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Find video protocols related to scientific articles indexed in Pubmed.
Characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identifies dormancy associated pathways.
Oncotarget
PUBLISHED: 10-11-2014
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Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM+/CD45- cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention.
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Targeting GPR30 with G-1: a new therapeutic target for castration-resistant prostate cancer.
Endocr. Relat. Cancer
PUBLISHED: 10-06-2014
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Castration-resistant prostate cancer (CRPC) is an advanced-stage prostate cancer (PC) associated with high mortality. We reported that G-1, a selective agonist of G protein-coupled receptor 30 (GPR30), inhibited PC cell growth by inducing G2 cell cycle arrest and arrested PC-3 xenograft growth. However, the therapeutic actions of G-1 and their relationships with androgen in vivo are unclear. Using the LNCaP xenograft to model PC growth during the androgen-sensitive (AS) versus the castration-resistant (CR) phase, we found that G-1 inhibited growth of CR but not AS tumors with no observable toxicity to the host. Substantial necrosis (approximately 65%) accompanied by marked intratumoral infiltration of neutrophils was observed only in CR tumors. Global transcriptome profiling of human genes identified 99 differentially expressed genes with 'interplay between innate and adaptive immune responses' as the top pathway. Quantitative PCR confirmed upregulation of neutrophil-related chemokines and inflammation-mediated cytokines only in the G-1-treated CR tumors. Expression of murine neutrophil-related cytokines also was elevated in these tumors. GPR30 (GPER1) expression was significantly higher in CR tumors than in AS tumors. In cell-based experiments, androgen repressed GPR30 expression, a response reversible by anti-androgen or siRNA-induced androgen receptor silencing. Finally, in clinical specimens, 80% of CRPC metastases (n=123) expressed a high level of GPR30, whereas only 54% of the primary PCs (n=232) showed high GPR30 expression. Together, these results provide the first evidence, to our knowledge, that GPR30 is an androgen-repressed target and G-1 mediates the anti-tumor effect via neutrophil-infiltration-associated necrosis in CRPC. Additional studies are warranted to firmly establish GPR30 as a therapeutic target in CRPC.
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Efficacy studies of an antibody-drug conjugate PSMA-ADC in patient-derived prostate cancer xenografts.
Prostate
PUBLISHED: 08-01-2014
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It is timely and important to develop new treatment modalities for advanced prostate cancer, because even the newly FDA approved treatments, despite providing significant survival benefits, do not constitute cure of this disease. Antibody drug conjugates (ADCs) represent a promising approach to cancer therapy. Prostate-specific membrane antigen (PSMA) is expressed in advanced prostate cancer and targeting this protein is used for imaging of advanced prostate cancer as well as development of targeting strategies. The objective of our studies was to evaluate the efficacy of PSMA ADC against a series of patient-derived prostate cancer xenografts (LuCaP 58, LuCaP 77, LuCaP 96CR, and LuCaP 105) with different characteristics, including varying levels of PSMA expression and responses to androgen suppression.
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AR-regulated TWEAK-FN14 pathway promotes prostate cancer bone metastasis.
Cancer Res.
PUBLISHED: 06-26-2014
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The recurrence of prostate cancer metastases to bone after androgen deprivation therapy is a major clinical challenge. We identified FN14 (TNFRSF12A), a TNF receptor family member, as a factor that promotes prostate cancer bone metastasis. In experimental models, depletion of FN14 inhibited bone metastasis, and FN14 could be functionally reconstituted with IKK?-dependent, NF?B signaling activation. In human prostate cancer, upregulated FN14 expression was observed in more than half of metastatic samples. In addition, FN14 expression was correlated inversely with androgen receptor (AR) signaling output in clinical samples. Consistent with this, AR binding to the FN14 enhancer decreased expression. We show here that FN14 may be a survival factor in low AR output prostate cancer cells. Our results define one upstream mechanism, via FN14 signaling, through which the NF?B pathway contributes to prostate cancer metastasis and suggest FN14 as a candidate therapeutic and imaging target for castrate-resistant prostate cancers.
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The role of the microenvironment-dormant prostate disseminated tumor cells in the bone marrow.
Drug Discov Today Technol
PUBLISHED: 05-23-2014
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Disseminated tumor cells (DTC) leave the primary tumor and reside in distant sites (e.g. bone) early in prostate cancer. Patients may harbor dormant DTC which develop into clinically overt metastasis years after radical prostatectomy. We will describe recent evidence suggesting high p38/ERK ratio, bone morphogenetic proteins, and tumor growth factor-beta 2 promote dormancy in solid tumors. Furthermore, we will discuss the possible regulation of dormancy by hematopoietic stem cell and vascular niches, and describe novel models recapitulating bone marrow metastatic latency and out- growth, 3D microvascular networks, and 3D biomatrix supportive niches in the studies of tumor cell dormancy.
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Complex MSH2 and MSH6 mutations in hypermutated microsatellite unstable advanced prostate cancer.
Nat Commun
PUBLISHED: 03-13-2014
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A hypermutated subtype of advanced prostate cancer was recently described, but prevalence and mechanisms have not been well-characterized. Here we find that 12% (7 of 60) of advanced prostate cancers are hypermutated, and that all hypermutated cancers have mismatch repair gene mutations and microsatellite instability (MSI). Mutations are frequently complex MSH2 or MSH6 structural rearrangements rather than MLH1 epigenetic silencing. Our findings identify parallels and differences in the mechanisms of hypermutation in prostate cancer compared with other MSI-associated cancers.
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Spheroid culture of LuCaP 147 as an authentic preclinical model of prostate cancer subtype with SPOP mutation and hypermutator phenotype.
Cancer Lett.
PUBLISHED: 03-07-2014
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LuCaP serially transplantable xenografts are valuable preclinical models of locally advanced or metastatic prostate cancer. For the first time, we recently succeeded in establishing and serially passaging spheroid cultures of several LuCaP xenografts. Here, we characterized in depth the molecular and cellular phenotype of LuCaP 147 cultures and found faithful retention of the characteristics of the original xenograft, including immunophenotype, genetic fidelity, gene expression profile and responsiveness to androgen. Furthermore, we demonstrated capabilities for high-throughput drug screening and that anti-cancer agents induced cell cycle arrest and apoptosis in spheroid cultures. Finally, we showed that cells formed tumors when re-introduced into mice, providing an authentic in vitro-in vivo preclinical model of a subtype of prostate cancer with a hypermutator phenotype and an SPOP mutation.
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Androgen receptor splice variants determine taxane sensitivity in prostate cancer.
Cancer Res.
PUBLISHED: 02-20-2014
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Prostate cancer growth depends on androgen receptor signaling. Androgen ablation therapy induces expression of constitutively active androgen receptor splice variants that drive disease progression. Taxanes are a standard of care therapy in castration-resistant prostate cancer (CRPC); however, mechanisms underlying the clinical activity of taxanes are poorly understood. Recent work suggests that the microtubule network of prostate cells is critical for androgen receptor nuclear translocation and activity. In this study, we used a set of androgen receptor deletion mutants to identify the microtubule-binding domain of the androgen receptor, which encompasses the DNA binding domain plus hinge region. We report that two clinically relevant androgen receptor splice variants, ARv567 and ARv7, differentially associate with microtubules and dynein motor protein, thereby resulting in differential taxane sensitivity in vitro and in vivo. ARv7, which lacks the hinge region, did not co-sediment with microtubules or coprecipitate with dynein motor protein, unlike ARv567. Mechanistic investigations revealed that the nuclear accumulation and transcriptional activity of ARv7 was unaffected by taxane treatment. In contrast, the microtubule-interacting splice variant ARv567 was sensitive to taxane-induced microtubule stabilization. In ARv567-expressing LuCap86.2 tumor xenografts, docetaxel treatment was highly efficacious, whereas ARv7-expressing LuCap23.1 tumor xenografts displayed docetaxel resistance. Our results suggest that androgen receptor variants that accumulate in CRPC cells utilize distinct pathways of nuclear import that affect the antitumor efficacy of taxanes, suggesting a mechanistic rationale to customize treatments for patients with CRPC, which might improve outcomes.
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Prostate cancer characteristics associated with response to pre-receptor targeting of the androgen axis.
PLoS ONE
PUBLISHED: 01-01-2014
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Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.
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Chemotherapy-induced monoamine oxidase expression in prostate carcinoma functions as a cytoprotective resistance enzyme and associates with clinical outcomes.
PLoS ONE
PUBLISHED: 01-01-2014
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To identify molecular alterations in prostate cancers associating with relapse following neoadjuvant chemotherapy and radical prostatectomy patients with high-risk localized prostate cancer were enrolled into a phase I-II clinical trial of neoadjuvant chemotherapy with docetaxel and mitoxantrone followed by prostatectomy. Pre-treatment prostate tissue was acquired by needle biopsy and post-treatment tissue was acquired by prostatectomy. Prostate cancer gene expression measurements were determined in 31 patients who completed 4 cycles of neoadjuvant chemotherapy. We identified 141 genes with significant transcript level alterations following chemotherapy that associated with subsequent biochemical relapse. This group included the transcript encoding monoamine oxidase A (MAOA). In vitro, cytotoxic chemotherapy induced the expression of MAOA and elevated MAOA levels enhanced cell survival following docetaxel exposure. MAOA activity increased the levels of reactive oxygen species and increased the expression and nuclear translocation of HIF1?. The suppression of MAOA activity using the irreversible inhibitor clorgyline augmented the apoptotic responses induced by docetaxel. In summary, we determined that the expression of MAOA is induced by exposure to cytotoxic chemotherapy, increases HIF1?, and contributes to docetaxel resistance. As MAOA inhibitors have been approved for human use, regimens combining MAOA inhibitors with docetaxel may improve clinical outcomes.
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Dietary protein restriction inhibits tumor growth in human xenograft models.
Oncotarget
PUBLISHED: 12-20-2013
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Purpose: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer. Experimental Design: To test our hypothesis we assessed the inhibitory effect of protein diet restriction on prostate and breast cancer growth, serum PSA and IGF-1 concentrations, mTOR activity and epigenetic markers, by using human xenograft cancer models. Results: Our results showed a 70% inhibition of tumor growth in the castrate-resistant LuCaP23.1 prostate cancer model and a 56% inhibition in the WHIM16 breast cancer model fed with a 7% protein diet when compared to an isocaloric 21% protein diet. Inhibition of tumor growth correlated, in the LuCaP23.1 model, with decreased serum PSA and IGF-1 levels, down-regulation of mTORC1 activity, decreased cell proliferation as indicated by Ki67 staining, and reduction in epigenetic markers of prostate cancer progression, including the histone methyltransferase EZH2 and the associated histone mark H3K27me3. In addition, we observed that modifications of dietary protein quality, independently of protein quantity, decreased tumor growth. A diet containing 20% plant protein inhibited tumor weight by 37% as compared to a 20% animal dairy protein diet. Conclusions: Our findings suggest that a reduction in dietary protein intake is highly effective in inhibiting tumor growth in human xenograft prostate and breast cancer models, possibly through the inhibition of the IGF/AKT/mTOR pathway and epigenetic modifications.
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Targeted Androgen Pathway Suppression in Localized Prostate Cancer: A Pilot Study.
J. Clin. Oncol.
PUBLISHED: 12-09-2013
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Ligand-mediated activation of the androgen receptor (AR) is critical for prostate cancer (PCa) survival and proliferation. The failure to completely ablate tissue androgens may limit suppression of PCa growth. We evaluated combinations of CYP17A and 5-?-reductase inhibitors for reducing prostate androgen levels, AR signaling, and PCa volumes.
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Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer.
Oncotarget
PUBLISHED: 12-03-2013
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BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.
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TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-07-2013
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Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC.
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Tumor necrosis factor receptor associated factor-4: an adapter protein overexpressed in metastatic prostate cancer is regulated by microRNA-29a.
Oncol. Rep.
PUBLISHED: 08-15-2013
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The tumor necrosis factor receptor (TNFR)-associated factor 4 (TRAF4) is a member of TRAF family proteins that act as major signal transducers of the TNF receptor and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF4 has been reported to be overexpressed in various human cancers. However, the exact mechanisms that regulate the expression of TRAF4 still remain elusive. The objective of the present study was to investigate the regulatory mechanism of TRAF4 expression in prostate cancer. We initially identified microRNA-29a (miR?29a) as a possible candidate to bind TRAF4 3 untranslated region (3UTR) by the algorithm, TargetScan. The expression of TRAF4 mRNA and protein was inversely associated with miR-29a expression in prostate cancer cell lines (LNCaP, DU145 and PC3). TRAF4 expression was reduced by the introduction of mimic miR-29a in LNCaP cells. Luciferase activity from the construct harboring wild-type TRAF4 3UTR was reduced by the mimic miR-29a and this reduction was diminished by introducing mutations at the predicted miR-29a binding site. On the other hand, TRAF4 was upregulated when transfected with the inhibitor of miR-29a in DU145 and PC3 cells. TRAF4 was significantly upregulated in patients with metastatic prostate cancer compared to those with localized prostate cancer. Furthermore, there was a significant inverse correlation between TRAF4 and miR-29a expression in tumor tissues from radical prostatectomy. Considered together, our results suggest that the tumor suppressor microRNA, miR-29a, is one of the regulators of TRAF4 expression in metastatic prostate cancer.
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Prostate cancer derived prostatic acid phosphatase promotes an osteoblastic response in the bone microenvironment.
Clin. Exp. Metastasis
PUBLISHED: 08-02-2013
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Approximately 90 % of patients who die of prostate cancer (PCa) have bone metastases, often promoting osteoblastic lesions. We observed that 88 % of castration-resistant PCa (CRPC) bone metastases express prostatic acid phosphatase (PAP), a soluble secreted protein expressed by prostate epithelial cells in predominately osteoblastic (n = 18) or osteolytic (n = 15) lesions. Additionally, conditioned media (CM) of an osteoblastic PCa xenograft LuCaP 23.1 contained significant levels of PAP and promoted mineralization in mouse and human calvaria-derived cells (MC3T3-E1 and HCO). To demonstrate that PAP promotes mineralization, we stimulated MC3T3-E1 cells with PAP and observed increased mineralization, which could be blocked with the specific PAP inhibitor, phosphonic acid. Furthermore, the mineralization promoted by LuCaP 23.1 CM was also blocked by phosphonic acid, suggesting PAP is responsible for the mineralization promoting activity of LuCaP 23.1. In addition, gene expression arrays comparing osteoblastic to osteolytic CRPC (n = 14) identified betacellulin (BTC) as a gene upregulated during the osteoblastic response in osteoblasts during new bone formation. Moreover, BTC levels were increased in bone marrow stromal cells in response to LuCaP 23.1 CM in vitro. Because new bone formation does occur in osteoblastic and can occur in osteolytic CRPC bone metastases, we confirmed by immunohistochemistry (n = 36) that BTC was highly expressed in osteoblasts involved in new bone formation occurring in both osteoblastic and osteolytic sites. These studies suggest a role for PAP in promoting the osteoblastic reaction in CRPC bone metastases and identify BTC as a novel downstream protein expressed in osteoblasts during new bone formation.
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Effects of androgen deprivation therapy and bisphosphonate treatment on bone in patients with metastatic castration-resistant prostate cancer: results from the University of Washington Rapid Autopsy Series.
J. Bone Miner. Res.
PUBLISHED: 06-28-2013
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Qualitative and quantitative bone features were determined in nondecalcified and decalcified bone from 20 predetermined bone sites in each of 44 patients who died with castration-resistant prostate cancer (CRPC), some of which received bisphosphonate treatment (BP) in addition to androgen-deprivation therapy (ADT). Thirty-nine of the 44 patients (89%) had evidence of bone metastases. By histomorphometric analysis, these bone metastases were associated with a range of bone responses from osteoblastic to osteolytic with a wide spectrum of bone responses often seen within an individual patient. Overall, the average bone volume/tissue volume (BV/TV) was 25.7%, confirming the characteristic association of an osteoblastic response to prostate cancer bone metastasis when compared with the normal age-matched weighted mean BV/TV of 14.7%. The observed new bone formation was essentially woven bone, and this was a localized event. In comparing BV/TV at metastatic sites between patients who had received BP treatment and those who had not, there was a significant difference (28.6% versus 19.3%, respectively). At bone sites that were not invaded by tumor, the average BV/TV was 10.1%, indicating significant bone loss owing to ADT that was not improved (11%) in those patients who had received BPs. Surprisingly, there was no significant difference in the number of osteoclasts present at the metastatic sites between patients treated or not treated with BPs, but in bone sites where the patient had been treated with BPs, giant osteoclasts were observed. Overall, 873 paraffin-embedded specimens and 661 methylmethacrylate-embedded specimens were analyzed. Our results indicate that in CRPC patients, ADT induces serious bone loss even in patients treated with BP. Furthermore, in this cohort of patients, BP treatment increased BV and did not decrease the number of osteoclasts in prostate cancer bone metastases compared with bone metastases from patients who did not receive BP.
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Establishment and serial passage of cell cultures derived from LuCaP xenografts.
Prostate
PUBLISHED: 06-06-2013
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LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long-term growth of LuCaP cells in vitro.
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5?-reductase inhibition suppresses testosterone-induced initial regrowth of regressed xenograft prostate tumors in animal models.
Endocrinology
PUBLISHED: 05-13-2013
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Androgen deprivation therapy (ADT) is the standard treatment for patients with prostate-specific antigen progression after treatment for localized prostate cancer. An alternative to continuous ADT is intermittent ADT (IADT), which allows recovery of testosterone during off-cycles to stimulate regrowth and differentiation of the regressed prostate tumor. IADT offers patients a reduction in side effects associated with ADT, improved quality of life, and reduced cost with no difference in overall survival. Our previous studies showed that IADT coupled with 5?-reductase inhibitor (5ARI), which blocks testosterone conversion to DHT could prolong survival of animals bearing androgen-sensitive prostate tumors when off-cycle duration was fixed. To further investigate this clinically relevant observation, we measured the time course of testosterone-induced regrowth of regressed LuCaP35 and LNCaP xenograft tumors in the presence or absence of a 5ARI. 5?-Reductase inhibitors suppressed the initial regrowth of regressed prostate tumors. However, tumors resumed growth and were no longer responsive to 5?-reductase inhibition several days after testosterone replacement. This finding was substantiated by bromodeoxyuridine and Ki67 staining of LuCaP35 tumors, which showed inhibition of prostate tumor cell proliferation by 5ARI on day 2, but not day 14, after testosterone replacement. 5?-Reductase inhibitors also suppressed testosterone-stimulated proliferation of LNCaP cells precultured in androgen-free media, suggesting that blocking testosterone conversion to DHT can inhibit prostate tumor cell proliferation via an intracrine mechanism. These results suggest that short off-cycle coupled with 5?-reductase inhibition could maximize suppression of prostate tumor growth and, thus, improve potential survival benefit achieved in combination with IADT.
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A gain-of-function mutation in DHT synthesis in castration-resistant prostate cancer.
Cell
PUBLISHED: 05-10-2013
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Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation that leads to a gain-of-function in 3?-hydroxysteroid dehydrogenase type 1 (3?HSD1), which catalyzes the initial rate-limiting step in conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3?HSD1 is a valid target for the treatment of CRPC.
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Dormancy in solid tumors: implications for prostate cancer.
Cancer Metastasis Rev.
PUBLISHED: 04-25-2013
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In cancer dormancy, residual tumor cells persist in a patient with no apparent clinical symptoms, only to potentially become clinically relevant at a later date. In prostate cancer (PCa), the primary tumor is often removed and many patients experience a prolonged period (>5 years) with no evidence of disease before recurrence. These characteristics make PCa an excellent candidate for the study of tumor cell dormancy. However, the mechanisms that constitute PCa dormancy have not been clearly defined. Additionally, the definition of tumor cell dormancy varies in the literature. Therefore, we have separated tumor cell dormancy in this review into three categories: (a) micrometastatic dormancy-a group of tumor cells that cannot increase in number due to a restrictive proliferation/apoptosis equilibrium. (b) Angiogenic dormancy-a group of tumor cells that cannot expand beyond the formation of a micrometastasis due to a lack of angiogenic potential. (c) Conditional dormancy-an individual cell or a very small number of cells that cannot proliferate without the appropriate cues from the microenvironment, but do not require angiogenesis to do so. This review aims to identify currently known markers, mechanisms, and models of tumor dormancy, in particular as they relate to PCa, and highlight current opportunities for advancement in our understanding of clinical cancer dormancy.
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Absolute quantification by droplet digital PCR versus analog real-time PCR.
Nat. Methods
PUBLISHED: 04-10-2013
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Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.
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Inhibition of CCL2 Signaling in Combination with Docetaxel Treatment Has Profound Inhibitory Effects on Prostate Cancer Growth in Bone.
Int J Mol Sci
PUBLISHED: 02-16-2013
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The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.
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Single cell transcriptomic analysis of prostate cancer cells.
BMC Mol. Biol.
PUBLISHED: 02-11-2013
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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).
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Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases.
J. Pathol.
PUBLISHED: 02-03-2013
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The complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumour cell survival in lethal prostate cancer metastases, we examined five survival proteins that operate within three survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic survivin, nuclear survivin, and stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of three or more proteins occurred in 81% of lethal prostate cancer metastases and BCL-2, cytoplasmic survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic survivin and MCL-1 had significantly higher expression in bone metastases (p?
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Prostate cancer cell phenotypes based on AGR2 and CD10 expression.
Mod. Pathol.
PUBLISHED: 01-25-2013
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The combination of expression patterns of AGR2 (anterior gradient 2) and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10(low)AGR2(high), CD10(high)AGR2(high), CD10(low)AGR2(low), and CD10(high)AGR2(low) were distinguished. AGR2(+) tumors were associated with longer recurrence-free survival and CD10(+) tumors with shorter recurrence-free survival. In high-stage cases, the CD10(low)AGR2(high) phenotype was associated with a ninefold higher recurrence-free survival than the CD10(high)AGR2(low) phenotype. The CD10(high)AGR2(high) and CD10(low)AGR2(low) phenotypes were intermediate. The CD10(high)AGR2(low) phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10(high)AGR2(low) phenotype in primary tumors is predictive of poor outcome; however, the CD10(low)AGR2(high) phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.
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A novel four-color fluorescence in situ hybridization assay for the detection of TMPRSS2 and ERG rearrangements in prostate cancer.
Cancer Genet
PUBLISHED: 01-24-2013
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Since the identification of the TMPRSS2-ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We report a novel four-color fluorescence in situ hybridization (FISH) assay that detects not only the typical TMPRSS2-ERG fusion but also alternative rearrangements of the TMPRSS2 or ERG gene. We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens, including cell lines, primary prostate cancer tissues, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients. When compared with either reverse transcription-polymerase chain reaction or the Gen-Probe method as the technical reference, analysis using the four-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% confidence interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting the TMPRSS2-ERG fusion. The TMPRSS2-ERG fusion was detected in 41% and 43% of primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Rearrangements other than the typical TMPRSS2-ERG fusion were confirmed by karyotype analysis and found in 7% of primary cancer and 13% of CRPC tumors. Successful karyotype analyses are reported for the first time on four of the xenograft samples, complementing the FISH results. Analysis using the four-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer.
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Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum.
J. Steroid Biochem. Mol. Biol.
PUBLISHED: 01-19-2013
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Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1?L of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.
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Characterization of osteoblastic and osteolytic proteins in prostate cancer bone metastases.
Prostate
PUBLISHED: 01-17-2013
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Approximately 90% of patients who die of Prostate Cancer (PCa) have bone metastases, which promote a spectrum of osteoblastic, osteolytic or mixed bone responses. Numerous secreted proteins have been reported to promote osteoblastic or osteolytic bone responses. We determined whether previously identified and/or novel proteins were associated with the osteoblastic or osteolytic response in clinical specimens of PCa bone metastases.
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HOXB13 G84E mutation in Finland: population-based analysis of prostate, breast, and colorectal cancer risk.
Cancer Epidemiol. Biomarkers Prev.
PUBLISHED: 01-04-2013
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A recently identified germline mutation G84E in HOXB13 was shown to increase the risk of prostate cancer. In a family-based analysis by The International Consortium for Prostate Cancer Genetics (ICPCG), the G84E mutation was most prevalent in families from the Nordic countries of Finland (22.4%) and Sweden (8.2%).
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Cabozantinib inhibits growth of androgen-sensitive and castration-resistant prostate cancer and affects bone remodeling.
PLoS ONE
PUBLISHED: 01-01-2013
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Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.
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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
PUBLISHED: 01-01-2013
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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.
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Circulating microRNA profiling identifies a subset of metastatic prostate cancer patients with evidence of cancer-associated hypoxia.
PLoS ONE
PUBLISHED: 01-01-2013
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MicroRNAs (miRNAs) are small (?22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1? signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.
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Reduced expression of the androgen receptor by third generation of antisense shows antitumor activity in models of prostate cancer.
Mol. Cancer Ther.
PUBLISHED: 10-25-2011
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The androgen receptor (AR) is a member of a unique class of transcription factors because it contains a ligand-binding domain that, when activated, results in nuclear translocation and the transcriptional activation of genes associated with prostate cancer development. Although androgen deprivation therapies are effective initially for the treatment of prostate cancer, the disease eventually relapses and progresses to castration-resistant prostate cancer (CRPC). Nonetheless, the AR still plays a critical role because late-stage investigational agents that deplete testosterone (abiraterone) or block ligand binding (MDV3100) can still control tumor growth in patients with CRPC. These findings indicate that downmodulation of AR expression may provide a complementary strategy for treating CRPC. In this article, we describe a novel, locked, nucleic acid-based antisense oligonucleotide, designated EZN-4176. When administered as a single agent, EZN-4176 specifically downmodulated AR mRNA and protein, and this was coordinated with inhibition of the growth of both androgen-sensitive and CRPC tumors in vitro as well as in animal models. The effect was specific because no effect on growth was observed with a control antisense oligonucleotide that does not recognize AR mRNA, nor on tumors derived from the PC3, AR-negative, tumor cell line. In addition, EZN-4176 reduced AR luciferase reporter activity in a CRPC model derived from C4-2b cells that were implanted intratibially, indicating that the molecule may control prostate cancer that has metastasized to the bone. These data, together with the continued dependency of CRPC on the AR signaling pathway, justify the ongoing phase I evaluation of EZN-4176 in patients with CRPC.
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A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.
Epigenetics
PUBLISHED: 10-01-2011
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DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERGs predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.
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Exome sequencing identifies a spectrum of mutation frequencies in advanced and lethal prostate cancers.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-26-2011
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To catalog protein-altering mutations that may drive the development of prostate cancers and their progression to metastatic disease systematically, we performed whole-exome sequencing of 23 prostate cancers derived from 16 different lethal metastatic tumors and three high-grade primary carcinomas. All tumors were propagated in mice as xenografts, designated the LuCaP series, to model phenotypic variation, such as responses to cancer-directed therapeutics. Although corresponding normal tissue was not available for most tumors, we were able to take advantage of increasingly deep catalogs of human genetic variation to remove most germline variants. On average, each tumor genome contained ~200 novel nonsynonymous variants, of which the vast majority was specific to individual carcinomas. A subset of genes was recurrently altered across tumors derived from different individuals, including TP53, DLK2, GPC6, and SDF4. Unexpectedly, three prostate cancer genomes exhibited substantially higher mutation frequencies, with 2,000-4,000 novel coding variants per exome. A comparison of castration-resistant and castration-sensitive pairs of tumor lines derived from the same prostate cancer highlights mutations in the Wnt pathway as potentially contributing to the development of castration resistance. Collectively, our results indicate that point mutations arising in coding regions of advanced prostate cancers are common but, with notable exceptions, very few genes are mutated in a substantial fraction of tumors. We also report a previously undescribed subtype of prostate cancers exhibiting "hypermutated" genomes, with potential implications for resistance to cancer therapeutics. Our results also suggest that increasingly deep catalogs of human germline variation may challenge the necessity of sequencing matched tumor-normal pairs.
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Resistance to CYP17A1 inhibition with abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants.
Clin. Cancer Res.
PUBLISHED: 08-01-2011
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Abiraterone is a potent inhibitor of the steroidogenic enzyme CYP17A1 and suppresses tumor growth in patients with castration-resistant prostate cancer (CRPC). The effectiveness of abiraterone in reducing tumor androgens is not known, nor have mechanisms contributing to abiraterone resistance been established.
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Hsp90 inhibitor 17-AAG inhibits progression of LuCaP35 xenograft prostate tumors to castration resistance.
Prostate
PUBLISHED: 07-31-2011
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Advanced prostate cancer is currently treated with androgen deprivation therapy (ADT). ADT initially results in tumor regression; however, all patients eventually relapse with castration-resistant prostate cancer. New approaches to delay the progression of prostate cancer to castration resistance are in desperate need. This study addresses whether targeting Heat shock protein 90 (HSP90) regulation of androgen receptor (AR) can inhibit prostate cancer progression to castration resistance.
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HIF induces human embryonic stem cell markers in cancer cells.
Cancer Res.
PUBLISHED: 06-28-2011
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Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIF?, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1?-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.
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LIV-1 promotes prostate cancer epithelial-to-mesenchymal transition and metastasis through HB-EGF shedding and EGFR-mediated ERK signaling.
PLoS ONE
PUBLISHED: 06-22-2011
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LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaP(E) (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaP(E) cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaP(E) cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.
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Androgen receptor variants occur frequently in castration resistant prostate cancer metastases.
PLoS ONE
PUBLISHED: 06-17-2011
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Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa).
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Lineage relationship of prostate cancer cell types based on gene expression.
BMC Med Genomics
PUBLISHED: 05-23-2011
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Prostate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells.
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Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab as a means of decreasing kidney retention.
Bioconjug. Chem.
PUBLISHED: 05-05-2011
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Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab, Fab, or engineered fragments) as cancer-targeting reagents for therapy with the ?-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab (107-1A4 Fab). Biodistributions of the five radioiodinated Fab conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab, then labeled (separately) with (125)I and (211)At. The radiolabeled Fab conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab, but the (211)At-labeled Fab reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab was cleared from kidney similar to earlier studies, the (211)At-labeled Fabwas not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab conjugated with closo-decaborate(2-) reagents are radioiodinated.
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Contribution of ARLTS1 Cys148Arg (T442C) variant with prostate cancer risk and ARLTS1 function in prostate cancer cells.
PLoS ONE
PUBLISHED: 04-26-2011
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ARLTS1 is a recently characterized tumor suppressor gene at 13q14.3, a region frequently deleted in both sporadic and hereditary prostate cancer (PCa). ARLTS1 variants, especially Cys148Arg (T442C), increase susceptibility to different cancers, including PCa. In this study the role of Cys148Arg substitution was investigated as a risk factor for PCa using both genetic and functional analysis. Cys148Arg genotypes and expression of the ARLTS1 were explored in a large set of familial and unselected PCa cases, clinical tumor samples, xenografts, prostate cancer cell lines and benign prostatic hyperplasia (BPH) samples. The frequency of the variant genotype CC was significantly higher in familial (OR?=?1.67, 95% CI?=?1.08-2.56, P?=?0.019) and unselected patients (OR?=?1.52, 95% CI?=?1.18-1.97, P?=?0.001) and the overall risk was increased (OR?=?1.54, 95% CI?=?1.20-1.98, P?=?0.0007). Additional analysis with clinicopathological data revealed an association with an aggressive disease (OR?=?1.28, 95% CI?=?1.05-?, P?=?0.02). The CC genotype of the Cys148Arg variant was also contributing to the lowered ARLTS1 expression status in lymphoblastoid cells from familial patients. In addition significantly lowered ARLTS1 expression was observed in clinical tumor samples compared to BPH samples (P?=?0.01). The ARLTS1 co-expression signature based on previously published microarray data was generated from 1587 cancer samples confirming the low expression of ARLTS1 in PCa and showed that ARLTS1 expression was strongly associated with immune processes. This study provides strong confirmation of the important role of ARLTS1 Cys148Arg variant as a contributor in PCa predisposition and a potential marker for aggressive disease outcome.
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Serum prosaposin levels are increased in patients with advanced prostate cancer.
Prostate
PUBLISHED: 04-05-2011
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We previously cloned prosaposin (PSAP) from metastatic castrate-resistant prostate cancer (mCRPCa) cells and demonstrated its genomic amplification and/or overexpression in metastatic PCa cell lines, xenografts, and lymph node metastases. The clinicohistopathological significance of serum PSAP levels and its tissue expression and association with predictive or prognostic variable in primary or advanced PCa are not known.
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Human prostate cancer harbors the stem cell properties of bone marrow mesenchymal stem cells.
Clin. Cancer Res.
PUBLISHED: 02-25-2011
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Prostate tumor cells frequently show the features of osteoblasts, which are differentiated from bone marrow mesenchymal stem cells. We examined human prostate cancer cell lines and clinical prostate cancer specimens for additional bone marrow mesenchymal stem cell properties.
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The miR-15a-miR-16-1 locus is homozygously deleted in a subset of prostate cancers.
Genes Chromosomes Cancer
PUBLISHED: 02-18-2011
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MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate the expression of protein coding genes. In this study, we screened highly informative prostate cancer cell lines and xenografts (n = 42) for miRNA gene copy number and expression changes. The expression profiling showed distinction between cell lines and xenografts as well as between androgen sensitive and independent models. Only a few copy number alterations that were associated with expression changes were identified. Most importantly, the miR-15a-miR-16-1 locus was found to be homozygously deleted in two samples leading to the abolishment of miR-15a, but not miR-16, expression. miR-16 is also expressed from another genomic locus. Mutation screening of the miR-15a-miR-16-1 gene in the model systems as well as clinical samples (n = 50) revealed no additional mutations. In conclusion, our data indicate that putative tumor suppressors, miR-15a and miR-16-1, are homozygously deleted in a subset of prostate cancers, further suggesting that these miRNAs could be important in the development of prostate cancer.
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Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases.
Am. J. Pathol.
PUBLISHED: 02-06-2011
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Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.
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Expression of SLCO transport genes in castration-resistant prostate cancer and impact of genetic variation in SLCO1B3 and SLCO2B1 on prostate cancer outcomes.
Cancer Epidemiol. Biomarkers Prev.
PUBLISHED: 01-25-2011
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Metastases from men with castration-resistant prostate cancer (CRPC) harbor increased tumoral androgens versus untreated prostate cancers. This may reflect steroid uptake by OATP (organic anion transporting polypeptide)/SLCO transporters. We evaluated SLCO gene expression in CRPC metastases and determined whether prostate cancer outcomes are associated with single nucleotide polymorphisms (SNP) in SLCO2B1 and SLCO1B3, transporters previously shown to mediate androgen uptake.
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Inhibition of angiopoietin-2 in LuCaP 23.1 prostate cancer tumors decreases tumor growth and viability.
Prostate
PUBLISHED: 06-29-2010
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Angiopoietin-2 is expressed in prostate cancer (PCa) bone, liver, and lymph node metastases, whereas, its competitor angiopoietin-1 has limited expression in these tissues. Therefore, we hypothesized that the inhibition of angiopoietin-2 activity in PCa will impede angiogenesis, tumor growth, and alter bone response in vivo.
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Expression of cancer/testis antigens in prostate cancer is associated with disease progression.
Prostate
PUBLISHED: 06-29-2010
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The cancer/testis antigens (CTAs) are a unique group of proteins normally expressed in germ cells but aberrantly expressed in several types of cancers including prostate cancer (PCa). However, their role in PCa has not been fully explored.
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Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant.
J. Clin. Invest.
PUBLISHED: 06-02-2010
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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.
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Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis and castration resistance.
Nat. Med.
PUBLISHED: 04-16-2010
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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.
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Osteoclast-derived matrix metalloproteinase-9 directly affects angiogenesis in the prostate tumor-bone microenvironment.
Mol. Cancer Res.
PUBLISHED: 03-23-2010
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In human prostate to bone metastases and in a novel rodent model that recapitulates prostate tumor-induced osteolytic and osteogenic responses, we found that osteoclasts are a major source of the proteinase, matrix metalloproteinase (MMP)-9. Because MMPs are important mediators of tumor-host communication, we tested the effect of host-derived MMP-9 on prostate tumor progression in the bone. To this end, immunocompromised mice that were wild-type or null for MMP-9 received transplants of osteolytic/osteogenic-inducing prostate adenocarcinoma tumor tissue to the calvaria. Surprisingly, we found that that host MMP-9 significantly contributed to prostate tumor growth without affecting prostate tumor-induced osteolytic or osteogenic change as determined by microcomputed tomography, microsingle-photon emission computed tomography, and histomorphometry. Subsequent studies aimed at delineating the mechanism of MMP-9 action on tumor growth focused on angiogenesis because MMP-9 and osteoclasts have been implicated in this process. We observed (a) significantly fewer and smaller blood vessels in the MMP-9 null group by CD-31 immunohistochemistry; (b) MMP-9 null osteoclasts had significantly lower levels of bioavailable vascular endothelial growth factor-A(164); and (c) using an aorta sprouting assay, conditioned media derived from wild-type osteoclasts was significantly more angiogenic than conditioned media derived from MMP-9 null osteoclasts. In conclusion, these studies show that osteoclast-derived MMP-9 affects prostate tumor growth in the bone microenvironment by contributing to angiogenesis without altering prostate tumor-induced osteolytic or osteogenic changes.
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Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells.
Anticancer Drugs
PUBLISHED: 02-05-2010
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Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.
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Genetic alterations and changes in expression of histone demethylases in prostate cancer.
Prostate
PUBLISHED: 02-04-2010
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Histone demethylases LSD1, JHDM2A, and GASC1 have been suggested to function as androgen receptor co-activators, and to be involved in prostate cancer (PC) progression. We aim to identify genetic alterations and changes in expression of these genes in PC.
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Bone morphogenetic protein 7 is expressed in prostate cancer metastases and its effects on prostate tumor cells depend on cell phenotype and the tumor microenvironment.
Neoplasia
PUBLISHED: 02-04-2010
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Bone morphogenetic protein (BMP) signaling is important in prostate development and prostate cancer (PCa) progression. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in PCa. In our studies, we have focused on BMP-7 because it is involved in prostate morphogenesis, and its expression is regulated by androgens. The objective of our study was to determine BMP-7 expression in PCa metastases and investigate the effects of BMP-7 on PCa cells. Our results show that BMP-7 is expressed in metastatic PCa and its levels are increased in castration-resistant PCa versus androgen-dependent PCa, whereas the expression of BMP-7 is decreased in primary PCa versus normal prostate. Our in vitro results show that BMP-7 inhibits proliferation of androgen-sensitive LNCaP cells, stimulates androgen receptor signaling, increases the expression of differentiation-associated genes, and decreases the levels of some wingless-regulated transcripts. Interestingly, these effects were not detected in C4-2 castration-resistant PCa cells. In vivo expression of BMP-7 in castration-resistant C4-2 cells did not alter proliferation when these cells were grown subcutaneously, but their growth was inhibited in the bone environment. In summary, our results show that BMP-7 is expressed at the highest level in advanced castration-resistant PCa cells and that the inhibitory effects of BMP-7 are dependent on the differentiation status of PCa cells and the tumor microenvironment. Further studies are needed to identify changes in BMP-7 signaling that lead to the loss of its control of proliferation during PCa progression.
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Estradiol suppresses tissue androgens and prostate cancer growth in castration resistant prostate cancer.
BMC Cancer
PUBLISHED: 01-24-2010
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Estrogens suppress tumor growth in prostate cancer which progresses despite anorchid serum androgen levels, termed castration resistant prostate cancers (CRPC), although the mechanisms are unclear. We hypothesize that estrogen inhibits CRPC in anorchid animals by suppressing tumoral androgens, an effect independent of the estrogen receptor.
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Molecular assays for the detection of prostate tumor derived nucleic acids in peripheral blood.
Mol. Cancer
PUBLISHED: 01-15-2010
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Prostate cancer is the second leading cause of cancer mortality in American men. Although serum PSA testing is widely used for early detection, more specific prognostic tests are needed to guide treatment decisions. Recently, the enumeration of circulating prostate epithelial cells has been shown to correlate with disease recurrence and metastasis following definitive treatment. The purpose of our study was to investigate an immunomagnetic fractionation procedure to enrich circulating prostate tumor cells (CTCs) from peripheral blood specimens, and to apply amplified molecular assays for the detection of prostate-specific markers (PSA, PCA3 and TMPRSS2:ERG gene fusion mRNAs).
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Transforming Growth Factor-{beta}-Stimulated Clone-22 Is an Androgen-Regulated Gene That Enhances Apoptosis in Prostate Cancer following Insulin-Like Growth Factor-I Receptor Inhibition.
Clin. Cancer Res.
PUBLISHED: 12-08-2009
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PURPOSE: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor 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 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. RESULTS: In castrate mice, the tumors were delayed in G(2) with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G(1) or G(2) delay. Transforming growth factor-beta-stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. 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 showed an increase in apoptosis and a delay in G(1). When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. CONCLUSIONS: These data indicate that IGF-I receptor 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. (Clin Cancer Res 2009;15(24):7634-41).
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DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-10-2009
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In metastatic prostate cancer (PCa) cells, imbalance between cell survival and death signals such as constitutive activation of phosphatidylinositol 3-kinase (PI3K)-Akt and inactivation of apoptosis-stimulated kinase (ASK1)-JNK pathways is often detected. Here, we show that DAB2IP protein, often down-regulated in PCa, is a potent growth inhibitor by inducing G(0)/G(1) cell cycle arrest and is proapoptotic in response to stress. Gain of function study showed that DAB2IP can suppress the PI3K-Akt pathway and enhance ASK1 activation leading to cell apoptosis, whereas loss of DAB2IP expression resulted in PI3K-Akt activation and ASK1-JNK inactivation leading to accelerated PCa growth in vivo. Moreover, glandular epithelia from DAB2IP(-/-) animal exhibited hyperplasia and apoptotic defect. Structural functional analyses of DAB2IP protein indicate that both proline-rich (PR) and PERIOD-like (PER) domains, in addition to the critical role of C2 domain in ASK1 activity, are important for modulating PI3K-Akt activity. Thus, DAB2IP is a scaffold protein capable of bridging both survival and death signal molecules, which implies its role in maintaining cell homeostasis.
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Novel biomarkers for prostate cancer including noncoding transcripts.
Am. J. Pathol.
PUBLISHED: 11-05-2009
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Levels of 27 transcripts were investigated as potential novel markers for prostate cancer, including genes encoding plasma membrane proteins (ADAM2, ELOVL5, MARCKSL1, RAMP1, TMEM30A, and TMEM66); secreted proteins (SPON2, TMEM30A, TMEM66, and truncated TMEFF2 (called POP4)); intracellular proteins (CAMK2N1, DHCR24, GLO1, NGFRAP1, PGK1, PSMA7, SBDS, and YWHAQ); and noncoding transcripts (POP1 (100 kb) from mRNA AK000023), POP2 (4 kb from mRNA AL832227), POP3 (50 kb from EST CFI40309), POP5 (intron of NCAM2, accession DO668384), POP6 (intron of FHIT), POP7 (intron of TNFAIP8), POP8 (intron of EFNA5), POP9 (intron of DSTN), POP10 (intron of ADAM2, accession DO668396), POP11 (87kb from EST BG194644), and POP12 (intron of EST BQ226050)). Expression of POP3 was prostate specific, whereas ADAM2, POP1, POP4, POP10, ELOVL5, RAMP1, and SPON2 had limited tissue expression. ELOVL5, MARCKSL1, NGFRAP1, PGK1, POP2, POP5, POP8, PSMA7, RAMP1, and SPON2 were significantly differentially expressed between laser microdissected malignant versus benign clinical samples of prostate tissue. PGK1, POP2, and POP12 correlated to clinical parameters. Levels of CAMK2N1, GLO1, SDBS, and TMEM30A transcripts tended to be increased in primary prostate cancer from patients who later had biochemical failure. Expression of GLO1, DHCR24, NGFRAP1, KLK3, and RAMP1 were significantly decreased in metastatic castration-recurrent disease compared with androgen-dependent primary prostate cancer. These novel potential biomarkers may therefore be useful in the diagnosis/prognosis of prostate cancer.
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Induction of neuronal apoptosis inhibitory protein expression in response to androgen deprivation in prostate cancer.
Cancer Lett.
PUBLISHED: 10-15-2009
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A mechanism for survival of prostate cancer cells in an androgen-deprived environment remains elusive. Here, we find that expression of neuronal apoptosis inhibitory protein (NAIP) was significantly increased in vivo and in vitro in response to androgen deprivation therapy (ADT). Increased expression of NAIP corresponded to increased DNA-binding activity of NF-kappaB that physically associated to previously uncharacterized kappaB-like sites in the NAIP locus. Importantly, expression of NAIP was significantly increased (p=0.04) in clinical samples of prostate cancer from patients receiving ADT. Expression of NAIP may be associated with enhanced survival of prostate cancer in response to castration.
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Comparative analyses of chromosome alterations in soft-tissue metastases within and across patients with castration-resistant prostate cancer.
Cancer Res.
PUBLISHED: 09-22-2009
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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 comparative genomic hybridization (array CGH), gene expression arrays, and fluorescence in situ hybridization (FISH). Bycluster 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 overrepresentation 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.
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Nemo-like kinase induces apoptosis and inhibits androgen receptor signaling in prostate cancer cells.
Prostate
PUBLISHED: 06-11-2009
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The mitogen-activated protein kinases (MAPKs) regulate cell growth, differentiation, and stress responses, and many critical signaling pathways are subject to cross-regulation by MAPK signaling. Previous studies have yielded evidence of cross-talk between the MAPK pathways and androgen receptor (AR) signaling, which plays a critical role in growth control of both normal prostate and prostate cancer (PCa). Objective of this study was to evaluate the expression of MAPK-like protein nemo-like kinase (NLK) in PCa and its effects on AR-mediated transcription.
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HE3235 inhibits growth of castration-resistant prostate cancer.
Neoplasia
PUBLISHED: 06-05-2009
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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 approximately 89% and dihydrotestosterone by approximately 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.
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Prostate cancer-associated gene expression alterations determined from needle biopsies.
Clin. Cancer Res.
PUBLISHED: 04-14-2009
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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.
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Osteoblast-derived factors induce an expression signature that identifies prostate cancer metastasis and hormonal progression.
Cancer Res.
PUBLISHED: 04-07-2009
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Identification of gene expression signatures associated with metastases provides a tool to discern mechanisms and potential therapeutic targets and may lead toward a molecular classification system in pathology. Prostate cancer (CaP) frequently metastasizes to the bone to form osteoblastic lesions. Correlative clinical data and in vitro evidence have led to the hypothesis that osteoblast-derived factors promote hormonal progression of CaP cells. Here, the gene expression signature of CaP exposed to osteoblast-derived factors was identified. This signature included known androgen-regulated genes, oncogenes, tumor suppressors, and genes whose products are involved in apoptosis and cell cycle. A comparative functional genomic approach involved the application of this responsive gene expression signature to clinical samples of human CaP, melanomas, and oral cancers. Cluster analysis revealed that this gene expression signature had specificity for CaP and could resolve clinical specimens according to stage (benign, localized, and metastatic) and androgen sensitivity with an accuracy of 100% and 80%, respectively. Together, these results suggest that factors derived from osteoblasts induce a more advanced phenotype of CaP and promotes hormonal progression.
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In vitro binding evaluation of 177Lu-AMBA, a novel 177Lu-labeled GRP-R agonist for systemic radiotherapy in human tissues.
Clin. Exp. Metastasis
PUBLISHED: 03-13-2009
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Members of the gastrin-releasing peptide (GRP) family and its analogs bombesin (BBN) have been implicated in the biology of several human cancers including prostate, breast, colon and lung. To date, three mammalian GRP/BBN receptor subtypes have been cloned and characterized: the neuromedin B receptor (NMBR), the GRP receptor (GRPR) and the BBN-receptor subtype 3 (BB(3)). The fourth BBN receptor subtype, BB(4), has only been identified in amphibian and at present no mammalian equivalent of this receptor has been described. GRPR analogs have been used as carriers to deliver drugs, radionuclides and cytotoxins to target various cancer types that are GRPR positive. We investigated the in vitro binding properties of (177)Lu-AMBA, a novel radiolabelled BBN analog currently undergoing clinical trial as systemic radiotherapy for hormone refractory prostate cancer (HRPC) patients. Pharmacological analyses of the (177)Lu-AMBA was determined using in vitro binding studies using membrane target system containing specific receptor subtypes. We investigated the distribution of binding sites for (177)Lu-AMBA by receptor autoradiography on human neoplastic and non-neoplastic tissues. Pharmacological characterizations of (177)Lu-AMBA shows, high affinity towards NMB and GRP receptors, while little or no affinity towards BB(3) receptor. Among the 40 different types of non-neoplastic tissues tested seven of them showed limited but specific binding of (177)Lu-AMBA. Fourteen of 17 primary prostate cancers, six of 13 primary breast cancers expressed binding sites for (177)Lu-AMBA. Furthermore, no apparent differences in (177)Lu-AMBA-binding sites expression were observed between matched pairs (primary vs. secondary) of prostate and breast cancer tissues. These data represent the molecular basis for clinical applications of (177)Lu-AMBA for diagnosis and treatment of GRP-R and NMB-R positive tumors.
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