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1.
Cell Res ; 32(3): 269-287, 2022 03.
Article in English | MEDLINE | ID: mdl-35046518

ABSTRACT

The emergence of SARS-CoV-2 variants and potentially other highly pathogenic sarbecoviruses in the future highlights the need for pan-sarbecovirus vaccines. Here, we discovered a new STING agonist, CF501, and found that CF501-adjuvanted RBD-Fc vaccine (CF501/RBD-Fc) elicited significantly stronger neutralizing antibody (nAb) and T cell responses than Alum- and cGAMP-adjuvanted RBD-Fc in mice. Vaccination of rabbits and rhesus macaques (nonhuman primates, NHPs) with CF501/RBD-Fc elicited exceptionally potent nAb responses against SARS-CoV-2 and its nine variants and 41 S-mutants, SARS-CoV and bat SARSr-CoVs. CF501/RBD-Fc-immunized hACE2-transgenic mice were almost completely protected against SARS-CoV-2 challenge, even 6 months after the initial immunization. NHPs immunized with a single dose of CF501/RBD-Fc produced high titers of nAbs. The immunized macaques also exhibited durable humoral and cellular immune responses and showed remarkably reduced viral load in the upper and lower airways upon SARS-CoV-2 challenge even at 108 days post the final immunization. Thus, CF501/RBD-Fc can be further developed as a novel pan-sarbecovirus vaccine to combat current and future outbreaks of sarbecovirus diseases.


Subject(s)
COVID-19 , Vaccines , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Macaca mulatta , Mice , Rabbits , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , T-Lymphocytes
2.
Prostate Cancer Prostatic Dis ; 24(2): 448-456, 2021 06.
Article in English | MEDLINE | ID: mdl-33009489

ABSTRACT

BACKGROUND: Precision medicine approaches for managing patients with metastatic castrate-resistant prostate cancer (mCRPC) are lacking. Non-invasive approaches for molecular monitoring of disease are urgently needed, especially for patients suffering from bone metastases for whom tissue biopsy is challenging. Here we utilized baseline blood samples to identify mCRPC patients most likely to benefit from abiraterone plus prednisone (AAP) or enzalutamide. METHODS: Baseline blood samples were collected for circulating tumor cell (CTC) enumeration and qPCR-based gene expression analysis from 51 men with mCRPC beginning treatment with abiraterone or enzalutamide. RESULTS: Of 51 patients (median age 68 years [51-82]), 22 received AAP (abiraterone 1000 mg/day plus prednisone 10 mg/day) and 29 received enzalutamide (160 mg/day). The cohort was randomly divided into training (n = 37) and test (n = 14) sets. Baseline clinical variables (Gleason score, PSA, testosterone, and hemoglobin), CTC count, and qPCR-based gene expression data for 141 genes/isoforms in CTC-enriched blood were analyzed with respect to overall survival (OS). Genes with expression most associated with OS included MSLN, ARG2, FGF8, KLK3, ESRP2, NPR3, CCND1, and WNT5A. Using a Cox-elastic net model for our test set, the 8-gene expression signature had a c-index of 0.87 (95% CI [0.80, 0.94]) and was more strongly associated with OS than clinical variables or CTC count alone, or a combination of the three variables. For patients with a low-risk vs. high-risk gene expression signature, median OS was not reached vs. 18 months, respectively (HR 5.32 [1.91-14.80], p = 0.001). For the subset of 41 patients for whom progression-free survival (PFS) data was available, the median PFS for patients with a low-risk vs high-risk gene expression signature was 20 vs. 5 months, respectively (HR 2.95 [1.46-5.98], p = 0.003). CONCLUSIONS: If validated in a larger prospective study, this test may predict patients most likely to benefit from second-generation antiandrogen therapy.


Subject(s)
Androstenes/therapeutic use , Benzamides/therapeutic use , Bone Neoplasms/secondary , Neoplastic Cells, Circulating/pathology , Nitriles/therapeutic use , Phenylthiohydantoin/therapeutic use , Prednisone/therapeutic use , Prostatic Neoplasms, Castration-Resistant/pathology , Transcriptome , Aged , Aged, 80 and over , Androgen Antagonists/therapeutic use , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Bone Neoplasms/blood , Bone Neoplasms/drug therapy , Bone Neoplasms/genetics , Follow-Up Studies , Humans , Lymphatic Metastasis , Male , Middle Aged , Neoplastic Cells, Circulating/metabolism , Prognosis , Prospective Studies , Prostatic Neoplasms, Castration-Resistant/blood , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Retrospective Studies , Survival Rate
3.
Anal Chem ; 91(10): 6585-6592, 2019 05 21.
Article in English | MEDLINE | ID: mdl-30994329

ABSTRACT

Development of a highly selective and sensitive imaging probe for accurate detection of myocardial hypoxia will be helpful to estimate the degree of ischemia and subsequently guide personalized treatment. However, an efficient optical approach for hypoxia monitoring in myocardial ischemia is still lacking. In this work, a cardiomyocyte-specific and nitroreductase-activatable near-infrared nanoprobe has been developed for selective and sensitive imaging of myocardial hypoxia. The nanoprobe is a liposome-based nanoarchitecture which is functionalized with a peptide (GGGGDRVYIHPF) for targeting heart cells and encapsulating a nitrobenzene-substituted BODIPY for nitroreductase imaging. The nanoprobe can specifically recognize and bind to angiotensin II type 1 receptor that is overexpressed on the ischemic heart cells by the peptide and is subsequently uptaken into heart cells, in which the probe is released and activated by hypoxia-related nitroreductase to produce fluorescence emission at 713 nm. The in vitro response of the nanoprobe toward nitroreductase resulted in 55-fold fluorescence enhancement with the limit of detection as low as 7.08 ng/mL. Confocal fluorescence imaging confirmed the successful uptake of nanoprobe by hypoxic heart cells and intracellular detection of nitroreductase. More significantly, in vivo imaging of hypoxia in a murine model of myocardial ischemia was achieved by the nanoprobe with high sensitivity and good biocompatibility. Therefore, this work presents a new tool for targeted detection of myocardial hypoxia and will promote the investigation of the hypoxia-related physiological and pathological process of ischemic heart disease.


Subject(s)
Boron Compounds/chemistry , Fluorescent Dyes/chemistry , Hypoxia/diagnostic imaging , Myocardial Ischemia/diagnostic imaging , Nitroreductases/analysis , Animals , Boron Compounds/toxicity , Cell Line , Cell Survival/drug effects , Drug Carriers/chemistry , Drug Carriers/toxicity , Fluorescent Dyes/toxicity , Limit of Detection , Liposomes/chemistry , Liposomes/toxicity , Male , Mice, Inbred ICR , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Peptides/chemistry , Peptides/metabolism , Peptides/toxicity , Rats , Receptor, Angiotensin, Type 1/metabolism
4.
ACS Omega ; 4(2): 2472-2481, 2019 Feb 28.
Article in English | MEDLINE | ID: mdl-30873507

ABSTRACT

Pyrvinium was identified as the first small molecule inhibitor of the androgen receptor (AR) DNA-binding domain (DBD). It was also among the first small molecules shown to directly inhibit the activity of AR splice variants (ARVs), which has important clinical implications in the treatment of castration-resistant prostate cancer. Important questions about pyrvinium's mechanism of action remain. Here, we demonstrate through mutational analysis that amino acids 609 and 612 are important for pyrvinium action. Nuclear magnetic resonance demonstrates a specific interaction between a soluble pyrvinium derivative and the AR DBD homodimer-DNA complex. Chromatin immunoprecipitation and electrophoretic mobility shift assay experiments demonstrate that, despite an interaction with this complex, pyrvinium does not alter the DNA-binding kinetics in either assay. AR immunoprecipitation followed by mass spectrometry was used to identify proteins whose interaction with AR is altered by pyrvinium. Several splicing factors, including DDX17, had reduced interactions with AR in the presence of pyrvinium. RNA sequencing of prostate cancer cells treated with pyrvinium demonstrated changes in splicing, as well as in several other pathways. However, pyrvinium did not alter the levels of ARVs in several prostate cancer cell lines. Taken together, our new data pinpoint the direct interaction between pyrvinium and AR DBD and shed light on the mechanism by which it inhibits AR transcriptional activity.

5.
Article in English | MEDLINE | ID: mdl-34926721

ABSTRACT

We sought to evaluate androgen receptor (AR) and PI3K pathway activity in ovarian cancer cell lines and tissue and determine if either pathway was correlated with growth of ovarian cancers. AR expression and activity were quantified using immunohistochemistry (IHC) and RT-qPCR in six ovarian cancer cell lines and 51 tissue samples. Phospho-mTOR and AKT expression were quantified by IHC as well. Cell growth was assessed in the presence of AR modulating drugs and metformin. We found that despite robust AR expression and activity, no cell line was dependent on androgen for growth. However, metformin inhibited activity in five of the six cell lines. Patient tissues had large variation in AR expression and activity, as well as in expression of phospho-mTOR and AKT, but none of these variables correlated with progression-free survival (PFS). AR expression and activity did not predict the dependence of ovarian cancer cell lines on androgens for growth, and AR expression and activity did not correlate with PFS. This result suggests that AR expression as a criterion for patient selection for clinical trials evaluating molecules targeting AR may not predict response for ovarian cancer patients.

6.
Urol Oncol ; 36(4): 162.e1-162.e6, 2018 04.
Article in English | MEDLINE | ID: mdl-29289429

ABSTRACT

BACKGROUND: With the advent of secondary androgen receptor (AR)-targeted therapies in metastatic castration resistant prostate cancer (PC), nonadenocarcinoma PCs are becoming more prevalent. Many of these cancers express neuroendocrine markers, which may provide biomarkers for emergence of this disease state. We aimed to quantify the expression of synaptophysin (Syp) on circulating tumor cells (CTCs) from serial samples of patients being treated with abiraterone acetate or enzalutamide. METHODS: CTCs were isolated from 44 patients with castration resistant PC before starting abiraterone or enzalutamide, at 4, 8, and 12 weeks on therapy, and at progression. Patients were stratified into 3 groups: de novo resistance, short response, and long response. CTCs were enumerated on the CellSearch platform and Syp expression was quantified using the open fluorescent channel on the platform. Correlative analyses were performed. RESULTS: A baseline CTC count of 5 or greater was associated with a more rapid time to progression and increasing CTC counts correlated with emergence of drug resistance. Syp was readily detectable on the surface of CTCs, and baseline percentage CTC Syp expression was significantly associated with time to progression. Furthermore, in evaluable patients, percent CTC Syp expression increased with the emergence of drug resistance. We also found that prior exposure to AR-targeted therapies was inversely associated with progression free survival. CONCLUSIONS: We have demonstrated that Syp can be quantified on CTCs and that Syp expression correlates with resistance to abiraterone and enzalutamide. Larger studies testing Syp as a biomarker of emergence of nonadenocarcinoma disease and as a marker of response to AR-targeted therapies are warranted.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor/metabolism , Neoplastic Cells, Circulating/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Synaptophysin/metabolism , Abiraterone Acetate/pharmacology , Abiraterone Acetate/therapeutic use , Aged , Aged, 80 and over , Androgen Receptor Antagonists/pharmacology , Androgen Receptor Antagonists/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzamides , Disease Progression , Disease-Free Survival , Drug Resistance, Neoplasm , Follow-Up Studies , Humans , Male , Middle Aged , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Phenylthiohydantoin/therapeutic use , Prostatic Neoplasms, Castration-Resistant/blood , Prostatic Neoplasms, Castration-Resistant/mortality , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Treatment Outcome
7.
Cancer ; 124(6): 1216-1224, 2018 03 15.
Article in English | MEDLINE | ID: mdl-29266182

ABSTRACT

BACKGROUND: Two androgen receptor (AR)-targeted therapies, enzalutamide and abiraterone acetate plus prednisone (abiraterone), have been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). Many patients respond to these agents, but both de novo and acquired resistance are common. The authors characterized resistant phenotypes that emerge after treatment with abiraterone or enzalutamide. METHODS: Patients who received abiraterone or enzalutamide in the course of routine clinical care were consented for serial blood collection. A proprietary system (CellSearch) was used to enumerate and enrich circulating tumor cells (CTCs). RNA-sequencing (RNA-seq) was performed on pools of up to 10 epithelial cell adhesion molecule (EpCAM)-positive/CD45-negative CTCs. The impact of gene expression changes observed in CTCs between patients who responded or were resistant to abiraterone/enzalutamide therapies was further explored in a model cell line system. RESULTS: RNA-seq data from CTCs identified mutations commonly associated with CRPC as well as novel mutations, including several in the ligand-binding domain of AR that could facilitate escape from AR-targeted agents. Ingenuity pathway analysis of differentially regulated genes identified the transforming growth factor ß (TGFß) and cyclin D1 (CCND1) signaling pathways as significantly upregulated in drug-resistant CTCs. Transfection experiments using enzalutamide-sensitive and enzalutamide-resistant LNCaP cells confirmed the involvement of SMAD family member 3, a key mediator of the TGFß pathway, and of CCND1 in resistance to enzalutamide treatment. CONCLUSIONS: The current results indicate that RNA-seq of CTCs representing abiraterone and enzalutamide sensitive and resistant states can identify potential mechanisms of resistance. Therapies targeting the downstream signaling mediated by SMAD family member 3 (SMAD3) and CCND1, such as cyclin-dependent kinase 4/cyclin-dependent kinase 6 inhibitors, could provide new therapeutic options for the treatment of antiandrogen-resistant disease. Cancer 2018;124:1216-24. © 2017 American Cancer Society.


Subject(s)
Androgen Antagonists/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Drug Resistance, Neoplasm/drug effects , Prostatic Neoplasms, Castration-Resistant/drug therapy , Protein Kinase Inhibitors/pharmacology , Abiraterone Acetate/pharmacology , Abiraterone Acetate/therapeutic use , Aged , Aged, 80 and over , Androgen Antagonists/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzamides , Biomarkers, Tumor/blood , Biomarkers, Tumor/metabolism , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Humans , Male , Middle Aged , Neoplastic Cells, Circulating/metabolism , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Phenylthiohydantoin/therapeutic use , Prostatic Neoplasms, Castration-Resistant/blood , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction/drug effects , Smad3 Protein/metabolism
8.
Oncotarget ; 8(8): 13818-13831, 2017 Feb 21.
Article in English | MEDLINE | ID: mdl-28099154

ABSTRACT

Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Vitamin K Epoxide Reductases/metabolism , Warfarin/pharmacology , Animals , Anticoagulants/pharmacology , Cell Line, Tumor , Humans , Immunohistochemistry , Immunoprecipitation , Male , Mass Spectrometry , Mice , Mice, Nude , Mutagenesis, Site-Directed , PPAR gamma/drug effects , PPAR gamma/metabolism , Polymerase Chain Reaction , Signal Transduction/drug effects
9.
Urol Oncol ; 35(3): 112.e13-112.e18, 2017 03.
Article in English | MEDLINE | ID: mdl-27889279

ABSTRACT

PURPOSE: Increasing evidence has demonstrated that men taking the anticoagulant warfarin have a lower risk of developing prostate cancer. This phenomenon is not observed in other cancers. We sought to determine if the target of warfarin, vitamin K epoxide reductase (VKOR), is expressed in benign and cancerous prostate tissues and if a functional single nucleotide polymorphism (SNP) in the VKOR gene is associated with prostate cancer risk. MATERIALS AND METHODS: The expression of VKOR was quantified by immunohistochemistry in an institutional series of 54 radical prostatectomy samples and metastatic biopsies, as well as in 40 other cancers and matched benign tissues on a tissue microarray. Genotyping of SNP rs2359612 was performed in a prospective series of 57 patients. RESULTS: VKOR is highly expressed in benign human prostate epithelial cells but is not expressed or expressed at very low levels in cancerous cells. This expression pattern is unique to prostate cancer. Additionally, the proportion of the carrier C allele of rs2359612 in the patients with prostate cancer was significantly higher than in the population, suggesting an association between this allele and the risk of having a diagnosis of prostate cancer. CONCLUSIONS: The expression of VKOR in benign prostate epithelial cells, along with the association between a functional VKOR SNP and prostate cancer risk, suggests a possible role for VKOR in mediating the effect of warfarin on prostate cancer risk. Larger multi-institutional cohort studies are warranted, as are molecular studies on the role of VKOR in prostate cancer development.


Subject(s)
Anticoagulants/pharmacology , Prostatic Neoplasms/pathology , Vitamin K Epoxide Reductases/metabolism , Warfarin/pharmacology , Aged , Alleles , Anticoagulants/therapeutic use , Biopsy , Genotyping Techniques , Humans , Immunohistochemistry , Male , Middle Aged , Polymorphism, Single Nucleotide , Prospective Studies , Prostate/pathology , Prostate/surgery , Prostatectomy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/prevention & control , Prostatic Neoplasms/surgery , Tissue Array Analysis , Vitamin K Epoxide Reductases/antagonists & inhibitors , Vitamin K Epoxide Reductases/genetics , Warfarin/therapeutic use
10.
Prostate ; 77(5): 530-541, 2017 04.
Article in English | MEDLINE | ID: mdl-27976414

ABSTRACT

BACKGROUND: Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. METHODS: Mice were castrated and implanted with capsules to achieve castrate, normal, or sub-physiological levels of T. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue. FACS was used to quantify the percentages of purported prostate stem and transit amplifying (TA) cells in mouse prostates. Prostate tissues were also stained for the presence of CD68+ cells and RNA was extracted from prostate tissue or specific cell populations to measure changes in transcript levels with low T treatment. RESULTS: Despite having significantly different levels of T and DHT in the serum, T and DHT concentrations in prostate tissue from different T treatment groups were similar. Low T treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining prostate androgen levels. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all T treatment groups, demonstrating that the mouse prostate can maintain functional levels of androgens despite low serum T levels. Low T increased the frequency of prostate stem and TA cells in adult prostate tissue and caused major transcriptional changes in those cells. Gene ontology analysis suggested that low T caused inflammatory responses and immunofluorescent staining indicated that low T treatment led to the increased presence of CD68+ macrophages in prostate tissue. CONCLUSIONS: Low T alters the AR signaling axis which likely leads to maintenance of functional levels of prostate androgens. Low T also induces quantitative and qualitative changes in prostate stem cells which appear to lead to inflammatory macrophage infiltration. These changes are proposed to lead to an aggressive phenotype once cancers develop and may contribute to the poor outcomes in men with low T. Prostate 77:530-541, 2017. © 2016 Wiley Periodicals, Inc.


Subject(s)
Prostate/cytology , Prostate/metabolism , Stem Cells/metabolism , Testosterone/metabolism , Animals , Male , Mice , Mice, Inbred C57BL , Orchiectomy/methods , Prostate/pathology , Stem Cells/pathology , Testosterone/blood
11.
Mol Cancer Res ; 13(1): 130-7, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25183163

ABSTRACT

UNLABELLED: Sarcomatoid metastatic renal cell carcinoma (mRCC) is associated with a poor prognosis, and the biology of the disease has been inadequately characterized. RNA sequencing (RNA-seq) was performed on adjacent benign, clear cell, and sarcomatoid components from clinical specimens with sarcomatoid mRCC. M phase and cell-cycle pathways were enriched in sarcomatoid versus adjacent clear cell components, suggesting greater cell proliferation. The expression of aurora kinase A (AURKA) was increased as part of these pathways, and its increased expression was validated by quantitative PCR (qPCR). Immunohistochemical (IHC) analysis revealed that AURKA levels were increased in sarcomatoid tissue compared with their benign or clear cell parts. The increase in AURKA correlated with increased mTOR pathway activity, as evidenced by increased expression of phosphorylated mTOR (S2448) and ribosomal protein S6K (T389). When AURKA was stably expressed in a RCC cell line (Renca), it resulted in increased expression and activity of mTOR, suggesting that overexpression of AURKA can activate the mTOR pathway. These results warrant the analysis of a larger clinical cohort and suggest that targeting AURKA and/or mTOR in patients with sarcomatoid mRCC should be explored. IMPLICATIONS: Comparative RNA-seq of adjacent sarcomatoid and clear cell histology of RCC indicates a proliferative phenotype and increased AURKA-dependent activation of mTOR signaling in sarcomatoid RCC, which could be targeted by available agents.


Subject(s)
Aurora Kinase A/biosynthesis , Carcinoma, Renal Cell/genetics , Prognosis , TOR Serine-Threonine Kinases/biosynthesis , Adult , Aged , Aurora Kinase A/genetics , Carcinoma, Renal Cell/pathology , Female , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , RNA/genetics , Signal Transduction , TOR Serine-Threonine Kinases/genetics
12.
Clin Genitourin Cancer ; 13(2): 130-6, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25450039

ABSTRACT

BACKGROUND: In this study, we aimed to determine the feasibility of identifying CTCs in patients with HRLPC, using a modified isolation procedure using the CellSearch (Veridex) platform, and to assess the expression of stem cell and epithelial-mesenchymal transition (EMT) markers on the CTCs. PATIENTS AND METHODS: Thirty-five patients with HRLPC who had chosen prostatectomy for definitive management were prospectively identified. After obtaining consent, four 30-mL blood draws were performed, 2 before surgery and 2 after surgery. The CTC-containing fraction was Ficoll-purified and transferred to a CellSave (Veridex) tube containing dilution buffer before standard enumeration using the CellSearch system. Loss of E-cadherin expression, a marker of EMT, and CD133, a putative prostate cancer stem cell marker, were characterized using the open channel of the CellSearch platform. CTC fragments were also enumerated. RESULTS: Using the modified methodology, CTCs were detectable in 49% of patients before surgery. Although no correlation between CTC count and biochemical recurrence (BR) was observed, the percentages of CD133 and E-cadherin-positive CTC fragments were associated with BR at 1 year. CONCLUSION: Our results suggest that further research into the development of CTCs as prognostic biomarkers in HRLPC is warranted.


Subject(s)
Antigens, CD/metabolism , Biomarkers, Tumor/metabolism , Cadherins/metabolism , Glycoproteins/metabolism , Neoplastic Cells, Circulating/pathology , Peptides/metabolism , Prostatic Neoplasms/pathology , AC133 Antigen , Aged , Cell Count , Cell Separation/methods , Epithelial-Mesenchymal Transition , Feasibility Studies , Humans , Male , Middle Aged , Neoplastic Cells, Circulating/metabolism , Phenotype , Prognosis , Prospective Studies , Prostatic Neoplasms/blood
13.
ACS Chem Biol ; 9(3): 692-702, 2014 Mar 21.
Article in English | MEDLINE | ID: mdl-24354286

ABSTRACT

Pyrvinium pamoate (PP) is a potent noncompetitive inhibitor of the androgen receptor (AR). Using a novel method of target identification, we demonstrate that AR is a direct target of PP in prostate cancer cells. We demonstrate that PP inhibits AR activity via the highly conserved DNA binding domain (DBD), the only AR inhibitor that functions via this domain. Furthermore, computational modeling predicts that pyrvinium binds at the interface of the DBD dimer and the minor groove of the AR response element. Because PP acts through the DBD, PP is able to inhibit the constitutive activity of AR splice variants, which are thought to contribute to the growth of castration resistant prostate cancer (CRPC). PP also inhibits androgen-independent AR activation by HER2 kinase. The antiandrogen activity of pyrvinium manifests in the ability to inhibit the in vivo growth of CRPC xenografts that express AR splice variants. Interestingly, PP was most potent in cells with endogenous AR expression derived from prostate or bone. PP was able to inhibit several other hormone nuclear receptors (NRs) but not structurally unrelated transcription factors. PP inhibition of other NRs was similarly cell-type selective. Using dual-energy X-ray absorptiometry, we demonstrate that the cell-type specificity of PP manifests in tissue-selective inhibition of AR activity in mice, as PP decreases prostate weight and bone mineral density but does not affect lean body mass. Our results suggest that the noncompetitive AR inhibitor pyrvinium has significant potential to treat CRPC, including cancers driven by ligand-independent AR signaling.


Subject(s)
Androgen Receptor Antagonists/pharmacology , Prostate/drug effects , Prostatic Neoplasms/metabolism , Pyrvinium Compounds/pharmacology , Receptors, Androgen/metabolism , Absorptiometry, Photon , Androgen Receptor Antagonists/adverse effects , Androgen Receptor Antagonists/chemistry , Androgen Receptor Antagonists/therapeutic use , Animals , Bone Density/drug effects , Cell Line, Tumor , Computational Biology , HEK293 Cells , Humans , Ligands , Male , Mice , Models, Biological , Molecular Docking Simulation , Prostate/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Pyrvinium Compounds/adverse effects , Pyrvinium Compounds/chemistry , Pyrvinium Compounds/therapeutic use , Xenograft Model Antitumor Assays
14.
J Mol Endocrinol ; 51(1): 143-53, 2013.
Article in English | MEDLINE | ID: mdl-23709748

ABSTRACT

Prostate cancer (PC) is both an age- and an androgen-dependent disease. Paradoxically, systemic levels of androgens decline with age as the risk of PC rises. While there is no correlation between systemic androgen levels and the risk of PC, systemic androgen levels do not reflect the levels of androgens in prostate tissue. In metastatic PC, changes in the androgen biosynthesis pathway during hormone therapy result in increased levels of androgens in cancer tissue and contribute to continued androgen receptor (AR) signaling. It is possible that similar changes occur in normal prostate tissue as androgen levels decline with age and that this contributes to tumorigenesis. In the present study, we sought to determine whether the rat prostate is able to maintain functional levels of androgens despite low serum testosterone levels. Rats were castrated and implanted with capsules to achieve castrate, normal, sub-physiological, and supra-physiological levels of testosterone. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of testosterone and dihydrotestosterone (DHT) in the serum and prostate tissue. Quantitative RT-PCR was used to quantify the expression of genes involved in the androgen/AR signaling axis. Despite significantly different levels of testosterone and DHT being present in the serum, testosterone and DHT concentrations in prostate tissue from different testosterone-treatment groups were very similar. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all the testosterone-treatment groups, demonstrating that the rat prostate can maintain a functional level of androgens despite low serum testosterone levels. Low-testosterone treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining functional androgen levels.


Subject(s)
Prostate/metabolism , Testosterone/metabolism , Androgens/blood , Androgens/metabolism , Animals , Biosynthetic Pathways/genetics , Gene Expression , Male , Prostate/pathology , Rats , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Steroids/biosynthesis , Testosterone/blood
15.
Endocr Res ; 37(4): 203-15, 2012.
Article in English | MEDLINE | ID: mdl-22591338

ABSTRACT

INTRODUCTION: Androgens regulate a wide array of physiological processes, including male sexual development, bone and muscle growth, and behavior and cognition. Because androgens play a vital role in so many tissues, changes in androgen signaling are associated with a plethora of diseases. How such varied responses are achieved by a single stimulus is not well understood. Androgens act primarily through the androgen receptor (AR), a hormone nuclear receptor that is expressed in a select variety of tissues. METHODS: In order to gain a better understanding of how the tissue-selective effects of androgens are achieved, we performed a comparison of microarray data, using previously published datasets and several of our own microarray datasets. These datasets were derived from clinically relevant, AR-expressing tissues dissected from rodents treated with the full androgen dihydrotestosterone (DHT). RESULTS: We found that there is a diverse response to DHT, with very little overlap of androgen regulated genes in each tissue. Gene ontology analyses also indicated that, while several tissues regulate similar biological processes in response to DHT, most androgen regulated processes are specific to one or a few tissues. Thus, it appears that the disparate physiological effects mediated by androgens begin with widely varying effects on gene expression in different androgen-sensitive tissues. CONCLUSION: The analysis completed in this study will lead to an improved understanding of how androgens mediate diverse, tissue-specific processes and better ways to assess the tissue-selective effects of AR modulators during drug development.


Subject(s)
Androgens/pharmacology , Dihydrotestosterone/pharmacology , Gene Expression Regulation/drug effects , Animals , Castration , Female , Gene Expression Profiling , Male , Ovary/drug effects , Prosencephalon/drug effects , Prostate/drug effects , Rats , Rats, Sprague-Dawley , Skin/drug effects
16.
Int J Cancer ; 129(6): 1425-34, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21128223

ABSTRACT

Formation and repair of platinum (Pt)-induced DNA adducts is a critical step in Pt drug-mediated cytotoxicity. Measurement of Pt-DNA adduct kinetics in tumors may be useful for better understanding chemoresistance and therapeutic response. However, this concept has yet to be rigorously tested because of technical challenges in measuring the adducts at low concentrations and consistent access to sufficient tumor biopsy material. Ultrasensitive accelerator mass spectrometry was used to detect [(14)C]carboplatin-DNA monoadducts at the attomole level, which are the precursors to Pt-DNA crosslink formation, in six cancer cell lines as a proof-of-concept. The most resistant cells had the lowest monoadduct levels at all time points over 24 hr. [(14)C]Carboplatin "microdoses" (1/100th the pharmacologically effective concentration) had nearly identical adduct formation and repair kinetics compared to therapeutically relevant doses, suggesting that the microdosing approach can potentially be used to determine the pharmacological effects of therapeutic treatment. Some of the possible chemoresistance mechanisms were also studied, such as drug uptake/efflux, intracellular inactivation and DNA repair in selected cell lines. Intracellular inactivation and efficient DNA repair each contributed significantly to the suppression of DNA monoadduct formation in the most resistant cell line compared to the most sensitive cell line studied (p < 0.001). Nucleotide excision repair (NER)-deficient and -proficient cells showed substantial differences in carboplatin monoadduct concentrations over 24 hr that likely contributed to chemoresistance. The data support the utility of carboplatin microdosing as a translatable approach for defining carboplatin-DNA monoadduct formation and repair, possibly by NER, which may be useful for characterizing chemoresistance in vivo.


Subject(s)
Antineoplastic Agents/metabolism , Carboplatin/therapeutic use , DNA Adducts/metabolism , DNA Repair , Carboplatin/administration & dosage , Carboplatin/chemistry , Carboplatin/metabolism , Cell Line, Tumor , DNA Damage , Drug Administration Schedule , Drug Resistance, Neoplasm , Glutathione/analysis , Humans , Inhibitory Concentration 50 , Mass Spectrometry
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