Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death

Telomere stability is important for cell viability, as cells with telomere DNA damage that is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice variant AR-V7, but full-length AR was found to be the predominant form of AR associated with telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this combined treatment approach to induce telomere DNA damage and inhibit the DDR was effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express AR splice variant

Class III β-tubulin expression as a predictor of docetaxel-resistance in metastatic castration-resistant prostate cancer

About half of the patients treated with docetaxel in the setting of metastatic castration-resistant prostate cancer (CRPC) are non-responders. Therefore, a marker of response would be beneficial for clinical decision-making. We evaluated class III β-tubulin (βIII-tubulin) expression as a predictor of resistance in this setting, which previously has been correlated with lack of response to taxanes in other cancers. Patients with CRPC were included if they were treated with at least 3 cycles of docetaxel between 1990 and 2011. βIII-tubulin expression was assessed by immunostaining, which was performed in tissue samples obtained either via biopsy or prostatectomy at the time of diagnosis. Rates of prostate-specific antigen (PSA) response and overall survival (OS) following docetaxel treatment were compared between patients with high (2+ or 3+ staining) vs. low (0 or 1+ staining) βIII-tubulin expression. Of 73 patients, 26 (35%) had a high expression of βIII-tubulin. A PSA decline of 10% or greater occurred in 65% of patients with a high βIII-tubulin expression vs. 89% with a low βIII-tubulin expression (p = 0.0267). The median OS for patients with a high βIII-tubulin expression was 17.4 (95% CI 8.7-21.0) months vs. 19.8 (95% CI 16.6-23.6) months for patients with a low expression (p = 0.039). Our results show that a high βIII-tubulin expression is a negative prognostic factor in metastatic CRPC patients treated with docetaxel

Averaged Differential Expression for the Discovery of Biomarkers in the Blood of Patients with Prostate Cancer

<div><h3>Background</h3><p>The identification of a blood-based diagnostic marker is a goal in many areas of medicine, including the early diagnosis of prostate cancer. We describe the use of averaged differential display as an efficient mechanism for biomarker discovery in whole blood RNA. The process of averaging reduces the problem of clinical heterogeneity while simultaneously minimizing sample handling.</p> <h3>Methodology/Principal Findings</h3><p>RNA was isolated from the blood of prostate cancer patients and healthy controls. Samples were pooled and subjected to the averaged differential display process. Transcripts present at different levels between patients and controls were purified and sequenced for identification. Transcript levels in the blood of prostate cancer patients and controls were verified by quantitative RT-PCR. Means were compared using a t-test and a receiver-operating curve was generated. The Ring finger protein 19A (RNF19A) transcript was identified as having higher levels in prostate cancer patients compared to healthy men through the averaged differential display process. Quantitative RT-PCR analysis confirmed a more than 2-fold higher level of RNF19A mRNA levels in the blood of patients with prostate cancer than in healthy controls (p = 0.0066). The accuracy of distinguishing cancer patients from healthy men using RNF19A mRNA levels in blood as determined by the area under the receiving operator curve was 0.727.</p> <h3>Conclusions/Significance</h3><p>Averaged differential display offers a simplified approach for the comprehensive screening of body fluids, such as blood, to identify biomarkers in patients with prostate cancer. Furthermore, this proof-of-concept study warrants further analysis of RNF19A as a clinically relevant biomarker for prostate cancer detection.</p> </div

Class III β-tubulin expression as a predictor of docetaxel-resistance in metastatic castration-resistant prostate cancer.

About half of the patients treated with docetaxel in the setting of metastatic castration-resistant prostate cancer (CRPC) are non-responders. Therefore, a marker of response would be beneficial for clinical decision-making. We evaluated class III β-tubulin (βIII-tubulin) expression as a predictor of resistance in this setting, which previously has been correlated with lack of response to taxanes in other cancers. Patients with CRPC were included if they were treated with at least 3 cycles of docetaxel between 1990 and 2011. βIII-tubulin expression was assessed by immunostaining, which was performed in tissue samples obtained either via biopsy or prostatectomy at the time of diagnosis. Rates of prostate-specific antigen (PSA) response and overall survival (OS) following docetaxel treatment were compared between patients with high (2+ or 3+ staining) vs. low (0 or 1+ staining) βIII-tubulin expression. Of 73 patients, 26 (35%) had a high expression of βIII-tubulin. A PSA decline of 10% or greater occurred in 65% of patients with a high βIII-tubulin expression vs. 89% with a low βIII-tubulin expression (p = 0.0267). The median OS for patients with a high βIII-tubulin expression was 17.4 (95% CI 8.7-21.0) months vs. 19.8 (95% CI 16.6-23.6) months for patients with a low expression (p = 0.039). Our results show that a high βIII-tubulin expression is a negative prognostic factor in metastatic CRPC patients treated with docetaxel

ATM Inhibition Potentiates Death of Androgen Receptor-inactivated Prostate Cancer Cells with Telomere Dysfunction.

Androgen receptor (AR) plays a role in maintaining telomere stability in prostate cancer cells, as AR inactivation induces telomere dysfunction within 3 h. Since telomere dysfunction in other systems is known to activate ATM (ataxia telangiectasia mutated)-mediated DNA damage response (DDR) signaling pathways, we investigated the role of ATM-mediated DDR signaling in AR-inactivated prostate cancer cells. Indeed, the induction of telomere dysfunction in cells treated with AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation (activation) of ATM and its downstream effector Chk2 and the presenceof phosphorylated ATM at telomeres, indicating activation of DDR signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation, induced PARP cleavage, abrogated cell cycle checkpoint activation and attenuated the formation of γH2AX foci at telomeres in AR-inactivated cells, suggesting that ATM inhibitor induces apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration-resistant prostate cancer

Role of androgen receptor in progression of LNCaP prostate cancer cells from G1 to S phase.

BACKGROUND: The androgen receptor (AR) plays a critical role in the proliferation of prostate cancer cells. However, its mechanism of action in proliferation remains unknown. An understanding of the mechanism of AR action in proliferation may lead to the development of effective strategies for the treatment of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report that pulse treatment of synchronized LNCaP cells with Casodex, an AR-antagonist, for 4 hours in mid-G(1) phase was sufficient to prevent cells from entering S phase. Since the assembly of pre-replication complex (pre-RC) in G(1) is required for the progression of cells from G(1) to S phase, the effect of Casodex during mid-G(1) suggested that the role of AR in proliferation might be to regulate the assembly of pre-RC. To test this possibility, we investigated the interaction between AR and Cdc6, an essential component of pre-RC in LNCaP cells. AR co-localized and co-immunoprecipitated with Cdc6, and Casodex treatment disrupted this interaction. AR-immunoprecipitate (AR-IP) also contained cyclin E and cyclin A, which play a critical role in pre-RC assembly and cell cycle entry into S phase, and DNA polymerase-α, PCNA, and ribonucleotide reductase, which are essential for the initiation of DNA synthesis. In addition, in cells in S phase, AR co-sedimented with components of the DNA replication machinery of cells that entered S phase. CONCLUSIONS/SIGNIFICANCE: Together, these observations suggest a novel role of AR as a component of the pre-RC to exert control over progression of LNCaP cells from G(1) to S phase through a mechanism that is independent of its role as a transcription factor