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1.  NanoVelcro Chip for CTC enumeration in prostate cancer patients 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.06.019.
Circulating tumor cells (CTCs) are one of the most crucial topics in rare cell biology and have become the focus of a significant and emerging area of cancer research. While CTC enumeration is a valid biomarker in prostate cancer, the current FDA-approved CTC technology is unable to detect CTCs in a large portion of late stage prostate cancer patients. Here we introduce the NanoVelcro CTC Chip, a device composed of a patterned silicon nanowire substrate (SiNW) and an overlaid polydimethylsiloxane (PDMS) chaotic mixer. Validated by two institutions participating in the study, the NanoVelcro Chip assay exhibits very consistent efficiency in CTC-capture from patient samples. The utilized protocol can be easily replicated at different facilities. We demonstrate the clinical utility of the NanoVelcro Chip by performing serial enumerations of CTCs in prostate cancer patients after undergoing systemic therapy. Changes in CTC numbers after 4–10 weeks of therapy were compared with their clinical responses. We observed a statistically significant reduction in CTCs counts in the clinical responders. We performed long-term follow up with serial CTC collection and enumeration in one patient observing variations in counts correlating with treatment response. This study demonstrates the consistency of the NanoVelcro Chip assay over time for CTC enumeration and also shows that continuous monitoring of CTC numbers can be employed to follow responses to different treatments and monitor disease progression.
doi:10.1016/j.ymeth.2013.06.019
PMCID: PMC3834112  PMID: 23816790
Nanomaterials; microfluidics; circulating tumor cell; prostate cancer; diagnostics
2.  Association between tumor-associated macrophage infiltration, high grade prostate cancer, and biochemical recurrence after radical prostatectomy 
Background: Tumor-associated macrophages (TAMs) are a key component of the inflammatory microenvironment. Their role in prostate cancer development and progression remains unclear. We examined whether the amount of TAMs in prostate cancer is: 1) higher than prostatic intraepithelial neoplasia (PIN) and benign tissue 2) associated with poorly differentiated disease, and 3) predictive of biochemical recurrence among surgically treated men. Methods: A tissue microarray (TMA) of prostatectomy specimens from 332 patients was stained for CD68, a TAM marker. A separate TMA was used for validation. Associations between mean TAMs in cancer cores and PSA recurrence were determined by Cox proportional hazards models after adjusting for age, preoperative PSA, race, body mass index, pathologic Gleason sum, seminal vesicle invasion, extracapsular extension, and margin status. Results: Mean TAM number was higher in cancer versus PIN and benign tissue (p<0.0001). Mean TAM number was higher in Gleason grade 4 cores vs. Gleason grade 3 cores (p=0.003). On multivariable analysis, no association was observed between mean TAM number per cancer core and biochemical recurrence in either cohort. Conclusion: Mean TAM number was higher in cancer cores vs. PIN and benign tissue, and higher in high grade prostate cancer supporting the potential role of TAMs in prostate cancer development. However, TAMs were not associated with biochemical recurrence after radical prostatectomy suggesting TAM counts do not provide independent prognostic value among surgically treated men. Further studies are required to elucidate the functional significance of TAMs in the prostate cancer microenvironment.
PMCID: PMC3816972  PMID: 24224130
Biochemical recurrence; cancer development; prostate; tumor associated macrophages; tissue microarray
3.  A Novel In Vitro Assay of Tumor-Initiating Cells in Xenograft Prostate Tumors 
The Prostate  2010;70(13):10.1002/pros.21171.
BACKGROUND
The field of prostate cancer has been stymied by the difficulty of cultivating patient-derived samples in the laboratory. In order to help circumvent this challenge, we sought to develop an in vitro assay of human prostate cancer initiation employing a prostate-associated mesenchymal feeder layer.
METHODS
Rat seminal vesicle mesenchyme (rSVM) harvested from male neonatal rats was plated in 12-well plates and then irradiated with 30 Gy after ~75% confluence. Single-cell suspensions of two human non-adherent prostate cancer xenograft lines (TRPC and LAPC9) were then plated on irradiated rSVM. At 3–4 weeks, three-dimensional solid structures, termed glandoids, were harvested and analyzed or transplanted singly into the renal capsule of immunodeficient mice. Animals were assessed for tumor formation 8–12 weeks after engraftment. Finally, clonality assays were performed to determine whether glandoids usually arise from a single cell and are therefore clonal in origin.
RESULTS
Glandoids form with reliable frequency (1/~300 plated cells), are constituted by relevant cell types (CK8+, CK5–, PSA+) and after implantation into immunocompromised mice, give rise to tumors that recapitulate original xenograft histology and cell composition; defining a glandoid as a tumor-initiating unit. In addition, assessment of red fluorescent protein (RFP)-labeled glandoids revealed either all red or non-red structures, with few areas of fusion, suggesting glandoids are clonal in origin.
CONCLUSIONS
The above assay describes an adjunct technique to readily cultivate cells from prostate cancer xenografts in vitro and as such provides a platform on which tumor-initiating cell studies and high-throughput drug discovery may be performed.
doi:10.1002/pros.21171
PMCID: PMC3808877  PMID: 20687210
prostate cancer; seminal vesicle mesenchyme; xenograft; tumor-initiating cell; cancer stem cell
4.  Combination of Rad001 (Everolimus) and Propachlor synergistically induces apoptosis through enhanced autophagy in prostate cancer cells 
Molecular cancer therapeutics  2012;11(6):1320-1331.
PI3K/AKT/mTOR pathway plays a key role in the tumorigenesis of many human cancers including prostate cancer. However, inhibitors of this pathway such as Rad001 have not shown therapeutic efficacy as a single agent. Through a high throughput screen of 5,000 widely used small molecules, we identified compounds that can synergize with Rad001 to inhibit prostate cancer cells. One of the compounds, Propachlor, synergizes with Rad001 to induce apoptosis of castration-resistant prostate cancer cells via enhanced autophagy. This enhanced autophagic cell death is accompanied by increased Beclin1 expression as well as upregulation of ATG5-ATG12 conjugate and LC3-2. Rad001 and Propachlor can also synergistically inhibit tumors in a xenograft animal model of prostate cancer. These findings provide a novel direction to develop combination therapies for advanced and metastatic prostate cancer that has failed the currently available therapies.
doi:10.1158/1535-7163.MCT-11-0954
PMCID: PMC3374015  PMID: 22491797
Rad001; Propachlor; synergism; autophagy; apoptosis
5.  Neuroendocrine differentiation of prostate cancer 
Asian Journal of Andrology  2013;15(3):328-332.
doi:10.1038/aja.2013.7
PMCID: PMC3739654  PMID: 23503426
6.  The PSA−/lo prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration 
Cell Stem Cell  2012;10(5):556-569.
SUMMARY
Prostate cancer (PCa) is heterogeneous and contains both differentiated and undifferentiated tumor cells, but the relative functional contribution of these two cell populations remains unclear. Here we report distinct molecular, cellular, and tumor-propagating properties of PCa cells that express high (PSA+) and low (PSA−/lo) levels of the differentiation marker PSA. PSA−/lo PCa cells are quiescent and refractory to stresses including androgen deprivation, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity. They preferentially express stem cell genes and can undergo asymmetric cell division generating PSA+ cells. Importantly, PSA−/lo PCa cells can initiate robust tumor development and resist androgen ablation in castrated hosts, and harbor highly tumorigenic castration-resistant PCa cells that can be prospectively enriched using ALDH+CD44+α2β1+ phenotype. In contrast, PSA+ PCa cells possess more limited tumor-propagating capacity, undergo symmetric division and are sensitive to castration. Together, our study suggests PSA−/lo cells may represent a critical source of castration-resistant PCa cells.
doi:10.1016/j.stem.2012.03.009
PMCID: PMC3348510  PMID: 22560078
prostate cancer; PSA; cancer stem cells; differentiation; asymmetric cell division; castration resistance
7.  Ptenloss and RAS/MAPK activation cooperate to promote EMT and metastasis initiated from prostate cancer stem/progenitor cells 
Cancer Research  2012;72(7):1878-1889.
PTEN loss or PI3K/AKT signaling pathway activation correlates with human prostate cancer progression and metastasis. However, in preclinical murine models, deletion of Pten alone fails to mimic the significant metastatic burden that frequently accompanies the end stage of human disease. To identify additional pathway alterations that cooperate with PTEN loss in prostate cancer progression, we surveyed human prostate cancer tissue microarrays and found that the RAS/MAPK pathway is significantly elevated both in primary and metastatic lesions. In an attempt to model this event, we crossed conditional activatable K-rasG12D/WT mice with the prostate conditional Pten deletion model. Although RAS activation alone cannot initiate prostate cancer development, it significantly accelerated progression caused by PTEN loss, accompanied by epithelial-to-mesenchymal transition (EMT) and macrometastasis with 100% penetrance. A novel stem/progenitor subpopulation with mesenchymal characteristics was isolated from the compound mutant prostates, which was highly metastatic upon orthotopic transplantation. Importantly, inhibition of RAS/MAPK signaling by PD325901, a MEK inhibitor, significantly reduced the metastatic progression initiated from transplanted stem/progenitor cells. Collectively, our findings indicate that activation of RAS/MAPK signaling serves as a potentiating second hit to alteration of the PTEN/PI3K/AKT axis and co-targeting both pathways is highly effective gin preventing the development of metastatic prostate cancers.
doi:10.1158/0008-5472.CAN-11-3132
PMCID: PMC3319847  PMID: 22350410
8.  Targeted Biopsy in the Detection of Prostate Cancer using an Office-Based MR-US Fusion Device 
The Journal of urology  2012;189(1):86-91.
Purpose
Targeted biopsy of lesions identified on MRI may enhance detection of clinically relevant prostate cancers (CaP). We evaluate CaP detection rates in 171 consecutive men using MR-US fusion prostate biopsy.
Materials and Methods
Subjects underwent targeted biopsy either for active surveillance (N=106) or persistently elevated PSA but negative prior conventional biopsy (N=65). Before biopsy, each man had a multiparametric MRI at 3.0-Tesla. Lesions on MRI were outlined in 3D and assigned increasing cancer suspicion levels (image grade 1–5) by a uroradiologist. The Artemis biopsy tracking system was used to fuse the stored MRI with real-time ultrasound (US), generating a 3D prostate model on-the-fly. Working from the 3D model, transrectal biopsy of target lesions and 12 systematic biopsies were performed under local anesthesia in the clinic.
Results
171 subjects (median age 65) underwent targeted biopsy. At biopsy, median PSA = 4.9 ng/ml and prostate volume = 48 cc. A targeted biopsy was three times more likely to identify cancer than a systematic biopsy (21% vs. 7%). CaP was found in 53% of men, 38% of whom had Gleason ≥7. 38% of men with Gleason ≥7 cancers were detected only on targeted biopsies. Targeted biopsy findings correlated with level of suspicion on MRI. 15 of 16 men (94%) with an image grade 5 target (highest suspicion) had CaP, including 7 with Gleason ≥7.
Conclusions
Prostate lesions identified on MRI can be accurately targeted using MR-US fusion biopsy by a urologist in clinic. Biopsy findings correlate with level of suspicion on MRI.
doi:10.1016/j.juro.2012.08.095
PMCID: PMC3561472  PMID: 23158413
Prostatic neoplasms; magnetic resonance imaging; ultrasonography; biopsy
9.  ERG–TMPRSS2 rearrangement is shared by concurrent prostatic adenocarcinoma and prostatic small cell carcinoma and absent in small cell carcinoma of the urinary bladder: evidence supporting monoclonal origin 
Prostatic carcinoma is a heterogeneous disease with frequent multifocality and variability in morphology. Particularly, prostatic small cell carcinoma is a rare variant with aggressive behavior. Distinction between small cell carcinoma of the prostate and urinary bladder may be challenging, especially in small biopsy specimens without associated prostatic adenocarcinoma or urothelial carcinoma. Recently, gene fusions between ETS genes, particularly ETS-related gene (ERG), and transmembrane protease, serine 2 (TMPRSS2) have been identified as a frequent event in prostate cancer. Thus, molecular methods may be helpful in determining the primary site of small cell carcinoma. Thirty cases of prostatic small cell carcinoma from the authors’ archives were studied, among which 13 had concurrent prostatic adenocarcinoma. Tricolor fluorescence in situ hybridization (FISH) was performed on formalin-fixed paraffin-embedded tissue sections with a probe cocktail for 3′/5′ ERG and TMPRSS2. Cases of small cell carcinoma of the bladder and conventional prostatic adenocarcinoma (25 each) were also tested as controls. ERG gene alterations were found only in prostate malignancies and not in benign prostatic tissue or bladder small cell carcinoma. TMPRSS2–ERG gene fusion was found in 47% (14/30) of prostatic small cell carcinoma. Of cases with concurrent prostatic adenocarcinoma, 85% (11/13) had identical findings in both components. In 20% of rearranged cases, the ERG abnormality was associated with 5′ ERG deletion. In 17% (5/30) of cases, gain of the 21q22 locus was present. Two cases showed discordant aberrations in the small cell carcinoma and adenocarcinoma, one with deletion of 5′ ERG and one with gain of chromosome 21q, both in only the adenocarcinoma component. Small cell carcinoma of the prostate demonstrates TMPRSS2–ERG rearrangement with comparable frequency to prostatic adenocarcinoma. In cases with concurrent adenocarcinoma and small cell carcinoma, the majority showed identical abnormalities in both components, indicating a likely common clonal origin. Discordant alterations were present in rare cases, suggesting that acquisition of additional genetic changes in multifocal tumors may be responsible for disease progression to a more aggressive phenotype. TMPRSS2–ERG fusion is absent in bladder small cell carcinoma, supporting the utility of FISH in distinguishing prostate from bladder primary tumors and identifying metastatic small cell carcinoma of unknown origin.
doi:10.1038/modpathol.2011.56
PMCID: PMC3441178  PMID: 21499238
ERG–TMPRSS2 rearrangement; fluorescence in situ hybridization; histogenesis; morphology; prostate; small cell carcinoma; urothelium
10.  Pathogenesis of prostatic small cell carcinoma involves the inactivation of the P53 pathway 
Endocrine-related cancer  2012;19(3):321-331.
Small cell neuroendocrine carcinoma (SCNC) of the prostate is a variant form of prostate cancer that occurs de novo or as a recurrent tumor in patients who received hormonal therapy for prostatic adenocarcinoma. It is composed of pure neuroendocrine (NE) tumor cells, but unlike the scattered NE cells in benign prostate and adenocarcinoma that are quiescent, the NE cells in SCNC are highly proliferative and aggressive, causing death in months. In this study, we provide evidence that interleukin 8 (IL8)–CXCR2–P53 (TP53) signaling pathway keeps the NE cells of benign prostate and adenocarcinoma in a quiescent state normally. While P53 appears to be wild-type in the NE cells of benign prostate and adenocarcinoma, immunohistochemical studies show that the majority of the NE tumor cells in SCNC are positive for nuclear p53, suggesting that the p53 is mutated. This observation is confirmed by sequencing of genomic DNA showing p53 mutation in five of seven cases of SCNC. Our results support the hypothesis that p53 mutation leads to inactivation of the IL8–CXCR2–p53 signaling pathway, resulting in the loss of an important growth inhibitory mechanism and the hyper-proliferation of NE cells in SCNC. Therefore, we have identified potential cells of origin and a molecular target for prostatic SCNC that are very different from those of conventional adenocarcinoma, which explains SCNC’s distinct biology and the clinical observation that it does not respond to hormonal therapy targeting androgen receptor signaling, which produces short-term therapeutic effects in nearly all patients with prostatic adenocarcinoma.
doi:10.1530/ERC-11-0368
PMCID: PMC3433057  PMID: 22389383
11.  Clinical Application of a 3D Ultrasound-guided Prostate Biopsy System 
Urologic oncology  2011;29(3):334-342.
Objectives
Prostate biopsy (Bx) has for three decades been performed in a systematic, but blind fashion using 2D ultrasound (US). Herein is described the initial clinical evaluation of a 3D Bx tracking and targeting device (Artemis, Eigen, Grass Valley, CA). Our main objective was to test accuracy of the new 3D method in men undergoing first and follow-up Bx to rule out prostate cancer (CaP).
Methods & Materials
Patients in the study were men ages 35-87 (66.1 +/- 9.9 yrs), scheduled for Bx to rule out CaP, who entered into an IRB-approved protocol. 218 subjects underwent conventional trans-rectal US (TRUS); the tracking system was then attached to the US probe; the prostate was scanned and a 3D reconstruction was created. All Bx sites were visualized in 3D and tracked electronically. In 11 men, a pilot study was conducted to test ability of the device to return a Bx to an original site. In 47 men, multi-parametric 3 Tesla MRI – incorporating T2-weighted images, dynamic contrast enhancement, and diffusion-weighted imaging – was performed in advance of the TRUS, allowing the stored MRI images to be fused with real-time US during biopsy. Lesions on MRI were delineated by a radiologist, assigned a grade of CaP suspicion, and fused into TRUS for biopsy targeting.
Results
3D Bx tracking was completed successfully in 180/218 patients, with a success rate approaching 95% among the last 50 men. Average time for Bx with the Artemis device was 15 minutes with an additional 5 minutes for MRI fusion and Bx targeting. In the tracking study, an ability to return to prior Bx sites (n=32) within 1.2 +/- 1.1 mm S.D. was demonstrated and was independent of prostate volume or location of Bx site. In the MRI fusion study, when suspicious lesions were targeted, a 33% Bx-positivity rate was found compared to a 7% positivity rate for systematic, non-targeted Bx (19/57 cores vs. 9/124 cores, p=0.03).
Conclusion
Use of 3D tracking and image fusion has the potential to transform MRI into a clinical tool to aid biopsy and improve current methods for diagnosis and follow-up of CaP.
doi:10.1016/j.urolonc.2011.02.014
PMCID: PMC3432280  PMID: 21555104
12.  PC3 Is a Cell Line Characteristic of Prostatic Small Cell Carcinoma 
The Prostate  2011;71(15):1668-1679.
BACKGROUND
The majority of the prostatic cancers are adenocarcinomas characterized by glandular formation and the expression of luminal differentiation markers androgen receptor (AR) and prostate-specific antigen (PSA). Most adenocarcinomas are indolent and androgen-dependent. Hormonal therapy that inhibits AR signaling produces symptomatic relief in patients with advanced and metastatic adenocarcinomas. Prostatic small cell neuroendocrine carcinoma (SCNC) is a variant form of prostate cancer (PC). In contrast to adenocarcinoma, the tumor cells of SCNC do not form glands and are negative for AR and PSA. SCNC is extremely aggressive and does not respond to hormonal therapy. The purpose of this study was to compare the important and relevant features of two most commonly used PC cell lines, LNCaP and PC3, with prostatic adenocarcinoma and SCNC.
METHODS
Xenograft tumors of LNCaP and PC3 were prepared and compared with human prostatic adenocarcinoma and SCNC for the expression of key signaling molecules by immunohistochemistry and Western blot analysis.
RESULTS
LNCaP cells express AR and PSA and their growth is inhibited by androgen withdrawal, similar to human prostatic adenocarcinoma. PC3 cells do not express AR and PSA and their proliferation is independent of androgen, similar to SCNC. Adenocarcinoma cells and LNCaP cells are negative for neuroendocrine markers and stem cell-associated marker CD44 while SCNC and PC3 cells are positive. LNCaP cells have identical cytokeratin profiles to adenocarcinoma while PC3 cells have cytokeratin profiles similar to SCNC.
CONCLUSION
LNCaP cells share common features with adenocarcinoma while PC3 cells are characteristic of SCNC.
doi:10.1002/pros.21383
PMCID: PMC3426349  PMID: 21432867
prostate cancer; small cell carcinoma; adenocarcinoma; PC3; LNCaP
14.  Prognostic value and function of KLF4 in prostate cancer: RNAa and vector-mediated overexpression identify KLF4 as an inhibitor of tumor cell growth and migration 
Cancer research  2010;70(24):10182-10191.
KLF4/GLKF4 is a transcription factor that can have divergent functions in different malignancies. The role of KLF4 in prostate cancer etiology remains unclear. We have recently reported that small double-stranded RNA (dsRNA) can induce gene expression by targeting promoter sequence in a phenomenon referred to as RNA activation (RNAa). In the present study, we examine KLF4 levels in prostate cancer tissue and utilize RNAa as a tool for gene overexpression to investigate its function. Expression analysis indicated that KLF4 is significantly downregulated in prostate cancer cell lines compared to non-tumorigenic prostate cells. Meta-analysis of existing cDNA microarray data also revealed that KLF4 is frequently depleted in prostate cancer tissue with more pronounced reduction in metastases. In support, tissue microarray analysis of tumors and patient-matched controls indicated downregulation of KLF4 in metastatic tumor samples. Logistic regression analysis found that tumors with a KLF4 staining score <5 had a 15-fold higher risk for developing metastatic prostate cancer (P = 0.001, 95% CI: 3.0–79.0). In vitro analysis indicated that RNAa-mediated overexpression of KLF4 inhibited prostate cancer cell proliferation and survival, as well as altered the expression of several downstream cell cycle-related genes. Ectopic expression of KLF4 via viral transduction recapitulated the RNAa results validating its inhibitory effects on cancer growth. Reactivation of KLF4 also suppressed migration and invasion of prostate cancer cells. These results suggest that KLF4 functions as an inhibitor of tumor cell growth and migration in prostate cancer and decreased expression has prognostic value for predicting prostate cancer metastasis.
doi:10.1158/0008-5472.CAN-10-2414
PMCID: PMC3076047  PMID: 21159640
KLF4; RNAa; tumor suppressor gene; prostate cancer; metastasis
15.  Purification and direct transformation of epithelial progenitor cells from primary human prostate 
Nature protocols  2011;6(5):656-667.
Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without prior immortalization via genetic influences such as SV40 T-antigen, limiting our knowledge of the events that can transform naïve human epithelium. Here we describe a system developed in our lab to directly transform freshly-isolated primary human prostate epithelial cells without prior culture or immortalization. Prostate tissue is obtained from patients, and benign tissue is separated from cancer. Benign and cancer tissue are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated based on differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. 8-16 weeks after transplantion into immune-deficient mice, the development of lesions histologically classified as benign prostate, prostatic intraepithelial neoplasia (PIN) and adenocarcinoma can be evaluated.
doi:10.1038/nprot.2011.317
PMCID: PMC3092477  PMID: 21527922
Prostate cancer; Human regeneration; Human prostate; Flow cytometry; Lentivirus
16.  Neurosteroid transport by the organic solute transporter OSTα-OSTβ 
Journal of neurochemistry  2010;115(1):220-233.
A variety of steroids, including pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) are synthesized by specific brain cells, and are then delivered to their target sites, where they exert potent effects on neuronal excitability. The present results demonstrate that [3H]DHEAS and [3H]PREGS are relatively high affinity substrates for the organic solute transporter, OSTα–OSTβ, and that the two proteins that constitute this transporter are selectively localized to steroidogenic cells in the cerebellum and hippocampus, namely the Purkinje cells and cells in the CA region in both mouse and human brain. Analysis of Ostα and Ostβ mRNA levels in mouse Purkinje and hippocampal cells isolated via laser capture microdissection supported these findings. In addition, Ostα-deficient mice exhibited changes in serum dehydroepiandrosterone (DHEA) and DHEAS levels, and in tissue distribution of administered [3H]DHEAS. OSTα and OSTβ proteins were also localized to the zona reticularis of human adrenal gland, the major region for DHEAS production in the periphery. These results demonstrate that OSTα-OSTβ is localized to steroidogenic cells of the brain and adrenal gland, and that it modulates DHEA/DHEAS homeostasis, suggesting that it may contribute to neurosteroid action.
doi:10.1111/j.1471-4159.2010.06920.x
PMCID: PMC2939961  PMID: 20649839
Organic solute transporter; neurosteroid transport; pregnenolone sulfate; dehydroepiandrosterone sulfate; Purkinje cells; CA region of the hippocampus
17.  The oncogenic gene fusion TMPRSS2: ERG is not a diagnostic or prognostic marker for ovarian cancer 
TMPRSS2:ERG is a gene fusion resulting from the chromosomal rearrangement of the androgen-regulated TMPRSS2 gene and the ETS transcription factor ERG, leading to the over-expression of the oncogenic molecule ERG. This gene rearrangement has been found in approximately half of all prostate cancers and ERG overexpression is considered as a novel diagnostic marker for prostate carcinoma. However, little is known about the role of the TMPRSS2:ERG gene fusion in ovarian cancer. The purpose of this study was to test ERG expression in ovarian cancer and its potential as a diagnostic marker for ovarian carcinoma progression. A tissue microarray containing 180 ovarian cancer tissues of various pathological types and grades were examined by immunohistochemical analysis for expression of ERG. We also used 40 prostate carcinoma tissues and 40 normal tissues for comparison in parallel experiments. ERG-positive expression was detected in 40% of the prostate tumor cancer, as well as in internal positive control endothelial cells, confirming over-expression of ERG in prostate cancer at relatively the same rate observed by others. In contrast, all of the ovarian tumor patient tissues of varying histologic types were ERG-negative, despite some positivity in endothelial cells. These results suggest that the oncogenic gene fusion TMPRSS2:ERG does not occur in ovarian cancer relative to prostate cancer. Therefore, development of ERG expression profile would not be a useful diagnostic or prognostic marker for ovarian cancer patient screening.
PMCID: PMC3209604  PMID: 22076164
ovarian cancer; prostate cancer; immunohistochemistry; ERG; androgens; TMPRSS2:ERG
18.  Cell Autonomous Role of PTEN in Regulating Castration-Resistant Prostate Cancer Growth 
Cancer cell  2011;19(6):792-804.
SUMMARY
Alteration of the PTEN/PI3K pathway is associated with late stage and castrate resistant prostate cancer (CRPC). However, how PTEN loss involves in CRPC development is not clear. Here we show that castration-resistant growth is an intrinsic property of Pten-null prostate cancer (CaP) cells, independent of cancer development stage. PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of Ar in the epithelium promotes the proliferation of Pten-null cancer cells, at least in part, by down-regulating androgen-responsive gene Fkbp5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy.
doi:10.1016/j.ccr.2011.05.006
PMCID: PMC3157296  PMID: 21620777
19.  CXCR2 Promotes Ovarian Cancer Growth through Dysregulated Cell Cycle, Diminished Apoptosis, and Enhanced Angiogenesis 
Purpose
Chemokine receptor CXCR2 is associated with malignancy in several cancer models; however, the mechanisms involved in CXCR2-mediated tumor growth remain elusive. Here, we investigated the role of CXCR2 in human ovarian cancer.
Experimental Design
CXCR2 expression was silenced by stable small hairpin RNA in ovarian cancer cell lines T29Gro-1, T29H, and SKOV3. Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, flow cytometry, electrophoretic mobility shift assay, and mouse assay were used to detect CXCR2, interleukin-8, Gro-1, cell cycle, apoptosis, DNA binding of NF-κB, and tumor growth. Immunohistochemical staining of CXCR2 was done in 240 high-grade serous ovarian carcinoma samples.
Results
Knockdown of CXCR2 expression by small hairpin RNA reduced tumorigenesis of ovarian cancer cells in nude mice. CXCR2 promoted cell cycle progression by modulating cell cycle regulatory proteins, including p21 (waf1/cip1), cyclin D1, CDK6, CDK4, cyclin A, and cyclin B1. CXCR2 inhibited cellular apoptosis by suppressing phosphorylated p53, Puma, and Bcl-xS; suppressing poly(ADP-ribose) polymerase cleavage; and activating Bcl-xL and Bcl-2. CXCR2 stimulated angiogenesis by increasing levels of vascular endothelial growth factor and decreasing levels of thrombospondin-1, a process likely involving mitogen-activated protein kinase, and NF-κB. Overexpression of CXCR2 in high-grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival (P < 0.001) and of early relapse (P = 0.003) in the univariate analysis.
Conclusions
Our data provide strong evidence that CXCR2 regulates the cell cycle, apoptosis, and angiogenesis through multiple signaling pathways, including mitogen-activated protein kinase and NF-κB, in ovarian cancer. CXCR2 thus has potential as a therapeutic target and for use in ovarian cancer diagnosis and prognosis.
doi:10.1158/1078-0432.CCR-10-0483
PMCID: PMC2930833  PMID: 20505188
20.  Invasive prostate carcinoma driven by c-Src and androgen receptor synergy 
Cancer research  2010;71(3):862-872.
Cellular Src (c-Src) integrates a large number of signal transduction pathways regulating cell division, migration, and other aspects of cell physiology. Mutations of Src kinase have not been described in human prostate cancer, but evidence for increased levels of expression accompanying cancer progression has been reported. We analyzed over-expression of c-Src in naïve mouse prostate epithelium and observed no change in tubule formation frequency or histological structure. However, when enhanced c-Src expression is coupled with enhanced expression of androgen receptor (AR), it results in a strong activation of Src kinase activity accompanied by activation of the MAPK pathway, and enhanced AR activity. Similar to the pathology induced by constitutively active c-Src(Y529F), the tubules progress to frank carcinoma with invasion and display markers of epithelial to mesenchymal transition. These combined results suggest that non-mutated Src kinase may play a more important role in the genesis and progression of prostate cancer than previously appreciated and that epigenetic changes that enhance the level of AR may select for enhanced expression of c-Src with accompanying activation and a strong drive to malignant progression.
doi:10.1158/0008-5472.CAN-10-1605
PMCID: PMC3032821  PMID: 21135112
Src kinase; Androgen receptor; Invasive prostate adenocarcinoma; Prostate carcinoma
21.  Thrombin Expression in Prostate: A Novel Finding 
Cancer investigation  2011;29(1):62-67.
Introduction
A variable repertoire of coagulation protein expression is observed in different cancers. We evaluated expression of thrombin in prostate tissue.
Methods
Detection of thrombin was performed using quantitative real-time PCR in fresh tissue and in situ hybridization (ISH) in archival prostate tissue and by immunohistochemistry of prostate tissue microarrays.
Results
(Pro)thrombin mRNA expression was detected in cancer tissue and localized to prostatic epithelium and stroma by ISH. Thrombin protein was detected in stroma of benign and malignant epithelium (p <.05).
Conclusions
Prostate tissue is a rich reservoir of thrombin. This may have potential for developing antithrombin-based cancer therapy.
doi:10.3109/07357907.2010.535057
PMCID: PMC3136353  PMID: 21166500
Prostatic neoplasms; Prothrombin; Coagulation
22.  Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis and castration resistance 
Nature medicine  2010;16(12):1414-1420.
The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. Comparing gene expression in isogenic androgen-dependent and CRPC xenografts, we found a reproducible increase in N-cadherin expression, which was also elevated in primary and metastatic tumors of individuals with CRPC. Ectopic expression of N-cadherin in nonmetastatic, androgen-dependent prostate cancer models caused castration resistance, invasion and metastasis. Monoclonal antibodies against the ectodomain of N-cadherin reduced proliferation, adhesion and invasion of prostate cancer cells in vitro. In vivo, these antibodies slowed the growth of multiple established CRPC xenografts, blocked local invasion and metastasis and, at higher doses, led to complete regression. N-cadherin–specific antibodies markedly delayed the time to emergence of castration resistance, markedly affected tumor histology and angiogenesis, and reduced both AKT serine-threonine kinase activity and serum interleukin-8 (IL-8) secretion. These data indicate that N-cadherin is a major cause of both prostate cancer metastasis and castration resistance. Therapeutic targeting of this factor with monoclonal antibodies may have considerable clinical benefit.
doi:10.1038/nm.2236
PMCID: PMC3088104  PMID: 21057494
23.  Ionizing radiation induces prostate cancer neuroendocrine differentiation through interplay of CREB and ATF2: Implications for disease progression 
Cancer research  2008;68(23):9663-9670.
Radiation therapy is a first line treatment for prostate cancer patients with localized tumors. Although some patients respond well to the treatment, approximately 10% of low-risk and up to 60% of high-risk prostate cancer patients experience recurrent tumors. However, the molecular mechanisms underlying tumor recurrence remain largely unknown. Here we show that fractionated ionizing radiation (IR) induces differentiation of LNCaP prostate cancer cells into neuroendocrine (NE)-like cells, which are known to be implicated in prostate cancer progression, androgen independent growth and poor prognosis. Further analyses revealed that two CRE-binding transcription factors, CREB and ATF2, function as a transcriptional activator and repressor, respectively, of NE-like differentiation and that IR induces NE-like differentiation by increasing the nuclear content of phospho-CREB and cytoplasmic accumulation of ATF2. Consistent with this notion, stable expression of a non-phosphorylatable CREB or a constitutively nuclear-localized ATF2 in LNCaP cells inhibits IR-induced NE-like differentiation. IR-induced NE-like morphologies are reversible, and three IR-resistant clones isolated from dedifferentiated cells have acquired the ability to proliferate and lost the NE-like cell properties. Also, these three IR-resistant clones exhibit differential responses to IR- and androgen depletion-induced NE-like differentiation. However, they are all resistant to IR-and the chemotherapeutic agent docetaxel-induced cell death, and to androgen depletion-induced growth inhibition. These results suggest that radiation therapy-induced NE-like differentiation may represent a novel pathway by which prostate cancer cells survive the treatment and contribute to tumor recurrence.
doi:10.1158/0008-5472.CAN-08-2229
PMCID: PMC3100895  PMID: 19047143
prostate cancer; neuroendocrine differentiation; ionizing radiation; ATF2; CREB
24.  Tyrosine Kinase ETK/BMX Is Up-Regulated in Bladder Cancer and Predicts Poor Prognosis in Patients with Cystectomy 
PLoS ONE  2011;6(3):e17778.
Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.
doi:10.1371/journal.pone.0017778
PMCID: PMC3049795  PMID: 21408190
25.  Identification of a cell-of-origin for human prostate cancer 
Science (New York, N.Y.)  2010;329(5991):568-571.
Prostate cancer induced in primary human prostate basal cells recapitulates disease initiation and progression in immunodeficient mice.
doi:10.1126/science.1189992
PMCID: PMC2917982  PMID: 20671189

Results 1-25 (32)