Tumor Biol. (2015) 36:967–972 DOI 10.1007/s13277-014-2709-z
RESEARCH ARTICLE
Androgen receptor signaling regulates growth of glioblastoma multiforme in men Xiaoming Yu & Yuhua Jiang & Wei Wei & Ping Cong & Yinlu Ding & Lei Xiang & Kang Wu
Received: 22 August 2014 / Accepted: 3 October 2014 / Published online: 15 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Although glioblastoma multiforme (GBM) is the most malignant primary human brain cancer with surprisingly high incidence rate in adult men than in women, the exact mechanism underlying this pronounced epidemiology is unclear. Here, we showed significant upregulated androgen receptor (AR) expression in the GBM tissue compared to the periphery normal brain tissue in patients. An expression of AR was further detected in all eight examined human GBM cell lines. To figure out whether AR signaling may play a role in GBM, we used high AR-expressing U87-MG GBM line for further study. We found that activation of transforming growth factor β (TGFβ) receptor signaling by TGFβ1 in GBM significantly inhibited cell growth and increased apoptosis. Moreover, application of active A R ligand 5αdihydrotestosterone (DHT) significantly decreased the effect of TGFβ1 on GBM growth and apoptosis, suggesting that AR signaling pathway may contradict the effect of TGFβ receptor signaling in GBM. However, neither total protein nor the phosphorylated protein of SMAD3, a major TGFβ receptor signaling downstream effector in GBM, was affected by DHT, suggesting that AR activation may not affect the SMAD3 X. Yu : Y. Jiang : W. Wei : P. Cong Department of Oncology, the Second Hospital of Shandong University, Jinan, China Y. Ding Department of General Surgery, the Second Hospital of Shandong University, Jinan, China L. Xiang Department of Pathology, School of Medicine, Shandong University, Jinan, China K. Wu (*) Department of Oncology, the People’s Hospital of Linqing, 317 Yao Kou Street, Xinhua Road, Linqing 252600, China e-mail: [email protected]
protein production or phosphorylation of TGFβ receptor and SMAD3. Finally, immunoprecipitation followed by immunoblot confirmed binding of pAR to pSMAD3, which may prevent the DNA binding of pSMAD3 and subsequently prevent its effect on cell growth in GBM. Taken together, our study suggests that AR signaling may promote tumorigenesis of GBM in adult men by inhibiting TGFβ receptor signaling. Keywords Androgen receptor signaling . 5α-Dihydrotestosterone . Glioblastoma multiforme . Transforming growth factor β receptor signaling . SMAD3
Introduction Glioblastoma multiforme (GBM) is the most malignant primary human brain cancer with an extremely low 5-year survival ratio in patients, largely because of its location in central nervous system which renders its growth and invasion considerably severe [1–4]. Although comprehension of the molecular regulation of GBM has potential clinical importance, our present knowledge about the tumorigenesis of GBM remains limited. Interestingly, the incidence rate of GBM is much greater in adult men than in women [5], while the exact mechanism underlying this pronounced epidemiology is unclear. Steroid hormones play a key role in brain development and differentiation [6–8]. Moreover, endogenous estrogens and other estrogenic compounds have been shown to be involved in various neurologic disorders, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and neoplasm [5]. However, a possible role of androgen receptor (AR) signaling in these setting has not been examined before. Androgens derive predominantly from the testis but also to a lesser extent from the adrenal glands. Androgens mediate
968
their effects via binding to AR [9]. Testicular testosterone and adrenal dehydroepiandrosterone (DHEA) or androstenedione can be converted into bioactive 5α-dihydrotestosterone (DHT) by the enzymes 5a-reductase, whose binding to the AR induces a conformational change that leads to the dissociation of chaperone and heat shock proteins and its subsequent interaction with co-regulatory molecules and importina, which facilitate nuclear translocation of AR-ligand complexes [9]. In the nucleus, the AR undergoes phosphorylation and dimerization, which permits chromatin binding to androgen-responsive elements (ARE) within androgenregulated target genes [9]. Transforming growth factor β (TGFβ) receptor signaling pathway is essential for many biological events [10–15]. There are two TGFβ receptors, types I and II. When a ligand binds to a type II receptor, it catalyzes the phosphorylation of a type I receptor and subsequently triggers phosphorylation of intracellular proteins SMAD2 and/or SMAD3, which form heteromeric complexes with SMAD4. The activated SMAD complexes then translocate to the nucleus, where they regulate the transcription of target genes [10]. Although TGFβ receptor signaling pathway has been shown to play a critical role during pathogenesis of many cancers including GBM [16–21], a relationship between AR and SMAD3 has only been shown in prostate cancer [22, 23]. Here, we showed a significant AR expression in the GBM tissue compared to the periphery normal brain tissue in patients. Expression of AR was further detected in all eight examined human GBM cell lines. Activation of TGFβ receptor signaling by TGFβ1 in GBM significantly inhibited cell growth and increased apoptosis in GBM cells, which was inhibited by active AR ligand DHT. However, neither total protein nor the phosphorylated protein of SMAD3, a major TGFβ receptor signaling downstream effector in GBM, was affected by DHT. Immunoprecipitation followed by immunoblot confirmed binding of pAR to pSMAD3, which may prevent the DNA binding of pSMAD3 and subsequently prevent its effect on cell growth in GBM.
Materials and methods
Tumor Biol. (2015) 36:967–972
Cell lines A172, LN-18, LN-229, M059, T-98G, U87-MG, U118MG, and U138-MG are human GBM cell line purchased from ATCC and were cultured in corresponding ATCC-recommended medium supplemented with 10 % fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Recombinant TGFβ1 and DHT were both purchased from Sigma (USA).
Immunoprecipitation and immunoblots/Western blot Proteins were extracted from fresh resected GBM tissue or periphery normal brain tissue (NBT) or cultured GBM cell lines using a detergent extraction buffer containing 1 % Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with the addition of protease inhibitor cocktail tablets. For immunoprecipitation (IP), extracted proteins were incubated with anti-pSMAD34 or anti-phosphorylated AR (pAR), at 4 °C overnight on a rocking platform. Protein A agarose was added and incubated at 4 °C for another 3 h. Immunoprecipitates were then washed three times with saline and resolved on 8 % SDS-PAGE gel. For immunoblots/Western blot, the polyvinylidene difluoride membrane carrying the transferred proteins was incubated at 4 °C overnight with anti-pAR or anti-pSMAD3 in TBST buffer containing 5 % nonfat dry milk. Immunodetection was accomplished by using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection system (GE Healthcare). Primary antibodies were anti-AR, anti-pAR, anti-SMAD3, anti-phosphorylated SMAD3 (pSMAD3), and anti-α-tubulin (all from Cell Signaling, Danvers, MA, USA). α-tubulin was used as a protein-loading control. Of note, total SMAD3 or AR was also used as a loading control.
Cell proliferation assay For assay of cell proliferation, the cells were seeded into 96well plate at 4000 cells per well and subjected to a Cell Proliferation Kit (MTT, Roche, USA), according to the manufacturer’s instruction.
Patient tissue specimens A total of 22 resected specimens from male GBM patients were collected for this study. All specimens had been histologically and clinically diagnosed at the Department of Oncology, the Second Hospital of Shandong University from 2008 to 2013. For the use of these clinical materials for research purposes, prior patient’s consents and approval from the institutional research ethics committee were obtained.
Apoptosis assay Cells were labeled with annexin V-FITC and propidium iodide (PI), and then examined with an apoptosis detecting kit (Invitrogen, USA) for apoptosis. Samples were analyzed by flow cytometry, and the results were analyzed by CellQuest software (Becton Dickinson, San Jose, CA, USA) as has been described before [24].
Tumor Biol. (2015) 36:967–972
969
examined ones (Fig. 1b), to study AR signaling in GBM. 5αDihydrotestosterone (DHT) is the bioactive form of AR ligand, and it binds to AR to induce nuclear translocation of the receptor complex followed by phosphorylation of AR. We found that activation of TGFβ receptor signaling through 10 ng/ml TGFβ1 treatment in U87-MG cells significantly inhibited cell growth in a MTT assay (Fig. 2a) and significantly increased apoptosis (Fig. 2b), consistent with previous reports. Moreover, application of 500 ng/ml DHT significantly decreased the effect of TGFβ1 on GBM growth (Fig. 2a) and apoptosis (Fig. 2b), while DHT alone did not affect growth and apoptosis of U87-MG cells (Fig. 2a, b), suggesting that AR signaling may contradict TGFβ receptor signaling in GBM.
Statistical analysis All statistical analyses were carried out using the SPSS 19.0 statistical software package. All values are depicted as mean± standard error from five individuals and are considered significant if p IB for pSMAD3
5 TGFβ1-/DHT+
4
TGFβ1-/DHT3
TGFβ1+/DHT+
2
TGFβ1+/DHT-
IP for pSMAD3> IB for pAR
NS
*
*
0
Relative DCF-DA levels (vs control)
b
1
2
Time (day)
3
*
+
+ -
+ +
TGFβ1 DHT
*
2 NS 1
0
-
+
+ -
+ +
TGFβ1 DHT
Fig. 2 AR activation abolished the inhibitory effect of TGFβ1 on growth of GBM cells. a Cell growth of U87-MG cells was evaluated in a MTT assay, with or without TGFβ1 treatment and with or without DHT treatment. b DCF-DA levels in U97-MG cells were quantified as an indicator of apoptosis, with or without TGFβ1 treatment and with or without DHT treatment. *p
RESEARCH ARTICLE
Androgen receptor signaling regulates growth of glioblastoma multiforme in men Xiaoming Yu & Yuhua Jiang & Wei Wei & Ping Cong & Yinlu Ding & Lei Xiang & Kang Wu
Received: 22 August 2014 / Accepted: 3 October 2014 / Published online: 15 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Although glioblastoma multiforme (GBM) is the most malignant primary human brain cancer with surprisingly high incidence rate in adult men than in women, the exact mechanism underlying this pronounced epidemiology is unclear. Here, we showed significant upregulated androgen receptor (AR) expression in the GBM tissue compared to the periphery normal brain tissue in patients. An expression of AR was further detected in all eight examined human GBM cell lines. To figure out whether AR signaling may play a role in GBM, we used high AR-expressing U87-MG GBM line for further study. We found that activation of transforming growth factor β (TGFβ) receptor signaling by TGFβ1 in GBM significantly inhibited cell growth and increased apoptosis. Moreover, application of active A R ligand 5αdihydrotestosterone (DHT) significantly decreased the effect of TGFβ1 on GBM growth and apoptosis, suggesting that AR signaling pathway may contradict the effect of TGFβ receptor signaling in GBM. However, neither total protein nor the phosphorylated protein of SMAD3, a major TGFβ receptor signaling downstream effector in GBM, was affected by DHT, suggesting that AR activation may not affect the SMAD3 X. Yu : Y. Jiang : W. Wei : P. Cong Department of Oncology, the Second Hospital of Shandong University, Jinan, China Y. Ding Department of General Surgery, the Second Hospital of Shandong University, Jinan, China L. Xiang Department of Pathology, School of Medicine, Shandong University, Jinan, China K. Wu (*) Department of Oncology, the People’s Hospital of Linqing, 317 Yao Kou Street, Xinhua Road, Linqing 252600, China e-mail: [email protected]
protein production or phosphorylation of TGFβ receptor and SMAD3. Finally, immunoprecipitation followed by immunoblot confirmed binding of pAR to pSMAD3, which may prevent the DNA binding of pSMAD3 and subsequently prevent its effect on cell growth in GBM. Taken together, our study suggests that AR signaling may promote tumorigenesis of GBM in adult men by inhibiting TGFβ receptor signaling. Keywords Androgen receptor signaling . 5α-Dihydrotestosterone . Glioblastoma multiforme . Transforming growth factor β receptor signaling . SMAD3
Introduction Glioblastoma multiforme (GBM) is the most malignant primary human brain cancer with an extremely low 5-year survival ratio in patients, largely because of its location in central nervous system which renders its growth and invasion considerably severe [1–4]. Although comprehension of the molecular regulation of GBM has potential clinical importance, our present knowledge about the tumorigenesis of GBM remains limited. Interestingly, the incidence rate of GBM is much greater in adult men than in women [5], while the exact mechanism underlying this pronounced epidemiology is unclear. Steroid hormones play a key role in brain development and differentiation [6–8]. Moreover, endogenous estrogens and other estrogenic compounds have been shown to be involved in various neurologic disorders, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and neoplasm [5]. However, a possible role of androgen receptor (AR) signaling in these setting has not been examined before. Androgens derive predominantly from the testis but also to a lesser extent from the adrenal glands. Androgens mediate
968
their effects via binding to AR [9]. Testicular testosterone and adrenal dehydroepiandrosterone (DHEA) or androstenedione can be converted into bioactive 5α-dihydrotestosterone (DHT) by the enzymes 5a-reductase, whose binding to the AR induces a conformational change that leads to the dissociation of chaperone and heat shock proteins and its subsequent interaction with co-regulatory molecules and importina, which facilitate nuclear translocation of AR-ligand complexes [9]. In the nucleus, the AR undergoes phosphorylation and dimerization, which permits chromatin binding to androgen-responsive elements (ARE) within androgenregulated target genes [9]. Transforming growth factor β (TGFβ) receptor signaling pathway is essential for many biological events [10–15]. There are two TGFβ receptors, types I and II. When a ligand binds to a type II receptor, it catalyzes the phosphorylation of a type I receptor and subsequently triggers phosphorylation of intracellular proteins SMAD2 and/or SMAD3, which form heteromeric complexes with SMAD4. The activated SMAD complexes then translocate to the nucleus, where they regulate the transcription of target genes [10]. Although TGFβ receptor signaling pathway has been shown to play a critical role during pathogenesis of many cancers including GBM [16–21], a relationship between AR and SMAD3 has only been shown in prostate cancer [22, 23]. Here, we showed a significant AR expression in the GBM tissue compared to the periphery normal brain tissue in patients. Expression of AR was further detected in all eight examined human GBM cell lines. Activation of TGFβ receptor signaling by TGFβ1 in GBM significantly inhibited cell growth and increased apoptosis in GBM cells, which was inhibited by active AR ligand DHT. However, neither total protein nor the phosphorylated protein of SMAD3, a major TGFβ receptor signaling downstream effector in GBM, was affected by DHT. Immunoprecipitation followed by immunoblot confirmed binding of pAR to pSMAD3, which may prevent the DNA binding of pSMAD3 and subsequently prevent its effect on cell growth in GBM.
Materials and methods
Tumor Biol. (2015) 36:967–972
Cell lines A172, LN-18, LN-229, M059, T-98G, U87-MG, U118MG, and U138-MG are human GBM cell line purchased from ATCC and were cultured in corresponding ATCC-recommended medium supplemented with 10 % fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Recombinant TGFβ1 and DHT were both purchased from Sigma (USA).
Immunoprecipitation and immunoblots/Western blot Proteins were extracted from fresh resected GBM tissue or periphery normal brain tissue (NBT) or cultured GBM cell lines using a detergent extraction buffer containing 1 % Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with the addition of protease inhibitor cocktail tablets. For immunoprecipitation (IP), extracted proteins were incubated with anti-pSMAD34 or anti-phosphorylated AR (pAR), at 4 °C overnight on a rocking platform. Protein A agarose was added and incubated at 4 °C for another 3 h. Immunoprecipitates were then washed three times with saline and resolved on 8 % SDS-PAGE gel. For immunoblots/Western blot, the polyvinylidene difluoride membrane carrying the transferred proteins was incubated at 4 °C overnight with anti-pAR or anti-pSMAD3 in TBST buffer containing 5 % nonfat dry milk. Immunodetection was accomplished by using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection system (GE Healthcare). Primary antibodies were anti-AR, anti-pAR, anti-SMAD3, anti-phosphorylated SMAD3 (pSMAD3), and anti-α-tubulin (all from Cell Signaling, Danvers, MA, USA). α-tubulin was used as a protein-loading control. Of note, total SMAD3 or AR was also used as a loading control.
Cell proliferation assay For assay of cell proliferation, the cells were seeded into 96well plate at 4000 cells per well and subjected to a Cell Proliferation Kit (MTT, Roche, USA), according to the manufacturer’s instruction.
Patient tissue specimens A total of 22 resected specimens from male GBM patients were collected for this study. All specimens had been histologically and clinically diagnosed at the Department of Oncology, the Second Hospital of Shandong University from 2008 to 2013. For the use of these clinical materials for research purposes, prior patient’s consents and approval from the institutional research ethics committee were obtained.
Apoptosis assay Cells were labeled with annexin V-FITC and propidium iodide (PI), and then examined with an apoptosis detecting kit (Invitrogen, USA) for apoptosis. Samples were analyzed by flow cytometry, and the results were analyzed by CellQuest software (Becton Dickinson, San Jose, CA, USA) as has been described before [24].
Tumor Biol. (2015) 36:967–972
969
examined ones (Fig. 1b), to study AR signaling in GBM. 5αDihydrotestosterone (DHT) is the bioactive form of AR ligand, and it binds to AR to induce nuclear translocation of the receptor complex followed by phosphorylation of AR. We found that activation of TGFβ receptor signaling through 10 ng/ml TGFβ1 treatment in U87-MG cells significantly inhibited cell growth in a MTT assay (Fig. 2a) and significantly increased apoptosis (Fig. 2b), consistent with previous reports. Moreover, application of 500 ng/ml DHT significantly decreased the effect of TGFβ1 on GBM growth (Fig. 2a) and apoptosis (Fig. 2b), while DHT alone did not affect growth and apoptosis of U87-MG cells (Fig. 2a, b), suggesting that AR signaling may contradict TGFβ receptor signaling in GBM.
Statistical analysis All statistical analyses were carried out using the SPSS 19.0 statistical software package. All values are depicted as mean± standard error from five individuals and are considered significant if p IB for pSMAD3
5 TGFβ1-/DHT+
4
TGFβ1-/DHT3
TGFβ1+/DHT+
2
TGFβ1+/DHT-
IP for pSMAD3> IB for pAR
NS
*
*
0
Relative DCF-DA levels (vs control)
b
1
2
Time (day)
3
*
+
+ -
+ +
TGFβ1 DHT
*
2 NS 1
0
-
+
+ -
+ +
TGFβ1 DHT
Fig. 2 AR activation abolished the inhibitory effect of TGFβ1 on growth of GBM cells. a Cell growth of U87-MG cells was evaluated in a MTT assay, with or without TGFβ1 treatment and with or without DHT treatment. b DCF-DA levels in U97-MG cells were quantified as an indicator of apoptosis, with or without TGFβ1 treatment and with or without DHT treatment. *p
