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Background: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints., Results: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq., Conclusion: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

Background: Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA)., Methods: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR., Results: Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue., Conclusions: We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients' blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.

Phosphodiesterase 4D7 was recently shown to be specifically over-expressed in localized prostate cancer, raising the question as to which regulatory mechanisms are involved and whether other isoforms of this gene family (PDE4D) are affected under the same conditions.We investigated PDE4D isoform composition in prostatic tissues using a total of seven independent expression datasets and also included data on DNA methylation, copy number and AR and ERG binding in PDE4D promoters to gain insight into their effect on PDE4D transcription.We show that expression of PDE4D isoforms is consistently altered in primary human prostate cancer compared to benign tissue, with PDE4D7 being up-regulated while PDE4D5 and PDE4D9 are down-regulated. Disease progression is marked by an overall down-regulation of long PDE4D isoforms, while short isoforms (PDE4D1/2) appear to be relatively unaffected. While these alterations seem to be independent of copy number alterations in the PDE4D locus and driven by AR and ERG binding, we also observed increased DNA methylation in the promoter region of PDE4D5, indicating a long lasting alteration of the isoform composition in prostate cancer tissues.We propose two independent metrics that may serve as diagnostic and prognostic markers for prostate disease: (PDE4D7 - PDE4D5) provides an effective means for distinguishing PCa from normal adjacent prostate, whereas PDE4D1/2 - (PDE4D5 + PDE4D7 + PDE4D9) offers strong prognostic potential to detect aggressive forms of PCa and is associated with metastasis free survival. Overall, our findings highlight the relevance of PDE4D as prostate cancer biomarker and potential drug target.

Small nucleolar RNAs (snoRNAs) are dynamically regulated in different tissues and affected in disease. SnoRNAs are processed further to stable smaller RNAs. We sequenced the small RNA transcriptome of prostate cancer (PCa) at different PCa stages and generated a quantified catalogue of 3927 small non-coding RNAs (sncRNAs) detected in normal and malignant prostate tissue. From these, only 1524 are microRNAs. The remaining 2401 sncRNAs represent stable sncRNAs species that originate from snoRNA, tRNA and other sncRNAs. We show that snoRNA-derived RNAs (sdRNAs) display stronger differential expression than microRNAs and are massively upregulated in PCa. SdRNAs account for at least one third of all small RNAs with expression changes in tumor compared to normal adjacent tissue. Multiple sdRNAs can be produced from one snoRNA in a manner related to the conservation of structural snoRNA motifs. Q-PCR analysis in an independent patient cohort (n=106) confirmed the processing patterns of selected snoRNAs (SNORD44, SNORD78, SNORD74 and SNORD81) and the cancer-associated up-regulation of their sdRNAs observed in sequencing data. Importantly, expression of SNORD78 and its sdRNA is significantly higher in a subset of patients that developed metastatic disease demonstrating that snoRNA and sdRNAs may present as novel diagnostic and/or prognostic biomarkers for PCa.

Current prostate cancer (PCa) biomarkers such as PSA are not optimal in distinguishing cancer from benign prostate diseases and predicting disease outcome. To discover additional biomarkers, we investigated PCa-specific expression of novel unannotated transcripts. Using the unique probe design of Affymetrix Human Exon Arrays, we identified 334 candidates (EPCATs), of which 15 were validated by RT-PCR. Combined into a diagnostic panel, 11 EPCATs classified 80% of PCa samples correctly, while maintaining 100% specificity. High specificity was confirmed by in situ hybridization for EPCAT4R966 and EPCAT2F176 (SChLAP1) on extensive tissue microarrays. Besides being diagnostic, EPCAT2F176 and EPCAT4R966 showed significant association with pT-stage and were present in PIN lesions. We also found EPCAT2F176 and EPCAT2R709 to be associated with development of metastases and PCa-related death, and EPCAT2F176 to be enriched in lymph node metastases. Functional significance of expression of 9 EPCATs was investigated by siRNA transfection, revealing that knockdown of 5 different EPCATs impaired growth of LNCaP and 22RV1 PCa cells. Only the minority of EPCATs appear to be controlled by androgen receptor or ERG. Although the underlying transcriptional regulation is not fully understood, the novel PCa-associated transcripts are new diagnostic and prognostic markers with functional relevance to prostate cancer growth.

Background: Forkhead box 2 (FOXF2) is a member of the large family of forkhead transcription factors and its expression pattern suggests a role in prostate cancer development. FOXF2 expression is stroma-specific and higher expressed in the prostate transition zone than the prostate peripheral zone. Moreover, expression of FOXF2 is decreased in prostate cancer., Methods: To identify the genes and pathways regulated by FOXF2, we compared microarray expression profiles of primary prostate stromal cells (PrSC) treated with control or small interfering RNA (siRNA) directed against FOXF2., Results: From our microarray analyses, we selected 190 differentially expressed genes, of which 104 genes were higher expressed in PrSC cells treated with FOXF2 siRNA and 86 were higher expressed in PrSC cells treated with negative control siRNA. Eight of the strongest differentially expressed genes were validated by RT-PCR. Genes down-regulated by FOXF2 included MT1E, MT1F, PDGFA, ITGB1, and PSG7 and genes up-regulated by FOXF2 included WASF2, BAMBI, and CXCL12. Ingenuity pathway analysis showed several pathways significantly regulated by FOXF2, including PPAR signaling, PDGF signaling, and extracellular matrix (ECM) signaling. GSEA analysis revealed that FOXF2 up-regulated genes were down-regulated in the same PrSC cells treated with transforming growth factor 3 (TGFbeta3)., Conclusions: The distinct expression pattern of FOXF2 in the prostate, its effect on expression of ECM signaling, and its opposing role in the TGFbeta3 pathway, suggests a role for FOXF2 in prostate homeostasis and stroma-epithelial interactions.

Background: The insulin-like growth factor (IGF) system is important for pituitary development and control, with each member of this axis having a specific temporal and spatial expression. Because IGF-binding protein-5 (IGFBP-5) is one of the most highly expressed binding proteins in the anterior pituitary (AP), it might be of special importance in this gland., Objective: The purpose of this study was to examine the temporal relationship between the expression of the IGFs and IGFBP-5 in the AP during postnatal development., Design and Methods: Quantitative reverse transcription polymerase chain reaction was used to study the mRNA levels of these proteins in the AP of male and female rats at 10, 20, 30, 40 and 60 days of age., Results: The highest mRNA levels of IGF-I and II occurred at 10 and 20 days of age with a dramatic decrease at 30 days (p < 0.0001). IGF-I, but not IGF-II, mRNA levels increased again during adulthood (60 days). The pattern of IGFBP-5 mRNA was inversely expressed, with maximum values occurring at 40 days. IGF-I mRNA levels were higher in males at 10 days, but higher in females at 20 days. The expression of IGF-II was higher in males both at 10 and 20 days. IGFBP-5 gene expression was higher in females at 40 days., Conclusion: The dramatic changes in the expression of IGF-I, IGF-II and IGFBP-5 throughout postnatal development suggest that these factors play important roles in the development of this gland and that their actions are highly interrelated., (Copyright 2002 S. Karger AG, Basel)

The effects of growth hormone (GH) in regulating the expression of the hepatic and renal GH and insulin-like growth factor (IGF) system were studied by administering a novel GH receptor antagonist (GHRA) (B2036-PEG) at different doses (0, 1.25, 2.5, 5 and 10 mg/kg/day) to mice for 7 days. No differences were observed in the groups with respect to body weight, food consumption or blood glucose. However, a dose-dependent decrease was observed in circulating IGF-I levels and in hepatic and renal IGF-I levels at the highest doses. In contrast, in the 5 and 10 mg/kg/day GHRA groups, circulating and hepatic transcriptional IGF binding protein-3 (IGFBP-3) levels were not modified, likely resulting in a significantly decreased IGF-I/IGFBP-3 ratio. Hepatic GH receptor (GHR) and GH binding protein (GHBP) mRNA levels increased significantly in all GHRA dosage groups. Endogenous circulatory GH levels increased significantly in the 2.5 and 5 mg/kg/day GHRA groups. Remarkably, increased circulating IGFBP-4 and hepatic IGFBP-4 mRNA levels were observed in all GHRA administration groups. Renal GHR and GHBP mRNA levels were not modified by GHRA administration at the highest doses. Also, renal IGFBP-3 mRNA levels remained unchanged in most GHRA administration groups, whereas IGFBP-1, -4 and -5 mRNA levels were significantly increased in the 5 and 10 mg/kg/day GHRA administration groups. In conclusion, the effects of a specific GHR blockade on circulating, hepatic and renal GH/IGF axis reported here, may prove useful in the future clinical use of GHRAs.

Insulin-like growth factors (IGF-I and -II) promote cellular mitosis and differentiation and have been implicated in fetal and placental growth. Together with the IGF receptors and IGF binding proteins (IGFBPs) they form a complex network, with tissue specific activity. This review will discuss the data generated to elucidate the functions of the IGF system during mouse development.