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OBJECTIVES: DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein-coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA-mediated cleavage of two mRNAs, Myl9 and Todr1. The goal of this study is to determine whether this pathway is conserved in human HSCs. METHODS: DROSHA and DICER were knocked down in human cord blood CD34+ HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5' phosphorylated RNAs. RESULTS: Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1. CONCLUSION: A miRNA-independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA-independent function that is essential for safeguarding the pluripotency of human HSCs.

It is now appreciated that long non-coding RNAs (lncRNAs) are important players in orchestrating cancer progression. In this study we characterized GHSROS, a human lncRNA gene on the opposite DNA strand (antisense) to the ghrelin receptor gene, in prostate cancer. The lncRNA was upregulated by prostate tumors from different clinical datasets. Transcriptome data revealed that GHSROS alters the expression of cancer-associated genes. Functional analyses in vitro showed that GHSROS mediates tumor growth, migration and survival, and resistance to the cytotoxic drug docetaxel. Increased cellular proliferation of GHSROS-overexpressing PC3, DU145, and LNCaP prostate cancer cell lines in vitro was recapitulated in a subcutaneous xenograft model. Conversely, in vitro antisense oligonucleotide inhibition of the lncRNA reciprocally regulated cell growth and migration, and gene expression. Notably, GHSROS modulates the expression of PPP2R2C, the loss of which may drive androgen receptor pathway-independent prostate tumor progression in a subset of prostate cancers. Collectively, our findings suggest that GHSROS can reprogram prostate cancer cells toward a more aggressive phenotype and that this lncRNA may represent a potential therapeutic target.

It is now appreciated that long non-coding RNAs (lncRNAs) are important players in orchestrating cancer progression. In this study we characterized GHSROS, a human lncRNA gene on the opposite DNA strand (antisense) to the ghrelin receptor gene, in prostate cancer. The lncRNA was upregulated by prostate tumors from different clinical datasets. Transcriptome data revealed that GHSROS alters the expression of cancer-associated genes. Functional analyses in vitro showed that GHSROS mediates tumor growth, migration and survival, and resistance to the cytotoxic drug docetaxel. Increased cellular proliferation of GHSROS-overexpressing PC3, DU145, and LNCaP prostate cancer cell lines in vitro was recapitulated in a subcutaneous xenograft model. Conversely, in vitro antisense oligonucleotide inhibition of the lncRNA reciprocally regulated cell growth and migration, and gene expression. Notably, GHSROS modulates the expression of PPP2R2C, the loss of which may drive androgen receptor pathway-independent prostate tumor progression in a subset of prostate cancers. Collectively, our findings suggest that GHSROS can reprogram prostate cancer cells toward a more aggressive phenotype and that this lncRNA may represent a potential therapeutic target.

Recent evidence suggests that numerous long non‑coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription‑quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA‑MB‑231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA‑MB‑231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.

Recent evidence suggests that numerous long non‑coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription‑quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA‑MB‑231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA‑MB‑231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.

Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated w

Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated w

Short tandem repeats (STRs) are repetitive sequences of a polymorphic stretch of two to six nucleotides. We hypothesized that STRs are associated with prostate cancer development and/or progression. We undertook RNA sequencing analysis of prostate tumors and adjacent non-malignant cells to identify polymorphic STRs that are readily expressed in these cells. Most of the expressed STRs in the clinical samples mapped to intronic and intergenic DNA. Our analysis indicated that three of these STRs (TAAA-ACTG2, TTTTG-TRIB1, and TG-PCA3) are polymorphic and differentially expressed in prostate tumors compared to adjacent non-malignant cells. TG-PCA3 STR expression was repressed by the anti-androgen drug enzalutamide in prostate cancer cells. Genetic analysis of prostate cancer patients and healthy controls (N > 2,000) showed a significant association of the most common 11 repeat allele of TG-PCA3 STR with prostate cancer risk (OR = 1.49; 95% CI 1.11-1.99; P = 0.008). A significant association was also observed with aggressive disease (OR = 2.00; 95% CI 1.06-3.76; P = 0.031) and high mortality rates (HR = 3.0; 95% CI 1.03-8.77; P = 0.045). We propose that TG-PCA3 STR has both diagnostic and prognostic potential for prostate cancer. We provided a proof of concept to be applied to other RNA sequencing datasets to identify disease-associated STRs for future clinical exploratory studies.

Short tandem repeats (STRs) are repetitive sequences of a polymorphic stretch of two to six nucleotides. We hypothesized that STRs are associated with prostate cancer development and/or progression. We undertook RNA sequencing analysis of prostate tumors and adjacent non-malignant cells to identify polymorphic STRs that are readily expressed in these cells. Most of the expressed STRs in the clinical samples mapped to intronic and intergenic DNA. Our analysis indicated that three of these STRs (TAAA-ACTG2, TTTTG-TRIB1, and TG-PCA3) are polymorphic and differentially expressed in prostate tumors compared to adjacent non-malignant cells. TG-PCA3 STR expression was repressed by the anti-androgen drug enzalutamide in prostate cancer cells. Genetic analysis of prostate cancer patients and healthy controls (N > 2,000) showed a significant association of the most common 11 repeat allele of TG-PCA3 STR with prostate cancer risk (OR = 1.49; 95% CI 1.11-1.99; P = 0.008). A significant association was also observed with aggressive disease (OR = 2.00; 95% CI 1.06-3.76; P = 0.031) and high mortality rates (HR = 3.0; 95% CI 1.03-8.77; P = 0.045). We propose that TG-PCA3 STR has both diagnostic and prognostic potential for prostate cancer. We provided a proof of concept to be applied to other RNA sequencing datasets to identify disease-associated STRs for future clinical exploratory studies.

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression.

BACKGROUND: Fusion transcripts are found in many tissues and have the potential to create novel functional products. Here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. We analyzed data from prostate (cancer) cells as previous studies have shown extensively that these cells readily undergo fusion transcription. RESULTS: We used the FusionMap program to identify high-confidence fusion transcripts from RNAseq data. The RNAseq datasets were from our (N = 8) and other (N = 14) clinical prostate tumors with adjacent non-cancer cells, and from the LNCaP prostate cancer cell line that were mock-, androgen- (DHT), and anti-androgen- (bicalutamide, enzalutamide) treated. In total, 185 fusion transcripts were identified from all RNAseq datasets. The majority (76%) of these fusion transcripts were 'read-through chimeras' derived from adjacent genes in the genome. Characterization of sequences at fusion loci were carried out using a combination of the FusionMap program, custom Perl scripts, and the RNAfold program. Our computational analysis indicated that most fusion junctions (76%) use the consensus GT-AG intron donor-acceptor splice site, and most fusion transcripts (85%) maintained the open reading frame. We assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. Our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. The abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that SINE repeats are involved in aberrant gene transcription. We found few instances of repetitive sequences at both fusion and non-fusion junctions. Finally, RT-qPCR was performed on RNA from both clinical prostate tumors and ad

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.