Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (Ϸ22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumorderived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.biomarker ͉ miR-141 ͉ plasma ͉ serum ͉ prostate cancer T he development of minimally invasive tests for the detection and monitoring of common epithelial malignancies could greatly reduce the worldwide health burden of cancer (1). Although conventional strategies for blood-based biomarker discovery (e.g., using proteomic technologies) have shown promise, the development of clinically validated cancer detection markers remains an unmet challenge for many common human cancers (2). New approaches that can complement and improve on current strategies for cancer detection are urgently needed.MicroRNAs (miRNAs) are small (typically Ϸ22 nt in size) regulatory RNA molecules that function to modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes (3, 4). We hypothesized that miRNAs could be an ideal class of blood-based biomarkers for cancer detection because: (i) miRNA expression is frequently dysregulated in cancer (5, 6), (ii) expression patterns of miRNAs in human cancer appear to be tissue-specific (7), and (iii) miRNAs have unusually high stability in formalin-fixed tissues (8-10). This third point led us to speculate that miRNAs may have exceptional stability in plasma and serum as well. We show here that miRNAs are in fact present in clinical samples of plasma and serum in a remarkably stable form. Furthermore, we establish proof-ofprinciple for blood-based miRNA cancer detection by using both a xenograft model system and clinical serum specimens from patients with prostate cancer. Our results lay the foundation for the development of miRNAs as a novel class of blood-based cancer biomarkers and raise provocative questions regarding the mechanism of stability and potential biological function of circulating miRNAs. Results Identification and Molecular Cloning of Endogenous miRNAs fromHuman Plasma. Prior reports have suggested that RNA from human plasma (the noncellular component of blood remaining after removing cells by centrifugation) is largely of low molecular weight (11). W...

Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than 100 prostate cancer (PrCa)-susceptibility loci. We meta-analyzed genotype data from a custom high-density array of 46,939 PrCa cases and 27,910 controls of European ancestry with previously genotyped data of 32,255 PrCa cases and 33,202 controls of European ancestry. Our analysis identified 62 novel loci associated (P < 5.0 × 10) with PrCa and one locus significantly associated with early-onset PrCa (≤55 years). Our findings include missense variants rs1800057 (odds ratio (OR) = 1.16; P = 8.2 × 10; G>C, p.Pro1054Arg) in ATM and rs2066827 (OR = 1.06; P = 2.3 × 10; T>G, p.Val109Gly) in CDKN1B. The combination of all loci captured 28.4% of the PrCa familial relative risk, and a polygenic risk score conferred an elevated PrCa risk for men in the ninetieth to ninety-ninth percentiles (relative risk = 2.69; 95% confidence interval (CI): 2.55-2.82) and first percentile (relative risk = 5.71; 95% CI: 5.04-6.48) risk stratum compared with the population average. These findings improve risk prediction, enhance fine-mapping, and provide insight into the underlying biology of PrCa.

Nearly half of patients with recurrent prostate cancer after radical prostatectomy have a long-term PSA response to SRT when treatment is administered at the earliest sign of recurrence. The nomogram we developed predicts the outcome of SRT and should prove valuable for medical decision making for patients with a rising PSA level.

resulting in the suppression of AR target genes and clinical remissions that generally last several years (1). However, ADT is not curative. PC recurs as castration-resistant prostate cancer (CRPC), typically with reactivated AR signaling. Second-generation AR pathway inhibitors (ARIs), such as enzalutamide (ENZ) and abiraterone (ABI), were designed to further repress AR signaling and are primarily used to treat CRPC. Although these agents extend survival, durable complete responses are rare and these therapies also eventually fail (2, 3). Typically, the vast majority of metastatic CRPC (mCRPC) tumors progress with rising prostate-specific antigen (PSA/KLK3) levels despite standard of care treatment. Moreover, most mCRPC tumors are adenocarcinomas, which have robust AR program activity (4). Though rigorous epidemiological data are lacking, recent studies report that a substantial number of mCRPC tumors progressing on ARIs have lost AR signaling (5). Paralleling increased use of ARIs has been an increase in the proportion of treatment-resistant CRPC metastases that have AR-null phenotypes, i.e. tumors with diffuse small cell or neuroendocrine (NE) characteristics (SCNPC) or the recently described double-negative (DNPC) phenotype that lacks both NE and AR activity (5). A contemporary study evaluating the histology and molecular characteristics of 202 men with mCRPC found that 17% of the evaluable tumors were classified as SCNPC and this phenotype was associated with short-Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole-genome RNA sequencing, gene set enrichment analysis, and immunohistochemistry. Our analyses revealed 5 mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: AR-high tumors (ARPC), AR-low tumors (ARLPC), amphicrine tumors composed of cells coexpressing AR and NE genes (AMPC), double-negative tumors (i.e., AR-/NE-; DNPC), and tumors with small cell or NE gene expression without AR activity (SCNPC). RE1 silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the 5 mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.

Prostate cancer is a highly heritable disease with large disparities in incidence rates across ancestry populations. We conducted a multiancestry meta-analysis of prostate cancer genome-wide association studies (107,247 cases and 127,006 controls) and identified 86 new genetic risk variants independently associated with prostate cancer risk, bringing the total to 269 known risk variants. The top genetic risk score (GRS) decile was associated with odds ratios that ranged from 5.06 [95% confidence interval (CI) 4.84–5.29] for men of European ancestry to 3.74 [95% CI 3.36–4.17] for men of African ancestry. Men of African ancestry were estimated to have a mean GRS that was 2.18-times higher [95% CI 2.14–2.22], and men of East Asian ancestry 0.73-times lower [95% CI 0.71–0.76], than men of European ancestry. These findings support the role of germline variation contributing to population differences in prostate cancer risk, with the GRS offering an approach for personalized risk prediction.

Modification of microRNA sequences by the 3′ addition of nucleotides to generate so-called “isomiRs” adds to the complexity of miRNA function, with recent reports showing that 3′ modifications can influence miRNA stability and efficiency of target repression. Here, we show that the 3′ modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3′ nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3′ modification is a biologically regulated process. To investigate the mechanism of 3′ nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3′ miRNA modification activity in human cells. Multiple enzymes, including MTPAP, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3′ nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes–MTPAP, ZCCHC6, and TUT1–have not previously been known to modify miRNAs. Collectively, our results indicate that 3′ modification observed in next-generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo.

Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene expression. Androgen levels and androgen-regulated gene expression (by microarray profiling, quantitative reverse transcription-PCR, and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the gonadotropin-releasing hormone antagonist, Acyline, versus placebo in healthy men. To assess the effects of long-term ADT, gene expression measurements were evaluated at baseline and after 3, 6, and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized prostate cancer. Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, and TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and prostatespecific antigen (PSA), were not suppressed after short-term castration or after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in prostate cancer samples at each time point of ADT. Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing prostate cancer cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment. [Cancer Res 2007;67(10):5033-41]

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