SPOP suppresses tumorigenesis by regulating Hedgehog/Gli2 signaling pathway in gastric cancer
© Zeng et al.; licensee BioMed Central Ltd. 2014
Received: 28 April 2014
Accepted: 2 September 2014
Published: 11 September 2014
Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown.
In this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells.
Immunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells.
Our findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing therapeutic agents of gastric cancer in future.
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 ,. 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 . Aberrant Hh pathway activation, driven by either mutation or ligand overexpression, is often associated with human cancers ,. 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) -. 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 ,. 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) . SuFu is antagonized by HIB (Hh-induced MATH and BTB domain protein), of which speckle-type POZ protein, SPOP, is the vertebrate homolog .
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) . SPOP is mostly studied in ubiquitin-dependent proteolysis, however, its role in tumorigenesis is largely unknown ,. Li C., et al. demonstrated that SPOP is responsible for full length Gli2 and Gli3 ubiquitination and proteolysis . 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 . Wang C., et al. showed that SPOP promotes full-length Gli2 and Gli3 degradation, which may indicate a potential tumor suppressor role of SPOP . 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.
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.
Correlation between SPOP expression and clinic pathological information
SPOP expression scoring
III + IV
Paraffin sections (5 μm) of formalin-fixed gastric cancer tissue and adjacent tissue arrays were de-waxed, rehydrated and incubated in 3% H2O2 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 . The widely accepted German semi-quantitative scoring system was used for assessing the staining intensity and area extent . 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).
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 .
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 . 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.
Cell migration was measured using a scratch assay . 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.
Colony formation assay
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.
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 . 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.
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.
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.
Clinical significance of SPOP expression in gastric cancer
We further analyzed the correlation between SPOP expression and clinical pathologic characteristics of patients. Since SPOP scoring ranged from 0 to 12, we defined the score less than 6 as low expression, and equal or more than 6 as high expression. As shown in Table 1, it is notable that SPOP was negatively correlated with lymph node metastasis, poor histopathologic differentiation and advanced TNM stages, regardless of age and gender. Taken together, these results indicate that SPOP is a potential oncogenic suppressor.
SPOP inhibits gastric cancer cell proliferation and migration
Repression of SPOP promotes proliferation and migration of MKN45 cells
SPOP inhibits Hh/Gli2 signaling activity
SPOP promotes apoptosis of gastric cancer cells
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 -. 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 ,. 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 . 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 . 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. 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 . 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 . 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 . 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 . 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.
We report herein that SPOP negatively regulates Hh/Gli2 signaling pathway mediated transcription through interfering Gli2 abundance in gastric cell lines, thus results in decreased tumor cell proliferation, invasion, migration and enhanced cell apoptosis. The identification of SPOP as a negative regulator of Gli2-mediated transcription may provide an alternative strategy for developing therapeutic agents for gastric cancer in future.
This work was supported in part by grants from the China National Basic Research Program (2010CB535001), the National Natural Science Foundation of China (81060095 and 31171359), the Natural Science Foundation of Jiangxi Province (20114BAB205035) and the National Science and Technology Major Projects program for “Major New Drugs Innovation and Development” of China (2011ZX09302-007-03).
- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin. 2011, 61: 69-90. 10.3322/caac.20107.View ArticlePubMedGoogle Scholar
- Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer statistics, 2009. CA Cancer J Clin. 2009, 59: 225-249. 10.3322/caac.20006.View ArticlePubMedGoogle Scholar
- Cohen MM: The Hedgehog signaling network. Am J Med Genet A. 2003, 123A: 5-28. 10.1002/ajmg.a.20495.View ArticlePubMedGoogle Scholar
- Evangelista M, Tian H, de Sauvage FJ: The Hedgehog signaling pathway in cancer. Clin Cancer Res. 2006, 12: 5924-5928. 10.1158/1078-0432.CCR-06-1736.View ArticlePubMedGoogle Scholar
- Long B, Zhu H, Zhu C, Liu T, Meng W: Activation of the Hedgehog pathway in chronic myelogeneous leukemia patients. J Exp Clin Cancer Res. 2011, 30: 8-10.1186/1756-9966-30-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Ingham PW, McMahon AP: Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001, 15: 3059-3087. 10.1101/gad.938601.View ArticlePubMedGoogle Scholar
- Kalderon D: Similarities between the Hedgehog and Wnt signaling pathways. Trends Cell Biol. 2002, 12: 523-531. 10.1016/S0962-8924(02)02388-7.View ArticlePubMedGoogle Scholar
- Ruel L, Rodriguez R, Gallet A, Lavenant-Staccini L, Therond PP: Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog. Nat Cell Biol. 2003, 5: 907-913. 10.1038/ncb1052.View ArticlePubMedGoogle Scholar
- Jiang J, Hui CC: Hedgehog signaling in development and cancer. Dev Cell. 2008, 15: 801-812. 10.1016/j.devcel.2008.11.010.View ArticlePubMedGoogle Scholar
- Merchant JL: Hedgehog signalling in gut development, physiology and cancer. J Physiol. 2012, 590: 421-432.PubMed CentralView ArticlePubMedGoogle Scholar
- Hooper JE, Scott MP: Communicating with Hedgehogs. Nat Rev Mol Cell Biol. 2005, 6: 306-317. 10.1038/nrm1622.View ArticlePubMedGoogle Scholar
- Kwon JE, La M, Oh KH, Oh YM, Kim GR, Seol JH, Baek SH, Chiba T, Tanaka K, Bang OS, Joe CO, Chung CH: BTB domain-containing speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase. J Biol Chem. 2006, 281: 12664-12672. 10.1074/jbc.M600204200.View ArticlePubMedGoogle Scholar
- Zhuang M, Calabrese MF, Liu J, Waddell MB, Nourse A, Hammel M, Miller DJ, Walden H, Duda DM, Seyedin SN, Hoggard T, Harper JW, White KP, Schulman BA: Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. Mol Cell. 2009, 36: 39-50. 10.1016/j.molcel.2009.09.022.PubMed CentralView ArticlePubMedGoogle Scholar
- Bunce MW, Boronenkov IV, Anderson RA: Coordinated activation of the nuclear ubiquitin ligase Cul3-SPOP by the generation of phosphatidylinositol 5-phosphate. J Biol Chem. 2008, 283: 8678-8686. 10.1074/jbc.M710222200.View ArticlePubMedGoogle Scholar
- Li C, Ao J, Fu J, Lee DF, Xu J, Lonard D, O’Malley BW: Tumor-suppressor role for the SPOP ubiquitin ligase in signal-dependent proteolysis of the oncogenic co-activator SRC-3/AIB1. Oncogene. 2011, 30: 4350-4364. 10.1038/onc.2011.151.PubMed CentralView ArticlePubMedGoogle Scholar
- Geng C, He B, Xu L, Barbieri CE, Eedunuri VK, Chew SA, Zimmermann M, Bond R, Shou J, Li C, Blattner M, Lonard DM, Demichelis F, Coarfa C, Rubin MA, Zhou P, O’Malley BW, Mitsiades N: Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover. Proc Natl Acad Sci U S A. 2013, 110: 6997-7002. 10.1073/pnas.1304502110.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang C, Pan Y, Wang B: Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors. Development. 2010, 137: 2001-2009. 10.1242/dev.052126.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang YY, Li L, Zhao ZS, Wang YX, Ye ZY, Tao HQ: L1 and epithelial cell adhesion molecules associated with gastric cancer progression and prognosis in examination of specimens from 601 patients. J Exp Clin Cancer Res. 2013, 32: 66-10.1186/1756-9966-32-66.PubMed CentralView ArticlePubMedGoogle Scholar
- Pan X, Zhou T, Tai YH, Wang C, Zhao J, Cao Y, Chen Y, Zhang PJ, Yu M, Zhen C, Mu R, Bai ZF, Li HY, Li AL, Liang B, Jian Z, Zhang WN, Man JH, Gao YF, Gong WL, Wei LX, Zhang XM: Elevated expression of CUEDC2 protein confers endocrine resistance in breast cancer. Nat Med. 2011, 17: 708-714. 10.1038/nm.2369.View ArticlePubMedGoogle Scholar
- Luo S, Zhang B, Dong XP, Tao Y, Ting A, Zhou Z, Meixiong J, Luo J, Chiu FC, Xiong WC, Mei L: HSP90 beta regulates rapsyn turnover and subsequent AChR cluster formation and maintenance. Neuron. 2008, 60: 97-110. 10.1016/j.neuron.2008.08.013.PubMed CentralView ArticlePubMedGoogle Scholar
- Bannazadeh Amirkhiz M, Rashtchizadeh N, Nazemieh H, Abdolalizadeh J, Mohammadnejad L, Baradaran B: Cytotoxic effects of alcoholic extract of dorema glabrum seed on cancerous cells viability. Adv Pharm Bull. 2013, 3: 403-408.PubMed CentralPubMedGoogle Scholar
- Liang CC, Park AY, Guan JL: In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007, 2: 329-333. 10.1038/nprot.2007.30.View ArticlePubMedGoogle Scholar
- Sasaki H, Hui C, Nakafuku M, Kondoh H: A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development. 1997, 124: 1313-1322.PubMedGoogle Scholar
- Onishi H, Katano M: Hedgehog signaling pathway as a therapeutic target in various types of cancer. Cancer Sci. 2011, 102: 1756-1760. 10.1111/j.1349-7006.2011.02010.x.View ArticlePubMedGoogle Scholar
- Xie K, Abbruzzese JL: Developmental biology informs cancer: the emerging role of the Hedgehog signaling pathway in upper gastrointestinal cancers. Cancer Cell. 2003, 4: 245-247. 10.1016/S1535-6108(03)00246-0.View ArticlePubMedGoogle Scholar
- Byun B, Tak H, Joe CO: BTB/POZ domain of speckle-type POZ protein (SPOP) confers proapoptotic function in HeLa cells. Biofactors. 2007, 31: 165-169. 10.1002/biof.5520310303.View ArticlePubMedGoogle Scholar
- Caro I, Low JA: The role of the Hedgehog signaling pathway in the development of basal cell carcinoma and opportunities for treatment. Clin Cancer Res. 2010, 16: 3335-3339. 10.1158/1078-0432.CCR-09-2570.View ArticlePubMedGoogle Scholar
- Yuan Z, Goetz JA, Singh S, Ogden SK, Petty WJ, Black CC, Memoli VA, Dmitrovsky E, Robbins DJ: Frequent requirement of Hedgehog signaling in non-small cell lung carcinoma. Oncogene. 2007, 26: 1046-1055. 10.1038/sj.onc.1209860.View ArticlePubMedGoogle Scholar
- Cui W, Wang LH, Wen YY, Song M, Li BL, Chen XL, Xu M, An SX, Zhao J, Lu YY, Mi XY, Wang EH: Expression and regulation mechanisms of Sonic Hedgehog in breast cancer. Cancer Sci. 2010, 101: 927-933. 10.1111/j.1349-7006.2010.01495.x.View ArticlePubMedGoogle Scholar
- Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D, Frese KK, Denicola G, Feig C, Combs C, Winter SP, Ireland-Zecchini H, Reichelt S, Howat WJ, Chang A, Dhara M, Wang L, Ruckert F, Grutzmann R, Pilarsky C, Izeradjene K, Hingorani SR, Huang P, Davies SE, Plunkett W, Egorin M, et al: Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science. 2009, 324: 1457-1461. 10.1126/science.1171362.PubMed CentralView ArticlePubMedGoogle Scholar
- Sheng T, Li C, Zhang X, Chi S, He N, Chen K, McCormick F, Gatalica Z, Xie J: Activation of the Hedgehog pathway in advanced prostate cancer. Mol Cancer. 2004, 3: 29-10.1186/1476-4598-3-29.PubMed CentralView ArticlePubMedGoogle Scholar
- Fan L, Pepicelli CV, Dibble CC, Catbagan W, Zarycki JL, Laciak R, Gipp J, Shaw A, Lamm ML, Munoz A, Lipinski R, Thrasher JB, Bushman W: Hedgehog signaling promotes prostate xenograft tumor growth. Endocrinology. 2004, 145: 3961-3970. 10.1210/en.2004-0079.View ArticlePubMedGoogle Scholar
- Katoh Y, Katoh M: Hedgehog signaling pathway and gastric cancer. Cancer Biol Ther. 2005, 4: 1050-1054. 10.4161/cbt.4.10.2184.View ArticlePubMedGoogle Scholar
- Ma X, Chen K, Huang S, Zhang X, Adegboyega PA, Evers BM, Zhang H, Xie J: Frequent activation of the Hedgehog pathway in advanced gastric adenocarcinomas. Carcinogenesis. 2005, 26: 1698-1705. 10.1093/carcin/bgi130.View ArticlePubMedGoogle Scholar
- Snijders AM, Huey B, Connelly ST, Roy R, Jordan RC, Schmidt BL, Albertson DG: Stromal control of oncogenic traits expressed in response to the overexpression of GLI2, a pleiotropic oncogene. Oncogene. 2009, 28: 625-637. 10.1038/onc.2008.421.PubMed CentralView ArticlePubMedGoogle Scholar
- Lauth M, Bergstrom A, Shimokawa T, Toftgard R: Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A. 2007, 104: 8455-8460. 10.1073/pnas.0609699104.PubMed CentralView ArticlePubMedGoogle Scholar
- Aza-Blanc P, Lin HY, Ruiz i Altaba A, Kornberg TB: Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities. Development. 2000, 127: 4293-4301.PubMedGoogle Scholar
- Zhang Q, Zhang L, Wang B, Ou CY, Chien CT, Jiang J: A Hedgehog-induced BTB protein modulates Hedgehog signaling by degrading Ci/Gli transcription factor. Dev Cell. 2006, 10: 719-729. 10.1016/j.devcel.2006.05.004.View ArticlePubMedGoogle Scholar
- Merchant M, Vajdos FF, Ultsch M, Maun HR, Wendt U, Cannon J, Desmarais W, Lazarus RA, de Vos AM, de Sauvage FJ: Suppressor of fused regulates Gli activity through a dual binding mechanism. Mol Cell Biol. 2004, 24: 8627-8641. 10.1128/MCB.24.19.8627-8641.2004.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.