Steroid receptor RNA activator (SRA) was first isolated as a steroid receptor co-activator that functioned as an RNA transcript. Later, we demonstrated that SRA needs to be translated in order to co-activate androgen receptor (AR). Here, we showed that three isoforms of human SRA enhanced AR activities. Small interfering RNA against SRA suppressed AR activities in PC-3 cells transfected with pSG5AR and in LNCaP cells that have an endogenous mutated-AR. Western blot showed that SRA protein was expressed at a higher level in PC-3 than in LNCaP cells, suggesting that SRA may be related to hormone-independent growth of prostate cancer.
Activation of the androgen receptor (AR) despite androgen ablation and anti-androgen therapy seems to play a pivotal role in hormone-refractory progression of prostate cancer. The above postulation is substantiated by the report by Linja et al.1 that AR was expressed at a higher level in all the hormone-refractory prostate cancer (HRPC) examined than untreated disease and that an amplification of AR was observed in one-third of HRPC.
The proposed mechanism by which AR is activated in HRPC includes the amplification of AR as mentioned above,1 mutation of AR that causes gain of function,2, 3, 4 and phosphorylation of AR through growth factor receptor tyrosine kinase and MAP kinase 5 or through IL-6, STAT3 and MAP kinase cascade.6
On the other hand, recent studies have isolated nuclear receptor co-activators such as TIF-1,7 CBP/p300,8, 9 SRC-1/TIF2,10, 11 ACTR,12 and RIP140.13 Among them, an increased expression of SRC-1 and TIF2 in recurrent prostate cancer has been reported, and the overexpression of these co-activators has been shown to increase AR activity at a physiological concentration of adrenal androgen.14 Another study on the possible association of nuclear receptor co-activators with tumor progression described that AIB1, ARA54, ARA70, CBP, cyclin D1, Her2/neu, BAG-1/M/L, SRC-1, SMRT, TIF2, ARA55, and FHL2 were expressed in human prostate cancer cell lines and prostate stroma cells with the exceptions of ARA55 and FHL2, but no significant differences in expression among the cells were observed.15
Recently, Lanz et al. reported that they have identified a new co-activator termed steroid receptor RNA activator (SRA), which functioned as an RNA transcript rather than a protein based on the experimental results that protein expression of SRA was unsuccessful and that the transfected SRA functioned in the presence of the de novo protein synthesis inhibitor cycloheximide.16 It was further shown to activate the progesterone receptor, the glucocorticoid receptor, and the estrogen receptors α and β.
On the other hand, we have serendipitously isolated from a rat prostate library a rat orthologue of SRA that has 78.2% nucleotide sequence identity with the reported human SRA, and have demonstrated that it functioned as a protein.17
Subsequently, Emberley et al. reported that they have cloned cDNA for three new isoforms of human SRA termed hSRA1, hSRA2, and hSRA3. They differed from the originally reported SRA by a 5′-extension, enabling to yield a stable SRA protein with a reticulocyte system. They further showed that an endogenous SRA protein was expressed in breast cancer cell lines by Western blot analysis.18 However, the functional properties of those isoforms have not been reported.
In the present study, we have investigated the mRNA and protein expression of these human SRA isoforms and the enhancing effect of them on androgen receptor activity using human prostate cancer cell lines. We have also studied the role of the endogenous SRA in prostate cancer cells by suppressing SRA expression using an RNA interference technique.
Materials and methods
Dual-luciferase reporter assay system and pRL-TK vector were purchased from Promega (Madison, WI, USA); pCMV-Script vector and PCR-script cloning kit were from Stratagene (La Jolla, CA, USA); FuGENE 6 Transfection Reagent was from Roche (Indianapolis, IN, USA); Pyrobest DNA polymerase was from TaKaRa BIO (Shiga, Japan). The prostate cancer cell lines, PC-3, DU-145, and LNCaP cells, were purchased from American Type Culture Collection (Rockville, MD, USA). MMTV-Luc reporter plasmid19 was a generous gift from NV Organon (Oss, The Netherlands). A human androgen receptor expression plasmid pSG5AR was generously provided by Dr Chawnshang Chang, University of Rochester (Rochester, NY, USA). An anti-androgen, flutamide, was a generous gift from Nippon Kayaku Corp (Tokyo, Japan).
Cloning of human steroid receptor RNA activator cDNA
Reverse transcription-polymerase chain reaction (RT-PCR) was carried out with 1 μg of total RNA isolated from LNCaP cells using Pyrobest DNA polymerase and a set of primers 5′-IndexTermATGACGCGCTGCCCCGCTGG-3′ (hSRA-F) and 5′-IndexTermTTATGAAGCCTGCTGGAAGC-3′ (hSRA-R). The PCR product was subcloned into the SrfI site of pCMV-script vector. Sequence analysis revealed that the amplified DNA has an identical sequence as hSRA3. An expression plasmid for hSRA1 and that for hSRA2 were constructed by site-directed mutagenesis PCR. In short, two sets of PCR were performed with hSRA3 as a template using a primer set hSRA-F and hSRA1-R (5′-IndexTermTCTCAGCACATCCTCCATCACAGCCTCAGACTCGACTGG-3′) and another primer set hSRA-R and hSRA1-F (5′-IndexTermCCAGTCGAGTCTGAGGCTGTGATGGAGGATGTGCTGAGA-3′). Then, further PCR was performed using the PCR products above as templates with primers hSRA-F and -R. The amplified DNA containing hSRA1 sequence was subcloned into the SrfI site of pCMV-script vector, the resulting construct termed as pCMV-hSRA1. pCMV-hSRA2 was constructed by the same method as pCMV-hSRA1.
Reverse transcription-PCR for detecting human steroid receptor RNA activator isoforms in prostate cancer cells
Reverse transcription-PCR was carried out with 1 μg of total RNA extracted from LNCaP, PC-3′, and DU-145 cells. The primers used were 5′-IndexTermGGGCCTCCACCTCCTTCAAGTA-3′ and 5′-IndexTermCACATCCTCCATCAGTCG-3′ for hSRA-3, and 5′-IndexTermGGGCCTCCACCTCCTTCAAGTA-3′ and 5′-IndexTermCTCAGCACATCCTCCATCAC-3′ for hSRA1 and hSRA2. Each PCR consisted of 40 cycles.
Cell culture and transient transfection
Human prostate cancer cell lines (DU-145 and PC-3 cells) and HeLa cells were maintained in Dulbecco's modified Eagle's medium (D-MEM) that contains 10% fetal calf serum (FCS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin. LNCaP cells were maintained in RPMI1640 supplemented with 10% FCS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. Cells (1.0 × 106 per each plate) were plated onto 6-cm culture plates, incubated for 24 h, and used for assay. PC-3, DU-145, and HeLa cells plated onto 6-cm plates were transiently transfected using FuGENE transfection reagent with 3 μg of DNA mixture containing pMMTV-luc reporter (1 μg), pSG5AR (960 ng), pCMV-hSRA (1 μg), and pRL-TK (40 ng) in accordance with the manufacturer's instructions and grown for an additional 24 h in D-MEM with 10% fetal bovine dialyzed (10 000 MW cutoff, Sigma, St. Louis, MO, USA) serum, penicillin and streptomycin. NCaP cells plated onto 6-cm culture plates were transiently transfected using FuGENE transfection reagent with pMMTV-luc (1 μg), pRL-TK (40 ng), and the negative control vector (NCV) or phSRAsiRNA1, 2, or 3 (1 μg) according to the manufacturer's instructions and grown for an additional 24 h in D-MEM with 10% fetal bovine dialyzed (10 000 MW cutoff, Sigma) serum, penicillin, and streptomycin. Then, 5α-dihydrotestosterone (DHT) with/without flutamide (50 μ M) was added and cells were cultured for another 24 h.
The luciferase assay was carried out using a Dual-luciferase reporter assay system (Promega) in accordance with the methods recommended by the manufacturer. In short, cells were rinsed with PBS and harvested following the addition of the lysis buffer by scraping with a rubber policeman. The cells were subjected to two freeze/thaw cycles for complete lysis, and the lysate was cleared by centrifugation for 1 min. After preparing Luciferase Assay Reagent II and Stop & Glo Reagent supplied in the kit, 20 μl of the cell lysate was added to 100 μl of the Luciferase Assay Reagent II in a luminometer tube and mixed. The firefly luciferase activity was measured using a luminometer with the program of a 2-s premeasurement delay followed by a 10-s measurement period for each assay. Then, 100 μl of Stop & Glo Reagent was added and the second measurement of the renilla luciferase activity was done. The luciferase activities were normalized using the activity of the renilla expression vector pRL-TK co-transfected with the reporter plasmid.
The DNA containing the small interfering RNA (siRNA) sequence that targets hSRA was synthesized by TaKaRa BIO, and it was subcloned into pcPURU6βi. The siRNA expression vector contains a sense sequence of the target, a loop sequence, and an antisense sequence of the target gene. Thus, the transcribed RNA forms a hairpin structure by annealing, which is designed to be recognized and cut by dicer to form a small double-stranded, interfering RNA (siRNA). The three targeting sequences for the siRNA, which are common among the three hSRA isoforms, are nucleotide numbers 687–706 (hSRA-siRNA1), 507–526 (hSRA-siRNA2), and 239–258 (hSRA-siRNA3), and the corresponding expression plasmids named phSRAsiRNA1, 2, and 3 were constructed, respectively. As a negative control, we used pcPURU6βI-stop that does not express the hairpin-structured RNA.
Immunoblot analysis was carried out according to the method of Towbin et al.,20 using 100 μg of the cell lysate for electrophoresis.
Anti-human steroid receptor RNA activator antibody
A rabbit polyclonal antibody (anti-hSRA antibody) raised against the 31-mer peptide IndexTermPGNKERGWNDPPQFSYGLQTQAGGPRRSLLC was obtained from TaKaRa BIO. Immunodetection of SRA protein was performed with the anti-hSRA antisera as a primary antibody and a goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibody as a secondary one.
In order to isolate hSRA cDNA from prostate cancer cells, RT-PCR was carried out with total RNA purified from LNCaP cells. The sequences of the primers used for the PCR were common among three hSRA isoforms, but the amplified cDNA from LNCaP cells had an identical sequence to hSRA3, which was subcloned into pCMV-script vector. Then, we have constructed an expression plasmid for hSRA1 (a splicing variant of hSRA3) and hSRA2 using site-directed mutagenesis PCR as described in Materials and methods section.
We investigated the difference in expression of each isoform among several prostate cancer cell lines. As shown in Figure 1a, mRNA of hSRA3 was expressed at a higher level in androgen-independent PC-3 and DU-145 cells than androgen-dependent LNCaP cells. On the other hand, expression levels of hSRA1 and hSRA2 were higher in LNCaP cells than those in PC-3 and DU-145 cells (Figure 1b). In order to investigate the existence of an endogenous hSRA protein in several prostate cancer cells, Western blot analysis was carried out using the anti-hSRA antibody raised against the 31-mer peptide. As shown in Figure 2, a doublet of the predicted size range was seen. As described by Chooniedass-Kothari et al. previously,21 the possible explanation for the lower band of the doublet is that it results from the alternative use of a downstream ATG as an initiation which is located close to the initiation codon. Western blot analysis showed that hSRA protein was expressed at a higher level in AR-negative PC-3 cells than in LNCaP cells that have a mutated AR. However, DU-145 cells, which are AR-negative, expressed hSRA protein at much lower levels (Figure 2).
The effects of hSRA3, hSRA2, and hSRA1 on transactivation activities of AR were measured using reporter assay with MMTV-luc as a reporter gene. As shown in Figure 3, activities of AR were enhanced by co-transfection of hSRA3 in PC-3, DU-145, and HeLa cells in a ligand-dependent manner. Co-transfection of hSRA2 and hSRA1 also enhanced transactivation activities of AR in HeLa cells (Figure 4). The results show that all the full-length isoforms of human SRA are capable of enhancing AR activities.
As we have shown the expression of endogenous hSRA protein in prostate cancer cells by Western blot analysis, we then investigated whether the endogenous hSRA in prostate cancer cells participates in an activation of AR, employing RNA interference technique. LNCaP cells were transfected with an expression vector of siRNAs, phSRAsiRNA1, phSRAsiRNA2, or phSRAsiRNA3, of which targeting sequences are common among the three isoforms, and reporter assays were performed with MMTV-luc as a reporter gene. As a negative control, pcPURU6βI-stop that does not express the hairpin-structured RNA was transfected. As shown in Figure 5, AR activities in LNCaP cells transfected with hSRA-siRNA1-, 2-, or 3-expressing vector were all suppressed compared with those with the negative control vector. The AR activities in PC-3 cells transfected with pSG5AR were also suppressed by transfection of these hSRA-siRNA-expressing plasmids. These results indicate that the endogenous SRA protein plays an important role in activating the wild-type AR transfected in PC-3 cells as well as the endogenous AR mutated at codon 877 in LNCaP cells.
Steroid receptor RNA activator was first isolated by Lanz et al.,16 who reported that it enhanced transactivation activities of steroid receptors as an RNA molecule rather than a protein. On the contrary, we have previously cloned from a rat prostate library a cDNA termed SRAP that has 78.2% nucleotide sequence similarity with the reported SRA, and we have demonstrated that it needs to be translated in order to function as a co-activator, showing that incorporation of mutations in SRAP that cause frameshift with an early termination and an insertion of a stop codon adjacent to the initiation codon of SRAP resulted in loss of enhancement of the transactivation activity.17 Thereafter, Emberley et al. reported that they have cloned cDNA for three isoforms of human SRA, hSRA1, 2, and 3, and that the originally reported SRA and our SRAP were partial clones missing the N-terminal region, emphasizing the necessity of characterizing the full-length SRA protein.18, 21 They have also successfully shown in vitro translation of these SRA isoforms using reticulocyte system,18 suggesting that the 5′-extension, that is, the N-terminal region, is required for the stable SRA protein expression.
In the present study, we demonstrated for the first time that the full-length human SRA isoforms were capable of enhancing AR activities in prostate cancer cells. Co-transfection of hSRA-siRNA-expressing plasmids suppressed both the native AR activity in PC-3 cells transfected with pSG5AR and the mutant AR activity in LNCaP cells, confirming the role of the endogenous hSRA as a relevant factor in AR-mediated transcription.
Intriguingly, the expression of SRA seems to be altered in human breast cancer tissue samples compared with normal tissue,22, 23 prompting us to extrapolate the possible involvement of this co-activator in progression of prostate cancer cells. We have previously shown that mRNA of SRA was expressed at a higher level in androgen-independent MAT-Lu and AT-2 cells than androgen-dependent G cells in Dunning rat prostate cancer cell lines.17 In the present study, we have shown that an endogenous hSRA protein was expressed in prostate cancer cells, and that the hSRA protein was expressed at a higher level in PC-3 than in LNCaP cells. However, DU-145 cells were shown to express the hSRA protein at much lower levels. Further study is required in order to associate hSRA and hormone-independent progression of prostate cancer.
The mechanisms by which SRA co-activates steroid receptors require further study. Deblois et al. showed that SRA enhanced the ER-α but not ER-β activity through the AF-1 domain and that a mutation at serine 118 of ER-α ceased the enhancing effect of SRA. They also demonstrated that an activation of MAP kinase induced enhancing effect of SRA and that an MEK1 inhibitor suppressed the effect of SRA, suggesting the involvement of phosphorylation of serine 118 in the ligand-independent activation of ER-α by SRA.24 On the other hand, Coleman et al.25 reported that SRA enhanced the AF-2 activities of both ER-α and ER-β, while it enhanced the activity of AF-1 of ER-α and that an incorporation of mutations at phosphorylation sites in AF-1 of ER-α did not alter the enhancing effect of SRA, implying that phosphorylation does not play a major role in SRA function in AF-1. Further study is necessary to elucidate the molecular mechanism of SRA protein by which the activities of nuclear receptors are enhanced.
We are presently examining the expression of SRA protein in clinical specimens of prostate cancer in order to study the role of this particular protein in hormone-refractory progression of prostate cancer.
steroid receptor RNA activator
human steroid receptor RNA activator
small interfering RNA
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We thank Dr Chawnshang Chang for providing pSG5AR. We also thank NV Organon and Nippon Kayaku Corp. for the materials. We thank Keiko Onishi and Sakae Masaki for technical assistance. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, and Osaka City University Medical Research Foundation.
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Kurisu, T., Tanaka, T., Ishii, J. et al. Expression and function of human steroid receptor RNA activator in prostate cancer cells: role of endogenous hSRA protein in androgen receptor-mediated transcription. Prostate Cancer Prostatic Dis 9, 173–178 (2006). https://doi.org/10.1038/sj.pcan.4500867
- androgen receptor
- hormone-refractory prostate cancer
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