Docetaxel-based therapy is one of the first-line options for castration-resistant prostate cancer (CRPC). However, a large proportion of CRPC patients show different extents of docetaxel resistance. The current study aims to investigate the role of testicular nuclear receptor 4 (TR4) in docetaxel resistance in CRPC. TR4 expression level in prostate biopsy samples from CRPC patients treated with docetaxel was measured by immunohistochemistry (IHC). Alternation of TR4 expression in prostate cancer (PCa) cell line PC3 was applied to find out the influence of TR4 on half-maximal inhibitory concentration (IC50), cell viability and cell apoptosis. Patients who failed to achieve prostate-specific antigen (PSA) response (<50% PSA reduction from baseline) after docetaxel-based chemotherapy had a comparatively higher TR4 expression than those who achieved PSA response (50% PSA reduction from baseline). Knocking down TR4 in PC3 cells led to a lower IC50 dose, poorer cell viability and more cell apoptosis when treated with docetaxel, whereas overexpression of TR4 in PC3 led to a higher IC50 dose, better cell viability and less cell apoptosis. TR4 enhances the chemo-resistance of docetaxel in CRPC. It may serve as a biomarker to determine the prognosis of docetaxel-based therapy and as a potential therapy target to combine with docetaxel to better suppress CRPC.
Prostate cancer (PCa) is the most common malignant cancer in the male genitourinary system among western developed countries.1 Though androgen deprivation therapy (ADT) has been proven to be effective in most patients, the progression to a castration-resistant state seems inevitable.2 The chemotherapeutic agent docetaxel is one of the first-line options for castration-resistant prostate cancer (CRPC). However, 52% of CRPC patients show different extents of drug resistance after docetaxel therapy.3 The mechanism of docetaxel resistance in CRPC remains unclear.
Testicular nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily.4 As an important transcription factor, TR4 can interact with several nuclear receptors and influence intracellular reactive oxygen species (ROS), oxidative stress resistance5,6 and DNA damage reparation via its various down stream target genes.7,8 In vivo studies of TR4 gene knockout mice (TR4−/−) demonstrated that they display impaired oxidative stress defense,5 subfertility9 and premature aging.6 However, the linkage between TR4 and PCa docetaxel resistance remains unclear.
In this study, we examined TR4 expression in CRPC biopsy samples from patients receiving docetaxel-based therapy. We also applied IC50, cell viability and cell apoptosis assay to investigate the connection between TR4 and docetaxel-resistance in PCa via in vitro experiments.
Materials and Methods
Reagents and cell cultures
The PC3 human PCa cells were obtained from American Type Culture Collection (ATCC, Beijing, China) and cultured in the recommended F-12 K Ham’s medium (Gibco, Shanghai, China) with 10% fetal bovine serum (FBS) (Gibco). The cells were maintained at 37 °C in 5% CO2. All the cells used for the following experiments were in the logarithmic phase of growth. The chemotherapeutic agent docetaxel (Hengrui Medicine, Lianyungang, China) was stored at −4 °C until its use.
Plasmids and cell infection
TR4-small interfering RNA (siRNA) was cloned in pLKO.1 plasmid and TR4 gene was cloned in pWPI plasmid. Lentivirus carrying either scramble/vector control (pLKO.1-scramble/pWPI-vector) or TR4-siRNA/TR4 (pLKO.1-TR4-siRNA/pWPI-TR4) plasmids was transfected into 293T cells with a mixture of pKLO.1/pWPI, PAX2 (virus packaging plasmid) and pMD2G (envelope plasmid) (4:3:2 ratio) using Lipofectamine 2000 (Invitrogen). The virus was then collected to infect PC3 cells. After viral infection, the media was replaced with normal culture media and sub cultured. Stable clones were selected. The selected positive clones were confirmed by quantative real-time PCR (qPCR) and western blot and then named as PC3-scramble/PC3-siTR4 and PC3-vector/PC3-TR4.
Total RNAs were extracted using TIANGEN Simple RNA kit (TIANGEN, Beijing, China) according to the manufacturer’s instruction. Complementary DNAs (cDNAs) were then reverse-transcribed from RNAs using TIANGEN Quantscript RT Kit (TIANGEN). These cDNAs were used for qPCR in ABI 7500 QPCR reaction apparatus (Applied Biosystems). The primers of TR4 and glyceraldehyde 3-phosphate dehydrogenase were designed by Primer Premier 5.0 and synthetized by Biosune Biological Technology. The sequences of TR4 primers are: forward: 5′-GGCTCTGAACCTGCCTCTG-3′, reverse: 5′-AGGATGAACTGCTGTTTGGG-3′. The sequences of GAPDH primers are: forward 5′-GGAGTCAACGGATTTGGT-3′, reverse: 5′-GTGATGGGATTTCCATTGAT-3′. qPCR reaction condition: Step1: 95 °C, 2 min; Step2: 95 °C, 30 s; 60 °C, 30 s; 68 °C, 1 min; 40 cycles; Step3: 72 °C, 10 min. The results were analyzed by delta–delta Ct method.
Cells were harvested and washed with cold phosphate-buffer saline (PBS) and lysed in RIPA+PMSF (radio-immunoprecipitation assay+phenylmethanesulfonyl fluoride). The protein concentration was determined using BCA Protein Assay Kit (KeyGEN BioTECH, Nanjing, China); samples were separated on 10% SDS-PAGE gel (Beyotime, Shanghai, China), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China), and nonspecific binding was blocked using 5% milk dissolved in TBST (a mixture of Tris-buffered saline and Tween 20). Membranes were incubated with primary antibodies (R&D) overnight at 4 °C, washed in TBST solution and incubated with secondary antibodies (R&D, Shanghai, China) for 2 h at room temperature.
Immunohistochemical staining (IHC)
We collected 10 biopsy samples from CRPC patients who did not achieve PSA response (<50% PSA reduction from baseline) after docetaxel-based therapy at Sir Run Run Shaw Hospital. Another 20 biopsy samples (1:2 ratio) from patients who achieved PSA response (50% PSA reduction from baseline) after docetaxel-based therapy was selected as control group, and matched pre-treatment for PSA level and Gleason score. IHC was then performed to evaluate TR4 expression in these core biopsy samples. The result was reported by two independent pathologists in Sir Run Run Shaw Hospital in a blinded manner.
Samples were first incubated overnight with rabbit anti-human TR4 monoclonal antibody (R&D) at 0 °C and then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (Epitomics, Hangzhou, China) at room temperature for 60 min. Using the DAB chromogenic method, slides were counterstained with hematoxylin and mounted with aqueous mounting media. Slides were then observed under a light microscope and photographs were taken.
TR4 was expressed mainly in the nucleus, staining from light yellow to brownish yellow. Using the Mattern’s method,10 TR4 level was classified into four grades according to staining depth: no staining for 0 point (−), mild yellow stain for 1 point (+), moderate staining brown for 2 points (++) and tan and severe staining for 3 points (+++). Percentage of positive cells: no staining for 0 point, stained cells <25% for 1 point, stained cells 25–50% for 2 points, stained cells >50% for 3 points. Depth points and percentage points were multiplied, 0–2 multiplied points was defined as negative and >2 multiplied points was defined as positive.
Cells were seeded on 24-well plates at a density of 4 × 104 per well. After the adhesion, various concentrations of docetaxel were added. There were three replicates for each concentration of docetaxel. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg ml−1, stock solution was prepared by dissolving the compound in PBS buffer; Amresco, Shanghai, China) was added at 1:10 ratio with medium at the end of 48 h incubation and then incubated for another 4 h. The supernatant was removed, and the insoluble formazan was dissolved in Dimethyl sulfoxide (DMSO, Amresco, Shanghai, China). Absorbance was read at 550 nm. Triplicate experiments were performed and mean values+s.e.m. were presented; calculation of IC50 was carried out by Graphpad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).
Cells were seeded on 24-well plates at a density of 2 × 104 per well. After the adhesion, 0.2 μg ml−1 docetaxel was added into each well for PC3-siTR4/PC3-scramble and 0.4 μg ml−1 for PC3-TR4/PC3-vector. MTT assay was used to read the absorbance at selected time points. Triplicate experiments were performed and mean values+s.e.m. were presented, cell viability (cell inhibition rate) was analyzed using Graphpad Prism 5.0 software.
Cells were seeded on six-well plates. After the adhesion, 0.2 μg ml−1 docetaxel was added into each well for PC3-siTR4/PC3-scramble and 0.4 μg ml−1 for PC3-TR4/PC3-vector, cell apoptosis was evaluated using Annexin V-FITC/APC Apoptosis Detection Kit (KeyGEN) at the end of 48 h following the manufacturer’s instructions. Triplicate experiments were performed and mean values+s.e.m. were presented
Values were expressed as mean±s.e.m. and statistical analysis was performed using one-tailed Fisher's exact test or Student’s t test. The results were considered statistically significant if P-value was <0.05.
TR4 expression might influence the efficacy of docetaxel in PCa patients
To study the potential role of TR4 in chemo-resistance of docetaxel, we selected 10 patients who failed to achieve PSA response and 20 matched patients who achieved PSA response after docetaxel-based therapy. IHC staining was then performed in biopsy samples from these 30 patients. The results showed that TR4 expression was significantly higher in biopsy samples from patients without PSA response compared to those with PSA response (Figure 1), suggesting that higher TR4 expression in PCa tissue might be related to poor outcome of docetaxel-based therapy.
TR4 affected IC50 of docetaxel in PCa cell line PC3
To confirm the results of in vivo human clinical data, we established in vitro cell lines to examine the influence of TR4 on IC50 when treated with docetaxel. We manipulated TR4 expression by either knockdown of TR4 or addition of TR4 via lentiviral system. We then treated these cells with docetaxel and used MTT assay to analyze IC50.
We found that TR4-knocked-down cells (PC3-siTR4), compared to the scramble control cells (PC3-scramble), were more sensitive to docetaxel with an IC50 of 0.2 and 0.34 μg ml−1, respectively (Figure 2b). In TR4-overexpressed cells (PC3-TR4), IC50 was 0.75 μg ml−1, while the IC50 of vector control cells was 0.43 μg ml−1 (Figure 2d). This alternation in IC50 suggested that higher TR4 expression led to increased chemo-resistance of docetaxel in PCa.
TR4 showed an impact on cell viability in PC3 cells treated with docetaxel as a long-term effect
We then applied cell viability assay to evaluate the long-term effect of TR4 on PCa cells treated with docetaxel. Our pre-experiments showed no differences in cell viability between PC3-siTR4 and PC3-scramble; the same occurs between PC3-TR4 and PC3-vector. However, docetaxel-treated PC3-siTR4 showed a higher percentage of cell deaths at each time point (0 h, 24 h, 48 h, 72 h, 96 h) than PC3-scramble (Figure 3a). Whereas PC3-TR4 showed a low percentage of cell deaths than PC3-vector (Figure 3b). These results indicate that in docetaxel-based therapy, the cells viability, a long-term effect of chemo-resistance, was enhanced by higher TR4 expression.
TR4 expression alters cell apoptosis of PCa cells treated with docetaxel
We also performed apoptosis assay using Annexin V-FITC/APC double staining. In TR4-knocked-down cells (PC3-siTR4), the apoptotic death is significantly more than scramble control (PC3-scramble) (Figure 4a). In contrast, the apoptotic death was significantly less in TR4-overexpressed cells (PC3-TR4) than vector control (PC3-vector) (Figure 4b). These results demonstrate TR4 enhanced the chemo-resistance of docetaxel possibly by suppressing cell apoptosis.
Together, the results from Figures 2, 3, 4 showed that targeting TR4 influenced the IC50, cell viability and cell apoptosis, suggesting TR4 enhanced the chemo-resistance of docetaxel in prostate cancer.
PCa is the third leading cancer-related cause of death among men of the Western world.11 Androgen deprivation therapy is applied as first-line treatment and has been reported to be effective in relieving disease-related symptoms as well as decreasing serum prostate-specific antigen. Despite these effects, a lot of patients still progress to CRPC.
In 2004, the TAX327 study reported that docetaxel extended the overall survival of CRPC patients for about 2.4 months in comparison with mitoxantrone, which showed no effect with respect to survival time.3 Since then, docetaxel-based therapies have remained first-line option for CRPC patients. Unfortunately, a great number of patients respond poorly to docetaxel owing to inherent or acquired drug resistance. There is a great need for biomarkers that can be used to determine the prognosis of CRPC patients receiving docetaxel-based therapy. Besides, there is also an urgent, unmet demand for new therapeutic agents to improve chemosensitivity to docetaxel in PCa patients.
TR4 was initially cloned from human and rat testes in 1994.12 Studies since then have reported the importance of TR4 in many physiological functions.5, 6,13 In this study, we aimed to investigate the role of TR4 in docetaxel resistance in prostate cancer. Our IHC results showed a higher TR4 expression level in patients with worse outcomes after docetaxel-based therapy, indicating that TR4 might be related to docetaxel resistance and might serve as a potential biomarker to determine the prognosis of CRPC patients receiving docetaxel-based therapy. Ideally, as with new therapeutics, the clinical utility of a predictive or prognostic tumor marker would be established with a high level of evidence generated in large, prospective trials.14 However, in our study, the sample size and observation time were limited. Large-scaled clinical studies following the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelines15 are required to further confirm this finding. By downregulating or upregulating TR4 in PC3 cells, our in vitro experiments found that TR4 enhanced chemo-resistance to docetaxel in PCa. On the basis of these results, we believe that targeting TR4 may serve as a potential therapeutic approach to increase docetaxel sensitivity in PCa. Although no specific ligands of TR4 have been found, our previous studies demonstrate that compounds such as metformin and polyunsaturated fatty acids (PUFAs)4,16,17 can regulate TR4 activity. We hope a specific and high-efficient TR4 suppressor would bring new options for battling CRPC.
In summary, this study provides clinical evidence of TR4 gene expression related to docetaxel resistance in prostate cancer for the first time. Our in vitro experiments also showed that the chemosensitivity of docetaxel in PCa cells can be influenced by altering TR4 expression. TR4 may serve as a biomarker to predict the prognosis of docetaxel-based therapy. A combination of docetaxel and TR4-targeting agent may become a new, potential approach to battle prostate cancer in the future.
This study is funded by The Qianjiang Talents Project of Zhejiang Province (grant number: 2011R10039), The Natural Science Foundation of Zhejiang Province (grant number: Y2110446), The National Natural Science Foundation of China (grant number: 30973001), Medical Platform Key Funding of Zhejiang Province (2014ZDA011), Medical General project of Zhejiang Province (2014KYB134) and The National Basic Research Program (973) of China (grant number: 2012CB518304).