Androgen-independent Progression Research Articles

Increased Hsp27 after Androgen Ablation Facilitates Androgen-Independent Progression in Prostate Cancer via Signal Transducers and Activators of Transcription 3–Mediated Suppression of Apoptosis

One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-naïve and post-hormone ablation-treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (>4-fold, P < or = 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate-specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub-G0-G1 fraction and caspase-3 cleavage >2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by Stat3 knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate-specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer.

Beyond simple castration: targeting the molecular basis of treatment resistance in advanced prostate cancer

Over the past 20 years, research on hormonal treatments for prostate cancer focused on maximizing androgen ablation through combination therapy. Unfortunately, maximal androgen ablation increases treatment-related side effects and expense and has not significantly prolonged time to androgen-independent (AI) progression. Intermittent androgen suppression (IAS) is based on the hypothesis that if tumor cells surviving androgen withdrawal can be forced along a normal pathway of differentiation by androgen replacement, then apoptotic potential might be restored, androgen dependence may be prolonged and progression to androgen independence may be delayed. Observations from animal model studies suggest that progression to androgen independence is delayed by IAS and this strategy is now being evaluated in phase III trials. Another strategy for improving therapies in advanced prostate cancer involves targeting genes that are activated by either androgen withdrawal or chemotherapy to delay or prevent the emergence of the resistant AI phenotype. Targeted inhibition of stress-associated increases in gene expression precipitated by androgen withdrawal or chemotherapy may enhance treatment-induced apoptosis and delay progression to AI disease. Proteins fulfilling these criteria include antiapoptotic members of the Bcl-2 protein family, clusterin, Hsp27, and IGFBP-2 and IGFBP-5. The purpose of this paper is to review the rationale and progress in using targeted gene therapies to enhance tumor cell death after androgen withdrawal or taxane chemotherapy. Antisense oligonucleotides offer one approach to target genes involved in cancer progression, especially those not amenable to small molecule or antibody inhibition. The current status and future direction of several antisense oligonucleotides that have potential clinical use in cancer are also reviewed.

Usefulness of Prostate-Specific Antigen Nadir as Predictor of Androgen-Independent Progression of Metastatic Prostate Cancer

The objective of this study was to analyze the value of the nadir level of prostate-specific antigen (PSA) to predict androgen-independent progression (AIP) in metastatic prostate cancer patients after androgen deprivation therapy. In a group of 185 metastatic prostate cancer patients who received androgen deprivation therapy serum PSA was determined every three months until AIP occurred. Multiple regression analysis was performed to define independent clinical and PSA-related predictors of AIP. AIP was assumed to be present after two consecutive increases in serum PSA after the PSA nadir. Independent predictors of the duration of AIP-free survival (less than 12 months versus more than 12 months) were the extent of bone involvement (six or fewer hot spots versus more than six) with an odds ratio (O.R.) of 3.95, Gleason score (7 or less versus more than 7) with an O.R. of 3.47, and PSA nadir (2 microg/L or less versus more than 2 microg/L) with an O.R. of 14.63. AIP was independently predicted by the extent of bone involvement with an O.R. of 1.72, Gleason score with an O.R. of 1.74, PSA nadir with an O.R. of 3.22, and time to reach the PSA nadir (9 months or less versus more than 9 months) with an O.R. of 2.84. When patients were stratified according to these predictors, those with three good prognostic factors had a median AIP-free survival of 58 months while those with two, one or no good prognostic factors had a median AIP-free survival of 19 months, 12 months and 7 months, respectively. We conclude that the PSA nadir seems to be a good predictor of AIP in patients with metastatic prostate cancer after androgen deprivation therapy. Time to PSA nadir, extent of bone involvement and Gleason score are also independent predictors. The combination of these prognostic factors allows to stratify metastatic prostate cancer patients for the prediction of AIP.

Optimising Hormone Therapy in Localised and Early Disease

Prostate cancer (PCa) is being diagnosed at an earlier age, and at an earlier stage of disease. Increasing numbers of relatively young patients (<60 years) with localised and early disease are receiving radical therapy. The addition of hormonal therapy in an adjuvant and/or neoadjuvant setting is widely used in clinical practice. The key issue when dealing with optimising hormone therapy in localised and early disease is to identify the patients who will benefit from this form of treatment, and optimise the timing of initiation as well as duration of the treatment. The current literature was reviewed to address this issue. Traditionally, most patients diagnosed with localised disease receive radical therapy, either radiation therapy or radical prostatectomy. However, many men have indolent PCa and may not require radical therapy. In contrast, patients with aggressive disease benefit from local therapy. As a consequence, the main clinical challenge is the stratification of patients with localised disease into good risk patients and intermediate-to-high risk patients in order to offer the patient a tailored treatment strategy. Patients diagnosed with good risk disease (stage T1c–T2a, Gleason score ≤6, small volume of disease on biopsy and prostate specific antigen (PSA) ≤10 ng/mL) can be closely monitored with periodic biopsies and measurement of the PSA doubling time (DT). Patients diagnosed with an aggressive form of disease (intermediate-to-high risk disease) need immediate curative therapy. Both radical prostatectomy and radiotherapy are currently used in combination with neoadjuvant and/or adjuvant hormonal therapy. Neoadjuvant therapy prior to radical prostatectomy has failed to demonstrate an improvement in PSA progression in numerous randomised trials. However, one trial showed a benefit in high risk patients (PSA >20 ng/mL) after 3 months of neoadjuvant therapy, based on retrospective stratification analysis. Overall, some practitioners recommend neoadjuvant therapy for patients with high risk localised PCa. Adjuvant hormonal therapy after surgery has not, to date, demonstrated an improvement in overall or disease specific survival, but does delay PSA progression. The significance of this remains uncertain. Neoadjuvant hormonal therapy prior to radiation therapy is recommended for patients with intermediate-to-high risk disease. The optimal duration of neoadjuvant hormone therapy is not yet defined. However, a 3 to 6 month course of neoadjuvant therapy prior to radiation is widely utilised. Adjuvant hormonal therapy after radiotherapy is also widely used in patients with intermediate-to-high risk disease. The optimal duration of adjuvant therapy is between 6 months and 3 years. For patients with biochemical failure, androgen deprivation therapy is appropriate for those whose clinical and pathologic parameters indicate that local recurrence is unlikely. For such patients who are at high risk (Gleason ≥8, or PSA DT <1 year), androgen deprivation should be implemented when the PSA is between 5 and 10 ng/mL. For lower risk patients, or those who have secondary failure after initial attempt at salvage radiation, there is no evidence that early androgen deprivation enhances survival or time to androgen independent progression. These patients should be monitored and treated upon clinical progression or rapid biochemical progression.

Molecular profile of androgen-independent prostate cancer xenograft LuCaP 23.1

After castration or therapeutic hormone deprivation, most cancer of the prostate (CaP) cells develop androgen-independent (AI) growth. In this work, we studied the effect of androgen depletion (castration) on the growth of experimental model LuCaP 23.1 xenograft. A total of 101 nude mice were implanted and analysed for their growth profile before experimental period 1 (11 weeks) and after castration experimental period 2 (15 weeks). For specific periods, tumors were harvested and assessed for molecular marker expression specific for CaP. Taking into account tumor dynamic growth, prior to castration we found 37 fast growing (FG) tumors (948.9 ± 76.9 mm 3) and 63 slow growing (SG) tumors (229.6 ± 18.4 mm 3). Real-time quantitative RT-PCR showed that in comparison to SGs, FGs contained elevated expression of epidermal growth factor receptor type 1 (HER1), urokinase plasminogen activator (uPA), thymidine phosphorylase (TP) and thymidilate synthase (TS) mRNAs expression and low levels of 5α-reductase 2 (5α-R2) mRNA. After castration all FG tumors progressed rapidly (by 5 weeks) to AI growth (FG-P). In SG castrated tumors, 66% of tumors showed retarded progression (by 12 weeks) to AI (SG-P), whereas 34% responded to castration (SG-R). Molecular analysis demonstrated distinct molecular profiles integrating different pathways associated with AI progression. The progressive tumors FG-P, and some tumors of SG-P subgroup, presented significantly high levels of HER1, epidermal growth factor receptor type 2 (HER2), TS, uPA, TP, tumor necrosis factor superfamily member 6 (FAS) and peptidylglycine α-amidating mono-oxygenase (PAM) mRNA all of which correlated with androgen receptor (AR) mRNA. The second subgroup of SG-P tumors showed a high expression of the anti-apoptotic gene Bcl-2. A third subgroup of SG-P tumors showed significant expression of hypoxia-related genes such as adrenomedullin (AM) after castration. LuCaP 23.1 xenograft represent a useful dynamic model to study pre-clinically new therapeutic molecules and evaluate non-randomized therapeutics protocols combining different target inhibition specific to each AI pathways.

Complete androgen ablation suppresses prostate stem cell antigen (PSCA) mRNA expression in human prostate carcinoma

Prostate stem cell antigen (PSCA) is a recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface protein belonging to the Thy-1/Ly-6 family of cell surface antigens. Prior data in prostate cancers indicated that PSCA is directly regulated by androgens and PSCA expression increases with high-tumor grade, advanced stage, extracapsular invasion, and androgen-independent progression. The effect of complete androgen ablation (CAA) on tumor PSCA mRNA expression has not been elucidated. The purpose of the present study was to investigate the variations in the expression levels of PSCA mRNA before and after CAA, and further evaluate the clinically prognostic value of PSCA in human prostate carcinoma. PSCA in situ hybridization (ISH) was performed on the cancerous pretreatment biopsy or transurethral resection of prostate (TURP) tissue of 42 men with primarily organ-confined prostate cancer before CAA, and on their tumor tissue from radical retropubic prostatectomy after CAA with bicalutamide and goserelin acetate for 3 months prior to undergoing radical prostatectomy. Tumor cytoplasmic staining of PSCA mRNA was evaluated by two independent pathologists and the differences of PSCA mRNA expression levels between the samples before and after CAA were analyzed using the Student's t-test. Thirty-six to forty months follow-up studies after radical retropubic prostatectomy were performed and aimed at assessing the correlation of PSCA mRNA expression level with local recurrences or metastases from the cancer. The percent of cells positive for PSCA mRNA by ISH labeling declined from 67.3% (0-89%)+/-9.4% before CAA to 33.8% (0-92%)+/-7.7% after CAA (P<0.001). Before CAA, 40 of 42 cases (95.2%) were positive for PSCA mRNA labeling, however, after CAA the percentage of positive reactivity of PSCA mRNA was decreased to 27 of 40 cases (67.5%), in which none was found with local recurrences or distant metastases after radical prostatectomy on follow-up. This decline in PSCA mRNA labeling was dependent on the original tumor grade with Gleason score of <or=6: 19.3%+/-4.7%, Gleason score of 7: 38.8%+/-7.2%, and a Gleason score of >or=8: 73.4%+/-13.8% (P<0.05, respectively). The rest 13 cases had the increased percentage of cells positive for PSCA mRNA after CAA, in which 3 cases were found with local recurrences and 4 cases with distant metastases from tumor on follow-up. Our data demonstrate that CAA for prostate cancer can suppress PSCA mRNA expression with a tumor grade dependence and the increased expression of PSCA mRNA after CAA may be a clinically adverse predictor for tumor recurrences or distant metastases.

Nuclear Factor-κB(NF-κB)/Interleukin-6 (IL-6) pathway activation confers resistance to docetaxel (D) in hormone-independent prostate cancer (HIPC)

9630 Background: The activation of the NF-κB/IL-6 pathway is associated with androgen-independent progression of prostate cancer (PC) and may be implicated in resistance to chemotherapy. We investigated the role of NF-κB/IL-6 in the response to D in human PC cell lines and in HIPC pts, and the inhibition of NF-κB as a strategy to increase chemosensitivity in PC cells. Methods: The hormone-dependent (LNCaP) and hormone-independent (PC-3, DU-145) PC cells were treated with D+/- PS-1145 (specific IκB kinase 2 inhibitor). Cells were tested for activated p65/ NF-κB (western blot using anti-p65NLS antibody), IL-6 in culture medium (ELISA) and cell viability (MTT). Pts with metastatic HIPC receiving D were tested for plasma IL-6 (ELISA) before the start and every 3–4 weeks until the end of D. Results: In vitro sensitivity towards D was lower for p65NLS expressing and IL-6 producing cells (PC3, DU145) versus LNCaP, with lower p65NLS and no IL-6 production. Media IL-6 increased in both PC-3 and DU-145 after D trea...

Changes in Chromogranin A Serum Levels During Endocrine Therapy in Metastatic Prostate Cancer Patients

Introduction: The concept of neuroendocrine (NE) differentiation in prostate cancer has become more widely recognized as its diagnostic, prognostic, and therapeutic usefulness. Patients and methods: We enrolled 38 patients with stage D prostate cancer who underwent endocrine therapy by medical or surgical castration and oral antiandrogen. According to PSA response, serum levels of CGA as a marker of NE differentiation were measured at the multiple points of time; (1) pre-treatment, (2) complete response (CR), (3) a nadir level of PSA, (4) PSA failure or hormone independent progression. We compared these serum values in relation to efficacy of endocrine therapy. Results: There was no correlation between serum PSA and CGA values. Patients consisted of 27 with CR and 11 without CR. Serum CGA increased as intervals of endocrine therapy became longer with positive correlation ( p < 0.05). Its velocity was higher in patients with PSA failure than in those without it (6.98 vs. 2.09 ng/ml/month, p = 0.011). Conclusion: During endocrine therapy in metastatic prostate cancer patients, serum CGA values were not related to serum PSA levels, and increased as treatment periods became longer. It is suggested that CGA velocity has potential to predict androgen independent progression after endocrine therapy.

227: Effect of Timing and Sequencing of Castration and Chemotherapy on Time to Androgen-Independent Progression in Shionogi Tumor Model

227: Effect of Timing and Sequencing of Castration and Chemotherapy on Time to Androgen-Independent Progression in Shionogi Tumor Model

The Progression of LNCaP Human Prostate Cancer Cells to Androgen Independence Involves Decreased FOXO3a Expression and Reduced p27KIP1 Promoter Transactivation

The progression of human prostate cancer from the initial androgen-dependent phase to androgen independence involves diminished apoptosis and a release from the cell cycle block triggered by androgen ablation therapy. FOXO transcription factors play a central role in promoting expression of proapoptotic and cell cycle regulatory genes (e.g., FasL and p27KIP1). Reduced FOXO function might, therefore, play a role in androgen-independent progression of human prostate cancer. Herein, we show that FOXO function is compromised in androgen-independent prostate cancer cells (LNAI) versus androgen-dependent LNCaP cells. The FOXO3a protein, the most highly expressed FOXO family member in prostate cancer cells, is hyperphosphorylated in LNAI cells. FOXO3a expression is also markedly reduced in these androgen-independent LNAI cells when compared with parental LNCaP cells. Together, reduced FOXO3a expression coupled to FOXO3a hyperphosphorylation would suppress FOXO transcriptional activity. Accordingly, activity of the FOXO-responsive p27KIP1 promoter is reduced 60% in these LNAI cells when compared with LNCaP cells. Moreover, mutation of a conserved FOXO response element suppresses p27KIP1 promoter activity, substantiating a regulatory role for this FOXO response element in p27KIP1 promoter transactivation. Finally, we show that the activity of a distinct FOXO-responsive promoter, the 3X-IRS promoter, is also reduced in LNAI cells. Collectively, these data show that reduced FOXO3a expression coupled to increased FOXO3a phosphorylation coincide with reduced FOXO-responsive promoter activity in androgen-independent LNAI cells when compared with androgen-dependent LNCaP cells. To the extent that this model reflects human disease, these data suggest that FOXO function may be compromised with androgen-independent progression of human prostate cancer.

Targeting Bcl-2 with oblimersen for patients with hormone refractory prostate cancer

Patients with metastatic hormone refractory prostate cancer (HRPC) have limited treatment options and new therapies are needed. Advances in the understanding of the molecular mechanisms implicated in prostate cancer progression have identified many potential therapeutic gene targets including Bcl-2, an important pro-survival regulator of apoptotic cell death. Bcl-2 is overexpressed in a variety of human malignancies including prostate cancer where it has also been associated with androgen independent progression and treatment resistance. Oblimersen is a phosphorothioate antisense oligonucleotide complimentary to the Bcl-2 mRNA and a potent inhibitor of Bcl-2 expression which in pre-clinical testing can significantly enhance the therapeutic effect of chemotherapy, hormone and radiation therapy. Clinical trials evaluating oblimersen in combination with chemotherapy in a variety of cancers have shown good tolerability and promising response rates. Randomized trials are required to determine if oblimersen can enhance the effectiveness of docetaxel in patients with HRPC.

Increased Insulin-Like Growth Factor I Receptor Expression and Signaling Are Components of Androgen-Independent Progression in a Lineage-Derived Prostate Cancer Progression Model

Apoptosis and inhibition of mitosis are primary mechanisms mediating androgen ablation therapy-induced regression of prostate cancer (PCa). However, PCa readily becomes androgen independent, leading to fatal disease. Up-regulated growth and survival signaling is implicated in development of resistance to androgen ablation therapy. We are testing the hypothesis that insulin-like growth factor (IGF) responsiveness is required for androgen-independent (AI) progression. Using the LNCaP human PCa progression model, we have determined that IGF-I-mediated protection from apoptotic stress and enhanced mitotic activity is androgen dependent in LNCaP cells but is androgen independent in lineage-derived C4-2 cells. Both cell lines exhibit androgen-responsive patterns of IGF-I receptor (IGF-IR) expression, activation, and signaling to insulin receptor substrate-2 and AKT. However, C4-2 cells express higher levels of IGF-IR mRNA and protein and exhibit enhanced IGF-I-mediated phosphorylation and downstream signaling under androgen-deprived conditions. In comparisons of naive and AI metastatic human PCa specimens, we have confirmed that IGF-IR levels are elevated in advanced disease. Together with our LNCaP/C4-2 AI progression model data, these results indicate that increased IGF-IR expression is associated with AI antiapoptotic and promitotic IGF signaling in PCa disease progression.

Molecular analysis integrating different pathways associated with androgen-independent progression in LuCaP 23.1 xenograft.

After therapeutic hormone deprivation, most prostate cancer (PrCa) cells develop androgen-independent (AI) growth. PrCa is highly heterogeneous and multifocal, suggesting that several molecular processes or pathways may be contributing to AI. The human LuCaP 23.1 xenograft model retains clinical hallmarks of PrCa, including heterogeneous growth, PSA production, androgen-responsiveness and progression to AI. In this work, we studied the effect of androgen depletion (castration) on the growth of LuCaP 23.1 xenografts. A total of 100 nude mice were implanted and analysed for their growth profiles before and after castration. By 11 and 15 weeks, tumours were harvested and assessed for molecular marker expression specific for PrCa. Prior to castration we found 37 fast growing (FG) tumours (948.9+/-76.9 mm(3)) and 63 slow growing (SG) tumours (229.6+/-18.4 mm(3)), a previously undescribed result for this PrCa model. Quantitative RT-PCR showed that in comparison to SGs, FGs contained high HER1, uPA and thymidilate synthetase (TS) expression with low levels of 5alpha-reductase 2 mRNA. All FG tumours progressed rapidly to AI growth 5 weeks after castration (FG-P). In SG castrated tumours, 66% of tumours (SG-P) showed retarded progression (by 12 weeks) to AI, whereas 34% responded to castration (SG-R). Molecular analysis permitted us to define distinct molecular profiles integrating different pathways associated with AI progression. FG-P, and a subgroup of SG-P tumours, presented significantly high levels of peptidylglycine alpha-amidating monooxygenase (PAM), HER1, HER2, TS, and uPA mRNA, all of which correlated with AR expression. The second subgroup of SG-P tumours showed overexpression of the antiapoptotic gene Bcl-2. A third subgroup of SG-P tumours showed significant expression of hypoxia-related gene (adrenomedullin) after castration. This work permitted to define distinct molecular profiles related to different AI growth in the LuCaP 23.1 xenograft.

Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer.

Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.

Intermittent androgen suppression for locally advanced and metastatic prostate cancer: Preliminary report of a prospective multicenter study

Objectives To clarify the effect of intermittent androgen suppression on the time to androgen-independent progression and changes in quality of life (QOL). Methods Patients with locally advanced or metastatic prostate cancer were treated with a combination of leuprolide acetate and flutamide for 36 weeks. When the serum prostate-specific antigen (PSA) levels at 24 and 32 weeks were less than 4.0 ng/mL, treatment was withheld until the PSA level reached 15 ng/mL or the pretreatment level. This cycle of on-treatment and off-treatment was repeated until PSA failure (three consecutive increases in PSA level greater than 4.0 ng/mL during the on-treatment period) or symptomatic progression was observed. Changes in QOL were assessed by a self-assessment questionnaire. Results Forty-nine patients (26 with T3N0M0, 8 with T2-T3N1M0, 2 with T4N0M0, and 13 with T2-T3N0M1) were enrolled. The mean follow-up period was 136.5 weeks. Thirty-one patients finished cycle 1, six finished cycle 2, and three finished cycle 3. The mean off-treatment duration in cycles 1, 2, and 3 was 46.1, 36.9, and 23.3 weeks, respectively. In the off-treatment period, statistically significant improvements in the QOL score were observed in the categories of potency (11.4 versus 2.4) and social/family well-being (20.3 versus 16.1) compared with those in the on-treatment period. PSA failure occurred in 6 patients (3 with T3N0M0 and 3 with T2-T3N1M0), and all patients were alive at last follow-up. Conclusions Our interim analysis indicated that QOL is remarkably improved during the off-treatment period. Intermittent androgen suppression would be a viable option for treatment of advanced prostate cancer, although a randomized controlled study is required to determine whether intermittent androgen suppression prolongs the time to androgen-independent cancer. We will continue follow-up in this study to a minimum of 3 years.

Glycogen synthase kinase-3beta activity is required for androgen-stimulated gene expression in prostate cancer.

Despite the specificity inferred by its name, glycogen synthase kinase (GSK)-3beta is an important kinase with a plethora of significant cellular targets, including cytoskeletal proteins and transcription factors, and its activity is regulated by phosphorylation on tyrosine/serine residues. As part of our efforts to dissect the molecular basis responsible for androgen-independent progression of prostate cancer, we investigated the role of GSK-3beta in androgen-stimulated gene expression in human prostate cancer cells. Pretreatment of prostate cancer cells harboring wild-type or mutant androgen receptor with the GSK-3beta inhibitors, lithium chloride (LiCl), RO318220, or GF109203X, inhibited R1881-stimulated androgen-responsive reporter activity in a dose- and time-dependent manner. In addition, the expression of two endogenous androgen-stimulated gene products, prostate-specific antigen and matrix metalloproteinase-2, was suppressed by the GSK-3beta inhibitors in those cells. Most importantly, knocking down GSK-3beta expression via a small interference RNA-mediated gene silencing approach also reduced R1881-stimulated gene expression, demonstrating the specificity of GSK-3beta involvement. Moreover, R1881 treatment of the cells increased phosphorylation status of GSK-3beta on tyrosine residue Y(216) but not on serine residue S(9). Pretreatment of the cells with phosphatidylinositol 3-kinase inhibitor LY294002 or wortmannin, which blocks androgen action in cells, abolished R1881-induced GSK-3beta Y(216) phosphorylation. However, the phosphatidylinositol 3kinase or GSK-3beta inhibitors did not block R1881-induced nuclear translocation of androgen receptor. Finally, knocking down the expression of Akt or beta-catenin, the two GSK-3beta-related signaling molecules, via siRNA-mediated gene silencing did not significant affect R1881-stimulated gene expression. These findings suggest that GSK-3beta activity is required for androgen-stimulated gene expression in prostate cancer cells.

Nadir prostate‐specific antigen best predicts the progression to androgen‐independent prostate cancer

The objective of our study was to analyze the value of prostate-specific antigen (PSA) levels before and after androgen suppression to predict the time to androgen-independent progression (AIP) in patients with advanced and metastatic prostate cancer. A series of 283 prostate cancer patients under androgen suppression as a single treatment was studied. The disease was locally advanced in 98 patients and metastatic in the remainder 185. AIP was defined after 2 consecutive increases of serum PSA after the nadir value. The mean follow-up before AIP was 29.2 months (3-198). AIP was detected in 205 patients (72.4%). In 152 patients (74.1%), the event was detected within 24 months, while in 53 patients (25.9%), it was observed beyond 24 months. The multivariate analysis showed that the nadir PSA and the time to reach the nadir PSA were the most significant predictors of the time to AIP. The odds ratio of having a biochemical response greater than 24 months was 20 times higher in patients that achieved an undetectable PSA level of 0.2 ng/mL or less. Moreover in those patients whose nadir PSA reached beyond 12 months after androgen suppression the odds ratio was 18 times higher. These results show that the ability to achieve an undetectable nadir PSA and the time to reach it are the most significant predictors of the time to AIP in patients with locally advanced and metastatic prostate cancer under androgen suppression as a single therapy.

Gene therapy for prostate cancer bone metastasis. Gene therapy targeting bone metastasis.

Cancer is not a single cell disease but involves complex interaction between cancer cells and their microenvironment (Ingber, 2002; Liotta and Kohn, 2001; Quaranta, 2002; Shekhar et al., 2003; Sung and Chung, 2002). It has been widely recognized that the growth, survival and invasion of cancer cells require the participation of vascular endothelial component of the host (Folkman, 2001; Monsky et al., 2002), the inflammatory and neuroendocrine cells and their cytokine repertoire (Aprikian et al., 1994; Coussens and Werb, 2002; Murakami et al., 2002), and the inductive fibromuscular stromal cells (Aumuller, 1989; Franks et al., 1970; Tuxhorn et al., 2002). Experimental model systems to determine the molecular and cellular basis of prostate cancer progression to androgen independence and metastasis to bone revealed that intimate interaction between cancer cells and prostate or bone stromal cells is required (Chung et al., 1989; Gleave et al., 1991; Gleave et al., 1992; Olumi et al., 1998; Olumi et al., 1999). Under the inductive influence of prostate or bone stromal cells, the human LNCaP prostate cancer cell line can be driven to express both androgen-independent (AI) and bone metastatic potential when co-cultured in vitro as 3- dimensional (3-D) prostate organoids or allowed to form chimeric tumors when co-inoculated in vivo in castrated hosts (Rhee et al., 2001; Thalmann et al., 1994; Wu et al., 1994). These experiments demonstrated a fundamental principle that governs prostate cancer progression, the reciprocal interaction between genetically modified prostate cancer cells and the relevant fibromuscular stromal environment under androgen-deprived and 3-dimensional (3-D) growth conditions. Only in the presence of such interactions can the AI and bone metastatic progression of human prostate cancer cells be achieved. Apparently, permanent phenotypic and genotypic changes are induced in both prostate cancer and interactive stromal compartments (Hyytinen et al., 1997; Pathak et al, 1997). The consequences of cellular interaction include changes observed in the stroma, which becomes “reactive” and drives further progression of human prostate cancer cells to the AI and metastatic state (Sung and Chung, 2002). To tackle the molecular mechanisms underlying the progression of prostate cancer cells to androgen independence and bone metastasis under the influence of fibromuscular stromal cells, we and others have proposed that both soluble factors and insoluble extracellular matrices (ECMs) are likely to be involved through cellular communication or “cross-talk” between these mosaic factors, their receptors and downstream signaling pathways (Sung and Chung, 2002; Tuxhorn et al., 2001; Varani et al., 1999; Wong and Wang, 2000). In this review, we will focus our discussion on three areas: first, the development of the gene therapy concept and strategies to co-target prostate cancer growth in bone; second, the application of local-regional and systemic gene therapy in human clinical trials; and third, a discussion of the future development of effective gene therapy approaches for the treatment of prostate cancer bone metastasis.KeywordsProstate CancerGene TherapyProstate Cancer CellHuman Prostate CancerMetastatic Prostate CancerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

YW1-05 Upregulation of Insulin like growth factor brinding protein 2 promotes Androgen independent progression in LNCaP model (Young Urologist Research Workshop)

YW1-05 Upregulation of Insulin like growth factor brinding protein 2 promotes Androgen independent progression in LNCaP model (Young Urologist Research Workshop)

Cotargeting tumor and stroma in a novel chimeric tumor model involving the growth of both human prostate cancer and bone stromal cells.

Stromal-epithelial interaction contributes to local prostate tumor growth, androgen-independent progression and distant metastasis. We have established in vitro coculture and in vivo chimeric tumor models to evaluate the roles of stromal cells isolated from either osteosarcoma or normal bone, a site where prostate cancer cells frequently metastasize, in contributing to the growth and survival of human prostate cancer cells. We have evaluated extensively the effects of toxic gene therapy using luciferase-tagged chimeric human prostate cancer models both in vitro and in vivo. In the in vitro cocultured cell model, we assessed cancer cell growth and residual cellular proteins after targeting either prostate cancer epithelial cells alone or both prostate cancer and bone stromal cells. In the in vivo animal model, we measured tumor volume and serum prostate-specific antigen (PSA) in mice bearing chimeric prostate tumors comprised of human prostate tumor cells and normal bone stromal cells. Our results demonstrated that: (1) The rate of human prostate cancer cell growth in vitro is accelerated by coculturing with human and rat osteosarcoma or normal mouse bone marrow stromal cell lines. No growth stimulation was noted when cocultured with a human prostate epithelial cell line. (2) Disabling the growth of normal bone stromal cells using transgenic targeting with a bystander gene, herpes simplex virus thymidine kinase (hsv-TK), plus the pro-drug ganciclovir (GCV) or acyclovir markedly depressed the growth of cocultured human prostate cancer cells in vitro and human prostate cancer-mouse normal bone stroma chimeric tumors in vivo. (3) By cotargeting both human prostate cancer and normal mouse bone stromal cells in vitro with an adenoviral construct, Ad-hOC-TK (a replication-defective Ad5 vector with the bystander transgene hsv-TK under the control of a human osteocalcin (hOC) promoter) plus GCV4, we observed greater inhibition of tumor cell growth than by targeting a single cell compartment with Ad-PSA-TK (a vector construct similar to Ad-hOC-TK except that the transgene expression is under regulation by a full-length human PSA promoter). These results, taken together, established a basic principle that cotargeting both tumor and its supporting stroma is more efficacious than targeting a single cell compartment in the treatment of human prostate cancer bone metastasis. This principle can be applied to other clinical conditions of cancer growth where stroma contribute to the overall growth and survival potential of the cancer.