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Despite being well recognized as the best biomarker for prostate cancer, pathophysiological roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histological analyses show the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumors specimens. By stably transfecting PSA-cDNA into various prostate cancer cell lines, we found PSA could promote the growth of AR-positive CWR22rv1 and high passage LNCaP (hormone refractory prostate cancer cells), but not that of AR-negative PC-3 and Du145 cells. Surprisingly, PSA’s protease activity is not crucial for PSA to stimulate growth and promote AR transactivaton. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating p53 pathway that result in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G1 phase. In vitro colony formation assay and in vivo xenografted tumors results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that in addition to be biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA-siRNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.
Prostate-specific antigen (PSA), a member of the kallikrein gene family is a serine protease with chymotrypsin-like activity that is expressed mainly in the prostate (1). Extensive evidence indicates that PSA and other tissue kallikrein members are involved in hormone-related tumorigenesis (2), including ovarian cancer (3), breast cancer (4), prostate cancer, lung adenocarcinoma (5), pancreatic ductal adenocarcinomas (6) and lymphoblastic leukemia (7). PSA has been known as the best biomarker for monitoring prostate cancer progression (8). In prostate tissue, PSA expression levels are correlated with clinical stage and cytological grade (9). In localized prostate tumors, T3 stage tumors produced higher tissue PSA than T2 stage tumors, while metastatic tumors have relatively low PSA expression (9). Early data suggested that PSA might promote prostate cancer growth and metastasis via its protease activity to digest IGFBP-3 (10) or hydrolyze several extracellular matrixes (11).
The androgen receptor (AR) is the primary regulator of PSA expression through androgen response elements (AREs) located in the PSA promoter (12). PC-3 cells with stably-transfected AR, but not parental PC-3 cells lacking AR, could express PSA (13). Therefore, during the hormone treatment sensitive stage, surgical or medical castration can significantly reduce PSA expression in prostate cancer tissue. But in the hormone-refractory cancer, although AR still exists (14), the abnormal PSA elevation may have little linkage with AR status. It has been reported that in addition to androgens, PSA expression may be induced by glucocorticoids (15), progestin (16), and Ets transcription factors (17). However, the significance of androgen/AR-independent PSA expression remains unclear.
Here, we find PSA may promote hormone refractory prostate cancer growth via enhancing ARA70-induced AR transactivation without involving its protease activity. Under the treatment of hydroxyflutamide (HF) or in the environment of Delta 5-androstenediol (Adiol), the increased tissue PSA may activate ARA70/AR transcription function, which may then result in tumor cell survival in the hormone refractory tissue. Therefore, targeting tissue PSA by PSA-siRNA or smaller molecules may be developed as alternative approaches to suppress prostate cancer growth.
All cell lines were obtained from the ATCC. The COS-1 and PC-3 cells were maintained at 37°C in DMEM (Gibco-BRL) supplemented with 10% charcoal deprived (CD) serum, 100 units/ml of penicillin, and 100 g/ml streptomycin under 5% CO2. High passage LNCaP cells were grown in RPMI-1640 (Life Technologies, Rockville, MD) with 10% CD serum, 100 units/ml of penicillin, and 100 μg/ml streptomycin under 5% CO2. 5 -Dihydrotestosterone (DHT) and delta5-androstenediol (Adiol) were obtained from Sigma and hydroxyflutamide (HF) was from Schering. For details of transfection and promoter reporter assay, please see our publications (18, 19). In brief, COS-1, PC-3, LNCaP and CWR22rv1 cells, grown in appropriate medium at 1-4 ×105 cells in 24-well plates, were transfected with indicated plasmids using SuperFect (Qiagen).
pSG5-AR, pSG5-ARA70N (N terminus), pSG5-ARA70f (full length), pGEX-GST-ARA70, MMTV-luc, pVP16-ARA70, and pGL-TK were constructed as described previously (18,19). Forward primer 5′-GCGGATCCGGGGAGCCCCAAGCTTACC-3′ and reverse primer 5′-CGTCTAGAGGGTGCTCAGGGGTTGGC-3′ were used to PCR amplify full-length PSA cDNA and cloned into BamHI- and XbaI-digested pcDNA3 vector. The mutant pCDNA-PSA (S213A) plasmid was generated by mutating the 213th amino acid residue from serine to alanine using QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). The siRNA target sites for PSA and ARA70 [GTGGATCAAGGACACCATC (753-771) and GAGGAGACACTTCAACAGC (384-402)] respectively, were selected by Oligoengine siRNA designing software. The negative controls for each of them were generated as scramble-siRNA. The positive controls were applied following "knock-back after knock down" strategy by generating pCDNA3-PSAkb plasmid and pSG5-ARA70kb plasmid, respectively. pCDNA3-PSAkb was generated from parental pCDNA3-PSA plasmid by mutating PSA-siRNA target sequence to GTGGATCAAAAACACCATC (753-771), while pSG5-ARA70kb was generated from parental pSG5-ARA70 plasmid by mutating ARA70-siRNA target sequence to GAGGAGACACCCCAACAGC (384-402). All the constructs were verified by DNA sequencing.
Hormone refractory prostate cancer specimens, defined by failure following HF treatment and acquired by TURP management of urethral obstruction, chosen for the study by evaluating the clinical records. They included 20 no hormone treatment 20 no hormone treatment, 4 hormone-refractory, and 15 HF treatment sensitive prostate cancer specimens. All samples were collected from the Department of Pathology of the Tianjin Institute of Urological Surgery and the Sir Run Shaw Hospital of Zhejiang University Medical School.
Samples were fixed in formalin and embedded in paraffin. The AR, ARA70 and PSA protein expression levels were determined by IHC method in 4 pairs of samples. The rabbit anti-PSA polyclonal antibody (DAKO, A0562), the mouse anti-ARA70 antibody, and the rabbit anti-AR (N20) antibody (Santa Cruz Biotechnology) were used in IHC staining. The bound primary antibody was recognized by the biotinylated secondary antibody (Vector), and visualized by VECTASTAIN ABC peroxidase system and peroxidase substrate DAB kit (Vector). The positive staining signals were semi-quantitated by Image J software.
CWR22rv1, high-passage LNCaP, PC-3 and DU145 cells were cultured to the mid- or late-logarithmic phase of growth. After trypsinization, the cells were resuspended and washed twice in 2.5% FBS medium without antibiotics. We then transferred 400 μl of the cell suspension (107 cells) into the electroporation cuvettes (VWR), set the voltages of the electroporator (Bio-RAD GENE PULSER II) to 300V and hinge capacity to 950 μF, added 20 μg of total plasmid DNA to each cuvette, and incubated for 5 min at RT. After pulse charge, the cells were incubated on ice for 5 min and transferred to a 35-mm culture dish. After culturing in complete medium for 72 h, the transfected cells were cultured in the appropriate selection medium and medium was changed every 3 days for 2-3 weeks until colonies of resistant cells formed.
For pCDNA3-PSA transfected cells, we used 1000 μg/ml neomycin for selection, while for pSuperior-siPSA or pSuperior-siARA70 transfected cells, we used 5 μg/ml puromycin.
Cells of stable transfected LNCaP, CWR22rv1, PC-3 and DU145 sublines were seeded in 24-well plates at a density of 5000 cells/well in media containing 10% CD-FBS with or without 1 nM DHT. At the indicated time point, medium was removed, serum-free medium containing MTT (0.5 mg/ml, Sigma) were then added into each well. Four hr after incubation at 37°C, cellular formazan product was dissolved with acidic isopropanol, and the absorbance at OD595 was measured by spectrophotometry (Beckman Du640B). We used 6 replicate wells for each sample at each time point. α-Antichymotrypsin (MP Biomedicals, Inc) at a concentration of 1000 ng/ml was used as a PSA proteinase inhibitor to treat the LNCaP cell sublines. For the cell cycle flow cytometry assay, we digested the cells by trypsin-EDTA, harvested as many as 1 x 106 cells, and fixed them in 70% ethanol at 4°C. After 12 hr, cells were centrifuged (1000g, 7 min, 4°C), resuspended in PBS containing 0.05 mg/ml RNase A (Sigma), and then incubated at room temperature for 30 min. After washing and staining with 10 mg/ml propidium iodide, cells were filtered through a 60 micrometer mesh, and 10,000 cells were analyzed by flow cytometry (FACSCalibur, BD Company) with MODFIT software (Verity Software House, Inc).
7-AAD (Sigma) was dissolved in acetone, diluted in phosphate-buffered saline (PBS) at a concentration of 200 μg/ml, and kept at 20°C without light until use. 100 μl of 7-AAD solution was added to 2.5 x 106 cells in 1 ml of PBS and incubated for 20 min at 4°C in the dark. Cells were then washed and total DNA was stained with 7-amino-actinomycin D (7-AAD; 20 μL per sample), followed by flow cytometric analysis using FACSort (Becton Dickinson). To assess apoptosis, 5x105 cells were resuspended in PBS, analyzed by flow cytometry. The total DNA content (7-AAD) was determined using Cell Quest (Becton Dickinson) and FCS Express software (De Novo Software). We challenged LN-vehicle and LN-PSA cells with 1 nM TPA and 1 nM DHT for 24 hr to induce apoptosis, and detected cell apoptosis by 7AAD flow cytometry. Simultaneously, we measured caspase-3 activity using Western blot by detecting its activated subunits, 17- and 12-kDa (Rabbit anti-Active Caspase-3, BD Biosciences).
Five g total RNA were extracted using Trizol and reverse transcribed into 20 μl cDNA immediately by the SuperScript III kit (Invitrogen) with oligo-dT primer. Real-time quantitative PCR was performed on iCycler IQ multicolor real-time PCR detection system with 1/5 μl cDNA amplified by SYBR Green PCR Master Mix. We designed primers by Beacon Designer 2 software as follows: NKX3.1 forward: 5′-ATGGTTCCAGAACAGACGCTAT-3′, reverse: 5′-TGCCCACGCAGTACAGGTAT-3′; PSP94 forward: 5′-TCCTGGGCAGCGTTGTGA-3′, reverse: 5′-TTGGGTGTTTGTTTCCTTTGAG-3′; PSMA forward: 5′-AAGGAAGGGTGGAGACCTAG-3′; reverse: 5′-ACTGAACTCTGGGGAAGGAC-3′; -Actin forward: 5′-TGTGCCCATCTACGAGGGGTATGC-3′, and reverse: 5′-GGTACATGGTGGTGCCGCCAGACA-3′. The β-Actin expression was used as internal control. We calculated δthreshold (CT) values by subtracting the control CT value from the corresponding β-actin CT from each time point. We confirmed the absence of nonspecific amplification products by agarose-gel electrophoresis.
10 athymic nude mice were castrated at 12 weeks old. One week following castration, we injected 5 ×107 high passage LNCaP and LN-siPSA cells into the left and right dorsal part of these nude mice, respectively. 12 weeks later, the nude mice developed obvious xenograft tumors. We harvested the tumors, measured the size and determined the PSA protein level.
We transformed the PGEX-GST-ARA70 plasmid into BL21-CodonPlus Competent cells (Stratagene) to express GST fusion ARA70 protein, and purified the fusion protein with glutathione-beads. We incubated the radiolabeled PSA protein for 2 hr with the GST-ARA70 fusion protein attached to glutathione beads. The protein complex beads were washed 4 times, resuspended in SDS-PAGE loading buffer, and resolved on 12% SDS-PAGE gel followed by autoradiography.
We incubated 500 μg protein from LNCaP cell lysates with 2 μg anti-ARA70 monoclonal antibody or normal mouse IgG for 4 hr at 4°C with agitation. Each sample was added with protein A/G plus agarose (50 μl), incubated for 1 hr, and washed three times with RIPA buffer. We resolved the complex on a 10% SDS-polyacrylamide gel, transferred to the membrane, and blotted with anti-ARA70, anti-AR, or anti-PSA monoclonal antibody, respectively. Immuno-responsive bands were developed by an alkaline phosphatase detection kit (Bio-RAD).
Cell were lysed in RIPA buffer, separated on SDS-10% PAGE gel, and then transferred to a polyvinylidene difluoride membrane. After blocking by 5% non-fat milk and 5% FBS in PBST buffer, we immunoblotted the membrane with the primary antibody, followed by incubation with AP-conjugated second antibody (Santa Cruz). Our laboratory generated the monoclonal anti-ARA70 antibody. The rabbit polyclonal anti-AR (N20) and mouse monoclonal anti-PSA antibodies, rabbit anti-cdk2, rabbit anti-cyclinD1, rabbit anti-p21, rabbit anti-PCNA, rabbit anti-RFC1, rabbit anti-bax, mouse anti-tubulin, and goat anti-βactin were from Santa Cruz. Anti-p53 monoclonal and anti-bcl2 monoclonal antibodies were from DAKO, Denmark. Rabbit anti-phospho-p53 (Ser392) antibody was from Cell Signaling Technology, Inc. We detected PSA protein amounts in equal amounts of total proteins from cell lysates and equal volumes of concentrated cultured media.
We determined the cell survival of LN-PSA and LN-siPSA in a colonogenic assay. Briefly, we plated cells (200 cells/well) in 6-well plates and cultured them in normal medium for 2 weeks. Then we fixed and stained the cells and 0.25% crystal violet in 80% methanol for 30 min, washed them with water, and counted the number of colonies that contained more than 50 cells. We determined the plating efficiency as the fraction of cells that were attached to the plate and grew into colonies larger than 1 mm diameter.
By histological analysis of Ki67 expression in the refractory prostate tumor specimens that were treated with HF, we found that the cell proliferation signals were higher in the hormone refractory tumors than those in the hormone sensitive tumors (Fig. 1a). Also, the cell apoptosis index of the hormone refractory tumors was lower than those of the hormone sensitive tumors using TUNEL assay (Fig. 1b). Tissue PSA levels are significantly higher in the hormone refractory tumors than those in hormone sensitive tumors (Fig. 1c). Higher PSA levels (Fig. 1c, iv) in the hormone refractory tissues are coincident with higher proliferation rates and lower apoptosis index in these tissues (P<0.05) (Fig. 1a and b, iv).
To test the PSA effects on the growth of prostate cancer cells, we stably transfected PSA cDNA into high passage number LNCaP (named LN-PSA), CWR22rv1 (named CWR-PSA), PC-3 (named PC3-PSA) and DU145 cells (named Du-PSA). Western blot was applied to examine the expression of ARA70 and PSA (Suppl Fig. 2a and b) in parental cells and over expression of PSA in LNCaP, CWR22rv1, PC-3, and DU145 cells. The growth rates of high passage number (n> 70) of LNCaP cells and CWR22rv1 cells are not sensitive to the androgen. Therefore, those two cell lines could represent AR-positive hormone refractory prostate cells. The cell viability assays by MTT showed that addition of PSA resulted in the increased number of living cells in AR-positive LNCaP and CWR22rv1 (Fig. 2a and b, left) cell lines, but not in AR-negative PC-3 and DU145 (Fig. 2c and d, left) cell lines with 1 nM DHT (human prostate DHT concentration after androgen deprivation therapy). The data of cell number differences between controlled groups were further interpreted into the different patterns of cell cycle distribution (Fig. 2a-d, middle) and cell death by flow cytometry analyses (Fig. 2a-d, right). Over-expression of PSA in LNCaP and CWR22rv1 cells (LN-PSA and CWR-PSA) resulted in the decreased G0-G1 phase from 69.1% to 54.7% and 49.2% to 40.4% and increased S phase from 18.8% to 29.4% and 37.5% to 44.2% (Fig. 2a and b, middle), respectively. However, ectopic PSA expression in PC3 and DU145 cells (PC3-PSA and Du-PSA in Figs. 2c and d, middle) had little influence on the cell cycle. Meanwhile, increased PSA expression in LN-PSA and CWR-PSA (Figs. 2a and b, right) cells resulted in the decreased cell death, while over-expression of PSA in PC3-PSA and Du-PSA (Fig. 2c and d, right) showed little change in the cell death. These results suggest the PSA effects on the cell cycle are evident by significant reduction in G0-G1 phase cell population. In contrast, the G0-G1 phase population of Du-vector vs Du-PSA is 53.5% vs 51.7%, which indicates little difference. The total amount of the cells reentering the cell-cycle, by counting percentage of cells into both S-phase and G2-M phases, is also similar between Du-vector and Du-PSA cells. Together with results from MTT assay, the overall cell number in proliferative (S and G2-M) and quiescent (G0-G1) phases of the cell cycle showed no significant difference between Du-vector and Du-PSA cells, suggesting that adding PSA had little influence on the cell proliferation of Du-145 cells. A similar conclusion was also reported previously by Denmeada, et al (11), showing PSA had little effect on the DU145 cell growth. The different PSA effects between different prostate cancer cell lines, which express AR differently, indicates that PSA’s growth stimulation activity could be an AR-dependent event.
To further confirm the PSA effects on the growth of AR-positive prostate cancer cells, we stably transfected PSA siRNA into high passage LNCaP (named LN-siPSA, Fig. 3a) and CWR22rv1 cells (named CWR-siPSA, Fig. 3c). Knockdown of endogenous PSA in LN-siPSA and CWR-siPSA cells resulted in retarded cell growth in MTT assays (Fig. 3a and c), consistent with increase in G0-G1 phase from 61.6% to 79.5%, and 47.8% to 61.7% (Fig. 3b and d, top), and increased cell death (Fig. 3d and h, bottom, respectively).
Our results revealed that the higher expression of PSA in human prostate refractory tumors may facilitate tumor cell survival from HF treatment by resistance to cell death and accelerating cell cycle. In contrast, we found PSA did not significantly alter the growth of AR-negative PC-3 and DU145 cell lines. Interestingly, we found PSA, unlike other general secretory proteins (20-(24), could be found in the cytosol outside of the Golgi’s apparatus (Suppl. Fig. 1c and d). We performed chromosome immunoprecipitation (ChIP) assay to analyze whether the PSA/ARA70/AR complex can bind onto the promoter of the AR target gene, and the results suggested that PSA/ARA70/AR complex might not form on the chromosome DNA. In contrast, using yeast two-hybrid screen, we found PSA inside the cell might function as an associated protein of ARA70 (data not shown). Using the co-immunoprecipitation assay, we also proved PSA/ARA70/AR form a complex (Suppl. Fig. 1a and b). Confocal microscope further clearly showed the existence of this AR-ARA70-PSA complex within the same cells (Suppl Fig. 1c). Together, these results suggested ARA70 might be required to coordinate for the formation of this complex.
To investigate whether PSA, as an ARA70 associated protein, could go through AR signals to increase cell growth; we applied AR functional study by MMTV-ARE luciferase assay. As shown in Fig. 4, addition of PSA could increase the AR transactivation in LNCaP (Fig. 4a) and CWR22rv1 cells (Fig. 4c), while suppression of endogenous PSA expression in LNCaP and CWR22rv1 cells reduced AR transactivation in the presence of 1 nM DHT (Fig. 4a and c), normalized by both positive (Fig. 4a lane 6) and negative control (Fig. 4a lane 4). Furthermore, the knockdown of ARA70 by siRNA could diminish the PSA-enhanced AR activity (Fig. 4a. lane 9 vs 3), suggesting that ARA70 is important for the PSA-enhanced AR transactivation.
To reduce the potential artificial effects due to transient transfection assays, we stably transfected either PSA-cDNA or PSA-siRNA into high passage LNCaP and CWR22rv1 cells. We then examined the PSA effects on AR transactivation in multiple sublines and results showed PSA could further enhance AR transactivation in both LNCaP and CWR22rv1 cells (data not shown). Using the stably transfected cell lines, we found PSA could also induce endogenous AR positive-regulated target genes, such as PSP94 (25) and Nkx3.1 (26), as well as suppress endogenous AR negative-regulated target genes such as PSMA (27,28) (Fig. 4b). In contrast, addition of PSA-siRNA into LNCaP cells results in opposite effects on AR target gene expressions (Fig. 4b). Western blot also shows that PSA and ARA70 are indeed silenced by PSA-siRNA and ARA70-siRNA (Fig. 3 and Suppl Fig. 2d).
As early studies suggested that the higher expression level of ARA70 could enhance the antiandrogen hydroxyflutamide (HF)- and Delta5-androstenediol (Adiol)-induced AR transactivation (29, 30), we tested whether PSA can cooperate with ARA70 to enhance HF- or Adiol-induced AR transactivation. As expected, we found PSA could enhance the ARA70-induced AR transactivation in the presence of 10 μM HF or 10 nM Adiol (Fig. 4d). This data revealed that increased tissue PSA in the hormone refractory stage (Fig. 1c, iv) could help tumor cell survival in the castration environment by activating AR transcription.
To further strengthen the above results demonstrating that PSA might need to go through the interaction with certain selective AR coregulators, such as ARA70, to induce AR transactivation, we tested whether reduced endogenous ARA70 (via siRNA) might interrupt the PSA-induced AR transactivation. As shown in both LNCaP and CWR22rv1 cells, knockdown of ARA70 by stably-transfected ARA70-siRNA results in the reduction of the PSA-induced AR transactivation (Fig. 4a, lanes 9 vs 3 in LNCaP; Fig. 4c. lanes 7 vs 2 in CWR22rv1), suggesting PSA might go through interaction with ARA70 to enhance its coactivity that results in the induction of AR transactivation. These data demonstrated that the existence of ARA70 is critical for PSA enhanced-AR transactivation.
Together, using several cell lines with either transient transfection or stable transfection of PSA, PSA-siRNA, or ARA70-siRNA to assay AR transactivation or AR endogenous target gene expression, we found PSA could promote cell growth that might go through the ARA70-induced AR transactivation.
We applied two different approaches to test whether our increased cell growth via increased PSA is protease activity dependent. We first added PSA protease inhibitor, α1-antichymotrypsin (1000 ng/ml) to the LN-PSA cells and parental control LNCaP cells and results showed α1-antichymotrypsin has limited influence on the PSA-induced cell growth in LN-PSA cells (Fig. 5a). We then mutated the essential protease domain (213 serine to 213 alanine) that inactivates the protease activity of PSA (31) and stably transfected this mutated mPSA-S213A cDNA into LNCaP cells (LN-mPSA), and showed that PSA expression levels are comparable between LN-PSA and LN-mPSA stable cells (Fig. 5b, top). The results again showed protease activity-null PSA still stimulates the growth of AR-positive prostate cancer cells (Fig. 5b). Interestingly, we also found that higher expression of wt PSA and mPSA could increase the LNCaP cell growth (Fig. 5b, bottom). Although mPSA is slightly less effective than wt PSA to enhance the prostate cancer cells growth, the difference in cell number between LN-PSA and LN-mPSA groups is not as dramatic as that between LN-mPSA and LN-vector, suggesting that PSA protease activity may have few, yet not significant, effects on LNCaP growth. Furthermore, we detected both cytosol PSA and secreted (into medium) PSA 24 hr after adding 10 nM DHT to the LNCaP cells with the passage number less than 50, which still respond to the DHT stimulation (Fig. 5c, top). However, adding these media with secreted PSA into LN-PSA cells results in little influence on the cell growth (Fig. 5c), suggesting the PSA induced cell growth effect might be elicited by the PSA existing inside the LN-PSA cells (named Tissue-PSA).Consistent with cell viability data, ectopic expression of the mutated PSA (mPSA-S213A) also enhances the ARA70-induced-AR transactivation in COS-1 cells (Fig. 5d, top) and LNCaP cells (Fig. 5d, bottom). These results clearly demonstrated that PSA, without its protease activity, could enhance ARA70-induced AR transactivation and stimulate cell growth.
Early studies suggested that androgen/AR might induce cell growth via modulation of p53-mediated cell growth arrest and apoptosis (32-34). Other studies also demonstrated that AR could modulate p53 expression (35) via several key factors, such as MDM2, HoxA5, and Egr-I. We therefore hypothesized that PSA enhanced ARA70-induced AR transactivation might result in the increased cell growth via modulation of p53-mediated cell growth arrest and apoptosis. We first challenged the LN-vector control cells and LN-PSA cells with 1 nM TPA and 1 nM DHT, a condition that was reported previously to accelerate cell apoptosis (36). Western blot analysis was also used to examine the apoptosis related markers and showed lower expression of p53 and bax and higher expression of bcl-2 in LN-PSA cells as compared to control LN-vector cells (Fig. 6a). Furthermore, lower expression of p53 was consistent with lower phosphorylation of p53 at Ser392 and lower activated form of caspase-3 (Fig. 6a) in LN-PSA cells treated by TPA as compared to LN-vector cells.
Under environmental changes, such as DNA damage or oxidative stress, p53 can be activated/stabilized to modulate a series of genes that facilitate cell cycle arrest and apoptosis (33). Functioning as a key downstream target of p53, the p21 might mediate G1 arrest via inhibition of cdks (33, 37). We found that LN-PSA cells with decreased G1 phase expressed a lower p21 and higher cdk2, cyclinD1, PCNA, and RFC1, while LN-siPSA cells with G1 arrest expressed a higher p21 and lower expression of cdk2, cyclinD1, PCNA, and RFC1 as compared to parental LNCaP cells (Fig. 6b). Together, these results demonstrate that PSA might go through the AR-p53 pathway to promote cell growth via the G1/S cell cycle checkpoint.
All the above data indicate that PSA can induce cell growth via ARA70/AR→ p53→cell apoptosis and G1 arrest, which suggests that PSA might become a new therapeutic target to treat the prostate cancer. To test this hypothesis, we applied different approaches to see if reducing endogenous PSA expression can result in the suppression of prostate cancer growth. Using the MTT cell viability assay, we first found that stably transfecting PSA-siRNA into high passage LNCaP cells (LN-siRNA) and CWR22rv1cells (CWR-siRNA) results in suppression of cell growth in the presence of 1 nM DHT (Fig. 3). Using colony formation assays we also found more colonies in LN-PSA cells, and less colonies in LN-siPSA cells, as compared to control LNCaP cells (Fig. 6c). Finally, in vivo tumor growth assays, using xenografted LNCaP (into the left flank) and LN-siPSA (into the right flank) cells in castrated nude mice also showed smaller tumors in LN-siPSA xenografts as compared to LNCaP xenografts. Four represented tumors from LNCaP and LN-siPSA were shown and quantitative tumor weight results are attached (Fig. 6d, n=10 for each group). Together, cell line MTT assays, colony formation assays, and in vivo xenograft tumor growth assays all demonstrate that targeting PSA via PSA-siRNA to reduce endogenous PSA expression is a potential new therapeutic approach to suppress prostate cancer cell growth.
Early studies suggested that PSA might modulate growth of PSA-producing cells and their surrounding cells (38,39) via its serine protease activity. PSA might promote the growth and invasion of prostate cancer via degradation of IGFBP-3, fibronectin, and laminin (2,10,40). Interestingly, Fortier et al presented evidence that PSA protein itself, without its protease activity, could also function as an endothelial cell-specific inhibitor of angiogenesis (41). Their findings, however, were countered by later findings from Isaacs et al showing the antiangiogenic effects of PSA are not significant enough in PSA-producing cells to appreciably effect tumor growth in vivo (11). Therefore, the significance of pathophysiological roles of PSA in prostate cancer, from the above studies, might depend predominantly, if not completely, on the protease activity from PSA. The development of inhibitors to block the protease activity of PSA might then have potential therapeutic advantages to battle the prostate cancer. Our findings here showing PSA, without involving its protease activity, can promote prostate cancer cell growth. Therefore, PSA may have two functions, one in invasion and one in proliferation, and that its protease activity could be important for one and not the other. These findings suggest targeting PSA itself, instead of just blocking its protease activity, might be needed to stop the PSA-induced prostate cancer progression.
An early study showed that 31 of 63 (49%) of the patients died of prostate cancer with their Tissue-PSA values increased (from 0.054 to 0.204 g Tissue-PSA/μg DNA) during androgen ablation treatment that includes either surgical or chemical castration. The average of their pre-treatment Tissue-PSA values were significantly lower as compared to other groups of patients (0.063 vs. 0.381 g Tissue-PSA/ g DNA) who were alive at the end of the observation period or died of causes other than prostate cancer (42). This is in agreement with our IHC staining (Fig. 1) showing the tissue PSA level was also higher in the hormone refractory samples than the hormone sensitive control group. The rationale for those patients that had increased tissue-PSA during treatment, yet failed to respond to androgen ablation therapy and died of prostate cancer, could be that the Tissue-PSA synthesis in these patients has become androgen insensitive (42) and other inducers, such as antiandrogens or Adiol, can then stimulate tissue-PSA synthesis (29,30).
One possible explanation for the above clinical observations and our experiments is that with expression of AR and ARA70 in those patients at the hormone refractory stage (14,43,44), antiandrogen HF or Adiol could induce AR transactivation (Fig. 4d), that results in the increased Tissue-PSA. The increased Tissue-PSA could then go through positive feed-back regulation to further enhance ARA70-induced AR transactivation that results in the suppression of p53 expression via modulation of MDM2/HoxA5/Egr-I signaling pathways (35). The consequence of AR suppression of p53 might resulting in the cell survival via the decrease of cell apoptosis via bax/bcl-2/caspase 3 signaling pathways, as well as in the decrease of cell G1 arrest via modulation of p21/cdk2/CyclinD1 signaling pathways (Fig. 6). Previous reports showed that increase in expression of the p53 and p21/WAF1 proteins is the early event during standard androgen withdrawal therapy (45). Besides, p53 was identified as a critical molecule in response to androgen deprivation in prostate from mouse model and LNCaP cells (35). In vitro studies on both LNCaP and LAPC4 cells indicated that AR promotes cell growth by abrogation of p53 mediated apoptosis (34). And mutant p53 can facilitate the androgen-independent growth of LNCaP cells (46). On the other hand, inhibition of p53 function diminishes AR-mediated signaling in prostate cancer cell lines (47). Also, a functional role for the wild type p53 gene in suppressing prostatic tumorigenesis was well documented (48, 49). And it was also documented that amplification of AR gene was associated with p53 mutation in hormone refractory prostate cancer (50). Taken together, AR-regulated p53 signaling pathway was significant for tumor progression, and PSA may play an important role in this pathway.
In summary, results from these studies show that tissue PSA, without involving its protease activity, can promote ARA70-AR mediated cell growth and facilitate refractory tumor development. This observation suggests that PSA might be treated as a new potential therapeutic target to battle the prostate cancer. Small molecules that can degrade PSA protein or siRNAs that can knockdown PSA expression, might be able to be developed in the future to inhibit the AR-mediated prostate cancer growth with fewer side effects from the AR influenced physiological functions in other tissues.
This work was supported by NIH grant DK60912, CA122295 and George Whipple Professorship Endowment.