Longitudinal analysis of androgen deprivation of prostate cancer cells identifies pathways to androgen independence

BACKGROUND: Following androgen ablation therapy, the majority of prostate cancer patients develop treatment resistance with a median time of 18-24 months to disease progression. METHODS: To identify molecular targets that promote prostate cancer cell survival and contribute to androgen independence, we evaluated changes in LNCaP cell gene expression during 12 months of androgen deprivation. At time points reflecting critical growth and phenotypic changes, we performed Affymetrix expression array analysis to examine the effects of androgen deprivation during the acute response, during the period of apparent quiescence, and following the emergence of a highly proliferative, androgen-independent prostate cancer cell phenotype (LNCaP-AI). RESULTS: We discovered alterations in gene expression for molecules associated with promoting prostate cancer cell growth and survival, and regulating cell cycle progression and apoptosis. Additionally, expression of AR co-regulators, adrenal androgen metabolizing enzymes, and markers of neuroendocrine disease were significantly altered. CONCLUSIONS: These findings contribute greatly to our understanding of androgen-independent prostate cancer. The value of this longitudinal approach lies in the ability to examine gene expression changes throughout the adaptive response to androgen deprivation; it provides a more dynamic illustration of genes which contribute to disease progression in addition to specific genes which constitute an androgen-independent phenotype.     

粘着斑蛋白、雄激素受体在前列腺癌中的表达及相关性研究

目的:检测粘着斑蛋白(VCL)和雄激素受体(AR)在良性前列腺增生(BPH)和前列腺癌(PCa)中的表达情况,并分析其与PCa不同临床分期、病理分级、PSA水平之间的相互关系。方法:采用免疫组化法检测18例BPH组织,38例PCa组织中VCL、AR的表达。并分析两者在BPH、PCa中的表达差异,在PCa不同临床分期、病理分级、初次PSA检测水平的表达差异,以及两者之间的相关性。结果:VCL在PCa中的表达较BPH组织增多,AR表达在BPH、PCa中无明显差异;VCL、AR在PCa组织中的表达与肿瘤临床分期、病理分级密切相关,与PSA值无明显关系;VCL、AR在PCa组织中的表达存在正相关。结论:VCL在BPH、PCa的表达具有差异性,可以作为前列腺良、恶性疾病鉴别诊断的一个潜在指标;AR、VCL随PCa进展表达逐步下降,两者存在正相关,两者结合可以成为PCa进展的诊断、预后评价指标。     

Effect of time of castration and tumour volume on time to androgen-independent recurrence in Shionogi tumours

The understanding of the timing and development of the recurrence of androgen‐independent prostate cancer is limited and has significant clinical implications. The paper by So et al. addresses, for the first time, the concept of recurrence after androgen ablation in an animal model. They show clearly in this well designed and conducted study that the recurrence of an androgen‐independent cancer depends on both the initial size of the tumour and the delay leading to castration. These results have significant clinical importance in the understanding and treatment of prostate cancer.

OBJECTIVE

To use the androgen‐dependent Shionogi tumour model in mice to help define the effects of the timing of androgen ablation on the development of androgen resistance in prostate cancer, as the timing of androgen ablation remains controversial.

MATERIALS AND METHODS

Groups of nine mice were castrated at 1, 3, 6, 10 and 14 days after tumour inoculation, with a similar‐sized group of mice castrated before tumour inoculation serving as a control. The time of first palpable tumour recurrence and tumour volume was monitored after castration.

RESULTS

All mice were observed for ≥ 80 days after castration. Only mice castrated at 10 and 14 days had palpable tumour at the time of castration. Mice castrated at 14 days had the highest rate of early tumour recurrence (all nine) while mice castrated before inoculation or at 1 and 3 days afterward had a significantly lower rate of tumour recurrence (four of nine; P < 0.01). Mice castrated at 14 and 10 days had tumour recurrence significantly earlier than mice in the other groups. When calcium‐channel blockers were administered to inhibit apoptosis, all mice had a similar time to recurrence and time to death regardless of the time of castration.

CONCLUSIONS

Large tumour volume and corresponding delay in castration reduced the time to androgen‐independent tumour recurrence and survival. Earlier androgen ablation, at the time of subclinical (impalpable) disease, significantly delayed the rate and time to androgen‐independent recurrence compared with delayed therapy when the tumour burden was high.

     

PC-3 cells with enhanced androgen receptor signaling: a model for clonal selection in prostate cancer

BACKGROUND: Two sublines of the human prostate cancer cell line, PC-3, which is widely used as a model of prostate cancer progression, have been reported: PC-3(AR-) that do not express androgen receptor (AR), and PC-3AR+ that have measurable AR RNA but little protein. METHODS: We assayed the geneotype, karyotype, AR expression, and physical characteristics of the two PC-3 sublines, and compared their ability to elicit a transactivation response from ectopic AR in the presence and absence of specific AR coregulators. RESULTS: PC-3(AR-) and PC-3AR+ cells are genotypically and karyotypically similar, but exhibit salient differences in their morphology, growth rate, and expression of AR RNA. Whereas endogenous AR expression in PC-3AR+ cells does not result in sufficient protein to confer androgen responsiveness in culture, ectopic AR consistently elicited a much greater transactivation response in PC-3AR+ than in PC-3(AR-) cells, without altered sensitivity to activation by native ligand or AR coregulators including GRIP1, BRCA1, and Zac1. Moreover, phenotypic differences of AR variants implicated in prostate cancer susceptibility and progression were only observed in PC-3AR+ cells. Higher levels of known AR coregulator proteins detected in PC-3AR+ compared with PC-3(AR-) cells likely contribute to these differences. CONCLUSIONS: These studies provide new evidence that the androgen-signaling axis can be sensitized in prostate cancer cells, and have important implications for the analysis and interpretation of AR structure and function in in vitro cell systems.     

The growth inhibitory effect of p21 adenovirus on androgen-dependent and -independent human prostate cancer cells

OBJECTIVE: To assess the potential of p21 as a gene therapy treatment for prostate cancer, by introducing p21 into both androgen-dependent (AD) and -independent (AI) human prostate cancer cell lines via a recombinant adenoviral vector, Ad5CMV-p21, carrying human p21 cDNA. MATERIALS AND METHODS: The LNCaP, DU145 and PC-3 human prostate cancer cell lines were cultured and infected with Ad5CMV-p21. Cell growth, cell-cycle progression and tumorigenicity were then assessed by thymidine incorporation into cellular DNA, and cell number, flow cytometry, and tumour growth after inoculating the cells into nude mice. RESULTS: Growth was inhibited in Ad5CMV-p21 viral-infected AD and AI prostate cancer cells. The effects were dose-dependent, regardless of the androgen status of the cell lines. Flow cytometric analysis showed that Ad5CMV-p21 arrested cell-cycle progression at G1/S with no appreciable effect on the levels of apoptotic cells. The tumorigenicity of cancer cells infected with Ad5CMV-p21 was greatly reduced in athymic mice. CONCLUSIONS: These results suggest that Ad5CMV-p21 may be a new therapeutic agent for human prostate cancer gene therapy.     

Androgen receptor expression in clinically localized prostate cancer： immunohistochemistry study and literature review

Aim： To evaluate androgen receptor （AR） expression in clinically localized prostate cancer （PCa）. Methods： Specimens were studied from 232 patients who underwent radical prostatectomy for clinically localized prostatic adenocarcinoma without neoadjuvant hormonal therapy or chemotherapy at our institution between November 2001 and June 2005. Immunohistochemical study was performed using an anti-human AR monoclonal antibody AR441. The mean AR density in the hot spots of different histological areas within the same sections were compared and the correlation of malignant epithelial AR density with clinicopathological parameters such as Gleason score, tumor, nodes and metastases （TNM） stage and pre-treatment prostate-specific antigen （PSA） value was assessed. Results： AR immunoreactivity was almost exclusively nuclear and was observed in tumor cells, non-neoplastic glandular epithelial cells and a proportion of peritumoral and interglandular stromal cells. Mean percentage of AR-positive epithelial cells was significantly higher in cancer tissues than that in normal prostate tissues （mean e SD, 90.0% ± 9.3% vs. 85.3% ±9.7%, P 〈 0.001）. The histological score yielded similar results. The percentage ofAR immunoreactive prostatic cancer nuclei and histological score were not correlated with existing parameters such as Gleason score, tumor, nodes and metastases stage and pre-treatment PSA value in this surgically treated cohort. Conclusion： The results of the present study suggest that there may be limited clinical use for determining AR expression （if evaluated in hot spots） in men with localized PCa.     

Transgelin induces apoptosis of human prostate LNCaP cells through its interaction with p53

The androgen receptor （AR） and its coregulators have important roles in the carcinogenesis of prostate cancer.p53 is an important tumour suppressor gerte,and the absence of a fundamental p53 response may predispose to cancer. Transgelin,known as an ARA54-associated AR inhibitor,can suppress AR function in LNCaP cells. In addition to these effects,we aimed to elucidate the proapoptotic effects of the protein on LNCaP and its underlying mechanisms,especially the interaction between transgelin and p53. Cell counting,flow cytometric analysis and terminal deoxynucleotidyl transferase-dUTP nick-end labelling assays were applied to measure the proapoptotic effect of transgelin. Using western blotting of p53 and double immunofluorescence staining of p53 with transgelin,we show that transfection of transgelin results in increasing cytoplasmic translocation of p53 and upregulation of p53 expression. We also found an interaction between transgelin and p53 in vivo by mammalian two-hybrid and coimmunoprecipitation assays. The activation of the mitochondria-associated apoptosis pathway was observed in LNCaP cells after transfection with transgelin. These results are indicative of p53-mediated mitochondria-associated apoptotic effects of transgelin on LNCaP cells in addition to its known suppressive effects on the AR pathway.     

Non‐steroidal antiandrogen monotherapy compared with luteinizing hormone‐releasing hormone agonists or surgical castration monotherapy for advanced prostate cancer: a Cochrane systematic review

To assess the effects of non‐steroidal antiandrogen monotherapy compared with luteinizing hormone‐releasing hormone agonists or surgical castration monotherapy for treating advanced hormone‐sensitive stages of prostate cancer. We searched the Cochrane Prostatic Diseases and Urologic Cancers Group Specialized Register (PROSTATE), the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Web of Science with Conference Proceedings, three trial registries and abstracts from three major conferences to 23 December 2013, together with reference lists, and contacted selected experts in the field and manufacturers. We included randomized controlled trials comparing non‐steroidal antiandrogen monotherapy with medical or surgical castration monotherapy for men in advanced hormone‐sensitive stages of prostate cancer. Two review authors independently examined full‐text reports, identified relevant studies, assessed the eligibility of studies for inclusion, extracted data and assessed risk of bias as well as quality of evidence according to the GRADE working group guidelines. We used Review Manager 5.2 for data synthesis and the fixed‐effect model as primary analysis (when heterogeneity was low with I² < 50%); we used a random‐effects model when confronted with substantial or considerable heterogeneity (when I² ≥50%). A total of 11 studies involving 3060 randomly assigned participants were included in the present review. Use of non‐steroidal antiandrogens resulted in lower overall survival times (hazard ratio [HR] 1.24, 95% confidence interval [CI] 1.05–1.48, six studies, 2712 participants) and greater clinical progression (1 year: risk ratio [RR] 1.25, 95% CI 1.08–1.45, five studies, 2067 participants; 70 weeks: RR 1.26, 95% CI 1.08–1.45, six studies, 2373 participants; 2 years: RR 1.14, 95% CI 1.04–1.25, three studies, 1336 participants), as well as treatment failure (1 year: RR 1.19, 95% CI 1.02–1.38, four studies, 1539 participants; 70 weeks: RR 1.27, 95% CI 1.05–1.52, five studies, 1845 participants; 2 years: RR 1.14, 95% CI 1.05–1.24, two studies, 808 participants), compared with medical or surgical castration. The quality of evidence for overall survival, clinical progression and treatment failure was rated as moderate according to GRADE. Use of non‐steroidal antiandrogens increased the risk for treatment discontinuation as a result of adverse events (RR 1.82, 95% CI 1.13–2.94, eight studies, 1559 participants), including events such as breast pain (RR 22.97, 95% CI 14.79– 35.67, eight studies, 2670 participants) and gynaecomastia (RR 8.43, 95% CI 3.19–22.28, nine studies, 2774 participants) The risk of other adverse events, such as hot flushes (RR 0.23, 95% CI 0.19–0.27, nine studies, 2774 participants) was decreased when non‐steroidal antiandrogens were used. The quality of evidence for breast pain, gynaecomastia and hot flushes was rated as moderate according to GRADE. The effects of non‐steroidal antiandrogens on cancer‐specific survival and biochemical progression remained unclear. Non‐steroidal antiandrogen monotherapy compared with medical or surgical castration monotherapy for advanced prostate cancer is less effective in terms of overall survival, clinical progression, treatment failure and treatment discontinuation resulting from adverse events. Evidence quality was rated as moderate according to GRADE; therefore, further research is likely to have an important impact on results for patients with advanced but non‐metastatic prostate cancer treated with non‐steroidal antiandrogen monotherapy.     

Long-term androgen-ablation causes increased resistance to PI3K/Akt pathway inhibition in prostate cancer cells

BACKGROUND: In advanced stages of prostate cancer, the phosphatidylinositol-3' kinase (PI3K)/Akt signaling cascade, one of the major survival pathways in the cell, is frequently constitutively activated due to mutation or loss of the tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Using cell culture models representing different tumor stages, we explored the effect of inhibition of this survival pathway on the induction of apoptosis. METHODS: Inhibition of the survival kinase Akt and induction of apoptosis was analyzed in androgen-insensitive DU145 and PC-3 cells, in androgen-responsive LNCaP, and in androgen-independent long-term androgen-ablated LNCaP-abl cells representing therapy-resistant prostate cancer cells. Activated Akt was determined by immunoblotting using a phospho-Akt specific antibody. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry and measurement of cleavage of the caspases substrate poly-ADP-ribose polymerase (PARP). RESULTS: IGF-1, EGF, and heregulin but not PDGF or activators of protein kinase A induced phosphorylation of Akt in DU145 cells and activation was completely blocked by the PI3K inhibitor LY294002. In the hormone-responsive prostate cancer cell line LNCaP that has a constitutively switched-on Akt kinase, LY294002 caused a dose- and time-dependent Akt inhibition, which was absent in long-term androgen-ablated LNCaP sublines. In agreement with the resistance to inhibition of the PI3K/Akt pathway, long-term androgen-ablated LNCaP sublines remained relatively resistant to induction of cell death by LY294002 or the cytotoxic drug etoposide. Inhibition of the PI3K/Akt pathway restored the sensitivity of long-term androgen-ablated cells to induction of apoptosis by a cytotoxic drug almost completely. CONCLUSION: These results suggest that long-term androgen ablation therapy for prostate cancer reinforces the PI3K/Akt pathway and impedes its inhibition thus contributing to increased resistance of tumor cells to induction of apoptosis. With regard to treatment of therapy-refractory prostate cancer, these findings suggest effectiveness of a combination of cytotoxic treatment and inhibition of the PI3K-Akt survival pathway in tumor cells after failure of androgen-ablation therapy.     

Importance of C16 ceramide accumulation during apoptosis in prostate cancer cells

AIM: Adenocarcinoma of the prostate is one of the most frequently diagnosed non-cutaneous cancers and the second leading cause of cancer-related deaths among men in the United States. To fully understand the role of ceramide during apoptosis induced by androgen ablation, we modified the levels of intracellular ceramide by pharmacological agents as well as through serum deprivation in androgen-dependent and independent cell lines. METHODS: Ceramide levels were modified using N-oleoylethanolamine (NOE), sphingosine-1-phosphate (S1P) as well as through serum deprivation, in LNCaP, DU145 and PC-3 prostate cancer cells. Various methods including nonyl acridine orange staining, propidium iodide staining/cell cycle analysis and lipid analysis were utilized. RESULTS: Our results demonstrate that the inhibition of acid ceramidase by NOE enhances the intracellular ceramide levels induced by androgen ablation in androgen-dependent LNCaP cells, and is accompanied by an increase in apoptotic cells. Sphingosine 1-phosphate had no effect in rescuing LNCaP cells from apoptosis induced by androgen ablation. Our results also show that serum deprivation causes intracellular ceramide accumulation and apoptosis in androgen-independent prostate cancer cells. CONCLUSIONS: Our studies indicate that the increase in intracellular ceramide itself, but not the balance between ceramide and S1P, determines whether LNCaP cells undergo apoptosis. Our results also show that the increase in intracellular ceramide strongly correlates with apoptosis induced by serum deprivation even in androgen-independent prostate cancer cell lines.