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1.  A New Therapy Paradigm for Prostate Cancer Founded on Clinical Observations 
Efficacy equivalent to that reported in other common adult solid tumors considered to be chemotherapy-sensitive has been reported with Docetaxel in patients with castrate-resistant prostate cancer. However, in contrast to other cancers, the expected increase in efficacy with the use of chemotherapy in earlier disease states has not been reported to date in prostate cancer. On the basis of these observations, we speculated that the therapy development paradigm used successfully in other cancers may not apply to the majority of prostate cancers. Several lines of supporting clinical and experimental observations implicate the tumor microenvironment in prostate carcinogenesis and resistance to therapy.
We conclude that a foundation to guide the development of therapy for prostate cancer is required. The therapy paradigm we propose accounts for the central role of the tumor microenvironment in bone and, if correct, will lead to microenvironment-targeted therapy.
PMCID: PMC3891669  PMID: 20145177
2.  Targeting Constitutively Activated β1 Integrins inhibits Prostate Cancer Metastasis 
Molecular cancer research : MCR  2013;11(4):405-417.
Disseminated prostate cancer (PCa) cells must survive in circulation for metastasis to occur. Mechanisms by which these cells survive are not well understood. By immunohistochemistry of human tissues, we found that levels of β1 integrins and integrin-induced autophosphorylation of FAK (pFAK-Y397) are increased in PCa cells in primary PCa and lymph node metastases, suggesting that β1 integrin activation occurs in metastatic progression of PCa. A conformation-sensitive antibody, 9EG7, was used to examine β1 integrin activation. We found that β1 integrins are constitutively activated in highly metastatic PC3 and PC3-mm2 cells, with less activation in low metastatic LNCaP and C4-2B4 cells. Increased β1 integrin activation as well as the anoikis resistance in PCa cells correlated with metastatic potential in vivo. Knockdown of β1 integrin abrogated anoikis resistance in PC3-mm2 cells. In agreement with β1 integrin activation, PC3-mm2 cells strongly adhered to type I collagen and fibronectin, a process inhibited by the β1 integrin neutralizing antibody mAb 33B6. mAb 33B6 also inhibited the phosphorylation of β1 integrin downstream effectors, focal adhesion kinase (FAK) and AKT, leading to a 3-fold increase in PC3-mm2 apoptosis. Systemic delivery of mAb 33B6 suppressed spontaneous metastasis of PC3-mm2 from the prostate to distant lymph nodes following intra-prostatic injection and suppressed metastasis of PC3-mm2 to multiple organs following intra-cardiac injection. Thus, constitutively activated β1 integrins play a role in survival of PC3-mm2 cells in circulation and represent a potential target for metastasis prevention.
PMCID: PMC3631285  PMID: 23339185
prostate cancer; metastasis; integrin; extracellular matrix; collagen; fibronectin
3.  Modeling A Lethal Prostate Cancer Variant with Small Cell Carcinoma Features 
Small-cell prostate carcinoma (SCPC) morphology predicts for a distinct clinical behavior, resistance to androgen ablation, and frequent but short responses to chemotherapy. We sought to develop model systems that reflect human SCPC and can improve our understanding of its biology.
Experimental Design
We developed a set of CRPC xenografts and examined their fidelity to their human tumors of origin. We compared the expression and genomic profiles of SCPC and large cell neuroendocrine carcinoma (LCNEC) xenografts to those of typical prostate adenocarcinoma xenografts. Results were validated immunohistochemically in a panel of 60 human tumors.
The reported SCPC and LCNEC xenografts retain high fidelity to their human tumors of origin and are characterized by a marked upregulation of UBE2C and other mitotic genes in the absence of AR, retinoblastoma (RB1) and cyclin D1 (CCND1) expression. We confirmed these findings in a panel of CRPC patients' samples. In addition, array comparative genomic hybridization of the xenografts showed that the SCPC/LCNEC tumors display more copy number variations than the adenocarcinoma counterparts. Amplification of the UBE2C locus and microdeletions of RB1 were present in a subset, but none displayed AR nor CCND1 deletions. The AR, RB1, and CCND1 promoters showed no CpG methylation in the SCPC xenografts.
Modeling human prostate carcinoma with xenografts allows in-depth and detailed studies of its underlying biology. The detailed clinical annotation of the donor tumors enables associations of anticipated relevance to be made. Futures studies in the xenografts will address the functional significance of the findings.
PMCID: PMC3923417  PMID: 22156612
castration resistance prostate cancer; small cell neuroendocrine carcinoma; retinoblastoma; UBE2C; cyclin D1
4.  When “Dueling Technologies” are Mistaken for Progress 
BJU international  2011;107(11):1699-1700.
PMCID: PMC3922611  PMID: 21592278
5.  In Silico Functional Profiling of Individual Prostate Cancer Tumors: Many Genes, Few Functions 
Cancer genomics & proteomics  2012;9(3):109-114.
Identification of genes that are differently expressed is a common approach used to analyze genetic mechanisms underlying cancer development. However, recent study results suggest that many such genes relate to a small number of biological functions. We hypothesized that analysis of these functions provides a better understanding of tumor biology than does actual identification of these genes does.
Materials and Methods
We re-analyzed publicly available gene expression data for paired samples of prostate tumor and adjacent normal tissue from the same patients to identify genes differently expressed in individual tumors and then used them to identify the functions.
We found significant interindividual variation in the type and the number of functions. After adjusting for redundancy and nonspecificity of the functional terms, we identified seven functions. Several of them showed a strong association with clinical traits, e.g. age at diagnosis, preoperative prostate-specific antigen concentration, Gleason grade, and biochemical recurrence. Actin cytoskeleton was the function most frequently associated with clinical traits. Of note, the association between function and clinical traits was much stronger than that between the genes differently expressed and those traits.
Different prostate tumors differ in their functional profiles. Functions of differently expressed genes are strongly associated with clinical traits. This suggests that analysis of functions of differently expressed genes may provide a better description of tumor biology than does analysis of the respective genes.
PMCID: PMC3922615  PMID: 22593245
Gene expression; prostate cancer; in silico; functional profiling; functionality index
6.  Dasatinib Combined With Docetaxel For Castration-Resistant Prostate Cancer: Results From a Phase 1/2 Study 
Cancer  2011;118(1):63-71.
To determine the potential efficacy of targeting both the tumor and bone microenvironment in patients with castration-resistant prostate cancer (CRPC), we conducted a phase 1/2 trial combining docetaxel with dasatinib, an oral SRC inhibitor.
In phase 1, 16 men received dasatinib 50–120 mg once daily (QD) and docetaxel 60–75 mg/m2 every 21 days (Q21D). In phase 2, 30 additional men received dasatinib 100 mg QD/docetaxel 75 mg/m2 Q21D. Efficacy endpoints included changes in prostate-specific antigen (PSA), measurable disease, bone scans, and markers of bone metabolism. Safety and pharmacokinetics were also studied.
Combination dasatinib and docetaxel therapy was generally well tolerated. Thirteen of 46 patients (28%) had a grade 3/4 toxicity. Drug–drug interactions and a maximum tolerated dose were not identified. Durable 50% PSA declines occurred in 26/46 patients (57%). Of 30 patients with measurable disease, 18 (60%) had a partial response. Fourteen patients (30%) had disappearance of a lesion on bone scan. In bone-marker assessments, 33/38 (87%) and 26/34 (76%) had decreases in urinary N-telopeptide or bone-specific alkaline phosphatase levels, respectively. Twenty-eight patients (61%) received single-agent dasatinib following docetaxel discontinuation and had stabilization of disease for an additional 1–12 months.
The high objective response rate and favorable toxicity profile are promising and justify randomized studies of docetaxel and dasatinib in CRPC. Parallel declines in levels of PSA and bone markers are consistent with co-targeting of epithelial and bone compartments of the cancer. Treatment with single-agent dasatinib following docetaxel cessation warrants further study.
PMCID: PMC3898168  PMID: 21976132
Dasatinib; Docetaxel; Prostate Cancer; Metastases; Bone
7.  Randomized Phase 3 Trial of Abiraterone Acetate in Men with Metastatic Castration-Resistant Prostate Cancer and No Prior Chemotherapy 
The New England journal of medicine  2012;368(2):138-148.
Abiraterone acetate, an androgen biosynthesis inhibitor, improves overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC) post-chemotherapy. Many mCRPC patients never receive chemotherapy and thus cannot benefit from abiraterone acetate; we evaluated this agent in mCRPC patients who had not received chemotherapy.
In this double-blind study, 1088 patients were randomized 1:1 to abiraterone acetate (1000 mg) plus prednisone (5 mg twice daily) or placebo plus prednisone. Co-primary end points were radiographic progression-free survival (rPFS) and OS. Secondary end points measured clinically relevant landmarks of mCRPC progression. Patient-reported outcomes included pain progression and quality of life.
The study was unblinded after a planned interim analysis (IA) at 43% of OS events. Treatment with abiraterone acetate-prednisone resulted in a 57% reduction in the risk of radiographic progression or death (hazard ratio [HR], 0.43; 95% confidence interval [CI]: 0.35 to 0.52; P<0.001; 13% OS events IA) and an estimated 25% decrease in the risk of death (HR, 0.75; 95% CI: 0.61 to 0.93; P=0.009; 43% OS events IA). Secondary end points supported superiority of abiraterone acetate-prednisone: time to cytotoxic chemotherapy initiation, opiate use for cancer-related pain, prostate-specific antigen progression (all P<0.001) and performance status deterioration (P=0.005). Self-reported time to pain progression and patient functional status degradation favored abiraterone acetate-prednisone (P=0.05 and P=0.003). Grade 3/4 mineralocorticoid-related adverse events and liver function test abnormalities were more common with abiraterone acetate-prednisone.
Abiraterone acetate produces OS and rPFS benefits, as well as significant delays in clinical deterioration and initiation of chemotherapy, in mCRPC.
PMCID: PMC3683570  PMID: 23228172
Abiraterone acetate; prednisone; metastatic castration-resistant prostate cancer; androgen; CYP17
8.  Neuroendocrine Prostate Cancer Xenografts with Large-cell and Small-cell Features Derived from a Single Patient’s Tumor: Morphological, Immunohistochemical and Gene Expression Profiles 
The Prostate  2010;71(8):10.1002/pros.21301.
Small-cell carcinoma (SCC) of the prostate is an AR-negative variant of prostate cancer found at progression in 10–20% of castrate-resistant disease. Its finding predicts a distinct clinical course and a poor prognosis. Large-cell neuroendocrine carcinoma (LCNEC) is a much rarer variant that behaves similarly to SCC. The biological mechanisms that drive these disease variants are poorly understood.
Eight tumor fragments from the salvage pelvic exenteration specimen of a patient with castrate-resistant prostate carcinoma were subcutaneously implanted into 6- to 8-week-old male CB17 SCID mice. Serial tissue sections and tissue microarrays of the resulting MDA PCa 144 xenograft lines were used for histopathologic and immunohistochemical characterization of the xenografts and their tissue of origin. RNA from two representative xenograft sublines was used for gene-expression profiling.
All eight fragments formed tumors: four of the MDA PCa 144 xenograft sublines had morphologic characteristics of SCC and four, of LCNEC. All retained high fidelity to their parent tumor tissue, which remained stable through serial passages. Morphological transitions in the specimen of origin suggested LCNEC represents an intermediate step between adenocarcinoma and SCC. Over 2,500 genes were differentially expressed between the SCC (MDA PCa 144-13) and the LCNEC (MDA PCa 144-4) sublines and enriched in “Nervous System Development” Gene Ontology subtree.
The eight xenograft models described represent the spectrum of neuroendocrine carcinomas in prostate cancer and will be valuable preclinical tools to study the pathogenesis of and therapy targets for this increasingly recognized subset of lethal prostate cancer.
PMCID: PMC3883511  PMID: 21456067
castrate-resistant; cancer; androgen-independent; neural development; array
9.  Integrated Hedgehog signaling is induced following castration in human and murine prostate cancers 
The Prostate  2012;73(2):10.1002/pros.22550.
The interplay between androgen and Hedgehog (Hh) signaling pathways may be associated with prostate cancer progression and resistance to therapy.
Tissue microarrays from prostatectomy specimens were derived from 53 patients treated preoperatively with androgen ablation (AA) with or without chemotherapy, and from 26 stage- and grade-matched controls. A previously characterized androgen-regulated human prostate cancer xenograft was used to conduct parallel murine studies. Expression of markers of interest was determined on both untreated and castrated tumors.
Four-month exposure to AA or AA with chemotherapy led to a uniform increase in Hh signaling as compared to controls, paired with an inverse trend of androgen receptor (AR) and CYP17 expression in clinically derived specimens. Changes in the expression profiles of Hh signaling were observed in the epithelium and stroma, in response to genotoxic stress of androgen ablation and chemotherapy. A reduced expression of KI67and increased bcl2 expression was observed in the malignant epithelial compartment.
To our knowledge, this is the first clinical evidence that Hh signaling is induced by AA or the combination of AA and chemotherapy and, by inference, contributes to castrate-resistant progression of prostate cancer as supported by parallel human and murine studies. These data are in agreement with previous reports that implicate Hh signaling in castrate-resistant progression of prostate cancer. Based on these findings, we are pursuing parallel clinical and murine investigations to determine if Hh signaling inhibition combined with AA will be more effective than AA alone.
PMCID: PMC3878994  PMID: 22753310
Prostate cancer; preoperative treatment; Hedgehog signaling; resistance to treatment; androgen ablation
10.  Modified Logistic Regression Models Using Gene Coexpression and Clinical Features to Predict Prostate Cancer Progression 
Predicting disease progression is one of the most challenging problems in prostate cancer research. Adding gene expression data to prediction models that are based on clinical features has been proposed to improve accuracy. In the current study, we applied a logistic regression (LR) model combining clinical features and gene co-expression data to improve the accuracy of the prediction of prostate cancer progression. The top-scoring pair (TSP) method was used to select genes for the model. The proposed models not only preserved the basic properties of the TSP algorithm but also incorporated the clinical features into the prognostic models. Based on the statistical inference with the iterative cross validation, we demonstrated that prediction LR models that included genes selected by the TSP method provided better predictions of prostate cancer progression than those using clinical variables only and/or those that included genes selected by the one-gene-at-a-time approach. Thus, we conclude that TSP selection is a useful tool for feature (and/or gene) selection to use in prognostic models and our model also provides an alternative for predicting prostate cancer progression.
PMCID: PMC3866878  PMID: 24367394
11.  Effects of Abiraterone Acetate on Androgen Signaling in Castrate-Resistant Prostate Cancer in Bone 
Journal of Clinical Oncology  2011;30(6):637-643.
Persistent androgen signaling is implicated in castrate-resistant prostate cancer (CRPC) progression. This study aimed to evaluate androgen signaling in bone marrow–infiltrating cancer and testosterone in blood and bone marrow and to correlate with clinical observations.
Patients and Methods
This was an open-label, observational study of 57 patients with bone-metastatic CRPC who underwent transiliac bone marrow biopsy between October 2007 and March 2010. Patients received oral abiraterone acetate (1 g) once daily and prednisone (5 mg) twice daily. Androgen receptor (AR) and CYP17 expression were assessed by immunohistochemistry, testosterone concentration by mass spectrometry, AR copy number by polymerase chain reaction, and TMPRSS2-ERG status by fluorescent in situ hybridization in available tissues.
Median overall survival was 555 days (95% CI, 440 to 965+ days). Maximal prostate-specific antigen decline ≥ 50% occurred in 28 (50%) of 56 patients. Homogeneous, intense nuclear expression of AR, combined with ≥ 10% CYP17 tumor expression, was correlated with longer time to treatment discontinuation (> 4 months) in 25 patients with tumor-infiltrated bone marrow samples. Pretreatment CYP17 tumor expression ≥ 10% was correlated with increased bone marrow aspirate testosterone. Blood and bone marrow aspirate testosterone concentrations declined to less than picograms-per-milliliter levels and remained suppressed at progression.
The observed pretreatment androgen-signaling signature is consistent with persistent androgen signaling in CRPC bone metastases. This is the first evidence that abiraterone acetate achieves sustained suppression of testosterone in both blood and bone marrow aspirate to less than picograms-per-milliliter levels. Potential admixture of blood with bone marrow aspirate limits our ability to determine the origin of measured testosterone.
PMCID: PMC3295561  PMID: 22184395
12.  Persistent, Biologically Meaningful Prostate Cancer After 1 Year of Androgen Ablation and Docetaxel Treatment 
Journal of Clinical Oncology  2011;29(18):2574-2581.
Clinicians are increasingly willing to treat prostate cancer within the primary site in the presence of regional lymph node or even limited distant metastases. However, no formal study on the merits of this approach has been reported. We used a preoperative clinical discovery platform to prioritize pathways for assessment as therapeutic targets and to test the hypothesis that the primary site harbors potentially lethal tumors after aggressive treatment.
Patients and Methods
Patients with locally advanced or lymph node–metastatic prostate cancer underwent 1 year of androgen ablation and three cycles of docetaxel therapy, followed by prostatectomy. All specimens were characterized for stage by accepted criteria. Expression of select molecular markers implicated in disease progression and therapy resistance was determined immunohistochemically and compared with that in 30 archived specimens from untreated patients with high-grade prostate cancer. Marker expression was divided into three groups: intracellular signaling pathways, stromal-epithelial interaction pathways, and angiogenesis.
Forty patients were enrolled, 30 (75%) of whom underwent prostatectomy and two (5%) who underwent cystoprostatectomy. Twenty-nine specimens contained sufficient residual tumor for inclusion in a tissue microarray. Immunohistochemical analysis showed increased epithelial and stromal expression of CYP17, SRD5A1, and Hedgehog pathway components, and modulations of the insulin-like growth factor I pathway.
A network of molecular pathways reportedly linked to prostate cancer progression is activated after 1 year of therapy; biomarker expression suggests that potentially lethal cancers persist in the primary tumor and may contribute to progression.
PMCID: PMC3138635  PMID: 21606419
13.  Phase II Study of Abiraterone Acetate Plus Prednisone in Chemotherapy-Naïve Metastatic Castration-Resistant Prostate Cancer Demonstrating Radiographic Flare Discordant With Serologic Measures of Response 
Abiraterone is an oral inhibitor of CYP17, essential for androgen biosynthesis. This multicenter study assessed its efficacy in patients with CRPC without prior exposure to chemotherapy or CYP17 targeted therapy, and assessed the frequency of interpretation of bone scans discordant with PSA and clinical response.
Patients and Methods
33 patients received abiraterone acetate 1000 mg daily with prednisone 5 mg twice daily in continuous 28-day cycles. Patients were evaluated monthly for efficacy and safety. Bone scan flare was defined as the combination, after 3 months of therapy, of an interpreting radiologist's report indicating “disease progression” in the context of a ≥50% decline in PSA, with scan improvement 3 months later.
A ≥ 50% PSA decline at week 12 was confirmed in 22/33 (67%) patients. PSA declines of ≥ 50% were seen in 26 (79%) patients. Undetectable PSA levels (≤ 0.1 ng/mL) occurred in 2 patients. Median time on therapy and time to PSA progression are 63 and 71 weeks, respectively. Twenty three patients were evaluable for bone scan flare. Progression was indicated in the radiologist's report in 12/23 (52 %), and 10/12 subsequently showed improvement. As prospectively defined, bone scan flare was observed in 10/23 (43.5%) evaluable patients or 10/33 (30%) enrolled patients. Adverse events were typically grade 1/2 and consistent with prior published abiraterone reports.
Clinical response to abiraterone acetate plus prednisone was frequent and durable in men with metastatic CRPC progressing on hormonal therapy with over half of patients on therapy > 1 year. Further investigation is needed to clarify the potential confounding effect of the frequently occurring bone scan flare phenomena on patient management and interpretation of clinical trial results.
PMCID: PMC3657705  PMID: 21632851
abiraterone acetate; castration-resistant prostate cancer; CRPC; hormone-resistant prostate cancer; therapy; efficacy
14.  Aberrant expression of katanin p60 in prostate cancer bone metastasis 
The Prostate  2011;72(3):291-300.
Katanin p60 is a microtubule-severing protein and is involved in microtubule cytoskeleton organization in both mitotic and non-mitotic processes. Its role in cancer metastasis is unknown.
Differential protein profiles of bone marrow aspirates were analyzed by chromatography, electropheresis and mass spectrometry. Expression of katanin p60 in primary and metastatic prostate cancer was examined by immunohistochemistry. Cellular function of katanin p60 was further examined in prostate cell lines.
In a proteomic profiling of bone marrow aspirates from men with prostate cancer, we found that katanin p60 was one of the proteins differentially expressed in bone metastasis samples. Immunohistochemical staining showed that katanin p60 was expressed in the basal cells in normal human prostate glands. In prostatic adenocarcinomas, in which the basal cells were absent, katanin p60 was expressed in the prostate cancer cells. In the specimens from bone metastasis, katanin p60 was detectable in the metastatic cancer cells. Strikingly, some of the metastatic cancer cells also co-expressed basal cell biomarkers including the tumor suppressor p53-homologous protein p63 and the high molecular weight cytokeratins, suggesting that the metastatic prostate cancer cells may have a basal cell-like phenotype. Moreover, overexpression of katanin p60 inhibited prostate cancer cell proliferation but enhanced cell migration activity.
Katanin p60 was aberrantly expressed during prostate cancer progression. Its expression in the metastatic cells in bone was associated with the re-emergence of a basal cell-like phenotype. The elevated katanin p60 expression may contribute to cancer cell metastasis via a stimulatory effect on cell motility.
PMCID: PMC3179562  PMID: 21681775
katanin; bone marrow; prostate cancer; metastasis
15.  Building a Statistical Model for Predicting Cancer Genes 
PLoS ONE  2012;7(11):e49175.
More than 400 cancer genes have been identified in the human genome. The list is not yet complete. Statistical models predicting cancer genes may help with identification of novel cancer gene candidates. We used known prostate cancer (PCa) genes (identified through KnowledgeNet) as a training set to build a binary logistic regression model identifying PCa genes. Internal and external validation of the model was conducted using a validation set (also from KnowledgeNet), permutations, and external data on genes with recurrent prostate tumor mutations. We evaluated a set of 33 gene characteristics as predictors. Sixteen of the original 33 predictors were significant in the model. We found that a typical PCa gene is a prostate-specific transcription factor, kinase, or phosphatase with high interindividual variance of the expression level in adjacent normal prostate tissue and differential expression between normal prostate tissue and primary tumor. PCa genes are likely to have an antiapoptotic effect and to play a role in cell proliferation, angiogenesis, and cell adhesion. Their proteins are likely to be ubiquitinated or sumoylated but not acetylated. A number of novel PCa candidates have been proposed. Functional annotations of novel candidates identified antiapoptosis, regulation of cell proliferation, positive regulation of kinase activity, positive regulation of transferase activity, angiogenesis, positive regulation of cell division, and cell adhesion as top functions. We provide the list of the top 200 predicted PCa genes, which can be used as candidates for experimental validation. The model may be modified to predict genes for other cancer sites.
PMCID: PMC3499550  PMID: 23166609
16.  Abiraterone and Increased Survival in Metastatic Prostate Cancer 
The New England journal of medicine  2011;364(21):1995-2005.
Biosynthesis of extragonadal androgen may contribute to the progression of castration-resistant prostate cancer. We evaluated whether abiraterone acetate, an inhibitor of androgen biosynthesis, prolongs overall survival among patients with metastatic castration-resistant prostate cancer who have received chemotherapy.
We randomly assigned, in a 2:1 ratio, 1195 patients who had previously received docetaxel to receive 5 mg of prednisone twice daily with either 1000 mg of abiraterone acetate (797 patients) or placebo (398 patients). The primary end point was overall survival. The secondary end points included time to prostate-specific antigen (PSA) progression (elevation in the PSA level according to prespecified criteria), progression-free survival according to radiologic findings based on prespecified criteria, and the PSA response rate.
After a median follow-up of 12.8 months, overall survival was longer in the abiraterone acetate–prednisone group than in the placebo–prednisone group (14.8 months vs. 10.9 months; hazard ratio, 0.65; 95% confidence interval, 0.54 to 0.77; P<0.001). Data were unblinded at the interim analysis, since these results exceeded the preplanned criteria for study termination. All secondary end points, including time to PSA progression (10.2 vs. 6.6 months; P<0.001), progression-free survival (5.6 months vs. 3.6 months; P<0.001), and PSA response rate (29% vs. 6%, P<0.001), favored the treatment group. Mineralocorticoid-related adverse events, including fluid retention, hypertension, and hypokalemia, were more frequently reported in the abiraterone acetate–prednisone group than in the placebo–prednisone group.
The inhibition of androgen biosynthesis by abiraterone acetate prolonged overall survival among patients with metastatic castration-resistant prostate cancer who previously received chemotherapy. (Funded by Cougar Biotechnology; COU-AA-301 number, NCT00638690.)
PMCID: PMC3471149  PMID: 21612468
Identifying genes associated with cancer development is typically accomplished by comparing mean expression values in normal and tumor tissues, which identifies differentially expressed (DE) genes. Interindividual variation (IV) in gene expression is indirectly included in DE gene identification because given the same absolute differences in means, genes with lower variance tend to have lower P values. We explored the direct use of IV in gene expression to identify candidate genes associated with cancer development. We focused on prostate (PCa) and lung (LC) cancers and compared IV in the expression level of genes shown to be cancer related with that in all other genes in the human genome. Compared with all those other genes, cancer-related genes tended to have greater IV in normal tissues and a greater increase in IV during the transition from normal to tumorous tissue. Genes without significantly different mean expression values between tumor and normal tissues but with greater IV in tumor than in normal tissue (note: the DE-based approach completely ignores those genes) had stronger associations with clinically important features like Gleason score in PCa or tumor histology in LC than all other genes were. Our results suggest that analyzing IV in gene expression level is useful in identifying novel candidate genes associated with cancer development.
PMCID: PMC3893106  PMID: 22809348
Gene expression; interindividual variation in gene expression; prostate cancer; lung cancer
International journal of radiation oncology, biology, physics  2012;85(1):10.1016/j.ijrobp.2012.08.036.
The aim of the study was to evaluate the prognostic value of prostate specific antigen (PSA) response to neoadjuvant androgen deprivation therapy (ADT) prior to dose-escalated radiation therapy (RT) and long-term ADT in high-risk prostate cancer.
Methods and Materials
We reviewed the charts of all patients diagnosed with high-risk prostate cancer and treated with a combination of long-term ADT (median, 24 months) and dose-escalated (median, 75.6 Gy) RT between 1990 and 2007. The association of patient, tumor and treatment characteristics with biochemical response to neoadjuvant ADT, and their effect on failure-free survival (FFS), time to distant metastasis (TDM), prostate cancer-specific mortality (PCSM) and overall survival (OS) were examined.
A total of 196 patients met the criteria for inclusion. Median follow up time for patients alive at last contact was 7.0 years (range, 0.5–18.1 years). Multivariate analysis identified the pre-radiation therapy PSA level (pre-RT PSA; <0.5 vs. ≥0.5 ng/ml) as a significant independent predictor of FFS (p=0.021), TDM (p=0.009), PCSM (p=0.039) and OS (p=0.037). On multivariate analysis, pre-treatment PSA (iPSA) and African-American race were significantly associated with failure to achieve a pre-RT PSA <0.5 ng/ml.
For high-risk prostate cancer patients treated with long-term ADT and dose-escalated RT, a pre-RT PSA level ≥0.5 ng/ml after neoadjuvant ADT predicts for worse survival measures. Both elevated iPSA and Afrian-American race are associated with increased risk of having a pre-RT PSA level ≥0.5 ng/ml. These patients should be considered for clinical trials testing newer more potent androgen depleting therapies such as abiraterone and MDV3100 in combination with radiation.
PMCID: PMC3840533  PMID: 23102837
pre-RT PSA; androgen deprivation therapy; radiation therapy; prostate cancer
19.  Androgen Regulation of 5α-Reductase Isoenzymes in Prostate Cancer: Implications for Prostate Cancer Prevention 
PLoS ONE  2011;6(12):e28840.
The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type–specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention.
PMCID: PMC3237548  PMID: 22194926
20.  Cadherin-11 increases migration and invasion of prostate cancer cells and enhances their interaction with osteoblasts 
Cancer research  2010;70(11):4580-4589.
Cell adhesion molecules have been implicated in the colonization of cancer cells to distant organs. Prostate cancer (PCa) has a propensity to metastasize to bone and cadherin-11, which is an osteoblast cadherin aberrantly expressed in PCa cells derived from bone metastases, has been shown to play a role in the metastasis of PCa cells to bone. However, the mechanism by which cadherin-11 is involved in this process is not known. Here, we show that expression of cadherin-11 in cadherin-11-negative C4-2B4 cells increases their spreading and intercalation into an osteoblast layer, and also stimulates C4-2B4 cell migration and invasiveness. Downregulation of cadherin-11 in cadherin-11-expressing metastatic PC3 cells decreases cell motility and invasiveness. Further, both the juxtamembrane (JMD) and β-catenin binding domains (CBS) in the cytoplasmic tail of cadherin-11 are required for cell migration and invasion, but not spreading. Gene array analyses showed that several invasion related genes, including MMP-7 and MMP-15, are upregulated in cadherin-11, but not in cad11-ΔJMD or cad11-ΔCBS, expressing C4-2B4 cells. These observations suggest that cadherin-11 not only provides a physical link between PCa cells and osteoblasts but also increases PCa cell motility and invasiveness that may facilitate the metastatic colonization of PCa cells in bone.
PMCID: PMC2923552  PMID: 20484040
cadherin-11; prostate cancer; osteoblast; adhesion; bone metastasis
21.  Morphologic Characterization of Preoperatively Treated Prostate Cancer: Toward a Post-Therapy Histologic Classification 
European urology  2009;57(6):1030-1038.
Preoperative treatment of prostate cancer (PCa) changes morphology of residual tumors so that the Gleason score is no longer valid.
To codify morphologic features of preoperatively treated PCa and identify potential classifiers predictive of outcome.
Design, setting, and participants
We performed a detailed morphologic evaluation of specimens obtained from 115 patients with high-risk PCa who had preoperative androgen ablation, alone or in combination with chemotherapy.
Included hierarchical clustering analysis of morphologic characteristics, associations with other pathologic parameters, and univariate and multivariate analyses in search for associations with disease outcome.
Results and limitations
Based on hierarchical clustering analysis, we categorized pretreated prostate cancer in three morphologically distinct groups: group A, characterized by a predominance of cell clusters, cell cords, and isolated cells; group B tumors, by intact and fused small glands; and group C tumors by any degree of cribriform growth pattern or intraductal tumor spread.
Univariate analysis identified associations between this grouping, pathologic tumor stage (p < 0.01) and residual tumor volume (p < 0.001). Presence of intraductal spread or cribriform pattern in biopsies was associated with group C tumors. The presence of cribriform or intraductal spread morphology and positive surgical margins were stronger predictors of biochemical relapse than pathologic stage on multivariate analysis. The number of specimens evaluated in this study was limited, and a prospective validation is warranted along with molecular studies to validate the proposed morphologic classifier.
If validated, this classification will introduce uniformity in the selection of tissue samples for biomarker studies, facilitate the comparison of trials among different institutions, and may provide a new prognostic tool for preoperatively treated PCa.
PMCID: PMC2962710  PMID: 19853370
22.  Pre-operative CTLA-4 blockade: tolerability and immune monitoring in the setting of a pre-surgical clinical trial 
CTLA-4 blockade is being explored in numerous clinical trials as an immune based therapy for different malignancies. Our group conducted the first pre-operative clinical trial with the anti-CTLA-4 antibody ipilimumab in 12 patients with localized urothelial carcinoma of the bladder.
Experimental Design
Six patients were treated with 3mg/kg/dose of anti-CTLA-4 and six patients were treated with 10mg/kg/dose of antibody. Primary endpoints of the study were safety and immune monitoring.
Most drug-related adverse events consisted of grade 1/2 toxicities. All patients had measurable immunologic pharmacodynamic effects, consisting of an increased frequency of CD4+ICOShi T cells in tumor tissues and the systemic circulation. To determine if CD4+ICOShi T cells could be a correlative marker for clinical outcome after treatment with anti-CTLA-4, a cohort of metastatic melanoma patients was studied retrospectively for frequency of CD4+ICOShi T cells and survival. Data from this small cohort of patients indicated that an increased frequency of CD4+ICOShi T cells, sustained over a period of 12 weeks of therapy, correlates with increased likelihood of clinical benefit consisting of overall survival.
Our trial demonstrates that anti-CTLA-4 therapy has a tolerable safety profile in the pre-surgical setting and that a pre-operative model can be used to obtain biological data on human immune responses, which can efficiently guide the monitoring of patients treated in the metastatic disease setting.
PMCID: PMC2919850  PMID: 20460488
23.  Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity 
Cancer biology & therapy  2009;8(3):245-252.
RNA-dependent protein kinase is an interferon-induced, double-stranded (ds), RNA-activated serine/threonine protein kinase involved in the eukaryotic response to viral infection. While PKR also functions in cellular differentiation, growth control and apoptosis, its role in human cancer remains poorly understood. To explore a role for PKR in human cancer, we evaluated PKR expression and function in a series of cancer cell lines from different tumor types. We observed that PKR protein expression is high in various cancer cells and low in normal cells. Knockdown of PKR protein expression by PKR siRNA induced cell death, indicating a PKR-dependent survival pathway under normal growth conditions. Inhibition of PKR signaling using a dominant negative adenoviral PKR mutant (Ad-Δ6PKR) also induced cancer cell apoptosis via a mechanism that blocks activation of AKT-mediated survival while simultaneously inducing ER stress. ER stress-mediated apoptosis was evidenced by unregulated expression of phosphorylated JNK (p-JNK), phosphorylated cJun (p-cJun), and caspase-4 and was significantly reduced in cancer cells treated with JNK and caspase-4 inhibitors. We further demonstrated that inhibition of PKR signaling via either siRNA or Ad-Δ6PKR sensitizes cancer cells to etoposide or cisplatin-mediated cell death. Our results suggest a rationale to develop therapeutic strategies that target PKR signaling in human cancer cells.
PMCID: PMC2854203  PMID: 19106640
PKR; gene therapy; adenovirus
24.  Selenium and Vitamin E: Cell Type– and Intervention-Specific Tissue Effects in Prostate Cancer 
Secondary analyses of two randomized, controlled phase III trials demonstrated that selenium and vitamin E could reduce prostate cancer incidence. To characterize pharmacodynamic and gene expression effects associated with use of selenium and vitamin E, we undertook a randomized, placebo-controlled phase IIA study of prostate cancer patients before prostatectomy and created a preoperative model for prostatectomy tissue interrogation.
Thirty-nine men with prostate cancer were randomly assigned to treatment with 200 μg of selenium, 400 IU of vitamin E, both, or placebo. Laser capture microdissection of prostatectomy biopsy specimens was used to isolate normal, stromal, and tumor cells. Gene expression in each cell type was studied with microarray analysis and validated with a real-time polymerase chain reaction (PCR) and immunohistochemistry. An analysis of variance model was fit to identify genes differentially expressed between treatments and cell types. A beta-uniform mixture model was used to analyze differential expression of genes and to assess the false discovery rate. All statistical tests were two-sided.
The highest numbers of differentially expressed genes by treatment were 1329 (63%) of 2109 genes in normal epithelial cells after selenium treatment, 1354 (66%) of 2051 genes in stromal cells after vitamin E treatment, and 329 (56%) of 587 genes in tumor cells after combination treatment (false discovery rate = 2%). Validation of 21 representative genes across all treatments and all cell types yielded Spearman correlation coefficients between the microarray analysis and the PCR validation ranging from 0.64 (95% confidence interval [CI] = 0.31 to 0.79) for the vitamin E group to 0.87 (95% CI = 0.53 to 0.99) for the selenium group. The increase in the mean percentage of p53-positive tumor cells in the selenium-treated group (26.3%), compared with that in the placebo-treated group (5%), showed borderline statistical significance (difference = 21.3%; 95% CI = 0.7 to 41.8; P = .051).
We have demonstrated the feasibility and efficiency of the preoperative model and its power as a hypothesis-generating engine. We have also identified cell type– and zone-specific tissue effects of interventions with selenium and vitamin E that may have clinical implications.
PMCID: PMC2734116  PMID: 19244175
25.  Phase III Trial of Androgen Ablation With or Without Three Cycles of Systemic Chemotherapy for Advanced Prostate Cancer 
Journal of Clinical Oncology  2008;26(36):5936-5942.
We conducted a phase III trial in patients with previously untreated metastatic prostate cancer to test the hypothesis that three 8-week cycles of ketoconazole and doxorubicin alternating with vinblastine and estramustine, given in addition to standard androgen deprivation, would delay the appearance of castrate-resistant disease.
Patients and Methods
Eligible patients had metastatic prostate cancer threatening enough to justify sustained androgen ablation and were fit enough for chemotherapy. The primary end point was time to castrate-resistant progression as shown by increasing prostate-specific antigen, new radiographic lesions, worsening cancer-related symptoms, or receipt of any other systemic therapy.
Three hundred six patients were registered; 286 are reported. Median time to progression was 24 months (95% CI, 18 to 39 months) in the standard therapy arm, and 35 months (95% CI, 26 to 44 months) in the chemohormonal group (P = .39). At median follow-up of 6.4 years, overall survival was 5.4 years (95% CI, 4.7 to 7.8 years) in the standard therapy arm versus 6.1 years (95% CI, 5.1 to 10.1 years; P = .41). Prostate-specific antigen kinetics at the time of androgen ablation and the nadir after hormone treatment were strongly correlated with survival. Chemotherapy significantly increased the burden of therapy, with 51% of patients experiencing an adverse event of grade 3 or worse, especially thromboembolic events.
There is no role for ketoconazole and doxorubicin alternating with vinblastine and estramustine before emergence of a castrate-resistant phenotype.
PMCID: PMC3864402  PMID: 19029421

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