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.
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.
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 Ac4ManNAz. 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.
Glycoprotein in a haystack: The metabolic labeling of human prostate cancer tissue slice cultures was employed to introduce azide‐labeled sialic acid into cell‐surface and secreted glycoproteins. 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.
Candidate gene-based studies have identified a handful of aberrant CpG DNA methylation events in prostate cancer. However, DNA methylation profiles have not been compared on a large scale between ...prostate tumor and normal prostate, and the mechanisms behind these alterations are unknown. In this study, we quantitatively profiled 95 primary prostate tumors and 86 benign adjacent prostate tissue samples for their DNA methylation levels at 26,333 CpGs representing 14,104 gene promoters by using the Illumina HumanMethylation27 platform. A 2-class Significance Analysis of this data set revealed 5912 CpG sites with increased DNA methylation and 2151 CpG sites with decreased DNA methylation in tumors (FDR < 0.8%). Prediction Analysis of this data set identified 87 CpGs that are the most predictive diagnostic methylation biomarkers of prostate cancer. By integrating available clinical follow-up data, we also identified 69 prognostic DNA methylation alterations that correlate with biochemical recurrence of the tumor. To identify the mechanisms responsible for these genome-wide DNA methylation alterations, we measured the gene expression levels of several DNA methyltransferases (DNMTs) and their interacting proteins by TaqMan qPCR and observed increased expression of DNMT3A2, DNMT3B, and EZH2 in tumors. Subsequent transient transfection assays in cultured primary prostate cells revealed that DNMT3B1 and DNMT3B2 overexpression resulted in increased methylation of a substantial subset of CpG sites that showed tumor-specific increased methylation.
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.
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.
Significance Differences in cell–cell interfacial energies can explain how multiple cell types sort into spatially organized tissues. However, this strategy of self-organization is not robust to heterogeneity or changes to the interfacial energies that drive correct cell positioning. Therefore, heterogeneous epithelial tissues such as the human mammary and prostate glands use a different strategy. First, disorganized aggregates form an adhesive interface at the tissue–ECM boundary that provides geometric constraints to self-organization. Second, only one cell type interacts appreciably with this interface. This strategy can explain how self-organization remains robust in vivo, provides generalizable rules for reconstituting tissues in vitro, and suggests how structure might break down during cancer progression.
Although numerous studies have implicated vitamin D in preventing prostate cancer, the underlying mechanism(s) remains unclear. Using normal human prostatic epithelial cells, we examined the role of ...mitogen-activated protein kinase phosphatase 5 (MKP5) in mediating cancer preventive activities of vitamin D. Up-regulation of MKP5 mRNA by 1,25-dihydroxyvitamin-D3 (1,25D) was dependent on the vitamin D receptor. We also identified a putative positive vitamin D response element within the MKP5 promoter that associated with the vitamin D receptor following 1,25D treatment. MKP5 dephosphorylates/inactivates the stress-activated protein kinase p38. Treatment of prostate cells with 1,25D inhibited p38 phosphorylation, and MKP5 small interfering RNA blocked this effect. Activation of p38 and downstream production of interleukin 6 (IL-6) are proinflammatory. Inflammation and IL-6 overexpression have been implicated in the initiation and progression of prostate cancer. 1,25D pretreatment inhibited both UV- and tumor necrosis factor alpha-stimulated IL-6 production in normal cells via p38 inhibition. Consistent with inhibition of p38, 1,25D decreased UV-stimulated IL-6 mRNA stabilization. The ability of 1,25D to up-regulate MKP5 was maintained in primary prostatic adenocarcinoma cells but was absent in metastases-derived prostate cancer cell lines. The inability of 1,25D to regulate MKP5 in the metastasis-derived cancer cells suggests there may be selective pressure to eliminate key tumor suppressor functions of vitamin D during cancer progression. These studies reveal MKP5 as a mediator of p38 inactivation and decreased IL-6 expression by 1,25D in primary prostatic cultures of normal and adenocarcinoma cells, implicating decreased prostatic inflammation as a potential mechanism for prostate cancer prevention by 1,25D.
Glycoproteins in focus: Metabolic labeling of glycans with azido sugars (see picture) in combination with two‐photon fluorescence lifetime imaging microscopy enables the visualization of specific ...glycoforms of endogenous proteins. This method can be utilized to detect glycosylated proteins in both cell culture and intact human tissue slices.
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.
Summary 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/m2 , 16·7 mg/m2 , 24·6 mg/m2 , 33 mg/m2 , 55 mg/m2 , and 83 mg/m2 ) 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/m2 cohort, three patients in the 16·7 mg/m2 cohort, three patients in the 24·6 mg/m2 cohort, four patients in the 33 mg/m2 cohort, three patients in the 55 mg/m2 , and six patients in the 83 mg/m2 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/m2 . 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/m2 was recommended as the targeted dose for phase 2 trials. Funding EpicentRx (formerly RadioRx).
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