Enhancement of Vitamin D Action in Prostate Cancer through Silencing of CYP24

Purpose: Clinical trials to develop vitamin D3-based therapies for advanced prostate cancer were hindered by the risk of hypercalcemia and the limited antitumor benefit. We aimed to define factors predictive of the tumor growth response to vitamin D3 in patients with prostate cancer and specifically examine the role of its major catabolic enzyme, CYP24A1 in prostate tumorigenesis. Experimental Design: Real-time PCR and immunostaining were performed using 36 benign and cancer biopsy samples and tissue microarrays with 112 prostate cancer cores derived from 56 patients. The functional role of CYP24A1 and the impact of selective inhibition of its expression on the anti-proliferative action of vitamin D3 were examined in human prostate cancer cell lines. Results: CYP24A1 mRNA was elevated in malignant human prostate tissues compared to benign lesions. High CYP24A1 protein levels were seen in poorly differentiated and highly advanced stages of prostate cancer (P<0.0001) and correlated with parallel increased staining of Ki67, a marker of proliferation (P<0.0002). While levels of CYP24A1 mRNA in prostate cancer cells inversely correlated with their respective growth response to vitamin D3, abrogation of CYP24A1 expression using specific siRNA significantly enhanced the vitamin D3-mediated growth inhibition. Conclusions: Increased CYP24A1 expression may restrict the vitamin D3-mediated growth inhibition in patients. The use of CYP24A1 siRNA renders cancer cells more sensitive to the anti-proliferative action of vitamin D3 and could be used in combination with therapeutic doses of vitamin D3 for prostate cancer treatment. INTRODUCTION Prostate cancer is the most commonly diagnosed malignancy in men. Treatments include radical prostatectomy followed by radiation or hormone ablation therapy. Unfortunately, durable clinical responses are not always obtained and as prostate carcinoma progresses, systemic metastasis to distant areas lead to patient morbidity and mortality. Therefore, development of effective therapeutic strategies are needed for the management of castrate-resistant prostate cancer. One promising alternative is the use of the hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or calcitriol, which exerts pro-differentiative, anti-proliferative, anti-angiogenic and pro-apoptotic effects on prostate cancer cells in vitro and in vivo (see for review (1)). Phase I and II trials of calcitriol, either alone or in combination with other therapeutic agents have been conducted in patients with castrateresistant prostate cancer (2-8). However, the clinical use of vitamin D3 based therapies was hampered by the risk of hypercalcemia and/or inadequate design for the optimal administration of calcitriol. In order to improve the clinical efficacy of exogenous vitamin D3 in the treatment of prostate cancer, it may be useful to define predictive factors for the growth suppressive response of vitamin D3 in patients. In studying the action of 1,25(OH)2D3 in prostate cancer, it is essential to examine the potential pathways that control local tissue levels of 1,25(OH)2D3. The cytochrome P450 enzyme, 24hydroxylase, encoded by CYP24A1 is central to the catabolism of 1,25(OH)2D3. While its constitutive expression levels are low, CYP24A1 is strongly induced by 1,25(OH)2D3 administration to produce less active vitamin D metabolites. Overexpression of CYP24A1 was reported in various human tumors including colon (9-11), cervical (12), ovarian (9, 12), esophageal (13), lung (14) and basal cell carcinomas (15). Changes of the expression or activity of vitamin D3 catabolic enzyme CYP24A1 may affect the bioavailability of 1,25(OH)2 vitamin D3 at the tumor site and abrogate the anti-proliferative action of vitamin D3. This may lead to failure of any vitamin D3 based therapy in patients. Therefore, determining local CYP24A1 expression levels would allow for individualized treatments and higher clinical efficacy of exogenous vitamin D3. To date, no study examined the gene expression levels of endogenous CYP24A1 in the human prostate. In this present article, we evaluated the in situ expression of CYP24A1 in human adenocarcinomas and benign prostate lesions. We analyzed its possible association with tumor cell proliferation and tested in human prostate cancer cell lines, whether the inhibition of CYP24A1 expression by RNA interference, could enhance the abrogating vitamin Dmediated growth control. MATERIALS AND METHODS Cell lines and chemicals Human prostate cancer cell lines (LnCAP, PC3 and DU145) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in RPMI, DMEM or MEM containing 10% fetal bovine serum, respectively. All cells were maintained in a humidified 37C incubator with 5% CO2. Calcitriol (Sigma-Aldrich, St. Louis, MO, USA) was reconstituted in 100% ethanol, stored at -20oC and protected from light. Transfection with small interfering RNA Cells were plated in 6-well plates (1 x 10/well) for RNA extraction or in 96 well plates (1-1.5 x 10/well) for cell proliferation assays. The next day, cells were transfected for 24 hours with 40 nM of nonspecific (Scramble) or siRNA for human CYP24A1 (Dharmacon, Lafayette, CO, USA) using the Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s procedure. Following transfection, the cells were treated with either vehicle (0.1% v/v ethanol) or calcitriol (10-100 nM), for 24 h (RNA extraction) or 6 days (for cell proliferation). Cell proliferation assays Briefly, 1-1.5 x 10 cells per well were seeded into 96-well plates and treated with or without CYP24A1 siRNA for 24 h, then cultured in the presence or absence of 1,25(OH)2D3 at concentrations ranging from 1 nM to 100 nM. Viable cell number was determined after 3, 6 or 9 days of treatment using the WST1 Cell proliferation (Roche Applied Science, IN, USA) according to the manufacturer’s instructions. The plates were read using a microplate spectrophotometer at a wavelength of 450 nm and corrected to 630 nm. Each independent experiment was performed at least three times. Creation of stable cell lines harboring CYP24A1 shRNA To generate stable prostate cancer cells expressing human CYP24A1 or non-targeting shRNA, cells were incubated with lentiviral transduction particles producing a non-target control or human CYP24A1 shRNA (Sigma-Aldrich, St. Louis, MO, USA), in presence of polybrene, according to the manufacturer’s protocol. Stable clones were selected with 1 μg/mL puromycin for three weeks. Quantitative RT-PCR analysis of CYP24A1 gene expression Total RNA was purified using the RNeasy Kit including an optional DNase I treatment according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). cDNA was prepared from 500 ng of total RNA by reverse transcription. Patients’ cDNA were prepared from well-documented cancer biopsy samples, normalized and assembled into ready-to-use gene expression panels (Origene, MD, USA). The supplier provided clinicopathological information for each patient. Real-time PCR was performed with TaqMan PCR Master Mix on an ABI StepOnePlus Realtime PCR System (Applied Biosystems, USA). PCR conditions were: 50oC for 2 min, 94oC for 2 min, followed by 40 cycles of 94oC for 15 s and 60oC for 30 s. For each experimental sample, the relative abundance value was normalized to the value derived from the endogenous control (beta-actin or GAPDH) of the same sample. Relative mRNA levels were quantified by the comparative ΔΔCT method. CYP24A1 Immunofluorescence Non-confluent prostate cells were fixed in paraformaldehyde 4% for 5 min on ice. Labeling with CYP24A1 antibody (Santa-Cruz, CA USA) was carried out in the presence of 0.1% Triton X-100, followed by Alexa Fluor 488 goat anti-rabbit IgG secondary antibody. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for cell nuclei visualization. Tissue Microarrays and CYP24A1 immunohistochemical detection Tissue microarrays containing 112 prostate cancer cores, derived from 56 patients with localized and metastatic prostate cancer disease and Gleason scores ranging from 4 to 10 (Folio, OH, USA) were used in this study. Slides of paraffin-embedded adenocarcinomas samples were deparaffinized and dehydrated. Antigen retrieval was performed by heat inactivation in 0.1 M sodium citrate for 30 min. CYP24A1 (Santa Cruz, CA, USA) or Ki67 (DAKO, CA USA) primary antibodies were used for immunodetection. The staining was performed using the avidin-biotin peroxidase system (ABC-peroxidase), and positive signals were visualized as brown precipitates using 3,3'diaminobenzidine tetra-hydrochloride. Control staining was conducted by omission of the primary antibody. Hematoxylin was used for counterstaining. Two investigators, blinded to the data, performed light microscopy (Axioplan 2, Carl Zeiss, USA) and scored semi-quantitatively the quantity of protein expression in the whole section (0=none, 1= moderate, 2=strong). Scoring for semi-quantitative analysis for Ki67 staining was evaluated by the percentage of cells showing positively labeled nuclei: 0 (0-1% positivity), 1 (1-5 % positivity), 2 (5-10% positivity), 3 (10-100% positivity). Statistical analysis The data were obtained from at least two different experiments and are presented as mean± S.E.M. All statistical analyses were performed using the one-way or two-way analysis of variance (ANOVA). Contingency tables with Chi-square analysis were done using GraphPad (La Jolla, CA, USA). Values were considered statistically significant if P < .05 (* if P< .05; ** if P< .01; *** if P< .001). RESULTS CYP24A1 Was Overexpressed In Patients With Prostate Cancer To assess the mRNA levels of endogenous CYP24A1 in adenocarcinomas of the human prostate, cDNAs obtained from pathologist-verified human prostate biopsies representing different pathological stages of prostate cancer as well as benign prostatic hyperplasia (BPH) were analyzed by quantitative PCR. Comparison of the CYP24A1 gene expression profile showed statistically significant mRNA overexpression (4 fold; P= 0.03, Wilcoxon rank-sum test) in prostate cancer samples (n= 29) compared with benign tissue (n=7) (Figure 1A). Consistently, malignant tissues displayed higher levels of CYP24A1 protein than the paired cancer-adjacent normal tissues (n=8) (Figure 1B). To gain insight into specific changes associated with cancer progression, we analyzed tissue samples from 112 prostate cancer cores, derived from 56 patients with localized and metastatic prostate cancer disease and Gleason scores ranging from 4 to 10. The percentage of immunoreactive cases with CYP24A1 was high (98% of cancer cores). However, the staining intensity varied between patients, ranging from intense immunoreactivity of the entire cytoplasm to more discrete granular staining. Importantly, high protein levels of CYP24A1 positively correlated with high-grade cancer (Figure 2; P< 0.0001, Pearson’s Chi-square) as well as with late stage tumor progression (Figure 2; P< 0.0001, Pearson’s Chi-square). Increased CYP24A1 staining intensity was indeed, frequently observed in cancer cores with Gleason scores higher than 7 and in lesions presenting with 3 or 4 TNM stages (Fig 2A-D). Next, to examine whether increased CYP24A1 expression reflects increased tumor proliferation, we determined its relationship to the expression of the proliferation marker Ki67. High expression of CYP24A1 positively correlated with parallel increased staining of Ki67 in the same human prostate cancer tissues (Figure 3; P<0.0002, Pearson’s Chi square). This observation supports the hypothesis that overexpression of CYP24A1 in prostate cancer cells may reduce the ability of 1,25dihydroxyvitamin D3 to inhibit proliferation. Collectively, we demonstrate for the first time, the changes of expression of CYP24A1 in human patients with prostate cancer and highlight the positive correlation between elevated CYP24A1 protein levels and tumor progression and proliferation status. Our observations provide support for the potential key role of the vitamin D catabolizing enzyme CYP24A1 in human prostate cancer. Inverse Correlation Between Basal Levels Of Human CYP24A1 mRNA And Growth Response To Vitamin D In Prostate Cancer Cells Since CYP24A1 is the main catabolic enzyme determining the biological half-life of vitamin D3, increased CYP24A1 expression in human prostate cancer tissues would presumably decrease the intra-tumor 1,25(OH)2D3 levels, effectively counteracting its anti-proliferative effects. As the steadystate levels of cellular vitamin D3 in prostate tumor tissue of patients is difficult to measure, we analyzed the expression levels of endogenous CYP24A1 in three human prostate cancer cell lines (LnCAP, PC3 and DU145) and monitored their respective cell proliferation in presence of vitamin D3. Consistent with the findings of previous reports on CYP24A1 enzymatic activity in these prostate cell lines (16), we found an inverse correlation between the constitutive cellular levels of CYP24A1 mRNA and the growth response to vitamin D3 (Figures 4A-D). Indeed, LnCAP, with the lowest constitutive expression levels of CYP24A1 (Figure 4A), is most responsive to the anti-proliferative effect of calcitriol (Figure 4B). In contrast, PC3, which displays the highest basal levels of CYP24A1 (Figure 4A), is the most resistant to the inhibitory effect of calcitriol on cell growth (Figure 4D). Thus, the differential prostate cell response to calcitriol is due to their inherent constitutive gene expression levels of 1,25(OH)2 vitamin D3 catabolic enzyme, CYP24A1. These data support the role of vitamin D3 as a paracrine hormone to inhibit the growth of the prostate and provide a rationale basis to the use of specific inhibitors of CYP24A1 to enhance vitamin D-based therapies for prostate cancer management. Specific Inhibition Of CYP24A1 Expression By siRNA Enhanced The Anti-Proliferative Effect Of 1,25(OH)2D3 In Prostate Cancer Cells. To investigate functional consequences of CYP24 overexpression, we used a siRNA-based approach. We chose to specifically target CYP24A1 expression with small interfering RNA (siRNA) in human prostate cancer cells that are resistant to vitamin D3 (PC3 and DU145) and examine whether the alteration of CYP24A1 gene expression would enhance the growth inhibition mediated by vitamin D3. We first assessed by qRT-PCR, the efficacy of inhibition of CYP24A1 expression. The relative levels of CYP24A1 mRNA were efficiently knocked down by siRNA in absence or presence of vitamin D3 (Figure 5A). When compared to cells transfected with the non-targeting siRNA, the abrogation of expression by CYP24A1 siRNA in DU145 and PC3 cells was about 90% and 80% in presence of vehicle (ethanol) and 60 and 74 % in presence of 10 nM vitamin D3, respectively (Figures 5A-B). The knockdown of CYP24A1 mRNA translated into reduced levels of CYP24A1 protein, as reflected by the attenuation of the punctuated staining seen in cells transfected with CYP24A1 siRNA compared to control cells transfected with the scrambled control construct (Figure 5B). To assess the cellular consequences of siRNA-mediated silencing of CYP24A1 gene expression, proliferation assays were performed. While PC3 and DU145 cells were not responsive to the growth inhibition mediated by vitamin D3 (Figures 2C and 2D), CYP24A1 siRNA significantly enhanced the anti-proliferative action of vitamin D3 (Figures 5C and 5D). Taken together, knockdown of CYP24A1 gene expression by siRNA renders prostate cancer cells more sensitive to the growth-suppressive effect of vitamin D3. To assess the impact of a sustained expression of CYP24A1 siRNA on the growth abrogation mediated by vitamin D3, we generated PC3 and DU145 cell lines stably expressing CYP24A1 shRNA or non-targeting shRNA. The random integration into the genome of the vector-based shRNA led to a significant knock down of the CYP24A1 expression in absence and presence of calcitriol (Figures 6A and 6C). Indeed, when compared to cells with non-targeting shRNA, relative CYP24A1 mRNA levels were reduced by 75% in PC3 and 85% in DU145 cells in presence of vehicle, and the knockdown was about 90% for PC3 and 60% for DU145 cells in presence of vitamin D3. In these clonal conditions, PC3 cells showed an improvement of their growth inhibitory response to vitamin D3, reaching significance at 100 nM (Figure 6B). In DU145 cells, a significant reduction of cell proliferation was already achieved at 10 nM of vitamin D3 (Figure 6D). Hence, the prolonged abrogation of CYP24A1 gene expression enhanced the 1,25(OH)2D3-mediated growth inhibition in prostate cancer cells and essentially changed vitamin D3 resistant cells into vitamin D3 sensitive cell lines.