Effector T cell chemokine IP-10 predicts cardiac recovery and clinical outcomes post-myocardial infarction.

Background and aims: Preclinical data suggest that activation of the adaptive immune system is critical for myocardial repair processes in acute myocardial infarction. The aim of the present study was to determine the clinical value of baseline effector T cell chemokine IP-10 blood levels in the acute phase of ST-segment elevation myocardial infarction (STEMI) for the prediction of the left ventricular function changes and cardiovascular outcomes after STEMI. Methods: Serum IP-10 levels were retrospectively quantified in two independent cohorts of STEMI patients undergoing primary percutaneous coronary intervention. Results: We report a biphasic response of the effector T cell trafficking chemokine IP-10 characterized by an initial increase of its serum levels in the acute phase of STEMI followed by a rapid reduction at 90min post reperfusion. Patients at the highest IP-10 tertile presented also with more CD4 effector memory T cells (CD4 TEM cells), but not other T cell subtypes, in blood. In the Newcastle cohort (n=47), patients in the highest IP-10 tertile or CD4 TEM cells at admission exhibited an improved cardiac systolic function 12 weeks after STEMI compared to patients in the lowest IP-10 tertile. In the Heidelberg cohort (n=331), STEMI patients were followed for a median of 540 days for major adverse cardiovascular events (MACE). Patients presenting with higher serum IP-10 levels at admission had a lower risk for MACE after adjustment for traditional risk factors, CRP and high-sensitivity troponin-T levels (highest vs. rest quarters: HR [95% CI]=0.420 [0.218-0.808]). Conclusion: Increased serum levels of IP-10 in the acute phase of STEMI predict a better recovery in cardiac systolic function and less adverse events in patients after STEMI.

Iron regulatory protein (IRP)-mediated iron homeostasis is critical for neutrophil development and differentiation in the bone marrow.

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. However, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory protein 1 (IRP1) and IRP2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

The S-phase-induced lncRNA SUNO1 promotes cell proliferation by controlling YAP1/Hippo signaling pathway.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Targeting LINC00673 expression triggers cellular senescence in lung cancer.

Aberrant expression of noncoding RNAs plays a critical role during tumorigenesis. To uncover novel functions of long non-coding RNA (lncRNA) in lung adenocarcinoma, we used a microarray-based screen identifying LINC00673 with elevated expression in matched tumor versus normal tissue. We report that loss of LINC00673 is sufficient to trigger cellular senescence, a tumor suppressive mechanism associated with permanent cell cycle arrest, both in lung cancer and normal cells in a p53-dependent manner. LINC00673-depleted cells fail to efficiently transit from G1- to S-phase. Using a quantitative proteomics approach, we confirm the modulation of senescence-associated genes as a result of LINC00673 knockdown. In addition, we uncover that depletion of p53 in normal and tumor cells is sufficient to overcome LINC00673-mediated cell cycle arrest and cellular senescence. Furthermore, we report that overexpression of LINC00673 reduces p53 translation and contributes to the bypass of Ras-induced senescence. In summary, our findings highlight LINC00673 as a crucial regulator of proliferation and cellular senescence in lung cancer.

MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs.

Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.

The cancer-associated microprotein CASIMO1 controls cell proliferation and interacts with squalene epoxidase modulating lipid droplet formation.

Breast cancer is a leading cause of cancer-related death in women. Small open reading frame (sORF)-encoded proteins or microproteins constitute a new class of molecules often transcribed from presumed long non-coding RNA transcripts (lncRNAs). The translation of some of these sORFs has been confirmed, but their cellular function and importance remains largely unknown. Here, we report the identification and characterization of a novel microprotein of 10 kDa, which we named Cancer-Associated Small Integral Membrane Open reading frame 1 (CASIMO1). CASIMO1 RNA is overexpressed predominantly in hormone receptor-positive breast tumors. Its knockdown leads to decreased proliferation in multiple breast cancer cell lines. Its loss disturbs the organization of the actin cytoskeleton, leads to inhibition of cell motility, and causes a G0/G1 cell cycle arrest. The proliferation phenotype upon overexpression is observed only with CASIMO1 protein expression, but not with a non-translatable mutant attributing the effects to the sORF-derived protein rather than a lncRNA function. CASIMO1 microprotein interacts with squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis and a known oncogene in breast cancer. Overexpression of CASIMO1 leads to SQLE protein accumulation without affecting its RNA levels and increased lipid droplet clustering, while knockdown of CASIMO1 decreased SQLE protein abundance and ERK phosphorylation downstream of SQLE. Importantly, SQLE knockdown mimicked the CASIMO1 knockdown phenotype and in turn SQLE overexpression fully rescued the effect of CASIMO1 knockdown. These findings establish CASIMO1 as the first functional microprotein that plays a role in carcinogenesis and is implicated in the cell lipid homeostasis.

The Long Noncoding RNA Cancer Susceptibility 9 and RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein L Form a Complex and Coregulate Genes Linked to AKT Signaling.

The identification of viability-associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability-associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up-regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)-mediated and siRNA pool-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9-depleted and HNRNPL-depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient-derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.

A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression.

Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage. Our analysis revealed two pairs of mRNA-lncRNA sense-antisense transcripts being inversely expressed upon DNA damage. The lncRNA NOP14-AS1 was strongly upregulated upon DNA damage, while the mRNA for NOP14 was downregulated, both in a p53-dependent manner. For another pair, the lncRNA LIPE-AS1 was downregulated, while its antisense mRNA CEACAM1 was upregulated. To test whether as expected the antisense genes would regulate each other resulting in this highly significant inverse correlation, we employed antisense oligonucleotides and RNAi to study transcript-dependent effects as well as dCas9-based transcriptional modulation by CRISPRi/CRISPRa for transcription-dependent effects. Surprisingly, despite the strong stimulus-dependent inverse correlation, our data indicate that neither transcript- nor transcription-dependent mechanisms explain the inverse regulation of NOP14-AS1:NOP14 or LIPE-AS1:CEACAM1 expression. Hence, sense-antisense pairs whose expression is strongly-positively or negatively-correlated can be nonetheless regulated independently. This highlights the requirement of individual experimental studies for each antisense pair and prohibits drawing conclusions on regulatory mechanisms from expression correlations.

The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells.

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.

The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance.

Little is known about the function of most non-coding RNAs (ncRNAs). The majority of long ncRNAs (lncRNAs) is expressed at very low levels and it is a matter of intense debate whether these can be of functional relevance. Here, we identified lncRNAs regulating the viability of lung cancer cells in a high-throughput RNA interference screen. Based on our previous expression profiling, we designed an siRNA library targeting 638 lncRNAs upregulated in human cancer. In a functional siRNA screen analyzing the viability of lung cancer cells, the most prominent hit was a novel lncRNA which we called Viability Enhancing LUng Cancer Transcript (VELUCT). In silico analyses confirmed the non-coding properties of the transcript. Surprisingly, VELUCT was below the detection limit in total RNA from NCI-H460 cells by RT-qPCR as well as RNA-Seq, but was robustly detected in the chromatin-associated RNA fraction. It is an extremely low abundant lncRNA with an RNA copy number of less than one copy per cell. Blocking transcription with actinomycin D revealed that VELUCT RNA was highly unstable which may partially explain its low steady-state concentration. Despite its extremely low abundance, loss-of-function of VELUCT with three independent experimental approaches in three different lung cancer cell lines led to a significant reduction of cell viability: Next to four individual siRNAs, also two complex siPOOLs as well as two antisense oligonucleotides confirmed the strong and specific phenotype. In summary, the extremely low abundant lncRNA VELUCT is essential for regulation of cell viability in several lung cancer cell lines. Hence, VELUCT is the first example for a lncRNA that is expressed at a very low level, but has a strong loss-of-function phenotype. Thus, our study proves that at least individual low-abundant lncRNAs can play an important functional role.

LIMT is a novel metastasis inhibiting lncRNA suppressed by EGF and downregulated in aggressive breast cancer.

Long noncoding RNAs (lncRNAs) are emerging as regulators of gene expression in pathogenesis, including cancer. Recently, lncRNAs have been implicated in progression of specific subtypes of breast cancer. One aggressive, basal-like subtype associates with increased EGFR signaling, while another, the HER2-enriched subtype, engages a kin of EGFR Based on the premise that EGFR-regulated lncRNAs might control the aggressiveness of basal-like tumors, we identified multiple EGFR-inducible lncRNAs in basal-like normal cells and overlaid them with the transcriptomes of over 3,000 breast cancer patients. This led to the identification of 11 prognostic lncRNAs. Functional analyses of this group uncovered LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT), a highly conserved lncRNA, which is depleted in basal-like and in HER2-positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. We also find that LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo In conclusion, lncRNAs dynamically regulated by growth factors might act as novel drivers of cancer progression and serve as prognostic biomarkers.

Rare Drosha splice variants are deficient in microRNA processing but do not affect general microRNA expression in cancer cells.

Drosha is a key enzyme in microRNA biogenesis, generating the precursor miRNA (pre-miRNA) by excising the stem-loop embedded in the primary transcripts (pri-miRNA). The specificity for the pri-miRNAs and determination of the cleavage site are provided by its binding partner DGCR8, which is necessary for efficient processing. The crucial Drosha domains for pri-miRNA cleavage are the middle part, the two enzymatic RNase III domains (RIIID), and the dsRNA binding domain (dsRBD) in the C-terminus. Here, we identify alternatively spliced transcripts in human melanoma and NT2 cell lines, encoding C-terminally truncated Drosha proteins lacking part of the RIIIDb and the entire dsRBD. Proteins generated from these alternative splice variants fail to bind to DGCR8 but still interact with Ewing sarcoma protein (EWS). In vitro as well as in vivo, the Drosha splice variants are deficient in pri-miRNA processing. However, the aberrant transcripts in melanoma cells do not consistently reduce mature miRNA levels compared with melanoma cell lines lacking those splice variants, possibly owing to their limited abundance. Our findings show that alternative processing-deficient Drosha splice variants exist in melanoma cells. In elevated amounts, these alternatively spliced transcripts could provide one potential mechanism accounting for the deregulation of miRNAs in cancer cells. On the basis of our results, the search for alternative inactive splice variants might be fruitful in different tumor entities to unravel the molecular basis of the previously observed decreased microRNA processing efficiency in cancer.

Transient cyclical methylation of promoter DNA.

Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor alpha (ERalpha) promoters could be partially methylated by treatment with deacetylase inhibitors, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERalpha and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERalpha and of pS2 transcripts.

Thanatop: a novel 5-nitrofuran that is a highly active, cell-permeable inhibitor of topoisomerase II.

A series of nitrofuran-based compounds were identified as inhibitors of estrogen signaling in a cell-based, high-throughput screen of a diverse library of small molecules. These highly related compounds were subsequently found to inhibit topoisomerase II in vitro at concentrations similar to that required for the inhibition of estrogen signaling in cells. The most potent nitrofuran discovered is approximately 10-fold more active than etoposide phosphate, a topoisomerase II inhibitor in clinical use. The nitrofurans also inhibit topoisomerase I activity, with approximately 20-fold less activity. Moreover, the nitrofurans, in contrast to etoposide, induce a profound cell cycle arrest in the G(0)-G(1) phase of the cell cycle, do not induce double-stranded DNA breaks, are not substrates for multidrug resistance protein-1 export from the cell, and are amenable to synthetic development. In addition, the nitrofurans synergize with etoposide phosphate in cell killing. Clonogenic assays done on a panel of human tumors maintained ex vivo in nude mice show that the most active compound identified in the screen is selective against tumors compared with normal hematopoietic stem cells. However, this compound had only moderate activity in a mouse xenograft model. This novel class of topoisomerase II inhibitor may provide additional chemotherapeutic strategies for the development of cytotoxic agents with proven clinical utility.

Relationships and distinctions in iron-regulatory networks responding to interrelated signals.

Specialized cDNA-based microarrays (IronChips) were developed to investigate complex physiological gene-regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (eg, oxidative stress, nitric oxide [NO] metabolism, or copper metabolism), and were immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H(2)O(2)) or NO (sodium nitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene-response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signal-specific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for iron-regulatory protein (IRP) and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron-loaded state in both cases, and the discrimination between the sources of iron loading. These findings also demonstrate the broad utility of gene-expression profiling with the IronChip to study iron metabolism and related human diseases.