Publications by authors named "Donna M Peehl"

114 Publications

MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1.

Mol Cancer Res 2021 Apr 12. Epub 2021 Apr 12.

Pharmacology, University of Colorado Anschutz Medical Campus

Prostate cancer (PCa) genomic subtypes that stratify aggressive disease and inform treatment decisions at the primary stage are currently limited. Previously, we functionally validated an aggressive subtype present in 15% of PCa characterized by dual deletion of MAP3K7 and CHD1. Recent studies in the field have focused on deletion of CHD1 and its role in androgen receptor (AR) chromatin distribution and resistance to AR-targeted therapy, however, CHD1 is rarely lost without co-deletion of MAP3K7. Here we show that in the clinically relevant context of co-loss of MAP3K7 and CHD1 there are significant, collective changes to aspects of AR signaling. While CHD1 loss mainly impacts the expansion of the AR cistrome, loss of MAP3K7 drives increased AR target gene expression. PCa cell line models engineered to co-suppress MAP3K7 and CHD1 also demonstrated increased AR-v7 expression and resistance to the AR-targeting drug enzalutamide. Furthermore, we determined that low protein expression of both genes is significantly associated with biochemical recurrence (BCR) in a clinical cohort of radical prostatectomy specimens. Low MAP3K7 expression, however, was the strongest independent predictor for risk of BCR over all other tested clinicopathologic factors including CHD1 expression. Collectively, these findings illustrate the importance of MAP3K7 loss in a molecular subtype of PCa that poses challenges to conventional therapeutic approaches. Implications: These findings strongly implicate MAP3K7 loss as a biomarker for aggressive prostate cancer with significant risk for recurrence that poses challenges for conventional androgen receptor-targeted therapies.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0913DOI Listing
April 2021

Resistance to Androgen Deprivation Leads to Altered Metabolism in Human and Murine Prostate Cancer Cell and Tumor Models.

Metabolites 2021 Feb 26;11(3). Epub 2021 Feb 26.

Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA 94143, USA.

Currently, no clinical methods reliably predict the development of castration-resistant prostate cancer (CRPC) that occurs almost universally in men undergoing androgen deprivation therapy. Hyperpolarized (HP) C magnetic resonance imaging (MRI) could potentially detect the incipient emergence of CRPC based on early metabolic changes. To characterize metabolic shifts occurring upon the transition from androgen-dependent to castration-resistant prostate cancer (PCa), the metabolism of [U-C]glucose and [U-C]glutamine was analyzed by nuclear magnetic resonance spectroscopy. Comparison of steady-state metabolite concentrations and fractional enrichment in androgen-dependent LNCaP cells and transgenic adenocarcinoma of the murine prostate (TRAMP) murine tumors versus castration-resistant PC-3 cells and treatment-driven CRPC TRAMP tumors demonstrated that CRPC was associated with upregulation of glycolysis, tricarboxylic acid metabolism of pyruvate; and glutamine, glutaminolysis, and glutathione synthesis. These findings were supported by C isotopomer modeling showing increased flux through pyruvate dehydrogenase (PDH) and anaplerosis; enzymatic assays showing increased lactate dehydrogenase, PDH and glutaminase activity; and oxygen consumption measurements demonstrating increased dependence on anaplerotic fuel sources for mitochondrial respiration in CRPC. Consistent with ex vivo metabolomic studies, HP [1-C]pyruvate distinguished androgen-dependent PCa from CRPC in cell and tumor models based on significantly increased HP [1-C]lactate.
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http://dx.doi.org/10.3390/metabo11030139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996870PMC
February 2021

The mA RNA demethylase FTO is a HIF-independent synthetic lethal partner with the VHL tumor suppressor.

Proc Natl Acad Sci U S A 2020 09 19;117(35):21441-21449. Epub 2020 Aug 19.

Department of Radiation Oncology, Stanford University, Stanford, CA 94305;

Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the mA RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide mA-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.
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http://dx.doi.org/10.1073/pnas.2000516117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474618PMC
September 2020

Elevated Tumor Lactate and Efflux in High-grade Prostate Cancer demonstrated by Hyperpolarized C Magnetic Resonance Spectroscopy of Prostate Tissue Slice Cultures.

Cancers (Basel) 2020 Feb 26;12(3). Epub 2020 Feb 26.

Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA 94158, USA.

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-C]pyruvate to [1-C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. P spectra demonstrated good viability and dynamic HP C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-C]pyruvate MR.
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http://dx.doi.org/10.3390/cancers12030537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139946PMC
February 2020

Trop2 is a driver of metastatic prostate cancer with neuroendocrine phenotype via PARP1.

Proc Natl Acad Sci U S A 2020 01 13;117(4):2032-2042. Epub 2020 Jan 13.

Department of Radiology, Stanford University, Stanford, CA 94304;

Resistance to androgen deprivation therapy, or castration-resistant prostate cancer (CRPC), is often accompanied by metastasis and is currently the ultimate cause of prostate cancer-associated deaths in men. Recently, secondary hormonal therapies have led to an increase of neuroendocrine prostate cancer (NEPC), a highly aggressive variant of CRPC. Here, we identify that high levels of cell surface receptor Trop2 are predictive of recurrence of localized prostate cancer. Moreover, Trop2 is significantly elevated in CRPC and NEPC, drives prostate cancer growth, and induces neuroendocrine phenotype. Overexpression of Trop2 induces tumor growth and metastasis while loss of Trop2 suppresses these abilities in vivo. Trop2-driven NEPC displays a significant up-regulation of PARP1, and PARP inhibitors significantly delay tumor growth and metastatic colonization and reverse neuroendocrine features in Trop2-driven NEPC. Our findings establish Trop2 as a driver and therapeutic target for metastatic prostate cancer with neuroendocrine phenotype and suggest that high Trop2 levels could identify cancers that are sensitive to Trop2-targeting therapies and PARP1 inhibition.
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http://dx.doi.org/10.1073/pnas.1905384117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994991PMC
January 2020

miR-22 Regulates Invasion, Gene Expression and Predicts Overall Survival in Patients with Clear Cell Renal Cell Carcinoma.

Kidney Cancer 2019 Aug 7;3(2):119-132. Epub 2019 Aug 7.

Department of Urology, School of Medicine, Stanford University, Stanford, California, USA.

Background: Clear cell renal cell carcinoma (ccRCC) is molecularly diverse and distinct molecular subtypes show different clinical outcomes. MicroRNAs (miRNAs) are essential components of gene regulatory networks and play a crucial role in progression of many cancer types including ccRCC.

Objective: Identify prognostic miRNAs and determine the role of miR-22 in ccRCC.

Methods: Hierarchical clustering was done in R using gene expression profiles of over 450 ccRCC cases in The Cancer Genome Atlas (TCGA). Kaplan-Meier analysis was performed to identify prognostic miRNAs in the TCGA dataset. RNA-Seq was performed to identify miR-22 target genes in primary ccRCC cells and Matrigel invasion assay was performed to assess the effects of miR-22 overexpression on cell invasion.

Results: Hierarchical clustering analysis using 2,621 prognostic genes previously identified by our group demonstrated that ccRCC patients with longer overall survival expressed lower levels of genes promoting proliferation or immune responses, while better maintaining gene expression associated with cortical differentiation and cell adhesion. Targets of 26 miRNAs were significantly enriched in the 2,621 prognostic genes and these miRNAs were prognostic by themselves. MiR-22 was associated with poor overall survival in the TCGA dataset. Overexpression of miR-22 promoted invasion of primary ccRCC cells and modulated transcriptional programs implicated in cancer progression including DNA repair, cell proliferation and invasion.

Conclusions: Our results suggest that ccRCCs with differential clinical outcomes have distinct transcriptomes for which miRNAs could serve as master regulators. MiR-22, as a master regulator, promotes ccRCC progression at least in part by enhancing cell invasion.
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http://dx.doi.org/10.3233/KCA-190051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839454PMC
August 2019

S100A10 Is a Critical Mediator of GAS6/AXL-Induced Angiogenesis in Renal Cell Carcinoma.

Cancer Res 2019 11 4;79(22):5758-5768. Epub 2019 Oct 4.

Department of Radiation Oncology, Stanford University, Palo Alto, California.

Angiogenesis is a hallmark of cancer that promotes tumor progression and metastasis. However, antiangiogenic agents have limited efficacy in cancer therapy due to the development of resistance. In clear cell renal cell carcinoma (ccRCC), AXL expression is associated with antiangiogenic resistance and poor survival. Here, we establish a role for GAS6/AXL signaling in promoting the angiogenic potential of ccRCC cells through the regulation of the plasminogen receptor S100A10. Genetic and therapeutic inhibition of AXL signaling in ccRCC tumor xenografts reduced tumor vessel density and growth under the renal capsule. GAS6/AXL signaling activated the expression of S100A10 through SRC to promote plasmin production, endothelial cell invasion, and angiogenesis. Importantly, treatment with the small molecule AXL inhibitor cabozantinib or an ultra-high affinity soluble AXL Fc fusion decoy receptor (sAXL) reduced the growth of a pazopanib-resistant ccRCC patient-derived xenograft. Moreover, the combination of sAXL synergized with pazopanib and axitinib to reduce ccRCC patient-derived xenograft growth and vessel density. These findings highlight a role for AXL/S100A10 signaling in mediating the angiogenic potential of ccRCC cells and support the combination of AXL inhibitors with antiangiogenic agents for advanced ccRCC. SIGNIFICANCE: These findings show that angiogenesis in renal cell carcinoma (RCC) is regulated through AXL/S100A10 signaling and support the combination of AXL inhibitors with antiangiogenic agents for the treatment of RCC.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015045PMC
November 2019

NMR quantification of lactate production and efflux and glutamate fractional enrichment in living human prostate biopsies cultured with [1,6- C ]glucose.

Magn Reson Med 2019 08 28;82(2):566-576. Epub 2019 Mar 28.

Department of Radiology and Biomedical Imaging, University of California, San Francisco, California.

Purpose: Image-guided prostate biopsies are routinely acquired in the diagnosis and treatment monitoring of prostate cancer, yielding useful tissue for identifying metabolic biomarkers and therapeutic targets. We developed an optimized biopsy tissue culture protocol in combination with [1,6- C ]glucose labeling and quantitative high-resolution NMR to measure glycolysis and tricarboxcylic acid (TCA) cycle activity in freshly acquired living human prostate biopsies.

Methods: We acquired 34 MRI-ultrasound fusion-guided prostate biopsies in vials on ice from 22 previously untreated patients. Within 15 min, biopsies were transferred to rotary tissue culture in 37°C prostate medium containing [1,6- C ]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative H high resolution magic angle spinning Carr-Purcell-Meiboom-Gill (CPMG) spectroscopy at 1°C with and without C decoupling. Lactate effluxed from the biopsy tissue was quantified in the culture medium using quantitative solution-state high-resolution NMR.

Results: Lactate concentration in low-grade cancer (1.15 ± 0.78 nmol/mg) and benign (0.74 ± 0.15 nmol/mg) biopsies agreed with prior published measurements of snap-frozen biopsies. There was substantial fractional enrichment of [3- C]lactate (≈70%) and [4- C]glutamate (≈24%) in both low-grade cancer and benign biopsies. Although a significant difference in tissue [3- C]lactate fractional enrichment was not observed, lactate efflux was significantly higher (P < 0.05) in low-grade cancer biopsies (0.55 ± 0.14 nmol/min/mg) versus benign biopsies (0.31 ± 0.04 nmol/min/mg).

Conclusion: A protocol was developed for quantification of lactate production-efflux and TCA cycle activity in single living human prostate biopsies, allowing metabolic labeling on a wide spectrum of human tissues (e.g., metastatic, post-non-surgical therapy) from patients not receiving surgery.
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http://dx.doi.org/10.1002/mrm.27739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150535PMC
August 2019

A spliced form of CD44 expresses the unique glycan that is recognized by the prostate cancer specific antibody F77.

Oncotarget 2018 Jan 16;9(3):3631-3640. Epub 2017 Dec 16.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Prostate cancer is the most common cancer occurring in men in the United States. The monoclonal antibody F77 that was originally developed in our laboratory recognizes mainly glycolipids as well as O-linked glycosylation on proteins in prostate cancer cells. We have identified a spliced form of glycoprotein CD44 as one critical protein expressing the F77 antigen. The F77-specific glycosylation occurs on multiple potential glycosylation sites on the CD44 protein encoded by the fourteenth exon. CD44 is a tumor stem cell marker and is known to induce tumor stemness and metastasis. Knockdown of CD44 or FUT1 genes dramatically reduced F77-induced apoptosis in prostate cancer cell lines. We developed an ELISA using both a CD44 antibody and F77 to identify the special form of glycosylated CD44 from prostate cancer cells as well as from serum samples of prostate cancer patients. These results reveal a CD44-dependent mechanism for F77 to induce tumor cell apoptosis, and a new strategy for the detection of glycosylated CD44 proteins secreted by prostate cancer cells.
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http://dx.doi.org/10.18632/oncotarget.23341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790488PMC
January 2018

Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics.

Angew Chem Int Ed Engl 2017 07 26;56(31):8992-8997. Epub 2017 Jun 26.

Department of Chemistry, Stanford University, Stanford, CA, 94305-4401, USA.

Sialylated glycans are found at elevated levels in many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to the cancer cell-surface sialylation are not well characterized, specifically in bona fide human disease tissue. Metabolic and bioorthogonal labeling methods have previously enabled the enrichment and identification of sialoglycoproteins from cultured cells and model organisms. Herein, we report the first application of this glycoproteomic platform to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of the azide-functionalized sialic acid biosynthetic precursor Ac ManNAz. The compound was metabolized to the azidosialic acid and incorporated into cell surface and secreted sialoglycoproteins. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance.
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http://dx.doi.org/10.1002/anie.201701424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675001PMC
July 2017

The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry.

Clin Epigenetics 2017 19;9. Epub 2017 Jan 19.

Department of Urology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305 USA.

Background: RRx-001, a dinitroazetidine derivative, is a novel anticancer agent currently in phase II clinical trials. It mediates immunomodulatory effects either directly through polarization of tumor associated macrophages or indirectly through vascular normalization and increased T-lymphocyte infiltration. With multiple additional mechanisms of action including upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis and epigenetic modulation, RRx-001 is being studied as a radio- and chemo-sensitizer to resensitize tumors to prior therapy and to prime tumors to respond to radiation, chemotherapy and immunotherapy in combination therapy studies. Here, we identified another mechanism, viral mimicry, which refers to the "unsilencing" of epigenetically repressed viral genes present in the tumor that provokes an immune response and may contribute to the anticancer activity of RRx-001.

Results: RRx-001 inhibited the growth of colon cancer cells (HCT 116) and decreased levels of the DNA methyltransferases DNMT1 and DNMT3a in a time and dose-dependent manner. Treatment of HCT 116 cells with 0.5 μM RRx-001 for 24 h significantly increased transcripts of interferon (IFN)-responsive genes and this induction was sustained for up to 4 weeks after transient exposure to RRx-001. ELISA assays showed that RRx-001 increased secretion of type I and III IFNs by HCT 116 cells, and these IFNs were confirmed to be bioactive. Transcription of endogenous retrovirus ERV-Fc2 and LTRs from the ERV-L family (MLT2B4 and MLT1C49) was induced by RRx-001. The induction of ERV-Fc2-env was through demethylation of ERV-Fc2 LTR as determined by methylation-specific polymerase chain reaction and combined bisulfite restriction analysis. Immunofluorescence staining with J2 antibody confirmed induction of double-stranded RNA.

Conclusions: Transient exposure of HCT 116 cells to low-dose RRx-001 induced transcription of silenced retroviral genes present in the cancer cell DNA with subsequent synthesis of IFN in response to this "pseudo-pathogenic" stimulus, mimicking an antiviral defense. RRx-001-mediated IFN induction may have the potential to improve the efficacy of immunotherapies as well as radiotherapy, standard chemotherapies and molecularly targeted agents when used in combination. The striking safety profile of RRx-001 in comparison to other more toxic epigenetic and immunomodulatory agents such as azacitidine makes it a leading candidate for such clinical applications.
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http://dx.doi.org/10.1186/s13148-017-0312-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270305PMC
July 2017

Cabozantinib inhibits tumor growth and metastasis of a patient-derived xenograft model of papillary renal cell carcinoma with MET mutation.

Cancer Biol Ther 2017 Nov 11;18(11):863-871. Epub 2016 Aug 11.

a Department of Urology , Stanford University School of Medicine , Stanford , CA , USA.

MET plays an important role in the development and progression of papillary renal cell carcinoma (pRCC). Evaluation of efficacy of MET inhibitors against pRCC has been hampered by limited preclinical models depicting MET abnormalities. We established a new patient-derived xenograft (PDX) model of pRCC carrying an activating mutation of MET and tested the ability of cabozantinib, an inhibitor of receptor tyrosine kinases including MET, to inhibit tumor growth and metastasis. Precision-cut, thin tissue slices from a pRCC specimen obtained by nephrectomy were implanted under the renal capsule of RAG2γC mice to establish first generation TSG-RCC-030. Histologic and genetic fidelity and metastatic potential of this model were characterized by immunohistochemistry, direct DNA sequencing and quantitative polymerase chain reaction (qPCR). The effect of cabozantinib on tumor growth and metastasis was evaluated. Whether measurements of circulating tumor DNA (ctDNA) by allele-specific qPCR could be used as a biomarker of tumor growth and response to therapy was determined. Subrenal and subcutaneous tumor grafts showed high take rates and metastasized to the lung. Both primary tumors and metastases expressed typical markers of pRCC and carried the same activating MET mutation as the parental tumor. Cabozantinib treatment caused striking tumor regression and inhibited lung metastasis in TSG-RCC-030. Plasma ctDNA levels correlated with tumor volume in control mice and changed in response to cabozantinib treatment. TSG-RCC-030 provides a realistic preclinical model to better understand the development and progression of pRCC with MET mutation and accelerate the development of new therapies for pRCC.
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http://dx.doi.org/10.1080/15384047.2016.1219816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710673PMC
November 2017

Endoscopic detection of cancer with lensless radioluminescence imaging and machine vision.

Sci Rep 2016 08 1;6:30737. Epub 2016 Aug 1.

Medical Physics, Stanford University School of Medicine, Stanford, CA 94305, USA.

Complete removal of residual tumor tissue during surgical resection improves patient outcomes. However, it is often difficult for surgeons to delineate the tumor beyond its visible boundary. This has led to the development of intraoperative detectors that can image radiotracers accumulated within tumors, thus facilitating the removal of residual tumor tissue during surgical procedures. We introduce a beta imaging system that converts the beta radiation from the radiotracer into photons close to the decay origin through a CdWO4 scintillator and does not use any optical elements. The signal is relayed onto an EMCCD chip through a wound imaging fiber. The sensitivity of the device allows imaging of activity down to 100 nCi and the system has a resolution of at least 500 μm with a field of view of 4.80 × 6.51 mm. Advances in handheld beta cameras have focused on hardware improvements, but we apply machine vision to the recorded images to extract more information. We automatically classify sample regions in human renal cancer tissue ex-vivo into tumor or benign tissue based on image features. Machine vision boosts the ability of our system to distinguish tumor from healthy tissue by a factor of 9 ± 3 and can be applied to other beta imaging probes.
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http://dx.doi.org/10.1038/srep30737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967900PMC
August 2016

Comprehensive Drug Testing of Patient-derived Conditionally Reprogrammed Cells from Castration-resistant Prostate Cancer.

Eur Urol 2017 03 6;71(3):319-327. Epub 2016 May 6.

Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland. Electronic address:

Background: Technology development to enable the culture of human prostate cancer (PCa) progenitor cells is required for the identification of new, potentially curative therapies for PCa.

Objective: We established and characterized patient-derived conditionally reprogrammed cells (CRCs) to assess their biological properties and to apply these to test the efficacies of drugs.

Design, Setting, And Participants: CRCs were established from seven patient samples with disease ranging from primary PCa to advanced castration-resistant PCa (CRPC). The CRCs were characterized by genomic, transcriptomic, protein expression, and drug profiling.

Outcome Measurements And Statistical Analysis: The phenotypic quantification of the CRCs was done based on immunostaining followed by image analysis with Advanced Cell Classifier using Random Forest supervised machine learning. Copy number aberrations (CNAs) were called from whole-exome sequencing and transcriptomics using in-house pipelines. Dose-response measurements were used to generate multiparameter drug sensitivity scores using R-statistical language.

Results And Limitations: We generated six benign CRC cultures which all had an androgen receptor-negative, basal/transit-amplifying phenotype with few CNAs. In three-dimensional cell culture, these cells could re-express the androgen receptor. The CRCs from a CRPC patient (HUB.5) displayed multiple CNAs, many of which were shared with the parental tumor. We carried out high-throughput drug-response studies with 306 emerging and clinical cancer drugs. Using the benign CRCs as controls, we identified the Bcl-2 family inhibitor navitoclax as the most potent cancer-specific drug for the CRCs from a CRPC patient. Other drug efficacies included taxanes, mepacrine, and retinoids.

Conclusions: Comprehensive cancer pharmacopeia-wide drug testing of CRCs from a CRPC patient highlighted both known and novel drug sensitivities in PCa, including navitoclax, which is currently being tested in clinical trials of CRPC.

Patient Summary: We describe an approach to generate patient-derived cancer cells from advanced prostate cancer and apply such cells to discover drugs that could be applied in clinical trials for castration-resistant prostate cancer.
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http://dx.doi.org/10.1016/j.eururo.2016.04.019DOI Listing
March 2017

Spheroid culture of LuCaP 136 patient-derived xenograft enables versatile preclinical models of prostate cancer.

Clin Exp Metastasis 2016 Apr 12;33(4):325-37. Epub 2016 Feb 12.

Department of Urology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

LuCaP serially transplantable patient-derived xenografts (PDXs) are valuable preclinical models of locally advanced or metastatic prostate cancer. Using spheroid culture methodology, we recently established cell lines from several LuCaP PDXs. Here, we characterized in depth the features of xenografts derived from LuCaP 136 spheroid cultures and found faithful retention of the phenotype of the original PDX. In vitro culture enabled luciferase transfection into LuCaP 136 spheroids, facilitating in vivo imaging. We showed that LuCaP 136 spheroids formed intratibial, orthotopic, and subcutaneous tumors when re-introduced into mice. Intratibial tumors responded to castration and were highly osteosclerotic. LuCaP 136 is a realistic in vitro-in vivo preclinical model of a subtype of bone metastatic prostate cancer.
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http://dx.doi.org/10.1007/s10585-016-9781-2DOI Listing
April 2016

A Protective Role for Androgen Receptor in Clear Cell Renal Cell Carcinoma Based on Mining TCGA Data.

PLoS One 2016 27;11(1):e0146505. Epub 2016 Jan 27.

Department of Urology, Stanford University School of Medicine, Stanford, California, United States of America.

Androgen receptor (AR) is expressed in normal murine and human kidneys of both genders, but its physiologic role is uncertain. Several studies showed loss of AR in renal cell carcinoma (RCC) in conjunction with increasing clinical stage and pathological grade, but others found that higher AR expression correlated with worse outcomes. Limited functional studies with renal cell lines suggested tumor-promoting activity of AR. In this study, we queried transcriptomic, proteomic, epigenetic and survival data from The Cancer Genome Atlas (TCGA) to evaluate AR expression and its association with overall survival in three subtypes of RCC (clear cell [ccRCC], papillary [pRCC], and chromophobe [chRCC]). We found that although there was no significant difference in AR mRNA expression in ccRCC of males vs. females, AR protein expression in ccRCC was significantly higher in male compared to female patients. More importantly, higher expression of AR at both transcript and protein levels was associated with improved overall survival in both genders with ccRCC, but did not predict survival of either gender with pRCC or chRCC. Genes whose transcript levels were associated with AR mRNA levels significantly overlapped between ccRCC and pRCC, but not with chRCC, suggesting a similar transcriptional program mediated by AR in ccRCC and pRCC. Ingenuity pathway analysis also identified overlapping pathways and upstream regulators enriched in AR-associated genes in ccRCC and pRCC. Hypermethylation of CpG sites located in the promoter and first exon of AR was associated with loss of AR expression and poor overall survival. Our findings support a tumor suppressor role for AR in both genders that might be exploited to decrease the incidence or progression of ccRCC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146505PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729482PMC
July 2016

Epigenetic effects of RRx-001: a possible unifying mechanism of anticancer activity.

Oncotarget 2015 Dec;6(41):43172-81

Department of Urology, Stanford University School of Medicine, Stanford, CA, USA.

RRx-001 is a novel aerospace-derived compound currently under investigation in several ongoing Phase II studies. In a Phase I trial, it demonstrated anti-cancer activity and evidence of resensitization to formerly effective therapies in heavily pre-treated patients with relapsed/refractory solid tumors. RRx-001 generates reactive oxygen and nitrogen species (ROS and RNS) and nitric oxide (NO), elicits changes in intracellular redox status, modulates tumor blood flow, hypoxia and vascular function and triggers apoptosis in cancer cells. We investigated the effect of RRx-001 on the epigenome of SCC VII cancer cells. RRx-001 at 0.5 and 2 μM significantly decreased global DNA methylation, i.e., 5-methylcytosine levels, in SCC VII cells. Consistently, 0.5-5 μM RRx-001 significantly decreased Dnmt1 and Dnmt3a protein expression in a dose- and time-dependent manner. In addition, global methylation profiling identified differentially methylated genes in SCC VII cells treated with 0.5, 2, and 5 μM RRx-001 compared to control cells. Twenty-three target sites were hypomethylated and 22 hypermethylated by >10% in the presence of at least two different concentrations of RRx-001. Moreover, RRx-001 at 2 μM significantly increased global acetylated histone H3 and H4 levels in SCC VII cells after 24 hour treatment, suggesting that RRx-001 regulates global acetylation in cancer cells. These results demonstrate that, in contrast to the traditional "one drug one target" paradigm, RRx-001 has multi(epi)target features, which contribute to its anti-cancer activity and may rationalize the resensitization to previously effective therapies observed in clinical trials and serve as a unifying mechanism for its anticancer activity.
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http://dx.doi.org/10.18632/oncotarget.6526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791224PMC
December 2015

Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study.

Lancet Oncol 2015 Sep 19;16(9):1133-1142. Epub 2015 Aug 19.

Sarah Cannon Research Institute, Tennessee Oncology, Nashville, TN, USA.

Background: Epigenetic alterations have been strongly associated with tumour formation and resistance to chemotherapeutic drugs, and epigenetic modifications are an attractive target in cancer research. RRx-001 is activated by hypoxia and induces the generation of reactive oxygen and nitrogen species that can epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation. The aim of this phase 1 study was to assess the safety, tolerability, and pharmacokinetics of RRx-001.

Methods: In this open-label, dose-escalation, phase 1 study, we recruited adult patients (aged >18 years) with histologically or cytologically confirmed diagnosis of advanced, malignant, incurable solid tumours from University of California at San Diego, CA, USA, and Sarah Cannon Research Institute, Nashville, TN, USA. Key eligibility criteria included evaluable disease, Eastern Cooperative Group performance status of 2 or less, an estimated life expectancy of at least 12 weeks, adequate laboratory parameters, discontinuation of all previous antineoplastic therapies at least 6 weeks before intervention, and no residual side-effects from previous therapies. Patients were assigned to receive intravenous infusions of RRx-001 at increasing doses (10 mg/m(2), 16·7 mg/m(2), 24·6 mg/m(2), 33 mg/m(2), 55 mg/m(2), and 83 mg/m(2)) either once or twice-weekly for at least 4 weeks, with at least three patients per dose cohort and allowing a 2-week observation period before dose escalation. Samples for safety and pharmacokinetics analysis, including standard chemistry and haematological panels, were taken on each treatment day. The primary objective was to assess safety, tolerability, and dose-limiting toxic effects of RRx-001, to determine single-dose pharmacokinetics, and to identify a recommended dose for phase 2 trials. All analyses were done per protocol. Accrual is complete and follow-up is still on-going. This trial is registered with ClinicalTrials.gov, number NCT01359982.

Findings: Between Oct 10, 2011, and March 18, 2013, we enrolled 25 patients and treated six patients in the 10 mg/m(2) cohort, three patients in the 16·7 mg/m(2) cohort, three patients in the 24·6 mg/m(2) cohort, four patients in the 33 mg/m(2) cohort, three patients in the 55 mg/m(2), and six patients in the 83 mg/m(2) cohort. Pain at the injection site, mostly grade 1 and grade 2, was the most common adverse event related to treatment, experienced by 21 (84%) patients. Other common drug-related adverse events included arm swelling or oedema (eight [32%] patients), and vein hardening (seven [28%] patients). No dose-limiting toxicities were observed. Time constraints related to management of infusion pain from RRx-001 resulted in a maximally feasible dose of 83 mg/m(2). Of the 21 evaluable patients, one (5%) patient had a partial response, 14 (67%) patients had stable disease, and six (29%) patients had progressive disease; all responses were across a variety of tumour types. Four patients who had received RRx-001 were subsequently rechallenged with a treatment that they had become refractory to; all four responded to the rechallenge.

Interpretation: RRx-001 is a well-tolerated novel compound without clinically significant toxic effects at the tested doses. Preliminary evidence of activity is promising and, on the basis of all findings, a dose of 16·7 mg/m(2) was recommended as the targeted dose for phase 2 trials.

Funding: EpicentRx (formerly RadioRx).
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http://dx.doi.org/10.1016/S1470-2045(15)00089-3DOI Listing
September 2015

Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001.

Oncotarget 2015 Aug;6(25):21547-56

Department of Radiology Stanford University Medical Center Stanford, CA 94304, USA.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. RRx-001 is a new anticancer agent that generates reactive oxygen and nitrogen species, and leads to epigenetic alterations in cancer cells. Here we report the RRx-001 mediated nuclear translocation of Nrf2 and the activation of expression of its downstream enzymes HO-1 and NQO1 in tumor cells. Inhibition of intrinsic Nrf2 expression by Nrf2-specific siRNA increased cell sensitivity to RRx-001. Molecular imaging of tumor cells co-expressing pARE-Firefly luciferase and pCMV-Renilla luciferase-mRFP in vitro and in vivo in mice revealed that RRx-001 significantly increased ARE-FLUC signal in cells in a dose- and time-dependent manner, suggesting that RRx-001 is an effective activator of the Nrf2-ARE signaling pathway. The pre-treatment level of ARE-FLUC signal in cells, reflecting basal activity of Nrf2, negatively correlated with the tumor response to RRx-001. The results support the concept that RRx-001 activates Nrf2-ARE antioxidant signaling pathways in tumor cells. Hence measurement of Nrf2-mediated activation of downstream target genes through ARE signaling may constitute a useful molecular biomarker for the early prediction of response to RRx-001 treatment, and thereby guide therapeutic decision-making.
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http://dx.doi.org/10.18632/oncotarget.4249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673285PMC
August 2015

Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor.

Prostate 2015 Oct 14;75(14):1601-9. Epub 2015 Jul 14.

Department of Radiology and Biomedical Imaging, University of California, San Francisco, California.

Background: Metabolic shifts in disease are of great interest for the development of novel therapeutics. In cancer treatment, these therapies exploit the metabolic phenotype associated with oncogenesis and cancer progression. One recent strategy involves the depletion of the cofactors needed to maintain the high rate of glycolysis seen with the Warburg effect. Specifically, blocking nicotinamide adenine dinucleotide (NAD) biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) inhibition depletes cancer cells of the NAD needed for glycolysis. To characterize this metabolic phenotype in vivo and describe changes in flux with treatment, non-invasive biomarkers are necessary. One such biomarker is hyperpolarized (HP) [1-(13) C] pyruvate, a clinically translatable probe that allows real-time assessment of metabolism.

Methods: We therefore developed a cell perfusion system compatible with HP magnetic resonance (MR) and positron emission tomography (PET) to develop translatable biomarkers of response to NAMPT inhibition in reduced volume cell cultures.

Results: Using this platform, we observed a reduction in pyruvate flux through lactate dehydrogenase with NAMPT inhibition in prostate cancer cells, and showed that both HP lactate and 2-[(18) F] fluoro-2-deoxy-D-glucose (FDG) can be used as biomarkers for treatment response of such targeted agents. Moreover, we observed dynamic flux changes whereby HP pyruvate was re-routed to alanine, providing both positive and negative indicators of treatment response.

Conclusions: This study demonstrated the feasibility of a MR/PET compatible bioreactor approach to efficiently explore cell and tissue metabolism, the understanding of which is critical for developing clinically translatable biomarkers of disease states and responses to therapeutics.
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http://dx.doi.org/10.1002/pros.23036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537380PMC
October 2015

Elevated serum microRNA levels associate with absence of high-grade prostate cancer in a retrospective cohort.

PLoS One 2015 14;10(4):e0124245. Epub 2015 Apr 14.

Department of Pathology, University of Illinois at Chicago, Chicago, IL, 60612, United States of America.

To reduce treatment of indolent prostate cancer (PCa), biomarkers are needed to improve identification of patients with a low-risk of having aggressive disease. Over-treatment of these patients occurs because of uncertainty in the aggressiveness of the entire tumor based on the biopsies, which do not accurately sample multifocal tumors. Circulating microRNAs (miRNAs) are stable serum markers and differential miRNA levels occur in men with PCa. The goal of this study was to identify circulating miRNAs that were associated with aggressive or indolent PCa. We measured circulating miRNAs in 150 patients prior to surgery and compared the miRNA levels to the pathology of the entire radical prostatectomy specimen. For this study we used an exceptionally well-characterized cohort of patients who had benign prostatic hyperplasia (BPH), low-grade or high-grade PCa. Low-grade was defined as patients with 100% Gleason grade 3 tumor as determined by step-wise sectioning. High-grade PCa patients had 30-90% Gleason grade 4+5 in the tumor. BPH patients had at least two biopsies negative for PCa. Twenty one miRNAs were selected for analysis. The miRNAs were quantified by RT-qPCR and analyzed by logistic regression. High levels of 14 miRNAs were exclusively present in the serum from patients with low-grade PCa or BPH, compared to men with high-grade PCa who had consistently low levels. The expression levels of the 14 miRNAs were combined into a "miR Score" which had a negative predictive value (NPV) of 0.939 to predict absence of high-grade PCa among PCa and BPH patients. Biochemical recurrence (BCR) was known for the PCa patients and a combined "miR Risk Score" accurately classified a subset of patients with low risk of BCR (NPV 0.941). In summary, measurement of serum miRNAs may have pre-surgical utility in combination with clinical risk calculators to identify patients with low risk of harboring aggressive PCa.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124245PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396984PMC
April 2016

Hepcidin regulation in prostate and its disruption in prostate cancer.

Cancer Res 2015 Jun 9;75(11):2254-63. Epub 2015 Apr 9.

Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-2465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4454355PMC
June 2015

Coordinate loss of MAP3K7 and CHD1 promotes aggressive prostate cancer.

Cancer Res 2015 Mar;75(6):1021-34

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Prostate cancer subtypes are poorly defined and functional validation of drivers of ETS rearrangement-negative prostate cancer has not been conducted. Here, we identified an ETS(-) subtype of aggressive prostate cancer (ERG(-)MAP3K7(del)CHD1(del)) and used a novel developmental model and a cell line xenograft model to show that cosuppression of MAP3K7 and CHD1 expression promotes aggressive disease. Analyses of publicly available prostate cancer datasets revealed that MAP3K7 and CHD1 were significantly codeleted in 10% to 20% of localized tumors and combined loss correlated with poor disease-free survival. To evaluate the functional impact of dual MAP3K7-CHD1 loss, we suppressed Map3k7 and/or Chd1 expression in mouse prostate epithelial progenitor/stem cells (PrP/SC) and performed tissue recombination experiments in vivo. Dual shMap3k7-shChd1 PrP/SC recombinants displayed massive glandular atypia with regions of prostatic intraepithelial neoplasia and carcinoma apparent. Combined Map3k7-Chd1 suppression greatly disrupted normal prostatic lineage differentiation; dual recombinants displayed significant androgen receptor loss, increased neuroendocrine differentiation, and increased neural differentiation. Clinical samples with dual MAP3K7-CHD1 loss also displayed neuroendocrine and neural characteristics. In addition, dual Map3k7-Chd1 suppression promoted E-cadherin loss and mucin production in recombinants. MAP3K7 and CHD1 protein loss also correlated with Gleason grade and E-cadherin loss in clinical samples. To further validate the phenotype observed in the PrP/SC model, we suppressed MAP3K7 and/or CHD1 expression in LNCaP prostate cancer cells. Dual shMAP3K7-shCHD1 LNCaP xenografts displayed increased tumor growth and decreased survival compared with shControl, shMAP3K7, and shCHD1 xenografts. Collectively, these data identify coordinate loss of MAP3K7 and CHD1 as a unique driver of aggressive prostate cancer development.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531265PMC
March 2015

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

Proc Natl Acad Sci U S A 2015 Feb 29;112(7):2287-92. Epub 2015 Jan 29.

University of California at Berkeley-University of California at San Francisco Graduate Program in Bioengineering, University of California, Berkeley, CA 94720; Pharmaceutical Chemistry, and Center for Systems and Synthetic Biology, University of California, San Francisco, CA 94143;

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.
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http://dx.doi.org/10.1073/pnas.1410776112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343104PMC
February 2015

Spheroid culture of LuCaP 147 as an authentic preclinical model of prostate cancer subtype with SPOP mutation and hypermutator phenotype.

Cancer Lett 2014 Sep 3;351(2):272-80. Epub 2014 Jul 3.

Department of Urology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

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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http://dx.doi.org/10.1016/j.canlet.2014.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112013PMC
September 2014

Nuclear KLLN expression associates with improved relapse-free survival for prostate carcinoma.

Endocr Relat Cancer 2014 Aug;21(4):579-86

Genomic Medicine InstituteDepartment of Anatomic PathologyCleveland Clinic, Cleveland, Ohio 44195, USADepartment of UrologyStanford University School of Medicine, Stanford, California 94305, USATaussig Cancer InstituteStanley Shalom Zielony Institute for Nursing ExcellenceCleveland Clinic, Cleveland, Ohio 44195, USADepartment of Genetics and Genome SciencesCASE Comprehensive Cancer CenterCase Western Reserve University School of Medicine, Cleveland, Ohio 44106, USAGenomic Medicine InstituteDepartment of Anatomic PathologyCleveland Clinic, Cleveland, Ohio 44195, USADepartment of UrologyStanford University School of Medicine, Stanford, California 94305, USATaussig Cancer InstituteStanley Shalom Zielony Institute for Nursing ExcellenceCleveland Clinic, Cleveland, Ohio 44195, USADepartment of Genetics and Genome SciencesCASE Comprehensive Cancer CenterCase Western Reserve University School of Medicine, Cleveland, Ohio 44106, USAGenomic Medicine InstituteDepartment of Anatomic PathologyCleveland Clinic, Cleveland, Ohio 44195, USADepartment of UrologyStanford University School of Medicine, Stanford, California 94305, USATaussig Cancer InstituteStanley Shalom Zielony Institute for Nursing ExcellenceCleveland Clinic, Cleveland, Ohio 44195, USADepartment of Genetics and Genome SciencesCASE Comprehensive Cancer CenterCase Western Reserve University School of Medicine, Cleveland, Ohio 44106, USAGenomic Medicine InstituteDepartment of Anatomic PathologyCleveland Clinic, Cleveland, Ohio 44195, USADepartment of UrologyStanford University School of Medicine, Stanford, California 94305, USATaussig Cancer InstituteStanley Shalom Zielony Institute for Nursing ExcellenceCleveland Clinic, Cleveland, Ohio 44195, USADepartment of Genetics and Genome SciencesCASE Comprehensive Cancer CenterCase Western Reserve University School of Medicine, Cleveland, Ohio 44106, USAGenomic Medicine InstituteDepartment of Anatomic PathologyCleveland Clinic, Cleveland, Ohio 44195, USADepartment of UrologyS

Men with organ-confined prostate cancer (CaP) are often treated with radical prostatectomy. Despite similar postoperative characteristics, a significant proportion of men with an intermediate risk of progression experience prostate-specific antigen (PSA)-defined failure, while others have relapse-free survival (RFS). Additional prognostic markers are needed to predict the outcome of these patients. KLLN is a transcription factor that regulates the cell cycle and induces apoptosis in cancer cells. We have shown that KLLN is an androgen-regulated gene and that loss of KLLN expression in primary CaP is associated with high Gleason score. In this retrospective study, we evaluated KLLN expression in the high-grade malignancy components from 109 men with intermediate risk CaP. Patients with nuclear KLLN-negative tumors had significantly higher preoperative serum PSA levels (12.24±2.37 ng/ml) and larger tumor volumes (4.61±0.71 cm(3)) compared with nuclear KLLN-positive patients (8.35±2.45 ng/ml, P=0.03, and 2.66±0.51 cm(3), P<0.0001, respectively). None of the nuclear KLLN-positive tumors had capsular penetration, whereas 34% of nuclear KLLN-negative tumors (P=0.004) had capsular penetration. Maintaining KLLN expression in tumor nuclei, but not in cytoplasm or stroma, associated with improved RFS after surgery (P=0.002). Only 7% of patients with nuclear KLLN-positive tumors had tumor recurrence, while 60% of patients in the KLLN-negative group developed PSA-defined failure with median relapse time of 6.6 months (P=0.0003). Our data suggest that KLLN expression may be used as a prognostic marker to predict outcome for intermediate risk patients, which could provide useful information for postoperative management.
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http://dx.doi.org/10.1530/ERC-14-0148DOI Listing
August 2014

Determination of carbohydrate structure recognized by prostate-specific F77 monoclonal antibody through expression analysis of glycosyltransferase genes.

J Biol Chem 2014 Jun 21;289(23):16478-86. Epub 2014 Apr 21.

From the Tumor Microenvironment Program, Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037,

This study reports the determination of the carbohydrate epitope of monoclonal antibody F77 previously raised against human prostate cancer PC-3 cells (Zhang, G., Zhang, H., Wang, Q., Lal, P., Carroll, A. M., de la Llera-Moya, M., Xu, X., and Greene, M. I. (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 732-737). We performed a series of co-transfections using mammalian expression vectors encoding specific glycosyltransferases. We thereby identified branching enzymes and FUT1 (required for Fucα1→2Gal linkage) as being essential for F77 antigen formation. When immortalized normal prostate 267B1 cells were transfected with FUT1 alone, cells showed weak expression of F77 antigen. By contrast, cells co-transfected with FUT1 plus either GCNT1, GCNT2, or GCNT3 (an enzyme required to form GlcNAcβ1→6Gal/GalNAc) showed robust F77 antigen expression, suggesting that F77 specifically binds to Fucα1→2Galβ1→4GlcNAcβ1→6Gal/GalNAc. RT-PCR for FUT1, GCNT1, GCNT2, and GCNT3 showed that F77-positive cell lines indeed express transcripts encoding FUT1 plus one GCNT. F77-positive prostate cancer cells transfected with siRNAs targeting FUT1, GCNT2, and GCNT3 showed significantly reduced F77 antigen, confirming the requirement of these enzymes for epitope synthesis. We also found that hypoxia induces F77 epitope expression in immortalized prostate RWPE1 cells, which express F77 antigen moderately under normoxia but at an elevated level under hypoxia. Quantitative RT-PCR demonstrated up-regulation of FUT1, GCNT2, and GCNT3 transcripts in RWPE1 cells under hypoxia, suggesting that hypoxia up-regulates glycosyltransferase expression required for F77 antigen synthesis. These results define the F77 epitope and provide a potential mechanism for F77 antigen synthesis in malignant prostate cancer.
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http://dx.doi.org/10.1074/jbc.M114.559047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047414PMC
June 2014

Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array.

J Biol Chem 2014 Jun 21;289(23):16462-77. Epub 2014 Apr 21.

From the Glycosciences Laboratory, Department of Medicine, Imperial College London, W12 0NN London, United Kingdom,

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.
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http://dx.doi.org/10.1074/jbc.M114.558932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047413PMC
June 2014

Development of a realistic in vivo bone metastasis model of human renal cell carcinoma.

Clin Exp Metastasis 2014 Jun 9;31(5):573-84. Epub 2014 Apr 9.

Department of Urology, Stanford University School of Medicine, Stanford, CA, 94305, USA.

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastases. The incidence of RCC is increasing and bone metastatic RCC merits greater focus. Realistic preclinical bone metastasis models of RCC are lacking, hampering the development of effective therapies. We developed a realistic in vivo bone metastasis model of human RCC by implanting precision-cut tissue slices under the renal capsule of immunodeficient mice. The presence of disseminated cells in bone marrow of tissue slice graft (TSG)-bearing mice was screened by human-specific polymerase chain reaction and confirmed by immunohistology using human-specific antibody. Disseminated tumor cells in bone marrow of TSG-bearing mice derived from three of seven RCC patients were detected as early as 1 month after tissue implantation at a high frequency with close resemblance to parent tumors (e.g., CAIX expression and high vascularity). The metastatic patterns of TSGs correlated with disease progression in patients. In addition, TSGs retained capacity to metastasize to bone at high frequency after serial passaging and cryopreservation. Moreover, bone metastases in mice responded to Temsirolimus treatment. Intratibial injections of single cells generated from TSGs showed 100 % engraftment and produced X-ray-visible tumors as early as 3 weeks after cancer cell inoculation. Micro-computed tomography (μCT) and histological analysis revealed osteolytic characteristics of these lesions. Our results demonstrated that orthotopic RCC TSGs have potential to develop bone metastases that respond to standard therapy. This first reported primary RCC bone metastasis model provides a realistic setting to test therapeutics to prevent or treat bone metastases in RCC.
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http://dx.doi.org/10.1007/s10585-014-9651-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351963PMC
June 2014

Phosphorylcholine-coated semiconducting polymer nanoparticles as rapid and efficient labeling agents for in vivo cell tracking.

Adv Healthc Mater 2014 Aug 25;3(8):1292-8. Epub 2014 Mar 25.

Molecular Imaging Program at Stanford, Department of Radiology, School of Medicine, Stanford University, CA, 94305, USA.

Despite the pressing need to noninvasively monitor transplanted cells in vivo with fluorescence imaging, desirable fluorescent agents with rapid labeling capability, durable brightness, and ideal biocompatibility remain lacking. Here, phosphorylcholine-coated near-infrared (NIR) fluorescent semiconducting polymer nanoparticles (SPNs) are reported as a new class of rapid, efficient, and cytocompatible labeling nanoagents for in vivo cell tracking. The phosphorylcholine coating results in efficient and rapid endocytosis and allows the SPN to enter cells within 0.5 h in complete culture medium apparently independent of the cell type, while its NIR fluorescence leads to a tissue penetration depth of 0.5 cm. In comparison to quantum dots and Cy5.5, the SPN is tolerant to physiologically ubiquitous reactive oxygen species (ROS), resulting in durable fluorescence both in vitro and in vivo. These desirable physical and physiological properties of the SPN permit cell tracking of human renal cell carcinoma (RCC) cells in living mice at a lower limit of detection of 10 000 cells with no obvious alteration of cell phenotype after 12 d. SPNs thus can provide unique opportunities for optimizing cellular therapy and deciphering pathological processes as a cell tracking label.
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http://dx.doi.org/10.1002/adhm.201300534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134769PMC
August 2014