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.

Averaged differential expression for the discovery of biomarkers in the blood of patients with prostate cancer.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Calmodulin protects androgen receptor from calpain-mediated breakdown in prostate cancer cells.

Although inactivation of the androgen receptor (AR) by androgen-ablation or anti-androgen treatment has been frontline therapy for disseminated prostate cancer for over 60 years, it is not curative because castration-resistant prostate cancer cells retain AR activity. Therefore, curative strategy should include targeted elimination of AR protein. Since AR binds to calmodulin (CaM), and since CaM-binding proteins are targets of calpain (Cpn)-mediated proteolysis, we studied the role of CaM and Cpn in AR breakdown in prostate cancer cells. Whereas the treatment of prostate cancer cells individually with anti-CaM drug or calcimycin, which increases intracellular Ca(++) and activates Cpn, led to minimal AR breakdown, combined treatment led to a precipitous decrease in AR protein levels. This decrease in AR protein occurred without noticeable changes in AR mRNA levels, suggesting an increase in AR protein turnover rather than inhibition of AR mRNA expression. Thus, CaM inactivation seems to sensitize AR to Cpn-mediated breakdown in prostate cancer cells. Consistent with this possibility, purified recombinant human AR (rhAR) underwent proteolysis in the presence of purified Cpn, and the addition of purified CaM to the incubation blocked rhAR proteolysis. Together, these observations demonstrate that AR is a Cpn target and AR-bound CaM plays an important role in protecting AR from Cpn-mediated breakdown in prostate cancer cells. These observations raise an intriguing possibility that anti-CaM drugs in combination with Cpn-activating agents may offer a curative strategy for the treatment of prostate cancer, which relies on AR for growth and survival.

Androgen receptor interacts with telomeric proteins in prostate cancer cells.

The telomeric complex, shelterin, plays a critical role in protecting chromosome ends from erosion, and disruption of these complexes can lead to chromosomal instability culminating in cell death or malignant transformation. We reported previously that dominant-negative mutants of one of the telomeric proteins called TIN2 cause death of androgen receptor (AR)-negative but not AR-positive prostate cancer cells, raising the question of a possible role of AR in the structural stability of telomeric complexes. Consistent with this possibility, in the present study, we observed that the AR antagonist Casodex (bicalutamide) disrupted telomeric complexes in AR-positive LNCaP cells but not in AR-negative PC-3 cells. Immunofluorescent studies revealed colocalization of TIN2 and AR. Reciprocal immunoprecipitation studies showed association of AR with telomeric proteins. Furthermore, telomeric proteins were overexpressed in prostate cancer cells compared with normal prostate epithelial cells, and sucrose density gradient analysis showed co-sedimentation of AR with telomeric proteins in a shelterin-like mega complex. Together, these observations suggest an allosteric role of AR in telomere complex stability in prostate cancer cells and suggest that AR-antagonist Casodex-mediated cell death may be due to telomere complex disruption.

Androgen regulated TRPM8 expression: a potential mRNA marker for metastatic prostate cancer detection in body fluids.

Identification of sensitive and specific biomarkers for early detection and prognosis of prostate cancer is essential for timely and appropriate treatment of the disease in individual patients. We identified an RNA transcript with sequence homology to TRPM8 (melastatin-related transient receptor potential member 8) that was overexpressed in tumor vs. patient-matched non-tumor prostate tissues by RT-PCR differential display (DD). Semi-quantitative RT-PCR analysis revealed that TRPM8 levels were higher in tumor than in non-tumor tissue from 31 of 40 (>75%) patients examined. Overexpression of TRPM8 was independent of changes in androgen receptor (AR) mRNA levels in tumor tissue. However, in studies with established cell lines, TRPM8 expression was detectable only in AR-positive, but not in AR-negative cells, and it was suppressed by steroid deprivation or anti-androgen bicalutamide (Casodex) treatment, suggesting the requirement of AR activity for TRPM8 expression in prostate cancer cells. TRPM8 mRNA was also detected in body fluids of men. Most importantly, its levels were significantly higher (p<0.001, n=18) in urine and blood of patients with metastatic disease than in those of healthy men. However, there was no significant difference (p>0.05, n=10) in its levels between prostate cancer patients with localized disease and healthy men. Together, these studies demonstrate that TRPM8 expression is androgen regulated in prostate cancer cells and that, while tissue TRPM8 mRNA levels can be used for detection of prostate cancer, urine and blood TRPM8 mRNA levels may prove to be useful for distinguishing metastatic disease from clinically localized prostate cancer at the time of diagnosis.

Identification of prostate cancer mRNA markers by averaged differential expression and their detection in biopsies, blood, and urine.

The advent of serum prostate-specific antigen (PSA) as a biomarker has enabled early detection of prostate cancer and, hence, improved clinical outcome. However, a low PSA is not a guarantee of disease-free status, and an elevated PSA is frequently associated with a negative biopsy. Therefore, our goal is to identify molecular markers that can detect prostate cancer with greater specificity in body fluids such as urine or blood. We used the RT-PCR differential display method to first identify mRNA transcripts differentially expressed in tumor vs. patient-matched nontumor prostate tissue. This analysis led to the identification of 44 mRNA transcripts that were expressed differentially in some but not all tumor specimens examined. To identify mRNA transcripts that are differentially expressed in most tumor specimens, we turned to differential display of pooled tissue samples, a technique we name averaged differential expression (ADE). We performed differential display of mRNA from patient-matched nontumor vs. tumor tissue, each pooled from 10 patients with various Gleason scores. Differentially expressed mRNA transcripts identified by ADE were fewer in number, but were expressed in a greater percentage of tumors (>75%) than those identified by differential display of mRNA from individual patient samples. Differential expression of these mRNA transcripts was also detected by RT-PCR in mRNA isolated from urine and blood samples of prostate cancer patients. Our findings demonstrate the principle that specific cDNA probes of frequently differentially expressed mRNA transcripts identified by ADE can be used for the detection of prostate cancer in urine and blood samples.

Androgen receptor regulates Cdc6 in synchronized LNCaP cells progressing from G1 to S phase.

We have shown previously that androgen receptor (AR) activity is required for the progression of cells from G(1) to S phase. In an attempt to elucidate the mechanism of androgen- and androgen-receptor-mediated proliferation of prostate cancer cells, we studied the effect of anti-androgen bicalutamide (Casodex) on the expression of cell-cycle regulatory genes in synchronized LNCaP cells progressing from G(1) to S phase. LNCaP cells were synchronized by isoleucine-deprivation. Expression of cell-cycle regulatory genes in S phase control cells versus Casodex-treated cells that fail to enter S phase was studied using a microarray containing cDNA probes for 111 cell-cycle specific genes. RT-PCR and Western-blots were used to validate microarray data. Casodex blocked synchronized LNCaP cells from entering S phase. Microarrays revealed downregulation of eight genes in cells prevented from entering into S phase by Casodex. Of these eight genes, only Cdc6, cyclin A, and cyclin B were downregulated at both the mRNA and protein level in Casodex treated cells as compared to control cells. The mRNA and protein levels of Cdc6 increased as synchronized LNCaP cells progressed from G(1) to S phase, and were attenuated in Casodex-treated cells failed to enter S phase. Cyclins A and B were detected when cells entered S phase, but not when they were in G(1) phase. Like Cdc6, the levels of both cyclins A and B were attenuated in Casodex-treated cells. AR may play an important role in the onset of DNA synthesis in prostate cancer cells by regulating the expression and stability of Cdc6, which is critically required for the assembly of the pre-replication complex(pre-RC).