Differentially Expressed Extracellular Vesicle-Contained microRNAs before and after Transurethral Resection of Bladder Tumors.

Bladder cancer (BC) is currently diagnosed and monitored by cystoscopy, a costly and invasive procedure. Potential biomarkers in urine, blood, and, more recently, extracellular vesicles (EVs), have been explored as non-invasive alternatives for diagnosis and surveillance of BC. EVs are nanovesicles secreted by most cell types containing diverse molecular cargo, including different types of small RNAs, such as microRNA (miRNA). In this study, we performed next-generation sequencing of EV-contained miRNA isolated from urine and serum of 41 patients with non-muscle invasive BC (27 stage Ta, 14 stage T1) and 15 non-cancer patients (NCP) with benign cystoscopy findings. MiRNA sequencing was also performed on serum supernatant samples for T1 patients. To identify potential BC-specific biomarkers, expression levels of miRNA in presurgery samples were compared to those at postsurgery check-ups, and to NCPs. Results showed that two miRNAs, urinary EV-contained miR-451a and miR-486-5p, were significantly upregulated in presurgery samples from T1 patients compared to postsurgery check-up samples. This was confirmed in a replica EV/RNA isolation and sequencing run of 10 T1 patients from the primary run; however, analyses revealed no differential expression of miRNAs in serum EVs, serum supernatant, or when comparing BC patients to NCPs. This is the first study to investigate EV-containing miRNA sequencing in pre- and postsurgery BC patient samples and our findings suggest that urinary EV-contained miR-451a and miR-486-5p may be potential biomarkers for recurrence-free survival of BC patients with stage T1 disease.

Myeloma-derived extracellular vesicles mediate HGF/c-Met signaling in osteoblast-like cells.

Multiple myeloma is an incurable cancer of antibody-producing plasma cells. Hepatocyte growth factor (HGF), a cytokine aberrantly expressed in half of myeloma patients, is involved in myeloma pathogenesis by enhancing myeloma growth and invasiveness, and may play a role in myeloma bone disease by inhibiting osteoblastogenesis. In this study, we investigated whether extracellular vesicles (EVs) may play a role in HGF signaling between myeloma cells and osteoblast-like target cells. EVs from the HGF-positive cell line JJN-3 and the HGF-negative cell line INA-6, and from bone marrow plasma and primary human myeloma cells, were isolated using sequential centrifugation techniques and the presence of HGF on the EV-surface was investigated with ELISA. EVs from both cell lines were added to an established bioassay where HGF is known to induce interleukin-11 secretion in osteoblast-like cells. Our results show that HGF was bound to the surface of JJN-3-derived EVs, while INA-6-derived EVs were negative for HGF. Only JJN-3-derived EVs induced IL-11 secretion in osteoblast-like recipient cells. When osteoblast-like cells were preincubated with a specific HGF-receptor (c-Met) inhibitor, no induction of interleukin-11 was observed. Downstream c-Met phosphorylation was demonstrated by immunoblotting. EVs isolated from bone marrow plasma and primary myeloma cells were HGF-positive for a subset of myeloma patients. Taken together, this work shows for the first time that HGF bound on the surface of myeloma-derived EVs can effectuate HGF/c-Met signaling in osteoblast-like cells. Myeloma-derived EVs may play a role in myeloma bone disease by induction of the osteoclast-activating cytokine interleukin-11 in osteoblasts.

Allelic mutations in noncoding genomic sequences construct novel transcription factor binding sites that promote gene overexpression.

The growth and survival factor hepatocyte growth factor (HGF) is expressed at high levels in multiple myeloma (MM) cells. We report here that elevated HGF transcription in MM was traced to DNA mutations in the promoter alleles of HGF. Sequence analysis revealed a previously undiscovered single-nucleotide polymorphism (SNP) and crucial single-nucleotide variants (SNVs) in the promoters of myeloma cells that produce large amounts of HGF. The allele-specific mutations functionally reassembled wild-type sequences into the motifs that affiliate with endogenous transcription factors NFKB (nuclear factor kappa-B), MZF1 (myeloid zinc finger 1), and NRF-2 (nuclear factor erythroid 2-related factor 2). In vitro, a mutant allele that gained novel NFKB-binding sites directly responded to transcriptional signaling induced by tumor necrosis factor alpha (TNFα) to promote high levels of luciferase reporter. Given the recent discovery by genome-wide sequencing (GWS) of numerous non-coding mutations in myeloma genomes, our data provide evidence that heterogeneous SNVs in the gene regulatory regions may frequently transform wild-type alleles into novel transcription factor binding properties to aberrantly interact with dysregulated transcriptional signals in MM and other cancer cells.

tRNA-Derived Fragments as Biomarkers in Bladder Cancer.

Bladder cancer (BC) diagnosis is reliant on cystoscopy, an invasive procedure associated with urinary tract infections. This has sparked interest in identifying noninvasive biomarkers in body fluids such as blood and urine. A source of biomarkers in these biofluids are extracellular vesicles (EVs), nanosized vesicles that contain a wide array of molecular cargo, including small noncoding RNA such as transfer RNA-derived fragments (tRF) and microRNA. Here, we performed small-RNA next-generation sequencing from EVs from urine and serum, as well as from serum supernatant. RNA was extracted from 15 non-cancer patients (NCPs) with benign findings in cystoscopy and 41 patients with non-muscle invasive BC. Urine and serum were collected before transurethral resection of bladder tumors (TUR-b) and at routine post-surgery check-ups. We compared levels of tRFs in pre-surgery samples to samples from NCPs and post-surgery check-ups. To further verify our findings, samples from 10 patients with stage T1 disease were resequenced. When comparing tRF expression in urine EVs between T1 stage BC patients and NCPs, 14 differentially expressed tRFs (DEtRFs) were identified. In serum supernatant, six DEtRFs were identified among stage T1 patients when comparing pre-surgery to post-surgery samples and four DEtRFs were found when comparing pre-surgery samples to NCPs. By performing a blast search, we found that sequences of DEtRFs aligned with genomic sequences pertaining to processes relevant to cancer development, such as enhancers, regulatory elements and CpG islands. Our findings display a number of tRFs that may hold potential as biomarkers for the diagnosis and recurrence-free survival of BC.

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination.

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses.

PSKH1, a novel splice factor compartment-associated serine kinase.

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in splicing factor compartments (SFCs) within the nucleus of interphase cells. Nuclear SFCs are considered mainly as storage sites for splicing factors, supplying splicing factors to active genes. The mechanisms controlling the interaction of the various spliceosome constituents, and the dynamic nature of the SFCs, are still poorly understood. We show here that endogenous PSKH1, a previously cloned kinase, is located in SFCs. Migration of PSKH1-FLAG into SFCs is enhanced during co-expression of T7-tagged ASF/SF2 as well as other members of the SR protein family, but not by two other non-SR nuclear proteins serving as controls. Similar to the SR protein kinase family, overexpression of PSKH1 led to reorganization of co-expressed T7-SC35 and T7-ASF/SF2 into a more diffuse nuclear pattern. This redistribution was not dependent on PSKH1 kinase activity. Different from the SR protein kinases, the SFC-associating features of PSKH1 were located within its catalytic kinase domain and within its C-terminus. Although no direct interaction was observed between PSKH1 and any of the SR proteins tested in pull-down or yeast two-hybrid assays, forced expression of PSKH1-FLAG was shown to stimulate distal splicing of an E1A minigene in HeLa cells. Moreover, a GST-ASF/SF2 fusion was not phosphorylated by PSKH1, suggesting an indirect mechanism of action on SR proteins. Our data suggest a mutual relationship between PSKH1 and SR proteins, as they are able to target PSKH1 into SFCs, while forced PSKH1 expression modulates nuclear dynamics and the function of co-expressed splicing factors.

A-kinase anchoring protein AKAP95 is a novel regulator of ribosomal RNA synthesis.

The RNA polymerase I transcription apparatus acquires and integrates the combined information from multiple cellular signalling cascades to regulate ribosome production essential for cell growth and proliferation. In the present study, we show that a subpopulation of A-kinase anchoring protein 95 (AKAP95) targets the nucleolus during interphase and is involved in regulating rRNA production. We show that AKAP95 co-localizes with the nucleolar upstream binding factor, an essential rRNA transcription factor. Similar to other members of the C2 H2 -zinc finger family, we show, using systematic selection and evolution of ligands by exponential enrichment and in vitro binding analysis, that AKAP95 has a preference for GC-rich DNA in vitro, whereas fluorescence recovery after photobleaching analysis reveals AKAP95 to be a highly mobile protein that exhibits RNA polymerase I and II dependent nucleolar trafficking. In line with its GC-binding features, chromatin immunoprecipitation analysis revealed AKAP95 to be associated with ribosomal chromatin in vivo. Manipulation of AKAP95-expression in U2OS cells revealed a reciprocal relationship between the expression of AKAP95 and 47S rRNA. Taken together, our data indicate that AKAP95 is a novel nucleolus-associated protein with a regulatory role on rRNA production.

Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro.

BACKGROUND: Multiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO)-receptor (EPOR) signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated. METHODS: Myeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this study. Transcript levels were analysed by quantitative PCR and cell surface levels of EPOR in primary cells by flow cytometry. Knockdown of EPOR by short interfering RNA was used to show specific EPOR signaling in the myeloma cell line INA-6. Flow cytometry was used to assess viability in primary cells treated with EPO in the presence and absence of neutralizing anti-EPOR antibodies. Gene expression data for total therapy 2 (TT2), total therapy 3A (TT3A) trials and APEX 039 and 040 were retrieved from NIH GEO omnibus and EBI ArrayExpress. RESULTS: We show that the EPOR is expressed in myeloma cell lines and in primary myeloma cells both at the mRNA and protein level. Exposure to recombinant human EPO (rhEPO) reduced viability of INA-6 myeloma cell line and of primary myeloma cells. This effect could be partially reversed by neutralizing antibodies against EPOR. In INA-6 cells and primary myeloma cells, janus kinase 2 (JAK-2) and extracellular signal regulated kinase 1 and 2 (ERK-1/2) were phosphorylated by rhEPO treatment. Knockdown of EPOR expression in INA-6 cells reduced rhEPO-induced phospo-JAK-2 and phospho-ERK-1/2. Co-cultures of primary myeloma cells with bone marrow-derived stroma cells did not protect the myeloma cells from rhEPO-induced cell death. In four different clinical trials, survival data linked to gene expression analysis indicated that high levels of EPOR mRNA were associated with better survival. CONCLUSIONS: Our results demonstrate for the first time active EPOR signaling in malignant plasma cells. EPO-mediated EPOR signaling reduced the viability of myeloma cell lines and of malignant primary plasma cells in vitro. Our results encourage further studies to investigate the importance of EPO/EPOR in multiple myeloma progression and treatment. TRIAL REGISTRATION: [Trial registration number for Total Therapy (TT) 2: NCT00083551 and TT3: NCT00081939 ].

Human Breast Milk miRNA, Maternal Probiotic Supplementation and Atopic Dermatitis in Offspring.

BACKGROUND: Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in infancy in a number of randomised trials. The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial involved a probiotic supplementation regime given solely to mothers in the perinatal period and demonstrated a ~40% relative risk reduction in the cumulative incidence of AD at 2 years of age. However, the mechanisms behind this effect are incompletely understood. Micro-RNAs (miRNA) are abundant in mammalian milk and may influence the developing gastrointestinal and immune systems of newborn infants. The objectives of this study were to describe the miRNA profile of human breast milk, and to investigate breast milk miRNAs as possible mediators of the observed preventative effect of probiotics. METHODS: Small RNA sequencing was conducted on samples collected 3 months postpartum from 54 women participating in the ProPACT trial. Differential expression of miRNA was assessed for the probiotic vs placebo and AD vs non-AD groups. The results were further analysed using functional prediction techniques. RESULTS: Human breast milk samples contain a relatively stable core group of highly expressed miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-3p. Functional analysis of these miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Although several miRNAs were found to be differentially expressed on comparison of the probiotic vs placebo and AD vs non-AD groups, none had an acceptable false discovery rate and their biological significance in the development of AD is not immediately apparent from their predicted functional consequences. CONCLUSION: Whilst breast milk miRNAs have the potential to be active in a diverse range of tissues and biological process, individual miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingestion in the perinatal period. TRIAL REGISTRATION: ClinicalTrials.gov NCT00159523.

Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing.

Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Calpha and Cbeta are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.

Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus.

We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.