A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer.

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5-7%, 1-2%, and 1-2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.

miRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4.

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.

Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl-tRNA synthetase 1.

Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.

The long non-coding RNA SPRIGHTLY and its binding partner PTBP1 regulate exon 5 skipping of SMYD3 transcripts in group 4 medulloblastomas.

Background: Although some of the regulatory genes, signaling pathways, and gene regulatory networks altered in medulloblastomas (MB) are known, the roles of non-coding RNAs, particularly long non-coding RNAs (lncRNAs), are poorly described. Here we report that the lncRNA SPRIGHTLY (SPRY4-IT1) gene is upregulated in group 4 medulloblastoma (G4 MB). Methods: SPRIGHTLY expression was assessed in MB subgroup patient-derived xenografts, cell lines, and patient samples. The effect of SPRIGHTLY hemizygous deletion on proliferation, invasion, apoptosis, and colony formation were assessed in vitro and on tumor growth in vivo. dChIRP pull-down assays were used to assess SPRIGHTLY-binding partners, confirmed by immunoprecipitation. SMYD3 ΔE5 transcripts were examined in cell lines and publicly available RNA-seq data. Pathway analysis was performed by phospho-kinase profiling and RNA-seq. Results: CRISPR/Cas9 deletion of SPRIGHTLY reduced cell viability and invasion and increased apoptosis in G4 MB cell lines in vitro. SPRIGHTLY hemizygous-deleted G4 MB cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells expressing both copies of SPRIGHTLY. SPRIGHTLY lncRNA bound to the intronic region of the SMYD3 pre-mRNA transcript. SPRIGHTLY also interacted with PTPB1 protein to regulate SMYD3 exon skipping to produce an aberrant protein. SPRIGHTLY-driven SMYD3 regulation enhanced the expression of EGFR pathway genes in G4 MB cell lines and activated cell coagulation/hemostasis-related gene expression, suggesting a novel oncogenic role in G4 MB. Conclusions: These results demonstrate the importance of SPRIGHTLY lncRNA as a promoter of G4 MB and the role of the SPRIGHTLY-SMYD3-PTPB1 axis as an important oncogenic regulator in MB.

Medulloblastoma cerebrospinal fluid reveals metabolites and lipids indicative of hypoxia and cancer-specific RNAs.

Medulloblastoma (MB) is the most common malignant brain tumor in children. There remains an unmet need for diagnostics to sensitively detect the disease, particularly recurrences. Cerebrospinal fluid (CSF) provides a window into the central nervous system, and liquid biopsy of CSF could provide a relatively non-invasive means for disease diagnosis. There has yet to be an integrated analysis of the transcriptomic, metabolomic, and lipidomic changes occurring in the CSF of children with MB. CSF samples from patients with (n = 40) or without (n = 11; no cancer) MB were subjected to RNA-sequencing and high-resolution mass spectrometry to identify RNA, metabolite, and lipid profiles. Differentially expressed transcripts, metabolites, and lipids were identified and their biological significance assessed by pathway analysis. The DIABLO multivariate analysis package (R package mixOmics) was used to integrate the molecular changes characterizing the CSF of MB patients. Differentially expressed transcripts, metabolites, and lipids in CSF were discriminatory for the presence of MB but not the exact molecular subtype. One hundred and ten genes and ten circular RNAs were differentially expressed in MB CSF compared with normal, representing TGF-ß signaling, TNF-α signaling via NF-kB, and adipogenesis pathways. Tricarboxylic acid cycle and other metabolites (malate, fumarate, succinate, α-ketoglutarate, hydroxypyruvate, N-acetyl-aspartate) and total triacylglycerols were significantly upregulated in MB CSF compared with normal CSF. Although separating MBs into subgroups using transcriptomic, metabolomic, and lipid signatures in CSF was challenging, we were able to identify a group of omics signatures that could separate cancer from normal CSF. Metabolic and lipidomic profiles both contained indicators of tumor hypoxia. Our approach provides several candidate signatures that deserve further validation, including the novel circular RNA circ_463, and insights into the impact of MB on the CSF microenvironment.

Mapping genetic variability in mature miRNAs and miRNA binding sites in prostate cancer.

MicroRNAs (miRNAs) regulate diverse cancer hallmarks through sequence-specific regulation of gene expression, so genetic variability in their seed sequences or target sites could be responsible for cancer initiation or progression. While several efforts have been made to predict the locations of single nucleotide variants (SNVs) at miRNA target sites and associate them with cancer risk and susceptibility, there have been few direct assessments of SNVs in both mature miRNAs and their target sites to assess their impact on miRNA function in cancers. Using genome-wide target capture of miRNAs and miRNA-binding sites followed by deep sequencing in prostate cancer cell lines, here we identified prostate cancer-specific SNVs in mature miRNAs and their target binding sites. SNV rs9860655 in the mature sequence of miR-570 was not present in benign prostate hyperplasia (BPH) tissue or cell lines but was detectable in clinical prostate cancer tissue samples and adjacent normal tissue. SLC45A3 (prostein), a putative oncogene target of miR-1178, was highly upregulated in PC3 cells harboring an miR-1178 seed sequence SNV. Finally, systematic assessment of losses and gains of miRNA targets through 3'UTR SNVs revealed SNV-associated changes in target oncogene and tumor suppressor gene expression that might be associated with prostate carcinogenesis. Further work is required to systematically assess the functional effects of miRNA SNVs.

Rapid Diagnosis of Prostate Cancer Disease Progression Using Paper Spray Ionization Mass Spectrometry.

The limitation of prostate specific antigen (PSA) for prostate cancer (PC) diagnosis is well-recognized. The Gleason score (GS) has been the most widely used grading system for prostate tumor differentiation and represents the best-established prognostic indicator for prostate cancer progression. However, a rapid and sensitive noninvasive diagnostic marker that differentiates GS-based prostate cancer disease progression is needed. As PC is becoming a leading cause of cancer related death for men in the U.S. and worldwide, an immediate need exists for an improved, sensitive, noninvasive, and rapid diagnostic test for PC screening. Here, we employed paper spray ionization-mass spectrometry (PSI MS)-based global metabolomics of urine liquid biopsies to distinguish between healthy (negative for any prostate specific health problems) and progressive PC states (low grade PC such as GS6 and high-grade PC such as GS7, GS8, and GS9). For PSI-MS-based direct untargeted metabolic investigation, a raw urine sample was directly pipetted onto a triangular paper substrate, without any additional sample preparation. Multivariate statistical analysis revealed distinct GS-specific metabolic signatures compared to a healthy control. Variable importance in projection from partial least-squares-discriminant analysis showed distinct metabolic patterns that were correlatively elevated with progressive disease and could serve as biomarkers for diagnosis of prostate cancer risk categorization.

The long noncoding RNA lnc-HLX-2-7 is oncogenic in Group 3 medulloblastomas.

BACKGROUND: Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the noncoding RNA genome, in particular long noncoding RNAs (lncRNAs), contributes to MB subgrouping. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential molecular marker and therapeutic target in Group 3 MBs. METHODS: Publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify lncRNAs that differentiate between MB subgroups. After characterizing a subset of differentially expressed lncRNAs in vitro and in vivo, lnc-HLX-2-7 was deleted by CRISPR/Cas9 in the MB cell line. Intracranial injected tumors were further characterized by bulk and single-cell RNA-seq. RESULTS: Lnc-HLX-2-7 is highly upregulated in Group 3 MB cell lines, patient-derived xenografts, and primary MBs compared with other MB subgroups as assessed by quantitative real-time, RNA-seq, and RNA fluorescence in situ hybridization. Depletion of lnc-HLX-2-7 significantly reduced cell proliferation and 3D colony formation and induced apoptosis. Lnc-HLX-2-7-deleted cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells. Pathway analysis revealed that lnc-HLX-2-7 modulated oxidative phosphorylation, mitochondrial dysfunction, and sirtuin signaling pathways. The MYC oncogene regulated lnc-HLX-2-7, and the small-molecule bromodomain and extraterminal domain family‒bromodomain 4 inhibitor Jun Qi 1 (JQ1) reduced lnc-HLX-2-7 expression. CONCLUSIONS: Lnc-HLX-2-7 is oncogenic in MB and represents a promising novel molecular marker and a potential therapeutic target in Group 3 MBs.

MicroRNA-211 Modulates the DUSP6-ERK5 Signaling Axis to Promote BRAFV600E-Driven Melanoma Growth In Vivo and BRAF/MEK Inhibitor Resistance.

MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells in vitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts in vivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.

Integrated RNA and metabolite profiling of urine liquid biopsies for prostate cancer biomarker discovery.

Sensitive and specific diagnostic and prognostic biomarkers for prostate cancer (PCa) are urgently needed. Urine samples are a non-invasive means to obtain abundant and readily accessible "liquid biopsies". Herein we used urine liquid biopsies to identify and characterize a novel group of urine-enriched RNAs and metabolites in patients with PCa and normal individuals with or without benign prostatic disease. Differentially expressed RNAs were identified in urine samples by deep sequencing and metabolites in urine were measured by mass spectrometry. mRNA and metabolite profiles were distinct in patients with benign and malignant disease. Integrated analysis of urinary gene expression and metabolite signatures unveiled an aberrant glutamate metabolism and tricarboxylic acid (TCA) cycle node in prostate cancer-derived cells. Functional validation supported a role for glutamate metabolism and glutamate oxaloacetate transaminase 1 (GOT1)-dependent redox balance in PCa, which could be exploited for novel biomarkers and therapies. In this study, we discovered cancer-specific changes in urinary RNAs and metabolites, paving the way for the development of sensitive and specific urinary PCa diagnostic biomarkers either alone or in combination. Our methodology was based on single void urine samples (i.e., without prostatic massage). The integrated analysis of metabolomic and transcriptomic data from these liquid biopsies revealed a glutamate metabolism and tricarboxylic acid cycle node that was specific to prostate-derived cancer cells and cancer-specific metabolic changes in urine.

Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

RNA half-life is closely related to its cellular physiological function, so stability determinants may have regulatory functions. Micro(mi)RNAs have primarily been studied with respect to post-transcriptional mRNA regulation and target degradation. Here we study the impact of the tumour suppressive melanoma miRNA miR-211 on transcriptome stability and phenotype in the non-pigmented melanoma cell line, A375. Using 5'-bromouridine IP chase (BRIC)-seq, transcriptome-wide RNA stability profiles revealed highly regulated genes and pathways important in this melanoma cell line. By combining BRIC-seq, RNA-seq and in silico predictions, we identified both existing and novel direct miR-211 targets. We validated DUSP3 as one such novel miR-211 target, which itself sustains colony formation and invasion in A375 cells via MAPK/PI3K signalling. miRNAs have the capacity to control RNA turnover as a gene expression mechanism, and RNA stability profiling is an excellent tool for interrogating functionally relevant gene regulatory pathways and miRNA targets when combined with other high-throughput and in silico approaches.

MicroRNA-211 Loss Promotes Metabolic Vulnerability and BRAF Inhibitor Sensitivity in Melanoma.

The clinical management of malignant melanoma remains a challenge because these tumors are intrinsically aggressive and prone to therapeutic resistance. MicroRNA (miR)-211 is an emerging melanoma oncogene. Melanoma metabolism adapts to promote survival, including in response to BRAFV600E inhibition, but how miR-211 participates in this process is unknown. Here, we generated miR-211 loss-of-function cell lines using CRISPR/Cas9 technology and show that miR-211 loss slowed growth and invasion in vitro, inhibited phosphoinositol-3-kinase signaling, and inhibited melanoma growth in vivo. miR-211 deficiency rendered melanoma cells metabolically vulnerable by attenuating mitochondrial respiration and tricarboxylic acid cycling. miR-211 was up-regulated by the BRAF inhibitor vemurafenib and in vemurafenib-resistant melanoma cells, with miR-211 loss rendering them more drug sensitive. miR-211 loss represents a "two-pronged" anticancer strategy by inhibiting both critical growth-promoting cell signaling pathways and rendering cells metabolically vulnerable, making it an extremely attractive and specific candidate combinatorial therapeutic target in melanoma.

The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules.

Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA's three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas.

MicroRNA-211 Regulates Oxidative Phosphorylation and Energy Metabolism in Human Vitiligo.

Vitiligo is a common chronic skin disorder characterized by loss of epidermal melanocytes and progressive depigmentation. Vitiligo has complex immune, genetic, environmental, and biochemical causes, but the exact molecular mechanisms of vitiligo development and progression, particularly those related to metabolic control, are poorly understood. In this study we characterized the human vitiligo cell line PIG3V and the normal human melanocyte line HEM-l by RNA sequencing, targeted metabolomics, and shotgun lipidomics. Melanocyte-enriched microRNA-211, a known metabolic switch in nonpigmented melanoma cells, was severely down-regulated in vitiligo cell line PIG3V and skin biopsy samples from vitiligo patients, whereas its predicted targets PPARGC1A, RRM2, and TAOK1 were reciprocally up-regulated. microRNA-211 binds to PGC1-α 3' untranslated region locus and represses it. Although mitochondrial numbers were constant, mitochondrial complexes I, II, and IV and respiratory responses were defective in vitiligo cells. Nanoparticle-coated microRNA-211 partially augmented the oxygen consumption rate in PIG3V cells. The lower oxygen consumption rate, changes in lipid and metabolite profiles, and increased reactive oxygen species production observed in vitiligo cells appear to be partly due to abnormal regulation of microRNA-211 and its target genes. These genes represent potential biomarkers and therapeutic targets in human vitiligo.

Correction: DNA Damage, Homology-Directed Repair, and DNA Methylation.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

The Long Noncoding RNA SPRIGHTLY Regulates Cell Proliferation in Primary Human Melanocytes.

The long noncoding RNA SPRIGHTLY (formerly SPRY4-IT1), which lies within the intronic region of the SPRY4 gene, is up-regulated in human melanoma cells compared to melanocytes. SPRIGHTLY regulates a number of cancer hallmarks, including proliferation, motility, and apoptosis. To better understand its oncogenic role, SPRIGHTLY was stably transfected into human melanocytes, which resulted in increased cellular proliferation, colony formation, invasion, and development of a multinucleated dendritic-like phenotype. RNA sequencing and mass spectrometric analysis of SPRIGHTLY-expressing cells revealed changes in the expression of genes involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage responses, and cell cycle. The proliferation marker Ki67, minichromosome maintenance genes 2-5, antiapoptotic gene X-linked inhibitor of apoptosis, and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes, whereas the proapoptotic tumor suppressor gene DPPIV/CD26 was down-regulated, followed by an increase in extracellular signal-regulated kinase 1/2 phosphorylation, suggesting an increase in mitogen-activated protein kinase activity. Because down-regulation of DPPIV is known to be associated with malignant transformation in melanocytes, SPRIGHTLY-mediated DPPIV down-regulation may play an important role in melanoma pathobiology. Together, these findings provide important insights into how SPRIGHTLY regulates cell proliferation and anchorage-independent colony formation in primary human melanocytes.

MicroRNA 211 Functions as a Metabolic Switch in Human Melanoma Cells.

MicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1α (HIF-1α) protein levels and decreased cell growth during hypoxia. HIF-1α protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1α protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations-a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.

Structure-Activity Relationship of Indole-Tethered Pyrimidine Derivatives that Concurrently Inhibit Epidermal Growth Factor Receptor and Other Angiokinases.

Antiangiogenic agents have been widely investigated in combination with standard chemotherapy or targeted cancer agents for better management of advanced cancers. Therapeutic agents that concurrently inhibit epidermal growth factor receptor and other angiokinases could be useful alternatives to combination therapies for epidermal growth factor receptor-dependent cancers. Here, we report the synthesis of an indole derivative of pazopanib using a bioisosteric replacement strategy, which was designated MKP101. MKP101 inhibited not only the epidermal growth factor receptor with an IC50 value of 43 nM but also inhibited angiokinases as potently as pazopanib. In addition, MKP101 effectively inhibited vascular endothelial growth factor-induced endothelial proliferation, tube formation, migration of human umbilical vein endothelial cells and proliferation of HCC827, an epidermal growth factor receptor-addicted cancer cell line. A docking model of MKP101 and the kinase domain of the epidermal growth factor receptor was generated to predict its binding mode, and validated by synthesizing and evaluating MKP101 derivatives. Additionally, a study of structure-activity relationships of indolylamino or indolyloxy pyrimidine analogues derived from MKP101 demonstrated that selectivity for epidermal growth factor receptor and other angiokinases, especially vascular endothelial growth factor receptor 2 depends on the position of substituents on pyrimidine and the type of link between pyrimidine and the indole moiety. We believe that this study could provide a basis for developing angiokinase inhibitors having high affinity for the epidermal growth factor receptor, from the pyrimidine scaffold.

Long noncoding RNAs as putative biomarkers for prostate cancer detection.

Prostate cancer is one of the leading causes of mortality among US males. There is an urgent unmet need to develop sensitive and specific biomarkers for the early detection of prostate cancer to reduce overtreatment and accompanying morbidity. We identified a group of differentially expressed long noncoding RNAs in prostate cancer cell lines and patient samples and further characterized six long noncoding RNAs (AK024556, XLOC_007697, LOC100287482, XLOC_005327, XLOC_008559, and XLOC_009911) in prostatic adenocarcinoma tissue samples (Gleason score >6.0) and compared them with matched normal (healthy) tissues. Interestingly, these markers were also successfully detected in patient urine samples and were found to be up-regulated when compared with normal (healthy) urine. AK024556 (SPRY4-IT1) was highly up-regulated in human prostate cancer cell line PC3 but not in LNCaP, and siRNA knockdown of SPRY4-IT1 in PC3 cells inhibited cell proliferation and invasion and increased cell apoptosis. Chromogenic in situ hybridization assay was developed to detect long noncoding RNAs in primary prostatic adenocarcinoma tissue samples, paving the way for clinical diagnostics. We believe that these results will set the stage for more extensive studies to develop novel long noncoding RNA-based diagnostic assays for early prostate cancer detection and will help to distinguish benign prostate cancer from precancerous lesions.