Showing papers by "Kenneth J. Pienta published in 2012"
TL;DR: The mutational landscape of a heavily treated metastatic cancer is described, novel mechanisms of AR signalling deregulated in prostate cancer are identified, and candidates for future study are prioritize.
Abstract: Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.
2,141 citations
TL;DR: The findings establish a comprehensive methylation signature that couples epigenomic and structural analyses, thereby offering insights into the genomic alterations in CRPC that are associated with a circumvention of hormonal therapy.
Abstract: Progression of primary prostate cancer to castration-resistant prostate cancer (CRPC) is associated with numerous genetic and epigenetic alterations that are thought to promote survival at metastatic sites. In this study, we investigated gene copy number and CpG methylation status in CRPC to gain insight into specific pathophysiologic pathways that are active in this advanced form of prostate cancer. Our analysis defined and validated 495 genes exhibiting significant differences in CRPC in gene copy number, including gains in androgen receptor (AR) and losses of PTEN and retinoblastoma 1 (RB1). Significant copy number differences existed between tumors with or without AR gene amplification, including a common loss of AR repressors in AR-unamplified tumors. Simultaneous gene methylation and allelic deletion occurred frequently in RB1 and HSD17B2, the latter of which is involved in testosterone metabolism. Lastly, genomic DNA from most CRPC was hypermethylated compared with benign prostate tissue. Our findings establish a comprehensive methylation signature that couples epigenomic and structural analyses, thereby offering insights into the genomic alterations in CRPC that are associated with a circumvention of hormonal therapy. Genes identified in this integrated genomic study point to new drug targets in CRPC, an incurable disease state which remains the chief therapeutic challenge.
129 citations
TL;DR: The robustness of the 384 hanging drop array plate performance is characterized in fluorescence‐ and colorimetric‐based assays through Z‐factor calculations and the versatility of the plate is demonstrated.
Abstract: We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through Z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types.
129 citations
TL;DR: With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis.
Abstract: Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis.
109 citations
TL;DR: It is found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP and that GAS6 regulates the expression of Axl.
Abstract: The receptor tyrosine kinase Axl is over-expressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our lab indicates that Axl and its ligand GAS6 may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer (PCa) cell lines PC3 and DU145 and has negligible levels of expression in a non-metastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial to mesenchymal transition in PCa cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were down-regulated. Moreover, CoCl2, a hypoxia mimicking agent, prevented GAS6 mediated down-regulation of Axl in these cell lines. Immunochemical staining of human PCa tissue microarrays demonstrated that Axl, GAS6 and Hif1-α (indicator of hypoxia) were all co-expressed in PCa and in bone metastases, compared to normal tissues. Together, our studies indicate that Axl plays a crucial role in PCa metastasis, and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression maintained leading to enhanced signaling.
99 citations
TL;DR: Cilengitide was well tolerated but had no detectable clinical activity, and CTCs are of questionable utility in non-metastatic prostate cancer.
Abstract: Background: Integrins mediate invasion and angiogenesis in prostate cancer bone metastases. We conducted a phase II study of Cilengitide, a selective antagonist of αvβ3 and αvβ5 integrins, in non-metastatic castration resistant prostate cancer with rising PSA. Methods: Patients were observed for 4 weeks with PSA monitoring, and then treated with 2,000 mg IV of cilengitide twice weekly until toxicity/progression. PSA, circulating tumor cells (CTCs) and circulating endothelial cells (CECs) were monitored each cycle with imaging performed every three cycles. Primary end point was PSA decline by ≥50%. Secondary endpoints were safety, PSA slope, time to progression (TTP), overall survival (OS), CTCs, CECs and gene expression. Results: 16 pts were enrolled; 13 were eligible with median age 65.5 years, baseline PSA 8.4 ng/mL and median Gleason sum 7. Median of three cycles was administered. Treatment was well tolerated with two grade three toxicities and no grade four toxicities. There were no PSA responses; 11 patients progressed by PSA after three cycles. Median TTP was 1.8 months and median OS has not been reached. Median pre- and on-treatment PSA slopes were 1.1 and 1.8 ng/mL/month. Baseline CTCs were detected in 1/9 patients. CTC increased (0 to 1; 2 pts), remained at 0 (2 pts) or decreased (23 to 0; 1 patient) at progression. Baseline median CEC was 26 (0–61) and at progression, 47 (15–148). Low cell counts precluded gene expression studies. Conclusions: Cilengitide was well tolerated but had no detectable clinical activity. CTCs are of questionable utility in non-metastatic prostate cancer.
80 citations
TL;DR: It is concluded that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.
78 citations
TL;DR: It is demonstrated that dysregulated expression of CtBP1 plays an important role in prostate cancer progression and may serve as a viable therapeutic target.
63 citations
TL;DR: Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTβRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction.
Abstract: We have examined whether Ad.sTβRFc and TAd.sTβRFc, two oncolytic viruses expressing soluble transforming growth factor-β receptor II fused with human Fc (sTGFβRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTβRFc and TAd.sTβRFc produced sTGFβRIIFc and viral replication; sTGFβRIIFc caused inhibition of TGF-β-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTβRFc, an E1– adenovirus, produced sTGFβRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×1010 viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTβRFc, TAd.sTβRFc, a...
62 citations
TL;DR: It is suggested that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines that can be reversed by inhibition of CCL2.
Abstract: A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.
62 citations
TL;DR: Some of the evidence and mechanisms by which DTCs act as molecular parasites of the HSC niche are described, which may provide new targets for niche-directed therapy, as well as insight into the perplexing clinical manifestations of metastatic PCa disease.
Abstract: The hematopoietic stem cell (HSC) niche in the bone marrow has been studied extensively over the past few decades, yet the bone marrow microenvironment that supports the growth of metastatic prostate cancer (PCa) has only been recently considered to be a specialized 'niche' as well. New evidence supports the fact that disseminated tumor cells (DTCs) of PCa actually target the HSC niche, displace the occupant HSCs and take up residence in the pre-existing niche space. This review describes some of the evidence and mechanisms by which DTCs act as molecular parasites of the HSC niche. Furthermore, the interactions between DTCs, HSCs and the niche may provide new targets for niche-directed therapy, as well as insight into the perplexing clinical manifestations of metastatic PCa disease.
TL;DR: Data from an in vivo murine model of human PCa cell metastasis to bone suggest that the differences in tumor prevalence after intravascular inoculation are a useful model to study the molecular basis of tumor dormancy and suggest that therapeutic manipulation of GAS6 levels may prove useful as a therapy for metastatic disease.
TL;DR: This work has shown that successful treatment of cancer with a single agent is rarely enough to cure a patient without strategically modifying the support systems conducive to survival of cancer.
Abstract: Ecosystems are interactive systems involving communities of species and their abiotic environment. Tumors are ecosystems in which cancer cells act as invasive species interacting with native host cell species in an established microenvironment within the larger host biosphere. At its heart, to study ecology is to study interconnectedness. In ecologic science, an ecologic network is a representation of the biotic interactions in an ecosystem in which species (nodes) are connected by pairwise interactions (links). Ecologic networks and signaling network models have been used to describe and compare the structures of ecosystems. It has been shown that disruption of ecologic networks through the loss of species or disruption of interactions between them can lead to the destruction of the ecosystem. Often, the destruction of a single node or link is not enough to disrupt the entire ecosystem. The more complex the network and its interactions, the more difficult it is to cause the extinction of a species, especially without leveraging other aspects of the ecosystem. Similarly, successful treatment of cancer with a single agent is rarely enough to cure a patient without strategically modifying the support systems conducive to survival of cancer. Cancer cells and the ecologic systems they reside in can be viewed as a series of nested networks. The most effective new paradigms for treatment will be developed through application of scaled network disruption. Clin Cancer Res; 18(10); 2801–8. ©2012 AACR.
TL;DR: HSC/HPCs are identified for the first time as novel targets for future therapy involved in the bone abnormalities of prostate Cancer Res; 10(3); 282–92.
Abstract: Prostate cancer (PCa) metastases and hematopoietic stem cells (HSCs) frequently home to the bone marrow where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMPs)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. Because it is not known, we examined if metastatic PCa cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPCs). HSC/HPCs isolated from mice bearing non-metastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line which produces mixed osteoblastic and osteolytic lesions in bone, HPCs but not HSCs were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts through an interleukin-6 (IL-6) mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of PCa.
TL;DR: A subpopulation of men that appear cured of prostate cancer (PCa) develop bone metastases many years after prostatectomy, indicating that PCa cells were present outside of the prostate at the time of prostatectom and remained dormant.
Abstract: A subpopulation of men that appear cured of prostate cancer (PCa) develop bone metastases many years after prostatectomy. This observation indicates that PCa cells were present outside of the prostate at the time of prostatectomy and remained dormant. Several lines of evidence indicate that there are disseminated tumor cells (DTCs) in the bone marrow at the time of prostatectomy. DTCs parasitize the bone microenvironment, where they derive support and impact the microenvironment itself. These DTCs appear to be a heterogeneous population of PCa cells; however, some of them appear to have some aspects of a cancer stem cell (CSC) phenotype as they can develop into clinically detectable metastases. The concept of CSC is controversial; however, several markers of CSC have been identified for PCa, which may represent cells of either basal or luminal origin. These DTCs have now been shown to compete for the hematopoietic stem cell niche in bone, where they may be placed in a dormant state. Interaction with a variety of host factors, including cytokine and cells, may impact the metastatic development and progression, including the dormant state. For example, myeloid cells have been shown to impact both the premetastatic niche and established tumors. Understanding the concepts of how PCa successfully parasitizes the bone microenvironment is paramount toward identifying therapeutic candidates to prevent or diminish PCa bone metastases.
TL;DR: This data indicates that fusions between the 5′‐untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases and drove tumor progression.
Abstract: BACKGROUND. Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5 0 -untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS. Here, we evaluated the expression levels of 5a-reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a 5a-reductase inhibitor, in TMPRSS2–ERG fusion-positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2–ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti-androgen in VCaP cell xenografts tumor model. RESULTS. Our data showed that 5a-reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre-treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti-androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model. CONCLUSIONS. Our findings suggest that dutasteride can inhibit ERG fusion-positive cell growth and in combination with anti-androgen, significantly reduce the tumor burden. Our study suggests that anti-androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS-positive prostate cancer.
TL;DR: It is reported that CCL2 and interleukin-6 (IL-6) serve as key molecules in EMT-mediated chemo-resistance in PCa cells using previously established paclitaxel-resistant cells, DU145-TxR and PC-3-txR, and synergistic effects were observed by adding both reagents.
Abstract: Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death in western countries. In China, the incidence has increased, unexpectedly and sharply, for the past 20 years. Chemo-resistance, in various types of tumor including PCa, is one of the critical problems in cancer therapy. In PCa, for example, the resistance to taxanes (DTX) accounts for tumor relapse and progression resulting in skeletal metastasis and high mortality. Epithelial-mesenchymal transition (EMT) has been suggested as a key process that dominates chemo-resistance through unknown mechanisms. In the current study, we reported that CCL2 and interleukin-6 (IL-6) serve as key molecules in EMT-mediated chemo-resistance in PCa cells using previously established paclitaxel-resistant cells, DU145-TxR and PC-3-TxR. Parental DU145 and PC-3 cells were used as controls. We observed that DU145-TxR and PC-3-TxR cells produced significantly higher amount of CCL2 and IL-6 compared to their parental cells, determined by cytokine array. In addition, DU145-TxR and PC-3-TxR cells demonstrated EMT phenotypes including hall maker changes in E-cadherin and Vimentin expression and transcriptional factor (Snail, ZEB1, and Slug) alterations. These changes were reversed partially by adding neutralizing antibodies against either CCL2 or interleukin-6. Interestingly, synergistic effects were observed by adding both reagents. Finally DU145-TxR, PC-3-TxR, or their parental cells were implanted into SCID mice and the mice were given DTX (10mg/kg, biweekly) and tumor growth was monitored for 4 weeks. Both DU145-TxR and PC-3-TxR tumors grew faster than the parental tumors. Tumor tissues were collected and total RNAs were isolated. Levels of CCL2 expression in these tumors were significantly higher in DU145-TxR and PC-3-TxR tumors. IHC analysis confirmed corresponding staining of EMT markers such as E-cadherin and Vimentin expression. Together we concluded that CCL2 and IL-6 regulates EMT-mediated chemo-resistance in prostate cancer. These results are of high impact of human prostate cancer chemotherapy. Supported by U.S. Department of Defense PC061231 (J. Zhang), National Natural Sciences Foundation Key Project (81130046, J. Zhang) and National Natural Science Foundation Project (81171993, Y. Lu). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 841. doi:1538-7445.AM2012-841
01 Jan 2012
TL;DR: The overexpression and mislocalization ofCtBP1 is observed in metastatic prostate cancer and the functional significance of CtBP1 in prostate cancer progression is demonstrated.
Abstract: Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are frequently altered in cancer. C-terminal binding protein 1 (CtBP1), a transcriptional corepressor that regulates the expression of tumor suppressors and genes involved in cell death, is known to play a role in multiple cancers. In this study, we observed the overexpression and mislocalization of CtBP1 in metastatic prostate cancer and demonstrated the functional significance of CtBP1 in prostate cancer progression. Transient and stable knockdown of CtBP1 in prostate cancer cells inhibited their proliferationand invasion. Expressionprofilingstudies ofprostatecancercelllinesrevealed thatmultiple
01 Jan 2012
TL;DR: It is found that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl, and in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.
Abstract: ThereceptortyrosinekinaseAxlisoverexpressedinavarietyofcancersandisknowntoplayaroleinproliferation andinvasion.PreviousdatafromourlaboratoryindicatethatAxlanditsligandgrowtharrest–specific6(GAS6)may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we foundthatAxlishighlyexpressedinmetastaticprostatecancercelllinesPC3andDU145andhasnegligiblelevelsof expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial–mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migrationand invasion.Interestingly,whenPC3andDU145cellsweretreatedwithGAS6,Axlproteinlevelswere downregulated.Moreover,CoCl2,ahypoxiamimickingagent,preventedGAS6-mediateddownregulationofAxlin thesecelllines.ImmunochemicalstainingofhumanprostatecancertissuemicroarraysshowedthatAxl,GAS6,and hypoxia-inducible factor-1a (Hif-1a; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling. Mol Cancer Res; 10(6); 703–12. � 2012 AACR.