European Journal of Cancer (2014) xxx, xxx– xxx
Available at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor q Amy Lee Han, Santosh Kumar, Jansina Y. Fok, Amit K. Tyagi, Kapil Mehta ⇑ Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, United States
KEYWORDS Castration-resistant prostate cancer Inflammation NF-jB Transglutaminase 2 Androgen receptor
q
Abstract Many studies have supported a role for inflammation in prostate tumour growth. However, the contribution of inflammation to the development of castration-resistant prostate cancer remains largely unknown. Based on observations that aberrant expression of the proinflammatory protein tissue transglutaminase (TG2) is associated with development of drug resistance and metastatic phenotype in multiple cancer types, we determined TG2 expression in prostate cancer cells. Herein we report that human prostate cancer cell lines with low expression of androgen receptor (AR) had high basal levels of TG2 expression. Also, overexpression of TG2 negatively regulated AR mRNA and protein expression and attenuated androgen sensitivity of prostate cancer cells. TG2 expression in prostate cancer cells was associated with increased invasion and resistance to chemotherapy. Mechanistically, TG2 activated nuclear factor (NF)-jB and induced epithelial–mesenchymal transition. TG2/NF-jB-mediated decrease in AR expression resulted from transcriptional repression involving cis-interaction of NF-jB in a complex with TG2 with the 50 -untranslated region of AR. Negative regulation of AR could be partially abrogated by repression of TG2 or NF-jB (p65/RelA) by gene-specific small interfering RNA. These results suggested that a novel pathway links androgen dependence with TG2-regulated inflammatory signalling and hence may make TG2 a novel therapeutic target for the prevention and treatment of castration-resistant prostate cancer. Ó 2014 Elsevier Ltd. All rights reserved.
Supported by S.K. Agarwal Donation Funds to The University of Texas MD Anderson Cancer Center.
⇑ Corresponding author: Address: Department of Experimental Therapeutics, Unit 1950, The University of Texas MD Anderson Cancer Center,
1515 Holcombe Blvd., Houston, TX 77030, United States. Tel.: +1 713 792 2649; fax: +1 713 745 4167. E-mail address:
[email protected] (K. Mehta). http://dx.doi.org/10.1016/j.ejca.2014.02.014 0959-8049/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
2
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
1. Introduction Prostate cancer is a common malignancy and the second leading cause of cancer-related deaths in the Western world [1]. Normal prostate cells and early-stage prostate cancer cells require the presence of androgens for growth and survival. Hence, androgen deprivation via surgical or chemical castration is a well-established treatment option for prostate cancer at various stages. Although hormonal ablation therapies initially induce remissions in most cases of prostate cancer, some cases progress and evolve to an androgen-independent state, known as castration-resistant prostate cancer (CRPC) [2]. CRPC is characterised by clinical and molecular heterogeneity and is the main cause of prostate cancer-associated mortality [1]. Treatment options for CRPC are extremely limited mainly because of the intrinsic chemoresistance acquired by the cancer cells during disease progression while the patient is receiving therapy. Understating the molecular mechanisms associated with progression of prostate cancer is critical to development of novel therapeutic approaches for CRPC. Androgen resistance mechanisms linked with the development of CRPC can be grouped into three categories. The first consists of DNA-based alterations, such as mutation or amplification of the androgen receptor (AR) gene. These occur in only a minority of patients [2]. The second mechanism is AR signalling that remains active even with castration levels of serum testosterone in patients without AR mutations or amplification. In these patients, alternative pathways can lead to AR signalling activation. For example, Her-2/neu-induced activation of AKT [3] or nuclear factor (NF)-jB [4] can promote AR activation. The third mechanism is complete bypass of AR pathways, allowing cancer cells to survive in the absence of androgen-dependent or -independent AR activation [2]. Indeed, large numbers of samples of metastatic tumours obtained from prostate cancer patients have exhibited lack of AR expression [5]. Moreover, tumour-initiating cells (TICs) isolated from human prostate tumours have lack of AR expression and had increased NF-jB activity [6]. Importantly, these TICs possessed stem cell characteristics and recapitulated the prostate tumour heterogeneity when injected into mice [6]. Investigators have proposed that inflammation can contribute to the progression of CRPC [7]. For example, androgen ablation therapy, which is accompanied by increased cytokine production owing to infiltration of immune cells in shrinking tumours, is known to support hormone-independent growth and survival of prostate cancer cells [8]. In addition to this effect of androgen ablation therapy, CRPC is resistant to chemotherapy and becomes metastatically competent. In light of these findings and recent observations that expression of proinflammatory protein, tissue transglutaminase (TG2) is
aberrantly upregulated in multiple drug-resistant and metastatic cancer cell types [9–11], we examined TG2 expression in hormone-dependent and -independent prostate cancer cell lines. Herein we present evidence that overexpression of TG2 promotes AR-independent growth and survival of prostate cancer cells by inducing constitutive activation of NF-jB. TG2 expression resulted in transcriptional repression of androgen receptor (AR) via interaction with the 50 -untranslated region of AR gene in a complex with NF-jB. Moreover, TG2 expression promoted the invasiveness of prostate cancer cells and their resistance to doxorubicin by inducing epithelial–mesenchymal transition (EMT). Taken together, these results suggest that aberrant expression of TG2 is a novel pathway that contributes to the progression of androgen-dependent prostate cancer to CRPC and thus may represent a promising target for treatment and prevention of this aggressive form of prostate cancer. 2. Materials and methods 2.1. Cell lines, vectors and reagents The prostate cancer cell lines LNCaP, V-CaP, DU 145, MDA-PCA-2b (A10), MDA-PCA-2a (A11), C4-2B and PC-3 were provided by Dr. Nora Navone (The University of Texas MD Anderson Cancer Center). Cells were maintained in Roswell Park Memorial Institute medium (RPMI 1640) or Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% foetal-calf serum (FCS), 2 mM L-glutamine and antibiotics. The MEM/F-12 medium was also supplemented with vitamins (Sigma–Aldrich, St. Louis, MO), non-essential amino acids (BioWhittaker, Walkersville, MD) and 1 mM/l sodium pyruvate (BioWhittaker). Full-length TG2 (TG2-WT) was subcloned into a pCDH lentiviral vector (System Biosciences, Mountain View, CA) as described previously [12]. Stable clones of TG2-transfected LNCaP cells were selected via growth in a puromycin-containing medium (1 lg/ml). For transient transfection of PC-3 cells with small interfering RNA (siRNA), SignalSilence TG2-specific and control siRNAs were purchased from Cell Signaling Technology (Danvers, MA). Anti-TG2 and anti-b-actin antibodies were purchased from Abcam (Cambridge, MA), and anti-E-cadherin, anti-N-cadherin and anti-fibronectin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000, Oligofectamine and Stealth siRNA (negative control) were obtained from Invitrogen (Carlsbad, CA). All media were purchased from Fisher Scientific (Pittsburgh, PA). 2.2. TG2 activity and expression Prostate cancer cells grown in 25-cm2 flasks were harvested at 80% confluence, washed twice with
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
phosphate buffered solution (PBS) and resuspended in 200 ll of buffer A (20 mM Tris–HCl, pH 7.4 with 1 mM ethylene diamine tetra-acetic acid (EDTA), 150 mM NaCl and 14 mM b-mercaptoethanol) containing freshly added 1 mM Phenylmethanesulfonyl fluoride (PMSF) and a protease inhibitor cocktail. Cell pellets were lysed via probe sonication with 8–10 pulses of 10 s each and used to measure the enzyme activity of TG2 by studying calcium-dependent conjugation of [3H]-putrescine into dimethylcasein as described previously [11]. 2.3. Western blotting For Western blot analysis prostate cancer cells were lysed (120 mM NaCl, 1% Triton X-100, 20 mM Tris– HCl, pH 7.5, 10% glycerol, 2 mM EDTA, protease inhibitor cocktail) for 30 min, and 30 lg of cellular protein was resolved on an 8% sodium monododecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. Proteins were transferred to a nitrocellulose membrane and probed with anti-TG2 (1:3000; Neomarkers, Fremont, CA), antib-actin (1:4000), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000), or anti-AR, anti-E-cadherin and anti-N-cadherin (all at 1:1000 dilution; Santa Cruz Biotechnology) antibodies. Antigen–antibody reactions were detected using the appropriate horseradish peroxidase-conjugated secondary antibody followed by visualisation using a chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). 2.4. Transfection of prostate cancer cells with siRNA/ short hairpin RNA 5
PC-3 cells (5 10 /well) were plated in six-well plates and allowed to adhere to the plates for 24 h. The next day, cell monolayers were transfected with TG2-specific or control siRNAs using Oligofectamine (Invitrogen) according to the manufacturer’s protocol. For stable transfection with short hairpin RNA (shRNA) PC-3 cells (104/well) were plated in 12-well tissue culture plates and allowed to adhere to the plates for 24 h. Monolayers of cells were then transfected with 5 lg/ml hexadimethrine bromide (Polybrene; Santa Cruz Labs, Santa Cruz, CA) in a culture medium containing 5 104 lentiviral particles containing TG2shRNA or control shRNA sequences following the manufacturer’s protocol. Cells were then prepared for further analyses. 2.5. Cell growth Prostate cancer cells in 96-well plates (2 103/well) were incubated overnight to allow the cells to attach to the wells. Either R1881 (0.1 nM) or bicalutamide (50 lM) was added to replicate wells and incubated
3
for 48 h. In a separate experiment, quadruplicate wells containing prostate cancer cells (2 103/well) were treated with increasing doses of doxorubicin (0–0.1 mg/ml) (Sigma) for 72 h. The number of viable cells remaining at the end of the incubation was determined using the tetrozolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. After 4 h of incubation with MTT, the medium was removed from wells and 200 ll of DMSO was added to dissolve formazan. The absorbance due to formazan was measured at 570 nm using a microplate reader. Cell survival was expressed as the percentage of surviving cells relative to that of untreated cells. 2.6. Immunofluorescence Prostate cancer cells were plated in a chamber slide with a polystyrene vessel (Fisher Scientific). Cells at 60–70% confluence were fixed with 4% formaldehyde in PBS for 10 min, permeabilised with 100% ice-cold methanol for 10 min and blocked for 1 h in a blocking solution (3% bovine serum albumin (BSA) and 1% goat serum). Cells were then incubated with the appropriate primary antibody in a blocking solution, followed by the secondary antibody (Alexa 488-conjugated antimouse, 1:200 dilution; Molecular Probes, Eugene, OR). Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI). Stained cells were then mounted on glass slides in a mounting buffer and viewed under an Eclipse fluorescence microscope (Nikon, Melville, NY). 2.7. Invasion assay The invasive behaviour of the cells in vitro was determined using Matrigel Transwell inserts. In brief, Transwell inserts with 12-lm pores were coated with 0.78 mg/ ml Matrigel in cold serum-free medium. Confluent cells were then trypsinised and washed with serum-free medium. Cell pellets were subsequently resuspended in serum-free medium, and 0.5 ml of the cell suspension (0.2 106 cells) was added to triplicate wells. After 48 h of incubation, cells that passed through the filter on the underside of the membrane were stained and counted under a light microscope. Five fields of cells were counted for each well, and the mean number of cells per field was calculated. Each experiment was performed in triplicate and repeated at least twice. 2.8. Electrophoretic mobility shift assay NF-jB activity was determined using an electrophoretic mobility shift assay (EMSA) as described previously [13]. In brief, nuclear extracts of 1.5 106 cells were incubated with an 32P-end-labelled NF-jB oligonucleotide probe (50 -TTG TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG30 ; boldface indicates NF-jB-binding sites) at 37 °C for
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
4
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
30 min, and the DNA–protein complex that formed was separated from the free oligonucleotide on 6.6% native polyacrylamide gels. The dried gels were visualised using a Storm 820 phosphorimager, and radioactive bands were quantitated using the ImageQuant software programme (GE Healthcare, Pittsburgh, PA). 2.9. RNA extraction, reverse transcription-PCR and quantitative reverse transcription-PCR Total RNA was extracted from prostate cancer cells using a mini-RNA isolation kit (QIAGEN, Valencia, CA). For reverse transcription-polymerase chain reaction (RT-PCR) 2.5 lg of total RNA was reverse-transcribed to cDNA using an RT2 First Strand Kit (SABiosciences, Valencia, CA). An equivalent volume (2 ll) of cDNA was used as the template for PCR analysis using gene-specific primers. Quantitative RT-PCR analysis of NF-jB target genes was performed using an RT2 Profiler PCR array (SABiosciences) according to the manufacturer’s protocol. The relative change in transcript level was calculated after normalisation according to GAPDH, b-actin and 18S ribosomal RNA. 2.10. Chromatin immunoprecipitation assay A chromatin immunoprecipitation (ChIP) assay was performed using an EZ-Magna ChIP kit (EMD Millipore, Billerica, MA) according to the manufacturer’s instructions. Briefly, prostate cancer cells were treated with 1% formaldehyde in a medium to induce protein cross-linking, lysed in buffer and sonicated to shear DNA to lengths ranging from 200 to 1000 bp. Lysates were centrifuged, and supernatants were precleared with protein-A agarose/salmon sperm DNA beads and incubated overnight with either an anti-p65 or anti-TG2 antibody (5 lg). Immunoprecipitates were pulled down using protein-A agarose/salmon sperm DNA beads and reverse-cross-linked with NaCl. PCR analysis was performed with phenol–chloroform–extracted DNA using the following primers for the AR promoter as described previously [14]: 38 to +246 bp (forward, 50 -GACCCGACTCGCAAACTGTT; reverse, 50 -CCTCCG AGTCTTTAGCAGCT) and 760 to 460 bp (forward, 50 -GGGTGATTTTGCCTTTGAGA; reverse, 50 -CATGACCAAGCCA GCAGATA). Primers corresponding to exon 1 of the AR gene (2403 to 2647 bp: forward, 50 -CCTGGCACACTCTCTTCACA-30 ; reverse, 50 -GG ATAGGGCACTCTGCTCAC-30 ) were used as negative controls. 2.11. Immunohistochemistry Archived prostate tumour samples in microarray were obtained from the MD Anderson Prostate Bank (provided by Dr. Nora Navone). Tissue array slides were deparaffinised and rehydrated using xylene and
graded ethanol followed by antigen retrieval in 0.1 M citrate buffer, pH 6. Endogenous peroxidase was blocked by incubating samples with 0.3% H2O2 in PBS for 15 min followed by 2 h of blocking in 5% goat serum in PBS. Samples were then incubated with a mouse antiTG2 monoclonal (clone CUB7401) or rabbit anti-AR polyclonal antibody (1:50) overnight at 4 °C. After washing, slides were incubated for 1 h in PBS containing an Alexa Fluor 488-conjugated goat anti-mouse or goat anti-rabbit antibody. Cells were then imaged under a Nikon fluorescence microscope. 2.12. Statistical analysis The statistical significance of the differences in the controls and tested samples was calculated using the Prism software programme (GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant. 3. Results 3.1. Prostate cancer cells with low AR expression have high basal levels of TG2 expression Western blot analysis revealed high basal levels of TG2 protein expression in two of the seven prostate cancer cell lines that we tested (Fig. 1A). Interestingly, both of these TG2-expressing cell lines lacked AR expression, whereas AR-expressing cell lines lacked TG2 expression. High enzymatic activity in DU 145 (21 nM/h/mg) and PC-3 (25 nM/h/mg) cells confirmed differential expression of TG2 in prostate cancer cell lines with low AR expression (Fig. 1B). The C4-2B cells, which express AR protein but do not depend on AR signalling for their growth or survival, did not have detectable TG2 activity or expression. These results suggested that TG2 expression enables prostate cancer cells to bypass AR signalling by activating alternative cell growth and survival signalling pathways. To confirm this, we stably transfected AR-dependent LNCaP cells with TG2 using a lentiviral construct (Fig. 1C). Treatment of control and vector-transfected LNCaP cells with the AR agonist R1881 (0.1 nM for 48 h) resulted in a 60–70% increase in cell number over that of control untreated cells (Fig. 1D). Conversely, treatment with the AR antagonist bicalutamide (50 lM for 48 h) caused a reduction in cell number of 60–70% below that of untreated control cells. Under identical conditions, TG2-transfected LNCaP cells did not exhibit noticeable changes in growth or survival in the presence or absence of R1881 or bicalutamide. TG2 expression alone had no effect on LNCaP cell growth as suggested by similar growth rates for control, vector-transfected and TG2-transfected cells (Fig. S1A). To authenticate the identity of TG2-transfected LNCaP cells, we performed
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
A
B AR
80kD -
TG2
37kD -
GAPDH
25 20 15 10 5 0
D
30
250 * Relative cell growth
Enzyme activity (nmol/hr/mg)
C
30 TG2 activity (nmol/h/mg)
110kD-
5
20
10
0 TG2
200
*
Control +R1881
*
+Bicalutamide
*P < 0.05
150
100
50
β-actin Cont. EV
TG2
LNCaP
0 Cont.
EV LNCaP
TG2
Fig. 1. Inverse correlation of tissue transglutaminase (TG2) expression with androgen receptor (AR) protein expression in prostate cancer cells. (A) AR protein expression in various prostate cancer cell lines as determined using Western blotting with an anti-AR polyclonal antibody. The nitrocellulose membrane was stripped and reprobed with anti-TG2 monoclonal and anti-b-actin antibodies to ensure even protein loading. (B) TG2 enzyme activity in cell extracts as determined by studying Ca2+-dependent incorporation of [3H]-putrescine into dimethylcasein as described in Section 2. (C) Expression of TG2 in control cells (Cont.) and LNCaP cells stably infected with a lentiviral vector alone (EV) or a vector containing a full-length TG2 coding sequence (TG2) as determined using Western blotting and according to enzymatic activity. (D) Effect of treatment with the androgen agonist R1881 and androgen antagonist bicalutamide on cell growth and viability. Cells were treated with R1881 (0.1 nM) or bicalutamide (50 lM) for 48 h. At the end of treatment, cell viability was determined using the tetrazoulium dye (MTT) reduction assay. Columns, means of six values from two independent experiments; bars, SD.
chromosomal karyotyping analysis and confirmed the presence of four marker chromosomes in all three LNCaP sublines (Fig. S1B). These results supported that TG2 expression can support the growth and survival of prostate cancer cells by generating AR-independent signalling pathways. 3.2. TG2 expression promotes EMT Overexpression of TG2 in prostate cancer cells was associated with visible changes in the cells’ morphology (Fig. 2A). TG2-deficient LNCaP cells (non-transfected or vector-infected only) grew into aggregates with close cell-to-cell contacts. In contrast, LNCaP-TG2 cells grew segregated from each other and appeared to be more spindle-shaped. PC-3 cells, which have high endogenous TG2 expression levels, closely resembled LNCaP-TG2 cells in their morphology. Loss of cell–cell adhesion is an important feature of cells undergoing EMT, which results from decreased E-cadherin expression. We next sought to determine the effect of TG2 expression on E-cadherin expression. Ectopic (LNCaP-TG2) as well as endogenous (PC-3) expression of TG2 in prostate
cancer cells was associated with decreased expression of E-cadherin at both the transcript (Fig. 2B) and protein (Fig. 2C and D) levels. In addition to loss of expression of E-cadherin, increased expression of N-cadherin, vimentin and fibronectin was associated with EMT. Interestingly, TG2 expression had no effect on N-cadherin expression in LNCaP cells, whereas PC-3 cells had high N-cadherin transcript and protein expression (Fig. 2B and C). Fibronectin and vimentin expression increased in TG2-transfected (LNCaP-TG2) and TG2expressing (PC-3) cells (Fig. 2B–D). These results suggested that TG2 expression promotes a partial EMT phenotype in prostate cancer cells. 3.3. TG2 expression promotes invasion and chemoresistance of prostate cancer cells EMT is associated with increased motility, invasion and drug resistance of cancer cells [15]. Therefore, we next examined the effect of TG2-induced EMT on the invasion of prostate cancer cells and their sensitivity to doxorubicin-induced death. TG2-overexpressing cells (PC-3 and LNCaP-TG2) were twofold to threefold more
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
6
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
A
LNCaP
B
LNCaP-TG2
RT-PCR E-Cadherin N-Cadherin Vimentin Fibronectin ZEB1 ZEB2 GAPDH
LNCaP-EV
C
Cont. EV TG2 LNCaP
PC-3
PC-3
D
Western Blot E-Cadherin
E-cadherin
N-Cadherin Vimentin Fn
Fibronectin TG2 β-Actin
TG2 Cont. EV TG2 PC-3 LNCaP EV
TG2
PC-3
LNCaP
Fig. 2. Induction of the epithelial–mesenchymal transition (EMT) phenotype in prostate cancer by tissue transglutaminase (TG2) expression. (A) Morphological changes in prostate cancer cells induced by TG2 expression. (B) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of EMT-related transcripts in control (Cont.), empty vector (EV)-infected LNCaP, TG2-transfected LNCaP and PC-3 cells. (C) Immunoblot analysis of EMT-related protein expression in control (Cont.), EV- and TG2-transfected LNCaP and PC-3 cells. The membranes were stripped and reprobed with anti-b-actin and anti-TG2 antibodies. (D) Immunofluorescence microscopic images of E-cadherin, fibronectin (Fn) and TG2 expression in EV- and TG2-transfected LNCaP cells and PC-3 cells. Cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) to visualise the nuclei.
invasive than their TG2-deficient counterparts (parental or empty vector [EV]-transfected LNCaP cells) (Fig. 3A). Increased invasion of these cells was evident in response to seeding in both 10% foetal calf serum and charcoal-stripped serum medium (Fig. 3B). Moreover, in LNCaP cells, TG2 expression was associated with a 10-fold increase in resistance to doxorubicininduced cell death (half-maximal inhibitory concentration, 50 ng/ml) over TG2 deficiency (half-maximal inhibitory concentration, 5 ng/ml) (Fig. 3C). Similarly, TG2 over expressing DU145 cells showed similar resistance to doxorubicin (data not shown), supporting that TG2 expression promotes cell survival and invasion independent of AR signalling.
(Fig. 4A) and transcript (Fig. 4B) level. Immunofluorescent staining of LNCaP and PC-3 cells for AR and TG2 confirmed these results (Fig. 4C). Moreover, transient downregulation of TG2 expression by siRNA could partially restore AR expression in PC-3 cells (Fig. 4D). Interestingly, a catalytically inactive mutant (C277S) of TG2 was able to induce similar silencing in AR expression in LNCaP cells (data not shown). These results suggested that TG2, independent of its enzymatic functions, can support AR-independent signalling pathways to promote the growth and survival of prostate cancer cells.
3.5. TG2 expression results in constitutive activation of NF-jB 3.4. TG2 represses AR expression As described above, prostate cancer cells have an inverse correlation between TG2 expression and AR expression (Fig. 1). We therefore determined the effect of TG2 expression on AR expression. The results we obtained were rather surprising. Overexpression of TG2 was associated with complete silencing of AR expression in prostate cancer cells at both the protein
According to a published report, TICs isolated from human prostate tumours lack AR expression and exhibit increased NF-jB activity [6]. In multiple cancer cell types, TG2 overexpression has been associated with constitutive activation of NF-jB [16]. Based on these observations, we next sought to determine the effect of TG2 expression on NF-jB activity in LNCaP cells. The EMSA results shown in Fig. 5A demonstrated
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
A
LNCaP
7
LNCaP-TG2
C 120
LNCaP LNCaP TG2
LNCaP-EV
B
PC-3
Number of invaded cells/field
120 + FCS
*
*P < 0.05
+ CSS
90
Relative Cell Viability (%)
100
PC3
80
60
40
20
*
0 control 0.00250.0050.01250.025 0.05 Doxorubicin, μg/ml
60
0.1
30 0 Cont.
EV
TG2
LNCaP
Fig. 3. Promotion of the invasiveness of prostate cancer cells by tissue transglutaminase (TG2) expression. (A) Comparison of the ability of control (Cont.; untransfected) and empty vector (EV)- and TG2-transfected LNCaP and PC-3 cells to invade through Matrigel Transwell inserts. Cells (2 104) were seeded in 10% FCS or charcoal-stripped serum (CSS) medium. The cells were incubated, and 48 h later, their membranes were fixed, stained and photographed as described in Section 2. (B) Graph of the number of cells that invaded through Matrigel Transwell inserts. The cells were counted under a light microscope, and the average numbers of invading cells ± SD in five random microscopic fields were plotted. (C) Effect of TG2 expression on doxorubicin-induced cell death in vector- and TG2-transfected LNCaP and PC-3 cells as determined using an MTT assay. The results shown are averages of the percent cell survival relative to that for control untreated cells in quadruplicate wells.
A
B
Western Blot
RT-PCR
AR
TG2
80kD -
TG2
AR
43kD -
β-actin
GAPDH
110kD -
Cont.
C
EV TG2 LNCaP
LNCaP-EV
Cont. EV TG2 PC-3 LNCaP
PC-3
LNCaP-TG2
PC-3
D
Western Blot PC-3
TG2
80kD -
AR
110kD -
TG2 AR Actin
Fig. 4. Downregulation of androgen receptor (AR) expression by tissue transglutaminase (TG2) expression. (A) TG2 expression in control (Cont.), empty vector (EV)-transfected and TG2-transfected LNCaP and PC-3 cells as determined using Western blotting. The membrane was stripped and reprobed with anti-AR and anti-b-actin antibodies. (B) Reverse transcription-polymerase chain reaction (RT-PCR) analysis validating the expression of AR in control (Cont.) and EV- and TG2-transfected LNCaP and PC-3 cells. (C) Immunofluorescence microscopic images of TG2 and AR protein expression in the nuclei of control and EV- and TG2-transfected LNCaP and PC-3 cells. Red, TG2; green, AR; blue, 40 ,6-diamidino2-phenylindole (DAPI). (D) Immunoblot analysis of the effect of TG2 repression induced by short hairpin RNA (shRNA) (TG2-shR) on AR protein expression in PC-3 cells. The membrane was stripped and reprobed with an anti-b-actin antibody to ensure equal protein loading. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
8
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
constitutive activation of NF-jB in prostate cancer cells overexpressing TG2 (PC-3 and LNCaP-TG2). Nuclear localisation of the p65/RelA subunit of NF-jB further confirmed the presence of TG2-induced NF-jB activity in these cells. Immunofluorescent staining and immunoblot studies revealed the presence of p65/RelA in the nuclei of TG2-expressing cells (Figs. 5B and S2A). Nuclear extracts from control and vector-infected LNCaP cells did not have p65/RelA protein expression (Fig. S2A). Importantly, nuclear localisation of p65/ RelA was accompanied by co-localisation of TG2, suggesting that TG2-activated NF-jB translocates to the nucleus in association with TG2. Next, we studied NF-jB target gene expression induced in response to TG2 expression in prostate cancer cells. Real-time PCR analysis of known NF-jB target genes revealed a strong, selective increase in Zeb1, Zeb2, cyclooxygenase (COX-2) and intracellular adhesion molecule (ICAM)-1 transcript expression in TG2overexpressing cells (Fig. 5C). TG2/NF-jB-induced increases in Zeb1 and Zeb2 expression were supported by RT-PCR data (Fig. 2B) and suggested that TG2induced EMT is related to increased expression of these transcription repressors.
3.6. The NF-jB/TG2 complex binds to the AR promoter and causes its transcriptional repression Based on our observation that TG2 expression induces constitutive activation of NF-jB (Fig. 5) and a report that tumour necrosis factor (TNF)-a-induced NF-jB activity negatively regulates AR expression [14], we next determined whether AR silencing by TG2 overexpression is mediated via a similar mechanism. We conducted a ChIP assay to examine the recruitment of TG2-regulated NF-jB to the NF-jB-binding sites in the AR promoter (Fig. S2C). TG2-overexpressing cells (LNCaP-TG2 and PC-3) exhibited specific association of NF-jB/p65 with chromatin fragments containing 38 to +246-bp and 460 to 760-bp sequences as revealed via PCR amplification of the immunoprecipitated DNA with primers specific for these regions of the AR promoter (Fig. 6A). Both of these fragments in the AR promoter are known to contain NF-jB-binding sites (Fig. S2C) [14]. Interestingly, TG2 was also recruited to these regions as confirmed by PCR amplification of DNA fragments immunoprecipitated with an anti-TG2 antibody (Fig. 6B). A ChIP assay with control IgG or a primer set designed to amplify the sequence
B A LNCaP LNCaP-EV
PC-3-cont.shRNA
LNCaP-TG2
PC-3-TG2.shRNA
NF-κB
C
Fig. 5. Induction of constitutive activation of nuclear factor (NF)-jB by tissue transglutaminase (TG2) in prostate cancer cells. (A) Results of electrophoretic mobility shift assay (EMSA) performed using a 32P-end-labelled, 45-mer double-stranded NF-jB oligonucleotide probe (50 -TTG TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG-30 ) with nuclear extracts prepared from empty vector (EV)transfected, TG2-transfected and tumour necrosis factor (TNF)-a-treated (10 nM for 30 min) LNCaP and PC-3 cells. (B) Localisation of p65/RelA protein (green) in nuclei (blue) of vector-infected and TG2-transfected LNCaP cells and in control and TG2-specific short hairpin RNA (shRNA)transfected PC-3 cells as determined using immunofluorescent staining. (C) Quantitative reverse transcription-polymerase chain reaction (RT-PCR) array showing relative changes in the expression of NF-jB target genes in EV- and TG2-transfected LNCaP and PC-3 cells. The results shown are from a representative experiment repeated at least twice with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
A
Anti-p65/RelA
- 38 bp +246 bp
+2403 bp +2667 bp
-760 bp - 460 bp
Input
Input
Input
B
9
Anti-TG2
- 38 bp +246 bp
+2403 bp +2667 bp
-760 bp -460 bp
Input
Input
Input
Fig. 6. Binding of tissue transglutaminase (TG2)-regulated nuclear factor (NF)-jB to the androgen receptor (AR) promoter. (A) Results of a chromatin immunoprecipitation (ChIP) assay performed using formaldehyde-fixed empty vector (EV)- and TG2-transfected LNCaP and PC-3 cell extracts with anti-p65/RelA and control anti-mouse IgG antibodies. (B) Results of a ChIP assay performed using formaldehyde-fixed EV- and TG2-transfected LNCaP and PC-3 cell extracts with TG2-specific or control anti-mouse IgG antibodies. Polymerase chain reaction (PCR) analysis was performed using these immunoprecipitates with a specific set of primers (described in Section 2 and shown in Fig. S2C) to amplify the AR promoter sequence containing the NF-jB-binding site at 38 to +246 bp (left panel) and 760 to 460 bp (right panel). Prior to immunoprecipitation, an aliquot from each sample was saved to determine the ‘input’ representing the PCR amplification of the genomic DNA without immunoprecipitation (PCR control).
from +2403 to +2667 bp failed to generate a PCR signal (Fig. 6A and B). These results suggested that TG2-regulated NF-jB, in association with TG2 itself, binds to the AR promoter at NF-jB response element sites and contributes to the observed TG2-mediated silencing of AR expression.
3.7. TG2 expression in tumour samples Next, we studied few selected prostate tumour samples (n = 15) to determine a relationship, if any, between TG2 and AR expression. The majority of the tumour samples (70%) lacked TG2 expression but exhibited
NF-κB
IκBα
TG2
p50 p65
IκBα
IκBα Proteasomal indpendent degradation
TG2
CRPC
TG2
EMT
p50 p65 TG2 p50 p65 p50 p65 TGM2 Promoter
AR
AR Promoter -38 or -760
to to
+246 bp -460 bp
?
܅
Zeb-1 & Zeb-2 TG2 p50 p65
HIF-1α
HIF-1α Promoter
Fig. 7. Schematic representation of tissue transglutaminase (TG2)-regulated pathways that play putative roles in the castration-resistant prostate cancer (CRPC) phenotype. Association of TG2 with IjBa results in rapid degradation of IjBa in a non-proteasomal pathway and liberates the p65/p50–nuclear factor (NF)-jB complex. In complex with the p65/RelA subunit of NF-jB, TG2 translocates to the nucleus, where it binds to the cognate NF-jB-binding sites on the androgen receptor (AR) promoter and causes transcriptional repression of AR. NF-jB also upregulates TG2 expression and creates a positive feedback loop in which TG2 activates NF-jB and NF-jB induces TG2 expression. As reported recently [13], TG2/ NF-jB also binds to the hypoxia-inducible factor (HIF)-1a promoter and induces its expression and results in the accumulation of HIF-1a protein (Fig. S3B) in cells even under normoxic conditions. HIF-1a in turn upregulates expression of the transcriptional repressors ZEB1 and ZEB2 resulting in activation of epithelial–mesenchymal transition (EMT). The combination of TG2/NF-jB-regulated silencing of AR expression and induction of the EMT phenotype increases cell survival, chemoresistance and invasiveness, which are the hallmarks of CRPC tumours.
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
10
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
strong nuclear immunofluorescent staining for AR protein (Fig. S3A, top panel). The remaining tumour samples had variable expression of TG2, ranging from strong and diffuse to more localised and restricted to islands of or isolated tumour cells. Importantly, TG2expressing tumour cells either completely lacked AR protein (Fig. S3A, bottom panel) or had decreased and punctated expression of AR protein in the nucleus (Fig. S3A, middle panel). These results implied that TG2 expression may not only affect AR transcriptional repression but also modulate AR distribution and, likely, bioavailability via mechanisms that remain to be determined. Future studies with larger groups of tumour samples are warranted to validate the correlation of TG2 with AR expression and its significance in the CRPC progression. 4. Discussion We report herein on a novel mechanism of promotion of a castration-resistant phenotype in prostate cancer cells. As outlined in Fig. 7, aberrant expression of the proinflammatory protein TG2 rendered androgendependent prostate cancer cells independent of AR signalling for their growth and survival. Stable expression of TG2 was associated with constitutive activation of the proinflammatory transcription factor NF-jB, occurrence of EMT, development of resistance to doxorubicin, increased invasiveness and transcriptional repression of AR, which are the hallmarks of CRPC [2]. We found that TG2 expression and activity were selectively upregulated in AR-deficient prostate cancer cells. All five AR-positive cell lines, lacked TG2 expression. A report by Birckbichler et al. [17] demonstrated that the basal expression of TG2 in benign and hyperplastic prostate glands was high but that its expression was generally lost in prostate adenocarcinoma cells [17]. In contrast with this but similar to our present results, Birckbichler and colleagues observed that ARdeficient DU 145 and PC-3 cells had high basal levels of TG2 expression. They could not explain why TG2 was not expressed in prostate adenocarcinomas but was in prostate carcinoma cell lines, and they did not speculate about the significance of elevated TG2 expression in AR-deficient prostate cells. On the basis of these observations and our previous findings that increased expression of TG2 in multiple cancer cell types is associated with increased cell survival and invasiveness [10– 12], we hypothesised that TG2 expression in prostate cancer cells can bypass AR-dependent cell growth and survival signalling. Indeed, stable expression of TG2 in androgen-dependent LNCaP cells rendered them resistant to treatment with the AR agonists (R1881) and antagonist (bicalutamide) (Fig. 1C and D). TG2 is a multifunctional 80-kD protein encoded by the stress response gene TGM2 [18]. Elevated expression
of TG2 is frequently observed in multiple drug-resistant and metastatic tumour cell types [10–12]. Although initially discovered as a cross-linking enzyme, understanding of TG2’s functions has markedly expanded in recent years. Specifically, TG2 can interact with several cytosolic, membranous and nuclear proteins [18]. This adapter/scaffold activity of TG2, which is independent of its enzymatic activity, is implicated to activate cell signalling pathways that play fundamental roles in cell survival, growth, migration and invasion [19]. Interestingly, increased expression of yet another member of transglutaminase family (TG4) has also been implicated in aggressive phenotype of prostate cancer. Thus, overexpression of TG4 resulted in increased cell-matrix adhesion, invasion and induction of the EMT in prostate cancer cells [20–22]. Increased expression of TG4 in prostate tumour samples is associated with higher Gleason score, increased levels of prostate-specific antigen and shorter biochemical recurrence-free survival after surgery [23]. An important and prominent example of TG2-dependent cell signalling pathway is the activation of NF-jB [13]. NF-jB belongs to a family of transcription factors that are important to inflammatory diseases and cancer development [24]. Under normal cellular conditions, NF-jB in the cytoplasm is inactive because of its association with IjBa. Exposure to stress stimuli results in ubiquitination of IjBa via a mechanism that involves IjB kinase (IKK)-dependent phosphorylation and proteasome-mediated degradation of IjBa. This results in release of NF-jB, which then translocates to the nucleus to activate gene expression [24]. Authors recently described an IKK-independent pathway of NF-jB activation that involves TG2-mediated degradation of IjBa [13,25]. These novel TG2-mediated IKK-independent mechanisms of NF-jB activation likely are involved in multiple aspects of cancer development. Chronic inflammation is implicated to play an important role in the progression of cancer, including prostate cancer [7,24]. For example, in an experimental model of prostate cancer, pretreatment of cancer cells with the proinflammatory cytokine TNF-a converted bicalutamide to an androgen agonist [26]. This suggested that TNF-a-regulated signalling can profoundly impact androgen-dependent prostate cells. Indeed, activation of NF-jB, which is an immediate response to treatment with TNF-a in many cells, takes place in a constitutive manner in CRPC tumours. Patients with elevated NFjB expression have worse prognoses than do patients without it [27]. NF-jB can regulate the expression of many genes and impact diverse processes, such as cell growth, differentiation, survival and invasion [24]. For example, NF-jB is known to induce the embryonically regulated process EMT, which is considered to play an essential role in conferring metastatic competence to and promoting chemoresistance of solid tumours,
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
including prostate cancer [24,27]. Our present data suggest that overexpression of TG2 in prostate cancer cells promotes constitutive activation of NF-jB (Fig. 5), accumulation of hypoxia-inducible factor (HIF)-1a protein (Fig. S3B) and induces EMT (Fig. 2). Thus, TG2overexpressing LNCaP-TG2 and PC-3 cells were more invasive than their TG2-deficient counterparts (Fig. 3). The impact of TG2-regulated NF-jB activation on downstream target genes revealed that Zeb1, Zeb2 and ICAM-1 are among the most commonly expressed genes in TG2-overexpressing cells (Fig. 5C). ZEB1 and ZEB2 are E-box-binding proteins that are known to not only regulate EMT but also promote stem cell-like features in prostate cancer cells [28,29]. Similarly, researchers have related ICAM-1 expression with increased invasion of prostate cancer cells and transition of them from an androgen-dependent to an androgen-refractory phenotype [30]. We previously demonstrated that TG2induced EMT is associated with acquisition of stem cell traits in mammary epithelial cells [31]. An increasing number of reports suggest that EMT is tightly linked with the biology of cancer stem cells and plays a critical role in the recurrence of prostate cancer and its progression to CRPC [29]. In view of these and our current findings that overexpression of TG2 promotes EMT and androgen refractoriness, TG2 may be a promising target for treatment of CRPC. We observed that TG2-induced NF-jB activation resulted in transcriptional repression of AR by binding to the functional NF-jB-binding sites located from 38 to +246 bp and from 760 to 460 bp upstream of the AR promoter. Interestingly, NF-jB recruitment at each cognate NF-jB-binding site was associated with a complex with TG2. Investigators recently observed a similar association between TG2 and NF-jB/p65 in transcriptional regulation of the HIF-1a gene in mammary epithelial cells [13]. In the present study, transient down regulation of either TG2 or NF-jB/p65 expression by gene-specific siRNA partially reversed downregulation of AR expression in PC-3 cells. We currently do not understand the significance of interaction between TG2 and NF-jB in transcriptional regulation of NFjB target genes. This interaction likely dictates functional regulation of NF-jB in terms of recruiting coactivators or repressors to the promoter sites of target genes. Indeed, many other posttranslational modifications, such as phosphorylation, acetylation and methylation, are known to affect this function of NF-jB. In general, NF-jB is a positive transcription regulator. However, depending on the composition of the NF-jB dimer and cis-interaction of NF-jB with other proteins, NF-jB can either induce or repress target gene expression. For example, progressive loss of AR mRNA expression in mice as they age is related to binding of the p50/p50 homodimer to an NF-jB-binding site in the AR promoter [32]. Similarly, transcriptional repression of
11
AR in prostate cancer cells was reported to result from interaction of p65/p50–NF-jB with the transcription factor B-Myb at a composite genomic element in the AR promoter [14]. Additional examples of NF-jBrepressible target genes include the growth arrest- and DNA damage-inducible gene GADD, the glutamate transporter gene EAAT-2 and the long-terminal repeat of latent human immunodeficiency virus [33–35]. Interaction of TG2 with NF-jB/p65 may promote recruitment of transcriptional repressors or prevent recruitment of transcription activators that are crucial for transcriptional regulation of AR. Nevertheless, functional assay results in our study clearly show that TG2 is an essential partner of NF-jB in transcriptional repression of the AR gene and promotion of AR-independent prostate cancer cell survival and growth. Conflict of interest statement Authors of the paper declare no conflict of interest, except that the University of Texas has filed a patent application related to transglutaminase inhibitors on which one of the authors (K.M.) is a co-inventor. Acknowledgements The authors thank Drs. B.B. Aggarwal for help with EMSAs and Mr. Norton Norwood for critical reading of and editorial help with this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2014.02.014. References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin 2013;63:11–30. [2] Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer 2001;1:34–45. [3] Craft N, Shostak Y, Carey M, Sawyers CL. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 1999;5:280–5. [4] Zhang L, Charron M, Wright WW, et al. Nuclear factor-jB activates transcription of the androgen receptor gene in Sertoli cells isolated from testes of adult rats. Endocrinology 2004;145: 781–9. [5] Shah RB, Mehra R, Chinnayian AM, et al. Androgen-independent prostate cancer is a heterogenous group of diseases: lessons from a rapid autopsy program. Cancer Res 2004;64:9209–16. [6] Rajasekhar VK, Studer L, Gerald W, Socci ND, Scher HI. Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-jB signaling. Nat Commun 2011;2:162. [7] Gueron G, Siervi AD, Vazquez E. Advanced prostate cancer: reinforcing the strings between inflammation and the metastatic behavior. Prostate Cancer Prostatic Dis 2012;15:213–21.
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
12
A.L. Han et al. / European Journal of Cancer xxx (2014) xxx–xxx
[8] Ammirante M, Luo JL, Grivennikov S, Nedospasov S, Karin M. B-cell-derived lymphotoxin promotes castration-resistant prostate cancer. Nature 2010;464:302–5. [9] Beninati S, Facchiano F, Piacentini M. Transglutaminases: future perspectives. Amino Acids 2013;44:1–9. [10] Verma A, Mehta K. Tissue transglutaminase-mediated chemoresistance in cancer cells. Drug Resist Updat 2007;10:144–51. [11] Mangala LS, Fok JY, Zorrilla-Calancha IR, Verma A, Mehta K. Tissue transglutaminase expression promotes cell attachment, invasion and survival in breast cancer cells. Oncogene 2007;26: 2459–70. [12] Kumar A, Xu J, Brady S, Gao H, et al. Tissue transglutaminase promotes drug resistance and invasion by inducing mesenchymal transition in mammary epithelial cells. PLoS One 2010;5:e13390. [13] Kumar S, Mehta K. Tissue transglutaminase constitutively activates HIF-1a promoter and nuclear factor-jB via a noncanonical pathway. PLoS One 2010;7:e49321. [14] Ko S, Shi L, Kim S, Song CS, Chatterjee B. Interplay of nuclear factor-kappaB and B-myb in the negative regulation of androgen receptor expression by tumor necrosis factor alpha. Mol Endocrinol 2008;22:273–86. [15] Gomes LR, Terra LF, Sogayar MC, Labriola L. Epithelialmesenchymal transition: implications in cancer progression and metastasis. Curr Pharm Biotechnol 2011;2011(12):1881–90. [16] Mann AP, Verma A, Sethi G, et al. Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel pathway. Cancer Res 2006;66:8788–95. [17] Birckbichler PJ, Bonner RB, Hurst RE, Bane BL, Pitha JV, Hemstreet GP. Loss of tissue transglutaminase as a biomarker for prostate adenocarcinoma. Cancer 2000;89:412–23. [18] Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140–56. [19] Agnihotri N, Kumar S, Mehta K. Tissue transglutaminase as a central mediator in inflammation-induced progression of breast cancer. Breast Cancer Res 2013;15:202. [20] Davies G, Ablin RJ, Mason MD, Jiang WG. Expression of the prostate transglutaminase (TGase-4) in prostate cancer cells and its impact on the invasiveness of prostate cancer. J Exp Ther Oncol 2007;6:257–64. [21] Jiang WG, Ye L, Sanders AJ, Ruge F, Kynaston HG, Ablin RJ, et al. Prostate transglutaminase (TGase4) enhances the adhesion of prostate cancer cells to extracellular matrix, the potential role of TGase-core domain. J Transl Med 2013;11:269. [22] Jiang WG, Ablin RJ. Prostate transglutaminase: a unique transglutaminase and its role in prostate cancer. Biomark Med 2011;5:285–91. [23] Cao Z, Wang Y, Liu ZY, Zhang ZS, et al. Overexpression of transglutaminase 4 and prostate cancer progression: a potential
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
predictor of less favorable outcomes. Asian J Androl 2013;15:742–6. DiDonato JA, Mercurio F, Karin M. NF-jB and the link between inflammation and cancer. Immunol Rev 2012;246: 379–400. Yakubov B, Chelladurai B, Schmitt J, Emerson R, Turchi JJ, Matei D. Extracellular tissue transglutaminase activates noncanonical NF-jB signaling and promotes metastasis in ovarian cancer. Neoplasia 2013;2013(15):609–19. Zhu P, Baek SH, Bourk EM, et al. Macrophage/cancer cell interactions mediate hormone resistance by a nuclear receptor derepression pathway. Cell 2006;124:615–29. Jain G, Cronauer MV, Schrader M, Mo¨ller P, Marienfeld RB. NF-jB signaling in prostate cancer: a promising therapeutic target? World J Urol 2012;30:303–10. Wu K, Bonavida B. The activated NF-kappaB-Snail-RKIP circuitry in cancer regulates both the metastatic cascade and resistance to apoptosis by cytotoxic drugs. Crit Rev Immunol 2009;29:241–54. Kong D, Banerjee S, Ahmad A, Li Y, Wang Z, Sethi S, et al. Epithelial to mesenchymal transition is mechanistically linked with stem cell signatures in prostate cancer cells. PLoS One 2010;5:e12445. Conrad F, Zhu X, Zhang X, et al. Human antibodies targeting cell surface antigens overexpressed by the hormone refractory metastatic prostate cancer cells: ICAM-1 is a tumor antigen that mediates prostate cancer cell invasion. J Mol Med 2009;87: 507–14. Kumar A, Xu J, Sung B, Kumar S, Yu D, Aggarwal BB, et al. Evidence that GTP-binding domain but not catalytic domain of transglutaminase 2 is essential for epithelial-to-mesenchymal transition in mammary epithelial cells. Breast Cancer Res 2012; 14:R4. Supakar PC, Jung MH, Song CS, Chatterjee B, Roy AK. Nuclear factor kappa B functions as a negative regulator for the rat androgen receptor gene and NF-kappa B activity increases during the age-dependent desensitization of the liver. J Biol Chem 1995;270:837–42. Zerbini LF, Wang Y, Czibere A, et al. NF-kappa B-mediated repression of growth arrest- and DNA-damage-inducible proteins 45alpha and gamma is essential for cancer cell survival. Proc Natl Acad Sci U S A 2004;101:13618–23. Sitcheran R, Gupta P, Fisher PB, Baldwin AS. Positive and negative regulation of EAAT2 by NF-kappaB: a role for N-myc in TNFalpha-controlled repression. EMBO J 2005;24:510–20. Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC. NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 2006;25:139–49.
Please cite this article in press as: Han A.L. et al., Tissue transglutaminase expression promotes castration-resistant phenotype and transcriptional repression of androgen receptor, Eur J Cancer (2014), http://dx.doi.org/10.1016/j.ejca.2014.02.014
