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1.  PPP2R2C loss promotes castration-resistance and is associated with increased prostate cancer-specific mortality 
Molecular cancer research : MCR  2013;11(6):568-578.
Metastatic prostate cancers generally rely on androgen receptor (AR) signaling for growth and survival, even following systemic androgen deprivation therapy (ADT). However, recent evidence suggests that some advanced prostate cancers escape ADT by utilizing signaling programs and growth factors that bypass canonical AR ligand-mediated mechanisms. We utilized an in vitro high-throughput RNAi screen to identify pathways in androgen-dependent prostate cancer cell lines whose loss of function promotes androgen ligand-independent growth. We identified 40 genes where knockdown promoted proliferation of both LNCaP and VCaP prostate cancer cells in the absence of androgen. Of these, 14 were down-regulated in primary and metastatic prostate cancer, including two subunits of the protein phosphatase 2 (PP2A) holoenzyme complex: PPP2R1A, a structural subunit with known tumor-suppressor properties in several tumor types; and PPP2R2C, a PP2A substrate-binding regulatory subunit that has not been previously identified as a tumor suppressor. We demonstrate that loss of PPP2R2C promotes androgen ligand depletion-resistant prostate cancer growth without altering AR expression or canonical AR-regulated gene expression. Furthermore, cell proliferation induced by PPP2R2C loss was not inhibited by the AR antagonist MDV3100, indicating that PPP2R2C loss may promote growth independently of known AR-mediated transcriptional programs. Immunohistochemical analysis of PPP2R2C protein levels in primary prostate tumors determined that low PPP2R2C expression significantly associated with an increased likelihood of cancer recurrence and cancer-specific mortality. These findings provide insights into mechanisms by which prostate cancers resist AR-pathway suppression, and support inhibiting PPP2R2C complexes or the growth pathway(s) activated by PPP2R2C as a therapeutic strategy.
doi:10.1158/1541-7786.MCR-12-0710
PMCID: PMC3687002  PMID: 23493267
PP2A; PPP2R2C; castration-resistant prostate cancer; androgen-pathway independence
2.  A Three-Marker FISH Panel Detects More Genetic Aberrations of AR, PTEN and TMPRSS2/ERG in Castration-Resistant or Metastatic Prostate Cancers than in Primary Prostate Tumors 
PLoS ONE  2013;8(9):e74671.
TMPRSS2/ERG rearrangement, PTEN gene deletion, and androgen receptor (AR) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the TMPRSS2/ERG, PTEN, and AR gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (P<0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (P<0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the AR copy number in primary tumors. AR copy number correlated well with the AR mRNA expression (rho = 0.52, P<0.001). Especially among TMPRSS2:ERG fusion-positive CRPC tumors, AR mRNA and ERG mRNA levels are strongly correlated (rho = 0.64, P<0.001). Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients.
doi:10.1371/journal.pone.0074671
PMCID: PMC3787014  PMID: 24098661
3.  AR Variant ARv567es Induces Carcinogenesis in a Novel Transgenic Mouse Model of Prostate Cancer12 
Neoplasia (New York, N.Y.)  2013;15(9):1009-1017.
Androgen deprivation therapy remains the primary treatment modality for patients with metastatic prostate cancer but is uniformly marked by progression to castration-resistant prostate cancer (CRPC) after a period of regression. Continued activation of androgen receptor (AR) signaling is attributed as one of the most important mechanisms underlying failure of therapy. Recently, the discovery of constitutively active AR splice variants (AR-Vs) adds more credence to this idea. Expression of AR-Vs in metastases portends a rapid progression of the tumor. However, the precise role of the AR-Vs in CRPC still remains unknown. ARv567es is one of the two AR variants frequently found in human CRPC xenografts and metastases. Herein, we developed a probasin (Pb) promoter-driven ARv567es transgenic mouse, Pb-ARv567es, to evaluate the role of ARv567es in both autonomous prostate growth and progression to CRPC. We found that expression of ARv567es in the prostate results in epithelial hyperplasia by 16 weeks and invasive adenocarcinoma is evident by 1 year of age. The underlying genetic cellular events involved a cell cycle-related transcriptome and differential expression of a spectrum of genes that are critical for tumor initiation and progression. These findings indicate that ARv567es could induce tumorigenesis de novo and signifies the critical role of AR-Vs in CRPC. Thus, the Pb-ARv567es mouse could provide a novel model in which the role of AR variants in prostate cancer progression can be examined.
PMCID: PMC3769880  PMID: 24027426
4.  Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B 
Nature medicine  2012;18(9):1359-1368.
Acquired resistance to anticancer treatments is a substantial barrier to reducing the morbidity and mortality that is attributable to malignant tumors. Components of tissue microenvironments are recognized to profoundly influence cellular phenotypes, including susceptibilities to toxic insults. Using a genome-wide analysis of transcriptional responses to genotoxic stress induced by cancer therapeutics, we identified a spectrum of secreted proteins derived from the tumor microenvironment that includes the Wnt family member wingless-type MMTV integration site family member 16B (WNT16B). We determined that WNT16B expression is regulated by nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB) after DNA damage and subsequently signals in a paracrine manner to activate the canonical Wnt program in tumor cells. The expression of WNT16B in the prostate tumor microenvironment attenuated the effects of cytotoxic chemotherapy in vivo, promoting tumor cell survival and disease progression. These results delineate a mechanism by which genotoxic therapies given in a cyclical manner can enhance subsequent treatment resistance through cell nonautonomous effects that are contributed by the tumor microenvironment.
doi:10.1038/nm.2890
PMCID: PMC3677971  PMID: 22863786
5.  Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation 
PLoS ONE  2013;8(5):e63563.
The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer growth, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control.
doi:10.1371/journal.pone.0063563
PMCID: PMC3660401  PMID: 23704919
6.  Single cell transcriptomic analysis of prostate cancer cells 
Background
The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR).
Results
Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient.
Conclusions
A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.
doi:10.1186/1471-2199-14-6
PMCID: PMC3599075  PMID: 23414343
Prostate cancer; Single-cell; Transcriptome; Disseminated tumor cells
7.  Androgen Receptor Gene Expression in Prostate Cancer is Directly Suppressed by the Androgen Receptor Through Recruitment of Lysine Specific Demethylase 1 
Cancer cell  2011;20(4):457-471.
SUMMARY
Androgen receptor (AR) is reactivated in castration resistant prostate cancer (CRPC) through mechanisms including marked increases in AR gene expression. We identify an enhancer in the AR second intron contributing to increased AR expression at low androgen levels in CRPC. Moreover, at increased androgen levels the AR binds this site and represses AR gene expression through recruitment of lysine specific demethylase 1 (LSD1) and H3K4me1,2 demethylation. AR similarly represses expression of multiple genes mediating androgen synthesis, DNA synthesis and proliferation, while stimulating genes mediating lipid and protein biosynthesis. Androgen levels in CRPC appear adequate to stimulate AR activity on enhancer elements, but not suppressor elements, resulting in increased expression of AR and AR repressed genes that contribute to cellular proliferation.
doi:10.1016/j.ccr.2011.09.001
PMCID: PMC3225024  PMID: 22014572
prostate cancer; androgen receptor; androgen deprivation therapy; H3K4 methylation; LSD1
8.  EPLIN Downregulation Promotes Epithelial-Mesenchymal Transition in Prostate Cancer Cells and Correlates With Clinical Lymph Node Metastasis 
Oncogene  2011;30(50):4941-4952.
Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling, and activation of β-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosing and treating metastasis at early stages.
doi:10.1038/onc.2011.199
PMCID: PMC3165108  PMID: 21625216
EPLIN; epithelial-mesenchymal transition; prostate cancer; lymph node metastasis; cytoskeleton
9.  Proteome and Transcriptome Profiles of a Her2/Neu-driven Mouse Model of Breast Cancer 
Proteomics. Clinical applications  2011;5(3-4):179-188.
Purpose
We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens.
Experimental design
Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias.
Results
In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate ≤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue.
Conclusions and clinical relevance
These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics.
doi:10.1002/prca.201000037
PMCID: PMC3069718  PMID: 21448875
Breast cancer; Her2; mouse; proteome; transcriptome
10.  IL-6 promotes prostate tumorigenesis and progression through autocrine cross-activation of IGF-IR 
Oncogene  2011;30(20):2345-2355.
As an established mediator of inflammation, IL-6 is implicated to facilitate prostate cancer progression to androgen independence through transactivation of the androgen receptor. However, whether IL-6 plays a causative role in de novo prostate tumorigenesis was never investigated. We now provide the first evidence that IL-6 can induce tumorigenic conversion and further progression to an invasive phenotype of non-tumorigenic benign prostate epithelial cells. Moreover, we find that paracrine IL-6 stimulates autocrine IL-6 loop and autocrine activation of IGF-IR to confer the tumorigenic property and that activation of STAT3 is critical in these processes. Inhibition of STAT3 activation or IGF-IR signaling suppresses IL-6-mediated malignant conversion and the associated invasive phenotype. Inhibition of STAT3 activation suppresses IL-6-induced upregulation of IGF-IR and its ligands IGF-I and IGF-II. These findings indicate IL-6 signaling cooperates with IGF-IR signaling in the prostate microenvironment to promote prostate tumorigenesis and progression to aggressiveness. Our findings suggest that STAT3 and IGF-IR may represent potential effective targets for prevention or treatment of prostate cancer.
doi:10.1038/onc.2010.605
PMCID: PMC3112005  PMID: 21258401
IL-6; STAT3; tumorigenesis; EMT; IGF-IR; prostate
11.  Differential Gene Expression in Benign Prostate Epithelium of Men with and without Prostate Cancer: Evidence for a Prostate Cancer Field Effect 
Background
Several malignancies are known to exhibit a “field-effect” whereby regions beyond tumor boundaries harbor histological or molecular changes that are associated with cancer. We sought to determine if histologically benign prostate epithelium collected from men with prostate cancer exhibits features indicative of pre-malignancy or field effect.
Methods
Prostate needle biopsies from 15 men with high grade(Gleason 8–10) prostate cancer and 15 age- and BMI-matched controls were identified from a biospecimen repository. Benign epithelia from each patient were isolated by laser capture microdissection. RNA was isolated, amplified, and used for microarray hybridization. Quantitative PCR(qPCR) was used to determine the expression of specific genes of interest. Alterations in protein expression were analyzed through immunohistochemistry.
Results
Overall patterns of gene expression in microdissected benign-associated benign epithelium (BABE) and cancer-associated benign epithelium (CABE) were similar. Two genes previously associated with prostate cancer, PSMA and SSTR1, were significantly upregulated in the CABE group(FDR <1%). Expression of other prostate cancer-associated genes, including ERG, HOXC4, HOXC5 and MME, were also increased in CABE by qRT-PCR, although other genes commonly altered in prostate cancer were not different between the BABE and CABE samples. The expression of MME and PSMA proteins on IHC coincided with their mRNA alterations.
Conclusion
Gene expression profiles between benign epithelia of patients with and without prostate cancer are very similar. However, these tissues exhibit differences in the expression levels of several genes previously associated with prostate cancer development or progression. These differences may comprise a field effect and represent early events in carcinogenesis.
doi:10.1158/1078-0432.CCR-10-0272
PMCID: PMC2992073  PMID: 20935156
Prostate cancer; gene regulation; carcinogenesis
12.  Coevolution of Prostate Cancer and Bone Stroma in Three-Dimensional Coculture: Implications for Cancer Growth and Metastasis 
Cancer research  2008;68(23):9996-10003.
Human bone stromal cells, after three-dimensional coculture with human prostate cancer (PCa) cells in vitro, underwent permanent cytogenetic and gene expression changes with reactive oxygen species serving as mediators. The evolved stromal cells are highly inductive of human PCa growth in mice, and expressed increased levels of extracellular matrix (versican and tenascin) and chemokine (BDFN, CCL5, CXCL5, and CXCL16) genes. These genes were validated in clinical tissue and/or serum specimens and could be the predictors for invasive and bone metastatic PCa. These results, combined with our previous observations, support the concept of permanent genetic and behavioral changes of PCa epithelial cells after being either cocultured with prostate or bone stromal cells as three-dimensional prostate organoids or grown as tumor xenografts in mice. These observations collectively suggest coevolution of cancer and stromal cells occurred under three-dimensional growth condition, which ultimately accelerates cancer growth and metastasis.
doi:10.1158/0008-5472.CAN-08-2492
PMCID: PMC3105756  PMID: 19047182
13.  The Expression of Osteoclastogenesis-Associated Factors and Osteoblast Response to Osteolytic Prostate Cancer Cells 
The Prostate  2010;70(4):412-424.
Background
Prostate cancer (PCa) has a propensity to metastasize to bone. Tumor cells replace bone marrow and can elicit an osteoblastic, osteolytic, or mixed bone response. Our objective was to elucidate the mechanisms and key factors involved in promoting osteoclastogenesis in PCa bone metastasis.
Methods
We cultured osteoblast-like MC3T3-E1 cells with conditioned medium (CM) from PC-3 and C4-2B cells. MC3T3-E1 mineralization decreased in the presence of PC-3 CM, whereas C4-2B CM had no effect on mineralization. Using oligo arrays and validating by real-time PCR, we observed a decrease in the expression of mineralization-associated genes in MC3T3-E1 cells grown in the presence of PC-3 CM. In addition, PC-3 CM induced the expression of osteoclastogenesis- associated genes IGFBP-5, IL-6, MCP-1, and RANKL while decreasing OPG expression in MC3T3-E1 cells. Furthermore, CM from MC3T3-E1 cells cultured in the presence of PC-3 CM, in association with soluble RANKL, increased osteoclastogenesis in RAW 264.7 cells. Investigation of PCa metastases and xenografts by immunohistochemistry revealed that the osteoclastic factor IL-6 was expressed in the majority of PCa bone metastases and to a lesser extent in PCa soft tissue metastases. In vitro it was determined that soluble IL-6R (sIL-6R) was necessary for IL-6 to inhibit mineralization in MC3T3-E1 cells.
Results
PC-3 cells inhibit osteoblast activity and induce osteoblasts to produce osteoclastic factors that promote osteoclastogenesis, and one of these factors, IL-6, is highly expressed in PCa bone metastases.
Conclusions
IL-6 may have an important role in promoting osteoclastogenesis in PCa bone metastasis through its’ interaction with sIL-6R.
doi:10.1002/pros.21075
PMCID: PMC2929015  PMID: 19866469
Bone metastasis; MC3T3-E1; PC-3; C4-2B; IL-6
14.  Variability in the androgen response of prostate epithelium to 5-alpha reductase inhibition: implications for prostate cancer chemoprevention 
Cancer research  2010;70(4):1286.
Androgens and the androgen receptor (AR) influence prostate carcinogenesis. Lowering intraprostatic dihydrotestosterone (DHT) by inhibiting 5-alpha-reductase (SRD5A) reduces prostate cancer (PCa) incidence, but is not uniformly effective. Mechanisms by which SRD5A inhibition influences PCa initiation and/or progression among different individuals have not been established. We sought to identify molecular alterations underlying the differential chemo-preventive activity of SRD5A inhibition. Men with clinically-localized PCa were randomized to prostatectomy alone (n=25) or 4 months treatment with the SRD5A-inhibitor dutasteride (0.5mg (n=26) or 3.5mg (n=24)) preceding prostatectomy. Serum and prostate androgens were measured using mass spectrometry. We evaluated benign epithelial gene expression using expression profiling and immunohistochemistry, and characterized tumor TMPRSS2-ERG fusion status using FISH. Dutasteride at 0.5 or 3.5mg decreased prostatic DHT by 93% (0.23ng/g; p<0.001) and 98.8% (0.04ng/g; p<0.001) vs. untreated patients (3.33ng/g). Despite significant and uniform suppression of tissue DHT, unsupervised clustering based on prostatic gene expression did not allow us to discriminate dutasteride-treated from untreated individuals. However, we could resolve subjects into distinct cohorts characterized by high or low expression of AR-regulated genes (irrespective of treatment dose) based solely on AR transcript expression. The high-dose dutasteride treatment group comprised significantly fewer cancers with TMPRSS2-ERG fusions. Despite substantial and uniform reductions in prostatic DHT, dutasteride was associated with highly variable alterations in benign epithelial gene expression. Segregation of subjects based on AR and androgen-regulated gene expression indicates patients are differentially sensitive to SRD5A inhibition. Tissue AR levels may serve as a pretreatment predictor of SRD5A chemo-preventive efficacy.
doi:10.1158/0008-5472.CAN-09-2509
PMCID: PMC2822890  PMID: 20124490
prostate cancer; chemo-prevention; 5-alpha-reductase; androgen receptor; dutasteride
15.  Transforming growth factor-β-stimulated clone-22 (TSC-22) is an androgen regulated gene that enhances apoptosis in prostate cancer following IGF-IR inhibition 
Purpose
Inhibition of IGF signaling using the human IGF-IR monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens.
Experimental Methods
The castrate-resistant human xenograft LuCaP 35V was implanted into intact or castrate SCID mice and treated with A12 weekly. After six weeks of tumor growth animals were sacrificed and tumors removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were performed.
Results
In castrate mice the tumors were delayed in G2 with no apoptosis; in contrast tumors from intact mice underwent apoptosis with either a G1 or G2 delay. TSC-22 was significantly elevated in tumors from the intact mice compared to castrate mice, especially in those tumors with the highest levels of apoptosis. In order to further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 demonstrated an increase in apoptosis and a delay in G1. When these cell lines were placed subcutaneously in SCID mice a decreased number of animals formed tumors and the rate of tumor growth was decreased compared to control tumors.
Conclusions
These data indicate that IGF-IR inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22.
doi:10.1158/1078-0432.CCR-09-0264
PMCID: PMC2795132  PMID: 19996218
castrate-resistant PCa; androgen; IGF-1R; A12; TSC-22
16.  The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression 
The Journal of Clinical Investigation  2010;120(12):4478-4492.
Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1–mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes.
doi:10.1172/JCI44239
PMCID: PMC2993601  PMID: 21099110
17.  Comparative analyses of chromosome alterations in soft-tissue metastases within and across patients with castration-resistant prostate cancer 
Cancer research  2009;69(19):7793-7802.
Androgen deprivation is the mainstay of therapy for progressive prostate cancer. Despite initial and dramatic tumor inhibition, most men eventually fail therapy and die of metastatic castration-resistant (CR) disease. Here, we characterize the profound degree of genomic alteration found in CR tumors using array CGH, gene expression arrays, and FISH. By cluster analysis, we show that the similarity of the genomic profiles from primary and metastatic tumors is driven by the patient. Using data adjusted for this similarity, we identify numerous high-frequency alterations in the CR tumors, such as 8p loss and chromosome 7 and 8q gain. By integrating array CGH and expression array data, we reveal genes whose correlated values suggest they are relevant to prostate cancer biology. We find alterations that are significantly associated with the metastases of specific organ sites, and others with CR tumors versus the tumors of patients with localized prostate cancer not treated with androgen deprivation. Within the high-frequency sites of loss in CR metastases, we find an over-representation of genes involved in cellular lipid metabolism, including PTEN. Finally, using FISH we verify the presence of a gene fusion between TMPRSS2 and ERG suggested by chromosome-21 deletions detected by array CGH. We find the fusion in 54% of our CR tumors, and 81% of the fusion-positive tumors contain cells with multiple copies of the fusion. Our investigation lays the foundation for a better understanding of and possible therapeutic targets for CR disease, the poorly responsive and final stage of prostate cancer.
doi:10.1158/0008-5472.CAN-08-3810
PMCID: PMC2771763  PMID: 19773449
Prostate cancer; castration-resistant metastases; metastatic prostate cancer; array CGH; expression; genomic alterations; TMPRSS2-ERG fusion
18.  The Effects of Aging on the Molecular and Cellular Composition of the Prostate Microenvironment 
PLoS ONE  2010;5(9):e12501.
Background
Advancing age is associated with substantial increases in the incidence rates of common diseases affecting the prostate gland including benign prostatic hyperplasia (BPH) and prostate carcinoma. The prostate is comprised of a functional secretory epithelium, a basal epithelium, and a supporting stroma comprised of structural elements, and a spectrum of cell types that includes smooth muscle cells, fibroblasts, and inflammatory cells. As reciprocal interactions between epithelium and stromal constituents are essential for normal organogenesis and serve to maintain normal functions, discordance within the stroma could permit or promote disease processes. In this study we sought to identify aging-associated alterations in the mouse prostate microenvironment that could influence pathology.
Methodology/Principal Findings
We quantitated transcript levels in microdissected glandular-adjacent stroma from young (age 4 months) and old (age 20–24 months) C57BL/6 mice, and identified a significant change in the expression of 1259 genes (p<0.05). These included increases in transcripts encoding proteins associated with inflammation (e.g., Ccl8, Ccl12), genotoxic/oxidative stress (e.g., Apod, Serpinb5) and other paracrine-acting effects (e.g., Cyr61). The expression of several collagen genes (e.g., Col1a1 and Col3a1) exhibited age-associated declines. By histology, immunofluorescence, and electron microscopy we determined that the collagen matrix is abundant and disorganized, smooth muscle cell orientation is disordered, and inflammatory infiltrates are significantly increased, and are comprised of macrophages, T cells and, to a lesser extent, B cells.
Conclusion/Significance
These findings demonstrate that during normal aging the prostate stroma exhibits phenotypic and molecular characteristics plausibly contributing to the striking age associated pathologies affecting the prostate.
doi:10.1371/journal.pone.0012501
PMCID: PMC2931699  PMID: 20824135
19.  Prostate Cancer-Associated Gene Expression Alterations Determined from Needle Biopsies 
Purpose
To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia.
Experimental Design
Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays comprised of 6200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative RT-PCR.
Results
Comparative analyses identified 954 transcript alterations associated with cancer (q value <0.01%) including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy utilization, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of AR expression changes was noted. In exploratory analyses, AR down regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy.
Conclusions
Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.
doi:10.1158/1078-0432.CCR-08-1982
PMCID: PMC2929837  PMID: 19366833
prostate cancer; needle biopsy; microdissection; gene expression
20.  Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant 
The Journal of Clinical Investigation  2010;120(8):2715-2730.
Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand.
doi:10.1172/JCI41824
PMCID: PMC2912187  PMID: 20644256
21.  Mitochondrial DNA Mutation Stimulates Prostate Cancer Growth in Bone Stromal Environment 
The Prostate  2009;69(1):1-11.
Background and Objectives
Mitochondrial DNA (mtDNA) mutations, inherited and somatically acquired, are common in clinical prostate cancer. We have developed model systems designed to study specific mtDNA mutations in controlled experiments. Because prostate cancer frequently metastasizes to bone we tested the hypothesis that mtDNA mutations enhance prostate cancer growth and survival in the bone microenvironment.
Methods
The pathogenic nucleotide position (np) 8993 mDNA mutation was introduced into PC3 prostate cancer cells by cybrid formation. Wildtype and mutant cybrids were grown as nude mouse subcutaneous xenografts with or without bone stromal cell co-inoculation. Cybrids were also grown in the intratibial space. Tumor growth was assayed by direct tumor measurement and luciferase chemiluminescence. Gene expression was assayed using cDNA microarrays confirmed by real time PCR, western blot analysis and immunohistochemistry.
Results
Cybrids with the 8993 mtDNA mutation grew faster than wildtype cybrids. Further growth acceleration was demonstrated in the bone microenvironment. A thirty-seven gene molecular signature characterized the growth advantage conferred by the mtDNA mutation and bone microenvironment. Two genes of known importance in clinical prostate cancer, FGF1 and FAK, were found to be substantially upregulated only when both mtDNA mutation and bone stromal cell were present.
Conclusions
The ATP6 np 8993 mtDNA mutation confers a growth advantage to human prostate cancer that is most fully manifest in the bone microenvironment. The identification of specific molecular alterations associated with mtDNA mutation and growth in bone may allow new understanding of prostate cancer bone metastasis.
doi:10.1002/pros.20854
PMCID: PMC2753601  PMID: 18850577
22.  HE3235 Inhibits Growth of Castration-Resistant Prostate Cancer12 
Neoplasia (New York, N.Y.)  2009;11(11):1216-1225.
Treatments for advanced prostate cancer (CaP) typically involve androgen deprivation therapy. However, most patients eventually develop castration-resistant CaP (CRPC) for which highly effective therapies are limited. We explored the efficacy of a novel agent, HE3235, in inhibiting growth of CRPC in preclinical models. Castrated male mice were implanted subcutaneously with LuCaP35V CaP xenografts in the presence and absence of 5′-androstenediol (AED) and treated with HE3235. To investigate the effect of HE3235 on CaP tumor in the bone, castrated mice were injected intratibially with C4-2B CaP cells and treated with HE3235. Serum prostate-specific antigen (PSA) levels, tumor volume, immunohistochemistry, gene expression, and levels of intratumoral androgens were analyzed. HE3235 significantly prolonged the tumor doubling time of LuCaP35V, decreased androgen receptor expression, and lowered levels of intratumoral testosterone by ∼89% and dihydrotestosterone by ∼63% in both the presence and the absence of AED. HE3235 inhibited tumor growth in the bone environment. Weights of tumored tibiae of HE3235-treated animals were lower than those of control (P = .031), and normalized PSA levels were also significantly decreased at the end of study by HE3235 treatment (P = .0076). HE3235 inhibits the growth of subcutaneous CRPC as well as CRPC in the bone environment. Our data show that HE3235 exhibits a wide range of effects, including alteration of androgen receptor signaling and reductions in levels of intratumoral androgens. Our results support ongoing clinical investigations into the effectiveness of HE3235 in the setting of CRPC and warrants further studies into the mechanisms behind the effects of HE3235.
PMCID: PMC2767223  PMID: 19881957
23.  Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate 
BMC Genomics  2008;9:246.
Background:
Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes.
Results:
Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance.
Conclusion:
Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.
doi:10.1186/1471-2164-9-246
PMCID: PMC2413246  PMID: 18501003
24.  Genetic background influences murine prostate gene expression: implications for cancer phenotypes 
Genome Biology  2007;8(6):R117.
Microarray analyses to quantitate transcript levels in the prostates of five inbred mouse strains identified differences in gene expression in benign epithelium that correlated with the differentiation state of adjacent tumors.
Background
Cancer of the prostate is influenced by both genetic predisposition and environmental factors. The identification of genes capable of modulating cancer development has the potential to unravel disease heterogeneity and aid diagnostic and prevention strategies. To this end, mouse models have been developed to isolate the influences of individual genetic lesions in the context of consistent genotypes and environmental exposures. However, the normal prostatic phenotypic variability dictated by a genetic background that is potentially capable of influencing the process of carcinogenesis has not been established.
Results
In this study we used microarray analysis to quantify transcript levels in the prostates of five commonly studied inbred mouse strains. We applied a multiclass response t-test and determined that approximately 13% (932 genes) exhibited differential expression (range 1.3-190-fold) in any one strain relative to other strains (false discovery rate ≤10%). Expression differences were confirmed by quantitative RT-PCR, or immunohistochemistry for several genes previously shown to influence cancer progression, such as Psca, Mmp7, and Clusterin. Analyses of human prostate transcripts orthologous to variable murine prostate genes identified differences in gene expression in benign epithelium that correlated with the differentiation state of adjacent tumors. For example, the gene encoding apolipoprotein D, which is known to enhance resistance to cell stress, was expressed at significantly greater levels in benign epithelium associated with high-grade versus low-grade cancers.
Conclusion
These studies support the concept that the cellular, tissue, and organismal context contribute to oncogenesis and suggest that a predisposition to a sequence of events leading to pathology may exist prior to cancer initiation.
doi:10.1186/gb-2007-8-6-r117
PMCID: PMC2394769  PMID: 17577413
25.  Inhibition of Androgen-Independent Prostate Cancer by Estrogenic Compounds Is Associated with Increased Expression of Immune-Related Genes1 
Neoplasia (New York, N.Y.)  2006;8(10):862-878.
Abstract
The clinical utility of estrogens for treating prostate cancer (CaP) was established in the 1940s by Huggins. The classic model of the anti-CaP activity of estrogens postulates an indirect mechanism involving the suppression of androgen production. However, clinical and preclinical studies have shown that estrogens exert growth-inhibitory effects on CaP under low-androgen conditions, suggesting additional modes whereby estrogens affect CaP cells and/or the microenvironment. Here we have investigated the activity of 17β estradiol (E2) against androgen-independent CaP and identified molecular alterations in tumors exposed to E2. E2 treatment inhibited the growth of all four androgen-independent CaP xenografts studied (LuCaP 35V, LuCaP 23.1AI, LuCaP 49, and LuCaP 58) in castrated male mice. The molecular basis of growth suppression was studied by cDNA microarray analysis, which indicated that multiple pathways are altered by E2 treatment. Of particular interest are changes in transcripts encoding proteins that mediate immune responses and regulate androgen receptor signaling. In conclusion, our data show that estrogens have powerful inhibitory effects on CaP in vivo in androgen-depleted environments and suggest novel mechanisms of estrogen-mediated antitumor activity. These results indicate that incorporating estrogens into CaP treatment protocols could enhance therapeutic efficacy even in cases of advanced disease.
PMCID: PMC1715921  PMID: 17032503
Prostate cancer; estrogen; estradiol; androgen independence; interferon-regulated genes

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