Inducible FGFR-1 activation leads to irreversible prostate adenocarcinoma and an epithelial-to-mesenchymal transition.

Fibroblast Growth Factor Receptor-1 (FGFR1) is commonly overexpressed in advanced prostate cancer (PCa). To investigate causality, we utilized an inducible FGFR1 (iFGFR1) prostate mouse model. Activation of iFGFR1 with chemical inducers of dimerization (CID) led to highly synchronous, step-wise progression to adenocarcinoma that is linked to an epithelial-to-mesenchymal transition (EMT). iFGFR1 inactivation by CID withdrawal led to full reversion of prostatic intraepithelial neoplasia, whereas PCa lesions became iFGFR1-independent. Gene expression profiling at distinct stages of tumor progression revealed an increase in EMT-associated Sox9 and changes in the Wnt signaling pathway, including Fzd4, which was validated in human PCa. The iFGFR1 model clearly implicates FGFR1 in PCa progression and demonstrates how CID-inducible models can help evaluate candidate molecules in tumor progression and maintenance.

Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression.

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-ß1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-ß1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-ß1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.

Determining prostate cancer-specific death through quantification of stromogenic carcinoma area in prostatectomy specimens.

We previously reported that reactive stroma grading in prostate cancer (PCa) is predictive of biochemical recurrence in prostatectomies and biopsies. In this study, we tested whether quantifying the percentage of reactive stromal grade 3 (RSG 3; stromogenic carcinoma pattern) in the entire tumor is predictive of PCa-specific death. Whole-mount prostatectomies operated by a single surgeon obtained between 1983 and 1998 were reviewed. Reactive stroma was evaluated as described previously, and areas of RSG 3 in the entire tumor were registered as percentages of total tumor. Statistical analysis was performed using Spearman, Kaplan-Meier, and Cox analyses. In all, 872 cases were evaluable. Quantification of RSG 3 percentage was an independent predictor of biochemical recurrence, analyzed as a continuous or grouped variable. Patients with higher RSG 3 percentages (larger tumor areas with RSG 3) had a significantly decreased biochemical recurrence-free survival than those with a lower RSG 3 percentage, even within the Gleason score 7 subset of patients. A nomogram introduced this new variable to the model. Furthermore, quantification of RSG 3 percentage was significantly predictive of PCa-specific death. Quantification of the RSG 3 (stromogenic carcinoma) area in PCa provides additional novel information on prognosis. These data substantiate the concept that the tumor microenvironment holds significant predictive information, as well as biological significance.

Prognostic value of Akt-1 in human prostate cancer: a computerized quantitative assessment with quantum dot technology.

BACKGROUND: Akt/protein kinase B signaling pathway has been implicated in tumorigenesis and progression. Previous studies showed the predictive potential of p-Akt-1, but total Akt-1 could provide more reliable information. We used image deconvolution, nanotechnology (quantum dots), and image analysis to improve Akt-1 quantification. DESIGN: This tissue microarray study included 840 radical prostatectomy cases. Slides were incubated with primary antibody against nonphosphorylated Akt-1 (Akt-1) followed by biotinylated secondary antibody and then by Qdot655 streptavidin conjugate. Slides were imaged under fluorescence microscopy and spectral deconvolution (Nuance) and quantified using plug-in image analysis software. Average intensity of Akt-1 signal was measured and subject to statistical analysis. Multivariate analysis (Cox regression) was applied to assess the prognostic value of Akt-1 for biochemical recurrence and prostate cancer-specific death. Akt-1 expression was also examined for correlations with Ki-67 index and apoptotic index in our database. RESULT: Akt-1 was inversely correlated with apoptotic index (rho = -0.203; P = 0.004) but not with Ki-67 index. The correlation between Akt and p-Akt is significant but weak (P = 0.0496; R(2) = 0.118). On multivariate analysis Akt-1 was independently predictive of biochemical recurrence [hazard ratio, 2.863 (95% confidence interval, 1.127-7.271); P = 0.0270]. Akt-1 level is also predictive of prostate cancer-specific death (P = 0.0376). CONCLUSION: High levels of Akt-1, assessed by quantum dots, deconvolution imaging, and image analysis, are associated with a higher risk of biochemical recurrence and prostate cancer-specific death.

Cytoplasmic accumulation of glycogen synthase kinase-3beta is associated with aggressive clinicopathological features in human prostate cancer.

BACKGROUND: Activation of glycogen synthase kinase-3 (GSK-3) is involved in the regulation of cell growth, differentiation, mobility, proliferation and survival. However, its clinicopathologic significance remains unclear in prostate cancer (PCa). MATERIALS AND METHODS: A tissue microarray was produced from 640 samples. Sections were immunostained with an antibody against the non-phosphorylated form of GSK-3(GSK-3beta) and were digitized. Spearman correlation test was processed for correlations between GSK-3beta and biological and clinicopathological variables. The prognostic value of GSK-3beta was analyzed by Cox Regression model. RESULTS: Cytoplasmic GSK-3beta was higher in PCa than in normal prostate (mean expression index 4.55 vs. 3.50, p<0.0001). Conversely, nuclear expression was higher in normal prostate than that in PCa (3.38 vs. 2.04, p<0.0001). Cytoplasmic levels of GSK-3beta were correlated with clinical stage (rho=0.095, p=0.0337), lymph node metastasis (rho=0.116, p=0.0096), extracapsular extension (rho=0.092, p=0.0392), and Gleason score (rho=0.167, p=0.0002). Increased cytoplasmic GSK-3beta expression was correlated with high Ki-67 labeling index (rho=0.319, p<0.0001), low apoptotic index by TUNEL (rho=-0.118, p=0.0134), high levels of androgen receptor (rho=0.292, p<0.0001) and p-Akt (rho=0.396, p<0.0001). Patients with higher cytoplasmic levels of GSK-3beta had a two-fold risk of biochemical recurrence-free survival compared to those with lower levels of GSK-3beta [HR 1.934 (1.020-3.667), p=0.043]. CONCLUSION: Cytoplasmic accumulation of GSK-3beta is potentially associated with a pro-survival mechanism that promotes PCa development and progression.

Cancer-related axonogenesis and neurogenesis in prostate cancer.

PURPOSE: Perineural invasion is the only interaction between cancer cells and nerves studied to date. It is a symbiotic relationship between cancer and nerves that results in growth advantage for both. In this article, we present data on a novel biological phenomenon, cancer-related axonogenesis and neurogenesis. EXPERIMENTAL DESIGN: We identify spatial and temporal associations between increased nerve density and preneoplastic and neoplastic lesions of the human prostate. RESULTS: Nerve density was increased in cancer areas as well as in preneoplastic lesions compared with controls. Two- and three-dimensional reconstructions of entire prostates confirmed axonogenesis in human tumors. Furthermore, patients with prostate cancer had increased numbers of neurons in their prostatic ganglia compared with controls, corroborating neurogenesis. Finally, two in vitro models confirmed that cancer cells, particularly when interacting with nerves in perineural invasion, induce neurite outgrowth in prostate cancer. Neurogenesis is correlated with features of aggressive prostate cancer and with recurrence in prostate cancer. We also present a putative regulatory mechanism based on semaphorin 4F (S4F). S4F is overexpressed in cancers cells in the perineural in vitro model. Overexpression of S4F in prostate cancer cells induces neurogenesis in the N1E-115 neurogenesis assay and S4F inhibition by small interfering RNA blocks this effect. CONCLUSIONS: This is the first description of cancer-related neurogenesis and its putative regulatory mechanism.

Bortezomib-mediated inhibition of steroid receptor coactivator-3 degradation leads to activated Akt.

PURPOSE: To assess the safety of administering bortezomib to patients undergoing a radical prostatectomy, to assess pathologic changes induced by bortezomib in prostate cancer specimen, and to verify alterations by the drug in proteasome protein targets. EXPERIMENTAL DESIGN: Bortezomib is a proteasome inhibitor that has shown activity in vitro and in vivo in prostate cancer. We performed a neoadjuvant clinical trial of bortezomib in men with prostate cancer at high risk of recurrence. The primary endpoints were to evaluate safety and biological activity. RESULTS: Bortezomib is generally safe in the preoperative setting. Antitumor activity was manifested by tumor cytopathic effect, drops in serum prostate-specific antigen in some patients, and increases in tumor apoptosis. This was associated with cytoplasmic entrapment of nuclear factor-kappaB. We found an unexpected increase in proliferation in treated tissues and in vitro. Bortezomib also increased SRC-3 levels and phosphorylated Akt, both in vitro and in treated prostate cancer tissues. Knockdown of SRC-3 blocked the increase in activated Akt in vitro. Combined treatment with bortezomib and the Akt inhibitor perifosine was more effective than either agent alone in vitro. CONCLUSION: These data suggest that combined therapies targeting the proteasome and the Akt pathway may have increased efficacy.

Reprint of: Stromogenic prostatic carcinoma pattern (carcinomas with reactive stromal grade 3) in needle biopsies predicts biochemical recurrence-free survival in patients after radical prostatectomy.

We previously reported that reactive stromal grading in radical prostatectomies is a predictor of recurrence and that reactive stromal grading 0 and 3 are associated with lower biochemical recurrence-free survival rates than reactive stromal grading 1 and 2. We explored the prognostic significance of reactive stromal grading in preoperative needle biopsies. At Baylor College of Medicine, 224 cases of prostatic carcinoma were diagnosed by needle biopsy. Reactive stromal grading was evaluated on hematoxylin-eosin (H&E)-stained sections on the basis of previously described criteria: grade 0, with 0% to 5% reactive stroma; grade 1, 6% to 15%; grade 2, 16% to 50%; grade 3, 51% to 100%, or at least a 1:1 ratio between glands and stroma. Kaplan-Meier and Cox proportional hazard analyses were used. Reactive stromal grading distribution was as follows: reactive stromal grading 0, 1 case (0.5%); reactive stromal grading 1, 149 cases (66.5%); reactive stromal grading 2, 59 cases (26.3%); reactive stromal grading 3, 15 cases (6.7%). Reactive stromal grading in biopsies was correlated with adverse clinicopathologic parameters in the prostatectomy. Patients with reactive stromal grading 1 and 2 had better survival than those with 0 and 3 (P = .0034). Reactive stromal grading was an independent predictor of recurrence (hazard ratio = 1.953; P = .0174). Reactive stromal grading is independent of Gleason 4 + 3 and 3 + 4 in patients with a Gleason score of 7. Quantitation of reactive stroma and recognition of the stromogenic carcinoma in H&E-stained biopsies is useful to predict biochemical recurrence in prostate carcinoma patients independent of Gleason grade and prostate-specific antigen.

Relaxin promotes prostate cancer progression.

PURPOSE: To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo. EXPERIMENTAL DESIGN: The relaxin and its receptor leucine-rich repeat containing G protein-coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP). RESULTS: The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals. CONCLUSIONS: Relaxin signaling plays a role in prostate cancer progression.

Targeting Aurora kinases for the treatment of prostate cancer.

Inappropriate expression of the Aurora kinases can induce aberrant mitosis, centrosome irregularities, and chromosomal instability, which lead to anueploidy and cell transformation. Here, we report that Aurora-A and Aurora-B are highly expressed in primary human and mouse prostate cancers and prostate cancer cell lines. In clinical samples, levels of Aurora-A and Aurora-B were significantly elevated in prostatic intraepithelial neoplasia lesions and prostate tumors when compared with the non-neoplastic samples. Interestingly, expression of Aurora-A in non-neoplastic prostates correlated with seminal vesicle invasion (rho = 0.275, P = 0.0169) and in prostate tumor with positive surgical margins (rho = 0.265, P = 0.0161). In addition, nuclear expression of Aurora-B in prostatic intraepithelial neoplasia lesions correlated with clinical staging of the tumor (rho = -0.4, P = 0.0474) whereas cytoplasmic expression in tumors correlated with seminal vesicle invasion (rho = 0.282, P = 0.0098). Cell lines and primary tumors derived from the TRAMP model were also found to express high levels of Aurora-A and Aurora-B. When human PC3, LNCaP, and mouse C1A cells were treated with the potent Aurora kinase inhibitor VX680, which attenuates phosphorylation of histone H3, cancer cell survival was reduced. VX680 could further reduce cell viability >2-fold when used in combination with the chemotherapy drug doxorubicin. Our findings support a functional relationship between Aurora kinase expression and prostate cancer and the application of small-molecule inhibitors in therapeutic modalities.

Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.

Caveolin-1 (cav-1) is a major scaffolding component of cell membrane invaginations (caveolae). It is involved in sequestering numerous effectors and signaling molecules and has antiapototic activities in prostate cancer. Perineural invasion (PNI) is associated with decreased apoptosis of cancer cells both in human tissues and the in vitro PNI model. We show here that stromal (perineurium) production of cav-1 is involved in a paracrine antiapoptotic loop in PNI. Transforming growth factor-beta1 is up-regulated in the cancer cells as they approach the nerve and is thought to up-regulate cav-1 in the perineurium of nerves with prostate cancer. Cav-1 is then secreted into the microenvironment and used by prostate cancer cells to inhibit apoptosis. In the in vitro PNI model, this phenomenon is partially reversed by neutralizing cav-1 antibodies or using ganglia from cav-1 knockout mice. Our results show a novel paracrine mechanism used by the prostate cancer in PNI to increase their proliferative activity and decrease apoptosis.

Stromogenic prostatic carcinoma pattern (carcinomas with reactive stromal grade 3) in needle biopsies predicts biochemical recurrence-free survival in patients after radical prostatectomy.

We previously reported that reactive stromal grading in radical prostatectomies is a predictor of recurrence and that reactive stromal grading 0 and 3 are associated with lower biochemical recurrence-free survival rates than reactive stromal grading 1 and 2. We explored the prognostic significance of reactive stromal grading in preoperative needle biopsies. At Baylor College of Medicine, 224 cases of prostatic carcinoma were diagnosed by needle biopsy. Reactive stromal grading was evaluated on hematoxylin-eosin (H&E)-stained sections on the basis of previously described criteria: grade 0, with 0% to 5% reactive stroma; grade 1, 6% to 15%; grade 2, 16% to 50%; grade 3, 51% to 100%, or at least a 1:1 ratio between glands and stroma. Kaplan-Meier and Cox proportional hazard analyses were used. Reactive stromal grading distribution was as follows: reactive stromal grading 0, 1 case (0.5%); reactive stromal grading 1, 149 cases (66.5%); reactive stromal grading 2, 59 cases (26.3%); reactive stromal grading 3, 15 cases (6.7%). Reactive stromal grading in biopsies was correlated with adverse clinicopathologic parameters in the prostatectomy. Patients with reactive stromal grading 1 and 2 had better survival than those with 0 and 3 (P = .0034). Reactive stromal grading was an independent predictor of recurrence (hazard ratio = 1.953; P = .0174). Reactive stromal grading is independent of Gleason 4 + 3 and 3 + 4 in patients with a Gleason score of 7. Quantitation of reactive stroma and recognition of the stromogenic carcinoma in H&E-stained biopsies is useful to predict biochemical recurrence in prostate carcinoma patients independent of Gleason grade and prostate-specific antigen.

Forkhead protein FKHR and its phosphorylated form p-FKHR in human prostate cancer.

In vitro studies suggest that the proapoptotic function of forkhead protein FKHR is probably inactivated by means of phosphorylation through the protein kinase B pathway. However, the clinical significance of FKHR in prostate cancer remains unclear. Six hundred forty radical prostatectomies were used for building tissue microarrays. Slides were stained with antibodies against FKHR and phosphorylated FKHR (p-FKHR). Correlations with clinicopathologic parameters were analyzed by Spearman rank test. Cox regression test and Kaplan-Meier test were used to determine the probability of disease recurrence, which is defined as a serum prostate-specific antigen (PSA) level greater than 0.4 ng/mL after radical prostatectomy. Nuclear FKHR level was higher in normal prostate than in benign prostatic hyperplasia and prostate cancer (P = .0000). Nuclear expression of FKHR was correlated with preoperative PSA level (rho = 0.108, P = .029), extracapsular extension (rho = 0.137, P = .005), and seminal vesicle invasion (rho = 0.101, P = .039). FKHR expression was not a significant indicator of biochemical failure by either univariate or multivariate analysis. Nuclear p-FKHR expression correlated with patients' age (rho = 0.179, P = .0003), Gleason score (rho = 0.130, P = .0083), extracapsular extension (rho = 0.227, P = .0000), clinical stage (Union Internationale Contre le Cancer system) (rho = 0.166, P = .0007), and lymph node status (rho = 0.101, P = .0401). Cytoplasmic p-FKHR correlated with patients' age (rho = 0.146, P = .0030) and clinical stage (rho = 0.117, P = .0180). Cytoplasmic p-FKHR was a significant indicator of biochemical recurrence (P = .0164; hazard ratio, 1.114-2.929). Nuclear p-FKHR strongly correlated with phosphorylated protein kinase B (rho = 0.368, P = .0000), androgen receptor (rho = 0.385, P = .0000), and Skp-2 (rho = 0.170, P = .0036). Our data suggest that the proapoptotic role of FKHR is probably regulated by several signaling pathways in prostate cancer.

Enhanced survival in perineural invasion of pancreatic cancer: an in vitro approach.

Pancreatic cancer (PanCa) is characterized by perineural invasion (PNI), early lymph node and liver metastasis, and poor prognosis. PNI is one of the important causes of local recurrence. Little is known about the mechanism of PNI in PanCa. We presented a novel model system that may shed light on the mystery of PNI in PanCa. In this study, mouse dorsal root ganglia (DRGs) and human PanCa cell line (MIA PaCa-2) were cocultured in Matrigel matrix (BD Biosciences, San Jose, CA) to build this PNI model. MIA PaCa-2 cell line alone (control 1) or DRG alone (control 2) was cultured with Matrigel matrix as controls. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were observed under inverted microscopy and then were photographed and quantitated with the Optimas imaging system (Optimas Corp., Bothell, MA). At day 14, both the experimental and control 2 samples were harvested and subjected to total RNA isolation and fixed in paraffin-embedded blocks. Slides cut from paraffin blocks were studied with Ki-67 immunostaining and TUNEL assay. Gene profiling was performed using complementary DNA microarray. Overexpressed target genes were verified by quantitative reverse transcriptase polymerase chain reaction. The results showed that reciprocity was observed between neurites and MIA PaCa colonies with 24 hours of coculture. Neurite outgrowth was stimulated in the presence of pancreatic carcinoma cells, which showed 2-fold more area than did control 2. After 72 hours, MIA PaCa colonies cocultured with DRG exhibited 58% more colony area than did control 1. The Ki-67 index of the DRG/MIA PaCa cells (mean, 5.02%) was significantly higher than that in control 1 (mean, 1.18%) (P < .05); in contrast, the apoptotic index in the DRG/MIA PaCa cells was significantly lower (mean, 0.45%) than that in the control 1 (mean, 1.85%) (P < .001). Prosurvival genes MALT1 and TRAF were increased 2-fold in DRG/MIA PaCa compared with controls. We demonstrated that neural-epithelial interaction is a mutually beneficial process for the growth of nerves and PanCa cells. It is possible that oncogenes and growth factors might act synergistically in promoting proliferation and/or inhibiting apoptosis, a survival strategy crucial to the development of PNI in PanCa.

Cellular interactions of the phosphorylated form of AKT in prostate cancer.

Phospho-Akt (P-Akt1) promotes proliferation and increased survival in vitro and plays an important role in prostate cancer (PCa) progression as well as the prediction of the probability of recurrence. In this study, the goal was to demonstrate the involvement and impact of P-Akt1 on cellular interactions, biomechanisms, and pathways in PCa. Tissue microarrays from 640 PCa patients were immunostained with various antibodies. Ki-67 was used to measure proliferation index, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling was used for apoptotic index. Increased expression of P-Akt1 was associated with an increased proliferation but inversely correlated with apoptotic index. Higher levels of P-Akt1 are associated with both higher levels of cytoplasmic p27 and higher levels of nuclear p27, suggesting an involvement in both cytoplasmic entrapment and phosphorylation of p27. P-Akt1 expression significantly correlated with nuclear and cytoplasmic staining of FHKR and GSK. The strongest correlations were found with the P- forms of both, suggesting enzyme kinetics in the latter. Here, phosphorylation is the principal method of FHKR and GSK inactivation. P-Akt1 correlated with nuclear transcription factor kappa B, suggesting a role in the inhibition through phosphorylation of nuclear transcription factor kappa B. The results of the current study are unique because of the scope of the markers and the size of the population used. In vitro- and in vivo-derived information of P-Akt1 and its downstream effectors demonstrates significant involvement in PCa. Our data suggest that PCa uses multiple mechanisms to regulate this pathway and substantiate the concept of redundancy in cancer pathway regulation. Consequently, new hypothesis-driven studies can be derived from this information.

Bcl-2 and bax expression and prostate cancer outcome in men treated with radiotherapy in Radiation Therapy Oncology Group protocol 86-10.

PURPOSE: Bcl-2 and bax are proteins with opposing roles in apoptosis regulation; yet abnormal expression of either has been associated with failure after radiotherapy (RT). In this study we examined bcl-2 and bax expression as predictive markers in men treated with radiotherapy +/- androgen deprivation on Radiation Therapy Oncology Group (RTOG) protocol 86-10. EXPERIMENTAL DESIGN: Suitable archival diagnostic tissue was obtained from 119 (26%) patients for bcl-2 analysis and 104 (23%) patients for bax analysis. Cox proportional hazards multivariate analysis was used to determine the relationship of abnormal bcl-2 and bax expression to the end points of local failure, distant metastasis, cause-specific mortality, and overall mortality. Bcl-2 overexpression was classified as any tumor cell cytoplasmic staining and altered bax expression was classified as greater or lesser cytoplasmic staining intensity of tumor cells as compared with adjacent normal prostate epithelium. RESULTS: The study cohort exhibited bcl-2 overexpression in 26% (n = 30) of cases and abnormal bax expression in 47% (n = 49) of cases. A borderline significant relationship was observed between abnormal bax expression and higher Gleason score (p = 0.08). In univariate and multivariate analyses, there was no statistically significant relationship seen between abnormal bcl-2 or bax expression and outcome. CONCLUSIONS: Abnormal bcl-2 and bax expression were not related to any of the end points tested. The cohort examined was comprised of patients with locally advanced disease and it is possible that these markers may be of greater value in men with earlier-stage prostate cancer.

The prolyl isomerase Pin1 is a novel prognostic marker in human prostate cancer.

Prostate cancer (PCa) is the most common male cancer in the United States. A major challenge that remains is to predict the clinical outcome in managing PCa patients. The prolyl isomerase Pin1 has been shown to be overexpressed in some human cancer tissues and thought to be an important player in several oncogenic pathways. However, the relationship between Pin1 expression and clinical outcome of cancer patients has not been explored. In this study, we investigated the role of Pin1 in human PCa progression and its clinicopathological significance. Immunohistochemical assessment with affinity-purified polyclonal Pin1-specific antibodies was performed on formalin-fixed paraffin sections of tissue microarray composed of 580 radical prostatectomy specimens. As determined by visual semiquantitation and confirmed by automated image analysis quantitation, Pin1 expression was positively correlated with clinical stage. Furthermore, Cox survival analysis results indicated that patients with a higher Pin1 expression had a significantly higher probability of recurrence than their counterparts with low Pin1 expression, as defined by a serum prostate-specific antigen level of > or =0.4 ng/ml on two consecutive occasions after radical prostatectomy. In addition, patients with high Pin1 expression had almost 4 times the risk of having earlier recurrence than those with low Pin1 expression; patients with a very high level had 8.1 times the risk of an earlier recurrence than a low Pin1 expresser. Pin1 was also an excellent predictor of recurrence in the subset of patients with Gleason score 6 or 7 when analyzed separately: a patient with high Pin1 expression had 8.6 times the risk of having earlier recurrence than one with low Pin1 expression. Pin1 expression is as good as or better than currently used postoperatively available clinicopathological parameters and potentially could be used in the preoperative setting to assist in choice of treatment. Thus, this study suggests a role for Pin1 expression as a potentially excellent prognostic marker in PCa and suggests that Pin1 may also serve as a novel therapeutic target for PCa.

Growth and survival mechanisms associated with perineural invasion in prostate cancer.

Perineural invasion (PNI) is the major mechanism of prostate cancer spread outside the prostate. Apoptotic and proliferation indices were determined in PNI cells using the PNI in vitro model and human PNI in tissue microarrays. RNA was extracted from the PNI model and controls and evaluated by cDNA microarray analysis. Differential expression of candidate genes was confirmed by real-time quantitative PCR, fluorescence, and immunohistochemistry using tissue microarrays. Genistein and BAY 11-7085 were added to the supernatant of cocultures and controls in microchamber cultures. The significance of nuclear factor kappaB (NFkappaB) nuclear translocation in human PNI was analyzed using Kaplan-Meier analysis. An increase in proliferation and a decrease in apoptosis were observed in human PNI cells and the PNI model as compared with controls. Three of 15 genes up-regulated in the cDNA microarray were involved in the apoptosis signaling pathway (NFkappaB), and its downstream targets defender against cell death 1 and PIM-2. The increase was corroborated by real-time quantitative PCR and immunofluorescence. NFkappaB nuclear translocation was seen in the in vitro model and human tissues, where strong nuclear expression was associated with a decrease in recurrence-free survival. Addition of genistein and BAY 11-7085 resulted in a decrease in NFkappaB, PIM-2 and defender against cell death 1 as well as a reversal of the inhibition of apoptosis. This is the first description of a biological mechanism and functional significance of PNI. Cancer cells in a perineural location acquire a survival and growth advantage using a NFkappaB survival pathway. Targeting PNI might help detain local spread of the tumor and influence survival.

Nuclear Receptor Corepressor 1 Expression and Output Declines with Prostate Cancer Progression.

PURPOSE: Castration therapy in advanced prostate cancer eventually fails and leads to the development of castration-resistant prostate cancer (CRPC), which has no cure. Characteristic features of CRPC can be increased androgen receptor (AR) expression and altered transcriptional output. We investigated the expression of nuclear receptor corepressor 1 (NCOR1) in human prostate and prostate cancer and the role of NCOR1 in response to antiandrogens. EXPERIMENTAL DESIGN: NCOR1 protein levels were compared between matched normal prostate and prostate cancer in 409 patient samples. NCOR1 knockdown was used to investigate its effect on bicalutamide response in androgen-dependent prostate cancer cell lines and transcriptional changes associated with the loss of NCOR1. NCOR1 transcriptional signature was also examined in prostate cancer gene expression datasets. RESULTS: NCOR1 protein was detected in cytoplasm and nuclei of secretory epithelial cells in normal prostate. Both cytoplasmic and nuclear NCOR1 protein levels were lower in prostate cancer than in normal prostate. Prostate cancer metastases show significant decrease in NCOR1 transcriptional output. Inhibition of LNCaP cellular proliferation by bicalutamide requires NCOR1. NCOR1-regulated genes suppress cellular proliferation and mediate bicalutamide resistance. In the mouse, NCOR1 is required for bicalutamide-dependent regulation of a subset of the AR target genes. CONCLUSIONS: In summary, we demonstrated that NCOR1 function declines with prostate cancer progression. Reduction in NCOR1 levels causes bicalutamide resistance in LNCaP cells and compromises response to bicalutamide in mouse prostate in vivo Clin Cancer Res; 22(15); 3937-49. ©2016 AACR.

High levels of phosphorylated form of Akt-1 in prostate cancer and non-neoplastic prostate tissues are strong predictors of biochemical recurrence.

Akt is a serine-threonine-kinase that phosphorylates proteins in several pathways regulating aspects of metabolism, apoptosis, and proliferation. Akt signaling promotes proliferation and increased cell survival and is thought to play an important role in prostate cancer progression. Tissue microarrays (640 patients) with triplicate cores of non-neoplastic prostate, BPH, and index tumor were immunostained with antibody to Phospho-Akt (Ser473), digitized, and quantified. The expression index (Intensity*Percentage) was used for statistical analysis. P-Akt-1 staining was found in both the non-neoplastic and cancer tissues, predominantly in cytoplasmic locations. High level P-Akt-1 is expressed almost exclusively in cancer. By Kaplan-Meier actuarial model, high expression of P-Akt-1 in prostate cancer was predictive of a higher probability of recurrence on univariate and multivariate analysis. Akt-1 expression was an independent prognostic indicator of biochemical recurrence-free survival when Gleason 6 and 7 patients were analyzed separately. Surprisingly, a high level of P-Akt-1 expression in non-neoplastic tissues is also an independent predictor of biochemical recurrence. This suggests that some patients might have an inherent predisposition to express a high level of P-Akt-1 and, therefore, to have an adverse prognosis. We conclude that P-Akt-1 is most likely involved in the progression of prostate cancer and is an excellent biomarker for biochemical recurrence.