Novel Therapeutic Targets For Castration-resistant Prostate Cancer Research Articles

Linc00963 Promote Cell Proliferation and Tumor Growth in Castration-Resistant Prostate Cancer by Modulating miR-655/TRIM24 Axis.

Previous studies have shown that both long intergenic non-coding RNA 00963 (Linc00963) and tripartite motif containing 24 (TRIM24) are activators of the PI3K/AKT pathway, and both are involved in the carcinogenesis and progression of prostate cancer. However, the regulatory mechanisms between Linc00963 and TRIM24 are still unclear. In this study, we aimed to elucidate the underlying relationship between Linc00963 and TRIM24 in castration-resistant prostate cancer (CRPC). We found that TRIM24, an established oncogene in CRPC, was positively correlated with Linc00963 in prostate cancer tissues. In addition, TRIM24 was positively regulated by Lin00963 in CRPC cells. Mechanistically, TRIM24 was the direct target of microRNA-655 (miR-655) in CRPC cells, and Linc00963 could competitively bind miR-655 and upregulate TRIM24 expression. Using gain- and loss-of- function assays and rescue assays, we identified that miR-655 inhibits TRIM24 expression and cell proliferation and colony forming ability in CRPC, and that Linc00963 promotes TRIM24 expression, cell proliferation, and colony forming ability of CRPC cells by directly suppressing miR-655 expression. We further identified that Linc00963 could promote tumor growth of CRPC cells by inhibiting miR-655 and upregulating TRIM24 axis in vivo. Taken together, our study reveals a new mechanism for the Linc00963/miR-655/TRIM24 competing endogenous RNA (ceRNA) network in accelerating cell proliferation in CRPC in vitro and in vivo, and suggests that Linc00963 could be considered a novel therapeutic target for CRPC.

Long Non-coding RNA SNHG17 Promotes Cell Proliferation and Invasion in Castration-Resistant Prostate Cancer by Targeting the miR-144/CD51 Axis.

Previously, we found that the expression of long non-coding RNA (lncRNA) small nucleolar RNA host gene 17 (SNHG17) was up-regulated in castration-resistant prostate cancer (CRPC) cells compared to that in hormone sensitive prostate cancer (HSPC) cells. Moreover, we found that CD51 was up-regulated in prostate cancer cells and promoted the carcinogenesis and progression of prostate cancer. However, the regulatory mechanism of SNHG17 and CD51 in the development of CRPC remains unclear. In the current study, we aimed to elucidate the expressions, functions, and underlying mechanism of SNHG17 and CD51 in CRPC. Our results further confirmed that both SNHG17 and CD51 were up-regulated in CRPC tissues and cells. In addition, we found that SNHG17 expression was positively correlated with CD51 expression in prostate cancer. Mechanically, SNHG17 functioned as a competing endogenous RNA (ceRNA) to up-regulate CD51 expression through competitively sponging microRNA-144 (miR-144), and CD51 was identified as a direct downstream target of miR-144 in CRPC. Functionally, down-regulation of SNHG17 or up-regulation of miR-144 inhibited the proliferation, migration, and invasion of CRPC cells, whereas up-regulation of SNHG17 and down-regulation of miR-144 promoted the proliferation, migration and invasion of CRPC cells in vitro and in vivo. Using gain and loss-of function assay and rescue assay, we showed that miR-144 inhibited cell proliferation, migration and invasion by directly inhibiting CD51 expression, and SNHG17 promoted cell proliferation, migration and invasion by directly enhancing CD51 expression in CRPC cells. Taken together, our study reveals the role of the SNHG17/miR-144/CD51 axis in accelerating CRPC cell proliferation and invasion, and suggests that SNHG17 may serve as a novel therapeutic target for CRPC.

The role of actinin-4 (ACTN4) in exosomes as a potential novel therapeutic target in castration-resistant prostate cancer

Prostate cancer is the second leading cause of cancer death in men in the United States. Several novel therapeutic agents have been developed for castration-resistant prostate cancer (CRPC), but the prognosis for patients with CRPC remains poor. The identification of novel therapeutic targets for CRPC is an urgent issue. Exosomes are small vesicles secreted by a variety of cells, and exosomes derived from cancer cells have been reported to circulate in the patient’s bodily fluids, promoting metastasis and invasion. We aimed to identify novel therapeutic targets for CRPC by proteomic analysis of serum exosomes. Exosomes were isolated by ultracentrifugation of sera from 36 men with metastatic prostate cancer: untreated (n = 8), well-controlled with primary androgen deprivation therapy (ADT) (n = 8), and CRPC (n = 20). We identified 823 proteins in the serum exosomes. Six proteins were increased in CRPC patients compared with untreated patients. In contrast, only ACTN4 was increased in the CRPC patients compared to the ADT patients. We focused on ACTN4 as a candidate for targeted therapeutics. ACTN4 was highly expressed in the prostate cancer cell line DU145 as well as exosomes from this line. RNA interference-mediated downregulation of ACTN4 significantly attenuated cell proliferation and invasion in DU145 cells. ACTN4 could be a potential therapeutic target for CRPC.

Aromatase-induced endogenous estrogen promotes tumour metastasis through estrogen receptor-α/matrix metalloproteinase 12 axis activation in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) following androgen deprivation therapy remains a major obstacle advanced prostate cancer management. Aromatase catalyzes estrogen from androgens, yet the role of aromatase-generated endogenous estrogen in CRPC is poorly understood. In this study, we assessed the expression and function of aromatase in CRPC. We found that aromatase expression was significantly increased in CRPC tissues and cell lines. In some prostate cancer cell lines, aromatase was predominantly expressed in CD44+ subsets. Bicalutamide treatment significantly increased aromatase expression, and CYP19A1 expression positively correlated with estrogen responses and epithelial-mesenchymal transition. Aromatase knockdown in PC3 cells reduced invasiveness and decreased metastasis-related gene expression. The aromatase inhibitor, letrozole, attenuated tumour metastasis in castrated PC3-xenograft mice. Mechanistically, aromatase-induced endogenous estrogen promoted estrogen receptor-α (ERα) binding to matrix metalloproteinase 12 (MMP12) promoter estrogen response element (ERE). MMP12 co-localized with CD44 on the cell membrane and MMP12 knockdown significantly reduced estradiol-induced PC3 invasion. Taken together, our findings indicated that increased endogenous estrogen, catalysed by elevated aromatase levels, enhanced MMP12 expression via ERα, participated in CRPC progression and promoted tumour metastasis. Thus, aromatase represents a potential novel therapeutic target for CRPC.

MicroRNA-144-3p inhibits cell proliferation and promotes apoptosis in castration-resistant prostate cancer by targeting CEP55.

MicroRNA-144-3p (miR-144-3p) has been implicated in the tumorigenesis of multiple types of cancer. However, its role in castration-resistant prostate cancer (CRPC) remains largely unknown. This study aimed to explore the biological role of miR-144-3p in the development of CRPC. RT-qPCR was performed to measure the expression levels of miR-144-3p in CRPC tissues. CRPC cells were transfected with miR-144-3p or NC. MTT and colony formation assays were used to determine cell growth; flow cytometry was used to measure apoptosis; a luciferase reporter assay was used to predict the target genes regulated by miR-144-3p. Finally, siCEP55 or siNC was transfected into DU145 cells, and the rates of cell proliferation and apoptosis were measured. Protein expression levels were confirmed by Western blot analysis. MiR-144-3p expression was significantly decreased in both CRPC tissues and cell lines compared with that from androgen-dependent prostate cancer (ADPC) tumors. Overexpression of miR-144-3p in CRPC cells effectively inhibited proliferation and colony formation and promoted apoptosis in these CRPC cells. Additionally, miR-144-3p directly targeted centrosomal protein 55 (CEP55) and suppressed CEP55 expression. Finally, CEP55 silencing remarkably suppressed proliferation and induced apoptosis of CRPC cells, indicating that miR-144-3p affects CRPC cell survival and proliferation by downregulating CEP55. MiR-144-3p serves as a tumor suppressor in CRPC cells by directly targeting CEP55, which appears to be a novel therapeutic target for CRPC.

Interleukin-6/signal transducer and activator of transcription 3 promotes prostate cancer resistance to androgen deprivation therapy via regulating pituitary tumor transforming gene 1 expression.

Prostate cancer can progress from androgen dependence to androgen deprivation resistance with some unknown mechanisms. The current study aims to explore the possible role of pituitary tumor transforming gene1 (PTTG1) in castration‐resistant prostate cancer (CRPC). Initially, we found that PTTG1 expression was significantly increased in androgen‐independent prostate cancer cell lines PC3, DU145 and CRPC specimens compared with that in androgen‐dependent prostate cancer cell line LNCaP and initial prostate cancer specimens. PTTG1 overexpression significantly enhanced the cell survival rate, clonality and tumorigenicity in LNCaP cells upon androgen‐deprivation therapy (ADT). While knockdown of PTTG1 expression significantly elevated the sensitivity of DU145 cells to ADT. The effects of PTTG1 overexpression on LNCaP cells may be ascribed to the induced EMT and increased CD44+ CD24‐ cancer stem cell population. Furthermore, we detected that PTTG1 expression was regulated by interleukin‐6 via activated signal transducer and activator of transcription 3 (STAT3) directly binding to the region −500 to +1 of PTTG1 promoter in LNCaP cells. In conclusion, our results elucidate that interleukin‐6/STAT3 activation can increase PTTG1 expression and, consequently, promote the resistance to ADT in CRPC by inducing EMT and increasing the cancer stem cell population, suggesting that PTTG1 may be a novel therapeutic target for CRPC.

Impact of novel miR-145-3p regulatory networks on survival in patients with castration-resistant prostate cancer

Background:Despite recent advancements, metastatic castration-resistant prostate cancer (CRPC) is not considered curative. Novel approaches for identification of therapeutic targets of CRPC are needed.Methods:Next-generation sequencing revealed 945–1248 miRNAs from each lethal mCRPC sample. We constructed miRNA expression signatures of CRPC by comparing the expression of miRNAs between CRPC and normal prostate tissue or hormone-sensitive prostate cancer (HSPC). Genome-wide gene expression studies and in silico analyses were carried out to predict miRNA regulation and investigate the functional significance and clinical utility of the novel oncogenic pathways regulated by these miRNAs in prostate cancer (PCa).Results:Based on the novel miRNA expression signature of CRPC, miR-145-5p and miR-145-3p were downregulated in CRPC. By focusing on miR-145-3p, which is a passenger strand and has not been well studied in previous reports, we showed that miR-145-3p targeted 4 key molecules, i.e., MELK, NCAPG, BUB1, and CDK1, in CPRC. These 4 genes significantly predicted survival in patients with PCa.Conclusions:Small RNA sequencing for lethal CRPC and in silico analyses provided novel therapeutic targets for CRPC.

RNF7 knockdown inhibits prostate cancer tumorigenesis by inactivation of ERK1/2 pathway

Development of castration resistance is a key contributor to mortality in patients with prostate cancer. High expression of RING finger protein 7 (RNF7) in cancer cells is known to play a key role in tumor progression. However, the role of RNF7 in prostate cancer progression is not well elucidated. In this study, we silenced RNF7 by shRNA interference in two castration resistant prostate cancer (CRPC) cell lines, DU145 and PC3. RNF7 knockdown attenuated proliferation and enhanced sensitivity of prostate cancer cells to cisplatin treatment. Invasive property of DU145 and PC3 cells was also attenuated by RNF7 silencing. The underlying mechanisms appear to be associated with accumulation of tumor suppressive proteins p21, p27 and NOXA, while inactivation of ERK1/2 by RNF7 knockdown. We demonstrated that RNF7 knockdown induced growth suppression of prostate cancer cells and inactivated ERK1/2 pathway, which suggested RNF7 might be a potential novel therapeutic target for CRPC.

Role of protein S in castration-resistant prostate cancer-like cells.

Understanding how castration-resistant prostate cancer (CRPC) cells survive the androgen-deprivation condition is crucial for treatment of this advanced prostate cancer (PCa). Here, we reported for the first time the up-regulation of protein S (PROS), an anticoagulant plasma glycoprotein with multiple biological functions, in androgen-insensitive PCa cells and in experimentally induced castration-resistant PCa cells. Overexpression of exogenous PROS in LNCaP cells reduced androgen deprivation-induced apoptosis and enhanced anchorage-dependent clonogenic ability under androgen deprivation condition. Reciprocally, PROS1 knockdown inhibited cell invasiveness and migration, caused the growth inhibition of castration-resistant tumor xenograft under androgen-depleted conditions, and potentiated Taxol (a widely prescribed anti-neoplastic agent)-mediated cell death in PC3 cells. Furthermore, PROS overexpression significantly stimulated AKT activation but failed to evoke oxidative stress in LNCaP cells under normal condition, suggesting that the malignance-promoting effects of the above-mentioned pathway may occur in the order of oxidative stress/PROS/AKT. The potential mechanism may be due to control of oxidative stress-elicited activation of PI3K-AKT-mTOR pathway. Taken together, our gain-of-function, loss-of-function analyses suggest that PROS may facilitate cell proliferation and promote castration resistance in human castration-resistant PCa-like cells via its apoptosis-regulating property. Future study emphasizing on delineating how PROS regulate cellular processes controlling transformation during the development of castration resistance should open new doors for the development of novel therapeutic targets for CRPC.

Somatic copy number alterations by whole‐exome sequencing implicates YWHAZ and PTK2 in castration‐resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.

Abstract B97: Somatic copy number alterations by whole-exome sequencing reveals YWHAZ and PTK2 as potential therapeutic targets in castration-resistant prostate cancer

Abstract Aim: Castration resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) with a poor prognosis, and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNA) by whole exome sequencing on five CRPC/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform. Methods: Genomic DNA was extracted from 5 CRPC/normal paired FFPE samples and sequenced using the SOLiD4 next generation sequencing platform. The sequencing output was mapped, sorted, filtered and annotated using human genome databases. Among a set of prominent amplified/deleted genes in PCa, the 8q12.2-24.22 region encoding PTK2 and YWHAZ was found to be amplified. The data was validated using fluorescence in-situ hybridization (FISH) assays with a PCa progression cohort containing 137 localized PCa samples, 105 primary tumors, 71 corresponding lymph node metastases and 39 CRPC samples. Additionally PTK2 and YWHAZ amplification status, mRNA expression (LightCycler) and protein expression (Western Blot) were determined in selected PCa cell lines. Within PC-3 cells, inhibition of PTK2 by the specific inhibitor TAE226 (Novartis), as well as knock down of YWHAZ were analyzed. The effect of PTK2 inhibition and YWHAZ knock down on cell proliferation (MTT assay) and cell-invasion (Matrigel invasion chambers) were determined. Results: Whole exome sequencing analysis identified regions of deletion and amplification, which included well-known genes such as PTEN, CMYC and AR. Furthermore, we identified PTK2 and YWHAZ on chromosome 8 to be amplified in the sequenced PCa samples. The FISH analysis of the region showed an increasing amplification frequency of PTK2 and YWHAZ with progression of the disease. PTK2 was amplified in 1% of the localized PCa and in 35% of the CRPC samples. YWHAZ was amplified in 4% of the localized PCa and in 48% of the CRPC samples. The FISH analysis revealed a high level amplification for PTK2/YWHAZ, in the PC-3 cells, which also exhibited a high PTK2/YWHAZ protein expression. PTK2 inhibition by TAE226 and YWHAZ knock down had a significant effect on cell proliferation and migration in the PC-3 cells in vitro. PC-3 cells treated with TAE226 or YWHAZ knocked down cells had a significantly reduced cell proliferation and cell migration ability compared to untreated cells. Conclusion: Our findings suggest that the inhibition of PTK2 and YWHAZ could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. The validated whole exome sequencing data shows that FFPE tissue could be a promising alternative for SCNA screening using next generation sequencing technologies. Citation Format: Roopika Menon, Kerstin Rüenauver, Mario Deng, Friedrich Kunze, Diana Boehm, Wenzel Vogel, Veit Scheble, Falko Fend, Glen Kristiansen, Nicolas Wernert, Nicole Oberbeckmann, Saskia Biskup, Mark Rubin, Zaki Shaikhibrahim, Sven Perner. Somatic copy number alterations by whole-exome sequencing reveals YWHAZ and PTK2 as potential therapeutic targets in castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B97.

Abstract C27: Whole-exome sequencing identifies potential therapeutic targets for castration-resistant prostate cancer

Abstract Aim: Castration resistant prostate cancer (CR-PCa) is the most aggresive form of prostate cancer (PCa) having a poor prognosis, and is a significant therapeutic challenge. The key to the development of novel therapeutic targets for CR-PCa is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to perform whole exome sequencing and gene copy number analysis on 5 CR-PCa/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform. Methods: Genomic DNA was extracted from 5 CR-PCa/normal paired FFPE samples. The DNA was subjected to targeted exon capture using the Agilent Sure Select kit. The captured DNA was sequenced using the SOLiD4 next generation sequencing platform. The sequencing output was mapped, sorted, filtered and annotated using well-known human genome databases. The results were further analyzed for SNPs and copy number variations. A set of amplified/deleted genes were validated using fluorescence in-situ hybridization (FISH) assays with a PCa progression cohort. The cohort consisted of 138 cases for localized cancer, 105 patients with primary PCa and corresponding LN metastasis, and 39 samples for castration resistant tumors. Results: Whole exome sequencing analysis identified focal regions of deletion, which included well-known tumor suppressors such as NKX3.1 and PTEN. Focal regions of amplification included well-known genes such as CMYC and AR that are known to play a role in PCa. Furthermore, we identified several amplified genes as druggable targets e.g. HDAC6, NTRK1, PLD1, SPHK1, and SIRT7. NTRK1 is a kinase that plays an active role in cell proliferation. HDAC6, PLD1, SPHK1 and SIRT7 regulate numerous complex cellular processes including signal transduction, transcription and apoptosis. Conclusions: This is the first study to use whole exome sequencing approaches on FFPE CR-PCa material to identity novel therapeutic targets. Validation studies would further shed light into the biological understanding of the disease and its plausible treatment options. Citation Format: Roopika Menon, Mario Deng, Diana Boehm, Falko Fend, Detlef Boehm, Saskia Biskup, Sven Perner. Whole-exome sequencing identifies potential therapeutic targets for castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C27.

Abstract 2715: Peroxiredoxin 3: A potential biological determinant of prostate cancer health disparities

Abstract Prostate Cancer (PCa) is the most frequently diagnosed male cancer and presents the greatest racial disparity of any cancer in the U.S., with high incidence and mortality among African American (AA) men. Eliminating these disparities requires a multi-prong approach that includes, in addition to aggressive social and public health interventions, a better understanding of the biology of prostate tumors from different ethnic groups, and the development of effective treatments for castration-resistant prostate cancer (CRPC). Our interest is to define the contribution of antioxidant and stress proteins induced by environmental factors to tumor aggressiveness and health disparities in PCa. We analyzed the expression of the peroxiredoxin (PRDX) protein family in prostate cell lines and prostate tissue microarrays (TMA), and observed a dramatic upregulation of PRDX3 in androgen responsive metastatic cell lines, particularly the AA-derived cell line MDA-PCa-2b. There was also a significant correlation between high PRDX3 tissue expression and PCa (when compared to normal tissues), advanced tumor stage, and elevated PSA (P<0.05). These observations prompted an analysis of PRDX3 protein expression in ethnicity PCa TMAs containing 150 cases from AA PCa patients and 150 cases from Caucasian (CC) PCa patients, plus normal prostate tissues. Although we did not observe significant differences in the percent of PRDX3-positive immunostained prostate tumor tissues when comparing AA vs CC tissues, high PRDX3 expression correlated with early age at diagnosis (P<0.01) and elevated Gleason score (P<0.05) in the AA group but not in the CC group (P=0.459 and P=0.3394, respectively). We also examined the association between PRDX3 prostate tumor tissue expression and PSA recurrence free survival in AA and CC patients for which disease progression data was available. PSA recurrence free survival in patients with low PRDX3 tumor expression was longer in the CC group than in the AA group (P<0.05, log rank test), suggesting a correlation with a more favorable prognosis for the CC patients. However, no statistically significant difference in PSA recurrence free survival was detected in patients with high PRDX3 expression, when both groups were compared (P=0.159). These results could be interpreted as that high PRDX3 expression in prostate tumors is associated with a less favorable prognosis regardless of ethnicity, whereas low PRDX3 expression is associated with a less favorable prognosis in AA patients compared to CC patients. The contribution of PRDX3 to these differences is unclear, but taken together the data suggest that the presence of PRDX3 (low or high) in prostate tumors from AA patients could be associated with a more aggressive disease phenotype. These results warrant further investigation of the significance of PRDX3 expression in prostate tumors, and exploiting PRDX3 as a novel therapeutic target for CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2715.

Identification of EP4 as a Potential Target for the Treatment of Castration-Resistant Prostate Cancer Using a Novel Xenograft Model

More effective therapeutic approaches for castration-resistant prostate cancer (CRPC) are urgently needed, thus reinforcing the need to understand how prostate tumors progress to castration resistance. We have established a novel mouse xenograft model of prostate cancer, KUCaP-2, which expresses the wild-type androgen receptor (AR) and which produces the prostate-specific antigen (PSA). In this model, tumors regress soon after castration, but then reproducibly restore their ability to proliferate after 1 to 2 months without AR mutation, mimicking the clinical behavior of CRPC. In the present study, we used this model to identify novel therapeutic targets for CRPC. Evaluating tumor tissues at various stages by gene expression profiling, we discovered that the prostaglandin E receptor EP4 subtype (EP4) was significantly upregulated during progression to castration resistance. Immunohistochemical results of human prostate cancer tissues confirmed that EP4 expression was higher in CRPC compared with hormone-naïve prostate cancer. Ectopic overexpression of EP4 in LNCaP cells (LNCaP-EP4 cells) drove proliferation and PSA production in the absence of androgen supplementation in vitro and in vivo. Androgen-independent proliferation of LNCaP-EP4 cells was suppressed when AR expression was attenuated by RNA interference. Treatment of LNCaP-EP4 cells with a specific EP4 antagonist, ONO-AE3-208, decreased intracellular cyclic AMP levels, suppressed PSA production in vitro, and inhibited castration-resistant growth of LNCaP-EP4 or KUCaP-2 tumors in vivo. Our findings reveal that EP4 overexpression, via AR activation, supports an important mechanism for castration-resistant progression of prostate cancer. Furthermore, they prompt further evaluation of EP4 antagonists as a novel therapeutic modality to treat CRPC.