression led to decreased endogenous Gli2 both in cytoplasm and nucleus, which provided another direct evidence of Gli2 turnover by SPOP. The biological function of Gli2 is suggested to translocate to the nucleus, where it acts as a transcriptional activator [
37]. Actually, we did find endogenous Gli2 expression within the nucleus by immunofluorescent staining, and diminished markedly when SPOP overexpressed. In other words, SPOP interferes Gli2 nucleus/cytoplasm shuttling thus further blocks Gli2 function as a transcription activator in tumorigenesis.
Our proposed degradation of Gli2 by SPOP is analogous to that of Ci by the HIB/Cul3 complex in
Drosophila[
38]. Like HIB, exogenous mammalian SPOP may recruit Cul3 from the cytoplasm along with degradation substrates, likely including Gli2. The molecular basis of Gli2 degradation by SPOP is affected by another inhibitory regulator for Gli proteins-SuFu, which is localized to both the cytoplasm and the nucleus. SuFu sequesters Gli proteins in the cytoplasms, and in the nucleus SuFu plays as a co-repressor of Gli proteins [
39]. SuFu and SPOP competitively interact with Gli2 and Gli3 proteins, and SPOP is likely to exhibit a lower binding affinity than SuFu to Gli2 and Gli3 [
17]. This might ensure the prompt activation and deactivation of Gli2 and Gli3 proteins in response to Hh signaling.
Limited studies suggest that SPOP also behaves in apoptosis. A study revealed that SPOP BTB protein serves as an adaptor of Daxx, which is a pro-apoptotic protein under various stress condition [
12]. Likewise, our data proved that SPOP knockdown by miR-SPOP transfection resulted in reduced expression of Caspase-3, cleaved Caspase-3, p16, p27, and p21 which are cell cycle inhibitors. Furthermore, we found that repressed SPOP promotes early mitosis through enhancing the expression of PCNA and Cyclin B1 respectively. These may indicate a function of SPOP besides E3 ligase adaptor.
Noted that in the control groups of our cultured AGS cell line and MKN45 cell line (Figure
2D,F and Figure
3C,E), under the same incubatory condition, the baseline cell ability of migration and proliferation were different from each other. Lower expression of SPOP may contribute to a more severe malignancy of AGS cells than MKN45 cells.
A recent published study of clear cell renal cell cancer (ccRCC) raises another question that SPOP acts as multiple regulators of cellular proliferation and apoptosis, including not only Gli2 but also tumor suppressor - PTEN, ERK phosphatases and pro-apoptotic molecule Daxx [
39]. Thus the total effect of SPOP on clear cell renal cell carcinoma is promoting tumorigenesis. However, in our gastric cancer cell line MKN45, different from ccRCC study, tumor suppressor PTEN was reduced and p-ERK was activated when SPOP was repressed (Figure
5B). These discrepancies indicate multiple roles of SPOP in tumors from different sources of tissues, and the molecular mechanisms are under investigation.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CZ and YW carried out the experiments and drafted the manuscript; QL was involved in the statistical analysis; JC contributed to the immunohistochemical staining; JZ performed the immunofluorescent staining, apoptosis related experiments; NL and TL reviewed the manuscript critically; SL managed the experimental design, reviewed the manuscript and gave funding support. All authors had read and approved the final manuscript.